JPH05271293A - Coccidiosis chicken vaccine - Google Patents

Abstract

PURPOSE: To provide new Eimeria proteins having immunmogenic properties.

CONSTITUTION: The proteins having ≥1 immunodetrminants of Eimeria antigens, the nucleic acid sequence to encode these proteins, the recombination vector molecules and vector virus contg. this nucleic acid sequence, the host cells transformed by the nucleic acid sequence or the vector molecules or infected by the vector virus and the method for developing the objective proteins consisting in culturing such host cells and the antibody or antiserum against the proteins described above. When such proteins are administrated to chickens, the chickens may be protected against the coccidiosis. The vector vaccine for the coccidiosis may be prepd. by using the DNA encoding the proteins.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

The present invention relates to the Eimeria antigen,
One or more immunogenic determinants, a nucleic acid sequence encoding the protein, a recombinant vector molecule or recombinant vector virus containing such a nucleic acid sequence, a host cell transformed with such a recombinant vector molecule, or
It is concerned with host cells infected with recombinant vector viruses, antibodies immunoreactive with the above proteins, and vaccines that protect birds from coccidiosis.

[0002] Coccidiosis is a subphylum of Apicomplexa,
It is a disease caused by protozoa, an intracellular parasite of the genus Eimeria. This parasite grows in the gastrointestinal tract and cells that form part of the digestive tract.

Due to the spread of intensive production, the damage caused by this parasite in the poultry industry has risen at a critical pace in recent decades. For example, Dutch poultry farmers suffer millions of guilders each year. That is, the loss in 1986 was about 13 million guilders, and in the same year, the United States suffered a loss of $ 300 million despite the use of coccidia-resistant drugs.

The pathogens of chicken coccidiosis can be divided into 9 different species. That is, Eimeria acer
vulina, E. maxima, E. tenella, E. necatrix,
E. brunetti, E. mitis, E. praecox, E. mivat
i and E. hagani . However, some suspect the existence of the last two species. This species is all chicken-hosted and exhibits a high degree of tissue specificity. However, the life cycles of the above species are similar.

The pathogenic effects of this species on chickens are different. The type of chicken is also relevant. As a result, broiler chickens are heavily damaged by parasitic species such as E. acervulina and E. maxima . This is because it infects most of the small intestine, and food digestion is the main part of it.

During the life cycle, the Eimeria parasite undergoes several stages. Those at the infectious stage (sporulating oocysts) are taken up in the mouth and enter the stomach of chickens, where as a result of the millstone action the cyst walls open. The four sporocysts contained in the oocysts are released and enter the duodenum, where they come into contact with bile and digestive enzymes. As a result, holes are made in the sporocyst cell walls, and the sporozoites (species) in them are released. This sporozoite is motile and seeks suitable host cells and epithelial cells, invades there, and reproduces. In some species, this first reproductive phase lasts 24 to 48 hours, forming tens to hundreds of merozoites. Each of these newly invades the host cell,
To reproduce. Depending on the species, such asexual reproduction cycle is repeated twice, and sometimes five times, and thereafter, intracellularly grows into male and female gametocytes. After insemination of the female mother cells by the male mother cells, zygotes are formed, which create a cyst wall around itself. The oocysts leave the host cell and are expelled with stool. When the temperature and humidity outside the chicken are relatively high and at the same time there is sufficient oxygen in the air, the oocysts form spores and enter the infection stage.

Therefore, no intermediate host is required for this parasite to transfer from chicken to chicken. Therefore, the infection pressure of the poultry farm rises sharply where the area used is highly occupied.

This parasitic species can be controlled in various ways.

In addition to performing appropriate controls, coccidiosis can also be eradicated with anticoccidial agents. This is often mixed with food and drinking water. However, in recent years, the efficacy of such drugs has also decreased. This is in part due to the high genetic capacity of this parasite to develop resistance to various pesticides. In addition, some of these drugs
It remains in the meat as a residue, which can form a consumer problem.

Therefore, immunological prophylaxis would be a far superior control method than conventional ones. Chickens that have survived a high enough infection,
It is well known that it is resistant to contact with syngeneic Eimeria . Resistance to Eimeria can also be induced by infecting chickens several times with low doses of oocysts or with weakened (nonpathogenic) species of oocysts. However, carrying out controlled dosing, especially in large numbers of broiler chickens, is in this case an almost unsolvable problem.

In the present invention, a purified protein of Eimeria antigen having one or more immunogenic determinants is used, which is substantially free of all parasitic species or other proteins normally associated therewith. Is. It can be used for the preparation of a vaccine for immunization of birds, in particular of chickens against cockroach.

The term "nucleic acid sequence" as used herein refers to a polymeric form of nucleotides of any length and relates to both ribonucleic acid and deoxyribonucleic acid. In principle,
The term refers to the primary structure of the molecule, and refers to double stranded, single stranded DNA as well as double stranded, single stranded RNA, and modified forms thereof.

In general, the term "protein" refers to a biologically active chain of amino acids, not a product of a particular length, and, if desired, in vitro or in vivo, eg, glycosylation, It can be modified by amidation, carboxylation or phosphorylation. Thus, in particular, peptides,
It includes oligopeptides and polypeptides.

The term "protein of the Eimeria antigen having one or more immunogenic determinants" refers to a protein having one or more epitopes and capable of eliciting an immune response against an Eimeria parasite of a host animal. ..

The term "molecular weight" is used herein as an estimate of the apparent size under the conditions described in the individual embodiments. The true molecular weight is possible only after sequencing the full length protein. For individual proteins, the apparent molecular weight by SDS-PAGE is due to the hydrophobic nature of the protein and the presence of oligosaccharides, lipids (acyl chains) and other interfering substituents.
It can make mistakes. Even the percentage of acrylamide gels can affect the relative mobility of the water-soluble marker proteins in the gel. An example is Frank, RN and Rodbard, D. (1975) Arc.
h. Biochem. Biophys. 171, 1-13. Aside from these restrictions, most of the SDS-PAGE (Western blot) electrophoresis performed for the application of the present invention was performed in a non-reducing state (thus beta.
Do not add mercaptoethanol or dithiothreitol). This is to improve the identification by Mabs.

[0016] In particular, the present invention is the Eimeria antigens, gives one or more immunogenic determinants protein with, here, Eimeria antigens, in SDS-PAGE, approximately,
It has a molecular weight of 200, 100, 50 or 20 kD, and this Eimer
ia antigen is monoclonal antibody E. ACER 11A-2A or
E. ACER 12B-2B, E.ACER 5F-2, E.ACER 10C-2A or
E.ACER 10E-2 binds specifically. An example of a hybridoma cell line producing the above monoclonal antibody is the European Collection of Animal Cell Cultures, Porton Down, UK.
(ECACC)), accession number, 91061223 (E.ACER 12B-2B),
And 91061222 (E.ACER 11A-2A), 91061219 (E.ACER5F-
2), 91061220 (E.ACER 10C-2A), and 9106221 (E.ACER
Deposited under 10E-2).

The Eimeria antigens disclosed above are characterized by their method of isolation. That is, the Eimeria acervulina parasite is extracted with 1.2% Triton X114 solution to obtain the antigen.

2A. The hydrophobic fraction obtained after phase separation in step 1 was 1. E.ACER 10C-2A Sepharose CL-4B binding immunoaffinity chromatography or E.ACER 10E-2 Sepharose CL-4B binding immunoaffinity chromatography or 2B. The hydrophilic fraction obtained after the phase separation in step 1 was used as E.ACER.
11A-2A Sepharose CL-4B coupled immunoaffinity chromatography or 2C. The hydrophilic fraction obtained after the phase separation in step 1 was used as E.ACER.
5F-2 Sepharose CL-4B binding immunoaffinity
Chromatography 3.1. Purified, 50, 100 or 200 kD Eimeria
Add 0.1M glycine / hydrochloric acid + 0.1% NP4O pH 2.
Elute at 6, 2. Purified 20 kD Eimeria protein was added to 25 mM T
ris / HCl + 0.5M NaCl + 0.1% NP4O 3M dissolved in pH 8.0
Elute with KSCN.

A preferred protein according to the invention is Eime
ria acervulina antigen Eam200, Eam100 or Eas100,
One or more immunogenic determinants of Eam45 or Eam20 (Example 2).

Eam200 is an Eimeria protein of about 200 kD extracted from Eimieria acervulina merozoite, and has immunoreactivity with the monoclonal antibody (Mab) E.ACER 11A-2A.

Eas100 is an Eimeria protein of about 100 kD extracted from Eimieria acervulina sporozoites and has immunoreactivity with Mab E.ACER 5F-2. Eam1
00 is the equivalent of merozoites.

Eam45 is an Eimeria protein of about 50 kD extracted from Eimieria acervulina merozoite, and has immunoreactivity with Mab E.ACER 10C-2A.

Eam20 is an Eimeria protein of about 20 kD extracted from Eimieria acervulina merozoite and has immunoreactivity with Mab E.ACER 10E-2.

Monoclonal antibody E.ACER 11A-2A, E.AC
ER 12B-2B is primarily against the Eam200 antigen. As shown in FIG. 1, E.ACER 12B-2B recognized this protein in both reduced and non-reduced forms. Lane 1 of panel B,
2 is shown. E-ACER 11A-2A recognized only the non-reduced form. This is as shown in Panel A, lanes 1 and 2. However, this one of Mab also, E. acervul
A set of MW 100-200 kD polypeptides contained in ina sporozoites and MW ± of E. tenella sporozoites.
A clear positive band of 130 kD was recognized. Lane 3, 5
As you can see.

Using fluorescence, the cross-reactivity to sporozoites was localized to the front end of sporozoites. Organs involved in invasion are localized here.

E. tenella second generation merozoites do not appear to bind to these Mabs, apparently due to the small amount of this protein at this stage.

Monoclonal antibody E.ACER 10C-2A anti-Eam
45 recognized only proteins of similar molecular weight in E.acervulina sporozoites, but no reaction to E.tenella was observed. As shown in FIG. 2A.

E.ACER 10E-2, anti-Eam20, also had a faint band (MW ± 20 k) only in sporozoites of the same species.
D) recognized. However, E.acervulina, and E. ten
No other species were tested except ella . See FIG. 2, panel B.

Monoclonal antibody E.ACER 5F-2 is E.ac
Against ervulina sporozoites, the same species also recognized the ± 100 kD protein in merozoites. Reactivity with other species was not tested.

Further, the present invention provides a purified E, as specified above.
Examples of proteins with one or more immunogenic determinants of imeria antigens are given. This example is SEQ ID NO: 2,
It is a protein consisting of the amino acid sequence shown in 6, 8 or 10.

Furthermore, the present invention provides an Eimeria protein having the amino acid sequence shown in SEQ ID NO: 4 and its functional variants. This protein is Eimeri
a Merozoite cDNA library was identified by screening with anti-Eam45 serum. This serum is
When it was used as a probe in a merozoite blot, it showed a positive reaction with a protein of about 100 kD (in addition to the positive reaction with a protein of about 50 kD) (Fig. 9).

