Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
  • C07K14/4355—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes
  • C07K14/43554—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes from Taenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

• This invention relates to protective antigens, to antigenic preparations containing such antigens and to the use of the preparations as vaccines for control and eradication of cysticercosis resulting from Taenia ovis infection m susceptible hosts such as ruminants.
• the Taenia ovis tapeworm exists in adult form in the small intestine of its primary host, the dog.
• the cystic stage is carried in the musculature of its secondary or intermediate hosts, notably sheep and goats.
• Current control measures include prevention of feeding of infected carcases to dogs and treatment of dogs with cestocidal drugs, notably praziquantel (Droncit, Bayer) to prevent transmission of the parasite to ruminants. These control measures are costly to implement and are not effective in eradicating T. ovis.
• T. ovis contains a large number of antigenic components, most of which are not lmmunologically effective against infection.
• T. ovis a host protective antigen for T. ovis (Howell M J and Hargreaves J J, Mol Biochem Parasitol 28 21-30 (1988)).
• a cDNA library was prepared using mRNA extracted from adult T. ovis tape worms.
• Recombmants expressing antigenic determinants as ⁇ -galactosidase fusion proteins were selected using antibodies in serum from sheep infected with T. ovis but trials of the host-protective nature of purified fusion proteins were not reported.
• T. ov s having a molecular weight of approximately 16 kDa as calculated by SDS PAGE.
• the present invention may broadly be said to consist of a purified antigenic polypeptide having an ammo acid sequence which comprises the amino acid sequence of Figure 5 and which is capable of generating a protective lmmunological response against T. ovis infection in a susceptible host, or a peptide fragment or variant of said polypeptide having substantially equivalent protective lmmunological activity thereto.
• the polypeptide has a molecular weight of about 16 kDa calculated by SDS-PAGE.
• the protective polypeptide or peptide fragment or variant of the invention is obtained by expression of the DNA sequence coding therefor in a host cell or organism.
• the invention consists of a composition of matter capable of generating a protective lmmunological response against T. ovis infection in a susceptible host which essentially consists of: (a) a polypeptide having the ammo acid sequence of Figure 5; (b) a peptide fragment of the polypeptide (a) having substantially equivalent protective lmmunological activity thereto; or
• the invention provides a DNA molecule which is selected from the group consisting of:
• the invention provides recombinant expression vectors which contain a DNA molecule as defined above, host cells transformed with such vectors and capable of expressing the polypeptide or peptide fragment or variant thereof which is encoded, and methods of producing an antigenic polypeptide or a peptide fragment or variant thereof comprising culturmg a host cell as defined above and recovering the expressed product.
• the invention consists in a vaccine against infection by a cestode parasite which comprises the antigenic polypeptide, peptide fragment or variant defined above in combination with an lmmunologically appropriate carrier and/or adjuvant therefor.
• the invention provides a recombinant viral vaccine which includes nucleic acid encoding an antigenic polypeptide, peptide fragment or variant as defined above and which is capable of expressing said encoded polypeptide, peptide fragment or variant m vivo m a host susceptible to infection by a cestode parasite.
• the invention may be said to consist in a method of protecting a susceptible host against infection by a cestode parasite, comprising administering to said host an amount of polypeptide, peptide fragment or variant defined above which is protective against such infection.
• the polypeptide, peptide fragment or variant is administered to said host in the form of a vaccine as defined above.
• the invention provides an antibody specific for the antigenic polypeptide, peptide fragment or variant defined above.
• Figure 1 is a silver stain of the 16 kDa protein contained within a fraction cut from an SDS PAGE gel which provides immunity to T. ovis infection.
• Lane 1 Pharmacia Low Molecular Weight markers
• Lane 2 T. ovis oncosphere proteins
• Lane 3 Proteins m Fraction A
• Lane 4 Proteins in Fraction B
• Lane 5 Proteins m Fraction C.
• Figure 2 is a silver stain of the 16 kDa protein contained within a defined fraction cut from an SDS PAGE gel which provides immunity to T. ov s infection.
