JPH03503054A - vaccine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] -2-two two L-2- ■H The present invention relates to parasitic nematode species, such as Trichinella, Ankylostoma, Strongyle, Nematode, Nematoda, Ostertasia, Trichophyton, Toxascaris, Lancinnata Genus Lia, Trichuris, Dirofilaria, Toxocara, Necator, Streptococcus, Sensitivity caused by species of the genus Strongyloides and the filamentous genera, especially the genus Tricholate and nematode. This study concerns the identification of antigens that induce protective immunity in the host against infectious diseases. like this Examples of common species include Trichinella, hookworm, Strongylus vulgaris, and Colubrifol. Mischilioid nematode, Torillus nematode, Ostertasia ostertagi, Pig field worm, Lyo Toxocara spp., Toxocara sp. larvae, American hookworm, Zubini hookworm, roundworm, human deworming, incineration, Strongyloides and vane Includes croft threadworms, particularly Colubliformis trichophytes and Torillus tricholates. Ru.

The invention also provides nucleotide sequences encoding these antigens as well as Recombinant DNA molecules containing nucleotide sequences and their nucleotide sequences It also relates to host cells expressing the .

The invention further provides antigens, nucleotide sequences, recombinant DNA molecules and hosts of the invention. It concerns the method of creation of the Lord.

The present invention provides antibodies raised against the antigens of the present invention and antibodies that act similarly to these antibodies. It is related to compounds.

Furthermore, the present invention provides for parasitic nematodes, such as Trichinella sp., Ankylostoma sp., Strongylus sp. genus Nematoda, genus Nematode, genus Ostertagia, genus Toxacaris, genus Lancinaria Genus, Trichuris, Dilophyllaria, Toxocara, Necator, Burning, Stro Infections by species of the genus Ngilloides and the filamentous genera, especially the genera Tricholate and nematode The present invention relates to a vaccine that induces protective immunity against. Examples of such species include the human indigenous hairworms, dog hookworm, horse strongylus vulgaris, sheep and goats; Colubliformis tricholate nematode, ovine and caprine tricholate nematode, bovine ostele Rutasia osteltaji, pig field indigenous or trichinella, cat liontoxa Scalis or Bacillus hookworm, dog hookworm or Trichuris purvis, dog larvae of the dog heartworm or human Toxocara spp. Infections with roundworms, human deworming, baked earth, Strongyloides or Bancroft heartworms, especially Col. Includes Buriformis tricholate nematode or Torillus tricholate nematode.

L-died Segmentless cylindrical insect with a long, spindle-shaped or pouch-like body covered with a cuticle Nematodes (nema-series; similar to oides) are naturally occurring in the soil where they live, It is almost ubiquitous in water and plants, and is host to a wide range of animals and plants. It is commonly associated with insect diseases.

Nematode parasites of mammals belong to the phylum Nematoda. Nematodes and hookworms (e.g. American hookworm) worms and hookworms), roundworms (e.g., common roundworms), sandy soils (e.g., human dewormers) ) and nematodes or nematodes (e.g. nematodes) and strongyloids, trichinella and phila Includes ria worms, or Bancroft heartworms. Other important nematode parasites include canine Hookworm (human infection), Strongylus vulgaris (horse infection), Colublipho Lumis trichophytes, Ostertadia circumcinctae (infection of sheep and goats) , Nematode Tortis (infection of sheep and goats), Ostertasia ostertagi, Hae Moncus bullasei (infection of cattle), pig soil (dyeing pigs), lion crested crest Squirrel or pinnacephalic hookworm (infection of dogs), Toxocara species (circulatory system infection of humans) and and heartworm (a circulatory system infection of cats and dogs).

Even when there are no symptoms, parasitic infections can persist in host animals for many reasons, including: Harmful. That is, these parasites deprive the host of food, damage organs, and can block ducts, produce substances toxic to the host, and invade other organisms. Provide an entrance. In other cases, the host may be a species grown for food. , and eating them can carry parasites and infect the animals that eat them. This is because that. If such parasites are discovered, these parasites can It is highly desirable to remove it as soon as possible.

More commonly, such infections have no symptoms. Mammal infections, especially parasitic Infection with live nematodes is particularly important for domestic pets, such as sheep, cows, horses, pigs, and goats. Horses, dogs, cats and birds, especially poultry, are a source of major economic losses (CSIR O/BAE Report - “Socio-economic developments and trends in the agricultural sector: Future research Perspectives), these animals must be driven to keep these infections under control. Must be treated regularly with insecticide chemicals. Otherwise, diseases like this The disease can cause anemia, diarrhea, dehydration, loss of appetite, and even death. Ru.

The only means currently available to control soil infections is through the use of anthelmintic chemicals. However, these chemicals are only effective against insects that are present at the time of treatment.

Therefore, animals are constantly exposed to infection and must be treated on an ongoing basis. 0 For example, anthelmintic treatment with diethylcarbamacine is effective for controlling dog heartworms. Therefore, it is necessary to perform it almost every day of the year or every other day. This is expensive and labor intensive. It is a labor intensive method. With the widespread use of anthelmintic chemicals, parasites have become resistant. As a result, new and more powerful chemical products must be developed. Law is clearly preferable.

Development of vaccines against parasitic nematodes for the prevention and treatment of field soil infections Many of the shortcomings inherent in handling supplies would be overcome. Protection from vaccines is certain persist for long periods of time, affect only vaccinated animals, and cause toxicity problems. Persistence of nematodes and residues will be minimized or avoided; therefore, using parasitic nematodes Several attempts have been reported to develop vaccines such as those described above. What a shame However, these attempts have met with limited success, and material availability and The large-scale use of these efforts by Foctor such as vaccine stability and hindered.

1 as described above by Dineen (1977) in J. One attempt involves the use of irradiated larval vaccines. Other trials like this Similar to the above, the utility of this method is due to the need to maintain viable nematodes for long periods of time. limited to.

Many factors prevent killed vaccine formulations from providing good anthelmintic protection. It is thought that it depends on the For example, J, T.

Hakama and Neilson (1975) found that parasitic nematodes affect the host's immune system. mechanisms that can secrete immunosuppressive or immunomodulatory products and that the host It is believed that people become more susceptible to infection. Deineen and Wagl and) (1982) that immunosuppressants or immunomodulators are used in killed vaccines. are believed to be present in crude preparations of parasitic nematodes. As shown in these review papers A second problem that has been raised is that parasitic nematodes may not be able to match their antigenic profile to that of the host. as a result of a vigorous immunological response in a natural infection. It is not induced by protective parasite antigens. Such a phenomenon is extinct It also occurs after vaccination with impure preparations of nematodes or their extracts.

from host animals using whole parasite homogenates and impure subfractions. Some researchers have shown that the proliferation of parasites is accelerated. For example, Rothwell and co-researchers (1974, 1977, 1979) , O'Donnell et al. (1985), Neilson and F. Van de Walle (1987), Silberman (Si lverman): US Patent No. 894, 603, Australian Patent No. 247, 354, Adams (1989), Eas (19B9), Munn and Greenw ood) (1987) (Australian Patent Application No. 77590/87), Connan (1965), Savin et al. (1988) and McGillivery et al. (198B).

In all of these studies, crude extracts of C. elegans were used to vaccinate the animals. and that any identified antigen or any individual component of the extract is protective. have not been identified as contributing to

Several reports have attempted to identify purified protective components. For example, sil Silberstein and Despov+ +1er) (1985), Hoetz et al. (1,985), Grande Grandea et al. (1989), Lucius et al. (1,9 88), Donelson et al. (1988), Nilssen et al. (1988).

However, no protection has been shown or proven for the listed ingredients. Not even there.

Purified and cloned subunits can only be used in one natural host/parasitic nematode system. Australian Patent Application No. 19998/88 not shown to be defensive Antigen derived from silk recombinant [)NT that has been demonstrated against tropomyosin in C. elegans in No. was shown to confer 50% protection in sheep against challenge with T. nematode challenge. It was done. For reasons that will become clear later herein, this antigen is referred to herein as The antigens of the present application are different from the antigens identified during in vitro incubation. Found in excreted/secreted fluids of nematodes after dilan.

C31RO/BAE Research Report “Socio-economic developments and trends in the agricultural sector: Future research prospects, 1, highlight the three most pressing issues in the Australian sheep industry. Intestinal parasites have been listed as one of the health challenges, and these infections can be better controlled. There are great expectations for the development of a vaccine.

Animals infected with parasitic nematodes develop immunity and become less susceptible to subsequent infections. has been established (see review by Rotbwell, 1989).

The infected host animal's immune system recognizes many parasite proteins during parasite infection It has been demonstrated (e.g., O'Donal et al., 1985) that the immune response Many of these have no functional significance in terms of resistance to reinfection. main stages protects the host animal from reinfection from the thousands of proteins present in the parasite. identify individual proteins capable of inducing an immune response in the host animal that That's true.

Recent advances in biotechnology, particularly in recombinant DNT technology, have commercially viable treatments against a range of economically important parasites of animals and domestic animals. Opportunities to produce cutin are realistically provided. This attempt raised a number of issues. The cause of the lack of effectiveness of killed vaccines using crude parasite preparations could be overcome. For example, vaccines produced by recombinant DNT technology Immunosuppressive or immune substances that may be present in crude extracts of parasitic nematode species It will not contain any infectious agents. However, it is first necessary to identify the antigen. .

Once identified and characterized, these proteins or protective epitopes can be In order to construct microorganisms that synthesize portions of these proteins containing recombinant D N? It is possible to use techniques and to protect animals from parasitic infections. Products synthesized by recombinant organisms are used in vaccines.

The inventors have detailed the excretory/secretory products obtained from adult Colubliformis trichophytes. to refine the ingredients from the mixture that can provide protection after vaccination of target animals. were produced and characterized at the molecular level.

Genuine skin of course The inventors have demonstrated that parasites can be infected by immunization with the excreted/secreted products of the parasitic nematode species. It was discovered that protective immunity can be induced against insect infection. Colubliformis Five molecules purified and characterized from the excretory-secretory fluid of adult hairworms are Tc. Ad ESAI, Tc Ad ESA2, Tc Ad ESA3, Tc They are named and described as Ad ESA4 and Tc Ad ESA5. The inventor is When administered with a vaccine, these proteins protect against infection with Trichophyllis colbriformis We found that this method induced a defensive response in guinea pigs.

Adult worms were collected from sheep 21 days after infection, washed and treated with RP containing antibiotics. It was maintained for 16 hours at 37°C in old 1640 medium. Colubliformis hairy nematode? The above medium containing excretory/secretory fluid obtained from a Diaflo membrane The mixture was concentrated and fractionated by absorption onto a lentil lectin-Sepharose 4B column.

