Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections

The present invention relates to the preparation of protective antigens by recombinant DNA technology for use as anthel intic agents and as protective immunogens in the control of diseases caused by helminth parasites.

Helminth parasites are responsible for a wide range of diseases and infestations of domestic animals which, leading as they do to loss of production and even animal mortality, are of considerable economic importance. Thus for example, the blood feeding ne atode Haemonchus infects the lining of the gastrointestinal tract of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated. Other genera of helminths of economic importance include Trichostrongylus and Nematodirus which cause enteritis in various animals, and trematodes.

Problems are also caused by nematodes such as hookworms (eg. Necator. Ancylostoma, Uncinaria and Bunostomum spp) and flukes (eg. Fasciola, Paramphistomum and Dicrocoelium) and their relatives which in addition to ruminants and domestic pets, also infect humans, frequently with fatal results.

Control of helminth parasites presently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques have a number of drawbacks however - frequent administration of drugs and pasture management are often not practical, and drug- resistant helminth strains are becoming increasingly widespread .

There is therefore a need in this field for an effective anti-helminth vaccine and many efforts have been concentrated in this area in recent years. However, as yet there are no commercially available molecular or sub-unit vaccines for the major helminth species, particularly for the gastrointestinal nematodes of ruminants, such as Haemonchus and Ostertagia.

Most promising results to date have been obtained with novel proteins isolated from Haemonchus, which have potential as protective antigens not only against Haemonchus but also against a range of other helminths. In particular the protein doublet H110D, found at the luminal surface of the intestine of H.contortus has been shown to confer protective immunity against haemonchosis in sheep.

H110D from H.contortus has an approximate molecular weight of 110 kilodaltons (kd) under reducing and non- reducing conditions, as defined by SDS-PAGE, and is described in W088/00835 and 090/11086. The term "HllOD" as used herein refers to the protein doublet H110D as defined in W088/00835 and W090/11086. Corresponding proteins have also recently been shown in other helminth species, eg. Necator americanus.

A number of methods for the purification of HllOD have been described in W088/00835 which suffice for the characterisation of the protein, and may be scaled up to permit production of the protein in experimentally and commercially useful quantities. There is however a need for an improved and convenient source from which to prepare not only HllOD but also related antigenic proteins, especially for a process based on recombinant DNA technology and expression of the proteins in suitably transformed prokaryotic or eukaryotic organisms.

The present invention seeks to provide such an improved procedure. Sequence determination of cDNAs for HllOD from Haemonchus contortus has been performed and

the predicted amino acid sequences have been found to display homology with a family of integral membrane aminopeptidases (systematic name: -amino acyl peptide hydrolase (microsomal) ) .

The mammalian integral membrane aminopeptidases are located in several tissues, eg. on the icrovillar brush border of intestines, and kidney. Their role in the kidney is unclear, but in the intestine their function is to cleave the small peptides which are the final products of digestion (for reviews, see Kenny & Maroux,1982; Kenny & Turner, 1987; Noren et al , 1986; Semenza, 1986) .

In one aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences which encode helminth aminopeptidase enzymes or antigenic portions thereof substantially corresponding to all or a portion of the nucleotide sequences as shown in Figures 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or sequences coding for helminth aminopeptidase enzymes which are substantially homologous with or which hybridise with any of said sequences.

A nucleic acid according to the invention may thus be single or double stranded DNA, cDNA or RNA.

Variations in the aminopeptidase-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages) , between similar strains of different geographical origin, and also within the same helminth. Such variations are included within the scope of this invention.

"Substantially homologous" as used herein includes those sequences having a sequence identity of approximately 50% or more, eg. 60% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By

"functionally equivalent" is meant nucleic acid sequences which encode polypeptides having aminopeptidase activities which are similarly immunoreactive ie. which raise host protective antibodies against helminths.

Nucleic acid molecules which hybridise with the sequences shown in Figures 2 , 3, 4 or 5 (composed of SEQ ID NOS: 1 to 15) or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. "Hybridisation" as used herein defines those sequences binding under non-stringent conditions (6 x SSC/50% formamide at room temperature) and washed under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SCC, 42°C) or conditions of higher stringency eg. 2 x SSC, 65°C (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2).

Methods for producing such derivative related sequences, for example by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modi ied nucleic acid has significant homology to the subject sequence, for example by hybridisation.

Provision of a nucleic acid molecule according to the invention thus enables recombinant aminopeptidase enzymes, or immunogenic fragments thereof, to be obtained in quantities heretofore unavailable, thereby permitting the development of anti-helminth vaccines.

In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides capable of raising protective antibodies against helminth parasites, which sequences incorporate one or more antigenic determinant-encoding regions from the aminopeptidase-encoding sequences as shown in Figures 2, 3, 4 or 5 (composed from SEQ ID NOS: 1 to 15) .

The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting an aminopeptidase enzyme or antigenic portions thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in Figure 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15), or a functionally- equivalent variant thereof other than a synthetic polypeptide corresponding to the protein doublet HllOD, or a synthetic polypeptide corresponding to any of the individual polypeptide sequences disclosed in WO90/11086.

Alternatively viewed, the invention also provides synthetic polypeptides comprising an amino acid sequence constituting an aminopeptidase enzyme or an antigenic portion thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in Figure 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or a functionally- equivalent variant thereof, substantially free from other Haemonchus contortus components.

The invention further extends to vaccine compositions for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic polypeptide as defined above, together with a pharmaceutically acceptable carrier. 090/11086 discloses a number of polypeptide or partial polypeptide sequences obtained by proteolytic digestion or chemical cleavage of the protein doublet HllOD as follows:

(a) Met Gly Tyr Pro Val Val Lys Val Glu Glu

Phe

-,

(b) Met Gly Phe Pro Val Leu Thr Val Glu Ser

(c) Met Gly/Phe Asn Phe Lys He Glu/Val Thr/Glu Ala Gly

(d) Met Lys Pro/Glu Thr/Val Lys Asp/Ala Thr/Lys Leu - He Thr

(e Met Leu Ala Leu Asp Tyr His Ser - Phe Val (f. Met Leu Ala Glu/Tyr Asp Gin/Ala Glu Asp Val

(g Met Gly Phe Pro Leu Val Thr Val Glu Ala Phe Tyr

(h Met Lys Thr Pro Glu Phe Ala Val/Leu Gin Ala Phe/Thr Ala Thr Ser/Gly Phe Pro (i Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/Leu Leu Asn/Gly

(j Lys - Thr Ser Val Ala Glu Ala Phe Asn (k Lys Ala Ala Glu Val Ala Glu Ala Phe Asp - He - Lys Gly

(1 Lys Ala Val Glu Val/Pro Ala Glu Ala Phe Asp Asp He Thr? Tyr - - Gly Pro Ser

( Lys - Glu Glu Thr Glu He Phe Asn Met (n Lys - - - Pro Phe Asn/Asp He Glu Ala

Leu

(o Asp Gin Ala Phe Ser Thr Asp Ala Lys

CP Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe -Ala Thr Ala Leu

(q Met Gly Phe Pro Val Leu Thr Val Glu Ser - Tyr? - Thr

(r Met Glu/Phe Asn Phe Leu He Glu/Val Thr/Glu Ala Gly - He Thr

(s Met Gly Phe Leu Val Thr Val Glu Ala Phe Tyr - Thr Ser

(t Met Lys Thr Pro Glu Phe Ala Val/Leu Gin Ala Phe/Thr Ala Thr Ser/Gly Phe Pro

(u Met Lys Pro/Glu Thr/Val Leu Asp/Ala Thr/Lys Leu - He Thr - Gly

(v Met Leu Ala Leu Asp Tyr His Ser - Phe Val Gly?

(w Met Leu Ala Glu/Tyr Asp Gin/Ala Glu Asp Val ⁽ x Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/Leu Leu Asn/Gly

⁽ y Lys - Thr Ser Val Ala Glu Ala Phe Asn

(z Lys Ala Ala Glu Val Ala Glu Ala Phe Asp - He - Lys Gly

Glu Val/Pro Ala Glu Ala Phe Thr? Tyr - - Gly Pro Ser Gin Thr Glu He Phe Asn Met Pro Phe Asn/Asp He Glu

(dd) Asp Gin Ala Phe Ser Thr Asp Ala Lys

Uncertainties are shown either by the form Phe/Gly, where the first three letter code represents the most likely correct amino acid based on the strength of the signal, or by a question mark; a sign "-" means an unknown residue.

The specific individual polypeptide sequences which are disclosed in WO09/11086 are disclaimed.

The term "polypeptide" as used herein includes both full length protein, and shorter peptide sequences.

"Functionally equivalent" as used above in relation to the polypeptide amino acid sequences defines polypeptides related to or derived from the above- mentioned polypeptide sequences where the amino acid sequence has been modified by single or multiple amino acid substitution, addition or deletion, and also sequences where the amino acids have been chemically modified, including by glycosylation or deglycosylation, but which nonetheless retain protective antigenic (immunogenic) activity. Such functionally-equivalent variants may occur as natural biological variations or may be prepared using known techniques, for example functionally equivalent recombinant polypeptides may be prepared using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.

Generally, the synthetic polypeptides according to the invention represent protective antigenic sequences. The term "protective antigen" as used herein defines those antigens capable of generating a host-protective (immunogenic) immune response ie. a response by the host

which leads to the generation of immune effector molecules, antibodies or cells which sterilise the fecundity of, damage, inhibit or kill the parasite and thereby "protect" the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.

The synthetic polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly described above operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Alternatively the polypeptides may be expressed by direct injection of a naked DNA molecule according to the invention into a host cell.

The synthetic polypeptide so expressed may be a fusion polypeptide comprising a portion displaying the immunogenicity of all or a portion of an aminopeptidase enzyme and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. For example, it may be desirable to produce a fusion protein comprising a synthetic aminopeptidase or other polypeptide according to the invention coupled to a protein such as /3-galactosidase, phosphatase, glutathione-S-transferase, urease, hepatitis B core antigen (Francis et al. , 1989) and the like. Most fusion proteins are formed by expression of a recombinant gene in which two coding sequences have been joined together with reading frames in phase. Alternatively, polypeptides can be linked in vitro by chemical means. All such fusion or hybrid derivatives of aminopeptidase-encoding nucleic acid molecules and their respective amino acid sequences are encompassed by

the present invention. Such suitable recombinant DNA and polypeptide expression techniques are described for example in Sambrook et al.. 1989. Alternatively, the synthetic polypeptides may be produced by chemical means, such as the well-known Merrifield solid phase synthesis procedure.

Further aspects of the invention include use of a nucleic acid molecule or a synthetic peptide or polypeptide as defined above, for the preparation of a vaccine composition for stimulating immune responses in a human or non-human, preferably mammalian animal against helminth parasite infections.

Alternatively viewed, the invention also provides a method of stimulating an immune response in a human or non-human, preferably mammalian, animal against a helminth parasite infection comprising administering to said animal a vaccine composition comprising one or more polypeptides encoded by a nucleotide sequence as defined above.

A vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture. Traditional vaccine formulations may comprise one or more synthetic polypeptides according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, QuilA, or more purified forms thereof, muramyl dipeptide, mineral oils, or Novasomes, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into a patient. Additional components such as preservatives may be included.

An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia virus, adenovirus, Salmonella) having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to

this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.

Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection, optionally at intervals eg. two injections at a 7-28 day interval.

As mentioned above, the amino acid translation of the nucleotide sequences depicted in Figures 2, 3, 4 or 5 show sequence homology with a family of integral membrane aminopeptidase enzymes. This was determined by searching various databases available in the Genetics Computer Group Sequence analysis software package, version 7.01, November 1991 (Devereux et al.. (1984)), using translations of the sequences shown in Figures 2, 3, 4 or 5. Two such comparisons are shown in Figure 6.

Expression of the aminopeptidase-encoding sequences according to the invention can, as mentioned above, be achieved using a range of known techniques and expression systems, including expression in prokaryotic cells such as E.coli and in eukaryotic cells such as yeasts or the baculovirus-insect cell system or transformed mammalian cells and in transgenic animals and plants. Particularly advantageously, the nucleotide sequences may be expressed using the transgenic nematode system such as the system for the nematode Caenorhabditis described for example in Fire, (1986) ; Fire et al.. (1989) ; Spieth et al.. (1988) ; Han et al. , (1990) .

A further aspect of the invention provides a method for preparing a synthetic polypeptide as defined above, which comprises culturing a eukaryotic or prokaryotic cell containing a nucleic acid molecule as defined above, under conditions whereby said polypeptide is expressed, and recovering said polypeptide thus produced„

Further aspects of the invention thus include cloning and expression vectors containing nucleotide sequences according to the invention. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codes) and transcriptional control elements (eg. promoter-operator regions, ribosomal binding sites, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.

Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include, as mentioned above, baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art.

A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature.

Transformed or transfected eukaryotic or prokaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above, form a further aspect of the invention.

Eukaryotic expression systems in general, and the nematode expression system in particular, have the advantage that post-translational processing, and particularly glycosylation can occur - in the case of the transgenic nematode system, a glycosylation corresponding to that found in the native protein may be expected. This represents an important aspect of the invention, since in many cases post-translational

processing is required for the recombinant protein to express optimum biological activity.

Mammalian cell expression systems, also have a number of advantages. Mammalian host cells provide good reproduction of the native form and protective epitopes of the antigen since a eukaryotic expression system will give rise to more similar glycosylation patterns, disulphide bonding and other post-translational modifications than E.coli which may produce an insoluble protein requiring refolding and having poor reproduction of the native form. In addition mammalian glycosylation is unlikely to induce an immune response which distracts from a protective anti-protein response. For protection of humans and domestic animals, it is thus preferable to use human or animal fibroblast or myeloma cell lines such as HeLa - a human cell line; BHK - baby hamster kidney cells; VERO, a monkey kidney cell line; FR3T3 , Fisher rat fibroblasts; NIH3T3, a mouse fibroblast cell line; C127I, a mouse mammary tumour cell line; CV-1, African green monkey kidney fibroblasts; 3T6, mouse embryo fibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster Ovary cell line; NSO NSI, SP2 and other mouse myeloma cell lines and rat myeloma cell lines such as YB2/0 and Y3.

Vectors appropriate for different classes of mammalian cell lines are well known in the art. In general, these will comprise a promoter and/or enhancer operably connected to a nucleotide sequence encoding the antigen or fragment thereof. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mouse metallothionein I promoter and mouse mammary tumour virus long terminal repeat. The vector preferably includes a suitable marker such as a gene for dihydrofolate reductase or glutamine synthetase. Vectors of those types are described in W086/05807, W087/04462, W089/01036 and W089/10404.

Transfection of the host cells may be effected using standard techniques, for example using calcium phosphate, DEAE dextran, polybrene, protoplast fusion, liposomes, direct microinjection, gene cannon or electroporation. The latter technique is preferred and methods of transfection of mammalian cell lines using electroporation are described by Andreason et al .. 1980. In general, linear DNA is introduced more readily than circular DNA.

In the case of the protein HllOD, it has been found to have a unique and unusual glycosylation pattern, which is thought to contribute to immunoactivity since many monoclonal antibodies so far obtained to HllOD from Haemonchus recognise carbohydrate epitopes which may be of importance in developing useful vaccines.

In particular the following glycosylation pattern for HllOD from Haemonchus has been demonstrated:

i. about 65% of oligosaccharides are N-linked, the remainder 0-1inked; ii. the major part (eg. about 48%) of the N-linked oligosaccharide is of the complex class; iii. substantially all (eg. greater than 95%) of the oligosaccharides are uncharged; iv. the relative molar content of the constituent monosaccharides is N-acetylgalactosamine 1.0, fucose 3.6, galactose 4.1, glucose 4.4, mannose 6.2 and N-acetylglucosamine 5.2; v. the oligosaccharides, other than the major oligosaccharide (designated oligosaccharide D) , are substantially resistant to degradation by a broad range of exo-glycosidases (eg. α-D-mannosidase, β- D-mannosidase, /3-D-glucosidase, /3-D-galactosidase, α-D-galactosidase, α-L-fucosidase, ?-D-xylosidase, /3-D-N-acetylglucosaminidase) .

Such oligosaccharides and glycoproteins containing them form a further aspect of this invention.

Oligosaccharide D of the Haemonchus HllOD glycoprotein is of the N-linked type and has a novel structure consisting of two fucose residues attached by an α-1,3 linkage and an α-1,2 linkage to a mannose (N- acetylglucosamine) ₂ core.

Another aspect of the invention thus provides an oligosaccharide having the structure:

Man 1

and more particularly the structure:

Fuc ol

αl Fuc

especially when linked to a protein, eg. a recombinant protein such as a helminth aminopeptidase protein or an antigenic fragment thereof, or when used to generate anti-idiotypic antigens for immunisation especially of very young animals.

Animal glycoproteins generally have fucose Q-1,6 linkages and the fucose α-1,3 linkage of the oligosaccharide of the present invention is an unusual feature.

This invention will now be described in more detail with particular reference to the protein HllOD from Haemonchus contortus. However, by a variety of techniques such as histochemistry and DNA hybridisation, HllOD equivalents have been observed in other parasite species. It is believed that the HllOD protein is a multigene complex and that in addition, the nucleotide sequences encoding it, may exhibit sequence variations between different strains and different life cycle stages of the helminth. Moreover there may exist multiple enzyme forms (isoenzymes) which may be differentially expressed at different stages, or in different strains. In this study DNA sequences, and thus the predicted amino acid sequences, have been determined from cDNA clones and PCR products obtained from mRNA corresponding to the HllOD gene by recombinant DNA technology from different sources, and at different parasitic stages of H. contortus life cycle.

Sequencing of cDNA and PCR products has enabled us to identify thre- '-losely related HllOD sequences which are here designa-. ^* -d Hll-1 (SEQ ID NO: 19) , Hll-2 (SEQ ID NO: 20) and Hll-3 (SEQ ID NO: 21) . Hll-1 comprises three contiguous and overlapping sequences, cDNA clone AustBl (SEQ ID NO: 6) , PCR product A-648 (SEQ ID NO: 9) and at the 3* end PCR product 014-178 (SEQ ID NO: 12) ; Hll-2 comprises the PCR products A-650 and 2.5kb (SEQ ID NOS: 10 and 7 respectively) ; Hll-3 comprises the PCR products 3.5kb and A-649 (SEQ ID NOS: 3 and 11 respectively) . The specific relationships between the individual sequenced cDNA and PCR product clones and Hll-1, -2 and -3 are summarised in Figure 1 and shown in detail in Figures 3, 4 and 5.

Differences and variations in the sequences obtained from the cDNA clones and PCR products have been observed, as can be seen in particular from Figures 2, 3, 4 and 5 (composed of SEQ ID NOS: 1 to 15 and 19 to 21) and as summarised in Table 1.

Table 1. Homologies of the deduced amino acid sequences obtained by translation of the nucleotide sequences shown in Figure 2.

% Similarity % Identity

77 63 79 65 82 69

The differences can be attributed to different mRNAs (of the multigene family) . In addition, the variations may be due, at least in part, to different variants of the HHOD-encoding sequence or mRNA present at different stages of the life cycle or in strains differing in geographical origin.

Table 2 additionally shows levels of identity and similarity between the corresponding predicted amino acid sequences and two published mammalian aminopeptidase sequences.

Table 2. Homologies of the HllOD amino acid sequences with rat aminopeptidase M (ApM) and mouse aminopeptidase A (ApA) .

