JPH05507207A - nematode 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] nematode vaccine Technical field The present invention relates to antigens that confer protective immunity against infection by parasitic nematodes.

The invention also provides vaccines and vaccines that confer protective immunity against infection by parasitic nematodes. This invention relates to antibodies that confer passive immunity against infection by parasitic nematodes.

Background technology Nematodes (newa - thin line; oides - similar) are elongated, spiny bodies covered with angles. An unsegmented roundworm with a cone-shaped or sac-like body; It is ubiquitous in soil, water and plants, and is found in a wide range of animals and plants. It is closely related to parasitic diseases of plants.

Mammalian roundworm parasites belong to the phylum Nematoda. Roundworms include hookworms (e.g. N. ecator affleriaanus and Aneylostowa, d uodenale), roundworms (e.g. Ascarfs, a common roundworm) umbricoides), whipworms (e.g. Trichuris trich iura), and enterobacteria or nematodes (e.g. Enterobjus ve rmicularus) and Strongyloides stercora , lis, Trichinella 5piralis (human and pig ), and the heartworm Wuchereria bancrofti.

Other important roundworm parasites include Aneylostowa caninuw (human (infectious diseases of horses), Strongylus vulgarls (infectious diseases of horses), Trichostrongylus colubrifor*is (sheep infectious diseases), Haemonchus contortus (infectious diseases of sheep and goats) Infectious disease)・Ostertagla ostertagi (infectious disease of cattle), Ascarjs 5uus (swine infection), Toxascarfs Ieo nfa or Llincinaria 5tenocephala (infection of dogs) ), Toxocara species (human circulatory system infections) and Diroftlar ia tmmitis (circulatory system infection of cats and dogs).

Even in the absence of symptoms, parasitic infections are harmful to the host animal in many ways; For example, they can deprive the host of food, damage organs or block ducts, and cause harm to the host. They may synthesize substances and provide entry routes for other organisms. in other cases The host may be a species bred for food, and the parasite may not be eaten. can be transmitted to infected animals. Such parasites It is highly desirable to remove them as soon as possible after they are discovered.

Furthermore, such infections are generally not asymptomatic.

Infectious diseases caused by mammalian insects, especially parasitic nematodes, are particularly common in livestock, beds, and animals. large animals such as sheep, cattle, horses, pigs, goats, dogs, cats and birds, especially poultry. This is a major cause of economic loss. These animals should be regularly treated with anthelmintics to prevent this. These infections must be kept under control, otherwise the disease can lead to anemia, diarrhea, It can cause dehydration, loss of appetite, and even death.

The only means currently available to prevent the spread of worm infections is through the use of anthelmintics. However, these compounds have no effect on parasites present at the time of treatment. It's just effective. Therefore, the treatment is continuous as the animal is constantly exposed to infection. For example, Dirofilarta i+amitis matahai To prevent heartworm growth, use diethyl chloride every day or every other day most of the year. Anthelmintic treatment with Carbamacin is required. . This is expensive and requires This is a time-consuming method. Widespread use of anthelmintic drugs causes parasites to become resistant therefore, new and more potent classes of drugs must be developed. No. Clearly, another method is desirable.

If a vaccine against parasitic nematodes is developed, it will be possible to prevent or treat nematode infections. Many of the shortcomings inherent in chemical treatment of rice will be overcome. This protection is certainly long-lasting. It will last for a long time, will only affect vaccinated animals, and will reduce the toxicity and toxicity of residues. and persistence problems would be greatly reduced or avoided. Therefore, No attempts to develop such vaccines using live nematodes have been reported in the prior art. Unfortunately, these attempts have had limited success, and material availability and Factors such as vaccine stability and vaccine stability preclude their reasonable use. Ru.

These previous attempts were published in International Patent Application No. PCT/^IJ8g100239 (W 089100163) and PCT/AU1191004i111 (v0901 It is described in the specification of No. 03433.

Advances in bacterial biotechnology, especially recombinant DNA technology, have made it possible for humans and families to Produce commercially viable vaccines against a range of economically important parasites of livestock Opportunities are realistically provided. According to this method, crude parasite preparations are used to Many of the issues raised to explain the lack of efficacy of dead vaccines remain to be resolved. It becomes.

For example, vaccines produced by recombinant DNA technology are made from crude strains of parasitic nematode species. It will not contain immunosuppressants or immunomodulators found in extracts. but However, it is first necessary to identify the antigen. Once identified and characterized Once done, recombinant DNA technology can be used to develop proteins or proteins containing protective epitopes. It constitutes a microorganism that synthesizes a part of this protein, and is synthesized by a recombinant organism. products can be used in vaccines to protect animals from infection by parasites. There will be.

PCT/AU88700239 states that it is nematode tropomyosin. The recombinant DNA-derived antigen shown is Haevronchus cont. It has been revealed that sheep have 50% protection against attacks by ortus. ing.

In PCT/AU8910041G, adult Trichstrongy Excretory/secretory antigens from P. luscolubrirora+fs were vaccinated. It has been shown to provide protection to guinea pigs. will be revealed later in this specification These antigens are different from the antigens identified herein for several reasons. .

As used herein, the term "adjuvant" refers to an immunizing agent for an immunizing composition. Represents an agent used to enhance the immune response of a given host.

As used herein, the term "parenteral" refers to subcutaneous, intraperitoneal or intramuscular injections. Includes injection or infusion techniques.

The term “homolog” refers to proteins that are clearly related in function. Proteins that are structurally related to the first protein or DNA sequence to some extent represents the protein or DNA sequence that encodes the protein. In the context of the present invention, the present invention H,contortυS-derived DNA encoding the antigen of Identification of specific NA sequences of other species of parasitic nematodes for use in diization experiments. It explains what can be done. Conditions under which hybridization experiments were conducted The fact is that the related DNA sequences are small in nucleotide sequence spanning 60 base pairs. Both are 50% homologous to the nucleotide sequence isolated from Il,contortus. This shows that they are the same. These related DNA segments have amino acid sequences. Also in the column are parasites related to protective antigens isolated from H. contortus. Encodes antigens in other species of live nematodes. Related proteins are found in other species Claimed to act as an effective immunogen to protect animals from parasitic infestations It is. These related DNA sequences are called homologous genes, and they are called homologous genes. antigens are called homologous antigens. In the context of the present invention, protective antigens also include genetic The coding polynucleotide and gene product are members of the 60 About 50% for nucleotides or 70% for 20 amino acids. It is clear that they share homology with the coding gene of the present invention and the protective antigen, respectively. It is being done. These related genes and gene products are also homologs of the present invention. It is.

Homologs of the invention are parasitic nematodes expressing in-human antigens as described below. Antigens obtained by isolation from life stages of parasitic nematodes as well as preparations from life stages of parasitic nematodes nucleotide sequences, such as genomic DNA mRNA.

cDNA synthesized from mRNA and having a sequence corresponding to the antigen coding sequence Antigens obtained by replication and expression of synthetic nucleotides created to It means to include.

It is expressed by a nematode or a cell line expressing a recombinant form of the antigen Also includes synthetic peptides created based on the known amino acid sequence of the antigen. [novel J or "hidden" antigens, i.e. valid and Can act as a protective immunogen but does not participate in naturally occurring immunity Preferably, the components of the parasite are identified. To identify which components the host were vaccinated with extracts from the parasite, the fractions that showed some protection were identified, and the protein This protective component can be refractionated using scientific chemistry techniques, and these small fractions can be used to protect animals. inoculate, identify these small fractions, and protect with this method, using pure parasite components. It is necessary to continue vaccinating animals until they are protected.

Whenever possible, natural hosts should be used in such experiments. laboratory model These animals cannot be natural hosts because animals are not natural hosts for parasites. It appears that the parasite can be rejected by a powerful mechanism. therefore, Antigens that protect laboratory animals from certain parasites may be ineffective in the parasite's natural host. It is possible that there is no such thing. Natural hosts develop immunity to parasites very slowly. This is even more likely in an epidemic situation.

The antigen characterized in this study is HaeIIlonchuscontor tus, but other species known to infect humans and livestock. Similar or related antigens from parasitic nematodes, ``homologs,'' can be identified. and these related antigens provide an effective vaccine against parasitism by C. elegans species. It is recognized that Species of parasites and the hosts they can infect include , such as halinosis, Hae + l1onchus contortus infection of goats, O8tertagia ostertagt infection in cattle, ^5carf in pigs s5uuI11 or Trichfnella 5piralis infection, Toxascaris Ieonina or Uncjnarta 5ten ocephala infections and Ancylostoma cantnum in dogs or Trichuris vulpts infections, as well as Toxocara spp. larvae to the human circulatory system and Dirof'1larja 1mmft1s Infections of the circulatory system of dogs or of the circulatory system of these and other species of animals. , infections of the genitourinary system, respiratory system, skin and subcutaneous tissues. this squirrel It should be noted that the chart is by no means complete.

A preparation that protects against infection by 11aemonchus contortus. Describe it. This protective component in the fraction contains low concentrations of zwitterionic surfactants. malt lectin is soluble in buffers containing Rafi is identified as not retained. Defenses created in this way The antigen for use is, for example, lentil lectin (LL) or O? tography or He1l X pomatia lectin (HpL) chromatography, reversed phase HPLC, service Is exclusion chromatography or in the presence of solubilizing surfactants Further purification can be achieved by other purification methods known to those skilled in the art.

According to a first aspect of the invention, a substantially purified nematode derived from a first species of parasitic nematode antigen, which is identical to nematode species 10 by vaccinating the host with this antigen. Protects the host from parasitism by a second nematode species, which may or may not be present This antigen is proteinaceous and has a p■ of 3.8. ~4.4, lentil lectin and He1ix pomatia It binds to cutin and has a molecular weight of approximately 45 kD as measured by reducing 5DS-PAGE. An antigen is provided. Typically, this antigen is at least It is 90% pure. This level of purity is important for formulations used for amino acid sequences. This is explained in relation to cleanliness.

The invention encompasses both glycosylated and non-glycosylated types of antigens.

Typically, the first parasitic nematode is Trichinella.

Ancylostoma SS Strongylus STrichostrong Ylus.

Haemonchus, Ostertagia, Ascarls% To xascaris.

Unclnarla 5 Trichuris, Diro “1laria 5 Toxocara.

Necator, Enterobius, Strongyloides and and Wuchereria species. Examples of such species include Tri chfnella 5piralls, Ancylostoma caniu m.

Strongylus vulgarfs STrlchostrongylu scolubrlformis SHaemonchus contortus , Ostertagia ostertagi, Ascaris sumS Toxascaris Ieoninas Uncinaria 5 tenoce phala STrichuris vulpis.

Dlrofilaria 1mm1tis 5Toxocara species, Necat oramericanus%Ancylosto11a duodenale , ^scarjslumbricoides STrichuris trich iura 5 Enterobiusvero + 1 eularus, Stro ngyloides 5tercoralis and Wuchereria b There is ancrofti.

Typically, the protection provided to the host is Trichinella.

Ancylostoma SS Strongylus STriehostron Ylus.

)1aemonchus, Ostertagfa SAscaris, To xascarissLlncfnarfa, TrictrLtris 5D irofilaria,, Toxocara.

Necator 5Enterobius, Strongyloides and Protection against parasitic nematodes selected from species of the genus Wuchereria.

Examples of such species include Trichinella 5piralis.

Ancylostoma canium, Strongylus vulga ris.

