WO1992004461A1 - Genetically engineered coccidiosis vaccine - Google Patents

Genetically engineered coccidiosis vaccine Download PDF

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Publication number
WO1992004461A1
WO1992004461A1 PCT/US1991/006431 US9106431W WO9204461A1 WO 1992004461 A1 WO1992004461 A1 WO 1992004461A1 US 9106431 W US9106431 W US 9106431W WO 9204461 A1 WO9204461 A1 WO 9204461A1
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glu
gly
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PCT/US1991/006431
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French (fr)
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James W. Jacobson
Robert L. Strausberg
Susan D. Wilson
Sharon H. Pope
Susan Lee Strausberg
Wolfgang Raether
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Genex Corporation
Hoechst Aktiengesellschaft
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Priority to JP3516046A priority Critical patent/JPH06504187A/en
Publication of WO1992004461A1 publication Critical patent/WO1992004461A1/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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/455—Eimeria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • 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

  • This invention is in the field of avian coccidiosis and is directed to recombinant antigenic proteins of avian coccidia and to the genes that encode the proteins. These antigenic proteins may be used in a vaccine against avian coccidia.
  • Coccidiosis is a disease of both invertebrates and vertebrates, including man, caused by intracellular parasitic protozoa which generally invade the epithelial cells lining the alimentary tract and the cells of associated glands.
  • the crowded conditions under which many domestic animals are raised have contributed to increased incidence of the disease.
  • Virtually every domestic animal is susceptible to infection, and distribution of the parasite is world-wide.
  • Coccidiosis is therefore the cause of signifi ⁇ cant economic loss throughout the world.
  • Eimeria contains the most economically important species. Various species of Eimeria infect a wide range of hosts, including mammals, but nine species have been recognized as being pathogenic to varying degrees in chickens: Eimeria acervulina. E. mivati. E. mitis. E. praecox. E. hagani, E. necatrix-, E. maxima, E. brunetti and E. tenella.
  • Eimeria are highly host specific, their life cycles are similar.
  • the developmental stages of the avian coccidia can be illustrated by the species Eimeria tenella. which proliferates in the cecum of the chicken.
  • the life cycle of the Eimeria species begins when the host ingests previously sporulated oocysts during ground feeding or by inhalation of dust Mechanical and chemical action in the gizzard and intestinal tract of the chicken ruptures the sporulated oocyst, liberating eight sporozoites.
  • the sporozoites are carried in the digestive contents and infect various portions of the intestinal tract by penetration of epithelial cells.
  • E. tenella has received the most attention.
  • E. tenella is an extremely pathogenic species, with death often occurring on the fifth or sixth day of infection.
  • chemotherapeutic agents poultry producers' attempts to control coccidiosis were limited to various management programs. These programs were directed toward attempts at sanitation through disinfection, or by mechanical removal of litter. Despite these efforts, sufficient oocysts usually remained to transmit the disease.
  • Another means of combating coccidia is drug treatment after the poultry is infected.
  • One drug that has been used is sulfanilamide which has shown anticoccidial activity against six species of coccidia.
  • medication may be started too late to be effective.
  • the best method for combating coccidia is preventive medication. Since the advent of the use of sulfonamide drugs, over forty compounds have been marketed for preventive medication against coccidia. There have been many problems with the use of such drugs, including anticoccidial contamination of layer flock feeds, inclusion of excessive anticoccidial drugs in the feed causing toxicity in the birds and omission of the anticoccidial from the feed resulting in coccidiosis outbreaks. A particularly frustrating problem has been the development of drug-resistant strains of coccidia.
  • the process involves the insertion of DNA (derived either from enzymatic digestion of cellular DNA or by reverse transcription of mRNA) into an expression vector.
  • DNA derived either from enzymatic digestion of cellular DNA or by reverse transcription of mRNA
  • expression vectors are derived from either plasmids or bacteriophage and contain: (1) an origin of replication functional in a microbial host cell; (2) genes encoding selectable markers, and (3) regulatory sequences including a promoter, operator, and a ribosome binding site which are functional in a microbial host cell and which direct the transcription and translation of foreign
  • eukaryotic proteins are often produced in prokaryotic cells as a fusion with sequences from the amino-terminus of a prokaryotic protein.
  • ⁇ -Galactosidase or the product of one of the E. coli tryptophan operon genes have been used successfully in this manner.
  • Expression vectors have also been developed for expression of foreign proteins in eukaryotic host cells, e.g., yeast and Chinese hamster ovary tissue culture cells.
  • Host cells transformed with expression vectors carrying foreign genes are grown in culture under conditions known to stimulate production of the foreign protein in the particular vector.
  • Such host cell/expression vector systems are often engineered so that expression of the foreign protein may be regulated by chemical or temperature induction.
  • Proteins which are secreted may be isolated from the growth media, while intracellular proteins may be isolated by harvesting and lysing the cells and separating the intracellular components. In this manner, it is possible to produce comparatively large amounts of proteins that are otherwise difficult to purify from native sources.
  • Such microbially produced proteins may be characterized by many well-known methods, including the use of monoclonal antibodies, hereinafter referred to as "MAbs," which are homogeneous antibodies that react specifically with a single antigenic determinant and display a constant affinity for that determinant, or by use of polyvalent antibodies, which may be derived from infected birds or other animals that have been immunized with life forms of Eimeria or with Eimeria protein, which react with a variety of different antigens and often with multiple determinants on a single antigen.
  • MAbs monoclonal antibodies
  • vaccinia has a long history of use as a vaccine and has been employed to virtually irradicate smallpox in humans. It now has been demonstrated that vaccinia virus can be effectively genetically engineered to express foreign antigens (Smith et al.. Nature 302:490-495 (1983); Panicali et al.. Proc. Natl. Acad. Sci. USA 80:5364-5368 (1983); Mackett et al.. J.
  • Fowl pox virus is very similar to vaccinia virus and many of the methods developed for vaccinia for the creation of recombinants expressing foreign antigens can be applied to fowl pox.
  • Attenuated fowl pox virus engineered to produce avian coccidia antigens thus is another method to produce an anticoccidial vaccine.
  • Live vaccines have the advantage of being inexpensive to produce and are characterized by the production of rapid immunity development
  • a second type of live vaccine results in the presentation of antigen in the gut where coccidia normally invades.
  • This method utilizes secretion or outer surface expression of the antigen by harmless bacteria introduced into the intestinal microbial population by incorporation in feed. Secretion is obtained by fusion of an antigen gene to the gene coding for a protein which is normally secreted, leaving the necessary secretion signal sequence intact Outer surface expression is achieved by fusion of the antigen genes to the genes that code for proteins normally localized on the outer surface.
  • This type of live vaccine is especially advantageous since manufacturing costs are minimal and the immune response stimulated is of a type particularly effective against coccidia invasion of the gut
  • a third type of live vaccine is the use of live recombinant bacteria expressing Eimeria antigens, which bacteria are injected subcutaneously or by other accepted routes, and which elicit an immune response to the expressed antigen (Miller et al. Infect Immun. 57:2014-2020 (1989). When compared to purified antigen or inactivated bacterial vaccines
  • bacteria the live bacterial vaccine resulted in a more protective immune response.
  • the subunit vaccines can also be used to raise an immune response against coccidiosis in ovo using techniques described by, for example, Hebrank U.S. Patent No. 4,681,063, Hebrank, EPC Patent Application
  • This invention relates to novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determi ⁇ nants, and to the genes that encode the antigenic peptides. It has now been found that particular polypeptides present in avian cells infected with coccidiosis, when purified and isolated, contain an antigenic determinant or determinants which can elicit an antibody response. This invention also relates to vaccines made using the novel antigenic proteins of avian coccidiosis and to methods of immunizing chickens against avian coccidia.
  • Figure 1 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-4c gene (Sequence ID No. 1).
  • Figure 2 shows the amino acid sequence of E. maxima antigen mc- 4c (Sequence ID No. 2).
  • Figure 3 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-5c gene (Sequence ID No. 3).
  • Figure 4 shows the amino acid sequence of E. maxima antigen mc- 5c (Sequence ID No.4).
  • Figure 5 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-30c gene (Sequence ID No.
  • Figure 6 shows the amino acid sequence of E. maxima antigen mc-
  • Figure 7 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima clone mc-35c gene (Sequence ID No. 7).
  • Figure 8 shows the amino acid sequence of E. maxima clone mc- 35c (Sequence ID No. 8).
  • Figure 9 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tg-3e gene (Sequence ID No. 9).
  • Figure 10 shows the amino acid sequence of E. tenella antigen tg- 3e (Sequence ID No. 10).
  • Figure 11 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-lle gene (Sequence ID. No. 11).
  • Figure 12 shows the amino acid sequence of E. tenella antigen tc- lle (Sequence ID No. 12).
  • Figure 13 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-23g gene (Sequence ID No. 13).
  • Figure 14 shows the amino acid sequence of E. tenella antigen tc- 23g (Sequence ID No. 14).
  • Figure 15 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-26h gene (Sequence ID No. 15).
  • Figure 16 shows the amino acid sequence of E. tenella antigen tc- 26h (Sequence ID No. 16).
  • Figure 17 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-30c gene (Sequence ID No. 17).
  • Figure 18 shows the amino acid sequence of E. tenella antigen tc-
  • Figure 19 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella clone tc-32c gene (Sequence ID No. 19).
  • Figure 20 shows the amino acid sequence of E. tenella clone tc-32c
  • Figure 21 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tc-33c gene (Sequence ID No. 21).
  • Figure 22 shows the amino acid sequence of E. tenella antigen tc- 33c (Sequence ID No. 22).
  • Figure 23 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-35c gene (Sequence ID. No. 23).
  • Figure 24 shows the amino acid sequence of E. tenella antigen tc-
  • Figure 25 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-37c gene (Sequence ID. No. 25).
  • Figure 26 shows the amino acid sequence of E. maxima antigen mc-37c (Sequence ID No. 26).
  • T is thymidyl
  • GLY is glycine
  • ALA is alanine
  • VAL valine
  • LEU leucine ILE is isoleucine
  • THR is threonine
  • PHE is phenylalanine
  • TRP is tyryptophan
  • CYS is cysteine
  • GLU glutamic acid
  • LYS is lysine
  • ARG is arginine
  • HIS histidine
  • GLN glutamine
  • ASN is asparagine.
  • the present invention relates to recombinant antigenic proteins, and fragments thereof containing antigenic determinants, that can elicit an antibody response against avian coccidiosis, and to the cloned genes that encode the antigenic proteins and fragments.
  • These antigenic proteins, and the fragments thereof containing antigenic determinants will bind with a specific monoclonal antibody or with polyvalent antibodies from infected chickens, or from other animals that have been immunized with life forms of Eimeria or Eimeria proteins, directed against an antige ⁇ nic protein of avian coccidia.
  • the antigenic proteins of this invention may be used for several applications: (1) the protein(s) can be used in an avian coccidia assay to detect antibodies against the coccidia; (2) antibodies may be prepared from the antigenic protein(s); (3) the protein(s) can be used for preparing vaccines against avian coccidiosis.
  • Antibodies directed against coccidial-antigens are used to identify, by immunological methods, transformed cells containing DNA encoding coccidial antigens.
  • the MAbs are used as a tool for identifying cells containing DNA sequences encoding coccidial antigens that are either species specific or common to all nine species.
  • Screening transformants with polyvalent chicken antiserum or chicken bile is used to identify DNA sequences encoding a wide spectrum of coccidial proteins which are antigenic in chickens upon infection.
  • Screening transformants with poly- valent rat antiserum is used to identify DNA sequences encoding coccidia proteins, which are antigenic when injected subcutaneously in rats and which may be antigenic in chickens. DNA sequences from the trans ⁇ formants thus identified then may be incorporated into a microorganism for large scale protein production.
  • the antigenic proteins, as native proteins or as hybrids with other proteins, may be used as vaccines to immunize birds to protect them from subsequent infection.
  • DNA sequences comprising the genes that encode antigenic proteins and fragments thereof may be used as DNA probes. These probes have a variety of uses, including screening a DNA library for additional genes that may encode antigenic determinants.
  • the DNA probe may be labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general almost any label useful in such methods can be applied to the present invention.
  • Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22:1243 (1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat No. 4,134,792); fluorescers (see Clin. Chem.
  • chromophores such as chemiluminescers and bioluminescers (see Clin. Chem. 25:512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as 3 H, 35 S, 32 P, 125 I and 14 C.
  • labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).
  • a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme.
  • an enzyme which acts upon a substrate to generate a product with a measurable physical property e.g., one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property.
  • Examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.
  • the term "antigenic” or “antigenic determinant” is meant immunologically cross-reactive antigenic determinants with which a given antibody will react Therefore, the antigenic peptides of this invention will include chemically synthesized peptides, peptides made by recombinant DNA techniques, and antibodies or fragments thereof which are anti-idiotypic towards the determinant of the peptides of this invention.
  • Several procedures may be used to construct a microorganism that produces an antigenic protein that binds with a monoclonal or polyvalent antibody that is directed against an antigenic protein of avian coccidia.
  • mRNA messenger RNA
  • cDNA complementary DNA
  • This route is referred to as the mRNA route.
  • the advantage to this route is that only "expressed" genes are cloned, reducing the number of individual transformants required to represent the entire population of genes.
  • the cloned DNA sequence is advantageously transferred to a suitable expression vector/host cell system for large scale production of the antigenic protein.
  • the DNA sequence that is to be isolated encodes an antigenic protein that will elicit an immune response when administered to chickens which will protect them from subsequent infections. It is not necessary to isolate a complete coccidial gene encoding such a protein, since those portions of the protein termed antigenic determinants are sufficient for triggering a protective immune response (Lerner, supra). This antigenic determinant should be on the surface of the folded microbially-produced protein to trigger the response (Lerner, supra).
  • the sequence may be isolated from the sporo- zoite life stage of the parasite. It has been demonstrated that part of the protective immune response in chickens is directed against the sporozoite.
  • Antigenic proteins isolated from other life stages also may be effective as vaccines.
  • MAbs or polyvalent antibodies which bind to various sporozoite proteins can be used to identify cloned DNA sequences encoding those proteins. Such proteins can be isolated and used to elicit a protective immune response in chickens.
  • Sporozoites can be obtained from oocysts by excystation using the method of Doran and Vetterling, Proc. Helminthol Soc. Wash. 34:59-65 (1967), and purified by the leucopak filter technique of Bontemps and Yvore, Ann. Rech. Vet 5:109-113 (1974). Although the method of
  • sporozoite mRNA may be isolated from intact sporulated oocysts, which contain the sporozoites.
  • Isolation of mRNA coding for the antigenic proteins of interest is advantageously accomplished by lysis of intact sporulated oocysts under conditions which minimize nuclease activity. This is accomplished using a modification of the procedure described by Pasternak et al. Molec.
  • RNA may be isolated by grinding the oocysts with glass beads in a solution containing guanadine thiocyanate, Sarkosyl, and Tris Buffer pH 8.0. Oocyst proteins are removed by phenol chloroform extraction. The total cellular RNA is separated from DNA by precipitation with lithium chloride. Oligo
  • (dT)-cellulose chromatography then can be used to isolate mRNA from the total RNA population.
  • Synthesis of cDNA may be accomplished using either a kit from
  • Moloney Leukemia virus reverse transcriptase and RNase H The kits are used according to the instructions provided by the manufacturer.
  • the poly r(A) tail of mRNA permits oligo(dT) (of about 12-18 nucleotides) to be used as a primer for cDNA synthesis or alternatively DNA oligonucleotides of random sequence can be used as a primer for cDNA synthesis.
  • the ds-cDNA prepared as described above is generally inserted into a suitable cloning vector, which is used for transforming appropriate host cells.
  • suitable cloning vectors include various plasmids and phages, but a bacteriophage lambda is preferred.
  • a cloning vector For a cloning vector to be useful for the expression of foreign proteins which are to be detected with antibodies, it should have several useful properties. Most importantly, it should have a cloning site within a gene which is expressed in the host being used. There should also be a means of controlling expression of the gene. The vector should be able to accept DNA of the size required for synthesis of the desired protein product and replicate normally. It is also useful to have a selectable property which allows identification of vectors carrying inserts. A cloning vector having such properties is the bacteriophage ⁇ gtll (ATCC 37194) (Young and Davis. Proc. Nat'l Acad. Sci. USA 80:1194-1198 (1983)).
  • This vector has a unique EcoRI site near the end of the bacterial gene coding for /3-galactosidase. That site can be used for insertion of foreign DNA to make hybrid proteins made up of /3-galactosidase and the foreign gene product
  • the expression of /3-galactosidase is under control of the lac promoter and can be induced by the addition of isopropyl- -D- thiogalactopyranoside (IPTG).