% Similarity % Identity Hll-l : ApM 55 32

Hll-l : ApA 55 31

Hll-2 : ApM 52 31

Hll-2 : ApA 54 31

Hll-3 : ApM 53 32

Hll-3 : ApA 52 30

Figure 1 shows a map of the H.contortus HllOD cDNA and PCR product clones sequenced and their relationships and relative positions along the HllOD mRNA;

Figure 2 shows the HllOD nucleotide sequences designated Hll-3, (SEQ ID NO: 21, derived from cloned PCR products SEQ ID NOS: 8 and 11 and cDNA clone MIAUΞ, SEQ ID NO: 5), Hll-2 (SEQ ID NO: 20, derived from cloned

PCR products SEQ ID NOS: 7 and 10) and Hll-l (SEQ ID NO: 19, derived from cloned PCR products SEQ ID NOS: 9 and 12 and cDNA clone AustBl, SEQ ID NO: 6) ;

Figure 3 shows the sequence Hll-3 (SEQ ID NO: 21) (shown in Figure 2) with alignment of the cDNA clones Ml and M1AUS (SEQ ID NOS: 1 and 5) ;

Figure 4 shows the sequence Hll-2 (SEQ ID NO: 20, shown in Figure 2) and the alignment of the cDNA clone B2 (SEQ ID NO: 4) ;

Figure 5 shows the sequence designated Hll-l (SEQ ID NO: 19) and alignment of the cDNA B1A and Aust Bl (SEQ ID NOS: 2 and 6 respectively);

Figure 6 shows a) the predicted amino acid sequences (SEQ ID NOS: 22, 23 and 24) derived from the DNA sequences Hll-l, Hll-2 and Hll-3 shown in Figure 2; bi) and ii) show the predicted amino acid sequence of Hll-3 compared with the published amino acid sequences of rat icrosomal aminopeptidase M (Watt et al. , 1989) and mouse microsomal aminopeptidase A (Wu et al . , 1990) respectively; identities are enclosed in boxes, dashes indicate spaces introduced to maximise the level of homology between the compared sequences. The conventional single letter code for amino acids is used. The horizontal line above the sequence indicates the position of the trans embrane region and the asterisks show the position of the zinc-binding motif. Levels of similarity are shown in Tables 1 and 2;

Figure 7 Shows the alignments of amino acid sequences (designated Pep A, Pep B, Pep C, Pep D and Pep E) obtained from CNBr and Lys-C fragments of HllOD as previously described (International patent application WO90/11086 and as listed earlier, polypeptide sequences (a) , (b) , (e) , (k) and (aa) , respectively) and three new sequences (SEQ ID NOS: 16, 17 and 18) obtained from HllOD following digestion by elastase or ther olysin with the translations of a) Hll-l, b) Hll-2 and c) Hll-

In a further aspect the invention also provides nucleic acid molecules comprising one or more nucleotide sequences which substantially correspond to or which are substantially complementary to one or more sequences selected from the sequences of clones Ml, B1A, B1A-3 ' , B2, M1AUS, AustBl, 014-015 (2.5PCR), 014-872 (3.5PCR clone 2), A-648 (5 ¹ end of Bl) , A-650 (5' end of 2.5PCR), A-649 (5' end of 3.5PCR) , 014-178 (3 ' end of AustBl clone 2), 014-178 (3 * end of AustBl clones 3 & 6), 014-872 (3.5PCR clone 10) and 014-872 (3.5PCR clone 19), Hll-l, Hll-2 and Hll-3 , SEQ ID NOS: 1 to 15 and 19 to 21 respectively as shown in Figures 2, 3, 4, and 5 or sequences which are substantially homologous with or which hybridise with any of the said sequences.

As mentioned above, comparison of the sequences of various of the clones mentioned above, against computer databases of known sequences, reveals substantial homology with the family of microsomal aminopeptidase enzymes (EC. 3.4.11.-). Enzymological activity and inhibitor studies performed with the HllOD protein and sub-fractions thereof confirm that the protein is in fact microsomal aminopeptidase (α-amino acyl peptide hydrolase (microsomal) ) . Such studies have further shown that both aminopeptidase A-like and aminopeptidase M-like activities are exhibited, and that each of the components of the HllOD doublet individually exhibit enzyme activity.

Studies with proteolytic digestion of HllOD have also been carried out. Using the enzyme elastase, it was found that H11QD may be partially cleaved, forming two fractions, a detergent-soluble fraction (which remained with the membrane) and a water-soluble fraction (which is designated H11S) . H11Ξ occurs in the form of a protein dimer which may be reduced to two components. Interestingly, it was found that only aminopeptidase M- like activity is associated with the water-soluble H11S fraction, whereas aminopeptidase A-like activity is only associated with the detergent-soluble fraction.

The following Example provides a description of the studies leading to determination of the sequences shown in Figures 1 to 7 , with reference to the following additional Figures in which:

Figure 8 shows Western blots of integral membrane proteins present in a detergent extract of Haemonchus contortus adults probed with affinity purified antibodies eluted from potential HllOD clones; a) antigens in a detergent extract of Haemonchus recognised by antiserum to the extract; b) antibodies eluted from a strip such as that shown in a) re-tested against a blot of the detergent extract confirm the success of the elution step; c) antibodies as in b) which bind to clone Ml expressed protein strongly recognise a region at 110 kd (and a relatively sharp band at about 205kd; d) there is no antibody binding when a non-recombinant is used to adsorb the serum;

Figure 9 shows a Northern blot of mRNA purified from 11, 15 and 23 day-old Haemonchus contortus probed with a) cDNA clone Ml (SEQ ID NO: 1) ; b) cDNA clone Ml AUS (SEQ ID NO: 5) ; c) cDNA clone BIA (SEQ ID NOS: 2 and 3) ; d) cDNA clone AustBl (SEQ ID NO: 6) ; e) cloned PCR product 014-872 (3.5-2, SEQ ID NO: 8) ; and f) cloned PCR product 014-015 (SEQ ID NO: 7) . The numbers 11, 15 and 23 indicate the age of the Haemonchus from which the mRNA was obtained;

Figure 10 shows Southern blots of Haemonchus contortus genomic DNA probed with cDNA clones M1AUS (SEQ ID NO: 5) , BIA (SEQ ID NOS: 2 and 3) and AustBl (SEQ ID NO: 6) and PCR products 014-872 (3.5-2, SEQ ID NO: 8) and 014-015 (SEQ ID NO: 7) ; a) blots were washed at a moderate stringency, b) blots were washed at a high stringency; for each probe, track 1 contained a Hindlll digest of ADNA as marker or was left blank, tracks 2 and 3 contained EcoRI and Hindlll digests respectively of Haemonchus genomic DNA;

Figure 11 shows Western blots of recombinant GST-MI and GST-B1A fusion proteins probed with affinity

purified antibodies to electrophoretically purified HllOD (H110DE) ;

Figure 12 shows Western blots of ConA HllOD antigen probed with antisera to ConA HllOD and to recombinant GST-MI and GST-B1A fusion proteins;

Figure 13 shows a) the results of analysis of HllOD protein and aminopeptidase enzyme activities in fractions obtained by ion exchange chromatography of ConA HllOD on a MonoQ column; b) SDS-PAGE of the fractions shown in Figure 13a) ;

Figure 14 shows a) the pH values at which fractions were obtained in a free-flow isoelectric focussing experiment; b) SDS-PAGE under reducing conditions of the fractions from 14a) in which the lower band of the HllOD doublet is found in Fraction 6 and the upper band in Fraction 16, with varying amounts of each in the intervening fractions; c) Western blots of the fractions shown in 14b) probed with i) monoclonal antibodies designated TS 3/19.7 and ii) affinity purified polyclonal anti-Mi antibodies; control antibodies gave no detectable reaction;

Figure 15 shows a) the pH values at which fractions were obtained in another free-flow isoelectric focussing experiment; b) SDS-PAGE under reducing conditions of fractions from 15a) used in enzyme assays, in which the lower band of the HllOD doublet is found in Fractions 4- 6 and the upper band in Fractions 16-18 with varying amounts of each band in the intervening fractions ; c) microsomal aminopeptidase specific activities of fractions shown in 15b) ;

Figure 16 shows protection of sheep by vaccination with separated upper (U) , lower (L) , reco bined (U+L) and intermediate doublet (D) bands from HllOD; a) parasite egg output, expressed as eggs per gram faeces, b) worm burden at post-mortem, relative to controls;

Figure 17 shows protection of sheep by vaccination with a water-soluble fragment (HllS) obtained from HllOD by digestion with elastase and H11A, the residual detergent-soluble HllOD. a) parasite egg output, expressed as eggs per gram faeces; b) worm burden at post-mortem, relative to controls (C) ;

Figure 18 shows examples of the relationship between inhibition of Ai) , Bi) aminopeptidase M-like and Aii) , Bii) aminopeptidase A-like activities of HllOD by antisera of individual sheep vaccinated with HllOD with levels of protection measured by Ai, ii) % reduction of worm burden at post-mortem and Bi, ii) % reduction reduction of faecal egg count; □ anti-HHOD, ■ anti- horse ferritin control;

Figure 19 shows the histochemical localisation of aminopeptidase enzyme activities in adult Haemonchus contortus - the light micrographs of cryo-sections of adult female Haemonchus contortus show aminopeptidase activity (red reaction product appears as dark band (arrowed) in these black and white photographs) associated only with the microvilli ( v) of the intestine (i) . None of the other tissues (eg. cuticle (c) , hypodermis (h) , genital tract (gt) , wall muscle (wm) ) show activity. In a) the substrate was L-leucine 4-methoxy-?-naphthylamide, in b) the substrate was L- glutamic acid α-(4-methoxy-3-naphthylamide) ;

Figure 20 shows a map of the 3.5 PCR product (clone 2) (SEQ ID NO: 8) sub-cloned into the baculovirus expression vector pBlueBacII ^* ;

Figure 21 shows a Western blot of extracts from baculovirus-infected insect Spodoptera frugiperda (Sf) 9 cells probed with anti-HHODN antibodies. Two cloned plaques, P3A and P4A expressed the full-length immuno- positive HllOD (arrowed) , the controls did not.

EXAMPLE

METHODS

CONSTRUCTION OF U.K. λGTll LIBRARY mRNA Isolation

Adult Haemonchus contortus (0.5 gm) of UK origin snap-frozen in liquid nitrogen were ground in liquid nitrogen using a pre-chilled mortar and pestle. The RNA was extracted from the grindate with 10 volumes of 4 M guanidine hydrochloride in 25 mM sodium citrate containing 0.5% w/v sarkosyl and 0.7% w/v 2- mercaptoethanol, followed by extraction with phenol and chloroform using the method of Chomczynski & Sacchi (1987) . Messenger RNA (mRNA) was prepared from this by affinity chromatography on oligo dT cellulose (twice) as described in Maniatis et al (1982) and the quality was assessed by in vitro translation using a rabbit reticulocyte lysate kit and ³⁵ S-methionine from Amersham International pic, according to the manufacturer's instructions. Polypeptides up to 120 kd were detected.

Complementary DNA Preparation

First strand complementary DNA (cDNA) was synthesized from 1 μg mRNA using random priming and avian reverse transcriptase and the second strand was synthesized using a replacement reaction with RNase H and E.coli DNA Polymerase I followed by repair of 3 ' overhangs using T4 DNA Polymerase, according to the method of Gubler & Hoffman (1983) . The yield of double- stranded (ds) cDNA was approximately 400 ng from 1 μg mRNA. The ds cDNA was examined by electrophoresis in a 1% agarose gel followed by autoradiography. The ds cDNA was in the size range 0.2-9.4 kilobases (Kb) , with the majority being in the range 0.5-2.3 Kb.

Cloning of cDNA in λgtn

Non-size selected cDNA was used to construct a

library in λgtll using the Amersham cDNA cloning system (kit no. RPN 1280, Amersham International pic) and in vitro packaging extracts (kit no. N334, Amersham International pic) as described in the manufacturer's instructions, and EcoRI linker oligonucleotides (5 -GGAATTCC) . The resulting library was plated on E.coli strain Y1090 in the presence of isopropylthio-?- D-galactoside (IPTG) and 5-bromo,4-chloro, 3-indolyl β-D- galactoside (X-gal) , under which conditions recombinant λgtll appear as clear ("white") plaques and wild-type non-recombinant λgtll as blue plaques. The library contained 90% white plaques and the cloning efficiency was calculated to be 4 x 10 ⁷ plaque forming units (pfu)/μg cDNA and a library titre of 2 x 10 ⁶ plaque forming units per ml. Analysis of the DNA from 20 recombinants picked at random revealed an average insert size of 0.51 Kb. However this mean was distorted by one clone with an insert of 3.5 Kb. The majority of the inserts were >300 base pairs (bp) . This unamplified λgtll library derived from UK worm mRNA was then immunoscreened.

PREPARATION OF ANTIBODY PROBES

Antiserum to Integral Membrane Proteins

Intestines were dissected from adult Haemonchus contortus (of UK origin) and homogenised in ice-cold phosphate buffered saline (PBS) , pH 7.4, containing 1 mM ethylenedia inetetraacetic acid (EDTA) and 1 mM phenyl ethylsulphonyl fluoride (PMSF) . The homogenate was centrifuged for 10 minutes using a microfuge and the pellet resuspended in the same buffer containing 0.1% v/v Tween 20 (Tween is a Trade mark) . After re- centrifugation, the pellet was resuspended in the same buffer containing 2% v/v Triton X-100 and extracted for two hours at A °C . This extract was centrifuged as above, to obtain a supernatant containing integral membrane

proteins (IMP) .

A sheep was hyperimmunised with IMP in Freund's Complete Adjuvant (FCA) by intramuscular injection of 50, 50, 120 and 130 μg of IMP given on weeks 0, 7, 11 and 15. Six weeks after the final injection, serum was harvested, and designated serum EE-068.

Preparation of integral membrane proteins by detergent extraction of Haemonchus contortus

An extract was prepared by homogenizing worms in 5- 10 volumes of PBS containing 1 mM EDTA and ImM PMSF. The suspension was centrifuged at 10,000 x g for 20 minutes at 4°C and the pellet washed in the same buffer containing 0.1% v/v Tween 20 then extracted with 5 volumes 2% v/v Triton X-100 as described above. The supernatant was re-centrifuged at 100,000 x g for 1 hour, and the resulting supernatant, which was enriched in HllOD but contained other IMP, was used in Western blotting experiments and for the preparation of non- denatured HllOD (see below) .

Preparation of HllOD and Affinity Purified Anti-HllODN

The extract enriched for HllOD, was subjected to affinity chromatography on ConA-agarose followed by ion exchange chromatography on MonoQ (as described in WO88/00835 and WO90/11086) . The purified HllOD was injected intramuscularly into lambs in FCA. Three doses of 100 μg were given at 3 week intervals. Serum collected from the lambs 4 weeks after the final injection was affinity purified by absorption to a column containing purified HllOD which had been coupled to cyanogen bromide activated Sepharose (Pharmacia) . Coupling of HllOD to the Sepharose, binding of antiserum and elution of anti—HllOD antibodies were according to the instructions supplied by Pharmacia. These affinity purified antibodies are designated anti-HHODN. The "N" distinguishes these antibodies from those raised to denatured, electrophoretically purified HllOD, which are

designated anti-HHODE.

Western Blotting

Western blotting was carried out using standard procedures (Johnstone et al. , 1982) .

ISOLATION AND CHARACTERISATION OF CLONES

Immunoscreening of the U.K. λgtll library

The method used to immunoscreen the library was essentially as described by Bowtell et al (1986) . Prior to use, the serum (EE-068) was depleted of anti-E.coli antibodies by absorption with lysates and whole cells of E.coli Y1090. The library was plated on E.coli Y1090 cells at a density of 10 ³ pfu per 90 mm diameter plate. Plates were overlaid with nitrocellulose filters impregnated with IPTG and incubated overnight. The filters were washed with TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.05% v/v Tween 20) and then blocked with 5% v/v horse serum in TBST for 2 hours. Serum EE-068 diluted 1 in 200 in TBST containing 5% horse serum was added and the filters incubated for 4 hours with gentle rocking. The filters were again washed in TBST, then incubated with horseradish peroxidase (HRP) -conjugated horse anti-sheep IgG diluted 1 in 500 in TBST containing 5% v/v horse serum for 2 hours. (Anti-serum to sheep IgG was raised in a horse, the anti-sheep IgG purified by affinity chromatography on a sheep IgG Sepharose column, and the antibodies conjugated to HRP by the method of Nakane & Kawaoi, 1974.) Filters were further washed in TBST and positive plaques detected using 0.6 mg/ml 3 , 3 ' -diaminobenzidine (DAB) and 0.1% v/v hydrogen peroxide. Twenty-five putative positives were picked and were rescreened with affinity purified anti-HHODN as described above. Following this secondary screen 5 recombinants were still positive, with the clone designated as Ml giving the strongest signal.

Affinity purification of antibody on recombinant phage

Confluent plates were prepared on E.coli Y1090 lawns by plating 10 ³ pfu of each of the antibody-positive λclones or non-recombinant λgtll negative control phage. The lawns were incubated for 4 hours at 42°C then overlaid with filters impregnated with IPTG and further incubated overnight at 37°C. The filters were removed from the plates and washed in TBST prior to being blocked with 5% v/v horse serum for 1 hour. The filters were then incubated with a 1 in 100 dilution of antiserum EE-068 for 6 hours, before being thoroughly rinsed with TBST. Bound antibodies were eluted from the filters by two applications of 2 ml of elution buffer (5 mM glycine, 500 mM NaCl, 0.2% Tween 20, pH 2.3) for 2 to 3 minutes each, neutralised by addition of 200 μl of 1 M tris-HCl, pH 7.4, diluted 1 in 200 and used to immunoscreen a Western blot of an HHOD-enriched extract.

DNA SEQUENCING OF THE Ml CLONE

Lambda DNA was isolated from the Ml clone according to the methods described in Maniatis et al (1982) . The 2.38 Kb KpnI-SstI fragment containing the 300 bp Ml fragment was isolated by gel electrophoresis, purified using a GENECLEAN kit (Stratagene) (GENECLEAN is a registered trade mark of BI0101) and subcloned into pBluescriptll SK ^* (Stratagene) . The EcoRI fragment was purified using the same methods and re-subcloned into the same vector.

The nucleotide sequence of the Ml insert was determined using a T7 Sequencing kit (Pharmacia, U.K.), using both the M13 forward and reverse primers.

PREPARATION OF AUSTRALIAN λGTll and λZAP cDNA LIBRARIES

mRNA Isolation

5 g adult Haemonchus contortus (Australian

McMaster susceptible strain) snap-frozen in liquid nitrogen were ground in liquid nitrogen and the RNA extracted using hot phenol by the method of Cordingley et al. (1983). Yield of total RNA was 10.35 mg. 1.3 mg of this RNA was used to prepare mRNA by affinity chromatography on oligo dT cellulose (2 sequential purifications) using the method described by Maniatis et al. (1982) . Yield of mRNA was 21.6 μg. Quality of mRNA was assessed by in vitro translation in rabbit reticulocyte lysate in the presence of ³⁵ S-methionine (Amersham) according to the supplier's instructions. The translation products obtained had clearly distinguished bands including bands >200 kd in size as demonstrated by electrophoresis on SDS-polyacrylamide gels followed by fluorography.

cDNA Synthesis and library preparation

1 μg mRNA was used to make cDNA by priming with oligo dT or random primers, using a cDNA synthesis kit from Amersham International pic following the manufacturer's instructions. Yield was 115 ng double stranded (ds) cDNA. The quality of the cDNA was examined by electrophoresis of the ³² P-labelled DNA on an alkaline agarose gel as described by the Amersham cDNA kit instructions. Size of the cDNA (by comparison with x-Hindlll markers, New England Biolabs) was from 150 bp to >10 Kb, with most of the products being in the size range 0.6-5 Kb. The oligo dT-pri ed and random-primed ds cDNAs were pooled and ligated to excess EcoRI 8-mer linkers ( 5 'GGAATTCC3 ' New England Biolabs, Catalogue No. 1018) which had been labelled with ₇ - ³² P-ATP and T4 polynucleotide kinase. The linkered cDNA was digested with EcoRI and excess linkers were removed by Sepharose B (Pharmacia) chromatography according to the methods described by Maniatis et al . (1982) . Fractions from the column were pooled in two lots, one containing cDNA iarger than 2 Kb and one of cDNA less than 2 Kb. Each pool was then ligated separately to 1 μg EcoRI cut,

phosphatased λZapH arms (Stratagene) and packaged separately using Gigapack Gold (Stratagene, registered trademark). The larger sized cDNA yielded 1.3 x 10 ⁵ recombinants and the smaller cDNA 1.4 x 10 ^s recombinants; these were pooled to yield a library of 2.7 x 10 ⁵ . The λZap library was amplified by plating on XLl-Blue cells (Stratagene) at 2 x 10 ⁴ pfu per 135 mm plate. The titre of the amplified library was 7 x 10 ⁷ pfu/ml.

A further 2 μg mRNA was used to make cDNA as described above, but using only oligo dT as primer. The yield of ds cDNA was 740 ng. This cDNA was treated with EcoRI methylase as described in Maniatis et al (1982) prior to addition of EcoRI linkers, and in this case 12- mer linkers (5*CCGGAATTCCGG3 ' New England Biolabs, Catalogue No. 1019) were used. Following digestion of the linkered cDNA with EcoRI, all fractions from a Sepharose 4B column which contained cDNA were pooled, and ligated to 2 μg EcoRI cut, phosphatased λgtll arms (Stratagene) . The ligation mix was split in two and packaged with two lots of Gigapack Gold (Stratagene) ; these were pooled to yield a λgtll library of 7 x lθ ⁶ pfu. The library was amplified by plating on ST9 cells at 5 x 10 ⁵ pfu per 135 mm plate. The titre of the amplified λgtll library was 4.5 x 10 ¹¹ pfu/ml.

