WO1998004702A2 - Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use - Google Patents

Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use Download PDF

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Publication number
WO1998004702A2
WO1998004702A2 PCT/IB1997/000981 IB9700981W WO9804702A2 WO 1998004702 A2 WO1998004702 A2 WO 1998004702A2 IB 9700981 W IB9700981 W IB 9700981W WO 9804702 A2 WO9804702 A2 WO 9804702A2
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gly
seq
protein
leu
asn
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PCT/IB1997/000981
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French (fr)
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WO1998004702A3 (en
Inventor
Bernhard Knapp
Erika Hundt
Karl-Heinz Schmidt
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Chiron Behring Gmbh & Co.
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Priority to JP10508651A priority Critical patent/JP2001502886A/en
Priority to EP97932967A priority patent/EP0918864A2/en
Priority to CA002259924A priority patent/CA2259924A1/en
Publication of WO1998004702A2 publication Critical patent/WO1998004702A2/en
Publication of WO1998004702A3 publication Critical patent/WO1998004702A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Proteins in particular membrane proteins, of Hel ⁇ cobacter pylori , their preparation and use
  • the present invention relates to novel proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, which are derived from Helicobacter pylori (H. pyl ori ) and which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five a ino acids, and to their preparation and use as pharmaceutical compositions, in particular as vaccines, or as a diagnostic agent. Based on these data, genes coding for these and related proteins were also isolated as shown in SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27.
  • Helicobacter pylori is a Gram-negative, microaerophilic, spiral bacterium which colonizes the mucosa of the human stomach.
  • the bacterium is the cause of chronic active gastritis and of peptic ulcer, in particular duodenal ulcer, and plays a role in the development of carcinomas of the stomach; consequently, Helicobacter pylori is an important human pathogen.
  • Immunization is the conventional way of preventing infectious diseases. It is therefore important to examine this option with regard to controlling an H. pylori infection.
  • Bacteriology 175, 674-683) and 63,000 Da are located in the outer membrane, which adhesins are candidates for an experimental vaccine which has the aim of inducing antibodies which prevent adhesion of the bacterium to the mucosal surface.
  • the outer membrane possesses porins of 30,000 Da (M.A. Tufano et al., 1994, Infection and Immunity 62, 1392-1399), 48,000 Da, 49,000 Da, 50,000 Da, 67,000 Da (M.M. Exner et al., 1995, Infection and Immunity 63, 1567-1572) and 31,000 Da (P.
  • a protein from Helicobacter pyl ori (H. pylori ) containing one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 and 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids.
  • the peptide sequences of the protein are N-terminal sequences.
  • the protein according to the first aspect of the present invention preferably contains a peptide sequence having the SEQ ID NO: 1 according to Table la and has a molecular weight of approx. 250 kD, or preferably contains a peptide sequence having the SEQ ID NO: 2 according to Table la and has a molecular weight of approx. 110 kD, or preferably contains a peptide sequence having the SEQ ID NO: 3 according to Table la and has a molecular weight of approx. 100 kD, or preferably contains a peptide sequence having the SEQ ID NO: 6 according to Table la and has a molecular weight of approx.
  • 60 kD or preferably contains a peptide sequence having the SEQ ID NO: 10 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 11 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 12 according to Table lb and has a molecular weight of from approx. 32 to approx. 36 kD, or preferably contains a peptide sequence having the SEQ ID NO: 14 according to Table lc and has a molecular weight of approx.
  • 30 kD or preferably contains a peptide sequence having the SEQ ID NO: 15 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 16 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 17 according to Table lc and has a molecular weight of approx. 25 kD, or preferably contains a peptide sequence having the SEQ ID NO: 18 according to Table lc and has a molecular weight of approx.
  • the protein according to the first aspect of the present invention is preferably a membrane protein or a protein which is firmly associated with the membrane. More preferably said protein is an integral membrane protein, in particular a Sarkosyl ® -insoluble integral membrane protein.
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods;
  • step (c) isolating the proteins, which have been separated in accordance with step (b) .
  • the proteins according to the second aspect of the present invention can be obtained by means of differential solubilization using Sarkosyl ® .
  • the proteins can also be obtained by means of separation by one or more SDS polyacrylamide gel electrophoreses, preferably by means of several SDS polyacrylamide gel electrophoreses having different polyacrylamide contents, more preferably wherein the polyacrylamide content of said gel electrophoreses is approximately 8%, 10% or 16%.
  • an antibody against one or more proteins according to the first or second aspects of the present invention and/or against one or more peptides according to the third aspect of the present invention is provided.
  • a polynucleotide encoding one or more proteins according to the first or second aspects of the present invention or one or more peptides according to the third aspect of the present invention.
  • a process for preparing the proteins according to the first or second aspects of the present invention characterized in that the following procedural steps are carried out: (a) isolating the proteins, by means of differential solubilization;
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods; and (c) isolating the proteins, which have been separated in accordance with step (b) .
  • the process is characterized in that the proteins are isolated in accordance with step (a) using Sarkosyl ® .
  • a seventh aspect of the present invention there is provided a process for preparing the peptides according to the third aspect of the present invention, characterized in that a chemical peptide synthesis is carried out.
  • an eighth aspect of the present invention there is provided a process for preparing the proteins according to the first or second aspects of the present invention or the peptides according to the third aspect of the present invention, characterized in that a polynucleotide according to the fifth aspect of the present invention is expressed.
  • a ninth aspect of the present invention there is provided the use of one or more proteins according to the first or second aspects of the present invention, one or more peptides according to the third aspect of the present invention, one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention for preparing a pharmaceutical composition or a diagnostic agent.
  • a pharmaceutical composition comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products.
  • said pharmaceutical composition is used as a vaccine.
  • a diagnostic agent comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products.
  • a protein from H. pylori containing one of the peptide sequences deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 and 27, or parts or homologues thereof having a minimum length of five amino acids.
  • a peptide selected from the C-terminal region of the peptide sequence of SEQ ID NO: 20 or homologue thereof.
  • said peptide is selected from RDPKFNLAHIEKEFEVWNWDYRA and EKHQKMMKDMHGKDMHHTKKKK, or parts or homologues thereof.
  • an antibody against one or more proteins according to the twelfth aspect of the present invention and/or against one or more peptides according to the thirteenth or fourteenth aspects of the present invention is provided.
  • a sixteenth aspect of the present invention there is provided a polynucleotide encoding one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention.
  • a host cell transformed with the polynucleotide according to the fifth or sixteenth aspects of the present invention.
  • an expression product expressed from the host cell according to the seventeenth aspect of the present invention is provided.
  • a pharmaceutical composition comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products.
  • said pharmaceutical composition is used as a vaccine. More preferably, when the pharmaceutical composition comprises a nucleotide sequence, said pharmaceutical composition is used as a DNA vaccine.
  • a diagnostic agent comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products.
  • a twenty-first aspect of the present invention there is provided the use of one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products for preparing a pharmaceutical composition or a diagnostic agent.
  • the present application describes the isolation and determination of, in all, 19 proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, which proteins are in a molecular weight range of from 17 kD to approx. 250 kD (Tables la-lc) .
  • membrane protein is generally understood to mean integral and peripheral membrane proteins and transmembrane proteins. Integral membrane proteins are proteins which are partially or entirely inserted into the cytoplasmic membrane. By contrast, peripheral membrane proteins only adhere to the surface of the membrane. Transmembrane proteins pass completely through the membrane (see, for example, B. Alberts et al. (eds).
  • the invention describes proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, in particular Sarkosyl ® -insoluble integral membrane proteins of H. pylori , which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five, preferably six amino acids, with these peptide sequences preferably constituting N-terminal sequences of the said proteins.
  • the novel peptides are particularly preferred which exhibit at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19.
  • those said parts are in particular preferred which contain an uninterrupted sequence of unambiguously specified amino acids.
  • part in the context of "part(s) of a sequence” in the present invention is defined herein as meaning a sequence of amino acids which can form a T-cell or B-cell epitope. Such an amino acid sequence is usually of a minimum of approximately four to eight amino acids.
  • homologue (s) in the context of the present invention is defined herein as meaning the same protein or peptide of a different strain of H. pylori but exhibiting the same function.
  • the actual amino acid sequences may not be identical between homologous proteins or peptides from different strains of H. pylori , the differences between the amino acid sequences merely represent strain-specific differences; the function of the homologues is identical.
  • the protein containing a peptide sequence having the SEQ ID NO: 1 according to Table la has a molecular weight of approx. 250 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 2 according to Table la has a molecular weight of approx. 110 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 3 according to Table la has a molecular weight of approx. 100 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 6 according to Table la has a molecular weight of approx.
  • the protein containing a peptide sequence having the SEQ ID NO: 10 according to Table lb has a molecular weight of approx. 42 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 11 according to Table lb has a molecular weight of approx. 42 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 12 according to Table lb has a molecular weight of from approx. 32 to approx. 36 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 14 according to Table lc has a molecular weight of approx.
  • the protein containing a peptide sequence having the SEQ ID NO: 15 according to Table lc has a molecular weight of approx. 28 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 16 according to Table lc has a molecular weight of approx. 28 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 17 according to Table lc has a molecular weight of approx. 25 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 18 according to Table lc has a molecular weight of approx. 25 kD
  • the protein containing a peptide sequence having the SEQ ID NO: 19 according to Table lc has a molecular weight of approx. 17 kD.
  • H. pylori strain No. ATCC 43504 is used, for example, as the starting material when isolating the proteins, with it being possible, in particular, to carry out the following procedural steps:
  • step (b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods, preferably by means of SDS polyacrylamide gel electrophoresis, with use being made, in particular, of polyacrylamide gels having differing polyacrylamide contents, in particular containing approx. 8, 10 or 16% polyacrylamide, and
  • step (c) isolating the proteins, which have been separated in accordance with step (b) , by means of known methods, for example by elution or by isolation on a membrane .
  • the proteins were first of all obtained using the method of Blaser et al. (see above).
  • the bacteria which had been disrupted in a glass bead homogenizer, were freed of intact bacteria by centrifugation at 5000 g; the supernatant was then centrifuged at 100,000 g.
  • the pellet was dissolved in Sarkosyl ® , and the Sarkosyl ® -insoluble fraction, which contains the integral membrane proteins in particular, was centrifuged off.
  • the pellet was resuspended in distilled water and fractionated by SDS polyacrylamide gel electrophoresis- (PAGE) .
  • the amino acids which are labelled Xaa in the sequence listing can be explained as follows:
  • the non-identifiable amino acids can be caused by interference due to impurities in the first sequencing step, a non-analysable amino acid, such as Cys or Trp, a modifiable amino acid which is missing in the elution programme, or an amino acid, such as Ser or Thr, which is difficult to determine, basically due to low sequence yields.
  • Different bands can also contain two proteins of very similar molecular weights in different quantities. This then results in two sequences which then also have to be assigned unambiguously on account of the different frequency of the individual amino acids.
  • the present invention also describes the peptides which are designated by the sequences according to SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five amino acids, in particular of six amino acids, which can be prepared, for example, by well-known chemical peptide synthesis
  • Bodanszky M. & Bodanszky, A. "The practice of peptide synthesis”. Springer Verlag, 1984) .
  • the novel peptides are particularly preferred which possess at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1 , 2 , 3, 6, 10, 11, 12, 14, 15, 16 and 19.
  • those said peptides are, in particular, preferred which contain an uninterrupted sequence of unambiguously determined amino acids, as is the case with the sequences from SEQ ID NO: 12, 14 and 15.
  • the present application also describes antibodies which can also be prepared by methods which are well known to the skilled person (see, for example, B.A. Diamond et al. (1981), The New England Journal of Medicine, 1344-1349) and which are directed against one or more of the novel proteins or peptides.
  • the skilled person is also familiar, from
  • nucleotide sequences which encode the peptides according to the sequence listing.
  • nucleotide sequences are preferred which occur most frequently in accordance with the rules for the frequency of use of the different codons in Helicobacter pylori .
  • These nucleotide sequences can be prepared, for example, by means of chemical polynucleotide synthesis (see, for example, E. Uhlmann & A. Peyman (1990), Chemical Reviews, 543-584, Vol. 90, No. 4) .
  • oligodeoxynucleotides which have been prepared in accordance with these rules can be employed for screening Helicobacter pylori gene libraries using known methods (J. Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Furthermore, taking the sequence data as a basis, peptides can be synthesized which are employed for obtaining antisera. Gene expression libraries can then be screened using these antisera. The clones resulting from these different screening methods can then be employed, by isolating and sequencing the inserted DNA fragments, for identifying DNA sequence segments which encode the N-terminally sequenced protein segments of the proteins.
  • DNA fragments do not contain the complete gene encoding any particular protein, these DNA fragments can be used to isolate the complete genes by screening other gene libraries. The genes which have been completely isolated in this manner can then be expressed, in accordance with the state of the art, in various well- known systems in order to obtain the corresponding protein.
  • oligonucleotides deduced from the N- terminal sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15, the genes corresponding to the SEQ ID NOS: 5, 8, 10, 12 and 15 were isolated and are specified as SEQ ID NOS: 20 (catalase) , 24 (50 kD membrane protein), 25 (42 kD protein), 26 (36/35/32 kD protein) and 23 (28 kD protein) .
  • the gene coding for Hop C could not be isolated using oligonucleotide 7.
  • oligonucleotide 7 hybridizes with an homologous gene specified as SEQ ID NO: 21 (Hop X) .
  • Two additional genes which belong to this family were able to be isolated and are specified as SEQ ID NO: 21 (Hop Y) and SEQ ID NO: 22 (Hop Z) .
  • Such vaccines may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection) .
  • These vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants") .
  • the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
  • Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) , such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components) , such as for example (a) those formulations described in
  • microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA) )
  • SAF containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion
  • MT monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon) , macrophage colony stimulating factor (M-CSF) , tumour necrosis factor (TNF)
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as StimulonTM (Cambridge Bioscience, Worcester, MA)
  • muramyl peptides include, but a r e n o t l i m i t e d t o , N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP) , N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2- (1 ' -2 * -dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy) - ethyla ine (MTP-PE), etc.
  • the immunogenic compositions typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the vaccine may be administered in conjunction with other immunoregulatory agents .
  • compositions in particular vaccines, and diagnostic agents which comprise one or more of the novel proteins and/or one or more of the novel peptides or one or more of the novel antibodies or one or more of the novel polynucleotides or one or more expression products of the novel polynucleotides.
  • a DNA vaccine can be prepared on the basis of the polynucleotides, or a diagnostic agent can be prepared on the basis of the polymerase chain reaction (PCR diagnosis), or an immunotest, for example a Western blot test or an enzyme immunotest (ELISA) can be prepared on the basis of the antibodies.
  • novel proteins or peptides, or their immunogenic moieties in particular when they contain an uninterrupted sequence of unambiguously determined amino acids, having a minimum length of five amino acids, preferably six amino acids and, in particular, in the case of the novel peptides having the SEQ ID NOS: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19 and peptides or proteins encoded by the DNA sequences of SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27, at least ten consecutive amino acids, can be used as antigens for immunizing mammals.
  • the two C-terminal regions Cl and C2 specific for H. pylori catalase (c.f. Example 6) can also be used as immunogens .
  • the antibodies which are formed by the immunization, or antibodies which are prepared by means of recombinant DNA methods can, inter alia, prevent adhesion of the bacteria to the mucosal surface, attract macrophages for the purpose of eliminating bacteria, and activate the complement system for the purpose of lysing the bacteria.
  • the following examples are intended to clarify the invention.
  • the H. pylori stain ATCC 43504 was passaged under microaerophilic conditions (BBL Jar/Campy Pak Plus, from Becton & Dickinson) on Columbia Agar plates containing 5% horse blood (incubation 48 h, 37°C) . Three plates were rinsed off when inoculating a 500 ml flow-spoiler flask (100 ml of Columbia broth, 7% FCS) ; during the incubation
  • the bacterial cultures are harvested in the phase of late logarithmic growth, washed in 10 mM Tris buffer (pH 7.4) and disrupted with glass beads in a ho ogenizer (Institut fiir Molekularbiologie und Analytik (IMA), Germany) at 4°C and 4000 rpm for 15 min. After that, the glass beads are removed by filtration and the bacterial suspension is centrifuged at 5000 g for 20 min in order to remove intact cells.
  • IMA Institute fiir Molekularbiologie und Analytik
  • the cell walls are pelleted out of the supernatant by centrifuging at 100,000 g for 60 minutes and at 4°C.
  • the resulting pellet is resuspended with a 1% solution of Sarkosyl ® in 7 mM EDTA, and the suspension is incubated at 37°C for 20 min.
  • the Sarkosyl ® -insoluble fraction which contains the integral membrane proteins, is pelleted by centrifugation at 50,000 g for 60 minutes and at 4°C and the pellet is resuspended in sterile distilled water; the suspension is then stored at -20°C.
  • the membrane protein fraction starting material is dissolved in 1.5% SDS, 2.5% mercaptoethanol, 5% glycerol and bromophenol blue in 63 mmol/1 Tris buffer, pH 6.8, and fractionated by SDS polyacrylamide gel electrophoresis.
  • Protein transfer from the SDS gel to the PVDF membrane is carried out in a BioRad (Munich) Trans Blot SD apparatus, under modified conditions.
  • the number of sequencing steps was 5 to 25 (depending on the quantity of substance available for sequencing) .
  • the oligonucleotides were deduced using the species-specific codon usage of Helicobacter pylori , which had been determined from 19 known H. pylori genes, and using the base inosine (I), which is capable of undergoing stable base pairing with the bases adenine (A) , cytosine (C) and thymine (T) with, in each case, two hydrogen bridges.
  • the degeneracy of the codon was kept as low as possible.
  • oligonucleotides which had been deduced from the peptide sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15 were labelled with digoxigenin (DIG) using a kit manufactured by Boehringer Mannheim (DIG Oligonucleotide 3'-End Labelling Kit) and employed for screening a H. pylori gene library which had been prepared using a kit manufactured by Stratagene (Predigested ZAP ExpressTM BamHI/CIAP Vector Cloning Kit) at 32°C under standard conditions.
  • DIG Oligonucleotides 1, 3 and 6 it was possible to identify clones which carry DNA fragments containing sequences which encode the peptide sequences of SEQ ID NOS: 5, 8 and 15.
  • Oligonucleotide 2 hybridized with a DNA fragment which encodes an homologous sequence of SEQ ID NO: 7.
  • oligonucleotides 4 and 5 it was only possible to isolate clones whose DNA fragments did not encode SEQ ID NOS: 10 and 12. This is why these oligonucleotides and the clones which had been isolated from the ⁇ ZAP Express gene library were employed in a Southern Blot analysis, which permitted the unequivocal identification of DNA fragments which hybridized with the oligonucleotides, but not with the DNA fragments resulting from the screening. With these DNA fragments, in each case one sub-gene library was prepared in the ⁇ ZAP Express vector, and each sub-gene library was screened with oligonucleotides 4 and 5. This allowed the identification of clones which carry DNA fragments encoding the sequences of SEQ ID NOS: 10 and 12.
  • SEQ ID NO: 20 describes the DNA sequence which encodes the catalase of H. pylori .
  • the nucleotide region 337 to 378 describes the hybridization site with oligonucleotide 1.
  • the catalase gene of H. pylori has been described in 1996 by Stefan Odenbreit, Bj ⁇ rn Wieland and Rainer Haas (J. Bacteriol. 178, 6960-6967) and is therefore not new. However, when comparing the amino acid sequences of the catalases of Escheri chia coJi, Bacillus firm ⁇ s, B. subtilis A, B.
