JPH07505787A - Polypeptides obtainable from Faschiola sp., vaccines, treatments and their DNA sequences - 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] Polypeptides obtainable from Faschiola sp.

and Vaccines, Treatment Methods and DNA Sequences The present invention generally relates to vaccines, treatment methods and DNA sequences thereof. Learn more about vaccines and policies that help reduce the spread of parasitic liver flukes. It concerns peptides. Furthermore, the present invention reduces the spread of liver sucking infections. It concerns how to

A detailed list of publications cited herein is compiled at the end of this description.

SEQ ID NOs for the nucleotide and amino acid sequences referred to herein The number (SEQ In NO.) and drawings are clearly indicated after the bibliography.

Throughout this specification, unless the context otherwise requires, the words “comprising” and “comprising” are used throughout the specification. "comprises" or "comprises" of variations thereof are not stated. means to include an element or whole or a group consisting of an element or whole. which consists of any other element or whole or consists of any other element or whole; This does not mean excluding other groups.

Australian Provisional Application No. PL7109 (No. 19 The cDNA clones “ICEA” and “ "IcEB" is herein referred to as "Fhcat" and "Fhcat2", respectively. has been newly named.

Trematode parasites, such as trematode parasites (trematodes), are a major cause of economic losses to livestock. as well as causing serious diseases in humans (Balloon and Hillier, 1 986). The major trematodes of economic or public health importance are Faschio La Hepati Force, Faschiola Giganti Force, Faschiola Magna, Sukis Tosoma povis, Scystosoma mati, Scystosoma mansoni , Schistosoma haematobium, Schistosoma japonicum, Paramphis Tomum microbotium, Nogantocotyl exbulanatum, dicro Coelium dendriticum, Eurytrema bankreaticum, Baragoni Mus westamani, Chronorchis nnenns and Obistorchis vii (Spichill, 1992). S4 Mansoni, S. Haematobiu Mu, S. Japonicum, C. Nensis, O. Vivellini, F. Hepati force; Human infections caused by F. is normal. Schistosoma species infect more than 200 million humans, and host populations The disease is caused by the deposition of parasitic eggs in the tissue, which triggers the granulomatous immune response. (McManus et al., 1993). The life cycle of all trematodes is that of an adult parasite. Accompanied by the release of eggs from. From these eggs, mira invade the intermediate host (mollusk) This leads to the release of a form that eventually infects a mammalian host. Nagaru.

Fasciola hepatitis is caused by infection with the trematode parasite Fasciora hepatitis. It will be rubbed. This infection causes hepatitis to lead to wasting, death and reduced wool and egg production. This is particularly important for industries involving invasive animals such as sheep and cattle, in that it brings about of economic importance (Balloon and Hillier, 1986). Reduces parasite spawning Vaccines that reduce transmission from one infected host to another can reduce the risk of In the case of Soma species, it would reduce human disease. However, with the current Vaccines to prevent fluke infections, specifically targeting the ability of internal parasites to reproduce It is not clear which vaccines are suitable for this purpose (Spitztil, 1992; 1993).

Until recently, control methods relied heavily on the use of anthelmintics, with limited success. It was In Australian Patent Application No. 50283/90, F. Glutathione S-transferase (GST) derived from adult T. Significant progress has been made in the development of liver-sucking vaccines by describing a vaccine comprising was seen. Although this vaccine is effective, it is especially effective against other aspects of the parasite's life cycle. It is important to continue the development of alternative liver vaccinal vaccines that target the human population.

In the research preparatory to the present invention, we have identified the Streptococcus larvae as a promising vaccine candidate. The protein proteases, especially the cathepsin proteases of trematodes, more specifically the fluke cathepsin proteases A study was carried out on the use of cathepsin protease derived from Aschiola sp. stop Finally, surprisingly, such protease-based vaccines have been shown to improve the survival of the insects. reduce the number of eggs in the feces and/or the number of eggs in the feces, thus reducing the ability of the insect to reproduce. discovered that it is effective. This vaccine also results in reduced egg viability. vinegar. Therefore, fewer viable eggs are shed by the animal. As a result, the contamination of pasture is reduced, so the vaccine according to the present invention This will reduce the prevalence of liver sucking type in plants. Additionally, this vaccine may be harmful. reduce the need to spray contaminated pasture with chemicals that can cause There is a huge environmental impact due to Even more surprisingly, cathepsin protein The enzyme contains one or more hydroxylated proline residues. See you.

Accordingly, one aspect of the invention provides a peptide comprising a certain sequence of amino acid residues. is particularly intended for isolated polypeptides, the sequence of which Amino acid residue groups as adjacent sequences: G　ln-X　aa-X　aa-Xaa-Xaa, -Xaa-Cys-Tr p-X aa-X aa-Xaa2 (SEQ ID NO: 23) In the formula, Xaa is any amino acid. Noic acid residue; Xaa is cxy or Glu; and Xaa2 is Ser.

Thr, Ala or Gly. The polypeptide contains Furthermore, it is characterized by the ability to induce a host immune response against helminths. It is.

Preferably, the double-width insect is a fluke. Preferably dust flukes are Fasciola species, Examples include, but are not limited to, the F hepati force, the F, nganti force, or the F magna. That doesn't mean it will happen. The most preferred species is F, hepatica. This Faschiola spp. may be mature or in the newly excysted larval stage. There may be.

In particular, the present invention provides an immune system that reduces worm viability and/or reduces the number of eggs in the feces. Polypeptides that are capable of eliciting a response in the host are of interest.

Generally, this polypeptide is a catebun protease, a catebun protease, etc. -like polypeptides or polypeptides with catebunbrotease-like properties. Ru. Catebunone protease or catebunone protease-like polypeptide is It is characterized by an active site defined by conserved residues in three regions in the molecule, among others: shall be. The cathepsin protease may be in the mature or precursor form.

Catebuns B and H differ in their overall sequence identity.

It consists of parts of sequences named L, S and G.

Preferably the host is a mammal. Preferably, the mammal is a domestic animal; For example, but not limited to, sheep or cattle species. most preferably , this immune response is a protective immune response against the insect's ability to reproduce.

Therefore, another aspect of the invention provides that cathepsins that can be isolated from Fasciola sp. comprising or derived from a protease and in a suitable host. to produce antibodies against cathepsin protease derived from Fasciola hepatitis. Peptides or polypeptides capable of stimulation are intended.

Preferably, the antibody response also reduces the viability of the worm and/or the number of eggs in the feces. It is something that brings.

The polypeptides of the present invention are conveniently referred to as F, hepatica, F, gigantica and Fasciola species selected from F magna, most preferably F, simple from hepatic force. a catabolic brotease or a part, fragment or derivative thereof that can be released; or the cathepsin protease or a part or fragment thereof. or a fusion molecule comprising a derivative, wherein the polypeptide is can stimulate antibody production against Faschiola sp. can. Such fusion molecules combine two or more identical or different cathep a fusion of a non-protease or a part, fragment or derivative thereof; or , one or more catebenbroteases or portions or fragments thereof. or a derivative and another protective molecule, e.g. including, but not limited to, fusions with transferase (GST). I can do it. The polypeptide may be present in immature (e.g., newly excysted larval stage) or or from mature Fasciola sp., although mature organisms are preferred. It is a good source of supply. For convenience, the following description of "F, Hepati force" is Fasci. be considered to include all species of Ora (e.g., F., Nogantei, F., Magna). and.

The term "polypeptide" is used in its broadest sense, and for convenience, Includes peptides, polypeptides, proteins, glycoproteins and fusion molecules. polypeptide is generally in isolated or recombinant or synthetic form. In isolated form, the port The repeptide has undergone at least one purification or isolation step. however, Preferably, the isolated molecule is in a form suitable for use in a vaccine and and/or at least 5% of the composition, preferably less than 5% of the composition. at least 20%, more preferably at least 35%, even more preferably at least at least 55-60%, more preferably at least 75-80%, or even more preferably or less (both correspond to 90-100%. The percentage of content is e.g. It is measured by weight, activity, antibody reactivity, or other means as appropriate.

The present invention relates to the use of non-naturally occurring amino acid residues or naturally occurring amino acid residues. Such derivatives, including derivatives incorporating chemical equivalents, congeners or analogs. It extends to non-natural (ie, synthetic) derivatives of polypeptides.

Preferably, the polypeptide is modified to include 3-hydroxy or 4-hydroxy. It has at least one proline residue that has become cyproline. More preferably, this The proline residue in is 3-hydroxyproline. Furthermore, the polypeptide is If the cathepsin protease is in its ripe size, this Therefore, the determined fraction of about 25-30 kDa, more preferably about 26-28 kDa It has molecular weight. However, the molecular weight of the polypeptide is whether it is a protease or a part, fragment or derivative thereof; It can change.

The peptide or polypeptide according to this aspect of the invention is particularly useful in liver vaccinations. It can be used as an active immunogen in immunotherapy. useful in this vaccine Catheproteases can be single molecules (including fusion molecules) , or a mixture of catephobroteases. Faschio isolated from Ra species (e.g. F, Hepati force, F, Giganti force or F, Magna). A mixture of proteases isolated from pond worms (e.g. trematodes) In some cases, preferably at least one of the proteases is hydroxylated. more preferably about 10-20%, even more preferably Approximately 20% of cathepsin proteases contain at least 1111 hydroxylated It has a proline residue. Since then, the name “Faschiola° species” has generally been against worms such as flukes containing the novel protease of the present invention Includes designation. A single catebump that the vaccine was isolated from Fasciola sp. when comprising a rotease or a fusion molecule thereof, then preferably at least more preferably at least 10-20% of the proline residues in position 3 or 4. It is hydroxylated at the 3-position, more preferably at the 3-position. Furthermore, the vaccine immunogenic fragments of cathep/7 protease individually or It may also comprise a fusion molecule between more than one such immunogenic fragment. Canada Additionally, such immunogenic fragments or fusion molecules may be combined with other proteins such as GST. It may also be fused with defense molecules.

Alternatively, the catephobrotease can be synthesized by recombinant means; In this case, the proline residue may or may not be hydroxylated. This embodiment According to comprising at least one unique recombinant cathepsin protease.

Recombinant cathepsin protease contains amino acids that are substantially identical to the naturally occurring sequence. You may have an array, or after making the following changes to the array, the corresponding The offspring are Fasciola species, such as F, Hepati force, F, Giganti force or F.

← Elicits an immune response against naturally occurring catebunone protease from beech One or more amino acid changes or deletions to the sequence, as far as possible and/or additions. Such an immune response preferably reduces the viability and and/or result in a decrease in egg numbers. created from recombinant molecules or natural Any fragment of a cathepsin protease, whether created from molecules present in Similar immunogenicity requirements are required for fragments or derivatives. Therefore In this specification, the term "catebunprotease" means that the molecule is The immune response to naturally occurring molecules from Fasciola species, such as Naturally occurring molecules, their recombinant forms and any mutants thereof, insofar as they are derived , is considered a designation for derivatives, fragments, homologs or analogs. "mosquito The term "tep/mbrotease" further refers to two or more canapsin proteins. Fusion molecules between theases or with other similar molecules related by amino acid sequence , the same < is extended to fusion molecules with other defense molecules such as GST.

Most preferably, the polypeptide for a Faschiola vaccine substantially corresponds to the SEQ ID NO. No. 2 (FIG. 9A) or SEQ ID NO. 12 (FIG. 9B) or SEQ ID NO. 24 (FIG. 12) Amino acid sequence disclosed or disclosed in SEQ ID NO: 21 or SEQ ID NO: 22 or the polypeptide has an N-terminal sequence such as F, can stimulate antibodies against catebunprotease from Schiola seeds. for the amino acid sequence or a region or part of the amino acid sequence, as long as at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70-80%, even more preferably less than 60%. Both have 90% similarity. Preferably, this antibody is used to improve insect viability and/or Reduces the number of eggs in feces.

Therefore, another aspect of the invention provides (i) a catebunon protease or Catebunbrotease-like molecules: (i) Can be isolated from Fasciola sp. and (ii) SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 24 All or part of the amino acid sequence disclosed in comprising an amino acid sequence having acid sequence homology; A polypeptide is provided.

In related embodiments, the polypeptide comprises (1) a cathepnon protease or is a cathepsin protease-like molecule: (11) can be isolated from Faschiola sp. and (ii) disclosed in SEQ ID NO: 21 or SEQ ID NO: 22 For all or part of the N-terminal amino acid sequence, a small amount (40% of the amino acid sequence) It comprises an amino acid sequence having homology to .

Preferably, the worm is a trematode as described above, most preferably F. It is a species of phaschiola like Ti force or F Giganti force.

In this context, "some" means at least 5, more preferably at least 5 It is 10 contiguous amino acid residues. Preferably the Fasciola species is F hepati It is power. Preferably Faschiola sp. is the mature form thereof, but the present invention provides new It is extended to catepenum proteases derived from the excysted larval stage of the parasite.

Furthermore, the present invention provides a naturally occurring cathepsin protease derived from Fasciola sp. derived from other animal or plant cells capable of eliciting an appropriate immune response against has cathepsin protease-like properties, such as the catepnon group proteases of It extends to any related polypeptides. The present invention also provides a polypropylene according to the present invention. encodes a peptide, and preferably SEQ ID NO: 1 (FIG. 9A) or SEQ ID NO: No. 11 (Fig. 9I3) or substantially similar to the whole or part thereof. Extended to cDNA, having a nucleotide sequence. What is “similar to actual purchase”? , has the same meaning as above. “a portion” in this context means at least 15 leotide, more preferably a contiguous chain of at least 25 nucleotides. Ru.

According to this embodiment: (i) Encodes cathepsin protease: (Tsuta 1) Can be isolated from Capelidae species and (iii) the entire nucleic acid sequence disclosed in SEQ ID NO: 1 or 11. or some or their complementary forms and low stringency conditions. hybridize under cy conditions), A nucleic acid molecule comprising a nucleotide sequence is provided.

The trematode is preferably a trematode, more preferably a species of Fasciola, such as F.

Hepati force, F Giganti force or F, Magna, but not limited to these. There isn't.

Nucleic acid molecules are - heavy or double-stranded, linear or covalently closed circular forms. can be RNA or DNA. Defining the level of stringency For the purpose of (which is hereby incorporated by reference), which is hereby incorporated by reference in Section 11. degree is considered high austerity. Low stringency is defined herein as 0.1-0.5v/vSD Defined as 2-3 hours at 37-45° C. in S. involved in hybridization Depending on the source and concentration of the nucleic acid, herein 0.25-0.5% w/v SDS Under moderate austerity conditions, considered at ≥45°C for 2-3 hours, or Sampurtsuk et al. Another stringency condition can be used, such as the high stringency condition disclosed by 89). Ru.