Screening of the Australian λgtll Library with Antisera to HllOD

Antisera were raised by injecting sheep with HllOD protein (of UK origin) which had been electro-eluted from polyacrylamide after electrophoresis in SDS according to the following method: ConA HllOD prepared as described in WO 83/00835 and WO 90/11086 was electrophoresed on SDS polyacrylamide gels (Laemmli 1970) to obtain electro-eluted HllOD. After electrophoresis, the area of the polyacrylamide gel containing HllOD was cut out, placed in an electroeluter (Atto) and elution carried out for 3 hours at 10 watts. The electroeluted HllOD (designated H110DE) was

concentrated on a Centripre 10 (Amicon) and buffer exchanged on a PD10 column (Pharmacia) into 50 mM ammonium bicarbonate/0.07% SDS, mixed with adjuvants and then injected into sheep. Immunoglobulins from the sera were precipitated with ammonium sulphate (Johnstone and Thorpe, 1982) . The precipitated antibodies were resuspended at 60 mg/ml in phosphate buffered saline, dialysed against phosphate buffered saline and diluted 1:10 in Tris buffered saline (TBS) containing 5% w/v low fat milk powder. 10 mg of ConA HllOD was made to 0.5% SDS, heated to 100°C for 3 minutes and dried onto a nitrocellulose filter. Following washes with TBS containing 0.2% v/v Tween 20 and 0.5% Triton X-100 (TBSTT) the filter was incubated for 1 to 2 hours at room temperature with the antibodies to H110DE. After washing the filter for 2 hours with TBSTT, the bound antibodies were eluted with 3 ml of 0. IM glycine, 0.15M NaCl pH 2.6 for 2 minutes and immediately adjusted to neutral pH by the addition of 75 μl of 1.5 M Tris pH 8.0. These affinity purified antibodies, designated anti-HHODE, were used to screen 5 x 10 ⁵ pfu of the Australian λgtll cDNA library as described above.

5 x 10 ⁵ recombinants from the λgtll library derived from Australian Haemonchus contortus were immunoscreened and three positives picked. Following further screening two of these recombinants were still positive and were designated BIA and B2.

Sequencing of BIA and B2 clones

The two clones were digested with EcoRI, yielding a single insert of approximately 500bp for BIA and three fragments, B2A (about 400bp) , B2B (about lOObp) and B2C (about lOObp) , for B2. These were subcloned into pBluescript SK ^* (Stratagene) and sequenced using a Sequenase 2.0 kit (United States Biochemicals) .

EXPRESSION OF CLONES Ml AND BIA

The Ml (SEQ ID NO: 1) and BIA (SEQ ID NOS: 2 and 3) inserts were expressed in E.coli, using a pGEX vector (Smith and Johnson 1988) . This vector expresses proteins at the C-terminus of Schistosoma iaponicum glutathione-S-transferase (GST) . The Ml and BIA EcoRI inserts were ligated to EcoRI-cut. phosphatased pGEXl and transformed into E.coli strain JM101 according to the methods described in Maniatis et al. 1982. Eight progeny were picked from each transformation and 2ml cultures were grown for 6 hours at 37°C. IPTG was added to induce fusion protein synthesis, and the incubation continued overnight. Cells were harvested by centrifugation, disrupted by boiling in sample buffer (Laemmli, 1974) , and the extracts analysed by SDS-PAGE and by Western blotting using affinity purified sheep antibodies specific for the SDS-denatured HllOD doublet (anti-HHODE - see above) . Bound antibodies were detected using alkaline-phosphatase conjugated rabbit anti-sheep IgG alkaline phosphatase conjugate (Jackson Immunoresearch) followed by colour development with 5- bromo,4-chloro,3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) . Cultures of immunopositive clones were grown and induced as above and disrupted by sonication. The sonicates were separated into soluble and insoluble fractions by centrifugation (Sorvall RC-2B centrifuge, HS4 rotor, 7000 rpm, 30 minutes, 4°C) . The insoluble pellets were resuspended in 8 M urea by sonication, and samples of fractions examined by SDS- PAGE. The fusion proteins were found to be in the insoluble inclusion body fraction. Each of these preparations was used to vaccinate 2 sheep three times at 150 μg fusion protein per dose in Freunds adjuvants. Positive control sheep were immunised with native ConA HllOD protein, and negative control sheep were immunised with solubilised protein from E.coli containing the pGEX vector without an Haemonchus insert. Sera from vaccinated sheep were analysed by Western

blotting against HllOD.

SCREENING OF THE AUSTRALIAN λZAP LIBRARY BY DNA HYBRIDISATION WITH Ml AND BIA INSERTS

Ml and BIA plasmid DNAs (cloned in pBluescript) were digested with EcoRI and the inserts isolated by electrophoresis in TBE (tris-borate-EDTA; 89 mM tris- borate, 89 mM boric acid, 2 mM EDTA pH approximately 8.3) buffer in 1% agarose gel, followed by purification using a GENECLEAN kit. The isolation and purification were repeated to avoid contamination of the probe with plasmid DNA sequences which would hybridise to λZAP sequences, causing unacceptable levels of background. The purified insert DNAs were labelled with α- ³² P-dCTP using a Nick Translation kit from Promega Biotech according to the manufacturer's instructions. Labelled DNA was separated from unincorporated label by spin column chromatography (Maniatis et al.. 1982) . Eight 135 mm plates of the λZAP library were plated at 10 ^s pfu/plate, and plaque lifts performed onto nitrocellulose filters (Maniatis et al.. 1982) . Following baking in a vacuum oven for two hours at 80°C, filters were prehybridised for two hours, then hybridised at 42°C overnight (as described below in the Southern Blot analysis section) . Four filters were screened with the Ml probe and four with the BIA probe. Filters were washed twice in 2 x SSC containing 0.5% SDS, once in 1 x SSC containing 0.5% SDS and once in 0.5 x SSC containing 0.5% SDS, all at 50°C, and autoradiographed. Potential positive plaques were picked, and re-screened with the probes. High titre phage stocks were prepared from confirmed positives (designated M1AUS for the Ml-hybridising clone and AustBl for the BlA-hybridising clone) and the clones rescued into pBLUESCRIPT according to the ΛZAP manufacturer's instruction manual (Stratagene) , using BB4 as the host E. coli strain. Plasmid DNA minipreps of

the resultant progeny were prepared by alkaline lysis (Maniatis et al. , 1982) and digested with EcoRI. Digests were analysed by agarose gel electrophoresis.

Sequencing of the M1AUS insert

DNA sequencing was carried out on purified pBLUESCRIPT plasmid DNA using the United States Biochemicals version 2.0 Sequenase kit, according to the manufacturer's instructions. For the first sequencing reactions primers from the ends of the vector sequence were used to prime the reactions. The sequencing data obtained from these reactions was used to design a second pair of primers and from the data generated with these second primers a third pair were designed. In this way the DNA was sequenced by ^λ walking along' from both the 5' and 3 ' ends.

Sequencing of the AustBl insert

This was carried out using Sequenase 2.0 T7 polymerase (USB Biochemicals) as descibed for the sequencing of the M1AUS insert.

POLYMERASE CHAIN REACTIONS

Preparation of cDNA mRNA (1 μg) from 11 day old post-infection U.K. H. contortus- prepared as described for adult UK worms, was mixed with T17 adaptor-primer

(5'GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3') in diethyl pyrocarbonate (DEPC) -treated water, then heated to 65°C for 5 minutes and immediately placed on ice. Methylmercury hydroxide was added to a final concentration of 28.6 mM and the mixture incubated at room temperature for 3 minutes. 2-mercaptoethanol was added to a final concentration of 14.2 mM and the mixture was placed on ice. To synthesize cDNA, RNAse Guard (Pharmacia) was added to 1 unit/μl, Reverse

Transcriptase buffer (Life Sciences) to 1 times concentration, dATP, dGTP, dCTP and dTTP each to 1 mM, and AMV Reverse Transcriptase (Life Sciences) to 2 units/μl (all given as final concentrations) . The reaction was incubated at 41°C for 75 minutes, then extracted with phenol and chloroform and purified by spun column chromatography (Maniatis et al , 1982) . The purified reaction mix was diluted 2.5-fold and stored at 4°C.

PCR Amplification of the cDNA Using MIAUS-Specific Primers

PCR reactions were carried out using a Programmable Thermal Cycler (M.J. Research Inc.). The reaction mix contained 1 μl out of the 250 μl diluted cDNA prepared as described above, 25 pmol of the first strand T17- adaptor-primer, 25 pmol of second strand amplification primer (either that based on positions 865-884 (5'ACGGGTGTTCGGTTTCCGTAT 3') or that based on positions 30-49 (5 'GCTGAATCTAACTCCAATCC 3') of the M1AUS sequence (SEQ ID NO: 5)) , 1 x Tag buffer (Northumbria Biologicals Ltd) and 0.5 mM each of dATP, dTTP, dGTP and dCTP, in a 100 μl reaction volume and covered with 40μl mineral oil to prevent evaporation. This mix was then heated in the thermal cycler to 95°C for 2 minutes then held at 72°C. Whilst at 72°C 2 units of Tag Polymerase (Northumbria Biologicals Ltd) was added and mixed gently with the other reactants. The following program was then carried out in the thermal cycler: Step 1 Anneal at 50°C for 5 minutes Step 2 Extend at 72°C for 40 minutes Step 3 Denature at 94°C for 40 seconds Step 4 Anneal at 50°C for 2 minutes Step 5 Extend at 72°C for 3 minutes Step 6 39 cycles of steps 3 to 5 Step 7 Final extension at 72°C for 15 minutes Step 8 Hold at ^■ . ^C, C

These conditions were established from Frohman et al. , (1988) .

Cloning of the PCR products

The PCR products from the above reactions were separated by electrophoresis in an agarose gel. Bands of DNA of approximately 2.5 and 3.5 kb were electroeluted onto glass fibre (Whatman) , phenol extracted and purified by G50 chromatography (Pharmacia) (Sambrook et al. , 1989) . The purified DNA was ligated into pT7Blue T- vector (Novagene) following the manufacturer's instructions.

Sequencing of the 2.5 kb and 3.5 kb PCR products

DNA sequencing was carried out with a Sequenase 2.0 kit (US Biochemicals) using the "oligonucleotide walking" technique described in the section on sequencing of M1AUS.

POLYMERASE CHAIN REACTIONS FOR THE 5" ENDS

Preparation of first strand cDNA

1 μg of mRNA from 11 day post-infection UK Haemonchus contortus prepared as described for adult worms was mixed with a constant primer

(5*AAIGAAAGCGGATGGCTTGAIGC 3') designed from a conserved region in AustBl and the 2.5kbPCR and 3.5kbPCR products (SEQ ID NOS: 6, 7 and 8 respectively) . The mixture was heated to 65°C for 5 min. , placed on ice and methyl mercury hydroxide added to a final concentration of 28.6 mM. The mixture was incubated at room temperature for 5 min. , then 2-mercaptoethanol added to a final concentration of 14.2 mM and the mixture placed on ice. First strand DNA was prepared using reagents from the 5 ' RACE system (Gibco/BRL) at a final concentration of 20 mM Tris/HCl pH 8.-;, 50 mM KC1, 2.5 mM MgCl.,, lOOμg/ l BΞA, 0.5 M of dATP,dCTP, dGTP, dTTP. 200 Units of

Superscript Reverse Transcriptase were added and the reaction was incubated at 42°C for 30 min. and then heated at 55°C for 5 min. RNAse H was added to a final concentration of 100 Unit/ml and the reaction incubated at 55°C for 10 min. and then placed on ice. The cDNA was purified through a Glassmax spin column (Gibco/BRL) and stored at -20°C.

C-Tailing of the cDNA

1/5 of the first strand cDNA was heated at 70°C for 5 min then chilled on ice for 1 min. Reagents from the 5 'RACE system (Gibco/BRL) were added to a final concentration of 10 mM Tris/HCl pH 8.4 , 25 mM KC1,1.25 mM MgCl, 50 ug/ml BSA, 0.2 mM dCTP. 500 Units/ml Terminal transferase were added and the reaction incubated at 37°C for 10 min, then heated at 70°c for 15 min and stored on ice.

PCR Amplification using AustBl, 2.5kbPCR and 3.5kbPCR specific primers

The PCR reactions were carried out in a programmable Thermal Cycler (M.J. Research Inc.) . For the 3' end one of 3 primers was used.

1. A primer specific for the 2.5 kb PCR product based on positions 374 to 394 (5'TGTTGTGGCTAATTTCGTCCA 3') .

2. A primer specific to the 3.5 kb product based on positions 1210 to 1229 (5' CATCTTIAGTTATCTGACCAG 3') .

3. A primer specific for the cDNA clone AustBl based on positions 357 to 377 (5' GACCATCGCTGATGAAGTCGG 3') .

For the 5' end of the reactions a common 'Anchor primer' (5 ' CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG3 ' ) was used. Each reaction mixture contained 4μl of the 50μl of C-tailed cDNA, 25 pMol of the appropriate 2 primers, lx Tag polymerase buffer (Boehringer/Mannheim) and 0.5 mM each of dATP,dCTP, dGTP and dTTP to a final volume of lOOμl. This mix was covered with 50μl of mineral oil and heated to 95°C in in the cycler for 2 min. The reaction mix was held at 80°C whilst 0.6 units

of Tag Polymerase were added and then put through the following programme:

1. Anneal at 50 °C for 5 min.

2. Extend at 72°C for 10 min.

3. Denature at 94°C for 45 sec.

4. Anneal at 50°C for 1 min.

5. Extend at 72 °C for 2.5 min.

6. 39 cycles of 3 to 5.

7. Extend at 72°C for 15 min.

8. Hold at 4°C.

Cloning of the 5' PCR products

The PCR products were separated by electrophoresis on an agarose gel and bands of the expected size, circa 1.3 kb, were cut out, the DNA purified using a GENECLEAN kit and ligated into PT7Blue T-Vector (Novagene) according to the manufacturer's instructions.

POLYMERASE CHAIN REACTION FOR THE PRODUCTION OF THE 3 ^» END OF AUSTB1

The first strand cDNA used was that described for the production of cDNA for use with MIAUS primers. A specific primer from 1414 to 1435

(5'TCTTGAAGAAATGAAAAAGCTT 3') in AustBl (SEQ ID NO: 6) was used with the T17 Adaptor primer used for the MIAUS PCR and the reactions carried out in a thermal cycler (M.J. Research Inc) . The reaction mixture consisted of 25 pMol of each primer, 2μl of cDNA, 1 x Taq Polymerase buffer (Boehringer Mannheim) , 0.5 mM dATP, dCTP, dGTP and dTTP in lOOμl. These were covered with 50ul mineral oil and heated to 95 °C for 2 min. 0.6μl of Taq polymerase was added and the same programme cycle carried out as for the 5' PCR described above.

Cloning and sequencing of the 3' products.

The products of the PCR were separated by

electrophoresis in an agarose gel and a band of the expected size, 1.3kb, cut out and the DNA purified using a GENECLEAN kit and ligated into PCR-Script (Stratagene) according to the manufacturer's instructions.

Sequencing of the cloned PCR products

The DNA from the PCR clones was sequenced using a Sequenase 2.0 kit (United States Biochemical) as instructed by the manufacturer. Oligonucleotide primers were used to "walk along" the DNA of the clones from both the 5' and 3' ends.

ANALYSIS OF ALL DNA SEQUENCES

Sequences were analysed using the GCG (Genetics Computer Group) Sequence Analysis Software Package, Devereux et al .. 1984.

NORTHERN AND SOUTHERN BLOT ANALYSES

Preparation of Northern Blots

Northern blots were performed in formaldehyde gels essentially as described in Maniatis et al . (1982) . mRNA samples (from 11-, 15- and 23-day old adult H.contortus) were treated with 17.5% v/v formaldehyde and 50% v/v forma ide in MOPS buffer (20 M 3-(N- morpholino)propanesulphonic acid, pH 7.0, 3 M sodium acetate, 1 M EDTA) at 65"C for 15 minutes, and cooled on ice. Gels were electrophoresed in MOPS buffer, and blotted onto Duralon membranes by capillary transfer as described in Sambrook et al . , (1989) .

Preparation of Southern Blots

Two gm of adult Haemonchus contortus which had been snap-frozen in liquid nitrogen were ground to a fine powder in liquid nitrogen. The powder was added slowly to 25 rr.i of lysis puffer (0.05 M Tris-HCl , pH 8, 0.1 M

EDTA, 1% w/v Sarkosyl, 0.05 mg/ml proteinase K (Boehringer Mannheim) ) and incubated for two hours at 65°C. The suspension was then extracted twice with one volume of phenol plus chloroform, twice with two volumes of chloroform, and ethanol precipitated. The precipitated genomic DNA was resuspended in 20 ml of Tris, EDTA buffer (TE, pH 8) overnight at 4°C on a rocking table, then dialysed against two changes of one litre of TE. RNA was removed by incubating with DNase- free RNase A Type 1 (Sigma) at a final concentration of 20 μg/ml, at 37°C for one hour, followed by one extraction with phenol-chloroform, one extraction with chloroform, and ethanol precipitation, as above. The precipitated genomic DNA pellet was washed twice with 70% v/v ethanol, and resuspended in one ml TE, as above.

Genomic DNA was digested with EcoRI or Hindlll (25μg of DNA in each digest) overnight at 37°c, then electrophoresed at 5μg per track on a 1% w/v agarose gel in Tris-acetate buffer. The gel was Southern blotted by capillary transfer as described in Maniatis et al. , (1982) onto Hybond-N membrane (Amersham International) . DNA was fixed onto the membrane using ultraviolet light, according to the manufacturer's recommendations.

Preparation of Probes pBLUESCRIPT plasmids containing the MIAUS, BIA or AustBl inserts were digested with EcoRI. pT7Blue plasmids containing 3.5kbPCR product inserts were digested with BamHl and those containing 2.5kbPCR product inserts were digested with BamHl and Xbal. Digests were electrophoresed, the inserts recovered and radioactively labelled with α- ³² P-dCTP by nick translation as described above under screening of the λZAP library.

Hybridization Conditions

For Southern blots

The membranes were cut into strips and pre-

hybridised in hybridisation buffer as described earlier, for 3 hours at 28°C. Genomic DNA Southern blot strips were hybridised to each of the above probes overnight at 28°C, washed twice at room temperature (24°C) then twice at 42°C, in 2 x SSC containing 0.1% w/v SDS (moderate stringency) and autoradiographed. Following development of the autoradiographs, strips were re-washed at a high stringency (0.1 x SSC, 0.1% w/v SDS at 65°C) and re- autoradiographed.

For Northern blots

For probes Ml, MIAUS and BIA (SEQ ID NOS: 1, 5 and 2 respectively)

The Northern blot of mRNA from 11, 15 and 23 day- old Haemonchus contortus was probed first with the Ml insert. The filter was prehybridised for 2 hours at 42°C in 2 x SSC (where 20 x SSC = 3 M NaCl, 0.3 M sodium citrate, pH 7.2) containing 5 x Denhardt's (0.1% w/v Ficoll 400 (Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin Fraction V (Sigma Chemical Corp) ) , 0.5% SDS (sodium dodecylsulphate) , 10% dextran sulphate, 0.1 mg/ml salmon testes DNA and 50% de-ionised formamide. The hybridisation to the probe was performed in the same buffer overnight at 42°C. The filters were washed twice for 30 minutes in 2 x SSC containing 0.5% SDS and 50% formamide, twice for 30 minutes in 2 x SSC containing 0.5% SDS and twice for 30 minutes in 2 x SSC. The first wash was at 42°C and all remaining washes were at 37°C. After autoradiography the blot was stripped by washing in boiling 0.1% SDS and re-autoradiographed to ensure removal of the probe. The same blot was then probed with the MIAUS insert, washed and autoradiographed. The blot was again stripped and checked and when clear was then probed with the BIA insert. After stripping again the blot was then probed as described below.

For probes AustBl, 2.5 kb and 3.5 kb PCR products (SEQ ID NOs: 6 , 1 and 8)

The Northern blot was hybridised with the AustBl insert using the conditions of moderate stringency as described for Southern blot hybridisation. After autoradiography the blot was stripped with boiling 0.1% SDS according to the membrane manufacturer's instructions (Amersham) , then probed with the 2.5 kb PCR insert (clone 2), stripped again and probed with the 3.5 kb PCR (clone 2) insert.

DIGESTION OF HllOD AND ASSAYS OF ENZYME ACTIVITY

Preparation of HllOD

Native HllOD (H110DN) was prepared according to the methods described in WO88/00835 and WO90/11086.

