WO1997040058A1 - Flea protease proteins, nucleic acid molecules, and uses thereof - Google Patents

Flea protease proteins, nucleic acid molecules, and uses thereof Download PDF

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Publication number
WO1997040058A1
WO1997040058A1 PCT/US1997/006121 US9706121W WO9740058A1 WO 1997040058 A1 WO1997040058 A1 WO 1997040058A1 US 9706121 W US9706121 W US 9706121W WO 9740058 A1 WO9740058 A1 WO 9740058A1
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WIPO (PCT)
Prior art keywords
seq
nucleic acid
flea
acid molecule
protease
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Application number
PCT/US1997/006121
Other languages
French (fr)
Inventor
Robert B. Grieve
Keith E. Rushlow
Shirley Wu Hunter
Glenn R. Frank
Gary L. Steigler
Patrick J. Gaines
Gary Silver
Original Assignee
Heska Corporation
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Publication date
Priority claimed from US08/639,075 external-priority patent/US6150125A/en
Priority claimed from US08/749,699 external-priority patent/US6210920B1/en
Application filed by Heska Corporation filed Critical Heska Corporation
Priority to AU28015/97A priority Critical patent/AU735717B2/en
Priority to EP97922303A priority patent/EP0900231A1/en
Priority to JP53813497A priority patent/JP2001510324A/en
Publication of WO1997040058A1 publication Critical patent/WO1997040058A1/en
Priority to US09/032,215 priority patent/US6204010B1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6408—Serine endopeptidases (3.4.21)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/641—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution

Definitions

  • the present invention relates to novel flea protease proteins and their use to reduce flea infestation of animals.
  • the present invention also relates to the use of anti-flea protease antibodies and other compounds that reduce flea protease activity to reduce flea infestation of animals .
  • Fleas which belong to the insect order Siphonaptera, are obligate ectoparasites for a wide variety of animals, including birds and mammals. Flea infestation of animals is of health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas cause and/or carry infectious agents that cause, for example, flea allergy dermatitis, anemia, murine typhus, plague and tapeworm. In addition, fleas are a problem for animals maintained as pets because the infestation becomes a source of annoyance for the pet owner who may find his or her home generally contaminated with fleas which feed on the pets. As such, fleas are a problem not only when they are on an animal but also when they are m the general environment of the animal.
  • the present invention relates to flea serine protease proteins, to flea ammopeptidase proteins, and to flea cysteine protease proteins; to flea serine protease, ammopeptidase and/or cysteine protease nucleic acid molecules, including those that encode such proteins; to antibodies raised agamst such proteins; and to compounds that inhibit flea serine protease, ammopeptidase and/or cysteine protease activities.
  • the present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included m the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.
  • One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene including a serine protease gene comprising a nucleic acid sequence including a nucleic acid molecule including SEQ ID NO: 9, SEQ ID NO-11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID N0:15, SEQ ID NO:17, SEQ ID N0:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID N0:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO-
  • the present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID.
  • a preferred nucleic acid sequence of the present invention includes a nucleic acid molecule comprising a nucleic acid sequence including SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28> SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO:131, SEQ ID
  • the present invention also includes an isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO
  • the present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention.
  • nucleic acid molecules recombinant molecules
  • recombinant viruses recombinant cells
  • Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation.
  • a therapeutic composition includes an excipient and a protective compound including: an isolated protein or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:
  • Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea protease activity, the method comprising: (a) contacting an isolated flea protease protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID N0:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO-.157, SEQ ID NO:161, SEQ ID
  • the present mvention also includes an isolated flea protease protem that cleaves an immunoglobulin, when the protem is incubated in the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C.
  • a preferred protease protem capable of cleaving immunoglbulm comprises an ammo acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO: 96.
  • Another embodiment of the present invention includes a method to identify a compound capable of inhibiting flea immunogloDulm proteinase protem activity, the method comprising: (a) contacting an isolated flea immunoglobulin proteinase protem with a putative inhibitory compound under conditions in which, m the absence of the compound, the protem has immunoglobulin proteinase activity; and (b) determining if the putative inhibitory compound inhibits the activity.
  • Fig. 1 is a scanned image depicting SDS-PAGE of DFP- labeled larval proteases m unfed larvae, fed 1st instar larvae and fed 3rd instar larvae.
  • the present invention includes the use of compounds that inhibit flea protease activity to protect a host animal from flea infestation.
  • proteases are significant components of the flea midgut and are good targets for lmmunotherapeutic and/or chemotherapeutic intervention to reduce flea burden ooth on the host animal and m the immediate (i.e., surrounding) environment of the animal.
  • the inventors have snown, for example, that the viability and/or fecundity of fleas consuming a blood meal is reduced when the blood meal contains compounds that reduce flea protease activity, probably because the compounds interfere with flea digestion and other functions.
  • Such compounds include flea protease vaccines, anti-flea protease antibodies, flea protease inhibitors, and/or compounds that suppress protease synthesis; such compounds are discussed in more detail below.
  • One embodiment of the present invention is a method to protect a host animal from flea infestation by treating the animal with a composition that includes a compound that reduces the protease activity of fleas feeding (includes fleas in the process of feeding as well as fleas having fed) from the treated animal thereby reducing the flea burden on the animal and in the environment of the animal.
  • composition of the present invention can include one or more compounds that target (reduced the activity of) one or more proteases in the flea.
  • the phrase "to protect an animal from flea infestation” refers to reducing the potential for flea population expansion on and around the animal (i.e., reducing the flea burden) .
  • the flea population size is decreased, optimally to an extent that the animal is no longer bothered by fleas.
  • a host animal as used herein, is an animal from which fleas can feed by attaching to and feeding through the skin of the animal. Fleas, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal m order to feed.
  • a certain percentage of a flea population can be on a host animal whereas the remainder can be in the environment surrounding the animal (i.e., in the environment of the animal) .
  • Such an environment can include not only adult fleas, but also flea eggs and/or flea larvae.
  • the environment can be of any size such that fleas in the environment are able to " jump onto and off of a host animal. As such, it is desirable not only to reduce the flea burden on an animal per se, but also to reduce the flea burden in the environment surrounding the animal.
  • a host animal is treated by administering to the animal a compound of the present invention in such a manner that the compound itself (e.g., a protease inhibitor, protease synthesis suppressor or anti-flea protease antibody) or a product generated by the animal in response to administration of the compound (e.g., antibodies produced in response to a flea protease vaccine, or conversion of an inactive inhibitor "prodrug" to an active protease inhibitor) ultimately enters the flea midgut.
  • An animal is preferably treated in such a way that the compound or product thereof enters the blood stream of the animal. Fleas are then exposed ro the compound when they feed from the animal.
  • flea protease inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas.
  • the treated animal mounts an immune response resulting in the production of antibodies agamst the protease (anti-flea protease antibodies) which circulate m the animal's blood stream and are taken up by fleas upon feeding.
  • the present invention also includes the ability to reduce larval flea infestation in that when fleas feed from a host animal that has been administered a therapeutic composition of the present mvention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the flea are excreted by the flea m feces, which is subsequently ingested by flea larvae. It is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
  • reducing proteolytic activity in flea midguts can lead to a number of outcomes that reduce flea burden on treated animals and their surrounding environments.
  • outcomes include, but are not limited to, (a) reducing the viability of fleas that feed from the treated animal, (b) reducing the fecundity of female fleas that feed from the treated animal, (c) reducing the reproductive capacity of male fleas that feed from the treated animal, (d) reducing the viability of eggs laid by female fleas that feed from the treated animal, (e) altering the blood feeding behavior of fleas that feed from the treated animal (e.g., fleas take up less volume per feeding or feed less frequently) , (f) reducing the viability of flea larvae, for example due to the feeding of larvae from feces of fleas that feed from the treated animal and/or (g) altering the development of flea larvae (e.g., by decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slow
  • One embodiment of the present invention is a composition that includes one or more compounds that reduce the activity of one or more flea proteases directly (e.g., an anti-flea protease antibody or a flea protease inhibitor) and/or indirectly (e.g., a flea protease vaccine) .
  • Suitable flea proteases to target include flea ammopeptidases, flea carboxypeptidases and/or flea endopeptidases.
  • Such proteases can include cytosolic and/or membrane-bound forms of a protease.
  • Preferred flea proteases to target include, but are not limited to, serine proteases, metalloproteases, aspartic acid proteases and/or cysteine proteases.
  • proteases include ammopeptidases, carboxypeptidases and/or endopeptidases.
  • Preferred flea proteases to target m include, but are not limited to, proteases that degrade hemoglobin, proteases mvolved m blood coagulation and/or lytic (anti-coagulation) pathways, proteases involved in the maturation of peptide hormones, proteases that inhibit complement or other host immune response elements (e.g., antibodies) and/or proteases mvolved in vitellogenesis.
  • proteases are known to those skilled in the art, including, but not limited to, ammopeptidases, such as leucine ammopeptidase and ammopeptidases B and M; astacin-like metalloproteases; calpams; carboxypeptidases, such as carboxypeptidases A, P and Y; cathepsms, such as cathepsms B, D, E, G, H, and L, chymotrypsms; cruzipams; meprins; papams; pepsins; reruns; thermolysms and trypsms.
  • ammopeptidases such as leucine ammopeptidase and ammopeptidases B and M
  • astacin-like metalloproteases calpams
  • carboxypeptidases such as carboxypeptidases A, P and Y
  • cathepsms such as cathepsms B, D, E, G, H, and L
  • a particularly preferred protease to target is a protease having a proteolytic activity that, when targeted with a composition of the present mvention, reduces flea burden without substantially harming the host animal.
  • a protease can be identified usmg, for example, methods as disclosed herem.
  • One aspect of the present invention is the discovery that a substantial amount of the proteolytic activity found in flea midguts is serine protease activity. Both in vi tro and in vi vo studies usmg a number of protease inhibitors substantiate this discovery, details of which are disclosed m the Examples.
  • a particularly preferred protease to target is a serine protease.
  • serine proteases include, but are not limited to, acrosms, bromelams, cathepsm G, chymotrypsins, coUagenases, elastases, factor Xa, ficms, kallikreins, papains, plasmins, Staphylococcal V8 proteases, thrombms and trypsins.
  • a preferred flea serine protease to target includes a protease having trypsin-like or chymotrypsin-like activity.
  • an enzyme having "like” proteolytic activity has similar activity to the referenced protease, although the exact structure of the preferred substrate cleaved may differ.
  • "Like” proteases usually have similar tertiary structures as their referenced counterparts.
  • Protease inhibitor studies disclosed in the Examples section also indicate that additional preferred proteases to target include aminopeptidases and/or metalloproteases. Examples of such proteases mclude exo- and endo- metalloproteases, digestive enzymes, and enzymes mvolved in peptide hormone maturation.
  • an ammopeptidase that is also a metalloprotease is leucine ammopept-dase .
  • Suitable compounds to include in compositions of the present mvention include, but are not limited to, a vaccine comprising a flea protease (a flea protease vaccine) , an antibody that selectively binds to a flea protease (an anti-flea protease antibody) , a flea protease inhibitor (a compound other than a vaccine or an antibody that inhibits a flea protease) , and a mixture of such compounds.
  • a mixture thereof refers to a combination of one or more of the cited entities.
  • Compositions of the present mvention can also include compounds to suppre-ss protease synthesis or maturation, such as, but not limited to, protease modulating peptides.
  • a preferred embodiment of the present invention is a flea protease vaccine and its use to reduce the flea population on and around an animal.
  • a flea protease vaccine can include one or more proteins capable of eliciting an immune response agamst a flea protease and can also include other components.
  • Preferred flea protease vaccines mclude a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease, with flea serine protease, flea metalloprotease and/or flea ammopeptidase vaccines being more preferred.
  • Examples of flea protease vaccines mclude soluble flea midgut preparations of the present invention as well as one or more isolated proteins of the present invention.
  • One embodiment of the present invention is a soluble flea midgut preparation.
  • a preparation mcludes primarily components naturally present m the lumen of a flea midgut and, depending on the method of preparation, can also include one or more peripheral midgut membrane proteins. Methods to preferentially include, or exclude, membrane proteins from such a preparation are known to those skilled m the art.
  • the present mvention mcludes the discovery that such a preparation has proteolytic activity, of which a substantial portion is serine protease activity.
  • At least about 70 percent of the proteolytic activity in a soluble flea midgut soluble preparation is serine protease activity, as can be indicated by the ability to inhibit at least about 70 percent of the proteolytic activity with 4-2-ammoethyl- benzenesulfonylfluo ⁇ de-hydrochloride (AEBSF) .
  • Serine protease activity can also be identified using other known inhibitors or substrates.
  • Other preferred inhibitors that can inhibit at least about 70 percent of the proteolytic activity of a soluble flea midgut preparation of the present invention include soybean trypsin inhibitor, 1,3- dnsopropylfluoro-phosphate or leupeptin.
  • a soluble flea midgut preparation of the present invention includes proteases that range m molecular weight from about 5 kilodaltons (kD or kDa) to about 20 ⁇ KD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a substantial portion of the serine proteases ranging m molecular weight from about 5 kD to about 60 kD, as determined by SDS-PAGE.
  • proteases that range m molecular weight from about 5 kilodaltons (kD or kDa) to about 20 ⁇ KD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a substantial portion of the serine proteases ranging m molecular weight from about 5 kD to about 60 kD, as determined by SDS-PAGE.
  • a substantial portion of protease activity in a soluble flea midgut preparation of the present invention has a pH activity optimum ranging from about pH 5 to about pH 10, preferably an activity optimum ranging from about pH 7 to about pH 9, and even more preferably an activity optimum of about pH 8. While not being bound by theory, such a pH optimum suggests that a large proportion of proteases in soluble flea midgut preparations of the present invention are serine proteases. It is also interesting to note that the pH of the flea midgut is also about pH 8.
  • proteases m soluble flea midgut preparations of the present invention exnibit a varied pattern of inhibition by protease inhibitors of a given type (e.g., serine protease inhibitors), as well as variances seen m molecular weights and pH optima of the proteases, suggest that there are a number of protease isoforms in such preparations.
  • protease inhibitors of a given type e.g., serine protease inhibitors
  • a soluble flea midgut preparation of the present mvention is preferably prepared by a method that mcludes tne steps of (a) disrupting a flea midgut to produce a mixture including a liquid portion and a solid portion and (b; recovering the liquid portion to obtain a soluble flea midgut preparation.
  • Such a method is a simplified version of methods disclosed m U.S. Patent No. 5,356,622, ibi d . It is to be noted that in accordance with the present mvention, methods disclosed m U.S. Patent No. 5,356,622, ibi d, can also be used to prepare soluble flea midgut preparations having similar proteolytic activities.
  • Flea midguts can be obtained (e.g., dissected from) from unfed fleas or from fleas that recently consumed a blood meal (i.e., blood-fed fleas) . Such midguts are referred to herein as, respectively, unfed flea midguts and fed flea midguts. Flea midguts can be obtained from either male or female fleas. As demonstrated in the Examples section, female flea midguts exhibit somewhat more proteolytic activity than do male flea midguts. Furthermore, fed flea midguts have significantly more proteolytic activity than do unfed flea midguts.
  • m flea midguts the synthesis and/or activation of proteases as well as other factors (e.g., enzymes, other proteins, co-factors, etc.) important m digesting the blood meal, as well as in neutralizing host molecules potentially damaging to the flea (e.g., complement, immunoglobulins, blood coagulation factors) .
  • unfed flea midguts may contain significant targets not found m fed flea midguts and vice versa.
  • the present application focuses primarily on flea midgut proteases, it is to be noted that the present invention also includes other components of soluble flea midgut preparations of the present mvention that provide suitable targets to reduce flea burden on an animal and in the environment of that animal; see also U.S. Patent No. 5,356,622, ibid.
  • Methods to disrupt flea midguts in order to obtain a soluble flea midgut preparation are known to those skilled in the art and can be selected according to, for example, the volume being processed and the buffers being used.
  • Such methods include any technique that promotes cell lysis, such as, but are not limited to, chemical disruption techniques (e.g., exposure of midguts to a detergent) as well as mechanical disruption techniques (e.g., homogenization, sonication, use of a tissue blender or glass beads, and freeze/thaw techniques) .
  • Methods to recover a soluble flea midgut preparation are also known to those skilled in the art and can include any method by which the liquid portion of disrupted flea midguts is separated from the solid portion (e.g., filtration or centrifugation) .
  • disrupted flea midguts are subjected to centrifugation, preferably at an acceleration ranging from about 10,000 x g to about 15,000 x g for several minutes (e.g., from about 1 min. to about 15 min.) .
  • the supernatant from such a centrifugation comprises a soluble flea midgut preparation of tne present mvention.
  • the present invention also mcludes an isolated protem that includes an ammo acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions (i.e., that hybridize under stringent nybridization conditions) with a nucleic acid molecule that encodes a protease present (i.e., the nucleic acid molecules hybridize with the nucleic acid strand that is complementary to the coding strand) m (i.e., can be found m) a flea midgut, such as a midgut from a blood-fed female flea, a midgut from a blood-fed male flea, a midgut from an unfed female flea or a midgut from an unfed male flea.
  • a preferred midgut protease is present m the lumen of the midgut.
  • an isolated protem of the present invention also referred to herein as an isolated protease protem, preferably is capable of eliciting an immune response against a flea midgut protease and/or has proteolytic activity.
  • an isolated, or biologically pure, protem is a protem that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the protem has been purified.
  • An isolated protease protem can be obtained from its natural source. Such an isolated protem can also be produced usmg recombinant DNA technology or chemical synthesis.
  • an isolated protem of the present mvention can be a full-length protem or any homologue of such a protem, such as a protem m which ammo acids have been deleted (e.g., a truncated version of the protem, such as a peptide) , inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl mositol) such that the homologue comprises a protem having an ammo acid sequence that is sufficiently similar to a natural flea midgut protease that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) the complement of a nucleic acid sequence encoding the corresponding natural flea midgut protease ammo acid sequence.
  • stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cl oning: A Labora tory Manual , Cold Spring Harbor Labs Press, 1989; Sambrook et al. , ibid. , is incorporated by reference herem m its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules havmg at least about 70 nucleic acid sequence identity v.ith the nucleic acid molecule being used to probe m the hyr>nd-.zat ⁇ or ⁇ reaction.
  • the minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protem.
  • the size of tne nucleic acid molecule encoding such a protem homologue is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) .
  • the minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to aoout 17 bases m length if they are AT-rich.
  • the minimal size of a nucleic acid molecule used to encode a protease protem homologue of the present invention is from aoout 12 to about 18 nucleotides in length.
  • nc limit other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof.
  • the minimal size of a protease protem homologue of the present invention is from about 4 to about 6 ammo acids in length, with preferred sizes depending on whether a full-length, multivalent (i.e., fusion protem having more than one domain each of which has a function) , or functional portions of such proteins are desired.
  • Protease protem homologues of the present mvention preferably have protease activity and/or are capable of eliciting an immune response agamst a flea midgut protease.
  • a protease protem homologue of the present invention can be the result of allelic variation of a natural gene encoding a flea protease.
  • a natural gene refers to the form of the gene found most often in nature.
  • Protease protein homologues can be produced using techniques known m the art including, but not limited to, direct modifications to a gene encoding a protein usmg, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
  • Isolated protease proteins of the present invention, including homologues can be identified m a straight-forward manner by the proteins' ability to effect proteolytic activity and/or to elicit an immune response against a flea midgut protease. Such techniques are known to those skilled in the art.
  • a preferred protease protein of the present invention is a flea serine protease, a flea metalloprotease, a flea aspartic acid protease, a flea cysteine protease, or a homologue of any of these proteases.
  • a more preferred protease protein is a flea serine protease, a flea metalloprotease or a homologue of either.
  • a flea cysteine protease or a homologue thereof Particularly preferred is a flea serine protease or a homologue thereof.
  • Preferred protease proteins of the present invention are flea protease proteins having molecular weights ranging from about 5 kD to about 200 kD, as determined by SDS-PAGE, and homologues of such proteins. More preferred are flea protease proteins having molecular weights ranging from about 5 kD to about 60 kD, as determined by SDS-PAGE, and homologues of such proteins.
  • flea serine protease proteins particularly those having molecular weights of about 26 kD (denoted PfSP26, now denoted PafSP-26K to distinguish from flea PfSP26 as described in Example 26) , about 24 kD (denoted PfSP24, now denoted PafSP-24K to distinguish from flea PfSP24 as described in Example 27), about 19 kD (denoted PfSP19, now denoted PafSP-19K to distinguish from flea PfSP19 as described m Example 32), about 6 kD (denoted PfSPo, now denoted PafSP-6K to distinguish from flea PfSP6 as described m Example 11), about 31 kD (denoted PfSP28), about 25 kD (denoted PlfSP-25Kl) from 1st instar larvae, about 25 kD (denoted PlfSP-25K3) from 3rd instar larvae, about 28 kD (denoted PlfSP
  • One preferred embodiment of the present invention is an isolated flea protease protem that mcludes an ammo acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene, with a flea ammopeptidase gene or with a flea cysteine protease gene.
  • a flea protease gene includes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
  • the inventors have discovered an extensive family of serine proteases, encoded by a family of serine protease genes. Such a gene family may be due to allelic variants (i.e., genes having similar, but different, sequences at a given locus in a population of fleas) and/or to, the existence of serine protease genes at more than one locus m the flea genome.
  • the present invention includes flea serine protease genes comprising not only the nucleic acid sequences disclosed herein (e.g., genes including nucleic acid sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121,
  • a preferred flea ammopeptidase gene mcludes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID NO: 112, which encode ammopeptidase proteins having ammo acid sequences including SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113. Additional preferred ammopeptidase genes include allelic variants of SEQ ID NO:110 and/or SEQ ID NO: 112.
  • a preferred flea cysteine protease gene includes nucleic acid sequence SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94, which encode a cysteine protease protem having ammo acid sequences including SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:77, and/or SEQ ID NO: 95.
  • cysteine protease genes include allelic variants of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94.
  • a preferred flea serine protease protem of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the followmg nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPl ⁇ , nfSP24, nfSP28, nfSP32, nfSP33 and nfSP40.
  • each of these nucleic acid molecules represent the entire coding region of a flea serine protease gene of the present invention (at least portions of which are also referred to by flea clone numbers, as described m the Examples) .
  • Nucleic acid molecules that contain partial coding regions or other parts of the corresponding gene are denoted by names that include the size of those nucleic acid molecules (e.g., nfSP40 42b ) .
  • Nucleic acid molecules containing apparent full length coding regions for which the size is known also are denoted by names that include the size of tnose nucleic acid molecules (e.g., nfSP40 841 ) .
  • the production, and at least partial nucleic acid sequence, of such nucleic acid molecules is disclosed in the Examples.
  • serine protease proteins are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18 B34 , nfSP18 77W nfSP18 225 , nfSP24 410 , nfSP24 1089 , nfSP24 774 , nfSP24 711 , nfSP28 P2 ,, nfSP32 qj3 , nfSP32 y33 , nfSP32 924 , nfSP32 699 , nfSP33 42b , nfSP33 71(( , nfSP33 1894 , nfSP33 1?00 , nfSP33 726 , nfSP40 841, nfSP5 806 , nfSPll 307 , nfSP8 sl5 , nfSP8 430, nfSP12 7
  • serine protease proteins include the following amino acid sequences: SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID
  • serine protease proteins are encoded by allelic variants of nucleic acid molecules encoding proteins that include the c_.ted ammo acid sequences. Also preferred are flea serine protease proteins including regions that have at least about 50 * :, preferably at least about 75%, and more preferably at least about 90% identity with flea serine protease proteins having ammo acid sequences as cited herem.
  • One embodiment of the present mvention is a flea serine protease that degrades immunoglobulin circulating m a host animal (i.e.-, flea immunoglobulin proteinase or IgGase) .
  • An example of a flea immunoglobulin proteinase is presented in the Examples section.
  • an immunoglobulin proteinase of the present mvention cleaves an immunoglobulin when the protem is incubated m the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C.
  • an immunoglobulin proteinase of the present mvention cleaves an immunoglobulin m about 300 microliters of 50 mM Tris-HCl, pH 8.0, for about 1 hour at about 37 C C.
  • Suitable immunoglobulin proteinase proteins of the present invention are capable of cleaving the hinge region of an immunoglobulin heavy chain.
  • the hinge region of an immunoglobulin is the flexible domain that " joins the Fab arms of the immunoglobulin to the Fc portion of the molecule.
  • a more preferred immunoglobulin proteinase protem m cludes a protem havmg a molecular weight ranging from about 25 kD to about 35 kD and more preferably having a molecular weight of about 31 kD, in its mature form.
  • An even more preferred immunoglobulin proteinase protem includes a protem comprising an ammo acid sequence including SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73 and/or SEQ ID NO: 96, which can be encoded by a gene comprising nucleic acid sequence SEQ ID NO: 66.
  • the proteinase activity of an immunoglobulin proteinase of the present invention cleaves an immunoglobulin m such a manner that the immunoglobulin maintains mtact heavy and light chain pairs, either as two Fab fragments or one Ffab') ⁇ fragment.
  • a Fab fragment refers to complete immunoglobulin light chains paired with the variable region and CHI domains of an immunoglobulin heavy chain.
  • a F(ab' ) fragment refers to two Fab fragments that remain linked by a disulfide bond. Both Fab and F(ab') 2 fragments are capable of binding antigen.
  • a preferred immunoglobulin proteinase protem of the present invention is capable of cleaving the hmge region of an immunoglobulin heavy chain at a site comprising an ammo acid sequence including D-C-P-K-C-P-P-P-E-M-L-G-G-P- S-I-F-I-F-P-P-K-P-K-D (SEQ ID NO: 104) , D-C-P-K-C-P-P-P-E-M- L-G-G-P-S-I-F-I-F-P-P-K-D-D-L-L-I-K-R-K-S-E-V (SEQ ID NO: 105) and/or D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K- P-K-D-T-L-S-I-S-R-T-P-
  • a preferred flea ammopeptidase protein of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP and/or nfAP2 (flea ammopeptidase full-length coding regions of a flea ammopeptidase gene of the present mvention) .
  • aminopeptidase proteins are encoded by a nucleic acid rrtolcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP 453 , nfAP 900r nfAP 73 ,, nfAP :580 , nfAP2 38 , and/or nfAP2 537. More preferred is an aminopeptidase protein encoded by a nucleic acid molcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP2 383 and/or nfAP2 537 .
  • aminopeptidase protein that includes amino acid sequence SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113, or an ammopeptidase protein encoded by an allelic variant of a nucleic acid molecule that includes SEQ ID NO:llC and/or SEQ ID NO: 112.
  • flea aminopeptidase proteins including regions that have at least about 50%, prefereably at least about 75%, and more preferably at least about 90% identity with flea ammopeptidase proteins having ammo acid sequences as cited herein.
  • a preferred flea cysteine protease protem of the present mvention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfCPl (a flea cysteine protease full-length coding region that includes nfCPl 57 , or nfCPl no (the production of which are described m the Examples) .
  • cysteine protease that includes ammo acid sequence SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 77, or a cysteine protease encoded by an allelic variant of a nucleic acid molecule that mcludes SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76, or SEQ ID NO: 94.
  • flea cysteine protease protem including regions that have at least about 50%, preferably at least about 75%, and more preferably at least about 90% identity with SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID N0:89, SEQ ID NO:92, SEQ ID NO:77, or SEQ ID NO: 95.
  • One embodiment of the present invention is an isolated protem having proteolytic activity that is substantially inhibited by a serine protease inhibitor, an ammopeptidase inhibitor and/or a cysteine protease inhibitor. Such inhibition can be measured by techniques known to those skilled m the art.
  • To be substantially inhibited means, for example, for a serine protease, that at least half of the proteolytic activity of the protease protem is inhibited by a serine protease inhibitor.
  • Preferred serine protease inhibitors include flea serpm proteins, and peptides or analogs thereof.
  • An isolated protem of the present invention can be produced m a variety of ways, including recovering such a protein from a flea midgut and producing such a protein recombinantly.
  • a flea midgut protease can be recovered by methods heretofore disclosed for obtaining a soluble flea midgut preparation.
  • a flea midgut protease protem can be further purified from a disrupted flea midgut by a number of techniques known to those skilled m the art, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis (e.g., standard, capillary and flow-through electrophoresis) , hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalm A chromatography, chromatofocusmg and differential solubilization.
  • a flea midgut protease is purified usmg protease inhibitor affinity chromatography, an example of which is disclosed m the Examples section.
  • Another embodiment of the present invention is a method to produce an isolated protem of the present invention using recombinant DNA technology.
  • Such a method includes the steps of (a) culturing a recombinant cell comprising a nucleic acid molecule encoding a protem of the present invention to produce the protem and (b) recovering the protem therefrom. Details on producing recombinant cells and culturing thereof are presented below.
  • the phrase "recovering the protem” refers simply to collecting the whole fermentation medium containing the protem and need not imply additional steps of separation or purification. Proteins of the present invention can be purified usmg a variety of standard protein purification techniques, as heretofore disclosed.
  • Isolated proteins of the present invention are preferably retrieved in "substantially pure” form.
  • substantially pure refers to a purity that allows for the effective use of the protem as a vaccine.
  • a vaccine for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating tne production of antibodies in a vaccinated animal.
  • Another embodiment of the present invention is an solated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a flea protease present in a flea midgut.
  • a nucleic acid molecule is also referred to herem as a flea protease nucleic acid molecule.
  • Particularly preferred is an isolated nucleic acid molecule that hybridizes under stringent conditions with a flea serine protease gene, with a flea aminopeptidase gene or with a flea cysteine protease gene. The characteristics of such genes are disclosed herein.
  • an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) .
  • isolated does not reflect the extent to which the nucleic acid molecule has been purified.
  • An isolated nucleic acid molecule can mclude DNA, RNA, or derivatives of either DNA or RNA.
  • a flea protease gene mcludes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post- translation control regions) as well as the coding region itself.
  • a nucleic acid molecule of the present invention can be an isolated natural flea protease nucleic acid molecule or a homologue thereof.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
  • the minimal size of a flea protease nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a correspondmg natural gene.
  • Flea protease nucleic acid molecules can also mclude a nucleic acid molecule encoding a hybrid protem, a fusion protem, a multivalent protem or a truncation fragment.
  • An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene.
  • the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity.
  • at least a portion of a nucleic acid sequence is an amount of a nucleic acid sequence capable of forming a stable hybrid with the corresponding gene under stringent hybridization conditions.
  • An isolated nucleic acid molecule of the present invention can also be produced usmg recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis.
  • Isolated flea protease nucleic acid molecules mclude natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a flea protease protem of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates.
  • a flea protease nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibi d. ) .
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • classic mutagenesis techniques and recombinant DNA techniques such as site-directed mutagenesis
  • chemical treatment of a nucleic acid molecule to induce mutations
  • Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., the ability of a homologue to elicit an immune response against a flea protease and/or to have proteolytic activity) and/or by hybridization with isolated flea protease nucleic acids under stringent conditions.
  • An isolated flea protease nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one flea protease protem of the present invention, examples of such proteins being disclosed herein.
  • nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding an flea protease protem.
  • One embodiment of the present invention is a flea protease nucleic acid molecule of the present mvention that is capable of hybridizing under stringent conditions to a nucleic acid strand that encodes at least a portion of a flea protease or a homologue thereof or to the complement of such a nucleic acid strand.
  • a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited.
  • nucleic acid molecules of the present invention for wnich a nucleic acid sequence has been determmed for one strand, that is represented by a SEQ ID NO, also comprises a complementary strand having a sequence that is a complement of that SEQ ID NO.
  • nucleic acid molecules of the present invention which can be either double-stranded or single-stranded, include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with either a given SEQ ID NO denoted herein and/or with the complement of that SEQ ID NO, which may or may not be denoted herein. Methods to deduce a complementary sequence are known to those skilled in the art.
  • a flea protease nucleic acid molecule that includes a nucleic acid sequence having at least about 65 percent, preferably at least about 75 percent, more preferably at least about 85 percent, and even more preferably at least about 95 percent homology with the corresponding region (s) of the nucleic acid sequence encoding at least a portion of a flea protease protein.
  • a flea protease nucleic acid molecule capable of encoding at least a portion of a flea protease that naturally is present in flea midguts and preferably is included in a soluble flea midgut preparation of the present invention. Examples of nucleic acid molecules of the present invention are disclosed in the Examples section.
  • a preferred flea serine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSPl9, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPl ⁇ , nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40.
  • nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18 534 , nfSP18 775 , nfSP18 225 , nfSP24 410 , nfSP24 10bQ , nfSP24,, 4 , nfSP24 7 ⁇ ; , nfSP28 7n , nfSP28 923 , nfSP32 q1 plausible nfSP32 924 , nfSP32 6qq , nfSP33 426/ nfSP33 77fl , nfSP33 1894 , nfSP33 li00 , nfSP33 72e/ nfSP40 ⁇ 4 plausible nfSP5 8 ⁇ 6 nfSPll 307 nfSP8 M5 nfSP8 436, nfSP12 758 , nfSP26 610 , nfSP27
  • nucleic acid molecules that include nfSP3, nfSP8, nf ⁇ P9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40 and even more nfSP18 53, , nfSPl ⁇ T-c, nfSP18 225 , nfSP24 410 , nfSP24 108y , nfSP24 774 , nfSP24 7n , nfSP28 923 , nfSP32 933 , nfSP40
  • nfSP34 390 , nfSP36 197 , nfSP38 341 , nfSP37 2t: , nfSP39,,- nfSP29 cl2 , nfSP30 641 , nfSP31 62b , nfSP32 433 , nfSP15 81! , nfSP19 8C ,, nfSP25 8b4 , nfSP21 5 hail, and/or nfSP40 717 , as well as other specific nucleic acid molecules disclosed in the Examples section.
  • flea serine protease nucleic acid molecules include at least one of the following sequences:SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28-, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO: 155, SEQ ID NO: 9
  • a preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP and/or nfAP2.
  • a more preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP 4Dl , nfAP 900> nfAP 732 , nfAP l380 , nfAP2 3b and/or nfAP2 53 - More preferred is an ammopeptidase nucleic acid molecule that includes nfAP 453 , nfAP 900> nfAP 732 , nfAP 1580 , nfAP2 38 , and/or nfAP2 ; , 7 .
  • Particularly preferred is a nucleic acid molecule that includes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID
  • a preferred flea cysteine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfCPl 57 ,, or nfCPl no9 (the production of which are described in the Examples) . More preferred is a cysteine protease nucleic acid molecule that includes nfCPlc- or nfCPl UQQ .
  • nucleic acid molecule that mcludes nucleic acid sequence SEQ ID NO:l, SEQ ID N0:3, SEQ ID NO:4, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO: 94, or allelic variants of such nucleic acid molecules.
  • nucleic acid molecule of a flea protease protem of the present mvention allows one skilled m the art to make copies of that nucleic acid molecule as well as to obtain a nucleic acid molecule including additional portions of flea protease protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or flea protease nucleic acid molecule homologues. Knowing a portion of an ammo acid sequence of a flea protease protem of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such a flea protease protem.
  • a desired flea protease nucleic acid molecule can be obtained in a variety of ways including screening appropriate expression libraries with antibodies which bind to flea protease proteins of the present invention; traditional cloning techniques usmg oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used) .
  • preferred cDNA libraries mclude cDNA libraries made from unfed whole fleas, fed whole fleas, fed flea midguts, unfed flea midguts, and flea salivary glands.
  • the present invention also mcludes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of a flea protease protem.
  • Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
  • the minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present mvention. Minimal size characteristics are disclosed herein.
  • oligonucleotide must also be sufficient for the use of the oligonucleotide m accordance with the present invention.
  • Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit flea protease production.
  • Such therapeutic applications include the use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug-based technologies.
  • the present invention therefore, includes such oligonucleotides and metnods to interfere witn the production of flea protease proteins by use of one or more of such technologies.
  • the present invention also includes a recombinant vector, which mcludes a flea protease nucleic acid molecule of the present invention inserted mto any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to flea protease nucleic acid molecules of the present invention.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of flea protease nucleic acid molecules of the present invention.
  • recombinant vector herein referred to as a recombinant molecule and described in more detail below, can be used m the expression of nucleic acid molecules of the present invention.
  • Preferred recombinant vectors are capable of replicating in the transformed cell.
  • Preferred nucleic acid molecules to include in recombinant vectors of the present invention are disclosed herein.
  • one embodiment of the present invention is a method to produce a flea protease protem of the present invention by culturing a cell capable of expressing the protem under conditions effective to produce the protem, and recovering the protem.
  • a preferred cell to culture is a recombinant cell that is capable of expressing the flea protease protem, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule mto a cell can be accomplished by any method by which a nucleic acid molecule can be inserted mto the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow mto a tissue, organ or a multicellular organism.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites withm a chromosome of the transformed (i.e., recombinant) cell m such a manner that their ability to be expressed is retained.
  • Preferred nucleic acid molecules with which to transform a host cell are disclosed herein.
  • Suitable host cells to transform include any cell that can be transformed and that can express the introduced flea protease protein. Such cells are, therefore, capable of producing flea protease proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.
  • Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells.
  • Preferred host cells include bacterial, yeast, insect and mammalian cells, with bacterial (e.g., E. coli ) and insect (e.g., Spodoptera) cells being particularly preferred.
  • a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
  • the phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) m recombinant cells of the present invention, including m bacterial, fungal, insect, animal, and/or plant cells.
  • nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present mvention.
  • a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function m at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled m the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, helminth, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda ( ⁇ ) (such as ⁇ p L and ⁇ p F and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionem, alpha mating factor, Pi chi a alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters) , baculovirus, Heli othi s zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actm, retroviral long terminal repeat, Rous sarcoma
  • transcription control sequences include tissue-specific promoters and enhancers as well as lymphokme-mducible promoters (e.g., promoters inducible by interferons or interleukins) .
  • Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding a flea protease protem.
  • Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed flea protease protem to be secreted from the cell that produces the protem.
  • Suitable signal segments include a flea protease protem signal segment or any heterologous signal segment capable of directing the secretion of a flea protease protem, including fusion proteins, of the present invention.
  • Preferred signal segments include, but are not limited to, flea protease, tissue plasmmogen activator (t- PA) , interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotem signal segments .
  • Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins.
  • Inclusion of a fusion sequence as part of a flea protease nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protem encoded by the nucleic acid molecule.
  • a fusion segment can function as a tool to simplify purification of a flea protease protem, such as to enable purification of the resultant fusion protem usmg affinity chromatography.
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool) .
  • Fusion segments can be joined to ammo and/or carboxyl termini of a flea protease protem.
  • Linkages between fusion segments and flea protease proteins can be constructed to be susceptible to cleavage to enable straight-forward recovery of the flea protease proteins.
  • Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protem including the fusion segment attached to either the carboxyl and/or amino terminal end of a flea protease protein.
  • a recombinant molecule of the present invention is a molecule that can mclude at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulatmg expression of the nucleic acid molecule (s) in the cell to be transformed.
  • a preferred recombinant molecule includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase and/or cysteine protease proteins, being more preferred.
  • a preferred recombinant cell includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase, and/or cysteine protease proteins, being more preferred. It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules mto one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shme-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protem production during fermentation.
  • the activity of an expressed recombinant protem of the present mvention may be improved by fragmenting, modifying, or derivatizing the resultant protem.
  • recombinant cells can be used to produce flea protease proteins of the present invention by culturing such cells under conditions effective to produce such a protem, and recovering the protem.
  • Effective conditions to produce a protem include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protem production.
  • An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing a flea protease protem.
  • Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • the medium may comprise complex nutrients or may be a defined minimal medium.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted m shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well withm the expertise of one of ordinary skill in the art.
  • resultant flea protease proteins may either remain withm the recombinant cell; be secreted mto the fermentation medium; be secreted mto a space between two cellular membranes, such as the periplasmic space m E. coli ; or be retained on the outer surface of a cell or viral membrane. Methods to purify such proteins are heretofore disclosed.
  • the present mvention also includes isolated anti-flea protease antibodies and their use to reduce flea infestation on a host animal as well as in the environment of the animal.
  • An anti-flea protease antibody is an antibody capable of selectively binding to a protease present m a flea midgut, including female and male fed midguts as well as female and male unfed midguts.
  • An anti- flea protease antibody preferably binds to the protease m such a way as to reduce the proteolytic activity of that protease.
  • Isolated antibodies are antibodies that have been removed from their natural milieu.
  • isolated does not refer to the state of purity of such antibodies.
  • isolated antibodies can mclude anti-sera containing such antibodies, or antibodies that have been purified to varying degrees.
  • selective binds to refers to the ability of such antibodies to preferentially bind to the protease agamst which the antibody was raised (i.e., to be able to distinguish that protease from unrelated components in a mixture.) . Binding affinities typically range from about 10 ' M to about 10 1 - M " .
  • Binding can be measured using a variety of methods known to those skilled m the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (e.g., ELISA) , immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., lbi a .
  • Antibodies of the present mvention can be either polyclonal or monoclonal antibodies.
  • Antibodies of the present invention mclude functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protem used to obtain the antibodies.
  • Antibodies of the present mvention also include chimeric antibodies that can bind to more than one epitope.
  • Preferred antibodies are raised m response to proteins that are encoded, at least m part, by a flea protease nucleic acid molecule of the present invention.
  • Anti-flea antibodies of the present invention mclude antibodies raised m an animal administered a flea protease vaccine of the present invention that exert their effect when fleas feed from the vaccinated animal's blood containing such antibodies.
  • Anti-flea antibodies of the present invention also include antibodies raised m an animal agamst one or more flea protease proteins, or soluble flea midgut preparations, of the present invention that are then recovered from the animal using techniques known to those skilled m the art.
  • Yet additional antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed for flea protease proteins of the present invention.
  • Antibodies produced agamst defined proteins can be advantageous because such antibodies are not substantially contaminated with antibodies agamst other substances that might otherwise cause interference m a diagnostic assay or side effects if used m a therapeutic composition.
  • Anti-flea protease antibodies of the present mvention have a variety of uses that are within the scope of the present invention. For example, such antibodies can be used m a composition of the present invention to passively immunize an animal m order to protect the animal from flea infestation. Anti-flea antibodies can also be used as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present mvention can be used to target cytotoxic agents to fleas m order to kill fleas. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled m the art.
  • a preferred anti-flea protease antibody of the present invention can selectively bind to, and preferentially reduce the proteolytic activity of, a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease.
  • More preferred anti-flea protease antibodies include anti-flea serine protease antibodies, anti-flea metalloprotease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies.
  • anti-flea serine protease antibodies particularly preferred are anti-flea serine protease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies, including those raised agamst flea serine protease proteins, flea ammopeptidase proteins or cysteine protease proteins of the present invention.
  • the present invention also includes the use of protease inhibitors that reduce proteolytic activity of flea proteases to reduce flea infestation of animals and the surrounding environment.
  • protease inhibitors are compounds that interact directly with a protease thereby inhibiting that protease's activity, usually by binding to or otherwise interacting with the protease 's active site.
  • Protease inhibitors are usually relatively small compounds and as such differ from anti- protease antibodies that interact with the active site of a protease.
  • Protease inhibitors can be used directly as compounds m compositions of the present invention to treat animals as long as such compounds are not harmful to the animals being treated.
  • Protease inhibitors can also be used to identify preferred types of flea proteases to target using compositions of the present mvention.
  • the inventors have shown herein the predominance of serine proteases m flea midguts, particularly in soluble flea midgut preparations, usmg protease inhibitors. Such knowledge suggests that effective reduction of flea infestation of an animal can be achieved using serine protease vaccines, anti-flea serine protease antibodies and other inhibitors of serine protease synthesis and activity that can be tolerated by the animal.
  • flea immunoglobulin proteinase activity disclosed herein can be targeted to reduce flea infestation. That other proteases are also present m flea midguts accordmg to the present mvention also suggests targeting such proteases. Methods to use protease inhibitors are known to those skilled in the art; examples of such methods are disclosed herein.
  • a protease inhibitor that can be used m a composition of the present invention to treat an animal is identified by a method including the following steps: (a) identifying candidate (i.e., putative, possible) inhibitor compounds by testing the efficacy of one or more protease inhibitors (l) in vi tro for their ability to inhibit flea protease activity and/or (ii) m a flea feeding assay for their ability to reduce the survival and/or fecundity of fleas by adding the inhibitors to the blood meal of a flea being maintained, for example, m a feeding system, such as that described by Wade et al., 1986, J. Med Entomol .
  • candidate compounds identified usmg the in vi tro assay may work "m the test tube” but may not work in vivo for a number of reasons, including the presence of interfering components m the blood meal that inhibit the activity of such compounds; e.g., although aprotinin can inhibit at least some flea serine proteases in vi tro, aprotinin does not work well m the presence of serum proteins, such as are found m the blood.
  • candidate inhibitor compounds identified by the flea feeding assays can include not only desired compounds but also compounds that reduce the viability and/or fecundity of fleas due to general toxicity (e.g., affecting the mitochondria of fleas) .
  • an inhibitor of a flea protease of the present invention is identified by a method comprising: (a) contacting an isolated flea protease protem comprising an ammo acid sequence including SEQ ID NO:10, SEQ ID N0:13, SEQ ID N0:16, SEQ ID N0:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ
  • a test kit can be used to perform such method.
  • a preferred test kit comprises an isolated flea protease protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:
  • protease inhibitors are used m the purification of corresponding proteases by, for example, affinity chromatography, m which, a protease inhibitor is incubated with a mixture containing a desired protease under conditions that the inhibitor forms a complex with the protease. The protease can then be recovered from the complex.
  • the protease inhibitor can be attached to a solid support and/or be labelled with, for example, a radioactive, fluorescent, or enzymatic tag that can be used to detect and/or recover the complex.
  • Suitable protease inhibitors to use in accordance with the present invention include serine protease inhibitors (including immunoglobulin proteinase inhibitors and serpms) , metalloprotease inhibitors, aspartic acid protease inhibitors, cysteine protease inhibitors and ammopeptidase inhibitors.
  • Preferred protease inhibitors include serine protease inhibitors, metalloprotease inhibitors, ammopeptidase inhibitors and cysteine protease inhibitors, particularly those that are broad spectrum inhibitors. More preferred are broad spectrum serine protease inhibitors.
  • protease inhibitors There is a wide variety of protease inhibitors, as is known to one skilled in the art.
  • Examples include, but are not limited to, AEBSF, aprotinin, bestatm, chloromethyl ketones TLCK (N ⁇ -p-tosyl-L-lysme chloromethyl ketone) and TPCK (N-tosyl-L-phenylalanme chloromethyl ketone), chymostatm, cystatm, 3 ' 4-d ⁇ chloro ⁇ socoumarm, E-64 (trans-epoxysuccmyl-L-leucylamido- (4-guan ⁇ dmo) butane) , EDTA (ethylenediammetetraacetic acid) , leupeptin, methyl ketones havmg a variety of leaving groups, oxidized L- leucmethiol, pepstatin, 1, 1O-orthophenanthrolme, phosphoramidon, soybean trypsm/chymotrypsm inhibitor and soybean trypsin inhibitor.
  • Preferred protease inhibitors for use m the present invention include AEBSF, bestatm, E-64 leupeptin, pepstatin, 1, 10-orthophenanthrolme, phosphoramidon, TLCK and TPCK, with AEBSF ⁇ a broad spectrum serine protease inhibitor) , bestatm (an inhibitor of leucine ammopeptidase) and 1, 10-orthophenanthrolme (a broad spectrum metalloprotease inhibitor) being particularly preferred.
  • Another preferred inhibitor of the present invention mcludes an inhibitor of an immunoglobulin proteinase of the present invention.
  • Suitable inhibitors of 6 ⁇ immunoglobulin proteinase activity are compounds that interact directly with an immunoglobulin proteinase protein's active site, thereby inhibiting that immunoglobulin proteinase' s activity, usually by binding to or otherwise interacting with or otherwise modifying the immunoglobulin proteinase' s active site.
  • Immunoglobulin proteinase inhibitors can also interact with other regions of the immunoglobulin proteinase protem to inhibit immunoglobulin proteinase activity, for example, by allosteric interaction.
  • Inhibitors of immunoglobulin protemases are usually relatively small compounds and as such differ from anti-immunoglobulm proteinase antibodies.
  • an immunoglobulin proteinase inhibitor of the present invention is identified by its ability to bind to, or otherwise interact with, a flea immunoglobulin proteinase protem, thereby inhibiting the activity of the flea immunoglobulin proteinase.
  • Preferred immunoglobulin proteinase inhibitors of the present invention include, but are not limited to, flea immunoglobulin proteinase substrate analogs, and other molecules that bind to a flea immunoglobulin proteinase
  • An immunoglobulin proteinase substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the active site of an immunoglobulin proteinase protem.
  • a preferred immunoglobulin proteinase substrate analog inhibits immunoglobulin proteinase activity.
  • Immunoglobulin proteinase substrate analogs can be of any inorganic or organic composition, and, as such, can be, but are not limited to, peptides, nucleic acids, and peptidomimetic compounds.
  • Immunoglobulin proteinase substrate analogs can be, but need not be, structurally similar to an immunoglobulin proteinase' s natural substrate as _.ong as they can interact with the active site of that proteinase protem. Immunoglobulin proteinase substrate analogs can be designed using computer-generated structures of immunoglobulin proteinase proteins of the present invention or computer structures of immunoglobulin protemases' natural substrates.
  • Substrate analogs can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or otner inorganic or organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., a flea immunoglobulin proteinase) .
  • molecules such as oligonucleotides, peptides, peptidomimetic compounds, or otner inorganic or organic molecules.
  • affinity chromatography techniques using the corresponding binding partner, (e.g., a flea immunoglobulin proteinase) .
  • a preferred immunoglobulin proteinase substrate analog is a peptidomimetic compound (i.e., a compound that is structurally and/or functionally similar to a natural substrate of an immunoglobulin proteinase of tne present invention, particularly to the region of the substrate that interacts with the proteinase active site, but that inhibits immunoglobulin proteinase activity upon interacting with the immunoglobulin proteinase active site) .
  • Another preferred flea immunoglobulin proteinase inhibitors of the present invention include antibodies that bind specifically to an immunoglobulin proteinase m such a manner that the proteinase activity of the immunoglobulin proteinase is inhibited.
  • Yet another preferred flea immunoglobulin proteinase inhibitor includes an inhibitor from the class of serine proteinase inhibitors. Suitable immunoglobulin proteinase inhibitor include serine proteinase inhibitors disclosed herein.
  • Protease inhibitors can be produced usmg methods known to those skilled m the art.
  • Protem- or peptide- based protease inhibitors such as cystatm or small peptides comprising a protease substrate, can be produced recombinantly and modified as necessary.
  • the present invention also includes the use of proteolytically active flea protease proteins of the present mvention to identify additional protease inhibitors, and preferably protease inhibitor compounds that can be included m a composition of the present invention to be administered to animals.
  • a method to identify a flea protease inhibitor includes the steps of (a) contacting (e.g., combining, mixing) an isolated flea protease protem with a putative (i.e., candidate) inhibitory compound under conditions in which, in the absence of the compound, the protein has proteolytic activity, and (b) determining if the putative inhibitory compound inhibits the proteolytic activity of the protein.
  • Putative inhibitory compounds to screen include organic molecules, antibodies (including functional equivalents thereof) and substrate analogs.
  • protease activity is known to those skilled in the art, as heretofore disclosed.
  • Particularly preferred for use in identifying inhibitors are flea serine protease proteins, flea aminopeptidase proteins and flea cysteine protease proteins of the present invention.
  • the present invention also includes inhibitors isolated by such a method, and/or test kit, and their use to inhibit any flea protease that is susceptible to such an inhibitor.
  • the present invention also includes mimetopes of compounds of the present mvention that can be used in accordance with methods as disclosed for compounds of the present invention.
  • a mimetope of a proteinaceous compound of the present invention e.g., a flea protease protein, an anti- flea protease antibody, a proteinaceous inhibitor of protease activity or synthesis
  • a mimetope of a flea protease protein is a compound that has an activity similar to that of an isolated flea protease protein of the present invention.
  • Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti- idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures) ; and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer- generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
  • the present invention includes therapeutic compositions, also referred to herein as compositions, that include a (i.e., at least one) compound of the present invention.
  • Preferred compounds to include in a composition of the present invention include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herein.
  • Such a therapeutic composition can protect an animal from flea infestation by reducing flea protease activity, thereby reducing flea burden on the animal and m the environment of the animal.
  • compositions of the present invention include at least one of the followmg compounds: an isolated flea serine protease protem or a mimetope thereof; an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene; an isolated antibody that selectively binds to a flea serine protease protem; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea ammopeptidase protem or a mimetope thereof; an isolated flea ammopeptidase nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea ammopeptidase gene; an isolated antibody that selectively binds to a flea ammopeptidase protem; an inhibitor of flea ammopeptidase activity identified by its ability to inhibit flea ammopeptidase activity; an isolated flea cyste
  • Another embodiment of the present invention is a therapeutic composition that includes a first compound that reduces flea protease activity and a second compound that reduces flea burden by a method other than by reducing flea protease activity.
  • the present invention also includes a method to protect an animal from flea infestation by administering to the animal such a composition.
  • the first compound of such a composition by effectively reducing flea protease activity m the midgut, enhances the activity of the second compound. While not being bound by theory, it is believed that a number of anti-flea treatments, particularly those that are proteinaceous, are not very effective because they are degraded m the flea midgut.
  • the present invention permits the effective use of such anti-flea treatments by reducing proteolytic degradation of such treatments by the flea midgut.
  • Preferred first compounds to include m such a composition include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herem, such compounds that target flea immunoglobulin proteinase activity.
  • a preferred therapeutic composition of the present invention comprises an excipient and a protective compound including: an isolated protem or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent nvbridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ
  • Suitable second compounds mclude any anti-flea agent (s) , including, but not limited to, proteinaceous compounds, insecticides and flea collars.
  • Preferred second compounds are proteinaceous compounds that effect active immunization (e.g., antigen vaccines), passive immunization (e.g., antibodies), or that otherwise inhibit a flea activity that when inhibited can reduce flea burden on and around an animal.
  • second compounds include a compound that inhibits binding between a flea membrane protein and its ligand (e.g., a compound that inhibits flea ATPase activity or a compound that inhibits binding of a peptide or steroid hormone to its receptor) , a compound that inhibits hormone (including peptide or steroid hormones) synthesis, a compound that inhibits vitellogenesis (including production of vitellm and transport and maturation thereof mto a major egg yolk protem) , a compound that inhibits fat body function, a compound that inhibits flea muscle action, a compound that inhibits the flea nervous system, a compound that inhibits the flea immune system and/or a compound that inhibits flea feeding.
  • a compound that inhibits binding between a flea membrane protein and its ligand e.g., a compound that inhibits flea ATPase activity or a compound that inhibits binding of a peptide or steroid hormone to its receptor
  • hormone including peptide or
  • an immunoglobulin proteinase of the present invention can also be used as a second compound in a therapeutic composition of the present invention to promote longevity of antibodies that bind specifically to selected flea proteins.
  • An immunoglobulin proteinase can be administered to an animal tc promote production of antibodies that bind specifically to the immunoglobulin proteinase, thereby inhibiting the activity of the proteinase.
  • An immunoglobulin proteinase can be administered to an animal either together with cr after administration of any desired flea protem to the animal.
  • a preferred immunoglobulin proteinase to include as a second compound in a therapeutic composition includes: an isolated protem or a mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising ammo acid sequence SEQ ID NO: 67, SEQ ID NO: 68 and/or SEQ ID NO: 69; and/or an isolated nucleic acid molecule that hybridizes under stringent conditions with a gene comprising a nucleic acid sequence including SEQ ID NO: 66 and other nucleic acid sequences encoding an immunoglobulin proteinase of the present mvention disclosed herein.
  • compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
  • a pharmaceutically acceptable excipient such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
  • compositions of the present mvention can be formulated m an excipient that the animal to be treated can tolerate.
  • excipients examples include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame on, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers mclude phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up m a suitable liquid as a suspension or solution for injection.
  • the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
  • the composition can also include an immunopotentiator, such as an adjuvant or a carrier.
  • adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; alummum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxce!- 1" , Inc.
  • Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention mto an animal.
  • a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel m si tu .
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible) .
  • a preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to reduce protease activity in fleas feeding from the animal over a period of time ranging from about 1 to about 12 months.
  • a controlled release formulation of the present invention is capable of effecting a treatment for preferably at least about 1 month, more preferably at least about 3 months and even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • a therapeutic composition of the present invention is administered to the animal in an effective manner such that the protease activity of fleas feeding from the blood stream of animals treated with the composition is reduced.
  • a treated animal is an animal that is competent to reduce the flea burden by reducing flea protease activity, or by reducing flea protease activity and at least one other flea activity.
  • the protease activity is reduced by at least about 50 percent, more preferably by at least about 70 percent and even more preferably by at least about 90 percent.
  • Methods to administer compositions to the animal in order to render the animal competent depend on the nature of the composition and administration regime.
  • Animals administered a protease vaccine with at least one booster shot usually become competent at about the same time as would be expected for any vaccine treatment. For example, animals administered a booster dose about 4 to 6 weeks after a primary dose usually become competent withm another about 3 to 4 weeks. Animals administered a composition including an anti-flea protease antibody or protease inhibitor become competent as soon as appropriate serum levels of the compound are achieved, usually with one to three days .
  • a composition of the present invention when administered to a host animal is able to reduce flea viability by at least about 50 percent withm at least about 21 days after the fleas begin feeding from the treated animal. (Note that fleas usually live about 40 days to about 50 days on one or more animals.)
  • a more preferred composition when administered to a host animal is able to reduce flea viability by at least about 65 percent withm at least about 14 days after the fleas begin feeding from the treated animal.
  • An even more preferred composition when administered to an animal is able to reduce flea viability by at least about 90 percent withm at least about 7 days after the fleas begin feeding from the treated animal.
  • a composition of the present invention when administered to a host animal is able to reduce flea fecundity (i.e., egg laying abi ⁇ ity) by at least about 50 percent, more preferably by at least about 70 percent, and even more preferably by at least about 90 percent, withm at least about 30 days after the fleas begin feeding from the treated animal. (Note that fleas usually do not begin laying eggs until about 7 days after taking a blood meal.)
  • compositions are administered to an animal in a manner such that the animal becomes competent to reduce flea protease activity in a flea that feeds from the competent; i.e., the animal becomes a treated animal.
  • a flea protease vaccine of the present mvention when administered to an animal m an effective manner, is able to elicit (i.e., stimulate) an immune response that produces an antibody titer in the blood stream of the animal sufficient to reduce flea protease activity.
  • an anti-flea protease antibody of the present invention when administered to an animal in an effective manner, is administered m an amount so as to be present m the animal's blood stream at a titer that is sufficient to reduce flea protease activity.
  • a protease inhibitor compound of the present invention when administered to an animal m an effective manner, is administered m a manner so as to be present m the animal's blood stream at a concentration that is sufficient to reduce flea protease activity.
  • Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of flea proteases.
  • compositions of the present invention can be administered to animals prior to or during flea infestation. It is to be noted that when vaccines of the present invention are administered to an animal, a time period is required for the animal to elicit an immune response before the animal is competent to inhibit protease activity of fleas feeding from that animal. Methods to obtain an immune response in an animal are known to those skilled in the art. Acceptable protocols to administer compositions m an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from flea infestation when administered one or more times over a suitable time period.
  • a preferred smgle dose of a protease vaccine or a mimetope thereof ranges from about 1 microgram ( ⁇ g, also denoted ug) to about 10 milligrams (mg) of the composition per kilogram body weight of the animal.
  • Booster vaccinations can be administered from about 2 weeks tc several years after the original administration. Booster vaccinations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from flea infestation.
  • a preferred administration schedule is one in which from about 10 ⁇ g to about 1 mg of the vaccine per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months.
  • a booster dose of a composition of the present invention is administered about 4 to 6 weeks after the primary dose, and additional boosters are administered about once or twice a year.
  • Modes of administration can include, but are not limited to, oral, nasal, topical, transdermal, rectal, and parenteral routes.
  • Parenteral routes can include, but are not limited to subcutaneous, intradermal, intravenous, and intramuscular routes.
  • a preferred single dose of an anti-flea protease antibody composition or a mimetope thereof ranges from about 1 ⁇ g to about 10 mg of the composition per kilogram body weight of the animal.
  • Anti- flea antibodies can be re-administered from about 1 hour to about biweekly for several weeks following the original administration.
  • Booster treatments preferably are administered when the titer of antibodies of the animal becomes insufficient to protect the animal from flea infestation.
  • a preferred administration schedule is one m which from about 10 ⁇ g to about 1 mg of an ant-.-f-.ea protease antibody composition per kg body weight of the animal is administered about every 2 to every 4 weeks. Suitable modes of administration are as disclosed herein and are known to those skilled in the art.
  • a nucleic acid molecule of the present invention can be administered to an animal m a fashion to enable expression of that nucleic acid molecule mto a protective protem (e.g., flea protease vaccine, anti-flea protease antibody, or proteinaceous protease inhibitor) or protective RNA (e.g., antisense RNA, ribozyme or RNA drug) in the animal to be protected from disease.
  • a protective protem e.g., flea protease vaccine, anti-flea protease antibody, or proteinaceous protease inhibitor
  • protective RNA e.g., antisense RNA, ribozyme or RNA drug
  • Nucleic acid molecules can be delivered to an animal m a variety of methods including, but not limited to, (a) direct injection (e.g., as "naked” DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Sci ence 247, 1465-1468) or (b) packaged as a recombinant virus particle vaccine or as a recombinant cell vaccine (i.e., delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine and a recombinant cell vaccine) .
  • direct injection e.g., as "naked” DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Sci ence 247, 1465-1468
  • packaged as a recombinant virus particle vaccine or as a recombinant cell vaccine i.e., delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine
  • a recombinant virus particle vaccine of the present invention includes a recombinant molecule of the present invention that is packaged m a viral coat and that can be expressed in an animal after administration.
  • the recombinant molecule is packaging-deficient.
  • a number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses.
  • a recombinant virus particle vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from disease caused by a parasite of the present invention.
  • a preferred single dose of a recombinant virus particle vaccine of the present invention is from about 1 x 10 4 to about 1 x 10' virus plaque forming units (pfu) per kilogram body weight of the animal.
  • Administration protocols are similar to those described herein for protein-based vaccines.
  • a recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention.
  • Preferred recombinant cells include Salmonella, E. coli , Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8, COS, MDCK and CRFK recombinant cells, with Salmonella recombinant cells being more preferred.
  • Such recombinant cells can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about IO 8 to about IO 12 bacteria per kilogram body weight. Administration protocols are similar to those described herein for protem-based vaccines .
  • Recombinant cell vaccines can comprise whole cells or cell lysates.
  • compositions of the present invention can be administered to any animal susceptible to flea infestation, including warm-blooded animals.
  • Preferred animals to treat include mammals and birds, with cats, dogs, humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail and turkeys as well as other furry animals, pets and/or economic food animals, bemg more preferred.
  • Particularly preferred animals to protect are cats and dogs.
  • compositions to treat flea infestation by any flea can be derived from any flea species.
  • Preferred fleas to target include fleas of the following genera: Ctenocephalides, Cyopsyll us, Diamanus ( Oropsylla) , Echi dnophaga, Nosopsyll us, Pulex, Tunga, and Xenopsylla, with those of the species Ctenocephalides cams, Ctenocephalides feli s, Diamanus montanus, Echidnophaga gallmacea, Nosopsyll us faciatus, Pulex irri tans, Pul ex simulans, Tunga penetrans and Xenopsylla cheopi s being more preferred.
  • fleas from which to protect animals mclude fleas of the species Ctenocephalides felis, Ctenocephalides cam s, and Pul ex species (e.g., Pulex irri tans and Pulex simulans ) . It is also wit.iin the scope of the present invention to administer compositions of the present mvention directly to fleas.
  • the present invention also includes the use of compositions of the present mvention to reduce infestation by other ectoparasites as well as the use of compositions including protease vaccines, anti-protease antibodies and compounds that inhibit protease synthesis and/or activity derived from any ectoparasite to reduce ectoparasite infestation, particularly controlled release formulations containing such compositions.
  • Preferred ectoparasites to target include arachnids, insects and leeches. More preferred ectoparasites to target include fleas; ticks, including both hard ticks of the family Ixodidae (e.g., Jxodes and Amblyomma) and soft ticks of the family Argasidae (e.g., Orni thodoros, such as 0. parkeri and 0.
  • flies such as midges (e.g., Culicoides) , mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis- causmg flies and bitmg gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas disease.
  • Even more preferred ectoparasites to target include fleas, mosquitos, midges, sandflies, blackflies, ticks and Rhodm us .
  • the following examples are provided for the purposes cf illustration and are not intended to limit the scope of tne present invention.
  • Example 1 includes a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al . , ibi d. , Borovsky, Arch Insect Bi ochem . and Phys . , 7:187-210, 1988, and related references. Examples 1 through 21, and the sequence information provided in the sequence listing therein, of related PCT Publication No. WO 96/11706, published April 25, 1996, are incorporated herein by this reference in their entirety.
  • Example 1 includes a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al . , ibi d. , Borovsky, Arch Insect Bi ochem . and Phys . , 7:187-210, 1988, and related references. Examples 1 through 21, and the sequence information provided in the
  • This example describes the determination of internal ammo acid sequence of a flea aminopeptidase.
  • the resulting pellet was resuspended and sonicated m 4 ml buffer comprising 20 mM NaAc, pH 6.0, 0.1% Brij , complete protease inhibitor cocktail (available from Pierce) and 0.25 mM bestatm; the sonicate was centrifuged at about 14,000 rpm for about 20 minutes. Both the pellet and supernatant were recovered. The pellet was re-sonicated and centrifuged as above, and the resulting supernatant was combined with the original supernatant.
  • the pooled supernatant was applied to a polyCAT cation exchange HPLC column and protem was eluted with a NaCl gradient ranging from 0M to IM NaCl in 20 mM NaAc, pH 6.0.
  • Fractions collected from the column were assayed by H-Leu-AMC fluorescence, and active fractions were pooled and applied to a C-1 reverse phase HPLC column (TMS 250, Toso Hass) . Proteins were eluted from the column using an acetonitrile gradient m 0.1% TFA m water, the gradient ranging between 20% and 100% acetonitrile.
  • Proteins contained m fractions from the column were analyzed by SDS-PAGE gel electrophoresis and silver staining. The results of tne gel electrophoresis indicated the presence of an about 95 kDa protem m some of the fractions. This protem correlates with the about 95 kDa protem described in Example 12 of related PCT Publication No. WO 96/11706 which was identified using membrane pellet from flea midgut lysates. To determine internal ammo acid sequence of the 95 kDa protem, those fractions containing the 95 kDa protem were pooled, dried and digested with BNPS-Skatole for about 72 hours at room temperature.
  • the BNPS-Skatole digest was separated by 18% Tris-glycme PAGE gel electrophoresis and blotted onto PVDF membrane. A major band of about 28 kDa was cut out and N-terminally sequenced using techniques as described in Example 7 of related PCT Publication No. WO 96/11706. A partial N-termmal ammo acid sequence of the internal peptide was obtained, namely LATTQFQATHARSAFPCFDEPAM (denoted herem SEQ ID NO:107) .
  • Manduca sexta and rat ammopeptidases having nucleic acid sequence 5' CCC AAA TTT TCC ATW GCN CCN GC 3' (N indicating any nucleotide; represented herein as SEQ ID NO: 108) was used m combination with primer M13 Reverse primer (SEQ ID NO: 87) to PCR amplify a portion of a flea ammopeptidase gene from a bovine blood-fed whole flea cDNA expression library as described m Example 8 of related PCT Publication No. WO 96/11706.
  • the resulting product of the PCR amplification was diluted about 1:50 and used as a template in a second, semi-nested PCR amplification usmg a primer APN3 m combination with degenerate primer APN1C, designed using SEQ ID NO: 107 (described in Example 1), having nucleic acid sequence 5' CAA TTY CAA GCT ACY CAT GC 3' (represented herein as SEQ ID NO: 109) .
  • the resulting PCR product, named nfAP2 383 was approximately 383-bp when visualized on a 1% agarose gel.
  • the PCR product nfAP2 383 was gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • the nucleotide sequence of nfAP2 38 is denoted SEQ ID NO: 110.
  • Translation of SEQ ID NO: 110 yielded a deduced flea ammopeptidase protem of about 127 ammo acids, denoted herem as PfAP2 ⁇ 27 , having ammo acid sequence SEQ ID NO: 111.
  • nfAP2 38 The PCR product nfAP2 38 , was labelled with "P and used as a probe to screen a bovine blood-fed whole flea phage expression library usmg standard hybridization techniques.
  • a single plaque purified clone was isolated, which included a 2100-nucleot ⁇ de insert, referred to herein as nfAP2 2100 .
  • Partial nucleic acid sequence was obtained using standard techniques from the 5' end of nfAP2 2100 , to yield a flea ammopeptidase nucleic acid molecule named nfAP2. )V . having nucleic acid sequence SEQ ID NO: 112.
  • nucleic acid molecule nfAP2 5 encodes a non-full-length flea ammopeptidase protem of about 178 ammo acids, referred to herein as PfAP ]78 , having ammo acid sequence SEQ ID NO: 113, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID N0:112.
  • SEQ ID NO:113 contains SEQ ID N0:107.
  • Flea ammopeptidase nucleic acid sequence SEQ ID NO: 112 was compared with additional nucleic acid sequences characterized from other organisms.
  • the nucleic acid sequence is about 50% identical to Manduca sexta ammopeptidase N nucleotides between corresponding regions of the two nucleic acid molecules.
  • Example 3 This example describes the cloning and sequencing of a flea cysteine protease nucleic acid molecule.
  • nfCPl s ⁇ A flea cysteine protease nucleic acid molecule, referred to herein as nfCPl s ⁇ was produced by PCR amplification using the following method.
  • Primer Cal3F (designed to obtain a calreticulin gene), having nucleic acid sequence 5' TTG GGA TAC ACT TTG ACT GTT AAC C 3', represented herein as SEQ ID NO: 97 was used in combination with the M13 universal primer, to PCR amplify, using standard techniques, a DNA fragment from a bovine blood-fed whole flea cDNA expression library as described above m
  • the isolated DNA fragment correlated with a cysteine protease nucleic acid sequence.
  • Sequence from this DNA fragment was used to design primer CyslR, having the nucleic acid sequence 5' GTG AGC AAC CAT TAT TTC CAT ATC 3', represented herein as SEQ ID NO: 98, which was used m a second PCR amplification m combination with the M13 reverse primer.
  • a third PCR amplification was performed usmg primer CyslF, having the nucleic acid sequence 5' CTT TCC TCA CAA TAC CAC CAA GGA AGC 3', represented herein as SEQ ID NO: 74, in combination with the M13 universal primer.
  • a fourth PCR amplification was performed using primer Cys2F, having the nucleic acid sequence 5' CTT GTA CGA TTG TCT CAA CAG GC 3', represented herein as SEQ ID NO: 76, m combination with the M13 universal primer.
  • the resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • a composite nucleic acid sequence representing a flea cysteine protease coding region was deduced, referred to herein as nfCPl 573 , was deduced and is denoted herein as SEQ ID NO:76.
  • SEQ ID NO: 76 suggests that nucleic acid molecule nfCPl 5 -> encodes a non- full-length flea cysteine protease protem of about 191 ammo acids, referred to herem as PfCPl ]91 , having ammo acid sequence SEQ ID NO: 77, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:76.
  • SEQ ID NO: 77 was found to be similar to the ammo acid sequence of P . sa ti vum cysteine protease. The most highly conserved region of contmuous similarity between SEQ ID NO: 77 and P.
  • sa ti vum cysteine protease ammo acid sequences spans from about ammo acid 71 through about ammo acid 165 of SEQ ID NO:77 and from about ammo acid 17 through about ammo acid 168 of the P. sa ti vum cysteine protease, there being about 42% identity between the two regions. Comparison of the nucleic acid sequence encoding ammo acids from about 205 through about 492 of nfCPl, , indicate that those regions are about 54- identical.
  • This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules. Additional serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706.
  • the actual primers used in PCR amplification of serine protease nucleic acid molecules from a bovme blood-fed flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) in combination with either M13 reverse primer (SEQ ID NO:87, or H57 primer (SEQ ID NO:99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • Two recombinant TA vector clones were isolated and found to correspond to previously cloned serine protease genes. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
  • a nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 5 (produced using primers cat try #2 and M13 reverse) , namely nfSP5 «Ct . is represented herem as SEQ ID NO:114.
  • SEQ ID NO: 116 and SEQ ID NO: 117 are both contained withm the sequence of the nucleic acid molecule nfSP5 806 .
  • a Genbank homology search revealed most homology between SEQ ID NO: 114 and a Gall us gall us trypsin gene, there being about 52% identity between correspondmg regions of the two nucleic acid molecules.
  • B. A nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 11 (produced using primers cat try #2 and M13 reverse) , namely nfSPll, 07 , is represented herein as SEQ ID NO: 118.
  • SEQ ID NO: 120 and SEQ ID NO: 121 are withm the sequence of the nucleic acio molecule nfSPll 3C - Translation of SEQ ID NO: 118 suggests tnat nucleic acid molecule nfSPll 30 - encodes a non-full- length flea serine protease protem of about 102 ammo acids, referred to herein as PfSPll 102 , having ammo acid sequence SEQ ID NO: 119, assuming the first codon spans from aoout nucleotide 1 through about nucleotide 3 of SEQ ID NO: 118. C.
  • Translation of SEQ ID NO: 122 suggests that nucleic acid molecule nfSP39 267 encodes a non-full-length flea serine protease protein of about 90 ammo acids, referred to herein as PfSP39 89 , having ammo acid sequence SEQ ID NO: 123, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:122.
  • Example 5 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules.