• Lane 1 Proteins in Fraction C1 ;
• Lane 2 Proteins in Fraction C2;
• Lane 3 Proteins in Fraction C3;
• Lane 4 Pharmacia Low Molecular Weight markers.
• Figure 3 is an immunoblot demonstrating that antibodies raised against the recombinant protein GST-16 recognise the native T. ovis oncosphere 16 kDa protein.
• Lane 1 Sheep antibody to T. ovis oncosphere antigens
• Lane 2 Sheep antibody to Fraction C2 antigens
• Lane 3 Sheep antibody to Fraction C3 antigens
• Lane 4 Rabbit antibody to T. ovis 16 kDa antigen
• Lane 5 Sheep antibody to GST-16 Fusion protein.
• Figure 4 shows the results obtained from analysis of GST-16 fusion protein by SDS-PAGE. Lane 1: Pharmacia Low Molecular Weight markers; Lane 5: GST-16 Fusion protein.
• Figure 5 represents the nucleotide sequence of T. ovis 16 cDNA and the predicted ammo acid sequence of the polypeptide encoded. DETAILED DESCRIPTION OF THE INVENTION
• the present invention is directed to the provision of an antigen which is host-protective against at least T. ovis infection.
• Hosts which are susceptible to T. ovis infection include ruminants. Accordingly, examples of hosts to which the invention has application are ovine and caprine hosts.
• T. ovis polypeptide As being involved in protection against T. ovis infection in a susceptible host.
• This T. ovis polypeptide is that having a molecular weight approximately 16 kDa as determined by SDS-PAGE.
• This polypeptide further has an ammo acid sequence which comprises the ammo acid sequence shown as SEQ ID NO. 3.
• the present invention also includes within its scope antigens derived from the native T. ovis polypeptide identified above where such derivatives have host-protective activity.
• These derivatives will normally be peptide fragments of the native polypeptide which include the protective epitope, but can also be functionally equivalent variants of the native polypeptide modified by well known techniques such as site-specific mutagenesis (see Adelman et al . , DNA 2 183 (1983)). For example, it is possible by such techniques to substitute amino acids in a sequence with equivalent ammo acids. Groups of ammo acids known normally to be equivalent are:
• the protective antigen of the invention can be produced by isolation from the native T. ovis oncosphere complement using conventional purification techniques. However, it is recognised that for production of the antigen commercial quantities, production by synthetic routes is desirable. Such routes include the stepwise solid phase approach described by Merryfield (J Amer Chem Soc 85 2149-2156 (1963)) and production using recombinant DNA techniques. The latter route in particular is being employed by the applicants. n a further aspect, the invention accordingly relates to the recombinant production of the antigenic polypeptide or peptide defined above.
• the production of the protective antigen of the invention by recombinant DNA techniques involves the transformation of a suitable host organism or cell with an expression vector including a DNA sequence coding for the antigen, followed by the culturing of the transformed host and subsequent recovery of the expressed antigen.
• Such techniques are described generally in Sambrook et al . , "Molecular Cloning", Second Edition, Cold Spring Harbour Press (1987).
• An initial step in the method of recombinantly producing the antigen involves the ligation of a DNA sequence encoding the antigen into a suitable expression vector containing a promoter and ribosome binding site operable in the host cell in which the coding sequence will be transformed.
• suitable expression vectors are plasmids which are double stranded DNA loops that replicate autonomously in the host cell.
• suitable vectors other than plasmids can be used in performing the invention.
• the host cell in which the DNA sequence encoding the polypeptide is cloned and expressed is a prokaryote such as E. coli .
• E. coli DH5 Ramaleigh E A et al . , Nucleic Acid Research 16 (4) 1563- 1575 (1988)
• E. coll K12 strain 294 ATCC 31446
• E. coll B E. coll X1776
• E. coli strain ST9 or E. coll JM 101 can be employed.
• prokaryotes can also be used, for example bacilli such as Bacillus subtilis and enterobacteriaceae such as Salmonella typhimurium, Serratia marcesans or the attenuated strain Bacille Calmette-Guerm (BCG) of Mycobacterium bov s.
• the host cell is a prokaryote
• expression or cloning vectors containing replication and control sequences which are derived from species compatible with the host cell are used.