Trichostrongylus C01ubrifOr+++iS, Ha effiOnChuScontortus, Ostertagia oste rtagi, Ascaris 5uui+%Toxascarls Ieonl na, 1 Jnclnaria 5 tenocephala.

Trlchuris vulpis SD! rolllarja 1mm1Hs , Toxocara species, Necator amerLcanusSAncyl ostoIIla duodenale.

Ascaris lumbricoides, Trichuris tric Hiura.

Enterobius vermicularus, Strongylot desstercora + ts and Wuchereria bancrof There is tf.

Preferably, the first and second nematode species are Haee+onchus.

selected from the genera Trichostrongylus and Ostertagia Ru.

More preferably, the first and second nematode species are selected from the genus Haemonchus. It will be done.

Preferred first and second nematode species are Haesonchus contortus STrfchostrongylus calubriformus and Os Tertagia circuitincta.

More preferably, the first and second nematode species are Haemonchus cont. Ortus.

The present inventors have demonstrated that the antigen of the first embodiment is a proteolytic protein of a high molecular weight nematode glycoprotein. It was determined that it appeared to be a cleavage product.

Caenorhabditis elegans glycoproteins react against the expression products of cloned genes. Antibodies can be prepared from natural sources by affinity chromatography using the human body. I can do it.

Glycosylated and non-glycosylated forms of macromolecular whole glycoproteins are also encompassed by the invention. hereinafter referred to as "antigen precursor".

A substantially purified form of the antigen precursor is also part of the first aspect of the invention.

Typically, the antigen precursor has the amino acid sequence shown in FIG.

According to a second aspect of the invention, a homolog of the antigen or antigen precursor of the first aspect is provided. Served.

Typically, this homolog is 20 amino acids relative to the amino acid sequence shown in Figure 8. are at least 70% homologous.

According to a third aspect of the invention, the antigen or antigen precursor of the first aspect, or the second excludes naturally occurring polynucleotide molecules encoding homologs of embodiments of Polynucleotide molecules are provided.

Typically, this polynucleotide molecule is a DNA molecule.

Preferably, the polynucleotide molecule is a cDNA molecule.

Preferred polynucleotide molecules of the invention have a configuration substantially as shown in FIG. 7 or 8. It is a cDNA molecule that has a sequence.

The present invention provides within its scope 60 nucleotides having the sequence shown in FIG. have at least 50% homology with each other and confer immunity against infection by parasitic nematodes. This includes DNA molecules that encode protective molecules that can be used to protect against cancer.

According to a fourth aspect of the present invention, a DNA molecule comprising the DNA molecule of the third aspect and vector DNA. A recombinant DNA molecule is provided.

Typically, the vector DNA molecule is a plasmid, phage or viral DNA. It consists of A.

Preferred vectors include λgtll, pUR290゜pUR291, pUR28 2. Based on pUK270, pUC8, pUC9, pZipNeoSSV40 vector, λgtlO1EMBL vector, pBR327, pBR329, or or pBR329 containing the par locus, baculovirus or vaccinia There is a virus.

According to a fifth aspect of the invention, at least one recombinant DNA according to the fourth aspect A transformed host having the molecule is provided.

Typically, this host includes bacteria, yeast, other fungi, insects, plants and mammalian organisms. selected from the cell line.

A preferred host cell is an E. coli K1.2 derivative.

According to a sixth aspect of the invention, the antigen or antigen precursor of the resistant aspect or the second There is provided an expression product of the transformed host of the fourth aspect having a homologue of the aspect. .

This expression product may be a fusion expression product.

According to a seventh aspect of the invention, an antigen, precursor, homolog or expression product of the invention A synthetic polypeptide corresponding to all or part of When infected, they are able to induce protective immunity against host invasion by parasitic nematodes. Synthetic polypeptides that can be used are provided.

According to a fourth aspect of the present invention, at least one antigen of the first aspect and/or or an antigen precursor, and/or a homolog of the second aspect, and/or a sixth aspect. Producing an effective amount of the expression product of the embodiment and/or the synthetic polypeptide of the seventh embodiment Pharmaceutically and/or veterinarily acceptable carriers, diluents, excipients and/or additives A vaccine comprising a dejuvant is provided. The vaccine of the invention also comprises: Mimics antigens, antigen precursors, homologs, expression products and/or synthetic polypeptides This can protect the host from infection by parasitic nematodes. Both can also contain one anti-idiotypic antibody. This active ingredient Pharmaceutically and/or veterinarily acceptable carriers, diluents, excipients and/or additives A juvant may be added.

Alternatively, the vaccine may be used to administer the transformed host of the fifth aspect to pharmaceutical and/or or with veterinary acceptable carriers, diluents, excipients and/or adjuvants. It may also be a whole cell vaccine containing both. This cell may be a living cell or a dead cell. It may be a cell.

The transformed cells express an expression product that is administered to the mucosa of the host to be vaccinated. For example, cell surface fusion products.

According to a ninth aspect of the invention, there is provided a method for preparing the antigen of the first aspect, comprising: (a) homogenizing young adults of a parasitic nematode species to produce a homogenate; (b) obtain a membranous material from this homogenate; (C) obtain a membranous material from this homogenate; (d) extracting with a buffer containing an ionic detergent to obtain a detergent extract; The detergent extract was chromatographed on a wheat malt lectin Sepharose column. , (e) collecting the effluent from the column; A method is provided comprising:

Preferably, the method comprises: R) Fractionation by isoelectric focusing and pr is 3.8 to 4.4, more preferably Collection of both parts in the range 4.0 to 4.3, Axis) Fractionated by gel filtration chromatography, with a molecular weight in the range of] 0 to 60 kD Collect fractions, and (h) lentil lectin and/or lelix po fractionation and collection of bound substances by n+atia lectin chromatography; Also included.

According to a tenth aspect of the invention, there is provided a method for preparing the vaccine according to the seventh aspect, comprising: at least one antigen and/or antigen precursor of the embodiment, and/or a second and/or the expression product of the sixth embodiment, and/or the seventh embodiment. and/or the transformed host of the ninth embodiment, and/or anti-idiotype antibodies in pharmaceutical and/or veterinary Can be mixed with pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants. A method of forming a spike is provided.

According to a tenth aspect of the invention, the host is infected with at least one parasitic nematode species. A method of protecting against infection, comprising: the antigen and/or antigen precursor of the first aspect; and/or a homolog of the second aspect and/or an expression product of the sixth aspect, and/or the synthetic polypeptide of the seventh aspect, and/or the synthetic polypeptide of the seventh aspect. A method is provided comprising administering to a host an effective amount of cutin.

According to a twelfth aspect of the invention, the antigen and/or antigen precursor of the first aspect, and/or a homolog of the second aspect and/or an expression product of the sixth aspect, and/or the synthetic polypeptide of the seventh aspect, and/or the synthetic polypeptide of the seventh aspect. Antibodies raised against cutin are provided. The antibodies of the present invention are monoclonal. It may be monoclonal or polyclonal. The present invention also relates to the antibody of the twelfth aspect. Other compounds exhibiting similar behavior can be added to the antigen and/or antigen precursor of the first aspect, and/or a homolog of the second aspect and/or an expression product of the sixth aspect, and/or the synthetic polypeptide of the seventh aspect and the structure and/or is provided by changing the function.

According to a thirteenth aspect of the invention, the antibody of the thirteenth aspect and a pharmaceutical and/or An antibody composition comprising a veterinarily acceptable carrier, diluent and/or excipient provided.

According to the Saturday and Sunday aspects of the present invention, there is provided a method for preparing antibodies according to the thirteenth aspect, comprising: The antigen and/or antigen precursor of the first aspect and/or the second aspect homologues of the embodiments, and/or expression products of the sixth embodiment, and/or immunizing with a synthetic polypeptide of an embodiment and/or a vaccine of a second embodiment A method is provided comprising:

According to a thirteenth aspect of the invention, there is a method for preparing the antibody composition of the thirteenth aspect, comprising: an effective amount of the antibody of the thirteenth aspect in a pharmaceutically and/or veterinarily acceptable carrier; A method is provided comprising mixing with a diluent and/or excipient.

According to a sixteenth aspect of the present invention, the present invention provides a method for treating a parasitic nematode in a host in need of treatment. A method of dynamically vaccinating, comprising: the antibody of the thirteenth aspect and/or the antibody of the thirteenth aspect; A method is provided comprising administering to a host an effective amount of an antibody composition of the embodiments.

According to a seventeenth aspect of the invention, there is provided a method for preparing a recombinant DNA molecule according to the fourth aspect. Accordingly, a method is proposed which consists of inserting a DNA molecule according to the third aspect into vector DNA. Served.

According to a tenth aspect of the present invention, there is provided a method for producing a transformed host according to the ninth aspect. to adapt the host for transformation, to provide a competent host, and to transform the competent host. There is provided a method comprising transforming a host with a recombinant DNA molecule of the fourth aspect. It will be done.

According to a nineteenth aspect of the invention, the antigens, antigen precursors, homologues, expression production of the invention A diagnosis consisting of a sample of a substance or synthetic polypeptide and/or an antibody of the invention A kit is provided.

According to a twentieth aspect of the invention, there is provided a method for biosynthesizing the expression product of the sixth aspect, comprising: A transformed host according to the fifth aspect is provided, and the host is cultured under suitable conditions to produce an expression product. A method comprising expressing a product and collecting the expression product from a transformed host. is provided.

According to a twentieth aspect of the present invention, the anti-antibiotics corresponds to a part of the antigen of the present invention; Diotypic antibodies can protect immunized hosts from invasion by parasitic nematode species. Anti-idiotypic antibodies that can be used are provided.

Changes in the amino acid and nucleotide sequences of a particular protein and this What can occur between different alleles of a gene(s) encoding a protein It recognized. Furthermore, once the sequence of a particular gene or protein is known, , a skilled person can manipulate these arrays using available techniques to They can be modified from the specific sequences obtained to determine which genes or proteins are involved. It is possible to provide a gene or protein that continues to function like a protein. Probably. These molecules are referred to herein as "homologs" and are also encompassed by the present invention. shall be provided.

In this context, "homolog" refers to the basic functional attributes, i.e. the protective properties of the antigen of the invention. A polypeptide that retains regulatory activity and is homologous to the antigen of the invention. this To illustrate, "homology" between two sequences refers to the derivation of the first sequence to the second sequence. It includes similarities that fall short of the sameness implied by. In particular, polypeptides and By comparing the amino acid sequences between antigens, approximately 7 When the polypeptide exhibits greater than 0% identity, it is considered "homologous" to the antigen of the invention. ”. Such sequence comparisons can be performed using known algorithms, such as Lippmann (L ipman) and Pearson, 5science, 227.1 435 (1985), which can be implemented by a computer. This can be done by using something that is easily realized.

According to the invention, homologues cannot be produced by conventional site-directed mutagenesis. This method does not render the resulting polypeptide biologically inactive. This is one way to routinely identify residues in molecules that can be modified. [1 ] Oligonucleotides having sequences containing desired nucleotide substitutions (mutations) synthesis of [fD the structural sequence of this oligonucleotide encoding the antigen of the invention] hybridization to a template with [iiD T4 DNA polymer Oligos consisting of extensions of oligonucleotides as primers using Nucleotide mutagenesis determines the effect of specific changes on the antigenic structural sequence It is preferable because it can be easily used.

Another example of an antigen homologue of the invention is a molecule corresponding to a part of an antigen or naturally occurring The part consisting of a part of the antigen that does not match the antigen and showing the protective activity of the antigen of the present invention. It is a child.