Unbound fraction (LL-) and bound fraction (LL"", methylman eluted with noside) each had only 2.3 protein bands, and even more Fractionation was performed by acrylamide gel electrophoresis and electroelution. Tc Ad ESAI , TcAd ESA2, and TcAdESA5. Tc Ad ESA3 was isolated from the lentil lecithin-binding fraction and two proteins called TcAd ESA4 further induce unbound flux. isolated from the section.

A total of five proteins were detected after intraperitoneal injection in guinea pigs, an ovine laboratory model. The substance confers immunity against infection with the trichophyte Colbriformis nematode.

Examples of antigens of the invention are purified protein Tc Ad ESAI, Tc Ad ESA 2, Tc Ad B5A3, Tc Ad ESA4 and Tc Ad ESA5 and when estimated by S[1S-PAGE, 30.37.17.11 and 30.37.17.11 respectively. and has a molecular weight of 81 kD.

According to a first embodiment of the invention, a second parasitic nematode species derived from a first parasitic nematode species; (these can be the same or different from the first nematode species) excreted/secreted proteins capable of inducing protective immunity against; or excreted/min Whole secreted proteins, parts, analogs, congeners, derivatives or combinations thereof It consists of a protein that can induce protective immunity in the host against infection by parasitic nematodes. An antigen consisting of a protein molecule is provided.

Preferably, excreted/secreted proteins have approximate molecular weight when estimated by 5O3-PAGE. has an amount of 11.17.30.37 or 81 kD.

Typically, the first parasitic nematode species are Trichinella, Ankylostomatoma, Strongyloides, and Trichinella. Nematode style, Nematode genus, Ostertasia genus, Field worm genus, Toxascaris genus, Lancinari genus Trichuria, genus Trichuris, genus Dirofilaria, genus Toxocara, genus Necator, Selected from species of the genus Longiloides and the filamentous genus. Examples of these types include trichinella, hookworm canis, strongilus vulgaris, colubliformis trichium, Trichotidium torsion, Ostertasia ostertagi, Pig clam, Liontoxascaris , Acute hookworm, Tricris purvis, Heartworm canis, Toxocara sp., American hookworm, There are hookworms, roundworms, human anthelmintics, pyrophytes, strongyloids, and bancroft heartworms.

Typically, the second parasitic nematode species is Trichinella, Ankylostoma, Strongyles, Nematode style, Nematode genus, Ostertasia genus, Trichopodia spp., Toxascaris spp., Lancina Lia, Trichuris, Dirofilaria, Toxocara, Necator, Octopus, Selected from species of the genus Strongyloides and Filamentosa. Examples of these types include , Trichinella canis, Hookworm canis, Strongylus vulgaris, Colubliformis hairy nematode , Tortis nematode, Ostertasia ostertagi, Pig field worm, Lion crested nematode squirrel, pinnacephalic hookworm, triclis pluvis, canine heartworm, Toxocara sp., American hookworm worms, hookworms, roundworms, human anthelminths, strongyloids, strongyloids, and bancroft heartworms. Ru.

Preferably, the first parasitic nematode species is Colubliformis nematode.

Preferably, the second parasitic nematode species is Colubliformis nematode or Tortis nematode. It's an insect.

According to a second embodiment of the invention, the amino acid sequence of the antigen of the first embodiment is a first nucleotide sequence that hybridizes with the first nucleotide sequence; a nucleotide sequence; or one or more base substitutions, insertions or deletions; a mutation that provides a nucleotide related to the first nucleotide sequence Ru.

The preferred nucleotide sequence of the present invention can be estimated by 5D5-PAGE. Excretion/min of the first embodiment with molecular weight 11.17.30.37 and 81 kD A nucleotide sequence encoding a secreted protein.

Preferably the nucleotide sequence is a DNA sequence. DNA included in the present invention Sequencing can be done, for example, by extracting total DNA from C. colubriformis cells and It can be produced by isolating the DNA sequence using standard methods. Or , DNA can be synthesized in vitro, synthetically, or biosynthetically, e.g., from an mRNA template. It can be manufactured using

According to a third embodiment of the invention, a DN encoding an antigen according to the first embodiment A method for selecting A or RNA sequences is provided. the method is one or more and which sequence is the antigen of the first embodiment. Hybridization with DNA or RNA sequences known to encode or by providing antiserum and expressing the antigen. The process consists of identifying the host-vector combinations that are present.

The sequences can be of natural origin, including RNA sequences, synthetic sequences, recombinant DNA sequences, etc. It can be the DNA sequence obtained from the offspring or a combination of these sequences.

Preferably used to identify and characterize DNA encoding an antigen. The method used is extraction of a+ RNA species from antigen-producing cells, double extraction of mRNA species, and conversion into chains (cDN^) and their autonomous replication foctors, e.g. plasmids. Involved in insertion into a host or phage vector. Following this, bacterial strains Transform host cells with the above factors and add any other cellular proteins. m to detect clones containing DNA encoding antigens different from the quality components. 0 synthetic RNA probes complementary to antigen encoding RNA or DNA sequences Screen the library produced.

According to a fourth embodiment of the invention, the DNA sequences and vectors of the third embodiment Recombinant DNA molecules consisting of 11NA are provided.

The DNA sequence can be a natural, synthetic or biosynthetic DNA sequence.

Preferred recombinant DNA molecules of the invention include proteins operably linked to the DNA sequence. Contains the current control array.

In one preferred form of the invention, the DNA sequence is E. coli β-galactosidase. are operated and combined. Other preferred control systems include tryptophan (Trp). Operon, Tra-T gene of E. coli, left promo of bacteriophage lambda promoter, Cupl promoter and hybrid promoters such as tac or viral promoters, such as the SV40 early promoter.

Preferably, the vector DNA is plasmid DNA. suitable plasmid vector The targets include pUR290Sp[Ic18, pYEUc114 and their inducers. Contains conductors.

Alternatively, the vector DNA may be a bacteriophage such as bacteriophage lambda. DNA and derivatives such as lambda gtll and lambda gtlO. can.

According to a fifth embodiment of the invention, a promoter, a translation initiation signal and a third A fusion gene is provided comprising a DNA sequence of an embodiment of the invention.

According to a sixth embodiment of the invention, a DNA consisting of the DNA sequence of the third embodiment Providing the insert and introducing the 1oNa insert into the cloning vector There is provided a method for producing a recombinant DNA molecule according to the fourth embodiment, comprising: .

Preferably, the DNA insert contains expression control sequences in a cloning vector. be introduced in the correct position and in the correct reading frame.

According to a seventh embodiment of the invention, at least one recombinant of the fourth embodiment A host transformed with a DNA molecule is provided.

Preferably, the transformed host is capable of expressing the antigen of the first embodiment. Ru.

Suitable hosts include bacterial cells, Satucharomyces cerevisiae strain CL13-ABSY8 6 yeasts, other fungi, vertebrate cells, insect cells, plant cells, human cells, cells, live viruses such as variola virus and baculovirus, and whole cells. Includes eukaryotes.

Suitable bacterial hosts include Escherichia coli and other enteric organisms, Pseudomonas and Bacillus. Includes species of the genus.

Preferred hosts are E. coli 12 derivatives, especially JM109 and Y1090.

According to an eighth embodiment of the invention, providing a host, making the host suitable for transformation. and introducing the recombinant DNA molecule of the fourth embodiment into a host. of the seventh embodiment, comprising: A method of transforming is provided.

According to a ninth embodiment of the invention, a seventh embodiment consisting of the antigen of the first embodiment Expression products of embodiments of transformed hosts are provided.

Preferably, the expression product is provided in substantially pure form.

Preferably, the expression product comprises a first polypeptide sequence homologous to the host; from a second polypeptide sequence that is an amino acid sequence encoding an antigen of an embodiment of Become.

More preferably, the first amino acid sequence is a portion of β-galactosidase or Total or Tra-T and the host cell is E. coli.

According to a tenth embodiment of the invention, a protein award consisting of the antigen of the first embodiment A method of biosynthesizing a product is provided. This method: such that the host is capable of expressing a protein product containing the antigen of the first embodiment; Transforming a host with the recombinant DNA molecule of the fourth embodiment; culturing the host for expression. and collecting the protein product; Consists of things.

According to an eleventh embodiment of the invention, the first embodiment contributes to a protective immune response. An epitope of the antigen is provided. This epitope has the sequence of a portion of an antigen Can be created artificially by synthesizing and manufacturing oligopeptides . This can be produced in bacteria or by chemical or chemical synthesis of natural or recombinant peptides. Results of immunochemical tests on protein fragments resulting from enzymatic cleavage It can be predicted from.

According to the twelfth embodiment of the present invention, the epitope of the eleventh embodiment Antibodies are provided. These antibodies or idiotypes are used for passive immunization of animals. It can be used for.

According to a thirteenth embodiment of the invention, for the variable region of the antibody of the twelfth embodiment Antibodies generated by this method, so-called anti-idiotype antibodies, are provided, and these antibodies are used to protect against antigens. epitope-like and can be used as an effective vaccine for active immunization in animals. I can do it.

According to a fourteenth embodiment of the invention, one or more of the first embodiment Effective amount of antigen, expression product of the ninth embodiment, epitope of the 11175th embodiment and/or the anti-idiotype antibody of the thirteenth embodiment and a pharmaceutically acceptable A vaccine consisting of possible excipients, carriers, adjuvants and/or diluents is proposed. Served.

Preferred vaccines include those suitable for injection or oral administration. Preferably , the injectable vaccine includes a pharmaceutically acceptable adjuvant.

According to embodiment U of the present invention, one or more antigens, antigens of the present invention host organisms, epitopes, anti-idiotypic antibodies and/or vaccines. By administering the vaccine, antibodies produced as a result of administering the vaccine to the host are presented. Served.

Such antibodies include polyclonal and monoclonal antibodies.

It is recognized that there are compounds that act in a similar manner to the antibodies of the fifteenth embodiment. Ru. Although these compounds are not antibodies, the presence of these compounds in the host can lead to antibodies. 0 Throughout the specification and claims of the present application, which can bring about a protective effect similar to Therefore, the description of the antibody in the fifteenth embodiment should be interpreted to cover these compounds. Should.

According to a sixteenth embodiment of the invention, at least one of the twelfth and/or Antibodies of embodiment 15 and pharmaceutically acceptable carriers, diluents and/or excipients An antibody composition comprising a formulation is provided.

According to a seventeenth embodiment of the invention, collecting excretory-secretory fluid from a parasitic nematode species; Hijack lectin chromatography using methyl mannoside as eluent fractionate the liquid; collect bound and unbound fractions and transfer to a 5O5-gel electrode. The first step consists of further fractionation by pneumophoresis; and electroelution of the antigen. Methods of producing embodiments of antigens are provided.