  • IPTG isopropyl- -D- thiogalactopyranoside
  • the ⁇ gtll phage contains 43.7 kb of DNA which is considerably smaller than wild type ⁇ . This allows insertion of pieces of DNA up to 8.3 kb in length, before the DNA becomes too large to fit inside the phage head.
  • the ds-cDNA can be conveniently inserted into the phage by addition of linkers containing an EcoRI restriction site and any convenient second restriction enzyme recognition site to the DNA and ligation into the EcoRI-cut ⁇ gtll DNA.
  • linkers containing an EcoRI restriction site and any convenient second restriction enzyme recognition site to the DNA and ligation into the EcoRI-cut ⁇ gtll DNA.
  • the DNA is packaged, in vitro, into ⁇ phage heads (Enquist and Sternberg, Methods in Enzvmology 68:281-298 (1979) and those phages are used to infect a suitable ⁇ -sensitive host With the proper choice of host, the phage may be screened as plaques or lysogens
  • yeast Old and Primrose, supra. pp. 62-68
  • filamentous fungi insect cells
  • mammalian cells U.S. 4,745,051 and 4,879,2366
  • the DNA described herein may be inserted into the above vectors by various techniques including homopolymeric tailing, blunt-end ligation or by use of linker molecules (Old and Primrose, supra, at p. 92).
  • the recombinant bacteriophages can be used to infect a suitable E. coli host which allows the formation of phage plaques on agar (or agarose) plates.
  • the plaques can be transferred to nitrocellulose membranes while being induced with IPTG.
  • the proper antibodies are then reacted with the filters.
  • the positive reactions are detected by reaction with either [ 125 I] Staphylococcus aureus Protein A or a second antibody conjugated with horseradish peroxidase, alkaline phosphatase, or vitamin B12.
  • the plaques containing cross-reactive antigens can then be detected by autoradiography, by detection of the conjugated enzyme, or, in the case of Vitamin B12 conjugates, by binding of streptavidin conjugate to one of the reporter enzymes.
  • the phages giving positive signals in the antibody-screening procedure can be shown to contain sequences coding for coccidial proteins by excision of the DNA originally inserted into the phage DNA and examination of the ability of that DNA to hybridize with coccidia mRNA or coccidia genomic DNA.
  • the nucleotide sequence of the cDNA insert is determined using the methods of Sanger et al. Proc. Natl Acad. Scl. USA 74:5463-5467 (1977); or Maxam and Gilbert, Proc. Natl. Acad.
  • Another method of cloning coccidial antigens begins with isolation of nuclear DNA from oocysts. This DNA is then broken into fragments of a size suitable for insertion into a cloning vector. To obtain such fragments, one can use mechanical shearing methods such as sonication or high-speed stirring in a blender to produce random breaks in the DNA. Intense sonication with ultrasound can reduce the fragment length to about 300 nucleotide pairs. (Old and Primrose, supra, p.
  • nuclear DNA may be partially digested with DNAsel, which gives random fragments, with restriction endonucleases, which cut at specific sites, or with mung bean nuclease in the presence of fo ⁇ namide, which has been shown with some related organisms (McCutchan, T.F., et al Science 225:625-628 (1984)) to produce DNA fragments containing intact genes.
  • nuclear DNA fragments may be inserted into any of the cloning vectors listed for the cloning of cDNA in the mRNA experimental method. If the nuclear DNA is digested with a restriction endonuclease, it can be inserted conveniently into a cloning vector digested with the same enzyme, provided the vector has only one recognition site for that enzyme. Otherwise, DNA fragments may be inserted into appropriate cloning vectors by homopolymeric tailing or by using linker molecules (Old and Primrose, supra, at p. 92).
  • genomic DNA expression libraries are constructed after digestion on nuclear DNA with any one of a number of restriction enzymes that yield blunt end DNA fragments.
  • the size of the DNA frag ⁇ ments can be controlled by reducing the time of digestion with the restriction enzyme.
  • Linkers such as an oligonucleotide encoding restriction site for EcoRI and Notl are ligated onto the blunt end DNA fragments and fragments are ligated into the EcoRI site of bacteriophage ⁇ gtll.
  • the resulting genomic expression library can be subjected to antibody screening as described for the mRNA route.
  • Plasmid DNA is isolated from transformants found to be "positive” by the above screening methods.
  • the nuclear DNA inserts of these plasmids are then subjected to DNA sequencing.
  • the cloned DNA sequence may be transferred to expression vectors engineered for high- level production of the antigenic protein.
  • the expression vectors are transformed into suitable host cells for production of the antigenic protein.
  • These host cells may include both prokaryotic and eukaryotic organisms.
  • the prokaryotic host cells include E. coli and B. subtilis.
  • the eukaryotic host cells include yeast, insect cells, and mammalian cells.
  • Coccidial antigens advantageously may be produced at high levels in E. coli as a fusion protein comprising the antigen and an amino terminal portion of the viral MS-2 polymerase. This fusion is accomplished by inserting a DNA sequence encoding a coccidial antigen into a plasmid vector, pEX32b, carrying the polymerase gene.
  • the expression of the fusion antigenic protein is highly regulated by temperature.
  • Host expression vector systems in which expression of foreign proteins is regulatable have the advantage of avoiding possible adverse effects of foreign protein accumulation as high cell densities are reached.
  • Some investigators have proposed that expression of gene fragments such as those encoding antigenic determinants may avoid the deleterious effects that expression of the entire antigenic protein would have on E. coli host cells. (Helfman et al. Proc. Natl Acad. Scl. USA 80:31-35 (1983)).
  • Coccidial antigens also may be produced in high levels as fusions at the carboxy-terminal of E. coli 3-galactosidase.
  • the coccidia antigen gene is transferred to a small plasmid such as pGX3217 which carries the gene for ampicillin resistance and which contains a /3-galactosidase gene with an EcoRI insertion site near the 3' end of the gene. Synthesis of the fused gene products is regulated by the ]ac promoter which is induced by addition of IPTG, the inducer of the lac operon.
  • An effective subunit vaccine against avian coccidiosis may consist of a mixture of antigen proteins derived from several species of Eimeria. Alternatively, production costs may be decreased by producing two or more antigen proteins as one fusion protein thus reducing the required number of fermentations and purifications.
  • Such a fusion protein would contain the amino acid sequence comprising an antigenic epitope of each antigen protein (or repetitions of those sequences) with variable amounts of surrounding nonantigenic sequence.
  • a hybrid gene designed to code for such a protein in E. coli would contain bacterial regulatory sequence (promoter/operator) and the 5' end of an E. coli gene (the ribosome binding site and codons for several amino acids) to ensure efficient translation initiation followed by the coding sequences for the antigenic epitopes all fused in the same reading frame.
  • E. coli cells transformed with the expression vector carrying a cloned coccidial antigen sequence are grown under conditions that promote expression of the antigenic polypeptide.
  • the antigenic protein is then tested for ability to elicit an immune response in chickens that will protect them from subsequent Eimeria infections.
  • the antigen protein may be presented to birds as a live E. coli expressing the protein, as killed E. coli expressing the protein or as purified protein.
  • the antigen may be combined with suitable carriers and adjuvants and administered to birds in their feed or by injection.
  • the cloned antigenic proteins used in vaccines above are tested for their ability to elicit an immune response in chickens that protects the birds from subsequent infection by any of the important species of Eimeria. including E. tenella. E.
  • cloned antigenic proteins which may be useful as vaccines to protect against coccidiosis
  • another useful alternative which may be derived from cloning antigen genes is the use of small, synthetic peptides in vaccines (see Lerner, supra). After the sequence of antigenic proteins is determined, it is possible to make synthetic peptides based on that sequence. The peptides are conjugated to a carrier protein such as hemocyanin or serum albumin and the conjugate then can be used to immunize against coccidia.
  • a carrier protein such as hemocyanin or serum albumin
  • expression libraries were prepared in the lambda vector, ⁇ gtll, using cDNA prepared from poryA mRNA isolated from E. maxima oocysts. The construction of the libraries is described in Genex Patent Application, PCT/US89/02918 incorporated herein by reference.
  • the cDNA expression library was screened with rat antiserum raised against E. maxima sporozoites.
  • the rat immune serum was prepared using the following protocol. Da
  • the library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transfered to nitrocellulose filters. Positive phage were identified after screening with the rat anti-E. maxima sporozoite antiserum. The cDNA inserts from the positive clones were cloned into bacteriophage M13 and subjected to sequence analysis. Ej. maxima antigen mc-4c was identified.
  • Antigen mc-4c is an analog of E. acerv ⁇ lina antigen ac-6b and E tenella antigen GX3262 (Miller etal. Infect. Immun. 57:2014-2020 (1989); Danforth et al. Poultry Sci. 68:1643-1652 (1989)). Only the carboxy terminal sequence of mc-4c was recovered so a cDNA expression library was screened with an 18 bp oligonucleotide complementary to the 5'- sequence of the original mc-4c clone. A second clone, ⁇ mc-36c, was identified that encodes all but the six amino terminal amino acids of the antigen. The sequence of mc-4c and the 21.7 Kd translation product that it encodes are shown in Figures 1 and 2 (Sequence ID Nos.l and 2).
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage
  • the carboxyl terminal sequence of antigen mc-5c is encoded by 1611 bp of open reading frame.
  • the sequence and its 59.2 Kd translation product are shown in Figures 3 and 4 (Sequence ID Nos.3 and 4).
  • a partial sequence of antigen mc-5c were expressed in pEX32b.
  • the terminal 227 amino acids encoded by an EcoRI fragment extending from the internal restriction site starting at base 928 to beyond the site of translation termination ( Figure 3) were inserted into pEX32b and expressed.
  • Antigen mc-5c is encoded in expression vector pGX5376.
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-30c was identified.
  • Antigen mc-30c had 510 bp of open reading frame fused in frame with the /3-galactosidase gene of ⁇ gtll. Within the open reading frame are two methionine codons (double underlined) followed by 321 bp of open reading frame ending with a termination codon ( Figure 5, Sequence ID No.5). Preceding the putative methionine initiation codons are two
  • the insert contains a complete gene encoding an Eimeria antigen consisting of 109 amino acids with a molecular weight of 12.2 Kd.
  • the amino acid sequence of the mc-30c antigen encoded by the entire open reading frame of mc- 30c is shown in Figure 6 (Sequence ID No.6) with the methionines at the putative initiation site underlined.
  • Antigen mc-30c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-30c is encoded in expression vector pGX5370 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-35c was identified.
  • Antigen mc-35c is encoded by 1461 bp of open reading frame fused in-frame with the /3-galactosidase sequence. The 5'-sequence consists of a 24 bp sequence encoding eight amino acids which is repeated with complete fidelity a total of 24 times.
  • This repeat is linked through a single glutamic acid residue to a second, different 24 bp sequence which is repeated 16 times.
  • the repeats are followed by 501 bp of open reading frame followed by a termination codon.
  • the nucleotide sequence of mc- 35c is shown in Figure 7 (Sequence ID No.7) and the amino acid sequence of mc-35c is shown in Figure 8 (Sequence ID No.8) with the first of each of the eight amino acid repeats underlined and the connecting glutamic acid residue double underlined.
  • Antigen mc-35c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-35c is encoded in expression vector pGX5367 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. tenella cDNA and genomic expression libraries in lambda phage were screened with chicken immune bile.
  • Immune bile was recovered from chickens infected with E. tenella oocysts seven days after the onset of infection. Before being used to screen the libraries, the immune bile was incubated with R coli proteins to inactivate antibodies cross-reactive with E. coli proteins. After incubation of the filters used to lift the lambda plaques from the growth plates with the immune bile, the filters were developed with goat anti-chicken IgA antibodies.
  • the positive plaques were visualized with donkey anti-goat antibodies conjugated with alkaline phosphatase. Phage that produce antigens cross-reactive with the immune bile were identified. The DNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis.
  • Antigen tg-3e was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tg-3e is encoded in expression vector pGX5390 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • E. tenella cDNA libraries were screened with the chicken anti-E. tenella immune bile. Phage that produce antigens cross-reactive with the immune bile were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis R tenella antigen tc-lle was identified.
  • Antigen tc-lle consists of a 417 bp open reading frame encoding 139 amino acids.
  • the sequence of tc-lle and the 13.9 Kd translation product that it encodes are shown in Figures 11 and 12 (Sequence ID
  • Antigen tc-lle was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tc-lle is encoded in expression vector pGX5394 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a temperature sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • the library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transferred to nitrocellulose filters using standard protocols (Berger et al. Methods in Enzvmologv, Vol 152, Academic Press, New York, NY (1987)). The filters were screened with the chicken anti-E. tenella sporozoite antiserum. Plaques recognized by chicken serum IgA antibodies were identified with goat anti-chicken IgA antibody followed by biotinylated donkey anti-goat IgG antibody. The positive plaques were visualized with an alkaline phosphatase-streptavidin conjugate using standard protocols (Berger et al.
  • Antigens tc-23g was expressed in E. coli after insertion into the plasmid expression vector pEX-32b using standard protocols (Berger et al. Methods in Enzymology. Vol. 152, Academic Press, New York, NY
  • the antigen is encoded in expression vector pGX5398 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the P L promoter.
  • the host strain pop2136 contains a tempera ⁇ ture sensitive repressor of P L and expression of the fusion protein is fully induced at 42°C.
  • Example 7 an E. tenella cDNA library was screened with chicken anti-E. tenella merozoites immune serum produced using the same injection regimen described in Example
  • a phage that produces an antigen cross-reactive with IgG antibodies in the immune serum was identified with biotinylated goat anti-chicken IgG antibody followed by visualization with an alkaline phosphatase-strep- tavidin conjugate.
  • the cDNA insert from the positive phage was cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74-5463-5467 (1977)). Following sequence analysis, E. tenella antigen tc-26h was identified.
  • the antigen consists of a peptide fragment which is composed of long strings of alanines and serines.
  • the nucleotide sequence of tc-26h and the 10 Kd translation product that it encodes are shown in Figures 15 and 16
  • Example 7 an E. tenella cDNA library was screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. fProc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigens to tc-30c, tc-32c, tc-33c and tc-35c were identified.
  • Antigen tc-30c consisted of 284 bp of open reading frame encoding an antigen fragment fused in-frame with the /3-galactosidase gene of ⁇ gtll. The nucleotide sequence of tc-30c and the 9.5 Kd translation product that it encodes are shown in Figures 17 and 18 (Sequence ID Nos.17 and 18).
  • Antigen tc-32c consisted of 261 bp of open reading frame encoding an antigen fragment The nucleotide sequence of tc-32c and the 8.8 Kd translation product that it encodes are shown in Figures 19 and 20 (Sequence ID Nos.19 and 20).
  • Antigen tc-33c consisted of 408 bp of open reading frame encoding the carboxy terminal sequence of an antigen.
  • the nucleotide sequence of tc-33c and the 12.5 Kd translation product that it encodes are shown in Figures 21 and 22 (Sequence ID Nos.21 and 22).
  • Antigen tc-35c consisted of 120 bp of open reading frame encoding the carboxy terminal sequence of an antigen. The nucleotide sequence of tc-35c and the 5.0 Kd translation product that it encodes are shown in
  • E. maxima cDNA libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified.
  • the cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Troc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigen mc-37c was identified.
  • Antigen mc-37c consists of 442 bp of open reading frame encoding an antigen fragment that is fused in-frame with the /3-galactosidase sequence of ⁇ gtll.
  • the nucleotide sequence of mc-37c and the 16.2 Kd peptide that it encodes are shown in Figures 25 and 26 (Sequence ID Nos. 25 and 26). While certain representative embodiments and details have been shown for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention.
  • ADDRESSEE Sterne, Kessler, Goldstein & Fox
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AGA TTA GTA GAT TTT TGT ATA CAA GAT TTT AAG AGA AAG AAT AGA TCT 624 Arg Leu Val Asp Phe Cys He Gin Asp Phe Lys Arg Lys Asn Arg Ser 195 200 205
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CAA CCA CCA CAC ATT CAT ACG GAG ACT CCT GCC ACA ACA CCA GTT GCA 240
  • MOLECULE TYPE DNA (genomic)
  • AGG CTC AAG CAA CAG CAA CAG CAG CAG CTT TGT CAG CAG CAG CCA GGG 384 Arg Leu Lys Gin Gin Gin Gin Gin Gin Gin Leu Cys Gin Gin Gin Pro Gly 115 120 125
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

This invention relates to novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determinants, and to the genes that encode the antigenic peptides. This invention also relates to vaccines made using the novel antigenic proteins of avian coccidiosis and to methods of immunizing chickens against avian coccidia.

Description

TITLE OF THE INVENTION
GENETICALLY ENGINEERED COCCIDIOSIS VACCINE
Cross-Reference to Related Applications
This application is a Continuation-in-part of US Ser. No. 07/581,694, filed September 12, 1990.
Field of the Invention
This invention is in the field of avian coccidiosis and is directed to recombinant antigenic proteins of avian coccidia and to the genes that encode the proteins. These antigenic proteins may be used in a vaccine against avian coccidia.