• the vector may also carry marking sequences which are capable of providing phenotypic selection in transformed cells.
• E. coli has commonly been transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al . , Gene 2 95 (1977)).
• pBR322 contains genes for ampicillm and tetracycline resistance and thus provides easy means for identifying transformed cells.
• the plasmid including the DNA to be expressed contains a promoter.
• Those promoters most commonly used in recombinant DNA construction for use with prokaryotic hosts include the ⁇ -lactamase
• eukaryotic microbes such as yeast may also be used.
• Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available.
• the plasmid YRp7 for example, (Stinchcomb et al . , Nature 282, 39 (1979); K gsman et al . , Gene 7, (1979); Tschemper et al . , Gene 10, 157 (1980)) is commonly used.
• This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow m tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)).
• the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
• Suitable promoting sequences m yeast vectors include the promoters for 3-phosphoglycerate k ase (Hitzeman et al . , J Biol Chem 255. 2073 (1980)) or other glycolytic enzymes (Hess et al . , J Adv Enzyme Reg 2 149 (1968); Holland et al., Biochemistry 17 4900 (1978).
• promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilisation.
• Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
• cultures of cells derived from multicellular organisms such as mammals and insects may also be used as hosts.
• any such cell culture is workable, whether from vertebrate or invertebrate culture.
• interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure (Tissue Culture, Academic Press, Kruse and Patterson, editors (1973)).
• useful host cell lines are VERO and HeLa cells and Chinese hamster ovary (CHO) cells.
• Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along w th any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional termination sequences.
• the control functions on the expression vectors are often provided by viral material.
• commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40(SV40).
• SV40 Simian Virus 40
• the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al . , Nature 273. 113, (1978)).
• SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hmdlll site toward the Bgll site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.
• An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g. Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
• the antigenic polypeptide or peptide fragment encoded can be produced often the form of a fusion protein by cultunng the host cells.
• the fusion protein including the polypeptide or peptide fragment is then recovered and purified as necessary. Recovery and purification can be achieved using any of those procedures known in the art, for example by adsorption onto and elution from an anion exchange resin.
• the carrier portion of the fusion protein can prove useful in this regard.
• the purification procedure adopted will of course depend upon the degree of purity required for the use to which the polypeptide or peptide is to be put. For most vaccination purposes, separation of the fusion protein from most of the remaining components of the cell culture is sufficient as the antigen can be incorporated into a vaccine in a relatively crude form. However, cases where a greater degree of purity is desired, the carrier component of the fusion protein can be cleaved from the antigenic component. As will again be apparent from the specific examples provided, this can be easily achieved through the provision of an appropriate enzyme cleavage site between the carrier component and the antigen.
• the first step is to obtain DNA encoding the desired product.
• DNA molecules comprise still a further aspect of this invention.
• the DNA molecule of the invention preferably comprises at least the nucleotide sequence shown as SEQ ID NO. 2 (nucleotides 10 to 369 of the Figure 5 sequence) . It is however most preferred that the DNA molecule comprise nucleotide sequence shown as SEQ ID NO. 1 (the entire nucleotide sequence of Figure 5) .
• the DNA molecule of the invention can be obtained contained within a DNA molecule isolated from an appropriate natural source or can be produced as intron-free cDNA using conventional techniques such as those used in the specific description set out hereinafter. cDNA is preferred.
• the invention also contemplates variants of the polypeptide which differ from the native amino acid sequences by the insertion, substitution or deletion of one or more ammo acids.
• the nucleotide sequence of the native DNA molecule is altered appropriately. Th s alteration can be made through elective synthesis of the DNA using an appropriate synthesizer such as the Applied Biosystems DNA Synthesizer or by modification of the native DNA by, for example, site specific or cassette mutagenesis.
• the DNA molecule is treated to be suitable for insertion together with the selected control sequence into the appropriate cloning and/or expression vector. To this end the DNA is cleaved, tailored and religated as required.