According to an eighteenth embodiment of the invention, a promoter, a translation initiation signal and a Embodiment 3 of the present invention provides a DNA sequence and includes a promoter, translation initiation signal and of the fusion gene of the fifth embodiment, which comprises manipulating and linking the fusion gene and the DNA sequence. A manufacturing method is provided.

According to the nineteenth embodiment of the invention, the first embodiment of at least one effective amount and/or the expression product of the nineteenth embodiment and/or the eleventh embodiment. the epitope of the embodiment and/or the anti-idiotype antibody of the thirteenth embodiment; Mixed with pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants There is provided a method for producing a vaccine according to a fourteenth embodiment, comprising:

According to a twentieth embodiment of the invention, the antigen of the first embodiment and/or the ninth embodiment and/or the epitope of the eleventh embodiment and/or the expression product of the embodiment of or the anti-idiotype antibody of the thirteenth embodiment and/or the fourteenth embodiment a fifteenth practice, comprising immunizing an immunocompetent host with a vaccine of Methods of producing antibodies of embodiments are provided.

According to a twenty-first embodiment of the invention, the antibody of the twelfth embodiment is used to respond to an immune response. of the anti-idiotypic antibody of the thirteenth embodiment, comprising immunizing a sexual host. A manufacturing method is provided.

According to a twenty-second embodiment of the invention, an effective amount of at least one of the twelfth and/or or the antibody of the fifteenth embodiment in a pharmaceutically acceptable carrier, diluent and/or The method for producing an antibody composition according to the sixteenth embodiment comprises mixing with an excipient. provided.

According to a twenty-third embodiment of the invention, the antigens, expression products, vaccines, etc. of the invention Vaccinate the host with pitopes and/or anti-idiotype antibodies to protect the host from infection by parasitic nematode species in need of protective treatment, consisting of A method is provided.

According to a twenty-fourth embodiment of the invention, an effective amount of at least one of the twelfth and/or or the antibody of the fifteenth embodiment and/or the antibody composition of the sixteenth embodiment. host that requires protective treatment, consisting of passively vaccinating the host using A method is provided for passively protecting the host against infection by parasitic nematode species.

carriers, excipients, diluents and/or antigens, expression products, epitopes and can be combined with adjuvants. / or the amount of anti-idiotype antibodies , will vary depending on the host treated and the particular mode of administration.

The particular dosage value for any particular host will depend on the particular antigen, expression product, epitope used, etc. topic, anti-idiotypic antibody and/or vaccine activity, age, body weight, total physical health, gender, diet, time of administration, route of administration, excretion rate, combination of drugs, treatment. Special infections to be treated or prevented and special infections being treated or prevented. It will also be understood that this will depend on a variety of factors, including severity.

The vaccines of the present invention can be administered orally or parenterally using any conventional, non-toxic, pharmaceutically acceptable method. A monomer containing the desired carriers, diluents, adjuvants and/or excipients that can The formulation can be administered in multiple dosages.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, are prepared using suitable dispersing agents or oleaginous suspensions. or may be formulated according to known techniques using wetting agents and suspending agents. Ru. Sterile injectable preparations may also be prepared in nontoxic parenterally acceptable diluents or solvents. It can also be an injection solution or suspension, for example a solution in 1,3-butanediol. . Acceptable carriers and solvents that may be employed include water, Ringer's solution and sodium chloride. Isotonic solutions are available. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. used. Contains synthetic triglycerides or diglycerides for the above purposes. Any bland fixed oil may be used. Furthermore, like oleic acid fatty acids are used in the preparation of injectables.

The term "pharmaceutically acceptable adjuvant" refers to a standard set of adjuvants for human administration. either a compound or a typical adjuvant used in animal vaccination. It means something. Suitable adjuvants are selected using techniques common in the art. can do.

Suitable adjuvants for animal and human vaccination include aluminum hydroxide and and oil-based emulsions, such as Marcol 52: Montanai Montanide 888 (Marcol is a trademark of Es5o, M (ontanide is a trademark of 5eppic of Paris) Other adjuvants suitable for use in the present invention include prokaryotes or eukaryotes. complete membrane proteins derived from human organisms, e.g. from expression products together with TraT. Conjugates include

Routes of administration, doses to be administered and frequency of injections can be determined using ordinary skill in the art. These are all factors that can be optimized as follows. Typically, the first work One or more "booster" vaccinations several weeks after vaccination This true effect is due to antigenic epitope, anti-idiotypic antibody or by generating a strong immunological response, such as a high titer of antibodies, against the expressed product. be.

Solid dosage forms for oral administration should be capsules, tablets, pills, powders and granules. Can be done. In such solid dosage forms, the antigen, epitope, anti-idiotype Antibodies and/or expression products are combined with at least one inert diluent, such as sucrose, Can be mixed with lactose or starch. Such dosage forms As in the practice, substances other than inert undiluted materials, such as magnesium stearate, It can also be configured by adding a lubricant. For Capsules, Tablets and Bills In some cases, the dosage form can also comprise a buffer. Tablets and tablets are also enteric coated. It can be manufactured using counterfeit products.

Liquid dosage forms for oral administration include very small particles, microcapsules, LTB conjugates, i.e. cholera or cholera B subunit as a conjugate in the art such as water. a pharmaceutically acceptable formulation containing commonly used inert diluents; It can be included in syrups, solutions, suspensions and elixirs. like this Compositions also include TraT as a wetting agent, emulsifying agent and suspending agent or conjugate. adjuvants such as and sugars such as sucrose, sorbitol, fructose, etc. Contains sweeteners, flavorings and flavors, polyethylene glycol, propylene glycol glycols such as oil, oils such as sesame oil, olive oil, soybean oil etc., alkyl Preservatives such as rose hydroxybenzoate, etc., as well as strawberry flavor, pepper - It can also consist of flavoring agents such as mint and the like.

zSara planning staff l theory Figure 1 shows the 5O5-PAGE analysis of the LL'' and LL- fractions.

Figure 2 shows 5DS-PAGE analysis of TaAd ESAI, 2.3.4 and 5. vinegar.

Figure 3 shows 505-PAGE analysis of Ta Ad ESAI before and after deglycosylation. show.

FIG. 4 shows the structure of TraT-Tc Ad ESAI.

Figure 5 shows the results used to express TcAd ESAI in Satucharomyces cerevisiae. The yeast expression vector pYEUc114 used is shown.

Figure 6 shows the detection of ESA-encoding sequences in C. elegans and C. elegans. .

of Noh and of Nucleotide sequences, fusion genes, recombinant DNA molecules and transformed hosts of the invention Primarily molecules such as those described by Maniatis et al. (1982) Created using standard techniques in biology.

In the preparation of the nucleotide sequences of the invention, the gene of interest and furthermore the It is recognized that cDNA copies made from genes are also supplied in low yields.

PCR (polymerase chain reaction) technology is used to facilitate detection and cloning. It can be used to amplify relevant DNA.

The expression products of the invention can be used to transform the transformed hosts of the invention into a standard suitable for a particular host. culture under standard conditions and isolate the expression product from the culture by standard techniques. The expression product that can be obtained by by standard techniques appropriate to the expression product that can be produced or produced and the particular host. It can be purified by

The vaccine of the invention comprises antigens, expression products, anti-idiotypic antibodies and/or epitope and a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. using standard drug preparation methods, preferably homogeneously mixing. Prepare.

Antigens, expression products, and anti-idiotype antibodies required to produce a single dosage form and/or the amount of the epitope, the infection to be treated or prevented, the treatment Varies depending on host and particular mode of administration. Specific administration of any particular host The amount depends on the antigen, expression product, anti-idiotype antibody and/or epithelial antibody used. Tope activity, age, host weight, general health, sex and diet, time of administration, It depends on the route of administration, rate of excretion, drug combination, and severity of the infection being treated.

The vaccine may contain any desired pharmaceutically acceptable carriers, diluents, excipients and/or excipients. Administer orally or parenterally in a unit dosage formulation containing the desired adjuvant. be able to.

Antibodies are raised in appropriate hosts using standard vaccination formats. host are antigens, expression products, epitopes, anti-idiotype antibodies and/or are vaccinated using vaccines.

Compounds that act similarly to the antibodies of the invention can be obtained by purifying naturally occurring compounds or by can be synthesized using standard techniques, including standard chemical or biosynthetic techniques. can be manufactured.

Antibody compositions are prepared by transferring the antibody to a pharmaceutically acceptable carrier using standard pharmaceutical preparation methods. , diluents and/or excipients, preferably by homogeneous mixing. Prepare.

The amount of antibody required to produce a single dosage form is determined by the amount of antibody required to produce a single dosage form. Varies depending on the intended host and the particular mode of administration. any particular host specific Dosage values depend on the activity of the antibody used, the host's age, weight, general health, gender and diet, time of administration, route of administration, excretion rate, combination of drugs and treatment. It depends on a variety of factors, including the severity of the infection involved.

Antibody compositions can be carried in any desired conventional, non-toxic, pharmaceutically acceptable carriers, diluents and administered orally or parenterally in unit dosage formulations containing be able to.

The invention will be further illustrated with reference to the following examples.

1 boat on fire Kolb 1 Formis ゛・　ESA　Quantity production Juvenile Merino/Border Leicester crossbreed, 12 months old, kept free of insects. Sheep were infected with 60,000 infective larvae of Colpriformis nematode. Ta. Twenty-one days after infection, sheep were stratified and bred with Bas. The nematodes were collected from the intestine by r+manization). Penicillin (10 RPM containing 0 units/ad) and streptomycin (100 I@/d) Wash the worms in 11640 medium and incubate in an incubator with 5% Cot. Incubated in the same medium as above for 16 hours at 7°C. Visually check insect survival examined and monitored, and typically more than 95% were alive and active. .

Worms and large pieces of necrotic tissue were removed from the medium by filtration or centrifugation.

The supernatant liquid thus obtained is called adult ESA (Tc Ad ESA). Ru.

A similar preparation, termed Tc L4 ESA, was collected from sheep 7-8 days after infection. It was made from the fourth stage larva of Colubliformis ciliary nematode. Then these extracts The components of were subjected to polyacrylamide gel electrophoresis (S) in the presence of sodium dodecyl sulfate. When analyzed by O5-PAGE), L4 and adult extracts have similar antigens. However, the adult extract produced more antigenic substances than the L4 extract, so it was prioritized. Use adult insect extract.

Implementation I L4 ESA and Mormon excretion/secretion antigen by ESA are actually Prepared from L4 and adult Colubliformis nematodes as described in Example 1. Manufactured. This material was prepared in terms of molarity using the method described by Otsdoner et al. (1985). Motts were used to vaccinate intraperitoneally. L4 or young imaginal line ESA obtained from insects provided highly significant protection (62-92% reduction in parasitic infections) in each experiment. (Tables 1 and 2).