Preparation of an elastase fragment (HllS) of HllOD

Adult Haemonchus were homogenized in 10 volumes of ice cold PBS/0.02% sodium azide and then centrifuged for 20 minutes at 13000 rpm. The pellet was resuspended in 10 volumes of PBS/azide, rehomogenised and the centrifugation repeated. Following resuspension in 50mM MOPS buffer pH 7.4 (the volume for suspension is 1ml for each 0.l7g of worms) and pre-warming at 37°C for 30 minutes, the pellet material was digested with elastase (800μl/20ml of suspension; lmg/ml fresh stock solution made up in ImM HCl/ 2mM Ca ^2* ) for one hour. The digestion was stopped by the addition of 3,4 dichloroisocoumarin (300μl of stock lOmM in DMSO/20ml of digest) - The mixture was centrifuged at 13000 rpm for 20 minutes and the pelleted material retained. The supernatant was ultracentrifuged at lOOOOOg for 1 hour 20 minutes. The resultant supernatant liquid was applied to a ConA column and the binding fractions obtained. For analysis, this fraction was run on an SDS- polyacrylamide gel and electrophoretically transferred to polyvmylidene difluoride membrane (Immobilon-P, Millipore; , lightly stained with Coomassie blue, the

105kd band excised and analysed in a gas phase amino acid sequenator. For vaccination studies, the ConA binding fractions were further purified by concentrating and applying to a Superose 12 (Pharmacia) gel filtration column and collecting those fractions containing aminopeptidase activity.

Thermolysin Digestion of HllOD

The HllOD doublet was purified by electroelution from a preparative scale 8% SDS-polyacrylamide gel to give H110DE, as described in WO90/11086 and by electroelution of a dimeric form, running at just over 200 kd (which yielded the characteristic doublet at 110 kd when re-run on SDS-PAGE) to give H110DE. Solutions of H110DE were concentrated to 200μl, calcium chloride was added to 5mM and the mixture warmed to 37°C. A freshly prepared solution of lmg/ml Thermolysin (in ImM HCl, 2mM CaCl ₂ ) was added in a ratio of O.lμg Thermolysin per μg H110DE. The mixture was incubated at 37°C for 120 minutes and the reaction then stopped by addition of 5μl of 0.5M EDTA.

The protein fragments were separated by 15% SDS- polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore) . Following staining with Coomassie blue the most intense discrete bands were excised and analysed in a gas phase amino acid sequenator.

Preparation of an HllOD fraction (H11A) enriched for aminopeptidase A activity

The pelleted material obtained after elastase treatment by centrifugation at 17000g for 20 min. (see above) was resuspended in PBS at 4°C and repelleted by centrifugation then resuspended in 1% Tween in PBS/azide and left (with stirring) for 1 hour. The suspension was centrifuged at 17,000g for 20 minutes and the supernatant removed. The pellet was repeatedly

extracted with 1% Thesit in PBS/azide. The supernatants after centrifugation at 17,000g for 20 minutes were combined and ultracentrifuged for 1 hour 20 minutes at 100,000g. The supernatant was applied to a ConA affinity column (Affigel, Biorad) and the bound material eluted and further fractionated by ion exchange chromatography on a MonoQ column.

Assays of enzyme activities α-amino acylpeptide hydrolase (microsomal) aminopeptidase activities in HllOD preparations were characterised by assays, in solution, using L-leucine, methionine, phenylalanine, α-glutamic acid and lysine p- nitroanilides (pNA) . All the amino acid p-nitroanilide substrates (except α-glutamic acid) were obtained from Sigma, Poole, Dorset UK, α-glutamic acid was from Fluka, Dorset, UK. Single hydrophobic (leucine- or phenyalanine-) and charged (α-glutamic acid-) amino acid pNA known to be substrates for mammalian aminopeptidase- M (ApM) and -A (ApA) respectively, were chosen to measure the effect of enzyme inhibitors and serum inhibition on HllOD aminopeptidase activities.

(a) Microplate assay

Micro-ELISA plate (Dynatech I mulon 1, Virginia, USA) wells were each filled with 250μl of either 50mM HEPES or MOPS pH 7 plus l-10μl of the fraction to be assayed. The plates were then pre-incubated at 37°C for 10 minutes prior to the addition of lOμl of 25mM amino acid p-nitroanilide substrate per well. The time zero optical density (OD) at 405nm was then measured using an ELISA plate reader and the plates were then incubated at 37°C for 15-30 minutes. The final OD reading was then taken as before and the OD change per minute per milligram of prote-m calculated.

(b) Inhibitor sensitivity

The method used for the enzyme assay was as described in (a) above except that the inhibitors were added to the 250ul of buffer plus lOμl of lmg/ml ConA

HllOD and pre-incubated for 10 minutes at 37°C prior to addition of the substrates (leucine-pNA or α-glutamic acid-pNA) . The percentage inhibition was calculated as follows

x-y X 100 = percentage inhibition, x

Where x = the ΔOD/min of the enzyme with no inhibitor added and y = ΔOD/min of the enzyme plus inhibitor. Nine compounds with differing enzyme class inhibition were tested individually: Amastatin, Bestatin (metalloprotease, aminopeptidase) , 1,10 phenanthroline, EDTA (metalloprotease) , phosphoramidon (metalloprotease, thermolysin, collagenase) , Aprotinin (serine protease) , Pepstatin (aspartic protease) , PMSF (serine and cysteine protease) and E64 (cysteine protease) . Amastatin, bestatin, 1,10 phenanthroline, PMSF and pepstatin were obtained from Sigma, Dorset, UK. All the other inhibitors were obtained from Boehringer-Mannheim. Each inhibitor was used at two concentrations equal to or greater than the maximum concentrations recommended by Boehringer-Mannheim. ( c) Assay of antiserum inhibition of enzyme

The assay was as described in (a) above except that two micro-ELISA plates were set up. In one plate lOμl of ConA HllOD (lmg/ml) plus lOμl of antiserum from each sheep per well were pre-incubated at 37°C for 15 minutes. The second plate, v/ith 250μl of HEPES/bicarbonate buffer plus lOμl of either phenylalanine-pNA or α-glutamic acid-pNA substrate per well was also pre-incubated at 37°C for 15 minutes. The ConA HllOD-serum mixtures were then added to the buffer-substrate mixtures in the second plate with a multi-pipette. The ΔOD/min was determined. For the purposes of the correlations, percentage inhibition was calculated using the formula in (b) above ^• .. ^• here x represents the mean :,0D/min for the wells wnich contained enzyme plus serum from negative control sheep (vaccinated with horse spleen ferritin)

and y = the ΔOD/min for the wells which contained enzyme plus sera from individual sheep vaccinated with HllOD. For the purposes of the correlations (Figure 18) , the percentage protection was calculated using the formula in (b) above where x = either the average faecal egg output per gram or the average worm burden of the controls, and y = either the faecal output per gram during the experiment or the worm burden of the individual sheep vaccinated with HllOD.

Localisation of Enzyme Activity by Histochemistry

Aminopeptidase activity was demonstrated on lOμm cryostat sections of adult Haemonchus contortus using L- leucine 4-methoxy-jS-naphthylamide and L-glutamic α- 4- methoxy-3-naphthylamide as substrates by the methods of Nachlas et al. (1957) , Nakane et al. (1974) and Lojda et al. (1980) .

EXPRESSION OF RECOMBINANT HllOD IN THE EUKARYOTIC BACULOVIRUS-INSECT CELL SYSTEM

Construction of expression plasmids

The 3.5K PCR fragments described above, were generated by amplification between an oligo dT adaptor (which contained a Sail site) and oligo 872, which represents base no. 's 30-49 in the MIAUS sequence, and were cloned into the pT7Blue vector. Clone pT73.5-2 is oriented with the vector polylinker BamHl site at its 5' end and the Hindlll, Xbal and Sail sites at the 3' end. The sequence at the 5 ' end is:

BamHl *! oligo 872 i—3.5K

5' GG ATC CGA TTG CTG AAT CTA ACT CCA ATC C 3 '

Leu Asn Leu Thr Pro He

The asterisk indicates the 3 ' dT/dA overhang used for cloning in the pT7 Blue Vector.

The 3.5 PCR clone 2 was digested with Hindlll (single site in the vector polylinker sequence at the 3 ' end of the 3.5K gene) and the ends filled in with deoxynucleotideε using DNA polymerase I (Klenow fragment) , according to Maniatis et al (1982) . A BamHl linker (5' CGGATCCG 3', New England Biolabs Catalog no. 1021) was ligated to the blunt ends, and clones with an extra BamHl site, allowing a full-length HllOD gene sequence to be excised as a BamHl fragment, selected. The BamHl fragment was isolated by electrophoresis on a 0.6% w/v agarose gel in tris-acetate buffer, followed by purification using GENECLEAN (BIO101) ; this procedure was carried out twice. The purified fragment was then ligated to BamHI-cut, phosphatased pBlueBac II (Invitrogen Corp.) , and clones carrying the fragment in the correct orientation (ie. with the 5' end of 3.5 PCR clone 2 placed under control of the baculovirus polyhedrin promoter) determined by digestion with Nhel and Xbal. The resultant plasmid was partially digested with BamHl and the ends filled in as described above. An Ncol linker containing an ATG (5 'CCCATGGG 3'; New England Biolabs Catalog no. 1040) was added and the mixture ligated. Clones which had the linker ligated at the 5' end BamHl site of 3.5 PCR clone 2 rather than the 3' site, were determined by digestion with Ncol. The resultant plasmid, designated pBB3.5-2(N) , is depicted diagrammatically in Figure 19.

This construction results in the insertion of an in-frame ATG at the 5' end of the 3.5 PCR clone 2 insert, to initiate translation. The sequence surrounding this initiating ATG is:

BamHl—Ncol link-BamHI *! oligo 872

5'GGATCCCC ATG GGG ATC CGA TTG CTG AAT CTA ACT CCA ATC

Met Gly He Arg Leu Leu Asn Leu Thr Pro He

The expressed protein will be missing amino acids 2-9 of the corresponding HllOD sequence, and will have 3 amino acids of linker sequence immediately following the ATG.

Generation of Recombinant Baculovirus Containing HllOD Sequences

The plasmid pBB3.5-2(N) was transfected into Spodoptera frugiperda (S_f9) cells (obtainable from Invitrogen Corp) , using linear Autographica californica nuclear polyhedrosis virus (ACNPV) DNA and cationic liposomes (Invitrogen Corp. transfection module) , according to the manufacturer's instructions. Cells were cultured in TC-100 medium (SIGMA) supplemented with foetal calf serum (CSL Ltd; heat-inactivated at 56°C for 45 minutes) and antibiotics (penicillin/streptomycin, gentamycin; CSL Ltd) . A control transfection, using a pBB3.5-2 (N) plasmid with the ATG inserted at the 3' end of the 3.5 PCR clone 2 sequence, was also carried out. Recombinant plaques were selected on the basis that the pBlueBac II vector also encodes E.coli 0-galactosidase (3-gal) , by including X-gal in the agarose overlay at the recommended level. A selection of blue plaques were picked and subjected to two further rounds of plaque purification, after which time infected monolayers showed no evidence of contaminating wild-type virus (which would be evidenced by the presence of nuclear polyhedra) . Purified viruses were designated 3.5-2-P2A, -P3A and -P4A, and were amplified by two sequential infections of S_f9 cells before use. A plaque purified from the control transfection was designated 3.5-2-rev.

Assessment of HllOD Expression in Insect Cells Infected With Recombinant Baculovirus

Monolayers of S_f9 cells (1 x 10 ⁶ cells in 25cm ² bottles) were infected with the 3.5-2 viruses, with wild-type (wt) virus, v/ith a control virus expressing β- gal, or were not infected. After four days growth at 26°C, monolayers were detached by gentle shaking, the

cells recovered by centrifugation (2000 rpm, 10 minutes) , and the cell pellets disrupted by three cycles of freeze-thawing. The lysates were resuspended in 500μl PBS, and 25μl aliquots assayed for ApM activity by the micro-well assay.

15 μl aliquots (3 x 10 ^A cell equivalents) of the above lysates were electrophoresed on denaturing 7.5% SDS-polyacrylamide gels. One gel was then stained with Coomassie blue to assess levels of expression. The other gel was Western blotted, and the blot probed with anti- H110DN (as described earlier) .

RESULTS

ANALYSIS OF IMMUNOPOSITIVE CLONES

Analysis of antibodies affinity purified on clone Ml

Affinity-purified antibodies specific for each of the 5 antibody-positive clones were prepared and used to probe a Western blot of HHOD-enriched extract. As shown in Figure 8, all 5 clones appeared to recognise the HllOD doublet. However, the reaction with clone Ml gave the strongest signal (Figure 8d) compared to the λgtll negative control blot (Figure 8e) . This clone was therefore investigated further.

Northern Blot Analysis With Clone Ml

Northern blot analysis of Haemonchus contortus mRNA probed with the Ml insert is shown in Figure 9. A single mRNA band was recognised, at approximately 3.5kb. This is of sufficient size to code for a protein of about HOkd.

Sequence analysis of clone Ml

Analysis of restriction digests of the DNA with EcoRI showed the Ml insert to be approximately 300bp. The DNA sequence or the Ml fragment was determined (SEQ ID NO: 1, and is shown in Figure 3. The fragment

comprises 295bp with an open reading frame starting at base number 3.

Northern blot analysis with clone BIA

Northern blot analysis of Haemonchus contortus mRNA probed with the BIA insert is shown in Figure 9c. As for Ml, a single mRNA band was recognised, at approximately 3.5kb.

Sequencing of clones BIA and B2

Clones of BIA were sequenced (SEQ ID NOS: 2 and 3) and the full sequence (SEQ ID NO: 2) is shown aligned to Hll-l (SEQ ID NO: 19) and AustBl (SEQ ID NO: 6) in Figure 5. The insert is 484 bp and has a full ORF from the first base. The 3 fragments of B2 resulting from digestion with EcoRI were sequenced and the complete sequence for B2 (SEQ ID NO: 4) is 581 bp. It is shown aligned with Hll-2 in Figure 4. The sequence has an ORF from position 3 to 213 bp, the stop codon and untranslated region matching that of the 2.5 kb PCR product sequence (SEQ ID NO: 7) .

Expression of Ml and BIA in E.coli

When subcloned into a GST expression vector, clones were obtained which expressed fusion proteins of 38-40 kd for Ml and of 45kd for BIA. These agree with the predicted sizes for these inserts, allowing for the molecular weight of glutathione-S-transferase. Both fusion proteins reacted very strongly on Western blots with affinity-purified antibodies to H110DE (Figure 11) . The fusion proteins were expressed as insoluble inclusion bodies.

Antibody Responses in Sheep Vaccinated with Ml-GST and B1A-GST Fusion Proteins

Antisera from sheep injected with the fusion proteins were tested by Western blotting against HllOD preparations. Both GST-MI and GST-B1A raised antibodies

which specifically recognised the HllOD doublet (Figure 12) . Sera from negative control sheep did not recognise the HllOD doublet.

ISOLATION AND CHARACTERISATION OF CLONES SELECTED BY HYBRIDISATION WITH Ml OR BIA INSERT DNA

The confirmed positive clone hybridising to the Ml probe was designated MIAUS (SEQ ID NO: 5) , and the clone hybridising to BIA was designated AustBl (SEQ ID NO: 6) . Restriction digestion of purified plasmid DNAs with EcoRI indicated an insert size of about 900 bp for MIAUS and of about 1.6Kb for AustBl. As shown in Figure 9b) and 9d) , on Northern blots, MIAUS and AustBl hybridised to the same-sized mRNA (about 3.5 kb) as did Ml and BIA.

Sequence analysis of MIAUS

Full sequencing of the MIAUS fragment was carried out using synthetic oligonucleotides to "walk" along the DNA from either end. Analysis of the sequence obtained revealed that the MIAUS insert was 948 bp, as shown in Figure 3. The sequence (SEQ ID NO: 5) begins with an ATG (which codes for methionine) and has an open reading frame (ORF) over the whole of its length. The sequence is 19 base pairs longer than the Ml sequence at the 5' end, and 634bp longer at the 3' end. The sequence common to the two clones (bases 20 to 314) were identical except for two nucleotide differences in a third codon position. Comparison of all possible reading frames to various databases showed that the reading frame starting with the ATG at base number one shared homology with the members of a family of microsomal aminopeptidases.

Sequence of AustBl

Full sequencing of the AustBl fragment was carried out using synthetic oligonucleotides to "walk" along the DNA from either end. The DNA sequence (SEQ ID NO: 6) is

shown in Figure 5. The clone is 1689 bp long and has an ORF from residue 2. This sequence forms part of Hll-l as shown in Figure 1. The amino acid translation of this sequence showed the zinc binding site motif characteristic of aminopeptidases.

PCR AMPLIFICATION OF THE cDNA OF THE HllOD mRNAs PCR using Ml AUS primers cDNA was synthesized from Haemonchus contortus mRNA using as primer oligo-dT containing an adaptor sequence to facilitate subsequent cloning and manipulation of the DNA. This cDNA was then used to amplify the MIAUS sequence by PCR, using as the 5' end primer a synthetic oligonucleotide based on positions 865-885. A PCR fragment of about 2.5 Kb was amplified. This is approximately the expected size of the fragment, based on the known size of the mRNA and on mammalian aminopeptidase cDNA sequences.

A second set of PCR reactions was performed using a primer near the 5' end of MIAUS (bases 30-49). Four bands were detected on an agarose gel. The largest of these, at 3.5 kb, corresponds to the predicted size for the PCR product.

Cloning and sequencing of 2.5 kb and 3.5 kb PCR products from MIAUS primers

The 2.5 kb and 3.5 kb PCR products were cloned and designated 2.5PCR (SEQ ID NO: 7) and 3.5PCR (SEQ ID NOS: 8, 14 and 15 for clone numbers 2, 10 and 19 respectively). On Northern blots 2.5PCR and 3.5PCR (clone 2, 3.5PCR-2) hybridised with mRNA of about 3.5 kb (Figure 9e, f) in the same pattern (with respect to age of Haemonchus used to obtain the mRNA) as Ml, BIA, MIAUS and AustBl.

Full sequencing of clones was carried out by Oligonucleotide walking'. As shown in Figure 1, the sequence for the 2.5 kb product (SEQ ID NO: 7) is part

of Hll-2 (SEQ ID NO: 20) and the sequence for the 3.5 kb product (SEQ ID NO: 8) is the major part of Hll-3 (SEQ ID NO: 21) . The amino acid translations of both these sequences (shown in Figure 6) contain the zinc binding motif His Glu Xaa Xaa His Xaa Trp (HEXXHXW) characteristic of microsomal aminopeptidases.

Sequencing of 5'end PCR clones cDNA was synthesised using a primer matching a conserved sequence in cDNA clone AustBl, 2.5PCR and 3.5PCR (SEQ ID NOS: 6, 7 and 8) which hybridises with the mRNA for these sequences about 1.3Kb from the 5' end. The cDNAs were C-tailed at the 5' end and then PCR reactions carried out with a universal Anchor (A) primer for the 5' end and three primers specific for each of the sequences AUSTB1, 2.5PCR and 3.5PCR-Clone 2 (SEQ ID NOS: 6, 7, 8) for the 3' end. The reactions each gave a product of the predicted size, just under 1.3kb: 1301bp (SEQ ID NO: 9) , 1280bp (SEQ ID NO: 10) and 1292 (SEQ ID NO: 11) respectively. All three sequences have an untranslated region at the 5" end (Figure 2) . All begin with the same 22bp sequence (5' GGTTTAATTACCCAAGTTTGAG 3 ' ) which is known as the Spliced Leader Sequence 1 (SL1) and is present in the untranslated 5' region of a wide variety of nematodes Huang et al . , 1990. In SEQ ID NOS: 9 and 10, the SL1 sequence is immediately before the initiating ATG. In SEQ ID NO: 11 there are 13bp between the SL1 and the initiating ATG. All three sequences have full ORFs.

Sequencing of AustBl 3 ^« end PCR clone

Using a specific primer matching positions 1414- 1438 in Aust Bl (SEQ ID NO: 6) , the PCR product gave a band as predicted of about 1.3kb. Sequencing of the cloned band yielded the sequences SEQ ID NOS: 12 and 13. They gave an ORF from l-615bp and a substantial untranslated region.