  • Certain flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen a bovine blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706), cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) .
  • Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
  • SEQ ID NO: 125 The nucleic acid sequence of a flea serine protease nucleic molecule correlating to flea clone 8, namely nfSP8 436 is represented herein as SEQ ID NO: 125.
  • SEQ ID NO: 127 is within the sequence of the nucleic acid molecule nfSP ⁇ 43b _ Translation of SEQ ID NO: 125 yields a protein of about 145 amino acids, denoted PfSP8 145 , having amino acid sequence SEQ ID NO:126, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 125.
  • a Genbank homology search revealed most homology between SEQ ID NO: 125 and an Anophel es gambiae trypsin precursor gene, there being about 48% identity between corresponding regions of the two nucleic acid molecules .
  • the nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 12, namely nfSP12 756 is represented herein as SEQ ID NO: 128.
  • SEQ ID NO: 130 and SEQ ID NO: 131 are both contained within the sequence of the nucleic acid molecule nfSP12 758 .
  • Translation cr SEQ ID NO: 128 yields a protem of about 246 ammo acids, denoted PfSP12 4 , having ammo acid sequence SEQ ID NO: 129, assuming an open reading frame m which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 128 and a stop codon spanning from about nucleotide 739 through about nucleotide 741 of SEQ ID NO: 128.
  • a Genbank homology search revealed most homology between SEQ ID NO: 128 and a rat trypsmogen gene, there being about 57? identity between corresponding regions of the two nucleic acid molecules.
  • Certain flea serine protease cDNA genes have been isolated from a cat blood-fed flea cDNA expression library by screening the library with the cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) probes.
  • the cat blood-fed flea library was produced in a similar manner as the bovme blood-fed flea library (described in Example 8 cf related PCT Publication No. WO 96/11706) except the fleas were fed on cat blood.
  • Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using methods described above.
  • the nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP26 Dl0 is represented herein as SEQ ID NO: 132.
  • Translation of SEQ ID NO: 132 yields a non-full-length sequence of about 185 ammo acids, denoted PfSP26 ]8C> , having ammo acid sequence SEQ ID NO: 133, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 132.
  • a Genbank homology search revealed most homology between SEQ ID NO: 133 and a Aedes aegypti trypsin protem sequence, there being about 48% identity between corresponding regions of the two ammo acid sequences.
  • SEQ ID NO: 134 The nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP27 386 is represented herem as SEQ ID NO: 134.
  • Translation of SEQ ID NO: 134 yields a protem of about 128 ammo acids, denoted PfSP27 128 , having amino acid sequence SEQ ID NO: 135, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 134.
  • Example 6 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules.
  • Certain serine protease cDNA nucleic acid molecules have been isolated from reverse transcriptase PCR amplification of mRNA isolated from cat blood-fed whole fleas.
  • the mRNA was isolated from fleas gathered over 72 hours after the initiation of feeding on cat blood. As such, the mRNA comprised a mixture of mRNA isolated at different time points over 72 hours.
  • the mRNA was isolated using ground-up fleas, extracting total flea RNA using Tri-Reagent (available from Molecular Research Center, Cincinnati, Ohio) and an Invitrogen Fast Track 1 '"' RNA isolation kit (available from Invitrogen, Inc. San Diego, CA) .
  • cDNA was synthesized using a Stratagene RT-PCR kit (available from Stratagene, Inc, San Diego, CA) .
  • Primers used for first-strand cDNA synthesis included an equal molar mixture of the followmg: 5'dT-2VT3' and 5'dT-2VC3' (as provided m a differential display kit, available from Operon Technologies, Inc. Alameda, CA) .
  • the actual primers used m the PCR amplification of the cDNA described above included cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • Recombinant TA vector clones were isolated and the nucleic acid molecules were subjected to nucleic acid sequencing using analysis as described above.
  • a nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP23 423 is represented herein as SEQ ID NO: 136.
  • nucleic acid molecule nfSP23 42 encodes a non- full-length flea serine protease protem of about 141 ammo acids, referred to herein as PfSP23 141 , having amino acid sequence SEQ ID NO: 137, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 136.
  • a Genbank homology search revealed most homology between SEQ ID NO: 136 and a Homo sapi ens plasmmogen precursor gene, there being about 51% identity between corresponding regions of the two nucleic acid molecules.
  • nucleic acid sequence of a flea serine protease nucleic molecule is represented herein as SEQ ID NO: 78.
  • Translation of SEQ ID NO:78. suggests that nucleic acid molecule nfSP24 41c encodes a non- full-length flea serine protease protein of about 136 ammo acids, referred to herein as PfSP24 136 , having ammo acid sequence SEQ ID NO: 79, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 78.
  • a Genbank homology search revealed most homology between SEQ ID NO:79 and an Anophel es gambiae chymotrypsm protem sequence, there being about 38% identity between corresponding regions of the two amino acid sequences.
  • SEQ ID NO: 82 Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP33 426 , is represented herein as SEQ ID NO: 82.
  • Translation of SEQ ID NO: 82 suggests that nucleic acid molecule nfSP33 42e encodes a non- full-length flea serine protease protein of about 142 ammo acids, referred to herein as PfSP33 :42 , having amino acid sequence SEQ ID NO: 83, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 82.
  • SEQ ID NO:138 Another nucleic acid sequence of one of the flea serine protease nucleic molecule, namely nfSP36, q7 , is represented herein as SEQ ID NO:138.
  • SEQ ID NO:138 represents a partial sequence of a PCR amplified nucleic acid molecule nfSP36 S00 .
  • nucleic acid molecule nfSP36 19 - encodes a non- full-length flea serine protease protem of about 65 ammo acids, referred to herem as PfSP36 65 , having ammo acid sequence SEQ ID NO: 139, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 138.
  • a Genbank homology search revealed most homology between SEQ ID NO: 139 and a Drosophila melanogaster easter protem sequence, there being about 42% identity between correspondmg regions of the two ammo acid sequences.
  • SEQ ID NO: 140 Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP38 34] , is represented herein as SEQ ID NO: 140.
  • Translation of SEQ ID NO: 140 suggests that nucleic acid molecule nfSP38, 41 encodes a non- full-length flea serine protease protem of about 113 ammo acids, referred to herein as PfSP38 113 , having ammo acid sequence SEQ ID NO: 141, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 140.
  • a Genbank homology search revealed most homology between SEQ ID NO: 141 and a rat trypsmogen protem sequence, there being about 30% identity between corresponding regions of the two ammo acid sequences.
  • F. A nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP34 390 , is represented herein as SEQ ID NO: 142.
  • nucleic acid molecule nfSP4 390 encodes a non- full-length flea serine protease protein of about 130 amino acids, referred to herein as PfSP34 130 , having ammo acid sequence SEQ ID NO: 143, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:
  • Genbank homology search revealed most homology between SEQ ID NO: 143 and a Drosophila mel anogaster Delta precursor protein sequence, there being about 33% identity between corresponding regions of the two ammo acid sequences.
  • Example 7 This example describes the cloning and sequencing of a flea serine protease nucleic acid molecule.
  • a serine protease cDNA nucleic acid molecule was isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706.
  • the actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed whole flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) in combination with M13 reverse primer (SEQ ID NO:87) .
  • the resulting PCR product was diluted 1:25 and used as a template in a second PCR reaction using the forward vector primer T3 in combination with the reverse primer (derived from the nucleic acid sequence of nfSP33 7 , & , described m Example 6) having the nucleic acid sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3', represented herein as SEQ ID NO: 100.
  • the resultant PCR product was gel purified and cloned mto the TA Vector 1 ⁇ . The clones were subjected to nucleic acid sequencing as described above.
  • a nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP33 77p is represented herein as SEQ ID NO: 84.
  • SEQ ID NO: 84 mcludes a portion of SEQ ID NO: 82
  • Translation of SEQ ID NO: 84 suggests that nucleic acid molecule nfSP33 778 encodes a non-full-length flea serine protease protem of about 259 amino acids, referred to herein as PfSP33 259 , havmg ammo acid sequence SEQ ID NO: 85, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 84.
  • a Genbank homology search revealed most homology between SEQ ID NO: 84 and a Drosophila serine protease stubble gene, there being about 54% identity between nucleotides 23 - 778 of SEQ ID NO: 84 and nucleotides 2324 - 3064 of the Drosophila serine protease stubble gene.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a cDNA clone of a flea serine protease was obtained usmg mRNA isolated from bovme blood-fed whole fleas.
  • the resulting cDNA was used as a template in PCR amplification using the primers cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR products were gel purified and cloned mto the TA VectorTM.
  • TA vector clone was isolated and the flea serine protease nucleic acid molecule and denoted nFS37 50C was subjected to nucleic acid sequencing as described in Sambrook et al . , ibid.
  • nucleic acid sequence of part of the flea serine protease nucleic molecule nFS37 500 is represented herem as SEQ ID NO: 144.
  • Translation of SEQ ID NO: 144 suggests that nucleic acid molecule nfSP37 z61 encodes a non-full-length sequence of a flea serine protease protem of about 87 ammo acids, referred to herein as
  • PfSP37 cr having ammo acid sequence SEQ ID NO: 145, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 144.
  • a Genbank homology search revealed most homology between SEQ ID NO: 145 and a chicken trypsmogen protem sequence, there being about 31% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the cloning and sequencing of certain larval flea serine protease nucleic acid molecules.
  • Certain serine protease cDNA nucleic acid molecules have been isolated from a mixed instar larval cDNA library produced using 1st, 2nd and 3rd instar larvae fed on cat blood, by PCR amplification.
  • the actual primers used in the PCR amplification included either cat-try #2 (SEQ ID NO: 86) m combination with either H57 primer (SEQ ID NO:99)or M13 reverse primer (SEQ ID NO:87) .
  • the resultant PCR products were gel purified and cloned into the TA Vector '1 '' 1 .
  • Three recombinant TA vector clones were isolated containing PCR products using cat-try #2 and M13 reverse as primers and one clone was isolated containing PCR products usmg cat-try #2 and H57 primers. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing as described above.
  • a nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP29 61z is represented herein as SEQ ID NO: 146.
  • Translation of SEQ ID NO: 146 suggests that nucleic acid molecule nfSP29 612 encodes a close to full-length flea serine protease protein of about 204 ammo acids, referred to herein as PfSP29 204 , having amino acid sequence SEQ ID NO: 147, assuming an open reading frame m which the first codon spans from about nucleotide 10 through about nucleotide 12 of SEQ IDNO: 146.
  • a Genbank homology search revealed most homology between SEQ ID NO: 146 and a rat trypsinogen gene, there being about 50% identity between corresponding regions of the two nucleic acid molecules.
  • SEQ ID NO: 148 Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP30 641 , is represented herein as SEQ ID NO: 148.
  • Translation of SEQ ID NO: 148 suggests that nucleic acid molecule nfSP30 64: encodes a non-full-length flea serine protease protein of about 213 amino acids, referred to herein as PfSP30 213 , having amino acid sequence SEQ ID NO: 149, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 148.
  • a Genbank homology search revealed most homology between SEQ ID NO: 148 and a Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • C Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP31 626 , is represented herein as SEQ ID NO: 150.
  • nucleic acid molecule nfSP31 62t encodes a non-full-length flea serine protease protein of about 208 amino acids, referred to herein as PfSP31 208 , having ammo acid sequence SEQ ID NO: 151, a assuming the f_.rst residue spans from about nucleotide 3 through about nucleotide 5 or from a putative start codon spanning from about nucleotide 6 to about nucleotide 8 of SEQ ID NO: 150.
  • a Genbank homology search revealed homology between SEQ ID NO: 150 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • Translation of SEQ ID NO: 80 suggests that nucleic acid molecule nfSP32 4 , 3 encodes a non-full-length flea serine protease protem of about 144 ammo acids, referred to herein as PfSP32 144 , having amino acid sequence SEQ ID NO: 81, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 80.
  • a Genbank homology search revealed most homology between SEQ ID NO: 80 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a bovme blood-fed whole flea cDNA library (prepared as described in Example 8 of related PCT Publication No. WO 96/11706' was immunoscreened with antiserum collected from a rabbit that was immunized with a collection of flea salivary gland products referred to as fspN (as described m PCT Publication No. WO 96.11271, entitled “NOVEL ECTOPARASITE SALIVA PROTEINS AND APPARATUS TO COLLECT SUCH PROTEINS", published April 18, 1996) . Immunoscreenmg was performed as follows.
  • New Zealand White rabbit antiserum developed agamst fspN flea saliva products was used m the immunoscreenmg protocols described in the picoBlueTM Immunoscreenmg Kit instruction manual, available from Stratagene, Inc.
  • the methods for preparation of the cDNA expression libraries for immunoscreenmg i.e., expression of the cDNA clones and procedures for transferring lambda phage plaques to membranes for immunoscreenmg, are described in the ZAP-cDNA Synthesis Kit instruction manual, also available from Stratagene, Inc., La Jolla, California.
  • a nucleotide sequence for a flea serine protease nucleic acid molecule named nfSP15 815 is denoted as SEQ ID NO: 152 and corresponds to SEQ ID NO: 154.
  • Translation of SEQ ID NO: 152 suggests that nucleic acid molecule nfSP15 8)C encodes a close to full-length flea serine protease protem of about 254 ammo acids, referred to herein as PfSP15 2C , 4 , having ammo acid sequence SEQ ID NO: 153, assuming an open reading frame in which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 152 and a stop codon spanning from about nucleotide 763 through about nucleotide 765 of SEQ ID NO: 152.
  • a Genbank homology search revealed homology between SEQ ID NO: 152 and an Anophel es gambiae trypsin gene,
  • This example describes the cloning and sequencing of additional flea serine protease nucleic acid molecules.
  • flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen an unfed flea cDNA expression library, nfSP8 299 (SEQ ID NO: 127) and nfSP19 359 (SEQ ID NO: 155) .
  • a clone that hybridized strongly to the probes was isolated and subjected to nucleic acid sequencing as described above.
  • SEQ ID NO: 156 The nucleic acid sequence of the flea serine protease nucleic molecule, namely nfSP19 855 , is represented herein as SEQ ID NO: 156.
  • SEQ ID NO: 155 is withm the sequence of the nucleic acid molecule nfSP19 855 Translation of SEQ ID NO: 156 yields an apparent full-length protem of about 253 ammo acids, denoted PfSP19 253 , having amino acid sequence SEQ ID NO: 157, assuming the first codon, an apparent start codon, spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 156.
  • a Genbank homology search revealed most homology between SEQ ID NO: 156 and an Aedes aegypti trypsin, there being about 53 identity between corresponding regions of both nucleic acid molecules .
  • SEQ ID NO: 158 The nucleic acid sequence of another flea serine protease nucleic molecule, namely nfSP25 8 €4 , is represented herein as SEQ ID NO: 158.
  • Translation of SEQ ID NO: 158 yields a protem of about 260 ammo acids, denoted PfSP25 260 , having ammo acid sequence SEQ ID NO: 159, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 158 and a stop codon spanning from about nucleotide 782 through about nucleotide 784 of SEQ ID NO: 159.
  • a Genbank homology search revealed most homology between SEQ ID NO: 159 and an Anophel es gambiae chymotrypsm protem sequence, there being about 34% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
  • a flea serine protease cDNA nucleic acid molecule has been isolated m a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, usmg nfSPll 25i (SEQ ID NO: 121) as a probe to screen an bovme blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706, .
  • a clone that hybridized strongly to the probe was isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described m Sambrook et al., ibid.
  • the nucleic acid sequence of the flea serine protease nucleic molecule namely nfSP21 595 , is represented herein as SEQ ID NO: 160.
  • Translation of SEQ ID NO: 160 yields a protem of about 198 ammo acids, denoted PfSP21 ⁇ 98 , having ammo acid sequence SEQ ID NO: 161, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 160 ⁇ nd a putative stop codon spanning from about nucleotide 596 to about nucleotide 598.
  • Genbank homology search revealed most homology between SEQ ID NO: 161 and Tachypl eus t ⁇ denta tus coagulation factor G protem sequence, there being about 45% identity between corresponding regions of the two ammo acid sequences.
  • This example describes the isolation and characterization of a 31 kD flea serine protease.
  • Guts from about 1500 fleas that had been fed on cat blood for about 24 hours were dissected m Gut Dissection Buffer (50 mM Tris 8.0, 100 mM CaCl 2 ) .
  • the guts were disrupted by freezing and thawing 4 times, followed by sonication.
  • the resulting extracts were clarified by centrifugation for 20 minutes at 14,000 rpm in a microfuge at 4 °C. The supernatant was recovered.
  • the gut supernatant was loaded onto a 3 ml column comprising p-aminobenzamidine cross-linked to Sepharose beads (Sigma) , previously equilibrated in Benzamidine Column Buffer (50 mM Tris 8.0, 100 mM CaCl 2 , 400 mM NaCl) . The supernatant was incubated on the column for about 10 min. Unbound protein was slowly washed off the column using Benzamidine Column Buffer until no protein was detectable by Bradford Assay (Bio Rad) .
  • Proteases bound to the benzamidine column were then eluted using 10 ml Benzamidine Column Buffer supplemented with 10 mM p-aminobenzamidine (brought to pH 8.0 with NaOH) .
  • Proteases in the eluant were concentrated and diafiltered into a volume of about 0.3 ml Gut Dissection Buffer using a Microcon 3 concentrator (Amicon) .
  • the membrane was stained with Coomassie Brilliant Blue. A dominant protem band of about 31 kDa was visualized. The membrane was then used for automated N-terminal sequencing (described in Example 7 of related PCT Publication No. WO 96/11706) .
  • a partial N-terminal amino acid sequence of the flea protease was determined to be IVGGEDVDISTCGWC (denoted SEQ ID NO: 68) .
  • This example describes the isolation and characterization of a 31 kD flea serine protease contained in a formulation having IgGase activity (i.e., ability to proteolyze immunoglobulin G proteins) .
  • Example 13 Cat blood-fed flea gut extracts were prepared and selected on a benzamidine column as described above in Example 13. IgG protease activity was assayed by incubating at 37°C, overnight, the benzamidine eluant with cat immunoglobulin G proteins (IgG) purified on Protein A sepharose. The ability of the flea gut benzamidine eluant to digest cat IgG was detected by resolving the samples by gel electrophoresis through a 14% SDS-PAGE gel and silver staining the gel using standard methods.
  • IgG immunoglobulin G proteins
  • the benzamidine eluant was then purified on a PolyPropylaspartamide hydrophobic interaction chromatography (HIC) column by applying the eluant to the column in buffer containing 0.1 M KP0 4 , pH 6.5 and 2 M (NH 4 ) SO,. Proteases bound to the column were eluted using an ammonium sulfate gradient of 2 M to 0 M in HIC column buffer. Column fractions were tested for IgG protease activity using the method described above.
  • HIC PolyPropylaspartamide hydrophobic interaction chromatography
  • Fractions containing IgG protease activity were pooled and applied to a PolyCat cation exchange column m 20 M sodium acetate, pH 6. The proteins were eluted using a sodium chloride gradient of 0 M to 1 M NaCl in 20 M sodium acetate. Fractions eluted from the column were tested for IgG protease activity and then each fraction was resolved by electrophoresis using SDS-PAGE. Fractions having the highest levels of IgG protease activity included a protem band that migrated at about 31 kDa on the SDS-PAGE gel. Weaker protease activity corresponded to an about 28 kDa band.
  • the 31 kDa protem present on the SDS-PAGE gel was used for N-terminal amino acid sequencing using the blotting method described above.
  • a partial N-terminal ammo acid sequence was determined to be IVGGEDVDIST (C)GWQI (S) FQ (S) ENLHF (C) GG (S) IIAPK (denoted herein as SEQ ID NO: 69) .
  • a comparison of SEQ ID NO: 69 and SEQ ID NO: 68 indicates a single residue difference between the two ammo acid sequences at residue 15 of each sequence (i.e., Q and C, respectively) . Since SEQ ID NO: 69 correlates with IgGase activity, the data suggests that the larval protem containing SEQ ID NO: 68 has IgGase activity.
  • This example describes the cloning and sequencing of a 31 kDa flea serine protease contained in a formulation having IgGase activity.
  • a flea protease nucleic acid molecule was isolated from a cat blood-fed whole flea library (described m Example 6) and a bovme blood-fed whole flea library (described m Example 8 of related PCT Publication No. WO 96/11706) by PCR amplification.
  • the actual primers used m the PCR amplification included FP31A primer designed using the N-termmal ammo acid sequence SEQ ID NO: 68, the primer having the nucleic acid sequence 5' GAA GAT GTW GAT ATT TCW ACA TGT GG 3' (SEQ ID NO: 101) used in combination with the M13 universal primer.
  • the resultant PCR products were gel purified and cloned into the TA Vector 1 *' and subjected to nucleic acid sequencing as described above.
  • a FP31B primer (5' GAA AAT GAA ATC CAC TTA AAC ATT ACG 3'), (represented herein as SEQ ID NO: 102) was designed using the DNA sequence of a DNA fragment from a bovme blood-fed cDNA library.
  • a flea protease cDNA nucleic acid molecule was isolated by PCR amplification of the cat blood-fed whole flea library and the bovine blood-fed whole flea library described above by PCR amplification. PCR amplification was performed using the FP31B primer in combination with M13 reverse primer.
  • PCR products were then diluted 1:25, and used as a template for a second PCR reaction using primer FP31C, having the sequence 5' CTC TTA TTG TAC GAG GGA TGC 3' (denoted herein SEQ ID NO: 103) in combination with T3 primer.
  • primer FP31C having the sequence 5' CTC TTA TTG TAC GAG GGA TGC 3' (denoted herein SEQ ID NO: 103) in combination with T3 primer.
  • the resulting nested PCR product was cloned into TA VectorTM and subjected to DNA sequencing.
  • SEQ ID NO: 66 The nucleic acid sequence of the resulting flea serine protease nucleic molecule, namely nfSP28 923 is represented herein as SEQ ID NO: 66.
  • Translation of SEQ ID NO: 66 yields a protein of about 267 amino acids, denoted PfSP28 267 , having amino acid sequence SEQ ID NO: 67, assuming an open reading frame in which the putative start codon spans from about nucleotide 8 through about nucleotide 10 of SEQ ID NO: 66 or from about nucleotide 11 through about nucleotide 13, and a stop codon spanning from about nucleotide 803 through about nucleotide 805 of SEQ ID NO: 66.
  • SEQ ID NO: 67 contains SEQ ID NO: 68 except Q is substituted for C, and SEQ ID NO: 69.
  • a Genbank homology search revealed most homology between SEQ ID NO: 66 and Bombix mori vitellin- degrading protease gene, there being about 53% identity between corresponding regions of the two nucleic acid sequences .
  • This example describes J H-DFP labelling of larval serine proteases.
  • Fig.l, lane B and fed 3rd instar larvae (Fig.l, lane C) produce serine proteases.
  • fed 1st instar larvae primarily produce a serine protease having a molecular weight of about 25 kD; and fed 3rd mstar larvae produce about serine proteases having molecular weights of about 25 kD, 28 kD and 31 kD.
  • the approximate size of standard molecular weight protem markers are shown in Fig.
  • This example describes the determination of partial N- terminal ammo acid sequences for several larval serine proteases.
  • the supernatant was rocked with the beads overnight at 4°C.
  • the beads were washed m about 45 ml Benzamidine Column Buffer to remove unbound protem.
  • the beads were then mixed 2 hours at 4°C with about 10 ml of Benzamidine Column Buffer containing 100 mM p-ammobenzamidme (pH 8.0 adjusted with NaOH) to elute proteins bound to the beads.
  • the eluted proteins were then collected. The elution process was repeated once more.
  • the eluted protem was concentrated by ultrafiltration with a Centriprep 10 concentrator (available from Amicon) .
  • the concentrate was diluted with Gut Dissection Buffer to a final volume of about 5 ml.
  • Partial N-termmal ammo acid sequence of proteins eluted from the beads was obtained using the method described m Example 11. Two proteins having molecular weights of about 25 kDa and about 26 kDa were identified on the Coomassie Brilliant Blue stained membranes. Partial N- termmal ammo acid sequence obtained for the protem having a molecular weight of about 25 kDa is IVGGVSVNINDYGYQLSLQSNGR, denoted herein as SEQ ID NO: 162. Partial N-termmal ammo acid sequence obtained for the protem having a molecular weight of about 26 kDa is IVGGHDTSIKQHPYQV, denoted herein as SEQ ID NO: 163.
  • Flea serine protease protem PfSPl 21 was m the following manner.
  • Flea serine protease nucleic acid molecule nfSPl 670 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector ⁇ P R /T 2 ori/S10HIS-RSET-A9 (the production of which is described in Tripp et al,
  • Example 7 The resultant recombinant molecule, referred to herein as pHisCro-nfSPl 670 , was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP1 670 .
  • the recombinant cell was cultured as described in Example 20 of related PCT
  • PfSPl 2U w as purified by nickel chelation chromatography followed by reverse phase high performance liquid chromatography (HPLC) .
  • Immunoblot analysis of the purified PfSPl 216 indicated that rabbit anti-flea protease antiserum, produced as described in example 14, selectively bound to PfSPl 21b .
  • Flea serine protease protein PfSP2 233 was produced in the following manner.
  • Flea serine protease nucleic acid molecule nfSP2-, 15 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector ⁇ P [ ⁇ /T * -o.r. ⁇ /S10HIS-RSET-A9 as described in Example 39A.
  • the resultant recombinant molecule referred to herem as pH ⁇ sCro-nfSP2-, t , was transformed mto E.
  • Flea serine protease protein PfSP13 225 was produced m the following manner.
  • Flea serine protease nucleic acid molecule nfSP13 700 produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned into expression vector ⁇ P R /T 2 or ⁇ /S10HIS-RSET-A9 as described in Example 18A.
  • the resultant recombinant molecule referred to herein as pH ⁇ sCro-nfSP13 700 , was transformed mto E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E.
  • PfSP13 22r Flea serine protease protein PfSP13 22r was purified by nickel chelation chromatography followed by reverse phase HPLC. Immunoblot analysis of the purified PfSP13 22b indicated that rabbit anti-flea protease antiserum, produced as described in Example 14 of related PCT Publication No. WO 96/11706, selectively bound to PfSP13 225 .
  • Flea serine protease protein PfSP20 222 was produced in the following manner.
  • An about 669-bp DNA fragment, referred to herein as nfSP20 569 was PCR amplified from flea serine protease clone 20 using the Xhol-site containing primer F27-S (sense) 5' GAG CTC TCG AGA ATC GTA GGA GGA CAC GAT AC 3' (SEQ ID NO: 164) and the EcoRI-site containing primer F20-A (antisense) 5' G GAC GAA TTC TTA AAC ACC AGA CAC TTC CTT G 3' (SEQ ID NO: 165) .
  • the PCR product nfSP20 669 was digested with Xhol and EcoRI restriction endonucleases, gel purified and subcloned into expression vector ⁇ P R /T 2 ori/S10HIS-RSET-A9 as described in Example 18A.
  • the resultant recombinant molecule referred to herein as pHisCro-nfSP20 669 , was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP20 669 .
  • the recombinant cell was cultured as described in Example 20 of related PCT Publication No. WO 96/11706.
  • This example describes that various flea serine protease nucleic acid molecules described in the foregoing examples can be obtained from multiple sources.
  • Nucleic acid molecules correspondmg to flea clone 4 have been obtained from a bovme blood-fed whole flea library (described in Example 8 of related PCT Publication
  • Nucleic acid molecules corresponding to flea clone 5 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 6 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library and an unfed whoie flea library.
  • Nucleic acid molecule correspondmg to flea clone 7 have been obtained from a bovme blood-fed whole flea library, and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 8 have been obtained from a bovme blood-fed whole flea library and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 12 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 13 have been obtained from a bovine blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 20 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library.
  • Nucleic acid molecules corresponding to flea clone 28 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
  • This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding a flea cysteine protease protem of the present which was described m Example 3. This example also provides the production of a cysteine protease protein in E . coli cells.
  • A. Additional Cysteine Protease Nucleic Acid Molecule The PCR products described in Example 3 were submitted to additional nucleic acid sequence analysis in order to obtain the nucleic acid sequence of additional portions of the coding region of the cysteine protease gene.
  • SEQ ID NO: 76 is contained within the sequence of the nucleic acid molecule nfCPl U09.
  • nucleic acid molecule nfCPl 1109 encodes a full- length flea cysteine protease protein of about 327 amino acids, referred to herein as PfCPl 327 , having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from about nucleotide 126 through about nucleotide 128 of SEQ ID NO:l and the termination codon spans from about nucleotide 1107 through about nucleotide 1109 of SEQ ID NO:l.
  • the complement of SEQ ID NO:l is represented herein by SEQ ID NO: 3.
  • the coding region encoding PfCPl 327 is represented by nucleic acid molecule nfCPl 984 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 4 and a complementary strand with nucleic acid sequence SEQ ID NO: 6.
  • the proposed mature protem, denoted herein as PfCPl 2 ⁇ contains about 226 ammo acids which is represented herein as SEQ ID NO: 8.
  • the nucleic acid molecule encoding PfCPl is denoted herem as nfCPl 68 , which is represented by SEQ ID NO:7.
  • the ammo acid sequence of PfCPl 3 ⁇ - (i.e., SEQ ID NO: 2) predicts that PfCPl 32 -, has an estimated molecular weight of about 42 kD and an estimated pi of about pi 8.84.
  • SEQ ID NO:l Comparison of nucleic acid sequence SEQ ID NO:l with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:l showed the most homology, i.e., about 55% identity, with the following three genes: a Drosophila cysteine protease gene, a BomJbyx cysteine protease gene and a Sarcophaga cysteine protease gene.
  • SEQ ID NO:2 i.e., the ammo acid sequence of PfCPl 3 _-
  • SEQ ID NO:2 showed the most homology, i.e., about 42- identity, with the following three proteins: a Drosophila cysteine protease protem, a BomJbyx cysteine protease protem and a Sarcophaga cysteine protease protein.
  • nfCPl b60 designed to encode an apparently mature cysteine protease protem
  • nfCPl b60 was PCR-amplifled from a flea mixed instar cDNA library produced usmg unfed 1st instar, bovme blood-fed 1st instar, bovme blood-fed 2 n ⁇ instar and bovme blood-fed 3- d instar flea larvae (this combination of tissues is referred to herein as mixed instar larval tissues for purposes of this example) .
  • Total RNA was extracted from mixed instar tissue using an acid- guanidmium-phenol-chloroform method similar to that described by Chomczynski et al .
  • RNA preparations were used in each RNA preparation.
  • Poly A+ selected RNA was separated from each total RNA preparation by oligo-dT cellulose chromatography using Poly(A) Quick® mRNA isolation kits (available from Stratagene Cloning Systems, La Jolla, CA) , according to the method recommended by the manufacturer.
  • a mixed instar cDNA expression library was constructed m lambda ( ⁇ ) Uni-ZAP XR vector (available from Stratagene Cloning Systems) using Stratagene' s ZAP-cDNA Synthesis Kit® protocol. About 6.34 ⁇ g of mixed instar poly A+ RNA were used to produce the mixed instar library.
  • the resultant mixed instar library was amplified to a titer of about 2.17 x 10 10 pfu/ml with about 97% recombmants.
  • the primers used in the PCR amplification were sense primer CysBS ' having the nucleotide sequence 5' GAT AAG GAT CCG TTA CCA GAT TCT TTC GAC TGG 3' (containing a BamHI-site; denoted SEQ ID NO: 64) and anti-sense primer CysHA having the nucleotide sequence 5' TTA TCA AGC TTC CAT TTA CAT GCC GTA AAA ATC 3' (containing a Hmdlll site; denoted SEQ ID NO: 65) .
  • nfCPl DD was submitted to nucleic acid sequence analysis to obtain a nucleic acid sequence of the coding strand, represented herein as SEQ ID NO: 94.
  • Translation of SEQ ID NO: 94 indicated that nfCPl bqr encodes a protem of about 220 ammo acids, called PfCPl 220 , having SEQ ID NO: 95. It is to be noted that this sequence analysis indicated that the stop codon was actually about 36 base pairs upstream from what had been predicted by SEQ ID NO:l; as such, the protem encoded by nfCPl 6c ⁇ is about 12 ammo acids shorter than would have been predicted by SEQ ID NO:l.
  • the nucleic acid molecule nfCPl 660 contains the coding region for PfCPl 220 .
  • Recombinant cell E. coli:pCro-nfCP1 6D0 was produced in tne following manner.
  • Nucleic acid molecule nfCPl b60 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector IambdaP R /T 2 o ⁇ /S10HIS-RSET-A9 (the production of which is described m Tripp et al, International PCT Publication No. WO 95/24198, published September 14, 1995; see m particular, Example 7), that was digested with BamHl and HindiII and dephosphorylated.
  • the resultant recombinant molecule referred to herem as pCro-nfCPl 6bC
  • pCro-nfCPl 6bC was transformed mto E. coli BL-21 competent cells 'available from Novagen, Madison, WI) to form recombinant cell E. coli:pCro-nfCPl 6b0 .
  • the recombinant cell was cultured as described m Example 20 of related PCT Publication No. WO 95/24198. About 1 ml of culture was collected prior to induction, and about 1 ml of culture was collected about 60 minutes following induction.
  • Example 21 This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding serine protease proteins of the present invention which are described herein and in the Examples section of related PCT Publication No.WO 96/11706.
  • a DNA probe labeled with 32 P comprising nucleotides from nfAP2 210C (described m Example 23 of related U.S. Patent Application Serial No. 08/639,075, filed April 24, 1996) was used to screen a bovine blood-fed whole flea cDNA library (described in Example 8 of related PCT Publication No.WO 96/11706) using standard hybridization techniques.
  • a clone was isolated having about a 459-nucleot ⁇ de insert, referred to herein as nfSP18 4- Struktur.
  • a nucleic acid sequence of the composite nucleic acid molecule produced using nucleic acid sequence from nfSP16 53 . and nfSP18. tq is referred to herein as nfSPl ⁇ , 7 , having a nucleic acid sequence of the coding strand which is denoted herem as SEQ ID NO: 9.
  • SEQ ID NO: 9 nucleic acid molecule nfSPl ⁇ -,-,- encodes a non-full- length flea serine protease protem of about 228 ammo acids, referred to herein as PfSP18 228 , having ammo acid sequence SEQ ID NO: 10, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 9 and the stop codon spans from about nucleotide 685 through about nucleotide 687 of SEQ ID NO: 9.
  • the complement of SEQ ID NO: 9 is represented herem by SEQ ID NO: 11.
  • the coding region encoding PfSP18 22 ⁇ is represented by nucleic acid molecule nfSP18 22 volunteer, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 12 and a complementary strand with nucleic acid sequence SEQ ID NO: 14.
  • the ammo acid sequence of PfSP18 228 (i.e., SEQ ID NO: 10) predicts that PfSP18 228 has an estimated molecular weight of about 25 kD and an estimated pi of about 9.09.
  • Comparison of nucleic acid sequence SEQ ID NO: 9 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 9 showed the most homology, i.e., about 51% identity, between SEQ ID NO: 9 and an Anophel es st ephensi trypsin 1 gene.
  • Comparison of ammo acid sequence SEQ ID NO: 10 i.e., the ammo acid sequence of PfSP18 22r
  • ammo acid sequences reported in GenBank indicates that SEQ ID NO: 10 showed the most homology, i.e., about 59? identity between S ⁇ Q ID NO: 10 and Vespa crabro protein.
  • flea serine protease nucleic molecule clone 24 (described in Example 6 was determined using primers designed from nfSP24 410 to amplify DNA from the bovine biood-fed whole flea cDNA library.
  • Sense primer Flea 24F having the nucleotide sequence 5' GGA CAA ACT GTT CAT TGC AG 3' (denoted SEQ ID NO: 46) was used in combination with the M13 universal primer in a first PCR reaction.
  • Anti-sense primer Flea 24R having the nucleotide sequence 5' CCC TCA TTT GTC GTA ACT CC 3' (denoted SEQ ID NO: 47) was used in combination with the M13 reverse primer in a second PCR reaction.
  • the resulting PCR products were each gel purified and cloned into the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • nfSP24 :089 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP24 :089 , was deduced and is denoted herein as SEQ ID NO: 15.
  • SEQ ID NO:78 is contained within the sequence of the nucleic acid molecule nfSP24 1089 _ Translation of SEQ ID NO: 15 suggests that nucleic acid molecule nfSP24 1089 encodes a full-length flea serine protease protein of about 258 amino acids, referred to herein as PfSP24 2f)8 , having amino acid sequence SEQ ID NO: 16, assuming an open reading frame in which the initiation codon spans from about nucleotide 33 through about nucleotide 35 of SEQ ID NO: 15 and the termination codon spans from about nucleotide 807 through about nucleotide 809 of SEQ ID NO: 15.
  • SEQ ID NO: 15 The complement of SEQ ID NO: 15 is represented herem by SEQ ID NO: 17.
  • the coding region encoding PfSP24 258 is represented by nucleic acid molecule nfSP24 774 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 18 and a complementary strand with nucleic acid sequence SEQ ID NO: 20.
  • the proposed mature protem, denoted herein as PfSP24 237 contains about 237 ammo acids which is represented herein as SEQ ID NO:22.
  • the nucleic acid molecule encoding PfSP24 23 -7 is denoted herein as nfSP24 7n , which is represented by SEQ ID NO: 21.
  • the ammo acid sequence of PfSP24 258 i.e., SEQ ID NO: 16 predicts that PfSP24 258 has an estimated molecular weight of about 28 kD and an estimated pi of about pi 6.70.
  • SEQ ID NO: 15 showed the most homology, i.e., about 51% identity between SEQ ID NO: 15 and an Anopheles stephensi trypsin 1 gene. Comparison of ammo acid sequence SEQ ID NO: 15
  • Anti- sense primer Flea 32R having the nucleotide sequence 5' GCA AAT CAG TTC CAG AAT CCA CTA ACC 3' was used m combination with the M13 reverse primer m a second PCR reaction.
  • the resultmg PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
  • SEQ ID NO:23 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to nerem as nfSP32 924 , was deduced and is denoted herein as SEQ ID NO:23.
  • SEQ ID NO: 80 is contained withm the sequence of the nucleic acid molecule nfSP32 924 Translation of SEQ ID NO:23 suggests that nucleic acid molecule nfSP32 9: consult encodes a full-length flea serine protease protem of about 268 ammo acids, referred to herein as PfSP32 268 , havmg ammo acid sequence SEQ ID NO: 24, assuming an open reading frame in which the initiation codon spans from about nucleotide 6 through about nucleotide 8 of SEQ ID NO: 23 and the termination codon spans from about nucleotide 810 through about nucleotide 812 of SEQ ID NO:23.
  • SEQ ID NO:25 The complement of SEQ ID NO:23 is represented herein by SEQ ID NO:25.
  • the coding region encoding PfSP32 268 is represented by nucleic acid molecule nfSP32 b0q , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 26 and a complementary strand with nucleic acid sequence SEQ ID NO.28.
  • the ammo acid sequence of PfSP32 26p i.e., SEQ ID NO: 24 predicts that PfSP32 268 has an estimated molecular weight of about 28.6 kD and an estimated pi of about pi 7.36.
  • Comparison of nucleic acid sequence SEQ ID NO:23 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 23 showed the most homology, i.e., about 52% identity between SEQ ID NO:23 and a Fusari um oxysporum preprotrypsm gene.
  • Comparison of ammo acid sequence SEQ ID NO: 24 (i.e., the ammo acid sequence of PfSP32 268 ) with ammo acid sequences reported in GenBank indicates that SEQ ID NO:24 showed the most homology, i.e., about 63% identity between SEQ ID NO:24 and a Bombyx mon vitellm -degrading protease precursor protem. D.
  • the remainder of flea serine protease nucleic molecule clone 33 was determined using primers designed from nfSP33-, 78 to amplify DNA from the flea mixed instar larvae cDNA library described above in Example 20.
  • Sense primer Flea 33F having the nucleotide sequence 5' CAG GGC GCT CTG CAG AAC GCA AC 3' (denoted SEQ ID NO: 50) was used in combination with the M13 universal primer m a first PCR reaction.
  • Anti-sense primer Flea 33R having the nucleotide sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3' was used m combination with the M13 reverse primer m a second PCR reaction.
  • the resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques .
  • nfSP33 1894 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP33 1894 , was deduced and is denoted herein as SEQ ID NO: 29.
  • SEQ ID NO: 84 and SEQ ID NO: 82 are contained withm the sequence of the nucleic acid molecule nfSP33, 894 _ Translation of SEQ ID NO:29 suggests that nucleic acid molecule nfSP33 1894 encodes a full-length flea serine protease protem of about 400 ammo acids, referred to herein as PfSP33 400 , having amino acid sequence SEQ ID NO: 30, assuming an open reading frame m which the initiation codon spans from about nucleotide 335 through about nucleotide 337 of SEQ ID NO:29 and the termination codon spans from about nucleotide 1535 through about nucleotide 1537 of SEQ ID NO:29.
  • SEQ ID NO: 31 The complement of SEQ ID NO:29 is represented herein by SEQ ID NO: 31.
  • the coding region encoding PfSP33 400 is represented by nucleic acid molecule nfSP33 1200 , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 32 and a complementary strand with nucleic acid sequence SEQ ID NO: 34.
  • the proposed mature protein, denoted herein as PfSP33 4 contains about 242 ammo acids which is represented herein as SEQ ID NO: 36.
  • the nucleic acid molecule encoding PfSP33. 4 is denoted herein as nfSP33 72b , which is represented by SEQ ID NO: 35.
  • the ammo acid sequence of PfSP33 40C i.e., SEQ ID NO:30 predicts that PfSP33 400 has an estimated molecular weight of about 44 kD and an estimated pl of about pi 7.59.
  • Comparison of nucleic acid sequence SEQ ID NO:29 with nucleic acid sequences reported m GenBank indicates that SEQ ID NO: 29 showed the most homology, i.e., about 48% identity between SEQ ID NO:29 and a Drosophil a melanogaster serine protease stubble gene.
  • Comparison of ammo acid sequence SEQ ID NO: 30 (i.e., the amino acid sequence of PfSP33 400 ) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 30 showed the most homology, i.e., about 63 identity between SEQ ID NO: 30 and a Drosophila melanogaster serine protease stubble protem.
  • Example 22 This example provides nucleic acid and deduced ammo acid sequence of another nucleic acid molecule encoding a serine protease protem of the present invention.
  • a serine protease cDNA nucleic acid molecules has been isolated in a manner similar to that described in Example 8 of related PCT Publication No.No.WO 96/11706.
  • the actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed flea cDNA expression library included cat-try #2 (SEQ ID NO: 86) m combination with H57 primer (SEQ ID NO: 99) .
  • the resultant PCR product was gel purified and cloned into the TA VectorTM.
  • a recombinant TA vector clone was isolated and subjected to nucleic acid sequencing.
  • SEQ ID NO: 37 A composite nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 40, namely nfSP40 428 was deduced and is denoted herem as SEQ ID NO: 37.
  • Translation of SEQ ID NO: 37 suggests that nucleic acid molecule nfSP40 428 encodes a non-full-length flea serine protease protem of about 142 ammo acids, referred to herein as PfSP40 142 , represented herem by SEQ ID NO: 38.
  • the complement of SEQ ID NO: 37 is represented herem by SEQ ID NO: 39.
  • the remainder of flea serine protease nucleic molecule clone 40 was determined using primers designed from nfSP40 4 _ 8 to amplify DNA from the cat blood-fed whole flea cDNA library.
  • Sense primer Flea 40F having the nucleotide sequence 5' GGC AAG TTT CGT TTC ACA ATA GG 3' (denoted SEQ ID NO: 52) was used in combination with the M13 universal primer in a first PCR reaction.
  • Anti-sense primer Flea 40R having the nucleotide sequence 5' TCC AAC CCT AAC TTT TAA ACC TTC 3' (denoted SEQ ID NO: 53) was used in combination with the M13 reverse primer m a second PCR reaction.
  • nfSP40 841 A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP40 841 , was deduced and is denoted herein as SEQ ID NO: 40.
  • SEQ ID NO: 37 is contained withm the sequence of the nucleic acid molecule nfSP40 841 Translation of SEQ ID NO: 40 suggests that nucleic acid molecule nfSP40 841 encodes a non-full-length flea serine protease protem of about 242 ammo acids, referred to herein as PfSP40 24i , havmg ammo acid sequence SEQ ID NO: 41, assuming an open reading frame in which the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 40 and tne termination codon spans from about nucleotide 728 through about nucleotide 730 of SEQ ID NO: 40.
  • SEQ ID NO:42 The complement of SEQ ID NO: 40 is represented herein by SEQ ID NO:42.
  • the coding region encoding PfSP40 242 is represented by nucleic acid molecule nfSP40 7 ⁇ , having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 43 and a complementary strand with nucleic acid sequence SEQ ID NO: 45.
  • the amino acid sequence of PfSP40 242 i.e., SEQ ID NO:41 predicts that PfSP40 242 has an estimated molecular weight of about 26 kD and an estimated pl of about pi 6.5.
  • SEQ ID NO: 40 Comparison of nucleic acid sequence SEQ ID NO: 40 with nucleic acid sequences reported m GenBank indicates that
  • SEQ ID NO: 40 showed the most homology, i.e., about 57% identity between SEQ ID NO: 40 and a Derma tophagoi des pteronyssmus Der P3 allergen gene.
  • Comparison of ammo acid sequence SEQ ID NO:41 (i.e., the ammo acid sequence of PfSP40_ 42 ) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 41 showed the most homology, i.e., about 40% identity between SEQ ID NO: 41 and a Bombyx mon vitellm-degradmg protease precursor protem.
  • nfSP24 714 (designed to encode an apparently mature serine protease protem) was PCR amplified from nfSP24 1089 usmg sense primer Flea 24 EF having the nucleotide sequence 5' CAC AGG ATC CAA TAA TTT GTG GTC AAA ATG C 3' (containing a BamHI-site; denoted SEQ ID NO: 54) and anti-sense primer Flea 24 ER havmg the nucleotide sequence 5' AAA AAG AAA GCT TCT TTA ATT TTC TGA CAT TGT CGT G 3' (containing a Hindlll; denoted SEQ ID NO: 55) .
  • nfSP24 714 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector lambdaP R /T-on/S10HIS-RSET-A9, that had been digested with BamHl and HindiII and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP24 714 , was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E.
  • the recombinant cell was cultured as described in Example 20 of related PCT Publication No.WO 95/24198.
  • nfSP32 b98 designed to encode an apparently mature serine protease protem
  • sense primer Flea 32 EF having the nucleotide sequence 5' GCG GGA TCC TAT TGT GGG TGG TGA AGC AGT G 3' (containing a BamHI-site; denoted SEQ ID NC:56) and anti-sense primer Flea 32 ER having the nucleotide sequence 5' GAC GGT ACC ATG TAT AAA ATA ATA TTA AAC TCC GG 3' (containing a Kpnl; denoted SEQ ID NO:57) .
  • nfSP32 698 was digested with BamHl and Kpn ⁇ restriction endonucleases, gel purified, and subcloned into expression vector 1 pTrcHisB (available from Ir.Vitrogen Corp., San Diego, CA) , that had been digested with BamHl and Kpnl and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pTrc-nfSP32 b98 , was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pTrc-nfSP32 698 .
  • the recombinant cell was cultured and protein production resolved by SDS- PAGE as described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the unmduced sample.
  • Flea serine protease protein PfSP33 400 was produced in the following manner.
  • An about 1200 bp nucleic acid molecule, referred to herein as nfSP33 1200 (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP33 1894 using sense primer Flea 33 EF having the nucleotide sequence 5' CCG GGA TCC TAT GTT AGC GAT CGT CCC GTC AAA C 3' (containing a BamHI-site; denoted SEQ ID NO: 58) and anti-sense primer Flea 33 ER having the nucleotide sequence 5' CCG GAA TTC TTA TCC CAT TAC TTT GTC GAT CC 3' (containing a EcoRI; denoted SEQ ID NO:59) .
  • nfSP33 1200 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned mto expression vector lambdaP R /T 2 ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP33 1200 , was transformed into £. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP33 1200 .
  • the recombinant cell was cultured using the method described above in Section A. D.
  • Flea serine protease protein PfSP40 242 was produced in the following manner.
  • An about 716 bp nucleic acid molecule, referred to herein as nfSP40 71b (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP40 841 using sense primer Flea 40 EF having the nucleotide sequence 5' GCG GGA TCC AAT AGT AGG AGG TGA AGA TGT AG 3' (containing a BamHI-site; denoted SEQ ID NO: 60) and anti-sense primer Flea 40 ER having the nucleotide sequence 5' CCG GAA TTC TTC TAA CAA ATT TTA TTT GAT TCC TGC 3' (containing a EcoRI; denoted SEQ ID N0:61) .
  • nfSP40 716 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T 2 ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP40 716
  • pCro-nfSP40 716 was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP40 7U - .
  • the recombinant cell was cultured and protein production resolved using the methods described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the uninduced samp1e.
  • This Example demonstrates the production of another serine protease protein of the present invention in E. coli cells.
  • nfSP28 71 An about 711 bp nucleic acid molecule, referred to herein as nfSP28 71 , (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP28 923 using sense primer Flea 28 F having the nucleotide sequence 5' GGA TCC AAT CGT TGG AGG TGA AGA TG 3' (containing a BamHI-site shown in bold; denoted SEQ ID NO: 62) and anti-sense primer Flea 28 R having the nucleotide sequence 5' GAA TTC GAA ATC CAC TTA AAC ATT AGC 3' (containing a EcoRI shown in bold; denoted SEQ ID NO: 63) .
  • nfSP28 7n was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaP R /T 2 ori/S10HIS-RSET-A9, that had been digested with BamHl and Xbal and dephosphorylated.
  • the resultant recombinant molecule referred to herein as pCro-nfSP28 ⁇ , was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E. coli :pCro-nfSP2 ⁇ n , .
  • the recombinant cell was cultured and protem production resolved using the methods described above m Example 20.
  • Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 36 kD protem in the induced sample but not m the unmduced sample.
  • Immunoblot analysis using a rabbit anti- flea midgut protease polyclonal antibody identified an about 38 kD protem in the induced sample.
  • Example 25 This Example demonstrates the production of another serine protease protem of the present invention m eukaryotic cells.
  • Recombinant molecule pBv-nfSP28 792 containing a flea serine protease nucleic acid molecule spanning nucleotides from about 11 through about 802 of SEQ ID NO: 66, operatively linked to baculovirus polyhedron transcription control sequences were produced m the following manner.
  • nfSP28- A PCR fragment of 792 nucleotides, named nfSP28-.
  • the N-termmal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
  • Bv-nfSP28-, 9Z the about 792 base pair PCR product
  • BamHl and Xbal subcloned mto BamHl and Xbal digested to produce the recombinant molecule referred to herein as pVL-nfSP28 79t .
  • the resultant recombinant molecule,pVL-nfSP28 792 was verified for proper insert orientation by restriction mapping.
  • the recombinant molecule was co-transfected with a linear Baculogold baculovirus DNA (available from Pharmmgen) mto S. frugiperda Sf9 cells (available from InVitrogen) to form the recombinant cells denoted 5.
  • frugiperda:pVL-nfSP28 -, 92 was cultured m order to produce a flea serine protease protem PfSP28 2b4 . Immunoblots of supematants from cultures of S .
  • frugiperda:pVL-nfSP28-, 92 cells producing the flea serine protease protem PfSP28 2b4 was performed using a cat anti- fSPFlea 26 polyclonal antibody which was produced as follows.
  • Recombinant Flea 28 protein (referred to herein as rSPFlea 28 protein) produced in E. coli described above in Example 24 was used to immunize cats.
  • the rSPFlea 28 protein was diluted to a concentration of about 1 mg/ml in PBS and emulsified in an equal volume of TiterMax research adjuvant (available from CytRx Corp., Norcross, GA) .
  • a series of cats were immunized each with about 50 ⁇ g of rSPFlea 28 protein in adjuvant by subcutaneous injection.
  • a second injection of the same dose of rSPFlea 28 protein in adjuvant was administered 32 days later.
  • Blood samples were obtained prior to immunization (pre-bleed) , 32 days and 47 days after the initial immunization.
  • Sera samples from the pre-immunization and Day 47 bleeds were used for subsequent immunoblot experiments. The latter is referred to as anti-fSPFlea 28 polyclonal antibody. Analysis of the immunoblots identified an about 33 kD protein and an about 36 kD protein.
  • This example describes the production of peptides from the 31 kD flea midgut serine protease and the generation of internal sequence data.
  • Midguts from about 30,000 cat blood-fed fleas were dissected as described m U.S. Patent No. 5,356,622, ibid.
  • m gut dissection buffer 50 mM Tris 8.0, 100 mM CaCl .
  • the guts (m three batches of about 10,000 each) were disrupted by a freeze-thaw cycle, followed by somcation.
  • the resulting extracts were clarified by centrifugation for 20 minutes at 3050 rpm m a swinging bucket centrifuge at 4°C.
  • the supematants were recovered, and adjusted to 400 mM NaCl m preparation for benzamidine column chromatography.
  • gut supematants were loaded into a 5 ml disposable column containing p-ammobenzamidme cross- linked to Sepharose beads (available from Sigma, St. Louis, MO) , previously equilibrated in benzamidine column buffer (50 mM Tris, pH 8.0, 100 mM CaCl 2 , 400 mM NaCl) and incubated with rocking overnight at 4°C. Unbound protem was slowly washed off the column using benzamidine column buffer until no protem was detectable by Bradford Assay (available from Bio-Rad Laboratories, Hercules, CA) .
  • Proteases bound to the benzamidine column were eluted using 4 ml benzamidine column buffer supplemented with 100 mM p-ammobenzamidme (brought to pH 8.0 with NaOH) .
  • Residual bound proteases were washed off with about 21 ml of additional benzamidine column buffer. The recovered proteases were then concentrated to a volume of about 2 ml using a Ultrafree 20 10-kD centrifugal concentrator
  • protease pools from the 3 preparations were combined for a total of about 30,000 gut equivalents m about 6 ml. Protem concentration was measured by Bradford assay and found to be about 0.5 mg/ml. About 150 ⁇ g of the isolated protease pool was resolved by polyacrylamide gel electrophoresis (PAGE) on a preparative-well 14% Tris-glycme gel (available from Novex, San Diego, CA) .
  • PAGE polyacrylamide gel electrophoresis
  • the proteins in the gel were visualized by staining for about 30 minutes in Coomassie brilliant blue stain (0.1% (w/v) Coomassie blue R, 40% (v/v) methanol, 10% (v/v) acetic acid) and oestammg for about 2.5 hours in 50% (v/v) methanol.
  • the band correspondmg to the 31-kD protease was excised with a razor blade.
  • the protem was electroeluted, concentrated, and partially digested for 24 hours with cyanogen bromide (CNBr) (Silver, et al. , 1995, J. Bi ol . Chem.
  • CNBr is known to cleave after methionine (M) residues under the conditions used for this digestion.
  • M methionine
  • the peptides in the sample were resolved by PAGE on an 18% Tris-glycme gel.
  • electrophoresis the separated protease peptides were electroblotted onto a PVDF membrane using a CAPS buffer (10 mM CAPS pH 11, 0.5 mM DTT, 10% (v/v) methanol) .
  • the membrane was stained with Coomassie Brilliant Blue and destamed with 50% (v/v) methanol. Three stained peptide bands were identified having apparent molecular weights of about 14 kD, 21 kD, and 22 kD, respectively. The portions of the membrane containing the 21 kD and 22 kD bands were excised separately. Peptides contained m each membrane segment were subjected to N- termmal ammo acid sequencing using a 473A Protem Sequencer (available from Applied Biosystems, Foster City, CA) using standard techniques.
  • N-termmal ammo acid sequence of the 21-kD peptide was H/R-V/P- G/A/S-Y/G-E/N-D/K-V/R-D/A-D-Y- D-F-D/P-V-A, denoted herein as SEQ ID NO: 70 and the N-terminal amino acid sequence of the 22-kD peptide to be I/Q-V-G-Y/G-E/N/T-D/M/P-V-K/D-I- N/S-M/T/N-F/C herein denoted as SEQ ID NO: 71.
  • the N- texmmal ammo acid sequence of the mtact 31-kD protease is either I-V-G-G- E-D-V-D-I-S-T-C-G-W-C (SEQ ID NO: 59, as disclosed m Example 34 m co-pending U.S. Patent Application Serial No. 08/639,075) , or IVGGEDVDIST(C)GWQI(S)FQ(S)ENLHF(C)GG(S) IIAPK (SEQ ID NO: 69, as disclosed in Example 35 in co-pending U.S. Patent Application Serial No. 08/639,075) .
  • SEQ ID NO: 68 contains a cysteine and SEQ ID NO: 69 contains a glutamine.
  • SEQ ID NO: 70 the sequences of both the 21-kD (SEQ ID NO:70) ano 22-kD (SEQ ID NO:71) peptides, though it is much stronger m the 22-kD peptide, leading to the conclusion that the SEQ ID NO: 71 represents the N-terminus of the 31- kD protease.
  • sequence for the 21-kD (SEQ ID NO:70) peptide is H/R-P-A/S-Y-N- K-R-A-D-Y-D-F-D-V-A, denoted herein as SEQ ID NO:72.
  • This sequence of ammo acids aligns with a stretch of deduced ammo acids from about residue 107 to residue 121 immediately following a methionine residue in SEQ ID NO: 67.
  • This example demonstrates that a 31-kD flea midgut serine protease contained in a formulation is able to proteolyze cat immunoglobulin G, A, and M proteins as well as bovme, dog, human, and rabbit immunoglobulin G proteins.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed fleas as follows. Cat blood-fed flea midgut extracts were prepared and selected on a benzamidine column as described above m Example 26. The benzamidine eluate was then further purified as described in Example 35 of co-pending U.S. Patent Application Serial No. 08/639,075 by PolyCAT A cation exchange chromatography (available from PolyLC, Inc., Columbia, MD) to isolate a protein band which migrated at about 31 kD on a silver stained SDS-PAGE gel.
  • a Sepharose or of purified dog, rabbit, or human IgG
  • This example describes the ability of a 31-kD flea midgut serine protease contained m a formulation to proteolyze cat immunoglobulin G at a specific site.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 26 and 27.
  • a band of about 33 kD was excised and subjected to N-termmal amino acid sequencing using techniques known to those skilled in the art.
  • a partial N- terminal ammo acid sequence of about 28 ammo acids was determined and is represented herem as SEQ ID NO: 73: X- P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K.
  • GenBank homology search using SEQ ID NO: 73 revealed most homology to Oryctolagus cani cul us gamma H-cham constant region 2, there being about 71 % identity over the 28 ammo acids.
  • the cleavage site was compared to that of a known protease, papain, as follows.
  • Cat immunoglobulin G 100 mg
  • papain purified from cat blood on Protein A sepharose
  • the reaction mixture was resolved on a 14% Tris-glycine SDS-PAGE gel, blotted onto PVDF membrane, stained with Coomassie R-250 and destained according to standard procedures.
  • a band of about 33 kD was excised and subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art.
  • a partial N-terminal amino acid sequence of about 25 amino acids was deduced and is represented herein as SEQ ID NO: 96: X-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-K-K-D-D-L-L-I .
  • This Example demonstrates the kinetics of cat IgG degrading activity in the midguts of fleas fed on live cats .
  • the fleas' midguts were removed as described in U.S. Patent No. 5,356,622, ibid. , homogenized by freeze-fracture and sonicated in a Tris buffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl 2 .
  • the extracts were centrifuged at about 14,000 x g for 20 min. and the soluble material recovered.
  • the soluble material was then diluted to a final concentration of about 1.2 midgut equivalents per microliter ( ⁇ l) of Tris buffer.
  • the proteins contained in 1 midgut equivalent of each timepomt were then resolved by SDS-PAGE under reducing conditions, and the proteins visualized by silver staining.
  • the flea chambers were removed and placed in a 28°C, 75% relative humidity growth incubator.
  • Fleas were subjected to dissection at time points of 0, 1, 2, 4, and 8 hr. following removal from the cats.
  • Midguts were homogenized, and the midgut contents were examined by silver stained SDS-PAGE and immunoblot analysis, as described in Section A.
  • the fleas fed for 1 hour had high molecular weight proteins, including the heavy chain and light chain of cat IgG detectable in their midguts at the 0 and 1 hour dissection timepoints, while the flea midguts evaluated at time points of 2 hours or greater had no detectable IgG heavy chain bands.
  • This example describes the ability of a 31-kD flea midgut serine protease contained in a formulation to proteolyze cat immunoglobulin G at a specific site.
  • the 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 14 and 15.
  • SEQ ID NO: 104 A partial N-termmal ammo acid sequence of about 25 ammo acids was determined and is represented herem as SEQ ID NO: 104: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- D. An additional 10 amino acids were also obtained beyond the last ammo acid of SEQ ID NO: 104.
  • SEQ ID NO: 105 D-C-P-K-C-P-P-P-E-M-L- G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K-S-E-V.
  • GenBank homology search using SEQ ID NO: 105 revealed most homology to Homo sapi en immunoglobulin gamma 3 heavy chain constant region 2 exon hinge IGHG3 gene (GenBank Accession No. X99549) , there being about 69% identity over the 35 amino acids.
  • SEQ ID NO: 105 Further alignments of SEQ ID NO: 105 with cat, rabbit, bovine and human IgG amino acid sequences indicated that purified cat blood-fed 31-kD flea midgut serine protease cleaved the cat IgG heavy chain about 6 amino acids just before the predicted C-terminal end of the IgG hinge region.
  • the first 6 amino acids, aspartic acid, cysteine, proline, lysine, cysteine and proline occur within the predicted hinge region while the remaining 29 amino acids of SEQ ID NO: 105, starting with the seventh amino acid proline, occur within the predicted constant heavy chain-2 region.
  • IgG (represented herein by SEQ ID NO: 106) is shown below:
  • SEQ ID NO:105 (continued) -D-D-L-L-I-K-R-K-S-E-V
  • SEQ ID NO:106 (continued) -D-T-L-S-I-S-R-T-P-E-V
  • SEQ ID NO: 105 Discrepancies between SEQ ID NO: 105 and SEQ ID NO: 106 are shown in bold. Applicants believe that the difference between SEQ ID NO: 105 and SEQ ID NO: 106 may be due to sequencing error of the last 10 amino acids of SEQ ID NO: 105.
  • ATGAAGTAAA AACCTTGCGT TGGTTTCCCC GGTCCCAGGA TCAGGAACAG TTGCACTTTA 120
  • GCC AAA AAT AAT AAT GCG GAT TTG ACG ATC ACT TAT TTA CTA TGT ACT 1079 Ala Lys Asn Asn Asn Ala Asp Leu Thr Ile Thr Tyr Leu Leu Cys Thr 305 310 315
  • MOLECULE TYPE protein
  • Asn Asn Asn Asn Ala Asp Leu Thr lie Thr Tyr Leu Leu Cys Thr Thr Phe 305 310 315 320 Lys Ile Asp Phe Tyr Gly Met
  • CAGGAACTTC ACCATGAGTT TGATTGGTCA ATCCCCATCC AGTAATTTTC AATTTTTCAC 420 CTCCGCGTAT AAAATCTTTG TGCAATTTGA TGGGACGAAC ATTTTTGCTA AGTTTTATAG 480
  • Met Asn Arg Trp lie Leu Thr Ala Ala His Cys Leu Thr Asp Gly Tyr 35 40 45
  • MOLECULE TYPE DNA (genomic)
  • CTTATNGACC AGAGGACCAC CAGAGTCACC CATGCATACA CCCTTTTGAG GTGCTTTGAA 180
  • AAA AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA TTA GTC 677 Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val 200 205 210 215
  • MOLECULE TYPE protein
  • MOLECULE TYPE DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATTTTCTGAC ATTGTCGTGT TAATCCAGTC CAAAAACGAT GTAATTCTTG TGTAAACGTC 60 AGGATAATAA CCACTTCCAC AAGGTTGCAT ACCCCAGGAT ACTAAACCGA CCAATGTATT 120 GTTTCCTTGG ACTAATGGAC CACCAGAATC ACCCTTACAA GATCCGGTTC CTTTTTTAGC 180 CTGTGCACAA ATTTGGCTTT CGAAAGTCTT TTTATAAATT GCGTTTTTGC AATCCTCATA 240
  • AGT CAA AAA TTA CAG GTC ATG ACA GCC AAA TCA CTA ACT TAT GAG GAT 480 Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser Leu Thr Tyr Glu Asp 145 150 155 160
  • CAG GCT AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA 576 Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro
  • TCT TCT TTA AAT TTG AAT GGA GGT TCT ATT CGA CCG GCT AGG TTA GTG 431 Ser Ser Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val 130 135 140

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Abstract

The present invention relates to flea serine protease proteins and flea cysteine protease proteins; to flea serine protease and cysteine protease nucleic acid molecules, including those that encode such proteins; to antibodies raised against such proteins; and to compounds that inhibit flea serine protease and/or cysteine protease activities. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included in the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.

Description

FLEA PROTEASE PROTEINS, NUCLEIC ACID MOLECULES,
AND USES THEREOF
Field of the Invention The present invention relates to novel flea protease proteins and their use to reduce flea infestation of animals. The present invention also relates to the use of anti-flea protease antibodies and other compounds that reduce flea protease activity to reduce flea infestation of animals . Background of the Invention
Fleas, which belong to the insect order Siphonaptera, are obligate ectoparasites for a wide variety of animals, including birds and mammals. Flea infestation of animals is of health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas cause and/or carry infectious agents that cause, for example, flea allergy dermatitis, anemia, murine typhus, plague and tapeworm. In addition, fleas are a problem for animals maintained as pets because the infestation becomes a source of annoyance for the pet owner who may find his or her home generally contaminated with fleas which feed on the pets. As such, fleas are a problem not only when they are on an animal but also when they are m the general environment of the animal. The medical and veterinary importance of flea infestation has prompted the development of reagents capable of controlling flea infestation. Commonly encountered methods to control flea infestation are generally focussed on use of insecticides in formulations such as sprays, shampoos, dusts, dips, or foams, or in pet collars. While some of these products are efficacious, most, at best, offer protection of a very limited duration. Furthermore, many of the methods are often not successful in reducing flea populations on the pet for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required); (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of flea populations resistant to the prescribed dose of pesticide. Additional anti-flea products include nontoxic reagents such as insect growth regulators (IGRs), including methoprene, which mimics flea hormones and affect flea larval development.
An alternative method for controlling flea infestation is the use of flea vaccines to be administered to animals prior to or during flea infestation. However, despite considerable interest m developing anti-flea reagents, no flea vaccine presently exists.
Summary of the Invention The present invention relates to flea serine protease proteins, to flea ammopeptidase proteins, and to flea cysteine protease proteins; to flea serine protease, ammopeptidase and/or cysteine protease nucleic acid molecules, including those that encode such proteins; to antibodies raised agamst such proteins; and to compounds that inhibit flea serine protease, ammopeptidase and/or cysteine protease activities. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitors. Also included m the present invention are therapeutic compositions comprising such proteins, nucleic acid molecules, antibodies, and/or inhibitors as well as the use of such therapeutic compositions to protect a host animal from flea infestation.
One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene including a serine protease gene comprising a nucleic acid sequence including a nucleic acid molecule including SEQ ID NO: 9, SEQ ID NO-11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID N0:15, SEQ ID NO:17, SEQ ID N0:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID N0:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO-117, SEQ ID NO:127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO:125, SEQ ID NO:118, SEQ ID N0:128, SEQ ID N0:152, SEQ ID N0:156, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:78, SEQ ID NO:158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO-.146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID N0:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO: 140, SEQ ID NO:122, SEQ ID NO:84 and/or SEQ ID NO: 45; an ammopeptidase gene comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 110 and/or SEQ ID NO: 112; and a cysteine protease gene comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94.
The present invention also includes a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID. NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO: 67, SEQ ID NO: 147', SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID N0:81, SEQ ID N0:83, SEQ ID N0:143, SEQ ID N0:139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID N0:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID N0:5, SEQ ID N0:8, SEQ ID NO:89, SEQ ID NO:92 and/or SEQ ID NO: 95, or with a nucleic acid sequence that is a complement of any of the nucleic acid sequences. A preferred nucleic acid sequence of the present invention includes a nucleic acid molecule comprising a nucleic acid sequence including SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28> SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO:131, SEQ ID NO:155, SEQ ID NO:114, SEQ ID NO:125, SEQ ID NO:118, SEQ ID NO:128, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:140, SEQ ID NO:122, SEQ ID NO:84, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:76, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:88, SEQ ID NO:90, SEQ ID N0:91, SEQ ID NO: 93 and/or SEQ ID NO: 94, and allelic variants thereof. The present invention also includes an isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO: 92 and SEQ ID NO: 95.
The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention.
Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Yet another embodiment of the present invention is a therapeutic composition that is capable of reducing hematophagous ectoparasite infestation. Such a therapeutic composition includes an excipient and a protective compound including: an isolated protein or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ID N0:9, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:l4, SEQ ID N0:15, SEQ ID N0:17, SEQ ID N0:18, SEQ ID NO:20, SEQ ID N0:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:155, SEQ ID NO:114, SEQ ID NO:125, SEQ ID NO:118, SEQ ID NO:128, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO: 160, SEQ ID NO:136, SEQ ID NO:78,- SEQ ID NO:158, SEQ ID NO: 132, SEQ ID NO-.134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO:84, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:76, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94; an isolated antibody that selectively binds to a protem encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ IL N0:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID NO:72, SEQ ID NO*73, SEQ ID NO:96, SEQ ID N0:115, SEQ ID NO:126, SEQ ID N0:119, SEQ ID N0:129, SEQ ID N0:153, SEQ ID NO:157, SEQ ID N0:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID N0:151, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO: 143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; an inhibitor of protease activity identified by its ability to mnibit the activity of a protem encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protem comprising an ammo acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID N0:159, SEQ ID NO:133, SEQ ID N0:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID N0:151, SEQ ID N0:81, SEQ ID N0:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID N0:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO: 111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; and a mixture thereof. Also included in the present invention is a method to reduce flea infestation, comprising the step of administering to the animal a therapeutic composition of the present invention.
Another embodiment of the present invention is a method to identify a compound capable of inhibiting flea protease activity, the method comprising: (a) contacting an isolated flea protease protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID N0:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO-.157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID N0:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ IE NO: 162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID N0:5, SEQ ID N0:8, SEQ ID NO:89, SEQ ID NO:92 and SEQ ID NO: 95 with a putative inhibitory compound under conditions in which, m the absence of said compound, the protem has proteolytic activity; and (b) determinmg if the putative inhibitory compound inhibits the activity. The present invention also mcludes a kit to to identify a compound capable of inhibiting flea protease activity.
The present mvention also includes an isolated flea protease protem that cleaves an immunoglobulin, when the protem is incubated in the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C. A preferred protease protem capable of cleaving immunoglbulm comprises an ammo acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO: 96. Another embodiment of the present invention includes a method to identify a compound capable of inhibiting flea immunogloDulm proteinase protem activity, the method comprising: (a) contacting an isolated flea immunoglobulin proteinase protem with a putative inhibitory compound under conditions in which, m the absence of the compound, the protem has immunoglobulin proteinase activity; and (b) determining if the putative inhibitory compound inhibits the activity.
Brief Description of the Drawings Fig. 1 is a scanned image depicting SDS-PAGE of DFP- labeled larval proteases m unfed larvae, fed 1st instar larvae and fed 3rd instar larvae.
Detailed Description of the Invention The present invention includes the use of compounds that inhibit flea protease activity to protect a host animal from flea infestation. The inventors have discovered that proteases are significant components of the flea midgut and are good targets for lmmunotherapeutic and/or chemotherapeutic intervention to reduce flea burden ooth on the host animal and m the immediate (i.e., surrounding) environment of the animal. The inventors have snown, for example, that the viability and/or fecundity of fleas consuming a blood meal is reduced when the blood meal contains compounds that reduce flea protease activity, probably because the compounds interfere with flea digestion and other functions. Compounds that reduce the amount and/or activity of flea proteases without substantially harming the host animal are mcluded m the present invention. Such compounds include flea protease vaccines, anti-flea protease antibodies, flea protease inhibitors, and/or compounds that suppress protease synthesis; such compounds are discussed in more detail below.
One embodiment of the present invention is a method to protect a host animal from flea infestation by treating the animal with a composition that includes a compound that reduces the protease activity of fleas feeding (includes fleas in the process of feeding as well as fleas having fed) from the treated animal thereby reducing the flea burden on the animal and in the environment of the animal.
It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a compound refers to one or more compounds. As such, the terms "a"
(or "an"), "one or more" and "at least one" can be used interchangeably herein. Thus, a composition of the present invention can include one or more compounds that target (reduced the activity of) one or more proteases in the flea.
As used herein, the phrase "to protect an animal from flea infestation" refers to reducing the potential for flea population expansion on and around the animal (i.e., reducing the flea burden) . Preferably, the flea population size is decreased, optimally to an extent that the animal is no longer bothered by fleas. A host animal, as used herein, is an animal from which fleas can feed by attaching to and feeding through the skin of the animal. Fleas, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal m order to feed. At any given time, a certain percentage of a flea population can be on a host animal whereas the remainder can be in the environment surrounding the animal (i.e., in the environment of the animal) . Such an environment can include not only adult fleas, but also flea eggs and/or flea larvae. The environment can be of any size such that fleas in the environment are able to "jump onto and off of a host animal. As such, it is desirable not only to reduce the flea burden on an animal per se, but also to reduce the flea burden in the environment surrounding the animal.