A functional variant of the protein specifically disclosed herein is a protein obtained from the above amino acid sequence, for example obtained by removing, inserting and / or substituting one or more amino acids, It carries one or more immunogenic determinants of the Eimeria antigen. That is, the above variant has one or more epitopes capable of inducing an immune response in the host animal.

It will be appreciated that natural variants of the particular proteins contained herein may be present in individual Eimeria parasites or strains thereof. This variant may be indicated as a difference of one or more amino acids in the overall sequence, ie the removal, substitution, insertion, inversion, addition of amino acid (s) in the sequence. Amino acid substitutions have been described that are not expected to substantially alter biological and immunological activity. Amino acid substitutions between related amino acids, or substitutions commonly found in evolution, include, in particular, Ser / Ala, Ser / Gl
y, Asp / Gly, Asp / Asn, Ile / Val (Dayhof, MD, Atlas
of protein sequence and structure, Nat. Biomed. R
es. Found., Washington DC, 1978, vol. 5, suppl.
3, see). Based on this knowledge, Lipman and Pear
son developed a fast and sensitive protein comparison method (Sci.
ence 227, 1435-1441, 1985), quantifying the functional similarity between homologous proteins.

In addition, immunogenic fragments of the proteins specifically disclosed herein, or functional variants thereof, are also included in the invention.

As used herein, the term "fragment" means a DNA or amino acid sequence consisting of a partial sequence of the nucleic acid sequence or protein of the present invention. The fragment is or encodes a polypeptide having one or more immunogenic determinants of the Eimeria antigen. Operable immunogenic polypeptide
The method for quantifying the fragment is outlined below. The fragment is produced by first enzymatically cleaving the precursor molecule. That is, restriction endonucleases are used for DNA and proteases for polypeptides. Other methods include chemical synthesis of fragments and expression of polypeptide fragments by DNA fragments.

Suitable immunogenic polypeptide fragments of the protein according to the invention are those which comprise the epitope (s), which is referred to in patent application W
O 86/06487, Geysen, HM et al. (Proc. Natl. Acad.
Sci. 81, 3998-4002, 1984), Geysen, HM et al.
(J. Immunol. Meth. 102, 259-274, 1987). This method is based on the so-called pepscan method, in which a series of partially overlapping peptides corresponding to a partial sequence of a complete polypeptide of interest are synthesized and their reactivity with an antibody is investigated.

In addition, several regions of the polypeptide having the above amino acid sequence can be made into epitopes based on theoretical considerations and structural agreement with currently known epitopes. This area was determined by Hopp and Woods
(Proc. Natl. Acad. Sci. 78, 3824-3828, 1981) and Chou and Fasman (Advances in Enzymol
ogy 47, 45-148, 1987) by matching the properties of the secondary structure.

T cell epitopes may be required, but this too is based on rationale, eg Be
rzofsky's amphipathic criterion (Science 235, 1059-62, 198
7) is obtained.

The present invention further provides isolated and purified nucleic acid sequences encoding the above proteins of Eimeria.

As is well known in the art, due to the degeneracy of the genetic code, the base of one codon is replaced with another codon, which still encodes the same amino acid. Such substitutions are possible. For example, the codon for the amino acid glutamic acid is
Both GAT and GAA. Therefore, the sequence number
For the expression of the protein having the amino acid sequence shown in 2, 4, 6, 8, or 10, SEQ ID NOs: 1, 3, 5, 7
It is obvious that derivative nucleic acid sequences having different codon composition different from the nucleic acid sequences shown in 9 and 9 can be used.

Therefore, the present invention provides SEQ ID NOs: 2, 4,
Provided is a nucleic acid sequence encoding at least a part of a protein having the amino acid sequence shown in 6, 8 or 10 and a functional variant thereof.

With the information provided in SEQ ID NOs: 1, 3, 5, 7 and 9, those skilled in the art will have immunological characteristics corresponding to the Eimeria proteins specifically disclosed herein.
It is possible to isolate and identify nucleic acid sequences encoding various functional variant proteins. For this purpose, commonly used Southern blotting and colony hybridization can be used (Experiments in M
olecular Biology, ed. RJ Slater, Clifton, US
A., 1986; Singer-Sam, J. et al., Proc. Natl., Aca
d. Sci. 80, 802-806, 1983; Maniatis, T. et al., Mo
lecular Cloning, A laboratory Manual, second editio
n, Cold Spring Harbor Laboratory Press, USA, 1989
). For example, a cDNA from a particular Eimeria strain
The library can be transferred to a single nitrocellulose filter or "blotted".
Now, on this filter, there is a specified indicator D
By hybridizing to an NA fragment or "probe", a specific Eimeria
The nucleic acid sequence can be identified. This probe is a (synthetic) poly or oligonucleotide sequence obtained from the nucleic acid sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9 under the specific conditions of salt concentration and temperature. And hybridizes with the homologous nucleic acid sequences present on the filter. After washing the filter, hybrids may be detected by autoradiography. The corresponding DNA fragment can now be eluted from the agarose gel and can also be used for the synthesis of functional variants of the polypeptide identified as SEQ ID NO: 2, 4, 6, 8 or 10. You can also do it.

Usually, the Eimeria cDNA library is
It can be constructed correctly by the method described in Example 3. Clone pGEM4Z Eam200, pGEM4Z Eam45 M1
(E), pGEM4Z Eam45 M3 (E), pGEM4Z Eam20 (E) or p
The insert obtained from GEM4Z Eam100E can be labeled with digoxigenin-dUTP by the random prime method. At this time, "DNA labeling / detection kit, supplied by Boehringer, Manheim (catalog number 1093657),
Follow the protocol attached to "Non-radioactive" exactly.

The filter containing the fixed DNA obtained from the above Eimeria cDNA library was treated with the above Maniatis et a
Prepare as described in l. and follow the manufacturer's instructions.
Freshly denatured protein (10 min at 95 ° C),
42 using the labeled Eimeria fragment as a probe
Incubate at 16 ° C for 16 hours. The filter is then washed as follows. 2xSSC, 0.1% (w / v) SDS (1 x SS
C means 0.015 mol / l sodium citrate, plus, 0.1
5 mol / l NaCl) at room temperature for 15 minutes twice, then 1xSS
C, 0.1% (W / V) SDS at 55 ° C for 15 minutes twice. For final identification, the filter was then filtered with PBS-tween (7.65 g /
l NaCl, 0.91 g / l Na 2 HPO 4. 2H 2 O, 0.21g / l KH 2 P
O ₄ , 0.05% (v / v) Tween 80, pH 7.3) at room temperature, 1
It was washed twice for 5 minutes. The filter was then loaded with PB of polyclonal goat anti-digoxigenin Fab fragment.
The mixture was reacted with a 1: 5000 dilution of S-tween and allowed to bind to alkaline phosphatase for 30 minutes at room temperature. Filter with PBS-tween at room temperature for 15 minutes, 4 times, 0.01 M Tri
After washing once with s-HCl pH 8.0, 0.15M NaCl for 15 minutes, the binding of alkaline phosphatase to the filter was 0.33 g / l nitroblue tetrazolium and 0.17 g / l.
l of 5-bromo-4-chloro-3-indolyl phosphoric acid, 0.
Detection was performed by incubating with a solution dissolved in 1M Tris HCl pH 9.6, 0.1M NaCl, 0.01M MgCl ₂ . The DNA that reacts with this probe can be used to express the encoded peptide as described below.

This polypeptide can then be assayed for the presence of one or more immunogenic determinants of the Eimeria antigenic protein using one of the following methods. Polypeptides can be purified from E. coli lysates by methods known in the art. For example, salt fractionation, ion exchange chromatography, hydrophobic interaction chromatography, metal chelate chromatography. The purified product is used to generate monospecific antibodies, as described below. The antibody is probed back onto a Western blot of parasitic material, eg merozoites or sporozoites. The positive signal binds the E. coli translation product directly to the parasitic protein.

Another possibility to carry out the above is to bind the antibody directly to a filter containing: That is, it is a single culture of recombinant phages expressing Eimeria DNA insert contained in E. coli.
This bound antibody was labeled by Osaki et al. (J. Immunological Methods
89, 213-219, 1986) and bound again by binding to a Western blot of Eimeria antigens, again by elution. For Eas100 and Eam45 clones, the following method was applied (Example 3, FIGS. 8 and 9).

The above-described hybridization method can also be used when only a small portion of the entire coding sequence is identified.
It may also be used to achieve full length clones. In particular,
The clones pGEM4Z Eam200, pGEM4Z Eam100E are cDNA
Alternatively, a genomic DNA library may be screened and used to see if there are additional coding sequences.

Another method for extending the DNA sequence is the "semi-specific" polymerase chain reaction outlined in Example 3.

Accordingly, the nucleic acid sequence encoding a functional variant of the proteins disclosed herein is one of the Eimeria antigens.
Encodes a polypeptide containing more than one immunogenic determinant,
Hybridizes with the DNA sequence shown in SEQ ID NO: 1, 3, 5, 7 or 9.

As another method, Eimeria cDNA was added to Huynh
et al. (In: D. Glover (ed.) DNACloning: A Practical
Approach, IRL Press Oxford, 49-78, 1985) and cloned into λgt11 phage and expressed in a bacterial host. Next, the recombinant phage was purified as above.
Polyclonal serum against Eimeria protein or SEQ ID NOs: 2, 4, 6, 8, 10 are screened to determine the corresponding immunological region of the variant polypeptide. Ei
The method for producing the polyclonal serum used here for the meria protein is described below.

The present invention further includes nucleic acid sequences that encode proteins of the Eimeria antigen that have one or more immunogenic determinants. In this antigen, the nucleic acid sequence is the DNA shown in SEQ ID NO: 1, 3, 5, 7, or 9, respectively.
Contains at least a portion of the array.

Nucleic acid sequences according to the invention can be used in certain Eimeria
Isolated from the strain, including polymerase chain method (PCR),
It may be amplified by recombinant DNA methods or it may be chemically synthesized in vitro by methods known in the art.

The nucleic acid sequences according to the invention can also be linked to replicable DNA sequences which are not naturally associated with or linked to them, so-called recombinant vector molecules. And this can also be used for transformation of an appropriate host. Useful recombinant vector molecules are preferably obtained from, for example, plasmids, bacteriophages, cosmids, or viruses.

The particular vectors or clone carriers used according to the invention for cloning nucleic acid sequences are known in the art, but in particular:
pBR322, various pUC, pGEM, plasmid vectors such as locus coeruleus, for example, λgt-Wes, Charon 28, M13
Bacteriophage, SV40,
Virus vectors such as adenovirus and polyoma virus (Rodriquez, RL and DT Denhardt, e
d., Vectors: A survey of molecular cloning vectors a
nd their uses, Butterworths, 1988; Lenstra, JA e
See also t al., Arch.Virol. 110, 1-24, 1990).
Can be mentioned. The methods used to construct recombinant vector molecules according to the present invention are known to those of ordinary skill in the art, and in particular Maniatis,
T. et al. (Molecular Cloning, A Laboratory Manual,
second edition; Cold Spring Harbor Laboratory, 19
89).

