PATENT DOCUMENT

Abstract:
The present invention provides one or more antigens from the fourth stage (L4) larvae of non-blood feeding parasitic nematodes, for raising an immune responses in an animals, in particular bovines. The invention further provides methods of making immunogenic and/or vaccine compositions.

Full Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to antigens capable of raising host immune responses to nematode parasites. In particular, the invention provides vaccines for use in protecting against and/or reducing instances of non-blood feeding nematode parasite infections in bovine hosts. 
       BACKGROUND OF THE INVENTION 
       [0002]      Ostertagia ostertagi  is the most economically important helminth parasite of cattle in temperate parts of the world (1). As with all gastrointestinal nematodoses of ruminants, ostertagiasis is controlled almost entirely by the use of anthelmintic drugs combined with pasture management. Unfortunately, reports of anthelmintic resistance in bovine gastrointestinal nematodes are becoming more frequent. For example, benzimidazole (BZ) resistance was identified in cattle in New Zealand (2) and South America (3) , and macrocyclic lactone (ML) resistance in New Zealand (4,5) , the Americas (6,7,8)  and Europe (9,10,11,12)  However, as no detailed surveys have been made, the extent of the problem remains unknown and probably underestimated. It is anticipated that anthelmintic resistance in cattle nematodes including  Ostertagia  is likely to follow the pattern experienced with sheep, where it has reached serious proportions (13,14,15,16)  although the pace at which it develops and spreads is likely to be slower. 
         [0003]    Alternative methods for controlling bovine ostertagiasis remain an attractive prospect, in part due to the threat of drug resistance and partly because of increasing consumer sensitivity to the possibility of chemical residues in meat and milk. One such possibility for control is by vaccination. Earlier attempts to do this, using either infection with irradiated larvae (17,18)  or immunisation with crude somatic or excretory/secretory products of the parasites (19,20)  were not successful. More recently promising results have been obtained using fractionated native excretory/secretory products of adult parasites, with reductions in faecal egg counts of up to 80% (21,22,23,24,25) . 
         [0004]    During the last 15 years or so substantial protection against the important blood sucking ovine nematode,  Haemonchus contortus,  has been achieved by immunising sheep with various antigens isolated from the intestinal membranes of adult parasites, a topic which has been reviewed extensively (26,27,28) . When the same gut antigen approach was tested against  Ostertagia,  some protection was conferred, but not at a level deemed to have practical potential (29) . However as the same  O. ostertagi  antigens cross-protected efficiently against  Haemonchus  in sheep, it was reasoned that the relative failure may have been because adult  Ostertagia  are not blood feeders and do not ingest sufficient antibody for the gut antigen approach to be highly effective against them. 
         [0005]    It was hypothesised that developing fourth stage (L4)  Ostertagia  might be more vulnerable to this type of vaccination than adult parasites. Firstly, since they inhabit and damage the gastric glands, L4s are likely to be continuously exposed to inflammatory exudate which is richer in host immunoglobulin than the mucous surface inhabited by adult worms, in other words, L4s in vaccinated calves would be expected to ingest a bigger dose of antibody per unit weight than adult worms. Second, because developing  Ostertagia  L4s grow very rapidly (30), increasing their mass about 20-fold in 10 days, they may be more sensitive to digestive interference than their slower metabolizing adult counterparts. 
       SUMMARY OF THE INVENTION 
       [0006]    In a first aspect, the present invention provides one or more antigens from the fourth stage (L4) larvae of non-blood feeding parasitic nematodes, for raising an immune response in an animal. 
         [0007]    Antigens from adult nematode parasites are used as vaccines for preventing, reducing or eliminating instances of animal infection. Antigens derived from adult parasitic nematodes, and in particular gut-derived antigens, can be used to raise protective host immune responses. Strategies of this type are particularly successful where the parasite is a blood-feeding nematode as they are routinely exposed to host immunoglobulin when taking blood meals. In contrast, parasites that do not feed on blood (the so-called “non-blood” feeders) are only occasionally (or in some cases never) exposed to blood and thus host circulating (humoral) antibodies to non-blood feeding parasite antigens are often ineffective. 
         [0008]    Without wishing to be bound by theory, the inventors hypothesise that in contrast to adult non-blood feeding nematodes, certain larval stages might be more regularly exposed to host immune factors including, for example, immunoglobulin. In particular, larvae which inhabit and damage gastric glands may frequently be exposed to host inflammatory exudate which comprises a variety of host inflammatory factors including immunoglobulin. 
         [0009]    The life cycle of a parasitic nematode comprises seven stages—an egg, four larval and two adult stages. The larval stages are commonly referred to as L1, L2, L3 and L4 (Borgsteede 1978) In non-blood feeding parasites, L4s inhabit host gastric glands, causing damage and inducing production of host inflammatory exudate. The L4s of all species which live in the true stomach (the abomasum in ruminants) do, but many species live in the intestines which obviously don&#39;t contain gastric glands. The intestines are lined by villi with glandular crypts between them. The L4s of the intestinal species reside in these crypts also causing local damage, so again are more likely than their adult counterparts to be exposed to inflammatory exudates. 
         [0010]    As such, antigens expressed by L4 stage nematode parasites may be used to raise protective host immune responses. Hereinafter, antigens expressed by L4 stage nematode parasites will be termed “L4 antigens”. 
         [0011]    In one embodiment, the invention relates to vaccines or vaccine compositions comprising one or more L4 antigens for raising immune responses in animals. Vaccines may be used prophylactically to prevent parasitic infection. In some cases the vaccines provided by this invention may be used to reduce infection or colonisation of a host by a nematode parasite. Animals exposed to the vaccines provided by this invention, may produce antibodies which bind to (or which exhibit affinity or specificity for) L4 antigens—such antibodies may otherwise be referred to as “protective” antibodies. 
         [0012]    The term “non-blood feeding nematode parasite” may include, for example nematode species from each of the following genera:  Ostertagia, Teladorsagia, Trichostrongylus, Nematodirus, Cooperia, Chabertia, Oesophagostomum  and  Ascaris  In particular, the invention relates to antigens derived from species belonging to any of the abovementioned genera and in one embodiment, the invention provides vaccines/vaccine compositions comprising antigens derived from L4 stage larvae of  Ostertagia ostertagi    
         [0013]    As such, the vaccines and vaccine compositions provided by this invention may be used to treat, prevent or reduce the symptoms of, diseases such as Bovine ostertagiasis. 
         [0014]    It should be understood that the term “antigens” may relate to, for example, proteins and/or peptides (including polypeptides and short peptide chains of one or more amino acids) including for example, glycoproteins and/or glycopeptides. In addition, the term “antigen” may relate to carbohydrate molecules. In one embodiment, the present invention relates to antigens expressed by L4 stage nematode parasites, such as, for example, antigens present in the cell membranes of L4 non-blood feeding nematode parasites. Antigens of this type may otherwise be known as “integral” membrane antigens. In one embodiment, the antigens for use in raising immune responses may comprise antigens which are specifically expressed by L4 stage parasites. Antigens specific to L4 stage larvae may not be expressed by nematodes at other lifecycle stages (for example L2, L3 and adult stages). Antigens specific to L4 stage  Ostertagia ostertagi  are shown in, for example  FIG. 2 . In certain embodiments, the antigens for use in this invention may comprise cell membrane antigens or antigens which are expressed by L4 stage nematodes and which are exposed to the host immune system. Such antigens may include antigens expressed on the surface or in the gut of L4 nematodes. It should also be understood that the term “antigens” relate to any fragments or immunogenic/antigenic fragments of the L4 antigens described herein. One of skill will appreciate that the term “antigens” may also encompass L4 proteins, polypeptides, peptides and/or carbohydrates which are otherwise known as “immunogens”. 
         [0015]    Antigens according to this invention may be obtained, purified or extracted from cell membrane preparations using a number of techniques. For example, cells obtained from nematodes may be subjected to lysis protocols (such as those involving Triton x-100) so as to fragment the cell membrane. Subsequent centrifugation techniques may be used to remove fragmented membrane debris and ultra-centrifugation to extract membrane proteins from supernates. 
         [0016]    Additionally, or alternatively, chromatography techniques such as, for example, those utilising affinity binding matrices, may be used to purify and/or extract membrane proteins from solution. In one embodiment, antigens for use in this invention may be purified/extracted using affinity matrices comprising concanavalin A (ConA)—a lectin which binds to certain structures present in, for example, carbohydrates, glycoproteins, and glycolipids. As such, “antigens” according to this invention may comprise proteins capable of binding ConA (ConA binding proteins) including, for example, glycoproteins proteins and/or peptides comprising a-linked mannose. 
         [0017]    In order to extract or purify antigens, a preparation of cell membrane proteins (for example proteins preparations prepared in accordance with the procedures outlined above), may be contacted with, for example, an affinity matrix under conditions which permit binding between the affinity matrix and antigens, for example (glycol) proteins/peptides, present in the membrane preparation. Antigens bound to an affinity matrix may be released or separated therefrom with the use of a suitable elution solution/buffer. In the case of an affinity matrix comprising ConA, antigens bound thereto may be separated or released by applying carbohydrate solutions. Additionally, material eluted from, for example, ConA affinity matrices, may be further subjected to procedures designed to remove carbohydrate. For example, material eluted from ConA affinity matrices may be contacted with Sephadex G-25 to remove carbohydrate. Antigen preparations of this type may be termed crude antigen preparations. Crude preparations of this type may be stored for prolonged periods of time at −20° C. or, more preferably at −80° C. 
         [0018]    Crude antigen preparations of the type described above may be further processed in order to yield antigen fractions comprising fewer and more highly purified (or cleaner)/concentrated antigens. By way of example, techniques such as, for example, anion exchange and/or gel filtration may be used to prepare one or more fractions of the crude antigen preparations described above. 
         [0019]    In one embodiment, the method of obtaining L4 antigens for use in raising animal immune responses is that shown in  FIG. 1 . 
         [0020]    The various cell membrane protein preparations and fractions thereof described herein may otherwise be referred to as “antigen pools”. Accordingly, the vaccines and vaccine compositions provided by this invention may comprise one or more of the antigen pools described herein. By way of example, vaccines for use in raising immune responses to the nematode parasite  Ostertagia ostertagi  may comprise one or more antigen pool(s) selected from the group consisting of (i) a pool comprising ConA binding proteins from a cell membrane preparation and (ii) fractions of ConA binding protein mixtures obtained by one or more rounds of anion exchange and/or gel filtration. In one embodiment, the vaccine or vaccine provided by this invention comprises one or more of the ConA, pool 1, pool 2 and/or pool 3 fractions described in the detailed description section (see part entitled “Preparation of Immunogens”) and in  FIGS. 1 and 5 . In one embodiment, the vaccine or vaccine composition provided by this invention comprises the antigen pool used in Trial 1, Trial 2, Trial 3 and/or Trial 4 as shown in Table 5 and  FIG. 1  and described in the detailed description section. 
         [0021]    In one embodiment, the antigens for use in this invention are derived from the L4 stage of the non-blood feeding bovine parasite,  Ostertagia ostertagi.  As such, the invention may provide one or more antigens from a fourth stage larvae of  Ostertagia ostertagi,  for raising an immune response in a bovine. Specifically, the invention may relate to ConA binding proteins or membrane (glyco)proteins and/or peptides from L4 stage  Ostertagia ostertagi,  for raising an immune response in a bovine. 
         [0022]    In one embodiment, the bovine is a neonatal or juvenile bovine—otherwise known as a calf. 
         [0023]    Antigens for use in raising animal immune responses may be obtained from whole or fragmented parasites harvested from donor animals. Donor animals may be naturally infected animals or animals which have been deliberately (or experimentally) infected with a particular parasite. For example, experimentally infected animals may be administered a dose of L3 stage parasites sometime before L4 stage parasites are harvested. One of skill will appreciate that the time between administration of an L3 dose and harvesting L4 parasites will vary depending, on for example, the life cycle of the parasite to be harvested. In the case of the non-blood feeding parasite,  Ostertagia ostertagi,  L3 stage larvae may be administered approximately 7 days before L4 larvae are harvested. 
         [0024]    In addition to providing proteins or peptide antigens from non-blood feeding L4 nematode parasites, the present invention relates to nucleic acid molecules encoding the same or fragments (preferably antigentic or immunogenic fragments) thereof. The nucleic acid may be DNA, RNA or a combination thereof and can include any combination of naturally occurring, chemically or enzymatically modified nucleotides. Furthermore, the nucleic acid may be double or single stranded. Within the scope of this invention are nucleic acid sequences that are substantially complementary to any of the L4 antigen sequences described herein. 
         [0025]    It should be understood that the term “substantially complementary” encompasses those nucleic acid molecules exhibiting a degree of sequence identity/homology with any of the L4 antigen nucleic acid sequences described herein—such as, for example, those presented in Tables 1 and 3 below. A sequence having a level of identity with a L4 antigen nucleic acid sequence of this invention may exhibit at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the full length L4 antigen nucleic acid sequence or any portion or fragment thereof. One of skill will appreciate that a level of sequence identity may be determined by comparing aligned nucleic acid sequences over a predetermined length so as to determine the number of positions at which an identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of nucleic acid bases in the length compared and multiplying the result by 100 to yield the percentage of sequence identity. 
         [0026]    The nucleic acid molecules provided by this invention may take the form of nucleic acid constructs or vectors such as, for example a cloning or expression cassettes/vectors. Vectors provided by this invention may be capable of directing the expression of nucleic acid sequences encoding L4 stage antigens in, for example, bacterial, fungal, mammalian and/or insect cells. 
         [0027]    Accordingly, a second aspect of this invention provides a vector, preferably an expression vector, comprising a nucleic acid sequence encoding any of the L4 antigens described herein (including, fragments, variants, analogues or derivatives thereof). Expression vectors suitable for use in this aspect of the invention may further comprise one or more promoter sequences capable of directing expression in prokaryotic or eukaryotic cells such as, for example, mammalian, fungal, bacterial, plant and/or insect cells. 
         [0028]    A vector provided by this invention may be circular or linear, single stranded or double stranded and can include DNA, RNA or a combination or modification thereof. Furthermore, vectors of this invention may be, for example, plasmids, cosmids or viral vectors (for example retroviral or bacteriophage vectors). Vectors provided by this invention may further comprise selection or marker elements, for example antibiotic resistance genes and/or optically detectable tags. A large number of suitable vectors are known and further information may be obtained from Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (ads.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988)—both of which are incorporated herein by reference. 
         [0029]    In addition to techniques in which L4 antigens are extracted or purified from membrane preparations from harvested parasitic organisms, antigens for use may be obtained using recombinant technology. In one embodiment, an expression vector comprising one or more nucleic acid sequences encoding L4 antigens may be used to produce recombinant L4 antigens. Accordingly, in a further aspect, the present invention provides host cells transfected or transformed with a vector as described herein. Eukaryotic or prokaryotic cells, such as, for example, plant, insect, mammalian, fungal and/or bacterial cells, may be transfected with one or more of the vectors described herein. One of skill in this field will be familiar with the techniques used to introduce heterologous or foreign nucleic acid sequences, such as expression vectors, into cells and these may include, for example, heat-shock treatment, use of one or more chemicals (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information regarding transformation/transfection techniques may be found in Current Protocols in Molecular Biology, Ausuble, F. M., ea., John Wiley &amp; Sons, N.Y. (1989) which is incorporated herein by reference. 
         [0030]    Another aspect of this invention relates to a host cell transformed with any one the nucleic acid constructs described herein. Suitable host cells include prokaryotic and/or eukaryotic cells. For example, bacterial, fungal, mammalian, plant and/or insect cells are all capable of being transformed with any of the nucleic acid constructs described herein. A host cell transformed with a nucleic acid construct provided by this invention may be referred to as a “transformant”. Where the vector comprises a selection/marker element, transformants may be selected by application of antibiotics to culture media. 
         [0031]    In view of the above, the present invention further provides a process for the production of a recombinant L4 protein or peptide for use in raising an immune response in an animal, said method comprising the step of (a) transforming a host cell with a nucleic acid sequence according to this invention or transfecting a host cell with a nucleic acid construct of the invention; (b) culturing the cells obtained in (a) under conditions in which expression of the protein takes place; and (c) isolating the expressed recombinant protein or peptide from the cell culture or/and the culture supernatant. 
         [0032]    Recombinant proteins/peptides produced according to the method described above may be partially purified from the host cell before being used as a vaccine. Where the polypeptide is secreted from the host cell, the cells may be separated from the media by centrifugation, the cells being pelleted and the media being the supernatant. In such a situation, the supernatant, which contains the secreted polypeptide, may be used directly as a vaccine, or in a vaccine composition. Alternatively, the polypeptide may be partially purified from this supernatant, for example using affinity chromatography. 
         [0033]    In one embodiment, purified L4 antigens/recombinant antigens may be admixed with another component, such as another polypeptide and/or an adjuvant, diluent or excipient. Vaccines or vaccine compositions provided by this invention may contain bacterial antigens used to control other diseases. For example, the vaccine or vaccine composition may be included within a multivalent vaccine which includes antigens against other bovine diseases. 
         [0034]    In a still further aspect, the present invention provides a bovine population treated or immunised with a vaccine or composition described herein. In one embodiment, the bovine population is a neonatal or juvenile bovine (i.e. calf) population. 
         [0035]    One of skill will appreciate that the vaccines described in this invention may take the form of subunit-type vaccines where by one or more proteinaceous L4 antigens are used to inoculate an animal. Additionally or alternatively, the vaccine may comprise a nucleic acid molecule (known as a DNA vaccine) encoding one or more L4 antigens to be expressed by the cells of an animal to be vaccinated. 
         [0036]    Suitable antigens for raising bovine immune responses to the non-blood feeding parasite  Ostertagia ostertagi  may include, for example, one or more antigens encoded by cDNA sequences having or comprising the following cDNA sequences (Tables 1 and 3): 
         [0000]    
       