Table 1 L4 ESA and lentil lectin affinity chromatography Prophylactic vaccination of guinea pigs using fractions derived from L4 ESA is related to Injected intraperitoneally with certain antigens (n indicates the number of guinea pigs in each group). 28 animals After 1 day, 2,000 larvae were challenged, and 13 days after challenge, they were killed by stratification. I counted the number of insects. LL” is a bound substance, which is eluted from the lentil lectin column. . LL- is a non-binding substance that passes through.

Adult ESA and lentil lectin affinity chromatography Protection of guinea pigs using insect ESA-derived fractions Vaccination was performed intraperitoneally with the relevant antigen (n is the number of guinea pigs in each group). animals were challenged with 2,000 larvae 28 days later. Thirteen days after antigen administration, the number of insects was counted by layer killing. Binds to lentil lectin column The bonded substance is LL'', and the unbound substance is LL-.

Real m Precept J “Easy 1υ3Jl” The supernatant was concentrated 40 times on a Guiaflo J (Amicon) YMIO membrane.

The concentrated liquid was prepared using Tris-buffered saline (TBS; 10s+M), Hi lentil lectin Sepharo equilibrated with 150mM NaCl pH 7,4) The sample was absorbed onto a Su-48 (Pharmacia) column (5XIC1). column Wash with TBSlood at a flow rate of 1 m/min to reduce the absorption of non-absorbing substances (2BOnm). The fractions containing (measured) were collected. specifically bound glycopeptides The column was eluted using a 78592% methyl mannoside solution. 2BOn Fractions containing substances that absorb at - were collected. Hibu lentil lectin binding (L Both the L'') and unbound (LL-) components were treated with 10 volumes of methanol. , the mixture was cooled at -20° C. for 16 hours and heated at 12.000×g for 15 minutes. It was spun out of solution by centrifugation.

When analyzed by 5OS-PAGE (Figure 1), the LL” fraction appears to be molecular weight 81.37.32 and 30 kilodaltons (kD>) and molecular weight standards Contains a Coomassie-stained band with substances of somewhat lower molecular weight when compared to Was. The LL-fraction is about 28-32.17 and 10-12 kg It contained several components including Dalton's major band.

1 boat ■Sat Himamelekun L4 and ESA's Mormo Noe±l Search 1 Prepared from L4 or adult ESA by lentil lectin chromatography. The material was used to vaccinate guinea pigs intraperitoneally (0° Don nal1 et al., 1985), both the bound material and the unbound fraction were These guinea pigs were highly susceptible to subsequent challenge with C. liformis. (Tables 1 and 2), thus reducing binding and spillage Both vaccines may have components that can induce a protective immune response after vaccination. It's obvious.

1 U ESA Heal Bean Lectin Mum Navi SDS Le 1 LL obtained from mouth adult ESA (100-500 μg of protein) and LL-fraction samples in Laemmli buffer (Laemmli, 1970 ) and on a 12.5% separating 5OS-polyacrylamide gel. It was subjected to electrophoretic separation. Proteins were visualized with Coomassie R-250 and electrolyzed. (Stearne et al., 1985).

The components described herein recovered from the LL” fraction are the apparent molecular Tc Ad ESAI with an amount of 30 ko; Tc A with an apparent molecular weight of 37 kll d ESA2 and TcAd ESA5 with an apparent molecular weight of 81 kD. (Figure 2), the components recovered from the LL-fraction and described herein are apparently Tc Ad ESA3 with a molecular weight of 17 kD and T with an apparent molecular weight of 11 kD c Ad ESA4 (Figure 2).

Western transfer fractionated with antibodies raised against purified Tc Ad FtSAl LL”32kD component and L because animals have shown cross-reactivity with these components. The L-28 to 30kD component (Figure 1) seems to be related to the LL''30kD antigen. (TcAd ESAI), these differences are due to the cloned DNA sequence. Analysis predicts that this component is extensively glycosylated; This is thought to be at least in part due to the different degree of glycosylation of TcAdESAI. Ru.

1 delivery 1- [by Mormon] Individual antigens electroeluted from SOS gels were quantified as described in Example 2. It was used for vaccination of motes. Guinea pigs are colpriformis trichophytes. challenged and were shown to be significantly protected against parasitic infection (Table 3). and 4).

Purified antigen, TcA, obtained from lentil lectin-binding fraction (LL'') d Works using ESAI, TcAd ESA2 and TcAd ESA5 Protection of guinea pigs by Chin vaccination Group Antigen Injection (i) n Number of bundles Protective χ vaccination was injected intraperitoneally with the relevant antigen.

Animals were challenged with 2,000 larvae 28 days later and 13 days after challenge. The number of bundles was counted in layers (n indicates the number of animals in each group).

-1-knee Purified antigen, Tc, obtained from lentil lectin non-binding fraction (LL-) Guinea pig vaccination with Ad ESA3 and Tc Ad ESA4 defense Vaccination was performed with antigen isolated from adult Colpriformis trichophyton ESA preparations. was injected intraperitoneally using Animals were infected with 2,000 infective larvae after 28 days. The cells were administered directly, and 13 days after the antigen administration, they were sacrificed and the number of bundles was counted.

Antibody electroeluted from 505-PAGE of both LL” and LL-fractions. In response to challenge infection with the nematode Colubliformis nematode, guinea pigs undergo parenchyma. It is clear from the results in Tables 3 and 4 that it can provide significant protection. In this study, LL” fraction Tc Ad ESAI, Tc Ad ESA2 and Tc Ad ESA5 components and LL-fraction Tc Ad ESA3 and The components of Tc Ad ESA4 are particularly relevant. Other Tc Ad ESA components also It has an effect and is relevant.

Guinea pig vaccine with alhydrogel-assisted TcAd ESAI Vaccination provides 63% protection. Deglucosylation of Tc Ad ESAI (In experiment 257, the number of bundles in the control was abnormal. ), indicating that the protein portion of this molecule may confer protection. Yes: Carbohydrates were clearly not a protective component.

! Service ■1 - Bev's Mi 1 Polypeftides isolated as described in Example 5 were purchased from Applied Biosys. TEM (Applied Biosystem+s) gas phase amino acid sequence analyzer The N-terminal amino acid sequence was analyzed. Purified protein to obtain internal sequence was digested with brotainase [37°C 1-night, 0. IM　NHJCO3(pH 7,8), with an enzyme/substrate ratio of 5−/wχ], the peptide was By HPLC using Aquapore RP-30 column , separated using a gradient from 0.1χTPA to 0.1χTFA/70χ acetonitrile. did. Some of the amino acid sequences obtained are shown in Table 5: the underlined sequences are cD Information for designing oligonucleotide probes suitable for isolation of NA clones It has been found to be particularly useful in providing

Some N-terminal and internal amino acid sequences obtained from Tc Ad ES^1-5 column Tc Ad, ESAI Amino acid terminal sequence: ANNKXQXDIEOLMPKYTc Ad ESA2 Tc Ad ESA3 Amino terminal sequence: KSDEEI IKDALSALTc Ad ESA4 Tryptic peptide: RLADDSDFGNYDSU MKGOWON Tc Ad ESA5 Amino terminal sequence: 5XSLKD In Tc Ad ESAI, reduction and carboxymethylation (0'Donal1 et al., 1973) later amino acid analysis showed the presence of two half-cystine residues. It was done. Tc Ad ESAI N-glycanase (Genzyme) Deglycosylation (this removes carbohydrates attached to asparagine) The apparent molecular weight decreased from 20 kD to 15 kD (Figure 3), which is in close agreement with the information provided by the cDNA sequence (see below).

In a similar procedure, when TcAd ESA2 is deglycosylated, SO5-PAG The apparent molecular weight analyzed by E was reduced from 37 kD to approximately 30 kD.

Disaster relief IL Tz Ad ESA　　　　　　　　：2--　　゛　-゛゛　-　E-Ki single base A cDNA-Eve-1- Metsenger RNA detects 4th stage larvae in the Colubliformis nematode nematode. Guanidine hydrochloride (6M), sodium acetate (0.2M, pH 5.2), and 2 - Triturated in a buffer containing mercaptoethanol (5 kM) and isolated from the larvae by sedimentation and fractionation on an oligo(dT)-cellulose column. did. L4 polyA mRNA is derived from Amersham ribo Nuclease) l/DNA Polymerase I Kit (Amersha+* cDNA A Synthesis System, 11RPN, 1256) as recommended by the manufacturer. It was used as a template for heavy chain cDNA synthesis. Added EcoRI linker Afterwards, the double ticDNA was bound to lambda gt11 and essentially Huin ( E. coli Y1090 cells were grown as described by ) Luynh et al. (1985). into live bacteriophage used to infect. using the above method A cDNA library consisting of 2X10' distinct recombinants was established using Ta. Adult worms in lambda gtlo containing 1.5 x 10' separate recombinants A similar technique was used to establish a cDNA library.

B 1st Gonu Leo ゛Plow゛ i) Tc Ad ESAI Amino acid sequence DIEQLMP is a degenerate oligonucleotide probe used to design.

ii) Tc Ad ESA2 The amino acid sequence NPPIKDTP uses deoxyinosine at the 4-fold heavy position. streetcar oligonucleotide probe ii) Tc Ad ESA3 The amino acid sequence DEEIIKDA is the streetcar oligonucleotide iv) Tc Ad E SA4 Amino acid sequence - MKGQWQN is an oligonucleotide probe To design 5’ TTTTGCCATTGICCTTCATCC^3” used.

v) Tc Ad ESA5 All oligonucleotides are the reverse of the DNA sequence encoding the selected amino acid sequence. It is a complement.

CcDNA-eve-1-, +e-lambda gtll and gtlO L4 and young adult cDNA libraries were respectively amplified and [1985] and Benton and Davis ( Detect duplicate filter lifts as described by Davis) [1977]. The synthetic oligonucleotides described above were used for screening.

D cDN^clone 1 A number of selected clones were cut again with EcoRI and digested with the same enzyme. It contained an insert that could be subcloned into M13mplB. sub The DNA sequence of the cloned insert was determined by Sanger et al. [1980 ] was determined using the method of

The DNA sequences of several clones of Tc Ad ESAI cDNA were determined and The results are summarized in Table 6. The DNA sequence encodes a 130 amino acid protein. It has a reading frame. The N-terminal amino acid sequence was isolated from Ad ESAI. (underlined part in Table 6), and T c Ad ESAI Armillaria ellea ) Two internal peptide sequences resulting from the digestion can also be identified. Tc Ad f ! Large intestine transformed with plasmid vector PTTQ18 containing SAI gene Strain TGI has been given the internal reference number BTA1689.

The DNA sequences obtained from some isolates show some variation in the translated amino acid sequence. It shows that there is. Amino acids that vary are double underlined in Table 6. Ru. The sequence corresponding to the mature protein has been determined. Putative N-terminal leader sequence Column array array has not yet been determined.