In accordance with the present invention, a host animal is treated by administering to the animal a compound of the present invention in such a manner that the compound itself (e.g., a protease inhibitor, protease synthesis suppressor or anti-flea protease antibody) or a product generated by the animal in response to administration of the compound (e.g., antibodies produced in response to a flea protease vaccine, or conversion of an inactive inhibitor "prodrug" to an active protease inhibitor) ultimately enters the flea midgut. An animal is preferably treated in such a way that the compound or product thereof enters the blood stream of the animal. Fleas are then exposed ro the compound when they feed from the animal. For example, flea protease inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas. In another embodiment, when a host animal is administered a flea protease vaccine, the treated animal mounts an immune response resulting in the production of antibodies agamst the protease (anti-flea protease antibodies) which circulate m the animal's blood stream and are taken up by fleas upon feeding. Blood taken up by fleas enters the flea midgut where compounds of the present invention, or products thereof, such as anti-flea protease antibodies, flea protease inhibitors, and/or protease synthesis suppressors, interact with, and reduce proteolytic activity m the flea midgut. The present invention also includes the ability to reduce larval flea infestation in that when fleas feed from a host animal that has been administered a therapeutic composition of the present mvention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the flea are excreted by the flea m feces, which is subsequently ingested by flea larvae. It is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
In accordance with the present invention, reducing proteolytic activity in flea midguts can lead to a number of outcomes that reduce flea burden on treated animals and their surrounding environments. Such outcomes include, but are not limited to, (a) reducing the viability of fleas that feed from the treated animal, (b) reducing the fecundity of female fleas that feed from the treated animal, (c) reducing the reproductive capacity of male fleas that feed from the treated animal, (d) reducing the viability of eggs laid by female fleas that feed from the treated animal, (e) altering the blood feeding behavior of fleas that feed from the treated animal (e.g., fleas take up less volume per feeding or feed less frequently) , (f) reducing the viability of flea larvae, for example due to the feeding of larvae from feces of fleas that feed from the treated animal and/or (g) altering the development of flea larvae (e.g., by decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slowing or blocking) molting, and/or otherwise inhibiting maturation to adults) .
One embodiment of the present invention is a composition that includes one or more compounds that reduce the activity of one or more flea proteases directly (e.g., an anti-flea protease antibody or a flea protease inhibitor) and/or indirectly (e.g., a flea protease vaccine) . Suitable flea proteases to target include flea ammopeptidases, flea carboxypeptidases and/or flea endopeptidases. Such proteases can include cytosolic and/or membrane-bound forms of a protease. Preferred flea proteases to target include, but are not limited to, serine proteases, metalloproteases, aspartic acid proteases and/or cysteine proteases. It is to be noted that these preferred groups of proteases include ammopeptidases, carboxypeptidases and/or endopeptidases. Preferred flea proteases to target mclude, but are not limited to, proteases that degrade hemoglobin, proteases mvolved m blood coagulation and/or lytic (anti-coagulation) pathways, proteases involved in the maturation of peptide hormones, proteases that inhibit complement or other host immune response elements (e.g., antibodies) and/or proteases mvolved in vitellogenesis. A number of proteases are known to those skilled in the art, including, but not limited to, ammopeptidases, such as leucine ammopeptidase and ammopeptidases B and M; astacin-like metalloproteases; calpams; carboxypeptidases, such as carboxypeptidases A, P and Y; cathepsms, such as cathepsms B, D, E, G, H, and L, chymotrypsms; cruzipams; meprins; papams; pepsins; reruns; thermolysms and trypsms. A particularly preferred protease to target is a protease having a proteolytic activity that, when targeted with a composition of the present mvention, reduces flea burden without substantially harming the host animal. Such a protease can be identified usmg, for example, methods as disclosed herem. One aspect of the present invention is the discovery that a substantial amount of the proteolytic activity found in flea midguts is serine protease activity. Both in vi tro and in vi vo studies usmg a number of protease inhibitors substantiate this discovery, details of which are disclosed m the Examples. As such a particularly preferred protease to target is a serine protease. Examples of serine proteases, include, but are not limited to, acrosms, bromelams, cathepsm G, chymotrypsins, coUagenases, elastases, factor Xa, ficms, kallikreins, papains, plasmins, Staphylococcal V8 proteases, thrombms and trypsins. In one embodiment, a preferred flea serine protease to target includes a protease having trypsin-like or chymotrypsin-like activity. It is appreciated by those skilled in the art that an enzyme having "like" proteolytic activity has similar activity to the referenced protease, although the exact structure of the preferred substrate cleaved may differ. "Like" proteases usually have similar tertiary structures as their referenced counterparts. Protease inhibitor studies disclosed in the Examples section also indicate that additional preferred proteases to target include aminopeptidases and/or metalloproteases. Examples of such proteases mclude exo- and endo- metalloproteases, digestive enzymes, and enzymes mvolved in peptide hormone maturation. One example of an ammopeptidase that is also a metalloprotease is leucine ammopept-dase .
Suitable compounds to include in compositions of the present mvention include, but are not limited to, a vaccine comprising a flea protease (a flea protease vaccine) , an antibody that selectively binds to a flea protease (an anti-flea protease antibody) , a flea protease inhibitor (a compound other than a vaccine or an antibody that inhibits a flea protease) , and a mixture of such compounds. As used herein, a mixture thereof refers to a combination of one or more of the cited entities. Compositions of the present mvention can also include compounds to suppre-ss protease synthesis or maturation, such as, but not limited to, protease modulating peptides. A preferred embodiment of the present invention is a flea protease vaccine and its use to reduce the flea population on and around an animal. A flea protease vaccine can include one or more proteins capable of eliciting an immune response agamst a flea protease and can also include other components. Preferred flea protease vaccines mclude a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease, with flea serine protease, flea metalloprotease and/or flea ammopeptidase vaccines being more preferred. Examples of flea protease vaccines mclude soluble flea midgut preparations of the present invention as well as one or more isolated proteins of the present invention.
One embodiment of the present invention is a soluble flea midgut preparation. Such a preparation mcludes primarily components naturally present m the lumen of a flea midgut and, depending on the method of preparation, can also include one or more peripheral midgut membrane proteins. Methods to preferentially include, or exclude, membrane proteins from such a preparation are known to those skilled m the art. The present mvention mcludes the discovery that such a preparation has proteolytic activity, of which a substantial portion is serine protease activity. Preferably at least about 70 percent of the proteolytic activity in a soluble flea midgut soluble preparation is serine protease activity, as can be indicated by the ability to inhibit at least about 70 percent of the proteolytic activity with 4-2-ammoethyl- benzenesulfonylfluoπde-hydrochloride (AEBSF) . Serine protease activity can also be identified using other known inhibitors or substrates. Other preferred inhibitors that can inhibit at least about 70 percent of the proteolytic activity of a soluble flea midgut preparation of the present invention include soybean trypsin inhibitor, 1,3- dnsopropylfluoro-phosphate or leupeptin. A soluble flea midgut preparation of the present invention includes proteases that range m molecular weight from about 5 kilodaltons (kD or kDa) to about 20υ KD, as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), with at least a substantial portion of the serine proteases ranging m molecular weight from about 5 kD to about 60 kD, as determined by SDS-PAGE. A substantial portion of protease activity in a soluble flea midgut preparation of the present invention has a pH activity optimum ranging from about pH 5 to about pH 10, preferably an activity optimum ranging from about pH 7 to about pH 9, and even more preferably an activity optimum of about pH 8. While not being bound by theory, such a pH optimum suggests that a large proportion of proteases in soluble flea midgut preparations of the present invention are serine proteases. It is also interesting to note that the pH of the flea midgut is also about pH 8. The findings that proteases m soluble flea midgut preparations of the present invention exnibit a varied pattern of inhibition by protease inhibitors of a given type (e.g., serine protease inhibitors), as well as variances seen m molecular weights and pH optima of the proteases, suggest that there are a number of protease isoforms in such preparations.
A soluble flea midgut preparation of the present mvention is preferably prepared by a method that mcludes tne steps of (a) disrupting a flea midgut to produce a mixture including a liquid portion and a solid portion and (b; recovering the liquid portion to obtain a soluble flea midgut preparation. Such a method is a simplified version of methods disclosed m U.S. Patent No. 5,356,622, ibi d . It is to be noted that in accordance with the present mvention, methods disclosed m U.S. Patent No. 5,356,622, ibi d, can also be used to prepare soluble flea midgut preparations having similar proteolytic activities.
Flea midguts can be obtained (e.g., dissected from) from unfed fleas or from fleas that recently consumed a blood meal (i.e., blood-fed fleas) . Such midguts are referred to herein as, respectively, unfed flea midguts and fed flea midguts. Flea midguts can be obtained from either male or female fleas. As demonstrated in the Examples section, female flea midguts exhibit somewhat more proteolytic activity than do male flea midguts. Furthermore, fed flea midguts have significantly more proteolytic activity than do unfed flea midguts. While not being bound by theory, it is believed that blood feeding induces m flea midguts the synthesis and/or activation of proteases as well as other factors (e.g., enzymes, other proteins, co-factors, etc.) important m digesting the blood meal, as well as in neutralizing host molecules potentially damaging to the flea (e.g., complement, immunoglobulins, blood coagulation factors) . It is also to be appreciated that unfed flea midguts may contain significant targets not found m fed flea midguts and vice versa. Furthermore, although the present application focuses primarily on flea midgut proteases, it is to be noted that the present invention also includes other components of soluble flea midgut preparations of the present mvention that provide suitable targets to reduce flea burden on an animal and in the environment of that animal; see also U.S. Patent No. 5,356,622, ibid.
Methods to disrupt flea midguts in order to obtain a soluble flea midgut preparation are known to those skilled in the art and can be selected according to, for example, the volume being processed and the buffers being used. Such methods include any technique that promotes cell lysis, such as, but are not limited to, chemical disruption techniques (e.g., exposure of midguts to a detergent) as well as mechanical disruption techniques (e.g., homogenization, sonication, use of a tissue blender or glass beads, and freeze/thaw techniques) .
Methods to recover a soluble flea midgut preparation are also known to those skilled in the art and can include any method by which the liquid portion of disrupted flea midguts is separated from the solid portion (e.g., filtration or centrifugation) . In a preferred embodiment, disrupted flea midguts are subjected to centrifugation, preferably at an acceleration ranging from about 10,000 x g to about 15,000 x g for several minutes (e.g., from about 1 min. to about 15 min.) . The supernatant from such a centrifugation comprises a soluble flea midgut preparation of tne present mvention.
The present invention also mcludes an isolated protem that includes an ammo acid sequence encoded by a nucleic acid molecule capable of hybridizing under stringent conditions (i.e., that hybridize under stringent nybridization conditions) with a nucleic acid molecule that encodes a protease present (i.e., the nucleic acid molecules hybridize with the nucleic acid strand that is complementary to the coding strand) m (i.e., can be found m) a flea midgut, such as a midgut from a blood-fed female flea, a midgut from a blood-fed male flea, a midgut from an unfed female flea or a midgut from an unfed male flea. A preferred midgut protease is present m the lumen of the midgut.
An isolated protem of the present invention, also referred to herein as an isolated protease protem, preferably is capable of eliciting an immune response against a flea midgut protease and/or has proteolytic activity. Accordmg to the present mvention, an isolated, or biologically pure, protem, is a protem that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protem has been purified. An isolated protease protem can be obtained from its natural source. Such an isolated protem can also be produced usmg recombinant DNA technology or chemical synthesis.
As used herein, an isolated protem of the present mvention can be a full-length protem or any homologue of such a protem, such as a protem m which ammo acids have been deleted (e.g., a truncated version of the protem, such as a peptide) , inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl mositol) such that the homologue comprises a protem having an ammo acid sequence that is sufficiently similar to a natural flea midgut protease that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) the complement of a nucleic acid sequence encoding the corresponding natural flea midgut protease ammo acid sequence. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cl oning: A Labora tory Manual , Cold Spring Harbor Labs Press, 1989; Sambrook et al. , ibid. , is incorporated by reference herem m its entirety. Stringent hybridization conditions typically permit isolation of nucleic acid molecules havmg at least about 70 nucleic acid sequence identity v.ith the nucleic acid molecule being used to probe m the hyr>nd-.zatιorι reaction. Formulae to calculate tne appropriate hybridization and wash conditions to achieve hybridization permitting 30" or less mismatch of nucleotides are disclosed, for example, m Memkoth et al . , 1984, Anal. Bi ochem . 138, 267-284; Memkoth et al . , ibid. , is incorporated by reference herein m its entirety.
The minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protem. As such, the size of tne nucleic acid molecule encoding such a protem homologue is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration) . The minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to aoout 17 bases m length if they are AT-rich. As such, the minimal size of a nucleic acid molecule used to encode a protease protem homologue of the present invention is from aoout 12 to about 18 nucleotides in length. There is nc limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof. Similarly, the minimal size of a protease protem homologue of the present invention is from about 4 to about 6 ammo acids in length, with preferred sizes depending on whether a full-length, multivalent (i.e., fusion protem having more than one domain each of which has a function) , or functional portions of such proteins are desired. Protease protem homologues of the present mvention preferably have protease activity and/or are capable of eliciting an immune response agamst a flea midgut protease.
A protease protem homologue of the present invention can be the result of allelic variation of a natural gene encoding a flea protease. A natural gene refers to the form of the gene found most often in nature. Protease protein homologues can be produced using techniques known m the art including, but not limited to, direct modifications to a gene encoding a protein usmg, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. Isolated protease proteins of the present invention, including homologues, can be identified m a straight-forward manner by the proteins' ability to effect proteolytic activity and/or to elicit an immune response against a flea midgut protease. Such techniques are known to those skilled in the art.
A preferred protease protein of the present invention is a flea serine protease, a flea metalloprotease, a flea aspartic acid protease, a flea cysteine protease, or a homologue of any of these proteases. A more preferred protease protein is a flea serine protease, a flea metalloprotease or a homologue of either. Also preferred is a flea ammopeptidase or a homologue thereof. Also preferred is a flea cysteine protease or a homologue thereof. Particularly preferred is a flea serine protease or a homologue thereof.
Preferred protease proteins of the present invention are flea protease proteins having molecular weights ranging from about 5 kD to about 200 kD, as determined by SDS-PAGE, and homologues of such proteins. More preferred are flea protease proteins having molecular weights ranging from about 5 kD to about 60 kD, as determined by SDS-PAGE, and homologues of such proteins. Even more preferred are flea serine protease proteins, particularly those having molecular weights of about 26 kD (denoted PfSP26, now denoted PafSP-26K to distinguish from flea PfSP26 as described in Example 26) , about 24 kD (denoted PfSP24, now denoted PafSP-24K to distinguish from flea PfSP24 as described in Example 27), about 19 kD (denoted PfSP19, now denoted PafSP-19K to distinguish from flea PfSP19 as described m Example 32), about 6 kD (denoted PfSPo, now denoted PafSP-6K to distinguish from flea PfSP6 as described m Example 11), about 31 kD (denoted PfSP28), about 25 kD (denoted PlfSP-25Kl) from 1st instar larvae, about 25 kD (denoted PlfSP-25K3) from 3rd instar larvae, about 28 kD (denoted PlfSP-28K3) and about 31 kD (denoted PlfSP-31K3), and flea ammopeptidase proteins, particularly those navmg molecular weights of about 95 kD (denoted PfAP-95K) as determined by SDS-PAGE, and homologues of such proteins.
One preferred embodiment of the present invention is an isolated flea protease protem that mcludes an ammo acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene, with a flea ammopeptidase gene or with a flea cysteine protease gene. As used herein, a flea protease gene includes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post-translation control regions) as well as the coding region itself.
The inventors have discovered an extensive family of serine proteases, encoded by a family of serine protease genes. Such a gene family may be due to allelic variants (i.e., genes having similar, but different, sequences at a given locus in a population of fleas) and/or to, the existence of serine protease genes at more than one locus m the flea genome. As such, the present invention includes flea serine protease genes comprising not only the nucleic acid sequences disclosed herein (e.g., genes including nucleic acid sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO:155, SEQ ID NO:114, SEQ ID NO:125, SEQ ID NO:118, SEQ ID NO:128, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO:78, SEQ ID NO:158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84 and/or nucleic acid sequences encoding proteins having amino acid sequences as disclosed herein (e.g., SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID N0:72, SEQ ID N0:73, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 13", SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID N0:151, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID N0:139, SEQ ID NO:145, SEQ ID N0:141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, and/or SEQ ID NO: 96, but also allelic variants of any of those nucleic acid sequences, as well as other nucleic acid molecules and ammo acid sequences disclosed in the examples section. (It should be noted that since nucleic acid sequencing technology is not entirely error-free, all sequences represented herem are at best apparent (i.e., deduced) nucleic acid or ammo acid sequences . )
A preferred flea ammopeptidase gene mcludes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID NO: 112, which encode ammopeptidase proteins having ammo acid sequences including SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113. Additional preferred ammopeptidase genes include allelic variants of SEQ ID NO:110 and/or SEQ ID NO: 112.
A preferred flea cysteine protease gene includes nucleic acid sequence SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94, which encode a cysteine protease protem having ammo acid sequences including SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:77, and/or SEQ ID NO: 95. Additional preferred cysteine protease genes include allelic variants of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76 and/or SEQ ID NO: 94.
A preferred flea serine protease protem of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the followmg nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPlβ, nfSP24, nfSP28, nfSP32, nfSP33 and nfSP40. As used herem, each of these nucleic acid molecules represent the entire coding region of a flea serine protease gene of the present invention (at least portions of which are also referred to by flea clone numbers, as described m the Examples) . Nucleic acid molecules that contain partial coding regions or other parts of the corresponding gene are denoted by names that include the size of those nucleic acid molecules (e.g., nfSP4042b) . Nucleic acid molecules containing apparent full length coding regions for which the size is known also are denoted by names that include the size of tnose nucleic acid molecules (e.g., nfSP40841) . The production, and at least partial nucleic acid sequence, of such nucleic acid molecules is disclosed in the Examples.
Particularly preferred serine protease proteins are encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18B34, nfSP1877W nfSP18225, nfSP24410, nfSP241089, nfSP24774, nfSP24711, nfSP28P2,, nfSP32qj3, nfSP32y33, nfSP32924, nfSP32699, nfSP3342b, nfSP3371((, nfSP331894, nfSP331?00, nfSP33726, nfSP40841, nfSP5806, nfSPll307, nfSP8sl5, nfSP8430, nfSP12758, nfSP26610, nfSP2738t,, nfSP23423, nfSP34390, nfSP36197, nfSP38m, nfSP37261, nfSP3926- nfSP29612, nfSP30M1, nfSP31626, nfSP32„3, nfSP15815, nfSP19855, nfSP25864, nfSP21Scι5, and/or nfSP407r.. Even more preferred serine protease proteins include the following amino acid sequences: SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, and/or SEQ ID NO: 96. Additional particularly preferred serine protease proteins are encoded by allelic variants of nucleic acid molecules encoding proteins that include the c_.ted ammo acid sequences. Also preferred are flea serine protease proteins including regions that have at least about 50*:, preferably at least about 75%, and more preferably at least about 90% identity with flea serine protease proteins having ammo acid sequences as cited herem.
One embodiment of the present mvention is a flea serine protease that degrades immunoglobulin circulating m a host animal (i.e.-, flea immunoglobulin proteinase or IgGase) . An example of a flea immunoglobulin proteinase is presented in the Examples section. Preferably, an immunoglobulin proteinase of the present mventioncleaves an immunoglobulin when the protem is incubated m the presence of the immunoglobulin m about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C. More preferably, an immunoglobulin proteinase of the present mvention cleaves an immunoglobulin m about 300 microliters of 50 mM Tris-HCl, pH 8.0, for about 1 hour at about 37CC. Suitable immunoglobulin proteinase proteins of the present invention are capable of cleaving the hinge region of an immunoglobulin heavy chain. The hinge region of an immunoglobulin is the flexible domain that "joins the Fab arms of the immunoglobulin to the Fc portion of the molecule. A more preferred immunoglobulin proteinase protem mcludes a protem havmg a molecular weight ranging from about 25 kD to about 35 kD and more preferably having a molecular weight of about 31 kD, in its mature form. An even more preferred immunoglobulin proteinase protem includes a protem comprising an ammo acid sequence including SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73 and/or SEQ ID NO: 96, which can be encoded by a gene comprising nucleic acid sequence SEQ ID NO: 66. Without being bound by theory, the proteinase activity of an immunoglobulin proteinase of the present invention cleaves an immunoglobulin m such a manner that the immunoglobulin maintains mtact heavy and light chain pairs, either as two Fab fragments or one Ffab')^ fragment. As used herein, a Fab fragment refers to complete immunoglobulin light chains paired with the variable region and CHI domains of an immunoglobulin heavy chain. As used herem, a F(ab' ) fragment refers to two Fab fragments that remain linked by a disulfide bond. Both Fab and F(ab')2 fragments are capable of binding antigen.
A preferred immunoglobulin proteinase protem of the present invention is capable of cleaving the hmge region of an immunoglobulin heavy chain at a site comprising an ammo acid sequence including D-C-P-K-C-P-P-P-E-M-L-G-G-P- S-I-F-I-F-P-P-K-P-K-D (SEQ ID NO: 104) , D-C-P-K-C-P-P-P-E-M- L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K-S-E-V (SEQ ID NO: 105) and/or D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K- P-K-D-T-L-S-I-S-R-T-P-E-V (SEQ ID NO: 106) . A preferred flea ammopeptidase protein of the present invention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP and/or nfAP2 (flea ammopeptidase full-length coding regions of a flea ammopeptidase gene of the present mvention) . Particularly preferred aminopeptidase proteins are encoded by a nucleic acid rrtolcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP453, nfAP900r nfAP73,, nfAP:580, nfAP238, and/or nfAP2537. More preferred is an aminopeptidase protein encoded by a nucleic acid molcule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfAP2383 and/or nfAP2537. Even more preferred is an aminopeptidase protein that includes amino acid sequence SEQ ID NO: 107, SEQ ID NO: 111 and/or SEQ ID NO: 113, or an ammopeptidase protein encoded by an allelic variant of a nucleic acid molecule that includes SEQ ID NO:llC and/or SEQ ID NO: 112. Also preferred are flea aminopeptidase proteins including regions that have at least about 50%, prefereably at least about 75%, and more preferably at least about 90% identity with flea ammopeptidase proteins having ammo acid sequences as cited herein.
A preferred flea cysteine protease protem of the present mvention is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule nfCPl (a flea cysteine protease full-length coding region that includes nfCPl57, or nfCPlno (the production of which are described m the Examples) . Even more preferred is a cysteine protease that includes ammo acid sequence SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95, SEQ ID NO: 77, or a cysteine protease encoded by an allelic variant of a nucleic acid molecule that mcludes SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 76, or SEQ ID NO: 94. Also preferred is a flea cysteine protease protem including regions that have at least about 50%, preferably at least about 75%, and more preferably at least about 90% identity with SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO:8, SEQ ID N0:89, SEQ ID NO:92, SEQ ID NO:77, or SEQ ID NO: 95.
One embodiment of the present invention is an isolated protem having proteolytic activity that is substantially inhibited by a serine protease inhibitor, an ammopeptidase inhibitor and/or a cysteine protease inhibitor. Such inhibition can be measured by techniques known to those skilled m the art. To be substantially inhibited means, for example, for a serine protease, that at least half of the proteolytic activity of the protease protem is inhibited by a serine protease inhibitor. Preferably at least about 70 percent, and even more preferably at least about 90 percent of the proteolytic activity of the protease protem is inhibited by a serine protease inhibitor. Preferred serine protease inhibitors include flea serpm proteins, and peptides or analogs thereof. An isolated protem of the present invention can be produced m a variety of ways, including recovering such a protein from a flea midgut and producing such a protein recombinantly. In one embodiment, a flea midgut protease can be recovered by methods heretofore disclosed for obtaining a soluble flea midgut preparation. A flea midgut protease protem can be further purified from a disrupted flea midgut by a number of techniques known to those skilled m the art, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis (e.g., standard, capillary and flow-through electrophoresis) , hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalm A chromatography, chromatofocusmg and differential solubilization. In one embodiment, a flea midgut protease is purified usmg protease inhibitor affinity chromatography, an example of which is disclosed m the Examples section.
Another embodiment of the present invention is a method to produce an isolated protem of the present invention using recombinant DNA technology. Such a method includes the steps of (a) culturing a recombinant cell comprising a nucleic acid molecule encoding a protem of the present invention to produce the protem and (b) recovering the protem therefrom. Details on producing recombinant cells and culturing thereof are presented below. The phrase "recovering the protem" refers simply to collecting the whole fermentation medium containing the protem and need not imply additional steps of separation or purification. Proteins of the present invention can be purified usmg a variety of standard protein purification techniques, as heretofore disclosed.
Isolated proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" refers to a purity that allows for the effective use of the protem as a vaccine. A vaccine for animals, for example, should exhibit no substantial toxicity and should be capable of stimulating tne production of antibodies in a vaccinated animal.
Another embodiment of the present invention is an solated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a flea protease present in a flea midgut. Such a nucleic acid molecule is also referred to herem as a flea protease nucleic acid molecule. Particularly preferred is an isolated nucleic acid molecule that hybridizes under stringent conditions with a flea serine protease gene, with a flea aminopeptidase gene or with a flea cysteine protease gene. The characteristics of such genes are disclosed herein. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) . As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can mclude DNA, RNA, or derivatives of either DNA or RNA. As stated above, a flea protease gene mcludes all nucleic acid sequences related to a natural flea protease gene such as regulatory regions that control production of a flea protease protem encoded by that gene (such as, but not limited to, transcription, translation or post- translation control regions) as well as the coding region itself. A nucleic acid molecule of the present invention can be an isolated natural flea protease nucleic acid molecule or a homologue thereof. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a flea protease nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a correspondmg natural gene. Flea protease nucleic acid molecules can also mclude a nucleic acid molecule encoding a hybrid protem, a fusion protem, a multivalent protem or a truncation fragment.
An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene. As used herein, the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity. For example, at least a portion of a nucleic acid sequence, as used herein, is an amount of a nucleic acid sequence capable of forming a stable hybrid with the corresponding gene under stringent hybridization conditions.
An isolated nucleic acid molecule of the present invention can also be produced usmg recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated flea protease nucleic acid molecules mclude natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the nucleic acid molecule's ability to encode a flea protease protem of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates.
A flea protease nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al., ibi d. ) . For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., the ability of a homologue to elicit an immune response against a flea protease and/or to have proteolytic activity) and/or by hybridization with isolated flea protease nucleic acids under stringent conditions.
An isolated flea protease nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one flea protease protem of the present invention, examples of such proteins being disclosed herein. Although the phrase "nucleic acid molecule" primarily refers to the physical nucleic acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding an flea protease protem. One embodiment of the present invention is a flea protease nucleic acid molecule of the present mvention that is capable of hybridizing under stringent conditions to a nucleic acid strand that encodes at least a portion of a flea protease or a homologue thereof or to the complement of such a nucleic acid strand. A nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited. It is to be noted that a double- stranded nucleic acid molecule of the present invention for wnich a nucleic acid sequence has been determmed for one strand, that is represented by a SEQ ID NO, also comprises a complementary strand having a sequence that is a complement of that SEQ ID NO. As such, nucleic acid molecules of the present invention, which can be either double-stranded or single-stranded, include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with either a given SEQ ID NO denoted herein and/or with the complement of that SEQ ID NO, which may or may not be denoted herein. Methods to deduce a complementary sequence are known to those skilled in the art. Preferred is a flea protease nucleic acid molecule that includes a nucleic acid sequence having at least about 65 percent, preferably at least about 75 percent, more preferably at least about 85 percent, and even more preferably at least about 95 percent homology with the corresponding region (s) of the nucleic acid sequence encoding at least a portion of a flea protease protein. Particularly preferred is a flea protease nucleic acid molecule capable of encoding at least a portion of a flea protease that naturally is present in flea midguts and preferably is included in a soluble flea midgut preparation of the present invention. Examples of nucleic acid molecules of the present invention are disclosed in the Examples section. A preferred flea serine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP3, nfSP8, nfSP9, nfSPIO, nfSPll, nfSPl9, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSPlδ, nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40. More preferred is a nucleic acid molecule that hybridizes under stringent hybridization conditions with at least one of the following nucleic acid molecules: nfSP18534, nfSP18775, nfSP18225, nfSP24410, nfSP2410bQ, nfSP24,,4, nfSP247ι;, nfSP287n, nfSP28923, nfSP32q1„ nfSP32924, nfSP326qq, nfSP33426/ nfSP3377fl, nfSP331894, nfSP33li00, nfSP3372e/ nfSP40 θ4„ nfSP5 8β6 nfSPll 307 nfSP8 M5 nfSP8436, nfSP12758, nfSP26610, nfSP273β6, nfSP23423, nfSP34390, nfSP36]97, nfSP38341, nfSP3726], nfSP39267 nfSP29612, nfSP3064], nfSP31b26, nfSP32433, nfSP158]6, nfSP19855, nfSP25θ64, nfSP21595, and/or nfSP40717, as well as other specific nucleic acid molecules disclosed in the Examples section. Even more preferred are nucleic acid molecules that include nfSP3, nfSP8, nfΞP9, nfSPIO, nfSPll, nfSP19, nfSP20, nfSP21, nfSP23, nfSP25, nfSP26, nfSP27, nfSP29, nfSP30, nfSP31, nfSP34, nfSP36, nfSP37, nfSP38, nfSP39, nfSP18, nfSP24, nfSP28, nfSP32, nfSP33 and/or nfSP40 and even more nfSP1853,, nfSPlδT-c, nfSP18225, nfSP24410, nfSP24108y, nfSP24774, nfSP247n, nfSP28923, nfSP32933, nfSP32933, nfSP32924, nfSP3269Q, nfSP33426, nr"SP33~,., nfSP33]H94, nfSP33-^on, nfSP3372t, nfSP408,., nfSP58:t, nfSPll3n-, nfSP851s, nfSP84U/ nfSP12-,5B, nfSP2661P, nfSP27_.tJe, nfSP2342.., nfSP34390, nfSP36197, nfSP38341, nfSP372t:, nfSP39,,- nfSP29cl2, nfSP30641, nfSP3162b, nfSP32433, nfSP1581!), nfSP198C,, nfSP258b4, nfSP215„, and/or nfSP40717, as well as other specific nucleic acid molecules disclosed in the Examples section.
Particularly preferred flea serine protease nucleic acid molecules include at least one of the following sequences:SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28-, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID NO:121, SEQ ID NO:131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO:136, SEQ ID NO:78, SEQ ID NO:158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO*.138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, and/or SEQ ID NO: 45, and complements thereof, as well as other specific nucleic acid molecules disclosed in the Examples section. Also preferred are allelic variants of such nucleic acid molecules.
A preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP and/or nfAP2. A more preferred flea ammopeptidase nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfAP4Dl, nfAP900> nfAP732, nfAPl380, nfAP23b and/or nfAP253- More preferred is an ammopeptidase nucleic acid molecule that includes nfAP453, nfAP900> nfAP732, nfAP1580, nfAP238, and/or nfAP2;,7. Particularly preferred is a nucleic acid molecule that includes nucleic acid sequence SEQ ID NO: 110 and/or SEQ ID NO: 112, a complement of any of such sequences, or allelic variants thereof.
A preferred flea cysteine protease nucleic acid molecule of the present invention is a nucleic acid molecule that hybridizes under stringent hybridization conditions with nfCPl57,, or nfCPlno9 (the production of which are described in the Examples) . More preferred is a cysteine protease nucleic acid molecule that includes nfCPlc- or nfCPl UQQ. Particularly preferred is a nucleic acid molecule that mcludes nucleic acid sequence SEQ ID NO:l, SEQ ID N0:3, SEQ ID NO:4, SEQ ID N0:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:93 and/or SEQ ID NO: 94, or allelic variants of such nucleic acid molecules.
Knowing a nucleic acid molecule of a flea protease protem of the present mvention allows one skilled m the art to make copies of that nucleic acid molecule as well as to obtain a nucleic acid molecule including additional portions of flea protease protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or flea protease nucleic acid molecule homologues. Knowing a portion of an ammo acid sequence of a flea protease protem of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such a flea protease protem. In addition, a desired flea protease nucleic acid molecule can be obtained in a variety of ways including screening appropriate expression libraries with antibodies which bind to flea protease proteins of the present invention; traditional cloning techniques usmg oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used) . To isolate flea protease nucleic acid molecules, preferred cDNA libraries mclude cDNA libraries made from unfed whole fleas, fed whole fleas, fed flea midguts, unfed flea midguts, and flea salivary glands. Techniques to clone and amplify genes are disclosed, for example, m Sambrook et al . , ibi d. The Examples section mcludes examples of the isolation of cDNA sequences encoding flea protease proteins of the present invention.
The present invention also mcludes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of a flea protease protem. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present mvention. Minimal size characteristics are disclosed herein. The size of the oligonucleotide must also be sufficient for the use of the oligonucleotide m accordance with the present invention. Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to inhibit flea protease production. Such therapeutic applications include the use of such oligonucleotides in, for example, antisense-, triplex formation-, ribozyme- and/or RNA drug-based technologies. The present invention, therefore, includes such oligonucleotides and metnods to interfere witn the production of flea protease proteins by use of one or more of such technologies.
The present invention also includes a recombinant vector, which mcludes a flea protease nucleic acid molecule of the present invention inserted mto any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to flea protease nucleic acid molecules of the present invention. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of flea protease nucleic acid molecules of the present invention. One type of recombinant vector, herein referred to as a recombinant molecule and described in more detail below, can be used m the expression of nucleic acid molecules of the present invention. Preferred recombinant vectors are capable of replicating in the transformed cell. Preferred nucleic acid molecules to include in recombinant vectors of the present invention are disclosed herein. As heretofore disclosed, one embodiment of the present invention is a method to produce a flea protease protem of the present invention by culturing a cell capable of expressing the protem under conditions effective to produce the protem, and recovering the protem. A preferred cell to culture is a recombinant cell that is capable of expressing the flea protease protem, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule mto a cell can be accomplished by any method by which a nucleic acid molecule can be inserted mto the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow mto a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites withm a chromosome of the transformed (i.e., recombinant) cell m such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a host cell are disclosed herein.
Suitable host cells to transform include any cell that can be transformed and that can express the introduced flea protease protein. Such cells are, therefore, capable of producing flea protease proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells. Preferred host cells include bacterial, yeast, insect and mammalian cells, with bacterial (e.g., E. coli ) and insect (e.g., Spodoptera) cells being particularly preferred. A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) m recombinant cells of the present invention, including m bacterial, fungal, insect, animal, and/or plant cells. As such, nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present mvention. As used herein, a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function m at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled m the art. Preferred transcription control sequences include those which function in bacterial, yeast, helminth, insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (λ) (such as λpL and λpF and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionem, alpha mating factor, Pi chi a alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters) , baculovirus, Heli othi s zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actm, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate ana nitrate transcription control sequences as well as other sequences capable of controlling gene expression m prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokme-mducible promoters (e.g., promoters inducible by interferons or interleukins) . Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding a flea protease protem.
Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed flea protease protem to be secreted from the cell that produces the protem. Suitable signal segments include a flea protease protem signal segment or any heterologous signal segment capable of directing the secretion of a flea protease protem, including fusion proteins, of the present invention. Preferred signal segments include, but are not limited to, flea protease, tissue plasmmogen activator (t- PA) , interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotem signal segments . Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins. Inclusion of a fusion sequence as part of a flea protease nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protem encoded by the nucleic acid molecule. Furthermore, a fusion segment can function as a tool to simplify purification of a flea protease protem, such as to enable purification of the resultant fusion protem usmg affinity chromatography. A suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool) . It is within the scope of the present invention to use one or more fusion segments. Fusion segments can be joined to ammo and/or carboxyl termini of a flea protease protem. Linkages between fusion segments and flea protease proteins can be constructed to be susceptible to cleavage to enable straight-forward recovery of the flea protease proteins. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protem including the fusion segment attached to either the carboxyl and/or amino terminal end of a flea protease protein.
A recombinant molecule of the present invention is a molecule that can mclude at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulatmg expression of the nucleic acid molecule (s) in the cell to be transformed. A preferred recombinant molecule includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase and/or cysteine protease proteins, being more preferred. Similarly, a preferred recombinant cell includes one or more nucleic acid molecules of the present invention, with those that encode one or more flea protease proteins, and particularly one or more flea serine protease, aminopeptidase, and/or cysteine protease proteins, being more preferred. It may be appreciated by one skilled in the art that use of recombinant DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules mto one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shme-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protem production during fermentation. The activity of an expressed recombinant protem of the present mvention may be improved by fragmenting, modifying, or derivatizing the resultant protem.
In accordance with the present mvention, recombinant cells can be used to produce flea protease proteins of the present invention by culturing such cells under conditions effective to produce such a protem, and recovering the protem. Effective conditions to produce a protem include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protem production. An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing a flea protease protem. Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. The medium may comprise complex nutrients or may be a defined minimal medium.
Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted m shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well withm the expertise of one of ordinary skill in the art. Depending on the vector and host system used for production, resultant flea protease proteins may either remain withm the recombinant cell; be secreted mto the fermentation medium; be secreted mto a space between two cellular membranes, such as the periplasmic space m E. coli ; or be retained on the outer surface of a cell or viral membrane. Methods to purify such proteins are heretofore disclosed.
The present mvention also includes isolated anti-flea protease antibodies and their use to reduce flea infestation on a host animal as well as in the environment of the animal. An anti-flea protease antibody is an antibody capable of selectively binding to a protease present m a flea midgut, including female and male fed midguts as well as female and male unfed midguts. An anti- flea protease antibody preferably binds to the protease m such a way as to reduce the proteolytic activity of that protease.
Isolated antibodies are antibodies that have been removed from their natural milieu. The term "isolated" does not refer to the state of purity of such antibodies. As such, isolated antibodies can mclude anti-sera containing such antibodies, or antibodies that have been purified to varying degrees. As used herein, the term "selectively binds to" refers to the ability of such antibodies to preferentially bind to the protease agamst which the antibody was raised (i.e., to be able to distinguish that protease from unrelated components in a mixture.) . Binding affinities typically range from about 10' M to about 101- M" . Binding can be measured using a variety of methods known to those skilled m the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (e.g., ELISA) , immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., lbi a .
Antibodies of the present mvention can be either polyclonal or monoclonal antibodies. Antibodies of the present invention mclude functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protem used to obtain the antibodies. Antibodies of the present mvention also include chimeric antibodies that can bind to more than one epitope. Preferred antibodies are raised m response to proteins that are encoded, at least m part, by a flea protease nucleic acid molecule of the present invention.
Anti-flea antibodies of the present invention mclude antibodies raised m an animal administered a flea protease vaccine of the present invention that exert their effect when fleas feed from the vaccinated animal's blood containing such antibodies. Anti-flea antibodies of the present invention also include antibodies raised m an animal agamst one or more flea protease proteins, or soluble flea midgut preparations, of the present invention that are then recovered from the animal using techniques known to those skilled m the art. Yet additional antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed for flea protease proteins of the present invention. Antibodies produced agamst defined proteins can be advantageous because such antibodies are not substantially contaminated with antibodies agamst other substances that might otherwise cause interference m a diagnostic assay or side effects if used m a therapeutic composition.
Anti-flea protease antibodies of the present mvention have a variety of uses that are within the scope of the present invention. For example, such antibodies can be used m a composition of the present invention to passively immunize an animal m order to protect the animal from flea infestation. Anti-flea antibodies can also be used as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present mvention can be used to target cytotoxic agents to fleas m order to kill fleas. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled m the art.
A preferred anti-flea protease antibody of the present invention can selectively bind to, and preferentially reduce the proteolytic activity of, a flea serine protease, a flea metalloprotease, a flea aspartic acid protease and/or a flea cysteine protease. More preferred anti-flea protease antibodies include anti-flea serine protease antibodies, anti-flea metalloprotease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies. Particularly preferred are anti-flea serine protease antibodies, anti-flea ammopeptidase antibodies, and anti-flea cysteine protease antibodies, including those raised agamst flea serine protease proteins, flea ammopeptidase proteins or cysteine protease proteins of the present invention. The present invention also includes the use of protease inhibitors that reduce proteolytic activity of flea proteases to reduce flea infestation of animals and the surrounding environment. As used herein, protease inhibitors are compounds that interact directly with a protease thereby inhibiting that protease's activity, usually by binding to or otherwise interacting with the protease 's active site. Protease inhibitors are usually relatively small compounds and as such differ from anti- protease antibodies that interact with the active site of a protease.
Protease inhibitors can be used directly as compounds m compositions of the present invention to treat animals as long as such compounds are not harmful to the animals being treated. Protease inhibitors can also be used to identify preferred types of flea proteases to target using compositions of the present mvention. For example, the inventors have shown herein the predominance of serine proteases m flea midguts, particularly in soluble flea midgut preparations, usmg protease inhibitors. Such knowledge suggests that effective reduction of flea infestation of an animal can be achieved using serine protease vaccines, anti-flea serine protease antibodies and other inhibitors of serine protease synthesis and activity that can be tolerated by the animal. For example, flea immunoglobulin proteinase activity disclosed herein can be targeted to reduce flea infestation. That other proteases are also present m flea midguts accordmg to the present mvention also suggests targeting such proteases. Methods to use protease inhibitors are known to those skilled in the art; examples of such methods are disclosed herein. In one embodiment, a protease inhibitor that can be used m a composition of the present invention to treat an animal is identified by a method including the following steps: (a) identifying candidate (i.e., putative, possible) inhibitor compounds by testing the efficacy of one or more protease inhibitors (l) in vi tro for their ability to inhibit flea protease activity and/or (ii) m a flea feeding assay for their ability to reduce the survival and/or fecundity of fleas by adding the inhibitors to the blood meal of a flea being maintained, for example, m a feeding system, such as that described by Wade et al., 1986, J. Med Entomol . 25, 186-190; and (b) testing the efficacy of the candidate inhibitor compounds in animals infested with fleas. Although one does not need both ir vi tro assay data and flea feeding assay data to determine which candidate compounds to administer to animals, evaluation of both sets of data is preferred since data from neither of the assays necessarily predicts data to be obtained from the other assay. For example, candidate compounds identified usmg the in vi tro assay may work "m the test tube" but may not work in vivo for a number of reasons, including the presence of interfering components m the blood meal that inhibit the activity of such compounds; e.g., although aprotinin can inhibit at least some flea serine proteases in vi tro, aprotinin does not work well m the presence of serum proteins, such as are found m the blood. Furthermore, candidate inhibitor compounds identified by the flea feeding assays can include not only desired compounds but also compounds that reduce the viability and/or fecundity of fleas due to general toxicity (e.g., affecting the mitochondria of fleas) . In a preferred embodiment, an inhibitor of a flea protease of the present invention is identified by a method comprising: (a) contacting an isolated flea protease protem comprising an ammo acid sequence including SEQ ID NO:10, SEQ ID N0:13, SEQ ID N0:16, SEQ ID N0:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID N0.151, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID N0:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID N0:5, SEQ ID N0:8, SEQ ID NO:89, SEQ ID NO: 92 and/or SEQ ID NO: 95 with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has proteolytic activity; and (b) determining if the putative inhibitory compound inhibits the activity. A test kit can be used to perform such method. A preferred test kit comprises an isolated flea protease protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID N0:151, SEQ ID N0:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID N0:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID N0:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and/or SEQ ID NO: 95, and a means for determining the extent of inhibition of the activity in the presence of a putative inhibitory compound. In another embodiment, protease inhibitors are used m the purification of corresponding proteases by, for example, affinity chromatography, m which, a protease inhibitor is incubated with a mixture containing a desired protease under conditions that the inhibitor forms a complex with the protease. The protease can then be recovered from the complex. The protease inhibitor can be attached to a solid support and/or be labelled with, for example, a radioactive, fluorescent, or enzymatic tag that can be used to detect and/or recover the complex. Suitable protease inhibitors to use in accordance with the present invention include serine protease inhibitors (including immunoglobulin proteinase inhibitors and serpms) , metalloprotease inhibitors, aspartic acid protease inhibitors, cysteine protease inhibitors and ammopeptidase inhibitors. Preferred protease inhibitors include serine protease inhibitors, metalloprotease inhibitors, ammopeptidase inhibitors and cysteine protease inhibitors, particularly those that are broad spectrum inhibitors. More preferred are broad spectrum serine protease inhibitors. There is a wide variety of protease inhibitors, as is known to one skilled in the art. Examples include, but are not limited to, AEBSF, aprotinin, bestatm, chloromethyl ketones TLCK (Nα-p-tosyl-L-lysme chloromethyl ketone) and TPCK (N-tosyl-L-phenylalanme chloromethyl ketone), chymostatm, cystatm, 3 ' 4-dιchloroιsocoumarm, E-64 (trans-epoxysuccmyl-L-leucylamido- (4-guanιdmo) butane) , EDTA (ethylenediammetetraacetic acid) , leupeptin, methyl ketones havmg a variety of leaving groups, oxidized L- leucmethiol, pepstatin, 1, 1O-orthophenanthrolme, phosphoramidon, soybean trypsm/chymotrypsm inhibitor and soybean trypsin inhibitor. Preferred protease inhibitors for use m the present invention include AEBSF, bestatm, E-64 leupeptin, pepstatin, 1, 10-orthophenanthrolme, phosphoramidon, TLCK and TPCK, with AEBSF {a broad spectrum serine protease inhibitor) , bestatm (an inhibitor of leucine ammopeptidase) and 1, 10-orthophenanthrolme (a broad spectrum metalloprotease inhibitor) being particularly preferred.
Another preferred inhibitor of the present invention mcludes an inhibitor of an immunoglobulin proteinase of the present invention. Suitable inhibitors of 6δ immunoglobulin proteinase activity are compounds that interact directly with an immunoglobulin proteinase protein's active site, thereby inhibiting that immunoglobulin proteinase' s activity, usually by binding to or otherwise interacting with or otherwise modifying the immunoglobulin proteinase' s active site. Immunoglobulin proteinase inhibitors can also interact with other regions of the immunoglobulin proteinase protem to inhibit immunoglobulin proteinase activity, for example, by allosteric interaction. Inhibitors of immunoglobulin protemases are usually relatively small compounds and as such differ from anti-immunoglobulm proteinase antibodies.
Preferably, an immunoglobulin proteinase inhibitor of the present invention is identified by its ability to bind to, or otherwise interact with, a flea immunoglobulin proteinase protem, thereby inhibiting the activity of the flea immunoglobulin proteinase.
Preferred immunoglobulin proteinase inhibitors of the present invention include, but are not limited to, flea immunoglobulin proteinase substrate analogs, and other molecules that bind to a flea immunoglobulin proteinase
(e.g., to an allosteric site) m such a manner that proteinase activity of the flea immunoglobulin proteinase is inhibited. An immunoglobulin proteinase substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the active site of an immunoglobulin proteinase protem. A preferred immunoglobulin proteinase substrate analog inhibits immunoglobulin proteinase activity. Immunoglobulin proteinase substrate analogs can be of any inorganic or organic composition, and, as such, can be, but are not limited to, peptides, nucleic acids, and peptidomimetic compounds. Immunoglobulin proteinase substrate analogs can be, but need not be, structurally similar to an immunoglobulin proteinase' s natural substrate as _.ong as they can interact with the active site of that proteinase protem. Immunoglobulin proteinase substrate analogs can be designed using computer-generated structures of immunoglobulin proteinase proteins of the present invention or computer structures of immunoglobulin protemases' natural substrates. Substrate analogs can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or otner inorganic or organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., a flea immunoglobulin proteinase) . A preferred immunoglobulin proteinase substrate analog is a peptidomimetic compound (i.e., a compound that is structurally and/or functionally similar to a natural substrate of an immunoglobulin proteinase of tne present invention, particularly to the region of the substrate that interacts with the proteinase active site, but that inhibits immunoglobulin proteinase activity upon interacting with the immunoglobulin proteinase active site) .
Another preferred flea immunoglobulin proteinase inhibitors of the present invention include antibodies that bind specifically to an immunoglobulin proteinase m such a manner that the proteinase activity of the immunoglobulin proteinase is inhibited. Yet another preferred flea immunoglobulin proteinase inhibitor includes an inhibitor from the class of serine proteinase inhibitors. Suitable immunoglobulin proteinase inhibitor include serine proteinase inhibitors disclosed herein.
Protease inhibitors can be produced usmg methods known to those skilled m the art. Protem- or peptide- based protease inhibitors, such as cystatm or small peptides comprising a protease substrate, can be produced recombinantly and modified as necessary.
The present invention also includes the use of proteolytically active flea protease proteins of the present mvention to identify additional protease inhibitors, and preferably protease inhibitor compounds that can be included m a composition of the present invention to be administered to animals. A method to identify a flea protease inhibitor includes the steps of (a) contacting (e.g., combining, mixing) an isolated flea protease protem with a putative (i.e., candidate) inhibitory compound under conditions in which, in the absence of the compound, the protein has proteolytic activity, and (b) determining if the putative inhibitory compound inhibits the proteolytic activity of the protein. Putative inhibitory compounds to screen include organic molecules, antibodies (including functional equivalents thereof) and substrate analogs. Methods to determine protease activity are known to those skilled in the art, as heretofore disclosed. Particularly preferred for use in identifying inhibitors are flea serine protease proteins, flea aminopeptidase proteins and flea cysteine protease proteins of the present invention.
The present invention also includes inhibitors isolated by such a method, and/or test kit, and their use to inhibit any flea protease that is susceptible to such an inhibitor.
It is to be appreciated that the present invention also includes mimetopes of compounds of the present mvention that can be used in accordance with methods as disclosed for compounds of the present invention. As used herein, a mimetope of a proteinaceous compound of the present invention (e.g., a flea protease protein, an anti- flea protease antibody, a proteinaceous inhibitor of protease activity or synthesis) refers to any compound that is able to mimic the activity of that proteinaceous compound, often because the mimetope has a structure that mimics the proteinaceous compound. For example, a mimetope of a flea protease protein is a compound that has an activity similar to that of an isolated flea protease protein of the present invention. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti- idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures) ; and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer- generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
The present invention includes therapeutic compositions, also referred to herein as compositions, that include a (i.e., at least one) compound of the present invention. Preferred compounds to include in a composition of the present invention include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herein. Such a therapeutic composition can protect an animal from flea infestation by reducing flea protease activity, thereby reducing flea burden on the animal and m the environment of the animal.
Particularly preferred therapeutic compositions of the present invention include at least one of the followmg compounds: an isolated flea serine protease protem or a mimetope thereof; an isolated flea serine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene; an isolated antibody that selectively binds to a flea serine protease protem; an inhibitor of flea serine protease activity identified by its ability to inhibit flea serine protease activity; an isolated flea ammopeptidase protem or a mimetope thereof; an isolated flea ammopeptidase nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea ammopeptidase gene; an isolated antibody that selectively binds to a flea ammopeptidase protem; an inhibitor of flea ammopeptidase activity identified by its ability to inhibit flea ammopeptidase activity; an isolated flea cysteine protease protem or a mimetope thereof; an isolated flea cysteine protease nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea cysteine protease gene; an isolated antibody that selectively binds to a flea cysteine protease protem; and an inhibitor of flea cysteine protease activity identified by its ability to inhibit flea cysteine protease activity. Another embodiment of the present invention is a therapeutic composition that includes a first compound that reduces flea protease activity and a second compound that reduces flea burden by a method other than by reducing flea protease activity. The present invention also includes a method to protect an animal from flea infestation by administering to the animal such a composition. The first compound of such a composition by effectively reducing flea protease activity m the midgut, enhances the activity of the second compound. While not being bound by theory, it is believed that a number of anti-flea treatments, particularly those that are proteinaceous, are not very effective because they are degraded m the flea midgut.
The present invention permits the effective use of such anti-flea treatments by reducing proteolytic degradation of such treatments by the flea midgut.
Preferred first compounds to include m such a composition include flea protease vaccines, anti-flea protease antibodies and/or protease inhibitors as disclosed herem, such compounds that target flea immunoglobulin proteinase activity.
A preferred therapeutic composition of the present invention comprises an excipient and a protective compound including: an isolated protem or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent nvbridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protem comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and/or SEQ ID NO: 95; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence including SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID N0:117, SEQ ID NO:127, SEQ ID N0:121, SEQ ID N0:131, SEQ ID N0:155, SEQ ID N0:114, SEQ ID NO:125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO:136, SEQ ID NO:78, SEQ ID NO:15δ, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO: 66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:βO, SEQ ID NO:62, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:140, SEQ ID NO:122, SEQ ID NO:84, SEQ ID NO:110, SEQ ID N0:112, SEQ ID NO:76, SEQ ID N0:1, SEQ ID NO: 3, SEQ ID N0:4, SEQ ID N0:6, SEQ ID N0:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and/or SEQ ID NO: 94; an isolated antibody that selectively binds to a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO:145, SEQ ID NO: 141, SEQ ID N0:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID N0:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and/or SEQ ID NO: 95; an inhibitor of protease activity identified by its ability to inhibit the activity of a protem encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protem comprising an ammo acid sequence including SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:δ5, SEQ ID NO:107, SEQ ID NOrlll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and/or SEQ ID NO: 95; and a mixture thereof. Suitable second compounds mclude any anti-flea agent (s) , including, but not limited to, proteinaceous compounds, insecticides and flea collars. Preferred second compounds are proteinaceous compounds that effect active immunization (e.g., antigen vaccines), passive immunization (e.g., antibodies), or that otherwise inhibit a flea activity that when inhibited can reduce flea burden on and around an animal. Examples of second compounds include a compound that inhibits binding between a flea membrane protein and its ligand (e.g., a compound that inhibits flea ATPase activity or a compound that inhibits binding of a peptide or steroid hormone to its receptor) , a compound that inhibits hormone (including peptide or steroid hormones) synthesis, a compound that inhibits vitellogenesis (including production of vitellm and transport and maturation thereof mto a major egg yolk protem) , a compound that inhibits fat body function, a compound that inhibits flea muscle action, a compound that inhibits the flea nervous system, a compound that inhibits the flea immune system and/or a compound that inhibits flea feeding.
According to the present invention, an immunoglobulin proteinase of the present invention can also be used as a second compound in a therapeutic composition of the present invention to promote longevity of antibodies that bind specifically to selected flea proteins. An immunoglobulin proteinase can be administered to an animal tc promote production of antibodies that bind specifically to the immunoglobulin proteinase, thereby inhibiting the activity of the proteinase. An immunoglobulin proteinase can be administered to an animal either together with cr after administration of any desired flea protem to the animal. A preferred immunoglobulin proteinase to include as a second compound in a therapeutic composition includes: an isolated protem or a mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising ammo acid sequence SEQ ID NO: 67, SEQ ID NO: 68 and/or SEQ ID NO: 69; and/or an isolated nucleic acid molecule that hybridizes under stringent conditions with a gene comprising a nucleic acid sequence including SEQ ID NO: 66 and other nucleic acid sequences encoding an immunoglobulin proteinase of the present mvention disclosed herein.
Compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier. For example, compositions of the present mvention can be formulated m an excipient that the animal to be treated can tolerate.
Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. δ o
Nonaqueous vehicles, such as fixed oils, sesame on, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers mclude phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up m a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, the composition can also include an immunopotentiator, such as an adjuvant or a carrier. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; alummum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (Vaxce!-1", Inc. Norcross, GA) ; Ribi adjuvants (available from Ribi ImmunoChem Research, Inc., Hamilton, MT) ; and saponms and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark) . Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, bacteria, viruses, oils, esters, and glycols.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention mto an animal. As used herein a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel m si tu . Preferred controlled release formulations are biodegradable (i.e., bioerodible) . A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to reduce protease activity in fleas feeding from the animal over a period of time ranging from about 1 to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment for preferably at least about 1 month, more preferably at least about 3 months and even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
In order to protect an animal from flea infestation, a therapeutic composition of the present invention is administered to the animal in an effective manner such that the protease activity of fleas feeding from the blood stream of animals treated with the composition is reduced. As such, a treated animal is an animal that is competent to reduce the flea burden by reducing flea protease activity, or by reducing flea protease activity and at least one other flea activity. Preferably, the protease activity is reduced by at least about 50 percent, more preferably by at least about 70 percent and even more preferably by at least about 90 percent. Methods to administer compositions to the animal in order to render the animal competent depend on the nature of the composition and administration regime. Animals administered a protease vaccine with at least one booster shot usually become competent at about the same time as would be expected for any vaccine treatment. For example, animals administered a booster dose about 4 to 6 weeks after a primary dose usually become competent withm another about 3 to 4 weeks. Animals administered a composition including an anti-flea protease antibody or protease inhibitor become competent as soon as appropriate serum levels of the compound are achieved, usually with one to three days .
In a preferred embodiment, a composition of the present invention when administered to a host animal is able to reduce flea viability by at least about 50 percent withm at least about 21 days after the fleas begin feeding from the treated animal. (Note that fleas usually live about 40 days to about 50 days on one or more animals.) A more preferred composition when administered to a host animal is able to reduce flea viability by at least about 65 percent withm at least about 14 days after the fleas begin feeding from the treated animal. An even more preferred composition when administered to an animal is able to reduce flea viability by at least about 90 percent withm at least about 7 days after the fleas begin feeding from the treated animal. In another preferred embodiment, a composition of the present invention when administered to a host animal is able to reduce flea fecundity (i.e., egg laying abi±ity) by at least about 50 percent, more preferably by at least about 70 percent, and even more preferably by at least about 90 percent, withm at least about 30 days after the fleas begin feeding from the treated animal. (Note that fleas usually do not begin laying eggs until about 7 days after taking a blood meal.) In accordance with the present invention, compositions are administered to an animal in a manner such that the animal becomes competent to reduce flea protease activity in a flea that feeds from the competent; i.e., the animal becomes a treated animal. For example, a flea protease vaccine of the present mvention, when administered to an animal m an effective manner, is able to elicit (i.e., stimulate) an immune response that produces an antibody titer in the blood stream of the animal sufficient to reduce flea protease activity. Similarly, an anti-flea protease antibody of the present invention, when administered to an animal in an effective manner, is administered m an amount so as to be present m the animal's blood stream at a titer that is sufficient to reduce flea protease activity. A protease inhibitor compound of the present invention, when administered to an animal m an effective manner, is administered m a manner so as to be present m the animal's blood stream at a concentration that is sufficient to reduce flea protease activity. Oligonucleotide nucleic acid molecules of the present invention can also be administered in an effective manner, thereby reducing expression of flea proteases.
Compositions of the present invention can be administered to animals prior to or during flea infestation. It is to be noted that when vaccines of the present invention are administered to an animal, a time period is required for the animal to elicit an immune response before the animal is competent to inhibit protease activity of fleas feeding from that animal. Methods to obtain an immune response in an animal are known to those skilled in the art. Acceptable protocols to administer compositions m an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from flea infestation when administered one or more times over a suitable time period. For example, a preferred smgle dose of a protease vaccine or a mimetope thereof ranges from about 1 microgram (μg, also denoted ug) to about 10 milligrams (mg) of the composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks tc several years after the original administration. Booster vaccinations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from flea infestation. A preferred administration schedule is one in which from about 10 μg to about 1 mg of the vaccine per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. In one embodiment, a booster dose of a composition of the present invention is administered about 4 to 6 weeks after the primary dose, and additional boosters are administered about once or twice a year. Modes of administration can include, but are not limited to, oral, nasal, topical, transdermal, rectal, and parenteral routes. Parenteral routes can include, but are not limited to subcutaneous, intradermal, intravenous, and intramuscular routes.
In another embodiment, a preferred single dose of an anti-flea protease antibody composition or a mimetope thereof ranges from about 1 μg to about 10 mg of the composition per kilogram body weight of the animal. Anti- flea antibodies can be re-administered from about 1 hour to about biweekly for several weeks following the original administration. Booster treatments preferably are administered when the titer of antibodies of the animal becomes insufficient to protect the animal from flea infestation. A preferred administration schedule is one m which from about 10 μg to about 1 mg of an ant-.-f-.ea protease antibody composition per kg body weight of the animal is administered about every 2 to every 4 weeks. Suitable modes of administration are as disclosed herein and are known to those skilled in the art.
According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal m a fashion to enable expression of that nucleic acid molecule mto a protective protem (e.g., flea protease vaccine, anti-flea protease antibody, or proteinaceous protease inhibitor) or protective RNA (e.g., antisense RNA, ribozyme or RNA drug) in the animal to be protected from disease. Nucleic acid molecules can be delivered to an animal m a variety of methods including, but not limited to, (a) direct injection (e.g., as "naked" DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Sci ence 247, 1465-1468) or (b) packaged as a recombinant virus particle vaccine or as a recombinant cell vaccine (i.e., delivered to a cell by a vehicle selected from the group consisting of a recombinant virus particle vaccine and a recombinant cell vaccine) .
A recombinant virus particle vaccine of the present invention includes a recombinant molecule of the present invention that is packaged m a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging-deficient. A number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses. W h e n administered to an animal, a recombinant virus particle vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from disease caused by a parasite of the present invention. A preferred single dose of a recombinant virus particle vaccine of the present invention is from about 1 x 104 to about 1 x 10' virus plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines.
A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells include Salmonella, E. coli , Mycobacterium, S. frugiperda, baby hamster kidney, myoblast G8, COS, MDCK and CRFK recombinant cells, with Salmonella recombinant cells being more preferred. Such recombinant cells can be administered in a variety of ways but have the advantage that they can be administered orally, preferably at doses ranging from about IO8 to about IO12 bacteria per kilogram body weight. Administration protocols are similar to those described herein for protem-based vaccines . Recombinant cell vaccines can comprise whole cells or cell lysates.
Compositions of the present invention can be administered to any animal susceptible to flea infestation, including warm-blooded animals. Preferred animals to treat include mammals and birds, with cats, dogs, humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits, raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail and turkeys as well as other furry animals, pets and/or economic food animals, bemg more preferred. Particularly preferred animals to protect are cats and dogs.
The present invention mcludes compositions to treat flea infestation by any flea. As such, compositions of the present mvention can be derived from any flea species. Preferred fleas to target include fleas of the following genera: Ctenocephalides, Cyopsyll us, Diamanus ( Oropsylla) , Echi dnophaga, Nosopsyll us, Pulex, Tunga, and Xenopsylla, with those of the species Ctenocephalides cams, Ctenocephalides feli s, Diamanus montanus, Echidnophaga gallmacea, Nosopsyll us faciatus, Pulex irri tans, Pul ex simulans, Tunga penetrans and Xenopsylla cheopi s being more preferred. Particularly preferred fleas from which to protect animals mclude fleas of the species Ctenocephalides felis, Ctenocephalides cam s, and Pul ex species (e.g., Pulex irri tans and Pulex simulans ) . It is also wit.iin the scope of the present invention to administer compositions of the present mvention directly to fleas. The present invention also includes the use of compositions of the present mvention to reduce infestation by other ectoparasites as well as the use of compositions including protease vaccines, anti-protease antibodies and compounds that inhibit protease synthesis and/or activity derived from any ectoparasite to reduce ectoparasite infestation, particularly controlled release formulations containing such compositions. Preferred ectoparasites to target include arachnids, insects and leeches. More preferred ectoparasites to target include fleas; ticks, including both hard ticks of the family Ixodidae (e.g., Jxodes and Amblyomma) and soft ticks of the family Argasidae (e.g., Orni thodoros, such as 0. parkeri and 0. t uπ ca ta ) ; flies, such as midges (e.g., Culicoides) , mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis- causmg flies and bitmg gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs, including those carrying Chagas disease. Even more preferred ectoparasites to target include fleas, mosquitos, midges, sandflies, blackflies, ticks and Rhodm us . The following examples are provided for the purposes cf illustration and are not intended to limit the scope of tne present invention.
Examples It is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al . , ibi d. , Borovsky, Arch Insect Bi ochem . and Phys . , 7:187-210, 1988, and related references. Examples 1 through 21, and the the sequence information provided in the sequence listing therein, of related PCT Publication No. WO 96/11706, published April 25, 1996, are incorporated herein by this reference in their entirety. Example 1
This example describes the determination of internal ammo acid sequence of a flea aminopeptidase.
About 10,200 cat blood-fed flea guts were dissected into 4 ml Gut Dissection Buffer (50 mM Tris-HCl, pH 8.0 and 100 mM CaCl2) . Flea gut extracts were prepared by sonicating the flea guts and centrifuging them at about 14,000 rpm for about 20 minutes. The resulting pellet was washed and briefly sonicated in 2ml Gut Dissection Buffer and centrifuged again at about 14,000 rpm for about 20 minutes. The resulting pellet was resuspended and sonicated m 4 ml buffer comprising 20 mM NaAc, pH 6.0, 0.1% Brij , complete protease inhibitor cocktail (available from Pierce) and 0.25 mM bestatm; the sonicate was centrifuged at about 14,000 rpm for about 20 minutes. Both the pellet and supernatant were recovered. The pellet was re-sonicated and centrifuged as above, and the resulting supernatant was combined with the original supernatant.
The pooled supernatant was applied to a polyCAT cation exchange HPLC column and protem was eluted with a NaCl gradient ranging from 0M to IM NaCl in 20 mM NaAc, pH 6.0. Fractions collected from the column were assayed by H-Leu-AMC fluorescence, and active fractions were pooled and applied to a C-1 reverse phase HPLC column (TMS 250, Toso Hass) . Proteins were eluted from the column using an acetonitrile gradient m 0.1% TFA m water, the gradient ranging between 20% and 100% acetonitrile. Proteins contained m fractions from the column were analyzed by SDS-PAGE gel electrophoresis and silver staining. The results of tne gel electrophoresis indicated the presence of an about 95 kDa protem m some of the fractions. This protem correlates with the about 95 kDa protem described in Example 12 of related PCT Publication No. WO 96/11706 which was identified using membrane pellet from flea midgut lysates. To determine internal ammo acid sequence of the 95 kDa protem, those fractions containing the 95 kDa protem were pooled, dried and digested with BNPS-Skatole for about 72 hours at room temperature. The BNPS-Skatole digest was separated by 18% Tris-glycme PAGE gel electrophoresis and blotted onto PVDF membrane. A major band of about 28 kDa was cut out and N-terminally sequenced using techniques as described in Example 7 of related PCT Publication No. WO 96/11706. A partial N-termmal ammo acid sequence of the internal peptide was obtained, namely LATTQFQATHARSAFPCFDEPAM (denoted herem SEQ ID NO:107) .
Example 2
This example describes the cloning and sequencing of another flea ammopeptidase nucleic acid molecule. Primer APN3 corresponding to a conserved region in
Manduca sexta and rat ammopeptidases, having nucleic acid sequence 5' CCC AAA TTT TCC ATW GCN CCN GC 3' (N indicating any nucleotide; represented herein as SEQ ID NO: 108) was used m combination with primer M13 Reverse primer (SEQ ID NO: 87) to PCR amplify a portion of a flea ammopeptidase gene from a bovine blood-fed whole flea cDNA expression library as described m Example 8 of related PCT Publication No. WO 96/11706. The resulting product of the PCR amplification was diluted about 1:50 and used as a template in a second, semi-nested PCR amplification usmg a primer APN3 m combination with degenerate primer APN1C, designed using SEQ ID NO: 107 (described in Example 1), having nucleic acid sequence 5' CAA TTY CAA GCT ACY CAT GC 3' (represented herein as SEQ ID NO: 109) . The resulting PCR product, named nfAP2383, was approximately 383-bp when visualized on a 1% agarose gel. The PCR product nfAP2383 was gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques. The nucleotide sequence of nfAP238, is denoted SEQ ID NO: 110. Translation of SEQ ID NO: 110 yielded a deduced flea ammopeptidase protem of about 127 ammo acids, denoted herem as PfAP2ι27, having ammo acid sequence SEQ ID NO: 111.
The PCR product nfAP238, was labelled with "P and used as a probe to screen a bovine blood-fed whole flea phage expression library usmg standard hybridization techniques. A single plaque purified clone was isolated, which included a 2100-nucleotιde insert, referred to herein as nfAP22100. Partial nucleic acid sequence was obtained using standard techniques from the 5' end of nfAP22100, to yield a flea ammopeptidase nucleic acid molecule named nfAP2.)V. having nucleic acid sequence SEQ ID NO: 112. Translation of SEQ ID NO: 112 suggests that nucleic acid molecule nfAP25,~ encodes a non-full-length flea ammopeptidase protem of about 178 ammo acids, referred to herein as PfAP]78, having ammo acid sequence SEQ ID NO: 113, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID N0:112. SEQ ID NO:113 contains SEQ ID N0:107.
Flea ammopeptidase nucleic acid sequence SEQ ID NO: 112 was compared with additional nucleic acid sequences characterized from other organisms. The nucleic acid sequence is about 50% identical to Manduca sexta ammopeptidase N nucleotides between corresponding regions of the two nucleic acid molecules.
Example 3 This example describes the cloning and sequencing of a flea cysteine protease nucleic acid molecule.
A flea cysteine protease nucleic acid molecule, referred to herein as nfCPls^ was produced by PCR amplification using the following method. Primer Cal3F (designed to obtain a calreticulin gene), having nucleic acid sequence 5' TTG GGA TAC ACT TTG ACT GTT AAC C 3', represented herein as SEQ ID NO: 97 was used in combination with the M13 universal primer, to PCR amplify, using standard techniques, a DNA fragment from a bovine blood-fed whole flea cDNA expression library as described above m Example 8 of related PCT Publication No. WO 96/11706. Surprisingly, the isolated DNA fragment correlated with a cysteine protease nucleic acid sequence. Sequence from this DNA fragment was used to design primer CyslR, having the nucleic acid sequence 5' GTG AGC AAC CAT TAT TTC CAT ATC 3', represented herein as SEQ ID NO: 98, which was used m a second PCR amplification m combination with the M13 reverse primer. A third PCR amplification was performed usmg primer CyslF, having the nucleic acid sequence 5' CTT TCC TCA CAA TAC CAC CAA GGA AGC 3', represented herein as SEQ ID NO: 74, in combination with the M13 universal primer. A fourth PCR amplification was performed using primer Cys2F, having the nucleic acid sequence 5' CTT GTA CGA TTG TCT CAA CAG GC 3', represented herein as SEQ ID NO: 76, m combination with the M13 universal primer. The resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques. A composite nucleic acid sequence representing a flea cysteine protease coding region was deduced, referred to herein as nfCPl573, was deduced and is denoted herein as SEQ ID NO:76. Translation of SEQ ID NO: 76 suggests that nucleic acid molecule nfCPl5 -> encodes a non- full-length flea cysteine protease protem of about 191 ammo acids, referred to herem as PfCPl]91, having ammo acid sequence SEQ ID NO: 77, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:76.
The nucleic acid and ammo acid sequences of the nfCPl57> nucleic acid molecule and PfCPlr protem, respectively, were compared to known nucleic acid and ammo acid sequences using a Genbank homology search. SEQ ID NO: 77 was found to be similar to the ammo acid sequence of P . sa ti vum cysteine protease. The most highly conserved region of contmuous similarity between SEQ ID NO: 77 and P. sa ti vum cysteine protease ammo acid sequences spans from about ammo acid 71 through about ammo acid 165 of SEQ ID NO:77 and from about ammo acid 17 through about ammo acid 168 of the P. sa ti vum cysteine protease, there being about 42% identity between the two regions. Comparison of the nucleic acid sequence encoding ammo acids from about 205 through about 492 of nfCPl, , indicate that those regions are about 54- identical.
Example 4
This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules. Additional serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706. The actual primers used in PCR amplification of serine protease nucleic acid molecules from a bovme blood-fed flea cDNA expression library (produced as described in Example 8 of related PCT Publication No. WO 96/11706) included cat-try #2 (SEQ ID NO: 86) in combination with either M13 reverse primer (SEQ ID NO:87, or H57 primer (SEQ ID NO:99) . The resultant PCR products were gel purified and cloned mto the TA Vector™. Two recombinant TA vector clones were isolated and found to correspond to previously cloned serine protease genes. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
A. A nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 5 (produced using primers cat try #2 and M13 reverse) , namely nfSP5«Ct. is represented herem as SEQ ID NO:114. SEQ ID NO: 116 and SEQ ID NO: 117 are both contained withm the sequence of the nucleic acid molecule nfSP5806. Translation of SEQ ID NO: 114 suggests that nucleic acid molecule nfSP5B0£ encodes a close to full-length flea serine protease protem of about 245 ammo acids, referred to herem as PfSP5/45, having ammo acid sequence SEQ ID NO: 115, assuming an open reading frame m which the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 114 and a stop codon spanning from about nucleotide 737 through about nucleotide 739 of SEQ ID NO:114. A Genbank homology search revealed most homology between SEQ ID NO: 114 and a Gall us gall us trypsin gene, there being about 52% identity between correspondmg regions of the two nucleic acid molecules. B. A nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 11 (produced using primers cat try #2 and M13 reverse) , namely nfSPll,07, is represented herein as SEQ ID NO: 118. SEQ ID NO: 120 and SEQ ID NO: 121 are withm the sequence of the nucleic acio molecule nfSPll3C- Translation of SEQ ID NO: 118 suggests tnat nucleic acid molecule nfSPll30- encodes a non-full- length flea serine protease protem of about 102 ammo acids, referred to herein as PfSPll102, having ammo acid sequence SEQ ID NO: 119, assuming the first codon spans from aoout nucleotide 1 through about nucleotide 3 of SEQ ID NO: 118. C. A nucleic acid sequence of the flea serine protease nucleic molecule corresponding to flea clone 39 (produced using primers cat try #2 and H57) , namely nfSP39267, is represented herein as SEQ ID NO: 122. Translation of SEQ ID NO: 122 suggests that nucleic acid molecule nfSP39267 encodes a non-full-length flea serine protease protein of about 90 ammo acids, referred to herein as PfSP3989, having ammo acid sequence SEQ ID NO: 123, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO:122.
Example 5 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules. A. Bovme Blood-Fed Library
Certain flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen a bovine blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706), cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) . Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described in Sambrook et al . , ibi d.