         
               
             
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   O. ostertagi  L4 cDNA library screen (with antiserum from protected 
               
               
                 calves immunised with pool 3 fraction from Trial 2 - see FIG. 1) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Clone1.1 
               
               
                 CTATCNTATTCATCGCGCGCANCCCGGGTATAACGAGGACGTCATGTCCGGCTTTGTNCCGCTTTCTTC 
               
               
                 ACCTTCTTCGTAGCGCTTTACGTCAGGAATGTGCCAACCATCGAAGACTTCGCTGTTCGATCGATCCCA 
               
               
                 AAGGAAGCTCAGGAGCTGACTGGTGAAGCGCTAGCGGAGATATGTGAACAGGCAGCAGCCGTTTCAAGG 
               
               
                 CCATGTATTCGCCAAATGTGGAGCGTCGTATGGCAAGTTTGATGAACACACTGGAATACCTCGAGAAAA 
               
               
                 GCAAGCGTCAATTGAAAATGAAAATGCCAGAAAAAGCCATAAACAACGACACGCTCCCTGAAAGTTTCG 
               
               
                 ACAGTCGGGAGCAATGGAAGGACTGCCCCTCTCTACAGTACGTTCGCGATCAGCCACACTGTGGCTCCT 
               
               
                 GTTGGGCTGTCTCGGCTGCGAGCGCTATGTCCGATCGACTCCGTATACAGACGAACGGCAAGAATAAGG 
               
               
                 TGATTCTCTCGGACACTGACATCCTTGCATGTTGTGGAGAATTTTGTGGACTTGGATGTGAAGGAGGAT 
               
               
                 ACCCCTCACAAGCCTGGGAATTTGCCCAAAGGAATGGCCGTGTGCAGTGGAGGATGGTATGGTGAAAAG 
               
               
                 GGTGTGTGTAAACCATATCCTCTCCATCCATGTGGAAAGCACGAAAATCAGACTTTCTATGGCGAATGT 
               
               
                 CCAGACCACACGTACAGAACTCCGGCGTGCAAGAAGTACTGCCAATATGGATACGACAAGCGCTATGAT 
               
               
                 AATGATAAAGTCTA 
               
               
                   
               
               
                 Clone2.1 
               
               
                 CATCGGCCNTAGGCGGGGGTATGAAGAATATCGACAAAGACGATACTTGCGTTATGTATTCTGTGCTGG 
               
               
                 CGTATGACGCAACCAGTGAAATTCACGAAACTATTGTGATGGTTCTCATAAAGAATGAGACGGGAAAAG 
               
               
                 TCAGATCTCACTACTTCAAGTATCAGGTGATAACTGATAAGACAACAAAGAAACAAAGCACTTGGATTG 
               
               
                 ACGACATGGACGCGCTTAATTTCATGTTAACGATAAGAAAGTGTAAGCTCGTCCCTTCTAGAGGTTAAA 
               
               
                 ATCCGTCTTGAATGAATGGACATGGAAATAAATTTTCGCAGCTGTAAGAAGG 
               
               
                   
               
               
                 Clone3.1 
               
               
                 ACCGGGAAATCGGCATCCGCGGGTTTAAGCTTTACACCGATTCCATCTATTGTGTCGCTGTTTGAGATT 
               
               
                 ATTCCGAAAAATGTGCATGAGGAGCAGAACAGAAAAGAGTATCTGGAGATGGTTACCGAGAAAATGGAG 
               
               
                 TGCTTGCTAGAAGATCTTCTTCACTGTCATGATCGAAATGTGAAAAGTCGTGGAAAGAAATCAGCCGTC 
               
               
                 TAGGTTTTAATACCAAACTTGCTTCCCAGGGAGGTCTTCCAGAAAATGCCCAACTTTCTCGAAAAAGTT 
               
               
                 TTTTTTTGCATGATGTANAGGTACATGCATGGAAGCCAGAATTGTGGCTCATCTTTCCTGAGCGGCCCT 
               
               
                 GTNNAATACC 
               
               
                   
               
               
                 Clone4.1 
               
               
                 TACAAATCGGAAGTGACCCCAGTNANTACAATGTTCTAGGCAAGCGATCATCGTCCAACTCCTCAAACA 
               
               
                 GCCCCAGCCAGGATCCACTACCACCGAGAACTCCTCTTCCGCTTGATTTCTGGATATTCCGGTAAAGCC 
               
               
                 CCATGGGTATTGAANTGGACTATACAACCTGCTCACATATATGTCAACACTGTTAGAGTCTGCTATGAT 
               
               
                 CGGTCTACTGAGGATCTATTGCCCAAAGTTATGTAATCTGTGCTATCGTCGTCTTTACATCAATTTTTA 
               
               
                 TTTGATCACTGTATGATATGGGAGGGATGTTTGAATAGTAGAGAAAGTGTAGTAGTATAAATAAATACC 
               
               
                 TCATACATACACAG 
               
               
                   
               
               
                 Clone6.1 
               
               
                 GCCGGGATCTGGCACTTACGGCCGGGGTNAGGAGGAATTGTTAAAATCCTTCAAAACAAGCGAGGAGAG 
               
               
                 CCACTAGATGATGATGAAATCAAGGCCATGTACAAGGCTAAGCCACCAATCGAGAACGGTGAAGTCGAC 
               
               
                 TACAAGGCATTCGCACATCTCATCACTACCGGAGCTCAAGACGAACTCACTGCCGCATAAACGTTCCTT 
               
               
                 CAATCATTTGTGTGTGTCTTTGATCGAATCGGATGAAAGAGAAATGGCCAG 
               
               
                   
               
               
                 Clone7.1 
               
               
                 GCGGGAATTTGGGNACTGANGGCGGGATAGCTAGCTNGCAACAACATGTGGATCAAAACCGTCACCCCT 
               
               
                 GAAGGAGAGGTTACCAAGTGTAGACTGGACAGATGTTTACCAGCAACGCCCGAAATGCTGTCGGTATTT 
               
               
                 CTGAACCAGGATACCTTACACATGAGGCGGTTCAGTGGTCAGAAACACAGGGACATTGGTACTTCCTTC 
               
               
                 CTAGAAAGGAGTCAAAGACTGTCTACGTAGAAGAAGAGGATGAGACGAAAGGCACGGATCTCCTGATTA 
               
               
                 CTGGAAATCCGGACCTTGACCAATTCGAAGTCAAGAGGATAGGAATACTGCGACCCGAACGCGGATATT 
               
               
                 CGGCATTCGATTTTATTCCTGGTACCGACGACAAGATCATCGTTGCCTTGAAGTCCAAAGAAGTGACCG 
               
               
                 ACGAGCCAGTCGAAACCTACATCACAGTATTTACCACTGACGGTCAACTCCTACTTGATGATCAGAAAC 
               
               
                 TCGACGGCAACTACAAATTTGAGGGACTCTACTTCATTTGAATTTTCCCTC 
               
               
                 GTCATGAAAAGTGTCAAATGTTGGCTAACAATAAATAATTTTATAGC 
               
               
                   