  • subtilis B rats, mice, cattle, humans, Staphylococcus violaceus, Haemophilus infl ⁇ enzae, B. fragilis, Pseudomonas mirabilis, B. pertussis and P. syringae with the amino acid sequence of H. pylori , it is possible to identify two C-terminal regions Cl (RDPKFNLAHIEKEFEVWNWDYRA) and C2 (EKHQKMMKDMHGKDMHHTKKKK) , which are specific to H. pyl ori catalase. These two peptides were synthesized using standard techniques, coupled to KLH and used for immunizing rabbits.
  • H. pylori catalase which had been produced by recombinant technique.
  • H. pyl ori- catalase-specific regions may conceivably be used for developing a vaccine which avoids the problem complex of autoimmune reactions or for the development of a diagnostic which reacts specifically with H. pylori catalase.
  • SEQ ID NO: 21 describes a nucleotide sequence which was identified by hybridization with the oligonucleotide 2.
  • the oligonucleotide hybridized with the sequence of nucleotide 1240 to 1284. This encodes a sequence which is homologous to the porin Hop C (Exner et al., 1995) and is identical with the published amino- terminal sequence EDDGGFFTVGYQLGQVMQDVQNPG in positions 1, 2, 3, 4, 9, 10, 11, 12, 14, 18 and 22.
  • the porins Hop A, Hop B, Hop C and Hop D have identical amino acids in 9 positions of the 20 N-terminal amino acids (Exner et al . , 1995). In 8 of these positions, there are identical positions also in the sequence described in the present publication; in the 9th position, a conserved amino acid exchange is present (Val - lie) . It can thus be assumed that the protein described in the present publication is equally part of this group of the porins; it was therefore termed Hop X.
  • the mature protein with 428 amino acids has a molecular weight of 47.3 kD and an isoelectric point of 10.0.
  • a further open reading frame was found upstream of the gene which encodes Hop X.
  • This further open reading frame encodes a protein which is homologous to Hop X (34% identity) and which was therefore termed Hop Y.
  • the gene region found to date encodes the 361 C- terminal amino acids of the protein. The gene region as yet outstanding is currently being isolated using stan- dard techniques .
  • SEQ ID NO: 22 describes a nucleotide sequence which was concomitantly isolated and sequenced during the screening process .
  • the amino acid sequence deduced encodes the 392 C-terminal residues of a protein which shows a high homology with Hop X (33% identity) and Hop Y (28% identity) and which was therefore termed Hop Z.
  • Hop Z The gene region which encodes the N-terminal portion of the protein is currently being isolated.
  • SEQ ID NO: 23 describes a DNA sequence which encodes a hitherto undescribed protein.
  • the nucleotide region 696 to 767 describes the hybridization site with the oligonucleotide 6.
  • the molecular weight is quite close to the molecular weight of 28 kD which had been determined by SDS gel electrophoresis.
  • the amino acid sequence deduced is homologous with the sequences of the proteins Hop X, Hop Y and Hop Z, for which the GCG Bestfit Programme determined identity values of 41%, 38% and 41%, respectively.
  • the 28 kD protein thus also seems to be part of the family of the porins or porin-like proteins.
  • SEQ ID NO: 24 describes a DNA sequence which encodes the non-heat-modifiable 50 kD membrane protein. This protein was first described by Exner et al., 1995, and an N-terminal sequence of the protein was determined. Using the approach described by us, we were then able to describe, with SEQ ID NO: 8, an N-terminal sequence which is identical to the sequence described by Exner et al. (1995) . With the aid of the oligonucleotide 3, which had been deduced using the method illustrated in Example 5 and had been used for screening a H. pylori gene library using the above-described methods, it was then possible to identify a DNA fragment which encodes the 50 kD membrane protein.
  • the amino acid residues 236 to 254 contain a hydrophobic region which is large enough to act as a transmembrane region. Based on such data and using standard methods for epitope analysis, it is possible to identify regions which might be presented on the surface of bacteria. Such regions might be used for developing a vaccine or a diagnostic.
  • SEQ ID NO: 25 describes a DNA sequence 2825 bp in size which was identified by means of hybridization with oligonucleotide 4, which was deduced from SEQ ID NO: 10. Oligonucleotide 4 hybridized with the nucleotide region 897 to 923 of the described sequence of SEQ ID NO: 25. The protein has no signal sequence.
  • the encoding region of SEQ ID NO: 25 codes for a protein of 399 amino acids with a molecular weight of 43.6 kD and an isoelectric point of 5.0.
  • a search for homologous sequences using the BLASTP program (S. F. Altschul et al., 1990, J. Mol. Biol. 215, 403-410) identified the 42 kD antigen of H.
  • elongation factor TU The maximum percentage of identity (89%) was found with the elongation factor TU from Wolinella succinogenes (W. Ludwig et al . , 1993, Antonie van Leeuwenhoek 64, 285- 305 ) .
  • SEQ ID NO: 26 describes a DNA sequence 2182 bp in size which hybridizes with oligonucleotide 5, which had been deduced from SEQ ID NO: 12. Oligonucleotide 5 hybridized with a Sau3AI fragment (position 1 to 575) of the gene library starting from position 524. The screening of different DNA libraries with specific oligonucleotides allowed the isolation of the complete gene described in SEQ ID NO: 26. An amino acid sequence which is identical to the one from SEQ ID NO: 12 can be deduced from SEQ ID NO: 26. Both protein sequencing and the hydrophobicity of the N-terminal sequence deduced allow the conclusion that the antigen has a signal sequence.
  • the mature protein consists of 328 amino acid residues with a molecular weight of 36.1 kD and an isoelectric point of 9.95. No homologous proteins were identified using the BLASTP program (S. F. Altschul et al., 1990) .
  • sequences described in SEQ ID NOS: 20 to 26 indicate nucleotide sequences which encode antigens of the H. pylori strain ATCC 43504. However, it is known for H. pylori that heterogeneity between identical antigens may exist amongst various strains. We therefore claim not only the sequences described in SEQ ID NOS: 21 to 26, but in addition also the sequences of other H. pylori strains which are homologous with the sequences described herein.
  • SEQ ID NO: 19 aligned with a very similar sequence using the TBLASTN program.
  • SEQ ID NO: 27 describes the nucleic acid sequence and deduced amino acid sequence from the coding region of a H. pylori gene (strain 26695) localised between position 843212 and 843691 of the genomic sequence. The protein has no signal sequence.
  • the N-terminal sequence of SEQ ID NO: 19 is highly homologous to the N-terminal region of the deduced amino acid sequence from amino acid residue 1 to 15. Only one different amino acid residue is present at position 4: the nucleotide sequence found by the alignment encodes a Ser residue in this position instead of an Asn residue determined by N-terminal sequencing. This can be explained by strain specific differences.
  • the identified nucleic acid sequence in SEQ ID NO: 27 codes for a protein of 159 amino acid residues with a molecular weight of 18.2 kD and an isoelectric point of 7.2. The molecular weight is very close to that of 17 kD determined from SDS polyacrylamide gel electrophoresis.
  • a search for homologous sequences using the BLASTP program shows that the
  • 17 kD antigen is very homologous to "hydroxymyristol-
  • Proteins in particular membrane proteins, of Heli cobacter pylori , their preparation and use
  • Xaa at positions 1, 5, 12, 14 and 16 are unknown amino acids. At position 8, Xaa is probably Gin, while at position 10 it is probably Ser, at position 11 it is probably Tyr and at position 15 it is probably Thr. Identification: unknown
  • Xaa is an unknown amino acid.
  • Xaa is He or Thr and at position 7 it is Ala or Lys.
  • Identification: unknown (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Xaa Lys Leu Xaa Pro Gin Xaa Gly Tyr Val Leu Met Tyr 1 5 10
  • Xaa at positions 1 and 10 are unknown ammo acids, and at position 4, Xaa is He or Thr and at position 7 it is Ala or Lys . Identification: unknown
  • Xaa at positions 1, 5 and 9 are unknown ammo acids, and at position 7 Xaa is Ala or Leu. Identification: unknown
  • Xaa at positions 1 and 11 are unknown, while at position 6, Xaa is probably Asn or Gin, at position 13 it is probably Thr and at position 14 it is probably He. Identification: unknown
  • Xaa at positions 1 and 4 are unknown, while at position 6 Xaa is Asn or Gin and at position 8 it is probably Pro. Identification: unknown
  • Xaa at positions 1 and 13 are unknown amino acids. Identification: unknown
  • Xaa at position 1 is an unknown ammo acid and at position 2 it is probably Trp. Identification: unknown
  • Xaa at positions 1, 2, 3, 6, 10 and 14 are unknown ammo acids, while at position 5, Xaa is Pro or Val and at position 7 it is probably Lys. Identification: unknown
  • Xaa at position 1 is an unknown ammo acid, while at position 5 Xaa is Pro or Lys.
  • Identification: unknown (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17; Xaa Gin Arg Met Xaa Gin Val Gly 1 5
  • Xaa at position 1 is an unknown amino acid, while at position 5 Xaa is Pro or Lys. Identification: unknown
  • Xaa at positions 1, 5 and 10 are unknown amino acids, while at position 11 Xaa is probably Gin and at position 15 it is probably Lys. Identification : unknown
  • PROPERTIES catalase from Heli cobacter pylori
  • GAG AAA GAG TTT GAA GTG TGG AAT TGG GAT TAC AGA
  • GCT GAT GAT AGC 1602 Glu Lys Glu Phe Glu Val Trp Asn Trp Asp Tyr Arg Ala Asp Asp Ser 410 415 420 425
  • PROPERTIES coding region coding for related proteins of
  • PROPERTIES protein HopZ of Helicobacter pylori
  • PROPERTIES 28 kD protein from Helicobacter pylori
  • AAA ATG GTG AAT GAC AAC GGC TTG CAA AAG CCT TTG ATA AAG TTC CCG 800
  • PROPERTIES 50 kD membrane protein from Helicobacter pylori
  • GCT ACT AAA ATG TGG CAA CAA TCA GGG CCA GGT GGT GTC ATT AAC CCT 1574 Ala Thr Lys Met Trp Gin Gin Ser Gly Pro Gly Gly Val He Asn Pro 100 105 110
  • PROPERTIES 42 kD protein from Helicobacter pylori
  • GCTAGAGCAT CAGCCTTCCA AGCTGAGGGT CGCGGGTTCG AGTCCCGTTT CCCGCTCCAT 660
  • GCA AAA GAA AAG TTT AAC AGA ACT AAG CCG CAT GTT AAT ATT GGA ACC 941
  • GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT ATT ATT GAA TAA 2090
  • PROPERTIES 36/35/32 kD protein from Helicobacter pylori
  • ATC GGT AAA AAA AGG TGG TTT GGG TTG CGC TAT TAC GGC TTT TTT GAT 883 He Gly Lys Lys Arg Trp Phe Gly Leu Arg Tyr Tyr Gly Phe Phe Asp 110 115 120
  • PROPERTIES 17 kD protein from Helicobacter pylori

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Abstract

The present invention relates to novel proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, which are derived from Helicobacter pylori (H. pylori) and which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables 1a-1c, or to parts or homologues thereof having a minimum length of five amino acids, and to their preparation and use as pharmaceutical compositions, in particular as vaccines, or as a diagnostic agent. Based on these data, genes coding for these and related proteins were also isolated as shown in SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27.

Description

Proteins, in particular membrane proteins, of Hel±cobacter pylori , their preparation and use
TECHNICAL FIELD OF THE INVENTION
The present invention relates to novel proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, which are derived from Helicobacter pylori (H. pyl ori ) and which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five a ino acids, and to their preparation and use as pharmaceutical compositions, in particular as vaccines, or as a diagnostic agent. Based on these data, genes coding for these and related proteins were also isolated as shown in SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27.
BACKGROUND OF THE INVENTION
Helicobacter pylori is a Gram-negative, microaerophilic, spiral bacterium which colonizes the mucosa of the human stomach. The bacterium is the cause of chronic active gastritis and of peptic ulcer, in particular duodenal ulcer, and plays a role in the development of carcinomas of the stomach; consequently, Helicobacter pylori is an important human pathogen.
Its helical shape and otility, due to from four to six flagellae, enables the bacterium to migrate through the gastric mucus in order to reach the boundary layer, which is virtually at neutral pH, between the mucus and the mucosa. Ammonium ions, which are produced during the enzymic cleavage of urea by bacterial urease, protect the pathogen from the aggressive gastric acid. The bacterium adheres to the endothelial cells of the stomach using specific adhesins. A consequence of chronic colonization of the mucosa can be an inflammatory granulocytic, and subsequently monocytic, infiltration of the epithelium which in turn, by way of inflammation mediators, contributes to the tissue destruction. Infection stimulates both a local and a systemic humoral immune response, without these responses being able to eliminate the pathogen effectively. Immunization is the conventional way of preventing infectious diseases. It is therefore important to examine this option with regard to controlling an H. pylori infection.
The development of a vaccine involves identifying factors which are crucial for virulence or structures which are accessible to the human immune system for the purpose of eliminating a pathogen. It is to be assumed that antigens of this nature are present in the outer membrane of the bacterium. Thus, adhesins of 19,600 Da
(P. Doig et al., 1992, J. of Bacteriology 174, 2539-
2547), 20,000 Da (D.G. Evans et al., 1993, J. of
Bacteriology 175, 674-683) and 63,000 Da (C. Lingwood et al., 1993, Infection and Immunity 61, 2474-2478) are located in the outer membrane, which adhesins are candidates for an experimental vaccine which has the aim of inducing antibodies which prevent adhesion of the bacterium to the mucosal surface. In addition, the outer membrane possesses porins of 30,000 Da (M.A. Tufano et al., 1994, Infection and Immunity 62, 1392-1399), 48,000 Da, 49,000 Da, 50,000 Da, 67,000 Da (M.M. Exner et al., 1995, Infection and Immunity 63, 1567-1572) and 31,000 Da (P. Doig et al., 1995, J. of Bacteriology 177, 5447-5452) molecular weight, and also iron-regulated outer membrane proteins of 77,000 Da, 50,000 Da and 48,000 Da (D.J. Worst et al.. 1995, Infection and Immunity 63, 4161-4165) molecular weight, erythrocyte-binding antigens of 59, 000 Da and 25,000 Da (J. Huang et al . , 1992, J. of Gen. Microbiol. 138, 1503-1513) molecular weight and proteins for binding laminin, collagen I and IV, fibronectin and vitronectin (I. Kondo et al . , 1993, European J. Gastroenterol . Hepatol . 5, 63-67). In addition, proteins of 19,000 Da (E.B. Drouet et al., 1991, J. of Clinical Microbiology 29, 1620-1624), 50,000 Da (M.M. Exner et al . , 1995, Infection and Immunity 63, 1567-1572) and 30,000 Da (J.
Bδlin et al . , 1995, J. of Clinical Microbiology 33, 381-
384) molecular weight, and also a 20,000 Da lipoprotein
(M. Kostrzynska et al., 1994, J. of Bacteriology 176,
5938-5948) and strain-specific, surface-located antigens of 51,000 Da, 60,000 Da and 80,000 Da (P. Doig and T.J.
Trust, 1994, Infection and Immunity 62, 4526-4533) have been described. The genes for the proteins of 20,000 Da
(HpaA) (Evans et al.) and 20,000 Da (lpp20) (M.
Kostrzynska et al.) molecular weight have now been isolated. N-terminal protein sequence data have been disclosed for the adhesins of 19,600 Da (P. Doig et al . , 1992) and 63,000 Da (C. Lingwood et al . ) molecular weight, for the porins of 48,000 Da, 49,000 Da, 50,000 Da, 67,000 Da (M.M. Exner et al.), 30,000 Da (M.A. Tufano, 1994) and 31,000 Da (P. Doig et al., 1995) molecular weight and for the 50,000 Da protein (M.M. Exner et al., 1995) .
SUMMARY OF THE INVENTION
According to a first aspect of the present invention there is provided a protein from Helicobacter pyl ori (H. pylori ) containing one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 and 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids. Preferably the peptide sequences of the protein are N-terminal sequences.
The protein according to the first aspect of the present invention preferably contains a peptide sequence having the SEQ ID NO: 1 according to Table la and has a molecular weight of approx. 250 kD, or preferably contains a peptide sequence having the SEQ ID NO: 2 according to Table la and has a molecular weight of approx. 110 kD, or preferably contains a peptide sequence having the SEQ ID NO: 3 according to Table la and has a molecular weight of approx. 100 kD, or preferably contains a peptide sequence having the SEQ ID NO: 6 according to Table la and has a molecular weight of approx. 60 kD, or preferably contains a peptide sequence having the SEQ ID NO: 10 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 11 according to Table lb and has a molecular weight of approx. 42 kD, or preferably contains a peptide sequence having the SEQ ID NO: 12 according to Table lb and has a molecular weight of from approx. 32 to approx. 36 kD, or preferably contains a peptide sequence having the SEQ ID NO: 14 according to Table lc and has a molecular weight of approx. 30 kD, or preferably contains a peptide sequence having the SEQ ID NO: 15 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 16 according to Table lc and has a molecular weight of approx. 28 kD, or preferably contains a peptide sequence having the SEQ ID NO: 17 according to Table lc and has a molecular weight of approx. 25 kD, or preferably contains a peptide sequence having the SEQ ID NO: 18 according to Table lc and has a molecular weight of approx. 25 kD, or preferably contains a peptide sequence having the SEQ ID NO: 19 according to Table lc and has a molecular weight of approx. 17 kD. The protein according to the first aspect of the present invention is preferably a membrane protein or a protein which is firmly associated with the membrane. More preferably said protein is an integral membrane protein, in particular a Sarkosyl®-insoluble integral membrane protein.
In a second aspect of the invention there are provided proteins according to the first aspect of the present invention, which can be obtained in accordance with the following procedural steps:
(a) isolating the proteins by means of differential solubilization;
(b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods; and
(c) isolating the proteins, which have been separated in accordance with step (b) .
Preferably the proteins according to the second aspect of the present invention can be obtained by means of differential solubilization using Sarkosyl®. The proteins can also be obtained by means of separation by one or more SDS polyacrylamide gel electrophoreses, preferably by means of several SDS polyacrylamide gel electrophoreses having different polyacrylamide contents, more preferably wherein the polyacrylamide content of said gel electrophoreses is approximately 8%, 10% or 16%.
In a third aspect of the present invention there is provided a peptide having the amino acid sequence according to SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids.
In a fourth aspect of the present invention there is provided an antibody against one or more proteins according to the first or second aspects of the present invention and/or against one or more peptides according to the third aspect of the present invention.
In a fifth aspect of the present invention there is provided a polynucleotide encoding one or more proteins according to the first or second aspects of the present invention or one or more peptides according to the third aspect of the present invention.
In a sixth aspect of the present invention there is provided a process for preparing the proteins according to the first or second aspects of the present invention, characterized in that the following procedural steps are carried out: (a) isolating the proteins, by means of differential solubilization;
(b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods; and (c) isolating the proteins, which have been separated in accordance with step (b) .
Preferably the process is characterized in that the proteins are isolated in accordance with step (a) using Sarkosyl®.
In a seventh aspect of the present invention there is provided a process for preparing the peptides according to the third aspect of the present invention, characterized in that a chemical peptide synthesis is carried out.
In an eighth aspect of the present invention there is provided a process for preparing the proteins according to the first or second aspects of the present invention or the peptides according to the third aspect of the present invention, characterized in that a polynucleotide according to the fifth aspect of the present invention is expressed.
In a ninth aspect of the present invention there is provided the use of one or more proteins according to the first or second aspects of the present invention, one or more peptides according to the third aspect of the present invention, one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention for preparing a pharmaceutical composition or a diagnostic agent.
In a tenth aspect of the present invention there is provided a pharmaceutical composition comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products. Preferably said pharmaceutical composition is used as a vaccine.
In an eleventh aspect of the present invention there is provided a diagnostic agent comprising one or more proteins according to the first or second aspects of the present invention and/or one or more peptides according to the third aspect of the present invention or one or more antibodies according to the fourth aspect of the present invention or one or more polynucleotides according to the fifth aspect of the present invention or their expression products.