• Cleavage is performed by treating with restriction enzyme(s) m a suitable buffer. Any of the large number of commercially available restriction enzymes can be used as specified by the manufacturer. After cleavage, the nucleic acid is recovered by, for example, precipitation with ethanol. Tailoring of the cleaved DNA is performed using conventional techniques. For example, if blunt ends are required, the DNA may be treated with DNA polymerase I (Klenow) , phenol and chloroform extracted, and precipitated by ethanol. Re-ligation can be performed by providing approximately equimolar amounts of the desired components, appropriately tailored for correct matching, and treatment with an appropriate ligase (eg T deliberately DNA ligase) .
• an appropriate ligase eg TRON DNA ligase
• the present invention provides a vaccine against T. ovis infection.
• a vaccine normally includes as the essential component a host protective amount of the polypeptide, peptide fragment or variant referred to above, together with an lmmunologically appropriate adjuvant or carrier.
• adjuvants examples include saponms (or derivative or related material), muramyldipeptide, trehalose dimycollate, Freund's complete adjuvant, Freund's incomplete adjuvant, other water m oil emulsions, double emulsions, dextran, diethylaminoethyl- dextran, potassium alum, aluminium phosphate, aluminium hydroxide, bentonite, zymosan, polyelectrolytes, retinol, calcium phosphate, protamine, sarcosine, glycerol, sorbitol, propylene glycol, fixed oils and synthetic esters of higher fatty acids. Saponms in particular have been found to be effective adjuvants.
• the vaccine may also be formulated to further include other host-therapeutic agents.
• therapeutic agents include anthelm tics or other vaccines, or immunostimulants such as mterferons or mterleukms.
• the protective antigen of the invention may also be treated in any conventional way to enhance its stability or to conserve or potentiate its lmmunogenic efficiency.
• the antigen may be treated with a suitable inhibitor, modifier, crosslinker or denaturant in such a way as to enhance its immunogenicity.
• the vaccine described above can be administered to the host by any of those methods known in the art.
• the preferred mode of administration of the vaccine is parenteral.
• the term "parenteral” is used herein to mean intravenous, intramuscular, mtradermal and subcutaneous injection. Most conveniently, the administration is by subcutaneous injection.
• the amount of the vaccine administered to the host to be treated will depend on the type, size and body-weight of the host as well as on the lmmunogenicity of the vaccine. Conveniently, the vaccine is formulated such that relatively small dosages of vaccine (1-5 ml) are sufficient to be protective.
• the vaccine may also be m the form of a live recombinant viral vaccine including nucleic acid encoding the polypeptide, peptide fragment or variant.
• the vaccine is administered to the host in this form and once within the host expresses the encoded polypeptide, peptide fragment or variant to induce a host-protective response.
• a number of such live recombinant viral vaccine systems are known.
• An example of such a system is the Vaccinia virus system (US Patent 4603112; Brochier et al . , Nature 354 520 (1991)).
• the invention provides a method of protecting a host susceptible to infection by a cestode parasite.
• the method of invention includes as its essential step the administration to the host of either the antigenic polypeptide or peptide fragment or variant per se, or of a vaccine as described above.
• the antigens of the invention are candidate protective antigens against at least the following cestode parasites other than T. ov s: E. ultilocularis, E. vogelii, E. granulosus, T. saginata, T. solium, T. mul ticeps and T. hydatigena .
• the use of the DNA molecule described above or a subsequence thereof as a probe is contemplated.
• the DNA molecule is used to identify by hybridisation DNA of a cestode parasite such as T. saginata, T. hydatigena or E. granulosus which encodes an lmmunogenic antigen of that parasite. In this way, further parasite antigens suitable for use m a vaccine can be identified.
• DNA amplification techniques such as the poly erase chain reaction (PCR) (Saiki et al . , Science 239 487 (1988)) can be employed to detect homologous DNA of other parasites, with the PCR primers being based upon the nucleotide sequence of SEQ ID NO. 2.
• PCR poly erase chain reaction
• antibody probes specific for the protective antigens of the invention can be used to screen the antigens expressed by organisms transformed by the DNA of the parasite in question. The location of a positive clone (one expressing an antigen recognised by the antibody) will allow identification of both the protective antigen itself and the DNA which encodes it.