Preferably the Fasciola species is F, Hepatica. Preferably Faschiola Although the species is a mature organism, the present invention deals with Fasciola in the newly excysted larval stage. Extended to living organisms.

The present invention further provides the categories of the present invention including fragments, derivatives, etc. to a composition of matter comprising a recombinant and valve recombinant form of Bunone protease. Expanded.

The present invention provides a method for administering a vaccine to an animal by administering a formulation of one or more categorical proteases to the animal. Inoculation reduces the number of eggs in the feces, which leads to a significant reduction in the reproductive capacity of the worms. It is based in part on the discovery that Egg viability is also reduced. This means that Fewer viable eggs are dropped into the environment by animals, reducing contamination of pasture. , thus resulting in a reduction in the prevalence of infection with this parasite. The present invention eliminates the parasite. This extends to captive animals that can carry or be infected, especially sheep and sheep. It is aimed at rebellious animals such as cows and cows.

According to this aspect of the invention, a method is contemplated for reducing the prevalence of parasitic worms; This method involves injecting catebnoids that can be isolated from the parasite into animals susceptible to infection by the parasite. an effective amount of a polypeptide derived from or comprising a proteinase; , for a sufficient period of time and for the animal to produce antibodies against the categorical protease. and under conditions. Preferably the trematode is a trematode, more preferably Preferably, F Hepati force, F. Fasciola like Giganti force or F Magna It is the seed of The Fasciola seeds are most preferably in a mature state.

The polypeptide is preferably a cateb/mbrotease as described above, with a small It may have at least one hydroxylated proline residue; It doesn't have to be. Vaccines are extracted, excreted, secreted, or purified or partially purified products, including products released or otherwise released. It may be a mixture of catepenbroteases. Such products are In the specification, it is referred to as "vomit" from the parasite. The present invention relates to the mode of action. Although not intended to be limited to one hypothesis, it is possible that the cateb/mbrotease It is said that antibodies against the worm reduce the number of eggs in feces, thus reducing the ability of the insect to reproduce. This is proposed in the present invention. The viability of expelled eggs is also reduced.

Catebnone protease can be administered orally, intravenously, subcutaneously, intracutaneously, intramuscularly, intraperitoneally, and suppositories. The agent may be administered by any suitable route, such as intranasal or intranasal administration.

The invention therefore provides immunization of one or more cathepsin proteases as described above. a sterically effective amount, as well as one or more carriers acceptable for veterinary use. and/or a diluent.

one or more cathepsin proteases or active immunogenic fragments thereof; The active ingredients of the vaccine composition comprising: is intended to exhibit superior activity in stimulating antibodies in target animals when ing. This change depends on the animal and cathepsin proteases, e.g. There is. For example, about 0.5 ug to about 20-g1 per animal and per dose, preferably preferably from about 0,651 g to about 101 mg, more preferably from about lag to about 1@H. The total amount of thepsin protease or cathepsin protease may be administered. can. Dosage regimen may be adjusted to provide the optimal therapeutic response. For example, how many Administer in divided doses daily, weekly, monthly, or other appropriate intervals. or the dose may be administered at a fixed rate as the exigencies of the situation dictate. can be reduced. The active compound can be administered orally, intravenously (if water soluble), Intramuscular, subcutaneous, nasal, intradermal or suppository routes or by implant (e.g. release) (using regulatory molecules). Depending on the route of administration, The active ingredient comprising one or more cateb/mbroteases Prevents the ingredient from the action of enzymes, acids and other natural conditions that may inactivate it. It may be necessary to coat it with a protective substance. For example, a low lipophilic cathepsin protein In the gastrointestinal tract by enzymes that can cleave peptide bonds, and by acid hydrolysis. It will probably be destroyed in the stomach. If this vaccine is administered by a route other than parenterally, For administration, catebu/mbrotease is treated with a substance that prevents its inactivation. may be coated with or administered with such materials. For example, The proteinase is administered in an adjuvant and is treated with an inhibitor of the enzyme. It can be administered simultaneously or in liposomes. Adjuvant in its broadest sense used in the treatment of any immune system such as interferon or other cytokines. including irritating compounds. Adjuvants contemplated herein include resolonol nonionic surfactants such as polyoxy/ethylene oleyl ether and Includes n-hexadecyl polyethylene ether and the like. For convenience, use this adj. Bunt is Freund's complete or incomplete adjuvant. Enzyme I Inhibitors include pancreatic trypsin inhibitor, neuisopropylfluorophosphat (DEP) and tranorol. Liposomes are water-in-oil-in-water CG F emulsion (CGF emuls) ions) and conventional liposomes.

The active compound may be glycerol, liquid polyethylene glycol, and/or It may also be administered as mixtures of these and dispersions made in oils. Normal γ retention Under the conditions and conditions of use, these preparations contain preservatives to prevent the growth of microorganisms. Contains.

Suitable dosage forms for injectable use are sterile aqueous solutions (if water soluble) or dispersions and and extemporaneous preparation of sterile injectable solutions or dispersions (exte+oporaneous sterile powder for preparation). in all cases , the dosage form must be sterile and must not be fluid to the extent that easy syringability exists. Must be. It must be stable under its manufacturing and storage conditions; and must be protected from the contaminating action of microorganisms such as bacteria and fungi. carrier For example, water, ethanol, polyols (e.g. glycerol, propylene glycerol) liquid polyethylene glycol, etc.), suitable mixtures thereof, and The solvent or dispersion medium may include vegetable oils and vegetable oils. For example, renotin By using a coating agent, the #4 dispersion can be improved by maintaining the required particle size. , and the use of surfactants allows proper fluidity to be maintained. Microorganism Prevention of the action of substances can be achieved by using various antibacterial and antifungal agents, such as parabens, chlorobutenes, etc. Can be caused by tanol, phenol, sorbic acid, tolumerosal, etc. can. Often contains osmotic agents, such as sugars or sodium chloride. It would be preferable to Sustained absorption of injectable compositions can be achieved by, for example, This can be brought about by using retarding substances.

Sterile injectable solutions contain the active compounds in the required amount, along with various others enumerated above, as required. It is manufactured by placing it in a suitable solvent with other ingredients and then filtering and sterilizing it.

Generally, dispersions include a basic dispersion medium and a variety of the sterilized active ingredients enumerated above. manufactured by placing it in a sterile medium containing the necessary other ingredients. will be built. For sterile powders for the manufacture of sterile injectable solutions, the preferred manufacturing method is , vacuum drying and freeze drying techniques, and the active ingredient with any further desired ingredients powders are produced from their solutions which have been previously filter sterilized.

Catebun proteases or mixtures thereof are preferably protected as described above. When the vaccine is administered, for example, with an inert diluent or an assimilable edible carrier. Administered by mouth or filled into hard or soft gelatin capsules or tablets It can be compressed or mixed directly into dietary foods. For therapeutic oral administration For purposes, the active compound is mixed with excipients and prepared into ingestible tablets, buccal tablets, and trowels. In the form of liquids, capsules, elixirs, suspensions, tablets, cannibals, etc. can be used. Such compositions and formulations contain at least 1% by weight of It should contain active compounds. Compositions and formulation percentages are of course However, it may range from about 5 to about 80% of the weight of the unit, as appropriate. vaccine group The amount of active compound in the composition is such that a suitable dosage will be obtained. To the present invention Such preferred compositions or formulations have an oral dosage unit form of about 0, sug and 20 sug. g of active compound.

Tablets, troches, gunpowder, capsules, etc. must further contain the following: Also available are gum gragacanth, gum arabic, corn starch or gelatin. Binders such as dicalcium phosphate, excipients such as corn starch, potato starch powder, decomposition agents such as alginic acid, lubricants such as magnesium stearate, and sweeteners such as sucrose, lactose or saccharin, or peppermint, wintergreen oil, Alternatively, flavoring agents such as cherry flavoring can be added. Dosage unit form is If it is a capsule, it may contain a liquid carrier in addition to the above type of material. can. Various other substances can be used as coatings or in dosage units in their own way. Sometimes it exists to modify the physical form of. For example, tablets, pills, Alternatively, the capsules can be coated with shellac, sugar or both. 7 0 knobs or elixirs contain the active compound, sugar as a flavoring agent, and as a preservative. of methyl and propyl parabens, color and cherry flavor or orange It may also contain fragrances such as fragrances. Pain theory, manufacture of any dosage unit form Any substance used in the It should be non-toxic in quality. In addition, the active compounds can be used in sustained release formulations and formulations. It is possible to incorporate it into Conveniently, the vaccine is administered to an animal such as a grain. Can be administered with feed. The vaccine composition may be mixed into a grain base or It is also possible to apply topically to the feed.

As used herein, "carrier and/or diluent suitable for veterinary use" Ha, Ren! and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents. , osmotic pressure regulators, absorption delaying agents, and the like. For pharmaceutically active substances The use of such media and materials is well known in the art. conventional media or In the vaccine composition, except to the extent that the substance is incompatible with the active ingredient, are intended to be used. Incorporating additional active ingredients into this composition You can also The invention extends to multivalent or multicomponent vaccines To the extent that the latter is specifically contemplated.

Vaccine compositions according to the invention contain one or more catebenproteases. In addition, other antigens, immune enhancers and /or one or more other active compounds such as drugs for veterinary use It can include. The vaccine composition further comprises one or more protease variants or (or partially purified F, including the exudate of F. hebatheica) You can stay there. The vaccine may alternatively or additionally contain 2 or between further cateb/mbroteases or their immunogenic fragments. or a fusion molecule of catebun protease or an immunogenic fragment thereof. and other defense molecules such as GST.

Catebunone protease or its antigenic fragments are incorporated into the intestinal flora. Using a bacterial expression system that expresses cathepnon protease in rare bacteria, It is also possible to transport the drug by means of in-vivo delivery methods. Alternatively, Viral expression systems can also be used. The present invention relates to catebunbrotease or extends to antibodies against its antigenic fragments. Antibodies are monoclonal or or polyclonal, such as F, Hepati force or F, Giganti force. Purification of catepentin protease or related molecules from Fasciola sp. It is useful when

The present invention includes catepenbrotease-reactive antibodies or other suitable antibodies. Applying the worm effluent to an affinity column consisting of a phagocytosis, preferably flukes, more preferably flukes, Chiora species, e.g. F., catebunos derived from hepatitis, or other biological samples. It extends to methods for purifying noproteases or related molecules. The column eluate is e.g. by 5DS-PAGE and/or ion exchange chromatography. Can be further processed. Alternatively, limit the exhalate or other biological sample. Concentrate by external filtration and use a molecular sieve to remove substances with the desired molecular weight. (.

Yet another aspect of the invention provides a screening method for screening antibodies against the above-mentioned cathepone proteases. A diagnostic assay for helminth infection in animals comprising This is for illustration purposes only. This assay can be used separately for monoclonal or polyclonal may include screening for catepentin protease antigens using local antibodies. . Although the most convenient test format is ELISA, the present invention can be used with any suitable assay format. It extends to.

The invention may be further described by reference to the following non-limiting drawings and/or examples. Explain.

drawing: Figure 1 represents cathepsin proteinase activity in adults of F. hepatica. This is a photo display. 50-fold concentration of adult ejecta (track a) and intact adult The ejected material (track b) was separated by 5DS-PAGE, and proteins were detected by silver staining. I visualized it. The proteolytic activity of intact adult vomit was determined by gelatin 5DS-PAGE. When dithiothreitol (2aM DDT) was added on the gel, lane (d) However, the amount was detected in the absence of DTT (Lane C).

Figure 2 shows a molecular sieve of the exudate of an adult F. hepatica. Concentrated effluent and 1 mg of the solution was applied to a gel permeation column (Example 1). Figure A: 280 stops (1,0AU Chromatographic profile of elution followed by PS). according to the order of elution The peaks are numbered. The area where the 1 minute fractions of Beaks 5 and 6 were collected is shown. vinegar.

Figure B: 5DS-PAGE analysis of both 1 minute portions of Beaks 5 and 6 (then silver stained) ).

5% of each aliquot was loaded in reducing sample buffer. Figure C: For each side Load 1% in reducing sample buffer for both Western blot analyses. F.

(using hepatica proteinase antiserum).

Figure 3 shows the anion '2 of catebun proteinase in the exudate of an adult F. hebatica. Figure 2 shows conversion chromatography. Pooling the material from peaks 5 and 6 in Figure 2, Concentrate and use 20!111 ethanolamine (pH 9,0; buffer MA) by FPLC. ) was applied to a medium mono Q column. Coat the column with buffer B (il buffer A+211NaCl ) Elute with buffer B supplied in a discontinuous gradient maintained at 5.5% and 13%. Ta. Panel A, chromatographic profile of protein elution from MonoQ column. melt The output was followed at 280 nm (1°QAUFS) and 13 fractions were collected as shown. Ta. Figure B: S D S - P A G E of fractions 1-13 with silver staining on i& analysis. Approximately 1% of each fraction was loaded in reduced sample 11iti fluid. Drawing C Western blot analysis for minutes 1-13 (using antiserum). Approximately 1% of each fraction was loaded in reducing sample buffer.

Figure 4 shows the gel fraction of mono0 fraction of catebun proteinase under non-reducing conditions. This is a photographic representation of the analysis. Load approximately 1% of fractions 2-5 from Figure 3A onto the gel. , separated by non-reducing 5DS-PAGE. S D S-P AG E gel medium The protein was detected by silver staining, and the proteinase was detected by Western blot. was detected using sheep antiserum.

Figure 5 shows purified cathepsin protein under non-reducing (NR) and reducing (R) conditions. Figure 2 is a photographic representation of 5DS-PAGE analysis of inase. Equally divided sample of protein Long Each lane was loaded and visualized by silver staining.

Figure 6 shows the EL of antibodies against F, hepatica cathepsin proteinase under investigation. Figure 2 is a graphical representation of an ISA analysis. -6 weeks (primary immunization 1f), -2 weeks (booster immunization) infection), −1 week (1 week before challenge), and 44 and 12 weeks after infection. Individual sera from blood draws were tested for anti-cathepsin proteinase activity by EL I. Measured by SA (see Example 1). Figure A shows an infected control sheep. The development of anti-tepnon proteinase titers is being followed. Maximum titer 10,0 00 was achieved by 1218 animals. Figure B shows the results over the 18-week trial. We followed the development of anti-cathepsin proteinase titers in immunized mice. It is something. A maximum titer of 225.000 was obtained by one animal at 12 weeks.