1. The nucleic acid sequence of a flea serine protease nucleic molecule correlating to flea clone 8, namely nfSP8436 is represented herein as SEQ ID NO: 125. SEQ ID NO: 127 is within the sequence of the nucleic acid molecule nfSPδ43b_ Translation of SEQ ID NO: 125 yields a protein of about 145 amino acids, denoted PfSP8145, having amino acid sequence SEQ ID NO:126, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 125. A Genbank homology search revealed most homology between SEQ ID NO: 125 and an Anophel es gambiae trypsin precursor gene, there being about 48% identity between corresponding regions of the two nucleic acid molecules .
2. The nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 12, namely nfSP12756 is represented herein as SEQ ID NO: 128. SEQ ID NO: 130 and SEQ ID NO: 131 are both contained within the sequence of the nucleic acid molecule nfSP12758. Translation cr SEQ ID NO: 128 yields a protem of about 246 ammo acids, denoted PfSP124 , having ammo acid sequence SEQ ID NO: 129, assuming an open reading frame m which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 128 and a stop codon spanning from about nucleotide 739 through about nucleotide 741 of SEQ ID NO: 128. A Genbank homology search revealed most homology between SEQ ID NO: 128 and a rat trypsmogen gene, there being about 57? identity between corresponding regions of the two nucleic acid molecules. B. Cat Blood-Fed Library
Certain flea serine protease cDNA genes have been isolated from a cat blood-fed flea cDNA expression library by screening the library with the cat-try #1 (SEQ ID NO: 124) and cat-try #2 (SEQ ID NO: 86) probes. The cat blood-fed flea library was produced in a similar manner as the bovme blood-fed flea library (described in Example 8 cf related PCT Publication No. WO 96/11706) except the fleas were fed on cat blood. Two clones that hybridized strongly to the probes were isolated and subjected to nucleic acid sequencing using methods described above.
1. The nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP26Dl0 is represented herein as SEQ ID NO: 132. Translation of SEQ ID NO: 132 yields a non-full-length sequence of about 185 ammo acids, denoted PfSP26]8C>, having ammo acid sequence SEQ ID NO: 133, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 132. A Genbank homology search revealed most homology between SEQ ID NO: 133 and a Aedes aegypti trypsin protem sequence, there being about 48% identity between corresponding regions of the two ammo acid sequences.
2. The nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP27386 is represented herem as SEQ ID NO: 134. Translation of SEQ ID NO: 134 yields a protem of about 128 ammo acids, denoted PfSP27128, having amino acid sequence SEQ ID NO: 135, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 134. Example 6 This example describes the cloning and sequencing of certain flea serine protease nucleic acid molecules.
Certain serine protease cDNA nucleic acid molecules have been isolated from reverse transcriptase PCR amplification of mRNA isolated from cat blood-fed whole fleas. The mRNA was isolated from fleas gathered over 72 hours after the initiation of feeding on cat blood. As such, the mRNA comprised a mixture of mRNA isolated at different time points over 72 hours. The mRNA was isolated using ground-up fleas, extracting total flea RNA using Tri-Reagent (available from Molecular Research Center, Cincinnati, Ohio) and an Invitrogen Fast Track1'"' RNA isolation kit (available from Invitrogen, Inc. San Diego, CA) . cDNA was synthesized using a Stratagene RT-PCR kit (available from Stratagene, Inc, San Diego, CA) . Primers used for first-strand cDNA synthesis included an equal molar mixture of the followmg: 5'dT-2VT3' and 5'dT-2VC3' (as provided m a differential display kit, available from Operon Technologies, Inc. Alameda, CA) .
The actual primers used m the PCR amplification of the cDNA described above included cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) . The resultant PCR products were gel purified and cloned mto the TA Vector™. Recombinant TA vector clones were isolated and the nucleic acid molecules were subjected to nucleic acid sequencing using analysis as described above. A. A nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP23423, is represented herein as SEQ ID NO: 136. Translation of SEQ ID NO: 136 suggests that nucleic acid molecule nfSP2342, encodes a non- full-length flea serine protease protem of about 141 ammo acids, referred to herein as PfSP23141, having amino acid sequence SEQ ID NO: 137, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 136. A Genbank homology search revealed most homology between SEQ ID NO: 136 and a Homo sapi ens plasmmogen precursor gene, there being about 51% identity between corresponding regions of the two nucleic acid molecules. B. Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP24410, is represented herein as SEQ ID NO: 78. Translation of SEQ ID NO:78. suggests that nucleic acid molecule nfSP2441c encodes a non- full-length flea serine protease protein of about 136 ammo acids, referred to herein as PfSP24136, having ammo acid sequence SEQ ID NO: 79, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 78. A Genbank homology search revealed most homology between SEQ ID NO:79 and an Anophel es gambiae chymotrypsm protem sequence, there being about 38% identity between corresponding regions of the two amino acid sequences.
C. Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP33426, is represented herein as SEQ ID NO: 82. Translation of SEQ ID NO: 82 suggests that nucleic acid molecule nfSP3342e encodes a non- full-length flea serine protease protein of about 142 ammo acids, referred to herein as PfSP33:42, having amino acid sequence SEQ ID NO: 83, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 82. A Genbank homology search revealed most homology between SEQ ID NO: 83 and a Drosophila serine protease stubble protein sequence, there being about 45% identity between corresponding regions of the two amino acid sequences. D. Another nucleic acid sequence of one of the flea serine protease nucleic molecule, namely nfSP36,q7, is represented herein as SEQ ID NO:138. SEQ ID NO:138 represents a partial sequence of a PCR amplified nucleic acid molecule nfSP36S00. Translation of SEQ ID NO: 138 suggests that nucleic acid molecule nfSP3619- encodes a non- full-length flea serine protease protem of about 65 ammo acids, referred to herem as PfSP3665, having ammo acid sequence SEQ ID NO: 139, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 138. A Genbank homology search revealed most homology between SEQ ID NO: 139 and a Drosophila melanogaster easter protem sequence, there being about 42% identity between correspondmg regions of the two ammo acid sequences. E. Another nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP3834], is represented herein as SEQ ID NO: 140. Translation of SEQ ID NO: 140 suggests that nucleic acid molecule nfSP38,41 encodes a non- full-length flea serine protease protem of about 113 ammo acids, referred to herein as PfSP38113, having ammo acid sequence SEQ ID NO: 141, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 140. A Genbank homology search revealed most homology between SEQ ID NO: 141 and a rat trypsmogen protem sequence, there being about 30% identity between corresponding regions of the two ammo acid sequences. F. A nucleic acid sequence of one of the flea serine protease nucleic molecules, namely nfSP34390, is represented herein as SEQ ID NO: 142. Translation of SEQ ID NO: 142 suggests that nucleic acid molecule nfSP4390 encodes a non- full-length flea serine protease protein of about 130 amino acids, referred to herein as PfSP34130, having ammo acid sequence SEQ ID NO: 143, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID
NO: 142. A Genbank homology search revealed most homology between SEQ ID NO: 143 and a Drosophila mel anogaster Delta precursor protein sequence, there being about 33% identity between corresponding regions of the two ammo acid sequences.
Example 7 This example describes the cloning and sequencing of a flea serine protease nucleic acid molecule.
A serine protease cDNA nucleic acid molecule was isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706. The actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed whole flea cDNA expression library (produced as described in Example 5) included cat-try #2 (SEQ ID NO: 86) in combination with M13 reverse primer (SEQ ID NO:87) . The resulting PCR product was diluted 1:25 and used as a template in a second PCR reaction using the forward vector primer T3 in combination with the reverse primer (derived from the nucleic acid sequence of nfSP337,&, described m Example 6) having the nucleic acid sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3', represented herein as SEQ ID NO: 100. The resultant PCR product was gel purified and cloned mto the TA Vector1^. The clones were subjected to nucleic acid sequencing as described above.
A nucleic acid sequence of a flea serine protease nucleic molecule, namely nfSP3377p is represented herein as SEQ ID NO: 84. As expected, SEQ ID NO: 84 mcludes a portion of SEQ ID NO: 82 Translation of SEQ ID NO: 84 suggests that nucleic acid molecule nfSP33778 encodes a non-full-length flea serine protease protem of about 259 amino acids, referred to herein as PfSP33259, havmg ammo acid sequence SEQ ID NO: 85, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 84. A Genbank homology search revealed most homology between SEQ ID NO: 84 and a Drosophila serine protease stubble gene, there being about 54% identity between nucleotides 23 - 778 of SEQ ID NO: 84 and nucleotides 2324 - 3064 of the Drosophila serine protease stubble gene.
Example 8
This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule. Using the method described in Example 5, a cDNA clone of a flea serine protease was obtained usmg mRNA isolated from bovme blood-fed whole fleas. The resulting cDNA was used as a template in PCR amplification using the primers cat-try #2 (SEQ ID NO: 86) used m combination with H57 primer (SEQ ID NO: 99) . The resultant PCR products were gel purified and cloned mto the TA Vector™. One recombinant
TA vector clone was isolated and the flea serine protease nucleic acid molecule and denoted nFS3750C was subjected to nucleic acid sequencing as described in Sambrook et al . , ibid.
The nucleic acid sequence of part of the flea serine protease nucleic molecule nFS37500, namely nfSP372oι, is represented herem as SEQ ID NO: 144. Translation of SEQ ID NO: 144 suggests that nucleic acid molecule nfSP37z61 encodes a non-full-length sequence of a flea serine protease protem of about 87 ammo acids, referred to herein as
PfSP37cr, having ammo acid sequence SEQ ID NO: 145, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 144. A Genbank homology search revealed most homology between SEQ ID NO: 145 and a chicken trypsmogen protem sequence, there being about 31% identity between corresponding regions of the two ammo acid sequences. Example 9
This example describes the cloning and sequencing of certain larval flea serine protease nucleic acid molecules. Certain serine protease cDNA nucleic acid molecules have been isolated from a mixed instar larval cDNA library produced using 1st, 2nd and 3rd instar larvae fed on cat blood, by PCR amplification. The actual primers used in the PCR amplification included either cat-try #2 (SEQ ID NO: 86) m combination with either H57 primer (SEQ ID NO:99)or M13 reverse primer (SEQ ID NO:87) . The resultant PCR products were gel purified and cloned into the TA Vector'1''1. Three recombinant TA vector clones were isolated containing PCR products using cat-try #2 and M13 reverse as primers and one clone was isolated containing PCR products usmg cat-try #2 and H57 primers. These newly cloned nucleic acid molecules were subjected to nucleic acid sequencing as described above.
A. A nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP2961z is represented herein as SEQ ID NO: 146. Translation of SEQ ID NO: 146 suggests that nucleic acid molecule nfSP29612 encodes a close to full-length flea serine protease protein of about 204 ammo acids, referred to herein as PfSP29204, having amino acid sequence SEQ ID NO: 147, assuming an open reading frame m which the first codon spans from about nucleotide 10 through about nucleotide 12 of SEQ IDNO: 146. A Genbank homology search revealed most homology between SEQ ID NO: 146 and a rat trypsinogen gene, there being about 50% identity between corresponding regions of the two nucleic acid molecules.
B. Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP30641, is represented herein as SEQ ID NO: 148. Translation of SEQ ID NO: 148 suggests that nucleic acid molecule nfSP3064: encodes a non-full-length flea serine protease protein of about 213 amino acids, referred to herein as PfSP30213, having amino acid sequence SEQ ID NO: 149, assuming the first codon spans from about nucleotide 3 through about nucleotide 5 of SEQ ID NO: 148. A Genbank homology search revealed most homology between SEQ ID NO: 148 and a Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules. C. Another nucleic acid sequence of one of the larval flea serine protease nucleic molecules isolated using cat-try #2 and M13 reverse primers, namely nfSP31626, is represented herein as SEQ ID NO: 150. Translation of SEQ ID NO: 150 suggests that nucleic acid molecule nfSP3162t encodes a non-full-length flea serine protease protein of about 208 amino acids, referred to herein as PfSP31208, having ammo acid sequence SEQ ID NO: 151, a assuming the f_.rst residue spans from about nucleotide 3 through about nucleotide 5 or from a putative start codon spanning from about nucleotide 6 to about nucleotide 8 of SEQ ID NO: 150. A Genbank homology search revealed homology between SEQ ID NO: 150 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
D. A nucleic acid sequence of a larval flea serine protease nucleic molecule isolated using cat-try #2 and H57 primers, namely nfSP32433, is represented herein as SEQ ID NO: 80. Translation of SEQ ID NO: 80 suggests that nucleic acid molecule nfSP324,3 encodes a non-full-length flea serine protease protem of about 144 ammo acids, referred to herein as PfSP32144, having amino acid sequence SEQ ID NO: 81, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 80. A Genbank homology search revealed most homology between SEQ ID NO: 80 and an Anophel es gambiae trypsin gene, there being about 52% identity between corresponding regions of the two nucleic acid molecules.
Example 10
This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule. A bovme blood-fed whole flea cDNA library (prepared as described in Example 8 of related PCT Publication No. WO 96/11706' was immunoscreened with antiserum collected from a rabbit that was immunized with a collection of flea salivary gland products referred to as fspN (as described m PCT Publication No. WO 96.11271, entitled "NOVEL ECTOPARASITE SALIVA PROTEINS AND APPARATUS TO COLLECT SUCH PROTEINS", published April 18, 1996) . Immunoscreenmg was performed as follows. New Zealand White rabbit antiserum developed agamst fspN flea saliva products was used m the immunoscreenmg protocols described in the picoBlue™ Immunoscreenmg Kit instruction manual, available from Stratagene, Inc. The methods for preparation of the cDNA expression libraries for immunoscreenmg, i.e., expression of the cDNA clones and procedures for transferring lambda phage plaques to membranes for immunoscreenmg, are described in the ZAP-cDNA Synthesis Kit instruction manual, also available from Stratagene, Inc., La Jolla, California.
A nucleotide sequence for a flea serine protease nucleic acid molecule named nfSP15815 is denoted as SEQ ID NO: 152 and corresponds to SEQ ID NO: 154. Translation of SEQ ID NO: 152 suggests that nucleic acid molecule nfSP158)C encodes a close to full-length flea serine protease protem of about 254 ammo acids, referred to herein as PfSP152C,4, having ammo acid sequence SEQ ID NO: 153, assuming an open reading frame in which the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 152 and a stop codon spanning from about nucleotide 763 through about nucleotide 765 of SEQ ID NO: 152. A Genbank homology search revealed homology between SEQ ID NO: 152 and an Anophel es gambiae trypsin gene, there being about 52 o identity between corresponding regions of the two nucleic acid molecules.
Example 11
This example describes the cloning and sequencing of additional flea serine protease nucleic acid molecules.
Certain flea serine protease cDNA nucleic acid molecules have been isolated in a manner similar to that described in Example 8 of related PCT Publication No. WO 96/11706, using two nucleic acid molecules as probes to screen an unfed flea cDNA expression library, nfSP8299 (SEQ ID NO: 127) and nfSP19359 (SEQ ID NO: 155) . A clone that hybridized strongly to the probes was isolated and subjected to nucleic acid sequencing as described above.
A. The nucleic acid sequence of the flea serine protease nucleic molecule, namely nfSP19855, is represented herein as SEQ ID NO: 156. SEQ ID NO: 155 is withm the sequence of the nucleic acid molecule nfSP19855 Translation of SEQ ID NO: 156 yields an apparent full-length protem of about 253 ammo acids, denoted PfSP19253, having amino acid sequence SEQ ID NO: 157, assuming the first codon, an apparent start codon, spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 156. A Genbank homology search revealed most homology between SEQ ID NO: 156 and an Aedes aegypti trypsin, there being about 53 identity between corresponding regions of both nucleic acid molecules .
B. The nucleic acid sequence of another flea serine protease nucleic molecule, namely nfSP258€4, is represented herein as SEQ ID NO: 158. Translation of SEQ ID NO: 158 yields a protem of about 260 ammo acids, denoted PfSP25260, having ammo acid sequence SEQ ID NO: 159, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 158 and a stop codon spanning from about nucleotide 782 through about nucleotide 784 of SEQ ID NO: 159. A Genbank homology search revealed most homology between SEQ ID NO: 159 and an Anophel es gambiae chymotrypsm protem sequence, there being about 34% identity between corresponding regions of the two ammo acid sequences.
Example 12
This example describes the cloning and sequencing of another flea serine protease nucleic acid molecule.
A flea serine protease cDNA nucleic acid molecule has been isolated m a manner similar to that described m Example 8 of related PCT Publication No. WO 96/11706, usmg nfSPll25i (SEQ ID NO: 121) as a probe to screen an bovme blood-fed flea cDNA expression library (produced as described m Example 8 of related PCT Publication No. WO 96/11706, . A clone that hybridized strongly to the probe was isolated and subjected to nucleic acid sequencing using the Sanger dideoxy chain termination method, as described m Sambrook et al., ibid.
The nucleic acid sequence of the flea serine protease nucleic molecule, namely nfSP21595, is represented herein as SEQ ID NO: 160. Translation of SEQ ID NO: 160 yields a protem of about 198 ammo acids, denoted PfSP21ι98, having ammo acid sequence SEQ ID NO: 161, assuming the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 160 ^nd a putative stop codon spanning from about nucleotide 596 to about nucleotide 598. A Genbank homology search revealed most homology between SEQ ID NO: 161 and Tachypl eus tπdenta tus coagulation factor G protem sequence, there being about 45% identity between corresponding regions of the two ammo acid sequences.
Example 13
This example describes the isolation and characterization of a 31 kD flea serine protease.
Guts from about 1500 fleas that had been fed on cat blood for about 24 hours were dissected m Gut Dissection Buffer (50 mM Tris 8.0, 100 mM CaCl2) . The guts were disrupted by freezing and thawing 4 times, followed by sonication. The resulting extracts were clarified by centrifugation for 20 minutes at 14,000 rpm in a microfuge at 4 °C. The supernatant was recovered. The gut supernatant was loaded onto a 3 ml column comprising p-aminobenzamidine cross-linked to Sepharose beads (Sigma) , previously equilibrated in Benzamidine Column Buffer (50 mM Tris 8.0, 100 mM CaCl2, 400 mM NaCl) . The supernatant was incubated on the column for about 10 min. Unbound protein was slowly washed off the column using Benzamidine Column Buffer until no protein was detectable by Bradford Assay (Bio Rad) .
Proteases bound to the benzamidine column were then eluted using 10 ml Benzamidine Column Buffer supplemented with 10 mM p-aminobenzamidine (brought to pH 8.0 with NaOH) . Proteases in the eluant were concentrated and diafiltered into a volume of about 0.3 ml Gut Dissection Buffer using a Microcon 3 concentrator (Amicon) .
About 120 μl of the concentrated eluant was further concentrated to a volume of about 30 μl. Proteases contained in this concentrate were resolved by gel electrophoresis on a 14% Tris-Glycine electrophoresis gel
(15 μl per lane = approximately 300 gut equivalents per lane) . After electrophoresis, the separated proteases were blotted onto a PVDF membrane using a CAPS buffer (10 mM
CAPS pH 11, 0.5 mM DTT) The membrane was stained with Coomassie Brilliant Blue. A dominant protem band of about 31 kDa was visualized. The membrane was then used for automated N-terminal sequencing (described in Example 7 of related PCT Publication No. WO 96/11706) . A partial N-terminal amino acid sequence of the flea protease was determined to be IVGGEDVDISTCGWC (denoted SEQ ID NO: 68) .
Example 14
This example describes the isolation and characterization of a 31 kD flea serine protease contained in a formulation having IgGase activity (i.e., ability to proteolyze immunoglobulin G proteins) .
Cat blood-fed flea gut extracts were prepared and selected on a benzamidine column as described above in Example 13. IgG protease activity was assayed by incubating at 37°C, overnight, the benzamidine eluant with cat immunoglobulin G proteins (IgG) purified on Protein A sepharose. The ability of the flea gut benzamidine eluant to digest cat IgG was detected by resolving the samples by gel electrophoresis through a 14% SDS-PAGE gel and silver staining the gel using standard methods. The marked decrease (compared with control samples lacking protease activity) of a 50 kDa band on the silver stained gel, representing cat IgG heavy chain, indicated that the benzamidine eluant contains IgG protease activity. The benzamidine eluant was then purified on a PolyPropylaspartamide hydrophobic interaction chromatography (HIC) column by applying the eluant to the column in buffer containing 0.1 M KP04, pH 6.5 and 2 M (NH4) SO,. Proteases bound to the column were eluted using an ammonium sulfate gradient of 2 M to 0 M in HIC column buffer. Column fractions were tested for IgG protease activity using the method described above. Fractions containing IgG protease activity were pooled and applied to a PolyCat cation exchange column m 20 M sodium acetate, pH 6. The proteins were eluted using a sodium chloride gradient of 0 M to 1 M NaCl in 20 M sodium acetate. Fractions eluted from the column were tested for IgG protease activity and then each fraction was resolved by electrophoresis using SDS-PAGE. Fractions having the highest levels of IgG protease activity included a protem band that migrated at about 31 kDa on the SDS-PAGE gel. Weaker protease activity corresponded to an about 28 kDa band. The 31 kDa protem present on the SDS-PAGE gel was used for N-terminal amino acid sequencing using the blotting method described above. A partial N-terminal ammo acid sequence was determined to be IVGGEDVDIST (C)GWQI (S) FQ (S) ENLHF (C) GG (S) IIAPK (denoted herein as SEQ ID NO: 69) . A comparison of SEQ ID NO: 69 and SEQ ID NO: 68 (described in Example 13) indicates a single residue difference between the two ammo acid sequences at residue 15 of each sequence (i.e., Q and C, respectively) . Since SEQ ID NO: 69 correlates with IgGase activity, the data suggests that the larval protem containing SEQ ID NO: 68 has IgGase activity.
Example 15
This example describes the cloning and sequencing of a 31 kDa flea serine protease contained in a formulation having IgGase activity. A flea protease nucleic acid molecule was isolated from a cat blood-fed whole flea library (described m Example 6) and a bovme blood-fed whole flea library (described m Example 8 of related PCT Publication No. WO 96/11706) by PCR amplification. The actual primers used m the PCR amplification included FP31A primer designed using the N-termmal ammo acid sequence SEQ ID NO: 68, the primer having the nucleic acid sequence 5' GAA GAT GTW GAT ATT TCW ACA TGT GG 3' (SEQ ID NO: 101) used in combination with the M13 universal primer. The resultant PCR products were gel purified and cloned into the TA Vector1*' and subjected to nucleic acid sequencing as described above.
A FP31B primer (5' GAA AAT GAA ATC CAC TTA AAC ATT ACG 3'), (represented herein as SEQ ID NO: 102) was designed using the DNA sequence of a DNA fragment from a bovme blood-fed cDNA library. A flea protease cDNA nucleic acid molecule was isolated by PCR amplification of the cat blood-fed whole flea library and the bovine blood-fed whole flea library described above by PCR amplification. PCR amplification was performed using the FP31B primer in combination with M13 reverse primer. The resulting PCR products were then diluted 1:25, and used as a template for a second PCR reaction using primer FP31C, having the sequence 5' CTC TTA TTG TAC GAG GGA TGC 3' (denoted herein SEQ ID NO: 103) in combination with T3 primer. The resulting nested PCR product was cloned into TA Vector™ and subjected to DNA sequencing.
The nucleic acid sequence of the resulting flea serine protease nucleic molecule, namely nfSP28923 is represented herein as SEQ ID NO: 66. Translation of SEQ ID NO: 66 yields a protein of about 267 amino acids, denoted PfSP28267, having amino acid sequence SEQ ID NO: 67, assuming an open reading frame in which the putative start codon spans from about nucleotide 8 through about nucleotide 10 of SEQ ID NO: 66 or from about nucleotide 11 through about nucleotide 13, and a stop codon spanning from about nucleotide 803 through about nucleotide 805 of SEQ ID NO: 66. SEQ ID NO: 67 contains SEQ ID NO: 68 except Q is substituted for C, and SEQ ID NO: 69. A Genbank homology search revealed most homology between SEQ ID NO: 66 and Bombix mori vitellin- degrading protease gene, there being about 53% identity between corresponding regions of the two nucleic acid sequences .
Example 16
This example describes JH-DFP labelling of larval serine proteases.
About 100 unfed larvae, 100 1st instar larvae, and 100 3rd Instar larvae were collected in 100 μl Gut Dissection Buffer (50 mM Tris 8.0, 100 mM CaCl2) . About 400 μl of water was added to the collected larvae, which were then sonicated. The sonicates were clarified by centrifugation at 15,000 RPM in an ΞS-34 rotor for 30 minutes at 4°C. The supernatant from ea-ch larval sonicate was recovered and concentrated to a volume of about 120 μl (1.2 μl per larval equivalent) . Samples containing about 25 larval equivalents (about
30μl) were labeled with about 2.5 μCi of 3H-dnsopropylfluorophosphate (DFP; available from New England Nuclear) and incubated at 4°C for 18 hours. Following the incubation period, 5 larval equivalents of each larval stage (about 6 μl) were run on a 14% Tris Glycine SDS-PAGE gel. The gel was then soaked in Entensify (available from New England Nuclear) to enhance the tritium signal for autoradiography, and dried. The dried gel was then exposed to X-ray film (Kodak XO-mat) for about 3 days at -70°C. The results indicate that gut extracts from unfed larvae do not contain detectable amounts of serine proteases (Fig.l, lane A), while both fed 1st instar larvae
(Fig.l, lane B) and fed 3rd instar larvae (Fig.l, lane C) produce serine proteases. In particular, fed 1st instar larvae primarily produce a serine protease having a molecular weight of about 25 kD; and fed 3rd mstar larvae produce about serine proteases having molecular weights of about 25 kD, 28 kD and 31 kD. The approximate size of standard molecular weight protem markers are shown in Fig.
Example 17
This example describes the determination of partial N- terminal ammo acid sequences for several larval serine proteases.
About 10,300 3rd Instar larvae were collected in Gut Dissection Buffer (50 mM Tris, pH 8.0, 100 mM CaCl2) . The larvae were homogenized by somcation and clarified by centrifugation at 15,000 rpm m an SS-34 rotor for 30 mm., at 4°C. The supernatant was recovered. The 3rd Instar supernatant was mixed with 5 ml of p-ammobenzamidme cross-linked to Sepharose beads (available from Sigma) equilibrated in Benzamidine Column Buffer (50 mM Tris pH 8.0, 100 mM CaCl2, 400 mM NaCl) . The supernatant was rocked with the beads overnight at 4°C. The beads were washed m about 45 ml Benzamidine Column Buffer to remove unbound protem. The beads were then mixed 2 hours at 4°C with about 10 ml of Benzamidine Column Buffer containing 100 mM p-ammobenzamidme (pH 8.0 adjusted with NaOH) to elute proteins bound to the beads. The eluted proteins were then collected. The elution process was repeated once more. The eluted protem was concentrated by ultrafiltration with a Centriprep 10 concentrator (available from Amicon) . The concentrate was diluted with Gut Dissection Buffer to a final volume of about 5 ml.
Partial N-termmal ammo acid sequence of proteins eluted from the beads was obtained using the method described m Example 11. Two proteins having molecular weights of about 25 kDa and about 26 kDa were identified on the Coomassie Brilliant Blue stained membranes. Partial N- termmal ammo acid sequence obtained for the protem having a molecular weight of about 25 kDa is IVGGVSVNINDYGYQLSLQSNGR, denoted herein as SEQ ID NO: 162. Partial N-termmal ammo acid sequence obtained for the protem having a molecular weight of about 26 kDa is IVGGHDTSIKQHPYQV, denoted herein as SEQ ID NO: 163.
Example 18
This Example demonstrates the production of certain flea serine protease proteins of the present invention. A. Flea serine protease protem PfSPl21. was m the following manner. Flea serine protease nucleic acid molecule nfSPl670, produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector λPR/T2ori/S10HIS-RSET-A9 (the production of which is described in Tripp et al,
International PCT Publication No. WO 95/24198, published
September 14, 1995; see, in particular, Example 7), that had been digested with Xhol and dephosphorylated. The resultant recombinant molecule, referred to herein as pHisCro-nfSPl670, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP1670. The recombinant cell was cultured as described in Example 20 of related PCT
Publication No. WO 96/11706. Flea serine protease protein
PfSPl2U was purified by nickel chelation chromatography followed by reverse phase high performance liquid chromatography (HPLC) . Immunoblot analysis of the purified PfSPl216 indicated that rabbit anti-flea protease antiserum, produced as described in example 14, selectively bound to PfSPl21b.
B. Flea serine protease protein PfSP2233 was produced in the following manner. Flea serine protease nucleic acid molecule nfSP2-,15, produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned mto expression vector λP[</T*-o.r.ι/S10HIS-RSET-A9 as described in Example 39A. The resultant recombinant molecule, referred to herem as pHιsCro-nfSP2-, t, was transformed mto E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHιsCro- nfSP2-π-. The recombinant cell was cultured as described m Example 20 of related PCT Publication No. WO 96/11706. Flea serine protease protem PfSP2,33 was purified by nickel chelation chromatography followed by reverse phase HPLC. Immunoblot analysis of the purified PfSP2233 indicated that rabbit anti-flea protease antiserum, produced as described m example 14 of related PCT Publication No. WO 96/11706, selectively bound to PfSP2233. C. Flea serine protease protein PfSP13225 was produced m the following manner. Flea serine protease nucleic acid molecule nfSP13700, produced as described in Example 20 of related PCT Publication No. WO 96/11706, was digested with Xhol restriction endonuclease, gel purified and subcloned into expression vector λPR/T2orα/S10HIS-RSET-A9 as described in Example 18A. The resultant recombinant molecule, referred to herein as pHιsCro-nfSP13700, was transformed mto E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHιsCro-nfSP1370C. The recombinant cell was cultured as described in Example 20 of related PCT Publication No. WO 96/11706. Flea serine protease protein PfSP1322r was purified by nickel chelation chromatography followed by reverse phase HPLC. Immunoblot analysis of the purified PfSP1322b indicated that rabbit anti-flea protease antiserum, produced as described in Example 14 of related PCT Publication No. WO 96/11706, selectively bound to PfSP13225.
D. Flea serine protease protein PfSP20222 was produced in the following manner. An about 669-bp DNA fragment, referred to herein as nfSP20569, and designed to encode an apparently mature serine protease protein, was PCR amplified from flea serine protease clone 20 using the Xhol-site containing primer F27-S (sense) 5' GAG CTC TCG AGA ATC GTA GGA GGA CAC GAT AC 3' (SEQ ID NO: 164) and the EcoRI-site containing primer F20-A (antisense) 5' G GAC GAA TTC TTA AAC ACC AGA CAC TTC CTT G 3' (SEQ ID NO: 165) . The PCR product nfSP20669 was digested with Xhol and EcoRI restriction endonucleases, gel purified and subcloned into expression vector λPR/T2ori/S10HIS-RSET-A9 as described in Example 18A. The resultant recombinant molecule, referred to herein as pHisCro-nfSP20669, was transformed into E. coli HB101 competent cells (available from Gibco BRL) to form recombinant cell E. coli :pHisCro-nfSP20669. The recombinant cell was cultured as described in Example 20 of related PCT Publication No. WO 96/11706. Immunoblot analysis of recombinant cell E. coli :pHisCro-nfSP20669 lysates using a T7 tag monoclonal antibody (available from Novagen, Inc.) directed agamst the fusion portion of the recombinant PHISCRO-PfSP20,^ fusion protem identified a protem of the appropriate size, namely an about 31-kD protem. Flea serine protease protem PfSP20,_ was purified by nickel chelation chromatography followed by reverse phase HPLC. Immunoblot analysis of the purified PfSP2022_ indicated that rabbit anti-flea protease antiserum, produced as described in example 14 of related PCT Publication No. WO 96/11706, selectively bound to PfSP20222.
Example 19
This example describes that various flea serine protease nucleic acid molecules described in the foregoing examples can be obtained from multiple sources.
Nucleic acid molecules correspondmg to flea clone 4 have been obtained from a bovme blood-fed whole flea library (described in Example 8 of related PCT Publication
No. WO 96/11706), a cat blood-fed whole f] ea library
(described in Example 5), an unfed whole flea library
(described in Example 8 of related PCT Publication No. WO 96/11706) , and a mixed instar whole flea library (described m Example 9) . Nucleic acid molecules corresponding to flea clone 5 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library. Nucleic acid molecules corresponding to flea clone 6 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library and an unfed whoie flea library. Nucleic acid molecule correspondmg to flea clone 7 have been obtained from a bovme blood-fed whole flea library, and a cat blood-fed whole flea library. Nucleic acid molecules corresponding to flea clone 8 have been obtained from a bovme blood-fed whole flea library and an unfed whole flea library. Nucleic acid molecules corresponding to flea clone 12 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library. Nucleic acid molecules corresponding to flea clone 13 have been obtained from a bovine blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library. Nucleic acid molecules corresponding to flea clone 20 have been obtained from a bovme blood-fed whole flea library, a cat blood-fed whole flea library, and an unfed whole flea library. Nucleic acid molecules corresponding to flea clone 28 have been obtained from a bovme blood-fed whole flea library and a cat blood-fed whole flea library.
Example 20
This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding a flea cysteine protease protem of the present which was described m Example 3. This example also provides the production of a cysteine protease protein in E . coli cells.
A. Additional Cysteine Protease Nucleic Acid Molecule The PCR products described in Example 3 were submitted to additional nucleic acid sequence analysis in order to obtain the nucleic acid sequence of additional portions of the coding region of the cysteine protease gene. A composite nucleic acid sequence representing a flea cysteine protease coding region, referred to herein as nfCPl1109, was deduced and is denoted herein as SEQ ID NO:l. SEQ ID NO: 76 is contained within the sequence of the nucleic acid molecule nfCPlU09. Translation of SEQ ID NO:l suggests that nucleic acid molecule nfCPl1109 encodes a full- length flea cysteine protease protein of about 327 amino acids, referred to herein as PfCPl327, having amino acid sequence SEQ ID NO:2, assuming an open reading frame in which the initiation codon spans from about nucleotide 126 through about nucleotide 128 of SEQ ID NO:l and the termination codon spans from about nucleotide 1107 through about nucleotide 1109 of SEQ ID NO:l. The complement of SEQ ID NO:l is represented herein by SEQ ID NO: 3. The coding region encoding PfCPl327, is represented by nucleic acid molecule nfCPl984, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 4 and a complementary strand with nucleic acid sequence SEQ ID NO: 6. The proposed mature protem, denoted herein as PfCPl2 ζ, contains about 226 ammo acids which is represented herein as SEQ ID NO: 8. The nucleic acid molecule encoding PfCPl is denoted herem as nfCPl68 , which is represented by SEQ ID NO:7. The ammo acid sequence of PfCPl3^- (i.e., SEQ ID NO: 2) predicts that PfCPl32-, has an estimated molecular weight of about 42 kD and an estimated pi of about pi 8.84.
Comparison of nucleic acid sequence SEQ ID NO:l with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:l showed the most homology, i.e., about 55% identity, with the following three genes: a Drosophila cysteine protease gene, a BomJbyx cysteine protease gene and a Sarcophaga cysteine protease gene. Comparison of ammo acid sequence SEQ ID NO:2 (i.e., the ammo acid sequence of PfCPl3_-) with ammo acid sequences reported in GenBank indicates that SEQ ID NO:2 showed the most homology, i.e., about 42- identity, with the following three proteins: a Drosophila cysteine protease protem, a BomJbyx cysteine protease protem and a Sarcophaga cysteine protease protein.
B. Production of Cysteine Protease Protem m E.
Figure imgf000132_0001
An about 660-bp nucleic acid molecule, referred to herein as nfCPlb60 (designed to encode an apparently mature cysteine protease protem) was PCR-amplifled from a flea mixed instar cDNA library produced usmg unfed 1st instar, bovme blood-fed 1st instar, bovme blood-fed 2nα instar and bovme blood-fed 3-d instar flea larvae (this combination of tissues is referred to herein as mixed instar larval tissues for purposes of this example) . Total RNA was extracted from mixed instar tissue using an acid- guanidmium-phenol-chloroform method similar to that described by Chomczynski et al . , 1987, Anal . Biochem. 1 62, p. 156-159. Approximately 5,164 mixed instar larvae were used in each RNA preparation. Poly A+ selected RNA was separated from each total RNA preparation by oligo-dT cellulose chromatography using Poly(A) Quick® mRNA isolation kits (available from Stratagene Cloning Systems, La Jolla, CA) , according to the method recommended by the manufacturer. A mixed instar cDNA expression library was constructed m lambda (λ) Uni-ZAP XR vector (available from Stratagene Cloning Systems) using Stratagene' s ZAP-cDNA Synthesis Kit® protocol. About 6.34 μg of mixed instar poly A+ RNA were used to produce the mixed instar library. The resultant mixed instar library was amplified to a titer of about 2.17 x 1010 pfu/ml with about 97% recombmants. The primers used in the PCR amplification were sense primer CysBS ' having the nucleotide sequence 5' GAT AAG GAT CCG TTA CCA GAT TCT TTC GAC TGG 3' (containing a BamHI-site; denoted SEQ ID NO: 64) and anti-sense primer CysHA having the nucleotide sequence 5' TTA TCA AGC TTC CAT TTA CAT GCC GTA AAA ATC 3' (containing a Hmdlll site; denoted SEQ ID NO: 65) . The resultmg PCR product nfCPlDD was submitted to nucleic acid sequence analysis to obtain a nucleic acid sequence of the coding strand, represented herein as SEQ ID NO: 94. Translation of SEQ ID NO: 94 indicated that nfCPlbqr encodes a protem of about 220 ammo acids, called PfCPl220, having SEQ ID NO: 95. It is to be noted that this sequence analysis indicated that the stop codon was actually about 36 base pairs upstream from what had been predicted by SEQ ID NO:l; as such, the protem encoded by nfCPl6c~ is about 12 ammo acids shorter than would have been predicted by SEQ ID NO:l. The nucleic acid molecule nfCPl660 contains the coding region for PfCPl220.
Recombinant cell E. coli:pCro-nfCP16D0 was produced in tne following manner. Nucleic acid molecule nfCPlb60 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector IambdaPR/T2oπ/S10HIS-RSET-A9 (the production of which is described m Tripp et al, International PCT Publication No. WO 95/24198, published September 14, 1995; see m particular, Example 7), that was digested with BamHl and HindiII and dephosphorylated. The resultant recombinant molecule, referred to herem as pCro-nfCPl6bC, was transformed mto E. coli BL-21 competent cells 'available from Novagen, Madison, WI) to form recombinant cell E. coli:pCro-nfCPl6b0. The recombinant cell was cultured as described m Example 20 of related PCT Publication No. WO 95/24198. About 1 ml of culture was collected prior to induction, and about 1 ml of culture was collected about 60 minutes following induction. These samples were then lysed m sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, resolved on a 14 °c Tris-glycme acrylamide gel and analyzed by immunoblot using a T7 (tag) antibody (available from Novagen) .
Example 21 This example provides additional nucleic acid and deduced ammo acid sequences of nucleic acid molecules encoding serine protease proteins of the present invention which are described herein and in the Examples section of related PCT Publication No.WO 96/11706. A. A DNA probe labeled with 32P comprising nucleotides from nfAP2210C (described m Example 23 of related U.S. Patent Application Serial No. 08/639,075, filed April 24, 1996) was used to screen a bovine blood-fed whole flea cDNA library (described in Example 8 of related PCT Publication No.WO 96/11706) using standard hybridization techniques. A clone was isolated having about a 459-nucleotιde insert, referred to herein as nfSP184-„.
A nucleic acid sequence of the composite nucleic acid molecule produced using nucleic acid sequence from nfSP1653. and nfSP18.tq is referred to herein as nfSPlδ ,7, having a nucleic acid sequence of the coding strand which is denoted herem as SEQ ID NO: 9. Translation of SEQ ID NO: 9 suggests that nucleic acid molecule nfSPlθ-,-,- encodes a non-full- length flea serine protease protem of about 228 ammo acids, referred to herein as PfSP18228, having ammo acid sequence SEQ ID NO: 10, assuming the first codon spans from about nucleotide 1 through about nucleotide 3 of SEQ ID NO: 9 and the stop codon spans from about nucleotide 685 through about nucleotide 687 of SEQ ID NO: 9. The complement of SEQ ID NO: 9 is represented herem by SEQ ID NO: 11. The coding region encoding PfSP1822θ, is represented by nucleic acid molecule nfSP1822„, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 12 and a complementary strand with nucleic acid sequence SEQ ID NO: 14. The ammo acid sequence of PfSP18228 (i.e., SEQ ID NO: 10) predicts that PfSP18228 has an estimated molecular weight of about 25 kD and an estimated pi of about 9.09.
Comparison of nucleic acid sequence SEQ ID NO: 9 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 9 showed the most homology, i.e., about 51% identity, between SEQ ID NO: 9 and an Anophel es st ephensi trypsin 1 gene. Comparison of ammo acid sequence SEQ ID NO: 10 (i.e., the ammo acid sequence of PfSP1822r) with ammo acid sequences reported in GenBank indicates that SEQ ID NO: 10 showed the most homology, i.e., about 59? identity between SΞQ ID NO: 10 and Vespa crabro protein.
B. The remainder of flea serine protease nucleic molecule clone 24 (described in Example 6 was determined using primers designed from nfSP24410 to amplify DNA from the bovine biood-fed whole flea cDNA library. Sense primer Flea 24F having the nucleotide sequence 5' GGA CAA ACT GTT CAT TGC AG 3' (denoted SEQ ID NO: 46) was used in combination with the M13 universal primer in a first PCR reaction. Anti-sense primer Flea 24R having the nucleotide sequence 5' CCC TCA TTT GTC GTA ACT CC 3' (denoted SEQ ID NO: 47) was used in combination with the M13 reverse primer in a second PCR reaction. The resulting PCR products were each gel purified and cloned into the TA Vector® System, and subjected to standard DNA sequencing techniques.
A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP24:089, was deduced and is denoted herein as SEQ ID NO: 15. SEQ ID NO:78 is contained within the sequence of the nucleic acid molecule nfSP241089_ Translation of SEQ ID NO: 15 suggests that nucleic acid molecule nfSP241089 encodes a full-length flea serine protease protein of about 258 amino acids, referred to herein as PfSP242f)8, having amino acid sequence SEQ ID NO: 16, assuming an open reading frame in which the initiation codon spans from about nucleotide 33 through about nucleotide 35 of SEQ ID NO: 15 and the termination codon spans from about nucleotide 807 through about nucleotide 809 of SEQ ID NO: 15. The complement of SEQ ID NO: 15 is represented herem by SEQ ID NO: 17. The coding region encoding PfSP24258, is represented by nucleic acid molecule nfSP24774, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 18 and a complementary strand with nucleic acid sequence SEQ ID NO: 20. The proposed mature protem, denoted herein as PfSP24237, contains about 237 ammo acids which is represented herein as SEQ ID NO:22. The nucleic acid molecule encoding PfSP2423-7 is denoted herein as nfSP247n, which is represented by SEQ ID NO: 21. The ammo acid sequence of PfSP24258 (i.e., SEQ ID NO: 16) predicts that PfSP24258 has an estimated molecular weight of about 28 kD and an estimated pi of about pi 6.70.
Comparison of nucleic acid sequence SEQ ID NO: 15 with nucleic acid sequences reported m GenBank indicates that
SEQ ID NO: 15 showed the most homology, i.e., about 51% identity between SEQ ID NO: 15 and an Anopheles stephensi trypsin 1 gene. Comparison of ammo acid sequence SEQ ID
NO: 16 (i.e., the ammo acid sequence of PfSP24253) with ammo acid sequences reported m GenBank indicates that SEQ ID
NO: 16 showed the most homology, i.e., about 59% identity between SEQ ID NO: 16 and an Anophel es gambiae chymotrypsm
II protem. C. The remainder of flea serine protease nucleic molecule clone 32 (described in Example 20 was determined using primers designed from nfSP324„ to amplify DNA from the cat-fed whole flea cDNA library. Sense primer Flea 32F having the nucleotide sequence 5' GGC TAG GTT AGT GGA TTC TGG 3' (denoted SEQ ID NO: 48) was used in combination with the M13 universal primer in a first PCR reaction. Anti- sense primer Flea 32R having the nucleotide sequence 5' GCA AAT CAG TTC CAG AAT CCA CTA ACC 3' (denoted SEQ ID NO: 49) was used m combination with the M13 reverse primer m a second PCR reaction. The resultmg PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques.
A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to nerem as nfSP32924, was deduced and is denoted herein as SEQ ID NO:23. SEQ ID NO: 80 is contained withm the sequence of the nucleic acid molecule nfSP32924 Translation of SEQ ID NO:23 suggests that nucleic acid molecule nfSP329:„ encodes a full-length flea serine protease protem of about 268 ammo acids, referred to herein as PfSP32268, havmg ammo acid sequence SEQ ID NO: 24, assuming an open reading frame in which the initiation codon spans from about nucleotide 6 through about nucleotide 8 of SEQ ID NO: 23 and the termination codon spans from about nucleotide 810 through about nucleotide 812 of SEQ ID NO:23. The complement of SEQ ID NO:23 is represented herein by SEQ ID NO:25. The coding region encoding PfSP32268, is represented by nucleic acid molecule nfSP32b0q, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 26 and a complementary strand with nucleic acid sequence SEQ ID NO.28. The ammo acid sequence of PfSP3226p (i.e., SEQ ID NO: 24) predicts that PfSP32268 has an estimated molecular weight of about 28.6 kD and an estimated pi of about pi 7.36. Comparison of nucleic acid sequence SEQ ID NO:23 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO: 23 showed the most homology, i.e., about 52% identity between SEQ ID NO:23 and a Fusari um oxysporum preprotrypsm gene. Comparison of ammo acid sequence SEQ ID NO: 24 (i.e., the ammo acid sequence of PfSP32268) with ammo acid sequences reported in GenBank indicates that SEQ ID NO:24 showed the most homology, i.e., about 63% identity between SEQ ID NO:24 and a Bombyx mon vitellm -degrading protease precursor protem. D. The remainder of flea serine protease nucleic molecule clone 33 was determined using primers designed from nfSP33-,78 to amplify DNA from the flea mixed instar larvae cDNA library described above in Example 20. Sense primer Flea 33F having the nucleotide sequence 5' CAG GGC GCT CTG CAG AAC GCA AC 3' (denoted SEQ ID NO: 50) was used in combination with the M13 universal primer m a first PCR reaction. Anti-sense primer Flea 33R having the nucleotide sequence 5' ATT CCT CGT GGT TCA GTC GCT C 3' (denoted SEQ ID NO: 51) was used m combination with the M13 reverse primer m a second PCR reaction. The resulting PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques .
A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP331894, was deduced and is denoted herein as SEQ ID NO: 29. SEQ ID NO: 84 and SEQ ID NO: 82 are contained withm the sequence of the nucleic acid molecule nfSP33,894_ Translation of SEQ ID NO:29 suggests that nucleic acid molecule nfSP331894 encodes a full-length flea serine protease protem of about 400 ammo acids, referred to herein as PfSP33400, having amino acid sequence SEQ ID NO: 30, assuming an open reading frame m which the initiation codon spans from about nucleotide 335 through about nucleotide 337 of SEQ ID NO:29 and the termination codon spans from about nucleotide 1535 through about nucleotide 1537 of SEQ ID NO:29. The complement of SEQ ID NO:29 is represented herein by SEQ ID NO: 31. The coding region encoding PfSP33400, is represented by nucleic acid molecule nfSP331200, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 32 and a complementary strand with nucleic acid sequence SEQ ID NO: 34. The proposed mature protein, denoted herein as PfSP334,, contains about 242 ammo acids which is represented herein as SEQ ID NO: 36. The nucleic acid molecule encoding PfSP33.4 is denoted herein as nfSP3372b, which is represented by SEQ ID NO: 35. The ammo acid sequence of PfSP3340C (i.e., SEQ ID NO:30) predicts that PfSP33400 has an estimated molecular weight of about 44 kD and an estimated pl of about pi 7.59.
Comparison of nucleic acid sequence SEQ ID NO:29 with nucleic acid sequences reported m GenBank indicates that SEQ ID NO: 29 showed the most homology, i.e., about 48% identity between SEQ ID NO:29 and a Drosophil a melanogaster serine protease stubble gene. Comparison of ammo acid sequence SEQ ID NO: 30 (i.e., the amino acid sequence of PfSP33400) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 30 showed the most homology, i.e., about 63 identity between SEQ ID NO: 30 and a Drosophila melanogaster serine protease stubble protem.
Example 22 This example provides nucleic acid and deduced ammo acid sequence of another nucleic acid molecule encoding a serine protease protem of the present invention.
A serine protease cDNA nucleic acid molecules has been isolated in a manner similar to that described in Example 8 of related PCT Publication No.No.WO 96/11706. The actual primers used in PCR amplification of the serine protease nucleic acid molecule from a cat blood-fed flea cDNA expression library (produced as described in Example 8 of related PCT Publication No.No.WO 96/11706) included cat-try #2 (SEQ ID NO: 86) m combination with H57 primer (SEQ ID NO: 99) . The resultant PCR product was gel purified and cloned into the TA Vector™. A recombinant TA vector clone was isolated and subjected to nucleic acid sequencing.
A composite nucleic acid sequence of a flea serine protease nucleic molecule corresponding to flea clone 40, namely nfSP40428 was deduced and is denoted herem as SEQ ID NO: 37. Translation of SEQ ID NO: 37 suggests that nucleic acid molecule nfSP40428 encodes a non-full-length flea serine protease protem of about 142 ammo acids, referred to herein as PfSP40142, represented herem by SEQ ID NO: 38. The complement of SEQ ID NO: 37 is represented herem by SEQ ID NO: 39.
The remainder of flea serine protease nucleic molecule clone 40 was determined using primers designed from nfSP404_8 to amplify DNA from the cat blood-fed whole flea cDNA library. Sense primer Flea 40F having the nucleotide sequence 5' GGC AAG TTT CGT TTC ACA ATA GG 3' (denoted SEQ ID NO: 52) was used in combination with the M13 universal primer in a first PCR reaction. Anti-sense primer Flea 40R having the nucleotide sequence 5' TCC AAC CCT AAC TTT TAA ACC TTC 3' (denoted SEQ ID NO: 53) was used in combination with the M13 reverse primer m a second PCR reaction. The resultmg PCR products were each gel purified and cloned mto the TA Vector® System, and subjected to standard DNA sequencing techniques. A composite nucleic acid sequence representing a flea serine protease coding region was deduced, referred to herein as nfSP40841, was deduced and is denoted herein as SEQ ID NO: 40. SEQ ID NO: 37 is contained withm the sequence of the nucleic acid molecule nfSP40841 Translation of SEQ ID NO: 40 suggests that nucleic acid molecule nfSP40841 encodes a non-full-length flea serine protease protem of about 242 ammo acids, referred to herein as PfSP4024i, havmg ammo acid sequence SEQ ID NO: 41, assuming an open reading frame in which the first codon spans from about nucleotide 2 through about nucleotide 4 of SEQ ID NO: 40 and tne termination codon spans from about nucleotide 728 through about nucleotide 730 of SEQ ID NO: 40. The complement of SEQ ID NO: 40 is represented herein by SEQ ID NO:42. The coding region encoding PfSP40242, is represented by nucleic acid molecule nfSP407π, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 43 and a complementary strand with nucleic acid sequence SEQ ID NO: 45. The amino acid sequence of PfSP40242 (i.e., SEQ ID NO:41) predicts that PfSP40242 has an estimated molecular weight of about 26 kD and an estimated pl of about pi 6.5. Comparison of nucleic acid sequence SEQ ID NO: 40 with nucleic acid sequences reported m GenBank indicates that
SEQ ID NO: 40 showed the most homology, i.e., about 57% identity between SEQ ID NO: 40 and a Derma tophagoi des pteronyssmus Der P3 allergen gene. Comparison of ammo acid sequence SEQ ID NO:41 (i.e., the ammo acid sequence of PfSP40_42) with ammo acid sequences reported m GenBank indicates that SEQ ID NO: 41 showed the most homology, i.e., about 40% identity between SEQ ID NO: 41 and a Bombyx mon vitellm-degradmg protease precursor protem.
Example 23
This Example demonstrates the production of serine protease proteins of the present mvention m E. coli cells . A. Flea serine protease protem PfSP24258 was produced m the following manner. An about 714 bp nucleic acid molecule, referred to herein as nfSP24714 (designed to encode an apparently mature serine protease protem) was PCR amplified from nfSP241089 usmg sense primer Flea 24 EF having the nucleotide sequence 5' CAC AGG ATC CAA TAA TTT GTG GTC AAA ATG C 3' (containing a BamHI-site; denoted SEQ ID NO: 54) and anti-sense primer Flea 24 ER havmg the nucleotide sequence 5' AAA AAG AAA GCT TCT TTA ATT TTC TGA CAT TGT CGT G 3' (containing a Hindlll; denoted SEQ ID NO: 55) . The resulting PCR product nfSP24714 was digested with BamHl and HindiII restriction endonucleases, gel purified, and subcloned mto expression vector lambdaPR/T-on/S10HIS-RSET-A9, that had been digested with BamHl and HindiII and dephosphorylated. The resultant recombinant molecule, referred to herein as pCro-nfSP24714, was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E.
Figure imgf000146_0001
The recombinant cell was cultured as described in Example 20 of related PCT Publication No.WO 95/24198. About 1 ml of culture was collected prior to induction, and about 1 ml of culture was collected about 60 minutes following induction. These samples were then lysed m sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and resolved on a 14% Tris-glycme acrylamide gel. Immunoblot analysis of the proteins using a T7 (tag) antibody (available from Novagen) showed expression of an about 36 kD protem m the induced sample but not m the unmduced sample. B. Flea serine protease protem PfSP32268 was produced m the following manner. An about 698 bp nucleic acid molecule, referred to herein as nfSP32b98 (designed to encode an apparently mature serine protease protem) was PCR amplified from nfSP329-M using sense primer Flea 32 EF having the nucleotide sequence 5' GCG GGA TCC TAT TGT GGG TGG TGA AGC AGT G 3' (containing a BamHI-site; denoted SEQ ID NC:56) and anti-sense primer Flea 32 ER having the nucleotide sequence 5' GAC GGT ACC ATG TAT AAA ATA ATA TTA AAC TCC GG 3' (containing a Kpnl; denoted SEQ ID NO:57) . The resulting PCR product nfSP32698 was digested with BamHl and Kpnτ restriction endonucleases, gel purified, and subcloned into expression vector 1 pTrcHisB (available from Ir.Vitrogen Corp., San Diego, CA) , that had been digested with BamHl and Kpnl and dephosphorylated. The resultant recombinant molecule, referred to herein as pTrc-nfSP32b98, was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pTrc-nfSP32698. The recombinant cell was cultured and protein production resolved by SDS- PAGE as described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the unmduced sample.
C. Flea serine protease protein PfSP33400 was produced in the following manner. An about 1200 bp nucleic acid molecule, referred to herein as nfSP331200 (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP331894 using sense primer Flea 33 EF having the nucleotide sequence 5' CCG GGA TCC TAT GTT AGC GAT CGT CCC GTC AAA C 3' (containing a BamHI-site; denoted SEQ ID NO: 58) and anti-sense primer Flea 33 ER having the nucleotide sequence 5' CCG GAA TTC TTA TCC CAT TAC TTT GTC GAT CC 3' (containing a EcoRI; denoted SEQ ID NO:59) . The resulting PCR product nfSP331200 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned mto expression vector lambdaPR/T2ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated. The resultant recombinant molecule, referred to herein as pCro-nfSP331200, was transformed into £. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP331200. The recombinant cell was cultured using the method described above in Section A. D. Flea serine protease protein PfSP40242 was produced in the following manner. An about 716 bp nucleic acid molecule, referred to herein as nfSP4071b (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP40841 using sense primer Flea 40 EF having the nucleotide sequence 5' GCG GGA TCC AAT AGT AGG AGG TGA AGA TGT AG 3' (containing a BamHI-site; denoted SEQ ID NO: 60) and anti-sense primer Flea 40 ER having the nucleotide sequence 5' CCG GAA TTC TTC TAA CAA ATT TTA TTT GAT TCC TGC 3' (containing a EcoRI; denoted SEQ ID N0:61) . The resulting PCR product nfSP40716 was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaPR/T2ori/S10HIS-RSET- A9, that had been digested with BamHl and EcoRI and dephosphorylated. The resultant recombinant molecule, referred to herein as pCro-nfSP40716, was transformed into E. coli BL-21 competent cells to form recombinant cell E. coli :pCro-nfSP407U- . The recombinant cell was cultured and protein production resolved using the methods described above in Section A. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 38 kD protein in the induced sample but not in the uninduced samp1e.
Example 24
This Example demonstrates the production of another serine protease protein of the present invention in E. coli cells.
A. Flea serine protease protein PfSP28237 was produced in the following manner. An about 711 bp nucleic acid molecule, referred to herein as nfSP2871, (designed to encode an apparently mature serine protease protein) was PCR amplified from nfSP28923 using sense primer Flea 28 F having the nucleotide sequence 5' GGA TCC AAT CGT TGG AGG TGA AGA TG 3' (containing a BamHI-site shown in bold; denoted SEQ ID NO: 62) and anti-sense primer Flea 28 R having the nucleotide sequence 5' GAA TTC GAA ATC CAC TTA AAC ATT AGC 3' (containing a EcoRI shown in bold; denoted SEQ ID NO: 63) . The resulting PCR product nfSP287n was digested with BamHl and EcoRI restriction endonucleases, gel purified, and subcloned into expression vector lambdaPR/T2ori/S10HIS-RSET-A9, that had been digested with BamHl and Xbal and dephosphorylated. The resultant recombinant molecule, referred to herein as pCro-nfSP28^ ,, was transformed mto E. coli BL-21 competent cells (available from Novagen, Madison, WI) to form recombinant cell E. coli :pCro-nfSP2δn, . The recombinant cell was cultured and protem production resolved using the methods described above m Example 20. Immunoblot analysis of the proteins using a T7 antibody showed expression of an about 36 kD protem in the induced sample but not m the unmduced sample. Immunoblot analysis using a rabbit anti- flea midgut protease polyclonal antibody (the production of which is described in Example 14 of related PCT Publication NO.WO 95/24198) identified an about 38 kD protem in the induced sample.
Example 25 This Example demonstrates the production of another serine protease protem of the present invention m eukaryotic cells.
Recombinant molecule pBv-nfSP28792, containing a flea serine protease nucleic acid molecule spanning nucleotides from about 11 through about 802 of SEQ ID NO: 66, operatively linked to baculovirus polyhedron transcription control sequences were produced m the following manner.
In order to subclone a flea serine protease nucleic acid molecule mto a baculovirus expression vector, a flea serine protease nucleic acid molecule-containing fragment was PCR amplified from nfSP2892,. A PCR fragment of 792 nucleotides, named nfSP28-.92, was amplified from nfSP28ς usmg a sense primer Flea 28 F3 having the nucleic acio sequence 5' -GCG GGA TTC TAT AAA TAT GAA ACT TTT GGT AGT TTT TGC -3' (SEQ ID NO: 62; BamHl site shown in bold) and an antisense primer Flea 28 R3 havmg the nucleic acid sequence 5' -GCT CTA GAC CAC TTA AAC ATT AGC ATA TTT TTC- 3' (SEQ ID NO: 63; Xbal site shown in bold) . The N-termmal primer was designed from the pol h sequence of baculovirus with modifications to enhance expression in the baculovirus system.
In order to produce a baculovirus recombinant molecule capable of directing the production of PfSP282b4, the about 792 base pair PCR product (referred to as Bv-nfSP28-,9Z) was digested with BamHl and Xbal and subcloned mto BamHl and Xbal digested to produce the recombinant molecule referred to herein as pVL-nfSP2879t .
The resultant recombinant molecule,pVL-nfSP28792, was verified for proper insert orientation by restriction mapping. The recombinant molecule was co-transfected with a linear Baculogold baculovirus DNA (available from Pharmmgen) mto S. frugiperda Sf9 cells (available from InVitrogen) to form the recombinant cells denoted 5. frugiperda:pVL-nfSP2δ79,. 5. frugiperda:pVL-nfSP28 -,92 was cultured m order to produce a flea serine protease protem PfSP282b4. Immunoblots of supematants from cultures of S . frugiperda:pVL-nfSP28-,92 cells producing the flea serine protease protem PfSP282b4 was performed using a cat anti- fSPFlea 26 polyclonal antibody which was produced as follows. Recombinant Flea 28 protein (referred to herein as rSPFlea 28 protein) produced in E. coli described above in Example 24 was used to immunize cats. The rSPFlea 28 protein was diluted to a concentration of about 1 mg/ml in PBS and emulsified in an equal volume of TiterMax research adjuvant (available from CytRx Corp., Norcross, GA) . A series of cats were immunized each with about 50μg of rSPFlea 28 protein in adjuvant by subcutaneous injection. A second injection of the same dose of rSPFlea 28 protein in adjuvant was administered 32 days later. Blood samples were obtained prior to immunization (pre-bleed) , 32 days and 47 days after the initial immunization. Sera samples from the pre-immunization and Day 47 bleeds were used for subsequent immunoblot experiments. The latter is referred to as anti-fSPFlea 28 polyclonal antibody. Analysis of the immunoblots identified an about 33 kD protein and an about 36 kD protein.
Example 26
This example describes the production of peptides from the 31 kD flea midgut serine protease and the generation of internal sequence data. Midguts from about 30,000 cat blood-fed fleas were dissected as described m U.S. Patent No. 5,356,622, ibid. m gut dissection buffer (50 mM Tris 8.0, 100 mM CaCl ) . The guts (m three batches of about 10,000 each) were disrupted by a freeze-thaw cycle, followed by somcation. The resulting extracts were clarified by centrifugation for 20 minutes at 3050 rpm m a swinging bucket centrifuge at 4°C. The supematants were recovered, and adjusted to 400 mM NaCl m preparation for benzamidine column chromatography. For each batch, gut supematants were loaded into a 5 ml disposable column containing p-ammobenzamidme cross- linked to Sepharose beads (available from Sigma, St. Louis, MO) , previously equilibrated in benzamidine column buffer (50 mM Tris, pH 8.0, 100 mM CaCl2, 400 mM NaCl) and incubated with rocking overnight at 4°C. Unbound protem was slowly washed off the column using benzamidine column buffer until no protem was detectable by Bradford Assay (available from Bio-Rad Laboratories, Hercules, CA) .
Proteases bound to the benzamidine column were eluted using 4 ml benzamidine column buffer supplemented with 100 mM p-ammobenzamidme (brought to pH 8.0 with NaOH) .
Residual bound proteases were washed off with about 21 ml of additional benzamidine column buffer. The recovered proteases were then concentrated to a volume of about 2 ml using a Ultrafree 20 10-kD centrifugal concentrator
(available from Millipore, Bedford, MA) . After concentration, the protease pools from the 3 preparations were combined for a total of about 30,000 gut equivalents m about 6 ml. Protem concentration was measured by Bradford assay and found to be about 0.5 mg/ml. About 150 μg of the isolated protease pool was resolved by polyacrylamide gel electrophoresis (PAGE) on a preparative-well 14% Tris-glycme gel (available from Novex, San Diego, CA) . After electrophoresis, the proteins in the gel were visualized by staining for about 30 minutes in Coomassie brilliant blue stain (0.1% (w/v) Coomassie blue R, 40% (v/v) methanol, 10% (v/v) acetic acid) and oestammg for about 2.5 hours in 50% (v/v) methanol. The band correspondmg to the 31-kD protease was excised with a razor blade. The protem was electroeluted, concentrated, and partially digested for 24 hours with cyanogen bromide (CNBr) (Silver, et al. , 1995, J. Bi ol . Chem. , 210, 13010-13016), except that a small amount of acetic acid was added to the sample after electroelution and concentration to lower the sample pH and therefore reduce autodigestion by the 31-kD protease. CNBr is known to cleave after methionine (M) residues under the conditions used for this digestion. After CNBr digestion, the peptides in the sample were resolved by PAGE on an 18% Tris-glycme gel. After electrophoresis, the separated protease peptides were electroblotted onto a PVDF membrane using a CAPS buffer (10 mM CAPS pH 11, 0.5 mM DTT, 10% (v/v) methanol) . The membrane was stained with Coomassie Brilliant Blue and destamed with 50% (v/v) methanol. Three stained peptide bands were identified having apparent molecular weights of about 14 kD, 21 kD, and 22 kD, respectively. The portions of the membrane containing the 21 kD and 22 kD bands were excised separately. Peptides contained m each membrane segment were subjected to N- termmal ammo acid sequencing using a 473A Protem Sequencer (available from Applied Biosystems, Foster City, CA) using standard techniques.
Although the results from the automated sequencing were difficult to interpret due to overlapping sequences, analysis of the chromatograms indicated the N-termmal ammo acid sequence of the 21-kD peptide to be H/R-V/P- G/A/S-Y/G-E/N-D/K-V/R-D/A-D-Y- D-F-D/P-V-A, denoted herein as SEQ ID NO: 70 and the N-terminal amino acid sequence of the 22-kD peptide to be I/Q-V-G-Y/G-E/N/T-D/M/P-V-K/D-I- N/S-M/T/N-F/C herein denoted as SEQ ID NO: 71. The N- texmmal ammo acid sequence of the mtact 31-kD protease is either I-V-G-G- E-D-V-D-I-S-T-C-G-W-C (SEQ ID NO: 59, as disclosed m Example 34 m co-pending U.S. Patent Application Serial No. 08/639,075) , or IVGGEDVDIST(C)GWQI(S)FQ(S)ENLHF(C)GG(S) IIAPK (SEQ ID NO: 69, as disclosed in Example 35 in co-pending U.S. Patent Application Serial No. 08/639,075) . These sequences vary at residue 15 m that SEQ ID NO: 68 contains a cysteine and SEQ ID NO: 69 contains a glutamine. These sequences can be identified m the sequences of both the 21-kD (SEQ ID NO:70) ano 22-kD (SEQ ID NO:71) peptides, though it is much stronger m the 22-kD peptide, leading to the conclusion that the SEQ ID NO: 71 represents the N-terminus of the 31- kD protease. If this sequence is subtracted from the sequence for the 21-kD (SEQ ID NO:70) peptide, then the resulting sequence for the 21-kD peptide is H/R-P-A/S-Y-N- K-R-A-D-Y-D-F-D-V-A, denoted herein as SEQ ID NO:72. This sequence of ammo acids aligns with a stretch of deduced ammo acids from about residue 107 to residue 121 immediately following a methionine residue in SEQ ID NO: 67.
These data confirm that the clone represented by nfSP28923
(SEQ ID NO: 66, as disclosed m Example 36 m co-pending U.S. Patent Application Serial No. 06/639,075) indeed encodes the 31-kD protease.
Example 27
This example demonstrates that a 31-kD flea midgut serine protease contained in a formulation is able to proteolyze cat immunoglobulin G, A, and M proteins as well as bovme, dog, human, and rabbit immunoglobulin G proteins.
The 31-kD flea midgut serine protease was purified from cat blood-fed fleas as follows. Cat blood-fed flea midgut extracts were prepared and selected on a benzamidine column as described above m Example 26. The benzamidine eluate was then further purified as described in Example 35 of co-pending U.S. Patent Application Serial No. 08/639,075 by PolyCAT A cation exchange chromatography (available from PolyLC, Inc., Columbia, MD) to isolate a protein band which migrated at about 31 kD on a silver stained SDS-PAGE gel.
A. The ability of the cat blood-fed 31-kD flea midgut serine protease to degrade immunoglobulin was demonstrated by measuring digestion of immunoglobulin heavy chain using a method similar to that described in Example 35 of co- pending U.S. Patent Application Serial No. Oδ/639,075.
Specifically, 1 μg samples of cat IgG, cat IgA, and cat IgM substrates (available from Bethyl Laboratories, Inc., Montgomery, Texas) were incubated with 500 cat blood-fed flea midgut equivalents of purified 31-kD flea midgut
serine protease in a total volume of 27 μl 0. IM Tris-HCl pH 8.0 at 37°C for 18 hours. The reaction mixtures were resolved by 14% Tris-glycine SDS-PAGE and the gel was silver stained using standard methods. The total disappearance (compared with control samples lacking addition of the purified 31-kD protein) of bands migrating at about 50, 60, and 80 kD on the silver stained gel in the lanes containing 31-kD protease-treated cat IgG, IgA, and IgM, respectively, indicated that the 31-kD flea midgut serine protease degraded the heavy chains of the various cat immunoglobulin isotypes.
B. The ability of the cat blood-fed 31-kD flea midgut serine protease to degrade IgG from several species was
demonstrated by incubating 1 μg samples of purified cat or bovme IgG (purified from cat and bovme blood on Protem
A Sepharose) , or of purified dog, rabbit, or human IgG
(each available from Sigma Chemical Co.) with 500 cat blood-fed flea midgut equivalents of the purified 31-kD flea midgut serine protease m a total volume of 27 μl 0. IM Tris-HCl pH 8.0 at 37°C for 18 hours. The reaction mixtures were resolved by 14% Tris-glycme SDS-PAGE and the gel was silver stained using standard methods. The total disappearance (compared with control samples lacking addition of the purified 31-kD protem) of bands migrating at about 50-55-kD on the silver stained gel in the lanes containing the 31-kD protease treated cat, bovme, dog, rabbit, and human IgG heavy chains, indicated that the 31- kD flea midgut serine protease can degrade IgG from various mammalian species.
Example 28
This example describes the ability of a 31-kD flea midgut serine protease contained m a formulation to proteolyze cat immunoglobulin G at a specific site. The 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 26 and 27.
To investigate cleavage site specificity of the purified 31-kD flea midgut serine protease, 10 μg of cat immunoglobulin G purified from cat blood on Protem A sepharose was incubated with 200 cat blood-fed flea midgut equivalents of purified 31-kD flea midgut serine protease in a total volume of 100 μl 0.2 M Tris-HCl pH 8.0 at 37°C for 18 hours. The reaction mixture was resolved by 14% Tris-glycme SDS-PAGE, blotted onto a PVDF membrane, stained with Coomassie R-250 and destained according to standard procedures. A band of about 33 kD was excised and subjected to N-termmal amino acid sequencing using techniques known to those skilled in the art. A partial N- terminal ammo acid sequence of about 28 ammo acids was determined and is represented herem as SEQ ID NO: 73: X- P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K. A GenBank homology search using SEQ ID NO: 73 revealed most homology to Oryctolagus cani cul us gamma H-cham constant region 2, there being about 71 % identity over the 28 ammo acids. Further alignments of SEQ ID NO: 73 with sheep, rat, rabbit, monkey, bovme, and human IgG ammo acid sequences indicated that the purified cat blood-fed 31-kD flea midgut serine protease cleaved the cat IgG heavy chain just before the predicted C-terminal end of the IgG hmge region. The predicted first amino acid cysteine and the second amino acid proline occur within the predicted hinge region while the remaining 26 amino acids of SEQ ID NO: 73, starting with the third amino acid proline, occur within the predicted constant heavy chain-2 region.
The further investigate the cleavage site specificity of the purified 31-kD flea midgut serine protease for cat IgG, the cleavage site was compared to that of a known protease, papain, as follows. Cat immunoglobulin G (100 mg) , purified from cat blood on Protein A sepharose, was incubated with 1 mg papain in lOOmM sodium acetate pH 5.5, 50 mM cysteine, 1 mM EDTA in a final volume of 150 μl at room temperature for 4.5 hours. The reaction mixture was resolved on a 14% Tris-glycine SDS-PAGE gel, blotted onto PVDF membrane, stained with Coomassie R-250 and destained according to standard procedures. A band of about 33 kD was excised and subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art. A partial N-terminal amino acid sequence of about 25 amino acids was deduced and is represented herein as SEQ ID NO: 96: X-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-K-K-D-D-L-L-I . This sequence was nearly identical to the one obtained from a 33-kD cat IgG cleavage product generated by the purified 31-kD flea midgut serine protease, the only difference being the substitution of a lysine (SEQ ID NO: 73) for a proline (SEQ ID NO: 96) at amino acid 19. Example 29
This Example demonstrates the kinetics of cat IgG degrading activity in the midguts of fleas fed on live cats . A. To determine the kinetics of cat IgG degradation m the guts of continuously feeding fleas, female fleas contained in chambers were fed on seven separate cats (i.e. one chamber per cat) as described in Example 21 of co- pending U.S. Patent Application Serial No. 08/639,075. Flea chambers were removed for dissections at timepoints of 15 mm., 30 mm., 1 hr., 2 hr., 4 hr., 6 hr. , 8 hr., and 17 hr.. After feeding on the cats, the fleas' midguts were removed as described in U.S. Patent No. 5,356,622, ibid. , homogenized by freeze-fracture and sonicated in a Tris buffer comprising 50 mM Tris, pH 8.0 and 100 mM CaCl2. The extracts were centrifuged at about 14,000 x g for 20 min. and the soluble material recovered. The soluble material was then diluted to a final concentration of about 1.2 midgut equivalents per microliter (μl) of Tris buffer. The proteins contained in 1 midgut equivalent of each timepomt were then resolved by SDS-PAGE under reducing conditions, and the proteins visualized by silver staining. The results indicated that IgG heavy chain levels were significantly lower at the 17 hour time point than m the 8 hour and earlier time points, and that light chain levels were reduced but not to the same extent as the heavy chain. The proteins contained in 5 gut equivalents of each timepomt were then resolved by SDS-PAGE gel under reducing conditions and were subjected to western blot analysis usmg alkaline phosphatase labeled goat anti-cat IgG (heavy plus light chain) antibody (available from Kirkegaard and
Perry Laboratories, Gaithersburg, MD) . The results indicated that cat IgG heavy chain was present in the midguts of continuously feeding fleas for at least 8 hours, but was not detected in the midguts of fleas allowed to feed continuously on a cat for 17 hours. Light chain was visible in all samples, though the amount visible m the 17 hour sample was significantly less than that visible in the
8 hour sample. These results suggest that even when fleas are continuously feeding on a cat, the levels of IgG- degrading proteases induced in the flea midguts at a time point of 17 hours is sufficient to degrade all detectable cat IgG ingested. These results suggest that when fleas are continuously feeding on a cat, the levels of IgG- degradmg proteases induced in the flea midguts are not sufficient to degrade all detectable cat IgG ingested for at least 8 hours.