BACKGROUND OF THE INVENTION
Coccidiosis is a disease of both invertebrates and vertebrates, including man, caused by intracellular parasitic protozoa which generally invade the epithelial cells lining the alimentary tract and the cells of associated glands. The crowded conditions under which many domestic animals are raised have contributed to increased incidence of the disease. Virtually every domestic animal is susceptible to infection, and distribution of the parasite is world-wide. Coccidiosis is therefore the cause of signifi¬ cant economic loss throughout the world.
The poultry industry suffers particularly severe losses, with coccidiosis being the most economically important parasitic disease of chickens. Since 1949, preventive anticoccidials have been used but have not been totally effective. Losses due to morbidity from coccidiosis, including reduced weight gains and egg production, and decreased feed conversion, persist. The cost of coccidiosis in broiler production has been estimated at 1/2 to 1 cent per pound. Based on an annual production of 3,000,000,000 broilers annually in the United States, losses would total between 60 and 120 million dollars. To this figure must be added the cost of anticoccidials estimated at 35 million dollars. These impressive figures emphasize the importance of reducing the incidence of coccidiosis in chickens. Of the nine genera of coccidia known to infect birds, the genus
Eimeria contains the most economically important species. Various species of Eimeria infect a wide range of hosts, including mammals, but nine species have been recognized as being pathogenic to varying degrees in chickens: Eimeria acervulina. E. mivati. E. mitis. E. praecox. E. hagani, E. necatrix-, E. maxima, E. brunetti and E. tenella.
Although the Eimeria are highly host specific, their life cycles are similar. The developmental stages of the avian coccidia can be illustrated by the species Eimeria tenella. which proliferates in the cecum of the chicken. The life cycle of the Eimeria species begins when the host ingests previously sporulated oocysts during ground feeding or by inhalation of dust Mechanical and chemical action in the gizzard and intestinal tract of the chicken ruptures the sporulated oocyst, liberating eight sporozoites. The sporozoites are carried in the digestive contents and infect various portions of the intestinal tract by penetration of epithelial cells.
Subsequent life stages involve asexual multiple fission, the infection of other epithelial cells, development of gametes, and fertilization to produce a zygote which becomes an oocyst which is passed out of the host with the droppings. The oocyst undergoes nuclear and cellular division resulting in the formation of sporozoites, with sporulation being dependent upon environmental conditions. Ingestion of the sporulated oocyst by a new host transmits the disease.
Of all species of Eimeria. E. tenella has received the most attention. E. tenella is an extremely pathogenic species, with death often occurring on the fifth or sixth day of infection. Before the use of chemotherapeutic agents, poultry producers' attempts to control coccidiosis were limited to various management programs. These programs were directed toward attempts at sanitation through disinfection, or by mechanical removal of litter. Despite these efforts, sufficient oocysts usually remained to transmit the disease.
One means of combating the hazards of coccidia is immunization. This method involves feeding to the poultry a small dose of oocysts of each of the species of coccidia during the first weeks of life. However, dosage control has proven difficult as birds ingest daughter oocysts, with some birds developing severe coccidiosis and others remaining susceptible.
Also, since this procedure produces mixed infections, sometimes adequate immunity does not develop to all species given. In addition, immunity development is too slow for use with broiler production.
Another means of combating coccidia is drug treatment after the poultry is infected. One drug that has been used is sulfanilamide which has shown anticoccidial activity against six species of coccidia. However, unless the drug treatment of the flock is quickly initiated after diagnosis of the disease, medication may be started too late to be effective.
Ideally, the best method for combating coccidia is preventive medication. Since the advent of the use of sulfonamide drugs, over forty compounds have been marketed for preventive medication against coccidia. There have been many problems with the use of such drugs, including anticoccidial contamination of layer flock feeds, inclusion of excessive anticoccidial drugs in the feed causing toxicity in the birds and omission of the anticoccidial from the feed resulting in coccidiosis outbreaks. A particularly frustrating problem has been the development of drug-resistant strains of coccidia. Moreover, there is a potential for drug residues being deposited in the meat Clearly, available methods for the control of coccidiosis have met with limited success, and the need for a safe, efficient, and inexpensive method of combating avian coccidiosis remains.
The development of an effective anticoccidial vaccine is a desirable solution to the problem of disease prevention. Vaccines produced by traditional methods will require extensive development There are reports of the production of attenuated strains through passage in embryos or cell culture. While this approach may eventually lead to successful vaccines, not all the important species of Eimeria have been adapted to growth in culture or embryos such that they are capable of completing their life cycle.
Genetic engineering methodology presents the opportunity for an alternative approach to vaccine development. It is known that genes encoding antigenic proteins of pathogenic organisms can be cloned into microorganisms. The antigenic proteins then can be expressed at high levels, purified, and used as vaccines against the pathogenic organism. These antigenic proteins have the advantage of being noninfectious and are potentially inexpensive to produce. Such "subunit vaccines" have been prepared from antigen genes for a number of viruses such as hepatitis, herpes simplex and foot and mouth disease virus. An alternate approach is to produce "synthetic vaccines", small chemically-synthesized peptides, whose sequence is chosen based upon the amino acid sequence translation of viral antigen DNA. The advantages of such "synthetic vaccines" over traditional vaccination with attenuated or killed pathogenic organisms have been summarized by Lerner in Nature 299:592-596 (1982).
It is now possible to produce foreign proteins, including eukaryotic proteins, in prokaryotic organisms such as gram positive or gram negative bacteria. The process involves the insertion of DNA (derived either from enzymatic digestion of cellular DNA or by reverse transcription of mRNA) into an expression vector. Such expression vectors are derived from either plasmids or bacteriophage and contain: (1) an origin of replication functional in a microbial host cell; (2) genes encoding selectable markers, and (3) regulatory sequences including a promoter, operator, and a ribosome binding site which are functional in a microbial host cell and which direct the transcription and translation of foreign
DNA inserted downstream from the regulatory sequences. To increase protein production and stability, eukaryotic proteins are often produced in prokaryotic cells as a fusion with sequences from the amino-terminus of a prokaryotic protein. β-Galactosidase or the product of one of the E. coli tryptophan operon genes have been used successfully in this manner. Expression vectors have also been developed for expression of foreign proteins in eukaryotic host cells, e.g., yeast and Chinese hamster ovary tissue culture cells.
Host cells transformed with expression vectors carrying foreign genes are grown in culture under conditions known to stimulate production of the foreign protein in the particular vector. Such host cell/expression vector systems are often engineered so that expression of the foreign protein may be regulated by chemical or temperature induction. Proteins which are secreted may be isolated from the growth media, while intracellular proteins may be isolated by harvesting and lysing the cells and separating the intracellular components. In this manner, it is possible to produce comparatively large amounts of proteins that are otherwise difficult to purify from native sources.
Such microbially produced proteins may be characterized by many well-known methods, including the use of monoclonal antibodies, hereinafter referred to as "MAbs," which are homogeneous antibodies that react specifically with a single antigenic determinant and display a constant affinity for that determinant, or by use of polyvalent antibodies, which may be derived from infected birds or other animals that have been immunized with life forms of Eimeria or with Eimeria protein, which react with a variety of different antigens and often with multiple determinants on a single antigen.
Alternate technology to the production of "subunit" or "synthetic" vaccines is the use of a fowl pox virus vector. The pox virus vaccinia has a long history of use as a vaccine and has been employed to virtually irradicate smallpox in humans. It now has been demonstrated that vaccinia virus can be effectively genetically engineered to express foreign antigens (Smith et al.. Nature 302:490-495 (1983); Panicali et al.. Proc. Natl. Acad. Sci. USA 80:5364-5368 (1983); Mackett et al.. J. of Virology 49:857-864 (1984)) and the engineered viruses can serve as a live vaccine against other viruses and infections besides smallpox. Fowl pox virus is very similar to vaccinia virus and many of the methods developed for vaccinia for the creation of recombinants expressing foreign antigens can be applied to fowl pox. Attenuated fowl pox virus engineered to produce avian coccidia antigens thus is another method to produce an anticoccidial vaccine. Live vaccines have the advantage of being inexpensive to produce and are characterized by the production of rapid immunity development
A second type of live vaccine results in the presentation of antigen in the gut where coccidia normally invades. This method utilizes secretion or outer surface expression of the antigen by harmless bacteria introduced into the intestinal microbial population by incorporation in feed. Secretion is obtained by fusion of an antigen gene to the gene coding for a protein which is normally secreted, leaving the necessary secretion signal sequence intact Outer surface expression is achieved by fusion of the antigen genes to the genes that code for proteins normally localized on the outer surface. (T. Silhavy, U.S. Patent No. 4,336,336.) This type of live vaccine is especially advantageous since manufacturing costs are minimal and the immune response stimulated is of a type particularly effective against coccidia invasion of the gut A third type of live vaccine is the use of live recombinant bacteria expressing Eimeria antigens, which bacteria are injected subcutaneously or by other accepted routes, and which elicit an immune response to the expressed antigen (Miller et al. Infect Immun. 57:2014-2020 (1989). When compared to purified antigen or inactivated bacterial vaccines
(bacterins), the live bacterial vaccine resulted in a more protective immune response.
The subunit vaccines can also be used to raise an immune response against coccidiosis in ovo using techniques described by, for example, Hebrank U.S. Patent No. 4,681,063, Hebrank, EPC Patent Application
No. 87306746.7, and Smith et al. EPC Patent Application No. 88303349.0.
SUMMARY OF THE INVENTION
This invention relates to novel recombinant antigenic proteins of avian coccidiosis, and fragments thereof containing antigenic determi¬ nants, and to the genes that encode the antigenic peptides. It has now been found that particular polypeptides present in avian cells infected with coccidiosis, when purified and isolated, contain an antigenic determinant or determinants which can elicit an antibody response. This invention also relates to vaccines made using the novel antigenic proteins of avian coccidiosis and to methods of immunizing chickens against avian coccidia.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-4c gene (Sequence ID No. 1).
Figure 2 shows the amino acid sequence of E. maxima antigen mc- 4c (Sequence ID No. 2). Figure 3 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-5c gene (Sequence ID No. 3).
Figure 4 shows the amino acid sequence of E. maxima antigen mc- 5c (Sequence ID No.4). Figure 5 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-30c gene (Sequence ID No.
5).
Figure 6 shows the amino acid sequence of E. maxima antigen mc-
30c (Sequence ID No. 6). Figure 7 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima clone mc-35c gene (Sequence ID No. 7). Figure 8 shows the amino acid sequence of E. maxima clone mc- 35c (Sequence ID No. 8).
Figure 9 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tg-3e gene (Sequence ID No. 9).
Figure 10 shows the amino acid sequence of E. tenella antigen tg- 3e (Sequence ID No. 10).
Figure 11 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-lle gene (Sequence ID. No. 11). Figure 12 shows the amino acid sequence of E. tenella antigen tc- lle (Sequence ID No. 12).
Figure 13 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-23g gene (Sequence ID No. 13). Figure 14 shows the amino acid sequence of E. tenella antigen tc- 23g (Sequence ID No. 14).
Figure 15 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-26h gene (Sequence ID No. 15). Figure 16 shows the amino acid sequence of E. tenella antigen tc- 26h (Sequence ID No. 16). Figure 17 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-30c gene (Sequence ID No. 17).
Figure 18 shows the amino acid sequence of E. tenella antigen tc-
30c (Sequence ID No. 18). Figure 19 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella clone tc-32c gene (Sequence ID No. 19).
Figure 20 shows the amino acid sequence of E. tenella clone tc-32c
(Sequence ID No. 20).
Figure 21 shows the nucleotide sequence of the 5'-3' strand of DNA encoding the E. tenella antigen tc-33c gene (Sequence ID No. 21).
Figure 22 shows the amino acid sequence of E. tenella antigen tc- 33c (Sequence ID No. 22).
Figure 23 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. tenella antigen tc-35c gene (Sequence ID. No. 23). Figure 24 shows the amino acid sequence of E. tenella antigen tc-
35c (Sequence ID No. 24).
Figure 25 shows the nucleotide sequence of the 5'-3' strand of cDNA encoding the E. maxima antigen mc-37c gene (Sequence ID. No. 25). Figure 26 shows the amino acid sequence of E. maxima antigen mc-37c (Sequence ID No. 26).
As is well known in the art, due to the degeneracy of the genetic code, the DNA sequences given in the Figures for the genes and antigenic peptides of this invention may be encoded by different DNA than those represented. Thus, knowledge of an amino acid sequence does not necessarily lead to a precise genetic sequence coding therefor. In all of the Figures with DNA and amino acid sequences the sequence is given as the 5' to 3' strand. The abbreviations have the following standard meanings: A is deoxyadenyl
T is thymidyl
G is deoxyguanyl
C is deoxycytosyl
GLY is glycine
ALA is alanine
VAL is valine
LEU is leucine ILE is isoleucine
SER is serine
THR is threonine
PHE is phenylalanine
TYR is tyrosine TRP is tyryptophan
CYS is cysteine
MET is methionine
ASP is aspartic acid
GLU is glutamic acid LYS is lysine
ARG is arginine
HIS is histidine
PRO is proline
GLN is glutamine ASN is asparagine. DFTAΠ FD DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention relates to recombinant antigenic proteins, and fragments thereof containing antigenic determinants, that can elicit an antibody response against avian coccidiosis, and to the cloned genes that encode the antigenic proteins and fragments. These antigenic proteins, and the fragments thereof containing antigenic determinants, will bind with a specific monoclonal antibody or with polyvalent antibodies from infected chickens, or from other animals that have been immunized with life forms of Eimeria or Eimeria proteins, directed against an antige¬ nic protein of avian coccidia.
The antigenic proteins of this invention may be used for several applications: (1) the protein(s) can be used in an avian coccidia assay to detect antibodies against the coccidia; (2) antibodies may be prepared from the antigenic protein(s); (3) the protein(s) can be used for preparing vaccines against avian coccidiosis.
Antibodies directed against coccidial-antigens are used to identify, by immunological methods, transformed cells containing DNA encoding coccidial antigens. The MAbs are used as a tool for identifying cells containing DNA sequences encoding coccidial antigens that are either species specific or common to all nine species. Screening transformants with polyvalent chicken antiserum or chicken bile is used to identify DNA sequences encoding a wide spectrum of coccidial proteins which are antigenic in chickens upon infection. Screening transformants with poly- valent rat antiserum is used to identify DNA sequences encoding coccidia proteins, which are antigenic when injected subcutaneously in rats and which may be antigenic in chickens. DNA sequences from the trans¬ formants thus identified then may be incorporated into a microorganism for large scale protein production. The antigenic proteins, as native proteins or as hybrids with other proteins, may be used as vaccines to immunize birds to protect them from subsequent infection.
In addition, the DNA sequences comprising the genes that encode antigenic proteins and fragments thereof may be used as DNA probes. These probes have a variety of uses, including screening a DNA library for additional genes that may encode antigenic determinants.
The DNA probe may be labeled with a detectable group. Such detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of immunoassays and in general almost any label useful in such methods can be applied to the present invention. Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. 22:1243 (1976)), enzyme substrates (see British Pat. Spec. 1,548,741), coenzymes (see U.S. Pat Nos. 4,230,797 and 4,238,565) and enzyme inhibitors (see U.S. Pat No. 4,134,792); fluorescers (see Clin. Chem. 25:353 (1979)); chromophores; luminescers such as chemiluminescers and bioluminescers (see Clin. Chem. 25:512 (1979)); specifically bindable ligands; proximal interacting pairs; and radioisotopes such as 3H, 35S, 32P, 125I and 14C. Such labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors). For example, a cofactor-labeled probe can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme. For example, one can use an enzyme which acts upon a substrate to generate a product with a measurable physical property.
Examples of the latter include, but are not limited to, beta-galactosidase, alkaline phosphatase and peroxidase.
As used herein, the term "antigenic" or "antigenic determinant" is meant immunologically cross-reactive antigenic determinants with which a given antibody will react Therefore, the antigenic peptides of this invention will include chemically synthesized peptides, peptides made by recombinant DNA techniques, and antibodies or fragments thereof which are anti-idiotypic towards the determinant of the peptides of this invention. Several procedures may be used to construct a microorganism that produces an antigenic protein that binds with a monoclonal or polyvalent antibody that is directed against an antigenic protein of avian coccidia. One such procedure can be divided into the following major stages, each of which is described more fully herein: (1) recovery and isolation of messenger RNA (mRNA) found in organisms of the genus Eimeria; (2) in vitro synthesis of complementary DNA (cDNA), using coccicidia mRNA as a template; (3) insertion of the cDNA into a suitable expression vector and transformation of bacterial cells with that vector; and, (4) recovery and isolation of the cloned gene or gene fragment. This route is referred to as the mRNA route. The advantage to this route is that only "expressed" genes are cloned, reducing the number of individual transformants required to represent the entire population of genes.