In a twelfth aspect of the present invention there is provided a protein from H. pylori containing one of the peptide sequences deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 and 27, or parts or homologues thereof having a minimum length of five amino acids.
In a thirteenth aspect of the present invention there is provided a peptide having the amino acid sequence deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 or 27, or parts or homologues thereof having a minimum length of five amino acids.
In a fourteenth aspect of the present invention there is provided a peptide selected from the C-terminal region of the peptide sequence of SEQ ID NO: 20 or homologue thereof. Preferably said peptide is selected from RDPKFNLAHIEKEFEVWNWDYRA and EKHQKMMKDMHGKDMHHTKKKK, or parts or homologues thereof.
In a fifteenth aspect of the present invention there is provided an antibody against one or more proteins according to the twelfth aspect of the present invention and/or against one or more peptides according to the thirteenth or fourteenth aspects of the present invention.
In a sixteenth aspect of the present invention there is provided a polynucleotide encoding one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention.
In a seventeenth aspect of the present invention there is provided a host cell transformed with the polynucleotide according to the fifth or sixteenth aspects of the present invention. In an eighteenth aspect of the present invention there is provided an expression product expressed from the host cell according to the seventeenth aspect of the present invention.
In a nineteenth aspect of the present invention there is provided a pharmaceutical composition comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products. Preferably said pharmaceutical composition is used as a vaccine. More preferably, when the pharmaceutical composition comprises a nucleotide sequence, said pharmaceutical composition is used as a DNA vaccine.
In a twentieth aspect of the present invention there is provided a diagnostic agent comprising one or more proteins according to the twelfth aspect of the present invention and/or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products.
In a twenty-first aspect of the present invention there is provided the use of one or more proteins according to the twelfth aspect of the present invention or one or more peptides according to the thirteenth or fourteenth aspects of the present invention or one or more antibodies according to the fifteenth aspect of the present invention or one or more polynucleotides according to the sixteenth aspect of the present invention or their expression products for preparing a pharmaceutical composition or a diagnostic agent.
DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE
The present application describes the isolation and determination of, in all, 19 proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, which proteins are in a molecular weight range of from 17 kD to approx. 250 kD (Tables la-lc) . The term membrane protein is generally understood to mean integral and peripheral membrane proteins and transmembrane proteins. Integral membrane proteins are proteins which are partially or entirely inserted into the cytoplasmic membrane. By contrast, peripheral membrane proteins only adhere to the surface of the membrane. Transmembrane proteins pass completely through the membrane (see, for example, B. Alberts et al. (eds). Membrane Proteins in "Molecular Biology of the Cell", 2nd ed., Garland Publishing, Inc., New York & London, 284-287, 1989). Two sequences were identified in one band in seven cases (SEQ ID NO: 2 and 3, 5 and 6, 7 and 8, 10 and 11, 13 and 14, 15 and 16, and 17 and 18), while it was only possible to identify one sequence in one band in a further five cases (SEQ ID NO: 1, 4, 9, 12 and 19) . Six N-terminal sequences from the 19 peptide sequences identified had already been described in earlier studies; these were the sequences for urease A and urease B (B.E. Dunn et al . , 1990, J. Biolog. Chem. 265, 9464-9469), for the exoenzyme S-like protein (C. Lingwood et al.), for the 50 kD membrane protein and for the porins hop B and hop C (M.M. Exner et al.). The only genes for these antigens which have so far been isolated are those for urease A and urease B (A. Labigne et al., 1991, J. Bacteriol. 173, 1920-1931). It was not possible to find the N-terminal sequences, which have already been described, of the membrane proteins of 19,600 Da (P. Doig et al., 1992), 48,000 Da, 67,000 Da (M.M. Exner et al . , 1995) and 31,000 Da (P. Doig et al., 1995) molecular weight among the 19 sequences which are described in accordance with the invention. Thus, the protein which is described by SEQ ID NO: 14 cannot be attributed, either, to the protein having the molecular weight of 31,000 Da (P. Doig et al., 1995). The remaining 13 amino terminal protein sequences of the 19 amino terminal protein sequences according to Tables la-lc have not been described. It is to be assumed that these sequences can be attributed to Helicobacter pyl ori proteins which have not previously been identified. It was surprising, therefore, that it was possible to demonstrate a large number of additional, novel H. pylori proteins in a Sarkosyl®-insoluble fraction. The proteins are very probably integral proteins of the outer membrane or proteins which are firmly associated with the membrane. They are therefore particularly suitable for use as candidates for developing a vaccine or a diagnostic agent.
The invention describes proteins, in particular membrane proteins or proteins which are firmly associated with the membrane, especially integral membrane proteins, in particular Sarkosyl®-insoluble integral membrane proteins of H. pylori , which contain one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five, preferably six amino acids, with these peptide sequences preferably constituting N-terminal sequences of the said proteins. The novel peptides are particularly preferred which exhibit at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19. In addition. those said parts are in particular preferred which contain an uninterrupted sequence of unambiguously specified amino acids.
The term "part" in the context of "part(s) of a sequence" in the present invention is defined herein as meaning a sequence of amino acids which can form a T-cell or B-cell epitope. Such an amino acid sequence is usually of a minimum of approximately four to eight amino acids.
The term "homologue (s) " in the context of the present invention is defined herein as meaning the same protein or peptide of a different strain of H. pylori but exhibiting the same function. Thus, although the actual amino acid sequences may not be identical between homologous proteins or peptides from different strains of H. pylori , the differences between the amino acid sequences merely represent strain-specific differences; the function of the homologues is identical.
In a particular embodiment, the protein containing a peptide sequence having the SEQ ID NO: 1 according to Table la has a molecular weight of approx. 250 kD, the protein containing a peptide sequence having the SEQ ID NO: 2 according to Table la has a molecular weight of approx. 110 kD, the protein containing a peptide sequence having the SEQ ID NO: 3 according to Table la has a molecular weight of approx. 100 kD, the protein containing a peptide sequence having the SEQ ID NO: 6 according to Table la has a molecular weight of approx. 60 kD, the protein containing a peptide sequence having the SEQ ID NO: 10 according to Table lb has a molecular weight of approx. 42 kD, the protein containing a peptide sequence having the SEQ ID NO: 11 according to Table lb has a molecular weight of approx. 42 kD, the protein containing a peptide sequence having the SEQ ID NO: 12 according to Table lb has a molecular weight of from approx. 32 to approx. 36 kD, the protein containing a peptide sequence having the SEQ ID NO: 14 according to Table lc has a molecular weight of approx. 30 kD, the protein containing a peptide sequence having the SEQ ID NO: 15 according to Table lc has a molecular weight of approx. 28 kD, the protein containing a peptide sequence having the SEQ ID NO: 16 according to Table lc has a molecular weight of approx. 28 kD, the protein containing a peptide sequence having the SEQ ID NO: 17 according to Table lc has a molecular weight of approx. 25 kD, the protein containing a peptide sequence having the SEQ ID NO: 18 according to Table lc has a molecular weight of approx. 25 kD, and the protein containing a peptide sequence having the SEQ ID NO: 19 according to Table lc has a molecular weight of approx. 17 kD.
The generally available H. pylori strain No. ATCC 43504 is used, for example, as the starting material when isolating the proteins, with it being possible, in particular, to carry out the following procedural steps:
(a) isolating the proteins by means of differential solubilization, in particular using Sarkosyl® (an N- lauroylsarcosine) in accordance with the method of Blaser et al. (1983, Infect. Immun. 42, 276-284),
(b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods, preferably by means of SDS polyacrylamide gel electrophoresis, with use being made, in particular, of polyacrylamide gels having differing polyacrylamide contents, in particular containing approx. 8, 10 or 16% polyacrylamide, and
(c) isolating the proteins, which have been separated in accordance with step (b) , by means of known methods, for example by elution or by isolation on a membrane .
For the purpose of isolating and characterizing the proteins according to the present invention, the proteins were first of all obtained using the method of Blaser et al. (see above). The bacteria, which had been disrupted in a glass bead homogenizer, were freed of intact bacteria by centrifugation at 5000 g; the supernatant was then centrifuged at 100,000 g. The pellet was dissolved in Sarkosyl®, and the Sarkosyl®-insoluble fraction, which contains the integral membrane proteins in particular, was centrifuged off. The pellet was resuspended in distilled water and fractionated by SDS polyacrylamide gel electrophoresis- (PAGE) . In this connection, it was found that SDS-PAGE, in contrast to HPLC, was a very effective method for separating Sarkosyl®-insoluble proteins. For this, the gels were pretreated with methionine in order to prevent oxidation of the methionine residues. After the run, the proteins were transferred from the SDS gel to a PVDF membrane
(Immobilon P®, from Millipore) , with 0.005% SDS being added to the cathode buffer in order to complete the transfer of the very insoluble proteins. For sequence analysis, the protein bands from four tracks, in each case, were cut out of the PVDF membrane and Ed an amino acid degradation was carried out in a 477A fluid-phase sequencer (Applied Biosystems, Inc. (ABI) ) to determine the amino acid sequence. While it is possible further to fractionate the proteins which run in one band, for example by means of isoelectric focusing or two- dimensional gel electrophoresis, this is not necessary for an unambiguous sequence analysis since the sequences can be assigned unambiguously on the basis of the different protein contents of the proteins which run in one band.
The amino acids which are labelled Xaa in the sequence listing can be explained as follows: The non-identifiable amino acids can be caused by interference due to impurities in the first sequencing step, a non-analysable amino acid, such as Cys or Trp, a modifiable amino acid which is missing in the elution programme, or an amino acid, such as Ser or Thr, which is difficult to determine, basically due to low sequence yields. Different bands can also contain two proteins of very similar molecular weights in different quantities. This then results in two sequences which then also have to be assigned unambiguously on account of the different frequency of the individual amino acids.
The present invention also describes the peptides which are designated by the sequences according to SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 16, 17, 18 or 19 according to Tables la-lc, or to parts or homologues thereof having a minimum length of five amino acids, in particular of six amino acids, which can be prepared, for example, by well-known chemical peptide synthesis
(Barani, G. & Merrifield, R. B. in "The Peptides:
Analysis, Synthesis and Biology" (Gross E., ed.). Vol. 2,
Academic Press, 1980, Johannes Meyenhofer Verlag;
Bodanszky, M. & Bodanszky, A. "The practice of peptide synthesis". Springer Verlag, 1984) . The novel peptides are particularly preferred which possess at least ten consecutive amino acids selected from the sequences having the SEQ ID NO: 1 , 2 , 3, 6, 10, 11, 12, 14, 15, 16 and 19. Furthermore, those said peptides are, in particular, preferred which contain an uninterrupted sequence of unambiguously determined amino acids, as is the case with the sequences from SEQ ID NO: 12, 14 and 15.
The present application also describes antibodies which can also be prepared by methods which are well known to the skilled person (see, for example, B.A. Diamond et al. (1981), The New England Journal of Medicine, 1344-1349) and which are directed against one or more of the novel proteins or peptides. The skilled person is also familiar, from
J. Sambrook et al. (1989, "Molecular Cloning, A Laboratory Manual", 2nd edn.. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), with methods for preparing polynucleotides which encode the novel proteins or peptides. In particular, the skilled person knows, on the basis of the genetic code, the nucleotide sequences which encode the peptides according to the sequence listing. In particular, the nucleotide sequences are preferred which occur most frequently in accordance with the rules for the frequency of use of the different codons in Helicobacter pylori . These nucleotide sequences can be prepared, for example, by means of chemical polynucleotide synthesis (see, for example, E. Uhlmann & A. Peyman (1990), Chemical Reviews, 543-584, Vol. 90, No. 4) .
For example, oligodeoxynucleotides which have been prepared in accordance with these rules can be employed for screening Helicobacter pylori gene libraries using known methods (J. Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", 2nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Furthermore, taking the sequence data as a basis, peptides can be synthesized which are employed for obtaining antisera. Gene expression libraries can then be screened using these antisera. The clones resulting from these different screening methods can then be employed, by isolating and sequencing the inserted DNA fragments, for identifying DNA sequence segments which encode the N-terminally sequenced protein segments of the proteins. If the inserted DNA fragments do not contain the complete gene encoding any particular protein, these DNA fragments can be used to isolate the complete genes by screening other gene libraries. The genes which have been completely isolated in this manner can then be expressed, in accordance with the state of the art, in various well- known systems in order to obtain the corresponding protein.
Using oligonucleotides deduced from the N- terminal sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15, the genes corresponding to the SEQ ID NOS: 5, 8, 10, 12 and 15 were isolated and are specified as SEQ ID NOS: 20 (catalase) , 24 (50 kD membrane protein), 25 (42 kD protein), 26 (36/35/32 kD protein) and 23 (28 kD protein) . The gene coding for Hop C could not be isolated using oligonucleotide 7. However, oligonucleotide 7 hybridizes with an homologous gene specified as SEQ ID NO: 21 (Hop X) . Two additional genes which belong to this family were able to be isolated and are specified as SEQ ID NO: 21 (Hop Y) and SEQ ID NO: 22 (Hop Z) .
Another approach is given by the recent access to the complete geno ic sequence of H. pylori on the internet which allowed, for example, the identification Of SEQ ID NO: 27. The novel proteins, peptides, antibodies and polynucleotides, and their expression products, can now be used, in accordance with methods known to the skilled person, for preparing a pharmaceutical composition , in particular a vaccine, or a diagnostic agent. Those regions of the proteins which, on the one hand, occur, if possible, in all H. pylori strains, and, on the other hand, bring about the formation of protective antibodies, are particularly suitable for preparing vaccines. A special preference is given to the regions which project from the surface of the bacteria.
Such vaccines may either be prophylactic (to prevent infection) or therapeutic (to treat disease after infection) . These vaccines comprise antigen or antigens, usually in combination with "pharmaceutically acceptable carriers," which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants") . Furthermore, the antigen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum) , such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components) , such as for example (a) those formulations described in
PCT Publ. No. WO 90/14837, including but not limited to
MF59 (containing 5% Squalene, 0.5% Tween 80, and 0.5%
Span 85 (optionally containing various amounts of MTP-PE
(see below), although not required) formulated into submicron particles using a microfluidizer such as Model HOY microfluidizer (Microfluidics, Newton, MA) ) , (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and
(c) RibiTM adjuvant system (RAS) , (Ribi Immunochem,
Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL) , trehalose dimycolate (TDM), and cell wall skeleton (CWS) , preferably MPL + CWS (DetoxTM) ; (3) saponin adjuvants, such as StimulonTM (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., gamma interferon) , macrophage colony stimulating factor (M-CSF) , tumour necrosis factor (TNF) , etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59 are preferred. As mentioned above, muramyl peptides include, but a r e n o t l i m i t e d t o , N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) , N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP) , N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2- (1 ' -2 * -dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy) - ethyla ine (MTP-PE), etc.
The immunogenic compositions (e.g., the antigen, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents .
The present invention describes, therefore, pharmaceutical compositions, in particular vaccines, and diagnostic agents which comprise one or more of the novel proteins and/or one or more of the novel peptides or one or more of the novel antibodies or one or more of the novel polynucleotides or one or more expression products of the novel polynucleotides. For example, according to the present invention, a DNA vaccine can be prepared on the basis of the polynucleotides, or a diagnostic agent can be prepared on the basis of the polymerase chain reaction (PCR diagnosis), or an immunotest, for example a Western blot test or an enzyme immunotest (ELISA) can be prepared on the basis of the antibodies. Furthermore, the novel proteins or peptides, or their immunogenic moieties, in particular when they contain an uninterrupted sequence of unambiguously determined amino acids, having a minimum length of five amino acids, preferably six amino acids and, in particular, in the case of the novel peptides having the SEQ ID NOS: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16 and 19 and peptides or proteins encoded by the DNA sequences of SEQ ID NOS: 20, 21, 22, 23, 24, 25, 26 and 27, at least ten consecutive amino acids, can be used as antigens for immunizing mammals. In this context, the two C-terminal regions Cl and C2 specific for H. pylori catalase (c.f. Example 6) can also be used as immunogens . The antibodies which are formed by the immunization, or antibodies which are prepared by means of recombinant DNA methods (see, for example, Winter G. & Milstein C. (1991) Nature, 293-299, Vol. 349), can, inter alia, prevent adhesion of the bacteria to the mucosal surface, attract macrophages for the purpose of eliminating bacteria, and activate the complement system for the purpose of lysing the bacteria. The following examples are intended to clarify the invention.
EXAMPLES
Example 1:
Culture of Helicobacter pylori
The H. pylori stain ATCC 43504 was passaged under microaerophilic conditions (BBL Jar/Campy Pak Plus, from Becton & Dickinson) on Columbia Agar plates containing 5% horse blood (incubation 48 h, 37°C) . Three plates were rinsed off when inoculating a 500 ml flow-spoiler flask (100 ml of Columbia broth, 7% FCS) ; during the incubation
(BBL Jar/Campy Pak Plus; 48 h, 37°C, 90 rpm) , the OD590 rose from 0.3 to 2.0. The bacteria were harvested by centrifugation at 10,000 rpm and washed twice with physiological sodium chloride solution. E am le 2 :
Isolation of Helicobacter pylori outer meinbrane proteins
The preparation of the outer membrane protein fraction, with the inner and outer membrane proteins being separated by means of differential solubilization with Sarkosyl® (Ciba-Geigy AG) , was carried out using the method of Blaser et al. In this method, the bacterial cultures are harvested in the phase of late logarithmic growth, washed in 10 mM Tris buffer (pH 7.4) and disrupted with glass beads in a ho ogenizer (Institut fiir Molekularbiologie und Analytik (IMA), Germany) at 4°C and 4000 rpm for 15 min. After that, the glass beads are removed by filtration and the bacterial suspension is centrifuged at 5000 g for 20 min in order to remove intact cells. The cell walls are pelleted out of the supernatant by centrifuging at 100,000 g for 60 minutes and at 4°C. The resulting pellet is resuspended with a 1% solution of Sarkosyl® in 7 mM EDTA, and the suspension is incubated at 37°C for 20 min. The Sarkosyl®-insoluble fraction, which contains the integral membrane proteins, is pelleted by centrifugation at 50,000 g for 60 minutes and at 4°C and the pellet is resuspended in sterile distilled water; the suspension is then stored at -20°C.
Example 3:
SDS polyacrylamide gel electrophoresis and blotting
Gel preparation, and the electrophoresis, were carried out in a BioRad (Munich) Protean II xi slab cell apparatus. The chemicals employed, and the polyacrylamide monomer (as a 30% solution containing 0.8% bisacryla ide) , were obtained from Oxford GlycoSystems (Oxford, UK) . In addition to a 10% standard gel, gels containing polyacrylamide contents of 8% and 16% were also especially employed for carrying out separations in the high-molecular weight and low-molecular weight ranges, respectively. The thickness of the gel was 1 mm. In order to eliminate undesirable oxidizing properties of the ammonium persulphate used for preparing the gel, all the wells of the gel were filled with a solution containing 50 pM of L-methionine/microlitre and left to stand overnight. After the solution has been sucked off on the following day, and after each of the wells has once again been filled with 10 microlitres of this solution in each case, a preliminary electrophoresis takes place. This preliminary treatment prevents the methionine residues of the protein from being oxidized and thereby enables a protein cleavage with BrCN (Met cleavage site) to be carried out if required. The membrane protein fraction starting material is dissolved in 1.5% SDS, 2.5% mercaptoethanol, 5% glycerol and bromophenol blue in 63 mmol/1 Tris buffer, pH 6.8, and fractionated by SDS polyacrylamide gel electrophoresis.
Protein transfer from the SDS gel to the PVDF membrane (Immobilon P®, from Millipore) is carried out in a BioRad (Munich) Trans Blot SD apparatus, under modified conditions.