               
               
                 Clone8.1 
               
               
                 TTAAACAGCTTCTACTACTGAAGCTTCGACCACTACTGTTCCGTATGAGGAGGACAACCAGATATGCCC 
               
               
                 ACATCACCGTGGCATGAAAGACACATTAAGAATAAGGGCATTAGTGGCTCACAATTATCGGAGGTCAAG 
               
               
                 GCTTGCTATGGGGCTTGTCAGAAACAGGAGAGGAAGAACACTGCCAACGGCGTCCAACATGAGACATTT 
               
               
                 GTCCTCAAAAAGAACATTTTCCACGACGACAGCGCACCACATCCACAGAGCAGTGATACCTCCTATCAC 
               
               
                 TGACAATTATTGAATACAATTGCACAAGTGAAGCATATGCCATACAAAATGCGGTGAGGTGCTCTGTAG 
               
               
                 TTCCACCTTCGACAATGCCTTCTTTTGTCCAAGAAAACCGCCATATGGTTTTAAAGTCTTTAGCAAACA 
               
               
                 CTAAGGAGAAAGCGCTTATAGTTCCGACTTCACGGCCGATCACGTCTGGTGAAATGCACTTTCACAATG 
               
               
                 CTATTGATTATGATATCATACATTTCAACTTATTGTGCTCGAAAAGTGACAGTTTCTGAGGGGCGAGAT 
               
               
                 GAGCTCAACTGCATAGCCACTAGTGCATCTTGCACAGCGGAAACACCACGAGTTATTCCTTCTGCCTGG 
               
               
                 CTACCGTACCAATGTTGCAT 
               
               
                   
               
               
                 Clone8.2 
               
               
                 GCCGGGAATTCGGNACCGCGGNTTTGCGAAGTGCTCCTGGAAGTTGCTGGTCGTGATGCCACTGAGGCC 
               
               
                 TTTGAGGACGTCGGTCATTCTACAGATGCTCGTGAGAATGAGGGAGCAATATCTTGTCGGAGATATTGC 
               
               
                 TGACGAGGAGAAGCAGCAATATTCGTATGATAAGAAGGAATGGGTGACCAGCCCCAGCGATAATAAACA 
               
               
                 AAGGGACTCGAACCCGTGGGCAGCATTGGACAAATACATCTATCCTGCTCTGTTCGCCATCGTCTTCGC 
               
               
                 CCTTATTTACTACCTTATCACAAACTAGATTTCTGCTTTTTGAAGTAGATTTGGGTTTTATTTCTTCAT 
               
               
                 GTTCCGATTTCTTGGATTGTCACTTAAATGTTTCACATTTGCATGTACCGGTATATCAGTTTTTATCCG 
               
               
                 TTGCACGATATATTATAGTTAAGGTTTGTGGTCTAACATTGTTAGGAATAAAAGT 
               
               
                   
               
               
                 Clone9.1 
               
               
                 CGATCGGCATTACGGCGGGTATATGGAACGAGGACTTGACCCCAAGCTGTGGAAGGACTTCTGGGATAT 
               
               
                 CTTCGAGAAGTTCCTGGAGAACCGCAAGCCACTAACTGCTGACCAGAAGGCTGCGCTTGATGCGATGGG 
               
               
                 CACAAGATTCAACGATGAAGCTCAGGAAGCGAACTGGCCGTCCTTGGACTTCCACACACATAAGAAACT 
               
               
                 CTCTTGGGAAATGCCTAGGTCTTGATGCGTCAGTGAATAAAGTATG 
               
               
                   
               
               
                 Clone10.2 
               
               
                 GCCGGGAATCGGCACTACGGCCGGGTTAGCGATGAAGAAAATCGAAGATCACAACACGCTTGTCTTCAT 
               
               
                 CGTTGATGTGCGAGCGAACAAGCACCAAATTCGGGCAGCTGTGAAGAAGTTGTACAAGTATTGAGGTGC 
               
               
                 AAAAGATTAACACACTTATCACTCCCCGTTATGGAGAAAAAGGCCTACATTCGCGTGTGACGACGGATT 
               
               
                 ATGATGCCTTAGATTGCGCCAATAAGATCGGAATCATATGAATTCTTGTTGTTTTGTTATGAATGGTTG 
               
               
                 ATAAATTTGGGTTAATTGAACAAGG 
               
               
                   
               
               
                 Clone11.2 
               
               
                 GGCCGGGGGTATCGGAAGGAAAGCATATTCAGACAAAAGCACGAGTCTAGCCCAATCAGACACTTCACT 
               
               
                 CTGATGGCATGGGCAACCACCGAATACATTGGATGCGCTGTGTCGCTCGCTCGTGCCCCAGGCGAGTGG 
               
               
                 TATATTGTATGTCACTACAGAAACGGAGGTAATACTTGTAAACGAACACGTTTACATGCCAGGGTCCCA 
               
               
                 CATGTTCAGACTGATCCCACAAGCTACCACTGTGGCGCGGACAAGTAGTAGCACGAAATCTTAAACAGC 
               
               
                 CTCCATGCTTATATANAGCTAATAAAAGGAAAATAAAGTTATAACGAGC 
               
               
                   
               
               
                 Clone12.2 
               
               
                 GCCGGGAATTGGCATTACNGCGGGTTGGGATTAGTTTGTGATGAAGTTTCATAACAGTGTCTATGTTTA 
               
               
                 TGATAAGTTGATGATGAGTGCTGTGTTTTTTTTAATATTTTTTGGTGTTAATTTGACTTTTTTTCCTTT 
               
               
                 GCATTTTGCTGGTTTGCACGGTTTTCCTCGTAAGTATATAGATTATCCGGATGTTTATTCTGTGTGAAA 
               
               
                 TGTTATATCTTCGTATGGTTCAATAGTAAGTGTTTTTGCTTTGTTTTTGGTTTTTGTATGTGTTGTTAG 
               
               
                 AATCTTTTTTTAGTTATCGCTTAGTGTTGGTTGATAATTTTGGTAAACAGCAGACCCGAGTATAGTTAT 
               
               
                 AGCAGTTATGTTTTGGGTCACAGTTATCAGAGTGATATTTATTTTAGAAGTGCTGTGTTAAAAAATT 
               
               
                   
               
               
                 Clone14.1 
               
               
                 CCGGGATCATCAATGAGCAATCCAGACGTTCTCAACTCGATCACTTCCAATCGCGTAAAGCAAGTGGTC 
               
               
                 AATGCTGGTGCGTCAGTGGCGCGTTGCATCAAAATGTGTTTCATCGAGAAAGAATAAAGACGGGTTTTG 
               
               
                 CTTCGACAAGAAAGGTTGTGAACCGAACATTGCGGACCGAAACGCAAAGCTTGCGATCAAACAGTGTCC 
               
               
                 ACGTTTGATTAATTGGAAGAAGGAGATCAGCGACTTGTGCATGTGTTCCAGTCAAGCAGGTGTTCATGG 
               
               
                 AATTTCGGACTACTGCGGAATGCTGAGCATAATGGGATAGAAGGAACATCTTGGTGATCATTTCGCGAT 
               
               
                 TGTTGCATGATTTGTACATTTATTGTATTTATACGACAGCGTAATCATCAAGAAACTAACTTCACTCGA 
               
               
                 TTTTCCTGATAAAATTTAACGAC 
               
               
                   
               
               
                 Clone14.2 
               
               
                 TTCAGCGTCGTGCTTTCGTTCGCGTCATTGGTGGCAGCCACTTGGGTACTCTTCAGCGACTATGTCCTT 
               
               
                 CTCACCGGTGATCATCCAGTGTGGCCTGGAGTAGCACTTTTCCTTACCAA 
               
               
                 TTTCATAATTTTTGCCTCATCTTGTGTTTACAAATTTGGCCGTACCGAAGAAATGTGGGGATAAATGTA 
               
               
                 ATATAATTTCAGTTGGTCCCCTATTTATTCCAGGATTTCGCTTTTTCTTTCCATCGGAGGCAGACACAG 
               
               
                 GCCATAATTTCTACTTACTTTTGTGAGGACATGTTCATACTTGTATTGGACTAAAGTATATTACG 
               
               
                   
               
               
                 Clone16.1 
               
               
                 GATCGATTCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCG 
               
               
                 ATTCGATTCGATCGATCGATCGATCGATCGATCGATCGATCATCATC 
               
               
                   
               
               
                 Clone17.1 
               
               
                 CATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATC 
               
               
                 GTCGTCGTCTCTCCCC 
               
               
                   
               
               
                 Clone18.1 
               
               
                 GATCGGATCGATCGGATCGGATCGATCGATCGATCGATCGATCGATCGACGTCGCGACGCGCGCGCGCG 
               
               
                 GGGCG 
               
               
                   
               
               
                 Clone 18.2 
               
               
                 ATCAGCGACTGGGGCGAAGACGGTTACTTCCGTATCGTACGAGGAGTGGACAACTGCGGTTTCCAGTCG 
               
               
                 GACGTCATCGCTGGGGACTTCCTTTGACGGTGTGATCGATCACCATAAATCTCATATGCATGAAATAAA 
               
               
                 TTGT 
               
               
                   
               
               
                 Clone19.1 
               
               
                 GATCGATTCCGATTCGATTCGGATTTCGGATTCGATTCGGATTCGGATTCGGATTGATTGGATTGAATT 
               
               
                 GAATTCGAATTGAATTTGAATTTGAATTTGAATTTGAATTTCGAATTTGAAATTTGAAAATTGAAAAAT 
               
               
                 TGAAAAA 
               
               
                   
               
               
                 Clone20.1 
               
               
                 TCGATCCGATCCGATCCGATCCGATCCGATCGATCGTCTCTCTCTCCCCC 
               
               
                   
               
               
                 Clone21.1 
               
               
                 GATCCGATCCGATCCGATCCGATCCGATCCGATCCGATTCCGATCCGATTCCGAATTCCCGATTCCCGA 
               
               
                 TTCCCGAATTCCCGAATTCCGATTCCGGATTCCGGATTCCGAATTCCGAATTCCGAATTCCGAATTCCC 
               
               
                 GAATTCCCGAATTTCCCCGAAA 
               
               
                   
               
               
                 Clone 21.2 
               
               
                 CTCACGGACGGGAAGCCGCAGCACATTTCACTTCACCTCATCGATCAGCGTTGTACAGAATACTCTCAT 
               
               
                 CACCACTCAGAGTCTTTAATAAAACAATATTTC 
               
               
                   
               
               
                 Clone22.1 
               
               
                 CATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCC 
               
               
                 GATCGATCGATCGATCGATCGTCGTCGTCGTCG 
               
               
                   
               
               
                 Clone 22.2 
               
               
                 AAATCATGTTATACTTCAGCAGAGCCGCAAACGATGAACGAGGATCACGCCGGCAGTATGGCTCGGATA 
               
               
                 GGATTGGAGCGTTATAGGAAAGATGGATGGTGTAATAAATACTATTACTCATGTCGTGCGATACTTGGC 
               
               
                 TTACCGCCAAAGGAACGAGCTCCTATCGGACCTAATGGCAAACGTCTGTGCCGCAAAAAACCGCTGTGA 
               
               
                 TTCGTCCCCTATTTGCGTATTTGTAGTGAAATACGAGCTGATTTTCGCTCCATAATGACTAGTTCGTTG 
               
               
                 AATATTTGTCATCGCTTTGCAGAATTTCACAGAATTTTTGCTTGCGCAGAAATAAATATTCCGCTCC 
               
               
                   
               
               
                 Clone23.1 
               
               
                 ATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGTCGTCGAT 
               
               
                 CGTCGTCGTCGTCGTCGTCGATCGATCGATCGA 
               
               
                   
               
               