An alternative procedure can be divided into the following major stages which will also be described more fully herein: (1) recovery and isolation of nuclear DNA found in organisms of the genus Eimeria: (2) fragmentation of the DNA and insertion into a suitable vector; (3) transformation into a suitable microbial host; (4) selection of transformants containing a gene which specifies the antigen of interest; and, (5) recovery and isolation of the cloned gene or gene fragment This route is referred to as the nuclear DNA route. The advantage to this route is that all genes are cloned, allowing the identification of genes not expressed at the time from which mRNA is isolated. These may include genes which are expressed during stages of the life cycle which are not easy to isolate. After recovery and isolation of the cloned gene that is derived from the procedures discussed above, the cloned DNA sequence is advantageously transferred to a suitable expression vector/host cell system for large scale production of the antigenic protein. The DNA sequence that is to be isolated encodes an antigenic protein that will elicit an immune response when administered to chickens which will protect them from subsequent infections. It is not necessary to isolate a complete coccidial gene encoding such a protein, since those portions of the protein termed antigenic determinants are sufficient for triggering a protective immune response (Lerner, supra). This antigenic determinant should be on the surface of the folded microbially-produced protein to trigger the response (Lerner, supra).
In the mRNA route, the sequence may be isolated from the sporo- zoite life stage of the parasite. It has been demonstrated that part of the protective immune response in chickens is directed against the sporozoite.
Antigenic proteins isolated from other life stages also may be effective as vaccines.
MAbs or polyvalent antibodies which bind to various sporozoite proteins can be used to identify cloned DNA sequences encoding those proteins. Such proteins can be isolated and used to elicit a protective immune response in chickens.
Sporozoites can be obtained from oocysts by excystation using the method of Doran and Vetterling, Proc. Helminthol Soc. Wash. 34:59-65 (1967), and purified by the leucopak filter technique of Bontemps and Yvore, Ann. Rech. Vet 5:109-113 (1974). Although the method of
Doran and Vetterling has been found suitable for obtaining sporozoites from oocysts, any method is suitable as long as the nucleic acids within the sporozoites remain intact. Also, sporozoite mRNA may be isolated from intact sporulated oocysts, which contain the sporozoites. mRNA Route
Isolation of mRNA coding for the antigenic proteins of interest is advantageously accomplished by lysis of intact sporulated oocysts under conditions which minimize nuclease activity. This is accomplished using a modification of the procedure described by Pasternak et al. Molec.
Biochem. Parisitol 3:133-142 (1982). Total RNA may be isolated by grinding the oocysts with glass beads in a solution containing guanadine thiocyanate, Sarkosyl, and Tris Buffer pH 8.0. Oocyst proteins are removed by phenol chloroform extraction. The total cellular RNA is separated from DNA by precipitation with lithium chloride. Oligo
(dT)-cellulose chromatography then can be used to isolate mRNA from the total RNA population. Synthesis of cDNA may be accomplished using either a kit from
Boehringer Mannheim which employs avian myeloblastosis virus reverse transcriptase and RNase H or a kit from Pharmacia which employs mouse
Moloney Leukemia virus reverse transcriptase and RNase H. The kits are used according to the instructions provided by the manufacturer. The poly r(A) tail of mRNA permits oligo(dT) (of about 12-18 nucleotides) to be used as a primer for cDNA synthesis or alternatively DNA oligonucleotides of random sequence can be used as a primer for cDNA synthesis.
For purposes of amplification and selection, the ds-cDNA prepared as described above is generally inserted into a suitable cloning vector, which is used for transforming appropriate host cells. Suitable cloning vectors include various plasmids and phages, but a bacteriophage lambda is preferred.
For a cloning vector to be useful for the expression of foreign proteins which are to be detected with antibodies, it should have several useful properties. Most importantly, it should have a cloning site within a gene which is expressed in the host being used. There should also be a means of controlling expression of the gene. The vector should be able to accept DNA of the size required for synthesis of the desired protein product and replicate normally. It is also useful to have a selectable property which allows identification of vectors carrying inserts. A cloning vector having such properties is the bacteriophage λgtll (ATCC 37194) (Young and Davis. Proc. Nat'l Acad. Sci. USA 80:1194-1198 (1983)). This vector has a unique EcoRI site near the end of the bacterial gene coding for /3-galactosidase. That site can be used for insertion of foreign DNA to make hybrid proteins made up of /3-galactosidase and the foreign gene product The expression of /3-galactosidase is under control of the lac promoter and can be induced by the addition of isopropyl- -D- thiogalactopyranoside (IPTG). The λgtll phage contains 43.7 kb of DNA which is considerably smaller than wild type λ. This allows insertion of pieces of DNA up to 8.3 kb in length, before the DNA becomes too large to fit inside the phage head. Because DNA is inserted into the gene for 3-galactosidase, transformants having inserts can easily be distinguished from those which do not by looking for /3-galactosidase activity. An indicator dye, 5-bromo-4-chloro-3-indolyl- ?-D-galactoside (Xgal), can be incorporated with agar plates. /3-galactosidase cleaves this molecule to give a blue product, thus allowing examination of the cultures for the presence of active 3-galactosidase. Those plaques having inserts are colorless on X-gal plates because the insertion of foreign DNA into the /3-galactosidase gene has eliminated its activity. The ds-cDNA can be conveniently inserted into the phage by addition of linkers containing an EcoRI restriction site and any convenient second restriction enzyme recognition site to the DNA and ligation into the EcoRI-cut λgtll DNA. After ligation of the cDNA into the phage DNA, the DNA is packaged, in vitro, into λ phage heads (Enquist and Sternberg, Methods in Enzvmology 68:281-298 (1979) and those phages are used to infect a suitable λ-sensitive host With the proper choice of host, the phage may be screened as plaques or lysogens
(colonies).
Aside from the E. coli bacteriophage λgtll system described, many other host vector combinations have been used successfully for the cloning of foreign genes in E. coli (Principles of Gene Manipulation. 2nd Ed., Old and Primrose, Univ. of California Press, 32-35, 46-47 (1981)).
The foregoing discussion has focused on cloning procedures in gram negative bacteria, e.g., E. coli. Alternatively, foreign genes may be cloned into plasmid vectors that will transform and replicate in a gram positive bacterium such as Bacillus subtilis (Old and Primrose, supra, pp.
51-53) or in a eukaryotic host cell such as yeast (Old and Primrose, supra. pp. 62-68) filamentous fungi, insect cells (U.S. 4,745,051 and 4,879,236) and mammalian cells. The DNA described herein may be inserted into the above vectors by various techniques including homopolymeric tailing, blunt-end ligation or by use of linker molecules (Old and Primrose, supra, at p. 92).
Many immunological methods for screening clone banks for those expressing a desired protein are known and include procedures described by Engvall and Pearlman, Immunochemistry 8:871-874 (1971); Koenen et al. The European Molecular Biology Organization Journal. Vol. 1, No.
4, pp. 509-512 (1982); Broome et al. Proc. Natl. Acad. Scl. USA
75:2746-2749 (1978); Villa-Komaroff et al. Proc. Natl. Acad. Scl. USA
75:3727-3731 (1978); Anderson et al. Methods in Enzvmologv 68:428-436 (1979); Clarke et al. Methods in Enzvmologv 68:436-442 (1979); Ehrlich et al. Methods in Enzvmologv 68:443-453 (1979); Kemp et al. Proc. Natl.
Acad. Scl. USA 78:4520-4524 (1981).
By the cloning procedures outlined, thousands of recombinant bacteriophage are generated which can be screened for production of coccidial antigens. Screening methods depend upon expression of the coccidial antigenic protein either alone or as a fusion protein with a bacterial gene. In the examples included herein, the coccidial antigens are produced as fusions with E. coli /3-galactosidase. The screening methods, therefore, depend on expression of the fusion product and detection of the product by reaction with antibodies, either monoclonal or polyvalent, directed against that antigen.
The recombinant bacteriophages can be used to infect a suitable E. coli host which allows the formation of phage plaques on agar (or agarose) plates. The plaques can be transferred to nitrocellulose membranes while being induced with IPTG. The proper antibodies are then reacted with the filters. After reaction of the primary antibodies with the filters, the positive reactions are detected by reaction with either [125I] Staphylococcus aureus Protein A or a second antibody conjugated with horseradish peroxidase, alkaline phosphatase, or vitamin B12. The plaques containing cross-reactive antigens can then be detected by autoradiography, by detection of the conjugated enzyme, or, in the case of Vitamin B12 conjugates, by binding of streptavidin conjugate to one of the reporter enzymes.
The phages giving positive signals in the antibody-screening procedure can be shown to contain sequences coding for coccidial proteins by excision of the DNA originally inserted into the phage DNA and examination of the ability of that DNA to hybridize with coccidia mRNA or coccidia genomic DNA. The nucleotide sequence of the cDNA insert is determined using the methods of Sanger et al. Proc. Natl Acad. Scl. USA 74:5463-5467 (1977); or Maxam and Gilbert, Proc. Natl. Acad.
Scl. USA 74:560-S64 (1977). Nuclear DNA Route
Another method of cloning coccidial antigens begins with isolation of nuclear DNA from oocysts. This DNA is then broken into fragments of a size suitable for insertion into a cloning vector. To obtain such fragments, one can use mechanical shearing methods such as sonication or high-speed stirring in a blender to produce random breaks in the DNA. Intense sonication with ultrasound can reduce the fragment length to about 300 nucleotide pairs. (Old and Primrose, supra, p. 20.) Alternatively, nuclear DNA may be partially digested with DNAsel, which gives random fragments, with restriction endonucleases, which cut at specific sites, or with mung bean nuclease in the presence of foπnamide, which has been shown with some related organisms (McCutchan, T.F., et al Science 225:625-628 (1984)) to produce DNA fragments containing intact genes.
These nuclear DNA fragments may be inserted into any of the cloning vectors listed for the cloning of cDNA in the mRNA experimental method. If the nuclear DNA is digested with a restriction endonuclease, it can be inserted conveniently into a cloning vector digested with the same enzyme, provided the vector has only one recognition site for that enzyme. Otherwise, DNA fragments may be inserted into appropriate cloning vectors by homopolymeric tailing or by using linker molecules (Old and Primrose, supra, at p. 92).
Advantageously, genomic DNA expression libraries are constructed after digestion on nuclear DNA with any one of a number of restriction enzymes that yield blunt end DNA fragments. The size of the DNA frag¬ ments can be controlled by reducing the time of digestion with the restriction enzyme. Linkers such as an oligonucleotide encoding restriction site for EcoRI and Notl are ligated onto the blunt end DNA fragments and fragments are ligated into the EcoRI site of bacteriophage λgtll. The resulting genomic expression library can be subjected to antibody screening as described for the mRNA route.
Plasmid DNA is isolated from transformants found to be "positive" by the above screening methods. The nuclear DNA inserts of these plasmids are then subjected to DNA sequencing. The cloned DNA sequence may be transferred to expression vectors engineered for high- level production of the antigenic protein. The expression vectors are transformed into suitable host cells for production of the antigenic protein. These host cells may include both prokaryotic and eukaryotic organisms. The prokaryotic host cells include E. coli and B. subtilis. The eukaryotic host cells include yeast, insect cells, and mammalian cells.
Coccidial antigens advantageously may be produced at high levels in E. coli as a fusion protein comprising the antigen and an amino terminal portion of the viral MS-2 polymerase. This fusion is accomplished by inserting a DNA sequence encoding a coccidial antigen into a plasmid vector, pEX32b, carrying the polymerase gene.
In the expression vector used the expression of the fusion antigenic protein is highly regulated by temperature. Host expression vector systems in which expression of foreign proteins is regulatable have the advantage of avoiding possible adverse effects of foreign protein accumulation as high cell densities are reached. Some investigators have proposed that expression of gene fragments such as those encoding antigenic determinants may avoid the deleterious effects that expression of the entire antigenic protein would have on E. coli host cells. (Helfman et al. Proc. Natl Acad. Scl. USA 80:31-35 (1983)).
Coccidial antigens also may be produced in high levels as fusions at the carboxy-terminal of E. coli 3-galactosidase. The coccidia antigen gene is transferred to a small plasmid such as pGX3217 which carries the gene for ampicillin resistance and which contains a /3-galactosidase gene with an EcoRI insertion site near the 3' end of the gene. Synthesis of the fused gene products is regulated by the ]ac promoter which is induced by addition of IPTG, the inducer of the lac operon.
An effective subunit vaccine against avian coccidiosis may consist of a mixture of antigen proteins derived from several species of Eimeria. Alternatively, production costs may be decreased by producing two or more antigen proteins as one fusion protein thus reducing the required number of fermentations and purifications. Such a fusion protein would contain the amino acid sequence comprising an antigenic epitope of each antigen protein (or repetitions of those sequences) with variable amounts of surrounding nonantigenic sequence. A hybrid gene designed to code for such a protein in E. coli would contain bacterial regulatory sequence (promoter/operator) and the 5' end of an E. coli gene (the ribosome binding site and codons for several amino acids) to ensure efficient translation initiation followed by the coding sequences for the antigenic epitopes all fused in the same reading frame.
E. coli cells transformed with the expression vector carrying a cloned coccidial antigen sequence are grown under conditions that promote expression of the antigenic polypeptide. The antigenic protein is then tested for ability to elicit an immune response in chickens that will protect them from subsequent Eimeria infections. The antigen protein may be presented to birds as a live E. coli expressing the protein, as killed E. coli expressing the protein or as purified protein. The antigen may be combined with suitable carriers and adjuvants and administered to birds in their feed or by injection. The cloned antigenic proteins used in vaccines above are tested for their ability to elicit an immune response in chickens that protects the birds from subsequent infection by any of the important species of Eimeria. including E. tenella. E. acervulina. E. brunetti. E. mivati. E. maxima. E. praecox. E. mitis. and E. necatrix. The cloning procedures de- scribed above may be repeated until DNA sequences encoding coccidial antigens that collectively protect chickens against coccidiosis are isolated and used as a vaccine by the methods above.
In addition to cloned antigenic proteins which may be useful as vaccines to protect against coccidiosis, another useful alternative which may be derived from cloning antigen genes is the use of small, synthetic peptides in vaccines (see Lerner, supra). After the sequence of antigenic proteins is determined, it is possible to make synthetic peptides based on that sequence. The peptides are conjugated to a carrier protein such as hemocyanin or serum albumin and the conjugate then can be used to immunize against coccidia.
It is contemplated that the procedures described may also be used to isolate antigenic proteins from other coccidia species that can be used in vaccines to protect other domestic animals from coccidiosis.
The following examples are supplied in order to illustrate, but not necessarily limit, the present invention.
Example 1
Identification of an E. maxima cDNA Clone Encoding Antigen mc-4c with Antiserum Raised Against
Eimeria maxima Sporozoites
In order to identify antigens from Eimeria maxima, expression libraries were prepared in the lambda vector, λgtll, using cDNA prepared from poryA mRNA isolated from E. maxima oocysts. The construction of the libraries is described in Genex Patent Application, PCT/US89/02918 incorporated herein by reference. The cDNA expression library was screened with rat antiserum raised against E. maxima sporozoites. The rat immune serum was prepared using the following protocol. Da
Figure imgf000025_0001
**Sporozoite sample is 8.0X106 sporozoites/ml The indicated vol is mixed 1:1 with Freund's Complete Adjuvant.
The library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transfered to nitrocellulose filters. Positive phage were identified after screening with the rat anti-E. maxima sporozoite antiserum. The cDNA inserts from the positive clones were cloned into bacteriophage M13 and subjected to sequence analysis. Ej. maxima antigen mc-4c was identified.
Antigen mc-4c is an analog of E. acervυlina antigen ac-6b and E tenella antigen GX3262 (Miller etal. Infect. Immun. 57:2014-2020 (1989); Danforth et al. Poultry Sci. 68:1643-1652 (1989)). Only the carboxy terminal sequence of mc-4c was recovered so a cDNA expression library was screened with an 18 bp oligonucleotide complementary to the 5'- sequence of the original mc-4c clone. A second clone, λmc-36c, was identified that encodes all but the six amino terminal amino acids of the antigen. The sequence of mc-4c and the 21.7 Kd translation product that it encodes are shown in Figures 1 and 2 (Sequence ID Nos.l and 2). Example 2
Identification of an E. maxima cDNA Clone Encoding
Antigen mc-5c with Antiserum Raised Against
Eimeria maxima Sporozoites
As described in Example 1, E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage
M13 and subjected to sequence analysis. Following sequence analysis, R tenella antigen mc-5c was identified.
The carboxyl terminal sequence of antigen mc-5c is encoded by 1611 bp of open reading frame. The sequence and its 59.2 Kd translation product are shown in Figures 3 and 4 (Sequence ID Nos.3 and 4).
A partial sequence of antigen mc-5c were expressed in pEX32b.