This amino acid sequence shows four potential sites of N-linked glycosylation. sequence, AsnX5er/Thr), which is the lentil antigen Antigen binding properties and antigen on S[1S-PAGE after treatment with N-glycanase This is consistent with changes in the mobility of

Finally, the molecular weight calculated from the indicated amino acids (15,300 Daltons) is N- Very consistent with that obtained with glycanase-treated antigen (Figure 3).

Ichimugi once- DNA sequence of cDNA encoding TcAd ESAl and complete mature protein AAA AAA AAA AAATc Ad ESA4 code code The DNA sequence of the clone is shown in Table 7.

The DNA sequence has an open reading frame that encodes a protein of 92 amino acids; It has only one glycosylation site.

Internal pBR322-based vector carrying the gene for TcAd ESA4 , E. coli strain TGI transformed with pBTA503 has internal reference number BTA169. 0 is given. Does not bind to the lentil lectin column and 5O5- The close agreement between the estimated molecular weight by PAGE and the sequence-based predicted molecular weight suggests that this protein This indicates that the protein is not glycosylated.

-JL Nishi- DNA sequence of cDNA encoding Tc Ad ESA4 and translated amino acid A AA AAA AAA AAA ATc Ad ESA3=1-do The DNA sequence of the partial clone is shown in Table 8. This DNA consists of 43 amino acid pairs. It has an open reading frame that encodes a petid. N-terminus obtained from natural protein Sequences corresponding to the amino acid sequences are underlined. Tc Ad shown in Table 8 Plasmid l)T? carrying the ESA3 gene fragment T3 (Phara acia) transformed E. coli strain DH5a F (BRL) with internal reference number 1691 is given.

−Table−■− DIiA sequence and translation amino acid of partial cDNA encoding TcAd ESA3 Noura Tachibaku TraT The TraT of a person's Tc Ad ESA's is large. It is an outer membrane riboprotein of certain strains of S. enterica.

The present inventor has developed antibiotic-resistant plasmid R100 (Og, 1taR, D6, etc.) TraT obtained from J. Bact, 1982, 819-827) We cloned a gene encoding TraT and introduced this gene into a gene encoding TraT. The protein under the control of the leftward promoter (PJ,) of cterophage lambda rasmid transformed. Cells with thermolabile repressor of PL, λc18 57 (Retaout E, et al., 1980. GeneS15.81-93) High levels of TraT can be obtained when incubated at 38-42°C. Wear.

The gene encoding Tc Ad ESAI is a new gene that is the first of TraT. encodes 30 amino acids (including a long signal sequence of 20 amino acids) fused to the 5″ position of the coding region of TraT, and then several amino acids are Occurs at restriction sites used for DNA manipulation and subsequently encodes Tc Ad ESAI A gene was generated that does this (Fig. 4).

Insertion of the Tra-T gene at this position will result in codons 31 and 3 of the TraT gene. 2 by creating a Pvull restriction site by site-specific mutagenesis. It has become possible. The Tc Ad ESAI gene is a 57Qbp Xa+n1 (Ec Cut the oRI site and insert IINA polymerase - Frenow fragment. and was obtained as a fragment of the old ndlff from .

In a suitable E. coli host, increasing the culture temperature will result in 0.00. . . 1 liter per TraT-Tc Ad ESA with an apparent molecular weight of 22kD up to 50μ I is produced.

If the expression level does not exceed the processing capacity of the cells, the signal sequence can be added to the fusion protein. The terminal cysteine can be further modified by cleaving it from the protein. This TraT-immune This modification is important because creating protofusions can confer self-auxiliary properties to the protein. It would be advantageous (International Patent Application PCT/AU87100107, Title of Invention: Exemption) epidemic reinforcement).

Tc Ad ESA TraTW person Tc Ad ESA4 code The gene was constructed using the same method as the construction of TcAd ESAI-TraT. The entire coding region of Tc AdESA4 (9 2 amino acids) are combined with Tc Ad ESA4 under the control of the lambda left promoter. It is expressed as

Code Tc Ad ESAI for roning to YEUC4 cDNA fragments are biotechnology The vector was inserted into pYEUc114, a yeast expression vector developed by the C5IRO department of (Fig. 5), this vector carried the Cup 1 gene (Metal code for tarothiocon). The associated promoter is present in yeast cells. The 0w4-inducible Cup1 promoter is copper-inducible when containing The Cup 1 gene cassette, which has a - and multiple cloning sites, is Laria Patent Application No. 15845/88 and Macreadie etc., described in Plasmid (P1ass+id), L, 147-150. Ru. EcoRI fragment containing the above Tc Ad ESAI cDN^ into pYEUc to replace most of the Cup 1-encoding sequence.

This results in the N-terminal 4 of the metallotiocone following the sequence shown in Table 6. A fusion protein consisting of two amino acids is synthesized. Recombinant plasmid (pY Satucharomyces cerevisiae cells (CL13-ABS Y86 strain, [α, ΔUra31eu2 his pral prbl prcl cps1l) was grown in minimal medium containing histidine and leucine. Ta. To induce the expression of TcAd ESAI, copper sulfate was added to the medium. It was set to 0.5+*M.

After 2 hours in the presence of copper, cells were harvested and treated with zymolyase to remove the yeast external wall. removed and then examined by 5O5-PAGE and Western method. Tc Ad The recombinant plasmid containing the DNA encoding ESAI is pYEUc30 It was named 84B.

1111 steps ゛ and　　　　　　　　　　　　　　　　　 Antigens expressed by recombinant E. coli cells should be purified for vaccination studies. Can be done. The following is illustrative of a method for isolating TcAd ESAI.

Bacterial cells containing the recombinant plasmid described in Example 9 were grown in a suitable medium at 28 grown at °C, and expression of TcAd ES^1 was induced by increasing the temperature to 42 °C. and induce the culture by incubating at this temperature for 4-6 hours. 4°C, Harvest the cells from the culture by centrifuging for 10 minutes at 10,000x g. The pellet in a suitable buffer, e.g. 50mM) Lis-HCl, 10mM EDT A, 50a+M resuspended in NaCl, pH 8.0 and as before Pellet the cells by centrifugation. The washed pellet is placed in a buffer, e.g. 50μH. Tris-HCl, 1a+M EDTA, 5+wM DTT, 0.1++M PM resuspended in SF, pH 8.0, Marton-Gau lin) Homogenize 6 times at 9000 ps i in a homogenizer. Then, the cell homogenate was centrifuged for 20 min at 4 °CCl2O, 0OOX g to remove the recombinant Dense inclusion bodies containing prey antigens are collected. The supernatant was decanted and pelleted. the kit in a solution suitable for solubilizing the proteins in the inclusion bodies, such as 8M urea. , 100mM NaPi, b+M EDTA, 40mM DTT, pH 8 , 5 and incubate for 4 hours at 37°C with stirring. Possible For the solubilized antigen, mix the above solution with “Guiaflo” Amicon YM30. passing through a membrane and then concentrating the eluate on a "Guiaflo" Amicon YMIO membrane. Then, the retenate beam can be recovered by Adjust the pH to 3.0 by adding phosphoric acid, dilute 1:1 with 8M urea, and adjust the Na concentration. to 50s+M, and 8M urea, 50wM, NaPi, 5mM S-Sepharose equilibrated with EOTA, 5mM 0TTS pH 3.0 0 recombinant antigen can be passed through the column in a high flow rate of 50-400mM Elute from the column with a gradient of NaPi. 21kDO recombinant Tc Ad The ESA antigen is collected and concentrated with a Guiaflo J Amicon YMIO membrane. , this concentration was taken as 0.1χ with respect to SDS, in 10,000 cassette-off dialysis bags, 8M urea, 50mM NaPi, 2mM DTT, 0.1χSDS, Dialyzed against pH 8.5 to reduce Na'' concentration to 50mM, and pH The following antigens listed in 8.5 are 15hM NaCl, 101M Tris-HCl, 0.0065M oxidized glutathione, 0.06+aM reduced glutathione, 0. 1χSOS.

Dialysis for 24 hours at room temperature against a solution containing pns,s and finally 150a+M NaC! , 10mM Tris-HCl, 0.1χ SDS, p) 17.4 can be dialyzed against for 24 hours at room temperature. The antigen recovered from the dialysis bag is 0.22 prior to formulation in a suitable adjuvant prior to vaccination of the main animal. - Can be sterilized with a filter.

The details of the purification protocol will vary for each antigen, but other recombinant antibodies can be Similar methods can be carried out to purify raw materials.

1゛　ETC　Ad　ESA 1 Yeast cells with pYEUc30B4E contain histidine and leucine. Cells were grown for 2 days in minimal medium for 2 hours. Cells were then placed in fresh medium for an additional 2 hours. After incubating for a while, steel sulfate was added to a concentration of 0.5 mM. further in Cubate was continued for 2 hours, cells were harvested by centrifugation, and Brau Cells were lysed using a cell homogenizer according to the manufacturer's instructions.

Simply put, cells are destroyed by shaking at high speed with glass beads. It will be done. The glass beads are allowed to sink under gravity to collect the cell lysate. 15 min of crude lysate ．． The supernatant and pellet obtained were centrifuged at 000 rpm■. The presence of one protein was tested. The protein is present only in the 15 krp pellet. It was seen.

This pellet was then combined with 8M urea, 2% SDS, 10mM EDTA and 2% Dissolved in 50s+M ammonium bicarbonate solution containing mercaptoethanol. This crude material was then purified to 1mM using a Sephadefex G75 column. Contains fiDTA, 0.1χ SDS and 0.1χ mercaptoethanol Fractionation was carried out by running a 50+wM ammonium bicarbonate solution. Tc Ad ESA I (non-glycosylated) containing substances of the expected molecular weight and Antiserum raised in rabbits against TcAd ESAI obtained from insect parasites (R Collect the fractions that react with 45) and use them in subsequent vaccination trials. did.

Imperial facility U To ’’ Tc Ad ESA 5295 Number of bundles ±5DI8% control 413±2 99 Vaccination 275±166 33 recombinant Tc Ad ESAI Vaccinate guinea pigs with the formulation and administer colbulifol as described above. The cells were challenged with C. elegans. The number of bundles in control animals was nonspecifically low and this characteristic It should be noted that in different experiments it was scattered. Since something like this happened In the previous case (see e.g. Table 3, Experiment 257), the observation can be made by repeating the experiment. (See, eg, Table 3, Experiment 236).