For example, to insert a nucleic acid sequence into a cloning vector according to the present invention, the gene and the desired cloning vehicle are identical to those used to prepare the complementary DNA ends. It is easily reached by cutting with the restriction enzyme (s).

Alternatively, the prepared restriction sites can be digested with single stranded DNA or by digesting with an appropriate D
It may be necessary to modify the blunt ends with NA polymerase by filling in the single-stranded ends. After that, blunt end ligation with an enzyme such as T4 DNA ligase may be performed.

If desired, any restriction site can be created by attaching a linker to this DNA terminus. Such linkers include specific oligonucleotide sequences that encode restriction site sequences. Vectors and nucleic acid sequences divided by restriction enzymes are also
It may be modified by homologous polymer ends.

As used herein, "transformation" refers to the introduction of a heterologous nucleic acid sequence into a host cell, regardless of the method used, eg by direct uptake or transduction. The heterologous nucleic acid sequence may be maintained by self-replication or integrated into the host genome. If desired, the recombinant vector molecule can also be provided with appropriate regulatory sequences compatible with the designated host. That is, this sequence regulates the expression of the inserted nucleic acid sequence. Besides microorganisms, cell cultures obtained from multicellular organisms may be used as hosts.

The recombinant vector molecule according to the invention is preferably used to select desired transformants, for example ampicillin or tetracycline resistance in pBR322, ampicillin resistance in pUC8 or the α-peptide of β-galactosidase. It is preferably capable of containing one or more marker activities.

Suitable host cells are microorganisms or cells which have been transformed with a nucleic acid sequence encoding a polypeptide or a recombinant vector molecule containing such a nucleic acid sequence and, if desired, as defined above. Such a microorganism or cell that can be used to express the above polypeptide encoded by a nucleic acid sequence. The host cell may be of prokaryotic origin, eg Escherichia
It may be a bacterium such as a coli, Bacillus subtilis, Pseudomonas, or one of eukaryotic origin, for example Sacc.
It may be a yeast such as haromyces cerevisiae,
It may be a higher eukaryotic cell such as an insect, plant or mammalian cell, including HeLa cells, Chinese hamster ovary (CHO) cells. For insect cells, Spodopte
ra frugiperda Sf9 cell line (Luckow et al., Biotechn
ology, 6, 47-55, 1988). Details of methods for cloning and expressing the nucleic acid sequences of the invention in eukaryotic cloning systems are described in Esser, K. et al. (Plasmids of
Eukaryotes, Springer-Verlag, 1986).

In general, prokaryotes are preferred for the construction of effective recombinant vector molecules of the present invention. For example, E. col
The iK12 strain is particularly preferable, for example, DH5 α or MC
It is a strain like 1061λ.

For expression, the nucleic acid sequences of the invention are introduced into an expression vector, ie the sequences are operably linked to expression control sequences. Such regulatory sequences include promoters, enhancers, operators, inducers, ribosome binding sites and the like. Thus, the recombinant vector molecule of the present invention comprises a nucleic acid sequence encoding the Eimeria protein identified above, operably linked to an expression control sequence, and comprising the DNA contained therein.
The sequence is such a vector molecule that allows the sequence to be expressed in transformed host cell (s).

It will be appreciated that the nucleotide sequence inserted into the selection site of this cloning vector is such that the transformed host has at least one or more immunogenic determinants of the Eimeria antigen. May contain nucleotides that are not part of the actual structural gene for the desired polypeptide, or that contain only one fragment of the complete structural gene for the desired protein. Well, it will be understood.

If the host cell is a bacterium, a typical and effective expression control sequence is the Trp promoter,
Operator (Goeddel, et al., Nucl. Acids Res. 8,
4057, 1980); lac promoter, operator (Chan
g, et al., Nature 275, 615, 1978); outer membrane protein promoter (Nakamura, K. and Inoue, M., EMBO J.
1, 771-775, 1982); bacteriophage lambda promoter, operator (Remaut, E. et al., Nucl. Acids R
es. 11, 4677-4688, 1983); α-amylase (B. subtili
s) Promoters and operators, terminal sequences, other expression enhancing and control sequences that are compatible with the host cell of choice. When the host cell is yeast, a typical effective expression control sequence is, for example, an α mating factor. In insect cells, the baculovirus polyhedrin or p10 promoters can be used (Smith, GE et al., Mol. Cell. Biol.
3, 2156-65, 1983). If the host cell is of mammalian origin, a typical, effective expression control sequence is the SV-40 promoter (Berman, PW et al., Scien
ce222, 524-527, 1983) or, for example, the metallothionein promoter (Brinster, RL, Nature 296, 39).
-42, 1982) or heat shock promoter (Voellm
y et al., Proc. Natl. Acad. Sci. USA 82, 4949-53,
1985). Alternatively, expression control sequences present in Eimeria can also be used. Roberts on how to maximize gene expression
See also and Lauer (Methods in Enzymology 68, 473, 1979).

As described above, the present invention is transformed with the above-mentioned nucleic acid sequence or recombinant expression vector molecule,
It comprises a host (s) capable of producing the Eimeria protein by expression of the nucleic acid sequence.

The immunization of birds against Eimeria infection is carried out, for example, by the addition of the protein according to the invention to the animal.
It is achieved by administering as a so-called subunit vaccine according to an immunological strategy. The subunit vaccine according to the invention comprises the protein in pure form, optionally in the presence of a pharmaceutically acceptable carrier. The protein may optionally be covalently linked to an unrelated protein. An irrelevant protein is, for example, one that favors purification of the fusion product. Examples include β-galactosidase, protein A,
Examples include prochymosin and blood coagulation factor Xa.

In some cases, the protein alone may have a poor ability to enhance prophylactic immunity. If possible, the small fragment should be conjugated to a carrier molecule, thereby enhancing its immunogenicity. Suitable carriers for this purpose include natural polymers (proteins such as hemocyanin in the key hole limpet, albumin, toxins) polyamino acids (polylysine,
Synthetic polymers such as polyalanine), macromolecules such as, amphipathic compound micelles such as saponin. Separately, this fragment, as its polymer,
If possible, it may be supplied as a linear polymer.

The proteins used in such subunit vaccines may be prepared by methods known in the art, for example by isolating the above polypeptides from Eimeria parasites or using recombinant DNA methods. Or by chemical synthetic methods.

If desired, the protein used in the vaccine according to the invention may be modified in vitro or in vivo, for example by glycosylation, amidation, carboxylation, phosphorylation.

An alternative to subunit vaccines is live vector vaccines. A nucleic acid sequence according to the present invention may be prepared by the recombinant DNA method using a microorganism (eg,
Bacteria or virus). In doing so, the insertion is such that the recombinant microorganism still replicates and expresses the polypeptide encoded by the inserted nucleic acid sequence,
Do so in such a manner as to elicit an immune response in the infected host animal.

A preferred embodiment of the present invention is a recombinant vector virus containing a heterologous nucleic acid sequence as described above, which DN
Such a vector virus, wherein the A sequence can be expressed in the host cell (s) infected with the recombinant vector virus or in the host animal. The term "heterologous" indicates that the nucleic acid sequence according to the invention is usually not naturally present in the vector virus.

Furthermore, the present invention provides for the infection of recombinant vector viruses and the expression of their nucleic acid sequences to produce Eimeri
a Host cell (s) or cell culture capable of producing a protein.

For example, the known method of in vivo homologous recombination can be used to insert a heterologous nucleic acid sequence, eg a nucleic acid sequence according to the invention, into the genome of a vector virus.

First, although it can be used to insert an insertion region of a vector genome, that is, a heterologous sequence,
However, a DNA fragment corresponding to such a region, which does not impair the essential functions of the vector necessary for infection and replication, is inserted into the cloning vector by the standard recombinant DNA method. Insertion regions have been reported for a large number of microorganisms (eg EP 80,80
6, EP 110,385, EP 83,286, EP 314,569, WO 88/02022,
WO 88/07088, US 4,769,330 and US 4,722,848
).

Secondly, if desired, the deletion can be introduced into the insertion region contained in the recombinant vector molecule obtained in the first procedure. This is done, for example, by using the recombinant vector molecule obtained in the first step with the appropriate exonuclease I
It can be carried out by digesting with II or by treating with a restriction enzyme.

Third, the heterologous nucleic acid sequence is inserted into the insertion region in the recombinant vector molecule of the first procedure or in place of the DNA removed from the recombinant vector molecule. The DNA sequence of the insertion region must have an appropriate length so that homologous recombination with the vector genome can be achieved. The appropriate cells may then be infected with the wild type vector virus or transformed with the vector genomic DNA. In the latter case, it is done in the presence of a recombinant vector molecule containing the insert linked to the appropriate vector DNA sequence. This is to cause recombination between the corresponding region of the recombinant vector molecule and the vector genome. Progeny of the recombinant vector may in turn be produced in cell culture and selected, eg, genetically or phenotypically, eg, by hybridization. This detects the enzymatic activity encoded by the gene interacting with the heterologous nucleic acid sequence or the antigenic heterologous polypeptide immunologically expressed by the recombinant vector.

Then, this recombinant microorganism is administered to chickens for immunization. This allows the microorganism to survive in the inoculated animal for some time and even replicate,
The polypeptide encoded by the inserted nucleic acid sequence according to the present invention is expressed in vivo to stimulate the immune system of the inoculated animal. Suitable vectors for the incorporation of the nucleic acid sequences according to the invention are derived from viruses such as vaccinia virus (EP 110,385, EP 83,286, US 4,769,330, US 4,722,84
8), pox virus such as bird pox virus (WO 88/02022), herpes virus such as HVT (WO 88/07088) or Marek's disease virus, adenovirus, or influenza virus, or It is obtained from bacteria, for example E. coli and certain Salmonella species. In this type of recombinant microorganism,
The polypeptide synthesized in the host animal may be exposed as a surface antigen. To proceed this way, fusion of the above-mentioned polypeptide with an OMP protein, for example the ciliary protein of E. coli, or a signal recognized by the microorganism, a synthetic terminal sequence is conceivable. It is also possible to immunize the above-mentioned immunogenic polypeptide, if desired, as a part of a larger whole by opening it inside the animal. In all these cases, 1
It is also possible that more than one immunogenic product is expressed and prophylaxis against various pathogens and / or against various antigens of a particular pathogen.

The vector vaccine according to the present invention is prepared by culturing a recombinant bacterium or host cell infected with a recombinant vector virus containing the nucleic acid sequence according to the present invention. That is, it allows recombinant bacteria, virus-containing cells, and / or recombinant vector viruses propagated in the cells to be recovered in pure form if desired and frozen in vaccine form if desired. Can be formed.