B. To determine the kinetics of cat IgG degradation in the guts of fleas fed for a specified time then removed from the cat, fleas (m chambers) were fed on cats as m Section A for periods of either 1 hour or 24 hours.
Following the 1 or 24-hour feeding periods, the flea chambers were removed and placed in a 28°C, 75% relative humidity growth incubator. Fleas were subjected to dissection at time points of 0, 1, 2, 4, and 8 hr. following removal from the cats. Midguts were homogenized, and the midgut contents were examined by silver stained SDS-PAGE and immunoblot analysis, as described in Section A. The fleas fed for 1 hour had high molecular weight proteins, including the heavy chain and light chain of cat IgG detectable in their midguts at the 0 and 1 hour dissection timepoints, while the flea midguts evaluated at time points of 2 hours or greater had no detectable IgG heavy chain bands. The results showed that when fleas were fed on a cat and then removed, they degraded the ingested cat IgG heavy chain nearly completely within 2 hours. The fleas fed on cats for 24 hours had no detectable IgG heavy or light chain proteins in midgut extracts at any of the timepoints. These results suggest that when no new cat IgG is ingested, as is the case when the fleas are removed from feeding, that the IgG-degrading proteases in the flea midgut fully degraded all cat IgG heavy chain in less than two hours.
Example 30
This example describes the ability of a 31-kD flea midgut serine protease contained in a formulation to proteolyze cat immunoglobulin G at a specific site. The 31-kD flea midgut serine protease was purified from cat blood-fed flea midgut extracts as described above in Examples 14 and 15.
To further investigate cleavage site specificity of the purified 31-kD flea midgut serine protease, 1.5 microgram of cat immunoglobulin G purified from cat blood on Protem A sepharose was incubated with 200 cat blood-fed flea midgut equivalents of purified 31-kD flea midgut serine protease in a total volume of 300 microliter of 50 mM Tris-HCl pH 8.0 at 37°C. About 10 microliter aliquots of the incubation mixture were removed at 1, 2, 4, 6, 8, 12, 16 and 24 hours after initiation of the incubation. Following removal, each aliquot was mixed with about 1 microliter of 20 mM p-aminobenzamidme and stored at -80°C. Each aliquot from the incubation mixture was resolved by 14% Tris-glycme SDS-PAGE, blotted onto a PVDF membrane, stained with Coomassie R-250 and destamed according to standard procedures. A band of about 34 kD was identified in the aliquot removed after 1 hour of incubation. The band was excised and subjected to N-termmal ammo acid sequencing usmg techniques known to those skilled in the art. A partial N-termmal ammo acid sequence of about 25 ammo acids was determined and is represented herem as SEQ ID NO: 104: D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- D. An additional 10 amino acids were also obtained beyond the last ammo acid of SEQ ID NO: 104. A combined ammo acid sequence of about 35 amino acids was determined and is represented herein as SEQ ID NO: 105: D-C-P-K-C-P-P-P-E-M-L- G-G-P-S-I-F-I-F-P-P-K-P-K-D-D-L-L-I-K-R-K-S-E-V. A GenBank homology search using SEQ ID NO: 105 revealed most homology to Homo sapi en immunoglobulin gamma 3 heavy chain constant region 2 exon hinge IGHG3 gene (GenBank Accession No. X99549) , there being about 69% identity over the 35 amino acids. Further alignments of SEQ ID NO: 105 with cat, rabbit, bovine and human IgG amino acid sequences indicated that purified cat blood-fed 31-kD flea midgut serine protease cleaved the cat IgG heavy chain about 6 amino acids just before the predicted C-terminal end of the IgG hinge region. The first 6 amino acids, aspartic acid, cysteine, proline, lysine, cysteine and proline, occur within the predicted hinge region while the remaining 29 amino acids of SEQ ID NO: 105, starting with the seventh amino acid proline, occur within the predicted constant heavy chain-2 region.
A comparison of SEQ ID NO: 105 and an amino acid sequence for portions of the hinge and CH2 regions of cat
IgG (represented herein by SEQ ID NO: 106) is shown below:
SEQ ID NO: 105 D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K- SEQ ID NO:106 D-C-P-K-C-P-P-P-E-M-L-G-G-P-S-I-F-I-F-P-P-K-P-K-
SEQ ID NO:105 (continued) -D-D-L-L-I-K-R-K-S-E-V SEQ ID NO:106 (continued) -D-T-L-S-I-S-R-T-P-E-V
Discrepancies between SEQ ID NO: 105 and SEQ ID NO: 106 are shown in bold. Applicants believe that the difference between SEQ ID NO: 105 and SEQ ID NO: 106 may be due to sequencing error of the last 10 amino acids of SEQ ID NO: 105.
SEQUENCE LISTING The following Sequence Listing is submitted pursuant to 37 CFR §1.821. A copy in computer readable form is also submitted herewith. Applicants assert pursuant to 37 CFR §1.621 (f) that the content of the paper and computer readable copies of SEQ ID NO:l through SEQ ID NO: 165 submitted herewith are the same.
(1) GENERAL INFORMATION: (x) APPLICANT: Grieve, Robert B.
Rushlow, Keith E.
Wu Hunter, Shirley
Frank, Glenn R.
Stiegler, Gary Games, Patrick J.
Silver, Gary
(n) TITLE OF INVENTION: FLEA PROTEASE PROTEINS, NUCLEIC ACID MOLECULES AND USES THEREOF
(m) NUMBER OF SEQUENCES: 165
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sheridan Ross P.C.
(B) STREET: 1700 Lincoln Street, Suite 3500
(C) CITY: Denver (D) STATE: Colorado
(E) COUNTRY: USA
(F) ZIP: 80203
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:
(C) CLASSIFICATION:
(vm) ATTORNEY/AGENT INFORMATION:
(A) NAME: Connell, Gary J.
(B) REGISTRATION NUMBER: 32,020 (C) REFERENCE/DOCKET NUMBER: 2618-25-C4-PCT
(IX) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 863-9700
(B) TELEFAX: (303) 863-0223 (2) INFORMATION FOR SEQ ID Nθ:l:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1109 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 126..1106
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
TACAATTAGG GTGACGATCT TTTAGACAAG CGAAATTGAT AACGAAGTTT TCGAAGTCGG 60
ATGAAGTAAA AACCTTGCGT TGGTTTCCCC GGTCCCAGGA TCAGGAACAG TTGCACTTTA 120
CCCCA ATG AGG GAA TTC GTG CAT CCC CAT TTT ACC GAA CAT ATT GAT 167 Met Arg Glu Phe Val His Pro His Phe Thr Glu His Ile Asp
1 5 10
GAA GAA TTC CAC CGA TTC ATC AAT AAA CAC GGA AAA ATT TAT AAT AAA 215 Glu Glu Phe His Arg Phe Ile Asn Lys His Gly Lys Ile Tyr Asn Lys 15 20 25 30 AAT GAA GAA CAT CAT TTC CGC AAA GAA ATT TTC AGA CTA AAC TTG AGG 263 Asn Glu Glu His His Phe Arg Lys Glu Ile Phe Arg Leu Asn Leu Arg 35 40 45
TAC ATT TTT TCT AAG AAT CGT GCA AAT TTG GGA TAC ACT TTG ACT GTT 311 Tyr Ile Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr Leu Thr Val 50 55 60
AAC CAT TTG GCT GAT CGT ACT GAA GCT GAA CTT AAG GCT TTG AGA GGA 359 Asn His Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala Leu Arg Gly 65 70 75
CAC AGA CCT TCC TCC GGT TAT AAT GGC GGT TTA CCC TTT CCT CAC AAT 407 His Arg Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe Pro His Asn 80 85 90
ACC ACC AAG GAA GCA AGA AAT TTA CCA GAT TCT TTC GAC TGG CGA ATT 455 Thr Thr Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp Trp Arg Ile 95 100 105 110
TAT GGA GCT GTT ACT CCA GTT AAA GAT CAA TCT GTT TGT GGT TCC TGC 503 Tyr Gly Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys 115 120 125
TGG TCT TTC GGA ACA ATT GGA GCA ATC GAA GGT GCA TAT TTC TTG AAA 551 Trp Ser Phe Gly Thr Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys 130 135 140
AAC GGC GGT AAT CTT GTA CGA TTG TCT CAA CAG GCT TTG ATT GAT TGT 599 Asn Gly Gly Asn Leu Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys 145 150 155 TCT TGG GGA TAT GGA AAT AAT GGT TGC GAC GGT GGC GAA GAT TTC CGC 647 Ser Trp Gly Tyr Gly Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg 160 165 170
GCC TAC CAA TGG ATG ATG AAA CAT GGA GGA ATC CCT ACT GAA GAG GAT 695 Ala Tyr Gin Trp Met Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp 175 180 185 190
TAT GGT GGT TAC TTG GGA CAA GAT GGT TAC TGC CAT GTC AAC AAC GTT 743 Tyr Gly Gly Tyr Leu Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val 195 200 205 ACT TTA GTT GCT CCC ATC ACA GGA TAT GTC AAC GTA ACT CGT AAC GAT 791 Thr Leu Val Ala Pro lie Thr Gly Tyr Val Asn Val Thr Arg Asn Asp 210 215 220
GTT GAC GCT ATG AAG GTT GCC CTT CTT AAA CAC GGT CCG ATT TCG GTG 839 Val Asp Ala Met Lys Val Ala Leu Leu Lys His Gly Pro Ile Ser Val 225 230 235
GCC ATT GAC GCA TCA CAC AAA ACA TCC AGT TTT TAC TCC AAC GGC GTT 887 Ala Ile Asp Ala Ser His Lys Thr Ser Ser Phe Tyr Ser Asn Gly Val 240 245 250
TAC TAC CAA CCG AAA TGT GGC AAT AAA AGA GGA CAA TTA GAC CAC GCC 935 Tyr Tyr Gin Pro Lys Cys Gly Asn Lys Arg Gly Gin Leu Asp His Ala 255 260 265 270
GTA TTA GTA GTC GGT TAT GGT GAA ATC AAC AGC GAA CCT TAC TGG TTG 983 Val Leu Val Val Gly Tyr- Gly Glu Ile Asn Ser Glu Pro Tyr Trp Leu 275 280 285 GTC AAG AAT CCT GGT CAA TTG TGG GGA AAC AAT GTT ATA TTT GAT GTC 1031 Val Lys Asn Pro Gly Gin Leu Trp Gly Asn Asn Val Ile Phe Asp Val 290 295 300
GCC AAA AAT AAT AAT GCG GAT TTG ACG ATC ACT TAT TTA CTA TGT ACT 1079 Ala Lys Asn Asn Asn Ala Asp Leu Thr Ile Thr Tyr Leu Leu Cys Thr 305 310 315
ACT TTT AAA ATT GAT TTT TAC GGC ATG TAA 1109
Tnr Phe Lys Ile Asp Phe Tyr Gly Met 320 325
(2) INFORMATION FOR SEQ ID NO:2: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 327 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Arg Glu Phe Val His Pro His Phe Thr Glu His Ile Asp Glu Glu 1 5 10 15
Phe His Arg Phe Ile Asn Lys His Gly Lys Ile Tyr Asn Lys Asn Glu 20 25 30 Glu His His Phe Arg Lys Glu Ile Phe Arg Leu Asn Leu Arg Tyr Ile 35 40 45
Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr Leu Thr Val Asn His 50 55 60 Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala Leu Arg Gly His Arg 65 70 75 80
Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe Pro His Asn Thr Thr 85 90 95
Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly 100 105 110
Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser 115 120 125
Phe Gly Thr Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly 130 135 140 Gly Asn Leu Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp 145 150 155 160
Gly Tyr Gly Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr
165 170 175
Gin Trp Met Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly 180 185 190
Gly Tyr Leu Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu 195 200 205
Val Ala Pro Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp 210 215 220 Ala Met Lys Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile 225 230 235 240
Asp Ala Ser His Lys Thr Ser Ser Phe Tyr Ser Asn Gly Val Tyr Tyr 245 250 255
Gin Pro Lys Cys Gly Asn Lys Arg Gly Gin Leu Asp His Ala Val Leu 260 265 270
Val Val Gly Tyr Gly Glu Ile Asn Ser Glu Pro Tyr Trp Leu Val Lys 275 280 285
Asn Pro Gly Gin Leu Trp Gly Asn Asn Val Ile Phe Asp Val Ala Lys 290 295 300 Asn Asn Asn Ala Asp Leu Thr Ile Thr Tyr Leu Leu Cys Thr Thr Phe 305 310 315 320
Lys Ile Asp Phe Tyr Gly Met 325 (2ι INFORMATION FOR SEQ ID NO: 3:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1109 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
TTACATGCCG TAAAAATCAA TTTTAAAAGT AGTACATAGT AAATAAGTGA TCGTCAAATC 60 CGCATTATTA TTTTTGGCGA CATCAAATAT AACATTGTTT CCCCACAATT GACCAGGATT 120
CTTGACCAAC CAGTAAGGTT CGCTGTTGAT TTCACCATAA CCGACTACTA ATACGGCGTG 180
GTCTAATTGT CCTCTTTTAT TGCCACATTT CGGTTGGTAG TAAACGCCGT TGGAGTAAAA 240
ACTGGATGTT TTGTGTGATG CGTCAATGGC CACCGAAATC GGACCGTGTT TAAGAAGGGC 300
AACCTTCATA GCGTCAACAT CGTTACGAGT TACGTTGACA TATCCTGTGA TGGGAGCAAC 360 TAAAGTAACG TTGTTGACAT GGCAGTAACC ATCTTGTCCC AAGTAACCAC CATAATCCTC 420
TTCAGTAGGG ATTCCTCCAT GTTTCATCAT CCATTGGTAG GCGCGGAAAT CTTCGCCACC 480
GTCGCAACCA TTATTTCCAT ATCCCCAAGA ACAATCAATC AAAGCCTGTT GAGACAATCG 540
TACAAGATTA CCGCCGTTTT TCAAGAAATA TGCACCTTCG ATTGCTCCAA TTGTTCCGAA 600
AGACCAGCAG GAACCACAAA CAGATTGATC TTTAACTGGA GTAACAGCTC CATAAATTCG 660 CCAGTCGAAA GAATCTGGTA AATTTCTTGC TTCCTTGGTG GTATTGTGAG GAAAGGGTAA 720
ACCGCCATTA TAACCGGAGG AAGGTCTGTG TCCTCTCAAA GCCTTAAGTT CAGCTTCAGT 780
ACGATCAGCC AAATGGTTAA CAGTCAAAGT GTATCCCAAA TTTGCACGAT TCTTAGAAAA 840
AATGTACCTC AAGTTTAGTC TGAAAATTTC TTTGCGGAAA TGATGTTCTT CATTTTTATT 900
ATAAATTTTT CCGTGTTTAT TGATGAATCG GTGGAATTCT TCATCAATAT GTTCGGTAAA 960 ATGGGGATGC ACGAATTCCC TCATTGGGGT AAAGTGCAAC TGTTCCTGAT CCTGGGACCG 1020
GGGAAACCAA CGCAAGGTTT TTACTTCATC CGACTTCGAA AACTTCGTTA TCAATTTCGC 1080
TTGTCTAAAA GATCGTCACC CTAATTGTA 1109
(2) INFORMATION FOR SEQ ID NO: 4:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 984 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA ( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..981
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : ATG AGG GAA TTC GTG CAT CCC CAT TTT ACC GAA CAT ATT GAT GAA GAA 48 Met Arg Glu Phe Val His Pro His Phe Thr Glu His Ile Asp Glu Glu 1 5 10 15
TTC CAC CGA TTC ATC AAT AAA CAC GGA AAA ATT TAT AAT AAA AAT GAA 96 Phe His Arg Phe Ile Asn Lys His Gly Lys Ile Tyr Asn Lys Asn Glu 20 25 30
GAA CAT CAT TTC CGC AAA GAA ATT TTC AGA CTA AAC TTG AGG TAC ATT 144 Glu His His Phe Arg Lys Glu Ile Phe Arg Leu Asn Leu Arg Tyr Ile 35 40 45
TTT TCT AAG AAT CGT GCA AAT TTG GGA TAC ACT TTG ACT GTT AAC CAT 192 Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr Leu Thr Val Asn His 50 55 60
TTG GCT GAT CGT ACT GAA GCT GAA CTT AAG GCT TTG AGA GGA CAC AGA 240 Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala Leu Arg Gly His Arg 65 70 75 80
CCT TCC TCC GGT TAT AAT GGC GGT TTA CCC TTT CCT CAC AAT ACC ACC 288
Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe Pro His Asn Thr Thr 85 90 95
AAG GAA GCA AGA AAT TTA CCA GAT TCT TTC GAC TGG CGA ATT TAT GGA 336 Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly 100 105 110
GCT GTT ACT CCA GTT AAA GAT CAA TCT GTT TGT GGT TCC TGC TGG TCT 384 Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser 115 120 125
TTC GGA ACA ATT GGA GCA ATC GAA GGT GCA TAT TTC TTG AAA AAC GGC 432 Phe Gly Thr Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly
130 135 140
GGT AAT CTT GTA CGA TTG TCT CAA CAG GCT TTG ATT GAT TGT TCT TGG 480 Gly Asn Leu Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp 145 150 155 160 GGA TAT GGA AAT AAT GGT TGC GAC GGT GGC GAA GAT TTC CGC GCC TAC 528 Gly Tyr Gly Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr 165 170 175
CAA TGG ATG ATG AAA CAT GGA GGA ATC CCT ACT GAA GAG GAT TAT GGT 576 Gin Trp Met Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly 180 185 190
GGT TAC TTG GGA CAA GAT GGT TAC TGC CAT GTC AAC AAC GTT ACT TTA 624 Gly Tyr Leu Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu 195 200 205
GTT GCT CCC ATC ACA GGA TAT GTC AAC GTA ACT CGT AAC GAT GTT GAC 672 Val Ala Pro Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp 210 215 220 GCT ATG AAG GTT GCC CTT CTT AAA CAC GGT CCG ATT TCG GTG GCC ATT 720 Ala Met Lys Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile 225 230 235 240
GAC GCA TCA CAC AAA ACA TCC AGT TTT TAC TCC AAC GGC GTT TAC TAC 768 Asp Ala Ser His Lys Thr Ser Ser Phe Tyr Ser Asn Gly Val Tyr Tyr
245 250 255
CAA CCG AAA TGT GGC AAT AAA AGA GGA CAA TTA GAC CAC GCC GTA TTA 816 Gin Pro Lys Cys Gly Asn Lys Arg Gly Gin Leu Asp His Ala Val Leu 260 265 270 GTA GTC GGT TAT GGT GAA ATC AAC AGC GAA CCT TAC TGG TTG GTC AAG 864 Val Val Gly Tyr Gly Glu Ile Asn Ser Glu Pro Tyr Trp Leu Val Lys 275 280 285
AAT CCT GGT CAA TTG TGG GGA AAC AAT GTT ATA TTT GAT GTC GCC AAA 912 Asn Pro Gly Gin Leu Trp Gly Asn Asn Val Ile Phe Asp Val Ala Lys 290 295 300
AAT AAT AAT GCG GAT TTG ACG ATC ACT TAT TTA CTA TGT ACT ACT TTT 960 Asn Asn Asn Ala Asp Leu Thr Ile Thr Tyr Leu Leu Cys Thr Thr Phe 305 310 315 320
AAA ATT GAT TTT TAC GGC ATG TAA 984 Lys Ile Asp Phe Tyr Gly Met
325
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 327 ammo acids ( B) TYPE : amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Arg Glu Phe Val His Pro His Phe Thr Glu His Ile Asp Glu Glu 1 5 10 15
Phe His Arg Phe Ile Asn Lys His Gly Lys Ile Tyr Asn Lys Asn Glu 20 25 30
Glu His His Phe Arg Lys Glu Ile Phe Arg Leu Asn Leu Arg Tyr Ile 35 40 45 Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr Leu Thr Val Asn His
50 55 60
Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala Leu Arg Gly His Arg 65 70 75 80
Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe Pro His Asn Thr Thr 85 90 95
Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly 100 105 110
Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser 115 120 125 Phe Gly Thr Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly 130 135 140
Gly Asn Leu Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp 145 150 155 160 Gly Tyr Gly Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr
165 170 175
Gin Trp Met Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly 180 185 190
Gly Tyr Leu Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu 195 200 205
Val Ala Pro Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp 210 215 220
Ala Met Lys Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile 225 230 235 240 Asp Ala Ser His Lys Thr Ser Ser Phe Tyr Ser Asn Gly Val Tyr Tyr
245 250 255
Gin Pro Lys Cys Gly Asn Lys Arg Gly Gin Leu Asp His Ala Val Leu 260 265 270
Val Val Gly Tyr Gly Glu Ile Asn Ser Glu Pro Tyr Trp Leu Val Lys 275 280 285
Asn Pro Gly Gin Leu Trp Gly Asn Asn Val Ile Phe Asp Val Ala Lys 290 295 300
Asn Asn Asn Ala Asp Leu Thr lie Thr Tyr Leu Leu Cys Thr Thr Phe 305 310 315 320 Lys Ile Asp Phe Tyr Gly Met
325
(2) INFORMATION FOR SEQ ID NO: 6:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 981 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CATGCCGTAA AAATCAATTT TAAAAGTAGT ACATAGTAAA TAAGTGATCG TCAAATCCGC 60
ATTATTATTT TTGGCGACAT CAAATATAAC ATTGTTTCCC CACAATTGAC CAGGATTCTT 120
GACCAACCAG TAAGGTTCGC TGTTGATTTC ACCATAACCG ACTACTAATA CGGCGTGGTC 180
TAATTGTCCT CTTTTATTGC CACATTTCGG TTGGTAGTAA ACGCCGTTGG AGTAAAAACT 240
GGATGTTTTG TGTGATGCGT CAATGGCCAC CGAAATCGGA CCGTGTTTAA GAAGGGCAAC 300 CTTCATAGCG TCAACATCGT TACGAGTTAC GTTGACATAT CCTGTGATGG GAGCAACTAA 360 AGTAACGTTG TTGACATGGC AGTAACCATC TTGTCCCAAG TAACCACCAT AATCCTCTTC 420
AGTAGGGATT CCTCCATGTT TCATCATCCA TTGGTAGGCG CGGAAATCTT CGCCACCGTC 480
GCAACCATTA TTTCCATATC CCCAAGAACA ATCAATCAAA GCCTGTTGAG ACAATCGTAC 540
AAGATTACCG CCGTTTTTCA AGAAATATGC ACCTTCGATT GCTCCAATTG TTCCGAAAGA 600 CCAGCAGGAA CCACAAACAG ATTGATCTTT AACTGGAGTA ACAGCTCCAT AAATTCGCCA 660
GTCGAAAGAA TCTGGTAAAT TTCTTGCTTC CTTGGTGGTA TTGTGAGGAA AGGGTAAACC 720
GCCATTATAA CCGGAGGAAG GTCTGTGTCC TCTCAAAGCC TTAAGTTCAG CTTCAGTACG 7 80
ATCAGCCAAA TGGTTAACAG TCAAAGTGTA TCCCAAATTT GCACGATTCT TAGAAAAAAT 840
GTACCTCAAG TTTAGTCTGA AAATTTCTTT GCGGAAATGA TGTTCTTCAT TTTTATTATA 900 AATTTTTCCG TGTTTATTGA TGAATCGGTG GAATTCTTCA TCAATATGTT CGGTAAAATG 960
GGGATGCACG AATTCCCTCA T 98 1
( 2 ) INFORMATION FOR SEQ ID NO : 7 :
( l ) SEQUENCE CHARACTERISTI CS :
(A) LENGTH : 68 1 base pai rs ( B ) TYPE : nucleic acid
( C ) STRANDEDNESS : singl e
( D ) TOPOLOGY : linear
( l l ) MOLECULE TYPE : cDNA
( IX ) FEATURE : (A) NAME /KEY : CDS
( B ) LOCATION : 1 . . 678
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TTA CCA GAT TCT TTC GAC TGG CGA ATT TAT GGA GCT GTT ACT CCA GTT 48 Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr Pro Val 1 5 10 15
AAA GAT CAA TCT GTT TGT GGT TCC TGC TGG TCT TTC GGA ACA ATT GGA 96 Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr Ile Gly 20 25 30
GCA ATC GAA GGT GCA TAT TTC TTG AAA AAC GGC GGT AAT CTT GTA CGA 144 Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu Val Arg 35 40 45
TTG TCT CAA CAG GCT TTG ATT GAT TGT TCT TGG GGA TAT GGA AAT AAT 192 Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly Asn Asn 50 55 60 GGT TGC GAC GGT GGC GAA GAT TTC CGC GCC TAC CAA TGG ATG ATG AAA 240 Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met Met Lys 65 70 75 80
CAT GGA GGA ATC CCT ACT GAA GAG GAT TAT GGT GGT TAC TTG GGA CAA 288 His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu Gly Gin 85 90 95 GAT GGT TAC TGC CAT GTC AAC AAC GTT ACT TTA GTT GCT CCC ATC ACA 336 Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro lie Thr 100 105 110
GGA TAT GTC AAC GTA ACT CGT AAC GAT GTT GAC GCT ATG AAG GTT GCC 384 Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys Val Ala 115 120 125
CTT CTT AAA CAC GGT CCG ATT TCG GTG GCC ATT GAC GCA TCA CAC AAA 432 Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser His Lys 130 135 140 ACA TCC AGT TTT TAC TCC AAC GGC GTT TAC TAC CAA CCG AAA TGT GGC 480 Thr Ser Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Gin Pro Lys Cys Gly 145 150 155 160
AAT AAA AGA GGA CAA TTA GAC CAC GCC GTA TTA GTA GTC GGT TAT GGT 528 Asn Lys Arg Gly Gin Leu Asp His Ala Val Leu Val Val Gly Tyr Gly 165 170 175
GAA ATC AAC AGC GAA CCT TAC TGG TTG GTC AAG AAT CCT GGT CAA TTG 576 Glu Ile Asn Ser Glu Pro Tyr Trp Leu Val Lys Asn Pro Gly Gin Leu 180 185 190
TGG GGA AAC AAT GTT ATA TTT GAT GTC GCC AAA AAT AAT AAT GCG GAT 624 Trp Gly Asn Asn Val Ile Phe Asp Val Ala Lys Asn Asn Asn Ala Asp 195 200 205
TTG ACG ATC ACT TAT TTA CTA TGT ACT ACT TTT AAA ATT GAT TTT TAC 672 Leu Thr Ile Thr Tyr Leu Leu Cys Thr Thr Phe Lys Ile Asp Phe Tyr 210 215 220 GGC ATG TAA 681
Gly Met 225
(2) INFORMATION FOR SEQ ID NO: 8:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 226 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr Pro Val
1 5 10 15
Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr Ile Gly 20 25 30
Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu Val Arg 35 40 45
Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly Asn Asn 50 55 60
Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met Met Lys 65 70 75 80 His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu Gly Gin 85 * 90 95
Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro Ile Thr 100 105 110 Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys Val Ala 115 120 125
Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser His Lys 130 135 140
Thr Ser Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Gin Pro Lys Cys Gly 145 150 155 160
Asn Lys Arg Gly Gin Leu Asp His Ala Val Leu Val Val Gly Tyr Gly 165 170 175
Glu Ile Asn Ser Glu Pro Tyr Trp Leu Val Lys Asn Pro Gly Gin Leu 180 185 190 Trp Gly Asn Asn Val Ile Phe Asp Val Ala Lys Asn Asn Asn Ala Asp 195 200 205
Leu Thr Ile Thr Tyr Leu Leu Cys Thr Thr Phe Lys Ile Asp Phe Tyr 210 215 220
Gly Met 225
(2) INFORMATION FOR SEQ ID NO: 9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 775 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..685
(D) OTHER INFORMATION: /note= "At pos. bp 559, change A to N. At pos. aa 187, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
TCA AAT CGG ATT GTT AAT GGA GTT AAT GCC AAA AAC GGT TCT GCT CCA 48 Ser Asn Arg Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro
1 5 10 15
TAT ATG GCT TCT CTA AGA GAT GTT ATG GAA ACC ATT TCT GTG GAG CAT 96 Tyr Met Ala Ser Leu Arg Asp Val Met Glu Thr Ile Ser Val Glu His 20 25 30 CGA TAT TGG ATG AAC CGC TGG ATT CTT ACT GCT GCC CAT TGC CTT ACT 144 Arg Tyr Trp Met Asn Arg Trp Ile Leu Thr Ala Ala His Cys Leu Thr 35 40 45
GAC GGT TAT CTA GAT ACA GTC TAC GTT GGT TCA AAT CAT CTT TCT GGC 192 Asp Gly Tyr Leu Asp Thr Val Tyr Val Gly Ser Asn His Leu Ser Gly 50 - 55 60
GAC GGA GAG TAC TAC AAT GTA GAA GAA CAA GTC ATC CAT GAT AAA TAT 240 Asp Gly Glu Tyr Tyr Asn Val Glu Glu Gin Val Ile His Asp Lys Tyr 65 70 75 80
TTT GGT CAA ACA ACC GGC TTC AAA AAT GAT ATT GCT CTC GTC AAA GTT 288 Phe Gly Gin Thr Thr Gly Phe Lys Asn Asp Ile Ala Leu Val Lys Val 85 90 95
TCT AGT GCT ATA AAA CTT AGC AAA AAT GTT CGT CCC ATC AAA TTG CAC 336 Ser Ser Ala Ile Lys Leu Ser Lys Asn Val Arg Pro Ile Lys Leu His
100 105 110
AAA GAT TTT ATA CGC GGA GGT GAA AAA TTG AAA ATT ACT GGA TGG GGA 384 Lys Asp Phe Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly Trp Gly 115 120 125 TTG ACC AAT CAA ACT CAT GGT GAA GTT CCT GAT GCT CTT CAA GAG TTA 432 Leu Thr Asn Gin Thr His Gly Glu Val Pro Asp Ala Leu Gin Glu Leu 130 135 140
CAG GTA GAA GCA CTT TCT AAC TCT AAA TGC AAG GCA ATT ACT GGT GTC 480 Gin Val Glu Ala Leu Ser Asn Ser Lys Cys Lys Ala Ile Thr Gly Val 145 150 155 160
CAT CTT CCT GCT CAT CTC TGC ACC TTC AAA GCA CCT CAA AAG GGT GTA 528 His Leu Pro Ala His Leu Cys Thr Phe Lys Ala Pro Gin Lys Gly Val 165 170 175
TGC ATG GGT GAC TCT GGT GGT CCT CTG GTC NAT AAG GGC AAG CAA GTT 576 Cys Met Gly Asp Ser Gly Gly Pro Leu Val Xaa Lys Gly Lys Gin Val
180 185 190
GGA GTC ACA TCT TTC GTC TGG GAA GGT TGT GCT TTG GGC AAC CCT GAT 624 Gly Val Thr Ser Phe Val Trp Glu Gly Cys Ala Leu Gly Asn Pro Asp 195 200 205 TTC TTT ACA AGA GTT TCG CTT TAT GTA GAC TGG GTC AAA AAG ATT CAA 672 Phe Phe Thr Arg Val Ser Leu Tyr Val Asp Trp Val Lys Lys Ile Gin 210 215 220
AAA GAA TAT AAA T GATATGTTGA TTGTCACTAA AATGCATCGA TTTGGATAAT 725
Lys Glu Tyr Lys 225
TTGGTTGTGA ATATAATTTT ATTTCTAGCA TCAAAAAAAA AAAAAAAAAA 775
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Ser Asn Arg Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro
1 5 10 15
Tyr Met Ala Ser Leu Arg Asp Val Met Glu Thr Ile Ser Val Glu His 20 25 30 Arg Tyr Trp Met Asn Arg Trp Ile Leu Thr Ala Ala His Cys Leu Thr 35 40 45
Asp Gly Tyr Leu Asp Thr Val Tyr Val Gly Ser Asn His Leu Ser Gly 50 55 60
Asp Gly Glu Tyr Tyr Asn Val Glu Glu Gin Val Ile His Asp Lys Tyr 65 70 75 80
Phe Gly Gin Thr Thr Gly Phe Lys Asn Asp Ile Ala Leu Val Lys Val 85 90 95
Ser Ser Ala Ile Lys Leu Ser Lys Asn Val Arg Pro Ile Lys Leu His 100 105 110 Lys Asp Phe Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly Trp Gly 115 120 125
Leu Thr Asn Gin Thr His Gly Glu Val Pro Asp Ala Leu Gin Glu Leu 130 135 140
Gin Val Glu Ala Leu Ser Asn Ser Lys Cys Lys Ala Ile Thr Gly Val 145 150 155 160
His Leu Pro Ala His Leu Cys Thr Phe Lys Ala Pro Gin Lys Gly Val 165 170 175
Cys Met Gly Asp Ser Gly Gly Pro Leu Val Xaa Lys Gly Lys Gin Val 180 185 190 Gly Val Thr Ser Phe Val Trp Glu Gly Cys Ala Leu Gly Asn Pro Asp 195 200 205
Phe Phe Thr Arg Val Ser Leu Tyr Val Asp Trp Val Lys Lys Ile Gin 210 215 220
Lys Glu Tyr Lys 225
(2) INFORMATION FOR SEQ ID NO:11:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 775 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: DNA (genomic) (XI) SEQUENCE DESCRIPTION: SEQ ID NO:ll: ττττττττττ TTTTTTTTGA TGCTAGAAAT AAAATTATAT TCACAACCAA ATTATCCAAA 60 TCGATGCATT TTAGTGACAA TCAACATATC ATTTATATTC TTTTTGAATC TTTTTGACCC 120 AGTCTACATA AAGCGAAACT CTTGTAAAGA AATCAGGGTT GCCCAAAGCA CAACCTTCCC 180 AGACGAAAGA TGTGACTCCA ACTTGCTTGC CCTTATNGAC CAGAGGACCA CCAGAGTCAC 240
CCATGCATAC ACCCTTTTGA GGTGCTTTGA AGGTGCAGAG ATGAGCAGGA AGATGGACAC 300
CAGTAATTGC CTTGCATTTA GAGTTAGAAA GTGCTTCTAC CTGTAACTCT TGAAGAGCAT 360
CAGGAACTTC ACCATGAGTT TGATTGGTCA ATCCCCATCC AGTAATTTTC AATTTTTCAC 420 CTCCGCGTAT AAAATCTTTG TGCAATTTGA TGGGACGAAC ATTTTTGCTA AGTTTTATAG 480
CACTAGAAAC TTTGACGAGA GCAATATCAT TTTTGAAGCC GGTTGTTTGA CCAAAATATT 540
TATCATGGAT GACTTGTTCT TCTACATTGT AGTACTCTCC GTCGCCAGAA AGATGATTTG 600
AACCAACGTA GACTGTATCT AGATAACCGT CAGTAAGGCA ATGGGCAGCA GTAAGAATCC 660
AGCGGTTCAT CCAATATCGA TGCTCCACAG AAATGGTTTC CATAACATCT CTTAGAGAAG 720 CCATATATGG AGCAGAACCG TTTTTGGCAT TAACTCCATT AACAATCCGA TTTGA 775
( 2 ) INFORMATION FOR SEQ ID NO : 12 :
( 1 ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 675 base pairs
( B ) TY PE : nucleic acid ( C ) STRANDEDNESS : single
( D ) TOPOLOGY : linear
( ii ) MOLECULE TYPE : DNA ( genomic )
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..675
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
ATT GTT AAT GGA GTT AAT GCC AAA AAC GGT TCT GCT CCA TAT ATG GCT 48 Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro Tyr Met Ala 1 5 10 15 TCT CTA AGA GAT GTT ATG GAA ACC ATT TCT GTG GAG CAT CGA TAT TGG 96 Ser Leu Arg Asp Val Met Glu Thr Ile Ser Val Glu His Arg Tyr Trp 20 25 30
ATG AAC CGC TGG ATT CTT ACT GCT GCC CAT TGC CTT ACT GAC GGT TAT 144 Met Asn Arg Trp Ile Leu Thr Ala Ala His Cys Leu Thr Asp Gly Tyr 35 40 45
CTA GAT ACA GTC TAC GTT GGT TCA AAT CAT CTT TCT GGC GAC GGA GAG 192 Leu Asp Thr Val Tyr Val Gly Ser Asn His Leu Ser Gly Asp Gly Glu 50 55 60
TAC TAC AAT GTA GAA GAA CAA GTC ATC CAT GAT AAA TAT TTT GGT CAA 240 Tyr Tyr Asn Val Glu Glu Gin Val Ile His Asp Lys Tyr Phe Gly Gin 65 70 75 80
ACA ACC GGC TTC AAA AAT GAT ATT GCT CTC GTC AAA GTT TCT AGT GCT 288 Thr Thr Gly Phe Lys Asn Asp Ile Ala Leu Val Lys Val Ser Ser Ala 85 90 95 ATA AAA CTT AGC AAA AAT GTT CGT CCC ATC AAA TTG CAC AAA GAT TTT 336 Ile Lys Leu Ser Lys Asn Val Arg Pro He Lys Leu His Lys Asp Phe 100 105 110
ATA CGC GGA GGT GAA AAA TTG AAA ATT ACT GGA TGG GGA TTG ACC AAT 384 Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly Trp Gly Leu Thr Asn 115 120 125 CAA ACT CAT GGT GAA GTT CCT GAT GCT CTT CAA GAG TTA CAG GTA GAA 432 Gin Thr His Gly Glu Val Pro Asp Ala Leu Gin Glu Leu Gin Val Glu 130 135 140
GCA CTT TCT AAC TCT AAA TGC AAG GCA ATT ACT GGT GTC CAT CTT CCT 480 Ala Leu Ser Asn Ser Lys Cys Lys Ala Ile Thr Gly Val His Leu Pro 145 150 155 160
GCT CAT CTC TGC ACC TTC AAA GCA CCT CAA AAG GGT GTA TGC ATG GGT 528 Ala His Leu Cys Thr Phe Lys Ala Pro Gin Lys Gly Val Cys Met Gly 165 170 175
GAC TCT GGT GGT CCT CTG GTC NAT AAG GGC AAG CAA GTT GGA GTC ACA 576 Asp Ser Gly Gly Pro Leu Val Xaa Lys Gly Lys Gin Val Gly Val Thr 180 185 190
TCT TTC GTC TGG GAA GGT TGT GCT TTG GGC AAC CCT GAT TTC TTT ACA 624 Ser Phe Val Trp Glu Gly Cys Ala Leu Gly Asn Pro Asp Phe Phe Thr 195 200 205 AGA GTT TCG CTT TAT GTA GAC TGG GTC AAA AAG ATT CAA AAA GAA TAT 672 Arg Val Ser Leu Tyr Val Asp Trp Val Lys Lys Ile Gin Lys Glu Tyr 210 215 220
AAA 675
Lys 225
(2) INFORMATION FOR SEQ ID NO: 13:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Ile Val Asn Gly Val Asn Ala Lys Asn Gly Ser Ala Pro Tyr Met Ala 1 5 10 15 Ser Leu Arg Asp Val Met Glu Thr Ile Ser Val Glu His Arg Tyr Trp
20 25 30
Met Asn Arg Trp lie Leu Thr Ala Ala His Cys Leu Thr Asp Gly Tyr 35 40 45
Leu Asp Thr Val Tyr Val Gly Ser Asn His Leu Ser Gly Asp Gly Glu 50 55 60
Tyr Tyr Asn Val Glu Glu Gin Val Ile His Asp Lys Tyr Phe Gly Gin 65 70 75 80 Thr Thr Gly Phe Lys Asn Asp Ile Ala Leu Val Lys Val Ser Ser Ala 85 90 95
Ile Lys Leu Ser Lys Asn Val Arg Pro Ile Lys Leu His Lys Asp Phe 100 105 110 Ile Arg Gly Gly Glu Lys Leu Lys Ile Thr Gly Trp Gly Leu Thr Asn 115 120 125
Gin Thr His Gly Glu Val Pro Asp Ala Leu Gin Glu Leu Gin Val Glu 130 135 140
Ala Leu Ser Asn Ser Lys Cys Lys Ala Ile Thr Gly Val His Leu Pro 145 150 155 160
Ala His Leu Cys Thr Phe Lys Ala Pro Gin Lys Gly Val Cys Met Gly 165 170 175
Asp Ser Gly Gly Pro Leu Val Xaa Lys Gly Lys Gin Val Gly Val Thr
180 185 190 Ser Phe Val Trp Glu Gly Cys Ala Leu Gly Asn Pro Asp Phe Phe Thr 195 200 205
Arg Val Ser Leu Tyr Val Asp Trp Val Lys Lys Ile Gin Lys Glu Tyr 210 215 220
Lys 225
(2) INFORMATION FOR SEQ ID NO: 14:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTTATATTCT TTTTGAATCT TTTTGACCCA GTCTACATAA AGCGAAACTC TTGTAAAGAA 60
ATCAGGGTTG CCCAAAGCAC AACCTTCCCA GACGAAAGAT GTGACTCCAA CTTGCTTGCC 120
CTTATNGACC AGAGGACCAC CAGAGTCACC CATGCATACA CCCTTTTGAG GTGCTTTGAA 180
GGTGCAGAGA TGAGCAGGAA GATGGACACC AGTAATTGCC TTGCATTTAG AGTTAGAAAG 240
TGCTTCTACC TGTAACTCTT GAAGAGCATC AGGAACTTCA CCATGAGTTT GATTGGTCAA 300 TCCCCATCCA GTAATTTTCA ATTTTTCACC TCCGCGTATA AAATCTTTGT GCAATTTGAT 360
GGGACGAACA TTTTTGCTAA GTTTTATAGC ACTAGAAACT TTGACGAGAG CAATATCATT 420
TTTGAAGCCG GTTGTTTGAC CAAAATATTT ATCATGGATG ACTTGTTCTT CTACATTGTA 480
GTACTCTCCG TCGCCAGAAA GATGATTTGA ACCAACGTAG ACTGTATCTA GATAACCGTC 540
AGTAAGGCAA TGGGCAGCAG TAAGAATCCA GCGGTTCATC CAATATCGAT GCTCCACAGA 600 AATGGTTTCC ATAACATCTC TTAGAGAAGC CATATATGGA GCAGAACCGT TTTTGGCATT 660 AACTCCATTA ACAAT 675
(2) INFORMATION FOR SEQ ID NO: 15:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1089 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 33..807
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GTTTTTAGTG CGGTGTTAAA TAAAAGTTTA AA ATG AAA CTC ATC ATC GTG CTA 53 Met Lys Leu Ile Ile Val Leu
1 5
GCA TTT GTT TTA GGA ATT TGT TCA GGT TCT CCA CAT TCA AGA ATA ATT 101 Ala Phe Val Leu Gly Ile Cys Ser Gly Ser Pro His Ser Arg Ile Ile
10 15 20 TGT GGT CAA AAT GCC AAA AAA AAT TCG GCT CCA TAC ATG GCA TCG GTT 149
Cys Gly Gin Asn Ala Lys Lys Asn Ser Ala Pro Tyr Met Ala Ser Val 25 30 35
CAA CTT TTA GAT AAA GTT GAA GGA GTC GAA AAA TTG TTT CAT TTT TGC 197 Gin Leu Leu Asp Lys Val Glu Gly Val Glu Lys Leu Phe His Phe Cys 40 45 50 55
GGA GGA GCA ATA GTT AAT GAT AGA TGG ATT TTG ACT GCT GCA CAT TGT 245 Gly Gly Ala Ile Val Asn Asp Arg Trp Ile Leu Thr Ala Ala His Cys 60 65 70
TTG AGA GGC AAA GAC CAC CTC CTG GAC AAA CTG TTC ATT GCA GTC GGC 293 Leu Arg Gly Lys Asp His Leu Leu Asp Lys Leu Phe Ile Ala Val Gly
75 80 85
CTG ACA AAT TTA GGT GAA GGA GGC ACC GTG TAT CCT GTA GAA AAA GGC 341 Leu Thr Asn Leu Gly Glu Gly Gly Thr Val Tyr Pro Val Glu Lys Gly 90 95 100 ATC ATG CAC GAA GAA TAT GAA CAT TAT GAC ATA GTC AAC GAT ATT GCA 389 Ile Met His Glu Glu Tyr Glu His Tyr Asp Ile Val Asn Asp Ile Ala 105 110 115
CTA ATC AAA GTC AAA TCT CCG ATA GAA TTC AAT GAA AAA GTA ACG ACT 437 Leu Ile Lys Val Lys Ser Pro Ile Glu Phe Asn Glu Lys Val Thr Thr 120 125 130 135
GTA AAA TTA GGT GAG GAT TAT GTT GGC GGA GAC GTC CAA CTT CGA TTG 485 Val Lys Leu Gly Glu Asp Tyr Val Gly Gly Asp Val Gin Leu Arg Leu 140 145 150
ACA GGA TGG GGA GTT ACG ACA AAT GAG GGA ATC GGA AGC CCG AGT CAA 533 Thr Gly Trp Gly Val Thr Thr Asn Glu Gly Ile Gly Ser Pro Ser Gin 155 160 165
AAA TTA CAG GTC ATG ACA GCC AAA TCA CTA ACT TAT GAG GAT TGC AAA 581 Lys Leu Gin Val Met Thr Ala Lys Ser Leu Thr Tyr Glu Asp Cys Lys 170 175 180
AAC GCA ATT TAT AAA AAG ACT TTC GAA AGC CAA ATT TGT GCA CAG GCT 629 Asn Ala Ile Tyr Lys Lys Thr Phe Glu Ser Gin Ile Cys Ala Gin Ala 185 190 195
AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA TTA GTC 677 Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Val 200 205 210 215
CAA GGA AAC AAT ACA TTG GTC GGT TTA GTA TCC TGG GGT ATG CAA CCT 725 Gin Gly Asn Asn Thr Leu Val Gly Leu Val Ser Trp Gly Met Gin Pro 220 225 230 TGT GGA AGT GGT TAT TAT CCT GAC GTT TAC ACA AGA ATT ACA TCG TTT 773 Cys Gly Ser Gly Tyr Tyr Pro Asp Val Tyr Thr Arg Ile Thr Ser Phe 235 240 245
TTG GAC TGG ATT AAC ACG ACA ATG TCA GAA AAT T AAAGAAAAAA 817
Leu Asp Trp Ile Asn Thr Thr Met Ser Glu Asn 250 255
AAATATCAAA ATAGTAGTTA AAATATTTTG TAGACTACTG TAAAAGTATT GAAATTAACA 877
AATATTTGTT TTGTATATAG GGCTGGTACC TAAAGACATT ATTATTTGTT AAAACTGATA 937
TTTATTTTTA TGACGAATTT CTATATTTAT AATATTATTT TATATGTATG TATTTGATTG 997
TATTTTAAAT ATGGACTGGT TGTTCCCAAT GTTATAAATT AAAATTATAA AAATACATCT 1057 ACTATTTTAT ACTAAAAAAA AAAAAAAAAA AA 1089
(2) INFORMATION FOR SEQ ID NO:16:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 258 amino acids
(B) TYPE : ammo acid ( D) TOPOLOGY : linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Lys Leu Ile Ile Val Leu Ala Phe Val Leu Gly Ile Cys Ser Gly 1 5 10 15 Ser Pro His Ser Arg Ile Ile Cys Gly Gin Asn Ala Lys Lys Asn Ser
20 25 30
Ala Pro Tyr Met Ala Ser Val Gin Leu Leu Asp Lys Val Glu Gly Val 35 40 45
Glu Lys Leu Phe His Phe Cys Gly Gly Ala Ile Val Asn Asp Arg Trp 50 55 60
Ile Leu Thr Ala Ala His Cys Leu Arg Gly Lys Asp His Leu Leu Asp 65 70 75 80 Lys Leu Phe Ile Ala Val Gly Leu Thr Asn Leu Gly Glu Gly Gly Thr 85 90 95
Val Tyr Pro Val Glu Lys Gly Ile Met His Glu Glu Tyr Glu His Tyr 100 105 110 Asp Ile Val Asn Asp Ile Ala Leu Ile Lys Val Lys Ser Pro Ile Glu 115 120 125
Phe Asn Glu Lys Val Thr Thr Val Lys Leu Gly Glu Asp Tyr Val Gly 130 135 140
Gly Asp Val Gin Leu Arg Leu Thr Gly Trp Gly Val Thr Thr Asn Glu 145 150 155 160
Gly Ile Gly Ser Pro Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser 165 170 175
Leu Thr Tyr Glu Asp Cys Lys Asn Ala Ile Tyr Lys Lys Thr Phe Glu 180 185 190 Ser Gin Ile Cys Ala Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly 195 200 205
Asp Ser Gly Gly Pro Leu Val Gin Gly Asn Asn Thr Leu Val Gly Leu 210 215 220
Val Ser Trp Gly Met Gin Pro Cys Gly Ser Gly Tyr Tyr Pro Asp Val 225 230 235 240
Tyr Thr Arg Ile Thr Ser Phe Leu Asp Trp Ile Asn Thr Thr Met Ser 245 250 255
Glu Asn
(2) INFORMATION FOR SEQ ID NO: 17:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1089 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:17:
TTTTTTTTTT TTTTTTTTTA GTATAAAATA GTAGATGTAT TTTTATAATT TTAATTTATA 60
ACATTGGGAA CAACCAGTCC ATATTTAAAA TACAATCAAA TACATACATA TAAAATAATA 120 TTATAAATAT AGAAATTCGT CATAAAAATA AATATCAGTT TTAACAAATA ATAATGTCTT 180
TAGGTACCAG CCCTATATAC AAAACAAATA TTTGTTAATT TCAATACTTT TACAGTAGTC 240
NACAAAATAT TTTAACTACT ATTTTGATAN TTTTTTNTCT TTAATTTTCT GACATTGTCG 300
TGTTAATCCA GTCCAAAAAC GATGTAATTC TTGTGTAAAC GTCAGGATAA TAACCACTTC 360
CACAAGGTTG CATACCCCAG GATACTAAAC CGACCAATGT ATTGTTTCCT TGGACTAATG 420 GACCACCAGA ATCACCCTTA CAAGATCCGG TTCCTTTTTT AGCCTGTGCA CAAATTTGGC 480 TTTCGAAAGT CTTTTTATAA ATTGCGTTTT TGCAATCCTC ATAAGTTAGT GATTTGGCTG 540
TCATGACCTG TAATTTTTGA CTCGGGCTTC CGATTCCCTC ATTTGTCGTA ACTCCCCATC 600
CTGTCAATCG AAGTTGGACG TCTCCGCCAA CATAATCCTC ACCTAATTTT ACAGTCGTTA 660
CTTTTTCATT GAATTCTATC GGAGATTTGA CTTTGATTAG TGCAATATCG TTGACTATGT 720 CATAATGTTC ATATTCTTCG TGCATGATGC CTTTTTCTAC AGGATACACG GTGCCTCCTT 780
CACCTAAATT TGTCAGGCCG ACTGCAATGA ACAGTTTGTC CAGGAGGTGG TCTTTGCCTC 840
TCAAACAATG TGCAGCAGTC AAAATCCATC TATCATTAAC TATTGCTCCT CCGCAAAAAT 900
GAAACAATTT TTCGACTCCT TCAACTTTAT CTAAAAGTTG AACCGATGCC ATGTATGGAG 960
CCGAATTTTT TTTGGCATTT TGACCACAAA TTATTCTTGA ATGTGGAGAA CCTGAACAAA 1020 TTCCTAAAAC AAATGCTAGC ACGATGATGA GTTTCATTTT AAACTTTTAT TTAACACCGC 1080
ACTAAAAAC 1089
( 2 ) INFORMATION FOR SEQ ID NO : 18 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 774 base pai rs (B) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..774
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
ATG AAA CTC ATC ATC GTG CTA GCA TTT GTT TTA GGA ATT TGT TCA GGT 48 Met Lys Leu Ile Ile Val Leu Ala Phe Val Leu Gly Ile Cys Ser Gly 1 5 10 15
TCT CCA CAT TCA AGA ATA ATT TGT GGT CAA AAT GCC AAA AAA AAT TCG 96 Ser Pro His Ser Arg Ile Ile Cys Gly Gin Asn Ala Lys Lys Asn Ser 20 25 30
GCT CCA TAC ATG GCA TCG GTT CAA CTT TTA GAT AAA GTT GAA GGA GTC 144 Ala Pro Tyr Met Ala Ser Val Gin Leu Leu Asp Lys Val Glu Gly Val
35 40 45
GAA AAA TTG TTT CAT TTT TGC GGA GGA GCA ATA GTT AAT GAT AGA TGG 192 Glu Lys Leu Phe His Phe Cys Gly Gly Ala Ile Val Asn Asp Arg Trp 50 55 60 ATT TTG ACT GCT GCA CAT TGT TTG AGA GGC AAA GAC CAC CTC CTG GAC 240 Ile Leu Thr Ala Ala His Cys Leu Arg Gly Lys Asp His Leu Leu Asp 65 70 75 80
AAA CTG TTC ATT GCA GTC GGC CTG ACA AAT TTA GGT GAA GGA GGC ACC 288 Lys Leu Phe Ile Ala Val Gly Leu Thr Asn Leu Gly Glu Gly Gly Thr 85 90 95 GTG TAT CCT GTA GAA AAA GGC ATC ATG CAC GAA GAA TAT GAA CAT TAT 336 Val Tyr Pro Val Glu Lys Gly Ile Met His Glu Glu Tyr Glu His Tyr 100 105 110
GAC ATA GTC AAC GAT ATT GCA CTA ATC AAA GTC AAA TCT CCG ATA GAA 384 Asp lie Val Asn Asp Ile Ala Leu Ile Lys Val Lys Ser Pro Ile Glu 115 120 125
TTC AAT GAA AAA GTA ACG ACT GTA AAA TTA GGT GAG GAT TAT GTT GGC 432 Phe Asn Glu Lys Val Thr Thr Val Lys Leu Gly Glu Asp Tyr Val Gly 130 135 140 GGA GAC GTC CAA CTT CGA TTG ACA GGA TGG GGA GTT ACG ACA AAT GAG 480 Gly Asp Val Gin Leu Arg Leu Thr Gly Trp Gly Val Thr Thr Asn Glu 145 150 155 160
GGA ATC GGA AGC CCG AGT CAA AAA TTA CAG GTC ATG ACA GCC AAA TCA 528 Gly lie Gly Ser Pro Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser 165 170 175
CTA ACT TAT GAG GAT TGC AAA AAC GCA ATT TAT AAA AAG ACT TTC GAA 576 Leu Thr Tyr Glu Asp Cys Lys Asn Ala Ile Tyr Lys Lys Thr Phe Glu 180 185 190
AGC CAA ATT TGT GCA CAG GCT AAA AAA GGA ACC GGA TCT TGT AAG GGT 624 Ser Gin lie Cys Ala Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly 195 200 205
GAT TCT GGT GGT CCA TTA GTC CAA GGA AAC AAT ACA TTG GTC GGT TTA 672 Asp Ser Gly Gly Pro Leu Val Gin Gly Asn Asn Thr Leu Val Gly Leu 210 215 220 GTA TCC TGG GGT ATG CAA CCT TGT GGA AGT GGT TAT TAT CCT GAC GTT 720 Val Ser Trp Gly Met Gin Pro Cys Gly Ser Gly Tyr Tyr Pro Asp Val 225 230 235 240
TAC ACA AGA ATT ACA TCG TTT TTG GAC TGG ATT AAC ACG ACA ATG TCA 768 Tyr Thr Arg Ile Thr Ser Phe Leu Asp Trp Ile Asn Thr Thr Met Ser 245 250 255
GAA AAT 774
Glu Asn
(2) INFORMATION FOR SEQ ID NO: 19: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 258 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Lys Leu Ile Ile Val Leu Ala Phe Val Leu Gly Ile Cys Ser Gly 1 5 10 15
Ser Pro His Ser Arg Ile Ile Cys Gly Gin Asn Ala Lys Lys Asn Ser 20 25 30 Ala Pro Tyr Met Ala Ser Val Gin Leu Leu Asp Lys Val Glu Gly Val 35 40 45
Glu Lys Leu Phe His Phe Cys Gly Gly Ala Ile Val Asn Asp Arg Trp 50 55 60 He Leu Thr Ala Ala His Cys Leu Arg Gly Lys Asp His Leu Leu Asp 65 70 75 80
Lys Leu Phe Ile Ala Val Gly Leu Thr Asn Leu Gly Glu Gly Gly Thr 85 90 95
Val Tyr Pro Val Glu Lys Gly Ile Met His Glu Glu Tyr Glu His Tyr 100 105 110
Asp Ile Val Asn Asp Ile Ala Leu Ile Lys Val Lys Ser Pro Ile Glu 115 120 125
Phe Asn Glu Lys Val Thr Thr Val Lys Leu Gly Glu Asp Tyr Val Gly 130 135 140
Gly Asp Val Gin Leu Arg Leu Thr Gly Trp Gly Val Thr Thr Asn Glu 145 150 155 160
Gly Ile Gly Ser Pro Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser 165 170 175
Leu Thr Tyr Glu Asp Cys Lys Asn Ala Ile Tyr Lys Lys Thr Phe Glu 180 " 185 190
Ser Gin Ile Cys Ala Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly 195 200 205
Asp Ser Gly Gly Pro Leu Val Gin Gly Asn Asn Thr Leu Val Gly Leu 210 215 220 Val Ser Trp Gly Met Gin Pro Cys Gly Ser Gly Tyr Tyr Pro Asp Val 225 230 235 240
Tyr Thr Arg Ile Thr Ser Phe Leu Asp Trp Ile Asn Thr Thr Met Ser 245 250 255
Glu Asn
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 774 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATTTTCTGAC ATTGTCGTGT TAATCCAGTC CAAAAACGAT GTAATTCTTG TGTAAACGTC 60 AGGATAATAA CCACTTCCAC AAGGTTGCAT ACCCCAGGAT ACTAAACCGA CCAATGTATT 120 GTTTCCTTGG ACTAATGGAC CACCAGAATC ACCCTTACAA GATCCGGTTC CTTTTTTAGC 180 CTGTGCACAA ATTTGGCTTT CGAAAGTCTT TTTATAAATT GCGTTTTTGC AATCCTCATA 240
AGTTAGTGAT TTGGCTGTCA TGACCTGTAA TTTTTGACTC GGGCTTCCGA TTCCCTCATT 300
TGTCGTAACT CCCCATCCTG TCAATCGAAG TTGGACGTCT CCGCCAACAT AATCCTCACC 360
TAATTTTACA GTCGTTACTT TTTCATTGAA TTCTATCGGA GATTTGACTT TGATTAGTGC 420 AATATCGTTG ACTATGTCAT AATGTTCATA TTCTTCGTGC ATGATGCCTT TTTCTACAGG 480
ATACACGGTG CCTCCTTCAC CTAAATTTGT CAGGCCGACT GCAATGAACA GTTTGTCCAG 540
GAGGTGGTCT TTGCCTCTCA AACAATGTGC AGCAGTCAAA ATCCATCTAT CATTAACTAT 600
TGCTCCTCCG CAAAAATGAA ACAATTTTTC GACTCCTTCA ACTTTATCTA AAAGTTGAAC 660
CGATGCCATG TATGGAGCCG AATTTTTTTT GGCATTTTGA CCACAAATTA TTCTTGAATG 720 TGGAGAACCT GAACAAATTC CTAAAACAAA TGCTAGCACG ATGATGAGTT TCAT 774
(2) INFORMATION FOR SEQ ID NO:21:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 711 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..711
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ATA ATT TGT GGT CAA AAT GCC AAA AAA AAT TCG GCT CCA TAC ATG GCA 48 Ile Ile Cys Gly Gin Asn Ala Lys Lys Asn Ser Ala Pro Tyr Met Ala 1 5 10 15 TCG GTT CAA CTT TTA GAT AAA GTT GAA GGA GTC GAA AAA TTG TTT CAT 96 Ser Val Gin Leu Leu Asp Lys Val Glu Gly Val Glu Lys Leu Phe His 20 25 30
TTT TGC GGA GGA GCA ATA GTT AAT GAT AGA TGG ATT TTG ACT GCT GCA 144 Phe Cys Gly Gly Ala Ile Val Asn Asp Arg Trp Ile Leu Thr Ala Ala 35 40 45
CAT TGT TTG AGA GGC AAA GAC CAC CTC CTG GAC AAA CTG TTC ATT GCA 192 His Cys Leu Arg Gly Lys Asp His Leu Leu Asp Lys Leu Phe Ile Ala 50 55 60
GTC GGC CTG ACA AAT TTA GGT GAA GGA GGC ACC GTG TAT CCT GTA GAA 240 Val Gly Leu Thr Asn Leu Gly Glu Gly Gly Thr Val Tyr Pro Val Glu 65 70 75 80
AAA GGC ATC ATG CAC GAA GAA TAT GAA CAT TAT GAC ATA GTC AAC GAT 288 Lys Gly Ile Met His Glu Glu Tyr Glu His Tyr Asp Ile Val Asn Asp 85 90 95 ATT GCA CTA ATC AAA GTC AAA TCT CCG ATA GAA TTC AAT GAA AAA GTA 336 Ile Ala Leu Ile Lys Val Lys Ser Pro Ile Glu Phe Asn Glu Lys Val 100 105 110
ACG ACT GTA AAA TTA GGT GAG GAT TAT GTT GGC GGA GAC GTC CAA CTT 384 Thr Thr Val Lys Leu Gly Glu Asp Tyr Val Gly Gly Asp Val Gin Leu 115 120 125
CGA TTG ACA GGA TGG GGA GTT ACG ACA AAT GAG GGA ATC GGA AGC CCG 432 Arg Leu Thr Gly Trp Gly Val Thr Thr Asn Glu Gly Ile Gly Ser Pro 130 135 140
AGT CAA AAA TTA CAG GTC ATG ACA GCC AAA TCA CTA ACT TAT GAG GAT 480 Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser Leu Thr Tyr Glu Asp 145 150 155 160
TGC AAA AAC GCA ATT TAT AAA AAG ACT TTC GAA AGC CAA ATT TGT GCA 528 Cys Lys Asn Ala Ile Tyr Lys Lys Thr Phe Glu Ser Gin Ile Cys Ala 165 170 175
CAG GCT AAA AAA GGA ACC GGA TCT TGT AAG GGT GAT TCT GGT GGT CCA 576 Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro
180 185 190
TTA GTC CAA GGA AAC AAT ACA TTG GTC GGT TTA GTA TCC TGG GGT ATG 624 Leu Val Gin Gly Asn Asn Thr Leu Val Gly Leu Val Ser Trp Gly Met 195 200 205 CAA CCT TGT GGA AGT GGT TAT TAT CCT GAC GTT TAC ACA AGA ATT ACA 672 Gin Pro Cys Gly Ser Gly Tyr Tyr Pro Asp Val Tyr Thr Arg Ile Thr 210 215 220
TCG TTT TTG GAC TGG ATT AAC ACG ACA ATG TCA GAA AAT 711
Ser Phe Leu Asp Trp Ile Asn Thr Thr Met Ser Glu Asn 225 230 235
(2) INFORMATION FOR SEQ ID NO:22:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 237 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ile Ile Cys Gly Gin Asn Ala Lys Lys Asn Ser Ala Pro Tyr Met Ala 1 5 10 15 Ser Val Gin Leu Leu Asp Lys Val Glu Gly Val Glu Lys Leu Phe His
20 25 30
Phe Cys Gly Gly Ala Ile Val Asn Asp Arg Trp Ile Leu Thr Ala Ala 35 40 45
His Cys Leu Arg Gly Lys Asp His Leu Leu Asp Lys Leu Phe Ile Ala 50 55 60
Val Gly Leu Thr Asn Leu Gly Glu Gly Gly Thr Val Tyr Pro Val Glu 65 70 75 80 Lys Gly Ile Met His Glu Glu Tyr Glu His Tyr Asp lie Val Asn Asp 85 90 95
Ile Ala Leu Ile Lys Val Lys Ser Pro Ile Glu Phe Asn Glu Lys Val 100 105 110 Thr Thr Val Lys Leu Gly Glu Asp Tyr Val Gly Gly Asp Val Gin Leu 115 120 125
Arg Leu Thr Gly Trp Gly Val Thr Thr Asn Glu Gly Ile Gly Ser Pro 130 135 140
Ser Gin Lys Leu Gin Val Met Thr Ala Lys Ser Leu Thr Tyr Glu Asp 145 150 155 160
Cys Lys Asn Ala Ile Tyr Lys Lys Thr Phe Glu Ser Gin Ile Cys Ala 165 170 175
Gin Ala Lys Lys Gly Thr Gly Ser Cys Lys Gly Asp Ser Gly Gly Pro 180 185 190 Leu Val Gin Gly Asn Asn Thr Leu Val Gly Leu Val Ser Trp Gly Met 195 200 205
Gin Pro Cys Gly Ser Gly Tyr Tyr Pro Asp Val Tyr Thr Arg Ile Thr 210 215 220
Ser Phe Leu Asp Trp Ile Asn Thr Thr Met Ser Glu Asn 225 230 235
(2) INFORMATION FOR SEQ ID NO:23:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 924 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic)
(IX) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 3..800
(D) OTHER INFORMATION: /note= "At pos. bp 132, change A to N; at pos. bp 202, change A to N; at pos. bp 780, change A to N. At pos. aa 44, 67 and 260, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: AA ATT TTA TTA CTG GTA TTA TTG GCA GTA TGC TTT GCT TCA GCT AAA 47
Ile Leu Leu Leu Val Leu Leu Ala Val Cys Phe Ala Ser Ala Lys 1 5 10 15
CGA GGT CCA CGA AAA CAT GTT CGC GAA ACA CAA AAA AGT CTT GCC TCT 95 Arg Gly Pro Arg Lys His Val Arg Glu Thr Gin Lys Ser Leu Ala Ser 20 25 30
GGG CGT ATT GTG GGT GGT GAA GCA GTG AGC ATT GAA NAC TAT GGA TGG 143 Gly Arg Ile Val Gly Gly Glu Ala Val Ser Ile Glu Xaa Tyr Gly Trp 35 40 45 CAA GTT TCT CTA CAA CGT TTT GGC AGT CAT TTC TGT GGA GGA TCT ATA 191 Gin Val Ser Leu Gin Arg Phe Gly Ser His Phe Cys Gly Gly Ser Ile 50 55 60
ATA TCC AGT ANA TGG ATT CTT TCA GCT GCT CAT TGC TTT TAT GGA ACG 239 Ile Ser Ser Xaa Trp Ile Leu Ser Ala Ala His Cys Phe Tyr Gly Thr 65 70 75
TTA TTT CCG ATT GGA TTC TCT GCG AGA GCC GGC AGC AGT ACT GTG AAT 287 Leu Phe Pro Ile Gly Phe Ser Ala Arg Ala Gly Ser Ser Thr Val Asn 80 85 90 95 TCA GGA GGA ACT GTG CAT ACA ATT TTG TAT TGG TAT ATT CAT CCA AAT 335 Ser Gly Gly Thr Val His Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn 100 105 110
TAT GAT TCA CAA AGT ACA GAC TTT GAT GTT TCT GTA GTT CGA CTA TTA 383 Tyr Asp Ser Gin Ser Thr Asp Phe Asp Val Ser Val Val Arg Leu Leu 115 120 125
TCT TCT TTA AAT TTG AAT GGA GGT TCT ATT CGA CCG GCT AGG TTA GTG 431 Ser Ser Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val 130 135 140
GAT TCT GGA ACT GAT TTG CCA GCC GGT GAG ATG GTT ACA GTA ACT GGA 479 Asp Ser Gly Thr Asp Leu Pro Ala Gly Glu Met Val Thr Val Thr Gly 145 150 155
TGG GGA CGA CTT TCG GAA AAT ACT TCT GTT CCC TCG CCA TCA ACT CTT 527 Trp Gly Arg Leu Ser Glu Asn Thr Ser Val Pro Ser Pro Ser Thr Leu 160 165 170 175 CAA GGA GTT ACA GTA CCA GTT GTA AGT AAT TCG GAA TGT CAA CAA CAA 575 Gin Gly Val Thr Val Pro Val Val Ser Asn Ser Glu Cys Gin Gin Gin 180 185 190
TTG CAA AAT CAG ACA ATC ACT GAC AAT ATG TTT TGT GCT GGT GAA TTA 623 Leu Gin Asn Gin Thr Ile Thr Asp Asn Met Phe Cys Ala Gly Glu Leu 195 200 205
GAA GGA GGA AAG GAC TCT TGT CAA GGA GAC AGT AGT GGT CCC ATG GTT 671 Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Ser Gly Pro Met Val 210 215 220
GAC AGC GAG GAT ACT CAA GTA GGA ATT GTA TCC TGG GGA ATA GGA TGT 719 Asp Ser Glu Asp Thr Gin Val Gly Ile Val Ser Trp Gly Ile Gly Cys 225 230 235
GCT AGA CCC AAT TTA CCA GGA GTT TAT ACG CGA ATT GCT TCA TCG CCA 767 Ala Arg Pro Asn Leu Pro Gly Val Tyr Thr Arg Ile Ala Ser Ser Pro 240 245 250 255 ATT AGA GAT TTC NTA AGA CGA ATA ACC GGA GTT TAATATTATT TTATACATTT 820 Ile Arg Asp Phe Xaa Arg Arg Ile Thr Gly Val 260 265
TTGACAAATA TGAGAACTAA TGAGAACTGT TGTATTGCTA TAATTCTTTG CAACATTGTG 880
CATGAATAAA TTATGAATAT AATTGTTAAA AAAAAAAAAA AAAA 924
(2) INFORMATION FOR SEQ ID NO:24: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ile Leu Leu Leu Val Leu Leu Ala Val Cys Phe Ala Ser Ala Lys Arg 1 5 10 15
Gly Pro Arg Lys His Val Arg Glu Thr Gin Lys Ser Leu Ala Ser Gly 20 25 30
Arg Ile Val Gly Gly Glu Ala Val Ser Ile Glu Xaa Tyr Gly Trp Gin 35 40 45
Val Ser Leu Gin Arg Phe Gly Ser His Phe Cys Gly Gly Ser Ile Ile 50 55 60 Ser Ser Xaa Trp Ile Leu Ser Ala Ala His Cys Phe Tyr Gly Thr Leu 65 70 75 80
Phe Pro Ile Gly Phe Ser Ala Arg Ala Gly Ser Ser Thr Val Asn Ser 85 90 95
Gly Gly Thr Val His Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn Tyr 100 105 110
Asp Ser Gin Ser Thr Asp Phe Asp Val Ser Val Val Arg Leu Leu Ser 115 120 125
Ser Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val Asp 130 135 140 Ser Gly Thr Asp Leu Pro Ala Gly Glu Met Val Thr Val Thr Gly Trp 145 150 155 160
Gly Arg Leu Ser Glu Asn Thr Ser Val Pro Ser Pro Ser Thr Leu Gin 165 170 175
Gly Val Thr Val Pro Val Val Ser Asn Ser Glu Cys Gin Gin Gin Leu 180 185 190
Gin Asn Gin Thr Ile Thr Asp Asn Met Phe Cys Ala Gly Glu Leu Glu 195 200 205
Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Ser Gly Pro Met Val Asp 210 215 220 Ser Glu Asp Thr Gin Val Gly Ile Val Ser Trp Gly Ile Gly Cys Ala 225 230 235 240