                 Clone 23.2 
               
               
                 AACCAGGGCGCTCTTGGCCTACGGACTGTTTGCAAGGCGTTCCGACAGTTCAAAGGATGC 
               
               
                 ATGGGAAAGGGATACTCGGCTTGCATAAACGCTGGTCACTTTGTTACCGCTTCAGTTCCG 
               
               
                 ATTTTCGAGTCCTATCAGTTTGTCAGCATCTTCAATCAAATGCATTATGTTTGTAGAGGA 
               
               
                 GGATTTCAGATTTATATGAGTAATGATGACTGCATGTCAAAAGCTTGGAGTGGGAGCACT 
               
               
                 GGAGATCAGCTGAACGCCTGTCGGTACAAGTTTGAAAAGAGTAGCGATGTCAGCGCAGAA 
               
               
                 GACGCTCAGTCCGTGAAGTACTTGGCCAACACTTACCTGACGTGTTTCGAAGACCAATTC 
               
               
                 AAGGAGGCTTGTGGTCTAAACTCCCGCGACACGCAATTCTGGGGTTGTGAATACGCGCGC 
               
               
                 GTCAATGTTTTCACTCGCTTTCCTCAGACTGACGTGGACTGTGTCTTACCCTACGCAGGC 
               
               
                 GGCATGATTGGATGAGAGCGAAGCCAATACTATTGTAAATGTTACTGTGTCAAGATATTG 
               
               
                 TGATAAGATTTGAAATAT 
               
               
                   
               
               
                 Clone24.1 
               
               
                 CGATCGATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGAT 
               
               
                 CGATCGATCGATCGTCGTCGTCGCGCC 
               
               
                   
               
               
                 Clone25.1 
               
               
                 CATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATC 
               
               
                 GATCGTCGTCGTCGTCCCC 
               
               
                   
               
               
                 Clone26.1 
               
               
                 GATCCGATTCCGATTCCGATTCCGATTCCGATTCCGATCCGATTCCGATTCCGATTCGATTCCGATTCG 
               
               
                 ATTCGATTCGATTCCGATTCCGATTCCGATCCGATCCGATCCGATCCGGAATCCGGAATCCGGAATCCG 
               
               
                 GAA 
               
               
                   
               
               
                 Clone27.1 
               
               
                 GATCCGATTCCGGATTCCGGATTCCGGATTCCGGATTCCGGATTCCGATCCGGATCCGGATCCGGATCC 
               
               
                 GATCCGATCCGATCCGATCCGATCCGATCCGATCCCGATCCCGATCCCGGATCCCGGATCCCGGATCCC 
               
               
                 GGATCCCGGA 
               
               
                   
               
               
                 Clone28.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCGATCCGATCCGATCCATCCGATCGATCCGTCCGATC 
               
               
                 GATCGATCGATCGAATCGATCGATCGAATCGATCGATCGA 
               
               
                   
               
               
                 Clone29.1 
               
               
                 CGATCGATCCGATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGAT 
               
               
                 CCGATCGATCGATCGATCGATCGATCGATCGTCGATCG 
               
               
                   
               
               
                 Clone 29.2 
               
               
                 GTGTAAGTCTGGTGGTAAATCATGTTATACTTCAGCAGAGCCGCAAACGATGAACGAGGATCACGCCGG 
               
               
                 CAGTATGGCTCGGATAGGATTGGAGCGTTATAGGAAAGATGGATGGTGTAATAAATACTATTACTCATG 
               
               
                 TCGTGCGATACTTGGCTTACCGCCAAAGGAACGAGCTCCTATCGGACCTAATGGCAAACGTCTGTGCCG 
               
               
                 CAAAAAACCGCTGTGATTCGTCCCCTATTTGCGTATTTGTAGTGAAATACGAGCTGATTTTCGCTCCAT 
               
               
                 AATGACTAGTTCGTTGAATATTTGTCATCGCTTTGCAGAATTTCACAGAATTTTTGCTTGCGCAGAAAT 
               
               
                 AAATATTCCGCTCCG 
               
               
                   
               
               
                 Clone30.1 
               
               
                 CGATCGATCCGATCCGATCCGATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGAT 
               
               
                 CGATCCGATCGATCGATCGATCGTCGATCGTCGTCG 
               
               
                   
               
               
                 Clone 30.2 
               
               
                 GCCGCAAACGATGAACGAGGATCACGCCGGCAGTATGGCTCGGATAGGATTGGAGCGTTATAGGAAAGA 
               
               
                 TGGATGGTGTAATAAATACTATTACTCATGTCGTGCGATACTTGGCTTACCGCCAAAGGAACGAGCTCC 
               
               
                 TATCGGACCTAATGGCAAACGTCTGTGCCGCAAAAAACCGCTGTGATTCGTCCCCTATTTGCGTATTTG 
               
               
                 TAGTGAAATACGAGCTGATTTTCGCTCCATAATGACTAGTTCGTTGAATATTTGTCATCGCTTTGCAGA 
               
               
                 ATTTCACAGAATTTTTGCTTGCGCAGAAATAAATATTCCGCTCC 
               
               
                   
               
               
                 Clone31.1 
               
               
                 GATCGATTCCGATTCCGATTCCGATTCCGATTCGATTCGATTCGATTCGATTCGTTCGTTCGATTCGAT 
               
               
                 TCGATGATTGATTGATTGATGATGATTGATTGATTGATTGA 
               
               
                   
               
               
                 Clone 31.2 
               
               
                 TTTGTGTAAAGATGTTATTTAATAGTTAGAATTATATAGGTAGCGAATAACTGTGAACTG 
               
               
                 TGTTAAAGTTAATTATTGATGACTCGGTGTTTCGGTGGTATTTATTTGTTTAGAAGTTTA 
               
               
                 TTTATCAAAAATTTGTTATAATTAGATTTTGTTTGTTGATTTGTGGGAATTAAAATTAAT 
               
               
                 AACACTGTGCTGTGTGTTTTTTGATATTTATTGTAAATGTTTTGTAACTTTGTGCAGGTG 
               
               
                 GGTTTTGGTGGTAAGTCAG 
               
               
                   
               
               
                 Clone32.1 
               
               
                 CGATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCG 
               
               
                 ATCGATCGATCGATCGTCTCGCCTCG 
               
               
                   
               
               
                 Clone 32.2 
               
               
                 GATGTTGAGTGTAAGTCTGGTGGTAAATCATGTTATACTTCAGCAGAGCCGCAAACGATG 
               
               
                 AACGAGGATCACGCCGGCAGTATGGCTCGGATAGGATTGGAGCGTTATAGGAAAGATGGATGGTGTAAT 
               
               
                 AAATACTATTACTCATGTCGTGCGATACTTGGCTTACCGCCAAAGGAACGAGCTCCTATCGGACCTAAT 
               
               
                 GGCAAACGTCTGTGCCGCAAAAAACCGCTGTGATTCGTCCCCTATTTGCGTATTTGTAGTGAAATACGA 
               
               
                 GCTGATTTTCGCTCCATAATGACTAGTTCGTTGAATATTTGTCATCGCTTTGCAGAATTTCACAGAATT 
               
               
                 TTTGCTTGCGCAGAAATAAATATTCCGCTCCG 
               
               
                   
               
               
                 Clone33.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCCGA 
               
               
                 TCGATCGATCGATCGTCGTCGTCGTCGTCGTCGTCGATCGA 
               
               
                   
               
               
                 Clone34.1 
               
               
                 GGCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGTCGTCGACGCGTCGCGACGACGACG 
               
               
                 ACGCGC 
               
               
                   
               
               
                 Clone35.1 
               
               
                 GATCGATTCCGGATCCGGATCCGGATCCGATCGATCGATCGTCGTCGCGCGACGACGCGCCCCCCCC 
               
               
                   
               
               
                 Clone 35.2 
               
               
                 ACCTCCCCTTCTGCTCAAAAGCGAAGCCGTCAACATTCAGAGCTGCTGATTGAACTCTCG 
               
               
                 TTCTCTTCTGCAAAGCATCTTACAGTGTATTTTCATACGCACCTACTGCGTAATTCTTCC 
               
               
                 TTCTTCTTATGTAAACTTGGTACCTATATAATCCATTCTCTTTCCCCGAATAAATATTAC 
               
               
                   
               
               
                 Clone36.1 
               
               
                 GTCGATCGATCGATCGATCGTCGTCGTCGTCGTCGTCGTCGTCGTCTCTCTCTCTCTCTC 
               
               
                   
               
               
                 Clone 36.2 
               
               
                 TTATATCCATAATCTGTTTATTCGTGTCTGTGCCCCTGTGAGTTCTTTTTTTGAGGTGAA 
               
               
                 ACATCTGGATTTGATCTGTCGTGTTCCTTCTATTGATGCGTTAACGCGACACAGAAATGA 
               
               
                 AATGTCACG 
               
               
                   
               
               
                 Clone37.1 
               
               
                 GGCGTCGTCGTCGTCGTCGTCGTCGTCGTCTCTCTCTCTCTCTCTCCCCC 
               
               
                   
               
               
                 Clone 37.2 
               
               
                 CATTACGGCCGGGGGAGCTGTGGGCGCTTTTTTGGACACTCATTCTTGAGTGCCATATCCATCGAAATT 
               
               
                 CACTATGCCCAGACCAATTGTAGCCACCCATCGTTTGTTAGTCGGTGCGACGGTGGCTCTTGAAACCAA 
               
               
                 ACGTTGTTTCTGAGCTAGATGCCTGCATGCGATGTACGTTCGTCTTACGGATGCCCATCATCTCTCTGT 
               
               
                 ATAAAATTCCATGATGCT 
               
               
                   
               
               
                 Clone38.1 
               
               
                 GTCGATCGATCGATCGATCGATCGATCGATCGTCGATCGATCGATCGATCGATCGATCGATCGATCGAT 
               
               
                 CGTCGTCGTCGATCGATCGTCG 
               
               
                   
               
               
                 Clone 38.2 
               
               
                 CGGCATTACGGCCGGGGTGGAAGAAGGGCGTGGTTCTCGACCTAACCATTTCAAGAAAGGGTCCGGCTC 
               
               
                 CGTAGTGCGCAAGGCCTTGCAGACCCTCGAAGCTATCAAATGGGTTGAGAAACATGCAGATGGCAAGGG 
               
               
                 TCGAGTCTTGTCAAAGCAGGGAAGAAAGGATTTGGATCGAATTGCAACCGAACTTCGTCAGCACGTTAA 
               
               
                 ACCGATTGAGCTCTAAGTTGTTTTCAGTGCATGTTGTTTTGTTATAAATGTTGCAATG 
               
               
                   
               
               
                 Clone39.1 
               
               
                 GGATCCGGGATTCCCGGGATTCCGGATTCCGGATTCCGGATTCCGGATTCCGGATTCGGATTCGATTCG 
               
               
                 ATTTCGATTCGATTCGATTCGATTCGATTCATTCATTCGTTCTTCATTCATTCGATTCGTTG 
               
               
                   
               
               
                 Clone 39.2 
               
               
                 ATTACGGCCGGGGCGTGTAGTCATTGACGTGTATCTTTTGAAACTTAACTTGTTATCTTT 
               
               
                 TGCTACATTGTTGTGCTGAATAATAAAGTAGTTTGAATTTTG 
               
               
                   
               
               
                 Clone 42.1 
               
               
                 TCGGCATTACGGCCGGGGTCCGTCGTCGACTCCAAAGCTACTAAGACTGGTCCAACCCTTCATGGAATT 
               
               
                 ATTGGTCGCAAATCCGGAACCGTTGATGGTTTTGATTACTCTGCTGCCAATAAAAACAAGGGAGTGGTA 
               
               
                 TGGACGCGAGAGACATTGTTTGAATACCTTCTGAACCCTAAGAAGTACATCCCTGGAACAAAGATGGTC 
               
               
                 TTCGCTGGATTGAAGAAAGTCGATGAACGAGCTGATCTCATAAAATACATTGAAGTTGAATCGGCGAAA 
               
               
                 CCTGTCAGTTAACCATAATGATTATTTAATTTGAGATATGTTCGTATAGGTTTTAGTGAAAGTTTTATA 
               
               
                 AAGATCTTGATATTTGCGCTGTTGCAGAAACGTTAGCGCTCGACTTAACCATTCGTTCATTATCTCATC 
               
               
                 TCAGCT 
               
               
                 GCCCTTTACCCGTATTGTAATACCAATTTTATAGTAGCAATGTCTCATTGAAGTGAATCT 
               
               
                 TCCACCGCG 
               
               
                   
               
               
                 Clone 45.1 
               
               
                 GTGCTCGGGAGCGCGCCATTGTGCTCGGGAAGCAGCGCCATTGTGCTCGCGCAAATGCATCGTCATGTG 
               
               
                 GGCCCGAAAAAGGACGATTGTGGGGTGCGAATACAAGCCAACCGGGAACTACTTTGGAGCTCCGATCTA 
               
               
                 CGAAGTAGGAGAACCGTGCTCGAAGTGCGACTGTGAGGGCTGCAAATGTAACAAGGACGATGGTCTTTG 
               
               
                 CGTTACACCGTAAATCCAGCTGGAAAGTCTTCCAAATAAACTTGAAAAG 
               
               
                   