Other homologues of the invention are fragments of the antigen that retain protective activity. Similarly, the present invention Within the range of <i> corresponds to a part of the amino acid sequence of the antigen, and (fi) corresponds to a part of the amino acid sequence of the antigen; There are synthetic polypeptides that retain the . Such synthetic polypeptides are preferably is 6-30 amino residues in length.

Whether a synthetic polypeptide that meets criterion (1) also satisfies criterion (11) is determined by It can be routinely determined by measuring protective activity in an appropriate host.

carriers, diluents, excipients and/or Antigens, antigen precursors, homologues, expression products and/or conjugated with adjuvants The amount of synthetic polypeptide is determined by the amount of infectious disease and disease being immunized against the host being treated. It will vary depending on the particular mode of administration.

The specific dosage level for any particular host will depend on the specific antigen, antibody used. progenitors, homologs, expression products and/or synthetic polypeptides, age, weight, general health, gender, diet, number of doses, route of administration, rate of excretion, drug concomitant use and It will vary depending on a variety of factors, such as the specific infectious disease state being prevented.

The vaccines of the invention can optionally be delivered parenterally or potentially by the mucosal route. Use conventional non-toxic pharmaceutically and/or veterinarily acceptable carriers, diluents and adjuvants. It can be administered in dosage unit formulations containing bundts and/or excipients.

Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, are prepared according to known techniques. may be formulated with suitable dispersing or wetting agents and suspending agents according to the can. Sterile injectable preparations may be prepared using non-toxic parenterally administrable diluents or solutions. Sterile injectable solutions or suspensions in vehicles, such as solutions in 1,3-butanediol. It's temo good. Acceptable vehicles and solvents that may be employed include water, There are Ringer's solutions and isotonic sodium chloride solutions. Furthermore, sterile fixed oils , solvents or suspending fixed oils may be employed. Furthermore, like oleic acid Other fatty acids are also used in injectable formulations.

Alum is currently the only adjuvant registered for human use. However, experimental studies are being conducted on other adjuvants for human use. These other adjuvants may be used in preparing compositions for human vaccination according to the invention. It is expected that it will be suitable for

Suitable adjuvants for animal vaccination include Freund's complete or incomplete adjuvants. Marcol (unsuitable for use on livestock), Marcol 52: Mon Montanide 888 (Marcol is a trademark of Esso) It is. Montanide is a trademark of 5EPPIC, Paris), squalane or squalane. Qualene, Adjuvant 65 (beannut oil, mannide) oil-based emers such as monooleate and aluminum monostearate) ion, aluminum hydroxide, aluminum phosphate, calcium phosphate and Mineral gels like alum, hexadecylamine, octadecylamine, litho Lecithin, dimethyldioctadecylammonium bromide, N,N-dioctade Sil-N', N'-bis(2-hydroxyethyl)propanediamine, methoxy Detergents such as cyhexadecylglycerol and pluronic polyols, such as oran, dextrans sulfate, polyacrylic acid and carbobol Polyanions, muramyl dipeptide, dimethylglycine, tuftsin and These include peptides and amino acids such as leno-10-sudimycolate; Not limited to these. Antigens, precursors, expression products and/or synthetic polymers of the invention After incorporation into liposomes or other microcarriers or polysaccharides, After conjugation to proteins or polymers or with Quit-A. "Iscoffls J (Immunostimulatory complex (Imfflun) ostimulating complexes) and administering them. Can also be done. Other adjuvants suitable for use in the invention include immunogens and Tr. There are conjugates consisting of prokaryotic integral membrane proteins such as aT. (PCT /A, U37100107). Route of administration, dosage and injection All frequencies can be optimized using techniques common in the art. It is a factor. Typically, one or more "boosters" are given several weeks after the first vaccination. Figure 1 shows the true effects of vaccination using SDS polyacrylic resin stained with silver. Shows gel filtration HPLC results for I EF5 and 6 on Mido. . Lanes 1-6 contain GFI-GF6, lane 7 indicates size in kilodaltons. The lanes contained low and high molecular weight standards from Bio-Rad. 8 includes IEF5 and 6 (Pre*GF HPL, C).

Figure 2 shows I EF5 and 6 on SDS polyacrylamide stained with silver. The results of lectin affinity chromatography are shown. Lane 1 is molecular weight Including standards. Lanes 2 and 3 are lentil lectin Sephas (LLS) Lane 4 contains the material that binds to and elutes from LLS, and lane 4 shows the initial binding to LLS and After release, it is combined with Helixpomatia lectin sepharose (HpLS). , contains the eluting material, lane 5 shows the initial binding to and elution from LLS. contains substances that do not bind to HpLS.

Figure 3 shows Concanavalin A-HRP and enzyme I as a Western plot overlaying reactive substances visualized by reaction Showing the results of lectin affinity chromatography for EF5 and 6 .

Lane 1 contains IEF5 and 6, lane 2 contains substances that do not bind to LLS, Lane 3 shows bound and eluted material from LLS, Lane 4 shows initial binding and LLS After elution from LS, lane 5 shows the initial Lane 6 contains substances that do not bind to HpLS after binding and elution from LLS. Lane 7 contains a high molecular weight standard of BRL. I'm reading.

Figure 4 shows that He1ix pomatia lectin-HRP and enzyme reaction IEF5 and Western plots with visualized reactive substances 6 shows the results of lectin affinity chromatography for No. 6. inside the lane The container is as shown in FIG.

Figure 5 shows the reverse phase HPLC for IEF5 and 6 on a PLRP-S column. Show the results. The gradient used for elution was 30% in water with 0.1% TFA. % acetonitrile to 60% acetonitrile in water with 0.1% TFA It consisted of The flow rate was 11/min, and the absorbance was observed at 220 nm. numbers The peak fractions indicated by were collected manually.

Figures 6(a) and 6(b) show silver-stained SDS polyacrylamide gels ( Reverse phase HPL for I EF5 and 6 as 9-22% gradient end) The results of C are shown. Numbers represent peak fractions shown in FIG. Lane S is bye Orad low and high molecular weight standards with sizes in kilodaltons. .

Figure 7 shows the cloned DNA sequence of pBTA879 and homologs of the protective light source. The derived amino acid sequence of the encoded cloned gene is shown.

Figure 8 shows the cloned DNA sequence of pBTA963 and homologues of the protective light source. The derived amino acid sequence of the encoded cloned gene is shown.

Figure 9 shows pBTA879 and pBTA879 showing the presence of homologous genes within the parasite genome. H, eontort hybridized to cDNA from TA963 Southern blotting of DNA from US. Lane 1 is Haemonc Probe with hus contortus D N A Sp B T A 963 Lane 2 is the HinfI digested product, and lane 2 is the HinfI digested product of Haen+onchus conto. Hinfl digestion probed with rtusD N A, p B T A 879 It is a thing.

Figure 10 shows T. colubrirormis with a 45 kD antigen probe. , Ostertagia circumcincta and Ostertag Southern blotting of DNA from Ia ostertagi 1. other This shows the presence of related genes in species of nematodes. Lane 1 is Haemonchus contortuspNA and HinfI digest. Lane 2 is T richylus colubrilormis DNA and This is a HinfI digest.

Lane 3 is Ostertagia ostertagiD N A and Hin f■ It is a digested product. Lane 4 is ostertagiacl rct+mcin ctaDNA and HtnfI digest. Lane 5 is plasmid pB TA963, Hinfl digest, positive control.

Figures 11(a) and 11(b) show recombinant 45kD antigen expressed in E. coli. 5DSPAGE gel and Western plot are shown. Figure 11(a) shows the large Expression of a 45 kDa antigen in S. enterica, and the sample was 12.5% SDS poly Electrophoresis was performed on acrylamide gel. Next, apply this gel to Komagi Brilliant Blue. -stained with Lane 1 is the uninduced control and lane 2 is the 1 hour post-induced control. lane 3 is 3 hours post-induction, lane 4 is prestained 5DSP This is an AGE standard. Figure 11(b) shows the Western representation of the expressed 45kDa antigen. The sample was electrophoresed in the same manner as in Figure 11(a), and in pairs, Nitrocell Plotted electrophoretically on a loin filter. This filter is 45kDa Rabbit generated for a peptide corresponding to the truncated N-terminus of the protein Serum was probed. Promega’Proto blot) using the alkaline phosphatase system (product number W3930) It developed color.

Figure 12 shows a sample of extracts from Il, coneortus and canine heartworm. A Stern plot, and antigens immunologically related to the antigens of the invention are those of other species. It has been shown to be expressed in produced nematodes. Lane 1 beam, + aunftfs drawing lane 2 is the H. eontortus extract, lane 3 is the high fraction of BRL. This is the quantum standard.

The best way to carry out the invention ) 1 young adults of aemonchus contortus were buffered with phosphate. Homogenize in saline solution (PBS) and centrifuge the homogenate to remove the nematodes. Membrane material was allowed to settle. The pellets will not infect sheep after two vaccinations. I found a way to protect myself. The protective fraction is then extracted with a number of different detergents. Ta. Zwtttergent 3-14 (Calbioga) Calbiochel) was found to be suitable, although other cleaning agents are available. It was effective. Extraction efficiency is determined by the amount of solubilization required to provide protection by the detergent used. The protective antigen with the highest specific activity (calculated by the number of micrograms of substance determined by the ability to solubilize (as determined by vaccination/challenge experiments), Insoluble residues that did not provide significant protection after the vaccination/challenge protocol are (42%, see Table 3). Extraction with Zwittergient 3-14 The process is not completely efficient and some of the protective antigens are present in the detergent-insoluble fraction. It is recognized that there are cases where it is discovered.

Recombinant DNA molecules and transformed host cells of the invention can be prepared using standard techniques of molecular biology. Prepare using

Expression products of the invention can be transformed into cells of the invention under standard conditions suitable for a particular host cell. to culture host cells and isolate the expression product from the culture using standard techniques. Therefore, it is obtained. The expression product may be used in impure form or Purification may be accomplished by standard techniques suitable for producing the product.

If appropriate, whole cells may be used in vaccines.

Synthetic polypeptides of the invention include antigens, antigen precursors, homologues and expression products of the invention. Prepared by standard techniques of peptide synthesis based on the known sequence of the product.

Homologues, expression products and synthetic polypeptides can be tested for protective activity by the methods described in the Examples below. can be tested for.

Using recombinant DNA techniques to provide large amounts of the protective antigens or homologues described herein. can be provided. Coating a protective antigen or a precursor or homolog of this protective antigen. This molecule can be assembled by inserting the DNA segment to be coded into any number of recombinant plasmid systems. can be synthesized in large quantities. Recombinant systems include E. coli, yeast and There are viruses such as baculovirus and vaccinia. Recombinant organisms are Recombinant antigens can be grown in large quantities in fermenters and prepared using standard methods, e.g. Potential in solutions containing urea and reducing agents such as DTT or mercaptoethanol. Solubilize, reduce, oxidize, t:r　jkg　fat　＞mother″″′E @(7 ) by 4-on exchange, filtration and/or gel permeation chromatography Purified and finally sterilized by filtration and optionally with numerous adjuvants such as oils It can be purified by af.