Sequence analysis of Cloned PCR products

Composites of the sequences described above, designated Hll-l, Hll-2 and Hll-3, are shown in Figure 2. The amino acid sequences predicted from these are shown in Figure 6a. The validity of the predicted translations of the DNA sequences presented is substantially confirmed by the matches with amino acid sequences determined by Edman degradation from CNBr and proteolytic cleavage fragments (Figure 7) . Thus 27 residues of the 29 residue N-terminal sequence of HllS (SEQ ID NO: 16) match Hll-2 from residues 61-90 (Figure 7b) . The matches of valine (V) at position 78 and glycine (G) at position 90 are characteristic of Hll-2 since Hll-3 has asparagine (N) at position 90 and Hll-l has leucine (L) at position 86 (which corresponds to position 78 in HI1-2) . Two residues of the HllS N- terminus amino acid sequence (SEQ ID NO: 16) do not match any of the three HllOD sequences presented here. To be particularly noted are the exact matches of the very similar, but distinctive sequences Pep A and B (previously described in WO90/11086) with Hll-2 and Hll-l respectively in the region of residues 540-555. Similarly in the 450-470 residue region, Pep D is an exact match for Hll-2, while the similar but distinct Pep E matches more closely Hll-3.

By way of example the translated amino acid sequence of one of the full-length sequences (Hll-3) is compared in Figure 6b with two sequences for mammalian microsomal aminopeptidases. The homology of the HllOD translation v/ith these aminopeptidases is shown by boxing identical amino acids. A characteristic motif of microsomal aminopeptidases is the amino acid sequence HEXXHXW, which functions as the zinc binding site (Jongeneel et al.. 1989; Vallee et al. , 1990) ; this is shown by asterisks in Figure 6. This sequence, which is shown to be present in the translations of Hll-l, Hll-2 and Hll-3, is conserved in all the microsomal aminopeptidases. Other features common to the

mammalian and Haemonchus microsomal aminopeptidases are the presence of a comparatively short intracellular region, a single transmembrane sequence adjacent to the N-terminus and several potential glycosylation sites. The levels of homology (similarities of 52-55% and identities of 30-32%) of Hll-l, -2 and -3 to mammalian microsomal aminopeptidases are shown in Table 2.

Southern Blot Analyses

The results of H.contortus genomic DNA Southern blots probed with various HllOD cDNA clones and PCR products are shown in Figure 10. All probes show multiple bands of hybridisation; this is typical of a multigene family. As expected, BIA and AustBl showed similar hybridisation patterns to each other, as did MIAUS and 3.5kbPCR. However, these patterns were noticeably different from each other and from that seen with the 2.5KbPCR probe, even under conditions of moderate stringency (Figure 10A) , reflecting the differing levels of homology between these three cDNAs.

Demonstration of Aminopeptidase Activities Associated With HllOD

Microsomal aminopeptidase activity was found to associate with those fractions containing HllOD, that is the supernatants from ultracentrifugation of Thesit extracts, ConA binding fraction (ConA HllOD) and the fractions containing HllOD obtained by ion exchange chromatography on a MonoQ column (Table 3) . The specific activities with all substrates tested increased as the purity of the HllOD increased.

TABLE 3 ENZYME ACTIVITIES OF FRACTIONS FROM A TYPICAL HllOD PREPARATION

Effects of inhibitors of mammalian aminopeptidases on H110D aminopeptidase activities

Addition of the inhibitor bestatin (which inhibits mammalian microsomal aminopeptidase) to ConA HllOD at the concentration recommended by the supplier (Boehringer Mannheim) reduced the activity against leucine-p-nitroanilide by approximately 70%. A series of experiments were performed to test inhibition of activity by a range of protease inhibitors. Those inhibitors which v/ere not specific for metalloproteases or aminopeptidases had no inhibitory effects on the reaction rate. Inhibitors that are known to affect metalloproteases or aminopeptidases did have an effect on reaction rates, as shown in Table 4. The most effective inhibitor was 5mM phenanthroline.

TABLE 4

Inhibition of HllOD aminopeptidase activities using various protease inhibitors

¹ A mammalian nminopeptidase-A substrate.

² Λ mammalian aminopeptidase-M substrate.

Sub-fractionation of HllOD

The distribution of activities associated with fractions from ion exchange chromatography of ConAHHOD on MonoQ are shown in Figure 13a and SDS-PAGE of the fractions in Figure 13b.

Further, enzymatic activity was associated with sub-fractions of HllOD separated by re-cycling free flow isoelectric focussing (Figures 14 and 15) . At lower pi values (pH 4.5) these sub-fractions contain only the larger of the bands which make up the HllOD doublet seen on SDS-PAGE and at higher values (pH 6.5) they contain only the smaller of bands which make up the HllOD doublet. Intermediate fractions contain both these bands. The smaller band may also be obtained as a separate fraction by ion exchange chromatography on MonoQ using the Pharmacia SMART ^R apparatus. All these sub-fractions bind sheep antibodies to HllOD affinity purified on the protein expressed by the the λgtll clone Ml whereas antibodies eluted from λgtll with no insert do not bind (Figure 14c) . All the sub-fractions bind mouse monoclonal antibodies designated TS 3/19.7 (Figure 14c) which also bind to the recombinant protein expressed by clone Ml. All the sub-fractions show microsomal aminopeptidase activity (Figure 15c) although this activity is comparatively low in the fractions obtained at the highest and lowest pis. This lower activity may be attributed to lowered protein concentrations, effects of extremes of pH during sub- fractionation or a requirement for the presence of both larger and smaller bands for maximal activity.

Vaccination with separated components of HllOD

The separated upper and lower bands obtained by free flow isoelectric focussing or by ion exchange chromatography induce the formation of protective antibodies when injected into sheep as exemplified in the following experiment. Thirty sheep approximately six months old were assigned to 5 groups of 6 so that

each group was matched for range of weights of sheep. Each animal was injected with a total of 150μg protein given in 3 equal doses as described in Munn et al. (1992) and Tavernor et al. (1992a, b) over a period of 54 days. The animals in group L were injected with the lower (smaller) band of the HllO Doublet, those in group U with the upper band, U+L with recombined upper and lower bands, D with the two (unseparated) bands obtained by free-flow isoelectric focussing at intermediate pH values and as a control (group C) horse spleen ferritin (an antigenic unrelated protein) . The sheep were challenged with 10,000 infective larvae three weeks after the third injection and the experiment terminated 29-31 days post infection. The outcome of the experiment is summarised in Figure 16. Injection of any of the sub-fractions reduced parasite egg output throughout the trial by some 90% and reduced total worm numbers by 63-84%, all showing a significant difference (p<0.05) to the controls using non-parametric statistical analyses. Reductions (70-88%) in the numbers of female worms were greater than the reductions in the numbers of male worms, and (except for the reduction in male worm numbers in the sheep injected with the recombined upper and lower bands where p<0.07) for both sexes the reductions were significant (p<0.05) .

Vaccination with HllS and HHA

A truncated, water-soluble form of HllOD (HllS; which retains its enzymic activity) may be obtained from the native molecule by treatment with elastase. This form was found to contain predominantly ApM-like enzyme activity and a Thesit extract of the elastase digested pellet (HHA) was enriched for ApA-like activity (see Table 5) .

Table 5

Ratio Aminopeptidase-M : Aminopeptidase-A (leucine-pNA) (α-glutamic acid-pNA)

HllOD 1.44 : 1

HllS 26.0 : 1

HHA 0.48 : 1

The following experiment shows that vaccination of sheep with either HllS or HHA gives protection against Haemonchus challenge or infection. Eighteen sheep approximately eight months old were assigned to 3 groups of 6 so that each group was matched for range of weights of sheep. Each animal was injected with a total of lOOμg protein given in 2 equal doses as described in Munn et al. (1992) and Tavernor et al. (1992a, b) over a period of 23 days. The animals in group A were injected with HHA, those in group S with HllS and those in group C with horse spleen ferritin (an antigenically unrelated protein) as a negative control. The sheep were then challenged with 10,000 infective larvae 25 days after the second injection and the experiment terminated at 34-36 days post infection. The outcome of the experiment is summarised in Figure 17. Injection of HllS reduced parasite egg output throughout the trial by 89% and reduced total worm numbers by 76%. Injection of HHA reduced parasite egg output throughout the trial by 98% and reduced total worm numbers by 84%. These showed a significant difference (p<0.05) from the controls using non-parametric statistical analyses.

Inhibition of HllOD aminopeptidase activities by antibodies

Solutions containing HllOD were incubated with sera from individual sheep injected with fractions containing HllOD or from control sheep. The solutions were then assayed for aminopeptidase activities using

phenylalanine and α-glutamic acid pNAs as substrates. The degree of inhibition of activity (maximally 80%) correlated with the level of protection shown by the individual sheep from which the sera were obtained (see Figure 18) .

Localisation of Enzyme Activity

Frozen sections of adult Haemonchus contortus were examined for aminopeptidase activity. As shown in Figure 19, aminopeptidase enzyme activities are localised to the lu inal surface of the intestine. HllOD protein is also specifically found in this location.

EXPRESSION OF HllOD (3.5 PCR CLONE 2) USING THE EUKARYOTIC BACULOVIRUS-INSECT CELL SYSTEM

Expression of aminopeptidase activity in insect cells

Infected cells were harvested and assayed for aminopeptidase activities using phe-, leu-, met- and lys-pNA as substrates. The assay was complicated by the observation that the insect cells possess an aminopeptidase activity with a marked preference for lys-linked amide bonds. However, cell extracts containing the expressed HllOD additionally cleaved leu-, met- and phe-pNA in that order of preference.

Molecular Weight and Immunoreactivity of the Expressed HllOD (3.5 PCR clone 2) Protein

Samples of infected or control cell extracts were electrophoresed on a 7.5% SDS-polyacrylamide gel, which was then stained v/ith Coomassie Blue. The 3.5-2-P3A and 3.5-2-P4A infected cell lysates both had a band at the same size as HllOD, HOkd, which migrated directly beneath the co-expressed 3-gal , which has a molecular weight of 120kd. This HOkd band was not present in any of the negative control lysates. (It was also absent from the P2A lysate, which did not express enzyme

activity) .

A duplicate gel was Western blotted and probed with anti-HHODN (Figure 21) . A very strong, specific positive immunoreaction was obtained to the HOkd band expressed by 3.5-2-P3A and 3.5-2-P4A, and to the native HllOD doublet in a control track containing ConA HllOD, while no reaction was seen in any of the negative control tracks.

References

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GENERAL INFORMATION:

APPLICANT:

NAME: Agricultural and Food Research Council

STREET: Babraham Hall

CITY: Babraham

STATE OR PROVINCE: Cambridgeshire

COUNTRY: United Kingdom

POSTAL CODE: CB2 AT

TELEPHONE: 223-832312

TELEFAX: 223-837952

TITLE OF INVENTION: Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections

NUMBER OF SEQUENCES: 24

COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE:

INFORMATION FOR SEQ ID NO: 1:

SEQUENCE CHARACTERISTICS:

LENGTH: 295 base pairs TYPE: nucleic acid STRANDEDNESS : double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CGCGGACATT GCTGAATCTA ACTCCAATCC GTCTTATTGT CGCATTATTT CTAGTAGCTG 60

CTGCAGTCGG CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT 120

CAGAAAAGCC AGGGAAGGAT GATACTGGTG GCAAGGACAA AGACAATTCT CCCTCTGCGG 180

CGGAACTACT TCTTCCAAGT AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT 240

ATCTACCTGG TTATGTGGAC TTCCCACCGG AGAAAAACCT CACATTCGAC GGGCG 295

INFORMATION FOR SEQ ID NO: 2:

SEQUENCE CHARACTERISTICS:

LENGTH: 484 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GCAATATCAG GATTCAATAC AATTTTGGAC TTTTTCGGCA GCGAACCCGA ATCTCAATGG 60

GCTTCGGAAT ACATGCGAAA ACTGATGAAG CCAATTTATG ACAAGAGTAG CATCAAGTTT 120

ATAGCGGAGA ACTACAAAAA AGATTCGCTT TTCTTCAAAA ATAATCTCCA AATAGCTGTT 180

ATTGACACAT ACTGTGGTCT TGGAGGCAAA GAATGTCTTG AAGAAATGAA AAAGCTTTTT 240

GACAAGGAGG TCATGAAATG TCAACCTGGT CAGCAAGCGA CCGACTGCGT AAAGGTAACT 300

GCTCCTCTCC GAAAAACTGT TTACTGCTAT GGGGTCCAGG AAGGCGGTGA TGAGGCATTC 360

GACAAGGTGA TGGAACTATA TAATGCGGAA CAAGTGCAGT TGGAGAAAGA CAGTCTACGT 420

GAAGCATTGG GATGCCATAA AGACGTTACA GCTCTAAAGG GACTTCTTAT GCTGGCTTTG 480

GATC 484

INFORMATION FOR SEQ ID NO: 3 :

SEQUENCE CHARACTERISTICS:

LENGTH: 216 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 3:

ACCTGGTCAG CAAGCGACCG ACTGCGTAAA GGTAACTGCT CCTCTCAAAA CTGTTTACTG 60

CTATGGGGTC CAGGAAGGCG GTGATGAGGC ATTCGACAAG GTGATGGAAC TATATAATGC 120

GGAACAAGTG CAGTTGGAGA AAGACAGTCT ACGTGAAGCA TTGGGATGCC ATAAAGACGT 180

TACAGCTCTA AAGGGACTTC TTATGCTGGC TTTGGA 216

INFORMATION FOR SEQ ID NO: 4:

SEQUENCE CHARACTERISTICS:

LENGTH: 581 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTIO : SEQ ID NO: 4 :

GGGAGGAAAT CACTGCGAGC TTGGAAACAG AACACAGAGC AGTTGATAAA GTGGTCGGCG 60

CTTGTTGCAC AGGAATTCGC TCCCAACAAC AAATAGATCA GCTGAAGAAG AATCTACAGA 120

AGAACAATGC GCAGGCTAAG AAGTTCCATA AAATTGCCTG GATCAAGAAA CATTTTCACA 180

GATTATCGGA ATTCTTCAAG AGAGCAAGAT CATAGCTTTT CACACTGAGC TCCAATTTTA 240

ACGTCTTCAA ACTAGGAGAC AGTTTTGCTG AAAAGTCAGT TTCACATTTT CCGTTTGAAT 300

GCCATCCATT CGAATACAAC CAACCCCATT TTAAGTACCT TTCATTCACA GTGATTACTA 360

AATTTCGAAT ATATTATGAA GCTTGTATCT TGAACGTTAT GATCGGTGAC TTTCAATTTA 420

TAGAGCTCAC TCTCCATTTT GTAGCTGTGA TGACTTGCAT TTAAGACCCA CCATTTACCA 480

'-GCCTATAATC TTTCCCCAAT ACATTCCAAA CTCCGATCAC CTCCACCGCT GACAATGCCC 540

AGATTTGTTT CTTTGTCTGC TATCCATCTA ACTGTTTCGA T 581

INFORMATION FOR SEQ ID NO: 5:

SEQUENCE CHARACTERISTICS:

LENGTH: 948 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 5:

ATGACGTCGC AGGGGAGAAC GCGGACATTG CTGAATCTAA CTCCAATCCG TCTTATTGTC 60

GCATTATTTC TAGTAGCTGC TGCAGTCGGC CTCTCTATTG GTCTCACCTA TTACTTTACT 120

CGCAAAGCGT TCGATACCTC AGAAAAGCCA GGGAAGGATG ATACTGGTGG CAAGGACAAA 180

GACAATTCTC CCTCTGCGGC GGAACTACTC CTTCCAAGTA ATATAAAACC ATTGTCTTAC 240

GACTTGACGA TCAAAACATA TCTACCTGGT TATGTGGACT TCCCACCGGA GAAAAACCTC 300

ACATTCGATG GGCGTGTGGA AATATCAATG GTTGTAATTG AGCCAACAAA GAGTATCGTA 360

CTCAATTCAA AGAAGATCTC TGTAATACCC CAAGAATGTG AACTGGTATC GGGCGATAAA 420

AAATTCGAAA TTGAAAGTGT AAAGGAGCAC CCAAGACTGG AAAAGGTTGA GTTTCTTATC 480

AAAAGCCAAC TGGAAAAAGA TTCACAAATC TTGCTCAAGT CGGCTTACAT CGGTCTCATC 540

AGCAACAGCC TTGGTGGAAT CTACCAGACC ACTTATACCA CCCCGGATGG CACCCCTAAG 600

ATCGCTGCAG TTTCACAAAA TGAGCCCATA GATGCTCGTC GAATGGTACC ATGCATGGAT 660

GAACCGAAAT ACAAAGCAAA CTGGACCGTT ACTGTCATTC ATCCAAAAGG CACCAAAGCC 720

GTCTCGAATG GAATCGAAGT GAACGGAGAT GGAGAGATCA GTGGTGATTG GATCACATCG 780

AAGTTCTTGA CTACTCCACG GATGTCATCC TACTTGTTGG CAGTTATGGT TTCAGAATTT 840

GAATACATCG AAGGTGAAAC AAAGACGGGT GTTCGGTTCC GTATATGGTC ACGCCCAGAG 900

GCAAAGAAGA TGACACAATA TGCTCTGCAA TCTGGTATCA AGTGCATA 948

INFORMATION FOR SEQ ID NO: 6:

SEQUENCE CHARACTERISTICS:

LENGTH: 1689 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 6:

INFORMATION FOR SEQ ID NO: 7 :

SEQUENCE CHARACTERISTICS:

LENGTH: 2472 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 7

CCAGAGAAAT ATCGTAATCC AAAATACGGG TTCAAGTGGG ATGTTCCCCT ATGGTATCAG 900 GAAGGCAATA GCAAAGAGGT GAAGCGAACA TGGCTAAAAA GAGATGAACC GCTGTACTTG 960 AACGTCAACA ATCGGGATAC ATCCCTTGTG GTGAACGCTG ATCGACATGG ATTTTATCGA 1O20 CAAAACTATG ATGCCAACGG TTGGAAAAAG ATAATCAAGC AGCTCAAGAA AGATCACAAG 1080 GTCTTCGGTC CAAGGACAAG GAACGCTATC ATAAGCGATG CATTTGCTGC AGCTACGATT 1140 GACGCAATCG ACTATGAAAC TGTATTCGAA CTACTTGAAT ATGCCAAAAA TGAAGAGGAA 1200 TTCTTGCCTT GGAAGGAAGC TCTGTCCGGC ATGTTCGCAG TTTTAAAGTT CTTCGGTAAT 1260 GAGCCGGAGA CAAAACCAGC TAGAGCTTAC ATGATGAGCA TATTAGAACC GATGTATAAT 1320 AAGAGCAGCA TTGATTACAT CGTCAAGAAT TATTTGGATG ATACGTTATT CACAAAAATT 1380 AATACTCAAA AGGATATCAT TGATGCATAT TGTTCCCTTG GATCAAAGGA CTGTATAAAG 1440 CAATATAAGG ATATCTTCTA CGATGAGGTT ATGCCCAAGT GTAAGGCCGG GGAAGCAGCA 1500 ACCAAATGCG TTAAGGTTTC CGCTCCTCTT CGAGCCAATG TTTACTGTTA TGGTGTACAG 1560 GAAGGTGGTG AAGAAGCTTT TGAAAAGGTG ATGGGGCTGT ATCTAGCAGA AGATGTTCAA 1620 CTGGAGAAGG GTATCCTGTT CAAAGCCTTG GCATGCCACA AAGATGTTAC AGCTCTAAAA 1680 GAACTTCTTT TGCGAGCCCT GGACCGTAAA TCGTCGTTTG TGCGTCTTCA GGATGTCCCT 1740 ACCGCTTTCC GTGCTGTATC TGAAAACCCT GTGGGCGAAG AATTCATGTT CAATTTCCTA 1800 ATGGAGAGAT GGGAGGAAAT CACTGCGAGC TTGGAAACAG AACACAGAGC AGTTGATAAA 1860 GTGGTCGGCG CTTGTTGCAC AGGAATTCGC TCCCAACAAC AAATAGATCA GCTGAAGAAT 1920 CTACAGAAGA ACAATGCGCA GGCTAAGAAG TTCGGCTCAT TCACCCAGGA AATCGAAAAA 1980 GGAGAACATA AAATTGCCTG GATCAAGAAA CATTTTCACA GATTATCGGA ATTCTTCAAG 2040 AGAGCAAGAT CATAGCTTTT CACACTGAGC TCCAATTTTA ACGTCTTCAA ACTAGGAGAC 2100 AGTTTTGCTG AAAAGTCAGT TTCACATTTT CCGTTTGAAT GCCATCCATT CGAATACAAC 2160 CAATAATACC ATTTTAAGTA CCTTTCATTC ACAGTGATTA CTGAATTTCG AATATATCAT 2220 GAAGCTTGTA TCTTGAACGT TATGATCGGT GACTTTCAAT TTATAGAGCT CACTCTCCAT 2280 TTTGTAGCTG TGATGACTTG CATTTAAGAC CCACCATTTA CCAGCCTAGA ATCTTTCCCC 2340 AATACATTCC AAACTCCGAT CACCTCCACC GCTGACAATG CCCAGATTTG TTTTTTTGTC 2400 TGCTATCCAT CTAACTGTTT CGATCGCCGG TTGTTTGTCA ATTGCTTATC TGATAAATAT 2460 TGACGTTGGT GT 2472