Arg Pro Asn Leu Pro Gly Val Tyr Thr Arg Ile Ala Ser Ser Pro Ile 245 250 255
Arg Asp Phe Xaa Arg Arg Ile Thr Gly Val 260 265
(2) INFORMATION FOR SEQ ID NO:25: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 924 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: ττττττττττ TTTTTTTAAC AATTANATTC ATAATTTATT CATGCACAAT GTTGCAAAGA 60
ATTATAGCAA NACAACAGTT CTCATTAGTT CTCATATTTG TCAAAAATGT ATAAAATAAT 120 ATTAAACTCC GGTTATTCGT CTTANGAAAT CTCTAATTGG CGATGAAGCA ATTCGCGTAT 180
AAACTCCTGG TAAATTGGGT CTAGCACATC CTATTCCCCA GGATACAATT CCTACTTGAG 240
TATCCTCGCT GTCAACCATG GGACCACTAC TGTCTCCTTG ACAAGAGTCC TTTCCTCCTT 300
CTAATTCACC AGCACAAAAC ATATTGTCAG TGATTGTCTG ATTTTGCAAT TGTTGTTGAC 360
ATTCCGAATT ACTTACAACT GGTACTGTAA CTCCTTGAAG AGTTGATGGC GAGGGAACAG 420 AAGTATTTTC CGAAAGTCGT CCCCATCCAG TTACTGTAAC CATCTCACCG GCTGGCAAAT 480
CAGTTCCAGA ATCCACTAAC CTAGCCGGTC GAATAGAACC TCCATTCAAA TTTAAAGAAG 540
ATAATAGTCG AACTACAGAA ACATCAAAGT CTGTACTTTG TGAATCATAA TTTGGATGAA 600
TATACCAATA CAAAATTGTA TGCACAGTTC CTCCTGAATT CACAGTACTG CTGCCGGCTC 660
TCGCAGAGAA TCCAATCGGA AATAACGTTC CATAAAAGCA ATGAGCAGCT GAAAGAATCC 720 ATNTACTGGA TATTATAGAT CCTCCACAGA AATGACTGCC AAAACGTTGT AGAGAAACTT 780
GCCATCCATA GTNTTCAATG CTCACTGCTT CACCACCCAC AATACGCCCA GAGGCAAGAC 840
TTTTTTGTGT TTCGCGAACA TGTTTTCGTG GACCTNGTTT AGCTGAAGCA AAGCATACTG 900
CCAATAATAC CAGTAATAAA ATTT 924
(2) INFORMATION FOR SEQ ID NO:26: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 699 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: DNA (genomic)
(IX) FEATURE:
(A) NAME /KEY: CDS
(B) LOCATION: 1..699
(D) OTHER INFORMATION: /note= "At pos. bp 31, change A to N; at pos. bp 101, change A to N; at pos. bp 679, change A to N. At pos. aa 11, 34 and 227, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATT GTG GGT GGT GAA GCA GTG AGC ATT GAA NAC TAT GGA TGG CAA GTT 48 He Val Gly Gly Glu Ala Val Ser Ile Glu Xaa Tyr Gly Trp Gin Val 1 5 10 15
TCT CTA CAA CGT TTT GGC AGT CAT TTC TGT GGA GGA TCT ATA ATA TCC 96 Ser Leu Gin Arg Phe Gly Ser His Phe Cys Gly Gly Ser Ile Ile Ser 20 25 30
AGT ANA TGG ATT CTT TCA GCT GCT CAT TGC TTT TAT GGA ACG TTA TTT 144 Ser Xaa Trp Ile Leu Ser Ala Ala His Cys Phe Tyr Gly Thr Leu Phe 35 40 45
CCG ATT GGA TTC TCT GCG AGA GCC GGC AGC AGT ACT GTG AAT TCA GGA 192 Pro Ile Gly Phe Ser Ala Arg Ala Gly Ser Ser Thr Val Asn Ser Gly 50 55 60
GGA ACT GTG CAT ACA ATT TTG TAT TGG TAT ATT CAT CCA AAT TAT GAT 240 Gly Thr Val His Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn Tyr Asp 65 70 75 80 TCA CAA AGT ACA GAC TTT GAT GTT TCT GTA GTT CGA CTA TTA TCT TCT 288 Ser Gin Ser Thr Asp Phe Asp Val Ser Val Val Arg Leu Leu Ser Ser 85 90 95
TTA AAT TTG AAT GGA GGT TCT ATT CGA CCG GCT AGG TTA GTG GAT TCT 336 Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val Asp Ser 100 105 110
GGA ACT GAT TTG CCA GCC GGT GAG ATG GTT ACA GTA ACT GGA TGG GGA 384 Gly Thr Asp Leu Pro Ala Gly Glu Met Val Thr Val Thr Gly Trp Gly 115 120 125
CGA CTT TCG GAA AAT ACT TCT GTT CCC TCG CCA TCA ACT CTT CAA GGA 432 Arg Leu Ser Glu Asn Thr Ser Val Pro Ser Pro Ser Thr Leu Gin Gly 130 135 140
GTT ACA GTA CCA GTT GTA AGT AAT TCG GAA TGT CAA CAA CAA TTG CAA 480 Val Thr Val Pro Val Val Ser Asn Ser Glu Cys Gin Gin Gin Leu Gin 145 150 155 160 AAT CAG ACA ATC ACT GAC AAT ATG TTT TGT GCT GGT GAA TTA GAA GGA 528 Asn Gin Thr Ile Thr Asp Asn Met Phe Cys Ala Gly Glu Leu Glu Gly 165 170 175
GGA AAG GAC TCT TGT CAA GGA GAC AGT AGT GGT CCC ATG GTT GAC AGC 576 Gly Lys Asp Ser Cys Gin Gly Asp Ser Ser Gly Pro Met Val Asp Ser 180 185 190
GAG GAT ACT CAA GTA GGA ATT GTA TCC TGG GGA ATA GGA TGT GCT AGA 624 Glu Asp Thr Gin Val Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Arg 195 200 205
CCC AAT TTA CCA GGA GTT TAT ACG CGA ATT GCT TCA TCG CCA ATT AGA 672 Pro Asn Leu Pro Gly Val Tyr Thr Arg Ile Ala Ser Ser Pro Ile Arg 210 215 220
GAT TTC NTA AGA CGA ATA ACC GGA GTT 699
Asp Phe Xaa Arg Arg Ile Thr Gly Val 225 230
(2) INFORMATION FOR SEQ ID NO:27: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 233 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Ile Val Gly Gly Glu Ala Val Ser Ile Glu Xaa Tyr Gly Trp Gin Val 1 5 10 15
Ser Leu Gin Arg Phe Gly Ser His Phe Cys Gly Gly Ser Ile Ile Ser 20 25 30
Ser Xaa Trp Ile Leu Ser Ala Ala His Cys Phe Tyr Gly Thr Leu Phe 35 40 45
Pro Ile Gly Phe Ser Ala Arg Ala Gly Ser Ser Thr Val Asn Ser Gly 50 55 60 Gly Thr Val His Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn Tyr Asp 65 70 75 80
Ser Gin Ser Thr Asp Phe Asp Val Ser Val Val Arg Leu Leu Ser Ser 85 90 95
Leu Asn Leu Asn Gly Gly Ser Ile Arg Pro Ala Arg Leu Val Asp Ser 100 105 110
Gly Thr Asp Leu Pro Ala Gly Glu Met Val Thr Val Thr Gly Trp Gly 115 120 125
Arg Leu Ser Glu Asn Thr Ser Val Pro Ser Pro Ser Thr Leu Gin Gly 130 135 140 Val Thr Val Pro Val Val Ser Asn Ser Glu Cys Gin Gin Gin Leu Gin 145 150 155 160
Asn Gin Thr Ile Thr Asp Asn Met Phe Cys Ala Gly Glu Leu Glu Gly 165 170 175
Gly Lys Asp Ser Cys Gin Gly Asp Ser Ser Gly Pro Met Val Asp Ser 180 185 190
Glu Asp Thr Gin Val Gly Ile Val Ser Trp Gly Ile Gly Cys Ala Arg 195 200 205
Pro Asn Leu Pro Gly Val Tyr Thr Arg Ile Ala Ser Ser Pro Ile Arg 210 215 220 Asp Phe Xaa Arg Arg Ile Thr Gly Val 225 230
(2) INFORMATION FOR SEQ ID NO:28:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 699 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
AACTCCGGTT ATTCGTCTTA NGAAATCTCT AATTGGCGAT GAAGCAATTC GCGTATAAAC 60
TCCTGGTAAA TTGGGTCTAG CACATCCTAT TCCCCAGGAT ACAATTCCTA CTTGAGTATC 120 CTCGCTGTCA ACCATGGGAC CACTACTGTC TCCTTGACAA GAGTCCTTTC CTCCTTCTAA 180
TTCACCAGCA CAAAACATAT TGTCAGTGAT TGTCTGATTT TGCAATTGTT GTTGACATTC 240
CGAATTACTT ACAACTGGTA CTGTAACTCC TTGAAGAGTT GATGGCGAGG GAACAGAAGT 300
ATTTTCCGAA AGTCGTCCCC ATCCAGTTAC TGTAACCATC TCACCGGCTG GCAAATCAGT 360
TCCAGAATCC ACTAACCTAG CCGGTCGAAT AGAACCTCCA TTCAAATTTA AAGAAGATAA 420 TAGTCGAACT ACAGAAACAT CAAAGTCTGT ACTTTGTGAA TCATAATTTG GATGAATATA 480
CCAATACAAA ATTGTATGCA CAGTTCCTCC TGAATTCACA GTACTGCTGC CGGCTCTCGC 540
AGAGAATCCA ATCGGAAATA ACGTTCCATA AAAGCAATGA GCAGCTGAAA GAATCCATNT 600
ACTGGATATT ATAGATCCTC CACAGAAATG ACTGCCAAAA CGTTGTAGAG AAACTTGCCA 660
TCCATAGTNT TCAATGCTCA CTGCTTCACC ACCCACAAT 699
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1894 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 335..1535 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
ACGCGACGGG CAGTCTCTTT CAGACCGCGG CCGAACGATG TTTTTGACGG TTTAATTTCA 60
ATTTTTGCAA CTTGTGACTT CGAATACACC CGTACGTGAC CTATCCACTT TACCATCGGC 120
CGACCGTGAA AGTTGTGTTT GTGCTATTGA AAATTTCGTG CTCAATAATA AATATTCAGT 180
TTTTTTGTCT AATCAGGATA TTTAAATTTA TGTGTACAAG TGTTAAACGC AATCTCGTCG 240 CTCTTCCTAA TTTTCTTGTT GCCAATGCTG GCCTCAGTGC ATCGGCCGAC GCAAATGTGC 300
TTGCAAAATA GAGAAATCCG GTGAAATCAC ACAT ATG TTA GCG ATC GTC CCG 352
Met Leu Ala Ile Val Pro 1 5
TCA AAC GGA GCG TTC GCA GAC CAT GCC AAC CTT GGT GGA GTT GAT GGT 400 Ser Asn Gly Ala Phe Ala Asp His Ala Asn Leu Gly Gly Val Asp Gly
10 15 20 CTT TCT GGT TTG ATT CTG GTC GCT GTT GCG ATA TCT TCG ATT GGA TAT 448 Leu Ser Gly Leu Ile Leu Val Ala Val Ala Ile Ser Ser Ile Gly Tyr 25 30 35
GCG GAC GCG GCG AAC GTT GCG CAG GAC GGA CAT CCG TCC AGC CAG CAA 496 Ala Asp Ala Ala Asn Val Ala Gin Asp Gly His Pro Ser Ser Gin Gin 40 45 50
GAG CAG GAG ATC CTG CTG CTG AAT GCC TTA GCT CGC AGG AAC GGA GCG 544 Glu Gin Glu lie Leu Leu Leu Asn Ala Leu Ala Arg Arg Asn Gly Ala 55 60 65 70 ACG GGG CAC CAA TTT GAC GTA GAT CAA GAT TCA ATT ATG GAT ATG CTA 592 Thr Gly His Gin Phe Asp Val Asp Gin Asp Ser Ile Met Asp Met Leu 75 80 85
GGA AGA ATG ATA CCT CAG ACT TGC CGG TAC AAA GGC GAA CGG TTC GAG 640 Gly Arg Met Ile Pro Gin Thr Cys Arg Tyr Lys Gly Glu Arg Phe Glu 90 95 100
TGC GGT TTG TCA ATT TCG TGC GTC CTG GGC GGC GGA AAA CCT CTT GAC 688 Cys Gly Leu Ser Ile Ser Cys Val Leu Gly Gly Gly Lys Pro Leu Asp 105 110 115
CTG TGC AGC GGC GGA ATG ATC TGG TCG TGC TGC GTC GAC AGG GAC ATT 736 Leu Cys Ser Gly Gly Met Ile Trp Ser Cys Cys Val Asp Arg Asp Ile 120 125 130
CGG CCT GAG CCG CAG CAC CAG GGC GCT CTG CAG AAC GCA ACT TGT GGA 784 Arg Pro Glu Pro Gin His Gin Gly Ala Leu Gin Asn Ala Thr Cys Gly 135 140 145 150 GAA TTG TAC ACG AGG TCT AAT AGA ATC GTA GGA GGT CAT TCA ACA GGA 832 Glu Leu Tyr Thr Arg Ser Asn Arg Ile Val Gly Gly His Ser Thr Gly 155 160 165
TTC GGG TCT CAT CCT TGG CAG GCG GCT TTG ATC AAA TCA GGA TTT TTG 880 Phe Gly Ser His Pro Trp Gin Ala Ala Leu Ile Lys Ser Gly Phe Leu 170 175 180
AGT AAA AAA TTA TCT TGC GGT GGC GCT TTA GTT AGC GAT CGA TGG GTT 928 Ser Lys Lys Leu Ser Cys Gly Gly Ala Leu Val Ser Asp Arg Trp Val 185 190 195
ATA ACT GCT GCA CAT TGC GTT GCC ACG ACA CCA AAT TCG AAC CTG AAG 976 He Thr Ala Ala His Cys Val Ala Thr Thr Pro Asn Ser Asn Leu Lys 200 205 210
GTG CGA TTG GGC GAA TGG GAC GTC CGC GAC CAC GAT GAG CGA CTG AAC 1024 Val Arg Leu Gly Glu Trp Asp Val Arg Asp His Asp Glu Arg Leu Asn 215 220 225 230 CAC GAG GAA TAC GCA ATC GAA CGC AAA GAA GTT CAT CCT TCA TAT TCA 1072 His Glu Glu Tyr Ala Ile Glu Arg Lys Glu Val His Pro Ser Tyr Ser 235 240 245
CCA ACC GAT TTC CGG AAT GAT GTA GCC TTA GTG AAA CTC GAT AGA ACT 1120 Pro Thr Asp Phe Arg Asn Asp Val Ala Leu Val Lys Leu Asp Arg Thr 250 255 260 GTT ATT TTC AAA CAA CAT ATT TTA CCT GTC TGC TTA CCT CAT AAG CAA 1168 Val Ile Phe Lys Gin His- lie Leu Pro Val Cys Leu Pro His Lys Gin 265 270 275
ATG AAA CTG GCT GGA AAA ATG GCA ACA GTC GCC GGA TGG GGA CGG ACG 1216 Met Lys Leu Ala Gly Lys Met Ala Thr Val Ala Gly Trp Gly Arg Thr 280 285 290
AGG CAC GGG CAG AGC ACT GTG CCG GCT GTC TTA CAA GAA GTC GAT GTC 1264 Arg His Gly Gin Ser Thr Val Pro Ala Val Leu Gin Glu Val Asp Val 295 300 305 310 GAG GTG ATT CCG AAT GAA AGA TGC CAG AGG TGG TTC CGT GCT GCG GGT 1312 Glu Val Ile Pro Asn Glu Arg Cys Gin Arg Trp Phe Arg Ala Ala Gly 315 320 325
CGA CGA GAA ACC ATT CAC GAT GTC TTT CTC TGC GCC GGA TAT AAA GAG 1360 Arg Arg Glu Thr Ile His Asp Val Phe Leu Cys Ala Gly Tyr Lys Glu 330 335 340
GGT GGT CGT GAT TCA TGC CAA GGT GAT TCT GGA GGT CCT CTA ATA ATG 1408 Gly Gly Arg Asp Ser Cys Gin Gly Asp Ser Gly Gly Pro Leu Ile Met 345 350 355
CAG ATT GAG GGT AGA AGG ACC CTT GTG GGT CTA GTT TCT TGG GGC ATT 1456 Gin Ile Glu Gly Arg Arg Thr Leu Val Gly Leu Val Ser Trp Gly Ile 360 365 370
GGA TGT GGT CGT GAG CAT TTA CCA GGC GTA TAT ACC AAT ATA CAA AAA 1504 Gly Cys Gly Arg Glu His Leu Pro Gly Val Tyr Thr Asn Ile Gin Lys 375 380 385 390 TTC ATA CCG TGG ATC GAC AAA GTA ATG GGA T AATTTTTATT CCATCGAGCT 1555 Phe Ile Pro Trp Ile Asp Lys Val Met Gly 395 400
TACCCAAAGT ATTTATTAAG TGTTAATCGA AAGTTCCAAT AATAAATTAA TTTAAAATTC 1615
TAAAGACGGG AATTTGAAAG ACCAAAAAGA CATACTTGTG ATTGTGTAAT TTTTATGATT 1675 AACTTTACAT CATCTGTGCT TAATTATTAA TTTGTATTAT TCTTGCAAAT ATTTCAAGAG 1735
TTACCGAAAA GTTTGCTAAT CGATAATGAT ATTTTAAGAA AAACAACTGC TGCTGATTCA 1795
GTCAATGTTA GAATAATTAT GTTTACTAAA TAATATTAAG TTCTGATTAG TAAATAAATA 1855
GCAAAATTAT CTAAATATAT ATAAAAAAAA AAAAAAAAA 1894
(2) INFORMATION FOR SEQ ID NO: 30: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(li) MOLECULE TYPE: protem (XI) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Leu Ala Ile Val Pro Ser Asn Gly Ala Phe Ala Asp His Ala Asn 1 5 10 15 Leu Gly Gly Val Asp Gly Leu Ser Gly Leu Ile Leu Val Ala Val Ala 20 - 25 30
Ile Ser Ser Ile Gly Tyr Ala Asp Ala Ala Asn Val Ala Gin Asp Gly 35 40 45 His Pro Ser Ser Gin Gin Glu Gin Glu Ile Leu Leu Leu Asn Ala Leu 50 55 60
Ala Arg Arg Asn Gly Ala Thr Gly His Gin Phe Asp Val Asp Gin Asp 65 70 75 80
Ser Ile Met Asp Met Leu Gly Arg Met Ile Pro Gin Thr Cys Arg Tyr 85 90 95
Lys Gly Glu Arg Phe Glu Cys Gly Leu Ser Ile Ser Cys Val Leu Gly 100 105 110
Gly Gly Lys Pro Leu Asp Leu Cys Ser Gly Gly Met Ile Trp Ser Cys 115 120 125 Cys Val Asp Arg Asp Ile Arg Pro Glu Pro Gin His Gin Gly Ala Leu 130 135 140
Gin Asn Ala Thr Cys Gly Glu Leu Tyr Thr Arg Ser Asn Arg Ile Val 145 150 155 160
Gly Gly His Ser Thr Gly Phe Gly Ser His Pro Trp Gin Ala Ala Leu 165 170 175
Ile Lys Ser Gly Phe Leu Ser Lys Lys Leu Ser Cys Gly Gly Ala Leu 180 185 190
Val Ser Asp Arg Trp Val Ile Thr Ala Ala His Cys Val Ala Thr Thr 195 200 205 Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp Val Arg Asp 210 215 220
His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu Arg Lys Glu 225 230 235 240
Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp Val Ala Leu 245 250 255
Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile Leu Pro Val 260 265 270
Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met Ala Thr Val 275 280 285 Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val Pro Ala Val 290 295 300
Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg Cys Gin Arg 305 310 315 320
Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp Val Phe Leu 325 330 335
Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser Cys Gin Gly Asp Ser 340 345 350 Gly Gly Pro Leu lie Met Gin Ile Glu Gly Arg Arg Thr Leu Val Gly 355 360 365
Leu Val Ser Trp Gly Ile Gly Cys Gly Arg Glu His Leu Pro Gly Val 370 375 380 Tyr Thr Asn Ile Gin Lys Phe Ile Pro Trp Ile Asp Lys Val Met Gly 385 390 395 400
(2) INFORMATION FOR SEQ ID NO:31:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1894 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( li ) MOLECULE TYPE : DNA ( genomic )
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 31 : ττττττττττ TTTTTTTATA TATATTTAGA TAATTTTGCT ATTTATTTAC TAATCAGAAC 60
TTAATATTAT TTAGTAAACA TAATTATTCT AACATTGACT GAATCAGCAG CAGTTGTTTT 120
TCTTAAAATA TCATTATCGA TTAGCAAACT TTTCGGTAAC TCTTGAAATA TTTGCAAGAA 180
TAATACAAAT TAATAATTAA GCACAGATGA TGTAAAGTTA ATCATAAAAA TTACACAATC 240
ACAAGTATGT CTTTTTGGTC TTTCAAATTC CCGTCTTTAG AATTTTAAAT TAATTTATTA 300 TTGGAACTTT CGATTAACAC TTAATAAATA CTTTGGGTAA GCTCGATGGA ATAAAAATTA 360
TCCCATTACT TTGTCGATCC ACGGTATGAA TTTTTGTATA TTGGTATATA CGCCTGGTAA 420
ATGCTCACGA CCACATCCAA TGCCCCAAGA AACTAGACCC ACAAGGGTCC TTCTACCCTC 480
AATCTGCATT ATTAGAGGAC CTCCAGAATC ACCTTGGCAT GAATCACGAC CACCCTCTTT 540
ATATCCGGCG CAGAGAAAGA CATCGTGAAT GGTTTCTCGT CGACCCGCAG CACGGAACCA 600 CCTCTGGCAT CTTTCATTCG GAATCACCTC GACATCGACT TCTTGTAAGA CAGCCGGCAC 660
AGTGCTCTGC CCGTGCCTCG TCCGTCCCCA TCCGGCGACT GTTGCCATTT TTCCAGCCAG 720
TTTCATTTGC TTATGAGGTA AGCAGACAGG TAAAATATGT TGTTTGAAAA TAACAGTTCT 780
ATCGAGTTTC ACTAAGGCTA CATCATTCCG GAAATCGGTT GGTGAATATG AAGGATGAAC 840
TTCTTTGCGT TCGATTGCGT ATTCCTCGTG GTTCAGTCGC TCATCGTGGT CGCGGACGTC 900 CCATTCGCCC AATCGCACCT TCAGGTTCGA ATTTGGTGTC GTGGCAACGC AATGTGCAGC 960
AGTTATAACC CATCGATCGC TAACTAAAGC GCCACCGCAA GATAATTTTT TACTCAAAAA 1020
TCCTGATTTG ATCAAAGCCG CCTGCCAAGG ATGAGACCCG AATCCTGTTG AATGACCTCC 1080
TACGATTCTA TTAGACCTCG TGTACAATTC TCCACAAGTT GCGTTCTGCA GAGCGCCCTG 1140
GTGCTGCGGC TCAGGCCGAA TGTCCCTGTC GACGCAGCAC GACCAGATCA TTCCGCCGCT 1200 GCACAGGTCA AGAGGTTTTC CGCCGCCCAG GACGCACGAA ATTGACAAAC CGCACTCGAA 1260 CCGTTCGCCT TTGTACCGGC AAGTCTGAGG TATCATTCTT CCTAGCATAT CCATAATTGA 1320
ATCTTGATCT ACGTCAAATT GGTGCCCCGT CGCTCCGTTC CTGCGAGCTA AGGCATTCAG 1380
CAGCAGGATC TCCTGCTCTT GCTGGCTGGA CGGATGTCCG TCCTGCGCAA CGTTCGCCGC 1440
GTCCGCATAT CCAATCGAAG ATATCGCAAC AGCGACCAGA ATCAAACCAG AAAGACCATC 1500 AACTCCACCA AGGTTGGCAT GGTCTGCGAA CGCTCCGTTT GACGGGACGA TCGCTAACAT 1560
ATGTGTGATT TCACCGGATT TCTCTATTTT GCAAGCACAT TTGCGTCGGC CGATGCACTG 1620
AGGCCAGHAT TGGCAACAAG AAAATTAGGA AGAGCGACGA GATTGCGTTT AACACTTGTA 1680
CACATAAATT TAAATATCCT GATTAGACAA AAAAACTGAA TATTTATTAT TGAGCACGAA 1740
ATTTTCAATA GCACAAACAC AACTTTCACG GTCGGCCGAT GGTAAAGTGG ATAGGTCACG 1800 TACGGGTGTA TTCGAAGTCA CAAGTTGCAA AAATTGAAAT TAAACCGTCA AAAACATCGT 1860
TCGGCCGCGG TCTGAAAGAG ACTGCCCGTC GCGT 1894
(2) INFORMATION FOR SEQ ID NO: 32:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..1200
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
ATG TTA GCG ATC GTC CCG TCA AAC GGA GCG TTC GCA GAC CAT GCC AAC 48 Met Leu Ala Ile Val Pro Ser Asn Gly Ala Phe Ala Asp His Ala Asn 1 5 10 15
CTT GGT GGA GTT GAT GGT CTT TCT GGT TTG ATT CTG GTC GCT GTT GCG 96 Leu Gly Gly Val Asp Gly Leu Ser Gly Leu Ile Leu Val Ala Val Ala 20 25 30
ATA TCT TCG ATT GGA TAT GCG GAC GCG GCG AAC GTT GCG CAG GAC GGA 144 Ile Ser Ser Ile Gly Tyr Ala Asp Ala Ala Asn Val Ala Gin Asp Gly 35 40 45
CAT CCG TCC AGC CAG CAA GAG CAG GAG ATC CTG CTG CTG AAT GCC TTA 192 His Pro Ser Ser Gin Gin Glu Gin Glu Ile Leu Leu Leu Asn Ala Leu 50 55 60 GCT CGC AGG AAC GGA GCG ACG GGG CAC CAA TTT GAC GTA GAT CAA GAT 240 Ala Arg Arg Asn Gly Ala Thr Gly His Gin Phe Asp Val Asp Gin Asp 65 70 75 80
TCA ATT ATG GAT ATG CTA GGA AGA ATG ATA CCT CAG ACT TGC CGG TAC 288 Ser Ile Met Asp Met Leu Gly Arg Met Ile Pro Gin Thr Cys Arg Tyr 85 90 95 AAA GGC GAA CGG TTC GAG TGC GGT TTG TCA ATT TCG TGC GTC CTG GGC 336 Lys Gly Glu Arg Phe Glu Cys Gly Leu Ser Ile Ser Cys Val Leu Gly 100 105 110
GGC GGA AAA CCT CTT GAC CTG TGC AGC GGC GGA ATG ATC TGG TCG TGC 384 Gly Gly Lys Pro Leu Asp Leu Cys Ser Gly Gly Met Ile Trp Ser Cys 115 120 125
TGC GTC GAC AGG GAC ATT CGG CCT GAG CCG CAG CAC CAG GGC GCT CTG 432 Cys Val Asp Arg Asp Ile Arg Pro Glu Pro Gin His Gin Gly Ala Leu 130 135 140 CAG AAC GCA ACT TGT GGA GAA TTG TAC ACG AGG TCT AAT AGA ATC GTA 480 Gin Asn Ala Thr Cys Gly Glu Leu Tyr Thr Arg Ser Asn Arg Ile Val 145 150 155 160
GGA GGT CAT TCA ACA GGA TTC GGG TCT CAT CCT TGG CAG GCG GCT TTG 528 Gly Gly His Ser Thr Gly Phe Gly Ser His Pro Trp Gin Ala Ala Leu 165 170 175
ATC AAA TCA GGA TTT TTG AGT AAA AAA TTA TCT TGC GGT GGC GCT TTA 576 Ile Lys Ser Gly Phe Leu Ser Lys Lys Leu Ser Cys Gly Gly Ala Leu 180 185 190
GTT AGC GAT CGA TGG GTT ATA ACT GCT GCA CAT TGC GTT GCC ACG ACA 624 Val Ser Asp Arg Trp Val Ile Thr Ala Ala His Cys Val Ala Thr Thr 195 200 205
CCA AAT TCG AAC CTG AAG GTG CGA TTG GGC GAA TGG GAC GTC CGC GAC 672 Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp Val Arg Asp 210 215 220 CAC GAT GAG CGA CTG AAC CAC GAG GAA TAC GCA ATC GAA CGC AAA GAA 720 His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu Arg Lys Glu 225 230 235 240
GTT CAT CCT TCA TAT TCA CCA ACC GAT TTC CGG AAT GAT GTA GCC TTA 768 Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp Val Ala Leu 245 250 255
GTG AAA CTC GAT AGA ACT GTT ATT TTC AAA CAA CAT ATT TTA CCT GTC 816 Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile Leu Pro Val 260 265 270
TGC TTA CCT CAT AAG CAA ATG AAA CTG GCT GGA AAA ATG GCA ACA GTC 864 Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met Ala Thr Val 275 280 285
GCC GGA TGG GGA CGG ACG AGG CAC GGG CAG AGC ACT GTG CCG GCT GTC 912 Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val Pro Ala Val 290 295 300 TTA CAA GAA GTC GAT GTC GAG GTG ATT CCG AAT GAA AGA TGC CAG AGG 960 Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg Cys Gin Arg 305 310 315 320
TGG TTC CGT GCT GCG GGT CGA CGA GAA ACC ATT CAC GAT GTC TTT CTC 1008 Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp Val Phe Leu 325 330 335
TGC GCC GGA TAT AAA GAG GGT GGT CGT GAT TCA TGC CAA GGT GAT TCT 1056 Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser Cys Gin Gly Asp Ser 340 345 350
GGA GGT CCT CTA ATA ATG CAG ATT GAG GGT AGA AGG ACC CTT GTG GGT 1104 Gly Gly Pro Leu Ile Met Gin Ile Glu Gly Arg Arg Thr Leu Val Gly 355 360 365
CTA GTT TCT TGG GGC ATT GGA TGT GGT CGT GAG CAT TTA CCA GGC GTA 1152 Leu Val Ser Trp Gly Ile Gly Cys Gly Arg Glu His Leu Pro Gly Val 370 375 380
TAT ACC AAT ATA CAA AAA TTC ATA CCG TGG ATC GAC AAA GTA ATG GGA 1200 Tyr Thr Asn Ile Gin Lys Phe Ile Pro Trp Ile Asp Lys Val Met Gly 385 390 395 400
(2) INFORMATION FOR SEQ ID NO:33:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 amino acids (B) TYPE: ammo acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Met Leu Ala Ile Val Pro Ser Asn Gly Ala Phe Ala Asp His Ala Asn 1 5 10 15
Leu Gly Gly Val Asp Gly Leu Ser Gly Leu Ile Leu Val Ala Val Ala 20 25 30
Ile Ser Ser Ile Gly Tyr Ala Asp Ala Ala Asn Val Ala Gin Asp Gly 35 40 45 His Pro Ser Ser Gin Gin Glu Gin Glu Ile Leu Leu Leu Asn Ala Leu 50 55 60
Ala Arg Arg Asn Gly Ala Thr Gly His Gin Phe Asp Val Asp Gin Asp 65 70 75 80
Ser Ile Met Asp Met Leu Gly Arg Met Ile Pro Gin Thr Cys Arg Tyr 85 90 95
Lys Gly Glu Arg Phe Glu Cys Gly Leu Ser Ile Ser Cys Val Leu Gly 100 105 110
Gly Gly Lys Pro Leu Asp Leu Cys Ser Gly Gly Met Ile Trp Ser Cys 115 120 125 Cys Val Asp Arg Asp Ile Arg Pro Glu Pro Gin His Gin Gly Ala Leu 130 135 140
Gin Asn Ala Thr Cys Gly Glu Leu Tyr Thr Arg Ser Asn Arg Ile Val 145 150 155 160
Gly Gly His Ser Thr Gly Phe Gly Ser His Pro Trp Gin Ala Ala Leu 165 170 175
Ile Lys Ser Gly Phe Leu Ser Lys Lys Leu Ser Cys Gly Gly Ala Leu 180 185 190 Val Ser Asp Arg Trp Val Ile Thr Ala Ala His Cys Val Ala Thr Thr 195 - 200 205
Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp Val Arg Asp 210 215 220 His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu Arg Lys Glu 225 230 235 240
Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp Val Ala Leu 245 250 255
Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile Leu Pro Val 260 265 270
Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met Ala Thr Val 275 280 285
Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val Pro Ala Val 290 295 300 Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg Cys Gin Arg 305 310 315 320
Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp Val Phe Leu 325 330 335
Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser Cys Gin Gly Asp Ser 340 345 350
Gly Gly Pro Leu Ile Met Gin Ile Glu Gly Arg Arg Thr Leu Val Gly 355 360 365
Leu Val Ser Trp Gly Ile Gly Cys Gly Arg Glu His Leu Pro Gly Val 370 375 380 Tyr Thr Asn Ile Gin Lys Phe Ile Pro Trp Ile Asp Lys Val Met Gly 385 390 395 400
(2) INFORMATION FOR SEQ ID NO: 34:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1200 base pairs (B) TYPE: nucleic acid
( C ) STRANDEDNESS : single
( D ) TOPOLOGY : linear
( ii ) MOLECULE TYPE : DNA ( genomic )
( xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 34 : TCCCATTACT TTGTCGATCC ACGGTATGAA TTTTTGTATA TTGGTATATA CGCCTGGTAA 60
ATGCTCACGA CCACATCCAA TGCCCCAAGA AACTAGACCC ACAAGGGTCC TTCTACCCTC 120
AATCTGCATT ATTAGAGGAC CTCCAGAATC ACCTTGGCAT GAATCACGAC CACCCTCTTT 180
ATATCCGGCG CAGAGAAAGA CATCGTGAAT GGTTTCTCGT CGACCCGCAG CACGGAACCA 240
CCTCTGGCAT CTTTCATTCG GAATCACCTC GACATCGACT TCTTGTAAGA CAGCCGGCAC 300 AGTGCTCTGC CCGTGCCTCG TCCGTCCCCA TCCGGCGACT GTTGCCATTT TTCCAGCCAG 360 TTTCATTTGC TTATGAGGTA AGCAGACAGG TAAAATATGT TGTTTGAAAA TAACAGTTCT 420
ATCGAGTTTC ACTAAGGCTA CATCATTCCG GAAATCGGTT GGTGAATATG AAGGATGAAC 480
TTCTTTGCGT TCGATTGCGT ATTCCTCGTG GTTCAGTCGC TCATCGTGGT CGCGGACGTC 540
CCATTCGCCC AATCGCACCT TCAGGTTCGA ATTTGGTGTC GTGGCAACGC AATGTGCAGC 600 AGTTATAACC CATCGATCGC TAACTAAAGC GCCACCGCAA GATAATTTTT TACTCAAAAA 660
TCCTGATTTG ATCAAAGCCG CCTGCCAAGG ATGAGACCCG AATCCTGTTG AATGACCTCC 720
TACGATTCTA TTAGACCTCG TGTACAATTC TCCACAAGTT GCGTTCTGCA GAGCGCCCTG 780
GTGCTGCGGC TCAGGCCGAA TGTCCCTGTC GACGCAGCAC GACCAGATCA TTCCGCCGCT 840
GCACAGGTCA AGAGGTTTTC CGCCGCCCAG GACGCACGAA ATTGACAAAC CGCACTCGAA 900 CCGTTCGCCT TTGTACCGGC AAGTCTGAGG TATCATTCTT CCTAGCATAT CCATAATTGA 960
ATCTTGATCT ACGTCAAATT GGTGCCCCGT CGCTCCGTTC CTGCGAGCTA AGGCATTCAG 1020
CAGCAGGATC TCCTGCTCTT GCTGGCTGGA CGGATGTCCG TCCTGCGCAA CGTTCGCCGC 1080
GTCCGCATAT CCAATCGAAG ATATCGCAAC AGCGACCAGA ATCAAACCAG AAAGACCATC 1140
AACTCCACCA AGGTTGGCAT GGTCTGCGAA CGCTCCGTTT GACGGGACGA TCGCTAACAT 1200
( 2 ) INFORMATION FOR SEQ ID NO : 35 :
( 1 ) SEQUENCE CHARACTERI STICS :
(A) LENGTH : 72 6 base pai rs
( B ) TYPE : nucl eic acid
( C ) STRANDEDNESS : single ( D ) TOPOLOGY : linear
( il ) MOLECULE TYPE : cDNA
( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..726 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
ATC GTA GGA GGT CAT TCA ACA GGA TTC GGG TCT CAT CCT TGG CAG GCG 48 Ile Val Gly Gly His Ser Thr Gly Phe Gly Ser His Pro Trp Gin Ala 1 5 10 15
GCT TTG ATC AAA TCA GGA TTT TTG AGT AAA AAA TTA TCT TGC GGT GGC 96 Ala Leu Ile Lys Ser Gly Phe Leu Ser Lys Lys Leu Ser Cys Gly Gly
20 25 30
GCT TTA GTT AGC GAT CGA TGG GTT ATA ACT GCT GCA CAT TGC GTT GCC 144 Ala Leu Val Ser Asp Arg Trp Val Ile Thr Ala Ala His Cys Val Ala 35 40 45 ACG ACA CCA AAT TCG AAC CTG AAG GTG CGA TTG GGC GAA TGG GAC GTC 192 Thr Thr Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp Val 50 55 60
CGC GAC CAC GAT GAG CGA CTG AAC CAC GAG GAA TAC GCA ATC GAA CGC 240 Arg Asp His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu Arg 65 70" 75 80
AAA GAA GTT CAT CCT TCA TAT TCA CCA ACC GAT TTC CGG AAT GAT GTA 288 Lys Glu Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp Val 85 90 95
GCC TTA GTG AAA CTC GAT AGA ACT GTT ATT TTC AAA CAA CAT ATT TTA 336 Ala Leu Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile Leu 100 105 110
CCT GTC TGC TTA CCT CAT AAG CAA ATG AAA CTG GCT GGA AAA ATG GCA 384 Pro Val Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met Ala 115 120 125
ACA GTC GCC GGA TGG GGA CGG ACG AGG CAC GGG CAG AGC ACT GTG CCG 432 Thr Val Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val Pro 130 135 140 GCT GTC TTA CAA GAA GTC GAT GTC GAG GTG ATT CCG AAT GAA AGA TGC 480 Ala Val Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg Cys 145 150 155 160
CAG AGG TGG TTC CGT GCT GCG GGT CGA CGA GAA ACC ATT CAC GAT GTC 528 Gin Arg Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp Val 165 170 175
TTT CTC TGC GCC GGA TAT AAA GAG GGT GGT CGT GAT TCA TGC CAA GGT 576 Phe Leu Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser Cys Gin Gly 180 " 185 190
GAT TCT GGA GGT CCT CTA ATA ATG CAG ATT GAG GGT AGA AGG ACC CTT 624 Asp Ser Gly Gly Pro Leu Ile Met Gin Ile Glu Gly Arg Arg Thr Leu 195 200 205
GTG GGT CTA GTT TCT TGG GGC ATT GGA TGT GGT CGT GAG CAT TTA CCA 672 Val Gly Leu Val Ser Trp Gly Ile Gly Cys Gly Arg Glu His Leu Pro 210 215 220 GGC GTA TAT ACC AAT ATA CAA AAA TTC ATA CCG TGG ATC GAC AAA GTA 720 Gly Val Tyr Thr Asn Ile Gin Lys Phe Ile Pro Trp Ile Asp Lys Val 225 230 235 240
ATG GGA 726
Met Gly
(2) INFORMATION FOR SEQ ID NO:36:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Ile Val Gly Gly His Ser Thr Gly Phe Gly Ser His Pro Trp Gin Ala 1 5 10 15 Ala Leu Ile Lys Ser Gly Phe Leu Ser Lys Lys Leu Ser Cys Gly Gly 20 25 30
Ala Leu Val Ser Asp Arg Trp Val Ile Thr Ala Ala His Cys Val Ala 35 40 45 Thr Thr Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp Val 50 55 60
Arg Asp His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu Arg 65 70 75 80
Lys Glu Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp Val 85 90 95
Ala Leu Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile Leu 100 105 110
Pro Val Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met Ala 115 120 125 Thr Val Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val Pro 130 135 140
Ala Val Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg Cys 145 150 155 160
Gin Arg Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp Val 165 170 175
Phe Leu Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser Cys Gin Gly 180 185 190
Asp Ser Gly Gly Pro Leu Ile Met Gin Ile Glu Gly Arg Arg Thr Leu 195 200 205 Val Gly Leu Val Ser Trp Gly Ile Gly Cys Gly Arg Glu His Leu Pro 210 215 220
Gly Val Tyr Thr Asn Ile Gin Lys Phe Ile Pro Trp Ile Asp Lys Val 225 230 235 240
Met Gly
(2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 428 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..428
(D) OTHER INFORMATION: /note= "At pos. bp 50, change A to N. At pos. aa 17, substitute Xaa." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GTA ACC AAA TAT GAA AAC GAT ATC GAA GGT TTA AAA GTT AGG GTT GGA 48 Val Thr Lys Tyr Glu Asn Asp Ile Glu Gly Leu Lys Val Arg Val Gly 1 5 10 15 ANC AAT GAG CAT AAC AAA GGT GGG CGT TTA TAC GAC ATT AAA GAA ATT 96 Xaa Asn Glu His Asn Lys Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile 20 25 30
AAA AAA CAT CCA AGA TAT AAC GAT CGA ACC AGA TAC GAT TTT GAT GTC 144 Lys Lys His Pro Arg Tyr Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val 35 40 45
GCT TTA TTA CGC ATT GCA AAG CCA ATT GCA TAC ACT GCT TGC ACT GTT 192 Ala Leu Leu Arg Ile Ala Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val 50 55 60
GTT CCT GTA GCA TTG GCA GAA ACT GGA AAA GAA GTT CCA GAA GGC GCA 240 Val Pro Val Ala Leu Ala Glu Thr Gly Lys Glu Val Pro Glu Gly Ala 65 70 75 80
CTC GTT AGT GTC ACA GGA TGG GGG GCT ACT ATG GTG GGC GGC CCA GCA 288 Leu Val Ser Val Thr Gly Trp Gly Ala Thr Met Val Gly Gly Pro Ala 85 90 95 TCA ACG CAT CTA AAA GGT GTT AAG GTT CCA ATC GTG TCA AAT GAA GAA 336 Ser Thr His Leu Lys Gly Val Lys Val Pro Ile Val Ser Asn Glu Glu 100 105 110
TGC AAC AAA AAT TAT ACC ATT CCT GGA GGT CTG GAT GAC AAA ATT TCA 384 Cys Asn Lys Asn Tyr Thr Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser 115 120 125
GAC AGC ATG TTT TGC GCT GGT TTC CCT GAA GGC GGA AAG GAC TC 428
Asp Ser Met Phe Cys Ala Gly Phe Pro Glu Gly Gly Lys Asp
130 135 140
(2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 142 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Val Thr Lys Tyr Glu Asn Asp Ile Glu Gly Leu Lys Val Arg Val Gly
1 5 10 15
Xaa Asn Glu His Asn Lys Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile 20 25 30 Lys Lys His Pro Arg Tyr Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val 35 40 45 Ala Leu Leu Arg lie Ala Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val 50 55 60
Val Pro Val Ala Leu Ala Glu Thr Gly Lys Glu Val Pro Glu Gly Ala 65 70 75 80 Leu Val Ser Val Thr Gly Trp Gly Ala Thr Met Val Gly Gly Pro Ala
85 90 95
Ser Thr His Leu Lys Gly Val Lys Val Pro Ile Val Ser Asn Glu Glu 100 105 110
Cys Asn Lys Asn Tyr Thr Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser 115 120 125
Asp Ser Met Phe Cys Ala Gly Phe Pro Glu Gly Gly Lys Asp 130 135 140
(2) INFORMATION FOR SEQ ID NO:39:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 428 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GAGTCCTTTC CGCCTTCAGG GAAACCAGCG CAAAACATGC TGTCTGAAAT TTTGTCATCC 60
AGACCTCCAG GAATGGTATA ATTTTTGTTG CATTCTTCAT TTGACACGAT TGGAACCTTA 120
ACACCTTTTA GATGCGTTGA TGCTGGGCCG CCCACCATAG TAGCCCCCCA TCCTGTGACA 180
CTAACGAGTG CGCCTTCTGG AACTTCTTTT CCAGTTTCTG CCAATGCTAC AGGAACAACA 240 GTGCAAGCAG TGTATGCAAT TGGCTTTGCA ATGCGTAATA AAGCGACATC AAAATCGTAT 300
CTGGTTCGAT CGTTATATCT TGGATGTTTT TTAATTTCTT TAATGTCGTA TAAACGCCCA 360
CCTTTGTTAT GCTCATTGNT TCCAACCCTA ACTTTTAAAC CTTCGATATC GTTTTCATAT 420
TTGGTTAC 428
(2) INFORMATION FOR SEQ ID NO: 40: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 841 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..728 (D) OTHER INFORMATION: /note= "At pos. bp 186, change A to N; at pos. bp 627, change A to N. At pos. aa 62 and 209, substitute Xaa."
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 40: A GAT CAT CGA ATA GTA GGA GGT GAA GAT GTA GAT ATT TCA ACT TGT 46
Asp His Arg Ile Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys 1 5 10 15
GGA TGG CAA GTT TCG TTT CAC AAT AGG AAA GGA CAT TTT TGT GGA GGG 94 Gly Trp Gin Val Ser Phe His Asn Arg Lys Gly His Phe Cys Gly Gly 20 25 30
TCC ATC ATT GGC AAA GAA TGG ATT CTA ACT GCT GCG CAT TGT GTA ACC 142 Ser lie Ile Gly Lys Glu Trp lie Leu Thr Ala Ala His Cys Val Thr 35 40 45
AAA TAT GAA AAC GAT ATC GAA GGT TTA AAA GTT AGG GTT GGA ANC AAT 190 Lys Tyr Glu Asn Asp Ile Glu Gly Leu Lys Val Arg Val Gly Xaa Asn 50 55 60
GAG CAT AAC AAA GGT GGG CGT TTA TAC GAC ATT AAA GAA ATT AAA AAA 238 Glu His Asn Lys Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile Lys Lys 65 70 75 CAT CCA AGA TAT AAC GAT CGA ACC AGA TAC GAT TTT GAT GTC GCT TTA 286 His Pro Arg Tyr Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val Ala Leu 80 85 90 95
TTA CGC ATT GCA AAG CCA ATT GCA TAC ACT GCT TGC ACT GTT GTT CCT 334 Leu Arg Ile Ala Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val Val Pro 100 105 110
GTA GCA TTG GCA GAA ACT GGA AAA GAA GTT CCA GAA GGC GCA CTC GTT 382 Val Ala Leu Ala Glu Thr Gly Lys Glu Val Pro Glu Gly Ala Leu Val 115 120 125
AGT GTC ACA GGA TGG GGG GCT ACT ATG GTG GGC GGC CCA GCA TCA ACG 430 Ser Val Thr Gly Trp Gly Ala Thr Met Val Gly Gly Pro Ala Ser Thr 130 135 140
CAT CTA AAA GGT GTT AAG GTT CCA ATC GTG TCA AAT GAA GAA TGC AAC 478 His Leu Lys Gly Val Lys Val Pro Ile Val Ser Asn Glu Glu Cys Asn 145 150 155 AAA AAT TAT ACC ATT CCT GGA GGT CTG GAT GAC AAA ATT TCA GAC AGC 526 Lys Asn Tyr Thr Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser Asp Ser 160 165 170 175
ATG TTT TGC GCT GGT TTC CCT GAA GGC GGA AAG GAC TCG TGT CAA GGA 574 Met Phe Cys Ala Gly Phe Pro Glu Gly Gly Lys Asp Ser Cys Gin Gly 180 185 190
GAT AGC GGT GGG CCT GTA GTG GAT GAA AAT AGG GTT CAG GTC GGA ATT 622 Asp Ser Gly Gly Pro Val Val Asp Glu Asn Arg Val Gin Val Gly Ile 195 200 205
GTG TNT TGG GGC GAA GGC TGT GCT TTA GCA GGA AAA CCA GGC GTT TAT 670 Val Xaa Trp Gly Glu Gly Cys Ala Leu Ala Gly Lys Pro Gly Val Tyr 210 215 220 GCA AAA GTT TCA CAT CCT GAC GTA AAA AGG TTT ATT GAA ACC GTA GCA 718 Ala Lys Val Ser His Pro Asp Val Lys Arg Phe Ile Glu Thr Val Ala 225 230 235
GGA ATC AAA T AAAATTTGTT AGAAAAAATG TAGACAAGTT GTATAAACTA 768 Gly Ile Lys 240
TCAATGAAAT TGTTTTATTT TTGGAAATAA AATATAATTT ATGAAAAAAA AAAAAAAAAA 828
AAAAAAAAAA AAA 841
(2) INFORMATION FOR SEQ ID NO: 41: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Asp His Arg Ile Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly 1 5 10 15
Trp Gin Val Ser Phe His Asn Arg Lys Gly His Phe Cys Gly Gly Ser 20 25 30 lie lie Gly Lys Glu Trp Ile Leu Thr Ala Ala His Cys Val Thr Lys 35 40 45
Tyr Glu Asn Asp Ile Glu Gly Leu Lys Val Arg Val Gly Xaa Asn Glu 50 55 60
His Asn Lys Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile Lys Lys His 65 70 75 80
Pro Arg Tyr Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val Ala Leu Leu 85 90 95
Arg Ile Ala Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val Val Pro Val 100 105 110 Ala Leu Ala Glu Thr Gly Lys Glu Val Pro Glu Gly Ala Leu Val Ser 115 120 125
Val Thr Gly Trp Gly Ala Thr Met Val Gly Gly Pro Ala Ser Thr His 130 135 140
Leu Lys Gly Val Lys Val Pro Ile Val Ser Asn Glu Glu Cys Asn Lys 145 150 155 160
Asn Tyr Thr Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser Asp Ser Met 165 170 175
Phe Cys Ala Gly Phe Pro Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp 180 185 190 Ser Gly Gly Pro Val Val Asp Glu Asn Arg Val Gin Val Gly Ile Val 195 200 205
Xaa Trp Gly Glu Gly Cys Ala Leu Ala Gly Lys Pro Gly Val Tyr Ala 210 215 220 Lys Val Ser His Pro Asp Val Lys Arg Phe Ile Glu Thr Val Ala Gly 225 230 235 240
Ile Lys
(2) INFORMATION FOR SEQ ID NO:42: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 841 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ( il ) MOLECULE TYPE : DNA ( genomic )
( xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 42 :
TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT CANAAATTAT ATTTTATTTC CAAAAATAAA 60
ACAATTTCAT TGANAGTTTA TACAACTTGT CTACATTTTT TCTAACAAAT TTTATTTGAT 120
TCCTGCTACG GTTTCAATAA ACCTTTTTAC GTCAGGATGT GAAACTTTTG CATAAACGCC 180 TGGTTTTCCT GCTAAAGCAC AGCCTTCGCC CCAANACACA ATTCCGACCT GAACCCTATT 240
TTCATCCACT ACAGGCCCAC CGCTATCTCC TTGACACGAG TCCTTTCCGC CTTCAGGGAA 300
ACCAGCGCAA AACATGCTGT CTGAAATTTT GTCATCCAGA CCTCCAGGAA TGGTATAATT 360
TTTGTTGCAT TCTTCATTTG ACACGATTGG AACCTTAACA CCTTTTAGAT GCGTTGATGC 420
TGGGCCGCCC ACCATAGTAG CCCCCCATCC TGTGACACTA ACGAGTGCGC CTTCTGGAAC 480 TTCTTTTCCA GTTTCTGCCA ATGCTACAGG AACAACAGTG CAAGCAGTGT ATGCAATTGG 540
CTTTGCAATG CGTAATAAAG CGACATCAAA ATCGTATCTG GTTCGATCGT TATATCTTGG 600
ATGTTTTTTA ATTTCTTTAA TGTCGTATAA ACGCCCACCT TTGTTATGCT CATTGNTTCC 660
AACCCTAACT TTTAAACCTT CGATATCGTT TTCATATTTG GTTACACAAT GCGCAGCAGT 720
TAGAATCCAT TCTTTGCCAA TGATGGACCC TCCACAAAAA TGTCCTTTCC TATTGTGAAA 780 CGAAACTTGC CATCCACAAG TTGAAATATC TACATCTTCA CCTCCTACTA TTCGATGATC 840
T 841
( 2 ) INFORMATION FOR SEQ ID NO : 43 :
( l ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 717 base pairs ( B ) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ( 11 ) MOLECULE TYPE : DNA ( genomic )
( ix ) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 1..717 (D) OTHER INFORMATION: /note= "At pos. bp 176, change A to
N; at pos. bp 617, change A to N. At pos. aa. 59 and 206, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
ATA GTA GGA GGT GAA GAT GTA GAT ATT TCA ACT TGT GGA TGG CAA GTT 48 He val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly Trp Gin Val
1 5 10 15
TCG TTT CAC AAT AGG AAA GGA CAT TTT TGT GGA GGG TCC ATC ATT GGC 96 Ser Phe His Asn Arg Lys Gly His Phe Cys Gly Gly Ser Ile Ile Gly 20 25 30 AAA GAA TGG ATT CTA ACT GCT GCG CAT TGT GTA ACC AAA TAT GAA AAC 144 Lys Glu Trp Ile Leu Thr Ala Ala His Cys Val Thr Lys Tyr Glu Asn 35 40 45
GAT ATC GAA GGT TTA AAA GTT AGG GTT GGA ANC AAT GAG CAT AAC AAA 192 Asp Ile Glu Gly Leu Lys Val Arg Val Gly Xaa Asn Glu His Asn Lys 50 55 60
GGT GGG CGT TTA TAC GAC ATT AAA GAA ATT AAA AAA CAT CCA AGA TAT 240 Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile Lys Lys His Pro Arg Tyr 65 70 75 80
AAC GAT CGA ACC AGA TAC GAT TTT GAT GTC GCT TTA TTA CGC ATT GCA 288 Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val Ala Leu Leu Arg Ile Ala
85 90 95
AAG CCA ATT GCA TAC ACT GCT TGC ACT GTT GTT CCT GTA GCA TTG GCA 336 Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val Val Pro Val Ala Leu Ala 100 105 110 GAA ACT GGA AAA GAA GTT CCA GAA GGC GCA CTC GTT AGT GTC ACA GGA 384 Glu Thr Gly Lys Glu Val Pro Glu Gly Ala Leu Val Ser Val Thr Gly 115 120 125
TGG GGG GCT ACT ATG GTG GGC GGC CCA GCA TCA ACG CAT CTA AAA GGT 432 Trp Gly Ala Thr Met Val Gly Gly Pro Ala Ser Thr His Leu Lys Gly 130 135 140
GTT AAG GTT CCA ATC GTG TCA AAT GAA GAA TGC AAC AAA AAT TAT ACC 480 Val Lys Val Pro Ile Val Ser Asn Glu Glu Cys Asn Lys Asn Tyr Thr
145 150 155 160
ATT CCT GGA GGT CTG GAT GAC AAA ATT TCA GAC AGC ATG TTT TGC GCT 528 Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser Asp Ser Met Phe Cys Ala
165 170 175
GGT TTC CCT GAA GGC GGA AAG GAC TCG TGT CAA GGA GAT AGC GGT GGG 576 Gly Phe Pro Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Gly Gly 180 185 190 CCT GTA GTG GAT GAA AAT AGG GTT CAG GTC GGA ATT GTG TNT TGG GGC 624 Pro Val Val Asp Glu Asn Arg Val Gin Val Gly Ile Val Xaa Trp Gly 195 200 205 GAA GGC TGT GCT TTA GCA GGA AAA CCA GGC GTT TAT GCA AAA GTT TCA 672 Glu Gly Cys Ala Leu Ala Gly Lys Pro Gly Val Tyr Ala Lys Val Ser
210 215 220
CAT CCT GAC GTA AAA AGG TTT ATT GAA ACC GTA GCA GGA ATC AAA 717 His Pro Asp Val Lys Arg Phe Ile Glu Thr Val Ala Gly Ile Lys 225 230 235
(2) INFORMATION FOR SEQ ID NO:44:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 ammo acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Ile Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly Trp Gin Val 1 5 10 15
Ser Phe His Asn Arg Lys Gly His Phe Cys Gly Gly Ser Ile Ile Gly 20 25 30
Lys Glu Trp Ile Leu Thr Ala Ala His Cys Val Thr Lys Tyr Glu Asn 35 40 45 Asp Ile Glu Gly Leu Lys Val Arg Val Gly Xaa Asn Glu His Asn Lys 50 55 60
Gly Gly Arg Leu Tyr Asp Ile Lys Glu Ile Lys Lys His Pro Arg Tyr 65 70 75 80
Asn Asp Arg Thr Arg Tyr Asp Phe Asp Val Ala Leu Leu Arg Ile Ala 85 90 95
Lys Pro Ile Ala Tyr Thr Ala Cys Thr Val Val Pro Val Ala Leu Ala 100 105 110
Glu Thr Gly Lys Glu Val Pro Glu Gly Ala Leu Val Ser Val Thr Gly 115 120 125 Trp Gly Ala Thr Met Val Gly Gly Pro Ala Ser Thr His Leu Lys Gly 130 135 140
Val Lys Val Pro Ile Val Ser Asn Glu Glu Cys Asn Lys Asn Tyr Thr 145 150 155 160
Ile Pro Gly Gly Leu Asp Asp Lys Ile Ser Asp Ser Met Phe Cys Ala 165 170 175
Gly Phe Pro Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Gly Gly 180 185 190
Pro Val Val Asp Glu Asn Arg Val Gin Val Gly Ile Val Xaa Trp Gly 195 200 205
Glu Gly Cys Ala Leu Ala Gly Lys Pro Gly Val Tyr Ala Lys Val Ser 210 215 220 His Pro Asp Val Lys Arg Phe Ile Glu Thr Val Ala Gly Ile Lys 225 230 235
(2) INFORMATION FOR SEQ ID NO: 45:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 717 base pairs
( B ) TYPE : nucleic acid
( C ) STRANDEDNESS : single
( D ) TOPOLOGY : linear
( il ) MOLECULE TYPE : DNA ( genomic ) ( xi ) SEQUENCE DESCRI PTION : SEQ ID NO : 45 :
TTTGATTCCT GCTACGGTTT CAATAAACCT TTTTACGTCA GGATGTGAAA CTTTTGCATA 60
AACGCCTGGT TTTCCTGCTA AAGCACAGCC TTCGCCCCAA NACACAATTC CGACCTGAAC 120
CCTATTTTCA TCCACTACAG GCCCACCGCT ATCTCCTTGA CACGAGTCCT TTCCGCCTTC 180
AGGGAAACCA GCGCAAAACA TGCTGTCTGA AATTTTGTCA TCCAGACCTC CAGGAATGGT 240 ATAATTTTTG TTGCATTCTT CATTTGACAC GATTGGAACC TTAACACCTT TTAGATGCGT 300
TGATGCTGGG CCGCCCACCA TAGTAGCCCC CCATCCTGTG ACACTAACGA GTGCGCCTTC 360
TGGAACTTCT TTTCCAGTTT CTGCCAATGC TACAGGAACA ACAGTGCAAG CAGTGTATGC 420
AATTGGCTTT GCAATGCGTA ATAAAGCGAC ATCAAAATCG TATCTGGTTC GATCGTTATA 480
TCTTGGATGT TTTTTAATTT CTTTAATGTC GTATAAACGC CCACCTTTGT TATGCTCATT 540 GNTTCCAACC CTAACTTTTA AACCTTCGAT ATCGTTTTCA TATTTGGTTA CACAATGCGC 600
AGCAGTTAGA ATCCATTCTT TGCCAATGAT GGACCCTCCA CAAAAATGTC CTTTCCTATT 660
GTGAAACGAA ACTTGCCATC CACAAGTTGA AATATCTACA TCTTCACCTC CTACTAT 7 17
( 2 ) INFORMATION FOR SEQ ID NO : 46 :
( 1 ) SEQUENCE CHARACTERISTI CS : (A) LENGTH : 20 base pai rs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:46: GGACAAACTG TTCATTGCAG 20
(2) INFORMATION FOR SEQ ID NO: 47:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
CCCTCATTTG TCGTAACTCC 20
(2) INFORMATION FOR SEQ ID NO: 48:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GGCTAGGTTA GTGGATTCTG G 21
(2) INFORMATION FOR SEQ ID NO: 49:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
GCAAATCAGT TCCAGAATCC ACTAACC 27
(2) INFORMATION FOR SEQ ID NO:50:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
CAGGGCGCTC TGCAGAACGC AAC 23
(2) INFORMATION FOR SEQ ID NO: 51: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:51:
ATTCCTCGTG GTTCAGTCGC TC 22
(2) INFORMATION FOR SEQ ID NO: 52:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 52 :
GGCAAGTTTC GTTTCACAAT AGG 23
(2) INFORMATION FOR SEQ ID NO:53:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
TCCAACCCTA ACTTTTAAAC CTTC 24
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(li) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
CACAGGATCC AATAATTTGT GGTCAAAATG C 31
(2) INFORMATION FOR SEQ ID NO: 55: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
AAAAAGAAAG CTTCTTTAAT TTTCTGACAT TGTCGTG 37
(2) INFORMATION FOR SEQ ID NO: 56:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
GCGGGATCCT ATTGTGGGTG GTGAAGCAGT G 31
(2) INFORMATION FOR SEQ ID NO: 57:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
GACGGTACCA TGTATAAAAT AATATTAAAC TCCGG 35
(2) INFORMATION FOR SEQ ID NO:58:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
CCGGGATCCT ATGTTAGCGA TCGTCCCGTC AAAC 34
(2) INFORMATION FOR SEQ ID NO: 59: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:59:
CCGGAATTCT TATCCCATTA CTTTGTCGAT CC 32
(2) INFORMATION FOR SEQ ID NO: 60:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
GCGGGATCCA ATAGTAGGAG GTGAAGATGT AG 32
(2) INFORMATION FOR SEQ ID NO: 61:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
CCGGAATTCT TCTAACAAAT TTTATTTGAT TCCTGC 36
(2) INFORMATION FOR SEQ ID NO: 62:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(li) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
GGATCCAATC GTTGGAGGTG AAGATG 26
(2) INFORMATION FOR SEQ ID NO: 63: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
GAATTCGAAA TCCACTTAAA CATTAGC 27
(2) INFORMATION FOR SEQ ID NO: 64:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
GATAAGGATC CGTTACCAGA TTCTTTCGAC TGG 33
(2) INFORMATION FOR SEQ ID NO: 65:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(Xi) SEQUENCE DESCRIPTION* SEQ ID NO: 65:
TTATCAAGCT TCCATTTACA TGCCGTAAAA ATC 33
(2) INFORMATION FOR SEQ ID NO: 66:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 923 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(IX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..802 (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
A TTA ACA ATG ATG AAA CTT TTG GTA GTT TTT GCG ATT TTC GCT CAA 46 Leu Thr Met Met Lys Leu Leu Val Val Phe Ala He Phe Ala Gin
1 5 10 15
ATC AGT TTT GTT TTT GGA AAT AAT GTA ACA GAA TTC GAT GAC CGA ATC 94 Ile Ser Phe Val Phe Gly Asn Asn Val Thr Glu Phe Asp Asp Arg Ile 20 25 30
GTT GGA GGT GAA GAT GTT GAT ATA TCA ACT TGT GGT TGG CAA ATT TCA 142 Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly Trp Gin He Ser 35 40 45
TTT CAA AGT GAA AAC CTT CAT TTT TGT GGA GGA TCA ATT ATT GCA CCA 190 Phe Gin Ser Glu Asn Leu His Phe Cys Gly Gly Ser Ile Ile Ala Pro 50 55 60
AAA TGG ATT CTA ACT GCT GCA CAC TGT GTT GAA TGG TTG AAA AAG CCG 238 Lys Trp Ile Leu Thr Ala Ala His Cys Val Glu Trp Leu Lys Lys Pre 65 70 75 CTC AAA GAC ATA ACC GTA CGT ATA GGA AGC AGT ATA CGT AAC AAA GGT 286 Leu Lys Asp Ile Thr Val Arg Ile Gly Ser Ser Ile Arg Asn Lys Gly 80 85 90 95
GGT CGA GTT CAT AAA GTA ATA GAT TTC CAC ATG CAT CCC TCG TAC AAT 334 Gly Arg Val His Lys Val Ile Asp Phe His Met His Pro Ser Tyr Asn 100 105 110
AAG AGG GCG GAT TAT GAT TTT GAC GTT GCT GTA CTA GAA CTT GAA AAA 382 Lys Arg Ala Asp Tyr Asp Phe Asp Val Ala Val Leu Glu Leu Glu Lys 115 * 120 125
CCA GTC TCA TAT ACG GTT TGT ACA GTA GTA TCA GTA GAT TTA GCC GAA 430 Pro Val Ser Tyr Thr Val Cys Thr Val Val Ser Val Asp Leu Ala Glu 130 135 140
AGT GGA ACT GAA GTT AAA CCT GGA GCA ATA CTT AGT GTC ACT GGA TGG 478 Ser Gly Thr Glu Val Lys Pro Gly Ala Ile Leu Ser Val Thr Gly Trp 145 150 155 GGT GCA ACT AAG GAA GGT GGT GGC GGA ACT TTG CAA CTA CAA GGT GTG 526 Gly Ala Thr Lys Glu Gly Gly Gly Gly Thr Leu Gin Leu Gin Gly Val 160 165 170 175
AAA GTT CCA GCT ATC TCT CCC AAA GAT TGT GCT AAG GGA TAT CCA CCT 574 Lys Val Pro Ala Ile Ser Pro Lys Asp Cys Ala Lys Gly Tyr Pro Pro 180 185 190
TCT GGA GGT AAA GAC AAA ATT ACA GAC AGC ATG TTA TGT GCT GGT CTT 622 Ser Gly Gly Lys Asp Lys Ile Thr Asp Ser Met Leu Cys Ala Gly Leu 195 200 205
CCT GAA GGA GGT AAA GAT TCC TGC CAA GGC GAC AGT GGC GGT CCA CTG 670 Pro Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Gly Gly Pro Leu 210 215 220
GTA GAT GAA AAT AGA AAG CAA GTA GGA GTG GTT TCT TGG GGT CAA GGA 718 Val Asp Glu Asn Arg Lys Gin Val Gly Val Val Ser Trp Gly Gin Gly 225 230 235 TGT GCC AGA CCA GGA AAA CCA GGA ATT TAT GCT AAA GTG TCA CAC CCC 766 Cys Ala Arg Pro Gly Lys Pro Gly He Tyr Ala Lys Val Ser His Pro 240 245 250 255 GAA ATC AGA AAA TTT ATT GAA AAA TAT GCT AAT GTT TAAGTGGATT 812
Glu Ile Arg Lys Phe Ile Glu Lys Tyr Ala Asn Val 260 265
TCATTTTCAA TATAATGTGA TTTAAGATAC TCTTTAATGT TATGATATGA ATTGTGATAA 872 ATTAAATAAT AAAGATTGAA GAAGTGATAA AAAAAAAAAA AAAAAAAAAA A 923
(2) INFORMATION FOR SEQ ID NO: 67:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Leu Thr Met Met Lys Leu Leu Val Val Phe Ala Ile Phe Ala Gin He 1 5 10 15 Ser Phe Val Phe Gly Asn Asn Val Thr Glu Phe Asp Asp Arg Ile Val
20 25 30
Gly Gly Glu Asp Val Asp lie Ser Thr Cys Gly Trp Gin Ile Ser Phe 35 40 45
Gin Ser Glu Asn Leu His Phe Cys Gly Gly Ser Ile Ile Ala Pro Lys 50 55 60
Trp Ile Leu Thr Ala Ala His Cys Val Glu Trp Leu Lys Lys Pro Leu 65 70 75 80
Lys Asp Ile Thr Val Arg Ile Gly Ser Ser He Arg Asn Lys Gly Gly 85 90 95 Arg Val His Lys Val Ile Asp Phe His Met His Pro Ser Tyr Asn Lys
100 105 110
Arg Ala Asp Tyr Asp Phe Asp Val Ala Val Leu Glu Leu Glu Lys Pro 115 120 125
Val Ser Tyr Thr Val Cys Thr Val Val Ser Val Asp Leu Ala Glu Ser 130 135 140
Gly Thr Glu Val Lys Pro Gly Ala Ile Leu Ser Val Thr Gly Trp Gly 145 150 155 160
Ala Thr Lys Glu Gly Gly Gly Gly Thr Leu Gin Leu Gin Gly Val Lys 165 170 175 Val Pro Ala Ile Ser Pro Lys Asp Cys Ala Lys Gly Tyr Pro Pro Ser
180 185 190
Gly Gly Lys Asp Lys Ile Thr Asp Ser Met Leu Cys Ala Gly Leu Pro 195 200 205
Glu Gly Gly Lys Asp Ser Cys Gin Gly Asp Ser Gly Gly Pro Leu Val 210 215 220 Asp Glu Asn Arg Lys Gin Val Gly Val Val Ser Trp Gly Gin Gly Cys 225 230 235 240
Ala Arg Pro Gly Lys Pro Gly Ile Tyr Ala Lys Val Ser His Pro Glu 245 250 255 He Arg Lys Phe Ile Glu Lys Tyr Ala Asn Val 260 265
(2) INFORMATION FOR SEQ ID NO: 68:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: Ile Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly Trp Cys 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 69:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: He Val Gly Gly Glu Asp Val Asp Ile Ser Thr Cys Gly Trp Gin Ile
1 5 10 15
Ser Phe Gin Ser Glu Asn Leu His Phe Cys Gly Gly Ser Ile Ile Ala 20 25 30
Pro Lys
(2) INFORMATION FOR SEQ ID NO:70:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: ammo acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: protem
(ix) FEATURE:
(A) NAME/KEY: Protem (B) LOCATION: 1..15 (D) OTHER INFORMATION: /note= "At pos. 1, Xaa = His or Arg; pos. 2, Val or Pro; pos. 3, Gly or Ala or Ser; pos. 4, Tyr or Gly; pos. 5, Glu or Asn; pos. 6, Asp or Lys; pos. 7, Val or Arg; pos. 8, Asp or Ala; pos. 13, Asp or Pro." (ix) FEATURE:
(A) NAME/KEY: Xaa = His or Arg
(B) LOCATION: 1
(ix) FEATURE:
(A) NAME/KEY: Xaa = Val or Pro (B) LOCATION: 2
(ix) FEATURE:
(A) NAME/KEY: Xaa = Gly or Ala or Ser
(B) LOCATION: 3
(ix) FEATURE: (A) NAME/KEY: Xaa = Tyr or Gly
(B) LOCATION: 4
(ix) FEATURE:
(A) NAME/KEY: Xaa = Glu or Asn
(B) LOCATION: 5 (ix) FEATURE:
(A) NAME/KEY: Xaa = Asp or Lys
(B) LOCATION: 6
(ix) FEATURE:
(A) NAME/KEY: Xaa = Val or Arg (B) LOCATION: 7
(IX) FEATURE:
(A) NAME/KEY: Xaa = Asp or Ala
(B) LOCATION: 8
(ix) FEATURE: (A) NAME/KEY: Xaa = Asp or Pro
(B) LOCATION: 13
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Tyr Asp Phe Xaa Val Ala 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protem
(B) LOCATION: 1..12 (D) OTHER INFORMATION: /note= "At pos. aa 1, Xaa = Ile or Gin; pos. 4, Tyr or Gly; pos. 5, Glu or Asn or Thr; pos. 6, Asp or Met or Pro; pos. 8, Lys or Asp; pos. 10, Asn or Ser; pos. 11, Met or Thr or Asn; pos. 12, Phe or Cys."
(ix) FEATURE: (A) NAME/KEY: Xaa = Ile or Gin
(B) LOCATION: 1
(ix) FEATURE:
(A) NAME/KEY: Xaa = Tyr or Gly
(B) LOCATION: 4 (ix) FEATURE:
(A) NAME/KEY: Xaa = Glu or Asn or Thr
(B) LOCATION: 5
(ix) FEATURE:
(A) NAME/KEY: Xaa = Asp or Met or Pro (B) LOCATION: 6
(ix) FEATURE:
(A) NAME/KEY: Xaa = Lys or Asp
(B) LOCATION: 8
(IX) FEATURE: (A) NAME/KEY: Xaa = Asn or Ser
(B) LOCATION: 10
(ix) FEATURE:
(A) NAME/KEY: Xaa = Met or Thr or Asn
(B) LOCATION: 11 (ix) FEATURE:
(A) NAME/KEY: Xaa = Phe or Cys
(B) LOCATION: 12
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:71:
Xaa Val Gly Xaa Xaa Xaa Val Xaa Ile Xaa Xaa Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO:72:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protem (B) LOCATION: 1..15
(D) OTHER INFORMATION: /note= "At pos. aa 1, Xaa = His or Arg; at pos. 3, Xaa = Ala or Ser."