• Such antibody probes can be either polyclonal or monoclonal and can be prepared by any of those techniques known m the art. For example, a suitable procedure by which polyclonal antibody probes can be prepared is set out in Example 3.
• monoclonal antibodies can be prepared in accordance with the procedure of Kohler and Milstein (Kohler G and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature 256 495-497 (1975)).
• Antibody binding fragments can be prepared by controlled protease digestion of whole immunoglobulin molecules as described by Tjissen P, Practice and Theory of Enzyme Immunoassavs in Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier, Amsterdam, New York, Oxford, 117-121 (1990).
• T. ovis oncospheres were prepared from bleach-hatched eggs as described previously (Harrison et al . , supra) and solubilised in 1% sodium dodecyl sulphate in 10 mM tris-HCl buffer pH 7.5 containing 1% dithiothreitol, 5 mM MgCl 2 and a cocktail of enzyme inhibitors, at a concentration of 4 x 10 6 oncosphere equivalents per mL. 4 mL of this antigen preparation were applied to a preparative 3mm thick SDS PAGE gel containing a 12-18% polyacrylamide gradient. After electrophoresis for 16h, the proteins were visualised by negative staining with CuCl 2 (Lee et al . , Analytical Biochem 166, 308-312, (1987)).
• Fractions were cut from the preparative gel with reference to Pharmacia Low Molecular Weight marker proteins run on the outside lanes of the same gel. Three fractions were cut as follows: Fraction A 67 kDa to 40 kDa Fraction B 40 kDa to 28 kDa Fraction C 28 kDa to 16 kDa
• the fractions were placed in separate containers and allowed to dry slightly before being homogenised with a glass rod. A small sample of each fraction was removed and boiled in the minimum volume of SDS PAGE sample buffer. The solubilised proteins were recovered from the gel particles by centrifugation and analysed by SDS PAGE. The protein content of each fraction is shown m Figure 1. The remainder of the homogenised polyacrylamide gel fractions were divided into two lots and blended with an equal volume of STM oil adjuvant (Bokhout et al . , Vet Immunol and I munopathol 2 491-500 (1981)).
• Example 2 23 million oncosphere equivalents were applied to a 3 mm preparative SDS PAGE gel containing a 12-18% polyacrylamide gradient. After electrophoresis for 17 h the proteins were visualised by the copper staining method described in Example 1. The region of the gel between 30 kDa and 14 kDa was located by reference to Pharmacia Low Molecular Weight marker proteins run on the same gel. Three fractions were cut as follows: Fraction C1 29-24 kDa Fraction C2 24-18 kDa Fraction C3 18-12 kDa • Gel fractions were allowed to dry slightly and were then homogenised with a glass rod. A sample was taken from each fraction, boiled in SDS PAGE sample buffer and centrifuged to remove gel particles. The solubilised proteins were analysed by SDS PAGE (see Figure 2) . The remainder of the homogenised fractions were divided into 2 lots and blended with an equal volume of STM adjuvant.
• Fraction C3 induced statistically-significant protection while Fraction C2 greatly reduced the number of cysts when compared to the control and Fraction C1. It is therefore clear that Fractions C2 and C3 contain protective antigens.
• a preliminary step in the technique used in the present invention involved the production of antibody specific to the 16 kDa antigen of the invention.
• such antibodies are commonly used as probes for screening the products of expression of a population of host organisms or cells transformed by an expression vector to allow identification of the organisms or cells expressing the required product.
• the specific antibody probes of the invention was formed as follows: Twelve million oncospheres solubilised in 1% SDS were separated on a 12-18% gradient SDS PAGE gel and stained with copper chloride as described. The 20-14 kDa region was located by reference to Pharmacia Low Molecular Weight Marker proteins run on the same gel.
• Strips containing the 16kDa protein and the regions immediately adjacent to the 16 kDa protein were excised, homogenised and dialysed against TBS to remove excess copper. After dialysis the gel particles and liquid mside the dialysis tubing were recovered, blended with STM oil adjuvant and injected into three rabbits on three occasions spread over two months. Two weeks after the third injection a 20mL blood sample was collected from each rabbit and the sera were analysed on Western Blots of native oncosphere antigens.