Figure 7 shows the purified excretory/secretory capacity of F. hepatica adult proteinase. is a photographic representation showing dimension 5DS-1” AGE analysis. Isoelectric focusing in the first dimension. over a pH range of 2.5-7.0 using 30 μg of purified protease. Really provided. Proteins were detected in the second dimension (15% protein/vSDS-PAGE) by silver staining. It was done. Lane (R) is a one-dimensional reduced 5DS- This is the result of PAGE.

Figure 8 shows the pH optimum of excreted/secreted cathepsin protease in F. hepatica adults. This is a graph display showing. A total of 15 and 30% g of enzyme was added to 100 liters of and lQQmM phosphate buffer, respectively. This assay is described in Example 2. Fluorogenic substrate (fluorogenic 5 ubstrat) e), carried out using Z-F RNMeC in the pH range shown.

Figure 9A: Peptide along with predicted amino acid and nucleotide sequences of FhCatl. A list of C's. The nucleotide sequence of Fhcatl (ICEA) cDNA is 1 It is 075 bases long. The predicted amino acid sequence is methionine (nucleotide 2 5) and ends with phenylalanine (nucleotide 1002). Direct N-terminus The sequence (N-ter■) also maps to the Fhca tl-order sequence.

The peptide was purified from F. hepatica efflux/mbrotease chymotli. Pusin (0) and endo-Glu-C (GC) /r + compound derived from .

Figure 9B Another cateb/mbrotease from liver fluke (Fhcat2[I CEB]) nucleotide sequence and predicted amino acid sequence.

Figure IQ: Fhcatl preproprotein, human preproprotein protein (HLIMC ATLP077] l), Promeline (P14518), human preprocateb Non-H (HU M CA TH, P 0966g), human preprocateb/n B (HUMCATB: PO7858), and Schistosoma thio Arranging the 4 amino acid sequences of Rukatebun (SchistoB: N21309) The receipt number of Chinoku Nonbank is shown in parentheses). Showing the similarity between these sequences We summarized the functionally conserved amino acids in order to

xed).

Figure 11 N-terminal sequence of excreted/secreted catebun proteinase from adult cuttlefish . Sequences can be determined by direct N-terminal amino acid sequencing or by endoGlu c was produced from peptides isolated from digests (Materials and Methods). Promera N (Lydon et al., 1989) and papain (Dress et al., 1971). Terminally, some mammalian thiolcatebnones (Uncatepnon S (weede) Lanoders et al., 1991), mouse catebunon B (Chan et al., 1986), human and mouse force T/N L (Mason et al., 1988: Noyosef et al., 1988 ) and trypanosome proteinases (Mottram et al., 1989 , Eakin et al., 1992).

Figure 12: Amino acid sequence of FhcatB1 sequence and human force Tebun B, Squis Tosoma mansonicatepsin B; Human force tebunun L; Fasciola hebate Lined up with Ca1l and 2. The gaps in the Fhcat Bl array are Indicates the sequence that should be recognized. Boldface indicates identical residues between sequences.

Example 1 Vaccine development 1 Materials and methods Parasite: Parasitizing Metachelia tomentosa into an intermediate host snail, Limnaea tomentosa. It was maintained by Original isolate from infected sheep in Lanka/Yar, UK (referred to herein as "Compton 2") was manufactured by Compton Buttock Labs. Local snail hosts and mammals purchased from Latries (Surrey, UK) It is maintained by parasitizing Merino sheep as hosts.

Gel analysis.

Various preparations of the exhalate and its extracts were run on 15% w/v SDS-PAGE. Proteins were analyzed under original conditions (Laemmli, 1970), and proteins were visualized by silver staining (Morris, 1970). Say, 1981).

Proteinase activity in adult vomitus was visualized using gelatin gel (Dalt N and Heffernan, 1989). Briefly, gelatin in separating gels Normal 5DS-PAGE (Laemmli, 1 A gel was prepared in the same manner as (970). Before electrophoresis, reduce enzyme activity during electrophoresis. The gel and running buffer were cooled to 4°C to allow for cooling. Sample non-reduced 5DS -PAGE samples were mixed with the loose sample solution and loaded onto the gel without denaturation. electric After pneumophoresis, the gel was washed with 25% V/V) 0.1 M containing Lydon X-100. Incubate twice for 30 minutes in sodium phosphate (pH 4,5). , its i&o, 1M sodium citrate (pH 4,5) (211M if appropriate) A final incubation of 1 hour at 37°C in dithiothreitol (DTT) containing Attached to bean tongue. The gel was mixed with 0.1% w/v Cumano Blue (50% V/V Meta). Nor, 40% v/v acetic acid, 10% v/vi distilled water) for 15 minutes, then Destained in 7% v/v acetic acid.

Prepare Western plots as described (Barnett, 1981); Bio-Rad Mini Transplot Model in pre-chilled (-20°C) transfer buffer. Electrotransfer was performed for 90 minutes at 150V at 4°C. sfer) was carried out. Blotted nitrocellulose membrane - and Schuell, d'Asossel, West Germany) plotted at 5% - PBS/Zween I blocked it out for an hour inside. After washing in PBS/Zween, The antiserum diluted in 100 ml of chlorine was applied to the membrane for 18 hours (on a shaking table) at 4°C. further cleaning After that, a species-specific 1g Western horseradish peroxidase conjugate (Sirenas 100 in PBS/Zween) was added to this plot for 2 hours (RT). Finally, the enzyme-immune complex was visualized by reaction with 4-chloro-1-naphthol. Sefston et al., 1990). Against F hepatica proteinase The sheep antiserum was prepared as follows. Rabbit anti-bromelain serum Victorian Institute of Animal Science, Atwood , Victoria 3049, obtained from Australia.

Preparation of antigen Fasciola hebatica proteinase is found in the vomit of mature insects. It was purified from An adult fluke collected from the liver of a sheep infected with Compton 2 strain. were washed three times in warm (37°C) PBS and treated with 1QmM glutamine (Flora 0.06% w/v NaHcOs (pH 7,5). 5% in buffered basal Eagle medium (Flow Laboratories, Melbourne). v/vc.

2 at 37°C for 2-4 hours. This medium is then decanted. It was frozen (-20°C) until required. Upon thawing, this ejectate is centrifuged. (10,000g, 30 minutes, 4℃) and filtered with a 045μ filter. Empty filtration followed by pre-equilibrated 10 kDa cut-off membrane (Minitan Brake). (NMWL, Millipore, Bedford, MA) for 8 hours at 8 psi. It was filtered and concentrated 40-50 times (Minitan Ultrafiltre Nozin Si) Stem, Tsusu-Tazuribore, Bedford, MA). During ultrafiltration, test The container receiving the water was kept above water. Typically, batches of 21 are 4040-5O The retentate was concentrated to 100% of the retained material. Batch-to-batch phase in protein concentration Due to the differences, some retentates are more dilute than other retentates and can be used by vacuum centrifugation. (Savant Instruments, Hicksville, N)') .

Molecular sieves were placed on a pre-calibrated Superose 6 column (Pharmacia). It was carried out by FPLC (Ffurmana). Column with 0.6M NaCL 10ml Pre-equilibrate by washing overnight in l Tris (pH 6,0) and prepare the sample for separation. was dialyzed in this buffer overnight. Microscope an equal sample (1 mg) of the concentrated ejectate. After heart separation and filtration, FPLC applied to a column at 4 ml/min. Elution was carried out for 80 minutes with elution being followed with a 28Qnm detector. 30kD P Collect the material eluting under water, pool it, and dialyze it against water for anion exchange. (-night, 4°C) to reduce the salt content and then by vacuum centrifugation (Savant). It was concentrated 5-10 times. This concentrate was further mixed with 20 ethanolamine (pH 9). , 0) overnight at 4°C. The MonoQ column (Farmana) also has the same buffer. Pre-equilibrated in solution overnight. Following microcentrifugation and filtration, up to 5 ml of The precipitate was loaded onto an anion exchange column. Elution of protein components is carried out using isocratic gradient for 5 minutes. step (buffer A20mII in ethanolamine (pH 9,0)), then buffer Supply salt onto the column at 14 mmol/min from B (buffer A+2) INacl). This occurred during a planned elution with a planned gradient. When buffer B is 5% Pause this gradient to elute the first major peak, then return to the normal program. I returned it.

After performing ion exchange, the first eluted substance in the peak was dialyzed against distilled water, and the true It was concentrated 5 times by empty centrifugation and then further dialyzed. This substance's radioactive iodine chloramine T method (Song and Yaramonisoto 1970) Therefore, it was manufactured. Radiolabeled tracer is mixed with dialysate They were mixed to give a specific activity of 5-10×10'cpm/ag protein. mixture Non-reduced 5DS-P, AG E sample 1! Denature in +1i solution and prepare for preparative use (pr 15% w/v SDS-PAGE gel. swimming Once the reaction is complete, remove the gel, wrap it in plastic wrap, add a fluorogenic marker, and X-ray film (XAR-5, Eastman-Kodanok, Roti) for 30-60 minutes at Westerly, NY). Using the marker as a guide, detect the 26kD radioactive band. Excised, fragmented, 10mM) 1% w/v SD in Lys (pH 8,0) Boiled in S for 30 minutes. This suspension was diluted to 0.1% w/v SDS and then I ran it overnight on RT. After the contents had settled, the supernatant was removed and the gel pieces were Washed with 0.1% w/v SDS in IoM Tris (pH 8,0).

The gel pieces were then resuspended in the same buffer and eluted for an additional 24 hours. This second melting The eluate was combined with the first eluate and then precipitated with 9 volumes of acetone at -20°C. Ta. Add pelletized precipitate to 1% w/v SDS, 20 ml Tris (pH 8,0). Re-dissolve in 200a1 and combine with re-solubilized precipitates from other gel runs. Ta. The combined material was acetone precipitated. from one batch of concentrated effluent. Since the entire contents are processed, all acetone precipitates were diluted with 1% w/v SDS, 10mM Resolubilized in Tris (pH 8.0) and then precipitated with large amounts of ethanol. Be The pelletized precipitate was resolubilized in 100% TCA, and water (4°C) was added to make 10% Diluted in v/v TCA and left on water for 1 hour. This TCA pellet was mixed with acid-ethyl alcohol. Wash twice with alcohol and then with ethanol to remove traces of salt and SDS. Ta. Finally, samples of this material were checked for purity and protein content was determined using non-reduced 5DS-PA. Evaluation was done by GE gel and silver staining.

production of antibodies Antiserum against cateb/proteinase was generated from adult vomitus and was thought to be pure. Five Merino castrated rams were immunized with approximately 70 μg of blotinase. It was generated by For the preparation of this antigen, purification methods include coles and lupus. Based on the method of Burno (1988). Discharge (from 8-48 hour culture of adult fluke) ) was clarified by centrifugation and cooled. Cooled discharge (300°C at 0-2°C) -400 ml) of cold ethanol (2.2 ml/min) while mixing the final ethanol The solution was added dropwise until a concentration of 60% v/v was reached. This solution was allowed to stand at -20°C for 18 hours. Equilibrate and then pelletize at 6300 g (30 minutes, 4°C). Next, the supernatant Make a final concentration of 75% v/v ethanol, equilibrate overnight at -20°C, and clear. (as above). Wash the pellet in 100% ethanol and resuspend in water. Ta. This substance is known to contain proteinase activity28. Silver staining for the kD band complex (Cols and Rubano, 1988) It was examined by 5DS-PAGE. This preparation is impure due to the presence of other substances. If you suspect that the material is and subjected to Kel purification. These steps are performed in the same way as the steps described in the previous section. did.

Ten such preparations were completed, with material containing only the 28kD complex approx. 500 μg was obtained.

For the primary immunization, approximately 250 μg of protein was delivered in complete Freund's adjuvant ( FCA) (Commonwealth Laboratories (C3L), Melbourne , Australia) and homogenized with L. and Merino. Five castrated rams were injected subcutaneously (S, C,). Approximately 30μg of 28kD protein Injected. With 40++g of purified 28kD material in incomplete FCA (C3L) A secondary immunization was performed 4 weeks later. serum from these animals for an additional 4 weeks. Afterward, the samples were collected and pooled in equal volumes to detect the presence of catheptan proteinase. It was used as a reagent.

Clinical trial protocol: Liver fluke-free liver fluke-free liver fluke-free liver fluke-free liver fluke-free liver fluke-free A castrated Reno ram (5 years old) was obtained. Individual animals were tested for fecal F, hepa F. hepatica extract analyzed by ELI SA (below) screened for low serological cross-reactivity to 10 animals 12 in water mixed with the same volume of FCA (CSL) 6 weeks before infection (-6 weeks). The cells were immunized subcutaneously (S, C,) with 0 gg of purified proteinase. 4 weeks After a period of time (-2 weeks), this same group was treated with purified antigen mixed with incomplete FCA (CSL). An additional subcutaneous (S, C,) immunization was given with 1 g of 90I. These animals and one more Two weeks after the booster treatment (week 0), 0 non-immunized mice were exposed to 300 mice in water. Attacked by placing Takerkaria in the rumen. The animal is liver fluke free It was managed outdoors in a pasture. -6 weeks (pre-bleed and immunization), -2a (booster immunization) Blood samples were taken at treatment 1 [), -1 week, 0 treatment (challenge), 4 weeks, and 12 weeks.

Animal health was measured using an automatic analyzer (Lonoyu, Basel, Switzerland) using Corbus MIR. Erythrocyte hemoglobin determined by Van Camben and Nono Yurstra, 19 Follow-up by assessing parasite-induced anemia by assessing did. Serum was collected and stored at -20°C until analysis.

At 14 weeks of challenge, fecal samples were collected from all sheep and then manually stratified to kill the liver. and gallbladder were obtained. Worms were collected from liver tissues, bile ducts, and gallbladder. Egg mass from the gallbladder ( egg mass) were also collected. described (Sefston et al., 1990) Fecal egg counts (FEC) were performed.