For the purposes of completing the protein transfer, 0.005% SDS is added to the cathode buffer, thereby counteracting too rapid an impoverishment of SDS in the gel. The use of six filter papers, which are soaked with this buffer, on the cathode side is found to give optimum results in this connection.
The blot was then stained with amidoblack using the protocol of R. Westermeier (Elektrophorese Praktikum (Electrophoresis Laboratory Manual) VCH Verlag Weinheim, 1990, ISBN 3-527-28172-X) . Example 4 :
N-terminal Edman degradation
The Edman amino acid degradation, and the determination of the PTH amino acids, were carried out in a 477 A liquid phase sequencer having an on-line 120A HPLC analyser (ABI) .
For the analyses, the corresponding bands from, in each case, four tracks were cut out of the PVDF blot membrane and sequenced after a washing step, as recommended by ABI.
The number of sequencing steps was 5 to 25 (depending on the quantity of substance available for sequencing) .
The Cys and Trp PTH amino acids cannot be detected under the conditions which were chosen.
Example 5 :
Deduction of oligonucleotides for screening gene libraries and for identifying DNA fragments via Southern Blot analysis
The following oligonucleotides were deduced from the resulting N-terminal sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15:
Figure imgf000027_0001
The oligonucleotides were deduced using the species-specific codon usage of Helicobacter pylori , which had been determined from 19 known H. pylori genes, and using the base inosine (I), which is capable of undergoing stable base pairing with the bases adenine (A) , cytosine (C) and thymine (T) with, in each case, two hydrogen bridges. When carrying out the deduction, the degeneracy of the codon was kept as low as possible.
Example 6:
Isolation and characterization of the genes using the oligonucleotides deduced from the peptide sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15
The oligonucleotides which had been deduced from the peptide sequences of SEQ ID NOS: 5, 7, 8, 10, 12 and 15 were labelled with digoxigenin (DIG) using a kit manufactured by Boehringer Mannheim (DIG Oligonucleotide 3'-End Labelling Kit) and employed for screening a H. pylori gene library which had been prepared using a kit manufactured by Stratagene (Predigested ZAP Express™ BamHI/CIAP Vector Cloning Kit) at 32°C under standard conditions. Using oligonucleotides 1, 3 and 6, it was possible to identify clones which carry DNA fragments containing sequences which encode the peptide sequences of SEQ ID NOS: 5, 8 and 15. Oligonucleotide 2 hybridized with a DNA fragment which encodes an homologous sequence of SEQ ID NO: 7.
Using oligonucleotides 4 and 5, it was only possible to isolate clones whose DNA fragments did not encode SEQ ID NOS: 10 and 12. This is why these oligonucleotides and the clones which had been isolated from the λZAP Express gene library were employed in a Southern Blot analysis, which permitted the unequivocal identification of DNA fragments which hybridized with the oligonucleotides, but not with the DNA fragments resulting from the screening. With these DNA fragments, in each case one sub-gene library was prepared in the λZAP Express vector, and each sub-gene library was screened with oligonucleotides 4 and 5. This allowed the identification of clones which carry DNA fragments encoding the sequences of SEQ ID NOS: 10 and 12.
Partial digestion of H. pylori DNA using the restriction enzymes Sau3AI, Alul and f-aelll gave a DNA which was used for establishing gene libraries in the vector λTriplex (Clontech) . These gene libraries were used as starting material for isolating the complete genes of the above-described DNA fragments using standard methods.
SEQ ID NO: 20 describes the DNA sequence which encodes the catalase of H. pylori . The nucleotide region 337 to 378 describes the hybridization site with oligonucleotide 1. The catalase gene of H. pylori has been described in 1996 by Stefan Odenbreit, Bjδrn Wieland and Rainer Haas (J. Bacteriol. 178, 6960-6967) and is therefore not new. However, when comparing the amino acid sequences of the catalases of Escheri chia coJi, Bacillus firmυs, B. subtilis A, B. subtilis B, rats, mice, cattle, humans, Staphylococcus violaceus, Haemophilus inflυenzae, B. fragilis, Pseudomonas mirabilis, B. pertussis and P. syringae with the amino acid sequence of H. pylori , it is possible to identify two C-terminal regions Cl (RDPKFNLAHIEKEFEVWNWDYRA) and C2 (EKHQKMMKDMHGKDMHHTKKKK) , which are specific to H. pyl ori catalase. These two peptides were synthesized using standard techniques, coupled to KLH and used for immunizing rabbits. These rabbits developed antibodies against the two peptides, which reacted in the Western Blot analysis with H. pylori catalase which had been produced by recombinant technique. These H. pyl ori- catalase-specific regions may conceivably be used for developing a vaccine which avoids the problem complex of autoimmune reactions or for the development of a diagnostic which reacts specifically with H. pylori catalase.
SEQ ID NO: 21 describes a nucleotide sequence which was identified by hybridization with the oligonucleotide 2. The oligonucleotide hybridized with the sequence of nucleotide 1240 to 1284. This encodes a sequence which is homologous to the porin Hop C (Exner et al., 1995) and is identical with the published amino- terminal sequence EDDGGFFTVGYQLGQVMQDVQNPG in positions 1, 2, 3, 4, 9, 10, 11, 12, 14, 18 and 22.
The porins Hop A, Hop B, Hop C and Hop D have identical amino acids in 9 positions of the 20 N-terminal amino acids (Exner et al . , 1995). In 8 of these positions, there are identical positions also in the sequence described in the present publication; in the 9th position, a conserved amino acid exchange is present (Val - lie) . It can thus be assumed that the protein described in the present publication is equally part of this group of the porins; it was therefore termed Hop X.
On the basis of the homology data and on the basis of the N-terminal sequence determined and on the basis of the hydrophobicity of the N-terminal protein sequence deduced from the nucleic acid sequence, it can be concluded that the protein deduced has a signal sequence. The mature protein with 428 amino acids has a molecular weight of 47.3 kD and an isoelectric point of 10.0.
A further open reading frame was found upstream of the gene which encodes Hop X. This further open reading frame encodes a protein which is homologous to Hop X (34% identity) and which was therefore termed Hop Y. The gene region found to date encodes the 361 C- terminal amino acids of the protein. The gene region as yet outstanding is currently being isolated using stan- dard techniques .
We have thus identified a gene region of H. pylori which encodes at least two porins which are connected in series. SEQ ID NO: 22 describes a nucleotide sequence which was concomitantly isolated and sequenced during the screening process . The amino acid sequence deduced encodes the 392 C-terminal residues of a protein which shows a high homology with Hop X (33% identity) and Hop Y (28% identity) and which was therefore termed Hop Z. The gene region which encodes the N-terminal portion of the protein is currently being isolated.
SEQ ID NO: 23 describes a DNA sequence which encodes a hitherto undescribed protein. The nucleotide region 696 to 767 describes the hybridization site with the oligonucleotide 6. On the basis of the N-terminal protein sequence which has been determined, in which it was not possible unequivocally to determine the amino acids in the first two positions, and on the basis of the hydrophobicity of the N-terminal protein sequence deduced from the nucleic acid sequence, it can be concluded that the protein deduced has a signal sequence of 17 amino acids. The mature protein of 231 amino acids has a molecular weight of 26.4 kD and an isoelectric point of 10.3. Thus, the molecular weight is quite close to the molecular weight of 28 kD which had been determined by SDS gel electrophoresis. The amino acid sequence deduced is homologous with the sequences of the proteins Hop X, Hop Y and Hop Z, for which the GCG Bestfit Programme determined identity values of 41%, 38% and 41%, respectively. The 28 kD protein thus also seems to be part of the family of the porins or porin-like proteins.
SEQ ID NO: 24 describes a DNA sequence which encodes the non-heat-modifiable 50 kD membrane protein. This protein was first described by Exner et al., 1995, and an N-terminal sequence of the protein was determined. Using the approach described by us, we were then able to describe, with SEQ ID NO: 8, an N-terminal sequence which is identical to the sequence described by Exner et al. (1995) . With the aid of the oligonucleotide 3, which had been deduced using the method illustrated in Example 5 and had been used for screening a H. pylori gene library using the above-described methods, it was then possible to identify a DNA fragment which encodes the 50 kD membrane protein. Using other standard methods, it was then possible to determine the nucleic acid sequence described in SEQ ID NO: 24, which encodes a mature protein of 499 amino acids which has a molecular weight of 56.3 kD and an isoelectric point of 9.75. Due to the data of the N-terminal sequencing procedures and the hydrophobicity of the N-terminal sequence, a signal sequence of 29 amino acids is assumed. The amino acid residues 236 to 254 contain a hydrophobic region which is large enough to act as a transmembrane region. Based on such data and using standard methods for epitope analysis, it is possible to identify regions which might be presented on the surface of bacteria. Such regions might be used for developing a vaccine or a diagnostic.
SEQ ID NO: 25 describes a DNA sequence 2825 bp in size which was identified by means of hybridization with oligonucleotide 4, which was deduced from SEQ ID NO: 10. Oligonucleotide 4 hybridized with the nucleotide region 897 to 923 of the described sequence of SEQ ID NO: 25. The protein has no signal sequence. The encoding region of SEQ ID NO: 25 codes for a protein of 399 amino acids with a molecular weight of 43.6 kD and an isoelectric point of 5.0. A search for homologous sequences using the BLASTP program (S. F. Altschul et al., 1990, J. Mol. Biol. 215, 403-410) identified the 42 kD antigen of H. pylori as the elongation factor TU. The maximum percentage of identity (89%) was found with the elongation factor TU from Wolinella succinogenes (W. Ludwig et al . , 1993, Antonie van Leeuwenhoek 64, 285- 305 ) .
SEQ ID NO: 26 describes a DNA sequence 2182 bp in size which hybridizes with oligonucleotide 5, which had been deduced from SEQ ID NO: 12. Oligonucleotide 5 hybridized with a Sau3AI fragment (position 1 to 575) of the gene library starting from position 524. The screening of different DNA libraries with specific oligonucleotides allowed the isolation of the complete gene described in SEQ ID NO: 26. An amino acid sequence which is identical to the one from SEQ ID NO: 12 can be deduced from SEQ ID NO: 26. Both protein sequencing and the hydrophobicity of the N-terminal sequence deduced allow the conclusion that the antigen has a signal sequence. The mature protein consists of 328 amino acid residues with a molecular weight of 36.1 kD and an isoelectric point of 9.95. No homologous proteins were identified using the BLASTP program (S. F. Altschul et al., 1990) .
The sequences described in SEQ ID NOS: 20 to 26 indicate nucleotide sequences which encode antigens of the H. pylori strain ATCC 43504. However, it is known for H. pylori that heterogeneity between identical antigens may exist amongst various strains. We therefore claim not only the sequences described in SEQ ID NOS: 21 to 26, but in addition also the sequences of other H. pylori strains which are homologous with the sequences described herein.
Example 7
Identification and isolation of genes from H. pylori corresponding to the peptide sequences listed in Tables la-lc using the access to the genomic sequence
The Institute for Genomic Research (TIGR) released the DNA sequence from H. pylori on 24th June 1997. This new information can be accessed on the internet at "www.tigr.org". Using the TBLASTN program (Altschul et al., 1997, Nucleic Acids Research 25, in press) the peptide sequences listed in Tables la-lc can be aligned to amino acid sequence data deduced from all six reading frames of the H. pylori strain 26695. Having access to the genomic DNA sequence, DNA sequences corresponding to the aligned amino acid sequences can be identified using GCG (Genetic Computer Group) programs. This approach is shown for SEQ ID NO: 19, for example. The sequence of SEQ ID NO: 19 aligned with a very similar sequence using the TBLASTN program. SEQ ID NO: 27 describes the nucleic acid sequence and deduced amino acid sequence from the coding region of a H. pylori gene (strain 26695) localised between position 843212 and 843691 of the genomic sequence. The protein has no signal sequence. The N-terminal sequence of SEQ ID NO: 19 is highly homologous to the N-terminal region of the deduced amino acid sequence from amino acid residue 1 to 15. Only one different amino acid residue is present at position 4: the nucleotide sequence found by the alignment encodes a Ser residue in this position instead of an Asn residue determined by N-terminal sequencing. This can be explained by strain specific differences. The identified nucleic acid sequence in SEQ ID NO: 27 codes for a protein of 159 amino acid residues with a molecular weight of 18.2 kD and an isoelectric point of 7.2. The molecular weight is very close to that of 17 kD determined from SDS polyacrylamide gel electrophoresis. A search for homologous sequences using the BLASTP program (S. F. Altschul et al., 1990) shows that the
17 kD antigen is very homologous to "hydroxymyristol-
[acyl carrier protein] dehydratase" from different bacteria. Table la
J :
Figure imgf000035_0001
Table lb
Figure imgf000036_0001
Table lc
O σ O
>
Figure imgf000037_0001
SEQUENCE LISTING
(1) GENERAL INFORMATION:
APPLICANT:
(A) NAME: Chiron Behring GmbH & Co.
(B) STREET: P.O. Box 16 30
(C) CITY: Marburg
(E) COUNTRY: Germany
(F) POSTAL CODE: D-35006
TITLE OF APPLICATION:
Proteins, in particular membrane proteins, of Heli cobacter pylori , their preparation and use
NUMBER OF SEQUENCES: 27
COMPUTER READABLE FORM:
(A) MEDIUM TYPE: floppy disk, 3^ inch, 1.44 MB
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PADAT sequence module version 1.0
(2) INFORMATION FOR SEQ ID NO: 1:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(vi) FURTHER INFORMATION:
Xaa at positions 1, 5, 12, 14 and 16 are unknown amino acids. At position 8, Xaa is probably Gin, while at position 10 it is probably Ser, at position 11 it is probably Tyr and at position 15 it is probably Thr. Identification: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Xaa Pro Asn Gly Xaa Tyr Met Xaa Arg Xaa Xaa Xaa lie Xaa 1 5 10
Xaa Xaa Gin 15
(2) INFORMATION FOR SEQ ID NO: 2:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
At position 1, Xaa is an unknown amino acid. At position 4, Xaa is He or Thr and at position 7 it is Ala or Lys. Identification: unknown (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Xaa Lys Leu Xaa Pro Gin Xaa Gly Tyr Val Leu Met Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO: 3:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
( v ) FRAGMENT TY PE : N-termmus
(vi) FURTHER INFORMATION:
Xaa at positions 1 and 10 are unknown ammo acids, and at position 4, Xaa is He or Thr and at position 7 it is Ala or Lys . Identification: unknown
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Xaa Gin Asp Xaa Phe Leu Xaa Glu Gly Xaa Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO: 4:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(Vl) FURTHER INFORMATION:
At position 1, Xaa is probably Met. Identification: urease B ( XI ) SEQUENCE DESCRI PTION: SEQ ID NO : 4 :
Xaa Lys Lys He Ser Arg Lys Glu Tyr Val Ser Met Tyr Gly
Figure imgf000041_0001
Pro
15
(2) INFORMATION FOR SEQ ID NO: 5:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: ammo ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(l ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-termmus
(VI) FURTHER INFORMATION:
Xaa at positions 1 and 10 are unknown ammo acids. Identification: 63 kD exoenzyme-like adhesin
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Xaa Val Asn Lys Asp Val Lys Gin Thr Xaa Ala Phe Gly Ala 1 5 10
Pro
15
(2) INFORMATION FOR SEQ ID NO: 6:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus (vi) FURTHER INFORMATION:
Xaa at positions 1, 5 and 9 are unknown ammo acids, and at position 7 Xaa is Ala or Leu. Identification: unknown
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Xaa Phe Gin Val Xaa Phe Xaa He Xaa Ala Met Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO: 7:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
At positions 2, 3 and 19, Xaa are unknown ammo acids, and at position 1 Xaa is probably Glu. Identification: Hop C
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Xaa Xaa Xaa Gly Gly Phe Phe Thr Val Gly Tyr Gin Leu Gly 1 5 10
Gin Val Met Gin Xaa Val 15 20
(2) INFORMATION FOR SEQ ID NO: 8:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: N-terminus
(Vi) FURTHER INFORMATION:
Xaa at positions 1, 2, 7 and 13 are unknown, and at position 8 Xaa is probably Asp and at position 9 it is probably Phe. Identification: 50 kD membrane protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Xaa Xaa Tyr Glu Val His Xaa Xaa Xaa He Asn Phe Xaa Lys 1 5 10
Val 15
(2) INFORMATION FOR SEQ ID NO: 9:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
Xaa at positions 1, 2 and 5 are unknown. Identification: Hop B
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Xaa Xaa Asp Gly Xaa Phe Met Thr Phe Gly Tyr Glu Leu Gly 1 5 10
Gin 15
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
Xaa at positions 1 and 11 are unknown, while at position 6, Xaa is probably Asn or Gin, at position 13 it is probably Thr and at position 14 it is probably He. Identification: unknown
(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Xaa Lys Glu Lys Phe Xaa Arg Thr Lys Pro Xaa Val Xaa Xaa 1 5 10
(2) INFORMATION FOR SEQ ID NO: 11:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-termmus
(VI) FURTHER INFORMATION:
Xaa at positions 1 and 4 are unknown, while at position 6 Xaa is Asn or Gin and at position 8 it is probably Pro. Identification: unknown
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Xaa Gly His Xaa Gin Xaa His Xaa Ala Gin 1 5 10
(2) INFORMATION FOR SEQ ID NO: 12:
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(Vi) FURTHER INFORMATION:
Xaa at position 1 is unknown, while at position 21 it is probably Thr. Identification: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Xaa Glu Lys Asn Gly Ala Phe Val Gly He Ser Leu Glu Val 1 5 10
Gly Arg Ala Asp Gin Lys Xaa 15 20
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(vi) FURTHER INFORMATION:
Identification: urease A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Met Lys Leu Thr Pro Lys Glu Leu Asp Lys Leu Met Leu His 1 5 10
Tyr Ala Gly Glu Leu Ala 15 20 (2 ) INFORMATION FOR SEQ ID NO : 14 :
( 1 ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 14 amino acids
(B ) TYPE : amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
Xaa at positions 1 and 13 are unknown amino acids. Identification: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Xaa Glu Phe Ala Gin Phe Val Gly Val Asn Tyr Gin Xaa Asn 1 5 10
(2) INFORMATION FOR SEQ ID NO: 15:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 ammo acids
(B) TYPE: ammo ac d
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(li) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
Xaa at position 1 is an unknown ammo acid and at position 2 it is probably Trp. Identification: unknown
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Xaa Xaa Ser Ala Ala Phe Val Gly Val Asn Tyr Gin Val Ser 1 5 10
Met He Gin Asn Gin Thr Lys Met Val Asn Asp 15 20 25 (2) INFORMATION FOR SEQ ID NO: 16:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
( i) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N- erminus
(Vl) FURTHER INFORMATION:
Xaa at positions 1, 2, 3, 6, 10 and 14 are unknown ammo acids, while at position 5, Xaa is Pro or Val and at position 7 it is probably Lys. Identification: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Xaa Xaa Xaa He Xaa Xaa Xaa Leu Tyr Xaa Leu Met Leu Xaa 1 5 10
Arg 15
(2) INFORMATION FOR SEQ ID NO: 17:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: am o acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(II) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(VI) FURTHER INFORMATION:
Xaa at position 1 is an unknown ammo acid, while at position 5 Xaa is Pro or Lys. Identification: unknown (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17; Xaa Gin Arg Met Xaa Gin Val Gly 1 5
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(vi) FURTHER INFORMATION:
Xaa at position 1 is an unknown amino acid, while at position 5 Xaa is Pro or Lys. Identification: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Xaa Leu Asn He Xaa Phe Ala 1 5
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: N-terminus
(Vi) FURTHER INFORMATION:
Xaa at positions 1, 5 and 10 are unknown amino acids, while at position 11 Xaa is probably Gin and at position 15 it is probably Lys. Identification : unknown
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 19 : Xaa Glu Gin Asn Xaa Gin Asn Leu Gin Xaa Xaa Phe Phe He 1 5 10
Xaa 15
(2) INFORMATION FOR SEQ ID NO: 20:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2310 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 334 to 1851 bp protein
(vi) PROPERTIES: catalase from Heli cobacter pylori
TTAAAAACAT CCAACAACTC TCCTTAAATT TAAAAATTCA AAAAATAAAA ATCAAAAAAA 60
AAAAAAAACA AAATCCGTCA ATGCATTGAT ATAAAATAGT ATAATAATTA TTATTAAAAC 120
CAGATTAAAA ATAAAATTTT GTCCTTAATC TTTCTTATTT TCATTAATTG TTACGAATAG 180
AAATACTTAA GGGGTTTTTT TAATTCTTAA AAAAGGATTT TTTAAGGAAA TTGAATCTTG 240
TTAGTCTTTG TATAACAAAT TATGTAATAA TCACCACAAG TGATCGGCTT AGTGTCAGAT 300
TACGAAGATT TAAGATCAAT TACAGGAAAA AAG ATG GTT AAT AAA GAT GTG AAA 354
Met Val Asn Lys Asp Val Lys
5 CAA ACC ACT GCT TTT GGC GCT CCC GTT TGG GAT GAC AAC AAT GTG ATT 402
Gin Thr Thr Ala Phe Gly Ala Pro Val Trp Asp Asp Asn Asn Val He 10 15 20
ACG GCC GGC CCT AGA GGT CCT GTT TTA TTA CAA AGC ACT TGG TTT TTG 450
Thr Ala Gly Pro Arg Gly Pro Val Leu Leu Gin Ser Thr Trp Phe Leu 25 30 35
GAA AAG TTA GCG GCG TTT GAC AGA GAA AGA ATC CCT GAA AGG GTG GTG 498
Glu Lys Leu Ala Ala Phe Asp Arg Glu Arg He Pro Glu Arg Val Val 40 45 50 55
CAT GCT AAA GGA AGC GGA GCT TAT GGC ACT TTC ACT GTG ACT AAA GAC 546
His Ala Lys Gly Ser Gly Ala Tyr Gly Thr Phe Thr Val Thr Lys Asp 60 65 70
ATC ACT AAA TAC ACT AAA GCG AAA ATT TTC TCT AAA GTG GGC AAA AAA 594
He Thr Lys Tyr Thr Lys Ala Lys He Phe Ser Lys Val Gly Lys Lys 75 80 85
ACC GAA TGC TTC TTC AGA TTT TCC ACT GTG GCT GGT GAA AGA GGC AGT 642
Thr Glu Cys Phe Phe Arg Phe Ser Thr Val Ala Gly Glu Arg Gly Ser 90 95 100
GCG GAT GCG GTA AGA GAC CCT AGA GGT TTT GCG ATG AAG TAT TAC ACT 690
Ala Asp Ala Val Arg Asp Pro Arg Gly Phe Ala Met Lys Tyr Tyr Thr 105 110 115
GAA GAA GGT AAC TGG GAT TTA GTA GGG AAC AAC ACG CCT GTC TTC TTT 738
Glu Glu Gly Asn Trp Asp Leu Val Gly Asn Asn Thr Pro Val Phe Phe 120 125 130 135
ATC CGT GAT GCG ATC AAA TTC CCT GAT TTC ATC CAC ACT CAA AAA CGA 786
He Arg Asp Ala He Lys Phe Pro Asp Phe He His Thr Gin Lys Arg 140 145 150
GAT CCT CAA ACC AAT TTG CCT AAC CAT GAC ATG GTA TGG GAT TTT TGG 834
Asp Pro Gin Thr Asn Leu Pro Asn His Asp Met Val Trp Asp Phe Trp 155 160 165
AGT AAT GTT CCT GAA AGC TTA TAC CAA GTA ACA TGG GTT ATG AGC GAT 882
Ser Asn Val Pro Glu Ser Leu Tyr Gin Val Thr Trp Val Met Ser Asp 170 175 180 AGA GGG ATT CCT AAA TCT TTC CGC CAC ATG GAT GGT TTT GGC AGT CAC 930
Arg Gly He Pro Lys Ser Phe Arg His Met Asp Gly Phe Gly Ser His 185 190 195
ACT TTC AGT CTT ATC AAC GCT AAA GGC GAA CGC TTT TGG GTG AAA TTC 978
Thr Phe Ser Leu He Asn Ala Lys Gly Glu Arg Phe Trp Val Lys Phe 200 205 210 215
CAC TTT CAC ACC ATG CAA GGC GTT AAG CAC TTG ACT AAC GAA GAA GCC 1026 His Phe His Thr Met Gin Gly Val Lys His Leu Thr Asn Glu Glu Ala 220 225 230
GCA GAA GTC AGA AAA TAT GAT CCT GAT TCC AAT CAA AGG GAT TTA TTC 1074 Ala Glu Val Arg Lys Tyr Asp Pro Asp Ser Asn Gin Arg Asp Leu Phe 235 240 245
AAT GCG ATC GCT AGA GGG GAT TTC CCA AAA TGG AAA TTA AGC GTT CAA 1122 Asn Ala He Ala Arg Gly Asp Phe Pro Lys Trp Lys Leu Ser Val Gin 250 255 260
GTG ATG CCA GAA GAA GAT GCT AAG AAG TAT CGA TTC CAT CCG TTT GAT 1170 Val Met Pro Glu Glu Asp Ala Lys Lys Tyr Arg Phe His Pro Phe Asp 265 270 275 280
GTG ACT AAA ATT TGG TAC CTC CAA GAT TAT CCG TTG ATG GAA GTG GGC 1218 Val Thr Lys He Trp Tyr Leu Gin Asp Tyr Pro Leu Met Glu Val Gly 285 290 295
ATT GTA GAG TTG AAT AAA AAT CCC GAA AAC TAT TTC GCA GAA GTG GAG 1266 He Val Glu Leu Asn Lys Asn Pro Glu Asn Tyr Phe Ala Glu Val Glu 300 305 310
CAA GCG GCA TTC AGT CCG GCT AAT GTC GTT CCT GGA ATT GGC TAT AGC 1314 Gin Ala Ala Phe Ser Pro Ala Asn Val Val Pro Gly He Gly Tyr Ser 315 320 325
CCT GAT AGG ATG TTA CAA GGG CGC TTG TTC TCT TAT GGG GAT ACA CAC 1362 Pro Asp Arg Met Leu Gin Gly Arg Leu Phe Ser Tyr Gly Asp Thr His 330 335 340 345
CGC TAC CGC TTA GGG GTT AAT TAC CCT CAA ATA CCG GTT AAT AAA CCA 1410 Arg Tyr Arg Leu Gly Val Asn Tyr Pro Gin He Pro Val Asn Lys Pro 350 355 360 AGA TGC CCG TTC CAC TCT TCT AGC AGA GAT GGT TAC ATG CAA AAT GGG 1458 Arg Cys Pro Phe His Ser Ser Ser Arg Asp Gly Tyr Met Gin Asn Gly 365 370 375
TAT TAC GGC TCT TTA CAA AAC TAT ACG CCT AGC TCA TTG CCT GGC TAT 1506 Tyr Tyr Gly Ser Leu Gin Asn Tyr Thr Pro Ser Ser Leu Pro Gly Tyr 380 385 390
AAA GAA GAT AAG AGC GCG AGA GAT CCT AAA TTC AAC TTA GCT CAT ATT 1554 Lys Glu Asp Lys Ser Ala Arg Asp Pro Lys Phe Asn Leu Ala His He 395 400 405
GAG AAA GAG TTT GAA GTG TGG AAT TGG GAT TAC AGA GCT GAT GAT AGC 1602 Glu Lys Glu Phe Glu Val Trp Asn Trp Asp Tyr Arg Ala Asp Asp Ser 410 415 420 425
GAT TAC TAC ACC CAA CCA GGT GAT TAC TAC CGC TCA TTG CCA GCT GAT 1650 Asp Tyr Tyr Thr Gin Pro Gly Asp Tyr Tyr Arg Ser Leu Pro Ala Asp 430 435 440
GAA AAA GAA AGG TTG CAT GAC ACT ATT GGA GAG TCT TTG GCT CAT GTT 1698 Glu Lys Glu Arg Leu His Asp Thr He Gly Glu Ser Leu Ala His Val 445 450 455
ACC CAT AAG GAA ATT GTG GAT AAA CAA TTG GAG CAT TTC AAG AAA GCT 1746 Thr His Lys Glu He Val Asp Lys Gin Leu Glu His Phe Lys Lys Ala 460 465 470
GAC CCC AAA TAC GCT GAG GGG GTT AAA AAA GCT CTT GAA AAA CAC CAA 1794 Asp Pro Lys Tyr Ala Glu Gly Val Lys Lys Ala Leu Glu Lys His Gin 475 480 485
AAA ATG ATG AAA GAC ATG CAT GGA AAA GAC ATG CAC CAC ACA AAA AAG 1842 Lys Met Met Lys Asp Met His Gly Lys Asp Met His His Thr Lys Lys 490 495 500 505
AAA AAG TAA CCCTTTTCTT TAAGCGTTCT TATTTTTTAG GAACGCTTTG 1891
Lys Lys
TCTTTCAAAA TTTAGGTTTT TGGATACTCA TCAGTCCTTT GGTGGTGTGT CCTATTTTTT 1951
CATTCATTCA ACGAATTTAA AAATTACAAT AAAGAGTTAT AGTTATGAAA CGAAGGGATT 2011 TTATTAAAAC GACTGCTTTA GGCGCTACAG GTGCTGTTTT AGGAGCACAG ATTTTGCAGG 2071
CAGAAGAAAG CAAAGGGAGT GTTGCAAAAT ATAAAATAGA AGCTCAATAC AGCATTGATT 2131
TTGATTCTGC AGAACACACT TCGCTTTTCA TTCCCATGCC GAGTGTTGTA GCGAGCAATG 2191
TGCATTTACA AGGCAATCAT GCCAGCTATA AAAGCATGCT CAATTTTGGA GTGCCTTATT 2251
TGCAAGTGGA TTTTTTAAAA AGCGCTCAAA AAAAGCAAGT CCATTTGTCT TATGAGATC 2310
( 2 ) INFORMATION FOR SEQ ID NO : 21 :
(i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 2639 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: ielicoJbacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 1 to 1086 bp protein HopY from 1099 to 1230 bp signal peptide of protein HopX from 1231 to 2517 bp mature protein HopX
(vi) PROPERTIES: coding region coding for related proteins of
Helicojbacter pylori (HopY and HopX)
GAT CTA TCC CAA CAA TAC GCT AAT CAG GGT GTC ATT AAG CCT TTG GTG 48
Asp Leu Ser Gin Gin Tyr Ala Asn Gin Gly Val He Lys Pro Leu Val
5 10 15
GTG GAT GTG GGG AAA GAA CAA ATC GGT ATT ACT GAT AGC ATG CTC TTG 96
Val Asp Val Gly Lys Glu Gin He Gly He Thr Asp Ser Met Leu Leu 20 25 30
GTG GCT CAA AAC ATC GTT TTA GCT TTA GGG CAA GTG GAT TTG AGC AAA 144
Val Ala Gin Asn He Val Leu Ala Leu Gly Gin Val Asp Leu Ser Lys 35 40 45
ATC CAA CAA AAT AAA AAT AAT GGT AAC GGA CAG CTA TAC GAA AAC ATC 192
He Gin Gin Asn Lys Asn Asn Gly Asn Gly Gin Leu Tyr Glu Asn He 50 55 60
ATG AAA GTC ATG CTT TTA GGT GCG GGC GGG ACT AAT GGA GCG TAT AAT 240
Met Lys Val Met Leu Leu Gly Ala Gly Gly Thr Asn Gly Ala Tyr Asn 65 70 75 80 GGC GTG AGT GTG GGC GAT ATT GCC ACA GGC ATG CAA AAT TTT TCT TCG 288
Gly Val Ser Val Gly Asp He Ala Thr Gly Met Gin Asn Phe Ser Ser 85 90 95
CAA ACG GGC TTG ATA GGG GCT AAT TCT ACG GTT AGC GAG CTC AAC GCT 336