INFORMATION FOR SEQ ID NO: 8:

SEQUENCE CHARACTERISTICS:

LENGTH: 3305 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GCTGAATCTA ACTCCAATCC GTCTTATTGT CGCATTATTT CTAGTAGCTG CTGCAGTCGG 60 CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC 120 AGGGAAGGAT GATACTGGTG GCAAGGACAA AGACAATTCT CCCTCTGCGG CGGAACTACT 180 CCTTCCAAGT AATATAAAAC CATTGTCTTA CGACTTGACG ATCAAAACAT ATCTACCTGG 240 TTATGTGGAC TTCCCACCGG AGAAAAACCT CACATTCGAT GGGCGTGTGG AAATATCAAT 300 GGTTGTAATT GAGCCAACAA AGAGTATCGT ACTCAATTCA AAGAAGATCT CTGTAATACC 360 CCAAGAATGT GAACTGGTAT CGGGCGATAA AAAACTCGAA ATTGAAAGTG TAAAGGAGCA 420 CCCAAGACTG GAAAAGGTTG AGTTTCTTAT CAAAAGCCAA CTGGAAAAAG ATCAACAAAT 480 CTTGCTCAAG GTCGGCTACA TCGGTCTCAT CAGCAACAGC TTTGGTGGAA TCTACCAGAC 540 CACTTATACC ACCCCGGATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT 600 AGATGCTCGT CGAATGGTAC CATGCATGGA TGAACCGAAA TACAAAGCAA ACTGGACCGT 660 TACTGTCATT CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA 720 TGGAGAGATC AGTGGTGATT GGATCACATC GAAGTTCTTG ACTACTCCAC GGATGTCATC 780 CTACTTGTTG GCAGTTATGG TTTCAGAATT TGAATACATC GAAGGTGAAA CAAAGACGGG 840 TGTTCGGTTC CGTATATGGT CACGCCCAGA GGCAAAGAAG ATGACACAAT ATGCTCTGCA 900 ATCTGGTATC AAGTGCATAG AATTCTACGA AGATTTCTTT GATATCAGAT TCCCTCTGAA 960 GAAACAAGAT ATGATTGCCC TTCCTGATTT CTCTGCCGGT GCCATGGAGA ATTGGGGCCT 1020 CATCACTTAC AGGGAAAACT CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA 1080 ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCTCAT CAGTGGTTCG GCGACTTGGT 1140 TACGATGAAG TGGTGGGATA ATTTGTGGTT GAATGAAGGT TTTGCAAGAT TCACAGAATT 1200 TATTGGAGCT GGTCAGATAA CTCAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA 1260 TGTACTTGAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG 1320 AATCGACAAA GCTGCAGAAG TTGAAGAAGC CTTTGATGAT ATCACATACG CCAAAGGAGC 1380 TTCTGTTCTT ACTATGCTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC 1440 GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1500 TGATGAAGTT GTCACTGACG TCGAAGGTCC AGACGGCAAA CCTATGAAAA CCACAGAGTT 1560

TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG AGTTTAACTC 1620 GACTACTTTG AAATTAACGC AAAGTCGATA TGAGGCGAAT AAAGACGCTG TGGAGAAAGA 1680 GAAGTACCGT CACCCGAAAT ACGGATTTAA ATGGGATATT CCACTGTGGT ATCAGGAAGG 1740 CGATAAGAAG GAGATAAAGC GAACATGGTT GAGAAGAGAT GAACCGCTTT ACTTGCATGT 1800 TAGTGATGCT GGCGCTCCCT TTGTGGTGAA CGCAGACCGC TATGGATTTT ATCGACAAAA 1860 TCATGACGCT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGATAATC ATGAGGTTTA 1920 CAGTCCCCGG ACAAGGAATG TCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC 1980 AATTGAGTAT GAGACTGTAT TTGAACTTCT GAATTATGCC GAAAAAGAAA CGGAATATCT 2040 ACCATTAGAA ATCGCAATGT CCGGGATCTC TTCGATTTTG AAATACTTCC CTACCGAGCC 2100 AGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAACCGATGT ATGAAAAAAG 2160 CAGTATCGAC TTCATTGCCA ATAACTACAG AAATGACAAG CTGTTTTTCC AAATCAACCT 2220 CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTCGGATCG CAAGACTGCA GGAAGAAATA 2280 TAAAAAACTT TTCGATGACG AAGTCATGAA CAAATGCAGG GATGGTCAAG CAGCAACCGA 2340 ATGCGTAAGA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 2400 CGGTGATTAT GCTTCCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TCGCCCTAGA 2460 AAAAGACTTC CTACGCCTAG CATTGGGATG TCATAAAGAT GTTACTGCTT TGAAAGGACT 2520 TCTCTTGCGG GCTCTGGACA GGAATTCGTC GTTCGTACGT ATGCAGGATA TCCCAAGTGC 2580 TTTCAACGAT GTAGCAGCAA ATCCTATTGG CGAAGAATTC ATTTTCAATT TCCTTATTGA 2640 GAGATGGCCA GATATCATTG AAAGTATAGG AACGAAGCAC ACATATGTTG AGAAAGTGAT 2700 ACCAGCCTGC ACTTCAGGAA TCCGCTCACA ACAGCAGATT GACCAGCTGA AGAATCTGCA 2760 GAAAAATGGC ATGAACGCTC GTCAATTCGG TGCATTCGAT AAAGCAATCG AACGAGCACA 2820 AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT TCAAGAAAGC 2880 CACCTTGTAA TTCGAATTAC ATTGCCAGTA ATCCAGATCT TAAAGTTCAT GAAGGAATAT 2940 GACAGGGAAC TGACTGTCTG TTGGTCACTG TTCCACTGAA TGGAAGTTTT TACCTACAAA 3000 AATTTTTATC GTTATATTTG CCTTCCGTGA GGGGTCATTG TTGTCACTTG AATAGTAAAC 3060 AAAGCTCAGT ATTGGCAACC GTAGAACAAT ATTACTTTCG CTTCATCAAA TTGTTATCTT 3120 CCCTATACCC TCTTCCTAAC TGAATTCGGA AATTTGTTCA TATTCGTTTG TAGTCTGTTG 3180 CTCAGAACAC TTTCTCCTCA ATAGCTTCTT GTTTGTTTTT TTTTTGATTG TATTGATCGT 3240 TTTACAATTG TATAGATTAG TTATCTTATA AATATTGATG GTTAAAAAAA AAAAAAAAAA 3300 AAAAA 3305

INFORMATION FOR SEQ ID NO: 9 :

SEQUENCE CHARACTERISTICS:

LENGTH: 1301 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTIO : SEQ ID NO: 9 :

INFORMATION FOR SEQ ID NO : 10 :

SEQUENCE CHARACTERISTICS :

LENGTH : 1280 base pairs TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear

SEQUENCE DESCRIPTION : SEQ ID NO : 10 :

GGTTTAATTA CCCAAGTTTG AGATGACGGC GGAGTGGCAG AAGCGTCGAA TCTTGGGCTT 60

CTCACCTATC AGCCTACTTT GTACATTATT TGTATTAGCT GCTGCCGTTG GACTCTCCAT 120

TGGTCTTACC TATTACTTCA CTCGTAAAGC ATTCGATACC ACACAAAAAG AACAGAAGGA 180

TGACAGTGGT GGTAAAGAAA AGGATAATTC TCCTTCTGCA GAAGAACTAC TTCTTCCAAC 240

GAACATAAAA CCAGTCTCGT ACGACTTGAA CATCAAAACA TATCTACCGG GTTACGTGAA 300

CTTTCCACCA GAAAAGAATC TCACATTTGA TGCCCATGTG GAGATTGCTA TGGTTGTGGT 360

TGAGCCTACA AATAGTATTG TGCTGAACTC GAAGAAAATC ACTTTGGCAC AAGGAGGATG 420

CGAACTGTTC TCAGGTAATC AGAAACTTGA CATCGAAAGT GTAAAGATGC AGGAAAGACT 480

TGACAAGCTT GAGATTACCC TCAAAAATCA GCTGCAAAAA GATCTGAAAA TCCTGCTCAA 540

GATCACTTAC ACCGGCCTTA TTAGCGACAC TCTCGGTGGG CTCTACCAGT CCATCTACAC 600

TGATAAGGAC GGAAAAACTA AGATCGTTGC TGTTTCACAA AATGAACCAT CAGACGCTCG 660

TCGTATAGCG CCATGCTTTG ACGAACCGAA GTACAAGGCA ACATGGACTG TCACCGTCGT 720

TCATCCCAAA GGTACAAAGG CTGCATCGAA CGGCATTGAA GCAAATGGAA AAGGGGAGCT 780

CAAGGGTGAT TGGATAACGT CTAAATTTAA AACTACCCCA CCGATGTCGT CCTATTTATT 840

GGCTATTATT GTTTGTGAAT TTGAATACAT TGAAGGATTT ACAAAAACAG GTGTACGGTT 900

CCGTATATGG TCTCGACCAG AGGCGATGGC AATGACGGGA TATGCCCTGG ATGCTGGCAT 960

CAGATGTCTG GAGTTCTATG AGAGATTCTT TGACATCAAA TTCCCTCTGG AAAAACAAGA 1020

TATGATTGCT CTACCTGATT TCACCGCTGG TGCTATGGAA AACTGGGGTC TTATCACTTA 1080

CAGAGAGGAT TCTCTTCTAT ACGATGAGAA AATTTATGCG CCGATGAATA AGCAGCGGGT 1140

TGCTCTCGTA GTTGCACACG AGCTTGCTCA TCAGTGGTTC GGCAATCTGG TCACATTGAA 1200

GTGGTGGGAT GATACGTGGT TGAACGAAGG TTTTGCGACA TTTGTTGAAT ATCTTGGAAT 1260

GGACGAAATT AGCCACAACA 1280

INFORMATION FOR SEQ ID NO : 11 :

SEQUENCE CHARACTERISTICS : LENGTH : 1293 base pairs TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear

SEQUENCE DESCRIPTION : SEQ ID NO : 11 :

GGTTTAATTA CCCAAGTTTG AGGGTCTCCA TCTAGATGAC GTCGCAGGGG AGAACGCGGA 60

CATTGCTGAA TCTAACTCCA ATCCGTCTTA TTGTCGCATT ATTTCTAGTA GCTGCTGCAG 120

TCGGCCTCTC TATTGGTCTC ACCΓATTACT TTACTCGCAA AGCGTΓCGAT ACCΓCAGAAA 180

AGCCAGGGAA GGATGATACT GGTGGCAAGG ACAAAGACAA TTCTCCCTCT GCGGCGGAAC 240

TACTCCΓTCC AAGTAATATA AAACCATTGT CΓTACGACTT GACGATCAAA ACATATCTAC 300

CTGGTTATGT GGACTTCCCA CCGGAGAAAA ACCTCACATT CGATGGGCGT GTGGAAATAT 360

CAATGGTTGT AATTGAGCCA ACAAAGAGTA TCGTACTCAA TTCAAAGAAG ATCTCTGTAA 420

TACCCCAAGA ATGTGAACTG GTATCGGGCG ATAAAAAACT CGAAATTGAA AGTGTAAAGG 480

AGCACCCAAG ACTGGAAAAG GTTGAGTTTC TTATCAAAAG CCAACTGGAA AAAGATCAAC 540

AAATCTTGCT CAAGGTCGGC TACATCGGTC TCATCAGCAA CAGCCTTGGT GGAATCTACC 600

AGACCACTTA TACCACCCCG GATGGCACCC CTAAGATCGC TGCAGTTTCA CAAAATGAGC 660

CCATAGATGC TCGTCGAATG GTACCATGCA TGGATGAACC GAAATACAAA GCAAACTGGA 720

CCGTTACTGT CATTCATCCA AAAGGCACCA AAGCCGTCTC GAATGGAATC GAAGTGAACG 780

GAGATGGAGA GATCAGTGGT GATTGGATCA CATCGAAGTT CTTGACTACT CCACGGATGT 840

CATCCTACTT GTTGGCAGTT ATGGTTTCAG AATTTGAATA CATCGAAGGT GAAACAAAGA 900

CGGGTGTTCG GTTCCGTATA TGGTCACGCC CAGAGGCAAA GAAGATGACA CAATATGCTC 960

TGCAATCTGG TATCAAGTGC ATAGAATTCT ACGAAGATTT CTTTGATATC AGATTCCCTC 1020

TGAAGAAACA AGATATGATT GCCCTTCCTG ATTTCTCTGC CGGTGCCATG GAGAATTGGG 1080

GCCTCATCAC TTACAGGGAA AACTCTTTGT TGTACGATGA CAGATTCTAT GCACCGATGA 1140

ATAAACAGCG AATTGCTCGC ATTGTTGCTC ATGAGCTTGC TCATCAGTGG TTCGGCGACT 1200

TGGTTACGAT GAAGTGGTGG GATAATTTGT GGTTGAATGA AGGTTTTGCA AGATTCACAG 1260

AATTCACTGG AGCTGGTCAG ATAACTCAAG ATG 1293

INFORMATION FOR SEQ ID NO: 12:

SEQUENCE CHARACTERISTICS :

LENGTH: 746 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 12:

INFORMATION FOR SEQ ID NO: 13:

SEQUENCE CHARACTERISTICS:

LENGTH: 1274 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 13

INFORMATION FOR SEQ ID NO : 14 :

SEQUENCE CHARACTERISTICS :

LENGTH : 3296 base pairs TYPE : nucleic acid STRANDEDNESS : double TOPOLOGY : linear

SEQUENCE DESCRIPTION : SEQ ID NO : 14 :

GCTGAATCTA ACTCCAATCC GTCTAATTTT TGCATTATTT CTAGTAGCTG CTGCAGTCGG 60

CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC 120

AGGGAAGGAT GATACTGGTG GCAAGGGCAA AGACAATTCT CCCTCTGCGG CGGAACTACT 180

TCTTCCAACC AATATAAAAC CATTGTCTTA CGATTTGACG ATCAAAACAT ATCTACCTGG 2 0

TTATGTGAAC TTCCCACCGG AGAAGAATCT CACATTCGAT GGGCGTGTGG AAATATCAAT 300

GGTTGTAATT GAGCCAACAA AGAGTATCGT GCTCAATTCA AAGAAGATCT CTGTGATACC 360

CCAAGAATGT GAACTGGTAT CGGGCGATAA AAAACACGAA ATTGAAAGTG TAAAGGAGCA 420

CCCAAGACTG GAAAAGGTCG AGTTTCTTCT TAAGAACCAA CTGGAAAAAG ATCAACAAAT 480

CTTGCTCAAG GTCGGCTATA TCGGCCTCAT CAGCAACAGT CTTGGAGGAA TCTACCAGAC 5 0

CACTTACACC ACCCCGAATG GCACCCCTAA GATCGCTGCA GTTTCACAAA ATGAGCCCAT 600

AGATGCTCGT CGAATGGTAC CATGCATGGA CGAACCGAAA TACAAAGCAA ACTGGACCGT 660

TACTGTCATT CATCCAAAAG GCACCAAAGC CGTCTCGAAT GGAATCGAAG TGAACGGAGA 720

TGGAGAGATC AGTGGTGATT GGATCACATC GAAGTTCTTG ACTACTCCAC GGATGTCATC 780

CTACTTGTTG GCAGTTATGG TTTCAGAATT TGAATACATT GAAGGTGAAA CAAGGACGGG 840

TGTCCGGTTC CGCATATGGT CACGCCCAGA GGCCAAGAAG ATGACAAAAC TTGCTTTGGA 900

TTATGGTATC AAATGCATAG AGTTCTACGA AGATTTCTTT GATATCAAAT TCCCTCTGAA 960

AAAACAAGAT ATGATCGCCC TTCCTGATTT CTCAGCAGGA GCCATGGAGA ACTGGGGTCT 1020

TATCACTTAC AGGGAAAACT CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA 1080

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCCCAT CAGTGGTTTG GGGACTTGGT 1140

TACAATGAAG TGGTGGGATA ATCTGTGGTT GAATGAAGGT TTTGCAAGAT TCACGGAATT 1200

CATTGGAGCT GGTCAGATAA CTAAAGATGA CGCCAGAATG AGGAACTACT TTCTGATTGA 1260

TGTACTTGAA CGCGCTTTGA AAGCTGATTC GGTAGCGTCA AGCCATCCAC TTTCCTTCAG 1320

AATCGACAAA GCTGCAGAAG TTGAAGAAGC CTTTGATGAT ATCACATACG CCAAAGGAGC 1380

TTCTGTTCTT ACTATGTTGA GAGCCTTGAT TGGAGAAGAA AAACATAAGC ATGCAGTATC 1440

GCAGTACCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1500

CGATGAAGTT GTCACTGACG TCGAAGGTCC AGACGGCAAA CCTATGAAAA CCACGGAATT 1560

TGCAAGTCAG TGGACGACTC AGATGGGCTT CCCAGTTATT TCCGTAGCAG AGTTTAACTC 1620

GACTACTTTG AAATTAACGC AAAGTCGATA TAAGGCGAAT AAAGACGCTG TGGAGAAAGA 1680

GAAGTACCGT CATCCGAAAT ACGGATTTAA ATGGGATATT CCATTGTGGT ATCAGGAAGG 1740

CGATAAGAAG GAGATAAAGC GAACATGGCT GAGAAGAGAT GAACCGCTTT ACTTGCATGT 1800

TAGTAATCCT GGTGCTCCAT TTGTGGTGAA CGCAGACCGC TATGGATTTT ATCGACAAAA 1860

TCATGACGCT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGACAATC ATGAGGTTTA 1920

TAGTCCTCGG ACAAGGAATG TCATCATTAG CGATGCGTTT GCTGCAGCCG CAACTGACGC 1980

AATTGAGTAT GAGACTGTTT TTGAACTTCT GAAATATGCC GAAAAAGAAA CGGAATACCT 2040

ACCGTTGGAA ATAGCAATGT CCGGGATCTC TTCGATTTTG AAGTACTTCG GTACCGAGCC 2100

GGAAGCAAAG CCAGCTCAAG TGTACATGAT GAACATATTG AAGCCGATGT ATGAAAAAAG 2160

CAGTATCGAG TTCATTACCA ATAACTACAG AAACGACACG CTGTTTTTCC AAATCAACCT 2220

CCAAAAGGAT GTCGTTGATA TGTTCTGCGC CCTTGGATCG CAAGACTGCA GGCAGAAATA 2280

TAAAAAACTT TTCGATGACG AAGTCATGGC GAAATGCAGG GATGGTCAAG CAGCAACCGA 2340

ATGCGTGAGA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 2400

CGGTGATTAT GCTTTCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TTGCCCTAGA 2460

AAAAGACTTC CTACGCCTAG CATTAGGATG TCACAAAGAT GTTACTGCTT TGAAAGGACT 2520

TCTCTTGCGG GCTCTGGACA GGAATTCGTC ATTCGTACGT ATGCAGGATA TCCCAAGTGC 2580

TTTCAACGAT GTAGCAGCAA ATCCTATTGG CGAAGAATTC ATTTTCAATT TCCTCATTGA 2640

GAGATGGCCA GATATCATTG AAAGTATAGG AACGAAGCAC ACATATGTTG AGAAAGTGAT 2700

ACCAGCCTGC ACTTCAGGAA TCCGCTCACA ACAGCAGATT GACCAGCTGA AGAATCTGCA 2760

GAAAAATGGC ATAAATGCTC GTCAATTTGG TGCATTCGAT AAAGCAATCG AACGAGCACA 2820

AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT TCAAGAAAGC 2880

CACCTTGTAG TTTGAATTAC GTCGCCATTA ATCCAGATCT TAAAGCTCGC TAAGGAATAT 2940

GTGGGAACTG ACTGTGTGTT GGTTACTGTT CCACTGAATG GAAGTTTTTA CCCACAAAAA 3000

TTTTTACCAT TTGCCTTCCA TGAGGGGTCA TTGTTGTCAC TTGAATAGTA AACAAAGCTC 3060

AGTATTAGGA CCCAGTGATC AATATTACTT TTGCTTCATC AAATTGTTAC CTTCTCTATA 3120

CCCTCTTCCT ACCTGAATTC AGAAATTTGT TCATATTCGT TTGTAGTCTG TTGCTCAGAA 3180

CACTTTCTCC TCGATAGCTT TTTGTTTGTT TTTCTTTTGA TTGTATTGAT CGTTTTACAA 3240

TTGTATAGAT TAGTTATCTG ATAAATATTG ATGGCTAAAA AAAAAAAAAA AAAAAA 3296

INFORMATION FOR SEQ ID NO: 15:

SEQUENCE CHARACTERISTICS:

LENGTH: 3319 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GCTGAATCTA ACTCCAATCC GTCTAATTTT TGCATTATTT CTAGTAGCCG CTGCAGTCGG 60

CCTCTCTATT GGTCTCACCT ATTACTTTAC TCGCAAAGCG TTCGATACCT CAGAAAAGCC 120

AGGGAAAGAT GATACTGGTG GTAAAGACAA AGATAATTCT CCCTCTGCGG CGGAACTACT 180

TCTTCCAACC AACATAAAAC CATTGTCTTA CGATTTGACA ATCAAAACAT ATCTACCTGG 240

TTATGTGAAC TTCCCACCGG AGAAGAATCT CACATTTGAT GGGCGTGTCG AAATTTCAAC 300

GGTTGTCATT GAGCCAACAA AGAGTATCGT GCTCAATTCA AAGAAGATCT CAGTAATACC 360

CCCTGAATGT GAACTGGTAT CGGGCGGTAA AAAACTCGAA ATCGAAAATG TAAAGGATCA 420

CCCAAGACTG GAAAAGGTTG AGTTTCTTCT TAAGAACCAA CTGGAAAAAG ATCAACAAAT 480

CTTGCTCAAG GTTGCCTACA TCGGCCTCAT CAGCAACAGC CTTGGCGGAA TCTACCAGAC 540

CACTTACACA ACCCCGGATG GCACCCCCAA GATCGCTACA GTGTCACAAA ATGAGCCCAT 600

AGATGCTCGT CGGATGGTGC CATGCATGGA TGAACCGAAA TACAAAGCGA ATTGGACCGT 660

TACTGTCATT CATCCAAAAG GTACAAAAGC CGTCTCGAAT GGCATCGAAA CGAACGGAGA 720

TGGAGAGATC AGTGGTGATT GGATTACGTC GAAGTTCTTG ACTACTCCGA GGATGTCATC 780

CTACTTGTTG GCAGTTATGG TATCAGAATT TGAATTTATC GAGGGTAAAA CAAAGACAGA 840

TGTTCGGTTC CGTATATGGT CACGCCCAGA GGCCAAGAAG ATGACAAAAC TTGCTTTGGA 900

TTATGGTATC AAATGCATAG AGTTCTACGA AGATTTCTTT GATATCAGAT TCCCCTTAAA 960

GAAACAAGAT ATGATCGCCC TTCCTGATTT CTCAGCAGGA GCCATGGAGA ACTGGGGTCT 1020

TATCACTTAC AGGGAAAACC CTTTGTTGTA CGATGACAGA TTCTATGCAC CGATGAATAA 1080

ACAGCGAATT GCTCGCATTG TTGCTCATGA GCTTGCCCAT CAGTGGTTTG GCGACTTGGT 1140

TACGATGAAG TGGTGGGATA ATCTGTGGTT GAATGAAGGT TTTGCAAGAT TCACAGAATT 1200

CATTGGAGCT GGTAAGATAA CTGAAGATGA CGCCAGAATG AGGAACTACT TCCTGATTGA 1260

TGTACTTGAA CGCGCGTTGA AAGCTGATTC CGTAGCGTCA AGCCATCCAC TTTCCTTCAG 1320

AATCGACAAA GCTGCAGAAG TTGAAGAAGC GTTTGATGAT ATCACATACG CCAAAGGAGC 1380

TTCTGTTCTT ACGATGCTGA GAGCGTTGAT CGGAGAAGAA AAACATAAGC ATGCGGTATC 1440

GCAGTATCTC AAGAAGTTCT CGTATAGCAA TGCAGAAGCG ACTGATCTAT GGGCAGTTTT 1500

CGATGAAGTT GTCACTGATG TCGAGGGTCC AGACGGCAAA CCTATGAAAA CCACGGAATT 1560

CGCAAGTCAG TGGACAACTC AGATGGGCTT TCCAGTAATT TCTGTGGCAG AGTTTAACTC 1620

GACTACTCTG AAACTAACGC AAAGTCGATA TAAGGCGAAT AAGGACGCTG TTGAGAAAGA 1680

GAAATACCGT CATCCGAAAT ACGGATTCAA GTGGGATATT CCATTGTGGT ATCAGGAAGG 1740

CGATAAGAAG GAGGTAAAGC GAGCATGGTT AAGAAGAGGT GAACCGCTTT ACTTGCATGT 1800

GAGTGATCCT GGCGCTCCAT TTGTGGTGAA TGCGGACCGC TATGGATTTT ACCGACAAAA 1860

CCACGACACT AATGGTTGGA AAAAGATAAT CAAGCAGCTC AAGGATAATC ATGAGGTTTA 1920

CAGTCCCCGG ACAAGGAATG CCATCATTAG CGATGCGTTT GCTGCGGCTG CAACTGACGC 1980

GATTGAGTAC GAGACTGTAT TCGAACTTCT GAAATATGCC GAAAAAGAAA CGGAATACCT 2040

ACCGTTGGAA ATAGCAATGT CTGGAATCTC TTCGATTTTG AAGTACTTCG GTACCGAGCC 2100

CGAGGCAAAG CCAGCTCAAA CATACATGAT GAACATATTG AAGCCGATGT ATGAGAAAAG 2160

CGATATCGAC TTCATTGCCA AAAACTACAA GGACGACAAG CTGTTTTTCC AAATCAACCT 2220

CCAAAAAGAT GTCATTGATA TGTTCTGCGC CCTTGGATCG CAAGACTGCA GGAAGAAATA 2280

TAAAAAACTT TTCGATGACA AAGTCATGGC GAAATGCAGG GATGGCCAAG CAGCAACCGA 2340

ATGCGTGAAA ATCGCCGCTC CTCTCCGATC AAGTGTTTAT TGTTATGGTG TGAAGGAAGG 2400

CGGTGATTAT GCTTTCGACA AGGTGATGGA GCTTTATACG GCCGAAACAC TCGCCCTAGA 2460

AAAAGACTTC CTACGCCTAG CATTAGGATG TCACAAAGAT GTTACCGCTT TGAAAGGACT 2520

TCTCCTGCGG GCTCTGGACA GGAATTCGTC GTTCGTACGA ATGCAGGATA TCCCAAGTGC 2580

TTTCAACGAT GTAGCAGCAA ATCCTATTGG CGAAGAATTC ATTTTCAATT TCCTTATTGA 2640

GAGATGGCCA GATATCGTTG AAAGTATAGG AACGAAACAC ACATATGTTG AAAAAGTGAT 2700

ACCAGCTTGC ACTTCAGGAA TCCGCTCACA ACAACAGATT GACCAGCTGA AGAATCTGCA 2760

GAAAAATGGC ATAAACGCTC GTCAATTTGG TGCATTCGAT AAAGCGATCG AACGAGCACA 2820

AAATAGGGTG GATTGGATTA AAAAACATTT CCAAAAATTA GCGGCTTTCT TCAAGAAAGC 2880

CACCTTGTAA TTCGTATTAC ATCACCATGA ATCCAGATCT TAAAACTCAC TAAGGAATGT 2940

GTGGGAACTG ACTGTCTGTT GCTTACTGTT CCACTGAATG GAAGTTTTTA CCCATAAAAA 3000

TTTTTACCAT TTGCCTTCCG TGAGGGGTCA TTGTTGTCAC TTGAATAGTA AACAAAGCTC 3060

AGTATTGGAC CCAGTGATCA ATATTACTTT CGCTTCATCG AATTGTTACC TTCTCTATAC 3120

CCTCGTCCTA CCTGAATTCA CACATTTGTT CATATTTGTT TGTAGTCTGT TGCTCAGAAC 3180

ACTTTCTCTT CGATAGCTTT TTGTTTGTTT TTCTTTTGAT TGTATTGATC GTTTTACAAT 3240

TGTATAGATT AGTTATCTGA TAAATATTGA TGGCTAAGGA AAAAAAAAAA AAAAAAAAAA 3300

AAAAAAAAAA AAAAAAAAA 3319

INFORMATION FOR SEQ ID NO: 16:

SEQUENCE CHARACTERISTICS:

LENGTH: 30 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Asp Asn Ser Pro Ser Ala Glu Glu Leu Leu Leu Pro Thr Asn lie 1 5 10 15

Lys Pro Val Ser Tyr Asp Leu Lys lie Ala Thr Tyr Leu Pro Gly 20 25 30

INFORMATION FOR SEQ ID NO: 17:

SEQUENCE CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 17:

Leu Tyr Leu Ala Glu Asp Val Gin Leu Xaa Lys Gly lie Leu Phe 1 5 10 15

INFORMATION FOR SEQ ID NO: 18:

SEQUENCE CHARACTERISTICS:

LENGTH: 15 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 18:

Leu Ala Tyr Asp Glu Lys Ser Tyr Ala Pro Asp Asn Lys Gin Tyr 1 5 10 15

INFORMATION FOR SEQ ID NO: 19:

SEQUENCE CHARACTERISTICS:

LENGTH: 3084 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 19

INFORMATION FOR SEQ ID NO: 20:

SEQUENCE CHARACTERISTICS:

LENGTH: 3358 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: 1inear

SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GGTTTAATTA CCCAAGTTTG AGATGACGGC GGAGTGGCAG AAGCGTCGAA TCTTGGGCTT 60

CTCACCTATC AGCCTACTTT GTACATTATT TGTATTAGCT GCTGCCGTTG GACTCTCCAT 120

TGGTCTTACC TATTACTTCA CTCGTAAAGC ATTCGATACC ACACAAAAAG AACAGAAGGA 180

TGACAGTGGT GGTAAAGAAA AGGATAATTC TCCTTCTGCA GAAGAACTAC TTCTTCCAAC 240

GAACATAAAA CCAGTCTCGT ACGACTTGAA CATCAAAACA TATCTACCGG GTTACGTGAA 300

CTTTCCACCA GAAAAGAATC TCACATTTGA TGCCCATGTG GAGATTGCTA TGGTTGTGGT 360

TGAGCCTACA AATAGTATTG TGCTGAACTC GAAGAAAATC ACTTTGGCAC AAGGAGGATG 420

CGAACTGTTC TCAGGTAATC AGAAACTTGA CATCGAAAGT GTAAAGATGC AGGAAAGACT 480

TGACAAGCTT GAGATTACCC TCAAAAATCA GCTGCAAAAA GATCTGAAAA TCCTGCTCAA 540

GATCACTTAC ACCGGCCΓTA TTAGCGACAC TCTCGGTGGG CTCTACCAGT CCATCTACAC 600

TGATAAGGAC GGAAAAACTA AGATCGTTGC TGTTTCACAA AATGAACCAT CAGACGCTCG 660

TCGTATAGCG CCATGCTTTG ACGAACCGAA GTACAAGGCA ACATGGACTG TCACCGTCGT 720

TCATCCCAAA GGTACAAAGG CTGCATCGAA CGGCATTGAA GCAAATGGAA AAGGGGAGCT 780

CAAGGGTGAT TGGATAACGT CTAAATTTAA AACTACCCCA CCGATGTCGT CCTATTTATT 840

GGCTATTATT GTTTGTGAAT TTGAATACAT TGAAGGATTT ACAAAAACGG GTGTTCGGTT 900

CCGTATATGG TCTCGACCAG AGGCGAAACG AATGACGGCA TACGCTTTGG ATGCTGGCAT 960

CAGATGCCTG GAGTTCTATG AGAAGTTCTT TGACATAAAA TTCCCTCTGG AAAAACAAGA 1020

TATGATTGCT CTTCCTGATT TCACCGCTGG TGCCATGGAA AACTGGGGCC TTATCACTTA 1080

TAGAGAGGAT TCTCTCCTAT ACGATGAAAA AATTTATGCA CCGATGAATA AACAGCGGGT 1140

TGCTCTCGTA GTTGCTCACG AGCTTGCTCA TCAGTGGTTC GGCAATCTGG TCACACTGAA 1200

GTGGTGGGAT GATACGTGGT TGAACGAAGG TTTTGCAACA TTTGTTGAGT ATCTTGGAAT 1260

GGACGAAATT AGCCACAACA ATTTCAGAAC GCAAGATTTC TTCTTGCTCG ATGGAATGGA 1320

TCGCGGAATG AGAGCTGACT CGGCAGCATC GAGCCATCCG CTTTCGTTTA GGATTGACAA 1380

AGCGGCAGAA GTTGCCGAAG CCTTTGACGA TATTTCATAC GCCAAGGGAG CGTCAGTTCT 1440

CACTATGCTA CGGGCTTTGA TTGGAGAGGA CAATTACAGG AATGCTGTTG TGCAATACCT 1500

CAAGAAGTTC TCCTACAGCA ATGCACAAGC AGCCGATCTG TGGAACGTCT TCAATGAAGT 1560

TGTCAAAGGT GTTAAGGGTC CTGACGGCAA CGTCATGAAA ATCGACCAAT TTACCGATCA 1620

GTGGACGTAT CAGATGGGTT ATCCTGTGGT TAAAGTAGAA GAATTTAATG CGACCGCCCT 1680

AAAGGTTACG CAGAGCCGGT ACAAGACAAA TAAAGACGCC TTGGAACCAG AGAAATATCG 1740

TAATCCAAAA TACGGGTTCA AGTGGGATGT TCCCCTATGG TATCAGGAAG GCAATAGCAA 1800

AGAGGTGAAG CGAACATGGC TAAAAAGAGA TGAACCGCTG TACTTGAACG TCAACAATCG 1860

GGATACATCC CTTGTGGTGA ACGCTGATCG ACATGGATTT TATCGACAAA ACTATGATGC 1920

CAACGGTTGG AAAAAGATAA TCAAGCAGCT CAAGAAAGAT CACAAGGTCT TCGGTCCAAG 1980

GACAAGGAAC GCTATCATAA GCGATGCATT TGCTGCAGCT ACGATTGACG CAATCGACTA 2040

TGAAACTGTA TTCGAACTAC TTGAATATGC CAAAAATGAA GAGGAATTCT TGCCTTGGAA 2100

GGAAGCTCTG TCCGGCATGT TCGCAGTTTT AAAGTTCTTC GGTAATGAGC CGGAGACAAA 2160

ACCAGCTAGA GCTTACATGA TGAGCATATT AGAACCGATG TATAATAAGA GCAGCATTGA 2220

TTACATCGTC AAGAATTATT TGGATGATAC GTTATTCACA AAAATTAATA CTCAAAAGGA 2280

TATCATTGAT GCATATTGTT CCCTTGGATC AAAGGACTGT ATAAAGCAAT ATAAGGATAT 2340

CTTCTACGAT GAGGTTATGC CCAAGTGTAA GGCCGGGGAA GCAGCAACCA AATGCGTTAA 2400

GGTTTCCGCT CCTCTTCGAG CCAATGTTTA CTGTTATGGT GTACAGGAAG GTGGTGAAGA 2460

AGCTTTTGAA AAGGTGATGG GGCTGTATCT AGCAGAAGAT GTTCAACTGG AGAAGGGTAT 2520

CCTGTTCAAA GCCTTGGCAT GCCACAAAGA TGTTACAGCT CTAAAAGAAC TTCTTTTGCG 2580

AGCCCTGGAC CGTAAATCGT CGTTTGTGCG TCTTCAGGAT GTCCCTACCG CTTTCCGTGC 2640

TGTATCTGAA AACCCTGTGG GCGAAGAATT CATGTTCAAT TTCCTAATGG AGAGATGGGA 2700

GGAAATCACT GCGAGCTTGG AAACAGAACA CAGAGCAGTT GATAAAGTGG TCGGCGCTTG 2760

TTGCACAGGA ATTCGCTCCC AACAACAAAT AGATCAGCTG AAGAATCTAC AGAAGAACAA 2820

TGCGCAGGCT AAGAAGTTCG GCTCATTCAC CCAGGAAATC GAAAAAGGAG AACATAAAAT 2880

TGCCTGGATC AAGAAACATT TTCACAGATT ATCGGAATTC TTCAAGAGAG CAAGATCATA 2940

GCTTTTCACA CTGAGCTCCA ATTTTAACGT CTTCAAACTA GGAGACAGTT TTGCTGAAAA 3000

GTCAGTTTCA CATTTTCCGT TTGAATGCCA TCCATTCGAA TACAACCAAT AATACCATTT 3060

TAAGTACCTT TCATTCACAG TGATTACTGA ATTTCGAATA TATCATGAAG CTTGTATCTT 3120

GAACGTTATG ATCGGTGACT TTCAATTTAT AGAGCTCACT CTCCATTTTG TAGCTGTGAT 3180

GACTTGCATT TAAGACCCAC CATTTACCAG CCTAGAATCT TTCCCCAATA CATTCCAAAC 3240

TCCGATCACC TCCACCGCTG ACAATGCCCA GATTTGTTTT TTTGTCTGCT ATCCATCTAA 3300

CTGTTTCGAT CGCCGGTTGT TTGTCAATTG CTTATCTGAT AAATATTGAC GTTGGTGT 3358

INFORMATION FOR SEQ ID NO: 21:

SEQUENCE CHARACTERISTICS: LENGTH: 3369 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

SEQUENCE DESCRIPTION: SEQ ID NO: 21

t o

INFORMATION FOR SEQ ID NO: 22:

SEQUENCE CHARACTERISTICS:

LENGTH: 977 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Met Thr Ala Glu Glu Ser Gin Glu Gin Glu Thr Gin Gin Pro Arg