               
               
                 Clone48.1 
               
               
                 CATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGAT 
               
               
                 CGTCGTCGTCGTCGTCGTCGTCGTCG 
               
               
                   
               
               
                 Clone49.1 
               
               
                 GATCCGATCCGATCCGATCCGATTCCGATTCCGATCCGATCCGATCCGATCCCGATCCCGATCCGATCC 
               
               
                 GATCCCGATCCCGATCCCCGATTCCCGATTCCCGATTCCCGATTCCGGATTCCCGGATTTCCCGGATTT 
               
               
                 CCCGGATTTCCCGGA 
               
               
                   
               
               
                 Clone50.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCC 
               
               
                 GATCGATCGATCGATCGATCCGATCCGATCCGATCGATCGATCGA 
               
               
                   
               
               
                 Clone51.1 
               
               
                 GATCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATCGATC 
               
               
                 GTCGTCGTCGTCGTCGTCCGCGCG 
               
               
                   
               
               
                 Clone 51.2 
               
               
                 GTTACAAAGGGAGTACAATATTTGAATGAAATCAAGGATTCGGTCGTTGCTGGATTCCAA 
               
               
                 TGGGCAACAAGAGAAGGAGTTCTGGCTGACGAACACATGCGCGGAATTCGTTTTGACATCCAAGATGTA 
               
               
                 ACACTTCACGCTGACGCTATCCACAGAGGTGGTGGCCAAATCATCCCAACTGCTCGTCGTGTAATTTAT 
               
               
                 GCATCTGTACTCACTGCTGCACCACGACTTCTGGAACCCGTTTACCTCGTTGAAATTCAATGTCCTGAG 
               
               
                 GTTGCCGTTGGTGGTATCTATGGTGTGCTCAATCGTCGAAGAGGACACGTGTTCGAAGAGTCACAGGTC 
               
               
                 ACCGGAACTCCTATGTTTGTTGTCAAAGCCTACCTTCCCGTCAACGAATCATTTGGTTTCACTGCCGAT 
               
               
                 CTTCGTTCGAATACCGGTGGTCAAGCTTTCCCTCAATGTGTGTTTGATCACTGGCAAGTTCTACCAGGA 
               
               
                 GACCCACTGGAGCCCGGTACTAAGCCTAACCAAGTTGTTCTGGAGACAAGGAAGCGTAAAGGACTCAAG 
               
               
                 GAGGGCGTGCCCGCTCTTGACAACTACCTTGACAAAATGTAAATCTATTGTTCCGGCTTGTTGTTACCG 
               
               
                 AAGTTATCTAATAAAAAAGGTTGTTGATGGAGCTGTTTCGCAGTTATTCGAAATTCCCGTTGTTTTATT 
               
               
                 TATGCAAGAGCTAAATAAAGTTGTATAGCT 
               
               
                   
               
               
                 Clone52.1 
               
               
                 GATCCGATCCCGATTCCCGGATTCCGGATTCCGGATTCCGGATCCGGATTCCGGATTCCGGATCCGGAT 
               
               
                 TCCGATTCCGATCCGGAATCCGGATCCGGATCCGGAATCGGAATCGGAAATCGGAATTCGGAATTCGGA 
               
               
                 ATTCGGAATTCGGAATTTCGGAA 
               
               
                   
               
               
                 Clone54.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATCG 
               
               
                 ATCGATCGATCGTCGTCGTCGATCGTCGTCG 
               
               
                   
               
               
                 Clone 54.2 
               
               
                 TATTCCTTAATTCTGGACCCGGAAACACTACCAGCCCCTTCAAATGCGAGTGGATGACCA 
               
               
                 TTAAGGATGACGTGCTCTATGTTGGCGGTCACGGCAATGTGTTCAGAAATAGAGCAGGAGAAATTGTGC 
               
               
                 ACAGCAACAACATGTGGATCAAAACCGTCACCCCGGAAGGAGAGGTTACCAATGTAGACTGGACAGATG 
               
               
                 TTTACAACAACGCCCGAAATGCTGTCGGTATTTCTGAACCAGGATACCTTACACATGAAGCGGTTCAGT 
               
               
                 GGTCAGAAACACAGGGACATTGGTACTTCCTTCCTAGAAAGGAGTCAAAGACTGTCTACGTAGAAGAAG 
               
               
                 AGGATGAGACAAAAGGCACGGATCTCCTGATTACTGGAAATCCGGACCTTGATCAATTCGAAGTCAAGA 
               
               
                 GGATAGGAATACTGCGACCCGAACGCGGATATTCGGCATTCGATTTTATTCCTGGTACCGACGACAAGA 
               
               
                 TCATCGTTGCCTTGAAGTCCAAAG 
               
               
                   
               
               
                 Clone55.1 
               
               
                 GCGTCGATCATCATCGATCACATCATCACACACAAAAAAAAAACCCCC 
               
               
                   
               
               
                 Clone56.1 
               
               
                 ATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGA 
               
               
                 TCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGA 
               
               
                   
               
               
                 Clone57.1 
               
               
                 GATCGGATCGGATCGATCGATCGACGAACGACGACGACACGAGATGTCGTCGCGCCGGA 
               
               
                   
               
               
                 Clone58.1 
               
               
                 GATCCGATTCCGATCCGATCCGATCCGATCGATCGTCGTCGTCGTCGTCC 
               
               
                   
               
               
                 Clone 58.2 
               
               
                 TTACGGCCGGGCTTCGGAGGTGCGATGAAGGGCATCTTGGGTTATACTGAAGACCAAGTTGTGTCGACT 
               
               
                 GACTTCTTGTCTGACACACGCTCATCCATTTTCGACGCGGGAGCGTGCATCTCTCTTAACCCGAACTTT 
               
               
                 GTCAAGCTCATTTCATGGTATGACAACGAGTACGGATACTCGCACCGTGTCGTCGATCTGCTTACCTAC 
               
               
                 ATTGCCAGCAAGGCCTAAAATGTTTGCCGTGTTTGCTGGTTTGCGCTCTCAATCAAAGTTGTGGTTCCC 
               
               
                 TAATGTTTCATAGAGTTAGTCACCACTATGAGCGTACATTTTTTCTGTAGTCTTGTAGGTTTCCTTTTT 
               
               
                 TCTTTGGTAGCATGTAATTTATGTAGAGCTTTTATGTAATAAAATTTTGTGATGTAAAAC 
               
               
                   
               
               
                 Clone59.1 
               
               
                 GATCCGATCCGATTCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGTCGAT 
               
               
                 CGTCTCTCGTCGATCGATCGACGACACA 
               
               
                   
               
               
                 Clone 59.2 
               
               
                 GGCATTACGGCCGGGGATCTGATTCGGAGGACCACAATGCCGAGACACTGGATCATCAATTCACTTTGG 
               
               
                 TGAAGAAAAGAACGAAACAATCCCACGTGATGCGTTACGGTAGTCTGGATATAGCCAAAGAGCCACCCC 
               
               
                 AAGAAGAGAAGGTGTCATGGCCATCAAGAGACGTCCAACTCATGCATCTGCAGATGCAGAAACTGTTCA 
               
               
                 ATCCTCAAGCAGCCGCTGTCGACATCGAAATCAACAGAATCCAGACGGATCGACAAAACATTGAGGCAG 
               
               
                 TTTTCACAAGTCTGATCAACCACCTCGTCGAAGATGGTAGCGAAAGGCGTCGTTTATTTGAGCAAAGGA 
               
               
                 GCGATATTGAAAATCTCGACTGCCATGACGATGTCGTGAGGGTATTCGATATGATTTGCATTGACGTAA 
               
               
                 ATAAGTATGACTATGCCCTGAAGTATGTGTATGTTCTGAACAACCTATGCACAAAGTTCAACGATTCGG 
               
               
                 CGAAGATCATCAAGGCAATGTGGACTATCTGCTCAAAGACGCGCTCAAAGTTCCTCTGAAGCATCTTCT 
               
               
                 TAATGAGCTCTGTCGATATTATTTCAGAATAAATATTCATGAAG 
               
               
                   
               
               
                 Clone60.1 
               
               
                 GATCCGATTCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGAATCCGATCGAATC 
               
               
                 GAATCGATCGATCGATCGATCGGATCGGATCGGATCGGATCGATCGA 
               
               
                   
               
               
                 Clone61.1 
               
               
                 GTCGTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGAATCGATCGA 
               
               
                 TCGAATCGAACGGAACGAACGACGGACGGATCCGGATCGGA 
               
               
                   
               
               
                 Clone 61.2 
               
               
                 CTCACGGCCGGGGGTTGCCAGCCAACCGATGATATCCCTAGAGCGAAGAAAGAGTCGAAGAGAAAGCAG 
               
               
                 AGGCGCCAATTGGCTCGTGGAGAGTATTGAACATGATTGTTGTTGTTGTTCAGTAAATATTTTGTTG 
               
               
                   
               
               
                 Clone62.1 
               
               
                 GTCGATCCGATCCGATTCGATCGTCGTCGTCGTCGTCGATCGACGATCGATCGATCGTCGATCGATCGT 
               
               
                 CGTCGTCGTCGTCGATCGA 
               
               
                   
               
               
                 Clone 62.2 
               
               
                 TCGGCATTACGGCCGGGGGGAGTACAGTTCGGAGCAGTAGCAGTACAATATGCTGTTCTGCTTCGTATG 
               
               
                 CCTTTCTCTCATCCTTAGCTCCGCTATCCAGAGTGGAGTCAATTCTATGTTTGCATCAGTGCCGCCTGG 
               
               
                 AGCTTCGGTAATGCAAGAATGCCGAGATCGGCTTCTCACATGCGAACACGATGCTAAAAATGGTTTCTG 
               
               
                 TGAAGACCTGAGCGACTACTACATATATTATTGCTGTAAAAGTTGTAGCCAACTTGAGTCAGTTGAAAA 
               
               
                 AAAAATGAAACAAGTTTCAATTCTATTCATCGATTTCAATAAACATTTCGCTTAC 
               
               
                   
               
               
                 Clone63.1 
               
               
                 GGATCCGATCCGATTCCGATTCCGATTCCGATCCGATCCGATCCGATTCCGATCCGATCCGATCCGATT 
               
               
                 CCGATCGATCGTTCGTTCGTTCGTCGTCGTCGTTCCGTTCCATTCCGA 
               
               
                   
               
               
                 Clone 63.2 
               
               
                 GCTTACGGCCGGGGGGCTCATCATTAATCCAGCACATTTCGCCATGATCTTCTACTTTGT 
               
               
                 CTGTGCACTTTTCCTTCTCAACGCATTCACAGCTGAGGGTGCTGCCACCGCGCCATGTGAGGATCAAGG 
               
               
                 AGGCGAGTCGTTCTGCCTTGGCCCAAAGCACGCCGGCCAGTGCAGCAGTCCGGACTTCCAGCCCATTGC 
               
               
                 ACAGCAGTTCTGTGCTAAAACTTGTGGTATTTGTCACTGAATAATCTGGAGGATATTCACTAATAAAGT 
               
               
                 TTCTCGGG 
               
               
                   
               
               
                 Clone64.1 
               
               
                 GGATCGGATCGGATCGGATCGGATCGATCGATCGATCGATCGATCGAATCGATCCGATCGATCGGATTC 
               
               
                 GGATTCGATTCGATTCGATCGATTCGAATTCGAAAATTCGAAAAATCGAAAAAATCGAAAAA 
               
               
                   
               
               
                 Clone65.1 
               
               
                 GATCGATCCGATTCCGATCCGAATCCGAATCCGAATCCGATCCGAATCCGAATCCGAATCCGAATCCGA 
               
               
                 ATCCGAATCCGGAATCCGGAATCCGAATCCGAATCCGAATCCGGAATCCGGAATCCGGAATCCGGAAAT 
               
               
                 CCGGAAATCCGGAAA 
               
               
                   
               
               
                 Clone66.1 
               
               
                 GTCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATTCCGATTCCGATTCCGGATTCCGGATTC 
               
               
                 CGATTCCGATTCCGATTTCCCGATTTCCCGAATTTCCCCGAATTTCCCCGGAATTTCCCCCGGAATTTC 
               
               
                 CCCCGGAATTTCCCCCGGGAAATTTCCCCCCGGGAAATTTCCCCCGGGAAAA 
               
               
                   