,”)’)f＞is・“4″fat″t″”゛■ Dragon 4・Separation・'Current product and/or or synthetic polypeptides and/or transformed hosts and/or antibodies. Otype antibodies can be prepared in pharmaceutically and/or veterinarily acceptable carriers, excipients and / or using adjuvants and standard methods of pharmaceutical and/or veterinary preparation. Mixed, preferably, J! #! 1°1 sieve on l''chi 161, with t' dosage form t' 6 anti-antigens, 5 homologs, expression products and/or synthesis required for growth. Polypeptide and/or transformed host and/or antiidiotype The amount of antibody will depend on the infection being vaccinated, the host being treated, and the particular mode of administration. It changes. The specific dosage level for any particular host depends on the age of the host, Body weight, general health, gender and diet, number of doses, dosage [ It varies depending on a variety of factors such as route, excretion rate, and drug formulation.

Vaccines can be prepared using conventional non-toxic pharmaceutically and/or veterinarily acceptable carriers; In unit dosage formulations optionally containing diluents, excipients and/or adjuvants. It may also be administered parenterally.

Antiidiotypes are antigens, precursors, expression products, homologues and/or antiidiotypes of the invention. Alternatively, the synthetic polypeptide can be inoculated into a suitable host and the antibodies produced can be used to to generate antibodies against the antigen-binding region of antibodies generated during vaccination. Therefore, it is generated.

Antibodies are raised using standard vaccination modalities in suitable hosts. to the host , antigens of the invention, antigen precursors, homologues, expression products, synthetic polypeptides and/or or vaccinate. An immune response occurs as a result of vaccination. child to observe the immune response of the patient, e.g. by measuring the concentration of antibodies produced. Can be done. , the antibody composition comprises an antibody that is pharmaceutically and/or veterinarily acceptable. suitable carriers, diluents and/or excipients and standards for pharmaceutical and/or veterinary preparations. It is prepared by mixing, preferably by homogeneous mixing using standard methods.

The amount of antibody required to produce a single dosage form depends on the infection being immunized and the treatment being used. This will vary depending on the host involved, and the particular mode of administration. specific to any particular host. Different dosage levels may vary depending on the age, weight, general health, sex and diet of the host, and the time of administration. various factors such as number, route of administration, rate of excretion, combination of drugs and severity of the infection being treated. varies depending on factors.

Antibody compositions can be prepared from conventional non-toxic pharmaceutically and/or veterinarily acceptable concentrations. , administered parenterally in unit dosage formulations optionally containing diluents and/or excipients. can passively protect the host from nematode infection.

Diagnostic kits contain expression products, antibodies, antigens, antigen precursors, homologues or synthetic polypeptides. peptide at a concentration suitable for the substance(s) to be detected. By combining with veterinary acceptable carriers, diluents and/or excipients. prepared. A positive control standard of known concentration of the substance to be detected is also prepared. Manufactured. Negative standards consist only of carrier, diluent and/or excipient. Ru.

The invention will be further illustrated with reference to the following examples, which should not be construed as limiting the scope of the invention. It's not something you can do.

Battle 1 Young H. contortus nematodes were collected from infected sheep carcasses. PB nematodes The cells were washed three times with S and homogenized in PBS. 500Xg of this homogenate Centrifugation was performed for 10 minutes to remove large unbroken biological material.

The supernatant was then centrifuged at an average of 120.000×g for 2 hours at 4°C. Beret After suspending the adjuvanted material in PBS, it was incubated at intervals of 4 weeks in the absence of adjuvant. Vaccinate the sheep twice at intervals, each vaccination using 1 kg of the sheep's weight. It contained the equivalent of about 50 mg fresh weight per serving. Second vaccination This sheep and five other unvaccinated control animals were tested 3 weeks after 10,000 infective larvae of Haeaonchus eontortus in I made him attack. 23.27.28.33.36 and 400th time since infection, in feces The number of eggs was counted for all sheep. The results (number of eggs/1g of feces) are shown in Table 1. vinegar. Table 1 Number of eggs in feces Eggs/gjt stool Animal number of days 23 27 29 33 36 401 L, 433 4.300 2,800 8,467 4,267 5,9002 700 3.633 3 ．． 400 7.367 7,267 8,6008 6 (104,4674,7 874,0334,3877,800486710,20011,40018, 33316, 5338, 10053, 2005, 3335, 1337, 3674 , 4,336,700 vaccinations administered 8 33 300 267 5 [172, 1331, 35076, 111171 4,4348,00022,4341f, 133 18.55Q8 33 33 0 33 1.333 8509 0 0 67 87 767 1.350 It is clear that the five vaccinated animals were well protected from infection ( p < 0 for vaccinated group vs. control group. 02).

Example 2 In a series of experiments, female guinea pigs were exposed to adult homozygotes of H. contortus. ate, and its homogenate (prepared essentially as described in Example 1) 120.000Xg pellets and supernatant were inoculated.

Guinea pigs are vaccinated intraperitoneally twice 3 or 4 weeks apart without adjuvant. 3 to 4 weeks after the second vaccination, 1,000 H. contor tus infective larvae. Five to six days after infection, the animals were sacrificed and I counted the number of insects.

Table 2 summarizes the results of these experiments. insect numbers from unvaccinated controls receiving the same challenge infection. Expressed as a percentage of what is recovered.

Table 2 Ryobu Defense % Experiment number Homogenate 81 183 Homogenate (group 3) 45,55.45 1B5120.000 x g Bere Kit 59 196120.000 x, g pellet 34*171120. 000 x g supernatant liquid 29 196120.000 x g supernatant liquid 15' The fraction derived from the homogenate of adult worms of 149H, contortus Can provide significant protection against Lumots, especially the 120,000xg Bere This is clearly the case with the particulate matter found in

Example 3 young adults of T. contortus were homogenized in Tris-buffered saline; A 120.000Xg pellet was prepared as described in Example 2. This beret 1 % (W/V) Zwittergient 5B-14 (Sigma) Sonicate and shake for 1 hr at 4 °C in Tris-buffered saline (TES) containing Therefore, it was resuspended. The extract was centrifuged for 30 minutes at an average of 5o, 000Xg and washed. Materials insoluble in the detergent were pelleted. The supernatant fraction was treated with wheat malt lectin-Sepha. The terminal N-terminus was Using the five fractions derived from the acetal-gluco ring, the same procedure as in Example 2 The cat was vaccinated. The results (Table 3) show that a significant portion of the protective substances are compatible with detergents. Although it is soluble, most of the protective antigen(s) do not bind to wheat malt lectin. clearly shows.

Fraction μg/animal protection % 5B-14 insoluble substance 800Hg 42% 5B-14 soluble substance 700Hg 71%+ 5B-14 soluble WGA 10 μg 22% 5B-14 soluble WGA-700H g 77% WGA- is a substance that does not bind wheat malt agglutinin.

5B-14 is Zwittergent (Calbiochem).

Contains in a granular preparation derived from a homogenate of H. contortus If quality is used to vaccinate guinea pigs or sheep, stimulate the immune response of these animals when they are infected with parasites. can reduce parasitism in The protective antigen is Zwittergent 5B- 14, and under the conditions used in the example above, wheat Does not bind to bud agglutinin.

flood The Zwittergient 5B-14 extract was treated with H,co as described in Example 3. ntortus young adults and in two experiments. was used to vaccinate sheep.

Animals were vaccinated twice 3 weeks apart, initially with complete Freund vaccination. in adjuvant, and the second time in incomplete Freund's adjuvant. Second time Three weeks after vaccination with H. contortus, sheep were given 10.000 doses of H. contortus. larvae were administered orally. Count the number of eggs in the feces for 1 week starting from 21 days after infection. The number of worms was counted twice every 4 weeks and the animals were sacrificed every 4 weeks (Tables 4a and 4). 4b). Zwittergient 5B-14 extract can be used to increase the number of eggs or insects in feces. used to vaccinate each animal in the experiment, as measured by number of Although the amount of protein present was extremely small (10±2.5+++g ), significantly protected sheep from infection, SDS polyacrylamide gel electrophoresis It is clear that the extract contained many components as determined by .

back 4a Table 4b For an experiment similar to Example 3, five times the amount of material used in the previous example was prepared. Tsuvu Ittergent 5B-14 extract (63 mg protein) was added to Pharmalite. (PharIIlaljte> 3-10 Umph 7 lines and 0,5% CHAP 4% Ultrodex (Ultrodex) containing S detergent (Calbiochem) x) Fractionated by preparative isoelectric focusing in resin (LKB). 1000 After electrophoresis at 0 Vh, the bed was divided into 30 fractions, and the substances in each fraction were was eluted in 0.1% CHAPS. The details of measuring the pH of both parts are as follows. The fractions were concentrated to 11 on a YMIO membrane. A portion of each fraction was stained with SDS Polya. Analyzed by acrylamide gel electrophoresis and stained with Comassi brilliant blue. Ta. A portion (approximately 1/8) of each fraction was visualized on an SDS gel. and pooled to obtain a total of 5 fractions, which was used as described in Example 2. The guinea pig was vaccinated. The number of insects (Table 5) indicates that most of the protective antigen(s) are in the pI range. contained in fractions 1 to 10 of the IEF gel, covering boxes 3.3 to 4.6. It shows. Contains other fractions, but the number of insects has decreased, and these Of interest in this application.

Table 5A Experiment number 229 Fraction pr Number of insects Defense % Control 467±190- 3B-14 extract 22B±6252%IEFI-10 3J-4, 13233 ±155 50% IEF 11-14 ty-5, 3341 ± 112 37% I EF 15-18 5.4-6.2 823±107 31%IEF 19-2 2 6.8-7.5 528±33 0%IEF 23-30 7.7-9.3 347±247 26% control 664±152- 9R-14 extract 254±111 62% IEFL-IQ 13-4.6 2 38±6764%IEF 11-14 4.7-5.3 274±287 59 %IEF 15-18 5.4-8.2 455±251 31%IEF 19 -22 1B-7,5474±427 28%IEF 23-30 7.7-9 ．． 3 356 ± 100 46% Second experiment in Example 4, the results are shown in Table 4b. For those shown, a group of sheep was pre-prepared with a wide range of IEF fractions as in the previous example. I was vaccinated. The results are shown in Table 50.

Table 50 :=:=! −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−1 Group average Adjuvantcon Troll Defense% Number of insects, number of eggs, number of insects in feces number of eggs Control 75600 2117' --3B-14 extract 9114 5 33 88 75IEF 1-10 26314 1440 65 321EF 11-14 27814 395 63 811EF 15-18 2427 1 503 88 761EF 19-22 16917 581 78 73 1EF 23-30 27329 958 64 55 Guinea pig vaccination and This data obtained in the attack experiment shows that IEF fractions 1 to 0 contain H, c Ontortus antigens that can protect sheep to a significant degree from challenge infection. This confirms that it is clear that there is. From these data, this experiment It is also clear that there are additional protective components in the other IEF fractions examined in are of interest for this application as well as for those contained in IEF fractions 1-10.

Example 6 Half of the initial loIEF fractions used in Example 5 were pooled in pairs and It was used for vaccination of children.

The number of insects (Table 6) indicates that the majority of protective substances include substances with a pI of 3.8 to 4.4. It is shown to be contained in fractions 5 and 6 from the original IEF gel.