Sweet potato friend Extension to other parasites TcAd ESA antigen created by recombinant DNA technology can be used for vaccination. Inducing a protective immune response against invasion by C. colubriformis nematodes in infected animals. I can do it. This immune response may also be influenced by other contributors, such as those cited elsewhere herein. Although it is also possible to provide protection against live nematode species, other parasitic nematode species are Tc Expressing a protein related to, but not identical to, the AdESA antigen, Layers can be considered. In most parasitic nematode species, genes can be cloned from these parasites. purifying and preparing components of parasitic substances for the purpose of testing the protective capacity of the components by It is impractical to obtain sufficient parasitic material to identify their structures in the product. , in these cases the only means by which the relevant antigen can be tested is Using recombinant DNA methods to isolate protein-encoding genes Related proteins are expressed in recombinant suspensions, and related proteins are expressed in this recombinant suspension. purified from recombinant organisms, vaccinated animals, and infected with parasitic nematodes. The method is to challenge the species with the following species. Obtaining enough parasite material to purify the antigen Even if possible, the protective antigen related to the TcAdESA antigen The above method uses molecular biology to clone genes for It is the preferred method for developing vaccines. This method is practicable In order to demonstrate the two species, virulent nematodes, which are especially gastric parasites of sheep and goats, and dogs. Remains related to Tc Ad ESAI and 4 are found in heartworm canis, which is a parasite of cats and cats. It proves that there is a gene.

Genomic DNA was obtained from Herrmann (Herr++ann) and Frisharaf (Fr- Purified from three parasites by the method described by John Schauf, 1987. Ta. 1trg of each DNA preparation contains restriction enzymes, BaaHI, PstI and Hin. dm (Promega). Use a marker of an appropriate size. Existing digested DNA was electrophoresed on a 0.4x agarose gel to determine its size. The DNA fragments were separated according to their characteristics. This gel was stained with ethidium bromide. After coloring and photographing the DNA, the DNA in the gel is transferred to the positively loaded nylon Hybridization as recommended by the membrane and manufacturer (A+*ersham) Transferred by alkaline transfer to membranes prepared for use. Tc Ad ESAI and The DNA fragment encoding 4 was obtained from the Boehringer-Mannheim The random labeling method described in the kit sold by Ringer Manheim) Therefore, it was labeled with 32p. This membrane was then treated with radioactive Tc at lQ'' cpm/d. 5xsSpE containing Ad ESA DNA, 5x Denhart (Denhart) hard ts) solution, 0.5χ SDS and 20n/d denatured Hering sperm D Incubated for 20 hours at 42°C in a solution of NA (see Naniatis et al., 1982). Incubated. The membrane is then washed to remove any non-specifically bound DNA. was removed and exposed to X-ray film. Figure 6 shows the torsion nematode and canis filamentum. Add Tc Ad ESA DNA fragment and hybrid to both insect DNA. This indicates that there are specific DNA sequences that form. This allows these The DNA sequence can be obtained from a genomic DNA library or a cDNA library. or polymerase chains from parasites of the above species Parasitic nematodes can be isolated by other recombinant DNA techniques such as reaction and by expansion. can be made from other species of parasitic nematodes, and vaccines against other species of parasitic nematodes Recombinant organisms can be constructed to synthesize these relevant genes for use. It is proven that there will be.

backyard example Scaling up to produce commercial vaccines The manufacturing and purification techniques described above The technique is being performed on a laboratory scale. To commercially produce the antigen of the present invention, Large scale fermentation of the transformed host is required.

Large M1. Peeling fermentation is carried out according to standard techniques; the particular technique chosen is It is suitable for transformed hosts used to produce antigens.

Two millet defeats and one The present invention is directed to parasitic nematodes, particularly colubriformis and torsion nematodes. It can be used as an effective vaccine to protect against parasitic infections in animals. Provide antigens that can be used

Antibodies raised against purified antigens isolated from C. colubriformis and The DNA sequences encoding these proteins have been Identifying relevant peptides and genes encoding antigens from foreign parasitic nematode species. It can be used to Identical DNA sequences and antibodies Related antigens encoding the above proteins within other nematode species that parasitize livestock can be used to identify proteins and genes, and these proteins For parasitic infections by the above species of nematodes, isolated from the parasite itself or Manufactured using recombinant DNA technology. expected to provide an effective vaccine . Parasite species and potential hosts include, for example: Trichinae engraftment or dog hookworm infection in humans, Strongylus vulgaris infection in horses, and human Columbriformis trichium nematode infection in rhododendrons, torsion trichophyte nematode infection in goats, male cows Tertasia ostertagi infection, pigs with trichinella infection, cats with leprosy. Ontoxascaris or Bacterium hookworm infection, Canis hookworm or Trichuris in dogs Pluvis infection and human circulatory system infection by Toxocara spp. larvae and canine filamentous infections. Infection of the circulatory system of dogs by insects, as well as the circulatory system, urogenital system, of the above and other species of animals, Infections of the respiratory system, skin and subcutaneous tissues. Please note that this list is by no means complete. Should.

M! / LL+ LL- Figure 1 T”e　Ad　ES＾ Figure 2 Figure 3 ■ Submission of translation of written amendment between departments of the letter of acknowledgment (Article 184-7, Paragraph 1 of the Patent Act) ■, derived from the parasitic stage of the first parasitic nematode species and selected from the first parasitic and filamentous main genus; The antigen according to claim 1, wherein any of claims 1 to 3 is selected.

9. The first nematode species are Trichinella, hookworm, Strongylus vulgaris, and Colubrych. formis trichophyte nematode, torsion tricholate nematode, ostertasia ostertagi, pig field soil, La iontoxascaris, occipital hookworm, triclis purvis, canis filiformis, toxoca Ra species, American hookworm, Zubini hookworm, roundworm, human dewormer, baked earth, fecal worm and bank Antigen according to claim 8, selected from lofted filamentous bundles.

10. The second nematode species are the genus Trichinella, the genus Ankylostoma, the genus Strongyloides, and the genus Trichinella. Needleworms, Ostertasia, Soil, Toxascaris, Lancinaria, Anthelmintic Genus, Dirofilaria, Toxocara, Necator, Burning, Strongillus Antibiotics according to any one of claims 1 to 9 selected from the genus Des and the filamentous genus. original.

11. The second nematode species are Trichinella, hookworm, Strongylus vulgaris, and Coluburi. Formis hairy nematode, Tortis hairy nematode, Ostertasia ostertagi, pig field soil, Lion Toxascaris, Pinocchia hookworm, Tricris purvis, Canis filiformis, Toxodonta Kara species, American hookworm, Zubini hookworm, roundworm, human dewormer, baked earth, fecal worm and van 11. The antigen according to claim 10, selected from Croft filamentous bundles.

12. Claims 1 to 11, wherein the first parasitic nematode species is Colubliformis trichophytes. The antigen according to any one of .

13. The nematode according to claims 1 to 12, wherein the second nematode species is Colubliformis nematode. The antigen according to any one of the above.

14° Aha Asn Asn Lys Gin Gin Thr Asp lie Glu Gln Leu Met Pr.

Lys Tyr Asn Ser Thr Phe Ala L ys Met Asn Gly Asn Tyr@5er Tyr Lys Leu Ile Trp Asp Asp S er　Met　Val　Ser　Asp　Ala@　Leu Gin Glu Ala Lys Glu Gln Tyr Ser Thr Asn Ala Thr Phe Lyslle Arg Arg Ar g Lys Val Phe Tie Lys Gay Asp 　Asn　Ala@　Thr Met Glu Glu Lys Val Glu Gly Ala Leu Lys Tyr Pro Val LeuArg Ala Asp Ly s　Phe　Leu　Arg　Arg　Leu　Leu　Trp　 Phe Thr g45 Tyr Ala Cys Asn Gly Tyr Tyr Asp Thr Lys Gly Gly His AspVal Leu Thr Va l Ala Cys Leu Tyr Arg Glu lie Asp Tyr@Lys Asn Ser His Tyr The antigen according to any one of claims 1 to 13, consisting of the amino acid sequence.

15° Met Leu Tyr Lys Lys Leu Arg Ser Gln Gly Asn Phe Arg LysAsn Asp Ser Ala T yr　Phe　Lys　Leu　Glu　Asn　Lys　Arg　Glu　L eu ys Gly Asp Asn Leu Pro Val Glu Glu Lys Val Arg Gln@Thr lle Glu Lys Phe Lys Asp Asp V al Ser Glu Ile Arg Arg@Leu Ala Asp Asp Ser Asp Phe Gly C ys Asn Gly Lys Glu Thr@Gay Gly Aha Met His lie Val Cys Phe Phe Gin Lys Asn Tyr AspTrp Met Lys Gly G Any of claims 1 to 13, consisting of the amino acid sequence in Trp Gln Asn. The antigen according to item 1.

16° Arg　Phe　Leu　Leu　Leu　Ala　Aha　Phe　Val　 Ala Tyr Ala Tyr AlaLys Ser Asp Glu G lu　Ile　Arg　Lys　Asp　Ala　Leu　Ser　Aha　L euAsp Val Val Pro Leu Gay Ser Thr P A chain consisting of the amino acids ro Glu Lys Leu Glu Asn Gly 14. The antigen according to any one of claims 1 to 13.

17. Tc Ad ESAI as defined above.

18, Tc Ad defined above ESA2゜19, Tc Ad defined above ESA3゜20, Tc Ad defined above ESA4゜21, defined above TcAd ESA5°22, the antigen according to any one of claims 1 to 21 a first nucleotide sequence encoding an amino acid sequence of the nucleotide sequence forms a hyperprint with the first nucleotide sequence or one or by mutation including multiple base substitutions, insertions, or deletions. hybridizes with a nucleotide sequence associated with a nucleotide sequence. Ru.

23. The nucleotide sequence is any one of claims 2 to 7 or claim 14. Claim 22 characterized by the amino acid sequence of the antigen according to any one of claims 21 to 22. The nucleotide sequence described.

24. The nucleotide sequence according to claim 22 or 23, wherein the nucleotide sequence is a DNA sequence. Nucleotide sequence.

25° GAA TTCGGG GGCAACACT TACAGT GCA AAACAAT AAG CAA CAG ACCGACATA G AA CAA CTCATG CCCAAA TAT AACTCG ACG TTCGCG AAGATG AAT GGA AAC TAT AGT TAT AAG CTG ATCTGG GAT 　GACAGCAsG GTA TCT GAT GCG CTG CAA GAA CA AAG GAG CAA TACAGT `CG AAT GCT ACCTTCAAG ATC-σII; T CGG AGA AAG GTG TTCAT8 AAG GGCG`T AACGCA ACG ATG GAG GAA AAA GTG GAG GGA GCT CTG AAG sACCCC GTCTTG AGA GCCGAT AAA TTT CTT CGCCGT CTT CTCTGG TTCACACACTACGCA TGCAAT GGA TAT TACGAT ACG AAA GGT GGA CACGATGTCCTG ACT GTCGC G TGT CTCTACAGA GAG ATCGAT TACA AA AATTCT CACTAT TAG AAA GCA GTCAAC AAA AACAGCAGA GTA AACTGACTG CACATT T CCGCA GTT TTT GAA TAA ATA CTT GAT GC Claim 24 consisting of A ACT CAAAAA AAA AAA The DNA sequence described in.