               
               
                 Clone 66.2 
               
               
                 GGGACAACAATGCTCGTCTATCTGTTGGTCGCTCTAATATTCCTCAACACCGTCACTGCA 
               
               
                 CAAGCTGATGCAACCGCGTGCAAAGACGCCGACCCAGGGGATCTCGCCACGCCCTGTGAAAACCTCAAG 
               
               
                 GACCAAGGTTTTTGCGACGATCTCGACATGCGCGACTACATGAACGACTACTGCAAAAAGACGTGCAAG 
               
               
                 TTTTGTATGCCTTAATGTGACTCTATCAAGTAAATTTCGGAGGGCTCATATCACTATGTCTTAAGTATC 
               
               
                 GGATAAATGTCTAGCAAT 
               
               
                   
               
               
                 Clone67.1 
               
               
                 GCGATCGATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCG 
               
               
                 ATCGATCGATCGATCGATCGATCGATCGATCGATCG 
               
               
                   
               
               
                 Clone 67.2 
               
               
                 CGGGGACCAAACGATCCATTCGGCAATTATAAGGTTCTCTCATACAAGGAAGGACGCCGATTCGAATAC 
               
               
                 GTCCATTACCCTTTCTTCGTTCTGCAATACGATGAGAGGAGGCAAACTTACAAAGCACACTACTTTGGA 
               
               
                 TACATAGAGGAGGAGGACAAGCAAACAAAGAAGTTCACTCGCAAAATAGGTCCGTTGACCGCTGAGGAA 
               
               
                 TTCACAACTAAATATAATCATTGCAACAAGTGGTGATAATGGTCGTCTACAACAAAACTTTGTCCACTT 
               
               
                 CGATGAAAATAAATTTCGCAGTTGAAT 
               
               
                   
               
               
                 Clone68.1 
               
               
                 GTCGATCGATCCGAATCGAATCGATCGATCGATCGTCGTCTCTCATCTCCCACCTCCATCATATAT 
               
               
                   
               
               
                 Clone69.1 
               
               
                 GTCGTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGAATCGATCGA 
               
               
                 TCGAATCGAACGGAACGAACGACGGACGGATCCGGATCGGA 
               
               
                   
               
               
                 Clone 69.2 
               
               
                 GCATTACGGCCGGGGTGTATTTAATAGTTAGAATTATAAAGGTAGAGTAAATAACTGTGA 
               
               
                 ACTGTGTTAAAGTTAATTATTAATGACTCGGTGTTTCGGTGATATTTATTTGTTTAGAAG 
               
               
                 TTTATTTATTTAAAAATTTATTATAATTAGATTTTGTTTGTTGATTTGTCGGAATTAAAA 
               
               
                 TTAACAATACTGTGCTGTGTGCTTTTTGATATTTATTGTAAATGTTTTGTAAACGTTTTG 
               
               
                 TAATTTTGTGTAGGTGGGTTTTGGTGGTAAGTTAC 
               
               
                   
               
               
                 Clone70.1 
               
               
                 TCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGA 
               
               
                 TCGATCGATCGATCGTCGTCGATCGTCGA 
               
               
                   
               
               
                 Clone 70.2 
               
               
                 TCGGCTTACGGCCGGGGTCCGGGTCGCAGTCGAAGGTGTCAGATGAGGACCTGGGAAGAGTGATGGGAA 
               
               
                 TTTGCCGATGTCTTAACCTTTCCTTCACTGAAGAACAAGTGTTGGCGATAATCGCGGTAATCGAAGCAG 
               
               
                 GAGCGAATCCATCTACGCTGGTGGATTGGTTGGCCGATATGGAAGAAGCAAAAGCGGGAGAGACAACTA 
               
               
                 GCTTGAATTTTTGAAAAACGATTGATCGTAGAAGGATATTATTTGTTTTATTTATGTTCACGATTATTT 
               
               
                 AAGAGAATATGTTGTGGCAGACGGGATGATCCTCTTTGATATTTATTGAGGGTCAATTATGTGAAGCAT 
               
               
                 ATTGTTGTTTGCGGATTTTTCGCAAATAAATGTCATTTC 
               
               
                   
               
               
                 Clone71.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCCGATCGATCGATCCGATCCGAT 
               
               
                 CCGATTCCGATTCCGATTCCGGATTCCGGATTCCGGATTCCGGATTTCCGGATTTCCGGATTTCCCGGA 
               
               
                   
               
               
                 Clone72.1 
               
               
                 GATCGATCGATCGATCGATCGATCGATCATCATCATCGATCGACGACGACGACGACGACGATCGATCGA 
               
               
                 TCGATCGATCGATCGATCGA 
               
               
                   
               
               
                 Clone73.1 
               
               
                 GATTCGATTCGATCGATCGTCGTCTCTCTCTTTTTTTTTTTTTTT 
               
               
                   
               
               
                 Clone74.1 
               
               
                 GATCGATCCGATCCGATCCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGA 
               
               
                 TCGATCGATCGTCGTCGTCGTCGTCGA 
               
               
                   
               
               
                 Clone75.1 
               
               
                 GATCGATCCGATCCGATCCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCTC 
               
               
                 ATCTCATCGATCGATCATCATCA 
               
               
                   
               
               
                 Clone76.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATCGATCG 
               
               
                 ATCGATCGATCGATCCGATCCGATTCCGAATTCCGAATTCCGAATTCCGAA 
               
               
                   
               
               
                 Clone77.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCG 
               
               
                 ATCCGATCCGATCGATCCGATCGATCCGATCCGATCGATCCGATCGA 
               
               
                   
               
               
                 Clone78.1 
               
               
                 CGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATC 
               
               
                 CGATCGATCGATCGATCGATCGTCGTCGTCGTCG 
               
               
                   
               
               
                 Clone 78.2 
               
               
                 GGCCGGGGACTTTACGATGTATATCGCATTGATTGCGGGGCAAACGTGACATTCAACCTGACTATCGGT 
               
               
                 GATCATGTCTACACTCTCGAGTCGGAAAATCTTATTGTCAAATTTGATGTT 
               
               
                 GATTTCTGTGCATTGGCAATATTCCCGATGCGCTCCGGCGGCTATGGACCCCAGTGGATTCTTGGCGAT 
               
               
                 CCGTTCATACGCCAGTACTGCAACATTCATGACATCGGCAAACAGCGAATTGGCTTTGCAAAACCAGTC 
               
               
                 AAGAAATAGCGATTTTGTGATGTTCTGATTAGATGGTATAAA 
               
               
                 TGCTTCAC 
               
               
                   
               
               
                 Clone79.1 
               
               
                 GTCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGA 
               
               
                 TCCGATCGGATCCGGATTCGGATTCGGGATTCCGGGATTCCGGGA 
               
               
                   
               
               
                 Clone 79.2 
               
               
                 TCGGCTTACGGCCGGGGTAGAAGGAAGTGAATTAGCTAAGGCTGAAGCACTTATACGAGCTGAGGTTGC 
               
               
                 TGACGCGTTGCTGAAGGCTGCCACTGGACAACAATAAATGTAACATAGGCCAATTTAGGATGGATTCTT 
               
               
                 TGCAAATTCAAAAACCACTCTTAATGGCAATTTTCTAAAATTTAATAGTGTTAACTTCATACTCGCCCT 
               
               
                 TGGTTATCGTACTATAGACTGATTGACGTTGAT 
               
               
                 GTAGTGAGAATAAATATCCTTCTATTATATAAAGCGCTAACTTTGT 
               
               
                   
               
               
                 Clone80.1 
               
               
                 ATCGATCCGATCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGA 
               
               
                 TTCCGATTCCGATTCCGATTCCGATTCCGATTTCCGATTTCCGATTTCCGATTTCCGGATTTCCGGAAT 
               
               
                 TTCCGGAATTTTCCCGGAA 
               
               
                   
               
               
                 Clone81.1 
               
               
                 GTCGATCGATCCGATCCGATCCGATCCGATCCGATCGATCCGATCCGATCGATCCGATCCGATCCGATC 
               
               
                 GATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGA 
               
               
                   
               
               
                 Clone82.1 
               
               
                 GATCCGATTCCGATTCCGATTCCGATTCCGGATTCCGATCCGATCCGATCCGATCCGATCCGTCGATCG 
               
               
                 ATCGATCGATCGATCGATCGATTCGATTCGATTCGATTCGATTCGATTTCGA 
               
               
                   
               
               
                 Clone83.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCG 
               
               
                 ATCGATCGATCGATTCGATCGATCGATCGATCGATCGATCGA 
               
               
                   
               
               
                 Clone 83.2 
               
               
                 AGGGCGTAAACGACGTGTAGACATGTAGATTATTTCGATTCCTTCTCCACGATGACCCCA 
               
               
                 TGTGAAATAAGCCCCTGTGCGATTCCATGTGCCTCTAGATTCTCATACTTGCAAGCCTTA 
               
               
                 CCAATTTCGGCGACCTAGGCGCCTTCTGACGGCGTCCGCTCTTCGATGTTGCTTTTTCCCAACTCCGTA 
               
               
                 AAACCAGCAACATCAGTGAACAGCTCGTGGGCGCTCTGCCTAGCTGCCACGCGCCGTCGATTACTTGTA 
               
               
                 TATGTCTTGTGAATATTACATTATTTACGGATATCATGTGGAAATAAATTATTG 
               
               
                   
               
               
                 Clone84.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCCGA 
               
               
                 TCGATCGATCGATCGATCGATCGATCGATCGATCCGATCGA 
               
               
                   
               
               
                 Clone85.1 
               
               
                 GGTCGATCGAATTCGAATTCGAATTCGAATTCGAATTCCGAATTCGAAAATTCGAAAAATCGAAAAATC 
               
               
                 GAAAAATCGAAAAATCGAAAATCCGGAAATCCGGAATCCCGGAATCCCGGAATCCCCGGAATCCCCGGA 
               
               
                 ATCCCGGAATTCCCGGAATTCCCGGAATTCCCGGAATCCCGGAA 
               
               
                   
               
               
                 Clone86.1 
               
               
                 ATCGATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGTCGATCGATCGT 
               
               
                 CGTCGTCGTCGTCGTCGTCGTCTC 
               
               
                   
               
               
                 Clone87.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATTCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATC 
               
               
                 CGATCCGATCCGATCCGATCCGATCCGATTCCGATTCCGGAATTCCGGAATTCCGGAATTCCGGAA 
               
               
                   
               
               
                 Clone88.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCGATCGATCGTCGTCGTCGTCGTCGTCTCGTCGACGCCCTCC 
               
               
                 CCG 
               
               
                   
               
               
                 Clone89.1 
               
               
                 GATCGATTCCGATCCGATCCGATCGATCCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATC 
               
               
                 GATCGATCGATCGATCGATCCGATCCGATCCGATCCGA 
               
               
                   
               
               
                 Clone90.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATTCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGAT 
               
               
                 CCGATTCCGATTCCGATTCCGATTTCCCGAATTCCCGAATTCCCGAATTCCCCGAATTCCCCGGAATTC 
               
               
                 CCCGGAATTCCCCGGAAA 
               
               
                   
               
               
                 Clone91.1 
               
               
                 GATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATC 
               
               
                 GTCGTCGTCGTCGTCGTCGTCGCC 
               
               
                   
               
               
                 Clone92.1 
               
               
                 CATCGATCGATCCGATCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCC 
               
               
                 GATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGA 
               
               
                   
               
               
                 Clone93.1 
               
               
                 GGATCGGATCCGGATCCGATCCGATCCGGGAATCCGGAATCCGAAATCCGAAATCCGAAATCCGAAAAT 
               
               
                 CGAAAACAAAACAAAACGAAATCGAATCGAATCCGAACCGACCGACCGACCGACGAC 
               
               
                   
               
               
                 Clone94.1 
               
               
                 GATCGATCGATCGATCGATCGATCGATCGATCGATCGTCGATCGATCGTCCGTCCTCTCGATCGTCGCG 
               
               
                 TCGTCGTCGTCGATCGA 
               
               
                   
               
               
                 Clone 94.2 
               
               
                 CGGCATTACGGCCGGGGAGAGAGGGTGATTCAACAGCTGTCAGAGTCCCCTCCTATACGCGCACTCGCT 
               
               
                 CGAGCAATGGTACGTGGAGGAAAAACAGTCCAGGACAAGCTCGGTAACACGGAGGTTGCTTCGCGACTG 
               
               
                 GAGAAGTTTACAAAGCTCTACCAAGAAGAATTCCAGAAAGCACTGAAAAAATAGCTCGAGTGAGGTGTA 
               
               
                 TGCAGTCATAGAATAATATGGGTAGTAATAAAGAATTCTGATT 
               
               
                   