(ix) FEATURE:
(A) NAME/KEY: Xaa = His or Arg (B) LOCATION: 1 (ix) FEATURE:
(A) NAME/KEY: "Xaa = Ala or Ser
(B) LOCATION: 3
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 72: Xaa Pro Xaa Tyr Asn Lys Arg Ala Asp Tyr Asp Phe Asp Val Ala 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 73:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH : 28 ammo acids ( B ) TYPE : ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein
(ix) FEATURE: (A) NAME/KEY: Protein
(B) LOCATION: 1..28
(D) OTHER INFORMATION: /note= "At pos. aa 1, Xaa = probably Cys . "
(ix) FEATURE: (A) NAME/KEY: Xaa = probably Cys
(B) LOCATION: 1
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Xaa Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile Phe Pro 1 5 10 15 Pro Lys Pro Lys Asp Asp Leu Leu Ile Lys Arg Lys
20 25
(2) INFORMATION FOR SEQ ID NO:74:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
CTTTCCTCAC AATACCACCA AGGAAGC 27
(2) INFORMATION FOR SEQ ID NO: 75:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
CTTGTACGAT TGTCTCAACA GGC 23
(2) INFORMATION FOR SEQ ID NO:76:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 573 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..573
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
CGA TCT TTT AGA CAA GCG AAA TTG ATA ACG AAG TTT TCG AAG TCG GAT 48 Arg Ser Phe Arg Gin Ala Lys Leu Ile Thr Lys Phe Ser Lys Ser Asp 1 5 10 15
GAA GTA AAA ACC TTG CGT TGG TTT CCC CGG TCC CAG GAT CAG GAA CAG 96 Glu Val Lys Thr Leu Arg Trp Phe Pro Arg Ser Gin Asp Gin Glu Gin
20 25 30
TTG CAC TTT ACC CCA ATG AGG GAA TTC GTG CAT CCC CAT TTT ACC GAA 144 Leu His Phe Thr Pro Met Arg Glu Phe Val His Pro His Phe Thr Glu 35 40 45
CAT ATT GAT GAA GAA TTC CAC CGA TTC ATC AAT AAA CAC GGA AAA ATT 192 His Ile Asp Glu Glu Phe His Arg Phe Ile Asn Lys His Gly Lys Ile 50 55 60
TAT AAT AAA AAT GAA GAA CAT CAT TTC CGC AAA GAA ATT TTC AGA CTA 240 Tyr Asn Lys Asn Glu Glu His His Phe Arg Lys Glu Ile Phe Arg Leu 65 70 75 80
AAC TTG AGG TAC ATT TTT TCT AAG AAT CGT GCA AAT TTG GGA TAC ACT 288 Asn Leu Arg Tyr Ile Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr 85 90 95
TTG ACT GTT AAC CAT TTG GCT GAT CGT ACT GAA GCT GAA CTT AAG GCT 336 Leu Thr Val Asn His Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala
100 105 110
TTG AGA GGA CAC AGA CCT TCC TCC GGT TAT AAT GGC GGT TTA CCC TTT 384 Leu Arg Gly His Arg Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe 115 120 125 CCT CAC AAT ACC ACC AAG GAA GCA AGA AAT TTA CCA GAT TCT TTC GAC 432 Pro His Asn Thr Thr Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp 130 135 140
TGG CGA ATT TAT GGA GCT GTT ACT CCA GTT AAA GAT CAA TCT GTT TGT 480 Trp Arg Ile Tyr Gly Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys 145 150 155 160
GGT TCC TGC TGG TCT TTC GGA ACA ATT GGA GCA ATC GAA GGT GCA TAT 528 Gly Ser Cys Trp Ser Phe Gly Thr He Gly Ala Ile Glu Gly Ala Tyr 165 170 175
TTC TTG AAA ACG GCG GTA ATC TGT ACG ATG TCT CAC AGC TTG ATG 573
Phe Leu Lys Thr Ala Val Ile Cys Thr Met Ser His Ser Leu Met 180 185 190
(2) INFORMATION FOR SEQ ID NO: 77: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 191 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
Arg Ser Phe Arg Gin Ala Lys Leu Ile Thr Lys Phe Ser Lys Ser Asp 1 5 10 15
Glu Val Lys Thr Leu Arg Trp Phe Pro Arg Ser Gin Asp Gin Glu Gin 20 25 30 Leu His Phe Thr Pro Met Arg Glu Phe Val His Pro His Phe Thr Glu 35 40 45
His Ile Asp Glu Glu Phe His Arg Phe Ile Asn Lys His Gly Lys Ile 50 55 60
Tyr Asn Lys Asn Glu Glu His His Phe Arg Lys Glu He Phe Arg Leu 65 70 75 80
Asn Leu Arg Tyr Ile Phe Ser Lys Asn Arg Ala Asn Leu Gly Tyr Thr 85 90 95
Leu Thr Val Asn His Leu Ala Asp Arg Thr Glu Ala Glu Leu Lys Ala 100 105 110 Leu Arg Gly His Arg Pro Ser Ser Gly Tyr Asn Gly Gly Leu Pro Phe 115 120 125
Pro His Asn Thr Thr Lys Glu Ala Arg Asn Leu Pro Asp Ser Phe Asp 130 135 140
Trp Arg Ile Tyr Gly Ala Val Thr Pro Val Lys Asp Gin Ser Val Cys 145 150 155 160
Gly Ser Cys Trp Ser Phe Gly Thr Ile Gly Ala Ile Glu Gly Ala Tyr 165 170 175
Phe Leu Lys Thr Ala Val He Cys Thr Met Ser His Ser Leu Met 180 185 190 (2) INFORMATION FOR SEQ "ID NO:78:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 410 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..408
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
TGG GTT GTT ACT GCT GCT CAT TGT TTG AGA GGC AAA GAC CAC CTC CTG 48 Trp Val Val Thr Ala Ala His Cys Leu Arg Gly Lys Asp His Leu Leu
1 5 10 15 GAC AAA CTG TTC ATT GCA GTC GGC CTG ACA AAT TTA GGT GAA GGA GGC 96 Asp Lys Leu Phe He Ala Val Gly Leu Thr Asn Leu Gly Glu Gly Gly 20 25 30
ACC GTG TAT CCT GTA GAA AAA GGC ATC ATG CAC GAA GAA TAT GAA CAT 144 Thr Val Tyr Pro Val Glu Lys Gly Ile Met His Glu Glu Tyr Glu His 35 40 45
TAT GAC ATA GTC AAC GAT ATT GCA CTA ATC AAA GTC AAA TCT CCG ATA 192 Tyr Asp Ile Val Asn Asp Ile Ala Leu Ile Lys Val Lys Ser Pro Ile 50 55 60
GAA TTC AAT GAA AAA GTA ACG ACT GTA AAA TTA GGT GAG GAT TAT GTT 240 Glu Phe Asn Glu Lys Val Thr Thr Val Lys Leu Gly Glu Asp Tyr Val 65 70 75 80
GGC GGA GAC GTC CAA CTT CGA TTG ACA GGA TGG GGA GTT ACG ACA AAT 288 Gly Gly Asp Val Gin Leu Arg Leu Thr Gly Trp Gly Val Thr Thr Asn 85 90 95 GAG GGA ATC GGA AGC CCG AGT CAA AAA TTA CAG GTC ATG ACA GCC AAA 336 Glu Gly Ile Gly Ser Pro Ser Gin Lys Leu Gin Val Met Thr Ala Lys 100 105 110
TCA CTA ACT TAT GAG GAT TGC AAA AAC GCA ATT TAT AAA AAA GAC TTT 384 Ser Leu Thr Tyr Glu Asp Cys Lys Asn Ala Ile Tyr Lys Lys Asp Phe 115 120 125
CGA AAG CCA AAT TTG TGC ACA GGC TA 410
Arg Lys Pro Asn Leu Cys Thr Gly 130 135
(2) INFORMATION FOR SEQ ID NO:79: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
Trp Val Val Thr Ala Ala His Cys Leu Arg Gly Lys Asp His Leu Leu
1 5 10 15
Asp Lys Leu Phe Ile Ala Val Gly Leu Thr Asn Leu Gly Glu Gly Gly 20 25 30
Thr Val Tyr Pro Val Glu Lys Gly Ile Met His Glu Glu Tyr Glu His 35 40 45
Tyr Asp He Val Asn Asp Ile Ala Leu Ile Lys Val Lys Ser Pro lie 50 55 60 Glu Phe Asn Glu Lys Val Thr Thr Val Lys Leu Gly Glu Asp Tyr Val 65 70 75 80
Gly Gly Asp Val Gin Leu Arg Leu Thr Gly Trp Gly Val Thr Thr Asn 85 90 95
Glu Gly Ile Gly Ser Pro Ser Gin Lys Leu Gin Val Met Thr Ala Lys 100 105 110
Ser Leu Thr Tyr Glu Asp Cys Lys Asn Ala He Tyr Lys Lys Asp Phe 115 120 125
Arg Lys Pro Asn Leu Cys Thr Gly 130 135
(2) INFORMATION FOR SEQ ID NO: 80:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 433 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle (D) TOPOLOGY: linear
(11) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..432 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
GTA ACT GCT GCA CAT TGC TTT TAT GGA ACG TTA TTT CCG ATT GGA TTC 48 Val Thr Ala Ala His Cys Phe Tyr Gly Thr Leu Phe Pro Ile Gly Phe 1 5 10 15
TCT GCG AGA GCC GGC AGC AGT ACT GTG AAT TCA GGA GGA ACT GTG CAT 96 Ser Ala Arg Ala Gly Ser Ser Thr Val Asn Ser Gly Gly Thr Val His
20 25 30
ACA ATT TTG TAT TGG TAT ATT CAT CCA AAT TAT GAT TCA CAA AGT ACA 144 Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn Tyr Asp Ser Gin Ser Thr 35 40 45 GAC TTT GAT GTT TCT GTA GTT CGA CTA TTA TCT TCT TTA AAT TTG AAT 192 Asp Phe Asp Val Ser Val Val Arg Leu Leu Ser Ser Leu Asn Leu Asn 50 55 60
GGA GGT TCT ATT CGA CCG GCT AGG TTA GTG GAT TCT GGA ACT GAT TTG 240 Gly Gly Ser Ile Arg Pro Ala Arg Leu Val Asp Ser Gly Thr Asp Leu 65 70 75 80
CCA GCC GGT GAG ATG GTT ACA GTA ACT GGA TGG GGA CGA CTT TCG GAA 288 Pro Ala Gly Glu Met Val Thr Val Thr Gly Trp Gly Arg Leu Ser Glu 85 90 95
AAT ACT TCT GTT CCC TCG CCA TCA ACT CTT CAA GGA GTT ACA GTA CCA 336 Asn Thr Ser Val Pro Ser Pro Ser Thr Leu Gin Gly Val Thr Val Pro
100 105 110
GTT GTA AGT AAT TCG GAA TGT CAA CAA CAA TTG CAA AAT CAG ACA ATC 384 Val Val Ser Asn Ser Glu Cys Gin Gin Gin Leu Gin Asn Gin Thr Ile 115 120 125 ACT GAC AAT ATG TTT TGT GCT GGT GAA TTA GAA GGA GGA AAG GAC TCT 432 Thr Asp Asn Met Phe Cys Ala Gly Glu Leu Glu Gly Gly Lys Asp Ser 130 135 140
T 433
(2) INFORMATION FOR SEQ ID NO:81: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 144 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
Val Thr Ala Ala His Cys Phe Tyr Gly Thr Leu Phe Pro Ile Gly Phe 1 5 10 15
Ser Ala Arg Ala Gly Ser Ser Thr Val Asn Ser Gly Gly Thr Val His 20 25 30 Thr Ile Leu Tyr Trp Tyr Ile His Pro Asn Tyr Asp Ser Gin Ser Thr 35 40 45
Asp Phe Asp Val Ser Val Val Arg Leu Leu Ser Ser Leu Asn Leu Asn 50 55 60
Gly Gly Ser Ile Arg Pro Ala Arg Leu Val Asp Ser Gly Thr Asp Leu 65 70 75 80
Pro Ala Gly Glu Met Val Thr Val Thr Gly Trp Gly Arg Leu Ser Glu 85 90 95
Asn Thr Ser Val Pro Ser Pro Ser Thr Leu Gin Gly Val Thr Val Pro 100 105 110 Val Val Ser Asn Ser Glu Cys Gin Gin Gin Leu Gin Asn Gin Thr Ile 115 120 125
Thr Asp Asn Met Phe Cys Ala Gly Glu Leu Glu Gly Gly Lys Asp Ser 130 135 140 (2) INFORMATION FOR SEQ ID NO: 82:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 426 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..426
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
GCC ACG ACA CCA AAT TCG AAC CTG AAG GTG CGT TTG GGC GAA TGG GAC 48
Ala Thr Thr Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp 1 5 10 15 GTT CGC GAC CAC GAT GAG CGA CTG AAC CAC GAG GAA TAC GCA ATC GAA 96 Val Arg Asp His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu 20 25 30
CGC AAA GAA GTT CAT CCT TCA TAT TCA CCA ACC GAT TTC CGG AAT GAT 144 Arg Lys Glu Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp 35 40 45
GTA GCC TTA GTG AAA CTC GAT AGA ACT GTT ATT TTC AAA CAA CAT ATT 192 Val Ala Leu Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile 50 55 60
TTA CCT GTC TGC TTA CCT CAT AAG CAA ATG AAA CTG GCT GGA AAA ATG 240 Leu Pro Val Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met 65 70 75 80
GCA ACA GTC GCC GGA TGG GGA CGG ACG AGG CAC GGG CAG AGC ACT GTG 288 Ala Thr Val Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val 85 90 95 CCG GCT GTC TTA CAA GAA GTC GAT GTC GAG GTG ATT CCG AAT GAA AGA 336 Pro Ala Val Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg 100 105 110
TGC CAG AGG TGG TTC CGT GCT GCG GGT CGA CGA GAA ACC ATT CAC GAT 384 Cys Gin Arg Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr Ile His Asp 115 120 125
GTC TTT CTC TGC GCC GGA TAT AAA GAG GGT GGT CGT GAT TCA 426
Val Phe Leu Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser 130 135 140
(2) INFORMATION FOR SEQ ID NO: 83: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 142 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
Ala Thr Thr Pro Asn Ser Asn Leu Lys Val Arg Leu Gly Glu Trp Asp 1 5 10 15
Val Arg Asp His Asp Glu Arg Leu Asn His Glu Glu Tyr Ala Ile Glu 20 25 30
Arg Lys Glu Val His Pro Ser Tyr Ser Pro Thr Asp Phe Arg Asn Asp 35 40 45
Val Ala Leu Val Lys Leu Asp Arg Thr Val Ile Phe Lys Gin His Ile 50 55 60 Leu Pro Val Cys Leu Pro His Lys Gin Met Lys Leu Ala Gly Lys Met 65 70 75 80
Ala Thr Val Ala Gly Trp Gly Arg Thr Arg His Gly Gin Ser Thr Val 85 90 95
Pro Ala Val Leu Gin Glu Val Asp Val Glu Val Ile Pro Asn Glu Arg 100 105 110
Cys Gin Arg Trp Phe Arg Ala Ala Gly Arg Arg Glu Thr He His Asp 115 120 125
Val Phe Leu Cys Ala Gly Tyr Lys Glu Gly Gly Arg Asp Ser 130 135 140
(2) INFORMATION FOR SEQ ID NO: 84:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 778 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(li) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..778 (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
T GAT ACC TCA GAA TTG CCG GTA CAA AGG CGA ACG GTT TCG AGT GCG 46
Asp Thr Ser Glu Leu Pro Val Gin Arg Arg Thr Val Ser Ser Ala 1 5 10 15
GTT TGT CAA TTT TCG TGC GTC CTG GGC GGC GGA AAA CCT CTT GAC CTG 94 Val Cys Gin Phe Ser Cys Val Leu Gly Gly Gly Lys Pro Leu Asp Leu
20 25 30
TGC AGC GGC GGA ATG ATC TGG TCG TGC TGC GTC GAC AGG GAC ATT CGG 142 Cys Ser Gly Gly Met Ile Trp Ser Cys Cys Val Asp Arg Asp He Arg 35 40 45 CCT GAG CCG CAG CAC CAG GGC GCT CTG CAG AAC GCA ACT TGT GGA GAA 190 Pro Glu Pro Gin His Gin Gly Ala Leu Gin Asn Ala Thr Cys Gly Glu 50 55 60
TTG TAC ACG AGG TCT AAT AGA ATC GTA GGA GGT CAT TCA ACA GGA TTC 238 Leu Tyr Thr Arg Ser Asn Arg Ile Val Gly Gly His Ser Thr Gly Phe 65 70 75
GGG TCT CAT CCT TGG CAG GCG GCT TTG ATC AAA TCA GGA TTT TTG AGT 286 Gly Ser His Pro Trp Gin Ala Ala Leu He Lys Ser Gly Phe Leu Ser 80 85 90 95
AAA AAA TTA TCT TGC GGT GGC GCT TTA GTT AGC GAT CGA TGG GTT ATA 334 Lys Lys Leu Ser Cys Gly Gly Ala Leu Val Ser Asp Arg Trp Val Ile 100 105 110
ACT GCT GCA CAT TGC GTT GCC ACG ACA CCA AAT TCG AAC CTG AAG GTG 382 Thr Ala Ala His Cys Val Ala Thr Thr Pro Asn Ser Asn Leu Lys Val
115 120 125
CGA TTG GGC GAA TGG GAC GTC CGC GAC CAC GAT GAG CGA CTG AAC CAC 430 Arg Leu Gly Glu Trp Asp Val Arg Asp His Asp Glu Arg Leu Asn His 130 135 140 GAG GAA TAC GCA ATC GAA CGC AAA GAA GTT CAT CCT TCA TAT TCA CCA 478 Glu Glu Tyr Ala Ile Glu Arg Lys Glu Val His Pro Ser Tyr Ser Pro 145 150 155
ACC GAT TTC CGG AAT GAT GTA GCC TTA GTG AAA CTC GAT AGA ACT GTT 526 Thr Asp Phe Arg Asn Asp Val Ala Leu Val Lys Leu Asp Arg Thr Val 160 165 170 175
ATT TTC AAA CAA CAT ATT TTA CCT GTC TGC TTA CCT CAT AAG CAA ATG 574 He Phe Lys Gin His Ile Leu Pro Val Cys Leu Pro His Lys Gin Met 180 . 185 190
AAA CTG GCT GGA AAA ATG GCA ACA GTC GCC GGA TGG GGA CGG ACG AGG 622 Lys Leu Ala Gly Lys Met Ala Thr Val Ala Gly Trp Gly Arg Thr Arg
195 200 205
CAC GGG CAG AGC ACT GTG CCG GCT GTC TTA CAA GAA GTC GAT GTC GAG 670 His Gly Gin Ser Thr Val Pro Ala Val Leu Gin Glu Val Asp Val Glu 210 215 220 GTG ATT CCG AAT GAA AGA TGC CAG AGG TGG TTC CGT GCT GCG GGT CGA 718 Val He Pro Asn Glu Arg Cys Gin Arg Trp Phe Arg Ala Ala Gly Arg 225 230 235
CGA GAA ACC ATT CAC GAT GTC TTT CTC TGC GCC GGA TAT AAA GAG GGT 766 Arg Glu Thr He His Asp Val Phe Leu Cys Ala Gly Tyr Lys Glu Gly 240 245 250 255
GGT CGT GAT TCA 778
Gly Arg Asp Ser
(2) INFORMATION FOR SEQ ID NO:85: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 259 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: Asp Thr Ser Glu Leu Pro Val Gin Arg Arg Thr Val Ser Ser Ala Val
1 5 10 15
Cys Gin Phe Ser Cys Val Leu Gly Gly Gly Lys Pro Leu Asp Leu Cys 20 25 30 Ser Gly Gly Met Ile Trp Ser Cys Cys Val Asp Arg Asp Ile Arg Pro 35 40 45
Glu Pro Gin His Gin Gly Ala Leu Gin Asn Ala Thr Cys Gly Glu Leu 50 55 60
Tyr Thr Arg Ser Asn Arg Ile Val Gly Gly His Ser Thr Gly Phe Gly 65 70 75 80
Ser His Pro Trp Gin Ala Ala Leu Ile Lys Ser Gly Phe Leu Ser Lys 85 90 95
Lys Leu Ser Cys Gly Gly Ala Leu Val Ser Asp Arg Trp Val Ile Thr 100 105 110 Ala Ala His Cys Val Ala Thr Thr Pro Asn Ser Asn Leu Lys Val Arg 115 120 125
Leu Gly Glu Trp Asp Val Arg Asp His Asp Glu Arg Leu Asn His Glu 130 135 140
Glu Tyr Ala Ile Glu Arg Lys Glu Val His Pro Ser Tyr Ser Pro Thr 145 150 155 160
Asp Phe Arg Asn Asp Val Ala Leu Val Lys Leu Asp Arg Thr Val Ile 165 170 175
Phe Lys Gin His Ile Leu Pro Val Cys Leu Pro His Lys Gin Met Lys 180 185 190 Leu Ala Gly Lys Met Ala Thr Val Ala Gly Trp Gly Arg Thr Arg His 195 200 205
Gly Gin Ser Thr Val Pro Ala Val Leu Gin Glu Val Asp Val Glu Val 210 215 220
Ile Pro Asn Glu Arg Cys Gin Arg Trp Phe Arg Ala Ala Gly Arg Arg 225 230 235 240
Glu Thr Ile His Asp Val Phe Leu Cys Ala Gly Tyr Lys Glu Gly Gly 245 250 255
Arg Asp Ser
(2) INFORMATION FOR SEQ ID NO: 86:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "probe" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: TAAWGGWCCA GARTCTCCTT GACA 24
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: GGAAACAGCT ATGACCATG 19
(2) INFORMATION FOR SEQ ID NO: 88:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 811 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..762
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15
GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG AAG 96 Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Lys 20 25 30 GAT CCG TTA CCA GAT TCT TTC GAC TGG AGA ATT TAT GGA GCT GTT ACT 144 Asp Pro Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr 35 40 45
CCA GTT AAA GAT CAA TCT GTT TGT GGT TCC TGC TGG TCT TTC GGA ACA 192 Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr 50 55 60
ATT GGA GCT ATC GAA GGT GCA TAT TTC TTG AAA AAC GGC GGT AAT CTT 240 Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu 65 70 75 80
GTA CGA TTG TCT CAA CAG GCT TTG ATT GAT TGT TCT TGG GGA TAT GGA 288 Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly
85 90 95 AAT AAT GGT TGT GAC GGT GGC GAG GAC TTC CGC GCT TAC CAA TGG ATG 336 Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met 100 105 110
ATG AAA CAT GGA GGA ATT CCT ACT GAA GAA GAT TAT GGT GGT TAC TTG 384 Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu 115 120 125
GGA CAA GAT GGT TAC TGC CAT GTC AAC AAC GTT ACT TTA GTT GCT CCC 432 Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro 130 135 140 ATC ACA GGG TAT GTC AAC GTA ACA CGT AAT GAC GTT GAC GCT ATG AAG 480 Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys 145 150 155 160
GTT GCC CTT CTT AAA CAC GGT CCA ATT TCG GTG GCC ATT GAC GCA TCA 528 Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser 165 170 175
CAC AAA ACA TTC AGT TTT TAC TCC AAC GGC GTT TAC TAC GAA CCG AAA 576 His Lys Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys 180 185 190
TGT GGC AAT AAA GAG GAC GAG TTG GAC CAT GCC GTA TTA GTA GTC GGT 624 Cys Gly Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly 195 200 205
TAT GGT GAA ATC AAC AAC GAA CCT TAC TGG TTG GTC AAG AAT TCC TGG 672 Tyr Gly Glu Ile Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp 210 215 220 TCG AAT TTG TGG GGA AAT GAT GGT TAT ATT TTG ATG TCC GCC AGA AAT 720 Ser Asn Leu Trp Gly Asn Asp Gly Tyr Ile Leu Met Ser Ala Arg Asn 225 230 235 240
AAT AAT TGC GGA GTT TTG ACT GAT CCA ACT TAT GTT ACT ATG 762
Asn Asn Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met 245 250
TAACTTACTT TTTAGGAATT TGATTTTTAC GGCATGTAAA TGGAAGCTT 811
(2) INFORMATION FOR SEQ ID NO: 89:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH : 254 ammo acids (B) TYPE : ammo acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15
Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Lys 20 25 30
Asp Pro Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr 35 40 45 Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr 50 . 55 60
Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu 65 70 75 80 Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly
85 90 95
Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met 100 105 110
Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu H5 120 125
Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro 130 135 140
Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys 145 150 155 160 Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser
165 170 175
His Lys Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys 180 185 190
Cys Gly Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly 195 200 205
Tyr Gly Glu Ile Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp 210 215 220
Ser Asn Leu Trp Gly Asn Asp Gly Tyr Ile Leu Met Ser Ala Arg Asn 225 230 235 240 Asn Asn Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met
245 250
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 823 base pairs - (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: CCATTTACAT GCCGTAAAAA TCAAATTCCT AAAAAGTAAG TTACATAGTA ACATAAGTTG 60
GATCAGTCAA AACTCCGCAA TTATTATTTC TGGCGGACAT CAAAATATAA CCATCATTTC 120
CCCACAAATT CGACCAGGAA TTCTTGACCA ACCAGTAAGG TTCGTTGTTG ATTTCACCAT 180
AACCGACTAC TAATACGGCA TGGTCCAACT CGTCCTCTTT ATTGCCACAT TTCGGTTCGT 240
AGTAAACGCC GTTGGAGTAA AAACTGAATG TTTTGTGTGA TGCGTCAATG GCCACCGAAA 300 TTGGACCGTG TTTAAGAAGG GCAACCTTCA TAGCGTCAAC GTCATTACGT GTTACGTTGA 360 CATACCCTGT GATGGGAGCA ACTAAAGTAA CGTTGTTGAC ATGGCAGTAA CCATCTTGTC 420
CCAAGTAACC ACCATAATCT TCTTCAGTAG GAATTCCTCC ATGTTTCATC ATCCATTGGT 480
AAGCGCGGAA GTCCTCGCCA CCGTCACAAC CATTATTTCC ATATCCCCAA GAACAATCAA 540
TCAAAGCCTG TTGAGACAAT CGTACAAGAT TACCGCCGTT TTTCAAGAAA TATGCACCTT 600 CGATAGCTCC AATTGTTCCG AAAGACCAGC AGGAACCACA AACAGATTGA TCTTTAACTG 660
GAGTAACAGC TCCATAAATT CTCCAGTCGA AAGAATCTGG TAACGGATCC TTATCTGGTA 720
ACGGATCCTT CTTATCGTCA TCGTCGTACA GATCCCGACC CATTTCCTGT CCACCAGTCA 780
TGCTAGCCAT ACCATGATGA TGATGATGAT GAGAACCCCG CAT 823
(2) INFORMATION FOR SEQ ID NO: 91: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 762 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (il) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..762
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT 48 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15
GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG AAG 96 Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Lys 20 25 30
GAT CCG TTA CCA GAT TCT TTC GAC TGG AGA ATT TAT GGA GCT GTT ACT 144 Asp Pro Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr 35 40 45
CCA GTT AAA GAT CAA TCT GTT TGT GGT TCC TGC TGG TCT TTC GGA ACA 192 Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr 50 55 60
ATT GGA GCT ATC GAA GGT GCA TAT TTC TTG AAA AAC GGC GGT AAT CTT 240 Ile Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu 65 70 75 80
GTA CGA TTG TCT CAA CAG GCT TTG ATT GAT TGT TCT TGG GGA TAT GGA 288 Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly 85 90 95
AAT AAT GGT TGT GAC GGT GGC GAG GAC TTC CGC GCT TAC CAA TGG ATG 336 Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met 100 105 110
ATG AAA CAT GGA GGA ATT CCT ACT GAA GAA GAT TAT GGT GGT TAC TTG 384 - Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu 115 120 125
GGA CAA GAT GGT TAC TGC CAT GTC AAC AAC GTT ACT TTA GTT GCT CCC 432 Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro 130 135 140
ATC ACA GGG TAT GTC AAC GTA ACA CGT AAT GAC GTT GAC GCT ATG AAG 480 He Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys 145 150 155 160
GTT GCC CTT CTT AAA CAC GGT CCA ATT TCG GTG GCC ATT GAC GCA TCA 528 Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser
165 170 175
CAC AAA ACA TTC AGT TTT TAC TCC AAC GGC GTT TAC TAC GAA CCG AAA 576 His Lys Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys 180 185 190 TGT GGC AAT AAA GAG GAC GAG TTG GAC CAT GCC GTA TTA GTA GTC GGT 624 Cys Gly Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly 195 200 205
TAT GGT GAA ATC AAC AAC GAA CCT TAC TGG TTG GTC AAG AAT TCC TGG 672 Tyr Gly Glu He Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp 210 215 220
TCG AAT TTG TGG GGA AAT GAT GGT TAT ATT TTG ATG TCC GCC AGA AAT 720 Ser Asn Leu Trp Gly Asn Asp Gly Tyr He Leu Met Ser Ala Arg Asn 225 230 235 240
AAT AAT TGC GGA GTT TTG ACT GAT CCA ACT TAT GTT ACT ATG 762 Asn Asn Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met
245 250
(2) INFORMATION FOR SEQ ID NO: 92:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 254 ammo acids (B) TYPE: ammo acid
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15
Gly Gly Gin Gin Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Lys 20 25 30
Asp Pro Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr 35 40 45 Pro Val Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr 50 55 60
He Gly Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu 65 70 75 80 Val Arg Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly 85 90 95
Asn Asn Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met 100 105 110 Met Lys His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu 115 120 125
Gly Gin Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro 130 135 140
Ile Thr Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys 145 150 155 160
Val Ala Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser 165 170 175
His Lys Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys 180 185 190 Cys Gly Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly 195 200 205
Tyr Gly Glu Ile Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp 210 215 220
Ser Asn Leu Trp Gly Asn Asp Gly Tyr Ile Leu Met Ser Ala Arg Asn 225 230 235 240
Asn Asn Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met 245 250
(2) INFORMATION FOR SEQ ID NO: 93:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 783 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
TTACATAGTA ACATAAGTTG GATCAGTCAA AACTCCGCAA TTATTATTTC TGGCGGACAT 60
CAAAATATAA CCATCATTTC CCCACAAATT CGACCAGGAA TTCTTGACCA ACCAGTAAGG 120
TTCGTTGTTG ATTTCACCAT AACCGACTAC TAATACGGCA TGGTCCAACT CGTCCTCTTT 180
ATTGCCACAT TTCGGTTCGT AGTAAACGCC GTTGGAGTAA AAACTGAATG TTTTGTGTGA 240 TGCGTCAATG GCCACCGAAA TTGGACCGTG TTTAAGAAGG GCAACCTTCA TAGCGTCAAC 300
GTCATTACGT GTTACGTTGA CATACCCTGT GATGGGAGCA ACTAAAGTAA CGTTGTTGAC 360 ATGGCAGTAA CCATCTTGTC CCAAGTAACC ACCATAATCT TCTTCAGTAG GAATTCCTCC 420
ATGTTTCATC ATCCATTGGT AAGCGCGGAA GTCCTCGCCA CCGTCACAAC CATTATTTCC 480
ATATCCCCAA GAACAATCAA TCAAAGCCTG TTGAGACAAT CGTACAAGAT TACCGCCGTT 540
TTTCAAGAAA TATGCACCTT CGATAGCTCC AATTGTTCCG AAAGACCAGC AGGAACCACA 600 AACAGATTGA TCTTTAACTG GAGTAACAGC TCCATAAATT CTCCAGTCGA AAGAATCTGG 660
TAACGGATCC TTATCTGGTA ACGGATCCTT CTTATCGTCA TCGTCGTACA GATCCCGACC 720
CATTTCCTGT CCACCAGTCA TGCTAGCCAT ACCATGATGA TGATGATGAT GAGAACCCCG 780
CAT 783
(2) INFORMATION FOR SEQ ID NO: 94: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 660 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..660
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: TTA CCA GAT TCT TTC GAC TGG AGA ATT TAT GGA GCT GTT ACT CCA GTT 48 Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr Pro Val 1 5 10 15
AAA GAT CAA TCT GTT TGT GGT TCC TGC TGG TCT TTC GGA ACA ATT GGA 96 Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr Ile Gly 20 25 30
GCT ATC GAA GGT GCA TAT TTC TTG AAA AAC GGC GGT AAT CTT GTA CGA 144 Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu Val Arg 35 40 45
TTG TCT CAA CAG GCT TTG ATT GAT TGT TCT TGG GGA TAT GGA AAT AAT 192 Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly Asn Asn 50 55 60
GGT TGT GAC GGT GGC GAG GAC TTC CGC GCT TAC CAA TGG ATG ATG AAA 240 Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met Met Lys 65 70 75 80 CAT GGA GGA ATT CCT ACT GAA GAA GAT TAT GGT GGT TAC TTG GGA CAA 288 His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu Gly Gin 85 90 95
GAT GGT TAC TGC CAT GTC AAC AAC GTT ACT TTA GTT GCT CCC ATC ACA 336 Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro Ile Thr 100 105 110
GGG TAT GTC AAC GTA ACA CGT AAT GAC GTT GAC GCT ATG AAG GTT GCC 384 Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys Val Ala 115 120 125
CTT CTT AAA CAC GGT CCA ATT TCG GTG GCC ATT GAC GCA TCA CAC AAA 432 Leu Leu Lys His Gly Pro Ile Ser Val Ala Ile Asp Ala Ser His Lys 130 135 140
ACA TTC AGT TTT TAC TCC AAC GGC GTT TAC TAC GAA CCG AAA TGT GGC 480 Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys Cys Gly 145 150 155 160
AAT AAA GAG GAC GAG TTG GAC CAT GCC GTA TTA GTA GTC GGT TAT GGT 528 Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly Tyr Gly
165 170 175
GAA ATC AAC AAC GAA CCT TAC TGG TTG GTC AAG AAT TCC TGG TCG AAT 576 Glu Ile Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp Ser Asn 180 185 190 TTG TGG GGA AAT GAT GGT TAT ATT TTG ATG TCC GCC AGA AAT AAT AAT 624 Leu Trp Gly Asn Asp Gly Tyr Ile Leu Met Ser Ala Arg Asn Asn Asn 195 200 205
TGC GGA GTT TTG ACT GAT CCA ACT TAT GTT ACT ATG 660
Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met 210 215 220
(2) INFORMATION FOR SEQ ID NO:95:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 220 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
Leu Pro Asp Ser Phe Asp Trp Arg Ile Tyr Gly Ala Val Thr Pro Val 1 5 10 15 Lys Asp Gin Ser Val Cys Gly Ser Cys Trp Ser Phe Gly Thr Ile Gly
20 25 30
Ala Ile Glu Gly Ala Tyr Phe Leu Lys Asn Gly Gly Asn Leu Val Arg 35 40 45
Leu Ser Gin Gin Ala Leu Ile Asp Cys Ser Trp Gly Tyr Gly Asn Asn 50 55 60
Gly Cys Asp Gly Gly Glu Asp Phe Arg Ala Tyr Gin Trp Met Met Lys 65 70 75 80
His Gly Gly Ile Pro Thr Glu Glu Asp Tyr Gly Gly Tyr Leu Gly Gin 85 90 95 Asp Gly Tyr Cys His Val Asn Asn Val Thr Leu Val Ala Pro Ile Thr
100 105 110
Gly Tyr Val Asn Val Thr Arg Asn Asp Val Asp Ala Met Lys Val Ala 115 120 125 Leu Leu Lys His Gly Pro He Ser Val Ala Ile Asp Ala Ser His Lys 130 . 135 140
Thr Phe Ser Phe Tyr Ser Asn Gly Val Tyr Tyr Glu Pro Lys Cys Gly 145 150 155 160 Asn Lys Glu Asp Glu Leu Asp His Ala Val Leu Val Val Gly Tyr Gly
165 170 175
Glu Ile Asn Asn Glu Pro Tyr Trp Leu Val Lys Asn Ser Trp Ser Asn 180 185 190
Leu Trp Gly Asn Asp Gly Tyr Ile Leu Met Ser Ala Arg Asn Asn Asn 195 200 205
Cys Gly Val Leu Thr Asp Pro Thr Tyr Val Thr Met 210 215 220
(2) INFORMATION FOR SEQ ID NO: 96:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
Xaa Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile Phe Ile Phe Pro
1 5 10 15
Pro Lys Lys Lys Asp Asp Leu Leu He 20 25
(2) INFORMATION FOR SEQ ID NO: 97:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
TTGGGATACA CTTTGACTGT TAACC 25
(2) INFORMATION FOR SEQ ID NO: 98:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (11) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
GTGAGCAACC ATTATTTCCA TATC 24
(2) INFORMATION FOR SEQ ID NO: 99:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
TGGGTWGTWA CWGCWGCWCA TTG 23
(2) INFORMATION FOR SEQ ID NO: 100:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
ATTCCTCGTG GTTCAGTCGC TC 22
(2) INFORMATION FOR SEQ ID NO: 101:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
GAAGATGTWG ATATTTCWAC ATGTGG 26
(2) INFORMATION FOR SEQ ID NO: 102:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(11) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: GAAAATGAAA TCCACTTAAA CATTACG 27
(2) INFORMATION FOR SEQ ID NO: 103:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 103: CTCTTATTGT ACGAGGGATG C 21
(2) INFORMATION FOR SEQ ID NO: 104:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 ammo acids
(B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
Asp Cys Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile 1 5 10 15
Phe He Phe Pro Pro Lys Pro Lys Asp 20 25
(2) INFORMATION FOR SEQ ID NO: 105:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
Asp Cys Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile
1 5 10 15 Phe Ile Phe Pro Pro Lys Pro Lys Asp Asp Leu Leu lie Lys Arg Lys 20 25 30
Ser Glu Val 35
(2) INFORMATION FOR SEQ ID NO: 106:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
Asp Cys Pro Lys Cys Pro Pro Pro Glu Met Leu Gly Gly Pro Ser Ile 1 5 10 15 Phe lie Phe Pro Pro Lys Pro Lys Asp Thr Leu Ser Ile Ser Arg Thr
20 25 30
Pro Glu Val 35
(2) INFORMATION FOR SEQ ID NO: 107: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
Leu Ala Thr Thr Gin Phe Gin Ala Thr His Ala Arg Ser Ala Phe Pro 1 5 10 15
Cys Phe Asp Glu Pro Ala Met 20
(2) INFORMATION FOR SEQ ID NO: 108:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (XI) SEQUENCE DESCRIPTION: SEQ ID NO:10£ CCCAAATTTT CCATWGCNCC NGC 23
(2) INFORMATION FOR SEQ ID NO: 109:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(ix) FEATURE:
(A) NAME/KEY: mιsc_feature
(B) LOCATION: 1..20
(D) OTHER INFORMATION: /label= primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: CAATTYCAAG CTACYCATGC 20
(2) INFORMATION FOR SEQ ID NO: 110:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 383 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 2..382
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
C CGT AGT GCT TTC CCT TGT TTC GAT GAA CCA GCA ATG AAG GCC CAT 46 Arg Ser Ala Phe Pro Cys Phe Asp Glu Pro Ala Met Lys Ala His 1 5 10 15
TTC GAA ATC AGC CTT ATA CAC CAT GAA AAA TTG AAA GCA ATT TCC AAT 94 Phe Glu Ile Ser Leu Ile His His Glu Lys Leu Lys Ala Ile Ser Asn 20 25 30
ATG GGT GTA GCA AAG GAA GAA AAC TTA GAT AAC AAC CGA AAA AGA ACA 142 Met Gly Val Ala Lys Glu Glu Asn Leu Asp Asn Asn Arg Lys Arg Thr
35 40 45
ACA TTC GAA CAA TCA GTT CTC ATG TCT CCA TAC CTG GTG GCG TTT ATT 190 Thr Phe Glu Gin Ser Val Leu Met Ser Pro Tyr Leu Val Ala Phe Ile 50 55 60 ATC TCA GAT TTC GAA TAT GTA GAA AAA ATT TCA GGA CCA GTG AAA TAC 238 Ile Ser Asp Phe Glu Tyr Val Glu Lys Ile Ser Gly Pro Val Lys Tyr 65 70 75
AGA ATA TAT ACT GAT CCT TTC TCG ATT GAT CAA GCT GAC TAT GCA TTG 286 Arg Ile Tyr Thr Asp Pro Phe Ser Ile Asp Gin Ala Asp Tyr Ala Leu 80 85 90 95
ACT ATG AGC CCC AAA AAT TTT AAC GGC TTT GGA ACA ACT CAC AGG TGT 334 Thr Met Ser Pro Lys Asn Phe Asn Gly Phe Gly Thr Thr His Arg Cys 100 105 110
AAA ATA TGT TTT GAA CAA GTT GGA CCA AGC AGC CAT TCC AGA TTT TGC 382 Lys Ile Cys Phe Glu Gin Val Gly Pro Ser Ser His Ser Arg Phe Cys 115 120 125
T 383
(2) INFORMATION FOR SEQ ID NO:111:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 127 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear (ll) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
Arg Ser Ala Phe Pro Cys Phe Asp Glu Pro Ala Met Lys Ala His Phe
1 5 10 15
Glu Ile Ser Leu Ile His His Glu Lys Leu Lys Ala Ile Ser Asn Met 20 25 30
Gly Val Ala Lys Glu Glu Asn Leu Asp Asn Asn Arg Lys Arg Thr Thr 35 40 45
Phe Glu Gin Ser Val Leu Met Ser Pro Tyr Leu Val Ala Phe Ile Ile 50 55 60 Ser Asp Phe Glu Tyr Val Glu Lys He Ser Gly Pro Val Lys Tyr Arg 65 70 75 80
Ile Tyr Thr Asp Pro Phe Ser Ile Asp Gin Ala Asp Tyr Ala Leu Thr 85 90 95
Met Ser Pro Lys Asn Phe Asn Gly Phe Gly Thr Thr His Arg Cys Lys 100 105 110
Ile Cys Phe Glu Gin Val Gly Pro Ser Ser His Ser Arg Phe Cys 115 120 125
(2) INFORMATION FOR SEQ ID NO: 112:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 537 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2. -535 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
A TAT ATT TTG GAA ATA AAA GGC TTC AAA TCT ACT TTG AGA GAT GAC 46
Tyr Ile Leu Glu He Lys Gly Phe Lys Ser Thr Leu Arg Asp Asp 1 5 10 15 ATG GCT GGC TTC TAC AAA AGT TCA TAC AAA ACG CCA AAA GGA GAA ACA 94 Met Ala Gly Phe Tyr Lys Ser Ser Tyr Lys Thr Pro Lys Gly Glu Thr 20 25 30
AGA TGG TTG GCT ACA ACC CAG TTT CAG GCA ACT CAT GCC CGT AGT GCT 142 Arg Trp Leu Ala Thr Thr Gin Phe Gin Ala Thr His Ala Arg Ser Ala 35 40 45
TTC CCT TGT TTC GAT GAA CCA GCA ATG AAG GCC CAT TTC GAA ATC AGC 190 Phe Pro Cys Phe Asp Glu Pro Ala Met Lys Ala His Phe Glu Ile Ser 50 55 60
CTT ATA CAC CAT GAA AAA TTG AAA GCA ATT TCC AAT ATG GGT GTA GCA 238 Leu Ile His His Glu Lys Leu Lys Ala Ile Ser Asn Met Gly Val Ala 65 70 75
AAG GAA GAA AAC TTA GAT AAC AAC CGA AAA AGA ACA ACA TTC GAA CAA 286 Lys Glu Glu Asn Leu Asp Asn Asn Arg Lys Arg Thr Thr Phe Glu Gin 80 85 90 95 TCA GTT CTC ATG TCT CCA TAC CTG GTG GCG TTT ATT ATC TCA GAT TTC 334 Ser Val Leu Met Ser Pro Tyr Leu Val Ala Phe He Ile Ser Asp Phe 100 105 110
GAA TAT GTA GAA AAA ATT TCA GGA CCA GTG AAA TAC AGA ATA TAT ACT 382 Glu Tyr Val Glu Lys Ile Ser Gly Pro Val Lys Tyr Arg Ile Tyr Thr 115 120 125
GAT CCT TTC TCG ATT GAT CAA GCT GAC TAT GCA TTG ACT ATG AGC CCC 430 Asp Pro Phe Ser Ile Asp Gin Ala Asp Tyr Ala Leu Thr Met Ser Pro 130 135 140
AAA AAT TTT AAC GGC TTT GGA ACA ACT CAC AGG TGT AAA ATA TGT TTT 478 Lys Asn Phe Asn Gly Phe Gly Thr Thr His Arg Cys Lys He Cys Phe 145 150 155
GAA CAA GTT GGA CCA AGC AGC CAT TCC AGA TTT TGC TGC CGG CGC AAT 526 Glu Gin Val Gly Pro Ser Ser His Ser Arg Phe Cys Cys Arg Arg Asn 160 165 170 175 GGA AAA TTT GG 537
Gly Lys Phe
(2) INFORMATION FOR SEQ ID NO: 113:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 178 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(il) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: Tyr Ile Leu Glu Ile Lys Gly Phe Lys Ser Thr Leu Arg Asp Asp Met 1 5 10 15
Ala Gly Phe Tyr Lys Ser Ser Tyr Lys Thr Pro Lys Gly Glu Thr Arg 20 25 30 Trp Leu Ala Thr Thr Gin Phe Gin Ala Thr His Ala Arg Ser Ala Phe 35 40 45
Pro Cys Phe Asp Glu Pro Ala Met Lys Ala His Phe Glu Ile Ser Leu 50 55 60
Ile His His Glu Lys Leu Lys Ala Ile Ser Asn Met Gly Val Ala Lys 65 70 75 80
Glu Glu Asn Leu Asp Asn Asn Arg Lys Arg Thr Thr Phe Glu Gin Ser 85 90 95
Val Leu Met Ser Pro Tyr Leu Val Ala Phe Ile Ile Ser Asp Phe Glu 100 105 110 Tyr Val Glu Lys Ile Ser Gly Pro Val Lys Tyr Arg Ile Tyr Thr Asp 115 120 125
Pro Phe Ser Ile Asp Gin Ala Asp Tyr Ala Leu Thr Met Ser Pro Lys 130 135 140
Asn Phe Asn Gly Phe Gly Thr Thr His Arg Cys Lys Ile Cys Phe Glu 145 150 155 160
Gin Val Gly Pro Ser Ser His Ser Arg Phe Cys Cys Arg Arg Asn Gly 165 170 175
Lys Phe
(2) INFORMATION FOR SEQ ID NO: 114:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 806 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..736 (D) OTHER INFORMATION: /note= "At pos. bp 656, change G to
S. At pos. aa 219, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
C TCG CAG TCT GCA CCC TTG GTG CTA GGC GTT CCT GAC TTT TGG AAC 46
Ser Gin Ser Ala Pro Leu Val Leu Gly Val Pro Asp Phe Trp Asn 1 5 10 15
AGG TTA GAT GGC AGA ATC GTT GGA GGA CAC GAT ACT AGC ATT GAC AAA 94 Arg Leu Asp Gly Arg Ile Val Gly Gly His Asp Thr Ser Ile Asp Lys 20 25 30
CAT CCT CAT CAA GTA TCT TTG ATT TAC ACA AAC CAC AAT TGT GGT GGT 142 His Pro His Gin Val Ser Leu Ile Tyr Thr Asn His Asn Cys Gly Gly 35 - 40 45
TCT TTA ATT GCC AAA AAC TGG GTT TTA ACA GCA GCT CAT TGC ATC AGC 190 Ser Leu Ile Ala Lys Asn Trp Val Leu Thr Ala Ala His Cys Ile Ser 50 55 60
TCA ACC TAC TAC AGA GTC CGG GTA GGA AGT TCA ATC AAG AAC AGT GGT 238 Ser Thr Tyr Tyr Arg Val Arg Val Gly Ser Ser Ile Lys Asn Ser Gly 65 70 75
GGT GTT GTT CAC AGC GTT AAA AAC CAA ATC AAG CAT CCA AAA TTC GGT 286 Gly Val Val His Ser Val Lys Asn Gin He Lys His Pro Lys Phe Gly 80 85 90 95
GAT TCG GCG ACA CTC GAC TTC GAT TTT GCA CTT CTG GAA TTG GAT GAA 334 Asp Ser Ala Thr Leu Asp Phe Asp Phe Ala Leu Leu Glu Leu Asp Glu 100 105 110 CCA GTT ACA GTA ACA AAA GAC GTC AAC ATC ATC AAA CTA GTA GAC CAA 382 Pro Val Thr Val Thr Lys Asp Val Asn He Ile Lys Leu Val Asp Gin 115 120 125
GAT GTA GAA TTA ACA CCT GGA ACT ATG TGC ACT GTT ACT GGA TGG GGA 430 Asp Val Glu Leu Thr Pro Gly Thr Met Cys Thr Val Thr Gly Trp Gly 130 135 140
TCA ACT GGA TCT GGT GGT CCA ATT ACA AAT GTT CTA CAA GAA GTC GAA 478 Ser Thr Gly Ser Gly Gly Pro Ile Thr Asn Val Leu Gin Glu Val Glu 145 150 155
GTT CCA TTT ATC GAC TTC AAC ACC TGC CGA AAA TCC TAC TCA ACC AGC 526 Val Pro Phe Ile Asp Phe Asn Thr Cys Arg Lys Ser Tyr Ser Thr Ser 160 165 170 175
TTA ACC GAC CGT ATG TTC TGC GCT GGA TTT TTG GGA ATT GGT GGT AAG 574 Leu Thr Asp Arg Met Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly Lys 180 185 190 GAC GCT TGT CAA GGT GAC TCT GGT GGC CCA GTT GTT GTC GAT GGT GTT 622 Asp Ala Cys Gin Gly Asp Ser Gly Gly Pro Val Val Val Asp Gly Val 195 200 205
CTT CAC GGA ATC GTA TCA TGG GGA CGT GGT TGC SCC CTT CCT GAC TAC 670 Leu His Gly Ile Val Ser Trp Gly Arg Gly Cys Xaa Leu Pro Asp Tyr 210 215 220
CCC GGA GTC TAC TCT AAG ATC TCA TAT GCC CGT GAC TGG ATT AAG GAA 718 Pro Gly Val Tyr Ser Lys Ile Ser Tyr Ala Arg Asp Trp Ile Lys Glu 225 230 235
AAT CAC TGG TGT TTA ATT TAATATTTAT TATACCAAAT AATTATATAT 766 Asn His Trp Cys Leu Ile 240 245
AAATATATAC TATTTTAAAT ACAAAAAAAA AAAAAAAAAA 806 (2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
Ser Gin Ser Ala Pro Leu Val Leu Gly Val Pro Asp Phe Trp Asn Arg 1 5 10 15 Leu Asp Gly Arg Ile Val Gly Gly His Asp Thr Ser He Asp Lys His
20 25 30
Pro His Gin Val Ser Leu Ile Tyr Thr Asn His Asn Cys Gly Gly Ser 35 40 45
Leu Ile Ala Lys Asn Trp Val Leu Thr Ala Ala His Cys Ile Ser Ser 50 55 60
Thr Tyr Tyr Arg Val Arg Val Gly Ser Ser Ile Lys Asn Ser Gly Gly 65 70 75 80
Val Val His Ser Val Lys Asn Gin Ile Lys His Pro Lys Phe Gly Asp 85 90 95 Ser Ala Thr Leu Asp Phe Asp Phe Ala Leu Leu Glu Leu Asp Glu Pro
100 105 110
Val Thr Val Thr Lys Asp Val Asn He lie Lys Leu Val Asp Gin Asp 115 120 125
Val Glu Leu Thr Pro Gly Thr Met Cys Thr Val Thr Gly Trp Gly Ser 130 135 140
Thr Gly Ser Gly Gly Pro Ile Thr Asn Val Leu Gin Glu Val Glu Val 145 150 155 160
Pro Phe Ile Asp Phe Asn Thr Cys Arg Lys Ser Tyr Ser Thr Ser Leu 165 170 175 Thr Asp Arg Met Phe Cys Ala Gly Phe Leu Gly Ile Gly Gly Lys Asp
180 185 190
Ala Cys Gin Gly Asp Ser Gly Gly Pro Val Val Val Asp Gly Val Leu 195 200 205
His Gly Ile Val Ser Trp Gly Arg Gly Cys Xaa Leu Pro Asp Tyr Pro 210 215 220
Gly Val Tyr Ser Lys He Ser Tyr Ala Arg Asp Trp Ile Lys Glu Asn 225 230 235 240
His Trp Cys Leu Ile 245 (2) INFORMATION FOR SEQ ID NO:116:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 157 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: GGAATTCGGC ACGAGTATCT GTGCCCTCGC AGTCTGCACC CTTGGCGTAG CGTTCCTGAC 60 TTTTGGAACA GGTTAGATGG CAGAATCGTT GGAGGACACG ATACTAGCAT TGATAACATC 120 CTCATGCAAG TATCTTTGAG TTTACACAAA CCACAAT 157
(2) INFORMATION FOR SEQ ID NO: 117:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 117: AAGATGTAGA ATTAACACCT GGAACTATGT GCACTGTTAC TGGATGGGGA TCAACTGGAT 60
CTGGTGGTCC AATTACAAAT GTTCTACAAG AAGTCGAAGT TCCATTTATC GACTTCAACA 120
CCTGCCGAAA ATCCTACTCA ACCAGCTTAA CCGACCGTAT GTTCTGCGCT GGATTTTTGG 180
GAATTGGTGG TAAGGACGCT TGCCAAGGCG ACTCCGGA 218
(2) INFORMATION FOR SEQ ID NO: 118: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 307 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (il) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..306
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: CAA ATA TTT AAG CAT GAA CCT GTG AAC GTG GTA TTA TTT CTT GTA GAA 48 Gin Ile Phe Lys His Glu Pro Val Asn Val Val Leu Phe Leu Val Glu 1 5 10 15
GAT CGC TTC AAT TTT GAA ATT ACT AAC GCG CGC CCT GTT GCC CTA CCA 96 Asp Arg Phe Asn Phe Glu Ile Thr Asn Ala Arg Pro Val Ala Leu Pro 20 25 30
GCA GAG AAC GAG GAA ACC GAA ACA GGG TCA CCA CTC ACG GTG ACG GGT 144 Ala Glu Asn Glu Glu Thr Glu Thr Gly Ser Pro Leu Thr Val Thr Gly 35 40 45
TGG GGA ACT ACA GAG AGT ACT GAA TCA TCA CAC CAC CTG AAA GAA GTT 192 Trp Gly Thr Thr Glu Ser Thr Glu Ser Ser His His Leu Lys Glu Val 50 55 60
GAA GTG AAC GCT GTA TCT AAT AGT GAA TGT CAA AAG GCC TAT GAA GAT 240 Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gin Lys Ala Tyr Glu Asp 65 70 75 80
CTT GCT ACT ATA TCA TCA CAT GAG ATA TGT GCA AGC GTT CCT GGT GGC 288 Leu Ala Thr Ile Ser Ser His Glu Ile Cys Ala Ser Val Pro Gly Gly 85 90 95 GGC AAA GAT TCT TGT CAA G 307
Gly Lys Asp Ser Cys Gin 100
(2) INFORMATION FOR SEQ ID NO: 119:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 102 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: Gin Ile Phe Lys His Glu Pro Val Asn Val Val Leu Phe Leu Val Glu 1 5 10 15
Asp Arg Phe Asn Phe Glu Ile Thr Asn Ala Arg Pro Val Ala Leu Pro 20 25 30
Ala Glu Asn Glu Glu Thr Glu Thr Gly Ser Pro Leu Thr Val Thr Gly 35 40 45
Trp Gly Thr Thr Glu Ser Thr Glu Ser Ser His His Leu Lys Glu Val 50 55 60
Glu Val Asn Ala Val Ser Asn Ser Glu Cys Gin Lys Ala Tyr Glu Asp
65 70 75 80 Leu Ala Thr Ile Ser Ser His Glu Ile Cys Ala Ser Val Pro Gly Gly
85 90 95
Gly Lys Asp Ser Cys Gin 100 (2) INFORMATION FOR SEQ ID NO: 120:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 162 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: GGTTGGGGAA CTACAGAGAG TACTGAATCA TCACACCACC TGAAAGAAGT TGAAGTGAAC 60 GCTGTATCTA ATAGTGAATG TCAAAGGCCT AATGAAGATC TTGCTACTAT ATCATCACAT 120 GAGATATGTG CAAGCGTTCC TGGTGGCGGC AAAGATTCTT GT 162
(2) INFORMATION FOR SEQ ID NO: 121:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 252 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: CGCGCGCCCT GTTGCCCTAC CAGCAGAGAA CGAGGAAACC GAAACAGGGT CACACTCACG 60
GTGACGGGTT GGGGAACTAC AGAGAGTACT GAATCATCAC ACCACCTGAA AGAAGTTGAA 120
GTGAACGCTG TATCTAATAG TGAATGTCAA AGGCCTAATG AAGATCTTGC TACTATATCA 180
TCACATGAGA TATGTGCAAG CGTTCCTGGT GGCGGCAAAG ATTCTTGTCA AGGAGACTCT 240
GGTGGTCCTT TA 252
(2) INFORMATION FOR SEQ ID NO: 122:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 267 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..267 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
CCG ACT ACC TTA GGC GGA TCT ACT GGT CAC AGC AAT GAT ATT GCC CTA 48 Pro Thr Thr Leu Gly Gly Ser Thr Gly His Ser Asn Asp He Ala Leu
1 5 10 15
ATC AAA GTC GAT AGA GAT ATC AAA TTC AGC AAA ACT GTC CAA CCT ATC 96 Ile Lys Val Asp Arg Asp Ile Lys Phe Ser Lys Thr Val Gin Pro Ile 20 25 30
AAA TTG CAC AAA AGT TTA ATA AAT GGA GGT GAA AAA TTG AAA ATT ACT 144 Lys Leu His Lys Ser Leu Ile Asn Gly Gly Glu Lys Leu Lys Ile Thr 35 40 45
GGA TGG GGA TTG ACG AAT CAA AGT CAT AGT GAT GAA CCA GAT GTT CTT 192 Gly Trp Gly Leu Thr Asn Gin Ser His Ser Asp Glu Pro Asp Val Leu 50 55 60
CAA GAG TTG CAT GTA AAA GCA CTT ACT GAT TCT GAG TGC GAG AAA GCT 240 Gin Glu Leu His Val Lys Ala Leu Thr Asp Ser Glu Cys Glu Lys Ala 65 70 75 80 ACA GGT GAA GAC CAT CCT ACA CAC CTT 267
Thr Gly Glu Asp His Pro Thr His Leu 85
(2) INFORMATION FOR SEQ ID NO: 123:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: Pro Thr Thr Leu Gly Gly Ser Thr Gly His Ser Asn Asp Ile Ala Leu 1 5 10 15
Ile Lys Val Asp Arg Asp Ile Lys Phe Ser Lys Thr Val Gin Pro Ile 20 25 30
Lys Leu His Lys Ser Leu Ile Asn Gly Gly Glu Lys Leu Lys He Thr 35 40 45
Gly Trp Gly Leu Thr Asn Gin Ser His Ser Asp Glu Pro Asp Val Leu 50 55 60
Gin Glu Leu His Val Lys Ala Leu Thr Asp Ser Glu Cys Glu Lys Ala 65 70 75 80 Thr Gly Glu Asp His Pro Thr His Leu
85 (2) INFORMATION FOR SEQ ID NO: 124:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "probe"
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 124: TAAWGGWCCW CCYGAATCTC CCTGGCA 27
(2) INFORMATION FOR SEQ ID NO: 125:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 436 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 2..436
(D) OTHER INFORMATION: /note= "At pos. bp 301, change A to W. At pos. bp 342, change C to Y. At pos. bp 431, change G to s. At pos. aa 100, 114 and 144, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: A GTT AGT CTT TCC AAT TCG ATC AGA CCT TCT TGT TTA TGG GCC AAT 46
Val Ser Leu Ser Asn Ser Ile Arg Pro Ser Cys Leu Trp Ala Asn 1 5 10 15
GAC GAG TTC GAC ACA GAT AGT TCA ATT GCT ACT GGT TGG GGA AAG ATA 94 Asp Glu Phe Asp Thr Asp Ser Ser Ile Ala Thr Gly Trp Gly Lys Ile 20 25 30
GAC TAT GCT GAG AGC AGA AGT GAT GAC CTA CTG AAA GTA GTA CTG AAA 142 Asp Tyr Ala Glu Ser Arg Ser Asp Asp Leu Leu Lys Val Val Leu Lys 35 40 45
ATT ATT GAT AAT AGG CAA TGC GCT CCC TTA TAC GTT GAT CAG ATT AAT 190 Ile Ile Asp Asn Arg Gin Cys Ala Pro Leu Tyr Val Asp Gin He Asn 50 55 60
AGA AGA AGA TTG AGA AAT GGA ATT GTA GAT ACA CAG ATG TGT GCA GGA 238 Arg Arg Arg Leu Arg Asn Gly Ile Val Asp Thr Gin Met Cys Ala Gly 65 70 75 GAA TTG GAT GGT GGC AAA GAC ACT TGC CAG GGA GAT TCA GGT GGG CCA 286 Glu Leu Asp Gly Gly Lys Asp Thr Cys Gin Gly Asp Ser Gly Gly Pro 80 85 90 95
TTG CAA ATA ACA AAW CAA AGC AAC AAA TGT ATC TTC TAC ATA GTG GGA 334 Leu Gin Ile Thr Xaa Gin Ser Asn Lys Cys He Phe Tyr Ile Val Gly 100 105 110
ATA ACA TYA TTC GGA AGG GGA TGT GGT GCT CCT AAT AGC CCC GGT GTT 382 Ile Thr Xaa Phe Gly Arg Gly Cys Gly Ala Pro Asn Ser Pro Gly Val 115 120 125
TAT ACT AGA GTC AGY AAG TAT GTT GAC TGG ATT GAA AGT GTT GTT TGG 430 Tyr Thr Arg Val Ser Lys Tyr Val Asp Trp Ile Glu Ser Val Val Trp 130 135 140
SCA AAT 436 Xaa Asn 145
(2) INFORMATION FOR SEQ ID NO: 126:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 145 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
(XI) SEQUENCE DE-SCRIPTION: SEQ ID NO: 126:
Val Ser Leu Ser Asn Ser He Arg Pro Ser Cys Leu Trp Ala Asn Asp 1 5 10 15
Glu Phe Asp Thr Asp Ser Ser Ile Ala Thr Gly Trp Gly Lys Ile Asp 20 25 30
Tyr Ala Glu Ser Arg Ser Asp Asp Leu Leu Lys Val Val Leu Lys Ile 35 40 45 Ile Asp Asn Arg Gin Cys Ala Pro Leu Tyr Val Asp Gin Ile Asn Arg 50 55 60
Arg Arg Leu Arg Asn Gly Ile Val Asp Thr Gin Met Cys Ala Gly Glu 65 70 75 80
Leu Asp Gly Gly Lys Asp Thr Cys Gin Gly Asp Ser Gly Gly Pro Leu 85 90 95
Gin Ile Thr Xaa Gin Ser Asn Lys Cys Ile Phe Tyr Ile Val Gly Ile 100 105 110
Thr Xaa Phe Gly Arg Gly Cys Gly Ala Pro Asn Ser Pro Gly Val Tyr 115 120 125 Thr Arg Val Ser Lys Tyr Val Asp Trp Ile Glu Ser Val Val Trp Xaa 130 135 140
Asn
145 (2) INFORMATION FOR SEQ ID NO: 127:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:
AATTCGGCAC GAGAGTTAGT CTTTCCAATT CGATCAGACC TTCTTGTTTA TGGGCCAATG 60 ACGAGTTCGA CACAGATAGT TCAATTGCTA CTGGTTGGGG AAAGATAGAC TATGCTGAGA 120
GCAGAAGTGA TGACCTACTG AAAGTAGTAC TGAAAATTAT TGATAATAGG CAATGCGVYC 180
CCTTATACGT TGATCAGATT AATAGAAGAA GATTGAGAAA TGGAATTGTA GATACACAGA 240
TGTGTGCAGG AGAATTGGAT GGTGGCAAAG ACACTTGCCA GGGAGATTCA GGTGGTCCT 299
(2) INFORMATION FOR SEQ ID NO: 128: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 758 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (il) MOLECULE TYPE: cDNA
(IX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..738
(D) OTHER INFORMATION: /note= "At pos. aa 241, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:
AAG GTA CTG ATC GTT TTA GCA GTC ATT GAA TTC GCA TCA GCG TCT TCA 48 Lys Val Leu Ile Val Leu Ala Val Ile Glu Phe Ala Ser Ala Ser Ser 1 5 10 15 ATC GGC TGG AGA ATC GTG GGT GGT GAA AAT GCT AAA GAA AAA TCG GTG 96 Ile Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Ser Val 20 25 30
CCC TAT CAA GTT TCA CTT CGA AAT GCT GAA AAC AAA CAT TTC TGT GGA 144 Pro Tyr Gin Val Ser Leu Arg Asn Ala Glu Asn Lys His Phe Cys Gly 35 40 45
GGA GCA ATT ATT GAC GAT TAT TGG GTT TTG ACT GCT GCT CAT TGC ATG 192 Gly Ala Ile Ile Asp Asp Tyr Trp Val Leu Thr Ala Ala His Cys Met 50 55 60
GGA CAA CGT TTT GAA GTC GTT GCC GGC GTG AAT AAA CTG GAT GAA GTA 240 Gly Gin Arg Phe Glu Val Val Ala Gly Val Asn Lys Leu Asp Glu Val 65 70 75 80
GGT GAA CGA TAT AGA ATA GAA AAA ACT ATT ACT GAC AAG TTT GAT GAA 288 Gly Glu Arg Tyr Arg Ile Glu Lys Thr Ile Thr Asp Lys Phe Asp Glu 85 90 95
CAA ACT GCC GCA AAT GAT TTG GCA CTT GTA AAA CTT CGG AAT AAA ATA 336 Gin Thr Ala Ala Asn Asp Leu Ala Leu Val Lys Leu Arg Asn Lys Ile 100 105 110
AAA TTC AGC GAT AAA GTG CAA AAA ATT CAA TTT GAA GAT AAA TAT ATC 384 Lys Phe Ser Asp Lys Val Gin Lys He Gin Phe Glu Asp Lys Tyr Ile 115 120 125
GGA GGC GGA GAG GAT GCT CGT TTG ACT GGA TGG GGA CGA TTG GGA AAA 432 Gly Gly Gly Glu Asp Ala Arg Leu Thr Gly Trp Gly Arg Leu Gly Lys 130 135 140
GAT TCA CCG CCA CCT AAT GAT TTA CAG GAA TTA AAT ACA TTT ACC ATC 480 Asp Ser Pro Pro Pro Asn Asp Leu Gin Glu Leu Asn Thr Phe Thr Ile 145 150 155 160 CCC CAA AGT GTT TGC AGA AGA ATG TTT AAT GAG GAT AAG ATT CCA ATC 528 Pro Gin Ser Val Cys Arg Arg Met Phe Asn Glu Asp Lys Ile Pro Ile 165 170 175
CAC GAT AGC CAA ATA TGC ACT TTT GCA GAT ATG GGC AAG GGT GCA TGT 576 His Asp Ser Gin Ile Cys Thr Phe Ala Asp Met Gly Lys Gly Ala Cys 180 185 190
AAG GGT GAT TCC GGT GGC CCC TTA GTC ATC AAT GGA CAA CTT CAT GGA 624 Lys Gly Asp Ser Gly Gly Pro Leu Val Ile Asn Gly Gin Leu His Gly 195 200 205
ATT GTT TCC TGG GGC ATT CCT TGC GCT GTC GGC AAG CCT GAT GTA TTC 672 Ile Val Ser Trp Gly Ile Pro Cys Ala Val Gly Lys Pro Asp Val Phe 210 215 220
ACA AGA GTT TCT CAT TAT GTC GAT TGG ATT AAA TCC AAA ATT GCC AAA 720 Thr Arg Val Ser His Tyr Val Asp Trp Ile Lys Ser Lys Ile Ala Lys 225 230 235 240 TAA AAT TGT TTA GTG TAT TAAAAAAAAA AAAAAAAAAA 758
Xaa Asn Cys Leu Val Tyr 245
(2) INFORMATION FOR SEQ ID NO: 129:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 246 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 129: Lys Val Leu Ile Val Leu Ala Val lie Glu Phe Ala Ser Ala Ser Ser 1 5 10 15
Ile Gly Trp Arg Ile Val Gly Gly Glu Asn Ala Lys Glu Lys Ser Val 20 25 30 Pro Tyr Gin Val Ser Leu Arg Asn Ala Glu Asn Lys His Phe Cys Gly 35 40 45
Gly Ala Ile Ile Asp Asp Tyr Trp Val Leu Thr Ala Ala His Cys Met 50 55 60 Gly Gin Arg Phe Glu Val Val Ala Gly Val Asn Lys Leu Asp Glu Val 65 70 75 80
Gly Glu Arg Tyr Arg He Glu Lys Thr Ile Thr Asp Lys Phe Asp Glu 85 90 95
Gin Thr Ala Ala Asn Asp Leu Ala Leu Val Lys Leu Arg Asn Lys Ile 100 105 110
Lys Phe Ser Asp Lys Val Gin Lys He Gin Phe Glu Asp Lys Tyr Ile 115 120 125
Gly Gly Gly Glu Asp Ala Arg Leu Thr Gly Trp Gly Arg Leu Gly Lys 130 135 140 Asp Ser Pro Pro Pro Asn Asp Leu Gin Glu Leu Asn Thr Phe Thr Ile 145 150 155 160
Pro Gin Ser Val Cys Arg Arg Met Phe Asn Glu Asp Lys Ile Pro Ile 165 170 175
His Asp Ser Gin Ile Cys Thr Phe Ala Asp Met Gly Lys Gly Ala Cys 180 185 190
Lys Gly Asp Ser Gly Gly Pro Leu Val Ile Asn Gly Gin Leu His Gly 195 200 205
Ile Val Ser Trp Gly Ile Pro Cys Ala Val Gly Lys Pro Asp Val Phe 210 215 220 Thr Arg Val Ser His Tyr Val Asp Trp Ile Lys Ser Lys He Ala Lys 225 230 235 240
Xaa Asn Cys Leu Val Tyr 245
(2) INFORMATION FOR SEQ ID NO:130: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 144 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: GTACTGATCG TTTTAGCAGT CATTGAATTC GCATCAGCGT CTTCAATCGG CTGGAGAATC 60 GTGGGTGGTG AAAATGCTAA AGAAAAATCG GTGCCCTATC AAGTTTCMCT TCGAAATGCT 120 GAAAACAAAC ATTTYTGTGG RGGR 144 (2) INFORMATION FOR SEQ ID NO: 131:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 225 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:
CCAATCCACG ATAGCCAATA TGCACTTTTG CAGATATGGG TCAAGGGTGC ATGTAAGGGT 60 GATTCCGGTG GCCCCTTAGT CATCAATGGA CAACTTCATG GAATTGTTTC CTGGGGCATT 120
CCTTGCGCTG TCGCAAGCCT GATGTATTCA CAAGAGTTTC TCATTATGTC GATTGGATTA 180
AATCCAAAAT TGCCAAATAA AATTGTTTAG AGTATTAAAA AAAAA 225
(2) INFORMATION FOR SEQ ID NO: 132:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 610 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..555
(D) OTHER INFORMATION: /note= "At pos. bp 133, change A to H. At pos. aa 45, substitute Xaa." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132:
GTT TTG ACA GCT GCT CAT TGT GTA GAA AAG TAT AAA TTG ACT GTT AGA 48 Val Leu Thr Ala Ala His Cys Val Glu Lys Tyr Lys Leu Thr Val Arg 1 5 10 15
GTT GGC AGC AGC GAT TTG GAA TCA GGA GGC AAA ATA CAT ACC ATT AAA 96 Val Gly Ser Ser Asp Leu Glu Ser Gly Gly Lys Ile His Thr Ile Lys
20 25 30
AAA ATC CAT GTT CAT CCA TAT TAC GAA CCG GTT GAC HAC GAT TTT GCC 144 Lys Ile His Val His Pro Tyr Tyr Glu Pro Val Asp Xaa Asp Phe Ala 35 40 45 TTG CTG GGA CTT GAT GAA CCA GTW TTC TTG AGC AAC AAA GTT CAA CTT 192 Leu Leu Gly Leu Asp Glu Pro Val Phe Leu Ser Asn Lys Val Gin Leu 50 55 60
GTG AAA CTT GTA GAA CAA GGT GTA GAT CTG GAT GAA GGA ACC TTT CTT 240 Val Lys Leu Val Glu Gin Gly Val Asp Leu Asp Glu Gly Thr Phe Leu 65 70 75 80
AAT GCC ACT GGA TGG GGT ACA ACA GCG ACC GAA GAT TTG GCT CCA GTT 288 Asn Ala Thr Gly Trp Gly Thr Thr Ala Thr Glu Asp Leu Ala Pro Val 85 90 95 CTT CAA TTA GTA ACA GTT CCA GTA GTC AAC ACA TAT ACT TGC AGC AAA 336 Leu Gin Leu Val Thr Val Pro Val Val Asn Thr Tyr Thr Cys Ser Lys 100 105 110
ATT TAC GAC TTT GGT ATC ACA CAA AGA ATG TTT TGC GCT GGT TAT ATG 384 He Tyr Asp Phe Gly Ile Thr Gin Arg Met Phe Cys Ala Gly Tyr Met 115 120 125
GAT GGA ACT CTT AAG GAC ATC TGC TCT GGA GAT TCA GGT AGT CCT GTG 432 Asp Gly Thr Leu Lys Asp Ile Cys Ser Gly Asp Ser Gly Ser Pro Val 130 135 140 GTG AAG GAT GGT ATC CAA TAT GGT GTG GTG TCT TGG GGA AAA GCT TGT 480 Val Lys Asp Gly Ile Gin Tyr Gly Val Val Ser Trp Gly Lys Ala Cys 145 150 155 160
GCC GAT CCA AGA TAT CCA AAT GTT TAT TCC AAA GTT AGC TAC GAA CGT 528 Ala Asp Pro Arg Tyr Pro Asn Val Tyr Ser Lys Val Ser Tyr Glu Arg 165 170 175
ATA TGG ATT AAA GAA GTG TCT GGA GTT TAAGTAGATG CATTCTTTAT 575
He Trp Ile Lys Glu Val Ser Gly Val 180 185
TATAATAAAA TTGTTTAATT AAAAAAAAAA AAAAA 610
(2) INFORMATION FOR SEQ ID NO: 133:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:
Val Leu Thr Ala Ala His Cys Val Glu Lys Tyr Lys Leu Thr Val Arg 1 5 10 15
Val Gly Ser Ser Asp Leu Glu Ser Gly Gly Lys Ile His Thr Ile Lys 20 25 30
Lys Ile His Val His Pro Tyr Tyr Glu Pro Val Asp Xaa Asp Phe Ala 35 40 45
Leu Leu Gly Leu Asp Glu Pro Val Phe Leu Ser Asn Lys Val Gin Leu 50 55 60 Val Lys Leu Val Glu Gin Gly Val Asp Leu Asp Glu Gly Thr Phe Leu 65 70 75 80
Asn Ala Thr Gly Trp Gly Thr Thr Ala Thr Glu Asp Leu Ala Pro Val 85 90 95
Leu Gin Leu Val Thr Val Pro Val Val Asn Thr Tyr Thr Cys Ser Lys 100 105 110
He Tyr Asp Phe Gly Ile Thr Gin Arg Met Phe Cys Ala Gly Tyr Met 115 120 125 Asp Gly Thr Leu Lys Asp Ile Cys Ser Gly Asp Ser Gly Ser Pro Val 130 135 140
Val Lys Asp Gly Ile Gin Tyr Gly Val Val Ser Trp Gly Lys Ala Cys 145 150 155 160 Ala Asp Pro Arg Tyr Pro Asn Val Tyr Ser Lys Val Ser Tyr Glu Arg
165 170 175
Ile Trp Ile Lys Glu Val Ser Gly Val 180 185
(2) INFORMATION FOR SEQ ID NO: 134: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 386 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..384
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134: AAR ATT GGT ACT AGC CAT AGG ATA CTT TAT GGT AGA GTG ATA GAC ATA 48 Lys Ile Gly Thr Ser His Arg Ile Leu Tyr Gly Arg Val Ile Asp Ile 1 5 10 15
AAA GAA ATT ATA ATG CAT CCA GAC TAT ACG TCT GTA TCA GGA AGT GGA 96 Lys Glu Ile Ile Met His Pro Asp Tyr Thr Ser Val Ser Gly Ser Gly 20 25 30
TAC GAT GTA GCA CTA TTG AAA CCG TCT ACA AAA ATT GTT TTT AAC TCA 144 Tyr Asp Val Ala Leu Leu Lys Pro Ser Thr Lys Ile Val Phe Asn Ser 35 40 45
AAA TCT ATC AAA CCT GTA AAG CTA ATT GAT GAA GGA ATC GAA ACG GCT 192 Lys Ser Ile Lys Pro Val Lys Leu Ile Asp Glu Gly Ile Glu Thr Ala 50 55 60
AAT GGT TCA ATA GCA ACC GTG GCA GGT TGG GGT AAA GTA GTG GAT GGT 240 Asn Gly Ser Ile Ala Thr Val Ala Gly Trp Gly Lys Val Val Asp Gly 65 70 75 80 TTT CCG TAC ATA CCT AAT TAT TTA TTG GCT GTA AAT GTA CCG ATT ATT 288 Phe Pro Tyr Ile Pro Asn Tyr Leu Leu Ala Val Asn Val Pro He Ile 85 90 95
GAT AGC GAT ACA TGC AAG TCG ATG AAT ATT GAA TAT CAA AAA TAT TTG 336 Asp Ser Asp Thr Cys Lys Ser Met Asn Ile Glu Tyr Gin Lys Tyr Leu 100 105 110
AAA CCA AAT ATG ATA TGC GCC GGA TAT GCA AAA GGT GGT AAA GAT TCT 384 Lys Pro Asn Met Ile Cys Ala Gly Tyr Ala Lys Gly Gly Lys Asp Ser 115 120 125
TG 386 (2) INFORMATION FOR SEQ ID NO:135:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 128 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 135:
Lys Ile Gly Thr Ser His Arg Ile Leu Tyr Gly Arg Val Ile Asp Ile 1 5 10 15 Lys Glu He Ile Met His Pro Asp Tyr Thr Ser Val Ser Gly Ser Gly
20 25 30
Tyr Asp Val Ala Leu Leu Lys Pro Ser Thr Lys Ile Val Phe Asn Ser 35 40 45
Lys Ser He Lys Pro Val Lys Leu lie Asp Glu Gly Ile Glu Thr Ala 50 55 60