Serology/ By ELI SA analysis of serum antibodies against whole Faschiola extract, animals were F hepatitis or other parasites that would result in high levels of cross-reactive antibodies. We investigated whether they had been exposed in advance. In this assay, polyvinyl Microtiter plate (Immunomakinsorb plate, Nanck, Denmark) h) in coating buffer (0, LM Na2COs, pH 9,5) at 1/2000 The cells were incubated overnight at 4°C using a diluted adult lysate (Wijfels et al., 1992). Overturned. Sheep serum was first plotted - PBS/) Vienna (5% sterile/V stock in PBS). Kimmilk powder, 005% v/v Tween 20 (Jig 7), pH 7,2) Diluted 1+500 and titrated on plates by serial 2-fold dilutions. 1 at 37℃ After an hour incubation/banning, the plates were washed with PBS/l-Vienna and plated. Lot - Donkey anti-sheep Ig diluted 1:2000 in PBS7/Tween Horseradish peroxidase conjugate (Sirenas, Melbourne, Aust) Laria) was applied for 1 hour at 37°C. Further wash and use substrate solution (62mM citric acid). Acid/76 Peng II NalHPO4, 15M2.2-Azinobis( 3-Ethylbenthiazole sulfonic acid (ABTS), 3% v/ vHtot) was added, and then the color development at 414 stops was automatically titrated. Measurements were made using a Ku Multiscan spectrophotometer (Flow Laboratories).

Animals with high serum titers (3-5 times the average) were excluded from use in clinical trials; Equivalent to 10% of any sheep population with no history of exposure to F, Hepati forces.

Throughout the trial, antibodies against F. hepatica proteinase were tested. Production was followed by ELISA. A very dilute solution of adult ejecta concentrate (subject Antigen derived from 1:16,000 solution in 11i buffer was added to a microtiter plate ( The mixture was adsorbed overnight (at 4°C) onto a glass plate (Nanku). 1 hour block process CRT lot-PBS/) Vienna), then the serum was diluted 1·1000 to 2-fold (pBS/) /) in Vienna) and incubated overnight at 4°C. anti-sheep Ig Conjugates were applied for 4 hours at RT and then read on ABTS. same dilution The titer can be determined by direct comparison with previous blood draws for each individual that have been similarly assayed over a range. It was determined. Titer is the highest serum dilution 0.1 absorbance unit above the previous blood draw of the same dilution. It was decided as.

Detectable host (sheep) immunoglobulin fragments present in untreated discharge Therefore, concentrated and ultrafiltered vomit should be used as antigen in preference to untreated vomit. used. Presumably, the ultrafiltered effluent contains reduced levels of these fragments. It would contain. Furthermore, using purified cathepsin proteinase as an antigen is superior to exhalate concentrate in terms of sensitivity or specificity in this assay. showed no benefit.

egg survival crab Collect the contents of the gallbladder and place the sample in a glass covered with Alfoil until further processing. Stored in a container at 4°C. Eggs are cleaned of bile contents by washing several times with tap water. separated from. The final water suspension was incubated for 14 days (in the dark) at 27-29°C. Cubated. This suspension was then exposed to an incandescent light source for 20 minutes. Evaluate survivability To evaluate the sample, add 10 drops of Lugol's iodine solution to the suspension sample 251. In addition, the contents were combined with phlegm, miracidium and viable valves (i.e., electrically focused). Eggs with no signs of differentiation are evaluated under a microscope: eggs with no signs of differentiation are considered non-viable. I did it.

2 Production of catebunon protease vaccine Identified in the vomit of adult F. hebatica catebenone proteinase.

5DS-PAGE analysis of gross specimens of adult liver flukes followed by silver staining revealed that the 28 A clear complex of 2-3 bands of 30 kDa was revealed (Figure 1, Tracks a and b). This complex or its components in the same preparation Sources of proteolytic activity detected at similar positions in the gelatin gel (Figure 1, Track C) It was thought that this may be the cause. This gelatinolytic activity (gelatinolytic activity) lytic activity) was significantly increased by the addition of dithiothreitol. This suggested the presence of a categorical proteinase (Fig. 1, track d). .

Putative catebunbrotinases were enriched using several chromatographic methods. Purified from the discharge. The 50x ultrafiltered concentrate was added to high salt Tris buffer (pH 6).

0), applied to a gel permeation column and found five main peaks (peaks 1-5) and It was partially resolved into a later eluting peak with lower M habit (peak 6, Figure 2A).

By using a high salt 1ffi solution and a slightly acidic pH, peaks 5 and 6 could be best separated from the earlier eluting peaks. peak 5 Reducing 5DS-PAGE analysis of the 1 minute fractions of It became clear that component D was present (Fig. 2B). peak instead of peak 6 The fraction derived from 5 contained a significantly higher amount of the 28 kD component. lower than Although molecular weight components (~1O-14kD) were also detected in the early fractions (1-5), , some of these may represent contaminating proteins from peak 4.

Derived from peaks 5 and 6 using Hino antihemolysis against catebunon proteinase Western blot analysis of the fractions revealed a 28kD immunodominant component in both fractions. The darkest staining corresponded to the fraction derived from peak 5 (Figure 201 1-5). The lower molecular weight components of 17kD, 15kD and 14kD are also was detected in most of the fractions by this antiserum, but it is likely that the substances in these fractions Identification in the later fractions was less easy due to lower amounts. these ingredients represents the degradation product of catebunon proteinase and is lower than that shown in Figure 2B. The molecular weight components seemed to match.

There was no clear difference in the antibody-reactive substances in peaks 5 and 6 (Figure 2). , these two fractions were pooled, and after appropriate dialysis and concentration, they were subjected to anion exchange chromatography. I attached it to the graph. Maintaining the salt gradient at 5.5%, buffer B (50 μL 10++11 NaC1) in tanolamine (pH 9,0) is a high salt elution component. This resulted in the elution of major protein groups that were well separated from each other (Figure 3A). 5DS-PA GE analysis revealed that the first major collection of 5-7 peaks contained almost no protein in the 28 kD region. It is clear that the most common staining occurs in the 14 and 15 kD regions. (Figure 3B, tracks 1-5). However, the waste of these fractions Tanplot analysis showed reactivity to cathepsin proteinase antiseptics (Figure 3C ) and consistent with the material collected in the gel permeation step (Figure 2C), 17.15 and a 14kD component was detected. Lower molecular weight components are cathepsin proteins. It appeared to represent a degradation product of inase. However, between these two techniques The difference in staining intensity between the 28kD component and other components does not seem to make sense. further research is needed.

The material in the main collection of fractions 2-5 of the anion exchange peak (Figure 3A) was When resolved on an original 5DS-PAGE gel and visualized by silver staining, there were still 14 Although low molecular weight components of 15kD and 15kD are detected, the 26-28kD region is more intensely stained. colored (left panel of Figure 4). This result was demonstrated by reduced 5DS-PAGE analysis. This suggests that a much larger amount of protein exists in the 28kD region than the 3B). However, when the non-reduced preparations were examined by Western blot, It was not found that the 14 and 15 kD components were antibody-reactive, and this time the 26 kD component and larger components were only detected by the antiserum. Molecular sieve and Yin Contaminant proteins are eluted with cathepsin proteinase by either on-exchange method. As a result, contaminants were removed from the reduced 5DS-PAGE gel. quality is reduced and denatured by fragments of cathepsin proteinase. This appears to have resulted in a product that became unclear. Reduced and non-reduced 5DS-P Changes in the migration of the major types of catheptan proteinases between AGE gels This suggests an internal/sulfide bond that binds some of this proteinase. type yields low molecular weight components upon reduction.

To confirm that the catebunone proteinase was purified, an ion exchange step was performed. Dialyze and concentrate the main elution peak of the Separated by 5DS-r'AGH.

Trial 1 of vaccination with F, m% J cateb/mproteinase of hepatitis. Catebun protein purified from the vomit of adult F, Hebatian force from 0 animals. The mice were immunized with a total of 21,011 g of zeolin (2 injections 4 weeks apart). 10, which is the control group None of the animals were immunized. After 2 weeks, both groups were treated with intrathecal injection. I attacked with 300 Metakel Ria of F Hepati power. fluke catebunompu Production of serum antibodies against roteinase was followed by ELISA. Figure 6 (Figure A ), all individuals in the infected control group showed cathepsin levels 12 weeks after infection. It produced low levels of antibodies against proteinases. Specific antibody proteins in immunized animals Rophiles are primary and secondary to immunization. condary) response was appropriately detected (Fig. 6, B). 4 weeks after challenge, immunity Nine out of 10 animals in the treatment group responded to phaschiolacatebun proteinase. increased antibody titers: half of this group showed significant increases. after infection At 8 weeks, these antibody levels were maintained in 8 out of 10 animals or Rose.

reproductive ability of insects Table 1 shows the insect burden (blind arm burdens) and final FEC of the study animals. Summarize.

The control group reported an insect burden over the dispersal range of 34-99 adults; yielding an arithmetic mean of °6 and a large coefficient of variation (C,V,) of 33%. Ta.

The spread of worm burden data is even more striking in the immunized group, with the average number of worms per sheep There were 793 adult flukes, and the standard deviation was 42 (c, v, = 53%).

Apparently, immunity due to fluke catebun proteinase under the regimen used The crop was unloaded after 14 weeks of infection with F6 Hepatica metachelaria. I'm not sure. However, FEC was significantly affected in the vaccinated group. I heard the sound. The average fecal eggs/excreted fecal dulam (206 ±86) was 70% lower than the control (700±377), and this difference Tukey's multiple range test test), it was statistically significant (p<0.05). insect load Similar to the data, the coefficients of variation were large, with different coefficients for the contralateral and vaccinated groups. 54% and 42%, but there was little overlap in the spread of the two groups.

When insect load and FEC are combined to determine fecal duram per egg/worm, The cutin inoculated group shows a 67% reduction in worm fecundity. However, the combination Large variations in these parameters introduced will result in a corresponding increase in the egg count. 68% and 56% for the paired and vaccinated groups, respectively. This results in variation coefficients C and V. The arithmetic mean was 3.10 eggs/g in the vaccinated group. /worm compared to 11.1 eggs/g/worm for the control. This difference is true It was significant (p<0.05) using the multiple range test.

egg viability Eggs from the gallbladders were collected and assessed for viability (Table 2). Control and vaccinated groups A comparison made over time showed that the average oviparous buoyancy in the vaccinated group was 38% ± 39%. , indicating a 56% reduction in egg viability from this group.

Example 2 Analysis of catebunbrotease in the vaccine of Example 1 1 Materials and methods kel analysis Sodium dobnol sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) Analysis was performed on 15% v/v gels and silver stained (Morris Say, 1981). Purified cathepsin protease 30 for two-dimensional gel analysis jg was applied to a first-dimensional isoelectric focusing gel (O'Farrell, 1975). Mix Almana Ampholyte 1ip82.5-5 and pH5-7 in a ratio of 12. (incorporate). Perform 5DS-PAGE in the second dimension using 15% reducing conditions, The gel was silver stained (Morrissey, 198],). sheep catepenum brotease anti Western blots with serum were performed as described in Example 1 above. all Fluke lysates were prepared as described by Winoufels et al. (1992).

Purification of F. hebatica catebunone protease cateb/mbrotease It was essentially purified from the exudate of an adult liver fluke according to the method described in 1.

Production of peptides Peptides were purified from several digests of F. hepatica proteinase. accomplished. Some peptides were treated with endobrotinase Glu-C (endo-G lu-C; Boehringer-Mannheim, Germany) from the digestate.

Peptides GC3,1,15,2,202 and 21.2 were purified as above. It was a digestion product of catebunon protease.

Approximately 20°C, 1 g of the cateprotease preparation was S-pyridylethylated and 2 %W/Il+endo-Glu-C (in acetone). 0.1M for digestion Conducted for 9 hours at 37°C in 100Ill of N84CO3 (pH 8.0, CO2) . Once the digestion was complete, the mixture was dried by vacuum centrifugation and diluted with 6i+HCl. Resolubilized in guaninium (in 1011I Tris(T)H8,O) and C18 Novavac reversed phase column (Waterzoom Millibore, Milford, MA) Injected. Peptides were analyzed by HPLC at 15 l/min (Waters 625LC system). A 5-60% acetonitrile (AcN) gradient (01% trifle) delivered by fluoroacetic acid (TFA)). Track the elution at 214 stops and check for high absorption peaks. The data were collected in a fixed time loop (a ti ed 1 loop). of the selected peak The contents were repurified in the same system using a 0-40% AcN (in 0.1% TFA) gradient. Manufactured.

Reduced and alkylated chymotryptic digestion to obtain large peptides It was applied to 100 gg of the purified protein that was not purified. Digestion was carried out for 4 hours, but otherwise It was performed as described by Winoyufels et al. (1992). The resulting peptide 5-60% AcN (0,1% C8 Novarback (Millipore Waters) reverse phase filter using a gradient (in TFA) Purification and refractionation using ram. This digestion results in peptides produced by chymotrypsin. peptides CT13.2 and CT11.3 were generated.

Two other chymotrypsin generated peptides CT21.2 and 0-13 ．． 3 was obtained from the digestion of the same preparation of cathepsin protease described in Example 1. I fell asleep. Chymotrypsin digestion was performed as previously described (Wijfels et al., 1999). 92). The peptide was purified similarly to the endo-Glu-C-digest.

Model 471A Protein Controller (Applied Biosystems) The N-terminal sequence was The purified peptide was dried by vacuum centrifugation before performing the l determination. PTH- Amino acids were added to 5% tetrahydrofuran (1 ml/min) delivered by HPLC. 50°11fiII in sodium acetate (pH 3,9)) and 100% AcN (ABI) gradient on a Spheri-5 isoPTH reverse phase column (ABr). I understand.

enzymatic assay A solution of purified catebunon protease was prepared based on the assay of Pallett (1980). (Assessed for proteolytic activity by fluorogenic assay. Reactions were performed in a 1 ml volume. with a final concentration of 25011M substrate, 2ml 1DTT, 1QQmil buffer and Proceed with 0.05% V/V Breez 35 and total purified enzyme 115-50n.

The mixture was incubated at 37°C for 25 minutes, then immediately 351m1 with emission and excitation wavelengths of 352 and 440n, respectively. ■By a fluorescence spectrophotometer (Perkin-Elmer model MPF-3) set to Fluorescence was measured. To determine the inhibitory effect of various reagents, the enzyme was added before the addition of the substrate. Breincubate with buffer, DTT and inhibitors for 10 min at RT. Ta. Substrate (N-CBZPhe-Arg 7-amino-4-methylcoumarin (Z- FRNMeC) and N-CBZ arginine 7-amido-4-methylcoumarin ( Z-RNMeC)) and inhibitory reagent (phenylmethylsulfonyl fluoride (P MSF), iodoacetamide (IAA), leupeptin, antipain, ap Rotinine, and E-64 (4-guanidino)butane)) were all purchased from Sigma, but EDTA was purchased from BD. Obtained from H.