Control 589±194- IEF l-1,03,3-4,8i83±8569%IEF l & 2 3 ．． 3-8.5 509±122 13%IEF 3 & 4 3.7-3.8 373±143 37%IEF 5 & 8 4.0-44 225±103 62% IEF 7 & 8’ 4.4-4.5 321±115 45% IE A portion of the F fraction was electrophoresed on an SDS polyacrylamide gel and stained with silver. As a result, a large number of components are visualized, and they are concentrated in the pI 3.8-4.4 fraction. It seems to be. Some of them are not sharp bands and are associated with glycoproteins. Conceivable. The appearance of these compounds compared to BRL high molecular weight protein semi-standards The molecular weight of is 100-120kD, 40-55kD, 14-20kD Possibly five-component clusters in the range and molecular weights not resolved by the gel. There was a substance with less than 15 kD. Furthermore, a protein with an apparent molecular weight of 32 to 36 kD Defensive Nyabu: Heavy beak, higher than adjacent, less defensive fractions. Due to its high concentration, it is not considered an antigen involved in a protective immune response. IEF fraction The serum from the sheep vaccinated in Example 4 was used as the indicator serum. It was also used in the Western plot. observed in silver-stained gels All components in the pI range of 3.8 to 4.4 react with sheep serum, so this experiment This method of vaccination can elicit an immune response in sheep.

These results demonstrate that potentially protective antigens can be isolated from young granular H. contortus. Isolated from adult worms and reconstituted at least partially with 1% Zwittergent 5B-14. can be solubilized, pI ranges from 3.8 to 4.4, and SDS gel electrophoresis The apparent molecular weight calculated by the method and compared to the BRL high molecular weight marker is 100 ~120kD.

40-55 kD, 14-20 kD or less than 15 kD It shows.

Example 7 IEF fractions 5 and 6 in 50Il1M sodium phosphate containing 0.6% CHAPS It was dialyzed against buffer pH 5,6 and concentrated up to 6 times on a YM-10 membrane. 0.　 2+al was added to Biosil (Bto-3tl) TSK-400 column (3 0X0, 75cm) (Piorad) with a flow rate of 0.21/min. The absorbance of the eluate was observed at 280 nm in a buffer solution, and the eluate was analyzed by HPLC gel filtration. and fractionated. Fractions were collected and pooled based on the absorbance curve of the eluate. lastly The fractions contained in the eluting peaks were pooled, and TSK-8W3 was purified using the conditions described above. Rechromatography was performed on a 000G gel filtration column (Toyo Soda).

Vaccinate the same guinea pigs as described in Example 2 using 6 pools at shipping cost. Ta. Each pool contained equivalents of 41 IEF5 and 6.

The number of insects (Table 7) indicates that most of the protective antigens are contained in GF5 and GF6; The approximate molecular weight range is shown to be <10 kD to 60 kD.

Fraction Molecular weight range Number of insects Protection % (kD) Control -1 436 ±123 --IEP 5 & 6 <10- >250 224±125 49GF l >250 369±104 15G F2 100->250 344 ±158 21GF 3 55-) 250 341±168 22CF4. 55 387±105 11GF 5 <10-60 233±1.41 47GF 6 (10-44237±17 0 4. A portion of the gel-filtrated pool was electrophoresed on an SDS polyacrylamide gel. When moved and stained with silver, numerous components were visualized, including GF5 and GF 6 (Figure 1). Piorad low molecular weight standard and ratio Their apparent molecular weight compared to in the 25-30 kD range, with perhaps 5 proteins in the 14-20 kD range It seems that there is. It does not split on this gel and migrates simultaneously to the buffer front. There are other ingredients as well.

The components retained in GF5 and GF6 can be further divided to a higher degree by several types of means. I was able to do that. The methods described in Examples 8 and 9 are for illustrative purposes only.

Example 8 I EF5 and 6 (6m, 100+aM) Lith/250d Sodium Chloride , pH 7, and lentil lectin-Sepha0-su 4B (Pharmacy). The mixture was stirred at 4°C for 16 hours with 0.51% of Pharaacia. Sefa The loose conjugates were removed by centrifugation at 3,000×g for 1 min, and the in 50mM) Lis-Saline buffer containing Targent 5B-14 (TSZ buffer). Washed four times and then eluted with the same buffer, which was then added to 30M methyl-α-D-manno. Pyranoside was added. Half of the fraction bound to lentil lectin was transferred to He1ix pomat1a lectin-sepha0-su 6MB (Pharmacia) and 1 at 4°C. After stirring for 6 hours and washing the conjugate four times with TSZ buffer, 0.2M N-acetyl Elution was performed with a TSZ buffer containing Le-D-galactosamine. A part of all fractions is S After three experiments on DS polyacrylamide gels, 1) staining with silver (Figure 1) or Western migration, and 2) Concanavalin A horseradish peroxidase (HPR) conjugate (Sigma) (Figure 3), or 3) Hetix porAat1 Staining was performed using a lectin-HRP (Sigma) (Figure 4). dyed with silver The gel shows that the major protein is of 45kD, which is lentil and He1. ix pomatla, but the latter binds to small amounts of contaminant proteins. It doesn't match. These observations are confirmed by two lectin plots ( Concanavalin A has the same sugar specificity as lentil lectin).

Guinea pigs were treated as described in Example 2 using samples of IEP 5 and 6 fractions. After vaccination, lectin affinity chromatography was performed as described above. It was fractionated by The number of insects at sacrifice (Table 8) was determined by the lectin-binding fraction. It indicated that it would be done. In this experiment, the other two parts were also somewhat protected. this is , removal of the 45 kD component from I EF5 and 6 by lectin is incomplete. This may be due to the fact that antigens are present in various forms with varying degrees of glycosylation. (However, substances of this molecular weight can exist in H The SDS gel profile of e1fx pomatia lectin-unbound fraction shows almost no (I can barely see it). The protection obtained in other fractions also It is also thought that this is due to the presence of other protective antigens. These other antigens are The details are interesting.

Ryobu Number of insects Protection% Control 462 ±320 0 IEF 5 and 6 273 ± 238 4 lectin binding (LL> 315 ±127 82 Lentil lectin unbound (LL-) 207 ±28 8 55LL”, He1ix poa+atia lectin Pa 221 ±150 52LL”, He1lx poll+atia lectin unbound 218 ±114 53 cases 9 I EF5 and 6 to 20w 1M Tris/IIIMEDTAS pH 7,5 After dialysis using a YM-10 membrane, it was concentrated 10 times. The amount of 0.31 acidified by adding fluoroacetic acid (TFA) to a concentration of 0.1%. After that, centrifugation was performed to remove the precipitate. The supernatant was diluted with 30% acetonitrile. PLRP-S reverse phase HPLC column equilibrated with 0.1% TFA (Polymer Laboratories) (PolyIIer Laboratories) for 30 minutes. Linear gradient until reaching 60% acetonitrile 10.1% TFA It was developed in The absorbance of the eluate was observed with 22OnIl (Figure 5), and the gradient Fractions were collected over the course of several days. Selected fractions were run on an SDS polyacrylamide gel. When processed and stained with silver (Figure 6), most of the proteins contained in the starting material were detected. It was shown that the particles were recovered and highly divided.

These data indicate that the antigen in the protective fraction is well separated by reverse-phase HPLC. However, upon further investigation, this substance was found to be It was found that vaccination was no longer protective in guinea pigs. perhaps By treating the antigen fraction with the solvent used in reverse-phase HPLC, the antigen is denatured. Therefore, it is thought that the structure of the antigen no longer resembles that of the original antigen.

Example 1O Remove detergents, concentrate samples, remove salts, and prepare antigens suitable for amino acid sequence analysis. For exchange into buffer, lentil lectin binding of approximately 45 kD, He1Lx pomatia-binding antigen with Polypore phenyl RP (30 X2, 1 mm) column by reverse phase HPLC, and it took 20 minutes to ．． 1% TFA/10% acetonitrile to 0.07% TFA/70% acetonitrile Dividing was performed using a rill gradient end (flow rate 200 μm/min).

When the 45kD antigen was eluted towards the center of the gradient, a small shoulder was detected. was obtained as the main peak with

Both parts of 2a were collected separately, and based on the obtained N-terminal amino acid sequence data, It was thought that they were composed of the same polypeptide.

The N-terminal amino acid sequences of these fractions were determined using Applied Biosystennis (Ap plied Btosystems) 470 A-type amino acid sequence analyzer in the gas phase Determined by sequencing. The following sequence was developed.

AF XPG SNNGM (T) DBIRQIF (V) (D) (K) (Y) () I) (D) (N) (S) (of (G) (P) (P) (P) (D) Additionally, a sample of the 45kD antigen was electrophoretically run on an SDS polyacrylamide gel. and purified, transferred to nitrocellulose and coated with Ponceau S. ) and cut the nitrocellulose containing the 45kD antigen to obtain endoplasts. Incubation was performed with roteinase Lys-C. produced by digestion The peptides were separated by reverse phase HPLC and sequenced by the same method as above. did. The following sequence was obtained.

(K)X X(P)(D)XEVEAN(T)A A YA(N)E(E)(Y ) (S) (K) D NEYR3LIAXX (ω (Q) conversion) X (S) (E) (H) (G) (K) (L) (D) (C) F A P K X (D) (G) (D) (G) (A) (A) (K) (H) NEY R (S) I conversion) (＾) (K) (P) X ( L) (N) X (K) X (P) Y (D) X (D) V X A (D) X X X (T) (P) (P) X (G) (K) (P) (T) (T) (D) (E) X indicates that the amino acid cannot be specified at a specific position. bracket Residues within the arc are less confident than others. In some cases, certain sites Since it was not possible to identify two or three residues in the If so, they are shown below each other. The first residue of each peptide is It is assumed that the

These sequences are H, contortus RNA (complementary DNA library ) or DNA (genomic library), or polymerase chain reaction (PC R) is used as a primer to encode the antigen in the generated gene library. Suitable for use as a hybridization probe for gene identification It is suitable for designing oligonucleotide sequences.

From the amino acid sequence described in Example 10, the gene(s) encoding the 45kD antigen was determined. For use in screening cDNA and genomic libraries to identify We prepared oligonucleotides that can Furthermore, this oligonucleotide used with oligo(dT) in R (Saiki et al., 1988). As a result, we were able to amplify DNA encoding a 45kD protein.

The following multi-degenerate primer (IIlultiply-degenerate primer mers) and the Applied Biosystem Model (Applied Biosystem Model). systema Model) 380A Designed and synthesized using an automated DNA synthesizer did. Additional nucleotides in addition to the nucleotides necessary to encode the required amino acid sequence. An otide was included on the 5' end of the oligonucleotide. These arrays are limited The DNA encoding the enzyme EcoRI and Smal sites was amplified by PCR. Assists in cloning into appropriate vectors. Oligo (dT) protein used in PCR A limer was also synthesized. This primer also contains EcoRI and SmaI restriction sites. I was reading.

Al12/301 GCGAA under CCGGG, GCA, m, CAT, CCG, GGG, AAC, AAC, AAC, GGG, ATG, ACG, GAC, GTCCAATTTA A.T. GCGAATTCCCGGG, OCA, T mouth’, CAT, CCG, GGG, T CC, AAC, AAC, GGG, ATG, ACG, GAC, f T CCA AAGA T T AA TA112/302 starts from the 5' end Identical to A, 112/301 except for the 6th codon. This is AAC/T to T/AC/GG/A/T/C to avoid mixed sequences found in the amino-terminal sequence. Gnar, i.e. asparagine (A, 112/301) or serine (A11 2/302) was explained.