The terminal 227 amino acids encoded by an EcoRI fragment extending from the internal restriction site starting at base 928 to beyond the site of translation termination (Figure 3) were inserted into pEX32b and expressed. Antigen mc-5c is encoded in expression vector pGX5376.
Example 3
Identification of an E. maxima cDNA Clone Encoding Antigen mc-30c with Antiserum Raised Against Eimeria maxima Sporozoites
As described in Example 1, E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-30c was identified. Antigen mc-30c had 510 bp of open reading frame fused in frame with the /3-galactosidase gene of λgtll. Within the open reading frame are two methionine codons (double underlined) followed by 321 bp of open reading frame ending with a termination codon (Figure 5, Sequence ID No.5). Preceding the putative methionine initiation codons are two
TATA boxes which are underlined. Thus it is possible that the insert contains a complete gene encoding an Eimeria antigen consisting of 109 amino acids with a molecular weight of 12.2 Kd. The amino acid sequence of the mc-30c antigen encoded by the entire open reading frame of mc- 30c is shown in Figure 6 (Sequence ID No.6) with the methionines at the putative initiation site underlined.
Antigen mc-30c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-30c is encoded in expression vector pGX5370 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the PL promoter. The host strain pop2136 contains a temperature sensitive repressor of PL and expression of the fusion protein is fully induced at 42°C.
Example 4 Identification of an E. maxima cDNA Clone Encoding
Antigen mc-35c with Antiserum Raised Against Eimeria maxima Sporozoites
As described in Example 1, E. maxima libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis, E. tenella antigen mc-35c was identified. Antigen mc-35c is encoded by 1461 bp of open reading frame fused in-frame with the /3-galactosidase sequence. The 5'-sequence consists of a 24 bp sequence encoding eight amino acids which is repeated with complete fidelity a total of 24 times. This repeat is linked through a single glutamic acid residue to a second, different 24 bp sequence which is repeated 16 times. The repeats are followed by 501 bp of open reading frame followed by a termination codon. The nucleotide sequence of mc- 35c is shown in Figure 7 (Sequence ID No.7) and the amino acid sequence of mc-35c is shown in Figure 8 (Sequence ID No.8) with the first of each of the eight amino acid repeats underlined and the connecting glutamic acid residue double underlined.
Antigen mc-35c was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen mc-35c is encoded in expression vector pGX5367 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the PL promoter. The host strain pop2136 contains a temperature sensitive repressor of PL and expression of the fusion protein is fully induced at 42°C.
Example 5
Identification of an E. tenella Genomic DNA Clone Encoding Antigen tg-3e with Immune Bile Recovered from Chickens Infected with Eimeria tenella
In order to identify antigens of E. tenella that elicite a mucosal immune response during parasitic infection of chickens, E. tenella cDNA and genomic expression libraries in lambda phage were screened with chicken immune bile. Immune bile was recovered from chickens infected with E. tenella oocysts seven days after the onset of infection. Before being used to screen the libraries, the immune bile was incubated with R coli proteins to inactivate antibodies cross-reactive with E. coli proteins. After incubation of the filters used to lift the lambda plaques from the growth plates with the immune bile, the filters were developed with goat anti-chicken IgA antibodies. The positive plaques were visualized with donkey anti-goat antibodies conjugated with alkaline phosphatase. Phage that produce antigens cross-reactive with the immune bile were identified. The DNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis.
Screening of an E. tenella genomic expression library identified antigen tg-3e which consists of 366 bp of open reading frame encoding
122 amino acids. The sequence of tg-3e and the 12.7 Kd translation product that it encodes are shown in Figures 9 and 10 (Sequence ID Nos. 9 and 10).
Antigen tg-3e was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tg-3e is encoded in expression vector pGX5390 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the PL promoter. The host strain pop2136 contains a temperature sensitive repressor of PL and expression of the fusion protein is fully induced at 42°C.
Example 6
Identification of an E. tenella cDNA Clone Encoding
Antigen tc-lle with Immune Bile Recovered from
Chickens Infected with Eimeria tenella
As described in Example 5, E. tenella cDNA libraries were screened with the chicken anti-E. tenella immune bile. Phage that produce antigens cross-reactive with the immune bile were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M13 and subjected to sequence analysis. Following sequence analysis R tenella antigen tc-lle was identified.
Antigen tc-lle consists of a 417 bp open reading frame encoding 139 amino acids. The sequence of tc-lle and the 13.9 Kd translation product that it encodes are shown in Figures 11 and 12 (Sequence ID
Nos. 11 and 12).
Antigen tc-lle was expressed in E. coli after insertion into the plasmid expression vector pEX-32b. Antigen tc-lle is encoded in expression vector pGX5394 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the PL promoter. The host strain pop2136 contains a temperature sensitive repressor of PL and expression of the fusion protein is fully induced at 42°C.
Example 7 Identification of an E. tenella cDNA Clone
Encoding Antigen tc-23g with Antiserum Raised Against Eimeria tenella Sporozoites
In order to identify antigens from Eimeria tenella. expression libraries were prepared in the lambda vector, λgtll, using cDNA prepared from polyA mRNA isolated from E. tenella oocysts. The construction of the libraries has been described previously (U.S. Ser. No. 07 215,162; Berger et al. Methods in Enzvmology. Vol. 152, Academic Press, New York, NY (1987)). The cDNA expression library was screened with chicken antiserum raised against E. tenella sporozoites. The chicken immune serum was prepared using the following protocol.
Figure imgf000031_0001
The library to be screened was plated on a host that allows lysis and plaque formation. Following induction of the antigens encoded by the phage, the plaques were transferred to nitrocellulose filters using standard protocols (Berger et al. Methods in Enzvmologv, Vol 152, Academic Press, New York, NY (1987)). The filters were screened with the chicken anti-E. tenella sporozoite antiserum. Plaques recognized by chicken serum IgA antibodies were identified with goat anti-chicken IgA antibody followed by biotinylated donkey anti-goat IgG antibody. The positive plaques were visualized with an alkaline phosphatase-streptavidin conjugate using standard protocols (Berger et al. Methods in Enzvmologv. Vol 152, Academic Press, New York, NY (1987)). The cDNA insert from the positive clone was cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. fProc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). The carboxy terminal sequence of E. tenella antigen tc-23g was identified. The nucleotide se- quence of tc-23g and the 20 Kd translation product that it encodes are shown in Figures 13 and 14 (Sequence ID Nos.13 and 14).
Antigens tc-23g was expressed in E. coli after insertion into the plasmid expression vector pEX-32b using standard protocols (Berger et al. Methods in Enzymology. Vol. 152, Academic Press, New York, NY
(1987)). The antigen is encoded in expression vector pGX5398 and is expressed as a fusion protein with 11 Kd of the MS-2 polymerase under control of the PL promoter. The host strain pop2136 contains a tempera¬ ture sensitive repressor of PL and expression of the fusion protein is fully induced at 42°C.
Example 8
Identification of E. tenella cDNA Clone Encoding Antigen tc-26h with Antiserum Raised Against Eimeria tenella Merozoites
Using the techniques described in Example 7, an E. tenella cDNA library was screened with chicken anti-E. tenella merozoites immune serum produced using the same injection regimen described in Example
7. A phage that produces an antigen cross-reactive with IgG antibodies in the immune serum was identified with biotinylated goat anti-chicken IgG antibody followed by visualization with an alkaline phosphatase-strep- tavidin conjugate. The cDNA insert from the positive phage was cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Proc. Natl. Acad. Sci. USA 74-5463-5467 (1977)). Following sequence analysis, E. tenella antigen tc-26h was identified. The antigen consists of a peptide fragment which is composed of long strings of alanines and serines. The nucleotide sequence of tc-26h and the 10 Kd translation product that it encodes are shown in Figures 15 and 16
(Sequence ID Nos.15 and 16). Example 9
Identification of E. tenella cDNA Clones
Encoding Antigens tc-30c. tc-32c. tc-33c and tc-35c with Antiserum Raised Against E. maxima Sporozoites
Using the techniques described in Example 7, an E. tenella cDNA library was screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. fProc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigens to tc-30c, tc-32c, tc-33c and tc-35c were identified. Antigen tc-30c consisted of 284 bp of open reading frame encoding an antigen fragment fused in-frame with the /3-galactosidase gene of λgtll. The nucleotide sequence of tc-30c and the 9.5 Kd translation product that it encodes are shown in Figures 17 and 18 (Sequence ID Nos.17 and 18). Antigen tc-32c consisted of 261 bp of open reading frame encoding an antigen fragment The nucleotide sequence of tc-32c and the 8.8 Kd translation product that it encodes are shown in Figures 19 and 20 (Sequence ID Nos.19 and 20).
Antigen tc-33c consisted of 408 bp of open reading frame encoding the carboxy terminal sequence of an antigen. The nucleotide sequence of tc-33c and the 12.5 Kd translation product that it encodes are shown in Figures 21 and 22 (Sequence ID Nos.21 and 22).
Antigen tc-35c consisted of 120 bp of open reading frame encoding the carboxy terminal sequence of an antigen. The nucleotide sequence of tc-35c and the 5.0 Kd translation product that it encodes are shown in
Figures 23 and 24 (Sequence ID Nos.23 and 24). Example 10
Identification of an E. maxima cDNA Clone
Encoding Antigen mc-37c with Antiserum
Raised Against E. maxima Sporozoites
Using the techniques described in Example 7, E. maxima cDNA libraries were screened with the rat anti-E. maxima sporozoite immune serum. Phage that produce antigens cross-reactive with the immune serum were identified. The cDNA inserts from the positive phage were cloned into bacteriophage M-13 and subjected to sequence analysis using the method of Sanger et al. (Troc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Following sequence analysis, E. tenella antigen mc-37c was identified. Antigen mc-37c consists of 442 bp of open reading frame encoding an antigen fragment that is fused in-frame with the /3-galactosidase sequence of λgtll. The nucleotide sequence of mc-37c and the 16.2 Kd peptide that it encodes are shown in Figures 25 and 26 (Sequence ID Nos. 25 and 26). While certain representative embodiments and details have been shown for the purpose of illustrating the invention, it will be apparent to those skilled in the art that various changes and modifications may be made therein without departing from the scope of the invention.
SEOUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: JACOBSON, JAMES
ST AUSBERG, ROBERT L WILSON, SUSAN POPE, SHARON STRAUSBERG, SUSAN LEE RAETHER, WOLFGANG
(ii) TITLE OF INVENTION: GENETICALLY ENGINEERED COCCIDIOSIS VACCINE
(iii) NUMBER OF SEQUENCES: 26
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Sterne, Kessler, Goldstein & Fox
(B) STREET: 1225 Connecticut Ave. NW Suite 300
(C) CITY: Washington
(D) STATE: D.C.
(E) COUNTRY: USA
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(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/581,694
(B) FILING DATE: 12-SEP-1990
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: GOLDSTEIN, JORGE A
(B) REGISTRATION NUMBER: 29,021
(C) REFERENCE DOCKET NUMBER: 0977.1590000
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 466-0800
(B) TELEFAX: (202) 833-8716
(2) INFORMATION FOR SEO ID NO:1: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 633 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..633
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GGT ATA ATT GGA GGA ATA ATT GGT GCT GCT GCT GCT GTA GAT GTA CCA 48
Gly He He Gly Gly He He Gly Ala Ala Ala Ala Val Asp Val Pro
1 5 10 15
GCA GAA GGA GAG AGA CAC CCT CGT GCT GCA GCA GGT ACA GAT TGG GGT 96
Ala Glu Gly Glu Arg His Pro Arg Ala Ala Ala Gly Thr Asp Trp Gly
20 25 30
GTA TGT ACA GCT AAA TTA GGG GAC ACA ATA AAG GAG ATA GAG ACA GTA 144
Val Cys Thr Ala Lys Leu Gly Asp Thr He Lys Glu He Glu Thr Val
35 40 45
ATA AAT AGT GTC TCC TTT ATT GCC GGT AGA TTA GCA AAT TGT CTC CGT 192
He Asn Ser Val Ser Phe He Ala Gly Arg Leu Ala Asn Cys Leu Arg
50 55 60
ATA GGT ATT GAG CAT CTA AGT AAG TGT ATA CTG TCT CCT TCT TCC TCC 240
He Gly He Glu His Leu Ser Lys Cys He Leu Ser Pro Ser Ser Ser
65 70 75 80