               
               
                 Clone95.1 
               
               
                 TCGTCGTCGTCGATCGTCGTCGTCGACCCCACACGACCCTCACACA 
               
               
                   
               
               
                 Clone 95.2 
               
               
                 TGATCCATGCGGTTGATGGCGACTGTCGTCTTGCCGTGCTCGCTGCGGTAGCCCACCCCATGGGTGTCC 
               
               
                 GGCATGAAATAGCTGTCCATCTCCACGAGGGTAAGGCGGCCCCGTACCATCTGTGCGGCCACGTGGCTT 
               
               
                 TCCACCGTGTCAAAGATGGCAAGCTCGCCGACGCGAATGCCATAAAGCGTCTCCAGATCCTCCAGCGGC 
               
               
                 ACCTTGAAAAAGGTAAACTGATCACCTTCAAAGTCCTGGTTCAGGGTGAAGCCCAGCATTGCTTCCGGC 
               
               
                 GGCAGGTTCTGTGCAGCAAGAACCTCGATCCACAGATCAACGTAGCAGTTTGTCTCCGGCCAAATCCGG 
               
               
                 TCCTGCGCATGCAGGGCATGCGGCCGGTAGGTCTGCGGATCGATATGGGGGAAAACAGCTTGCATGCCG 
               
               
                 CCGGTCAGCCCCACAATTCCTGGCGAACCTGTTCCGGCCAGGCTTCGATATCGAGGCCATGATGGTGGA 
               
               
                 ACAGGGCAAGCGCAATCCGCTCCAGGCCAAATCCGACACAGGCAGTATGGGCGGTCGTGCCATCAGCGA 
               
               
                 ATTGAATGCCCCATTTCGTGCCGAAATGATCCTGATGATAGTTGAAGCTTATGCATGCGGCCGCAAG 
               
               
                   
               
               
                 Clone96.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCG 
               
               
                 ATCCGATCGATCCGATCGATCCGATCCGATCCGATCCGATCCGATCCGA 
               
               
                   
               
               
                 Clone 96.2 
               
               
                 ACTTGAGGGGAGGCGCCAAGAAGGTTGTCATCTCCGCACCATCAGCAGATGCCCCGATGTTTGTTATGG 
               
               
                 GTGTAAACAACGAGACTTACAACGCTGCCAACAACCACATTATCAGGAACCCG 
               
               
                   
               
               
                 Clone97.1 
               
               
                 CATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGATCG 
               
               
                 ATCGATCGATCGATCGTCGATCGTCGTCG 
               
               
                   
               
               
                 Clone98.1 
               
               
                 GATCCGATCCGATTCCGATCCGATCCGATCCGATCGTCGTCGTCTCTCCC 
               
               
                   
               
               
                 Clone 98.2 
               
               
                 GACCAGAACGCCACCACTACACAATGTTGGAGTACAAATGAGTACACCGCATTTTACTGA 
               
               
                 CAGGTATCCCTATGTGCGTTATAGCTACGGAAATACGGACACCTCCTTAGGCATCGCTACTCAATCCGA 
               
               
                 GTCTGTATACGCTAGAAGTACTGCCGTTCGAGATATTGGTACGAAACGGTGGTTGGAAGGCAAGTTGAC 
               
               
                 CGCGTACAACCCATCTCAATTTCAACATCGAGCGGACTATAGACCAACATACGAACGTCCACATGTGCC 
               
               
                 ACAGAGAAGCTACATAAGGTACATGCCTGTTGACGACGCCGTCGATATGTATAAGAAGAGATGCATGAC 
               
               
                 TGTTGGGACCCTGTCAAAGTACTGGCTATCCCCTGCCACGTGGGCCTCTCGAAGAGACAAGGAATTGAA 
               
               
                 TCTGTCATCGTCGCTGAGTCGTGGAAATTACACCTACACCAACAAATATAACAGATTCAGCAGCCGTCT 
               
               
                 ATACTAACTGCAGAAACACTGCCCTTACATCATTTGGTCTATCAGCTATCAAACGTTTCCGACCTTTCA 
               
               
                 TTATGTTCGATCGTTTGCTCATATCTTAACGGAGGAATACTATTGAATGAATCTTTTAT 
               
               
                   
               
               
                 Clone99.1 
               
               
                 ATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGA 
               
               
                 TCGATCGATCGATCGATCGATCGATCCGGA 
               
               
                   
               
               
                 Clone 99.2 
               
               
                 GGAAGAAGTGCGGTGAAGAAGGTGGAGCGAAATGCTGCGAAGGAAAACCATGCTGCAAGTAATTTCGCC 
               
               
                 CGAGATGTCGAACGGTGGACGTGGTCATCATGGTCGCCTAGCTATCTGCGATCTACACCACTGATCAAC 
               
               
                 AGCGATAATTCCCTTGTGCAGCTGTAATTTCGTATTTAAATTTCCATATTTGTCCGTTTGTTTGTGTTC 
               
               
                 AGTGTGAGTGTGAAAGCGCGATAATTGTGTTTTTAAGTGTGTATTCCTCATCGGCATAGTCGAATAAAA 
               
               
                 TTTTCTGC 
               
               
                   
               
               
                 Clone100.1 
               
               
                 GATCCGATTCCGATTCCGATTCCGATCCGATCCGATCCGATCCGATCCGATCGATCCGATCCGATCGAT 
               
               
                 CGATCGATCGATCGATCGATCGATCGATCGATCGATCCGATCCGA 
               
               
                   
               
               
                 Clone101.1 
               
               
                 ATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCCGATCGATCGATC 
               
               
                 GATCGTCGTCGATCGTCGTCGTCGTCGATCG 
               
               
                   
               
               
                 Clone102.1 
               
               
                 ATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCG 
               
               
                 ATCGATCGTCGTCTCGTCGTCC 
               
               
                   
               
               
                 Clone103.1 
               
               
                 GATTCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATTCCGATT 
               
               
                 CCCGATTTCCGATTTCCGATTTCCGGATTTTCCGGAATTTCCGGATTTTCCGGAATTTCCGGAATTTCC 
               
               
                 CGGGAATTTTCCCGGGAAATTTTCCCGGGAATTTTTTCCCGGGGAAATTTTTCCCGGGGAAA 
               
               
                   
               
               
                 Clone104.1 
               
               
                 CGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATC 
               
               
                 CGATCGATCGATCGATCGATCGATCGATCGTCGTCG 
               
               
                   
               
               
                 Clone105.1 
               
               
                 GGGACGTTTCGATCCATCGAATTCGATTCGTCTCGACGAACACGCGTCTCATCACGTCTTCATAGTCTT 
               
               
                 GAGA 
               
               
                   
               
               
                 Clone106.1 
               
               
                 GGGCAATTTTCAAATTCAAATTCAAATTCAAATCGGATTCGGTTTCGATCATCGATTTCATTCATTTCG 
               
               
                 TCTCATTTGTTATGTTGATTGGTCGAGTGTTA 
               
               
                   
               
               
                 Clone 106.2 
               
               
                 TATTTTGTTAAGTTGATTGGGTCAGTGTTTAGTGGAAGAGCCTTTTACTACACTAAGTCC 
               
               
                 TATTTTTTCGTTTATTTATTTTTTTATTATTTTTATTATGATATTAATATTTAATTTTAG 
               
               
                 TAAAAAATTGTTTATT 
               
               
                   
               
               
                 Clone107.1 
               
               
                 GGGGGATCCGGATTCCGATTTCCCGGAAATTCCGGATTTCCGAATTTCCCGGATTCCCGATTCCCGATT 
               
               
                 CCGGATTCCGATTCCGGATTCCGATCCGATTCCGATTCCGATCCGATCCGATCGATCCGGTCCGATCCG 
               
               
                 AATCGATCGTCG 
               
               
                   
               
               
                 Clone108.1 
               
               
                 GAAAAAAAAAAAAAAAAA 
               
               
                   
               
               
                 Clone109.1 
               
               
                 GGGACTTCATCCGGATCGATTCATCCTCATCCTCGGATTCGATCGTCCATCCGAACGACCACATCGACG 
               
               
                 ATCGAATCACGTCGATCGATCCGA 
               
               
                   
               
               
                 Clone 109.2 
               
               
                 GACGGGGATTTACGGAATGTTACTACTGGATTGAGTCTCCAAAAGGAACCACAATCGAAGTGAAATTAG 
               
               
                 CGGATTACCCATGGGGTTATGTTGGTTCAGGATGCAGTGTTGCTGGTTTCGAGCTCAAAACCAACAAGA 
               
               
                 ACCAAACACTTACTGGCTACAGGTTCTGCACTCCTGAGGGTGTTGGACATGTGTTTCAATCTTACACAA 
               
               
                 ATCGTGTGCCAGTGATTACGTACAGCAGCTCTCTTTATAATTTCATAACCACTGAACTCGAATATCGAT 
               
               
                 ACGTTCCTGGACGTCCATCTGCCTAAACGATTTCTGCATATGGACACTATTATGGCCATCAGAGTGATA 
               
               
                 AAGTGCTGAAGTGACTTTTCTGTTGCTAAACTTCGCGGTTTAATAAAGTTTTCGC 
               
               
                   
               
               
                 Clone110.1 
               
               
                 GCATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGAT 
               
               
                 CGATCGATCGATCGATCGATCGTCTCGTCG 
               
               
                   
               
               
                 Clone111.1 
               
               
                 GATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATC 
               
               
                 GATCGATCGATCGATCGATCGTCGATCGTCG 
               
               
                   
               
               
                 Clone112.1 
               
               
                 GGGGGAATCGGATTTTCCGAAAATTTCCCCCGAATTTTTCCGGGATTCCGGATTCCGGATCCCCGGATT 
               
               
                 TCGGATTTCGAAATTCGATCCGAATTCGATTTTCGATTCAATCGAACGCGGTCGTCACAGTACGTCG 
               
               
                   
               
               
                 Clone113.1 
               
               
                 GGGACGATCGATCCGATTTCCGATTCCGGATCCCGGATCCCGATTCCCGATTCCGGATCCCGGAATTCC 
               
               
                 GAATCCCGGAATCCGGAATTTCCGGATTTCCGGATTCCCGATTTCCGAAATTTTCCGGATTTTCCGGAT 
               
               
                 TTCCGGATTTTCCGGATTTTTCCGAATTTCCGAAAATTTTTCCGGA 
               
               
                   
               
               
                 Clone114.1 
               
               
                 GCCTCATCGATCGATCATCATCATCATCATCGTCTCATCTCTCGTCGTCTCTC 
               
               
                   
               
               
                 Clone115.1 
               
               
                 GGAATTATCTTTGGAAAAAAAAAAAAAACAAATCGATCGTCCGTCCGTCCGTCCGCCGTCGATCGTCTC 
               
               
                 ATCGATCGATCGATCGACGACGATCGA 
               
               
                   
               
               
                 Clone116.1 
               
               
                 GGGCCGTCACGTCGATCATCGACGCAACGTCGCTGTCGTGTCATCAGTCGTGACACG 
               
               
                   
               
               
                 Clone117.1 
               
               
                 GGGCTGGCACGGAATTCCTTCACTCACGATCGTGTTGTTTGGAGATAGCTGTGAGTGCTGCG 
               
               
                   
               
               
                 Clone 117.2 
               
               
                 AAATACTAGAATGTGTTGTTTTTGGGAGAATAGCTGGTGGAAGTGCTGCGTCTATTAATT 
               
               
                 ATGGCCATGAAGAACTTTGAGTTTCCAGCACTTACAATTTCAAAATGTGATCTGTCACGA 
               
               
                 GTTTTTACGTAGCCGGTTGTTTCATAATCAGGCCATTATAATTGTTTGGGCAGCATAGTA 
               
               
                 TTTTATTGTTCACTTTTTGACATGTTTGGCTTTGTGTGTTGTGAATACTGACAATAAAGT 
               
               
                 AATTCGTAC 
               
               
                   
               
               
                 Clone118.1 
               
               
                 GGGAATGTTGACAGTGAAGAGTAATTGTTCTTGTTATCATAATGTGTTTAAAAAAAAAACGCG 
               
               
                   
               
               