26° ACT ACA AGA CCCCAA TTG TACACG AAA　TTCTTCCAACGAA　GAA　AACAGCCTA　A AT CTG AGA TGG AACCACATA TGT C GCAGCATG CTCTACAAG AAA TTG AGA AGCCAG　GGA　AAT　TTT　CGCAAA　AAT　 GAT　TC`　GCA TAT TTCAAG CTCGAA AACAAG AGG GA A　CTG　AAG　GGA　GACAAT　CsA CCA GTG GAG GAG AAA GTA CGCCAA ACT ATT GAA AAA TTCAAf GAT GAT　GTA　AGCGAA　ATCAGA　CGT　CTT　 GCT GAT GAT TCG GAT TTs GGA TGCAACGGCAAA GAA ACCGGG GGT GCA ATG CACATT GTG TGT TTCTTCCAG A AG AAT TAT GACTGG ATG AAA GGA CAA TGG CAA AAbTGA TTT TTCTGA AGT ACT TGT TGG ATT CTT CGT AGA ATCGAT GC` CAA AAT ACCTTT TTT GGG AGA CAA CTT CGCAT AAA CTT CTCGAT @GAA AAA AAA AAA 8 20. The DNA sequence of claim 19 consisting of.

27° CGG TTCCTT CTT CTA GCA GCG TTC GTC, GCCTAT GCG TAT GCA `AG TCA GAT GAA GAA ATCCGA AAA GAT GCA CTA TCT GCT CTG fAT GTA GTT CCA CTG GGT TCG ACT CCCGAA According to claim 24, consisting of AAA, CTG, GAA, AAT, and GGC. The DNA sequence of

28. DNA encoding the antigen according to any one of claims 1 to 21, or A method of selecting an RNA sequence, the method comprising selecting one or more DNA or provides an RNA sequence, and which sequence is defined in any one of claims 1 to 21. hybridizes with a DNA or RNA sequence known to encode the antigen described above. Alternatively, provide an antiserum against the antigen and It consists of identifying the expressing host-vector combination.

29, the DNA sequence and vector D according to any one of claims 24 to 27 A recombinant DNA molecule consisting of NA.

30, further comprising an expression control sequence operably linked to said DNA sequence. The recombinant 13NA molecule described in .

31, the expression control sequence is the β-galactosidase gene of Escherichia coli, tryptophan Peron, Tra-T gene of E. coli, left-sided promoter of bacteriophage lambda promoter, tac promoter, Cupl promoter and SV40 early promoter 31. The recombinant DNA molecule according to claim 30, which is selected from the group consisting of:

32. Any of claims 29 to 31, wherein the vector DNA is plasmid DNA. The recombinant DNA molecule according to item 1.

33. The above plasmid DNA is pUR290, pUc18, pYEUc114 33. A recombinant DNA molecule according to claim 32, selected from: and derivatives thereof.

34. Claim 29, wherein the vector DNA is bacteriophage DNA; or The recombinant DNA molecule according to 30.

35. The bacteriophage DNA described above produces lambda gtlO and lambda gtll. 35. selected from bacteriophage lambda and derivatives thereof comprising recombinant DNA molecules.

36, pYEUc30B4E as defined above.

37, a promoter, a translation initiation signal, and any one of claims 24 to 27 A fusion gene consisting of the DNA sequence described in section.

38. A method for preparing a recombinant DNA molecule according to any one of claims 29 to 35. 28. A method, wherein the method comprises using a DNA according to any one of claims 24 to 27. providing a DNA insert consisting of the sequence and introducing the DNA insert into vector DNA. It consists of entering.

39. The DNA insert is in the correct location and correct readability with respect to the expression control sequences. 39. The method according to claim 38, wherein the method is introduced into a cloning vector in frame.

40, at least one recombinant D according to any one of claims 29 to 36 A transformed host transformed with an NA molecule.

41, wherein the host is capable of expressing the antigen according to any one of claims 1 to 21; 41. The transformed host according to claim 40.

42, the host is a bacterial cell, Satucharomyces cerevisiae strain CL13-ABSY8 6 including yeast, other fungi, animal cells, insect cells, plant cells, human cells, human Synthetic cells, live viruses such as variola virus or baculovirus, or whole viruses. 42. The transformed host according to claim 40 or 41, which is a eukaryote.

43. The above host is E. coli, other intestinal organisms other than E. coli, Pseudomonas species, or 43. The transformed host according to any one of claims 40 to 42, which is Hasillus sp.

44, 12 induction into E. coli where the above host is selected from JM109 and Y1090 44. The transformed host according to claim 43, which is a transformed host.

45, BTA defined above 1689° 46, BTA defined above 169 1°47, BTA as defined above 1690°48, any of claims 40 to 47 A method for transforming a host which provides the transformed host according to item 1 above, wherein The method provides a host, creates a host suitable for transformation, and comprises from introducing the recombinant DNA molecule according to any one of 36 into the host. Ru.

49. Claims 40 to 49, consisting of the antigen according to any one of claims 1 to 21. 48. An expression product of the transformed host according to any one of 47.

50. The expression product of claim 49 of a substantially purified trait.

51, a first polypeptide sequence homologous to said host and the host of claims 1 to 21. a second polypeptide sequence which is an amino acid sequence encoding an antigen according to any one of the preceding paragraphs; 51. An expression product according to claim 49 or 50, consisting of a sequence of

52, the first polypeptide sequence is all or part of β-galactosidase or 52. The expression product of claim 51, wherein is Tra-T and the host is E. coli.

53. A protein product consisting of the antigen according to any one of claims 1 to 21. 22. A method for biosynthesizing a host according to any one of claims 1 to 21. claims 29 to 36 for expressing a protein product containing an antigen according to transforming the host with a recombinant DNA molecule according to any one of the following: consists of cultivating, expressing, and harvesting the protein product.

54. Any one of claims 1 to 21, wherein the epitope is responsible for a protective immune response. epitopes of the antigens described in .

55. An antibody raised against the epitope of claim 54.

56. An anti-idiotype antibody raised against the variable region of the antibody according to claim 55. .

57. An effective amount of one or more of any one of claims 1 to 21. an antigen according to any one of claims 49 to 52, an expression product according to any one of claims 49 to 52, and/or the anti-idiotype antibody according to claim 56. consisting of a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. vaccine.

58. A vaccine according to claim 57, suitable for oral administration or in injectable form.

59, Tc Ad ESAI, Tc Ad ES8 defined above, Tc A d ESA3, Tc Ad ESA4, Tc Ad ESA5 or these antigens an effective amount of the antigen selected from all or some combination of Vaccines consisting of acceptable carriers, diluents, excipients and/or adjuvants .

60. One or more antigens according to any one of claims 1 to 21; The expression product according to any one of claims 49 to 52, the epitope according to claim 54 tope, the anti-idiotype antibody of claim 56 and/or claim 57 Vaccine in a host by administering to the host the vaccine according to any one of Paragraph 59. Antibodies produced as a result of Chin inoculation.

61, an effective amount of at least one antibody according to claim 55 or 60 and a pharmaceutically acceptable amount of An antibody composition comprising an acceptable carrier, diluent and/or excipient.

62. A method for preparing the antigen according to any one of claims 1 to 21, comprising: The method involves collecting excretory/secretory fluid from parasitic nematode species during the parasitic stage and using methane as the eluent. The above liquid is analyzed by chromatography using lentil lectin using rumanoside. The bound and unbound fractions were collected and analyzed by 5DS-gel electrophoresis. further fractionation and electroelution of the antigen.

63. A method for preparing a fusion gene according to claim 37, the method comprising: -, a translation initiation signal and the DN according to any one of claims 24 to 27. A sequence and manipulate the promoter, translation initiation signal and DNA sequence. It consists of creating and combining.

64, a method for preparing the vaccine according to any one of claims 57 to 59, , the method comprises an effective amount of at least one of the following: an antigen and/or an expression product according to any one of claims 49 to 52; and/or the epitope according to claim 54 and/or the antibody according to claim 56. Idiotypic antibodies and pharmaceutically acceptable carriers, diluents, excipients and/or It consists of mixing with an adjuvant.

65, a method for preparing the antibody according to claim 55 or 60, the method comprising: The antigen according to any one of claims 1 to 21, any of claims 49 to 52 or the expression product of claim 1, the epitope of claim 54, or the expression product of claim 56. or the vaccine according to any one of claims 57 to 59. consists of immunizing an immunocompetent host with Chin.

66. A method for preparing an anti-idiotype antibody according to claim 56, the method comprising: Immunization with the anti-epitope antibody according to claim 55 or the antibody composition according to claim 61. consists of immunizing an infectiously-competent host.

67. A method for preparing the antibody composition of claim 61, the method comprising: or an effective amount of at least one antibody according to 60 in a pharmaceutically acceptable carrier; It consists of mixing with diluents and/or excipients.

68. A method for preventing infection of a host that requires protection against infection by parasitic nematodes, The method comprises at least one antigen according to any one of claims 1 to 21; an expression product according to any one of claims 49 to 52; an epitope according to claim 54; anti-idiotype antibody according to claim 56, and/or from claim 57. comprising vaccinating a host with a vaccine according to any one of Items 59 to 59.

69, to passively protect the host in need of treatment against infection by parasitic nematodes. 61. A method comprising at least one antibody according to claim 55 or 60. and/or passively vaccinating a host with the antibody composition of claim 61. It consists of things.