TCC TCC TCC TTC CCT TCC TCC TCC TCC TCT TGC CCA TGC CTA CTA GAT 288
Ser Ser Ser Phe Pro Ser Ser Ser Ser Ser Cys Pro Cys Leu Leu Asp
85 90 95
AAG AAT GAT ATT AAT GAG GGT ATA GAG GCA GGG AGA CAG GGG ACA GAG 336
Lys Asn Asp He Asn Glu Gly He Glu Ala Gly Arg Gin Gly Thr Glu
100 105 110
TGT CTC CTT AGG GGG GGT AAA TTA TTT ATT GAG GTA TTA ATA GAT GCA 384
Cys Leu Leu Arg Gly Gly Lys Leu Phe He Glu Val Leu He Asp Ala
115 120 125
AGT AAG ATA GCA GCA ACA AGG TGT CTC CTA TTA GCA GCA AGC AGC AAG 432
Ser Lys He Ala Ala Thr Arg Cys Leu Leu Leu Ala Ala Ser Ser Lys
130 135 140 GAT ATT ATT ATT AGG CAG CTA CCT TAT ACA CAA GAT AAA TTA TAT AAG 480 Asp He He He Arg Gin Leu Pro Tyr Thr Gin Asp Lys Leu Tyr Lys 145 150 155 160
ACT TAT TCT TCT TTC CTT AGG GGA TAC CAG AGC 528 Thr Tyr Ser Ser Phe Leu Arg Gly Tyr Gin Ser 165 170
Figure imgf000037_0001
CTT GGT GGT GCT GCT GCT GCA CCT TAT TCT GCT GCA GCA CAG CAG CAA 576 Leu Gly Gly Ala Ala Ala Ala Pro Tyr Ser Ala Ala Ala Gin Gin Gin 180 185 190
CCT AGC TAT GGT GCT CCT CCT GCT AGT CAG CAA CCA TCA GGG GGA TTC 624 Pro Ser Tyr Gly Ala Pro Pro Ala Ser Gin Gin Pro Ser Gly Gly Phe
195 200 205
TTC TGG TAA 633 Phe Trp 210
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Gly He He Gly Gly He He Gly Ala Ala Ala Ala Val Asp Val Pro 1 5 10 15
Ala Glu Gly Glu Arg His Pro Arg Ala Ala Ala Gly Thr Asp Trp Gly 20 25 30
Val Cys Thr Ala Lys Leu Gly Asp Thr He Lys Glu He Glu Thr Val 35 40 45
He Asn Ser Val Ser Phe He Ala Gly Arg Leu Ala Asn Cys Leu Arg 50 55 60
He Gly He Glu His Leu Ser Lys Cys He Leu Ser Pro Ser Ser Ser 65 70 75 80
Ser Ser Ser Phe Pro Ser Ser Ser Ser Ser Cys Pro Cys Leu Leu Asp 85 90 95 Lys Asn Asp He Asn Glu Gly He Glu Ala Gly Arg Gin Gly Thr Glu 100 105 110
Cys Leu Leu Arg Gly Gly Lys Leu Phe He Glu Val Leu He Asp Ala 115 120 125
Ser Lys He Ala Ala Thr Arg Cys Leu Leu Leu Ala Ala Ser Ser Lys 130 135 140
Asp He He He Arg Gin Leu Pro Tyr Thr Gin Asp Lys Leu Tyr Lys 145 150 155 160
Thr Tyr Ser Ser Phe Leu Arg Gly Tyr Gin Ser Ser Ala Ala Ala Arg 165 170 175
Leu Gly Gly Ala Ala Ala Ala Pro Tyr Ser Ala Ala Ala Gin Gin Gin 180 185 190
Pro Ser Tyr Gly Ala Pro Pro Ala Ser Gin Gin Pro Ser Gly Gly Phe 195 200 205
Phe Trp 210
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1611 base pairs
(B) TYPE: nucleic acid
(C) STRAHDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
Figure imgf000038_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ACG GAT ACA GAG AGG TTA GTA GGG GAT GCA GCA AAG AAT CAG GTA GCA 48
Thr Asp Thr Glu Arg Leu Val Gly Asp Ala Ala Lys Asn Gin Val Ala
1 5 10 15
AGG AAT CCA GAG AAT ACA GTA TTT GAT GCA AAG AGG TTG ATA GGT AGG 96
Arg Asn Pro Glu Asn Thr Val Phe Asp Ala Lys Arg Leu He Gly Arg 20 25 30 AAG TAT GAC GAT CCA GCT GTA CAG GCA GAC ATG AAG CAT TGG CCC TTT 144 Lys Tyr Asp Asp Pro Ala Val Gin Ala Asp Met Lys His Trp Pro Phe 35 40 45
ATA GTA AAA GGA GGA CCA GGA GGT AAA CCA TTA ATA GAA GTA AAT TAC 192 He Val Lys Gly Gly Pro Gly Gly Lys Pro Leu He Glu Val Asn Tyr 50 55 60
CAA GGA ATT AAG AAA ACA TTT CAT CCT GAG GAG ATA AGT GCT ATG GTA 240
Gin Gly He Lys Lys Thr Phe His Pro Glu Glu He Ser Ala Met Val
65 70 75 80
TTA ATG AAG ATG AAG GAG ATT GCT GAA CAA TTT ATA GGT AAA GAA ATA 288 Leu Met Lys Met Lys Glu He Ala Glu Gin Phe He Gly Lys Glu He 85 90 95
AAA GAA GCA GTA ATA ACA GTA CCT GCA TAT TTT AAT GAT TCA CAG AGA 336 Lys Glu Ala Val He Thr Val Pro Ala Tyr Phe Asn Asp Ser Gin Arg 100 105 110
CAG GCA ACA AAG GAT GCA GGT ACT ATA GCG GGT CTG AAT GTA TTA CGT 384 Gin Ala Thr Lys Asp Ala Gly Thr He Ala Gly Leu Asn Val Leu Arg 115 120 125
ATA ATA AAT GAG CCA ACA GCA GCA GCA ATA GCT TAT GGG TTG GAT AAG 432 He He Asn Glu Pro Thr Ala Ala Ala He Ala Tyr Gly Leu Asp Lys 130 135 140
AAG GGA CAA GGG GAG ATG AAT GTT CTT ATC TTT GAT ATG GGG GGA GGG 480 Lys Gly Gin Gly Glu Met Asn Val Leu He Phe Asp Met Gly Gly Gly 145 150 155 160
ACC TTT GAT GTC TCC TTG CTT ACT ATA GAG GAT GGC ATC TTC GAG GTC 528 Thr Phe Asp Val Ser Leu Leu Thr He Glu Asp Gly He Phe Glu Val 165 170 175
AAG GCT ACA GCA GGA GAT ACA CAT CTA GGG GGA GAA GAC TTT GAT AAT 576 Lys Ala Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn 180 185 190
AGA TTA GTA GAT TTT TGT ATA CAA GAT TTT AAG AGA AAG AAT AGA TCT 624 Arg Leu Val Asp Phe Cys He Gin Asp Phe Lys Arg Lys Asn Arg Ser 195 200 205
AAG GAT CCA AGT AAT AAT AGT AGG GCA TTA AGG AGA CTA CGT ACA CAG 672 Lys Asp Pro Ser Asn Asn Ser Arg Ala Leu Arg Arg Leu Arg Thr Gin 210 215 220
TGC GAG AGA GCA AAG AGG ACA CTA AGT AGT AGT ACA CAA GCT ACT ATA 720 Cys Glu Arg Ala Lys Arg Thr Leu Ser Ser Ser Thr Gin Ala Thr He 225 230 235 240 GAG ATT GAT AGT TTA TAT GAG GGT ATT GAT TAC TCT GTC TCC TTA TCT 768 Glu He Asp Ser Leu Tyr Glu Gly He Asp Tyr Ser Val Ser Leu Ser 245 250 255
AGG GCT AGA TTC GAG GAA TTC TGT ATG GAT TAT TTC CGT AAC TCT CTT 816 Arg Ala Arg Phe Glu Glu Phe Cys Met Asp Tyr Phe Arg Asn Ser Leu 260 265 270
ATA CCC GTA GAA AAA GTA CTA AAA GAT AGT AAT ATA GAT AAG AGG AGT 864 He Pro Val Glu Lys Val Leu Lys Asp Ser Asn He Asp Lys Arg Ser 275 280 285
GTA CAT GAG GTA GTA TTA GTA GGA GGG AGT ACA AGA ATA CCT AAG ATA 912 Val His Glu Val Val Leu Val Gly Gly Ser Thr Arg He Pro Lys He 290 295 300
CAA CAA CTT ATA CAA GAA TTC TTT AAT GGT AAG GAA CCA TGT AGA TCT 960 Gin Gin Leu He Gin Glu Phe Phe Asn Gly Lys Glu Pro Cys Arg Ser 305 310 315 320
ATT AAT CCT GAT GAG GCT GTT GCT TAT GGT GCA GCT GTA CAG GCA GCT 1008 He Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gin Ala Ala 325 330 335
ATA TTA AAA GGA GTA AAT AGT ACA CAA GTA CAA GAT TTA TTA TTA TTA 1056 He Leu Lys Gly Val Asn Ser Thr Gin Val Gin Asp Leu Leu Leu Leu 340 345 350
GAT GTT GCT CCT TTA TCA TTA GGA TTA GAG ACA GCA GGA GGA GTA ATG 1104 Asp Val Ala Pro Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met 355 360 365
ACA AAA TTA ATA GAA AGG AAC ACA ACA ATA CCA ACA AAA AAA TCA CAA 1152
Thr Lys Leu He Glu Arg Asn Thr Thr He Pro Thr Lys Lys Ser Gin 370 375 380
ATA TTC ACA ACA TAT GCA GAT AAT CAA CCA GGT GTA CTT ATT CAG GTG 1200 He Phe Thr Thr Tyr Ala Asp Asn Gin Pro Gly Val Leu He Gin Val 385 390 395 400
TAT GAA GGG GAA AGG GCT ATG ACT AAA GAT AAT AAT TTA CTA GGA AAG 1248 Tyr Glu Gly Glu Arg Ala Met Thr Lys Asp Asn Asn Leu Leu Gly Lys 405 410 415
TTC CAT CTA GAT GGT ATA CCT CCT GCA CCT AGA GGT GTA CCA CAA ATA 1296 Phe His Leu Asp Gly He Pro Pro Ala Pro Arg Gly Val Pro Gin He 420 425 430
GAG GTT ACC TTT GAT ATA GAT GCC AAT GGT ATT ATG AAT GTT ACT GCT 1344 Glu Val Thr Phe Asp He Asp Ala Asn Gly He Met Asn Val Thr Ala 435 440 445 ACA GAG AAA AAT ACA GGA AAA AGT AAC CAA ATA ACT ATA ACT AAT GAT 1392 Thr Glu Lys Asn Thr Gly Lys Ser Asn Gin He Thr He Thr Asn Asp 450 455 460
AAG GGT AGA CTT AGT CAA GGA GAA ATA GAT AGA ATG GTT GCT GAG GCA 1440 Lys Gly Arg Leu Ser Gin Gly Glu He Asp Arg Met Val Ala Glu Ala 465 470 475 480
GAA AAG TAT AAG GCA GAG GAT GAG GCT AAT AAA CAA AGG ATA GAG GCA 1488 Glu Lys Tyr Lys Ala Glu Asp Glu Ala Asn Lys Gin Arg He Glu Ala 485 490 495
AAA AAT AAT CTA GAG AAT TAT TGT TAT AGT ATG AGG AGT ACA CTT GAT 1536 Lys Asn Asn Leu Glu Asn Tyr Cys Tyr Ser Met Arg Ser Thr Leu Asp 500 505 510
GAG GAA AAG GTT AAG GAT AAA ATT AGT AAG GAA GAT AAG GAT ACT GCT 1584 Glu Glu Lys Val Lys Asp Lys He Ser Lys Glu Asp Lys Asp Thr Ala 515 520 525
GCT GCT GCT ATA CAG AAA ACC TCT TGA 1611
Ala Ala Ala He Gin Lys Thr Ser 530 535
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 536 amino acids
(B) TYPE: amino acid (0) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Thr Asp Thr Glu Arg Leu Val Gly Asp Ala Ala Lys Asn Gin Val Ala 1 5 10 15
Arg Asn Pro Glu Asn Thr Val Phe Asp Ala Lys Arg Leu He Gly Arg 20 25 30
Lys Tyr Asp Asp Pro Ala Val Gin Ala Asp Met Lys His Trp Pro Phe 35 40 45
He Val Lys Gly Gly Pro Gly Gly Lys Pro Leu He Glu Val Asn Tyr 50 55 60
Gin Gly He Lys Lys Thr Phe His Pro Glu Glu He Ser Ala Met Val 65 70 75 80 Leu Met Lys Met Lys Glu He Ala Glu Gin Phe He Gly Lys Glu He 85 90 95
Lys Glu Ala Val He Thr Val Pro Ala Tyr Phe Asn Asp Ser Gin Arg 100 105 110
Gin Ala Thr Lys Asp Ala Gly Thr He Ala Gly Leu Asn Val Leu Arg 115 120 125
He He Asn Glu Pro Thr Ala Ala Ala He Ala Tyr Gly Leu Asp Lys 130 135 140
Lys Gly Gin Gly Glu Met Asn Val Leu He Phe Asp Met Gly Gly Gly 145 150 155 160
Thr Phe Asp Val Ser Leu Leu Thr He Glu Asp Gly He Phe Glu Val 165 170 175
Lys Ala Thr Ala Gly Asp Thr His Leu Gly Gly Glu Asp Phe Asp Asn 180 185 190
Arg Leu Val Asp Phe Cys He Gin Asp Phe Lys Arg Lys Asn Arg Ser 195 200 205
Lys Asp Pro Ser Asn Asn Ser Arg Ala Leu Arg Arg Leu Arg Thr Gin 210 215 220
Cys Glu Arg Ala Lys Arg Thr Leu Ser Ser Ser Thr Gin Ala Thr He 225 230 235 240
Glu He Asp Ser Leu Tyr Glu Gly He Asp Tyr Ser Val Ser Leu Ser 245 250 255
Arg Ala Arg Phe Glu Glu Phe Cys Met Asp Tyr Phe Arg Asn Ser Leu 260 265 270
He Pro Val Glu Lys Val Leu Lys Asp Ser Asn He Asp Lys Arg Ser 275 280 285
Val His Glu Val Val Leu Val Gly Gly Ser Thr Arg He Pro Lys He 290 295 300
Gin Gin Leu He Gin Glu Phe Phe Asn Gly Lys Glu Pro Cys Arg Ser 305 310 315 320
He Asn Pro Asp Glu Ala Val Ala Tyr Gly Ala Ala Val Gin Ala Ala 325 330 335
He Leu Lys Gly Val Asn Ser Thr Gin Val Gin Asp Leu Leu Leu Leu 340 345 350 Asp Val Ala Pro Leu Ser Leu Gly Leu Glu Thr Ala Gly Gly Val Met 355 360 365
Thr Lys Leu He Glu Arg Asn Thr Thr He Pro Thr Lys Lys Ser Gin 370 375 380
He Phe Thr Thr Tyr Ala Asp Asn Gin Pro Gly Val Leu He Gin Val 385 390 395 400
Tyr Glu Gly Glu Arg Ala Met Thr Lys Asp Asn Asn Leu Leu Gly Lys 405 410 415
Phe His Leu Asp Gly He Pro Pro Ala Pro Arg Gly Val Pro Gin He 420 425 430
Glu Val Thr Phe Asp He Asp Ala Asn Gly He Met Asn Val Thr Ala 435 440 445
Thr Glu Lys Asn Thr Gly Lys Ser Asn Gin He Thr He Thr Asn Asp 450 455 460
Lys Gly Arg Leu Ser Gin Gly Glu He Asp Arg Met Val Ala Glu Ala 465 470 475 480
Glu Lys Tyr Lys Ala Glu Asp Glu Ala Asn Lys Gin Arg He Glu Ala 485 490 495
Lys Asn Asn Leu Glu Asn Tyr Cys Tyr Ser Met Arg Ser Thr Leu Asp 500 505 510
Glu Glu Lys Val Lys Asp Lys He Ser Lys Glu Asp Lys Asp Thr Ala 515 520 525
Ala Ala Ala He Gin Lys Thr Ser 530 535
(2) INFORMATION FOR SEQ 10 NO:5:
(i) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 513 base pairs
(B) TYPE: nucleic acid
(C) STRANOEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..513 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAA GAA GCA GCA GAT ACA TCA GCA TGG GAT ACA TCC GTC AAG GAA TGG 48
Glu Glu Ala Ala Asp Thr Ser Ala Trp Asp Thr Ser Val Lys Glu Trp 1 5 10 15
CTT GTT GAT ACA GGA AGA GTT TGT GCA GGT GCA GTA GCA AGC TTA GCA 96
Leu Val Asp Thr Gly Arg Val Cys Ala Gly Ala Val Ala Ser Leu Ala 20 25 30
GAT GAA GGT CGT ATA TTT GGT TGT GCA ATA GAT AAT GAG AAT GAA TCT 144
Asp Glu Gly Arg He Phe Gly Cys Ala He Asp Asn Glu Asn Glu Ser
35 40 45
GCA TGG GAG AAA TTA ATA AAA AAT AAT TAT AAA ATA GAA ATG ATG AAA 192
Ala Trp Glu Lys Leu He Lys Asn Asn Tyr Lys He Glu Met Met Lys
50 55 60
GAA GAT GGA GAA ATT GAA TTA ATT GAT TGT TAT GAA CAT GAA TTA CTT 240
Glu Asp Gly Glu He Glu Leu He Asp Cys Tyr Glu His Glu Leu Leu 65 70 75 80
AGA CAT GCA ATT GTT GAT GGT AAA GCA CCT AAT GGT GTA TAT ATT GGA 288
Arg His Ala He Val Asp Gly Lys Ala Pro Asn Gly Val Tyr He Gly 85 90 95
GGT ATT AAA TAT AAA CTT GCT GAA GTT AAA CGT GAT TTT ACT TAT AAT 336
Gly He Lys Tyr Lys Leu Ala Glu Val Lys Arg Asp Phe Thr Tyr Asn
100 105 110
GAT CAG AAC TAT GAT ATA GCT ATA CTA GGT AAG AAT AAA GGA GGA GGT 384
Asp Gin Asn Tyr Asp He Ala He Leu Gly Lys Asn Lys Gly Gly Gly
115 120 125
TTT CTT ATA AAA ACA CCT AAT GAT AAT GTA GTT GTT GCC CTT TAT GAC 432
Phe Leu He Lys Thr Pro Asn Asp Asn Val Val Val Ala Leu Tyr Asp
130 135 140
GAA GAA AAA GAA CAA AAT AAG GCA GAT GCA TTA ACT ACT GCA TTA GCA 480
Glu Glu Lys Glu Gin Asn Lys Ala Asp Ala Leu Thr Thr Ala Leu Ala
145 150 155 160
TTT GCT GAA TAC CTA TAT CAA GGA GGA TTC TAA 513
Phe Ala Glu Tyr Leu Tyr Gin Gly Gly Phe 165 170
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 170 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Glu Ala Ala Asp Thr Ser Ala Trp Asp Thr Ser Vat Lys Glu Trp 1 5 10 15
Leu Val Asp Thr Gly Arg Val Cys Ala Gly Ala Val Ala Ser Leu Ala 20 25 30
Asp Glu Gly Arg He Phe Gly Cys Ala He Asp Asn Glu Asn Glu Ser 35 40 45
Ala Trp Glu Lys Leu He Lys Asn Asn Tyr Lys He Glu Met Met Lys 50 55 60
Glu Asp Gly Glu He Glu Leu He Asp Cys Tyr Glu His Glu Leu Leu 65 70 75 80
Arg His Ala He Val Asp Gly Lys Ala Pro Asn Gly Val Tyr He Gly 85 90 95
Gly He Lys Tyr Lys Leu Ala Glu Val Lys Arg Asp Phe Thr Tyr Asn 100 105 110
Asp Gin Asn Tyr Asp He Ala He Leu Gly Lys Asn Lys Gly Gly Gly 115 120 125
Phe Leu He Lys Thr Pro Asn Asp Asn Val Val Val Ala Leu Tyr Asp 130 135 140
Glu Glu Lys Glu Gin Asn Lys Ala Asp Ala Leu Thr Thr Ala Leu Ala 145 150 155 160
Phe Ala Glu Tyr Leu Tyr Gin Gly Gly Phe 165 170
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1464 ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
Figure imgf000046_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 48
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
1 5 10 15
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 96
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
20 25 30
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 144
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
35 40 45
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 192
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
50 55 60
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 240
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
65 70 75 80
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 288
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
85 90 95