Gin Thr Gly Leu He Gly Ala Asn Ser Thr Val Ser Glu Leu Asn Ala 100 105 110
TTG ATT AAG AGC GGG ATT TCT TTA GAT CGT GAG ACT TTG AGG TTA GGG 384
Leu He Lys Ser Gly He Ser Leu Asp Arg Glu Thr Leu Arg Leu Gly 115 120 125
AGT TTT ATT GAA AAA AAT ATT TGT AGC AGT GCA TCG TCT TGT TTT ACT 432
Ser Phe He Glu Lys Asn He Cys Ser Ser Ala Ser Ser Cys Phe Thr 130 135 140
GGG AGT CAG CTT ATC TAT AAG AAA GGG CTA GAT AGA ACC ATA AAC ATC 480
Gly Ser Gin Leu He Tyr Lys Lys Gly Leu Asp Arg Thr He Asn He 145 150 155 160
ATT AAT GCG GTA TTA GGT CAG TTT GAA TCT TCG GCT AGT TCT CTT TAT 528
He Asn Ala Val Leu Gly Gin Phe Glu Ser Ser Ala Ser Ser Leu Tyr 165 170 175
AAG ATT TCT TAT ATC CCT AAC CTC TTT TCG CTC AAA GAT TAC CAG TCA 576
Lys He Ser Tyr He Pro Asn Leu Phe Ser Leu Lys Asp Tyr Gin Ser 180 185 190
GCG AGC ATG AAC GGC TTT GGG GCT AAG ATG GGT TAT AAA CAA TTT TTC 624
Ala Ser Met Asn Gly Phe Gly Ala Lys Met Gly Tyr Lys Gin Phe Phe 195 200 205
ACC CAT AAG AAA AAT ATT GGC TTA AGG TAT TAC GGG TTT TTG GAT TAT 672
Thr His Lys Lys Asn He Gly Leu Arg Tyr Tyr Gly Phe Leu Asp Tyr 210 215 220
GGC TAT GCG AAT TTT GGC GAT ACG AAT TTA AAA GTG GGA GCG AAT CTT 720
Gly Tyr Ala Asn Phe Gly Asp Thr Asn Leu Lys Val Gly Ala Asn Leu 225 230 235 240
GTT ACT TAT GGG GTA GGA ACG GAT TTT TTA TAC AAC GTG TAT GAA CGC 768
Val Thr Tyr Gly Val Gly Thr Asp Phe Leu Tyr Asn Val Tyr Glu Arg 245 250 255 TCT AGA AGG AGG GAA AGG ACT ACA ATC GGC CTT TTC TTT GGC GCT CAA 816
Ser Arg Arg Arg Glu Arg Thr Thr He Gly Leu Phe Phe Gly Ala Gin 260 265 270
ATT GCA GGG CAA ACT TGG AGC ACT AAT GTA ACG AAC TTA TTG AGC GGG 864
He Ala Gly Gin Thr Trp Ser Thr Asn Val Thr Asn Leu Leu Ser Gly 275 280 285
CAA AGG CCT GAT GTC AAG TCT AGT TCG TTC CAA TTC TTG TTT GAT TTG 912
Gin Arg Pro Asp Val Lys Ser Ser Ser Phe Gin Phe Leu Phe Asp Leu 290 295 300
GGC GTG CGC ACC AAC TTT GCA AAA ACC AAT TTC AAC AAG CAC AGG CTA 960
Gly Val Arg Thr Asn Phe Ala Lys Thr Asn Phe Asn Lys His Arg Leu 305 310 315 320
GAC CAA GGG ATA GAA TTT GGG GTG AAA ATC CCT GTT ATC GCT CAT AAA 1008 Asp Gin Gly He Glu Phe Gly Val Lys He Pro Val He Ala His Lys 325 330 335
TAT TTC GCA ACC CAA GGC TCA AGC GCG AGC TAT ATG AGG AAT TTT AGC 1056 Tyr Phe Ala Thr Gin Gly Ser Ser Ala Ser Tyr Met Arg Asn Phe Ser 340 345 350
TTC TAT GTG GGC TAT TCA GTC GGT TTT TAA GGAAGGCTCT TG ATG AAA 1104 Phe Tyr Val Gly Tyr Ser Val Gly Phe Met Lys
355 360
AAT ACC AAT ACA AAA GAG ATA AAG AAT ACA AGA ATG AAA AAA GGT TAT 1152 Asn Thr Asn Thr Lys Glu He Lys Asn Thr Arg Met Lys Lys Gly Tyr -40 -35 -30
AGT CAA TAC CAT GCG CTC AAA AAA GGG CTT TTA AAA ACT GCT CTG CTT 1200 Ser Gin Tyr His Ala Leu Lys Lys Gly Leu Leu Lys Thr Ala Leu Leu -25 -20 -15
TTT AGC CTT CCC TTA AGC ATG GCG TTA GCT GAA GAC GAT GGC TTT TAT 1248 Phe Ser Leu Pro Leu Ser Met Ala Leu Ala Glu Asp Asp Gly Phe Tyr -10 -5 5
ATG GGA GTG GGC TAT CAA ATC GGC GGT GCG CAA CAA AAC ATC AAT AAC 1296 Met Gly Val Gly Tyr Gin He Gly Gly Ala Gin Gin Asn He Asn Asn 10 15 20
AAA GGC AGC ACC CTA AGG AAT AAT GTC ATT GAT GAT TTC CGC CAA GTG 1344 Lys Gly Ser Thr Leu Arg Asn Asn Val He Asp Asp Phe Arg Gin Val 25 30 35
GGC GTG GGT ATG GCA GGG GGT AAC GGG CTT TTA GCT TTA GCG ACA AAC 1392 Gly Val Gly Met Ala Gly Gly Asn Gly Leu Leu Ala Leu Ala Thr Asn 40 45 50
ACG ACC ATG GAC GCT CTT TTA GGG ATA GGC AAC CAA ATT GTC AAT ACC 1440 Thr Thr Met Asp Ala Leu Leu Gly He Gly Asn Gin He Val Asn Thr 55 60 65 70
AAT ACA ACT GTT GGC AAC AAC AAC GCA GAG TTA ACC CAG TTT AAA AAA 1488 Asn Thr Thr Val Gly Asn Asn Asn Ala Glu Leu Thr Gin Phe Lys Lys 75 80 85
ATA CTC CCC CAA ATT GAG CGA CGC TTT GAG ACG AAT AAA AAC GCT TAT 1536 He Leu Pro Gin He Glu Arg Arg Phe Glu Thr Asn Lys Asn Ala Tyr 90 95 100
AGC GTT CAA GCC TTG CAA GTG TAT TTG AGT AAT GTG CTT TAT AAC TTG 1584 Ser Val Gin Ala Leu Gin Val Tyr Leu Ser Asn Val Leu Tyr Asn Leu 105 110 115
GTT AAT AAT AGT AAT AAT GGC AGT AAT AAT GGA GTC GTT CCT GAA TAT 1632 Val Asn Asn Ser Asn Asn Gly Ser Asn Asn Gly Val Val Pro Glu Tyr 120 125 130
GTA GGG ATT ATA AAA GTT CTC TAT AAT TCT CAA AAT GAA TTC AGT CTC 1680 Val Gly He He Lys Val Leu Tyr Asn Ser Gin Asn Glu Phe Ser Leu 135 140 145 150
TTA GCC ACG GAG AGT GTG GCG CTT TTA AAC GCG CTT ACA AGG GTG AAT 1728 Leu Ala Thr Glu Ser Val Ala Leu Leu Asn Ala Leu Thr Arg Val Asn 155 160 165
CTG GAT AGC AAT TCG GTG TTT TTA AAA GGG CTA TTA GCC CAA ATG CAG 1776 Leu Asp Ser Asn Ser Val Phe Leu Lys Gly Leu Leu Ala Gin Met Gin 170 175 180
CTT TTT AAT GAC ACT TCT TCA GCA AAG CTA GGC CAG ATC GCA GAA AAC 1824 Leu Phe Asn Asp Thr Ser Ser Ala Lys Leu Gly Gin He Ala Glu Asn 185 190 195
TTG AAT AAG AGT GGT GGT GCA GGG GCC ATG CTT CAA AAG GAT GTG AAA 1872 Leu Asn Lys Ser Gly Gly Ala Gly Ala Met Leu Gin Lys Asp Val Lys 200 205 210
ACC ATC TCG GAT CGA ATC GCT ACT TAC CAA GAG AAT CTA AAA CAA CTA 1920 Thr He Ser Asp Arg He Ala Thr Tyr Gin Glu Asn Leu Lys Gin Leu 215 220 225 230
GGA GGG ATG CTG AAT AAT TAC GAT GAG CCT TAC TTG CCC CAA TTT GGG 1968 Gly Gly Met Leu Asn Asn Tyr Asp Glu Pro Tyr Leu Pro Gin Phe Gly 235 240 245
CCA GGC AAA AGC TCT CAG CAT GGG GTT ATT AAT GGC TTT GGC ATT CAA 2016 Pro Gly Lys Ser Ser Gin His Gly Val He Asn Gly Phe Gly He Gin 250 255 260
GTG GGC TAT AAG CAA TTT TTT GGG AGC AAG AGG AAT ATA GGC TTA CGG 2064 Val Gly Tyr Lys Gin Phe Phe Gly Ser Lys Arg Asn He Gly Leu Arg 265 270 275
TAT TAC GCT TTC TTT GAT TAT GGC TTT ACG CAA TTG GGC AGT CTT AAT 2112 Tyr Tyr Ala Phe Phe Asp Tyr Gly Phe Thr Gin Leu Gly Ser Leu Asn 280 285 290
AGC GCT GTT AAA GCG AAC ATC TTT ACT TAT GGC GCT GGC ACG GAC TTT 2160 Ser Ala Val Lys Ala Asn He Phe Thr Tyr Gly Ala Gly Thr Asp Phe 295 300 305 310
TTA TGG AAT ATC TTT AGA AGG GTT TTT AGC GAT CAG TCT TTG AAT GTG 2208 Leu Trp Asn He Phe Arg Arg Val Phe Ser Asp Gin Ser Leu Asn Val 315 320 325
GGG GTG TTT GGG GGC ATT CAA ATA GCG GGT AAC ACT TGG GAT AGC TCT 2256 Gly Val Phe Gly Gly He Gin He Ala Gly Asn Thr Trp Asp Ser Ser 330 335 340
TTA AGA GGC CAA ATT GAA AAC TCG TTT AAA GAA TAC CCC ACT CCC ACG 2304 Leu Arg Gly Gin He Glu Asn Ser Phe Lys Glu Tyr Pro Thr Pro Thr 345 350 355 AAT TTC CAA TTT TTA TTT AAT TTG GGC TTA AGG GCT CAT TTT GCC AGC 2352 Asn Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg Ala His Phe Ala Ser 360 365 370
ACC ATG CAC CGC CGG TTT TTG AGC TCG TCT CAA AGC ATT CAG CAT GGT 2400 Thr Met His Arg Arg Phe Leu Ser Ser Ser Gin Ser He Gin His Gly 375 380 385 390
ATG GAA TTT GGC GTG AAA ATC CCG GCT ATC AAT CAA AGG TAT TTG AAA 2448 Met Glu Phe Gly Val Lys He Pro Ala He Asn Gin Arg Tyr Leu Lys 395 400 405
GCG AAT GGG GCT GAT GTG GAT TAC AGG CGT TTG TAT GCG TTC TAT ATC 2496 Ala Asn Gly Ala Asp Val Asp Tyr Arg Arg Leu Tyr Ala Phe Tyr He 410 415 420
AAC TAC ACG ATA GGT TTT TAA GCTCTTTTTA GGGCTTATAA AGAGGTTCTT 2547
Asn Tyr Thr He Gly Phe 425
TACTTTTTTT TGGTATTCTA ACAAGCTTTT AAACCATCCA ATCTACTTTG TTTTAAGGAT 2607
AATATTTTAT GGCAGATGTC GTTGTGGGGA TC 2639
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1710 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 1 to 1179 bp mature peptide (without N- Terminus)
(vi) PROPERTIES: protein HopZ of Helicobacter pylori
ATC AAA AAC GCC CAA GAA ATC GTC GCA CAA GCT CAA AGC CTT AAC AAC 48
He Lys Asn Ala Gin Glu He Val Ala Gin Ala Gin Ser Leu Asn Asn
5 10 15
CCG CAA AAC AAT CAA AAC GCG CCG CAA GAT TTC AAT CCT TAC ACC TCT 96
Pro Gin Asn Asn Gin Asn Ala Pro Gin Asp Phe Asn Pro Tyr Thr Ser 20 25 30
GCT GAT AGG GCT TTC GCT CAA AAC ATG CTC AAT CAC GCG CAA GCG CAA 144
Ala Asp Arg Ala Phe Ala Gin Asn Met Leu Asn His Ala Gin Ala Gin 35 40 45
GCC AAG ATG CTT GAA CTA GCC AAT CAA ATC AAA ACC AAT CTT AGC GCT 192
Ala Lys Met Leu Glu Leu Ala Asn Gin He Lys Thr Asn Leu Ser Ala 50 55 60
ATC CCG CAA CAT TTC ACC AAA GAT TAC TTG GCA GCT TGC CGC AAT GGG 240
He Pro Gin His Phe Thr Lys Asp Tyr Leu Ala Ala Cys Arg Asn Gly 65 70 75 80
GGT GGG ACA TTA CCT GAT GCA GGG GTT ACT AAC AAC ACT TGG GGA GCC 288
Gly Gly Thr Leu Pro Asp Ala Gly Val Thr Asn Asn Thr Trp Gly Ala 85 90 95
GGT TGC GCC TAT GTG GAA GAG ACC ATA ACG GCT TTA AAC AAC AGC CTT 336
Gly Cys Ala Tyr Val Glu Glu Thr He Thr Ala Leu Asn Asn Ser Leu 100 105 110
GTG CAT TTT GGC ACT CAA GCC GAG CAA ATC AAG CAA TCT GAG TTG CTG 384
Val His Phe Gly Thr Gin Ala Glu Gin He Lys Gin Ser Glu Leu Leu 115 120 125
GCG CGC ACG ATA TTT GAT TTT AAA GGC AGC CTT AAG GAT TTA AAC AGC 432
Ala Arg Thr He Phe Asp Phe Lys Gly Ser Leu Lys Asp Leu Asn Ser 130 135 140
ACT TAT AAC AGC ATC ACC ACG ACC GCT TCA AAC ACG CCC AAT TCC CCA 480
Thr Tyr Asn Ser He Thr Thr Thr Ala Ser Asn Thr Pro Asn Ser Pro 145 150 155 160
TTC CTT AAA AAT TTG ATA AGC CAA TCC ACT AAC CCT AAT AAC CCC GGG 528
Phe Leu Lys Asn Leu He Ser Gin Ser Thr Asn Pro Asn Asn Pro Gly 165 170 175
GGC TTA CAG GCC GTT TAT CAA GTC AAC CAA AGC GCT TAT TCG CAA TTA 576
Gly Leu Gin Ala Val Tyr Gin Val Asn Gin Ser Ala Tyr Ser Gin Leu 180 185 190
TTA AGC GCC ACG CAA GAA TTA GGG CAT AAC CCT TTC AGA CGC TTT GGA 624
Leu Ser Ala Thr Gin Glu Leu Gly His Asn Pro Phe Arg Arg Phe Gly 195 200 205
TTA ATC AGC TCT CAA ACC AAC AAT GGT GCC ATG AAT GGG ATC GGT GTG 672
Leu He Ser Ser Gin Thr Asn Asn Gly Ala Met Asn Gly He Gly Val 210 215 220
CAA ATA GGG TAT AAA CAA TTT TTT GGT GAA AAG AGA AAA TGG GGG GCT 720
Gin He Gly Tyr Lys Gin Phe Phe Gly Glu Lys Arg Lys Trp Gly Ala 225 230 235 240
AGG TAT TAC GGC TTT TTT GAC TAT AAC CAT GCT TAT ATC AAA TCC AGC 768
Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr He Lys Ser Ser 245 250 255
TTT TTC AAC TCC GCC TCT GAT GTG TTC ACT TAT GGG GTA GGA ACA GAT 816 Phe Phe Asn Ser Ala Ser Asp Val Phe Thr Tyr Gly Val Gly Thr Asp 260 265 270
GTC CTC TAT AAC TTT ATC AAC GAT AAA GCC ACC AAA AAC AAT AAG ATT 864
Val Leu Tyr Asn Phe He Asn Asp Lys Ala Thr Lys Asn Asn Lys He 275 280 285
TCT TTT GGG GTG TTT GGG GGG ATT GCT TTA GCT GGC ACT TCG TGG CTC 912
Ser Phe Gly Val Phe Gly Gly He Ala Leu Ala Gly Thr Ser Trp Leu 290 295 300
AAT TCT CAA TAC GTG AAT TTA GCG ACC TTC AAT AAT TTC TAT AGC GCT 960
Asn Ser Gin Tyr Val Asn Leu Ala Thr Phe Asn Asn Phe Tyr Ser Ala 305 310 315 320
AAA ATG AAT GTG GCG AAT TTC CAA TTC TTA TTC AAC TTG GGC TTG AGA 1008 Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe Asn Leu Gly Leu Arg 325 330 335
ATG AAT CTG GCT AAA AAC AAA AAG AAA GCG AGC GAT CAT GCG GCT CAA 1056 Met Asn Leu Ala Lys Asn Lys Lys Lys Ala Ser Asp His Ala Ala Gin 340 345 350
CAT GGC GTG GAA CTA GGC GTG AAG ATC CCC ACG ATC AAC ACG AAT TAC 1104 His Gly Val Glu Leu Gly Val Lys He Pro Thr He Asn Thr Asn Tyr 355 360 365
TAT TCT TTG CTA GGC ACT CAA CTA GAA TAC CGC AGA CTC TAT AGC GTG 1152 Tyr Ser Leu Leu Gly Thr Gin Leu Glu Tyr Arg Arg Leu Tyr Ser Val 370 375 380
TAT TTG AAT TAT GTG TTT GCG TAT TAA AAGCTTGCGT TAAACCCTTT 1199
Tyr Leu Asn Tyr Val Phe Ala Tyr 385 390
GTGGAACTCC CTTTTTAAGG GGTTTCTTTT GAAGCCTTTT TTTTGAACCT TTTTTTGGGG 1259
GTCAAGCGTA AAATCCACCC CTATCCCTTT AAGAAAATAA AATAAAACTT TAAGAACTTT 1319
AAGAACTTTA AGAAAATGCG TTTTACAACA AAATAAGATC TAAAACAATA AAACAAAACC 1379
CCATTTTTTA ACAATGAAAT TTTTTAAACA AAAAAGCATT AAATCCTAAT AAGGTTTGTT 1439 AGATCTTGAT AAAAACAAAG CTTTTTTAAA ACCCCAAAAA CAATACTAAC CAATAACCAA 1499
AACGCATCTA TTGTGATCCT TATAGCATAA AACCAAGTTT TTATTTAAGC AAAAGCTGTT 1559
ATGCCGTTTT AAGAGCGTTT CGTTTCTATG AAAACCGCAA TATTTTTCAA TTATTCTTGA 1619
CAAGCGTTAA AAAAAATTGT ATCATTATCT TTTTGTGAGA CCCGTTAGCT CAGTTGGTAG 1679
AGCAATTCCC TTTTAAGGAA TGGGAGCGGC C 1710
(2) INFORMATION FOR SEQ ID NO: 23:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2078 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR T PE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 642 to 692 bp signal peptide from 693 to 1388 bp mature peptide
(vi) PROPERTIES: 28 kD protein from Helicobacter pylori
GATCCATCGC TAAAAGCATT AATTTTGGGC TGGTGTATGG CATGGGGAGT AAGAAATTGA 60
GCGAAACTTT AAACATTTCT TTAAATGAGG CTAAAAGCTA CATAGAAGCG TATTTCAAGC 120
GATTCCCTAG CATCAAAGAT TATTTAAACC GCATGAAAGA AGAGATTTTA AAAACTTCTA 180
AAGCCTTCAC TTTGCTTGGG CGTTATCGGG TGTTTGATTT TACCGGCGCA AATGACTATA 240
TCAAGGGCAA TTATTTGCGA GAGGGCGTGA ATGCGATTTT TCAAGGGAGT GCCAGCGATT 300
TATTGAAATT AGGCATGCTC AAAGTGAGCG AGCGTTTCAA AAATAACCCT TCGGTGAGGC 360
TGCTTTTGCA AGTGCATGAC GAATTGATTT TTGAAATTGA AGAAAAAAAC GCATTAGAGT 420
TGCAGCAAGA AATCCAACGC ATTCTTAATA ATGAAGTGTA TCCTTTGAGG GTGCCACTAG 480
AAACGAGCGC GTTTGTGGCG AATCGTTGGA ATGAACTAAA AGGTTAGTTT TTTAATTGAG 540
TTTAAGAAAA AATATATTAT TATTTCTTTA TAAGTAATAC TTAGCTATTT TAAGCTAATA 600
TAATATAGAG CGATTATCAA AAAATAAAGG GAAAAGACTG A ATG TTG AAA AGA ATT 656
Met Leu Lys Arg He - 15
ATA TTA TTA GGG GCT TTG GGT GTT TTA GCG AAC GCT GAA GAG AGC GCG 704
He Leu Leu Gly Ala Leu Gly Val Leu Ala Asn Ala Glu Glu Ser Ala -10 -5
GCT TTT GTG GGA GTC AAT TAC CAG GTG AGC ATG ATA CAA AAT CAG ACT 752
Ala Phe Val Gly Val Asn Tyr Gin Val Ser Met He Gin Asn Gin Thr 5 10 15 20
AAA ATG GTG AAT GAC AAC GGC TTG CAA AAG CCT TTG ATA AAG TTC CCG 800
Lys Met Val Asn Asp Asn Gly Leu Gin Lys Pro Leu He Lys Phe Pro 25 30 35
CCT TAT GCA GGA GCG GGT TTT GAA GTG GGC TAT AAA CAA TTT TTT GGC 848
Pro Tyr Ala Gly Ala Gly Phe Glu Val Gly Tyr Lys Gin Phe Phe Gly 40 45 50
AAG AAA AAA TGG TTT GGT GCG CGT TAT TAT GGG TTT TTT GAC TAC GCG 896
Lys Lys Lys Trp Phe Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Ala 55 60 65
CAC AAC CGC TTT GGC GTG ATG AAA AAG GGT ATC CCG GTG GGC GAG AGC 944
His Asn Arg Phe Gly Val Met Lys Lys Gly He Pro Val Gly Glu Ser 70 75 80
GGG TTT ATT TAC AAT AGT TTT AGT TTT GGA GGG AAC ACT TTA ATG GAA 992
Gly Phe He Tyr Asn Ser Phe Ser Phe Gly Gly Asn Thr Leu Met Glu 85 90 95 100
AGG GAT TCC TAT CAA GGG CAA TAC TAT GTC AAT TTA TTC ACT TAT GGT 1040 Arg Asp Ser Tyr Gin Gly Gin Tyr Tyr Val Asn Leu Phe Thr Tyr Gly 105 110 115
GTG GGG CTA GAT ACG CTG TGG AAT TTT GTG AAT AAA GAA AAC ATG GTT 1088 Val Gly Leu Asp Thr Leu Trp Asn Phe Val Asn Lys Glu Asn Met Val 120 125 130
TTT GGT TTT GTG GTA GGA ATC CAA TTA GCT GGG GAT AGT TGG GCA ACG 1136 Phe Gly Phe Val Val Gly He Gin Leu Ala Gly Asp Ser Trp Ala Thr 135 140 145
AGC ATC AGT AAA GAG ATC GCC AGC TAT GCA AAA CAC CAC AGC AAT TCC 1184 Ser He Ser Lys Glu He Ala Ser Tyr Ala Lys His His Ser Asn Ser 150 155 160
AGT TAT AGC CCG GCC AAT TTC CAG TTT TTA TGG AAG TTT GGG GTC CGC 1232 Ser Tyr Ser Pro Ala Asn Phe Gin Phe Leu Trp Lys Phe Gly Val Arg 165 170 175 180
ACC CAT ATC GCT AAA CAC AAT AGC CTA GAA TTA GGG ATT AAA GTG CCT 1280 Thr His He Ala Lys His Asn Ser Leu Glu Leu Gly He Lys Val Pro 185 190 195
ACG ATC ACG CAC CGG CTT TTC TCT CTT ACC AAC GAA AAG GGA TAC ACC 1328 Thr He Thr His Arg Leu Phe Ser Leu Thr Asn Glu Lys Gly Tyr Thr 200 205 210
TTA CAG GCT GAT GTG CGC CGA GTT TAT GCG TTT CAA ATC AGT TAC TTG 1376 Leu Gin Ala Asp Val Arg Arg Val Tyr Ala Phe Gin He Ser Tyr Leu 215 220 225