Immunological screening of cDNA library λZAP F to batika cDNA The library was divided into 50,000 plaque-forming units per 150 L-broth agar plates. plated on a lawn of Enoerychia coli B B4 cells at a density of (pfu) I did it. Approximately 5xlO’pfu was purchased from Promega (Madison, USA). Using the Protoplot method described in the Protoplot Technical Manual The F hepatitis was screened for expression of IcE. The library is Pooled sheep antiserum raised against purified ICE of adult F. was used for screening at a dilution of 1/600.

Filter: 10mM Trif, HCl, pH 8.0, 150ml NaCL 0.05% v/v) in a buffer containing Vienna 20.1% v/v gelatin. Blocked. - pick up plaques identified as positive in the next screening; Replate at lower density and use sheep antiserum until individual positive plaques are identified. Then I re-screened it.

Isolation and sequencing of cDNA inserts Phagemid DNA containing cDNA inserts from positive λZAP phage clones (phargcmid DNA) from Stratanone (U. Noyola, USA) Isolation by in vivo excision of pBluescript phagemid under recommended conditions did. Phagemid DNA was obtained by the method of Birnboim and Dolly (1979). I extracted it.

After denaturing the plasmid DNA by treatment with NaOH, the D NA sequencing was performed by the chain termination method (Sanger et al., 1977). I did it.

2 Analysis of catebunon protease Heterogeneity of F-hepatica proteinase protease Excretion/secretion of F-hepatica The heterogeneous nature of catebunon proteases in gel penetration profile first observed. Under conditions of high salt and slightly acidic pH, most carbon Thevenone protease elutes below one peak, followed by a second, less important peak. It is accompanied by a small fraction that elutes slightly later, resulting in a brighter peak. Major categories Collect only the latter fraction of the Aase peak and most of the less important peaks, and An attempt was made to obtain a catebun protease that does not contain . These two parts are When analyzed by reducing 5DS-PAGE, a significant 26 kD A protein complex has been revealed. Fraction 1 contained another low molecular weight component. 2 Both aliquots containing only the 6kD component were pooled and subjected to Western blot and reduction analysis. Further characterization by 5DS-PAGE under It was clear. Some of the reduced preparations contained the 28kD potent cabrenod and the more stained There are thin 14, 15, 17 and 20 kD components, whereas the non-reducing material is It was relatively homogeneous, with a major complex at 26 kD.

Two-dimensional 5DS-PAGE analysis of purified excretory/secretory protease was performed. Now, the 28 kD is divided into several constituent components, with at least three closely migrated components. There were a significant component that was moving and four components that were less important with higher pl (Fig. 7 ). Lower molecular weight components (at 20 and 15 kD) were also detected, but these the same pl as the three most prominent constituents, which means that they are Fragments of the catepenlum protease type, which are oligomers linked together, or or an autoproteolysis product of the intact polypeptide. Ru.

Enzymatic characterization of F-hepatite cathepnon protease Purified protease protein Two fluorogenic substrates (N-CBZ Among Arg NMeC and N-CBZ Phe ArgNMeC), N-C Only BZ Phe Arg NMeC was found to be sensitive, and this was used for all enzymatic characterizations. N-CBZ Arg NMeC has fermentation There was no detectable activity even at high concentrations. cathepsin protease When determining the optimum pH, activity was significantly influenced by the ions of the buffer used. p A H maximum of 72 was achieved in 100 mM phosphate, and the activity was observed over a wide pH range (pH 6, 5-90, Figure 4). On average of 46#M, this cathepsia (1 00 Lilli Phosphate, pH 7.45). Use half the enzyme concentration in Tris buffer , the pH maximum was not measured, and the activity increased under alkaline conditions (Fig. 8).

The tn toxicity assay shows that catebunone proteases are present in very low concentrations of this class of proteases. Although highly sensitive to classical inhibitors of teases (Table 3), IA A is the least effective, with only 74% inhibition at 10100p degree. It was revealed. PMSF has very little inhibitory effect, whereas abrotinin and and EDTA did not result in any decrease in activity. DTT is the absolute for activity It was found that the requirements were (Table 3).

71 homologous adult F hepatitis expression cDNA ligation for thiol tep/n Braries were screened with sheep anti-brotease serum. High level Bell reactivity was observed, and even after prolonged absorption of this antiserum with E. coli, Approximately 0.1-1% of the library was positive. 9 randomly selected positives The sex clones were purified and their cDNA inserts were partially sequenced. those It was found that all of the sequences at their 3° ends were identical. Max clone insert , Fhcatl was sequenced and analyzed on both strands and found that nucleotides 25 to 1 The open reading frame extended to 002 is clearly visible. It became clear (Fig. 9A). The predicted open reading frame is estimated to be 326 amino acids long. It encodes a protein and is a member of the thiol catebunone family of catebunone proteases. -Has homology to family members (E, C, 3, 4, 22, -) ( Figure 10). This putative Fhcatl preproprotein is similar to human prebrocatebenone. and 39% homology to prebrocatepun H. . Human preprocathepsin B (23%) and Schistosoma derived (20%, Figure 10) and Haemonchus-derived (20%) catebunbrotease. Homology was separated.

Another cDNA located during cDNA library screening The A sequence, the nucleotide and predicted amino acid sequences of Fhcat2 are shown in Figure 9B.

It can be seen that Fhcat2 has 87% homology to Fhcatl.

Homology to mammalian thiol proteins suggests that the putative Fhcatl prep Amino acids 1-17 of the protein encode the pre region, Mino acids 18-107 encode the Pro region. child The bleed and pro regions of the protein are cleaved and removed, resulting in a mature protease with a length of 219 amino acids. ze is formed. Residues 126-136 of the predicted Fhca t1 mature protein are , thiolcathepsin consensus pattern Q-x(3)-[GE]-x-C- W-x(2)-[5TAG] followed by thiol cathepsins. The best homology was found when comparing with the mature polypeptide, and cathepsin L and and H were 54% and 44% (Figure 10). Surprisingly, good condition High homology was obtained in various plant thiol cathepsins (promelain: 39% (Figure 10), barley alinyline: 47%, actininonni 42%, papa In, 35%).

The cloned cDNA is expressed in F. hepatica excreted/secreted protein. To ensure that the purified peptide is representative of the from the chymotryptic and endo-Glu-C digests of proteinase. Made it happen. A series of peptides consisting of (CT21.2, GC15,2 and CT13° 3) partially overlap with each other, and these adjacent sequences are part of the putative Fhcatl polypeptide. It is aligned with positions 188-206 of Tido (Fig. 9A). Peptide CT13.3 is One polymorphic site was shown at position 204 where both Tyr and Cys were detected.

The second series of peptides (CT13.2 and CTII, 3) also When mapping to positions 277-291 of the predicted amino acid sequence of cDNA, the adjacent were in contact (Fig. 9A). CT11.3 is a highly conserved protein containing especially Asn residues. This is the catalytic triad of thiolcathepsin. tic triad) (Fig. 9A and Mini-Sill et al., 1999). 91).

F, N-terminal sequence of hepatica cathepsin proteinase purified catebunon protea Direct N-terminal sequencing of the enzyme allows us to identify other members of the thiolcatepneum family. A single major sequence, representative of the N-terminus, was revealed (Figure 11). End- Glu-C and chymotryptic digests also target the N-terminus of thiol catebens. (Figure 11), and the predicted Fhcat1 polypeptide Peptides (GC20,2, GC3, 1) was produced. However, the peptide sequence and the direct N-terminal sequence are not identical. There wasn't. In both cases, a highly conserved prologue close to the N-terminus There was a Similarly, conserved in cathepsin L and H subfamilies The DWR motif is intact in the two putative F, hebatheca N-terminal sequences. there were.

Besides these conservations, the sequence differs in several respects. End-Glu -C peptide, (GC20,2) is conserved, corresponding to the N-terminus of promelain. It has two residues before the proline. This peptide has Glu at position 9. However, exactly in this region, chicken cathepsin L&- is on the straight line. It appears to be adjacent to the peptide GC3,1, which is aligned with the peptide GC3,1 (Fig. 11). This GC20 , 2 sequence is 78% identical to the N-terminus of bromelain, but catebun L and H It also has a similar level of homology with the N-terminus. However, the direct N-terminal sequence , has only one residue (valine) in front of the proline at position 2, and this is where most of the thiol This is a characteristic shared by the lucateb/n class. Therefore, this sequence The thiol force after the devione deviates from most of the N-terminal sequence, resulting in promelain (61%) and a low level of homology to the Catebun L subfamily. This will result in a loss.

Excretion of F. hebatica/Hydroxyprotein in secreted catebun proteinase Rin Amino acid sequencing of peptides GC15,2 and GC20,2 revealed that respectively producing anomalous products in cycles 4 and 3, and any other phenylthio Hydantoin amino acid derivatives (PTH-amino acids) were also absent. Fasci When carrying out N-terminal sequencing of purified preparations of Auritheranones, significant amounts of PTH were detected. - Along with proline, a small amount of equally eluting product was also detected. other thiols Good homology of these sequences to regions in catebunones (Figs. 10 and 11 ) suggested the presence of proline at these sites, so these peptides We investigated the possibility of modified proline within the compound.

The simplest proline modification occurs in collagen, where proline is Hydroxylated at either C3 or C4 to form 3-hydroxyprolyl, respectively (3-Hyp) or 4-hydroxyproline (4-Hyp). centre Abnormal PTH amino acids derived from aschiolacathepsin protease are true Human type III collagen to determine whether proline is hydroxylated in 3-Hyp-PT) (derived from the CB6 peptide of the C1 and α, chains of and 4-Hyp-PTH were compared by chromatography. Is it proline? (3-Hyp and 4-) (partial hydroxylation to yp results in sequence: G P/3 α1 collagen with -Hyp P/4-HypGLAGPP P/4-Hyp This was demonstrated in the chain CB6 peptide. When this peptide is subjected to Edman degradation, In cycle 2, a small amount of 3-Hyp-PTH is accompanied by a large amount of PTH-proline. whereas the two major adducts of 4-H, p-PTH were detected in cycle 3. and 9 are unique. Similarly, we found that α, CB6 peptide (G 3-Hyp/PRGAPG), in the second cycle of sequencing However, the more important modification from proline to 3-Hyp was detected.

The chromatogram of collagen-induced 3-Hyp-PTH was Aligned with chromatograms of unidentified Edman degradation products derived from peptides. equivalent elution was evident. These purified fasciola peptides In both cases, PTH-proline is also a less important product, including total proline. 16% and 12% of the cases were detected. However, purified fasciolacatep/n N-terminal sequencing of proteases shows that hydroxylation near the N-terminus can occur in two different ways. Only 12 of the prolines were found in the hydroxylated form in a sequencing run and only 21% of cases, indicating that it was an event of low importance. these days Both 3-Hyp and proline behave similarly during Edman degradation. There were some things I expected to do. Importantly, 4-Hyp-PTH It was not detected in any amino acid sequencing of the Chiola product.

Table 1.2F. Worm burden and final FEC for individual animals Reproductive ability of insects (FEC/insects) (c, v, = variation coefficient, geo, +5ea n = Geometric mean) Table 2 Control and Stein protease vaccination eggs Viability%.

*Statistical data for the control group also includes eggs exhibiting dormancy (i.e. 0% viability). This excludes animals that are exposed to water.

Table 3 F, hepatic protein protein protein by known protease inhibitors Inhibition of enzymes Example 3 Efficacy of recombinant vaccines Page 26 except that 120I1g of protein was given per dose ("F, purification product of Hepatica The preparations were performed as described in "Tep/Nproteinase Vaccination Trial"). We conducted a clinical trial of a replacement vaccine. A group of 10 sheep has a natural degenerative force (Group A). ), natural active force tepnon (group B), Fhcat2 (group 0), Fhcatl (Group D) and Fhca tl+2 (Group E). G group were given PBS alone, while group E served as an uninfected control.

1 Naturally active and naturally modified cathepsin broth derived from Fasciola hepatica Purification of aase Adult flukes were isolated from experimentally infected sheep and washed with PBS (3 x 10 minutes). ) and then incubated for 2 hours at 37°C in RPMI. This extract is 2 It was frozen at 0°C and thawed at 4°C when necessary. Centrifuge the extract at 10,000xg Separate to remove eggs and particulate matter, then filter through a 0.45 micron filter did. Next, a minitan concentrator with a 10,000 Dalton molecular weight cutoff membrane (Millipore) to reduce the filtrate volume from approximately 2 liters to approximately 100 ml. I did it. This solution was applied to a microphone using a 10,000 Dalton molecular weight cutoff membrane. Using a Loprodicon concentrator, further concentrate and simultaneously 50 mII Tris pH 7. , 5,111 NaCl (buffer 1) was dialyzed to 1 liter. Buffer 1 and Using a Pharmacia FPLC equipped with a Superose 12 column and a flow rate of 0.25 Gel filtration chromatography was performed at haze l/min and l++1 fractions were collected. child Both aliquots were subjected to 5DS-PAGE and Western blot on reducing and non-reducing gels. Analyzed using rotting, as well as cymogram analysis. According to these standards Both aliquots showing homogeneity were used for preparation of antigen for vaccination. to group A The antigen to be presented was incubated for 30 minutes in 1% oxidation/vSDS and then It was setone precipitated and resuspended in PBS.

2. Production and maturation of clones expressing recombinant Fhcat t1 and Fhcat2 The DNA encoding catep/n was combined with the following primers + ICEF 5゜A. CAGCTCGAGGATCCGGCTGTACCCGACA3' (SEQ ID NO. 1.9) + ICER5°CTCGAGGATCCTATCACGG8AATC GTGC3' (SEQ ID NO: 20) containing l''hcatl and Fhcat2. was amplified from the plasmid. The PCR product was cut with BaaHl and then pE Bawl of T15b (Novaan, USA) E. coli strain BL21 (DE3) pLyss transformed into. Following transformation, lII cells were treated with ampinoline (20 μg/■l ) and chloramphenicol (25ug/sl) on broth agar plates. plated on top. Express Fhcat using the recombinant clone by the following method. We screened them for their abilities. Pick up individual colonies and ampinoli (20mg/ml), chloramphenicol (25ag/sl) and l+ The cells were used to inoculate 1 liter of a culture containing MIPTG and grown for 3 hours at 37°C. cells were then collected by centrifugation. Next, the cell pellet was added to sample Ill solution ( Laemmli, 1970) and loaded onto a 15% Soda/V polyacrylamide gel. The mixture was heated at 95° C. for 5 minutes before being heated. After electrophoresis, the gel was stained with Cumano blue. did. A clone expressing a large amount of protein of approximately 3QkDa was selected. sheep anti Western blot using cathepsin antiserum revealed that selected clones were recombinant F It was confirmed that hcat was expressed.