Host cells transformed with the recombinant vector molecules according to the invention can also be cultured under conditions which favor the expression of the polypeptide encoded by the nucleic acid sequence. The vaccine is a crude culture specimen,
It may be prepared using a host lysate or a host cell extract. However, in another embodiment, a purer purified product of the polypeptide according to the present invention is prepared into a vaccine depending on its intended use. In order to purify the produced polypeptide, the host cell transformed with the recombinant vector according to the present invention is cultivated to a suitable volume, and the produced polypeptide is secreted from the cell or its protein. If so, isolate from the culture. The polypeptide secreted into the culture medium can be isolated and purified by standard methods, for example, salt fractionation method, centrifugation, ultracentrifugation, chromatography, gel filtration, or immunoaffinity chromatography. On the other hand, the intracellular polypeptide is obtained by first recovering the above cells, destroying the cells with, for example, sonication or other mechanical disruption means, for example, a French press, and then the polypeptide is Separated from other intracellular components,
Can be formed into a vaccine. Cell disruption can also be performed chemically (eg, EDTA, detergents such as Triton X114), or using enzymatic means such as lysozyme digestion.

Antibodies or antisera directed against the polypeptides according to the invention have potential utility in passive immunotherapy, diagnostic immunoassays and production of anti-idiotype antibodies.

Using the Eimeria protein having the above-mentioned characteristics, it is possible to produce polyclonal antibodies as well as monospecific monoclonal antibodies. If polyclonal antibodies are desired, methods for producing and treating polyclonal sera are known in the art (eg, Mayer and Walter, eds., Immunochemical
Methods in Cell and Molecular Biology, Academic P
ress, London, 1987). In short, a selected mammal, eg, rabbit, is infused (multiple times) with the immunogen.
That is, about 20 μg to about 80 μg per immunization
Inject the protein. Immunization is given by an acceptable adjuvant, generally an equal volume of immunogen and adjuvant. Acceptable adjuvants include Freund's complete, Freund's incomplete, alum precipitate, or a water-in-oil suspension, although Freund's complete adjuvant is preferred for initial immunization. Freund's incomplete adjuvant is preferred for all booster immunizations. The first immunization is to administer approximately 1 ml of suspension to multiple subcutaneous sites on the back of the rabbit. Booster immunizations containing an equal volume of immunogen are given at approximately 1 month intervals and continued until a sufficient concentration of antibody appears in the individual rabbit serum. Blood is collected and serum is
Isolated by methods known in the art.

Monospecific antibodies against the immunogen were prepared from multispecific antisera prepared by immunizing rabbits with purified protein as described above, Hall et al. (Nature 311, 379).
-387, 1984) and purified by affinity. Monospecific antibody, as used herein, is defined as a single antibody species or multiple antibody species with homologous binding characteristics for the relevant antigen. Homologous binding, as used herein, refers to the ability of multiple antibody species to bind to a particular antigen or epitope.

Monoclonal antibodies reactive with Eimera immunogens were pure mice, preferably Balb / c
May be prepared by immunizing with a suitable protein. Mice contain an equal volume of acceptable adjuvant, 0.5
About 100 ng to about 10 μg of immunogen per ml dose
Immunize by injection into the abdominal cavity. Such acceptable immunogens include Freund's complete, Freund's incomplete, alum precipitate, and water-in-oil suspension. The mice are immunized intravenously with a booster immunization with an equal amount of the immunogen without adjuvant about 14 days, 21 days, and 63 days after the primary immunization. Approximately 3 days after the final booster immunization, individual mice are serologically tested for the presence of anti-immunogen antibody. Antibody-producing mouse spleen cells were isolated and treated with rodent bone marrow cells, such as SP-2 / 0, and techniques known in the art (Kohler and Milstein, Nature 256; 4).
95-497, 1975). Hybridoma cells are selected by growing them in a suitable cell culture medium, for example Dulbecco's Modified Eagle's Solution (DMEM), in the presence of hypoxanthine, thymidine and aminopterin. Antibody-producing hybridomas, preferably MacPherson (So
ft Agar Techniques, Tissue Culture Methods and App
lications, Kruse and Paterson, eds., Academic Pres
s, 276, 1973) and the discontinuous colonies are transferred to individual wells of a culture plate and cultured in an appropriate culture medium. Antibody-producing cells are identified by screening with an appropriate immunogen. Immunogen-positive hybridoma cells are maintained by methods known in the art. This hybridoma is cultured in vitro, and after assembling the hybridoma by a procedure known in the art, a mouse ascites fluid is prepared to produce a specific anti-monoclonal antibody.

The anti-idiotype antibody is an immunoglobulin having an "internal image" of the antigen of the pathogen to be protected and can be used as an immunogen in a vaccine (Dr.
eesman et al., J. Infect. Disease 151, 761, 1985)
. Methods for raising anti-idiotypic antibodies are known in the art (MacNamara et al., Science 2
26, 1325, 1984).

The vaccine according to the invention can be administered in the usual, active immunization regimens. That is, in a manner compatible with the dosage form, and as a prophylactically effective amount,
Single or repeated administration, the prophylactically effective amount is the amount of immune antigens or recombinant microorganisms that induces immunity in birds against challenge with malignant Eimeria parasites. The amount is sufficient to express the above antigen. Immunity is defined as eliciting a significantly higher level of protection in the post-vaccination chicken population compared to the non-vaccinated population.

In the case of live virus vector vaccines,
The dose rate per chicken is constant at 10 ⁵ -10 ⁸ pfu.

A typical subunit, according to the present invention,
The vaccine comprises 1 μg-1 mg of the protein according to the invention.

Vaccine can be administered, for example, intradermally, subcutaneously,
It can be performed intramuscularly, intraperitoneally, intravenously, orally, or intranasally.

In addition, the vaccine may also contain an aqueous solvent or suspension, and water, often with other ingredients. The addition of this component is to increase activity and / or shelf life. This ingredient is salt, p
H-buffers, stabilizers (such as skim milk or casein hydrolysates), suspensions, adjuvants for improving immune response (eg oils, muramyl dipeptide, aluminum hydroxide, saponin, multiple shades) Ions, amphiphiles) and preservatives.

Vaccines according to the invention may also include immunogens for other pathogens of chickens, or may include nucleic acid sequences encoding such immunogens. For example, Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious cyst disease virus (IBD)
V), chicken anemia (CAA), reovirus, avian retrovirus, chicken adenovirus, turkey rhinotracheitis virus, E. E. coli and other Eimeria species.
This allows the production of multivalent vaccines.

The present invention also relates to "immunochemical reagents". This reagent comprises the protein according to the invention.

The term "immunochemical reagent" means that the protein according to the invention is bound to a suitable support, or
It is supplied together with the labeling substance.

The supports used are, for example, the inner walls of microwells or cuvettes, tubes or capillaries, membranes, filters, test strips or particles, for example latex particles,
Surfaces of red blood cells, dye sols, metal sols or metal compounds as sol particles.

The labeling substance used is a radioisotope, a fluorescent substance, an enzyme, a dye sol, a metal sol, and a metal compound as sol particles.

The nucleic acid sequences according to the invention can also be used to design specific probes for hybridization experiments. That is, it can be used to detect Eimeria-related nucleic acids in any type of tissue.

The present invention also includes a test kit containing the above nucleic acid sequence, which is effective in the diagnosis of Eimeria infection.

The present invention also relates to test kits used in immunoassays. And this kit comprises at least one immunochemical reagent according to the invention.

The immunochemical reaction obtained using this test kit is preferably a sandwich reaction, an agglutination reaction, a competition reaction, or an inhibition reaction, if possible.

To carry out the sandwich reaction, the test kit comprises, for example, a peptide according to the invention bound to a solid phase support and a labeled polypeptide according to the invention,
It may consist of any of the labeled anti-antibodies.
This support is, for example, the inner wall of a micro test well.

[0100]

【Example】

Example 1 Preparation and isolation of parasites E. acervulina Houghton strain was obtained from AFRC Houghton Laboratories and subcultured to coccidia-uninfected chickens.

Preparation of parasites and their fractions. The E. tenella parasite is maintained and the oocysts are maintained by Long et al. (Fol. Vet. Lat.
6 , 201-217, 1976). Sporozoites are from Wisher & Rose (Parasitology 8
8 , 515-519, 1984) and isolated and purified as described in Larsen et al. (J. Parasitol. 70 , 597-601, 1).
984), the nylon / wool refining method was carried out.

Merozoites were inoculated as follows and collected 72 hours later (in addition to Jenkins and Dame, Mol. Bioc.
hem. Parasitol. 25 , 155-164, 1987). Four to six week old chickens were orally infected with 1-5x10 ⁶ sporulated oocysts. 72 hours after the inoculation, the chickens were killed, the duodenum was removed to the Meckel's diverticulum, and stored in ice-cold phosphate buffer saline (0.04M PBS pH 7.3). The duodenum was cut longitudinally and washed with ice-cold PBS. Next, this intestinal tract was cut into 5 cm pieces, containing 100-200 U / ml penicillin and 100-200 μg streptomycin / ml
Suspended in Hanks-BSS at 37 ° C for 15-30 minutes.

The supernatant was removed and filtered through 120, 60, and 35 mesh stainless steel sieve. This solution was centrifuged at 130 g for 8 minutes. Collect the centrifugation supernatant and
The merozoite was concentrated by centrifugation at 500 g at 4 ° C. for 10 minutes.

The concentrated pellet was resuspended in 25 mM Tris-HCl pH 8.0 containing 150 mM NaCl and purified on DE-52 (Whatman) equilibrated with the same buffer. The merozoites eluted in the unbound fraction. Yield is 1 infected chicken
It was about 1x10 ⁹ merozoites per wing.

Example 2 Purification of Eimeria Antigen A. Method Triton X114 extraction Bordier (Bordier, C., J. Biol. Chem. 256 , 1604-160
7, 1981), the material is: -preconcentrated Triton X114 (TX114) (see below) -10 mM Tris / HCl-150 mM NaCl pH 7.4 (TBS) -100 mM phenylmethylsulfonyl fluoride (PMSF) dissolved in isopropanol. 5x10 ⁸ E. in 1 ml of 6% sucrose solution (sucrose cushion) TBS in TBS containing -0.06% TX114.
Acer vulina merozoite was homogenized. The mixture was mixed with 1 mM PMSF and 10% (v / v) preconcentrated TX1.
Added to 14.

Proteins were mechanically sheared at 0 ° C.
Extracted for at least 2 hours. The insoluble substance was pelletized by centrifugation at 12,000 g and 4 ° C. for 10 minutes using an Eppendorf centrifuge. The supernatant containing the dissolved substance is
It was overlaid on an equal volume sucrose cushion and incubated at 40 ° C. for 10 minutes.

After centrifuging at 400 g for 10 minutes (at room temperature), the upper phase containing the hydrophilic substance was taken out, re-extracted, layered on the same sucrose cushion and centrifuged as above.

The lower phase fraction was stored separately from the remaining upper phase fraction.

If the aqueous phase had to be completely removed from the hydrophobic material, the extraction was repeated once again.