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 336
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
100 105 110
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 384
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
115 120 125
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 432
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
130 135 140
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 480
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
145 150 155 160
GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 528
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro
165 170 175 GAA GAT GAT AAG GTT GAG AAA CCT GAA GAT GAT AAG GTT GAG AAA CCT 576 Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 180 185 190
GAA GAA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 624 Glu Glu Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 195 200 205
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 672 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 210 215 220
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 720 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 225 230 235 240
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 768 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 245 250 255
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 816 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 260 265 270
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 864 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 275 280 285
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 912 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 290 295 300
GGT CCA CCT CAA AGG CCT GGA CAT GGT CCA CCT CAA AGG CCT GGA CAT 960 Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 305 310 315 320
GAT GGG GGA CAC CCA AGT AGG GGA TCA GGC CGA GGA GGT CTC ATC CCA 1008 Asp Gly Gly His Pro Ser Arg Gly Ser Gly Arg Gly Gly Leu He Pro 325 330 335
AAG AGA TTT GCA GGC AGA CCA GAC AGA GGC AGC GAG CAG AAT CAA GAG 1056 Lys Arg Phe Ala Gly Arg Pro Asp Arg Gly Ser Glu Gin Asn Gin Glu 340 345 350
GAG GAA CAA TCA GGA GGC CAG AAT TCA ACC CGC AGT GCG GAG TCA GAT 1104 Glu Glu Gin Ser Gly Gly Gin Asn Ser Thr Arg Ser Ala Glu Ser Asp 355 360 365
GGT GAG CAG TAT GAT CAG CAA ATA GAT GAC CAC CAG TCT CAG CTT GCT 1152 Gly Glu Gin Tyr Asp Gin Gin He Asp Asp His Gin Ser Gin Leu Ala 370 375 380 GAG GAC CTG GAA CTC GCA GCG AAG GAA GCC AGA CAA GCA GCG TTC CGA 1200 Glu Asp Leu Glu Leu Ala Ala Lys Glu Ala Arg Gin Ala Ala Phe Arg 385 390 395 400
CTG CGC CGC GCG GCA GCT GCA GCG CGC AAG GCC GCC GCC GCA GCC CGT 1248 Leu Arg Arg Ala Ala Ala Ala Ala Arg Lys Ala Ala Ala Ala Ala Arg 405 410 415
GAA CGA GTG GAG GCA ACC GGT TTT GAA GGC GGC GCT GAG CCC TCT CCT 1296 Glu Arg Val Glu Ala Thr Gly Phe Glu Gly Gly Ala Glu Pro Ser Pro 420 425 430
CCT ACC AGC TCC ACT AGT TCA GGG GAC TCC GGC GCA GAC AGC TCC GAT 1344 Pro Thr Ser Ser Thr Ser Ser Gly Asp Ser Gly Ala Asp Ser Ser Asp 435 440 445
GAT AGG GAA GGA CGA GGA AAT GAT TCG GCT GAG CAA CAG GAG AGA AGC 1392 Asp Arg Glu Gly Arg Gly Asn Asp Ser Ala Glu Gin Gin Glu Arg Ser 450 455 460
GGT CAC CAG AGT TCC AAC GGC GAG TCT AGC CGT GGG GCC CTG AGC CGC 1440 Gly His Gin Ser Ser Asn Gly Glu Ser Ser Arg Gly Ala Leu Ser Arg 465 470 475 480
AGT CTC CGG GGA TAT CGC ATG TAA 1464 Ser Leu Arg Gly Tyr Arg Met 485
(2) INFORMATION FOR SEQ ID NQ:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Glu Asp Asp Lys Vat Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 1 5 10 15
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 20 25 30
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 35 40 45
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 50 55 60 Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 65 70 75 80
Glu Asp Asp Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 85 90 95
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 100 105 110
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 115 120 125
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 130 135 140
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 145 150 155 160
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 165 170 175
Glu Asp Asp Lys Val Glu Lys Pro Glu Asp Asp Lys Val Glu Lys Pro 180 185 190
Glu Glu Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 195 200 205
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 210 215 220
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 225 230 235 240
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 245 250 255
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 260 265 270
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 275 280 285
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 290 295 300
Gly Pro Pro Gin Arg Pro Gly His Gly Pro Pro Gin Arg Pro Gly His 305 310 315 320
Asp Gly Gly His Pro Ser Arg Gly Ser Gly Arg Gly Gly Leu He Pro 325 330 335 Lys Arg Phe Ala Gly Arg Pro Asp Arg Gly Ser Glu Gin Asn Gin Glu 340 345 350
Glu Glu Gin Ser Gly Gly Gin Asn Ser Thr Arg Ser Ala Glu Ser Asp 355 360 365
Gly Glu Gin Tyr Asp Gin Gin He Asp Asp His Gin Ser Gin Leu Ala 370 375 380
Glu Asp Leu Glu Leu Ala Ala Lys Glu Ala Arg Gin Ala Ala Phe Arg 385 390 395 400
Leu Arg Arg Ala Ala Ala Ala Ala Arg Lys Ala Ala Ala Ala Ala Arg 405 410 415
Glu Arg Val Glu Ala Thr Gly Phe Glu Gly Gly Ala Glu Pro Ser Pro 420 425 430
Pro Thr Ser Ser Thr Ser Ser Gly Asp Ser Gly Ala Asp Ser Ser Asp 435 440 445
Asp Arg Glu Gly Arg Gly Asn Asp Ser Ala Glu Gin Gin Glu Arg Ser 450 455 460
Gly His Gin Ser Ser Asn Gly Glu Ser Ser Arg Gly Ala Leu Ser Arg 465 470 475 480
Ser Leu Arg Gly Tyr Arg Met 485
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 366 base pairs
(B) TYPE: nucleic acid
(C) STRAHOEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
Figure imgf000050_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9: GAA TTC CAT TGT GTT GGA CTC CAC TCG CTT GCG GAG GAC CCT GGA TTT 48 Glu Phe His Cys Val Gly Leu His Ser Leu Ala Glu Asp Pro Gly Phe 1 5 10 15
GCT GCT TCA GCC AGT AGA GGC CAG CAG CAG 96 Ala Ala Ser Ala Ser Arg Gly Gin Gin Gtn 20 25
Figure imgf000051_0001
CTG CAG CCG GAA GCA GCC CAT GCA GAG CAG CTA CTG CGG CAC ACT GCA 144 Leu Gin Pro Glu Ala Ala His Ala Glu Gin Leu Leu Arg His Thr Ala 35 40 45
GCG GCA TGG GAA GAA GCT GAT ACT CAG CTT CTG CTG AAG TTG CTG CTG 192 Ala Ala Trp Glu Glu Ala Asp Thr Gin Leu Leu Leu Lys Leu Leu Leu 50 55 60
CAA CCA CCA CAC ATT CAT ACG GAG ACT CCT GCC ACA ACA CCA GTT GCA 240
Gin Pro Pro His He His Thr Glu Thr Pro Ala Thr Thr Pro Val Ala
65 70 75 80
CTA AAA GAT AAG CGT GTC CAC GTG GGT GGT GCT GCG GCC TCA GCA GCC 288 Leu Lys Asp Lys Arg Vat His Val Gly Gly Ala Ala Ala Ser Ala Ala 85 90 95
GCA GCA GCG TCT GCA GTC TCA TCA ACA GCA AGA GGA AAG GCG CTT GTG 336 Ala Ala Ala Ser Ala Val Ser Ser Thr Ala Arg Gly Lys Ala Leu Val 100 105 110
AAC CCC CTC GTG TCC AAC ACA ATG GAA TTC 366
Asn Pro Leu Val Ser Asn Thr Met Glu Phe
115 120
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 122 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
( i) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Glu Phe His Cys Val Gly Leu His Ser Leu Ala Glu Asp Pro Gly Phe 1 5 10 15
Ala Ala Ser Ala Ser Arg Gly Gin Gtn Gin Pro Gin His Ala Leu Leu 20 25 30
Leu Gin Pro Glu Ala Ala His Ala Glu Gin Leu Leu Arg His Thr Ala 35 40 45 Ala Ala Trp Glu Glu Ala Asp Thr Gin Leu Leu Leu Lys Leu Leu Leu 50 55 60
Gin Pro Pro His He His Thr Glu Thr Pro Ala Thr Thr Pro Val Ala 65 70 75 80
Leu Lys Asp Lys Arg Val His Vat Gly Gly Ala Ala Ala Ser Ala Ala 85 90 95
Ala Ala Ala Ser Ala Val Ser Ser Thr Ala Arg Gly Lys Ala Leu Val 100 105 110
Asn Pro Leu Val Ser Asn Thr Met Glu Phe 115 120
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 417 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
Figure imgf000052_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ACT CTC GCT GCA GCA CCC GCG CCC TCG GCT GCT GCA CCA GCA GCA GCA 48
Thr Leu Ala Ala Ala Pro Ala Pro Ser Ala Ala Ala Pro Ala Ala Ala
1 5 10 15
GCA GCA GCA GCG CCG CCA GCA GCA GCA CCA GCA GCA GCA CCT GCA GCA 96
Ala Ala Ala Ala Pro Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala
20 25 30
GCA GCA GCG GGG CCG GAT GGG GAC AGC AAC AGC GAA GGC GCA GCA AGC 144
Ala Ala Ala Gly Pro Asp Gly Asp Ser Asn Ser Glu Gly Ala Ala Ser
35 40 45
GGG GTG GAG GGC GGC GGG GGC GGC TGG GAG CCT CTG GTG CAT GCA GCA 192
Gly Val Glu Gly Gly Gly Gly Gly Trp Glu Pro Leu Val His Ala Ala
50 55 60
CAA TGG CGA GAT GAT ATG GGC CAA ATA ATT CCA GCA GCA AGA ATT GCG 240 Gln Trp Arg Asp Asp Met Gly Gin He He Pro Ala Ala Arg He Ala 65 70 75 80
288
Figure imgf000053_0001
CAA GCT GCC AGA GAC AGA GCC CTG AGA AGG GCC CCC GGC ACC CAC GTA 336 Gin Ala Ala Arg Asp Arg Ala Leu Arg Arg Ala Pro Gly Thr His Val 100 105 110
AGG CTC AAG CAA CAG CAA CAG CAG CAG CTT TGT CAG CAG CAG CCA GGG 384 Arg Leu Lys Gin Gin Gin Gin Gin Gin Leu Cys Gin Gin Gin Pro Gly 115 120 125
TGG GAG CAA CAG AGC CAG CAG CGG CTA AAG GGC 417 Trp Glu Gin Gin Ser Gin Gin Arg Leu Lys Gly 130 135
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 139 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Thr Leu Ala Ala Ala Pro Ala Pro Ser Ala Ala Ala Pro Ala Ala Ala 5 10 15
Ala Ala Ala Ala Pro Pro Ala Ala Ala Pro Ala Ala Ala Pro Ala Ala
20 25 30
Ala Ala Ala Gly Pro Asp Gly Asp Ser Asn Ser Glu Gly Ala Ala Ser
35 40 45
Gly Val Glu Gly Gly Gly Gly Gly Trp Glu Pro Leu Val His Ala Ala
50 55 60
Gin Trp Arg Asp Asp Met Gly Gin He He Pro Ala Ala Arg He Ala
65 70 75 80
Phe Leu Lys Asp Leu Gin Asp Ala Ala Leu Val Ala Ala Thr Met Thr
85 90 95
Gin Ala Ala Arg Asp Arg Ala Leu Arg Arg Ala Pro Gly Thr His Val
100 105 110 Arg Leu Lys Gin Gin Gin Gin Gin Gin Leu Cys Gin Gin Gin Pro Gty 115 120 125
Trp Glu Gin Gin Ser Gin Gin Arg Leu Lys Gly 130 135
(2) INFORMATION FOR SEQ ID HO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 585 base pairs
(B) TYPE: nucleic acid
(C) STRAHDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix)
Figure imgf000054_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GCC CCC CCT GCT GCT GCT GCA GCT CTC GCC GCA AAG CCA AAC TGC CCA 48
Ala Pro Pro Ala Ala Ala Ala Ala Leu Ala Ala Lys Pro Asn Cys Pro
1 5 10 15
GCT TTT GGC CCC GCG GGC CCC TCG GGC TCT GCA CAC CAG CGA AGG GAG 96
Ala Phe Gly Pro Ala Gly Pro Ser Gty Ser Ala His Gin Arg Arg Glu
20 25 30
GCT CTG GGG CGT TTG GGG AGG CCG CGG GAC TCT TGG GGT GTA CGT ACA 144
Ala Leu Gty Arg Leu Gly Arg Pro Arg Asp Ser Trp Gly Val Arg Thr
35 40 45
CCG CAA TGG GGG CCC CAG AAC TCG CCC CAC AGC CGC CCC AGC CCG AGG 192
Pro Gin Trp Gly Pro Gin Asn Ser Pro His Ser Arg Pro Ser Pro Arg
50 55 60
GAA GCT CCC CCG AGG CTG CTG CTG CTG CTG CTG CTG CGC CCC CAG AAG 240
Glu Ala Pro Pro Arg Leu Leu Leu Leu Leu Leu Leu Arg Pro Gin Lys
65 70 75 80
CTG CAG CAG ATC AAA AAA ATA AGA GTG ACA GTG AAG CTC CCC GTT CGC 288
Leu Gin Gin He Lys Lys He Arg Val Thr Val Lys Leu Pro Val Arg
85 90 95
CGC GGC CAG TCA TTC CAC TTT CGG GGC TTG CTG CTC TTG CTG CTG ACG 336
Arg Gly Gin Ser Phe His Phe Arg Gty Leu Leu Leu Leu Leu Leu Thr
100 105 110 CAG GGC GAA AAG CTG AAT ACC CAG CCA GCT CCT GTC TAT GAA GGA AGC 384 Gin Gly Glu Lys Leu Asn Thr Gin Pro Ala Pro Val Tyr Glu Gly Ser 115 120 125
GGC ACA GTG GGG GCT GGC GGA AGC CCA GCA GCA GCA GCA GCA GCA GCA 432 Gly Thr Val Gly Ala Gly Gly Ser Pro Ala Ala Ala Ala Ala Ala Ala 130 135 140
GCA GGA GCA GCA GCA GCA GGC TCT TCT CCA AGG CCC TAC GGT GGC CCG 480 Ala Gly Ala Ala Ata Ala Gly Ser Ser Pro Arg Pro Tyr Gly Gly Pro 145 150 155 160
CCC GGC GCA GGG CCG TCA CCA GTG GTT GGG GGC GTC CGC GTT ATG CCG 528 Pro Gly Ala Gly Pro Ser Pro Val Val Gly Gly Val Arg Val Met Pro 165 170 175
ACA GCC GAC GCA GAA GTC CAG CGG ATC CTA GCC GAA AGA CTG AAG AAG 576 Thr Ala Asp Ala Glu Val Gin Arg He Leu Ala Glu Arg Leu Lys Lys 180 185 190
ACT GGG TGA 585 Thr Gly
195
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Ala Pro Pro Ala Ala Ala Ala Ala Leu Ala Ala Lys Pro Asn Cys Pro 1 5 10 15
Ala Phe Gly Pro Ala Gly Pro Ser Gty Ser Ala His Gin Arg Arg Glu 20 25 30
Ala Leu Gly Arg Leu Gty Arg Pro Arg Asp Ser Trp Gly Val Arg Thr 35 40 45
Pro Gin Trp Gly Pro Gin Asn Ser Pro His Ser Arg Pro Ser Pro Arg 50 55 60
Glu Ala Pro Pro Arg Leu Leu Leu Leu Leu Leu Leu Arg Pro Gin Lys 65 70 75 80 Leu Gin Gin He Lys Lys He Arg Val Thr Val Lys Leu Pro Val Arg 85 90 95
Arg Gly Gin Ser Phe His Phe Arg Gly Leu Leu Leu Leu Leu Leu Thr 100 105 110
Gin Gly Glu Lys Leu Asn Thr Gin Pro Ala Pro Val Tyr Glu Gly Ser 115 120 125
Gly Thr Val Gly Ala Gly Gly Ser Pro Ala Ala Ala Ala Ala Ala Ala 130 135 140
Ala Gty Ala Ala Ala Ala Gly Ser Ser Pro Arg Pro Tyr Gly Gty Pro 145 150 155 160
Pro Gly Ala Gly Pro Ser Pro Val Val Gly Gly Val Arg Val Met Pro 165 170 175
Thr Ala Asp Ala Glu Vat Gin Arg He Leu Ala Glu Arg Leu Lys Lys 180 185 190
Thr Gly
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 316 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: COS
(B) LOCATION: 1..316
(xi) SEQUENCE DESCRIPTION: SEQ ID HO:15:
GCT GCT GCT GCT TCT TCC CGA GAA GCT GCT GCT GCT GAG GCT TCG CGG 48
Ala Ala Ala Ala Ser Ser Arg Glu Ala Ala Ala Ala Glu Ala Ser Arg
1 5 10 15
CGA GAC CTC CTT GCT GCT GCT GCT TCT TCG CGG CGA GAG CTT GCT GCT 96
Arg Asp Leu Leu Ala Ala Ala Ala Ser Ser Arg Arg Glu Leu Ala Ala 20 25 30 GCT GCT TCG TTG CGA GAA GCT GCT GCT GCT CTT GCT GCT GCT GCT GCG 144 Ala Ala Ser Leu Arg Glu Ala Ala Ala Ala Leu Ala Ala Ala Ala Ala 35 40 45
CGG CCC CTG GAG TCT GCT GCT GAT GCG TCA GCG GAT TCG TCT CTG GAG 192 Arg Pro Leu Glu Ser Ala Ala Asp Ala Ser Ala Asp Ser Ser Leu Glu 50 55 60