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Claims (64)

[Claims] 1. derived from a first parasitic nematode species, the same or different from the first parasitic nematode species; induce protective immunity against infection of the host by a second parasitic nematode species that can excreted/secreted proteins obtained; or all, parts, or analogs of said excreted/secreted proteins; protein, homolog, derivative, or a combination thereof (the protein proteinaceous molecules can confer protective immunity to the host against infection by parasitic nematodes) Antigen consisting of. 2. Approximate molecular weight 11, 17, 30, 37 or An antigen consisting of the excreted/secreted protein according to claim 1 having 81 kD. 3. The first nematode species are Trichinella, Ankylostoma, Strongyle, Tricholate, and Needle. Genus Insecta, Genus Ostertasia, Genus Arachnidae, Genus Toxascaris, Genus Uncinaria, Genus Trichuris. , Scirofilaria spp., Toxocara spp., Necator spp., Pinworm spp., Strongyloidea 3. The antigen according to claim 1 or 2, which is selected from the genus Moroccans and the genus Filiaria. 4. The first parasitic nematode species are Trichinella, hookworm, Strongylus vulgaris, and Kolb. liformis tricholate nematode, torsion tricholate nematode, ostertasia ostertagi, swine worm , lion toxascaris , occipital hookworm , triclis vulpis , canine heartworm , ibis Socara species, American hookworm, Zubini hookworm, roundworm, human whipworm, pinworm, fecal worm, and barium worm. 4. The antigen according to claim 3, which is selected from P. nigra. 5. The first nematode species are Trichinella, Ankylostoma, Strongyle, Tricholate, and Needle. Genus Insecta, Genus Ostertasia, Genus Arachnidae, Genus Toxascaris, Genus Uncinaria, Genus Trichuris. , Scirofilaria spp., Toxocara spp., Necator spp., Pinworm spp., Strongyloidea The antigen according to any one of claims 1 to 4, which is selected from the genus Traciformes and the genus Filiaria. . 6. The second parasitic nematode species are Trichinella, hookworm, Strongylus vulgaris, and Kolb. liformis tricholate nematode, torsion tricholate nematode, ostertasia ostertagi, swine worm , lion toxascaris , occipital hookworm , triclis vulpis , canine heartworm , ibis Socara species, American hookworm, Zubini hookworm, roundworm, human whipworm, pinworm, fecal worm, and barium worm. 6. The antigen according to claim 5, which is selected from P. nigra. 7. 7. The nematode of claims 1 to 6, wherein the first parasitic nematode species is Colubliformis nematode. The antigen according to any one of the above. 8. Any one of claims 1 to 7, wherein the second nematode species is Colubliformis nematode. The antigen according to item 1. 9. According to any one of claims 1 to 8, consisting of the amino acid sequence [there is a sequence]. antigens. 10. [There is an array] The antigen according to any one of claims 1 to 8, consisting of the amino acid sequence. 11. [There is an array] The antigen according to any one of claims 1 to 8, consisting of the amino acid. 12. Tc Ad ESA1 defined above. 13. Tc Ad ESA2 defined above. 14. Tc Ad ESA3 defined above. 15. Tc Ad ESA4 defined above. 16. TcAd ESA5 defined above. 17. Encoding the amino acid sequence of the antigen according to any one of claims 1 to 16 a first nucleotide sequence of said first nucleotide sequence, wherein said nucleotide sequence is identical to said first nucleotide sequence; hybridizes with the leotide sequence or has one or more nucleotide sequences. The first nucleic acid is mutated by mutation including multiple base substitutions, insertions or deletions. Associated with the otide sequence. 18. The nucleotide sequence is according to claim 2 or any one of claims 9 to 16. 18. The nucleotide sequence according to claim 17 encoding the amino acid sequence of the antigen according to claim 17. . 19. Claim 17 or 18, wherein the nucleotide sequence is a DNA sequence. Nucleotide sequence. 20. [There is an array] 20. The DNA sequence of claim 19 consisting of. 21. [There is an array] 20. The DNA sequence of claim 19 consisting of. 22. [There is an array] 20. The DNA sequence of claim 19 consisting of. 23. DNA encoding the antigen according to any one of claims 1 to 16, or A method of selecting an RNA sequence, the method comprising selecting one or more DNA or provides an RNA sequence, and which sequence is defined in any one of claims 1 to 16. hybridizes with a DNA or RNA sequence known to encode the antigen described above. Alternatively, provide an antiserum against the antigen and It consists of identifying the expressing host-vector combination. 24. DNA sequence according to any one of claims 19 to 22 and vector D A recombinant DNA molecule consisting of NA. 25. Claim 24 further comprising an expression control sequence operatively linked to said DNA sequence. The recombinant DNA molecule described in . 26. The expression control sequence is the β-galactosidase gene of E. coli, tributophane Peron, Tra-T gene of E. coli, left-sided promoter of bacteriophage lambda promoter, tac promoter, Cup1 promoter and SV40 early promoter 26. The recombinant DNA molecule according to claim 25, selected from the group consisting of: 27. Claim 25 or 24, wherein the vector DNA is plasmid DNA. recombinant DNA molecules. 28. The above plasmid DNA is pUR290, pUC18, pYEUC114 and 28. A recombinant DNA molecule according to claim 27, selected from: and derivatives thereof. 29. Claim 25, wherein the vector DNA is a bacteriophage DNA. 26. The recombinant DNA molecule according to 26. 30. The bacteriophage DNA described above produces lambda gt10 and lambda gt11. 30. The bacteriophage lambda and its derivatives comprising: recombinant DNA molecules. 31. pYEUC30B4E as defined above. 32. A promoter, a translation initiation signal and any one of claims 19 to 22 A fusion gene consisting of the DNA sequence described in section. 33. A method for preparing a recombinant DNA molecule according to any one of claims 24 to 29. 23. A method, wherein the method comprises using a DNA according to any one of claims 19 to 22. providing a DNA insert consisting of the sequence and introducing the DNA insert into vector DNA. It consists of entering. 34. the DNA insert is in the correct position and correct reading with respect to the expression control sequences; 34. The method according to claim 33, wherein the method is introduced into a cloning vector in frame. 35. At least one recombinant D according to any one of claims 24 to 31 A transformed host transformed with an NA molecule. 36. Claims that the host is capable of expressing the antigen according to any one of claims 1 to 16. 36. The transformed host according to item 35. 37. The above host is a bacterial cell, Saccharomyces cerevisiae strain CL13-ABSY8 Yeast, other fungi, vertebrate cells, insect cells, plant cells, human cells, humans, including 6 tissue cells, live viruses such as variola virus or baculovirus, or whole 37. The transformed host according to claim 35 or 36, which is a eukaryote. 38. If the above host is E. coli, other intestinal organisms other than E. coli, Pseudomonas species or 38. The transformed host according to any one of claims 35 to 37, which is a Bacillus species. 39. E. coli K12 induction in which the host is selected from JM109 and Y1090 The transformed host according to claim 38, which is a transformed host. 40. BTA 1689 as defined above. 41. BTA 1691 as defined above. 42. BTA 1690 as defined above. 43. A host that provides the transformed host according to any one of claims 35 to 42. a transformation method, wherein the method provides a host and a suitable host for transformation. and the recombinant DNA segment according to any one of claims 24 to 31. It consists of introducing the offspring into the host. 44. Claims 35 to 35, consisting of the antigen according to any one of claims 1 to 14. 43. An expression product of the transformed host according to any one of 42. 45. 45. The expression product of claim 44, which is a substantially purified trait. 46. a first polypeptide sequence homologous to said host and the host of claims 1 to 16; a second polypeptide sequence which is an amino acid sequence encoding an antigen according to any one of the preceding paragraphs; 46. An expression product according to claim 44 or 45, consisting of a sequence. 47. The first polypeptide sequence is all or part of β-galactosidase or 47. The expression product of claim 46, wherein is Tra-T and the host is E. coli. 48. A protein asset organism comprising the antigen according to any one of claims 1 to 16. 17. A method for biosynthesizing a host according to any one of claims 1 to 16. Claims 24 to 3 capable of expressing a protein product containing an antigen according to claim 24 to 3. transforming the host with a recombinant DNA molecule according to any one of 1. It consists of culturing, expressing, and harvesting the protein product. 49. 17. Any one of claims 1 to 16, wherein the epitope is responsible for a protective immune response. epitopes of the antigens described in . 50. 50. An antibody raised against the epitope of claim 49. 51. An anti-idiotypic antibody raised against the variable region of the antibody of claim 50. . 52. An effective amount of one or more of any one of claims 1 to 16. an antigen according to any one of claims 44 to 47, an expression product according to any one of claims 44 to 47, according to claim 49 and/or the anti-idiotype antibody according to claim 51. consisting of a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant. vaccine. 53. 53. A vaccine according to claim 52, suitable for oral administration or injectable form. 54. Tc Ad ESA1, Tc Ad ESA2, Tc A defined above d ESA3, Tc Ad ESA4, Tc Ad ESA5 or these antigens an effective amount of the antigen selected from all or some combination of Vaccines consisting of acceptable carriers, diluents, excipients and/or adjuvants . 55. One or more of the antigens according to any one of claims 1 to 16; The expression product according to any one of claims 44 to 47, the expression product according to claim 49 Tope, the anti-idiotypic antibody of claim 51 and/or claim 52 Vaccine in a host by administering the vaccine according to any one of Paragraph 54 to the host. Antibodies produced as a result of Chin inoculation. 56. an effective amount of at least one antibody according to claim 50 or 55; An antibody composition comprising an acceptable carrier, diluent and/or excipient. 57. A method for preparing the antigen according to any one of claims 1 to 16, comprising: The method involves collecting excretory/secretory fluid from a parasitic nematode species and using methylmannose as the eluent. The above liquid was fractionated by lentil lectin chromatography using Combined and unbound fractions were collected and further fractionated by SDS-gel electrophoresis. , and electroeluting the antigen. 58. 33. A method for preparing a fusion gene according to claim 32, wherein the method comprises -, a translation initiation signal, and the DNA according to any one of claims 19 to 22. Providing sequences and manipulating promoters, translation initiation signals and DNA sequences and then combine them. 59. 55. A method for preparing a vaccine according to any one of claims 52 to 54, , the method comprises an effective amount of at least one of the following: an antigen and/or an expression product according to any one of claims 44 to 47; and/or the epitope according to claim 49 and/or the antibody according to claim 51. idiotypic antibodies and pharmaceutically acceptable carriers, diluents, excipients and/or It consists of mixing with an adjuvant. 60. 56. A method for preparing an antibody according to claim 50 or 55, wherein the method comprises administering an effective amount of The antigen according to any one of claims 1 to 16, any one of claims 44 to 47 The expression product according to claim 1, the epitope according to claim 49, the expression product according to claim 51 or the vaccine according to any one of claims 52 to 54. consists of immunizing an immunocompetent host with Chin. 61. 52. A method for preparing an anti-idiotypic antibody according to claim 51, comprising: Immunization with the anti-epitope antibody according to claim 50 or the antibody composition according to claim 56. consists of immunizing an infectiously-competent host. 62. 57. A method for preparing an antibody composition according to claim 56, wherein the method comprises: or an effective amount of at least one antibody according to 55 in a pharmaceutically acceptable carrier; It consists of mixing with diluents and/or excipients. 63. A method for preventing infection of a host that requires protection against infection by parasitic nematodes, the method comprising: The method comprises at least one antigen according to any one of claims 1 to 16; the expression product according to any one of claims 44 to 47; the epitope according to claim 49; anti-idiotypic antibody according to claim 51 and/or from claim 52 vaccinating a host with a vaccine according to any one of 54. 64. passively protects the host in need of treatment against infection by parasitic nematodes 56. A method comprising at least one antibody according to claim 50 or 55. and/or passively vaccinating a host with the antibody composition of claim 56. It consists of things.