All fractions were stored frozen at -70 ° C until used for analysis.

Preconcentration of Triton X114 20 ml Triton X114 (Serva) was made up to 1 liter with cold TBS pH 7.4, mixed and incubated at 0-4 ° C.
After complete dissolution, the solution was transferred to a 40 ° C water bath. Phase separation was complete after 16 hours. The upper phase was removed and instead an equal amount of TBS was added. This operation was repeated twice. The final lower phase, called "preconcentrated TX114",
This was stored in a 100 ml bottle at 4 ° C. Final TX114
The concentration is about 20%.

Monoclonal antibody E. acervul in Balb / c mice was repeatedly inoculated intraperitoneally with 10 ⁶ -10 ⁷ merozoites.
An antibody against ina merozoite was obtained.

Spleen cells from each individual and myeloma P3X63Ag 8.
6.5.3 is integrated, and Scheonherr et al. (Develop. Biol. St.
and. 50 , 235-242, 1982).

Screening was performed by immunofluorescence on dried and acetone-fixed merozoites. The highly concentrated monoclonal antibody solution was prepared in vitro using the dialysis module as a culture vessel and continuously exchanging the culture solution. This method is described by Schonherr and van Ge
lder (Animal Cell Biotechnology 3, 337-355).

Immunoaffinity Chromatography- Activation of Affinity Matrix Sepharose CL-4B (Pharmacia) was activated with 50 mg / ml of cyanogen bromide (CNBr) dissolved in distilled water. Activation is
Performed in a well-ventilated hood. The mixture is stirred with a slowly rotating magnetic stirrer and the pH is
Maintained at 10.5-11.0 for 30 minutes at room temperature with 4M NaOH.
After completion of the reaction, separate Sepharose on a glass filter.
0ml cold water, 500ml cold 0.2M NaHCO3 (binding buffer)
Washed by. This gel was immediately used for immunoglobulin binding.

-Binding of Monoclonal IgG to Sepharose Monoclonal antibody was used as a highly concentrated supernatant,
It was extensively dialyzed against 0.2M NaHCO3 (binding buffer). The buffer was also exchanged using a PD10 column (Pharmacia). The method followed the manufacturer's protocol. CNBr-activated Sepharose CL-4B was added to a final concentration of 2-5 mg / ml MoAb IgG and the mixture was vortexed overnight at 4 ° C. or at room temperature for 2 hours. Unbound fraction was removed and BCA reagent (Pierce)
Was used to measure the protein. 10 Sepha Roses
Wash twice, then 1 volume of 1M ethanolamine / HCl pH
Inverted and mixed with 8.5 at room temperature for 2 hours. 200 ml 0.1M
Tris / HCl -0.5M NaCl pH 8.0 and 0.1M acetic acid -0.5M NaCl pH
The non-covalent substances were removed by washing four times with alternating 4.0 changes. The gel was stored at 4 ° C in PBS containing 0.05% azide.

Affinity purification buffers A) 25mM Tris / HCl + 0.5M NaCl + 0.1% Nonidet P40
(NP40) pH 8.0 B) 0.1M glycine / HCl + 0.1% NP40 pH 2.6 C) 0.1M Tris / HCl + 0.5M NaCl + 0.1% NP40 pH 8.0 D) 3M KSCN dissolved in solution A E) 1M Tris / HCl pH 8.0 F) 10 mM Tris / HCl + 150 mM NaCl pH 8.0 7 ml Sepharose-IgG was transferred to a Pharmacia C10 / 20 column equipped with a cooling jacket. A 10-fold diluted TX114 hydrophobic extract (pellet) dissolved in buffer A was applied to the column at 0.5 ml / min and the temperature was 16-20 at 8 ° C.
Circulated again for hours. After washing the column with 5-10 column volumes of buffer A, the flow direction was reversed and then 7.5 ml buffer B), 5 ml buffer C), 10 ml buffer A), 7.5 ml buffer D). , And 40 ml buffer A).

The acidic fraction (1 ml) was immediately neutralized with 0.1 ml buffer E). The KSCN fraction was dialyzed overnight against buffer F). All fractions were analyzed by SDS-PAGE and immunoblotting, and the protein content was analyzed by B
It was analyzed by the CA method.

SDS-polyacrylamide gel electrophoresis
Analysis (SDS-PAGE) and immunoblotting SDS-PAGE was performed on a 12% acrylamide gel using Laemmli's buffer system (Laemmli, UK, Nature 227, 680-685, 1970). Western blotting was performed by Vermeulen et al. (Vermeulen, AN et.
al., J. Exp.Med. 162 , 1460-1476, 1985). 25-fold dilution as blotting buffer
Laemmli's lower layer electrophoresis buffer was used. Blots in Bio-Rad Trans-Blot cells
It occurred at 90V for 1.5 hours.

Nitrocellulose (0.25 μm, Schleicher
and Scheull) were dissolved in PBS (0.9% saline)
It was blocked with 0.1% NFMP (skim milk powder (OXOID)) dissolved in 0.01M phosphate pH 7.3) for 30 minutes.

The serum and the alkaline phosphatase-conjugated antiserum (Zymed) were incubated for 1.5 hours. Positive binding was detected using BCIP / NBT as substrate.

Polyclonal Antibody Rabbit 8275 (K8275) antibody was found in rabbits (New Zealand White) in incomplete Freund's adjuvant suspension.
E. acervulina 72 hours were obtained by immunization with two intradermal injections at 4 week intervals.

The monospecific antibody was anti-Eimeria in serum.
Obtained in rabbits preselected due to the absence of antibody.

To rabbits 5706 and 5792, 55-100 μg of affinity-purified Eam45, emulsified with incomplete Freund's adjuvant (water in oil), was added.
Injected once (4-5 weeks apart).

For rabbit 5796, the same protocol
Affinity purified Eam20 was injected.

The same protocol for rabbit 5794
The TX114 hydrophobic extract before affinity purification was injected. This fraction contained Eam45, Eam20 and several other proteins.

Monospecific antibodies against Eas100 were found in chickens,
The purified protein dissolved in 100 mM Tris-HCl + 150 mM NaCl + 0.1% NP40 pH8.0 and emulsified in incomplete Freund's adjuvant was obtained by injecting subcutaneously into the neck 3 times at 14-day intervals. Eleven days after the last immunization procedure, chickens were exsanguinated and serum was collected (serum of chicken 323 was used for subsequent analysis).

B. Results Extraction Figure 3 by TX114 shows TX114 extraction, various fractions obtained after phase separation. Samples were electrophoresed, blotted onto nitrocellulose and probed with a mixture of monoclonal antibodies.
This mixture had specificity for Eam200, Eam100, Eam45, and Eam20 proteins, and each had a relative molecular weight of 180-210 kD (average 200 kD) when measured in the non-reduced state.
D), 95-105kD (average 100kD), 45-55kD (average 50kD)
), And 18-22kD (20kD on average).

The Eam200 and Eam100 proteins appear to be hydrophilic. They were not found in the hydrophobic pellet (lane 5). vice versa,
Eam45 and Eam20 were not present in the hydrophilic fraction.

Eam45, Eam20 immunoaffinity cross
The matography monoclonal antibody E.ACER 10C-2A was attached to Sepharose and conjugated to the Eam45 protein. On the other hand, Eam20
E.ACER 10E-2 was used to bind to.

The binding efficiency was above 90% for both MoAbs with minimal leakage of MoAb from the column.

The "Eam20" column was connected to the "Eam45" column. This allows the latter unbound fraction to bind to the former matrix. Both columns were eluted separately.

FIG. 4 shows an SDS-PAGE / immunoblot of 10C-2A (Eam45) matrix fractions. This figure was obtained from an experiment separate from FIG. The blot was probed with rabbit K8275 antibody. Eam45 mainly has a pH of 2.
It appears to elute in 6 (lanes 12 to 14), but some remains and co-elutes with KSCN (lane 1).
6 to 18). However, the latter fraction also contained other low molecular weight material, probably unrelated to the Eam45 antigen.

FIG. 5 is a similar blot diagram, but with Ea
From a 10E-2 column that binds the m20 sample.

Lane 3 did not bind to the 10C-2A column and thus contained a sample that was the starting material for the 10E-2 adsorbent. This fraction is believed to contain no Eam45 sample. The band marked at 29 kD is artifactual and belongs to the Eam20 protein. This artifact was induced by the presence of Triton X114 in the electrophoretic specimen.

Lane 4 contains the unbound fraction of the 10E-2 column, from which this MoAb was Eam20.
Its high efficiency of absorbing all samples is shown.

Some of the sample had a pH of 2.6 (lane 10
To 12), but mostly with 3M KSCN (lanes 14 to 17). This fraction contained no non-specifically bound sample.

Monoclonal antibodies against Eam45 and Eam20 recognized surface proteins on merozoites in vivo.

Although the apparent molecular weight of Eam45 measured on SDS-PAGE was 45-55 kD, it was determined that its identity was unchanged, referring to previous reports. Eam4
The molecular weight of 5 was assumed here to be around 50 kD. On reducing gel, Eam45 shows 55 kD.

All anti-Eam45 sera showed a positive reaction around 50 and 100 kD when examined on merozoite blot.

From sporozoites, E. acervulina 100kD
Protein Purification For purification of the E. acervulina 100kD protein, sporozoites were extracted with TX114 according to the protocol described above. Eas100 was detected exclusively in the hydrophilic phase. It was then bound to CNBr-activated Sepharose-4B.
It was bound to the immunoaffinity column of MoAb E.ACER 5F-2. The binding and elution conditions are as described above.

Eas100 eluted as a double band at acidic pH. The fraction containing Eas100 is shown in Figure 6 (lane 4). The blot was then post-treated with rabbit anti-E.acer sporozoite antibody.

No other parasite-derived bands were found in this fraction. The only contaminating band (MW> 200k
D) is likely due to IgG leakage from the substrate.

This sample was used to obtain chicken antibodies against Eas100.

Using the antibody of chicken 323 for 72 hours E. acer
cDNA obtained from vulina merozoite mRNA (Example 3)
The library was screened.

The antibody selections on the positive clones responded to Eas100 as expected and also to proteins of the same size in E. acervulina merozoites. Immunoblotted and affinity purified Eam100 (MoA
b E.ACER 16B-2B) is the MoA used to purify E.ACER 5F-2, a sporozoite-equivalent Eas100.
It showed a positive reaction with b. Therefore, both proteins have a correlation.

Immunoaffinity of Eam200 derived from merozoites
Chromatography monoclonal antibody E.ACER 11A-2A was attached to Sepharose to bind Eam200 protein.

The binding efficiency was 90% or more, and the leakage of MoAb from the column was minimal, but detectable.

The hydrophobic fraction of TX114 extract containing Eam200, Eam100 was bound to the column according to the protocol used in Eam45, Eam20 above.

As shown in FIG. 7, the purified Eam200 was released from the column by acidic elution (lane 4).