Purification of recombinant Fhcat protein A clone encoding the mature type of Fhca t 1 or 2 was treated with ampicillin ( 20gg/ml) and chloramphenicol (25μg/gl). Grow overnight in 10 ml of broth. These were then treated with ampinoline (20 μg/ ml), chloramphenicol (25ag/■1) and 1*1llPTG Inoculated into 200 mt of broth containing C. and grown for 3 hours at 37.degree. this cell Collected by centrifugation (10,000 g, 10 min), then the cell pellet was Resuspend in Qml of PBS, 0.1% v/v Triton X-100. Freeze/thaw Following this, cells were lysed and insoluble materials were centrifuged (10,000g, 10 minutes). Collected by. This insoluble material was then resuspended in 8M urea in 1x binding a buffer. The mixture was clouded and heated at 65°C for 10 minutes. Insoluble material was removed by centrifugation and the supernatant was saved. The recombinant protein was purchased from the manufacturer (Nova Table 4). His Bind Resin (LLis, Bind) as described by Ann, USA) , Re5in) was applied to a modified condition containing 8Il urea. It was purified from the supernatant using the same method. The eluted material (approximately 10×1) was collected in 1 liter at 4℃. Dialyzed twice against Tutle's PBS. During dialysis the recombinant protein precipitates from solution Ru. The dialyzed solution is then sonicated to form a fine suspension and the protein concentration was measured.

3゜Result Fecal egg count data were obtained in infected i & 14 and 15 week sheep. Flatten the number of eggs per group (10 sheep per group) with mean ± SD and percent decline and p-value. Enumerate. The groups in this trial are: Group Vaccine A Natural denatured adult cathepsin protease (ACP) B Natural active ACP CFhcat2 D　Fhcatl E Fhcatl+2 F Infection control G. Non-infected control The data show that vaccination with naturally active catepuncture proteases in sheep increases the risk of infection. & leads to a 67-75% reduction in fecal egg count (FEC) at 14-15 weeks It is shown that. This result is significant (p<,01). Denatured cathepsin pro Vaccination with Tease or Fhcat2 also reduced FEC at 14 weeks. Yes! (59%) (P<0.013), Fhcat2 is no longer active. led to a significant 51% decrease in FEC at week 15 (p<,036). this As such, vaccination with recombinant Fhcat2 protein was performed to F. Its effectiveness is very similar to that of Tenyou catepnon protease.

Example 4 1 Data on the number of eggs in feces (eggs per fecal duram) 2 ND, not measured; P> 0.05 Table 5 Data on the number of eggs in the stool of 11 animals (eggs per fecal duram) 2ND, not measured: P> 0.05 14 and 15 weeks post-infection from vaccinated animals as shown on page 39. Summary of Fecal Egg Counts from Recombinant Vaccine Trials in Weeks Example 5 Cloning of catebunon B protease from larval F. hepatica F. hepatica Mosquito larvae were excysted in vitro (Carmona et al., 1993) and Ultraspeck Manufacturer (Biotyphoid Laboratories, Hyuston, Texas) Total RNA was extracted as recommended by. Oligo-dT using MMLV reverse transcriptase cDNA second strand synthesis was carried out using ). Catepun B cDNA was amplified using the N-terminal amino acid sequence shown in Example 4. (SEQ ID NO: 21) using oligonucleotide and oligo dT. carried out. The amplified cathepsin B cDNA was transferred to pCR-script SK (+ ) Cloning kit (Stratagene, Ujola, USA) It has become an online version. One clone (FhcatBl) was obtained. Isolate plasmid DNA Then, the DNA sequence was determined as described on page 30 above. DNA translation and prediction Amino acid sequence alignments were performed using Starden software and Geetsbyte (Gen The sequence was carried out using the sequence from the bank).

As shown in Figure 12, the FhcatB1 sequence is a member of the catebunon B family. Polypeptides showing high similarity to rotease are predicted. In particular, human The cateb/in B sequence from S. and S. mansoii is similar to FhcatBl.

FhcatBl is directed against the human and F, hepatic force-derived catebun L sequences. indicates lower similarity.

Those skilled in the art will appreciate that variations and modifications to the invention described herein will be apparent to those skilled in the art, unless otherwise indicated. You will understand that it can be decorated. The present invention covers all such changes and modifications. It should be understood that this includes Furthermore, the present invention is directed to Including all specified steps, features, compositions and compounds individually or collectively and any and all of any two or more of such steps or features. This includes combinations.

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R.W. Mason, J.E. Walker and F.D., Northguchi Tubu, 198. 6° N-terminal amino acid sequence of the heavy chain and U chain of human Thevenone L. mouse maklov cDNA clones of major catebunbroteases derived from the phagicel line. relationship to the tone. Biochemical Journal, Vol. 240, pp. 373-377.

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Sequence list (1) General information: (1) Applicant, Daratec Proprietary Limited Inventor, Milner; Panasothio, Spisothil and Vijufels (1, Name of invention: Faschio Polypeptides and vaccines obtainable from La species, treatment methods and their DNA sequences. Number of column (iji) arrays: 24 (iv) Contact information: (^) Recipient Davis Collison Cape (B) Street: Little Corinth Street, No. 1 (C) City, Melbourne (D) State of Victoria (E) Country: Australia (F)ZIP・3000 (V) Computer decoded format: (^) Media type, floppy disk (B) Computer: [BMPC compatible (C) operating system] System: PC-DO5/MS-DO3 (D) Software: Patentin Lily Base #1.0, version 11.25 (vi) This application data: (^) Application number: PCT-International (B) Application date: February 4, 1994 (vii) Priority application data: (A) Application number/AU PL7109 (B) Application mill + February 5, 1993 (ν1ii) Patent attorney/agent information: (^) Name, Hughes, E Jong L (C) case/reference number: EJH/E K (ix) Telephone contact information: (^) Telephone: (613) 254 2777 (B) Telefacsimile + (61 3) 254 2770 (2) Information on array number 1 (i) Characteristics of array: (^) Length: 1075 base pairs (B) type, nucleic acid (C) Number of strands: 1 strand (D) Topology: linear (11) Type of sequence: DNA (1x) Characteristics of array・ (^) Name/Symbol representing characteristics: CD5 (B) Location of existence 25. ．． 1002 (xl) Sequence, SEQ ID NO. 1: AAA 1075 (2) Information on sequence number 2 (i) Characteristics of array: (^) Length: 326 amino acids (B) type diamino acid (D) Topology: linear (ii) Type of sequence: protein (xl) Sequence: Sequence number 2: Squad ArB L@u Ph@ILe CLu Ale Vat L@u Th r　VaL　(:Ly　VaL　CLu　GLr　5at1　5　t.

Amn Asp Asp Lsu Trp Hts GLn Trp Lls ArB Mac Tyr Asn LN5 GLu τyr Asn, C; Ly ALa Asp Asp Gin His Aa Arl Asn Ile Trp CLu LysAsn ValLys HIS 11@C+Ln G Lu Has Asn L@u ArB His Asp Lsu (:Ly CLu Vat τ■u Tyr　Thr　L@u　GLy　Ldinu　Asn GLn　Ph@Thr Asp Met Dinghr Ph @ CLu (:tu Pl Lys ALa Lys Dingyr CLu Thr CLu Hat S@r ^;8 ALa Sat Asp mouth * Lay Sar’rhs CLy V aL l, ;OTyr CLu ALa Asn ↑sn ArB ALa V aL Pro Sag Lys Ha^spτ "p↑r! CLu Sat C Ly　Tyr　VaAτhr　to Lu　VaL　Lys　Asg　to iLn　C Ly^Waste Cys Gmi Sir Cys Trp^La 7:; Satτhr Thr　CLy　ThX　M@t　(Lu and ty CLn■ Mixed...Ly・A mn CL-Visual Thr Set CLn Sur Ph*%...CLu GL n　GLn　CLu1$3　160 VaIAsp Cys Smt (,:< Pro Trp CLy Amn Asn　GLy　Cys　GLy　(1<　CLu0°c　C1u　A sn AA8 Tyr Gin Tyr Lsu L, Ks Gin Ph* Gly　CLu　C4u　Thr　L■ 5ir Set Tgr Pro Dyr τh "ALa VIA CLu CLy (iLn Cys A5! Tyr Asn Ly■ GLn L@u Gly VaL ALa Lys Val Thr GLy Tyr　Tyr　Thr　VaL　His　S@r　CLuS@tCLuVaL C1uLauLysAsnL@uVaL に Ly^11^rg^rCustomer Pro^L1 ^L1225 2:10 23$ 240 VaL ALa VaL Asp VaL CLu S@r Asp Ph@H ag　M@Cτyr　Arg　S@r　CLK　It@2mu5 Tyr to ln Sat CLn Thr Cys S@r Pro L@u Arg VaL Asn His ＾1m VaL L・UALa VaL ( :Ly　Tyr　(:Ly　Thr　(:Ln　CL5　(:Ly　Thr　＾ $9 Tyr TiN n@ Vat kys Asn Sag Trp GLy Thr Tyr Tag GLy (:L u Arg Idly “g’ 11- Ar Dew Hat OLm %J ASn ArgoLyAsn Cys (,1y11°゛1°57; CLu　ALa　S°゛1°u　SrgM@t　VaL　ALa　Arg　Ph @Pr.

Ko25 (2) Information on sequence number 3 (i) Characteristics of array: (A) Length: 13 amino acids CB) type diamino acid (C) Chain number knee heavyweight (D) Topology/linear (ii) Type of sequence; protein (xi) Sequence: Sequence number 3: VaL Pro to Lu Asp 11-Asp Trp Arg CLy Tgr Tyr Tyr VaL (2) Information on sequence number 4 (i) Characteristics of array: (^) Length = 10 amino acids (B) type diamino acid (C) Number of knee heavy chains (D) Topology: linear (11) Type of sequence: protein (xi) Sequence, SEQ ID NO: 4: ALa VaL Pro Asp Lys ILa Asp Try Arc CLu(2) Information on sequence number 5 (i) Characteristics of array: (^) Length: 6 amino acids (B) type diamino acid (C) Number of chains knee heavy chain (D) Topology: linear (11) Type of sequence: protein (xi) Sequence: SEQ ID NO: 5; Ssr GLy Tyr VaL Thr CLu (2) Information on sequence number 6 (i) Characteristics of array: (^) Length: 8 amino acids (B) type/amino acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: protein (xl) Sequence/SEQ ID NO: 6; CLy Leu C1u Thr CLu S@r Ser Tyr (2) Array Number 7 information (1) Characteristics of array.

(B) type, amino acid (C) Number of chains, - heavy chain (D) Topology/linear (ii) Sequence species a: protein (i) Sequence: Sequence number 7: S@rS@tTyrProTyrThr^LaVaLC1u (2) SEQ ID NO: 8 information (i) Characteristics of array: (^) Length = 9 amino acids (B) type diamino acid (C) Number of chains knee heavy chain (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence: Sequence number 8: Thr　All　Vat　(Lu　CLy　CLn　Cys　ArB　Tyr( 2) Information on sequence number 9 (i) Characteristics of array: (^) Length: 9 amino acids (B) type diamino acid (C) Number of strands - single strand (D) Topology double layer (it) Sequence type: protein (xl) Sequence, SEQ ID NO. 9: CLy Thr to ln GLy C1y Thr Asp Tyr τrp ( 2) Information on sequence number 10 (】) Characteristics of arrays: (^) Length: 6 amino acids (B) type diamino acid (C) Number of IJI--heavy chain (D) Topology/linear (ii) Type of sequence, protein (xl) Sequence/Sequence number 10: ILaVre Lys Ain S@r Trp (2) Information on SEQ ID NO: 11 (i) Properties of sequences.

(^) Length: 1041 base pairs (B) type/nucleic acid (C) Number of chains, - heavy chain (D) Topology: linear (if) Sequence type: DNA (^) Name/Symbol representing characteristics: CD5 (B) Location: 8. ．． 985 (xi) Sequence/Sequence number 11: TGAATAATAA ATTTCACτCCCGAAAA 1061(2) Arrangement Information on column number 12 (^) Length: 326 amino acids (B) type diamino acid (D) Toboron: linear (ii) Sequence type/protein (xi) Sequence: Sequence number 12° Hsc＾rg Lau VaL rLa Lau Thr Leu Lau 1 1＠VJL Gly VaL Phs＾La s＠rAin Asp Amp La3 Trp His CLn Trp Lys Arg ILa Tyc Asn LNg GLu DingyrLys Gly Ala Asp Asp Asp Hf Arg Arg Asn ILa Dingrp GLu GLn A sn　VaLLysTosIl*(:LnCLuHisAsnL@u＾rg+4 isAspL@uC1yL*uVatThrSo 55 60 Tyc Lyz Lau to Ly La11 Asn Gin Phe Thr Asp? I@c dinghr f’ha GLu CLu oh@ Lys ALa Lys Tyr Leu Thr GLu Mac Pro ＾chi8　ALa　S@r　(:Lu　Lau　L;u　S@■ 811 C1y IL Stirrup Pr Tyr Lys ALa Asn Lgs＾rg ALa Val Pro↑sg Arg tf^sp Trp Ar (CLu S@r GLy Tyc Vi Thr CLu Vat Lys Asg CLn CLy to LyCys (, ly Sar Cys Dingrp ALa Ph@Sar Thr Thr GLy ALa Mac GLu Gly G1■ 130 1 piece 5 140 Tyr Met Lys Asn C1u Lys Thr Sar ILa Sar Ph@Ser GLu Gin Gln LeuVal^sp Cy s Ser GLy Pro Phe C1y Asn Tyr to Ly Cy *^sn　GLy　CLy　Lau8ReniLu　Asn↑Aa　Tyr　Glu Tyr Leu LNs＾rg Ph＠Gly Lau Yutaka Thr GLuS @r S@r Tyr Pro Tyr＾rg ALa VaL Glu GL y to Ln Cys Arg Tyr Asn GLulq5 200 205 (,Ln　L@u　GLy　VmL^La　Lys　Vat　Thr　GLy Tyrτyr Thr VaL His Met GLy210 215 22 G ＾sp　CLu　VaL　CLu　Lau　Gin　Asn　L@u　VaL GLy Cys Arg Arg Pro ALa ALa225 230 2 Go5 240 VsL Ala L@u Aip Vat (:Lu Ssr Asp l’h @Hog Mac Tyr＾rg Ser G:K IL*Tyr CLn S er　Can　Thr　Cys　S@r　Pro　M! *rg L@u As n His (:L2; Vat La■ Ala Vil ly Tyc ly IIs CLn Ass (:ly Ding hr Amp Tyr 几g ILa VaL Ly ＾sn Ser T rp (:Ly Thr Trp Trp CLy lu Asp ty Tyr　He　Ar1Fhaζ　VaL290　295　)0G 2g! Lys Arg C1y Asn Met Cys Gly IIs ^(a Sir Lau Ale Sat VaL P, rAg MaCVaL Ala Gin Ph@Pr.