(b) RNA isolation and cDNA library construction Total RNA was Using the RNA extraction kit (catalog number XY-016-00-01) purchased from 1. Contortusl g (wet weight). Denitrified (e xsheathed)　L　15 days after infection with stage 3 parasites, Larvae were obtained from the abomasum of sheep, quickly frozen in liquid nitrogen, and then stored at -70°C. Ta. To extract RNA, the frozen insects were pulverized under liquid nitrogen, and 7 ml was extracted. solution (containing guanidine thiocyanate, N-lauryl sarcosine and EDTA) Buffered aqueous solution; density at 25°C - 1,15 g/II) plus 13X5 2X 1.25s + I C5TFA (CsTFA) in 1s polyaromer tube buffered aqueous solution containing; density at 25°C - 1,51 g/gl) layered on each other. This gladiend is manufactured by L8-70 Beclvan. ) at 15°C at 31.00 Orpm using a 5W50.10-tar in an ultracentrifuge. Centrifuged for 6 hours. After centrifugation, the RNA pellet was soaked in TE buffer (1011 M Tris-HCl, pH 7.5, 1mM EDTA) and incubated with ethanol at -25°C. It was reprecipitated from the pool. Next, the precipitated RNA was dissolved in TE again, and the oligo-(d T) - Cellulose column (Pharmacia mRNA purification kit, catalog number XY -012-00-02) as described by the manufacturer. It was further purified by

The purified poly(A)+RNA obtained was purified using the Pharmacia cDNA synthesis kit ( Construct a cDNA library using catalog number XY-003-00-03) I used it to do this. Briefly, polya purified from 325 μg of total RNA. Denylated RNA was added to Moloney's murine leukemia cells in the presence of oligo(dT). virus reverse transcriptase (Moloney Murlne LeukaemiaVIr) It was treated with US Reverse Transcrlptase). second strand Synthesis was performed using RNase H and E. coli DNA polymerase I. 2 water tract cDNA was treated with the Flenow fragment of DNA polymerase and EcoRI /Notl adapter. The cDNA was then transformed into a T4 polynucleotide kit. This was treated with Nasalase to phosphorylate it, and digested with EcoRI to dephosphorylate it. In vitro, infectious bacteriophage age particles (flotoclone lambda gt 10 system and baccagene scissors) Stem, packaged in Promega according to manufacturer's instructions did. The packaged cDNA was transferred to E. coli C600Hfl and incubated with 10 mM MgSO4. Cultured on Luria agar plates using Luria) whelk agar containing did. A total of 6 x 107 pru were obtained, of which 98% were recombinants. flat The average insert size was 2.0 kbp.

(C) Preparation of DNA probes for screening recombinant libraries A 45 kD antigen-specific double-stranded DNA probe was prepared using PCR. for The method used was based on the method described by Saikj et al. (1985). A cloned form of Taq polymerase (Burkisix Parkin Elmer Cetus was used.

2 μg of total RNA was added to 1100n oligo(dT) PCR primer in 6 μl of water. annealed by heating at 70°C for 5 minutes and then allowing to cool to room temperature. Ta. The annealed RNA-oligo (dT) was then treated with 50IIM Tris-HCl. (pH 8,3), 75a+MKCL 1.0mMM g Cl 2.5mM permidine, 10 dDTT, 200 units of reverse in the presence of 1 unit of RNasein. Incubate with transcriptase (BRL) for 1 hour at 37°C in a final volume of 25 μl. It was done. A similar reaction without reverse transcriptase was performed as a negative control for the PCR reaction. Used as a roll.

PCR was performed on cDNA produced as described above. The reaction mixture is , 1 μg of total RNA, either A112/301 or A112/302, respectively. 1 μM and 1 μM oligo(dT) PCR primers, 2 dNTPs of each 00μM, 50mM KCI, 10mM l-Lis-HC1 (pH 9,0), 2mM MgCl 2.0.01% gelatin, 0.01% Triton X-100 and and 2 units of Taq polymerase with a total volume of 100 μm. It contained the first stranded cDNA. Denaturation at 94°C for 1 minute, denaturation at 40”C. It consisted of a 2 minute annealing and a 3 minute extension at 72°C.

A sample of each PCR reaction was analyzed on a 0.8% agarose gel at the end of the reaction. Ta.

For reactions containing primer A112/301 and oligo(dT), approximately 650 bp of A unique band was observed.

Several other bands were present, but these bands were detected using oligo(dT) only. It was also seen in the reaction. Primer A 1i 2/302㎲mL1, approx. No 650 bp band was observed. Negative control omitting reverse transcriptase No bands were observed in the reaction.

The approximately 650 bp PCR product was digested with EcoRI and electrophoresed on an agarose gel. purified by conventional techniques (Manjatis et al., 1982). ) using Bee Bluescript Niskei (pBluescript jpt 5K )) (Stratagene), ditheoxy linkage Stopping Method (Amersham Microtiter Plate Sea Fencing Kit) Amersham Microtitre Plate Sequence g Kit), catalog number RPN1590). clone From the sequence analysis of both ends of It was confirmed that the ends contained a poly(A) stretch sequence. Furthermore , 18 immediately downstream from the 3' end of the region corresponding to primer A112/301 14 of the nucleotides were predicted from the amino acid sequence of the purified protein. It corresponded to. This is 86% homology at the amino acid level (N-terminal sequencing). (18 out of 21 amino acids generated from Amino acid sequence obtained from purified antigen and Differences between the amino acid sequence predicted from the DNA sequence of the PCR clone Ambiguity and/or PCR uptake in protein amino-terminal sequence analysis filter error, but this is a dubious explanation given the large number of differences. Ru. PCR clones were grown, DNA was isolated, digested with ECoRI, and incubated. In this example, the insert was purified and used as a hybridization probe ( The cDNA library described in b) was screened.

Approximately 10 pf’u, 2×10” cpm/all probe, 5×SSP E, 5x Denhardt's solution, 0.5% (lI wt/vol) SDS and 20 μg/l Incubate the library at 55°C in a solution containing 11 sheared, denatured salmon sperm DNA. Screen by hybridization of nitrocellulose filter replicates I nged. Filters were washed with 0.5X SSC at 60°C, followed by 0.1% SDS. and autoradiography, 16 positive plaques were identified. It was. Of these, eight were used for the next purification and analysis. EcoRI insert was isolated from purified phage DNA and subdivided into P. Bluescript SK-. I took a loan and did further analysis. The sequence of one of these clones, pBTA879, is , shown in FIG.

Consisting of 1336 (!l) nucleotides followed by the translation stop codon TGA There is a single long transcription open reading frame. The open reading frame of this clone is It extends from the 5' end of the tube. Methionine start codon contained in this region of the sequence This clone does not appear to represent the complete coding region.

A detailed study of the amino acid sequence derived from the cDNA sequence (Figure 7) reveals that Regions of homology to the amino acid sequence of the purified 45kD protein were identified using cDNA clones. Starting at nucleotide 65 from the 5' end of the sample, the predicted amino acids 11 are It can be seen that this occurs over 16 residues. Additionally, nucleotide 773 of the sequence There is a second region of homology with the N-terminal sequence starting with . 19 amino acids Of these, 14 residues are identical to those determined from the protein sequence. a The level of homology at the amino acid level was 73.7%. Within the molecule, there are repeat domains It seems possible that there is. Both of these regions that are homologous are marked by double underlining. It is shown in FIG.

Endoproteinase Lys-C peptide sequence derived from purified protein and Other regions within the predicted amino acid sequences that share homology are shown in Figure 7 (− (by underlining). All of these regions are located at the carboxy-terminus of the molecule. within half, all in the part that is carboxy-terminal to amino acid 253 .

Additionally, peptide sequences that were hypothesized to be highly susceptible to proteinase digestion and There is a region immediately preceding the sequence starting at nucleotide 773 that is similar.

The N-terminus of the amino acid sequence for those starting at nucleotide 65 is highly hydrophobic; Contains the same amino acids recognized by signal peptidases.

The most likely explanation for these analyzes is the cDNA clone described in Figure 7. is related to the original glycoprotein isolated from H. contortus. encode glycoproteins that are not identical. cDNA clone is complete does not encode a protein of long length and has at least one complete signal peptide , there are additional amino acids present in the full-length original molecule over those shown in Figure 7. It is possible that there can be

To isolate a cDNA clone encoding the full-length original 45 kD antigen. Next, the cDNA library was screened with the fragment isolated from pBTA879. -ning. The cDNA library described in (b) of this example also Screening was performed using the EcoRI band of pBTA879 as an oxidation probe. -ning. Approximately 105 pfu were washed with SSC using the same conditions as above. , 0.1% SDS and autoradiography, 16 positive results were obtained. A plaque was identified. Of these, 11 EcoRI inserts were purified. , was subcloned into B Bluescript SK- for further analysis. these The sequence of one of the clones, pBTA963, is shown in FIG. This is 1320 nucleotide and the transcriptional open reading frame followed by the translation stop codon TAA. There is.

Also, although this clone does not contain an initiating methionine, the 5' sequence of this clone Therefore, 16 of the 18 amino acids encoded are identical to the original N-terminal amino acid sequence. be.

This region of homology begins at base 50 or amino acid 12, and the second repeat begins at base 7. You can find that it starts with 22. Both regions are double underlined. other pep A sequence can also be located in the translated amino acid sequence from pBTA963. Ru. These are much higher in terms of peptide sequence than seen in pBTA879. have homology. In most cases this homology is 100%.

Other features of the two protein sequences are very similar. Both are hydrophobic lees. Both have peptidase-sensitive regions toward the center of the molecule. range, and they are similar in molecular weight and overall charge. however, They both note that homology may be limited in some places, especially where the peptide sequence is derived. However, the overall amino acid sequence homology is only about 54%.

To ensure that the cloned gene codes for the purified antigen, the EcoRI fragment isolated from the original PCR clone to isolate the bacterial strain induced by IPTG. inserted into an expression vector. The gene product was isolated, purified and used to vaccinate sheep. It was used to Using serum obtained from these sheep, H.contort Western plot of 45 kD glycoprotein and homogenate of US I was disappointed. In all cases, the apparent molecular weight in IEF fractions 1 to 10 was 45 kD. It was found that the antigen specifically reacted with the antiserum. predicted from the DNA sequence Antibodies raised against a 24 amino acid long peptide with a sequence of It reacted with the 45kD antigen in F fractions 1-10.

Whole parasite homogenates and recombinant antigens were divided with serum from vaccinated sheep. A detailed examination of the Western plots of each wide range of IEF fractions In fact, higher molecular weight parasite components that specifically react with post-vaccination serum have been detected. I discovered something. These higher molecular weight components are derived from the same gene as the isolated antigen. It is thought to be a product of the same type. They are specifically located in rEF fractions 2.3 and 4, This seems to obscure isoelectric focusing. Sharp when fractionated by IEF This phenomenon of not converging as a specific band is characteristic of glycoproteins and is a variable phenomenon. carbohydrate residues make the charge on the molecule non-uniform, making the number pl of the molecule non-uniform. It is.

Therefore, the original 45 kD antigen isolated from the parasite is identical throughout the shell of the molecule. It is a member of a class of proteins that have similar amino acid sequences. This is both Ec Transfection of H. contortus genomic DNA using the oRI insert (Figure 9). This is further supported by a hybrid blotting analysis.

These show that pBTA963 hybridized with many bands at various intensities. This indicates that the

The bands at 1480.890 and 740 base pairs show the strongest hybridization. is on. However, the same plot screened with pBTA879 890 and 890, which weakly bind to this probe with a band of 1350 base pairs. It illuminates the 740 bands.