AGG GAT TTT TAA CCCCTTTTTA GATACAATCG CACCTAAAAT CAATTTAAAG 1428
Arg Asp Phe 230
GTGTGAAATG GTGAATTTAG AAAATTTAGA CTGGAAAAAT TTAGGCTTTA GCTACATTAA 1488
AACGGATTTT CGCTTCATCG CTACTTATAA AAACGGCTCT TGGTCGCATG GCGGATTGGT 1548
GAGCGAAAAT GTGCTACAAA TCAGCGAAGG CTCGCCGGTC TTGCACTACG GGCAGGCTTG 1608
TTTTGAAGGC TTGAAGGCTT ACCGCTCTCA AAAGGGGAAG GCTTTACTTT TTCGCCCTTT 1668
AGAAAACGCC AAACGCTTGC AAACTTCATG CGAAAGACTG CTCATGCCCA AAGTGAGCGA 1728
AGAGCTGTTT TTAAGGGCAT GCGCTGAAGT AGTCAAAGCG AATCAAAAAT GGCTCGCTCC 1788
TTATAAAAGC GGGGCGAGTT TGTATTTGCG CCCTTTTGTC ATAGGCGTAG GGGATAATTT 1848
GGGGGTGAAG CCGGCTAATG AATACCTTTT TATCGTGTTT TGCGCGCCTG TGGGGGCGTA 1908
TTTTAAGGGG GGTATAGAAA AAGGAGGGGC TAGGTTTATC ACTACGATTT TTGATAGGGC 1968
CGCGCCTAAA GGCACCGGAG GGGTGAAAGT GGGGGGGAAT TACGCTGCAA GCCTGTTAGC 2028 CCATAAAATA GCCACAGAGC AGGGCTATGA TGATTGCATT TATTTAGATC 2078
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3144 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR T PE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 1149 to 1235 bp signal peptide from 1236 to 2735 bp mature peptide
(vi) PROPERTIES: 50 kD membrane protein from Helicobacter pylori
GATCGGCAGG CAAATACACA TCTTTATTGC AACCCATTCC TTCGTATTTT TCAACTTTCA 60
AGGTCCCCAC CAATAATTCC TTATGCTTGC CTTTCCAAAG CGCGGTCGTG TCGTTGGTGG 120
CATCATTTTT ATTCGCAAAT ACCAGATACA TTTGGTATTC TATGGGCTTA GTTTTAAGGT 180
GTTGTTGGAA TGAAGAAAGC AGATAATTTG AATCTTTTTG CTTTAATTCT TGGGGGTTAA 240
GATACTTAAT GCCCTCTTTA GGCACAAATT TCCATCTCGC AGGCAATAAT TTTTCTTTCT 300
TATCTTTAAA CCTGAACGCA TGCACGCTAT AATAGGGCGT GTTAGCCACG CTTGAGCTAA 360
TCCCTATCGT TTTGGTGTAA GCGGCAAAAT TCTTATAAGA GGGGACTTCT TCATAAAGCT 420
TTTTGATTCT TGCTTCATCC ACCTTGCCAT TTTTAGGGAT TCTCATCTCA AAGAATTGGG 480
CGAATTCGTT AGGGTTTTTG GCAAAATTGA TTTCTGTATT GAGCATCACC ATTGTCCAGC 540
TAGCGTTTTG ATTTTCTAAT TTTAACGCCA TTCCCCTAAC TTTGCTTTTA TCGTCCATTG 600
CCACGCCTCC TAAAGAATAC CTTACAGATG CAGGGATTTC TTTTTCATTG AGTAATGGCA 660 CATCTAAATC CTTTTTTGCT TGCGCATTAG GGAGGAACAC GCCTTTAGCA CAAAACCCCT 720
TAGTGTGGTT GATTTTCATT TTAGGCTCTT TGGCGTTGAG CTTGTAGAAA ATATCCGCAA 780
TCTCTTCAGC GCTCACTTCA TGGGCTTTTA AAAAACCCAA GCTAAAAACC AAACACAAGC 840
TCAAACCAAT TTTTTTCATT GTTTCGCTCC TTAATTATTA TTTTTATATA AAACAACGCT 900
TTCTATTGTA TCAGTAAATT CCCTATTGAG CCTTAAAAAA GCCTTTTTTT CAATATATTA 960
TTAGGTCTTA AAAAATTATC CTATCCATTT GCATGCTCAT GAGCAAGCTT TTAAGGATAA 1020
ACTTGTGTTT TAAATTTTGT GATTTTTAAG AAAAATTAGC TTGATTTTAA ACTAATTCTA 1080
TATTCTTTTA TGCTACAATT ATTTCTACAG AGTAATTTAT CTATTCTCAG GTAAAGTAAG 1140
GAAGAGGA ATG AAA TTA AAG AAA CGA AAA GTT GCG GCT ACA TTG CTA AAG 1190 Met Lys Leu Lys Lys Arg Lys Val Ala Ala Thr Leu Leu Lys -25 -20
CGT TTT ACC TTA CCA CTA TTG TTC ACT ACG GGT TCA TTA GGG GCG GTT 1238 Arg Phe Thr Leu Pro Leu Leu Phe Thr Thr Gly Ser Leu Gly Ala Val -15 -10 -5
ACT TAT GAA GTG CAT GGG GAT TTT ATC AAC TTC TCC AAA GTG GGT TTT 1286 Thr Tyr Glu Val His Gly Asp Phe He Asn Phe Ser Lys Val Gly Phe 5 10 15
AAC CAT TCG CCC ATT AAC CCT GTT AAA GGT ATC TAT CCC ACA GAG ACT 1334 Asn His Ser Pro He Asn Pro Val Lys Gly He Tyr Pro Thr Glu Thr 20 25 30
TTT GTT AAC CTT ACG GGT AAG CTA GAG GGT TCT GTG CAT TTA GGT AGG 1382 Phe Val Asn Leu Thr Gly Lys Leu Glu Gly Ser Val His Leu Gly Arg 35 40 45
GGA TGG ACC GTG AAT TTA GGC GGT GTT TTG GGC GGA CAG GCT TAT GAT 1430 Gly Trp Thr Val Asn Leu Gly Gly Val Leu Gly Gly Gin Ala Tyr Asp 50 55 60 65
GGC ACT AAG TAT GAT AGG TGG GCG AAG GAT TTT ACC CCC CCA AGC TAT 1478 Gly Thr Lys Tyr Asp Arg Trp Ala Lys Asp Phe Thr Pro Pro Ser Tyr 70 75 80 TGG GAT AAA ACT TCT TGC GGC ACT GAT TCT ATG AGC CTT TGT ATG AAT 1526 Trp Asp Lys Thr Ser Cys Gly Thr Asp Ser Met Ser Leu Cys Met Asn 85 90 95
GCT ACT AAA ATG TGG CAA CAA TCA GGG CCA GGT GGT GTC ATT AAC CCT 1574 Ala Thr Lys Met Trp Gin Gin Ser Gly Pro Gly Gly Val He Asn Pro 100 105 110
AGA GGT ATT GGT TGG GAA TAT ATG GGT GAG TGG AAC GGC TTG TTC CCT 1622 Arg Gly He Gly Trp Glu Tyr Met Gly Glu Trp Asn Gly Leu Phe Pro 115 120 125
AAC TAC TAT CCG GCT AAC GCC TAC TTG CCT GGT GGC TCA AGG CGT TAT 1670 Asn Tyr Tyr Pro Ala Asn Ala Tyr Leu Pro Gly Gly Ser Arg Arg Tyr 130 135 140 145
CAA GTC TAT AAA GCA AAT TTG ACC TAT GAC AGC GAC AGA GTC CAT ATG 1718 Gin Val Tyr Lys Ala Asn Leu Thr Tyr Asp Ser Asp Arg Val His Met 150 155 160
GTA ATG GGG CGT TTT GAT ATT ACC GAG CAG GAG CAA ATG GAT TGG ATT 1766 Val Met Gly Arg Phe Asp He Thr Glu Gin Glu Gin Met Asp Trp He 165 170 175
TAC CAA TTG TTC CAA GGG TTT TAT GGG ACT TTC AAG CTC ACT AAG AAT 1814 Tyr Gin Leu Phe Gin Gly Phe Tyr Gly Thr Phe Lys Leu Thr Lys Asn 180 185 190
ATG AAA TTC TTG CTC TTT AGT GGT TGG GGT CGT GGT ATC GCT GAT GGT 1862 Met Lys Phe Leu Leu Phe Ser Gly Trp Gly Arg Gly He Ala Asp Gly 195 200 205
CAG TGG TTG TTC CCT ATC TAT CGT GAA AAG CCT TGG GGG GTT CAT AAA 1910 Gin Trp Leu Phe Pro He Tyr Arg Glu Lys Pro Trp Gly Val His Lys 210 215 220 225
GCG GGT ATT ATT TAT CGC CCT ACA AAG AAT TTG ATG ATC CAC CCT TAT 1958 Ala Gly He He Tyr Arg Pro Thr Lys Asn Leu Met He His Pro Tyr 230 235 240
GTG TAT CTT ATC CCA ATG GTA GGC ACA TTG CCC GGT GTT AAA GTA GAG 2006 Val Tyr Leu He Pro Met Val Gly Thr Leu Pro Gly Val Lys Val Glu 245 250 255
TAT GAT ACC AAT CCG GAA TTT AGC GGT AGG GGC ATT AGG AAT AAA ACG 2054 Tyr Asp Thr Asn Pro Glu Phe Ser Gly Arg Gly He Arg Asn Lys Thr 260 265 270
ACT TTC TAT GCG TTG TAT GAC TAT CGT TGG AAT AAC GCT GAA TAC GGT 2102 Thr Phe Tyr Ala Leu Tyr Asp Tyr Arg Trp Asn Asn Ala Glu Tyr Gly 275 280 285
CGT TAT GCG CCC GCT CGT TAT AAC ACT TGG GAT CCG TTC TTG GAT AAT 2150 Arg Tyr Ala Pro Ala Arg Tyr Asn Thr Trp Asp Pro Phe Leu Asp Asn 290 295 300 305
GGT AAG TGG CGT GGC TTG CAA GGT CCT GGC GGT GCG ACG CTT CTT TTG 2198 Gly Lys Trp Arg Gly Leu Gin Gly Pro Gly Gly Ala Thr Leu Leu Leu 310 315 320
CGC CAC CAT ATA GAT ATT AAC AAC TAT TTT GTG GTT GGT GGT GCT TAT 2246 Arg His His He Asp He Asn Asn Tyr Phe Val Val Gly Gly Ala Tyr 325 330 335
CTC AAC ATT GGT AAC CCT AAC ATG AAC TTA GGT ACT TGG GGT AAC CCT 2294 Leu Asn He Gly Asn Pro Asn Met Asn Leu Gly Thr Trp Gly Asn Pro 340 345 350
GTG GCT CTT GAT GGT ATC GAA CAA TGG GTC GGT AGT ATC TAC AGC TTA 2342 Val Ala Leu Asp Gly He Glu Gin Trp Val Gly Ser He Tyr Ser Leu 355 360 365
GGG TTT GCG GGG ATT GAC AAC ATT ACC GAT GCT GAT GCG TTC ACC GAG 2390 Gly Phe Ala Gly He Asp Asn He Thr Asp Ala Asp Ala Phe Thr Glu 370 375 380 385
TAT GTT AAA GGT GGA GGC AAG CAT GGT AAG TTC AGT TGG AGC GTT TAT 2438 Tyr Val Lys Gly Gly Gly Lys His Gly Lys Phe Ser Trp Ser Val Tyr 390 395 400
CAG CGC TTC ACC ACT GCA CCA AGG GCT TTG GAA TAT GGT ATC GGT ATG 2486 Gin Arg Phe Thr Thr Ala Pro Arg Ala Leu Glu Tyr Gly He Gly Met 405 410 415
TAT CTA GAT TAT CAG TTC AGC AAG CAT GTT AAA GCG GGT CTC AAA CTC 2534 Tyr Leu Asp Tyr Gin Phe Ser Lys His Val Lys Ala Gly Leu Lys Leu 420 425 430
GTA TGG TTA GAG TTC CAA ATT CGT GCG GGT TAC AAC CCT GGA ACC GGT 2582 Val Trp Leu Glu Phe Gin He Arg Ala Gly Tyr Asn Pro Gly Thr Gly 435 440 445
TTC CTT GGG CCA AAC GGT CAG CCG CTT AAC TTG AAT ACT GGT TTG TTT 2630 Phe Leu Gly Pro Asn Gly Gin Pro Leu Asn Leu Asn Thr Gly Leu Phe 450 455 460 465
GAG TCT TCA GCG TTC GCG CAA GGC CCT CAA AAC ATG GGC GGT ATC GCA 2678 Glu Ser Ser Ala Phe Ala Gin Gly Pro Gin Asn Met Gly Gly He Ala 470 475 480
AAA AGC ATC ACT CAA GAC AGA AGC CAT TTG ATG ACA CAC ATT AGT TAT 2726 Lys Ser He Thr Gin Asp Arg Ser His Leu Met Thr His He Ser Tyr 485 490 495
AGT TTC TAA GAGAGTTCTC CCCCTATCTC TTAGATATGC CTTTTTTGTA TTTTTATTTT 2785 Ser Phe
AATATCTTTG GGAGTTAGAG TTTTGGAAAT TAAGAAATAT TTTTCTTACT TTCTATTTTT 2845
TTTGCTTTTT TCTAGTCTCT TTTTATCCAA ACTTCAAGCT TATAAATTCA ACATGAGCAT 2905
TGTTGGAAAG GTGAGCAGCT ATACCAAGTT TGGCTTTAAC AACCAAAGAT ACCAGCCTTC 2965
TAAAGACATT TATCCTACAG GTAGCTACAC TTCTTTGCTC GGCGAATTGA ATTTGAGCAT 3025
GGGTTTATAC AAGGGTTTGA GGGCGGAAGT GGGGGCTATG ATGGCAGCGC TCCCCTATGA 3085
CTCTACCGCC TATCAAGGCA ACAATATCCC TAACGGCCAG CCCGGCTCTA GGACCGATC 3144 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2825 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 891 to 2090 bp protein
(vi) PROPERTIES: 42 kD protein from Helicobacter pylori
GATCGGAAGC GAGAGTGATA AAAGGCATTA GGAGAGTTAG AGACATCACT TCCACAAAAG 60
AAGAAAAAAC CGCCATCAGC ACAAGCAGGA AAAAAACCAT TTTTTCCTTG AAAGTGATGA 120
GCATATAGAT TTGCTTTAAA GAACGCAAAA AGTATTTTAA AGTAGAGATT TTATGTTTTT 180
TTTTCGCCAT AATTTAAGTG TCCATAAATT CTTTTATATG TAATAAGCTT GAGCTGTGTT 240
AAGCCAAATT GAGCTAGATT ATAGCTAAAT TTTAACCATG CTCTGTGCCA TACGAATAAT 300
TTAGCTTTCT GCCATCATTT CTTGACAAGT CAAGTATAAA ACTGCTATAA TCCCAAGTCT 360
TTAATTTGTT TAATTTGTTG CTGGCTTAGC TCAGTTGGTA GAGCAGCTGC CTTGTAAGCA 420
GCAGGTCGGG GGTTCAAGTC CCTTAGCCAG CTCCAGTTGA AATGTTATTG TGCAAAGTTT 480
TTGGTGAGAT ACTCAAGTGG CCAACGAGGG CAGACTGTAA ATCTGCTGAC TATGTCTTCC 540
GTGGTTCGAA TCCACGTCTC ACCACCATTT TGTTTTATAG ATGCGGGAAT AGCTCAGTTG 600
GCTAGAGCAT CAGCCTTCCA AGCTGAGGGT CGCGGGTTCG AGTCCCGTTT CCCGCTCCAT 660
TTTTAGGATA ACATTTTAGT TTTTGAGGCG CCTATATAGC TCAGAGGCAG AGCACTTCCT 720 TGGTAAGGAA GAGGTCGGCG GTTCAATTCC GCTTATAGGC TCCAGTTTAT AATCTCTTGA 780
ATGGCGATAA GACAAAAATG TCTTAAATTT TGTGGTAGCA TTTAGGAATA CTTAGGATTT 840
TGTTTAGTAT AATTCTAAAA TCCATTTCAA AAAATTAAGG AGAAATACAA ATG 893
Met
GCA AAA GAA AAG TTT AAC AGA ACT AAG CCG CAT GTT AAT ATT GGA ACC 941
Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn He Gly Thr 5 10 15
ATT GGG CAT GTA GAC CAT GGT AAA ACG ACT TTG AGT GCA GCG ATT TCA 989
He Gly His Val Asp His Gly Lys Thr Thr Leu Ser Ala Ala He Ser 20 25 30
GCG GTG CTT TCT TTG AAA GGT CTT GCA GAA ATG AAA GAC TAT GAT AAT 1036 Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp Asn 35 40 45
ATT GAT AAC GCC CCT GAA GAA AAA GAA AGA GGG ATC ACT ATC GCT ACT 1085 He Asp Asn Ala Pro Glu Glu Lys Glu Arg Gly He Thr He Ala Thr 50 55 60 65
TCC CAC ATT GAA TAT GAG ACT GAA AAC AGA CAC TAT GCG CAT GTG GAT 1133 Ser His He Glu Tyr Glu Thr Glu Asn Arg His Tyr Ala His Val Asp 70 75 80
TGC CCA GGA CAC GCT GAC TAT GTA AAA AAC ATG ATC ACC GGT GCG GCA 1181 Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met He Thr Gly Ala Ala 85 90 95
CAA ATG GAC GGA GCG ATT TTG GTT GTT TCT GCA GCT GAT GGC CCT ATG 1229 Gin Met Asp Gly Ala He Leu Val Val Ser Ala Ala Asp Gly Pro Met 100 105 110
CCT CAA ACC AGA GAG CAT ATC TTA TTG TCT CGT CAA GTA GGC GTG CCT 1277 Pro Gin Thr Arg Glu His He Leu Leu Ser Arg Gin Val Gly Val Pro 115 120 125
CAC ATC GTT GTT TTC TTA AAC AAA CAA GAC ATG GTA GAT GAC CAA GAA 1325 His He Val Val Phe Leu Asn Lys Gin Asp Met Val Asp Asp Gin Glu 130 135 140 145 TTG TTA GAG TTG GTA GAA ATG GAA GTG CGC GAA TTG TTG AGC GCG TAT 1373 Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala Tyr 150 155 160
GAA TTC CCT GGT GAT GAC ACT CCT ATC GTA GCG GGT TCA GCT TTA AGA 1421 Glu Phe Pro Gly Asp Asp Thr Pro He Val Ala Gly Ser Ala Leu Arg 165 170 175
GCT TTA GAG GAA GCA AAG GCT GGT AAT GTG GGT GAA TGG GGT GAA AAA 1469 Ala Leu Glu Glu Ala Lys Ala Gly Asn Val Gly Glu Trp Gly Glu Lys 180 185 190
GTG CTT AAG CTC ATG GCT GAA GTG GAT GCC TAT ATC CCT ACT CCA GAA 1517 Val Leu Lys Leu Met Ala Glu Val Asp Ala Tyr He Pro Thr Pro Glu 195 200 205
AGA GAC ACT GAA AAA ACT TTC TTG ATG CCG GTT GAA GAT GTG TTC TCT 1565 Arg Asp Thr Glu Lys Thr Phe Leu Met Pro Val Glu Asp Val Phe Ser 210 215 220 225
ATT GCG GGT AGA GGG ACT GTG GTT ACA GGT AGG ATT GAA AGA GGT GTG 1613 He Ala Gly Arg Gly Thr Val Val Thr Gly Arg He Glu Arg Gly Val 230 235 240
GTG AAA GTA GGC GAT GAA GTG GAA ATC GTT GGT ATC AGA GCT ACA CAA 1661 Val Lys Val Gly Asp Glu Val Glu He Val Gly He Arg Ala Thr Gin 245 250 255
AAA ACG ACT GTA ACC GGT GTG GAA ATG TTT AGA AAA GAG CTA GAA AAA 1709 Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu Lys 260 265 270
GGT GAG GCC GGC GAT AAT GTG GGC GTG CTT TTG AGA GGA ACT AAA AAA 1757 Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys Lys 275 280 285
GAA GAA GTA GAA CGC GGT ATG GTT CTA TGC AAA CCA GGT TCT ATC ACT 1805 Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser He Thr 290 295 300 305
CCG CAC AAG AAA TTT GAG GGA GAA ATT TAT GTC CTT TCT AAA GAA GAA 1853 Pro His Lys Lys Phe Glu Gly Glu He Tyr Val Leu Ser Lys Glu Glu 310 315 320
GGC GGG AGA CAC ACT CCA TTC TTC ACC AAT TAC CGC CCG CAA TTC TAT 1901 Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gin Phe Tyr 325 330 335
GTG CGC ACG ACT GAT GTG ACT GGC TCT ATC ACC CTT CCT GAA GGC GTA 1949 Val Arg Thr Thr Asp Val Thr Gly Ser He Thr Leu Pro Glu Gly Val 340 345 350
GAA ATG GTT ATG CCT GGC GAT AAT GTG AAA ATC ACT GTA GAG TTG ATT 1997 Glu Met Val Met Pro Gly Asp Asn Val Lys He Thr Val Glu Leu He 355 360 365
AGC CCT GTT GCG TTA GAG TTG GGA ACT AAA TTT GCG ATT CGT GAA GGC 2045 Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala He Arg Glu Gly 370 375 380 385
GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT ATT ATT GAA TAA 2090
Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn He He Glu 390 395
TATTAGCAAA AAGAGTTACC ATAAAGGGTC ATTATGAAAG TTAAAATAGG GTTGAAGTGT 2150
TCTGATTGTG AAGATATCAA TTACAGCACA ACCAAGAACG CTAAAACTAA CACTGAAAAA 2210
CTGGAGCTTA AGAAGTTCTG CCCAAGGGAA AACAAACACA CTCTTCATAA AGAAATCAAA 2270
TTGAAGAGCT AGTTCTTTCT TTTGTGTTGT GATTGAAAAG GAGGGGAGGT TAGGTCAGTA 2330
GCTCCAATGG TAGAGCGTCG GTCTCCAAAA CCGGTTGTTG GGGGTTCGAG TCCCTCCTGG 2390
CCTGCCATCT ACTAATTTAT TCTATCAAAT TTTTGTTTCA ATTGGATTGT TTTTGAATTT 2450
TTTAATTTTA GTTTAAGCTA TTTTGGATAA AATTGAAAAT TCTTTTAATG TATAAATATT 2510
AAGTTTAAGT GAGGGCGAAA AGAAACTATG GATAAATGGC TCATGCAATA TAAATTAGCT 2570
AGAGAAGAGC TTTCTAAAGT GATATTTCCT ATTAAGGAGC AGATACGCAA CGCGCTTGTT 2630
TCTGTTTTGG TGGTGGTGAG TGCTATCACG CTGTTTTTAG CTTTGTTGGA TTTTTCTCTG 2690
GGGGCTTTTA TCTCTAGTGT TCTATAGGTT GGTGGCTTTA AATAAGGAGA ATAATGATGG 2750 ATTGGTATGC CATACAAACT TATTCAGGGA GCGAGCAGTC CGTTAAGAAA GCGATTGAGA 2810
ATCTAGCGAA CGATC 2825
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2182 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: ATCC 43504
(v) FEATURES: from 344 to 520 bp signal peptide from 521 to 1507 bp mature protein
(vi) PROPERTIES: 36/35/32 kD protein from Helicobacter pylori
GATCGCTCTT TGAGTGATTC CTGTATTCGC TTTATTGGCA AACTCTTCGC CAAACATTTT 60
CTTCACATTA GGGAAAATTA CCCCATCAAA AAACAAGTAG CCAATAAAAA TAATGGCGCA 120
CAATAAATGA ACAACCAACA CATAAGGATA AATCGCATCC ATTTAAAATC CTTTATTCAT 180
GGGAAAATTA AAGAGTTTTT AATCTACTAT AAAAGGGTTT TATTGTCAAG TATCCCACTA 240
TTATGGGAAT TTTAGGGGTG GTTTTTGTTT GACTTTTAAG ATTGCAATTA GCTATAATAA 300
AATAATTAAA AAAGTAACAC TTAAGCGGAG ACCCTAGAGA GTG ATG CTC AAT TTT 355
Met Leu Asn Phe
ATG ACA AAG AAG AAA AAT AGA ATG CAA GAT TGC AAA ATG GTT GGT AAA 403
Met Thr Lys Lys Lys Asn Arg Met Gin Asp Cys Lys Met Val Gly Lys -55 -50 -45 -40