Example 3 Preparation of cDNA library of E. acervulina merozoites, immunological screening, DNA sequence analysis. Method RNA Isolation For isolation of RNA, a pellet of 10 ⁹ merozoites was
Resuspended in 0.5 ml H ₂ O. 0.5 ml of solution A (Tris 1
0 mM, sodium acetate 75 mM, EDTA 2 mM, SDS 1% (pH 7.
2)), 1 ml of solution B (5M guanidine monoisothiocyanate, EDTA 10 mM, Tris 50 mM (pH 7.5)), and
After adding 2 g glass beads (0.5 mm), the suspension was stirred for 1 minute. 4 ml of solution B and 0.4 ml of β-mercaptoethanol were added, and the test tube was placed in a water bath (60 ° C) for 15
Set a minute. After adding 5 ml of phenol, the tube was heated at 60 ° C. for another 15 minutes and cooled to room temperature (RT). 2.5 ml of this suspension (0.1M sodium acetate
pH 5.2, 10mM Tris (pH 7.5), 1mM EDTA), and 5ml
Chloroform-isoamyl alcohol (24: 1)
Stir and mix with and then centrifuge (5 rpm at 6000 rpm
Minutes) to separate the phases. The aqueous phase was again mixed with 20 ml phenol / chloroform / isoamyl alcohol (25: 24: 1).
), It was mixed on a roller drum for 10 minutes and extracted. After centrifugation at 6000 rpm for 5 minutes, 2 volumes of ethanol was added to precipitate the nucleic acid, and the mixture was centrifuged (10
Min). The pellet was washed with 70% ethanol and A ⁺ RNA was isolated by the method described by Maniatis et al. (Maniatis, T. et al., Molecular Cloning, A labora.
tory Manual, second edition, Cold Spring Harbor La
boratory Press, USA, 1989). About 10 μg of poly A ⁺ RNA was isolated from 10 ⁹ merozoites.

Construction of cDNA Library Poly A ⁺ RNA was converted to cDNA by MMLV reverse transcriptase. For this, 0.5 μg poly A ⁺ mRNA was added to 10 μg.
Dissolved in 1 H ₂ O, heated at 65 ° C. for 10 minutes, then cooled rapidly on ice. cDNA synthesis is performed by Pharmacia (Ph
armacia) cDNA synthesis kit. To obtain a blunt-ended DNA molecule, cDNA was added to 1 μl of Klenow DNA polymerase (7 U / μl) 3
It was treated at 7 ° C. for 20 minutes and then incubated with 1 μl of T4 DNA polymerase (7.5 U / μl) at 37 ° C. for 10 minutes. Extract with an equal volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and centrifuge (13000).
Biofuge for 5 minutes at rpm, then add 1 volume of ammonium acetate and 4 volumes of ethanol to give cDN
A was precipitated. The pellet was washed with 70% ethanol and dissolved in 82 μl H ₂ O. 10 μl of binding buffer (Tris 500 mM (pH 8.0), MgCl ₂ 100 mM, DTT 100 mM,
ATP 100mM, 50% (w / v) Polypropylene glycol 8000
) 5 μl of EcoRI receptor solution (Pharmacia cDNA)
A synthesis kit) and 3 μl of T4 DNA ligase (7U /
μl) and incubated at 12 ° C. overnight,
The EcoRI receptor was attached to the cDNA. The reaction was stopped by heating (65 ° C. for 10 minutes). This cDNA
Was phosphorylated by adding 10 μl of ATP (10 mM) and 2 μl of polynucleotide kinase (7 U / μl) and incubating at 37 ° C. for 1 hour. This cDNA was added to 1 volume of phenol / chloroform /
Extracted with isoamyl alcohol (25: 24: 1), Biogel A
Purified on -15 m column. The cDNA containing the fraction was
1 volume sodium acetate (3M sodium acetate (pH 5.6)),
Two volumes of ethanol were precipitated by addition. The pellet was washed with 70% ethanol and 20 μl T10E0.1 (Tri
It was dissolved in s 10 mM (pH 7.6), EDTA 0.1 mM). This cDN
A molecule is designated as λgt10, or λgt11 DNA
oning techniques: by a method described in A practical approach, 1984).

Use antisera against Eimeria protein
Screening of the Lambda gt11 cDNA library was performed. The lambda gt11 cDNA library was screened with the antibodies obtained against the Eimeria parasite-derived protein. Either mouse monoclonal antibodies or rabbit or chicken monospecific antisera were used. Prior to use, the antibody was applied to 1x Tris salt (Tris-HCl 10 mM, NaCl 150 mM, pH
8.0) + 0.05% Tween 20 + 10% fetal calf serum (FCS) and diluted on the filter for 2 hours at room temperature. Next, the filter is applied 4 times for 10 minutes each time.
50 ml 1x Tris salt + 0.05% Tween for each filter
Washed with 20. For the second antibody incubation, a complex of goat anti-mouse or goat anti-rabbit or rabbit anti-chicken antibody and alicariphosphatase was used (1x
Tris salt + 0.05% Tween 20 + 10% FCS diluted 1: 7500), incubated for 30 minutes at room temperature, then the filters were washed as above. Color reaction in advance
0.33 mg / ml nitroblue tetrazolium and 0.17 mg / ml
5-Bromo-4-chloro-3-indolylphosphoric acid dissolved in Tris-HCl 100 mM, NaCl 100 mM, MgCl2 10 mM (pH
9.6). After incubation for 30 minutes at room temperature, the reaction was stopped.

Immunoreactive positive clones were purified by plating the isolated plaques a few more times and performing the immunological screening described above.

Characteristics of Lambda gt11 cDNA Clone Preparation of the phage strain and DNA extraction were performed by standard methods (Maniatis, T. et al., Molecular Cloning, A labor.
atory Manual, second edition, Cold Spring Harbor La
boratory Press, USA, 1989). After digestion with restriction endonucleases, the DNA was 89 mM Tris, 89 mM boric acid,
It was analyzed by electrophoresis on an agar gel dissolved in 2 mM EDTA, pH 8.3.

Antibody selection experiments were performed by Osaki, LS et al. (J.
Immunological Methods 89, 213-219, 1986) and isolated lambda gt11cDN.
This was the final test to identify the protein encoded by the A clone. Dilute the phage strain to 5x10 ⁴ pfu / μl and
μl was incubated with 200 μl of E. coli Y1090− cells and plated. A nitrocellulose filter saturated with IPTG for 2.5 hours was placed on the plate and incubated for 5.5 hours, then the filter was turned over and the incubation was continued for another 2 hours. Plates with filters were stored overnight at 4 ° C. after which the filters were washed with 1 × Tris salt for 20 minutes and blocked at room temperature for 2 hours with 20% FCS in 1 × Tris salt. After washing with Tris salt for 5 minutes at room temperature,
The filter was air dried. Antibody preparations were purified by caprylic acid precipitation and 1x Tris salt + 20% FCS + 0.05% N
Diluted 1: 150 with P4O. Each filter was incubated with 15 ml of serum for 60 minutes at room temperature. The filters were washed 3 times for 10 minutes with 1x Tris salt + 0.05% NP40. Bound IgG was treated with 5 ml of 0.2 M glycine HCl.
Elute with (pH 2.8) for 1 minute at room temperature, 150 μl 2M
Rapidly neutralized with Tris, 0.2 ml PBS Tween (25x), 1 ml FCS (all dishes used for elution procedure were first blocked by adding 1x Tris salt + 10% FCS for 1 hour at room temperature).
This eluate was used on Eimeria merozoid or sporozoite western blots to identify the corresponding proteins.

Lambda g by hybridization
Screening of t10 cDNA The 200 bp insert obtained from the lambda gt11 / Eam20 clone was digested with digoxigenin-by the random prime method.
Labeled with dUTP. This is Boehringer, Mannheim
Published by (Catalog number 1093657), "DNA labeling"
and the detection kit, non-radioactive ”was followed exactly. The lambda g above.
Fixed D from t10 E. acervulina cDNA library
Filters containing NA were prepared as described by Maniatis et al. (Supra) and freshly denatured (10 min at 95 ° C).
Labeled E. acervulina cDNA fragment at 42 ° C 16
Time probed. The method followed the manufacturer's instructions.

The filter was washed as follows.

2xSSC, 0.1% (w / v) SDS (1 x SSC is 0.01
5 mol / l sodium citrate, pH 7.0+ 0.15 mol / l NaC
l) at room temperature twice for 15 minutes, 1 x SSC, 0.1% (w / v) SDS,
15 minutes twice at 60 ° C, 0.1 x SSC, 0.1% (w / v) SDS,
30 minutes twice at 60 ℃, once every 15 minutes, PBS-tween (7.65g / l
_{NaCl, 0.91g / l Na 2 HPO} 4. 2H 2 O, 0.21g / l KH 2 PO
₄ , 0.05% (v / v) Tween 80, pH 7.3) at room temperature for 15 minutes twice.

Next, the filter was treated with PBS-tween at 1: 5000.
Diluted anti-digoxigenin Fa
It was reacted with the b fragment and allowed to complex with alkaline phosphatase for 30 minutes at room temperature. Filter at room temperature,
15 minutes 4 times with PBS-tween, 0.01M Tris-HCl pH 8.0,
After washing with 0.15M NaCl once for 15 minutes, binding of alkaline phosphatase to the filter was performed with 0.33 g / l nitroblue tetrazolium and 0.17 g / l 5-bromo-4-chloro-3-indolyl phosphate. M Tris-HCl pH9.6,
Detection was performed by incubating with a solution dissolved in 0.1M NaCl and 0.01M MgCl ₂ . Positive plaques were purified by plating the isolated plaques an additional 2-3 times and hybridizing as above.

Extended DNA sequences by "semi-specific" PCR
The first clone isolated from the lambda gt11 library by immunological screening or the clone isolated from lambda gt10 by hybridization analysis did not contain the full length reading frame of the respective protein, Further DNA sequences were generated by the polymerase chain reaction method.

To this end, the primary cDNA library of lambda gt11 was amplified. 5x10 ⁴ pfu, 60
Incubated with 0 μl E. coli Y1090- cells and plated. After overnight incubation, collect the upper layer of agar in a test tube, add 5 ml of phage dilution buffer (Tris (pH 7.6) 10 mM, MgCl2 10 mM, NaCl 100 mM, gelatin 1 mg / ml) and incubate at 4 ° C for 16 hours. did. The suspension was clarified by centrifugation, and the supernatant was used for immediate PCR reaction. Blakely and Carman
The method of (Bio Techniques 10, 53-55 (1991)) was used with modifications. 2.5 μl of supernatant containing about 10 ¹⁰ pfu / μl was added to 10 μl of dNTP stock solution (each dNTP 20 mM).
μl buffer (Tris 150 mM (pH 7.6), KCl 600 mM,
MgCl ₂ 25 mM included), 1 μg of each primer, 3 μl of DMS
O was added. 2.5 U of Taq polymerase (Cetus / Perkin-Elmer) was added to 100 μl of the final mixture.