(2) Information on sequence number 13 (1) Characteristics of array: (^) Length: 326 amino acids (B) type diamino acid (C) Number of chains, - heavy chain (D) Topology/linear (ii) Type of sequence: protein (xl) Sequence; SEQ ID NO: 13: Mec＾rg Liu Ph@He Lau Ala VaL Leu Thr VaL GLy Vat Lau GLy 5arAsn Asp Asp Lau Trp HLs GLn Trp IIs Arg Mac Tyc Ain Lgs GLu τyrAsn (:ly ALa Asp Asp Gin Hrs Arg Arg Asn rL eyelid τrp Glu Lys A sn　VaLLysH1slie(:LnClu)lisAsnL@uPraH isAspLeu to LyL@uVatdinghrτyr Thr Leu Gly Leu Ain (:Ln Phe T?+r Asp Metτhr Ph@ Glu　(Lu　g；Lys　ALa　Lys　Tyr　Lau　Thr　GL u Mat SIr Arl ALA SIr Asp ILs Lau 5i rHLI (:Ly VaL Proτyr GLu ALa Asn↑gn Arl^La Val Pro Asg Lys ILeAsp Trp↑r! CLu Sat (:Ly Tyr VaA The CLu VaL Ly s Aug GLn GLy ArnCys CLy SIr Cy Award Trp ALa Ph@Sirτhr Thr GLy Thr NeCC1u C1y G1nTyr Mac Lys Asn to Lu Ar3 Thr 5sr ILe Sor P, h,; Sir CLu GLn GLn p■■ VaL Asp Cys Sat? P Pro Trp GLy Asn↑s n　Gly　Cys　SIr　C1y　CL<　LuuMat　Glu　Asn ^[1　Tyr　Ln　Tyr　Leu　Lys　C1n　Ph@GLy Lau Glu Thr CLuS@r SIr T(, r f’ro Ty r Thr ALa VaA to Lu CLy GLn Cys Act Dingy r　Asn　L凾■ (:lnL@u にLyVJLALaLyiVatτhr(:1yTyrTyrT hrVaLOOfS@rC1ySer(:1uValC1uLsenLysAsnLa uVaLCLyALa＾rgArgPro＾La＾ [Chang VaL　ALa　riak Asp Vat CLu SIr Asp Ph Eyelid MC5Met Tyr ArB Sir ctr + LaTyr C1n Sur CLII Thr Cys Sir Pro Lau ArB VaL Asn)lis Alm 　VaL　LNext■ A1m Vat Gly Tyr (:Ly Thr CLo! nily T hr Asp Tyr Tr!　IIoVaL　Lye^sn　SEX　Trp CLy Thr Tyr Trp Ly CLu Arg GLy TIF , El＠＾r＠ Mac＾t1＾r＠ Asn ArB CLy Asn M at Cys Ly ILs Ala Ssr Lou Ala Ser L au Pr.

]G0 Ko10 315 Ko20 Met　VaL　^L1^r@　Phe　Pr.

Ko25 (2) Information on sequence number 14 (i) Characteristics of array: (^) Length: 333 amino acids (B) type/amino acid (C) Several chains, one by one (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence: SEQ ID NO: 14: Mat Asn Pro Thr Lsu ILe Lau Ala Ala P: *Cy〥〥　Lou　GLy　ILe　All La5erAla　Thr　La u Thr Ph @ Asp His Sar Lau to Lu Ala Gi n Trp Dhr Lys τ "pLys A(a Net I(is As n ArB Lou Tar cry Nsc Asn GLu Gju Ga y　Trp　Ar■ ＾rg ALm Vat Trp GLu Lys Asn Mat Lye Mat 1111 Glu Leu His Asn G1■ ni Lu Tyr Arg GLu (1111Lys HIs Sat Ph@ Thr Hat^la Mat ssn ALa Ph5 ly Asp Ms c Thr Ssr (L+a C1u Phs Ar GLn Val H at　Asn　GLy　Phs　G1■ Asn＾rg Ly Tsumugi Pro Arg Lys GLy Lys VaL Ph @CLn to Lu Pro Lau PheτyrGlu xla Pro A rB Set VaL Asp τ "p Ar Dew GLu Ly Surprise Gly T yr　VaL　Thr　Pr.

115 12G +25 VaL Lys Asn Gln C1y C1n Cys GLy Sir Cys Trp ALm Ph@S@r Ala Thr ly Ala Lo u GLu CLy CLn Mat Ph @ ArB Lye Dinghr CL y Arg Lau ILe 5ar @uSetGLuC; lnAsnLeu ValAspCymSer to LyPro(LnCLyAsnGLuC1yCy s Asn CLy CLy Lau Msc Asp Tyr Ala Ph @ Gin Tyr VaL in ^5p180 185 19G ^sn Gly Gly Leu Asp 5er Glu (lu SIr Tyr Pea Tyr clu^L1τhr CLuCLu SIr Cys Lys Tyr Asn Pro Lye Dyr SIr VaL Ala Asn Asp The GLyPh @ VaL^sp ILe Pro Ly s　GLn　CLu　Lys　ALa　Lau　Mat　Lys＾La　VaL ALa225 2] 0 235 240 Thr VaL Nily Pro Justs Mat VaL ALa fl@A sg ALa Gly His GLu Sir Ph*Leu Ph@Tyr Lys GLu C1y Its Tyr Ph@niLu Pro Asp Cys Smt Sir CLu^sp de ec Sales HLs GLy VaL L su】VaL　CLyτyr　GLy　Ph:　Glu　SatτhrGLu Ser Asp Asn Asn Lyt Dingyr Trp Leu Vat Lys Asn Sir Dingrp Gty GLu290295ko00 CLu260 CLy MC (Ly (Ly Tyr Vat Lys Mac ^Lm Lys Asp Arg^r1^＄nko05 Go10 Ko15 320 'rhs Cys ni Ly ti@ALa S@r^Lm ALa Sur 搗 Pro Thr VaLCLu^La VaL ALa Leu Tyr^sn Pro VaL Sat Ph @ ALa Ph GLu VaL Thr (: Ln　Asp　Ph@Met　M@Cτyr＾rg　Thr　GAN　ILa　 Tyr Sat sar Band Sir Cys His Lys Thr Pro Asp Lys VaL Asn +4is ALa VaL Lau ALa VaL GLy Ty■ 275 2g0 285 CLy Glu Lyt Asn CLy 11 @ Pro Tyeτrp I lm VaL Ly Asn Sir Trp CLyPro CLn Trp C1y Met Asn cry Tyr Ph@Lau H@GLu A+IB GLy Lys A snow n3Q5 310 315 Ko20 M@CCys GLy Lau ALa ALa Cys Ala Sir T N5 Pro Its Pro Lau Va: 25 (11) Type of sequence: protein Mac Dingrp Gin Lau Dingrp All Sar Lau Cys Cys Liu Lau VaL Lau ALa A1n1 5 to 15 ^1a　Arg　Sir^rg　Pro　Ser　Phe　Pra　V al Ser Asp (: Lu Lau VaL AsnTyr VaL A sn Lys Arg Asn Thr Thr Trp to Ln ALa C Ly　14is　Asn　Phe　Ty■ Ain Vil AGP Met 5er Tyr Leu Lyi ArB Lsu Cys Ly Dinghr Phe Lau GLyC1y Pro L ys Pro Pro Gin Arg ViL Met Ph@Thr CL u Asp Lau Lys L@uPro Ala Ser Phe Asp ALa At Ward C1u (:ln Trp Pra in Cy Dining Pro Thr　IlmLys　(、Lu　ILa　AHA　Asp　Ln　ly　 Ser　Cgs　cLy　Ser　Cys　Trp↑P　Ph@GLyAll　 VaL　GLu　ALa　ILa　S@rAsp　Ar81LkaiCys　rLMoth Him Thr Asn ALa H45Vat Sar Val (:Lu VaL　S＠r＾14　CLu　Asp　Lau　Lsu　Thr　Cys　C ys GLy 5ar130 135 L4G M@c Cys Gly Asp CLy Cys Asn GLy CLy Tyr　Pro　ALa　GLu　ALa 工rp＾5nPh@Trp Thr Arg Lys 1. y Lau Vat Sar Gig cry La u Dingyr ni Lu Ss: HL■ νml CLy Cys↑’g Proτyr Met Its Pro Pr o Cys Glu His＾easy VaL AsnCLy Ser Ina Pro P ro Cys Thr C1u (Ly Asp Thr Pro Lys Cys 5irLys IlmCys CLu Pro GLy Tyr Sa r Pro Thr Tyr Ly Gin Asp Lys HLsTyr To Ly Tyr Asn Sat TSS Sar VaL Sir ALa Sir GLu Lys Amp Ilm Kai ALa to Lu ILe Tyr L, ys^sn GLy Pro VaL GLu GLy Ala Ph@ S@r　VmLτyrSir　Asp　Phe　Leu　Lau　Tyr　Ly s Sur GLy VaL Tyr Gin His Val Thr GL y Lu Mac Met CLy GLy His ALa Ilm ArB H@Lau GLy”g CLy Vat (Lu275 28G Asn　g　Thr　Pro　Tyr　Trp　L;u　VaL　ALa　A sn Ser TXjE xsn Thr Asp TrprAy ASP A sn　CLy　PhkaiPh＊　Lys　Els　Lau　Arg　ct7　GL n^sp His Cys Gly305 Goto 3L5 320 fl@GLu Sir GLu Va [VaL ALm ni Ly rL@Pro Arg Thr Asp (:Ln Tyr Trp (2) Information of SEQ ID NO. 18 Information (A) Length, 340 amino acids (C) Number of chains, - heavy chain (D) Topology linear (11) Sequence type, protein (xl) Sequence: Sequence number 18゜ Mac Lau Thr Sar Els Lau Cys Its ALa Sar　Lau　ILa　Thr　Phs　Leu　GLul　5　10　15 ALm　His　IlmSer　VaL　Lys　Asn　(:Lu　Lys　 Phs GLu Pro Lau Sar Asp^k ILe rle Ssr Tyr Ilm Asn ni) Iis Pro Asn ALa (L Lu to yτrp Arg^Lm ys Ser Agn ArB Ph@H Is! +er　Lau　Asp　Asp　Ala　ArB　ILs　(:Ln 　Met　Cky ^LM Arg^rg CLu to lu Pro Asp Lsu Arg A rIIts Arg＾rg Pro Thrχ3L＾sp 14is Asn Asp Trp Asn Vat (:Lu Ilm Pro Sur^sn Phe ASP Sir^rgLys Lys Trp Pro GLy Cy s　Lys　! +@r I-Zan P1 Otori Dinghr Ila ArB ↑sg (:L n Sir＾rg Cys C1y Sar Cys Trp Sur Ph@ GLy ALa VaL GLu ALa Mac S・c AspArg S ar　Cys　Ila　GLn　Sar　C1y　GLy　Lys　CLn　A sn Company V GLu Lau Sar ALaVat Asp Leu L*uτ hr Cys Cys CLu Sar Cys Gly Lau CLy C ys CLu Gly (:ly IlmLau Gly Pro Ala Tr p Asp Tyr Trg VaL Lys CLu C1y ILa Vm LThr ALa Sar Sir Lys GLu Asn His Dhr GLy Cys Glu Pro Tyr Pro Ph・Pro Lys Cys C1u His His Thr Lys Gly Lys Tyr Pro Pro Cys GLy 5srLys Ilm Dingyr Asn T hr Pro ArB Cys Lys Gin Ding hr Cys GLn A rg Lys TyrLys Thr Pro Tyr Thr (:Ln A sp　Lys　His　Arg　(:ly　Lys　Sar　Sar　dyr A Tang ■ 225 230 235 2'1O VaL Lys Agn Asp 01u Lys All [1@Gin Lys GLu ry@H@e Lys Dingyr CLyPro Val G lu Ala Ser Phe Thr VaL Tyr to lu Asp P he Lau Asn Tyr LysSer CLy Its Tyr Ly s Hts He Thr GLy GLu ALa Lau Gly GLy Hf Ala+L* Arg 11 @ 11 @ ly Trp Gly V aL CLu Asn Lys Thr Pro Dingyr Trp Lau11 e Ala Asn Ser Trp Asn CLu Asp Trp GL y　(:Lu　Asn　GLy　Tyr　Ph＠　＾r■ 3Q5 310 315 320 1ie　VaL　ArB　C1y　Ar! Amp GLu Cys Ser Ilm Lu Ser GLu VaL rL@^La (2) SEQ ID NO: 19 information (A) Length: 29 base pairs (D) Topolon-0 linear (ii) Type of sequence: DNA (D) Topology @Fate shape (11) Type of sequence: protein (xl) Sequence, SEQ ID NO: 21: L@u Pro Glu 5*r Phs Asp ALa ＾tz Gin( 2) Information on sequence number 22 (i) Characteristics of array (A) Length: 9 amino acids (B) type diamino acid (C) Number of chains knee heavy chain (D) Topology, linear (11) Type of sequence: protein (xi) Sequence: Sequence number 22. VaL Pre^1j5ae Phe Asp Ala Arg CLn (2) Information on array number 23 (1) Characteristics of array: (^) Length, 11 amino acids (B) type/amino acid (C) Number of chains, - heavy chain (D) Topology, vertical shape (11) Type of sequence: peptide (xi) Sequence: Sequence number 23゜ (2) Information on sequence number 24 (]) Characteristics of arrays: (A) Length: 235 amino acids (B) type; amino acid (C) Number of chains knee heavy chain (D) Topology: lI chain (11) Types of sequences/proteins (xl) Sequence: Sequence number 24゜ Arg Sur cln Dingrp Pea CLn Cyz Trp Tnr Ha　S@t　CLu　11-　Arg　Asp　G1nALa　Sur　Cy s　GLy　s@r　Cys　Trp　Ala　Ala　Gly　Gly　Th r　5ar　Ala　Mac　5ur20　25　ko0 Asp Arg V=l Cyl Its HLI S@r A5n cry (:Ln Mac＾rg story 6＾rg Lsu Pr.