AAT TTT AAT CGT AAG GAA TCT GTT TTG ATA GCT CAA TCT TTA GAA ATT 451
Asn Phe Asn Arg Lys Glu Ser Val Leu He Ala Gin Ser Leu Glu He -35 -30 -25
TCT AAA AAA GGC TCG GTA ATT TTA GGC GCT CTT TTG AGT TCG TTA TGG 499 Ser Lys Lys Gly Ser Val He Leu Gly Ala Leu Leu Ser Ser Leu Trp -20 -15 -10
CTG ACA AAC CCC TTA AAT GCC CAT GAA AAG AAT GGC GCG TTT GTG GGG 547
Leu Thr Asn Pro Leu Asn Ala His Glu Lys Asn Gly Ala Phe Val Gly -5 5
ATT AGC TTG GAA GTG GGT AGG GCT GAT CAA AAG ACC AAC GCT TAT AGA 595
He Ser Leu Glu Val Gly Arg Ala Asp Gin Lys Thr Asn Ala Tyr Arg 10 15 20 25
AAC GGC GAG TTG TTT CAA GTG CCT TTT GGC GAT GTT TCA GCC AAT GAT 643
Asn Gly Glu Leu Phe Gin Val Pro Phe Gly Asp Val Ser Ala Asn Asp 30 35 40
GAT GGC AAA GTC CCT AAC GGG CAG ACC GGT GGC TGT CAG CCA GCT TCA 691
Asp Gly Lys Val Pro Asn Gly Gin Thr Gly Gly Cys Gin Pro Ala Ser 45 50 55
GGG ACG CCA GGA ACG CCA GGC TAT ACT AAA GCT AAT TGC GTG GTC AAT 739
Gly Thr Pro Gly Thr Pro Gly Tyr Thr Lys Ala Asn Cys Val Val Asn 60 65 70
TGG ACT TCT CGC ACC ATG CTT AGC ACC AAT AAA AAC ATT CCT GGC CGT 787 Trp Thr Ser Arg Thr Met Leu Ser Thr Asn Lys Asn He Pro Gly Arg 75 80 85
AAC CAG CCG ATG TAT GGG CTA GGT GTG ATG ACG GGC TAT AAG CAT TTT 853 Asn Gin Pro Met Tyr Gly Leu Gly Val Met Thr Gly Tyr Lys His Phe 90 95 100 105
ATC GGT AAA AAA AGG TGG TTT GGG TTG CGC TAT TAC GGC TTT TTT GAT 883 He Gly Lys Lys Arg Trp Phe Gly Leu Arg Tyr Tyr Gly Phe Phe Asp 110 115 120
TAT GGG CAT ACC AAT TTC TCT AAC TCC AGG GCC GCT AAC GCT ATA TCG 931 Tyr Gly His Thr Asn Phe Ser Asn Ser Arg Ala Ala Asn Ala He Ser 125 130 135
CCT TTC TAT TTG AGC GAT CAA AAA GCG GAC ATG TAT ACT TAT GGT TTT 979 Pro Phe Tyr Leu Ser Asp Gin Lys Ala Asp Met Tyr Thr Tyr Gly Phe 140 145 150 GGC ACA GAC ATG CTT TTT AAC ATT ATA GAT AAG CCT AAA GCC ACG GCC 1027
Gly Thr Asp Met Leu Phe Asn He He Asp Lys Pro Lys Ala Thr Ala 155 160 165
GGG TTT TTT GTG GGC GTG AAT TTT GCG GGT AAC ACT TGG ACC AAT AAT 1075
Gly Phe Phe Val Gly Val Asn Phe Ala Gly Asn Thr Trp Thr Asn Asn
170 175 180 185
CGT GTG GGG TAT TTT AAG GAC GGG TAT GTT TAT GGC GTC AAT ACG GAT 1123
Arg Val Gly Tyr Phe Lys Asp Gly Tyr Val Tyr Gly Val Asn Thr Asp
190 195 200
GCT GAC GCT TAC ATG ACT AAC GCT GAT GGC ACA ATC ACA TGC GGG GAC 1171
Ala Asp Ala Tyr Met Thr Asn Ala Asp Gly Thr He Thr Cys Gly Asp 205 210 215
ACG ACG CCG GCG AGT TGT GAT GTG GGG ATT AAT CCT AAT AGC GTC TAT 1219
Thr Thr Pro Ala Ser Cys Asp Val Gly He Asn Pro Asn Ser Val Tyr 220 225 230
ACC ACA GGA AAA TTG AAC GCT AAA GTG AAT CAC ACG ATT TTC CAA TTT 1267
Thr Thr Gly Lys Leu Asn Ala Lys Val Asn His Thr He Phe Gin Phe 235 240 245
TTA GTG AAT GTG GGC ATT AGA ACT AAT ATT TTT GAA CAC CAT GGC ATT 1315
Leu Val Asn Val Gly He Arg Thr Asn He Phe Glu His His Gly He
250 255 260 265
GAG TTT GGT ATC AAA ATC CCC ACG CTC CCT AAT TAC TTT TTC AAA GGC 1363
Glu Phe Gly He Lys He Pro Thr Leu Pro Asn Tyr Phe Phe Lys Gly
270 275 280
TCT ACT ACC ATA AGA GCG AAA AAA CAA GGC CCG CTA GAG AAT GGC CAA 1411
Ser Thr Thr He Arg Ala Lys Lys Gin Gly Pro Leu Glu Asn Gly Gin 285 290 295
CCA ACC ACT ATC ACC GGA GCA GAA ACC AAT TTC AGC TTA ACC CAA ACC 1459 Pro Thr Thr He Thr Gly Ala Glu Thr Asn Phe Ser Leu Thr Gin Thr 300 305 310
TTA CGC CGT CAA TAT TCT ATG TAT TTG CGC TAT GTT TAT ACT TTT TGA 1507 Leu Arg Arg Gin Tyr Ser Met Tyr Leu Arg Tyr Val Tyr Thr Phe 315 320 325 ATTTGGTAGG GTTTTTAGGC AGGGCTTATA GCTTATATAT GGATATATGA AAGCTTGATT 1567
TGTCAAGCTT TAGGGTTGTC ATTGAGTTGC AATAACTCTG TGCTGTTTTC TACTTTTTTG 1627
ATAAAATCAT TAATGGCATA ACAGCGTATG TTAATATTGT CTTTGAAATG GGCAAATCCC 1687
GCATATTCTT TGGCGTCATC ATGGATATTT GGAGCTACAA AAACACTAAA TTTTTCTCTA 1747
ATATCAGTGC TATTTTTAAT CAATTCTTTT AAATGTCTGG CAATAGGTAT CATTTCCAAG 1807
GTACTTTGAC TTCTATCTCT AATCAAGCTC ACTTCTATAT AACTTTGGGC TTTTGTGTCC 1867
ATAGCTACAA TATCAGGTTT GTTACCGCTT GCTGTGTATA CGGGCAAGCC TTCATCATCG 1927
CTTTTATAAT TGGGTATCAC GCTTAAATTT TCAAAATGTT GTTTCAAGAA AATAGCGCTT 1987
AAAAATTCTA AGCGTAAAGG TTTATCAATG AGTCTTAAAA AACTATCTTT TGATTCTTGC 2047
TTGTTGCAAG TAATGAGTAA TTCTTGCTTG ATAAAATCTT TAGTATAAGT GGTTGCTAGT 2107
TCATTCAATT TGCTTGTTTT AACGCTCTCA TCAGCGCTGA TTGGAGTAAC GCTAACAAGA 2167
AAGCTATCCA CGATC 2182
(2) INFORMATION FOR SEQ ID NO: 27:
(I) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 480 bp
(B) TYPE: nucleotide with deduced protein
(C) STANDEDNESS: single
(D) TOPOLOGY: linear
(II) MOLECULAR TYPE: Genomic DNA
(iii) ORIGIN OF ORGANISM: Helicobacter pylori Direct experimental origin
(iv) NAME OF CELL LINE: Strain 26695
(v) FEATURES: from 843691 bp to 843212 bp mature protein
(vi) PROPERTIES: 17 kD protein from Helicobacter pylori
ATG GAA CAA AGC CAT CAA AAC TTG CAA TCT CAA TTT TTT ATA GAG CAT 843644 Met Glu Gin Ser His Gin Asn Leu Gin Ser Gin Phe Phe He Glu His 5 10 15
ATC TTA CAA ATT CTA CCT CAC CGC TAT CCC ATG CTT TTA GTG GAT AGA 843596 He Leu Gin He Leu Pro His Arg Tyr Pro Met Leu Leu Val Asp Arg 20 25 30
ATT ATA GAG TTA CAA GCC AAT AAA AAA ATT GTC GCT TAT AAG AAT ATC 843548 He He Glu Leu Gin Ala Asn Lys Lys He Val Ala Tyr Lys Asn He 35 40 45
ACT TTT AAT GAA GAC GTG TTT AAC GGG CAT TTC CCT AAT AAG CCC ATT 843500 Thr Phe Asn Glu Asp Val Phe Asn Gly His Phe Pro Asn Lys Pro He 50 55 60
TTC CCG GGC GTT TTG ATC GTA GAG GGC ATG GCG CAA ACG GGA GGG TTT 843452 Phe Pro Gly Val Leu He Val Glu Gly Met Ala Gin Thr Gly Gly Phe 65 70 75 80
TTA GCC TTC ACT AGC TTG TGG GGG TTT GAC CCT GAA ATC GCC AAA ACA 843404 Leu Ala Phe Thr Ser Leu Trp Gly Phe Asp Pro Glu He Ala Lys Thr 85 90 95 AAA ATC GTG TAT TTC ATG ACG ATT GAT AAG GTT AAA TTC CGC ATC CCT 843356 Lys He Val Tyr Phe Met Thr He Asp Lys Val Lys Phe Arg He Pro 100 105 110
GTA ACC CCA GGC GAC AGA TTA GAA TAC CAT TTA GAA GTC TTA AAG CAT 843308 Val Thr Pro Gly Asp Arg Leu Glu Tyr His Leu Glu Val Leu Lys His 115 120 125
AAG GGC ATG ATC TGG CAA GTG GGT GGC ACG GCT CAA GTG GAT GGC AAA 843260 Lys Gly Met He Trp Gin Val Gly Gly Thr Ala Gin Val Asp Gly Lys 130 135 140
GTG GTC GCT GAA GCC GAA TTG AAA GCC ATG ATT GCA GAG AGA GAT TAA 843212 Val Val Ala Glu Ala Glu Leu Lys Ala Met He Ala Glu Arg Asp 145 150 155

Claims

1. A protein from Heiicojbacter pylori (H. pylori ) containing one of the peptide sequences selected from SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 and 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids.
2. A protein according to Claim 1, characterized in that the peptide sequences are N-terminal sequences.
3. A protein according to Claim 1 or 2, characterized in that the protein containing a peptide sequence having the SEQ ID NO: 1 according to Table la has a molecular weight of approx. 250 kD, the protein containing a peptide sequence having the SEQ ID NO: 2 according to Table la has a molecular weight of approx. 110 kD, the protein containing a peptide sequence having the SEQ ID NO: 3 according to Table la has a molecular weight of approx. 100 kD, the protein containing a peptide sequence having the SEQ ID NO: 6 according to Table la has a molecular weight of approx. 60 kD, the protein containing a peptide sequence having the SEQ ID NO: 10 according to Table lb has a molecular weight of approx. 42 kD, the protein containing a peptide sequence having the SEQ ID NO: 11 according to Table lb has a molecular weight of approx. 42 kD, the protein containing a peptide sequence having the SEQ ID NO: 12 according to Table lb has a molecular weight of from approx. 32 to approx. 36 kD, the protein containing a peptide sequence having the SEQ ID NO: 14 according to Table lc has a molecular weight of approx. 30 kD, the protein containing a peptide sequence having the SEQ ID NO: 15 according to Table lc has a molecular weight of approx. 28 kD, the protein containing a peptide sequence having the SEQ ID NO: 16 according to Table lc has a molecular weight of approx. 28 kD, the protein containing a peptide sequence having the SEQ ID NO: 17 according to Table lc has a molecular weight of approx. 25 kD, the protein containing a peptide sequence having the SEQ ID NO: 18 according to Table lc has a molecular weight of approx. 25 kD, and the protein containing a peptide sequence having the SEQ ID NO: 19 according to Table lc has a molecular weight of approx. 17 kD.
4. A protein according to any one of Claims 1 to 3, characterized in that the protein is a membrane protein or a protein which is firmly associated with the membrane .
5. A protein according to any one of Claims 1 to 4, characterized in that the protein is an integral membrane protein, in particular a Sarkosyl^-insoluble integral membrane protein.
6. A protein according to any one of Claims 1 to 5, which can be obtained in accordance with the following procedural steps:
(a) isolating the proteins by means of differential solubilization;
(b) separating the proteins, which have been isolated in accordance with step (a), by means of gel electrophoretic methods; and
(c) isolating the proteins, which have been separated in accordance with step (b) .
7. A protein according to Claim 6, characterized in that the protein can be obtained by means of differential solubilization using Sarkosyl®.
8. A protein according to Claim 6 or 7, characterized in that it can be obtained by means of separation by one or more SDS polyacrylamide gel electrophoreses .
9. A protein according to Claim 8, characterized in that it can be obtained by means of several SDS polyacrylamide gel electrophoreses having different polyacrylamide contents.
10. A protein according to Claim 8 or 9, characterized in that the polyacrylamide content is approximately 8%, 10% or 16%.
11. A peptide having the amino acid sequence according to SEQ ID NO: 1, 2, 3, 6, 10, 11, 12, 14, 15, 16, 17, 18 or 19 according to Tables la-lc, or parts or homologues thereof having a minimum length of five amino acids .
12. An antibody against one or more proteins according to any one of Claims 1 to 10 and/or against one or more peptides according to Claim 11.
13. A polynucleotide encoding one or more proteins according to any one of Claims 1 to 10 or one or more peptides according to Claim 11.
14. A process for preparing the proteins according to any one of Claims 1 to 5, characterized in that the following procedural steps are carried out:
(a) isolating the proteins, by means of differential solubilization;
(b) separating the proteins, which have been isolated in accordance with step (a) , by means of gel electrophoretic methods; and
(c) isolating the proteins, which have been separated in accordance with step (b) .
15. A process according to Claim 14, characterized in that the proteins are isolated in accordance with step
(a) using Sarkosyl®.
16. A process for preparing the peptides according to Claim 11, characterized in that a chemical peptide synthesis is carried out.
17. A process for preparing the proteins according to any one of Claims 1 to 10, or the peptides according to
Claim 11, characterized in that a polynucleotide according to Claim 13 is expressed.
18. The use of one or more proteins according to any one of Claims 1 to 10, one or more peptides according to Claim 11, one or more antibodies according to Claim 12 or one or more polynucleotides according to Claim 13 for preparing a pharmaceutical composition or a diagnostic agent.
19. A pharmaceutical composition comprising one or more proteins according to any one of Claims 1 to 10 and/or one or more peptides according to Claim 11 or one or more antibodies according to Claim 12 or one or more polynucleotides according to Claim 13 or their expression products.
20. A pharmaceutical composition according to Claim 19, characterized in that the pharmaceutical composition is used as a vaccine.
21. A diagnostic agent comprising one or more proteins according to any one of Claims 1 to 10 and/or one or more peptides according to Claim 11, one or more antibodies according to Claim 12 or one or more polynucleotides according to Claim 13 or their expression products.
22. A protein from H. pylori containing one of the peptide sequences deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 and 27, or parts or homologues thereof having a minimum length of five amino acids.
23. A peptide having the amino acid sequence deduced from SEQ ID NO: 21, 22, 23, 24, 25, 26 or 27, or parts or homologues thereof having a minimum length of five amino acids.
24. A peptide selected from the C-.terminal region of the peptide sequence of SEQ ID NO: 20 or homologue thereof.
25. A peptide according to Claim 24, wherein said peptide is selected from RDPKFNLAHIEKEFEVWNWDYRA and EKHQKMMKDMHGKDMHHTKKKK, or parts or homologues thereof.
26. An antibody against one or more proteins according to Claim 22 and/or against one or more peptides according to any one of Claims 23 to 25.
27. A polynucleotide encoding one or more proteins according to Claim 22 or one or more peptides according to any one of Claims 23 to 25.
28. A host cell transformed with the polynucleotide of Claim 13 or 27.
29. An expression product expressed from the host cell according to Claim 28.
30. A pharmaceutical composition comprising one or more proteins according to Claim 22 and/or one or more peptides according to any one of Claims 23 to 25, or one or more antibodies according to Claim 26, or one or more polynucleotides according to Claim 27 or one or more of their expression products.
31. A pharmaceutical composition according to Claim 30, characterized in that the pharmaceutical composition is used as a vaccine.
32. A pharmaceutical composition according to Claim 30 or 31, characterized in that when the pharmaceutical composition comprises a nucleotide sequence, said pharmaceutical composition is used as a DNA vaccine.
33. A diagnostic agent comprising one or more proteins according to Claim 22 and/or one or more peptides according to any one of Claims 23 to 25, or one or more antibodies according to Claim 26, or one or more polynucleotides according to Claim 27 or one or more of their expression products.
34. The use of one or more proteins according to Claim 22, one or more peptides according to any one of Claims 23 to 25, one or more antibodies according to Claim 26, one or more polynucleotides according to Claim 27 or one or more of their expression products as a pharmaceutical composition or as a diagnostic agent.
PCT/IB1997/000981 1996-07-26 1997-07-25 Proteins, in particular membrane proteins, of helicobacter pylori, their preparation and use WO1998004702A2 (en)

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