One of each primer set is extended E
It is specific for any imeria sequence, eg, Eam20, Eam45, Eam100. The second primer in each set is
The “generic” primer is homologous to the 3 ′ end of the β-galactosidase gene of lambda gt11 (lambda gt
11 primers (reverse transcription), 24 MER # 1222 (New Englan
d Biolabs).

The PCR fragment was purified by gel electrophoresis and cloned in the TA Cloning Kit (Invitrogen) vector exactly according to the manufacturer's instructions. The sequence of the obtained clone was determined. Individual DNA
To correct the PCR-derived error present in the clones, its sequence was determined in at least 3 independent clones for each extended DNA fragment.

DNA Sequence Analysis Inserts from the above λgt10, λgt11 clones were subcloned into the pGEM-4Z vector (Promega) for sequencing. The sequencing reaction was performed by the dideoxy method (Bankier &
Barrell, Techniques in the Life Sciences (Biochemi
stry) 85: Techniques in Nucleic Acids Biochem. 1-3
4; 1983). The sequencing primer is Applie
Synthesized on a Biosystems 380B instrument using β-cyanoethylphosphoramadite chemistry.

B. Results A clone encoding the Eam200 reading frame (part of it) was isolated by the mouse monoclonal antibody used to screen the lambda gt11 cDNA library. One of the 2.10 ⁵ independent clones was positive. Various mouse for Eam200
Among the monoclonal antibodies, for example, E.ACER 12B-2A, EA
The reaction between CER 12C-2B, E.ACER 12B-2B and the clone selected for future analysis was considered sufficient and conclusive evidence to confirm the identity of the reading frame contained in this clone. .. Fusion protein encoded by a lysogenic strain of lambda gt11 / Eam200 and antibody E.ACER 12B-2B
Is shown in FIG. Partial sequences of Eam200 are shown in SEQ ID NOs: 1 and 2. As can be seen from this table, the total length of the insert is 1491 bp, of which 1341 bp encodes the protein.

Isolation of a clone encoding this protein was performed using the monospecific anti-Eam45 serum obtained from rabbit 5706 (see Example 2). Screened
5.10 from ⁴ plaques, the two clones were isolated.
Inserts of this clone, Eam45 M1 and Eam45 M3
These were 817 bp and 786 bp, respectively.
Both inserts were expressed in E. coli. Eam45 M1 is about 13k
It is encoded as a D protein and Eam45 M3 is encoded as a 24 kD protein. Both expression products reacted with monospecific rabbit anti-Eam45 sera (data not listed here) on Western blots. In the antibody selection experiment, the antibody eluted from clone M3 reacted with Eam45 protein derived from merozoite (Fig. 9). When the same kind of experiment was carried out with clone M1, no reaction was observed.

Eam45 M1, M3 extended clones were prepared by PCR as described in the method above. For M1,
The extended PCR fragment is 127 bp, 84 in M3.
Addition of 5 bp was observed. Therefore, the total sequence obtained for M1 was 944 bp and for M3 1631 bp. This sequence, called Eam45 M1E and Eam45 M3E, respectively, is shown in SEQ ID NO: 3 (MIE), 5 (M3E). The first ATG in M1E was at positions 82 to 84 and in M3E was at 505 to 507. Both ATGs are
It is preceded by a frame upstream stop codon, and therefore
It appeared to represent the true start codon. Eam45 M1E
The primary amino acid sequences encoded by M3E and M3E are shown in SEQ ID NOs: 4 (M1E) and 6 (M3E). A monospecific, anti-Eam20 from rabbit 5796 (see Example 2) was used to isolate a clone encoding this protein. All clones isolated from the lambda gt11 expression library had inserts smaller than 200 bp. Therefore, the insert of one of this clones was used as a probe to screen the lambda gt10 library. 2.10 ⁵ out of ⁵ plaques were positive.
The longest insert obtained was 579 bp with a coding capacity of 11 kD. From this, an extended clone was generated using PCR with the added 221 bp. Eam20
The total sequence obtained for is therefore 800 bp. This clone was named Eam20E and its sequence is shown in SEQ ID NO: 7. The reading frame of Eam20E is completely open from the first nucleotide, but at the first ATG (positions 80 to 8 of SEQ ID NO: 7).
It is certain that 2) represents the start codon. The protein coding sequence of Eam20E (SEQ ID NO: 8) is therefore preferably read from Met at position 27.

Monospecific serum (323) was used to isolate clones encoding the Eam100 protein. This was obtained from chickens immunized with immunoaffinity purified Eas100. Eas100 was purified by affinity chromatography using immobilized monoclonal antibody E.ACER5F-2, which was used to obtain antibodies from chickens by the method described in Example 2. Using this antibody, E.
Lambda gt11 from acervulina merozoite mRNA
The cDNA library was screened. One of 2.105 clones screened was positive. From various sera, it was revealed that the antibody selected by this clone reacted with 100 kD protein existing in both merozoite and sporozoite on Western blot (FIG. 8). From this, it was revealed that the reading frame of this clone actually encodes this 100 kD protein (a part thereof). The insert of this clone was 1259 bp long and had a coding capacity of 34 kD (data not shown). From this, an extended clone was generated by PCR with the added 1116 bp. Therefore, Eam100
The total sequence obtained for is 2375 bp. Eam this
It is named 100E and is shown in SEQ ID NO: 9. The amino acid sequence deduced from this is shown in SEQ ID NO: 10.

Example 4 Immunization of Chickens with Affinity Purified Antigen A. Method 6x10 ¹⁰ E. avervulina 72 hours, hydrophobic and hydrophilic merozoites as starting materials, the antigens were separated by TX114 extraction (Example 2). Individual antigens were purified by immunoaffinity chromatography.

[0171]

[Table 1]

Protein concentration was determined by both cinchoninic acid methods (Pi
erce Chemicals) according to the manufacturer's prescription.

Immunization Schedule Purified antigen was mixed with Quil A (Superfos Biosector A / S) such that each dose contained 100 micrograms of Quil A in a 0.5 ml total volume. A group of 20 white leghorns was housed in independent cages from hatching to prime. Chickens were immunized with 0.5 ml Quil A / antigen subcutaneously injected into the neck three times at weekly intervals. This antigen dose is shown in the table above.

Challenge 10 days after the third inoculation, chickens were individually dosed with 200-30
0 fresh sporulating E. acervulina oocysts were administered. The released oocysts were counted from stool specimens collected 4 to 7 days after infection.

Immunological parameters Antibody titers Serum was immunized prior to immunization, prior to challenge, per immunization.
Collected 7 days after challenge. E. acerv per serum
The antibody titer against the ulina merozoite antigen was examined. An ELISA test was used for this. Add to this 0.1 ml of 50 mM
1x10 5 suspended in carbonate / bicarbonate buffer pH 9.6
Of merozoites from each well of the microassay plate,
Coated by heating at 50 ° C. overnight. Wash the plate,
Blocked with bovine serum albumin and mixed with various serum dilutions
Incubate at 7 ° C for 1 hour, wash several times, then
Peroxidase-labeled rabbit anti-chicken IgG (H + L) and 37 ℃
Incubated for 1 hour. After washing appropriately, positive binding was detected using urea-TMB substrate and absorption was
It was measured at 450 nm. The antibody titer was represented by ² log (terminal dilution).

Lymphocyte Stimulation Prior to challenge, peripheral blood cells were collected from 10 chickens in each group. Peripheral blood leukocytes from whole blood at room temperature for 6
Isolated by centrifugation at 4g for 6 minutes. The raw skin coat was removed, the remaining cells and plasma were remixed and agitated again.
The white blood cells collected through the two steps were counted by a hemocytometer, and the concentration was measured by RPMI 1640 (Dutch standard).
The cells were adjusted to 1 × 10 ⁷ cells / ml.

E. acervulina merozoites (4x10 ⁸ )
Suspend in 6.7 ml RPMI 1640 with Bracket Micro Tip
Sonicated with an nson sonicator. At position 6 for 3x20 seconds with intercooling. This was diluted to make it suitable for the concentration used for stimulation. 96 wells,
Round-bottom tissue culture plate, 0.05 ml cell suspension, 0.150 m
l Inoculate with the antigen suspension, and
It was cultured at 64 ° C. for 64 hours. Then add 0.5 microcurie of ³ H-thymidine to each well, and after 8 hours,
The cells were filtered through a glass fiber filter (Pharmacia / LK
B) collected. At this time, 96-well cell collector (Skat
ron Norway) was used. Saturate the filter with scintillation fluid (LKB BetaScint) and use Betaplate 1205
(Pharmacia / LKB Sweden).

B. Results Immunological parameters Antibody titers Table 2 shows that E. acervulina merozoite antigen
The average antibody titer before challenge of each group examined by LISA is shown. All antigens elicited high antibody titers, with a minimum difference of 30 compared to controls.

[0179]

[Table 2]

Lymphocyte Stimulation All groups of PBLs were treated with E. acerv at three different concentrations.
The ulina merozoite antigen, ie, ⁵ × 10 ⁵ , 1 × 10 ⁶ , and 3 × 10 ⁶ sonicated merozoites per well, respectively, were stimulated. The optimal concentration was determined for each group.

Table 3 shows the mean Dcpm (antigen stimulated well cpm, minus, unstimulated control cpm). All antigens appear to induce detectable T-cells in peripheral blood upon challenge. Generally, 6 or 7 out of 10 have reacted,
It was 0 in the control.

[0182]

[Table 3]

Zygospore production Table 4 shows the average number of shed oocysts as a percentage of control for each group. Controls received Quil A Adjuvant only. Eam200, Eam100, Eam45, Eam20 reduced oocyst release.

[0184]

[Table 4]

Sequence list (1) General information (i) Applicant (A) Name-AKZO, NV (B) Town name-Velperweg 76 (C) City-Arnhem (E) Country-Netherlands (F) Mail No. -6842 BM (ii) Title of invention --- Chicken vaccine for coccidiosis (iii) Number of sequences -10 (vii) Previous application data (A) Application number --- EP 91.201.523.7 (B) Registration date- -June 18, 1991 (C) Classification

[0186]

[Table 5]

[0187]

[Table 6]

[0188]

[Table 7]

[0189]

[Table 8]

[0190]

[Table 9]

[0191]

[Table 10]

[0192]

[Table 11]

[0193]

[Table 12]

[0194]

[Table 13]

[0195]

[Table 14]

[0196]

[Table 15]

[0197]

[Table 16]

[0198]

[Table 17]

[0199]

[Table 18]

[0200]

[Table 19]

[0201]

[Table 20]

[0202]

[Table 21]

[0203]

[Table 22]

[0204]

[Table 23]

[Brief description of drawings]

Figure 1: Mabs for various Eimeria species and stages
s Electrophoretic photograph showing recognition of E.ACER 11A-2A (panel A) and E.ACER 12B-2B (panel B). Lane 1--E. Acervulina merozoites, non-reducing SDS
-PAGE (NR) Lane 2--E. Acervulina merozoite, reduction Lane 3--E. Acervulina sporozoite, NR Lane 4--E. Tenella, second generation merozoite, NR lane 5--E. Tenella sporozoite, NR Arrow Indicates the position of the molecular weight marker. Beta galactosidase (MW = 116kD), down arrow. Myosin (MW = 200
kD), up arrow (not shown in lane 3).