TCT GCT GCT GCT GCT GCT TCG TCG CGG TCC TCG CCT GCT GCT GCT GCT 240
Ser Ala Ala Ala Ala Ala Ser Ser Arg Ser Ser Pro Ala Ala Ala Ala
65 70 75 80
GCA TCG CGG GAC TCT GCT GCT GCT GCT GCT TCC TCC TCA GAG TCT TCT 288 Ala Ser Arg Asp Ser Ala Ala Ala Ala Ala Ser Ser Ser Glu Ser Ser 85 90 95
GCT GCT GCT GCC GTG TCG TCA GAG TCT T 316 Ala Ala Ala Ala Val Ser Ser Glu Ser 100 105
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Ala Ala Ala Ala Ser Ser Arg Glu Ala Ala Ala Ala Glu Ala Ser Arg
1 5 10 15
Arg Asp Leu Leu Ala Ala Ala Ala Ser Ser Arg Arg Glu Leu Ala Ala 20 25 30
Ala Ala Ser Leu Arg Glu Ala Ala Ala Ala Leu Ala Ala Ala Ala Ala 35 40 45
Arg Pro Leu Glu Ser Ala Ala Asp Ala Ser Ala Asp Ser Ser Leu Glu 50 55 60
Ser Ala Ala Ala Ala Ala Ser Ser Arg Ser Ser Pro Ala Ala Ala Ala 65 70 75 80
Ala Ser Arg Asp Ser Ala Ala Ala Ala Ala Ser Ser Ser Glu Ser Ser 85 90 95 Ala Ala Ala Ala Val Ser Ser Glu Ser 100 105
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 284 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
Figure imgf000058_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GGT AGC AGC AGC GCG CCC CCG TCC GCA GCA GCA GCA GCA GCA GCA GCA 48
Gly Ser Ser Ser Ala Pro Pro Ser Ala Ala Ata Ala Ala Ala Ala Ala
1 5 10 15
AGA CCA GCA GCA GCA AGG GCG CAG CCG CGA GGC CGC TCG GGA GAA AGA 96
Arg Pro Ala Ala Ala Arg Ala Gin Pro Arg Gly Arg Ser Gly Glu Arg
20 25 30
TTT GAA AGC CGA GGT GGA GAT ACA CCT GAA GAG AGA GCT GAG GAT ACA 144
Phe Gtu Ser Arg Gly Gly Asp Thr Pro Glu Glu Arg Ala Glu Asp Thr
35 40 45
CCT GAA GAG CAG CAA GCA GCA GAA GAC CTG GAG CTG GCA GCA AAA GAG 192
Pro Glu Gtu Gin Gin Ala Ala Glu Asp Leu Glu Leu Ala Ala Lys Glu
50 55 60
GCC CGT GAA GCA GCA AAG AAG CTC CGC AGC GCA GCA GCA GCA GCA CGC 240
Ala Arg Gtu Ala Ala Lys Lys Leu Arg Ser Ala Ala Ala Ala Ala Arg
65 70 75 80
AGC GCA GCA GCA GCA GCA CGC AGC CAG GCC GAG ACA GAA GCG GG 284
Ser Ala Ala Ala Ala Ala Arg Ser Gin Ala Glu Thr Glu Ala
85 90
(2) INFORMATION FOR SEQ ID HO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 94 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Gly Ser Ser Ser Ala Pro Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 15
Arg Pro Ala Ala Ala Arg Ala Gin Pro Arg Gly Arg Ser Gly Glu Arg 20 25 30
Phe Gtu Ser Arg Gly Gly Asp Thr Pro Glu Gtu Arg Ala Glu Asp Thr 35 40 45
Pro Glu Glu Gin Gin Ala Ata Glu Asp Leu Glu Leu Ala Ala Lys Glu 50 55 60
Ala Arg Glu Ala Ala Lys Lys Leu Arg Ser Ala Ala Ala Ala Ala Arg 65 70 75 80
Ser Ala Ala Ala Ala Ala Arg Ser Gin Ala Glu Thr Glu Ala 85 90
(2) INFORMATION FOR SEQ ID HO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 261 base pairs
(B) TYPE: nucleic acid
(C) STRAN0EDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix)
Figure imgf000059_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
CCC CAC AGC CGC AGC AGC AGC ACG GAA GCA GGC GAG GAG AGG GAG GAA 48
Pro His Ser Arg Ser Ser Ser Thr Glu Ala Gly Glu Glu Arg Glu Glu
1 5 10 15
GAA CGC AGC AGC AGC AGC ACC ACC ACC ACC ACT GCT GCT CCA CGA GCT 96
Glu Arg Ser Ser Ser Ser Thr Thr Thr Thr Thr Ala Ala Pro Arg Ala 20 25 30
TCC ACA CTA GCA GCA ACT CTC GTC GGC AGC GGC GTC ACG GAA GCA GCA 144 Ser Thr Leu Ala Ala Thr Leu Val Gly Ser Gly Val Thr Glu Ala Ala 35 40 45
AGC AGC AGC AGC AGC AGC ACA AGA GCA GCT GAG GAG GAG CGC AGC AGC 192 Ser Ser Ser Ser Ser Ser Thr Arg Ala Ala Glu Glu Glu Arg Ser Ser 50 55 60
AGC AGC ACC CGG GCC GCC GAG CAC GAG CGC AGC AGC AGC ACT GCA GCA 240
Ser Ser Thr Arg Ala Ala Glu His Glu Arg Ser Ser Ser Thr Ala Ala
65 70 75 80
GAA CAC AGC AGC AGC AGC ACC 261 Glu His Ser Ser Ser Ser Thr 85
(2) INFORMATION FOR SEQ ID NO-.20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N :20:
Pro His Ser Arg Ser Ser Ser Thr Gtu Ala Gly Glu Glu Arg Glu Glu 1 5 10 15
Glu Arg Ser Ser Ser Ser Thr Thr Thr Thr Thr Ala Ala Pro Arg Ala 20 25 30
Ser Thr Leu Ala Ala Thr Leu Val Gly Ser Gly Val Thr Glu Ala Ala 35 40 45
Ser Ser Ser Ser Ser Ser Thr Arg Ala Ala Gtu Glu Glu Arg Ser Ser 50 55 60
Ser Ser Thr Arg Ala Ala Glu His Glu Arg Ser Ser Ser Thr Ala Ala 65 70 75 80
Gtu His Ser Ser Ser Ser Thr 85
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 base pairs
(B) TYPE: nucleic acid
(C) STRANOEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: COS
(B) LOCATION: 1.-411
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
48
Figure imgf000061_0001
96
Figure imgf000061_0002
144
Figure imgf000061_0003
ACC CGC GCA GCT GAA GAA GAG AGC AGC AGC AGC AGC ACC CGC AGC AGA 192 Thr Arg Ala Ala Glu Glu Glu Ser Ser Ser Ser Ser Thr Arg Ser Arg 50 55 60
240
Figure imgf000061_0004
AGC GGA GTC ACC GAA GCA GCA GAG GGC AGC AGC AGC AGC AGC AGC AGC 288 Ser Gly Val Thr Gtu Ala Ala Glu Gly Ser Ser Ser Ser Ser Ser Ser 85 90 95
336
Figure imgf000061_0005
AGC AGC AGC AGC ACA AGA GCA GCA GAG CAT GAG CGC 384
Ser Ser Ser Ser Thr Arg Ala Ala Glu His Glu Arg
Figure imgf000061_0006
120 125
AGC AGC AGC TCC ACT AGA GCC GTC TA 411
Ser Ser Ser Ser Thr Arg Ala Val
130 135
(2) INFORMATION FOR SEQ ID t*0:22: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 136 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ser Thr Ala Ala Glu His Ser Ser Ser Ser Thr Ser Thr He Ala Ala 1 5 10 15
Thr Leu Val Gly Ser Gly Val Thr Glu Ala Ala Glu Glu Glu Ser Ser 20 25 30
Ser Ser Ser Thr Arg Ala Ala Glu Glu Glu Gly Ser Ser Ser Ser Ser 35 40 45
Thr Arg Ala Ala Glu Glu Glu Ser Ser Ser Ser Ser Thr Arg Ser Arg 50 55 60
Ser Ser Ser Thr Ser Thr Thr Thr Val Ala Pro Ala Leu Leu Phe Gly 65 70 75 80
Ser Gly Val Thr Glu Ala Ala Glu Gly Ser Ser Ser Ser Ser Ser Ser 85 90 95
Thr Glu Ala Ala Glu Arg He Ser Ser Ser Ser Ser Thr Arg Ala Ala 100 105 110
Glu Arg Glu Tyr Ser Ser Ser Ser Thr Arg Ata Ala Glu His Gtu Arg 115 120 125
Ser Ser Ser Ser Thr Arg Ala Val 130 135
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 123 base pairs
(B) TYPE: nucleic acid
(C) STRAN0E0NESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix)
Figure imgf000062_0001
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
TTC TTC AAA CGT TTT GAT GAC GTA GAC TTT GAC GAA CAA CAA GAT GCT 48
Phe Phe Lys Arg Phe Asp Asp Val Asp Phe Asp Glu Gin Gin Asp Ala 1 5 10 15
GTT CAT GAA GAT CGT CAT ATT TTC TAC TTA TCA AAT ATT GAA AAT AAC 96
Val His Glu Asp Arg His He Phe Tyr Leu Ser Asn He Glu Asn Asn 20 25 30
GTT CGC GAA TAT CAC AGA CCA GAG TA 123
Val Arg Gtu Tyr His Arg Pro Glu 35 40
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Phe Phe Lys Arg Phe Asp Asp Val Asp Phe Asp Glu Gin Gin Asp Ala 1 5 10 15
Val His Glu Asp Arg His He Phe Tyr Leu Ser Asn He Gtu Asn Asn 20 25 30
Val Arg Glu Tyr His Arg Pro Glu 35 40
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 442 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
( i) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..442 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATG AAC GTC CTG CTT GCG GAG GCT GAT GTA CCT TAC AAA ATT GTC AAA 48
Met Asn Val Leu Leu Ala Glu Ala Asp Val Pro Tyr Lys He Val Lys
1 5 10 15
GAG ATG TCT GAG GTT AAC CCA GAA ATC AGC TCT TAC GAT GTT ATC CTT 96
Glu Met Ser Glu Val Asn Pro Glu He Ser Ser Tyr Asp Val He Leu
20 25 30
GTT GTT GGA GCT AAC GAT ACC GTC AAT CCT GCT GCA CTG GAG CCA GGA 144
Val Val Gly Ala Asn Asp Thr Val Asn Pro Ala Ala Leu Glu Pro Gty
35 40 45
TCA AAG ATA TCC GGA ATG CCT GTC ATT GAG GCA TGG AAG GCT AGA CGC 192
Ser Lys He Ser Gly Met Pro Val He Glu Ala Trp Lys Ala Arg Arg
50 55 60
GTA TTC GTA CTG AAG CGC TCC ATG GCA GCT GGA TAT GCC AGC AAT GAG 240
Val Phe Val Leu Lys Arg Ser Met Ala Ala Gty Tyr Ala Ser Asn Glu
65 70 75 80
AAT CCT CTG TTC CAC CTG GAG AAC ACT CGC ATG CTC TTT GGA AAT GCC 288
Asn Pro Leu Phe His Leu Glu Asn Thr Arg Met Leu Phe Gly Asn Ala
85 90 95
AAG AAT ACA ACT TCT GCT GTG TTC GCT CGT GTC AAC GCG AAG GCA GAA 336
Lys Asn Thr Thr Ser Ala Val Phe Ala Arg Val Asn Ala Lys Ala Glu
100 105 110
CAA ATG CCA CCC TCC GCT GCT CGT GAT GAC CTC GAA TCC GGT CTT CTT 384
Gin Met Pro Pro Ser Ala Ala Arg Asp Asp Leu Glu Ser Gly Leu Leu
115 120 125
GAG TTC GAA AGA GAA GAG CGC GTT GAT CAA TCT TCT TGG CCT TAT CCC 432
Glu Phe Glu Arg Glu Glu Arg Val Asp Gin Ser Ser Trp Pro Tyr Pro
130 135 140
AGA CTG GGT G 442
Arg Leu Gly
145
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Asn Val Leu Leu Ala Glu Ala Asp Val Pro Tyr Lys He Val Lys 1 5 10 15
Glu Met Ser Glu Val Asn Pro Glu He Ser Ser Tyr Asp Val He Leu 20 25 30
Val Val Gly Ata Asn Asp Thr Val Asn Pro Ala Ala Leu Glu Pro Gly 35 40 45
Ser Lys He Ser Gly Met Pro Val He Glu Ala Trp Lys Ala Arg Arg 50 55 60
Val Phe Vat Leu Lys Arg Ser Met Ala Ala Gly Tyr Ala Ser Asn Glu 65 70 75 80
Asn Pro Leu Phe His Leu Glu Asn Thr Arg Met Leu Phe Gly Asn Ata 85 90 95
Lys Asn Thr Thr Ser Ala Val Phe Ala Arg Val Asn Ala Lys Ala Glu 100 105 110
Gin Met Pro Pro Ser Ala Ala Arg Asp Asp Leu Glu Ser Gly Leu Leu 115 120 125
Glu Phe Glu Arg Glu Glu Arg Val Asp Gin Ser Ser Trp Pro Tyr Pro 130 135 140
Arg Leu Gly 145

Claims

What Is Claimed Is:
1. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 1.
2. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 2.
3. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 3.
4. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 4.
5. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 5.
6. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 6.
7. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 7.
8. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 8.
9. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 9. 10. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No.
10.
11. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 11.
12. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 12.
13. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 13.
14. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 14.
15. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 15.
16. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 16.
17. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 17.
18. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 18.
19. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 19. 20. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No.
20.
21. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 21.
22. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 22.
23. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 23.
24. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 24.
25. A cloned DNA molecule comprising the nucleotide sequence as shown in Sequence ID No. 25.
26. An antigenic protein comprising the amino acid sequence as shown in Sequence ID No. 26.
27. An expression vector comprising the nucleotide sequence of any of claims 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25 under the control of a regulatory region capable of directing expression of said nucleotide sequence.
28. The expression vector of claim 27 wherein said vector is selected from the group consisting of plasmids, bacteriophages, viruses, or hybrids thereof.
29. A host cell or organism transformed by the expression vector of claim 27 selected from the group consisting of bacteria, yeast fungi, insect and mammalian cells.
30. A vaccine for immunizing birds against avian coccidiosis comprising the antigenic protein of any of claims 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 combined with a carrier or adjuvant
31. A method of immunizing birds against avian coccidiosis comprising administering to said birds an effective amount of the vaccine of claim 30.
32. The method of claim 31 wherein said administration is by injection or by mixing the antigenic protein with feed.
33. A vaccine for immunizing birds against avian coccidiosis comprising a live microorganism transformed by the expression vector of claim 27.
34. A method of immunizing birds against avian coccidiosis which comprises administering to said birds the vaccine of claim 33.
PCT/US1991/006431 1990-09-12 1991-09-05 Genetically engineered coccidiosis vaccine WO1992004461A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US58169490A 1990-09-12 1990-09-12
US581,694 1990-09-12

Publications (1)

Publication Number Publication Date
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Country Link
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JP (1) JPH06504187A (en)
WO (1) WO1992004461A1 (en)

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WO2005010040A1 (en) * 2003-07-15 2005-02-03 Barros Research Institute Eimeria tenella antigen for immunotherapy of coccidiosis
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US6365717B1 (en) * 1992-03-13 2002-04-02 Akzo Nobel, N.V. Peptides and nucleic acid sequences related to the Epstein Barr virus
US7507804B2 (en) 1992-03-13 2009-03-24 Akzo Nobel, N.V. Peptides and nucleic acid sequences related to the Epstein Barr Virus
EP0775746A2 (en) 1995-07-03 1997-05-28 Akzo Nobel N.V. Coccidiosis poultry vaccine
EP2311859A1 (en) * 2003-07-04 2011-04-20 Bayer Animal Health GmbH Uses of a novel Eimeria gene and corresponding protein
WO2005010040A1 (en) * 2003-07-15 2005-02-03 Barros Research Institute Eimeria tenella antigen for immunotherapy of coccidiosis
US7393534B2 (en) 2003-07-15 2008-07-01 Barros Research Institute Compositions and methods for immunotherapy of cancer and infectious diseases
US8257714B2 (en) 2003-07-15 2012-09-04 Michigan State University Compositions and methods for immunotherapy of cancer and infectious diseases

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EP0548252A4 (en) 1995-02-15
EP0548252A1 (en) 1993-06-30

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