1 5 10 15

Lys Asn Thr Val Leu Arg Leu Thr Pro lie Lys Ser Leu Phe Ala

20 25 30

Leu Leu Val Val Ala Ala Ala Val Gly Leu Ser lie Gly Leu Thr

35 40 45

Tyr Tyr Phe Thr Arg Lys Ala Phe Asp Thr Thr Gly Gly Asn Gly

50 55 60

Lys Gly Asp Gin Pro lie Val Asp Asp Asn Ser Pro Ser Ala Glu

65 70 75

Glu Leu Arg Leu Pro Thr Thr ie Lys Pro Leu Thr Tyr Asp Leu

80 85 90

Val lie Lys Thr Tyr Leu Pro Asn Tyr Val Asn Tyr Pro Pro Glu

95 100 105

Lys Asp Phe Ala lie Asp Gly Thr Val Val He Ala Met Glu Val

110 115 120

Val Glu Pro Thr Lys Ser He Val Leu Asn Ser Lys Asn He Pro

125 130 135

Val He Ala Asp Gin Cys Glu Leu Phe Ser Asn Asn Gin Lys Leu

140 145 150

Asp He Glu Lys Val Val Asp Gin Pro Arg Leu Glu Lys Val Glu

155 160 165

Phe Val Leu Lys Lys Lys Leu Glu Lys Asn Gin Lys He Thr Leu

170 175 180

Lys He Val Tyr He Gly Leu He Asn Asp Met Leu Gly Gly Leu

185 190 195

Tyr Arg Thr Thr Tyx Thr Asp Lys Asp Gly Thr Thr Lys He Ala

200 205 210

Ala Cys Thr His Met Glu Pro Thr Asp Ala Arg Leu Met Val Pro

215 220 225

Cys Phe Asp Glu Pro Thr Phe Lys Ala Asn Trp Thr Val Thr Val

230 235 240

He His Pro Lys Gly Thr Ser Ala Val Ser Asn Gly He Glu Lys

245 250 255

Gly Glu Gly Glu Val Ser Gly Asp Trp Val Thr Thr Arg Phe Asp

260 265 270

Pro Thr Pro Arg Met Pro Ser Tyr Leu He Ala Leu Val He Ser

275 280 285

Glu Phe Lys Tyr He Glu Asn Tyr Thr Lys Ser Gly Val Arg Phe

290 295 300

Arg He Pro Ala Arg Pro Glu Ala Met Lys Met Thr Glu Tyr Ala

305 310 315

Met He Ala Gly He Lys Cys Leu Asp Tyr Tyr Glu Asp Phe Phe

320 325 330

Gly He Lys Phe Pro Leu Pro Lys Gin Asp Met Val Ala Leu Pro

335 340 345

Asp Phe Ser Ser Gly Ala Met Glu Asn Trp Gly Leu He Thr Tyr

350 355 360

Arg Glu Gly Ser Val Leu Tyr Asp Glu Asn Leu Tyr Gly Pro Met

365 370 375

Asn Lys Glu Arg Val Ala Glu Val He Ala His Glu Leu Ala His

380 385 390

Gin Trp Phe Gly Asn Leu Val Thr Met Lys Trp Trp Asp Asn Leu

395 400 405

Trp Leu Asn Glu Gly Phe Ala Ser Phe Val Glu Tyr He Gly Ala

410 415 420

Asp Phe He Ser Asp Gly Leu Trp Glu Met Lys Asp Phe Phe Leu

425 430 435

Leu Ala Pro Tyr Thr Ser Gly He Thr Ala Asp Ala Val Ala Sex

440 445 450

Ser His Pro Leu Ser Phe Arg He Asp Lys Ala Ala Asp Val Ser

455 460 465

Glu Ala Phe Asp Asp He Thr Tyr Arg Lys Gly Ala Ser Val Leu

470 475 480

Gin Met Leu Leu Asn Leu Val Gly Asp Glu Asn Phe Lys Gin Ser

485 490 495

Val Ser Arg Tyr Leu Lys Lys Phe Ser Tyr Asp Asn Ala Ala Ala

500 505 510

Glu Asp Leu Trp Ala Ala Phe Asp Glu Thr Val Gin Gly He Thr

515 520 525

Gly Pro Asn Gly Gly Pro Leu Lys Met Ser Glu Phe Ala Pro Gin

530 535 540

Trp Thr Thr Gin Met Gly Phe Pro Val Leu Thr Val Glu Ser Val

545 550 555

Asn Ala Thr Thr Leu Lys Val Thr Gin Lys Arg Tyr Arg Gin Asn

560 565 570

Lys Asp Ala Lys Glu Pro Glu Lys Tyr Arg His Pro Thr Tyr Gly

575 580 585

Phe Lys Trp Asp Val Pro Leu Trp Tyr Gin Glu Asp Glu Gin Gin

590 595 600

Val Lys Arg Thr Trp Leu Lys Arg Glu Glu Pro Leu Tyr Phe His

605 610 615

Val Ser Asn Ser Asp Ser Ser Val Val Val Asn Ala Glu Arg Arg

620 625 630

Ala Phe Cys Arg Ser Asn Tyr Asp Ala Asn Gly Trp Arg Asn He

635 640 645

Met Arg Arg Leu Lys Gin Asn His Lys Val Tyr Gly Pro Arg Thr

650 655 660

Arg Asn Ala Leu He Ser Asp Ala Phe Ala Ala Ala Ala Val Glu

665 670 675

Glu Met Asn Tyr Glu Thr Val Phe Glu Met Leu Lys Tyr Thr Val

680 685 690

Lys Glu Glu Asp Tyr Leu Pro Trp Lys Glu Ala He Ser Gly Phe

695 700 705

Asn Thr He Leu Asp Phe Phe Gly Ser Glu Pro Glu Ser Gin Trp

710 715 720

Ala Ser Glu Tyr Met Arg Lys Leu Met Lys Pro He Tyr Asp Lys

725 730 735

Ser Ser He Lys Phe He Ala Glu Asn Tyr Lys Lys Asp Ser Leu

740 745 750

Phe Phe Lys Asn Asn Leu Gin He Ala Val He Asp Thr Tyr Cys

755 760 765

Gly Leu Gly Gly Lys Glu Cys Leu Glu Glu Met Lys Lys Leu Phe

770 775 780

Asp Lys Glu Val Met Lys Cys Gin Pro Gly Gin Gin Ala Thr Asp

785 790 795

Cys Val Lys Val Thr Ala Pro Leu Arg Lys Thr Val Tyr Cys Tyr

800 805 810

Gly Val Gin Glu Gly Gly Asp Glu Ala Phe Asp Lys Val Met Glu

815 820 825

Leu Tyr Asn Ala Glu Gin Val Gin Leu Glu Lys Asp Ser Leu Arg

830 835 840

Glu Ala Leu Gly Cys His Lys Asp Val Thr Ala Leu Lys Gly Leu

845 850 855

Leu Met Leu Ala Leu Asp Arg Asn Ser Ser Phe Val Arg Leu Gin

860 865 870

Asp Ala His Asp Val Phe Asn He Val Ser Arg Asn Pro Val Gly

875 880 885

Asn Glu Leu Leu Phe Asn Phe Leu Thr Glu Arg Trp Glu Glu He

890 895 900

Leu Glu Ser Leu Ser He Arg His Arg Ser Val Asp Arg Val He

905 910 915

Lys Ala Cys Thr Aig Gly Leu Arg Sei Aig Glu Gin Val Gin Gin

920 925 930

Leu Lys Asn Leu Tyr Lys Asn Asp Lys Arg Ala Arg Glu Tyr Gly

935 940 945

Ala Phe Gly Gly Ala He Glu Arg Ser Glu His Arg Val Lys Trp

950 955 960

He Glu Lys His Phe Arg Lys Leu Ala Ala Phe Phe Lys Lys Ser

965 970 975 Asn Sex

INFORMATION FOR SEQ ID NO: 23:

SEQUENCE CHARACTERISTICS:

LENGTH: 972 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 23 :

Met Thr Ala Glu Trp Gin Lys Arg Arg He Leu Gly Phe Ser Pro

1 5 10 15

He Ser Leu Leu Cys Thr Leu Phe Val Leu Ala Ala Ala Val Gly

20 25 30

Leu Ser He Gly Leu Thr Tyr Tyr Phe Thr Arg Lys Ala Phe Asp

35 40 45

Thr Thr Gin Lys Glu Gin Lys Asp Asp Ser Gly Gly Lys Glu Lys

50 55 60

Asp Asn Ser Pro Ser Ala Glu Glu Leu Leu Leu Pro Thr Asn He

65 70 75

Lys Pro Val Ser Tyr Asp Leu Asn He Lys Thr Tyr Leu Pro Gly

80 85 90

Tyr Val Asn Phe Pro Pro Glu Lys Asn Leu Thr Phe Asp Ala His

95 100 105

Val Glu He Ala Met Val Val Val Glu Pro Thr Asn Ser He Val

110 115 120

Leu Asn Ser Lys Lys He Thr Leu Ala Gin Gly Gly Cys Glu Leu

125 130 135

Phe Ser Gly Asn Gin Lys Leu Asp He Glu Ser Val Lys Met Gin

140 145 150

Glu Arg Leu Asp Lys Leu Glu He Thr Leu Lys Asn Gin Leu Gin

155 160 165

Lys Asp Leu Lys He Leu Leu Lys He Thr Tyr Thr Gly Leu He

170 175 180

Sex Asp Thr Leu Gly Gly Leu Tyr Gin Ser He Tyr Thr Asp Lys

185 190 195

Asp Gly Lys Thr Lys He Val Ala Val Ser Gin Asn Glu Pro Ser

200 205 210

Asp Ala Arg Arg He Ala Pro Cys Phe Asp Glu Pro Lys Tyr Lys

215 220 225

Ala Thr Trp Thr Val Thr Val Val His Pro Lys Gly Thr Lys Ala

230 235 240

Ala Ser Asn Gly He Glu Ala Asn Gly Lys Gly Glu Leu Lys Gly

245 250 255

Asp Trp He Thr Ser Lys Phe Lys Thr Thr Pro Pro Met Ser Ser

260 265 270

Tyr Leu Leu Ala He He Val Cys Glu Phe Glu Tyr He Glu Gly

275 280 285

Phe Thr Lys Thr Gly Val Arg Phe Arg He Trp Ser Arg Pro Glu

290 295 300

Ala Lys Arg Met Thr Ala Tyr Ala Leu Asp Ala Gly He Arg Cys

305 310 315

Leu Glu Phe Tyr Glu Lys Phe Phe Asp He Lys Phe Pro Leu Glu

320 325 330

Lys Gin Asp Met He Ala Leu Pro Asp Phe Thr Ala Gly Ala Met

335 340 345

Glu Asn Trp Glv Leu He Thr Tyr Arg Glu Asp Ser Leu Leu Tyr

350 355 360

Asp Glu Lvs He Tyr Ala Pro Met Asn Lys Gin Arg Val Ala Leu

365 370 375

Val Val Ala His Glu Leu Ala His Gin Tip Phe Gly Asn Leu Val

380 385 390

Thr Leu Lys Trp Trp Asp Asp Thr Trp Leu Asn Glu Gly Phe Ala

395 400 405

Thr Phe Val Glu Tyr Leu Gly Met Asp Glu He Ser His Asn Asn

410 415 420

Phe Arg Thr Gin Asp Phe Phe Leu Leu Asp Gly Met Asp Arg Gly

425 430 435

Met Arg Ala Asp Ser Ala Ala Ser Ser His Pro Leu Ser Phe Arg

440 445 450

He Asp Lys Ala Ala Glu Val Ala Glu Ala Phe Asp Asp He Ser

455 460 465

Tyr Ala Lys Gly Ala Ser Val Leu Thi Met Leu Arg Ala Leu He

470 475 480

Gly Glu Asp Asn Tyr Arg Asn Ala Val Val Gin Tyr Leu Lys Lys

485 490 495

Phe Ser Tyr Ser Asn Ala Gin Ala Ala Asp Leu Tip Asn Val Phe

500 505 510

Asn Glu Val Val Lys Gly Val Lys Gly Pro Asp Gly Asn Val Met

515 520 525

Lys He Asp Gin Phe Thr Asp Gin Trp Thr Tyr Gin Met Gly Tyr

530 535 540

Pro Val Val Lys Val Glu Glu Phe Asn Ala Thr Ala Leu Lys Val

545 550 555

Thr Gin Ser Arg Tyr Lys Thr Asn Lys Asp Ala Leu Glu Pro Glu

560 565 570

Lys Tyr Arg Asn Pro Lys Tyr Gly Phe Lys Trp Asp Val Pro Leu

575 580 585

Trp Tyr Gin Glu Gly Asn Ser Lys Glu Val Lys Arg Thr Trp Leu

590 595 600

Lys Arg Asp Glu Pro Leu Tyr Leu Asn Val Asn Asn Arg Asp Thr

605 610 615

Ser Leu Val Val Asn Ala Asp Arg His Gly Phe Tyr Arg Gin Asn

620 625 630

Tyr Asp Ala Asn Gly Trp Lys Lys He He Lys Gin Leu Lys Lys

635 640 645

Asp His Lys Val Phe Gly Pro Arg Thr Arg Asn Ala He He Ser

650 655 660

Asp Ala Phe Ala Ala Ala Thr He Asp Ala He Asp Tyr Glu Thr

665 670 675

Val Phe Glu Leu Leu Glu Tyr Ala Lys Asn Glu Glu Glu Phe Leu

680 685 690

Pro Trp Lys Glu Ala Leu Ser Gly Met Phe Ala Val Leu Lys Phe

695 700 705

Phe Gly Asn Glu Pro Glu Thr Lys Pro Ala Arg Ala Tyr Met Met

710 715 720

Ser He Leu Glu Pro Met Tyr Asn Lys Ser Ser He Asp Tyr He

725 730 735

Val Lys Asn Tyr Leu Asp Asp Thr Leu Phe Thr Lys He Asn Thr

740 745 750

Gin Lys Asp He He Asp Ala Tyr Cys Ser Leu Gly Ser Lys Asp

755 760 765

Cys He Lys Gin Tyr Lys Asp He Phe Tyr Asp Glu Val Met Pro

770 775 780

Lys Cys Lys Ala Gly Glu Ala Ala Thr Lys Cys Val Lys Val Ser

785 790 795

Ala Pro Leu Arg Ala Asn Val Tyr Cys Tyr Gly Val Gin Glu Gly

800 805 810

Gly Glu Glu Ala Phe Glu Lys Val Met Gly Leu Tyr Leu Ala Glu

815 820 825

Asp Val Gin Leu Glu Lys Gly He Leu Phe Lys Ala Leu Ala Cys

830 835 840

His Lys Asp Val Thr Ala Leu Lys Glu Leu Leu Leu Arg Ala Leu

845 850 855

Asp Arg Lys Ser Ser Phe Val Arg Leu Gin Asp Val Pro Thr Ala

860 865 870

Phe Arg Ala Val Ser Glu Asn Pro Val Gly Glu Glu Phe Met Phe

875 880 885

Asn Phe Leu Met Glu Arg Trp Glu Glu He Thr Ala Ser Leu Glu

890 895 900

Thr Glu His Arg Ala Val Asp Lys Val Val Gly Ala Cys Cys Thr

905 910 915

Gly He Arg Ser Gin Gin Gin He Asp Gin Leu Lys Asn Leu Gin

920 925 930

Lys Asn Asn Ala Gin Ala Lys Lys Phe Gly Ser Phe Thr Gin Glu

935 940 945

He Glu Lys Gly Glu His Lys He Ala Trp He Lys Lys His Phe

950 955 960

His Arg Leu Ser Glu Phe Phe Lys Arg Ala Arg Ser

965 970

INFORMATION FOR SEQ ID NO: 24:

SEQUENCE CHARACTERISTICS:

LENGTH: 972 amino acids TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Met Thr Ser Gin Gly Arg Thr Arg Thr Leu Leu Asn Leu Thr Pro

1 5 10 15

He Arg Leu He Val Ala Leu Phe Leu Val Ala Ala Ala Val Gly

20 25 30

Leu Ser He Gly Leu Thr Tyr Tyr Phe Thr Arg Lys Ala Phe Asp

35 40 45

Thr Ser Glu Lys Pro Gly Lys Asp Asp Thr Gly Gly Lys Asp Lys

50 55 60

Asp Asn Ser Pro Ser Ala Ala Glu Leu Leu Leu Pro Ser Asn He

65 70 75

Lys Pro Leu Ser Tyr Asp Leu Thr He Lys Thr Tyr Leu Pro Gly

80 85 90

Tyr Val Asp Phe Pro Pro Glu Lys Asn Leu Thr Phe Asp Gly Arg

95 100 105

Val Glu He Ser Met Val Val He Glu Pro Thr Lys Ser He Val

110 115 120

Leu Asn Ser Lys Lys He Ser Val He Pro Gin Glu Cys Glu Leu

125 130 135

Val Ser Gly Asp Lys Lys Leu Glu He Glu Ser Val Lys Glu His

140 145 150

Pro Arg Leu Glu Lys Val Glu Phe Leu He Lys Ser Gin Leu Glu

155 160 165

Lys Asp Gin Gin He Leu Leu Lys Val Gly Tyr He Gly Leu lie

170 175 180

Ser Asn Ser Phe Gly Gly He Tyr Gin Thr Thr Tyr Thr Thr Pro

185 190 195

Asp Gly Thr Pro Lys He Ala Ala Val Ser Gin Asn Glu Pro He

200 205 210

Asp Ala Arg Arg Met Val Pro Cys Met Asp Glu Pro Lys Tyr Lys

215 220 225

Ala Asn Trp Thr Val Thr Val He His Pro Lys Gly Thr Lys Ala

230 235 240

Val Ser Asn Gly He Glu Val Asn Gly Asp Gly Glu He Ser Gly

245 250 255

Asp Trp He Thr Ser Lys Phe Leu Thr Thr Pro Arg Met Ser Ser

260 265 270

Tyr Leu Leu Ala Val Met Val Ser Glu Phe Glu Tyr He Glu Gly

275 280 285

Glu Thr Lys Thr Gly Val Arg Phe Arg He Trp Ser Arg Pro Glu

290 295 300

Ala Lys Lys Met Thr Gin Tyr Ala Leu Gin Ser Gly He Lys Cys

305 310 315

He Glu Phe Tyr Glu Asp Phe Phe Asp He Arg Phe Pro Leu Lys

320 325 330

Lys Gin Asp Met He Ala Leu Pio Asp Phe Ser Ala Gly Ala Met

335 340 345

Glu Asn Trp Gly Leu He Thr Tyr Arg Glu Asn Ser Leu Leu Tyr

350 355 360

Asp Asp Arg Phe Tyi Ala Pio Met Asn Lys Gin Arg He Ala Arg

365 370 375

He Val Ala His Glu Leu Ala His Gin Trp Phe Gly Asp Leu Val

380 385 390

Thr Met Lys Trp Trp Asp Asn Leu Trp Leu Asn Glu Gly Phe Ala

395 400 405

Arg Phe Thr Glu Phe He Gly Ala Gly Gin He Thr Gin Asp Asp

410 415 420

Ala Arg Met Arg Asn Tyr Phe Leu He Asp Val Leu Glu Arg Ala

425 430 435

Leu Lys Ala Asp Ser Val Ala Ser Ser His Pro Leu Ser Phe Arg

440 445 450

He Asp Lys Ala Ala Glu Val Glu Glu Ala Phe Asp Asp He Thr

455 460 465

Tyr Ala Lys Gly Ala Ser Val Leu Thr Met Leu Arg Ala Leu He

470 475 480

Gly Glu Glu Lys His Lys His Ala Val Ser Gin Tyr Leu Lys Lys

485 490 495

Phe Ser Tyr Ser Asn Ala Glu Ala Thr Asp Leu Trp Ala Val Phe

500 505 510

Asp Glu Val Val Thr Asp Val Glu Gly Pro Asp Gly Lys Pro Met

515 520 525

Lys Thr Thr Glu Phe Ala Ser Gin Trp Thr Thr Gin Met Gly Phe

530 535 540

Pro Val He Ser Val Ala Glu Phe Asn Ser Thr Thr Leu Lys Leu

545 550 555

Thr Gin Ser Arg Tyr Glu Ala Asn Lys Asp Ala Val Glu Lys Glu

560 565 570

Lys Tyr Arg His Pro Lys Tyr Gly Phe Lys Trp Asp He Pro Leu

575 580 585

Trp Tyr Gin Glu Gly Asp Lys Lys Glu He Lys Arg Thr Trp Leu

590 595 600

Arg Arg Asp Glu Pro Leu Tyr Leu His Val Ser Asp Ala Gly Ala

605 610 615

Pro Phe Val Val Asn Ala Asp Arg Tyr Gly Phe Tyr Arg Gin Asn

620 625 630

His Asp Ala Asn Gly Trp Lys Lys He He Lys Gin Leu Lys Asp

635 640 645

Asn His Glu Val Tyr Ser Pro Arg Thr Arg Asn Val He He Ser

650 655 660

Asp Ala Phe Ala Ala Ala Ala Thr Asp Ala He Glu Tyr Glu Thr

665 670 675

Val Phe Glu Leu Leu Asn Tyr Ala Glu Lys Glu Thr Glu Tyr Leu

680 685 690

Pro Leu Glu He Ala Met Ser Gly He Ser Ser He Leu Lys Tyr

695 700 705

Phe Pro Thr Glu Pro Glu Ala Lys Pro Ala Gin Thr Tyr Met Met

710 715 720

Asn He Leu Lys Pro Met Tyr Glu Lys Ser Ser He Asp Phe He

725 730 735

Ala Asn Asn Tyr Arg Asn Asp Lys Leu Phe Phe Gin He Asn Leu

740 745 750

Gin Lys Asp Val He Asp Met Phe Cys Ala Leu Gly Ser Gin Asp

755 760 765

Cys Arg Lys Lys Tyr Lys Lys Leu Phe Asp Asp Glu Val Met Asn

770 775 780

Lys Cys Arg Asp Gly Gin Ala Ala Thr Glu Cys Val Arg He Ala

785 790 795

Ala Pro Leu Arg Sex Ser Val Tyr Cys Tyr Gly Val Lys Glu Gly

800 805 810

Gly Asp Tyx Ala Ser Asp Lys Val Met Glu Leu Tyr Thi Ala Glu

815 820 825

Thr Leu Ala Leu Glu Lys Asp Phe Leu Arg Leu Ala Leu Gly Cys

830 835 840

His Lys Asp Val Thr Ala Leu Lys Gly Leu Leu Leu Arg Ala Leu

845 850 855

Asp Arg Asn Ser Ser Phe Val Arg Met Gin Asp He Pro Ser Ala

860 865 870

Phe Asn Asp Val Ala Ala Asn Pro He Gly Glu Glu Phe He Phe

875 880 885

Asn Phe Leu He Glu Arg Trp Pro Asp He He Glu Ser He Gly

890 895 900

Thr Lys His Thr Tyr Val Glu Lys Val He Pro Ala Cys Thr Ser

905 910 915

Gly He Arg Ser Gin Gin Gin He Asp Gin Leu Lys Asn Leu Gin

920 925 930

Lys Asn Gly Met Asn Ala Arg Gin Phe Gly Ala Phe Asp Lys Ala

935 940 945

He Glu Arg Ala Gin Asn Arg Val Asp Trp He Lys Lys His Phe

950 955 960

Gin Lys Leu Ala Ala Phe Phe Lys Lys Ala Thr Leu

965 970