Figure 2: Mabs for various Eimeria species and stages
s Electrophoretic photograph showing recognition of E.ACER 10C-2A (panel A) and E.ACER 10E-2 (panel B). Lane 1--E. Acervulina merozoites, non-reducing SDS
-PAGE (NR) Lane 2--E. Acervulina merozoite, reduction Lane 3--E. Acervulina sporozoite, NR Lane 4--E. Tenella, second generation merozoite, NR lane 5--E. Tenella sporozoite, NR Arrow Indicates the position of the band of recognition positive.

FIG. 3: Western blot (non-reducing PAGE) of various fractions of TX114 extract of E. acervulina merozoite.
Is a photograph of electrophoresis. The blot is Eam200 ("20
0), Eam100 (“100”), Eam45 (“5
0 "), Eam20 (" 20 ") was recognized by the Mab conjugate. Lane 1-insoluble sample (concentrated) Lane 2--total dissolved sample Lane 3--hydrophilic fraction (aqueous phase) ) Lane 4--sucrose fraction (contact phase) Lane 5--hydrophobic fraction (detergent phase)

FIG. 4 is an electrophoretic photograph showing a Western blot (non-reducing PAGE) of various fractions obtained by immunoaffinity purification using E.ACER 10C-2A. The blot was probed with K8275 polyclonal rabbit serum. Lane 2--Molecular weight marker Lane 3--TX114 Hydrophobic fraction Lane 4-10- Fraction obtained from washing step after binding Lane 11-14-Acid elution fraction (pH 2.6) Lane 15-18 --3M KSCN elution lane 19--unbound fraction

FIG. 5 is an electrophoretic photograph showing a Western blot (non-reducing PAGE) of various fractions obtained by immunoaffinity purification using E.ACER 10E-2. The blot was probed with K8275 polyclonal rabbit serum. Lane 2--Molecular weight marker Lane 3--TX114 hydrophobic fraction, provided by E.ACER 10C-2
After passing the A column. Lane 4--Unbound Fraction Lane 5-9-- Fraction obtained from the washing step after binding Lane 10-12-Acid Elution Fraction (pH 2.6) Lane 14-18-3-M KSCN Elution

FIG. 6 is a photograph of electrophoresis showing a Western blot (non-reducing PAGE) of various fractions obtained by immunoaffinity purification when E.ACER 5F-2 was used. Blots were probed with polyclonal rabbit serum directed against E. acervulina sporozoites (K802). Lane 1-TX114 hydrophilic fraction of sporozoites Lane 2--non-bound fraction Lane 3-5-acidic elution fraction (pH 2.6) Lane 6-7--3M KSCN elution arrow Double arrow of Eas100. Bands are shown.

FIG. 7 is an electrophoretic photograph showing a Western blot (non-reducing PAGE) of various fractions obtained by immunoaffinity purification using E.ACER 11A-2A. The blot was probed with a set of monoclonal antibodies reactive with Eam200, Eam100, Eam45, Eam20. Lane 1-Molecular weight marker Lane 2--TX114 hydrophilic fraction Lane 3--unbound fraction Lane 4--acidic elution fraction (pH 2.6) Immediately above the Eam200 band, in Lanes 3 and 4, the light IgG.
I can see the band. This is due to the Mab leaking from the column.

FIG. 8: E. acervulina protein (non-reducing PAGE)
Electrophoresis photograph showing the reaction of the Eam100-selected antibody clone in the Western blot of. All other strips contain merozoite proteins, except strip 5 which contains E. acervulina sporozoite proteins. The strip reacted with: 1. E. acervulina Antiserum for total protein from merozoites (obtained from rabbit K8275). 2. Immunoaffinity purified Monospecific antiserum to Eam100 (chicken 323). 3. Antibodies selected from the Lambda gt11 / Eam100 clone obtained from chicken 323 antiserum. 4. Antibodies selected from the Lambda gt11 / Eam100 clone obtained from rabbit K8275 antiserum. 5. Antibody selected from the Lambda gt11 / Eam100 clone, obtained from rabbit K802 antiserum. Same as 6.5. 7. Antibodies selected by wild-type lambda gt11, obtained from chicken 323 antiserum. 8. Antibodies selected from wild-type lambda gt11 obtained from rabbit K802 antiserum. 9. Antibodies to all sporozoite proteins (Rabbit K80
Obtained from 2). 10. Monoclonal antibody E.ACER 5F-2

FIG. 9: E. acervulina protein (non-reducing PAGE)
Electrophoresis photograph showing the reaction of Eam45 (M3) -selected antibody clone in Western blot of. All strips contain merozoite proteins. The strip reacted with: 1. Lambda gt11 / Eam45 (M3) obtained from rabbit K5706 antiserum
Antibody selected by clone. 2. Lambda gt11 / Eam45 (M3) obtained from rabbit K5794 antiserum
Antibody selected by clone. 3. Lambda gt11 / Eam45 (M3) obtained from rabbit K5796 antiserum
Antibody selected by clone. 4. Lambda gt11 / Eam45 (M3) obtained from rabbit K8275 antiserum
Antibody selected by clone. 5. Immunoaffinity purified Monospecific antiserum against Eam45 (obtained from rabbit K5706). 6. Antiserum against the TX114 hydrophobic extract from merozoites (rabbit K5794). 7. Immunoaffinity purified Monospecific antiserum against Eam45 (obtained from rabbit K5792). 8. Antibodies selected from wild type lambda gt11 obtained from rabbit K5706 antiserum. 9. Antibodies obtained from rabbit K5794 antiserum, selected by wild-type lambda gt11. 10. Antibodies selected from wild-type lambda gt11 obtained from rabbit K5796 antiserum. 11. Antibodies selected from wild type lambda gt11 obtained from rabbit K8275 antiserum. 12. Monoclonal antibody E.ACER 10C-2A

FIG. 10 is an electrophoretic photograph showing Western blot analysis of lambda gt / Eam200 expression products. Expression products from lysogenic strains of Lambda gt / Eam200 were (reduced) SDS-PAGE
Run on gel, blot, monoclonal antibody EA
Probed with CER 12B-2B (lane 2). As a control, the lambda gt11 expression product was run in lane 1.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 15/86 C12P 21/02 A 8214-4B // (C12P 21/02 C12R 1:90) C07K 99:00 (72) Inventor Jacobs Johannus Kotsk The Netherlands, 6546 De Her Nieimegen, Meuseatskele 11-28 (72) The inventor Arnoldus Nikolaas Welmeulen The Netherlands, 5431 Her Har・ Quaker, Cole Hoendel Weld ・ 34

Claims (23)

[Claims] 1. A protein having one or more immunogenic determinants of an Eimeria antigen. 2. The protein of claim 1, which is Eimer
The ia antigen is a. It has a molecular weight of about 200 kD on SDS-PAGE and
Monoclonal antibody deposited as 061222
An antigen that binds to E.ACER 11A-2A, b. An antigen that has a molecular weight of about 100 kD in SDS-PAGE and is deposited as 91061219 at the European animal cell culture collection facility, which binds to the monoclonal antibody E.ACER 5F-2,
c. Monoclonal antibody E.ACER 10C-, which has a molecular weight of about 50 kD on SDS-PAGE and has been deposited as 91061220 at the European animal cell culture collection facility.
An antigen that binds to 2A, d. It has a molecular weight of about 20 kD on SDS-PAGE and is selected from the group consisting of antigens that bind to monoclonal antibody E.ACER10E-2, which has been deposited as 91061221 at a European animal cell culture collection facility. Said protein. 3. The protein according to claim 1, wherein at least a part of the amino acid sequence shown in SEQ ID NO: 2,
Alternatively, the above protein characterized in that it consists of a functional variant thereof. 4. The protein according to claim 1, wherein at least a part of the amino acid sequence shown in SEQ ID NO: 4,
Alternatively, the above protein characterized in that it consists of a functional variant thereof. 5. The protein according to claim 1, which is at least a part of the amino acid sequence shown in SEQ ID NO: 6,
Alternatively, the above protein characterized in that it consists of a functional variant thereof. 6. The protein according to claim 1, wherein at least a part of the amino acid sequence shown in SEQ ID NO: 8,
Alternatively, the protein comprises a functional variant thereof. 7. The protein according to claim 1, wherein at least a part of the amino acid sequence shown in SEQ ID NO: 10,
Alternatively, the above protein characterized in that it consists of a functional variant thereof. 8. A nucleic acid sequence encoding a protein according to claims 1-7. 9. A nucleic acid sequence according to claim 8, characterized in that said nucleic acid sequence comprises at least part of the DNA sequence shown in SEQ ID NO: 1. 10. A nucleic acid sequence according to claim 8, characterized in that the nucleic acid sequence comprises at least part of the DNA sequence shown in SEQ ID NO: 3. 11. A nucleic acid sequence according to claim 8, characterized in that the nucleic acid sequence comprises at least part of the DNA sequence shown in SEQ ID NO: 5. 12. A nucleic acid sequence according to claim 8, characterized in that the nucleic acid sequence comprises at least part of the DNA sequence shown in SEQ ID NO: 7. 13. A nucleic acid sequence according to claim 8, characterized in that the nucleic acid sequence comprises at least part of the DNA sequence shown in SEQ ID NO: 9. 14. A recombinant vector molecule comprising a nucleic acid sequence according to claims 8-13. 15. A recombinant vector molecule according to claim 14, characterized in that its nucleic acid sequence is operably linked to an expression control sequence. 16. A recombinant vector virus harboring a heterologous nucleic acid sequence according to claims 8-13. 17. A nucleic acid sequence according to claims 8 to 13,
Alternatively, a host cell transformed with a recombinant vector molecule according to claims 14 to 15 or infected with a recombinant vector virus according to claim 16. 18. A method for expressing a protein according to claims 1 to 7, which comprises culturing the host cell according to claim 17. 19. An antibody or antiserum immunoreactive with the protein according to claims 1-7. 20. A vaccine for preventing birds against coccidiosis, the recombinant vector virus according to claim 16, the host cell according to claim 17, the protein according to claims 1 to 7, or claim 18.
Vaccine comprising a protein prepared by the method according to 1. and a pharmaceutically acceptable carrier therefor. 21. Preparation of a coccidiosis vaccine comprising the steps of culturing an infected host cell according to claim 17, recovering a recombinant vector virus, and prescribing the viruses into a drug having immunological activity. Law. 22. A protein according to claims 1 to 7,
Alternatively, a method for preparing a coccidiosis vaccine, which comprises formulating the protein prepared by the method of claim 18 into a drug having immunological activity. 23. A method for preventing birds against coccidiosis, which comprises administering the vaccine according to claim 20 to the birds.