Western plot shows that some proteins exceed 65kD. It is thought that it may have a molecular weight. IEF fractions of Examples 4 and 5 herein The protection afforded to sheep and guinea pigs vaccinated with 2.3 and 4 is here: It is also possible that higher molecular weight forms of the described protective antigens exist.

DNA or protein sequences with significant homology to that of this clone is Scene Bank (Genbank), EMBL or PIR convenience store. I couldn't find it even after searching the database.

Example 12 Homologous genes related to protective antigen genes are present in other species of parasitic nematodes DNA hybridization (or Southern blot analysis) was performed using standard techniques (matrix (Niatis et al., 1982), and other species of parasitic nematodes are H,C0ntO Determine whether or not there is a J gene homologous to that encoding the rtuS protective antigen. Established. EcoRI of cDNA clone 1BTA963 described in Example 11 (Figure 8) Hive the insert to DNA isolated from members of other species of parasitic nematodes. It was used as a redization probe. This probe is used for homologous species, i.e. Several restriction fragments in the DNA isolated from H. contortus cooperate. As expected, Osterta gia circuIIlcfncta, Ostertagfa oste rtagi and Trichostrongylus colubrifor II Hybridization also occurs with specific restriction fragments of DNA isolated from lis. It was done. After thorough cleaning of these plots, the level of homology was approximately 70 % (Figure 10).

This clearly applies to other species of parasitic nematodes, and by extension to all species of parasitic nematodes. This indicates that there are genes closely related to genes encoding protective antigens. Ru.

Can isolate and synthesize related or homologous antigens from other species of parasitic nematodes We were able to create a recombinant organism that does this. We discovered that these related antigens is an effective immunogen to protect vaccinated animals against infection by parasites of other species. I believe that it works in many ways. Furthermore, related substances isolated from a wide range of parasitic nematodes We have isolated a series of antigens and found that they are effective in protecting animals from infection by these widely charged parasites. were able to provide a protective immunogen.

It has already been shown that this method works (International Application Section PCT/A0881 00239 specification). This patent application specifies that the guinea pig is T, colu T, c based on the ability of the antigen to protect against challenge infection by S. brirormis A method for purifying this antigen from homogenate of S. olubrirormis is described. It is. The amino acid sequence information about this antigen has been determined, and this gene Antigens isolated from recombinant DNA libraries can be encoded. next , T, hybridize DNA encoding S. colubriformis. The “homologous” gene from H. contortus was used as a control probe. We identified introduced organisms that coded. Next, synthesize the Il,contortus antigen. We constructed a recombinant organism that uses this antigen and used it to vaccinate sheep and guinea pigs. and attack tests were conducted. t(,contortus recombinant antigen is H,Contor Protects vaccinated sheep against infection by T. tus and T. colubriro. guinea pigs were protected from challenge infection with S. rmis.

This indicates that the DNA sequence shown in the hybridization experiment described above is Assuming homology, clones encoding protective antigens from certain parasitic nematodes The transformed DNA sequences can be used to encode homologous gene products from other species of parasitic nematodes. identify a clone to code, design a recombinant organism that expresses the homologous gene, and use this showed that cutin can be used to protect against other species of parasitic nematodes. are doing. A natural extension of the results provided here is to do this with the DNA sequences of the invention. This is thought to be the case.

The nucleotide sequence of the homologous gene and the amino acid sequence of the homologous antigen are those of the first target species. may not be identical to the same thing, but at least over a length of at least 60 base pairs 50% and preferably this relationship is greater than 70% over this same area. and at least 70% homology across 20 amino acids at the amino acid level. It should be understood that In most cases, the degree of homology between the two sufficient to unambiguously identify gene or protein relatedness.

Example 13 Expression of 45kD in E. coli A number of systems have been used to infect recombinant 45kD antigens, e.g. complement cells, viruses. can be expressed in insect cells, yeast or bacteria. For example, the gene is large It was expressed in Ih bacterium. Complete cDNA fragment derived from pBTA963 It was isolated as a 6 base pair BamHI/Hindl I fragment and Subcloned into expression vector pBTA954.

pBTA954 contains the lac promoter, an open vector for the β-galactosidase gene. It is a pUR-based vector with starting amino acids and multiple cloning sites. Ru. When expression was induced by adding 1mM IPTG, this resulted in a 45kD protein. A fusion with the 11 N-terminal amino acids encoded by the vector fused to The combined protein was purified. The apparent molecular weight of this fusion protein is approximately 61 kD. (Fig. 11a). The fusion protein consists of a truncated N of the 45 kD protein. − was recognized by rabbit serum raised against a peptide corresponding to the terminus (Fig. 1 1b). This serum was previously analyzed for the dominant proteins in the original extract IEF fraction 1. It has been shown that humans can recognize different qualities.

The fusion protein is purified by standard techniques known in the art and Combined with appropriate adjuvants, it can be used to vaccinate hosts such as sheep to reduce parasite susceptibility. I was able to protect myself from dye.

Example 14 against the 45kD antigen and Dlrolilarja iamitis protein. Immunological cross-reactivity between antibodies Prophylaxis of rabbits with a synthetic peptide derived from the sequence of the N-terminal region of the 45kD antigen Inoculated. Using antisera from these rabbits, D, Iaaltlg and H , Western plots of extracts from adults of P. contortus were probed. Ta.

from each species after development with a second antibody conjugated to alkaline phosphatase. The two types of antigens were shown to be reactive. The characteristics of 45kD and 27kD are H , 52 and 32kD, 1auait is extract (Figure 12). This result is 1. DNA hybridization Using a method of This supports other observations that it is present and expressed in

The manufacturing and purification techniques described so far are performed on a laboratory scale. The anti-inflammatory agent of the present invention For commercial production of raw materials, large-scale fermentation of transformed hosts is required.

Large-scale fermentation is carried out by standard techniques, and the particular technique chosen depends on the production of the antigen. It is suitable as a transformed host used for production.

Deposit of microorganisms BTA2033 strain of Escherichia coli JM10 containing plasmid pBTA879 was adopted by the Australian Government on 29 January 1992 pursuant to the Budapest Treaty. Austral Analytical Laboratories (Austral janGove) rnment Analytlcal Laboratories) 1, Sua Kin Street, Binpur 2073, Niney South Wales, Aust It was deposited with Laria under registration number N92/4387.

The genotype of E. coli JMIOI is Δ(pro-1ac). F’　1acl qΔM15. traD1. λ-0pBTA879 is a gastrointestinal nematode Haemon 140 encoding part of the antigenic protein from Chus contortus pBLUESCRI PT-5K-my containing a 0 base pair EcoRI insert eggplant (Stratagene, San Diego, California, USA).

BTA2125 strain, an E. coli 5URE strain containing plasmid pBTA963, The Australian Government was established on 29 January 1992 under the terms of the Budapest Treaty. National Analytical Laboratories (Aus tra I tanGov) ernment Analytical Laboratores) 1. Akinoo Street, Binpur 2073, New South Wales, Aus. It was deposited with Tralia under registration number N92/4388.

The genotype of E. coli 5URESBTA2125 strain is E. coli SURETM strain (STR). Latagene, San Diego, CA, USA), genotype + mcrA.

Δ (mrr, hsd RMS mcrBC).

endAl, 5upE44. thi-1, λ-1gyrA96. relAl , Iac, recB, recJ, 5bcC, umuC: :Tnr (kanR ).

uvrC, IF’, proAB, 1acIqZΔM15°TnlO(tetR )).

The lasmid pBTA963 is used in the gastrointestinal nematode Haeionchus controller. A 1386 base pair EcoRI clone encoding part of the antigenic protein from tus pBLUEscRIPT-SK-minus (Stratagene, Surg. Diego, California, USA).

industrial use The present invention provides antigens, vaccines and drugs suitable for protecting animals from infection by parasitic nematodes. and antibodies.

literature Manlatls, T., Fr. ltsch, EF) and Sa IIlbrook, J. ) (Supervision) (1982) Molecular Cloning Near Fist Laboratory Manual ・CSH◆Laboratory (Molecular atoning: A laboratory manualC8+I Laboratory), code Cold Spring Harbor.

Saiki, RK (Saiki, RK), Gelfand, DH ( Gelfand, DH), Storfels, Schaaf ・Scharf, SJ, Higuchi, R ()tiguchi, R), Horn, GT, Mullis, Cavy (M uftis KB) and Ehrlich HA ( (1988), polymerization of specific primer DNA using a thermostable DNA polymerase. Amplification (Primerd reflected an+prirtcation orD NA vtth a thermostable DNA polyIIlera se), 5science, 239.487-491゜dan22 123456S Fig　4゜ rig pull 17° Ka Z O shoulder 1 core (:R with sword Z Jiman door 1 tou (Suspicious Ribo double I...Chopsticks I7 Toad Trp Th e Engineering 1a Ayuh Gly (ym enter sn TYt 8 sn Pro Gl y　Gly　Mp　Ain　Ayua　tyr　嘯兯池@l/a↓ , 6114 513 622 531 640 649 Otsu de Ji Z HXE Ichibe In the 1st part, I'm lazy to go π, and I'm lazy in the Z button, and I'm sweating in the Z, and I'm sweating next time - Val on IRC lEτ Gyo Y Alp pro LJ Ro The Ding h [＾sp ＾1 Todoroki Enter sp cys hys cys person ↓=@Giy C mouth nζ/,.

.../F+ 57 c m-ts-g Jgsg! ! 7 6] g 685 594 F a3-2 〒ga = de work ζ; で=でツCysVALCysSatGinGluGlu入上1MutuCys! 1 Spea de to G Luryeτhe de to Lau Yundatan pair'' 555 employees = 42 pairs It's over, you've got it, you've got a lot of power. Lau Ajnτhr kLh evil x MET Lea L, ys kKqτrp Gly Mn AJn Mw’: So 2, I'm cursing the violet, and I'm tactful and sweaty, fuji Z, and αF Uada πL work number CτhrME F enters. n Mal 'Nia τ dinghr τh; ru au Sat Lys Qτ Ser Gin Mu au ＾ 工１τtpG 工 n Sa■ lxsg Engineering 2Q Fue 2 Engineering 6 1 Suyu 5 1234 124 twins ζZ easily possible W No. Engineering 1e GLy Cys Vai Van Val Pra Cys Ding:p 5ee-5er rrp The@Val Yal =；1≠Banshiten Hikagi Jz Banbikyoga π)=Stomach pa in the first cut =czyAJPproa ysdingMLysλsp入上1^5pcy寞C1nCyltwoaNiaCyxTh eL:'fsS@t2! DaLma・ 64 73 82 91. 100 109xiz1a11901992011 z.

Sword Zk Te de ET Phe Gly Val, prO in sp Glu in s * Xle t, su　he? ItτGLu vil the ■K＾[9 44th 451 460 469 4th floor 48th [3rd 9th ζ dinner xc AC A otsu stone tsuba sigma=get Z6 stone f te Xi? Dede-KGIY Va"h GI Y l! is Qr Tht Gin VaL Person 11 Trp Gin Sa [　Sat　JJ9　Lyt　Xl■@Gly　Cys 496 SO55145235315418 x3 u〒suteεdte℃=Xx This is X Terra ACA is here Ala Val Giu Trp Cys Pr mouth Mr Ding Ding hr Leu Val Ala (ys G Engineering U Tyr Person 4!1@Pro ＾1晟 GAA GACTGT CλA τG 8CT QC; CTGζ ＾Cτ 3 x 3 Cys Gln Cys Thr Gly Cys Thr cy≦5er Lys Asp GLu Ala LsP@Cys 11e C mouth nse/.