• This antibody was subsequently used to screen the T. ovis cDNA library.
• T. ovis oncosphere ⁇ gtll cDNA library (Johnson et al . , Nature 338 585-587 (1989)) was screened using a 1/50 dilution of rabbit ant ⁇ -16 kDa serum following standard methods (Glover, DNA Cloning: A Practical Approach; IRL Press, Oxford (1985)). Positive clones were detected using alkaline phosphatase (AP) conjugated goat anti-rabbit IgG or 125 I-labelled protein A. Positive clones were subjected to two further rounds of purification and screening, followed by high titre phage stock preparation.
• AP alkaline phosphatase
• Antibodies specific for individual clones were affinity purified from the rabbit ant ⁇ -16 kDa serum by low pH elution from nitrocellulose filters impregnated with ⁇ gt11 - expressed fusion protein. These antibodies were used in plaque arrays to determine which clones were related (sibling analysis) and in lmmunoblots to confirm that antibody to the recombinant protein recognised the 16 kDa oncosphere protein.
• DNA from the selected positive clone was prepared from liquid cultures and restriction digested with Eco R1. The insert DNA was recovered after electrophoresis in 1% agarose gel and was ligated to pGEX-IT plasmid DNA which had been prepared as described below.
• the vector pGEX-1T was constructed to allow m- frame expression of subcloned DNA together with the thrombin cleavage site to enable subsequent removal of the GST fusion partner.
• Plasmid DNA from pGEX-2T was cut with EcoRI and BamHI and the linear plasmid isolated by agarose gel electrophoresis followed by purification using a GENECLEAN Kit (Bio 101).
• pUC19 plasmid DNA was also cut with EcoRI and BamHI and the small linker fragment purified by electrophoresis on a 20% polyacrylamide gel in tris-Borate-EDTA buffer.
• the linker band was visualised with ethidium bromide staining, cut out and DNA eluted into d.H 2 0 overnight.
• the linker was ligated to the linear pGEX-2T which was then used to transform E. coli JM101.
• Resultant colonies were grown m L-broth and plasmid DNA isolated. Colonies containing the linker sequence were identified by the appearance of new restriction sites for Kpn 1 and Sac 1 in the plasmid DNA.
• the new plasmid was named pGEX-1T.
• pGEX-1T plasmid DNA was cut with EcoRI . treated with phosphatase and ligated to the insert cDNA's derived from the selection of ⁇ gt11 clones described above. Ligations were transformed into E. coli JM101 and clones expressing the GST-16 kDa fusion protein were identified by colony immunoassay or by immunoblottmg of protein extracts from the bacteria.
• Soluble fusion protein was isolated from bacterial extracts by affinity purification with glutathione-agarose (Smith and Johnson, Gene 67 31-40 (1988)). Analysis of the fusion protein by SDS PAGE indicated a relative electrophoretic mobility corresponding to a molecular weight of approximately 40 kDa ( Figure 4) .
• the nucleotide sequence of caesium chloride purified pGEX-1T-16 DNA was determined by direct dideoxy chain-termination sequencing using primers corresponding to pGEX-1 forward and reverse sequences flanking the insert site (Maniatis et al . , 1989, supra). Both coding and non-coding DNA strands were sequenced in this manner.
• the DNA sequence and predicted amino acid sequence are shown in Figure 5.
• the calculated molecular weight of the T. ovis portion of the fusion protein is 13,386 Da based on the predicted am o acid sequence.
• Serum samples from sheep immunised with GST-16 were analysed by immunoblotting against native oncosphere antigens.
• Fig.3 Antibodies to the recombinant GST-16 recognised the 16 kDa native antigen indicating that the recombinant protein shares one or more epitopes with the native 16 kDa protein.
• a polypeptide antigen of T. ovis which is effective in generating a protective immunological response against T. ovis infection in susceptible hosts. It has been established that vaccination with this polypeptide stimulates a degree of immunity against challenge infection with T. ovis eggs.
• the invention also provides a recombinant method for expression of the antigen by which commercial quantities can be obtained.

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