Ala ALa Asp Pro L@u S@r Cys Fallen Xaa Xaa Xaa Cgs Gly Gin CLy Cysa55 ::z Val C1y Tyr HNs 7 ALa Xaa Trp As p TKr Trp Xaa Xaa Xaa X5aXaaXaaXaaXx aXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXa aXaa Xaa Xaa Xam Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Thr Pro Pro Cytloo 1051 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu GLu ALa Cys CLn Thr CLy Tyr AsnLys Ding yr GLu Glu Asp Lys Ph threat Tyr to Ly Asn S er　S@r　Tyr　Asn　l/a■ 1 office lady 35 140 C1y Asn Thr to Lu Herχaa Xaa Xaa Xaa I t@Mac Gin GLu IIs Lys AsnC1y Pro Va L GLu Vat Thr Ph@ALa Xaa fL@Ph@Gin A sp　Ph＠Rain VaL165　X7Q Tyr　Arg　Sir　GLy　IL@Tyr　His　His　Vat　A La Xaa GLy Lys Ph@rL@ni1y＾rg His↑:a V at At Iris Met HLI Intestine Trp GLy Val CLu Agn G Ly VaL Stone Tyr Trg L@u Squad ^La Asn Sir Trp ^sn to Lu GLu TJ to Ly GLu Asn GlyTyr Ph e Arg Mat VaL Ar3 ly Arg Asn Glu Th rr fraction cooo-cx)-6, L-6 + ≧ Fixed translation g Music sheet included copy Tomiro ta 3 1010 ゞ0σ BA’J 1. j’10 　\Size -″ ~ c+′I + Ri-f-dimension -1 (, AU C+<-1, 8 1ba 5. -LL13 V+3 'L/'lj: J to Q to - Blasphemy - U Ro =　og　＜＠　ﾃ〃　1 is　-Tomoe　““Snake　-ni　1=blasphemy-self=3-3-ko knee threshold bow Ko-yumi ~ Mi-yumi -3-Scolding-Meichiyun 1-mo,=-≦>'4-2Cj3. Chang to w c: + w w-cp L+ (C-Yo-Go-Self-Subject-People-Ichinomi-Teeth Pi-Sen-g--Go Ichigo Ichime -+5 z me! Illδ Snake −8− ni delicious ← Ro Ro Ku Ro ＜＜＜ ``i2 100 koku; 122g Town Council Maro: Tomimaru Yari♀ro 8 Shu ρgar-~He-1'F) -! Ma + + size - no - 1 - 1 - 1 - 1 - 1 - 1 - ≦ = Blasphemy - Snake - Painting - Minister - Self - Derogatory thighs - Minister - Minister - ← T-li a+-ri -〇->G-2-303x accumulation<8 (i, i>u Cjj, i J (w=C!5L,E-,2(!j,323'::303-Me, one, one, one, one, one, one, one, one, one, one sorrow) Ikomi jizu-■-Baichiji cloudy-9-te beduku C■　o　Oro　. the law of nature (-(Juj;) YO "blasphemy 1 -ni 2; 2 death:>3 VII, 3 L/'l blasphemy C8 く り く　- 　.　I-(←　o　ト=；、：　0、 3′:′3:! :5ゞ訃　(i:　C)3　)9　(j3　　　　03　 1 ~ number = story and scolding for a day = 丞 M-C2F-f%Jri C0 size-〇〇'jl+-+NL+10j? ? O'l On lj) ω -- ~ CD 9m wN-1 -ma -■ Ma [F] -■～　■　-m　-mm　-　-　-　-　-　-E　E5　,≦　E8　E8　E is Yo 8- = Kumi 8- Shuichi 1 gosu = Goichiki Ichiri and Ichiha Ichi. ---( ’=’;:. 508ωba 5kkuri-〇　--. ← ← し　■　I-Cj　<　w　(.　Q　←-Goichi 8=Low death-mi 1713.g -fl-i・Kakufuki-g=Lee Xun-Kiichibeichisozu=Mezuichimekaku-B-Jo-Me 3 o-ig zu cps-mili-1koda c&l-taichi-shiichiichi-(((its Wealth = VI IZ Q-:i: Engineering jN > HfN N H2Z Z mouth o.

O >> H >> :), jZCL1! Σ l l Ill 1〉cut ψ lot← 0- Sea ΣΣ 111 countries!蓼　←11−ΣΣZZUI＞＞＞%%gl+al+a% %IIIE-111Ct):ecdZZZ＞＞wrb’:sζ RororoφqΩ 11 嘗ψ11 20 Roni 〇〇 cut 0 mouths 0000ψ Sonokuni ψ cut dark bacteria fungus 00 -0 Seki-C (1): xa-E-E-E’ Caa: M”ENG CIJIJ3 >>>C bE-'+ GC5C5oz×===mouth >> 1-1 >> C:l >> pooO Lolo ku-ku! -ku (1:WZ 二菰 11111:It　小((〉nn　＞,a ><et>>QCJ<<< (C/l?Ql) l:l C3(3C5(!1c5 c5 g++z: exCl, OIO+>>> (41a, Cbl-1:>J + tu+＞＞＞-Shi 22 WO1-I cut ○O←be ψku bebe knee-><<1 11acIak:＞1-IHO＞＞＞JJ　＞C:)＜＞＞＞　工匡nee JJ　 SCb:5ens'sCd? Q, WOIOI CdCtlCtl (/14.d: >Hl-1>>>C3C5QQelC5田-←-Kozono〉〉〉H〉et c<a<a fossa 2s O 匡口>ψ×==2 Mountain--Mountain Mountain Mountain Bulky Works Work Work Dark Rozono Kouyami E->>>>> 00 (jC) 2Ct ml,) OQZZ QC5C3C 5C5C5 million O 　×Mantobe(、+1−1Σ−〇匡ヱ Yami Oguzonons〉Country 0111　”;sΣ< OO匡ΣbeCountry-mouth Darkness Darkness←←0(J(Jlt+＋＋＋＞＞＞WCdCt )(/IQ-111n　22!2:1j(jQトH←1嘗I　CountryCountry 4.2J　 C) I j J(J(J(nffltntF3flffiQωl■1 ΣΣ0Σ”: ta <)1h Rototo Fables 22 Compilation 4 (JCJIII +++-+JJ!>:)M QiO'O' Pebebe xxg to 1L:I ;;-C:: ≦ = Engineering =: Suga 1 嘗　-(=瓢：＜Ctツ１）Cd　：e：：：e（＋－）C/Icノ　） ::>1|1>>> Country 11+1Ltl+%≦! 〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉〉 1 suggestion""old ゛1・-〇"-N.

PC'IVAII 94100051 International search report 1-+-mtxmaN.

1’LTIAU 94++1101151rnr Pct, l5A12+01 c+w++m+a++++e cs seguchi 1hee+Xmy 19921 Continuation of ccopm front page (51) Int, C1,' Identification symbol Internal office reference number Cl2N 15109 ZNA (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , 0A (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN.

TD, TG), AT, AU, BB, BG, BR, BY.

CA, CH, CN, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, LK, LU, LV, MG, MN, MW, NL, No, NZ, PL, PT.

R○, RU, SD, SE, SK, UA, US, UZ, VFI (72) Inventor: Spitchle, Terrance William Australia, 3759 65 Main Road, Panton Hill, Victoria (72) Inventor Wijufels, Jean Lewis Australia, 4068 77 Billy Road, Indall, Taringa, Queensland

Claims (52)

[Claims] 1. A polypeptide comprising a sequence of amino acid residues, the sequence comprising: Groups of amino acid residues as connected sequences: Gln-Xaa-Xaa-Xaa-Xaa1-Xaa-Cys-Trp-Xaa -Xaa-Xaa2 In the formula, Xaa is any amino acid residue, Xaa1 is Gly or is Glu and X8a2 is Ser, Thr, Ala or Gly: furthermore, the peptide induces a host immune response against the helminth. An isolated polypeptide characterized in that it has the ability to 2. The isolated polypeptide of claim 1, wherein the polypeptide is a cathepsin protease. polypeptide. 3. 3. The isolated polypeptide of claim 1 or 2, wherein the helminth is a trematode. . 4. 4. The isolated polypep of claim 3, wherein the trematode is a species of Fasciola. Chido. 5. 5. The isolated trematode of claim 4, wherein the trematode is Fasciola hepatica. polypeptide. 6. Claim 4 or 5, wherein said polypeptide is derived from said Faschiola species. The isolated polypeptide described. 7. 7. The polypeptide according to claim 6, wherein said polypeptide is derived from a mature state of Faschiola sp. isolated polypeptides. 8. the polypeptide is derived from newly excysted larval stage Fasciola; 7. The isolated polypeptide of claim 6. 9. 2. The isolated polypeptide of claim 1, wherein said host is a mammal. 10. 10. The isolated polypeptide of claim 9, wherein said mammal is a livestock animal. 11. 11. An isolated pot according to claim 10, wherein the domestic animal is of the sheep or bovine species. Ripeptide. 12. 12. The isolated immune response of claim 1 or 11, wherein the immune response is a protective immune response. polypeptide. 13. 13. The isolated polypeptide of claim 12, wherein said immune response is a humoral immune response. peptide. 14. the polypeptide substantially comprises the amino acid sequence disclosed in SEQ ID NO: 2; 3. The isolated polypeptide of claim 2, consisting of. 15. the polypeptide substantially comprises the amino acid sequence disclosed in SEQ ID NO: 12; 3. The isolated polypeptide of claim 2, consisting of. 16. The polypeptide has an N-terminal sequence substantially as disclosed in SEQ ID NOs: 21 and 22. 3. The isolated polypeptide of claim 2, comprising a sequence. 17. Recombinant, isolated polypeptide. 18. Antigenicity of the polypeptide according to claim 1 or 14 or 15 or 16 Fragment, portion, derivative or analog. 19. (i) is a cathepsin protease or cathepsin protease-like molecule; the law of nature: (ii) Concatenated amino acid sequence Gln-Xaa-Xaa-Xaa-Xaa1-X Contains an amino acid sequence containing aa-Cys-Trp-Xaa-Xaa-Xaa2 consisting of: (iii) isolable from helminths; and (iv) substantially consisting of SEQ ID NO. No. 2 or the entire amino acid sequence disclosed in SEQ ID No. 12 or SEQ ID No. 24. an amino acid sequence having at least 50% amino acid sequence homology to or to a part thereof; A polypeptide comprising a sequence. 20. 20. The isolated polypeptide of claim 19, wherein the helminth is a trematode. 21. 21. The isolated polype of claim 20, wherein the trematode is a species of Fasciola. Petite. 22. 21. The isolated trematode of claim 20, wherein the trematode is Fasciola hepatica. polypeptide. 23. 21. The isolated trematode of claim 20, wherein the trematode is Fasciola gigantica. polypeptide. 24. 23. The isolated polype of claim 22, wherein Fasciola sp. is in a mature state. Petite. 25. The polypeptide is derived from the newly excysted larval stage of Fasciora 23. The isolated polypeptide of claim 22. 26. Furthermore, it is characterized by being able to induce an immune response in the host. , the isolated polypeptide of claim 19. 27. 27. The isolated polypeptide of claim 26, wherein said host is a mammal. 28. 28. The isolated polypeptide of claim 27, wherein said mammal is a livestock animal. 29. 29. The isolated poultry plant of claim 28, wherein the domestic animal is of the sheep or bovine species. Ripeptide. 30. 30. The unit of claim 29, wherein the immune response is a protective immune response against helminth infection. Separated polypeptide. 31. An isolated or is a recombinant polypeptide. 32. having substantially the amino acid sequence disclosed in SEQ ID NO: 12 or SEQ ID NO: 24. An isolated or recombinant polypeptide. 33. substantially the N-terminal amino acid sequence disclosed in SEQ ID NO: 21 or SEQ ID NO: 22. An isolated or recombinant polypeptide having a sequence. 34. (i) encodes a cathepsin protease; (ii) is isolated from helminth species; and (iii) the nucleic acid sequence disclosed in SEQ ID NO: 1 or 11. hybridize under low stringency conditions with all or part of the sequences or their complementary forms. A nucleic acid molecule comprising a sequence of nucleotides that contains 35. 35. The nucleic acid molecule according to claim 34, wherein the helminth is a trematode. 36. 36. The nucleic acid molecule of claim 35, wherein the trematode is a Fasciola species. 37. 37. The Fasciola species is Fasciola hepatica. Nucleic acid molecule. 38. 37. The nucleic acid molecule of claim 36, wherein the Faschiola sp. is in a mature state. 39. 37. The nucleus of claim 36, wherein the Fasciola sp. is in a newly excysted larval stage. acid molecule. 40. A nucleic acid molecule having a sequence of nucleotides substantially as disclosed in Sequence No. 1. 41. A nucleic acid molecule having a sequence of nucleotides substantially as disclosed in SEQ ID NO: 11 . 42. The cathepsymph according to claim 1 or 19 is administered to an animal susceptible to infection with the parasite. an effective amount of a polypeptide comprising or derived from a rotease; sufficient time and conditions to develop an immune response against the cathepsin protease. A method of reducing the infestation of helminth parasites, the method comprising administering to a patient. 43. 44. The method of claim 43, wherein the helminth is a trematode. 44. 43. The method of claim 42, wherein the immune response is a protective immune response. 45. 44. The method of claim 43, wherein the Fasciola species are mature. 46. 44. A species according to claim 43, wherein the species of Phaschiola is in a newly excysted larval stage. Method. 47. 44. The method of claim 43, wherein the helminth is a species of Fasciola. 48. 48. The Phaschiola species of claim 47, wherein the Phaschiola species is Phaschiola hepatica. Method. 49. 43. The method of claim 42, wherein the host is a mammal. 50. 43. The method of claim 42, wherein the mammal is a livestock animal. 51. 51. The method of claim 50, wherein the livestock is of the sheep or bovine species. 52. A polypeptide according to claim 1 or 19 and one or more veterinarians. A vaccine comprising an acceptable carrier and/or diluent for medical use. Composition.