17F + 51 ship mar-m-aabJ CλGτGAAAC＾＾入＾G(GG(eC(Gλ＾DingdingC13g6Ddan2m abstract a substantially purified antigen from a parasitic nematode of the second species, the antigen being vaccination with a nematode species that is the same or different from the first nematode species. an antigen capable of protecting the host from parasitism by a second nematode species, which may be , this antigen is proteinaceous, pl is in the range of 3,8 to 4,4, and lenti Binds to lulectin and nelix poa+atia lectin, reducing 5DS - A patent characterized by a molecular weight of approximately 45 kD as determined by PAGE. Copy and translation of the written amendment) Submitted document 184M071111

Claims (51)

[Claims] 1. a substantially purified antigen from a first species of parasitic nematode, which By vaccinating with an antigen that is the same as or different from the first nematode species, It is an antigen that can protect the host from parasitism by a second nematode species that may be present. Therefore, this antigen is proteinaceous, has a pI in the range of 3.8 to 4.4, and has a low binds to antillectin and Helix pomatia lectin, and reduces S An anti-inflammatory agent characterized by having a molecular weight of about 45 kD as measured by DS-PAGE. original. 2. 2. The antigen of claim 1, wherein the antigen is at least 90% pure. 3. An antigen according to claim 1 in non-glycosylated form. 4. The first parasitic nematode species are Trichinella, Ancylostoma , Strongylus, Trichostrongylus, Haemonc hus, Ostertagia, Ascaris, Toxascaris, Un cinaria, Trichuris, Dirofilaria, Toxoca ra, Necator, Enterobius, Strongyloides and Any one of claims 1 to 3 selected from species of the genus Wuchereria and Wuchereria. The antigen according to item 1. 5. The first parasitic nematode species is Trichinella spiralis, An cylostoma, canium, Strongylus vulgaris , Trichostrongylus colubriformis, Haem onchus contortus, Ostertagia ostertag i, Ascaris sum, Toxascaris leonina, Un cinariastenocephala, Trichuris vulpis , Dirofilariaimmitis, Toxocara sp., Necato r americanus, Ancylostoma duodenale, A scaris umbricoides, Trichuris trichi ura, Enterobius vermicularus, Strongyl oides stercoralis, Wuchereria bancrof Claim selected from ti and Ostertagia circumcinta The antigen according to item 4. 6. A second nematode species is Trichinella, Ancylostma, Str. ongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxacaris, Uncina ria, Trichuris, Dirofilaria, Toxocara, N. ecator, Enterobius, Strongyloides and Wuc The antigen according to claim 1, selected from species of the genus Hereria. 7. A second nematode species is Trichinella spiralis, Ancyl Ostoma canium, Strongylus vulgaris, Tr icostrongylus colubriformis, Haemonc hus contortus, Ostertagia ostertagi, A scaris sum, Toxascaris leonina, Uncin ariastenocephala, Trichuris vulpis, Di rofilariaimmitis, Toxocara species, Necator a mericanus, Ancylostoma duodenale, Asca ris lumbricoides, Trichuris trichiura , Enteroblus vermicularus, Strongyloid es stercoralis, Wuchereria bancrofti and and Ostertagia Circumcincta. Antigen according to paragraph 6. 8. Claims wherein the first and second nematode species are selected from the genus Haemonchus. The antigen according to item 1 above. 9. The first and second nematode species are Haemonchus contortus. , the antigen according to claim 8. 10. 10. A method according to any one of claims 1 to 9 in substantially purified form. Antigen precursor of an antigen. 11. Antigen precursor according to claim 10, having a purity of at least 90%. . 12. The anti-inflammatory agent according to claim 10 or 11 in non-glycosylated form. Proto-precursor. 13. has the amino acid sequence substantially as shown in FIG. 8 and is glycosylated or The non-glycosylated form of the antigen precursor, as it naturally occurs One that excludes precursors. 14. An amino acid consisting of amino acids 12 to 440 of the amino acid sequence substantially shown in FIG. It has a amino acid sequence and can be used in glycosylated or non-glycosylated form. original, excluding antigens as they naturally occur. 15. of the antigen or antigen precursor according to any one of claims 1 to 14. Homolog. 16. At least over 20 amino acids for the amino acid sequence shown in Figure 8. 16. A homolog according to claim 15, which is also 70% homologous. 17. The amino acid sequence substantially as shown in FIG. 7 or the amino acid sequence shown in FIG. 16. A homologue according to claim 15, consisting of amino acids 17-454 of. 18. polynucleotides excluding naturally occurring polynucleotide molecules A molecule, the antigen or antigen precursor according to any one of claims 1 to 17. A polynucleotide molecule that encodes a protein or homologue. 19. 19. A polynucleotide molecule according to claim 18, which is a DNA molecule. 20. A polynucleotide molecule according to claim 19, which is a cDNA molecule. . 21. A polynucleotide having a nucleotide sequence substantially as shown in FIG. 7 or FIG. octide molecule and substantially nucleotides 65-1381 of FIG. Polynucleotide having a nucleotide sequence corresponding to leotide 50-1339 19. A polynucleotide molecule according to claim 18, selected from: 22. Excluding naturally occurring DNA molecules, the sequence shown in Figure 8 and at least Both have 50% homology and can provide immunity against infection by parasitic nematodes. A DNA molecule that encodes a protective molecule. 23. Claims in which the polynucleotide molecules are DNA molecules and vector DNA A recombinant DNA molecule according to any one of paragraphs 18 to 22. 24. the vector DNA consists of plasmid, phage or viral DNA; A recombinant DNA molecule according to claim 23. 25. Vector DNA is λgt11, pUR290, pUR291, pUR28 2. Based on pUK270, pUC8, pUC9, pZipNeo, SV40 vector, λgt10, EMBL vector, pBR327, pBR329, or or pBR329 containing the par locus, baculovirus or vaccinia 25. A recombinant DNA molecule according to claim 24, selected from viruses. 26. pBTA879 and pBTA963 as defined above. 27. At least one recombinant according to any one of claims 23 to 26 A transformed host having a genetic DNA molecule. 28. The host is selected from bacteria, yeast, other microorganisms, insects, plants and mammalian cell lines. 28. The transformed host according to claim 27, which is selected. 29. Transformation according to claim 28, wherein the host is an E. coli K12 derivative. host. 30. BTA2033 and BTA2125 as defined above. 31. The antigen, antigen precursor, or antigen according to any one of claims 1 to 17 28. An expression product of a transformed host according to claim 27, consisting of a homolog. 32. The expression product of claim 31, wherein the expression product is a fused expression product. current product. 33. Antigen, precursor or homologue according to any one of claims 1 to 17 or of an expression product according to any one of claims 31 or 32. Synthetic polypeptides corresponding in whole or in part, when administered to a host animal , capable of inducing protective immunity against infection of host animals by parasitic nematodes; Synthetic polypeptide. 34. At least one antigen according to any one of claims 1 to 17 , a precursor or a homologue, or according to any one of claims 31 or 32. and/or the synthetic polypeptide according to claim 33. an effective amount of pharmaceutically and/or veterinarily acceptable carriers, diluents and/or or a vaccine comprising with an adjuvant. 35. Pharmaceutically and/or veterinarily acceptable carriers, diluent excipients and/or or an adjuvant, the form according to any one of claims 27 to 30. A whole cell vaccine, in live or dead form, comprising a transformed host. 36. Claims for administration by the transformed host to the mucous membrane of the host to be vaccinated expressing the antigen, precursor or homolog according to any one of paragraphs 1 to 17; 36. The whole cell vaccine according to claim 35, which is capable of 37. A method for preparing the antigen according to claim 1, comprising: (a) homogenizing young adults of a parasitic nematode species to produce a homogenate; (b) obtain a membranous material from this homogenate; (c) obtain a membranous material from this homogenate; (d) extracting with a buffer containing an ionic detergent to obtain a detergent extract; The extract was chromatographed on a wheat malt lectin Sepharose column. (e) collecting the effluent from the column; A method consisting of things. 38. Fractionation by preparative isoelectric focusing and pI of 3.8 to 4.4, more preferred collection of fractions, preferably in the range 4.0 to 4.3; Fractionated by gel filtration chromatography, fractions with molecular weights in the range of 10 to 60 kD and lentil lectin or Helix pomatia lectin. Claim 3 also includes chromatographic fractionation and collection of bound substances. The method described in Section 7. 39. At least one type of antigen according to any one of claims 1 to 17 , an antigen precursor or a homologue, an expression product according to claim 31 or 32. and/or an effective amount of the synthetic polypeptide according to claim 33. and/or veterinary acceptable carriers, diluents, excipients and/or Preparation of the vaccine according to claim 34, comprising mixing with a vaccine. Law. 40. A method of protecting a host from infection by at least one parasitic nematode species. Therefore, at least one type of antigen according to any one of claims 1 to 17 , an antigen precursor or a homologue, an expression product according to claim 31 or 32. , the synthetic polypeptide according to claim 33, and/or the synthetic polypeptide according to claim 33. A method consisting of administering to a host an effective amount of the vaccine described in paragraph 34 or 35. Law. 41. The antigen, antigen precursor, or antigen according to any one of claims 1 to 17 coffin complex, expression product according to claim 31 or 32, claim 33 The synthetic polypeptide according to claim 34 or the vaccine according to claim 34 or 35 Antibodies raised against Chin. 42. At least one antibody according to claim 41 can be used for pharmaceutical and/or or together with a veterinary acceptable carrier, diluent and/or excipient. , antibody composition. 43. The antigen, antigen precursor, or antigen according to any one of claims 1 to 17 homologues, expression products according to claim 31 or 32, claim 33 and/or the synthetic polypeptide according to claim 34 or 35. claim 4, comprising vaccinating an immunocompetent host with the vaccine described above. A method for preparing the antibody according to item 1. 44. An effective amount of the antibody according to claim 41 can be administered in a pharmaceutical and/or veterinary manner. The claim consists of mixing with a scientifically acceptable carrier, diluent and/or excipient. A method for preparing the antibody composition according to claim 42. 45. Inserting the DNA molecule according to claim 19 or 20 into vector DNA. 24. A method for preparing a recombinant DNA molecule according to claim 23, comprising: 46. Adapt the host for transformation, provide a competent host, and Transforming a host with a recombinant DNA molecule according to claim 23. A method for preparing a transformed host according to claim 27. 47. The antigen, antigen precursor, or antigen according to any one of claims 1 to 17 homologs, expression products according to claim 31 or 32 or claim 32; The synthetic polypeptide according to claim 33 and/or the synthetic polypeptide according to claim 41 A diagnostic kit comprising an antibody sample. 48. A transformed host according to claim 27 is provided, and this host can be used for expression production. The expression product is harvested from the transformed host by culturing it under conditions suitable for expressing the product. Biosynthesis of the expression product according to claim 31 or 32, consisting of assembling Enacted law. 49. Corresponding to a part of the antigen according to claim 1 and Antibodies that can protect immunized hosts from invasion by parasitic nematode species Tidiotypic antibodies. 50. The anti-idiotypic antibody according to claim 49 can be used as a pharmaceutical and/or or with veterinary acceptable carriers, diluents, excipients and/or adjuvants. A vaccine containing both. 51. A method of passively defending a host against invasion by parasitic nematode species, at least one antibody according to claim 41 or claim 42. 2. A method comprising administering to a host an effective amount of the antibody composition described above.