Asn Gly Ser Ile Ala Thr Val Ala Gly Trp Gly Lys Val Val Asp Gly 65 70 75 80
Phe Pro Tyr lie Pro Asn Tyr Leu Leu Ala Val Asn Val Pro Ile Ile 85 90 95
Asp Ser Asp Thr Cys Lys Ser Met Asn Ile Glu Tyr Gin Lys Tyr Leu
100 105 110
Lys Pro Asn Met Ile Cys Ala Gly Tyr Ala Lys Gly Gly Lys Asp Ser 115 120 125
(2) INFORMATION FOR SEQ ID NO: 136: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (11) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..423
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136: GTA GTT ACA GCT GCA CAT TGT GTC ACC GTT GGA GGA CAT AAC CAA GTC 48 Val Val Thr Ala Ala His Cys Val Thr Val Gly Gly His Asn Gin Val
1 5 10 15
GTA GCT GTT GTA GGA ACC AAC AAA TTG AGC TCC GGA GGC ACC ACA TAC 96 Val Ala Val Val Gly Thr Asn Lys Leu Ser Ser Gly Gly Thr Thr Tyr 20 25 30
AAA GCT GAA CGT GTT GTT GTA CAC GAA CGT TAT GGC AAT GCT GAT ATT 144 Lys Ala Glu Arg Val Val Val His Glu Arg -Tyr Gly Asn Ala Asp Ile 35 40 45
GAC AAC GAT CTT GCC TTG ATC AAG TTG ACC CAA GAT GTC GTA TTC ACT 192 Asp Asn Asp Leu Ala Leu Ile Lys Leu Thr Gin Asp Val Val Phe Thr 50 55 60
GAC CGC GTA CAG CCC GTC ACC GTA TCC AGA ACT ACA GTC AAA GGA GGA 240 Asp Arg Val Gin Pro Val Thr Val Ser Arg Thr Thr Val Lys Gly Gly 65 70 75 80
GAA ACC TTG AGA ATC ACT GGA TGG GGT TAC ACC AAC CAC GGT GGC CCA 288 Glu Thr Leu Arg Ile Thr Gly Trp Gly Tyr Thr Asn His Gly Gly Pro 85 90 95 GTT CTG CCC GAC AGT TTG CAA GAA CTT CAT GTA ACC GCC CAG ACC CCA 336 Val Leu Pro Asp Ser Leu Gin Glu Leu His Val Thr Ala Gin Thr Pro 100 105 110
AGC ACA TGC CAA AAA TAC ACA CCA GCC GCC ACA CAA TTG TGC ACT TTC 384 Ser Thr Cys Gin Lys Tyr Thr Pro Ala Ala Thr Gin Leu Cys Thr Phe 115 120 125
TTG AAG ACT GGA CAA GGA GTT TGC AAC GGT GAT TCT GGA 423
Leu Lys Thr Gly Gin Gly Val Cys Asn Gly Asp Ser Gly 130 135 140
(2) INFORMATION FOR SEQ ID NO: 137: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(li) MOLECULE TYPE: protem (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:
Val Val Thr Ala Ala His Cys Val Thr Val Gly Gly His Asn Gin Val 1 5 10 15
Val Ala Val Val Gly Thr Asn Lys Leu Ser Ser Gly Gly Thr Thr Tyr 20 25 30 Lys Ala Glu Arg Val Val Val His Glu Arg Tyr Gly Asn Ala Asp Ile 35 40 45
Asp Asn Asp Leu Ala Leu Ile Lys Leu Thr Gin Asp Val Val Phe Thr 50 55 60
Asp Arg Val Gin Pro Val Thr Val Ser Arg Thr Thr Val Lys Gly Gly 65 70 75 80 Glu Thr Leu Arg Ile Thr Gly Trp Gly Tyr Thr Asn His Gly Gly Pro 85 90 95
Val Leu Pro Asp Ser Leu Gin Glu Leu His Val Thr Ala Gin Thr Pro
100 105 110 Ser Thr Cys Gin Lys Tyr Thr Pro Ala Ala Thr Gin Leu Cys Thr Phe 115 120 125
Leu Lys Thr Gly Gin Gly Val Cys Asn Gly Asp Ser Gly 130 135 140
(2) INFORMATION FOR SEQ ID NO: 138: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..197
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: GT GGT GGG CTG CCT TCT TCA ATA ACA TTA GCG CGA GTC AGA CTC GGC 47
Gly Gly Leu Pro Ser Ser He Thr Leu Ala Arg Val Arg Leu Gly 1 5 10 15
GAA CAT AAT GAT CAA TCG GGT ATA GAT TGC GAG GAC GAT GTT TGC GCA 95 Glu His Asn Asp Gin Ser Gly Ile Asp Cys Glu Asp Asp Val Cys Ala 20 25 30
GAA CCT GTC CAA GAT TTC GAT CCT GTG AAA ATA ATT CCA CAT CCA GAA 143 Glu Pro Val Gin Asp Phe Asp Pro Val Lys Ile Ile Pro His Pro Glu 35 40 45
TAC AAA GAC GAA CTA TTT AAA CAT GAT ATA GCT CTG ATA AAA TTG GTA 191 Tyr Lys Asp Glu Leu Phe Lys His Asp Ile Ala Leu Ile Lys Leu Val 50 55 60
GAA AAT 197
Glu Asn 65
(2) INFORMATION FOR SEQ ID NO:139:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 ammo acids
Figure imgf000269_0001
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
Gly Gly Leu Pro Ser Ser He Thr Leu Ala Arg Val Arg Leu Gly Glu 1 5 10 15 His Asn Asp Gin Ser Gly Ile Asp Cys Glu Asp Asp Val Cys Ala Glu 20 25 30
Pro Val Gin Asp Phe Asp Pro Val Lys Ile Ile Pro His Pro Glu Tyr 35 40 45 Lys Asp Glu Leu Phe Lys His Asp Ile Ala Leu Ile Lys Leu Val Glu 50 55 60
Asn 65
(2) INFORMATION FOR SEQ ID NO: 140: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 341 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..341
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: GA AAA TCT CGA ATT GGG CGA CAG CTT AAA ATT CAC TAT GGA AAT AAC 47
Lys Ser Arg Ile Gly Arg Gin Leu Lys Ile His Tyr Gly Asn Asn 1 5 10 15
GAC TGG CAC TTT GGC TTC GTT AGT ATT GTA AAA AAG GCT ATT ATT CAT 95 Asp Trp His Phe Gly Phe Val Ser Ile Val Lys Lys Ala Ile Ile His 20 25 30
CCA AAT TAC AAC CCA GTG ACA TTT GAT AGT GAT GTG GCC CTT CTG AAG 143 Pro Asn Tyr Asn Pro Val Thr Phe Asp Ser Asp Val Ala Leu Leu Lys 35 40 45
CTG CAC TCT CCA ATT ACC TTC ACA AAT GGC GTT CAT AAA GTG TCG CTG 191 Leu His Ser Pro Ile Thr Phe Thr Asn Gly Val His Lys Val Ser Leu 50 55 60
GTC GAA AAA GGT CAA GAT CCT GTA CCT TAT TCA CCT GCG ATG ATC ACT 239 Val Glu Lys Gly Gin Asp Pro Val Pro Tyr Ser Pro Ala Met Ile Thr 65 70 75 GGC TGG GGC CAT ACA ATG GAA GGT GAT ACT AGT ATT TCG CAA ATT TTA 287 Gly Trp Gly His Thr Met Glu Gly Asp Thr Ser Ile Ser Gin Ile Leu 80 85 90 95
CAA GGA GCT GTG GTC CCA ATC GTA AAC AGA AAT GAT TGT CCG AAT TAT 335 Gin Gly Ala Val Val Pro Ile Val Asn Arg Asn Asp Cys Pro Asn Tyr 100 105 110
GGA CTC 341
Gly Leu (2) INFORMATION FOR SEQ ID NO: 141:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:
Lys Ser Arg Ile Gly Arg Gin Leu Lys Ile His Tyr Gly Asn Asn Asp 1 5 10 15 Trp His Phe Gly Phe Val Ser Ile Val Lys Lys Ala Ile Ile His Pro
20 25 30
Asn Tyr Asn Pro Val Thr Phe Asp Ser Asp Val Ala Leu Leu Lys Leu 35 40 45
His Ser Pro Ile Thr Phe Thr Asn Gly Val His Lys Val Ser Leu Val 50 55 60
Glu Lys Gly Gin Asp Pro Val Pro Tyr Ser Pro Ala Met Ile Thr Gly 65 70 75 80
Trp Gly His Thr Met Glu Gly Asp Thr Ser Ile Ser Gin Ile Leu Gin 85 90 95 Gly Ala Val Val Pro He Val Asn Arg Asn Asp Cys Pro Asn Tyr Gly
100 105 110
Leu
(2) INFORMATION FOR SEQ ID NO: 142: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 390 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..390
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142: GTA TAT GGC AGA GCA ACT CCA TCA CTG TTC ACC GTT GTA TCA GGC GCA 48 Val Tyr Gly Arg Ala Thr Pro Ser Leu Phe Thr Val Val Ser Gly Ala 1 5 10 15
CTT TAC TTA ACT GAA GGT GGA GAA CAT CAT GCT GTT GCT TCC ATC AAA 96 Leu Tyr Leu Thr Glu Gly Gly Glu His His Ala Val Ala Ser Ile Lys 20 25 30
TAT CAC GAA AAG TAC AGC CCA AAC ACT TTG GAC AAT GAT GTG GCA GTT 144 Tyr His Glu Lys Tyr Ser Pro Asn Thr Leu Asp Asn Asp Val Ala Val 35 40 45 TTG AAG TTG AAA CAG CCA TTG ACT TTC AAT GCT AAC CAG AAA CCT GTC 192 Leu Lys Leu Lys Gin Pro Leu Thr Phe Asn Ala Asn Gin Lys Pro Val 50 55 60
GCC TTG GCC TCA AAG GAT ACA CCT GGA GAC CTC AAA TGC AAA TTC TCT 240 Ala Leu Ala Ser Lys Asp Thr Pro Gly Asp Leu Lys Cys Lys Phe Ser 65 70 75 80
GGT TGG GGA TTA GAC GCA TAT CCA AGT GAT GTT TTA CCA AAT CAC TTA 288 Gly Trp Gly Leu Asp Ala Tyr Pro Ser Asp Val Leu Pro Asn His Leu 85 90 95 CAA AAA ATG GAT GTT CTG ACC TAC AAT AAC GCT GAC TGC CAA AAG TTC 336 Gin Lys Met Asp Val Leu Thr Tyr Asn Asn Ala Asp Cys Gin Lys Phe 100 105 110
CAT AAT GCT GGA CCT AAA TCT AAC ACA ATC TAC CCA GGA ATG TTG TGC 384 His Asn Ala Gly Pro Lys Ser Asn Thr Ile Tyr Pro Gly Met Leu Cys 115 120 125
GGA TTC 390
Gly Phe 130
(2) INFORMATION FOR SEQ ID NO: 143: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 130 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:
Val Tyr Gly Arg Ala Thr Pro Ser Leu Phe Thr Val Val Ser Gly Ala 1 5 10 15
Leu Tyr Leu Thr Glu Gly Gly Glu His His Ala Val Ala Ser Ile Lys 20 25 30 Tyr His Glu Lys Tyr Ser Pro Asn Thr Leu Asp Asn Asp Val Ala Val 35 40 45
Leu Lys Leu Lys Gin Pro Leu Thr Phe Asn Ala Asn Gin Lys Pro Val 50 55 60
Ala Leu Ala Ser Lys Asp Thr Pro Gly Asp Leu Lys Cys Lys Phe Ser 65 70 75 80
Gly Trp Gly Leu Asp Ala Tyr Pro Ser Asp Val Leu Pro Asn His Leu 85 90 95
Gin Lys Met Asp Val Leu Thr Tyr Asn Asn Ala Asp Cys Gin Lys Phe 100 105 110 His Asn Ala Gly Pro Lys Ser Asn Thr Ile Tyr Pro Gly Met Leu Cys 115 120 125 Gly Phe 130
(2) INFORMATION FOR SEQ ID NO: 144:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 261 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: smgle
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..261
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144:
CCG ACT ACC TTA GGC GGA TCT ACT GGT CAC AGC AAT GAT ATT GCC CTA 48 Pro Thr Thr Leu Gly Gly Ser Thr Gly His Ser Asn Asp Ile Ala Leu 1 5 10 15
ATC AAA GTC GAT AGA GAT ATC AAA TTC AGC AAA ACT GTC CAA CCT ATC 96 He Lys Val Asp Arg Asp Ile Lys Phe Ser Lys Thr Val Gin Pro Ile 20 25 30 AAA TTG CAC AAA AGT TTA ATA AAT GGA GGT GAA AAA TTG AAA ATT ACT 144 Lys Leu His Lys Ser Leu Ile Asn Gly Gly Glu Lys Leu Lys Ile Thr 35 40 45
GGA TGG GGA TTG ACG AAT CAA AGT CAT AGT GAT GAA CCA GAT GTT CTT 192 Gly Trp Gly Leu Thr Asn Gin Ser His Ser Asp Glu Pro Asp Val Leu 50 55 60
CAA GAG TTG CAT GTA AAA GCA CTT ACT GAT TCT GAG TGC GAG AAA GCT 240 Gin Glu Leu His Val Lys Ala Leu Thr Asp Ser Glu Cys Glu Lys Ala 65 70 75 80
ACA GGT GAA GAC CAT CCT ACA 261 Thr Gly Glu Asp His Pro Thr
85
(2) INFORMATION FOR SEQ ID NO: 145:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:
Pro Thr Thr Leu Gly Gly Ser Thr Gly His Ser Asn Asp Ile Ala Leu 1 5 10 15 He Lys Val Asp Arg Asp Ile Lys Phe Ser Lys Thr Val Gin Pro lie 20 25 30
Lys Leu His Lys Ser Leu Ile Asn Gly Gly Glu Lys Leu Lys Ile Thr 35 40 45 Gly Trp Gly Leu Thr Asn Gin Ser His Ser Asp Glu Pro Asp Val Leu 50 55 60
Gin Glu Leu His Val Lys Ala Leu Thr Asp Ser Glu Cys Glu Lys Ala 65 70 75 80
Thr Gly Glu Asp His Pro Thr 85
(2) INFORMATION FOR SEQ ID NO: 146:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 612 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA
(IX) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..612
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 146:
GTT TGC AAG ATG GCA ACA ACT CTG TTT TCT CTT GCC ATA GTG TTG TTT 48 Val Cys Lys Met Ala Thr Thr Leu Phe Ser Leu Ala Ile Val Leu Phe 1 5 10 15 ATC TCT ACT TCA GAA GAA TCA GCA CAT ATT TCG CAA GGA TCT CGA ATA 96 He Ser Thr Ser Glu Glu Ser Ala His Ile Ser Gin Gly Ser Arg Ile 20 25 30
TTA GGA GGT AGA AAT GCA AAA CTC GGA GAT GCT CCT TAT CAA GTA TCA 144 Leu Gly Gly Arg Asn Ala Lys Leu Gly Asp Ala Pro Tyr Gin Val Ser 35 40 45
CTA AGA GAT AAT TTT GGA CAT TTT TGT GGA GGT TCT ATC ATT AGT GAA 192 Leu Arg Asp Asn Phe Gly His Phe Cys Gly Gly Ser Ile Ile Ser Glu 50 55 60
AAT TTT GTG ATT ACA GCA GCT CAT TGC CTT GAT GGA TAC ACA GTG AGC 240 Asn Phe Val Ile Thr Ala Ala His Cys Leu Asp Gly Tyr Thr Val Ser 65 70 75 80
AAA TTT AAA GTA GCA ACT GGT ACA ATC GAG TAT GGT AAA GGA GGT GAC 288 Lys Phe Lys Val Ala Thr Gly Thr Ile Glu Tyr Gly Lys Gly Gly Asp 85 90 95 GAA TAT AAA GTC ATC AAC TTT GTT GTC CGG GAT GAC TTT CAA TAT GTC 336 Glu Tyr Lys Val Ile Asn Phe Val Val Arg Asp Asp Phe Gin Tyr Val 100 105 110
AAA TTA GAA AAT GAT ATT GCT ATA GTA CAA ATA GAT GGA TCT TTT AAA 384 Lys Leu Glu Asn Asp Ile Ala Ile Val Gin Ile Asp Gly Ser Phe Lys 115 - 120 125
TTT AAT GAC TAT GTA AAG CCT ATA AAA TTG CCA AAT CAA GAT ACT AAA 432 Phe Asn Asp Tyr Val Lys Pro Ile Lys Leu Pro Asn Gin Asp Thr Lys 130 135 140
GTT GGC GCG GAT GTT GTC CTA ACA GGA TGG GGA AAA ATG GAA GGT GGT 480 Val Gly Ala Asp Val Val Leu Thr Gly Trp Gly Lys Met Glu Gly Gly 145 150 155 160
AAA AAT CCA GAA ACT CTA CAA ATC TTG AAC TTA AAA ACA ATT GAT CAA 528 Lys Asn Pro Glu Thr Leu Gin Ile Leu Asn Leu Lys Thr Ile Asp Gin
165 170 175
GGA GAA TGC AAA CAA GCT TTG GCA GAA GTA AAC ACA GTT CTT CCA AGT 576 Gly Glu Cys Lys Gin Ala Leu Ala Glu Val Asn Thr Val Leu Pro Ser 180 185 190 CAA ATT TGT ACC TAT GTT GGT GTT GGC AAA GGA GCT 612
Gin Ile Cys Thr Tyr Val Gly Val Gly Lys Gly Ala 195 200
(2) INFORMATION FOR SEQ ID NO: 147:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 204 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(li) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: Val Cys Lys Met Ala Thr Thr Leu Phe Ser Leu Ala Ile Val Leu Phe
1 5 10 15
Ile Ser Thr Ser Glu Glu Ser Ala His Ile Ser Gin Gly Ser Arg Ile 20 25 30
Leu Gly Gly Arg Asn Ala Lys Leu Gly Asp Ala Pro Tyr Gin Val Ser 35 40 45
Leu Arg Asp Asn Phe Gly His Phe Cys Gly Gly Ser Ile Ile Ser Glu 50 55 60
Asn Phe Val Ile Thr Ala Ala His Cys Leu Asp Gly Tyr Thr Val Ser 65 70 75 80 Lys Phe Lys Val Ala Thr Gly Thr He Glu Tyr Gly Lys Gly Gly Asp
85 90 95
Glu Tyr Lys Val Ile Asn Phe Val Val Arg Asp Asp Phe Gin Tyr Val 100 105 110
Lys Leu Glu Asn Asp Ile Ala Ile Val Gin Ile Asp Gly Ser Phe Lys 115 120 125 Phe Asn Asp Tyr Val Lys Pro Ile Lys Leu Pro Asn Gin Asp Thr Lys 130 135 140
Val Gly Ala Asp Val Val Leu Thr Gly Trp Gly Lys Met Glu Gly Gly 145 150 155 160 Lys Asn Pro Glu Thr Leu Gin Ile Leu Asn Leu Lys Thr Ile Asp Gin
165 170 175
Gly Glu Cys Lys Gin Ala Leu Ala Glu Val Asn Thr Val Leu Pro Ser 180 185 190
Gin Ile Cys Thr Tyr Val Gly Val Gly Lys Gly Ala 195 200
(2) INFORMATION FOR SEQ ID NO: 148:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 641 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 3..641
(D) OTHER INFORMATION: /note= "At pos. bp 91, change G to N; at pos. bp 385, change A to M; at bp 404, change A to W. At pos. aa 30, 128 and 134, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: CA AAC ATG AAA CTT TAT GTT TTT ACA TTG ATC ATC GCA GCC GCT GTT 47
Asn Met Lys Leu Tyr Val Phe Thr Leu Ile Ile Ala Ala Ala Val 1 5 10 15
GCT GCA CCA ACA AAC GAA ATC GAT TGG ATA GGC TTT CCC GAA GNT TTT 95 Ala Ala Pro Thr Asn Glu Ile Asp Trp Ile Gly Phe Pro Glu Xaa Phe 20 25 30
CCT CGT GTA GTT GGT GGC CAG ACT GCC AAA CCT CAC CAG TTC CCC TGG 143 Pro Arg Val Val Gly Gly Gin Thr Ala Lys Pro His Gin Phe Pro Trp 35 40 45
CAG GTT TCC CTG CAA AGG TCC GGA AAG CAT TTG TGC GGC GGT TCC ATC 191 Gin Val Ser Leu Gin Arg Ser Gly Lys His Leu Cys Gly Gly Ser Ile 50 55 60
TTG AAC GAC AGG TGG GTC TTG ACA GCC GCG CAC TGC ATC AGC GGA ACT 239 Leu Asn Asp Arg Trp Val Leu Thr Ala Ala His Cys Ile Ser Gly Thr 65 70 75 GAA AAT TAC GAG GCT GTA GTC GGA AAA CAC GAT TTG TCG AAA AGC GAA 287 Glu Asn Tyr Glu Ala Val Val Gly Lys His Asp Leu Ser Lys Ser Glu 80 85 90 95
TCA TCT GAG CAA CGT TGC GCC TAC AAG AGG ACC ATC GTC CAC TCG TCC 335 Ser Ser Glu Gin Arg Cys Ala Tyr Lys Arg Thr Ile Val His Ser Ser 100 105 110
TTC ACT GGA AGG GTG GGT CCT TAC GAT GTC GCT TTG ATT GAA TTA GAA 383 Phe Thr Gly Arg Val Gly Pro Tyr Asp Val Ala Leu Ile Glu Leu Glu 115 120 125
AMA CCT TTC AAA TTG AAC GAW AAA TGT AAG CCA ATC AGA CTT CCA TTG 431 Xaa Pro Phe Lys Leu Asn Xaa Lys Cys Lys Pro He Arg Leu Pro Leu 130 135 140
AAA GAT GAA GCA CAT TCT GGA CAA GTG ACG CTT TCT GGT TGG GGA TCT 479 Lys Asp Glu Ala His Ser Gly Gin Val Thr Leu Ser Gly Trp Gly Ser 145 150 155
ACT TCA ACT ACC ATC TTC CCC ACT TAC CCA AAT GAA CTA CAG TAT GTT 527 Thr Ser Thr Thr Ile Phe Pro Thr Tyr Pro Asn Glu Leu Gin Tyr Val 160 165 170 175
GAC AAA CCG ATT GTT CCA TAT ACT GAT TGC GAA AAT GCT ATG GGC GGA 575 Asp Lys Pro Ile Val Pro Tyr Thr Asp Cys Glu Asn Ala Met Gly Gly 180 185 190
CCA GGA GCA TCT CCT CTT GAT CCT TTG AAC ATC TGC ACT GGT CCC TTG 623 Pro Gly Ala Ser Pro Leu Asp Pro Leu Asn Ile Cys Thr Gly Pro Leu 195 200 205
ACT GGT GGC ATC AGT GCT 641
Thr Gly Gly Ile Ser Ala 210
(2) INFORMATION FOR SEQ ID NO: 149: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:
Asn Met Lys Leu Tyr Val Phe Thr Leu Ile Ile Ala Ala Ala Val Ala 1 5 10 15
Ala Pro Thr Asn Glu Ile Asp Trp Ile Gly Phe Pro Glu Xaa Phe Pro 20 25 30 Arg Val Val Gly Gly Gin Thr Ala Lys Pro His Gin Phe Pro Trp Gin 35 40 45
Val Ser Leu Gin Arg Ser Gly Lys His Leu Cys Gly Gly Ser Ile Leu 50 55 60
Asn Asp Arg Trp Val Leu Thr Ala Ala His Cys Ile Ser Gly Thr Glu 65 70 75 80
Asn Tyr Glu Ala Val Val Gly Lys His Asp Leu Ser Lys Ser Glu Ser 85 90 95 Ser Glu Gin Arg Cys Ala Tyr Lys Arg Thr Ile Val His Ser Ser Phe 100 . 105 110
Thr Gly Arg Val Gly Pro Tyr Asp Val Ala Leu Ile Glu Leu Glu Xaa 115 120 125 Pro Phe Lys Leu Asn Xaa Lys Cys Lys Pro Ile Arg Leu Pro Leu Lys 130 135 140
Asp Glu Ala His Ser Gly Gin Val Thr Leu Ser Gly Trp Gly Ser Thr 145 150 155 160
Ser Thr Thr He Phe Pro Thr Tyr Pro Asn Glu Leu Gin Tyr Val Asp 165 170 175
Lys Pro Ile Val Pro Tyr Thr Asp Cys Glu Asn Ala Met Gly Gly Pro 180 185 190
Gly Ala Ser Pro Leu Asp Pro Leu Asn Ile Cys Thr Gly Pro Leu Thr 195 200 205 Gly Gly Ile Ser Ala
210
(2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 626 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA
(IX) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 3..626
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:
TG ACT ATG AAC TTT GCA TTG TGG TTT GTG ACT CTT GTG TCC ATC ACC 47
Thr Met Asn Phe Ala Leu Trp Phe Val Thr Leu Val Ser Ile Thr 1 5 10 15
AGC GCT GAT CCA ATT CGT GTT TCC TCA ATT GGA AAT GCT AAT ATG GAA 95 Ser Ala Asp Pro He Arg Val Ser Ser Ile Gly Asn Ala Asn Met Glu 20 25 30
TCT CGC GTT GTT GGT GGC GAA AAC GCA GAA GTA GGA GCT GCT CCT TAC 143 Ser Arg Val Val Gly Gly Glu Asn Ala Glu Val Gly Ala Ala Pro Tyr
35 40 45
CAA GTT TCT TTG AAA TAC AAT AAT GGA GCT CAT TTT TGC GGA GGT GTC 191 Gin Val Ser Leu Lys Tyr Asn Asn Gly Ala His Phe Cys Gly Gly Val 50 55 60 GTG ATA ACC AAA ACT TGG GTG CTA ACT GCT GCA CGT TGT ATT CAC GAG 239 Val Ile Thr Lys Thr Trp Val Leu Thr Ala Ala Arg Cys Ile His Glu 65 70 75
GAG GAA CCC GAC AGA TTC ACA GTG GTT GTT GGT ACC AAC ACT TTA AAT 287 Glu Glu Pro Asp Arg Phe Thr Val Val Val Gly Thr Asn Thr Leu Asn
80 85 90 95
GCT GGA GGA GAA GGT TAC AAC GTT AAA CAG ATA GTT ATT CAT ATG CAA 335
Ala Gly Gly Glu Gly Tyr Asn Val Lys Gin Ile Val Ile His Met Gin 100 105 110
TTC AAC CAA GTT TAT CTT CTG AAC GAT ATT GGT TTG ATC GAA ACC GAA 383 Phe Asn Gin Val Tyr Leu Leu Asn Asp Ile Gly Leu Ile Glu Thr Glu 115 120 125
TCA CCA ATA CAA TTT CAT GAT CTG GTT AAG CCA ATC TCA GTC CCC AAT 431 Ser Pro Ile Gin Phe His Asp Leu Val Lys Pro Ile Ser Val Pro Asn 130 135 140
ATG CAT GTT GAA GAT GGC ACG AGA GTT ACT CTT TTT GGA TGG GGA AAT 479 Met His Val Glu Asp Gly Thr Arg Val Thr Leu Phe Gly Trp Gly Asn 145 150 155 TTA ACG GCT GAA GGA CAT ATG CCA AAC CAT TTA CAA ACA ATT GAT TTG 527 Leu Thr Ala Glu Gly His Met Pro Asn His Leu Gin Thr Ile Asp Leu 160 165 170 175
TTA ACA ATA AAT CTG AGT GAA TGC TCC CGA TTA TTA CCT GAA CCA AGT 575 Leu Thr Ile Asn Leu Ser Glu Cys Ser Arg Leu Leu Pro Glu Pro Ser 180 185 190
ATG ATC AGT ACA AAA CAC ATT TGC ACC TTT GTT TCA TAT GGA AAA GGA 623 Met lie Ser Thr Lys His Ile Cys Thr Phe Val Ser Tyr Gly Lys Gly 195 200 205
CTT 626 Leu
(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:
Thr Met Asn Phe Ala Leu Trp Phe Val Thr Leu Val Ser Ile Thr Ser 1 5 10 15
Ala Asp Pro He Arg Val Ser Ser He Gly Asn Ala Asn Met Glu Ser 20 25 30
Arg Val Val Gly Gly Glu Asn Ala Glu Val Gly Ala Ala Pro Tyr Gin 35 40 45 Val Ser Leu Lys Tyr Asn Asn Gly Ala His Phe Cys Gly Gly Val Val 50 55 60
Ile Thr Lys Thr Trp Val Leu Thr Ala Ala Arg Cys Ile His Glu Glu 65 70 75 80 Glu Pro Asp Arg Phe Thr Val Val Val Gly Thr Asn Thr Leu Asn Ala 85 90 95
Gly Gly Glu Gly Tyr Asn Val Lys Gin Ile Val Ile His Met Gin Phe 100 105 110 Asn Gin Val Tyr Leu Leu Asn Asp He Gly Leu Ile Glu Thr Glu Ser 115 120 125
Pro He Gin Phe His Asp Leu Val Lys Pro Ile Ser Val Pro Asn Met 130 135 140
His Val Glu Asp Gly Thr Arg Val Thr Leu Phe Gly Trp Gly Asn Leu 145 150 155 160
Thr Ala Glu Gly His Met Pro Asn His Leu Gin Thr Ile Asp Leu Leu 165 170 175
Thr Ile Asn Leu Ser Glu Cys Ser Arg Leu Leu Pro Glu Pro Ser Met 180 185 190 Ile Ser Thr Lys His Ile Cys Thr Phe Val Ser Tyr Gly Lys Gly Leu 195 200 205
(2) INFORMATION FOR SEQ ID NO:152:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 815 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: cDNA
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..762
(D) OTHER INFORMATION: /note= "At pos. bp 454, change G to V; at 456, G to V; at 457, A to M; at 460, A to R; at 470, G to S; at 493, A to R. At pos. aa 120, 136, 152, 153, 154, 157 and 165, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152:
ATT TTA TTA AGC GCA TTA TTT GCA AGT GTA ATT TGC TCC TTT AAC GCG 48 Ile Leu Leu Ser Ala Leu Phe Ala Ser Val Ile Cys Ser Phe Asn Ala 1 5 10 15 GAA GTA CAA AAT CGA ATC GTT GGT GGC AAT GAT GTA AGT ATT TCA AAA 96 Glu Val Gin Asn Arg Ile Val Gly Gly Asn Asp Val Ser Ile Ser Lys 20 25 30
ATT GGG TGG CAA GTA TCT ATT CAA AGT AAT AAC CAA CAT TTC TGT GGT 144 lie Gly Trp Gin Val Ser Ile Gin Ser Asn Asn Gin His Phe Cys Gly 35 40 45
GGT TCA ATC ATT GCT AAA GAT TGG GTA CTG ACT TCT TCT CAA TGC GTC 192 Gly Ser Ile Ile Ala Lys Asp Trp Val Leu Thr Ser Ser Gin Cys Val 50 55 60
GTG GAC AAA CAA AGT CCA CCG AAG GAT TTA ACT GTT CGT GTT GGA ACT 240 Val Asp Lys Gin Ser Pro Pro Lys Asp Leu Thr Val Arg Val Gly Thr 65 70 75 80
AGC ACT CAC AAT GAT GGA GGA AAA GTG TAT GAT GTT ATT GAA ATT ATA 288 Ser Thr His Asn Asp Gly Gly Lys Val Tyr Asp Val He Glu Ile Ile 85 90 95
AAA CAT CCG AAA TAT AAT AAA GCA GTG CCA GAT GAT TTT GAT GTT GCA 336 Lys His Pro Lys Tyr Asn Lys Ala Val Pro Asp Asp Phe Asp Val Ala 100 105 110
CTT TTA CGG ATC AAA GAG CCA ATC ATT TAC TCC ATG CAC AGT AAC TCC 384 Leu Leu Arg Ile Lys Glu Pro Xaa Ile Tyr Ser Met His Ser Asn Ser 115 120 125
TGT AAA ATT AAT ACA ATC GGG AAA GAA GTA CCG AAG GGA ACA ACT TTG 432 Cys Lys Ile Asn Thr Ile Gly Xaa Glu Val Pro Lys Gly Thr Thr Leu 130 135 140 AGT GTA ACT GGA TGG GGC GCM VCV MAG RAA TGG GGG CSC AAT TTC GCC 480 Ser Val Thr Gly Trp Gly Ala Xaa Xaa Xaa Trp Gly Xaa Asn Phe Ala 145 150 155 160
AAA GTT ACA AGA RAG TTA AAG TTA AAG CTA CTC AAG TCA AGA ATG CAA 528 Lys Val Thr Arg Xaa Leu Lys Leu Lys Leu Leu Lys Ser Arg Met Gin 165 170 175
GAA CAG TCT GCT ATT AAC AGT GAC ATC ATT TCT GAC AGT ATG ATG TGC 576 Glu Gin Ser Ala He Asn Ser Asp Ile Ile Ser Asp Ser Met Met Cys 180 . 185 190
GCT GGT TTT CCT CAA GGA CAA AAA GAT ACT TGT CAT GGG GAT AGC GGT 624 Ala Gly Phe Pro Gin Gly Gin Lys Asp Thr Cys His Gly Asp Ser Gly 195 200 205
GGC ACT GTA GAT AAA AAA CAG GTT CAA GTA GGA GTT ATA TCC TGG AGG 672 Gly Thr Val Asp Lys Lys Gin Val Gin Val Gly Val Ile Ser Trp Arg 210 215 220 CGA GGA TGC GCG CGA CCT GGA TAT CCT GGC GTA TAT ACA AAA TTG AGC 720 Arg Gly Cys Ala Arg Pro Gly Tyr Pro Gly Val Tyr Thr Lys Leu Ser 225 230 235 240
CAC CCG GAA ATC CAA CAG TTT ATT AAA AAC AAT GTA AAA CTT 762
His Pro Glu Ile Gin Gin Phe Ile Lys Asn Asn Val Lys Leu 245 250
TAAATCATAA AACTGTATGA AAATAACAAT AACAATTACG GGAAAAAAAA AAA 815
(2) INFORMATION FOR SEQ ID NO: 153:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 254 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 153: He Leu Leu Ser Ala Leu Phe Ala Ser Val Ile Cys Ser Phe Asn Ala 1 5 10 15
Glu Val Gin Asn Arg Ile Val Gly Gly Asn Asp Val Ser Ile Ser Lys 20 25 30 Ile Gly Trp Gin Val Ser lie Gin Ser Asn Asn Gin His Phe Cys Gly 35 40 45
Gly Ser Ile Ile Ala Lys Asp Trp Val Leu Thr Ser Ser Gin Cys Val 50 55 60
Val Asp Lys Gin Ser Pro Pro Lys Asp Leu Thr Val Arg Val Gly Thr 65 70 75 80
Ser Thr His Asn Asp Gly Gly Lys Val Tyr Asp Val Ile Glu Ile He 85 90 95
Lys His Pro Lys Tyr Asn Lys Ala Val Pro Asp Asp Phe Asp Val Ala 100 105 110 Leu Leu Arg Ile Lys Glu Pro Xaa Ile Tyr Ser Met His Ser Asn Ser 115 120 125
Cys Lys Ile Asn Thr Ile Gly Xaa Glu Val Pro Lys Gly Thr Thr Leu 130 135 140
Ser Val Thr Gly Trp Gly Ala Xaa Xaa Xaa Trp Gly Xaa Asn Phe Ala 145 150 155 160
Lys Val Thr Arg Xaa Leu Lys Leu Lys Leu Leu Lys Ser Arg Met Gin 165 170 175
Glu Gin Ser Ala Ile Asn Ser Asp Ile Ile Ser Asp Ser Met Met Cys 180 185 190 Ala Gly Phe Pro Gin Gly Gin Lys Asp Thr Cys His Gly Asp Ser Gly 195 200 205
Gly Thr Val Asp Lys Lys Gin Val Gin Val Gly Val Ile Ser Trp Arg 210 215 220
Arg Gly Cys Ala Arg Pro Gly Tyr Pro Gly Val Tyr Thr Lys Leu Ser 225 230 235 240
His Pro Glu Ile Gin Gin Phe Ile Lys Asn Asn Val Lys Leu 245 250
(2) INFORMATION FOR SEQ ID NO:154:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 177 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: CACGAGATTT TATTAAGCGC ATTATTTGCA AGTGTAATTT GCTCCTTTAA CGCGGAAGTA 60
CAAAATCGAA TCGTTGGTGG CAATGATGTA AGTATTTCAA AAATTGGGTG GCAAGTATCT 120
ATTCAAAGTA ATAAACAACA TTTCTGTGGT GGTTCAATCA TTGCTAAAGA TGGGTCC 177
(2) INFORMATION FOR SEQ ID NO: 155: (1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ll) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:
GGGTTCGAAT TTGTGGATCG AAAAGGCAGA TATTACGATG TAGAAAGATT TGTGATGCAC 60
CATAATTATA CTGGAAAGAT AGTTGCCAAT GTCGCTGATA TAGGTCTAAT AAAACTAGCA 120
GAAGATATAA AATTCAGTGA CAAGGTACAA CCTGTAAAAA TTCATCAAAC TCAAATCAAG 180 GGCGGAGAGA TTTGCAAAGC TACTGGATGG GGCAGGTTGG GTGCTGATCA GCCTGTACCA 240
AATAAATTAC AACAATTGGA GACAATTGCT ATTAGTGATG AGAAATGTTA TGCAGATACA 300
GGGTTTTTAG AACCTACATC TCAAATATGT GTATTCAGTG CATTTGGAAA AGGAGTTGT 359
(2) INFORMATION FOR SEQ ID NO: 156:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 855 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: cDNA (IX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..759
(D) OTHER INFORMATION: /note= "At pos. bp 693, change C to N. At pos. aa 231, substitute Xaa." (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:
ATG GCT TAT ATT ATA TTA GTT ACT TTA ATT AGT TTG GGA TCG TTG GTT 48 Met Ala Tyr Ile Ile Leu Val Thr Leu Ile Ser Leu Gly Ser Leu Val 1 5 10 15
TCT TCC GAA TAC CTT TCG TTT TCT ACT GAT CCT CGG ATA ATT GGT GGT 96 Ser Ser Glu Tyr Leu Ser Phe Ser Thr Asp Pro Arg Ile Ile Gly Gly
20 25 30 GAA GAT GCT CCC GAG GGT TCT GCA CCA TAT CAG GTT TCA TTA AGA AAT 144 Glu Asp Ala Pro Glu Gly Ser Ala Pro Tyr Gin Val Ser Leu Arg Asn 35 40 45
TCT GAC TTG CAG CAT TTT TGT GGT GGT TCC ATC CTA AAC AAA CGA TGG 192 Ser Asp Leu Gin His Phe Cys Gly Gly Ser Ile Leu Asn Lys Arg Trp 50 55 60
ATT TTA ACA GCA GCA CAT TGT CTC GAA CCT GGT TTT TTA AAT TCT GTA 240 Ile Leu Thr Ala Ala His Cys Leu Glu Pro Gly Phe Leu Asn Ser Val 65 70 75 80 TAC ATG GGT TCG AAT TTG TTG GAT CGA AAA GGC AGA TAT TAC GAT GTA 288 Tyr Met Gly Ser Asn Leu Leu Asp Arg Lys Gly Arg Tyr Tyr Asp Val 85 90 95
GAA AGA TTT GTG ATG CAC CAT AAT TAT ACT GGA AAG ATA GTT GCC AAT 336 Glu Arg Phe Val Met His His Asn Tyr Thr Gly Lys Ile Val Ala Asn 100 105 110
GTC GCT GAT ATA GGT CTA ATA AAA CTA GCA GAA GAT ATA AAA TTC AGT 384 Val Ala Asp Ile Gly Leu Ile Lys Leu Ala Glu Asp He Lys Phe Ser 115 120 125
GAC AAG GTA CAA CCT GTA AAA ATT CAT CAA ACT CAA ATC AAG GGC GGA 432 Asp Lys Val Gin Pro Val Lys Ile His Gin Thr Gin Ile Lys Gly Gly 130 135 140
GAG ATT TGC AAA GCT ACT GGA TGG GGC AGG TTG GGT GCT GAT CAG CCT 480 Glu Ile Cys Lys Ala Thr Gly Trp Gly Arg Leu Gly Ala Asp Gin Pro 145 150 155 160 GTA CCA AAT AAA TTA CAA CAA TTG GAG ACA ATT GCT ATT AGT GAT GAG 528 Val Pro Asn Lys Leu Gin Gin Leu Glu Thr Ile Ala Ile Ser Asp Glu 165 170 175
AAA TGT TAT GCA GAT ACA GGG TTT TTA GAA CCT ACA TCT CAA ATA TGT 576 Lys Cys Tyr Ala Asp Thr Gly Phe Leu Glu Pro Thr Ser Gin Ile Cys 180 185 190
GTA TTC AGT GCA TTT GGA AAA GGA GTT TGT TTT GGA GAT TCT GGT GGT 624 Val Phe Ser Ala Phe Gly Lys Gly Val Cys Phe Gly Asp Ser Gly Gly 195 200 205
CCA TTA GTT TAC AAA GGT GAA CAA GTA GGA GTT GCA TCA TTC ATC ATG 672 Pro Leu Val Tyr Lys Gly Glu Gin Val Gly Val Ala Ser Phe Ile Met 210 215 220
ATC ACT TGT GGT GGT GGC AGN CCA GAT GTA TTT GTT AGA GTA CTC GAT 720 He Thr Cys Gly Gly Gly Xaa Pro Asp Val Phe Val Arg Val Leu Asp 225 230 235 240 TAT CAG GAT TGG ATA AAT TCA TTT ATT TCT GGA GAT AAC TAGTCTTTAA 769 Tyr Gin Asp Trp Ile Asn Ser Phe Ile Ser Gly Asp Asn 245 250
TGTAAAATGA ACTATTATAA TATATATTTT TTATTCTTAT AAAATATATA CATTTTATTA 829
CGCACAAAAA AAAAAAAAAA AAAAAA 855 (2) INFORMATION FOR SEQ ID NO: 157:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 253 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:
Met Ala Tyr Ile Ile Leu Val Thr Leu Ile Ser Leu Gly Ser Leu Val 1 5 10 15 Ser Ser Glu Tyr Leu Ser Phe Ser Thr Asp Pro Arg Ile Ile Gly Gly
20 25 30
Glu Asp Ala Pro Glu Gly Ser Ala Pro Tyr Gin Val Ser Leu Arg Asn 35 40 45
Ser Asp Leu Gin His Phe Cys Gly Gly Ser Ile Leu Asn Lys Arg Trp 50 55 60
Ile Leu Thr Ala Ala His Cys Leu Glu Pro Gly Phe Leu Asn Ser Val 65 70 75 80
Tyr Met Gly Ser Asn Leu Leu Asp Arg Lys Gly Arg Tyr Tyr Asp Val 85 90 95 Glu Arg Phe Val Met His His Asn Tyr Thr Gly Lys Ile Val Ala Asn
100 105 110
Val Ala Asp Ile Gly Leu Ile Lys Leu Ala Glu Asp Ile Lys Phe Ser 115 120 125
Asp Lys Val Gin Pro Val Lys Ile His Gin Thr Gin Ile Lys Gly Gly 130 135 140
Glu Ile Cys Lys Ala Thr Gly Trp Gly Arg Leu Gly Ala Asp Gin Pro 145 150 155 160
Val Pro Asn Lys Leu Gin Gin Leu Glu Thr Ile Ala Ile Ser Asp Glu 165 170 175 Lys Cys Tyr Ala Asp Thr Gly Phe Leu Glu Pro Thr Ser Gin lie Cys
180 185 190
Val Phe Ser Ala Phe Gly Lys Gly Val Cys Phe Gly Asp Ser Gly Gly 195 200 205
Pro Leu Val Tyr Lys Gly Glu Gin Val Gly Val Ala Ser Phe Ile Met 210 215 220
Ile Thr Cys Gly Gly Gly Xaa Pro Asp Val Phe Val Arg Val Leu Asp 225 230 235 240
Tyr Gin Asp Trp Ile Asn Ser Phe Ile Ser Gly Asp Asn 245 250 (2) INFORMATION FOR SEQ ID NO: 158:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 864 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: cDNA
(IX) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 2..781
(D) OTHER INFORMATION: /note= "At pos. bp 456, change G to K; at pos. bp 504, change A to R. At pos. aa 152 and 168, substitute Xaa."
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 158: T ACA AAA CCT ATT ACA ATT CAA AAG TTG TTC CAA ATG ATG GCA AAT 46
Thr Lys Pro Ile Thr Ile Gin Lys Leu Phe Gin Met Met Ala Asn 1 5 10 15
TTT GTG CTA TTC ACC TTA CTA GCC TTA GTA TCA GTA GCA TGT TCC AAA 94 Phe Val Leu Phe Thr Leu Leu Ala Leu Val Ser Val Ala Cys Ser Lys 20 25 30
TAT ATT GAT CCA AGA ATC ATT GGA GGC GAA GAT GCT CCT GAA GGC TCG 142 Tyr Ile Asp Pro Arg Ile Ile Gly Gly Glu Asp Ala Pro Glu Gly Ser 35 40 45
GCT CCG TAT CAA GTT TCA CTG AGA AAT CGG GAC CTA GAG CAT TTC TGT 190 Ala Pro Tyr Gin Val Ser Leu Arg Asn Arg Asp Leu Glu His Phe Cys 50 55 60
GGT GGC TCC ATC TTA AAC AAA CGT TGG ATT GTG ACG GCT GCA CAT TGC 238 Gly Gly Ser Ile Leu Asn Lys Arg Trp Ile Val Thr Ala Ala His Cys 65 70 75 CTA AAA CCT GGC ATT TTA AAA TCC GTC TAT ATG GGA TCA AAC TCA TTA 286 Leu Lys Pro Gly Ile Leu Lys Ser Val Tyr Met Gly Ser Asn Ser Leu 80 85 90 95
GAT GGC AAT GGT ACA TAC TAC GAC GTC GAA CGT TTT GTG ATG CAT CAT 334 Asp Gly Asn Gly Thr Tyr Tyr Asp Val Glu Arg Phe Val Met His His 100 105 110
AAA TAT ACA CCA AAA ATT ACT GTC AAC TAT GCT GAT ATT GGT CTA ATA 382 Lys Tyr Thr Pro Lys Ile Thr Val Asn Tyr Ala Asp Ile Gly Leu Ile 115 120 125
AAA GTG ACA AAA GAC ATT ATA TTC AGT GAC AAA GTT CAA CCA ATC AAA 430 Lys Val Thr Lys Asp Ile Ile Phe Ser Asp Lys Val Gin Pro Ile Lys 130 135 140
ATA GCA AAA AAA ATA TCA AGG GTG GKG AAT CTG CAA GGC CAC TGG TTG 478 He Ala Lys Lys Ile Ser Arg Val Xaa Asn Leu Gin Gly His Trp Leu 145 150 155 GGG TCG ATT GGC GGA TGG GGC CCC CRG TAC CAA ACG AAT TGC AAC AAG 526 Gly Ser He Gly Gly Trp Gly Pro Xaa Tyr Gin Thr Asn Cys Asn Lys 160 165 170 175
GTG GAA ACC ACT GCA ATA ACA AAT GAA AAG TGC TAC GAA TTG TCT CAA 574 Val Glu Thr Thr Ala Ile Thr Asn Glu Lys Cys Tyr Glu Leu Ser Gin 180 185 190
TTC GTT GAG CCA ACT TCG CAA ATA TGT ACA TTA AGG GAA TTT TTA AGA 622 Phe Val Glu Pro Thr Ser Gin Ile Cys Thr Leu Arg Glu Phe Leu Arg 195 200 205
GGC ATT TGC TTT GGT GAT TCT GGT GGA CCA CTG GTT TAC AAA GGT GAA 670 Gly Ile Cys Phe Gly Asp Ser Gly Gly Pro Leu Val Tyr Lys Gly Glu 210 215 220
CTG GTT GGC GTT TCT TCG TTT GTC TTG TAC ACT TGC GGA GCT GGA CGC 718 Leu Val Gly Val Ser Ser Phe Val Leu Tyr Thr Cys Gly Ala Gly Arg 225 230 235 CCA GAT GTT TTT GTT AAA GTG CGT GAT TTC CAA TCT TGG ATC AAT TCT 766 Pro Asp Val Phe Val Lys Val Arg Asp Phe Gin Ser Trp Ile Asn Ser 240 245 250 255
GAA ATT AGA AAA AAA TAAATAGATT TCAATCATGA TTTGTTGTAA TAAAAAATGG 821 Glu Ile Arg Lys Lys 260
TTAAATAAAG GCAGCATAAT TTAAAAAAAA AAAAAAAAAA AAA 864
(2) INFORMATION FOR SEQ ID NO: 159:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:
Thr Lys Pro Ile Thr He Gin Lys Leu Phe Gin Met Met Ala Asn Phe 1 5 10 15
Val Leu Phe Thr Leu Leu Ala Leu Val Ser Val Ala Cys Ser Lys Tyr 20 25 30
He Asp Pro Arg Ile He Gly Gly Glu Asp Ala Pro Glu Gly Ser Ala 35 40 45 Pro Tyr Gin Val Ser Leu Arg Asn Arg Asp Leu Glu His Phe Cys Gly 50 55 60
Gly Ser Ile Leu Asn Lys Arg Trp Ile Val Thr Ala Ala His Cys Leu 65 70 75 80
Lys Pro Gly Ile Leu Lys Ser Val Tyr Met Gly Ser Asn Ser Leu Asp 85 90 95
Gly Asn Gly Thr Tyr Tyr Asp Val Glu Arg Phe Val Met His His Lys 100 105 110 Tyr Thr Pro Lys Ile Thr Val Asn Tyr Ala Asp Ile Gly Leu Ile Lys 115 120 125
Val Thr Lys Asp Ile Ile Phe Ser Asp Lys Val Gin Pro Ile Lys Ile 130 135 140 Ala Lys Lys Ile Ser Arg Val Xaa Asn Leu Gin Gly His Trp Leu Gly 145 150 155 160
Ser He Gly Gly Trp Gly Pro Xaa Tyr Gin Thr Asn Cys Asn Lys Val 165 170 175
Glu Thr Thr Ala Ile Thr Asn Glu Lys Cys Tyr Glu Leu Ser Gin Phe 180 185 190
Val Glu Pro Thr Ser Gin Ile Cys Thr Leu Arg Glu Phe Leu Arg Gly 195 200 205
Ile Cys Phe Gly Asp Ser Gly Gly Pro Leu Val Tyr Lys Gly Glu Leu 210 215 220 Val Gly Val Ser Ser Phe Val Leu Tyr Thr Cys Gly Ala Gly Arg Pro 225 230 235 240
Asp Val Phe Val Lys Val Arg Asp Phe Gin Ser Trp Ile Asn Ser Glu 245 250 255
Ile Arg Lys Lys 260
(2) INFORMATION FOR SEQ ID NO: 160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 595 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 2..595
(D) OTHER INFORMATION: /note= "At pos. bp 509, change C to Y; at pos. 556, change C to Y; at pos. 557, change C to Y; at 561, change C to Y; at 573, change C to Y. At pos. aa. 170, 186, 187, 190 and 191, substitute Xaa."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:
T GGT TCC ATC TTG AAC AAA CGT TGG ATT GTA ACA GCT GCA CAT TGC 46
Gly Ser He Leu Asn Lys Arg Trp Ile Val Thr Ala Ala His Cys 1 5 10 15
CTA AAC GCT GGC ATT TTA AAA TCC GTC TAT TTG GGA TCA AAC TCA TTA 94 Leu Asn Ala Gly Ile Leu Lys Ser Val Tyr Leu Gly Ser Asn Ser Leu
20 25 30
GAT GGC GAT GGT ACA TAC TAC GAC GTC GAA CGT TTT GTG ATG CAT GAT 142 Asp Gly Asp Gly Thr Tyr Tyr Asp Val Glu Arg Phe Val Met His Asp 35 40 45
AAA TAT ACA CCA AGA ATC ACT GTC AAC TAT GCT GAT ATT GGT CTA ATA 190 Lys Tyr Thr Pro Arg He Thr Val Asn Tyr Ala Asp Ile Gly Leu Ile 50 55 60
AAA GTG GCA AAA GAC ATT GTA TTC GGT GAC AAA GTC CAA CCG ATC AAA 238 Lys Val Ala Lys Asp Ile Val Phe Gly Asp Lys Val Gin Pro Ile Lys 65 70 75
ATT AGC AAG AGA AAC ATC AAG GGT GGT GAA ATT TGC AAG GCA ACT GGT 286 He Ser Lys Arg Asn Ile Lys Gly Gly Glu Ile Cys Lys Ala Thr Gly 80 85 90 95
TGG GGT CTA TTA GGT TCT GTG GAC TCA GTA CCA AAC GAA TTA CAA CAA 334 Trp Gly Leu Leu Gly Ser Val Asp Ser Val Pro Asn Glu Leu Gin Gin 100 105 110 GTA GAA ACC ACT GCA ATA ACA GAC GAA AAG TGC TTT GAA TTG ACT CAA 382 Val Glu Thr Thr Ala Ile Thr Asp Glu Lys Cys Phe Glu Leu Thr Gin 115 120 125
TTC ATT GAC CCA ACT TCG CAA ATA TGT ACA TTC AGG GAA TTT GGT AGA 430 Phe Ile Asp Pro Thr Ser Gin Ile Cys Thr Phe Arg Glu Phe Gly Arg 130 135 140
GGC ATT TGC TTT GGT GAT TCT GGT GGA CCA CTA GTT TAC AAA AAT GAA 478 Gly Ile Cys Phe Gly Asp Ser Gly Gly Pro Leu Val Tyr Lys Asn Glu 145 150 155
CTT GTT GGC ATT ACA TCG ATG CAC TTA TAC YCC TGC AGA GGT GGC AGG 526 Leu Val Gly Ile Thr Ser Met His Leu Tyr Xaa Cys Arg Gly Gly Arg 160 165 170 175
CCA GAT ATT TTT TGT GAA AGT GCG AGA TTY YCA AYC CTG GAT TAA AYT 574 Pro Asp He Phe Cys Glu Ser Ala Arg Phe Xaa Xaa Leu Asp Xaa Xaa 180 185 190 CTG AAA TTG AAA AAA ATT AAA 595
Leu Lys Leu Lys Lys Ile Lys 195
(2) INFORMATION FOR SEQ ID NO: 161:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 198 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ll) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161: Gly Ser Ile Leu Asn Lys Arg Trp Ile Val Thr Ala Ala His Cys Leu 1 5 10 15
Asn Ala Gly Ile Leu Lys Ser Val Tyr Leu Gly Ser Asn Ser Leu Asp 20 25 30 Gly Asp Gly Thr Tyr Tyr Asp Val Glu Arg Phe Val Met His Asp Lys 35 40 45
Tyr Thr Pro Arg Ile Thr Val Asn Tyr Ala Asp He Gly Leu Ile Lys 50 55 60 Val Ala Lys Asp Ile Val Phe Gly Asp Lys Val Gin Pro Ile Lys Ile 65 70 75 80
Ser Lys Arg Asn Ile Lys Gly Gly Glu Ile Cys Lys Ala Thr Gly Trp 85 90 95
Gly Leu Leu Gly Ser Val Asp Ser Val Pro Asn Glu Leu Gin Gin Val 100 105 110
Glu Thr Thr Ala Ile Thr Asp Glu Lys Cys Phe Glu Leu Thr Gin Phe 115 120 125
Ile Asp Pro Thr Ser Gin Ile Cys Thr Phe Arg Glu Phe Gly Arg Gly 130 135 140 Ile Cys Phe Gly Asp Ser Gly Gly Pro Leu Val Tyr Lys Asn Glu Leu 145 150 155 160
Val Gly Ile Thr Ser Met His Leu Tyr Xaa Cys Arg Gly Gly Arg Pro 165 170 175
Asp Ile Phe Cys Glu Ser Ala Arg Phe Xaa Xaa Leu Asp Xaa Xaa Leu 180 185 190
Lys Leu Lys Lys Ile Lys 195
(2) INFORMATION FOR SEQ ID NO: 162:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ll) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:
Ile Val Gly Gly Val Ser Val Asn Ile Asn Asp Tyr Gly Tyr Gin Leu 1 5 10 15
Ser Leu Gin Ser Asn Gly Arg 20
(2) INFORMATION FOR SEQ ID NO: 163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: (D) TOPOLOGY: linear
(11) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:
Ile Val Gly Gly His Asp Thr Ser Ile Lys Gin His Pro Tyr Gin Val 1 5 10 15
Ser
(2) INFORMATION FOR SEQ ID NO: 164:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:
GAGCTCTCGA GAATCGTAGG AGGACACGAT AC 32
(2) INFORMATION FOR SEQ ID NO: 165:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:
GGACGAATTC TTAAACACCA GACACTTCCT TG 32
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:
1. An isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene selected from the group consisting of a serine protease gene comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 12, SEQ ID N0:14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID
NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO: 37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO: 43, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID
NO-116, SEQ ID NO-117, SEQ ID NO:127, SEQ ID NO: 121, SEQ ID
NO: 131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID
NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID
NO: 132, SEQ ID NO: 134, SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID
NO:148, SEQ ID NO:150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO_:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84 and SEQ ID NO: 45; an aminopeptidase gene comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO: 110 and SEQ ID NO: 112; and a cysteine protease gene comprising a nucleic acid molecule selected from the group consisting of SEQ ID NO:l, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID N0:76 and SEQ ID NO: 94.
2. An isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO:157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO: 113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95.
3. An isolated protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO: 139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO: 123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO: 92 and SEQ ID NO: 95.
4. A therapeutic composition that, when administered to an animal, reduces flea infestation, said therapeutic composition comprising a protective compound selected from the group consisting of: an isolated protein or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID N0:27, SEQ ID N0:30, SEQ ID N0:33, SEQ ID N0:36, SEQ ID N0:38, SEQ ID N0:41, SEQ ID N0:44, SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID N0:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID N0:115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO:157, SEQ ID N0:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO:131, SEQ ID NO:155, SEQ ID NO:114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID N0:150, SEQ ID NO:80, SEQ ID NO:62, SEQ ID NO: 142, SEQ ID NO: 138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO:122, SEQ ID NO:84, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:76, SEQ ID N0:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94; an isolated antibody that selectively binds to a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO: 33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 6 SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; an inhibitor of protease activity identified by its ability to inhibit the activity of a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID N0:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO:141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; and a mixture thereof.
5. A method to reduce flea infestation comprising treating an animal with a therapeutic composition comprising as a protective compound selected from the group consisting of: an isolated protein or mimetope thereof encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ
ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID
NO: 30, SEQ ID NO: 33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID
NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID
NO:119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID
NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID
NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID
NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID
NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID
NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID
NO: 113, SEQ ID NO: 77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; an isolated nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:120, SEQ ID NO:130, SEQ ID NO:154, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:127, SEQ ID N0:121, SEQ ID N0:131, SEQ ID NO: 155, SEQ ID NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO:128, SEQ ID NO:152, SEQ ID NO:156, SEQ ID NO:160, SEQ ID NO:136, SEQ ID NO: 78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO:134, SEQ ID NO:66, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO:144, SEQ ID NO:140, SEQ ID NO:122, SEQ ID NO: 84, SEQ ID NO:110, SEQ ID NO: 112, SEQ ID NO:76, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94; an isolated antibody that selectively binds to a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO: 36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO:126, SEQ ID NO: 119, SEQ ID NO:129, SEQ ID NO: 153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO:95; an inhibitor of protease activity identified by its ability to inhibit the activity of a protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule having a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO:161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO:141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID N0:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID N0:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; and a mixture thereof.
6. A method to produce a flea protease protein, said method comprising culturing a cell capable of expressing said protein, said protein being encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a gene comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID N0:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID N0:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 120, SEQ ID NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID NO:114, SEQ ID NO:125, SEQ ID NO:118, SEQ ID NO:128, SEQ ID NO:152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO:78, SEQ ID NO:158, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:142, SEQ ID NO:138, SEQ ID NO: 144, SEQ ID NO: 140, SEQ ID NO: 122, SEQ ID NO: 84, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:76, SEQ ID NO:l, SEQ ID N0:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94.
7. A method to identify a compound capable of inhibiting flea protease activity, said method comprising: (a) contacting an isolated flea protease protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID
NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO:129, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:161, SEQ ID NO: 137, SEQ ID NO:79, SEQ ID NO: 159, SEQ ID
NO: 133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO:139, SEQ ID NO:145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO: 85, SEQ
ID NO:107, SEQ ID NO: 111, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:89, SEQ ID NO: 92 and SEQ ID NO: 95 with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has proteolytic activity; and (b) determining if said putative inhibitory compound inhibits said activity.
8. A test kit to identify a compound capable of inhibiting flea protease activity, said test kit comprising an isolated flea protease protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO: 133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO:77, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95, and a means for determining the extent of inhibition of said activity in the presence of a putative inhibitory compound.
9. An isolated flea protease protein that cleaves an immunoglobulin, when said protein is incubated in the presence of said immunoglobulin in about 100 microliters of about 0.2M Tris-HCl for about 18 hours at about 37°C.
10. A method to identify a compound capable of inhibiting flea immunoglobulin proteinase protein activity, said method comprising:
(a) contacting an isolated flea immunoglobulin proteinase protein with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has immunoglobulin proteinase activity; and
(b) determining if said putative inhibitory compound inhibits said activity.
11. The invention of Claim 1, wherein said serine protease gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO: 41, SEQ ID NO:44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO: 96, SEQ ID NO: 115, SEQ ID NO: 126, SEQ ID NO: 119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 137, SEQ ID NO: 79, SEQ ID NO: 159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:67, SEQ ID NO:147, SEQ ID NO: 149, SEQ ID NO:151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO:123, SEQ ID NO:68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO: 69 and SEQ ID NO: 85, and wherein said aminopeptidase gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 107, SEQ ID NO: 111 and SEQ ID NO: 113, and wherein said cysteine protease gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92, SEQ ID NO: 95 and SEQ ID NO:77.
12. The invention of Claims 1 or 2, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a flea protease protein selected from the group consisting of a larval serine protease protein, an adult serine protease protein, a larval aminopeptidase protein, an adult aminopeptidase protein, a larval cysteine protease protein and an adult cysteine protease protein.
13. The invention of Claims 1 or 2, wherein said nucleic acid molecule is a flea nucleic acid molecule.
14. The invention of Claims 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of Ctenocephalides, Ceratophyll us, Di amanus, Echidnophaga ,
Nosopsyll us, Pul ex, Tunga , Oropsylla , Orchopeus and Xenopsylla nucleic acid molecules.
15. The invention of Claims 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of Ctenocephalides felis, Ctenocephalides canis,
Ceratophyllus pulicidae, Pulex irritans, Oropsylla
(Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex simulans nucleic acid molecules.
16. The invention of Claims 1 or 2, wherein said nucleic acid molecule comprises a Ctenocephalides felis nucleic acid molecule.
17. The invention of Claim 1, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with a nucleic acid molecule selected from the group consisting of nfSP18534, nfSP18775, nfSP18225, nfSP24410, nfSP241089, nfSP24774, nfSP24711, nfSP28711, nfSP28923, nfSP32933, nfSP32924, nfSP32699, nfSP33426, nfSP33778, nfSP331894, nfSP331200, nfSP33726, nfSP40841, nfSP40717, nfSP5806, nfSPll307, nfSP851β nfSP843br nfSP12758, nfSP2661t7 nfSP27 38/6 nfSP23 4£3 nfSP34 Λ0 nfSP36197, nfSP38341, nfSP37261, nfSP39267 nfSP29612, nfSP30641, nfSP31626, nfSP32433, nfSP15815, nfSP19855, nfSP25864, nfSP21595, nfAP2383, nfAP2537, nfCPl573 and nfCPl1109.
18. The invention of Claim 1, wherein said nucleic acid molecule comprises a nucleic acid molecule selected from the group consisting of nfSP18534, nfSP18775, nfSP18225, nfSP24410, nfSP241089, nfSP24774, nfSP24711, nfSP28711, nfSP28923, nfSP32933, nfSP32924, nfSP32ε99, nfSP33426, nfSP33778, nfSP331894, nfSP331200, nfSP33726, nfSP40841, nfSP40717, nfSP5806, nfSPll307, nfSP851s, nfSP8436, nfSP12758, nfSP26610, nfSP27386, nfSP23423, nfSP34390, nfSP36197, nfSP38341, nfSP3726:, nfSP39267 nfSP29 6I2 nfSP30641, nfSP31626, nfSP32433, nfSP15815, nfSP198SS, nfSP258b4, nfSP21595, nfAP2383, nfAP2537, nfCPl573 and nfCPln09.
19. The invention of Claims 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID N0:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID NO:115, SEQ ID NO:126, SEQ ID NO:119, SEQ ID NO: 129, SEQ ID NO:153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO:149, SEQ ID NO: 151, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO: 163, SEQ ID NO: 162, SEQ ID NO: 69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID NO:lll, SEQ ID NO:113, SEQ ID NO: 77, SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule encoding any of said amino acid sequences.
20. The invention of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID
NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:34, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO: 43 and SEQ ID NO: 45, SEQ ID NO: 120, SEQ ID
NO: 130, SEQ ID NO: 154, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID
NO: 127, SEQ ID NO: 121, SEQ ID NO: 131, SEQ ID NO: 155, SEQ ID
NO: 114, SEQ ID NO: 125, SEQ ID NO: 118, SEQ ID NO: 128, SEQ ID
NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 136, SEQ ID NO:78, SEQ ID NO: 158, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID
NO: 66, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID
NO: 80, SEQ ID NO: 82, SEQ ID NO:142, SEQ ID NO: 138, SEQ ID
NO:144, SEQ ID NO:140, SEQ ID NO:122, SEQ ID NO:84, SEQ ID
NO:110, SEQ ID NO:112, SEQ ID NO:76, SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO: 88,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 93 and SEQ ID NO: 94; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule having any of said nucleic acid sequences .
21. The invention of Claim 1, wherein said nucleic acid molecule comprises an oligonucleotide.
22. The invention of Claim 3, wherein said protein, when administered to an animal elicits an immune response against a flea protease selected from the group consisting of a flea serine protease, a flea aminopeptidase, and a flea cysteine protease.
23. The invention of Claim 12, wherein said protein, when administered to an animal elicits an immune response against a flea protease selected from the group consisting of a flea serine protease, a flea aminopeptidase, and a flea cysteine protease.
24. A recombinant molecule comprising a nucleic acid molecule as set forth in Claims 1 or 2 operatively linked to a transcription control sequence.
25. A recombinant virus comprising a nucleic acid molecule as set forth in Claims 1 or 2.
26. A recombinant cell comprising a nucleic acid molecule as set forth in Claims 1 or 2, said cell being capable of expressing said nucleic acid molecule.
27. The invention of Claim 2, wherein said nucleic acid molecule hybridizes under stringent hybridization conditions with the complement of a nucleic acid sequence encoding said protein.
28. The invention of Claim 3, wherein said protein is selected from the group consisting of: a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:38, SEQ ID N0:41, SEQ ID NO: 44, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:96, SEQ ID N0:115, SEQ ID NO:126, SEQ ID N0:119, SEQ ID NO: 129, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:137, SEQ ID NO:79, SEQ ID NO:159, SEQ ID NO:133, SEQ ID NO: 135, SEQ ID NO: 67, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 143, SEQ ID NO: 139, SEQ ID NO: 145, SEQ ID NO: 141, SEQ ID NO: 123, SEQ ID NO: 68, SEQ ID NO:163, SEQ ID NO:162, SEQ ID NO:69, SEQ ID NO:85, SEQ ID NO:107, SEQ ID N0:111, SEQ ID N0:113, SEQ ID NO:77, SEQ ID N0:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 89, SEQ ID NO: 92 and SEQ ID NO: 95; and a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein comprising any of said amino acid sequences.
29. The invention of Claim 3, wherein said protein is used to identify an inhibitor selected from the group consisting of an inhibitor of flea serine protease activity, an inhibitor of aminopeptidase activity, and an inhibitor of flea cysteine protease activity.
30. The invention of Claim 29, wherein said inhibitor, when administered to an animal, is capable of reducing flea infestation.
31. An isolated antibody that selectively binds to a protein as set forth in Claim 3.
32. The invention of Claims 4, 5 or 52, further comprising a component selected from the group consisting of an excipient, an adjuvant, a carrier, and a mixture thereof.
33. The invention of Claims 4 or 5, wherein said composition comprises a controlled release formulation.
34. The invention of Claims 4 or 5, wherein said composition further comprises a compound that reduces flea burden by a method other than by reducing flea protease activity.
35. The invention of Claim 5, wherein said animal is selected from the group consisting of adult fleas, flea larvae and animals susceptible to flea infestation.
36. The invention of Claim 5, wherein larval flea infestation is reduced by flea larvae ingesting adult flea feces comprising said therapeutic composition.
37. The invention of Claim 5, wherein larval flea infestation is reduced by flea larvae ingesting adult flea feces, said feces comprising anti-flea protease antibodies elicited in a host animal in response to administration of one or more of said isolated flea protease proteins, said adult flea having fed from said host animal after said administration.
38. The invention of Claim 5, wherein said animal is selected from the group consisting of mammals and birds.
39. The invention of Claim 5, wherein said animal is selected from the group consisting of cats and dogs.
40. The invention of Claim 5, wherein said fleas are of a genus selected from the group consisting of Ctenocephalides, Cyopsyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla .
41. The invention of Claim 5, wherein said fleas are of a species selected from the group consisting of Ctenocephalides felis, Ctenocephalides canis, Pulex irri tans and Pulex simulans .
42. The invention of Claim 5, wherein said composition further comprises a compound that reduces flea burden by a method other than by reducing flea protease activity.
43. The invention of Claim 9, wherein said protease protein cleaves immunoglobulin heavy chain.
44. The invention of Claim 9, wherein said protease protein cleaves the hinge region of an immunoglobulin heavy chain.
45. The invention of Claim 9, wherein the mature form of said protein has a molecular weight ranging from about
25 kD to about 35 kD.
46. The invention of Claim 45, wherein the mature form of said protein has a molecular weight of about 31 kD.
47. The invention of Claim 9, wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:96.
48. The invention of Claim 9, wherein said flea protease protein comprises an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a flea serine protease gene that cleaves immunoglobulins.
49. The invention of Claim 48, wherein said serine protease gene comprises nucleic acid sequence SEQ ID NO: 66.
50. The invention of Claim 48, wherein said serine protease gene comprises a nucleic acid sequence that encodes an amino acid sequence selected from the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:96.
51. The invention of Claim 9, wherein said protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with a nucleic acid molecule selected from the group consisting of nfSP28711 and nfSP28923.
52. The invention of Claim 9, wherein said protein comprises an amino acid sequence encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with nucleic acid molecule SEQ ID NO: 66.
53. The invention of Claim 9, wherein said protem comprises an ammo acid sequence encoded by a nucleic acid molecule selected from the group consisting of nfSP28711 and nfSP28923.
54. The invention of Claim 9, wherem said protein is produced by a method comprising culturing a cell expressing nucleic acid molecule nfSP28711
55. A therapeutic composition comprising an excipient and a protective compound selected from the group consisting of a nucleic acid molecule of Claim 48 and a protem of Claim 47.
56. The invention of Claim 55, wherein said composition further comprises a component selected from the group consisting of an adjuvant, a carrier, and a mixture thereof.
57. The invention of Claim 55, wherem said composition further comprises a compound that reduces hematophagous ectoparasite burden by a method other than by reducing flea immunoglobulin proteinase activity.
58. The mvention of Claim 55, wherem said composition is administered to an animal to reduce ectoparasite infestation of said animal.
PCT/US1997/006121 1995-06-07 1997-04-24 Flea protease proteins, nucleic acid molecules, and uses thereof WO1997040058A1 (en)

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JP53813497A JP2001510324A (en) 1996-04-24 1997-04-24 Flea protease proteins, nucleic acid molecules, and uses thereof
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US08/749,699 US6210920B1 (en) 1991-12-13 1996-11-15 Flea protease proteins, nucleic acid molecules, and uses thereof
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