                 Clone119.1 
               
               
                 GGGTATACCTGTGGCGGATTCATTTTGATATTTGGCAAACTAATACAATTATGCAAAAAAAAAAAAAAA 
               
               
                 AATG 
               
               
                   
               
               
                 Clone120.1 
               
               
                 GGGATTCACGTCGGCTCATCCAGCTTGGCTCGACAGTGAATCGACCACGACAGTGTCATCCAACACA 
               
               
                   
               
               
                 Clone 120.2 
               
               
                 GGGCACAAGAATCGACCACGACAGTGTCATCCAACACAGTGCCGGTAACTATCCCGATCAAGGTACCCC 
               
               
                 GGGCGTTTCCAGAAACTGGATCGTTCGGATCATTCGGATCTTATGGAAAGGCTGGTCTTGCATGCTAAT 
               
               
                 CAAATAAAGCATTTGGTTTAC 
               
               
                   
               
               
                 Clone121.1 
               
               
                 GCTCATCTCGATCGTCATCATCATCATCGCATCCCTCCTTA 
               
               
                   
               
               
                 Clone122.1 
               
               
                 GGCCCTTCACTCGATGTCACACATCATCGCATAGCTTATGTTATATTTGATAAGTTAGAAG 
               
               
                   
               
               
                 Clone123.1 
               
               
                 GGCATTCGATCCGATCGATCCGGGATCCGATCCGATCCGATTCCGGATTCCGATCCGATTCCGGGATCC 
               
               
                 GATCCGGATCCCGGGAATTCCGAATTCCGGGATTTCCGGGATTCCGGAATCCCGAAAAATTCCCGGATT 
               
               
                 TCCCCCCCGGATTTCCCGGGGATTTCCCGGGGGGGGGAA 
               
               
                   
               
               
                 Clone124.1 
               
               
                 GATCATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCGA 
               
               
                 TCGATCGATCGATCGATCGATCGATCGATC 
               
               
                   
               
               
                 Clone125.1 
               
               
                 GGGATACTGGATCAGACAGCACACCATTCCTCTGTCTTCAGTAACCTATAGAGATATAA 
               
               
                   
               
               
                 Clone 125.2 
               
               
                 AGGCACATCCCATTTCCTCTGTTCTTAAGTAAAACCCTATAGAGAATATAACTAACTTGA 
               
               
                 TAAAAGACAGTGCTTCTTATTCAAGA 
               
               
                   
               
               
                 Clone126.1 
               
               
                 GGTACGAAAGGAGAACCATCGCAACAACGAACGACTCGTAGTGTTCCGGTCGTCGCACTGCAGTG 
               
               
                   
               
               
                 Clone 126.2 
               
               
                 GCAAAACATACGAAGACTGTAAGTGTTCCGGCTGCACTTGCAGTGTAGAAGAAGCGCTCT 
               
               
                 GTGTTGCTCCTTAACACCTATGTCAATAAATTTTCATCAATAAAAAAAAAAAAAAAAAAA 
               
               
                 AAAAAAAAAATTG 
               
               
                   
               
               
                 Clone127.1 
               
               
                 GGGCGAATCATCGAATCGCGACCGCGCAATTCGTCACGTCGACTTCGTGACGCGTAGTACTGCACG 
               
               
                   
               
               
                 Clone 127.2 
               
               
                 CAATGTGAGCGTAGTACTTGCAAGCGCCCATTTGCTATCATAGCACTGATTTCTCTCGTC 
               
               
                 TTTTTCTCAGTTATTGAAGGTTTATATTCTATCCGTGTGGTTTCCTTAATATTTAGTCAA 
               
               
                 CCAAAAGAGTGTTATGCTCAACACAGTCGTTGACGGATTGGTGGTTGTTCATTCTTCGCT 
               
               
                 GCCGACCTCGCGAATGTCTCGAGAAGAACCAACTGCTTCCGTTCATCCCCTCATGCAAGTCATATGTCA 
               
               
                 TACAGTGTGTTATGAGATTATTGTGATGAATAAAGAGTTG 
               
               
                   
               
               
                 Clone128.1 
               
               
                 GGGGGGATCCAAAATCGGAATCCGGAAATCCCGAATCCGGGAATCCCGAAATCCGGATCCCCGAATCGG 
               
               
                 AATCCCGATTCGGGATTCCGGAAATCGGAAATCGGTCCCGATTCGGTCCCGGATCCCGAATCGGAATCG 
               
               
                 GAAATCGGAAATCGGGAATCGGGAAACGGGAA 
               
               
                   
               
               
                 Clone 128.2 
               
               
                 GTAAATCTGACATCCAAAACAACGATCGATCACTCTCATCTCTCATCATCTACATGTTCT 
               
               
                 TCTCCTCGCTTTCGACACTTCTTGAACAATTTCGCTATTGTGTGAGTGTGTAAAACGTGG 
               
               
                 CT 
               
               
                   
               
               
                 Clone129.1 
               
               
                 GGGACGATCGGATCCTCGATCCGATTCGGATCCATCGTCCATCCAATCACACCATCCGGTCGTCGGATC 
               
               
                 GATCGTCGTTCGTCGCCAACCACCGA 
               
               
                   
               
               
                 Clone130.1 
               
               
                 GGATCGTCCGTCGTCCGTCATCGATCACGCTCGCGTGTGCGTACACAC 
               
               
                   
               
               
                 Clone131.1 
               
               
                 GGGGGAGTCATCCGAACGATCGGTCGTCGACGTCGTCGGACATTCGGTTCACGCTATTGATGCTGATCA 
               
               
                 CGATCGTC 
               
               
                   
               
               
                 Clone132.1 
               
               
                 GGTGTCCATCGTCCGATCATCGCACGATCGACGTCGCTCTCCTCCGTGC 
               
               
                   
               
               
                 Clone133.1 
               
               
                 GCTCATCGATCGAATCGAATCGATCCGATCCGATTCCGATCCGATCCGAATTCCGAATCCGAATCCCGA 
               
               
                 TTCCCGATTCCGAATTCCGGATTTCCCGGGGATTCCCGGAATTTTTCCCGGAATTCCCGGGGAATTCCC 
               
               
                 GGAATTTCCCGGGAATTCCCCGGAA 
               
               
                   
               
               
                 Clonel34.1 
               
               
                 GGTAACGATCGATCGATCGATCATCCGTCCATCGATCATCGTCTCATCGTCGATCACGACGTCGATCTC 
               
               
                 GACATCATCG 
               
               
                   
               
               
                 Clone 134.2 
               
               
                 GGCGGCATGCGGCGTGTCCTTGTTTAACGGAACGGAATGAAGAAGAGAAAAAGCTTTCTTAAAGTAACG 
               
               
                 AGGCCAATAACGGATCGCAATGAAGATCTAATAGAGCTGTACGGCTACTTCTCTGATTTCCTTCAGTAT 
               
               
                 ACAAAAAATGTTGTTAGTATCCGCAATCGTCTAGGGGAAACGATCTTATTAAGAAAGTAGAAAGAAATA 
               
               
                 GCTAACACCTATTTCGTTTCTTGATTTTTTGAA 
               
               
                 CCACTGGCGTAAATGCTCTTACACATGTTTCCCATGAAACCAGGGTTTTCCGTTATCCTA 
               
               
                 GTCTTCCTCATAATCAAATGATCTGGTAXTTCCTGGATGXCGTTTCCTCTTTCTACCAAG 
               
               
                 ACCXTCATCXTCXXATXACXACAACGCC 
               
               
                   
               
               
                 Clone135.1 
               
               
                 ATCGATCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCCGATCGATCGATCGATCG 
               
               
                 ATCGATCGATCGATCGATCGATCGATCGTC 
               
               
                   
               
             
          
         
       
     
         [0037]    In a further embodiment, the antigens and/or vaccine/vaccine compositions provided by this invention may comprise proteins and/or peptides comprising sequences encoded by any one of the nucleic acid sequences shown in Table 1. 
         [0038]    Table 2, below, identifies some specific proteins/peptides encoded by cDNA sequences comprising the nucleic acid sequences detailed in Table 1. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                   
               
               
                 0. ostertagi L4 cDNA library screen 
               
             
          
           
               
                 Clone 
                 I.D. 
               
               
                   
               
             
          
           
               
                 1.1 
                 Cathepsin B6 
               
               
                 2.1 
                 Ser/Thr protein phosphatase (Wormbase) 
               
               
                 3.1 
                 GAL-1 
               
               
                 4.1 
                 no hit 
               
               
                 6.1 
                 Myosin 
               
               
                 7.1 
                 ?apyrase (T. circ. 40.3 kDa protein) ?thrombospondin (wormbase) 
               
               
                 8.1 
                 ASP-like protein ? 
               
               
                 8.2 
                 ? Cytochrome b5 
               
               
                 9.1 
                 Globin-like host-protective protein 
               
               
                 10.2 
                 T. circ similar to Cadherin 
               
               
                 11.2 
                 ASP-like protein ? 
               
               
                 12.2 
                 NADH dehydrogenase ND4L 
               
               
                 14.1 
                 G-protein coupled receptor (Wormbase) 
               
               
                 14.2 
                   Haemonchus  est contains THR repeat element 
               
               
                 18.1 
                 Cathepsin B 
               
               
                 18.2 
                 Cysteine PRotease related family member (cpr-6) 
               
               
                 19.1 
                 NADH ubiquinone oxidoreductase 
               
               
                 21.2 
                 Unknown 
               
               
                 22.2 
                 Putative amino acid permease 
               
               
                 23.1 
                 unknown 
               
               
                 29.1 
                 Unknown 
               
               
                 30.2 
                 Unknown 
               
               
                 31.1 
                   O. ostertagi  est 
               
               
                 31.2 
                 Unknown 
               
               
                 32.2 
                 Unknown 
               
               
                 35.2 
                 Prolyl Carboxy Peptidase like family member (pcp-4) 
               
               
                 36.2 
                 Amine transmembrane transporter activity 
               
               
                 37.2 
                 Unknown 
               
               
                 38.1 
                 Ribosomal protein 
               
               
                 38.2 
                 Ribosomal protein S19S 
               
               
                 42.1 
                 Putative cytochrome c 
               
               
                 45.1 
                 C-type single domain activation associated secreted protein ASP3 
               
               
                   
                 precursor 
               
               
                 51.2 
                 Putative Elongation Factor 
               
               
                 52.1 
                 ubiquitin 
               
               
                 54.2 
                 APYrase family member (apy-1) 
               
               
                 58.2 
                 Glyceraldehyde-3-phosphate dehydrogenase 
               
               
                 59.2 
                 Legumain 
               
               
                 60.1 
                 T. circ adult cDNA 
               
               
                 61.1 
                   O.ost  L4 est 
               
               
                 61.2 
                 60S ribosomal protein 
               
               
                 62.1 
                 metallo panstimulin 
               
               
                 62.2 
                 Unknown 
               
               
                 63.1 
                 T. circ est 
               
               
                 63.2 
                 Unknown 
               
               
                 65.1 
                 Ribosomal protein 
               
               
                 66.2 
                 Formate dehydrogenase 
               
               
                 67.2 
                 17 kDa ES antigen protein 
               
               
                 68.1 
                   O. ostertagi  putative ES protein 
               
               
                 69.1 
                   H. sapiens  chromosome 4 
               
               
                 69.2 
                 Unknown 
               
               
                 70.1 
                 unknown 
               
               
                 70.2 
                 Unknown 
               
               
                 75.1 
                   O. ostertagi  library 
               
               
                 77.1 
                 T. circ est 
               
               
                 78.2 
                 Aspartyl protease precursor 
               
               
                 79.1  
                   O. ostertagi  similar to ATP synthase 
               
               
                 79.2  
                 ATPase, F1 complex, epsilon/delta subunit 
               
               
                 80.1  
                   O. ostertage  cytochrome c oxidase 
               
               
                 81.1  
                 unknown 
               
               
                 83.2 
                 Neuropeptide-Like Protein family member (nlp-42) 
               
               
                 87.1 
                 Antigenic glycoprotein precursor 30kDa 
               
               
                 89.1  
                 unknown 
               
               
                 91.1 
                 unknown 
               
               
                 92.1 
                 lumen protein? Receptor 
               
               
                 94.2 
                 Unknown 
               
               
                 95.1 
                 unknown 
               
               
                 95.2 
                 Isoleucyl tRNA Synthetase family member (irs-2) 
               
               
                 96.2 
                 Glyceraldehyde-3-phosphate dehydrogenase 
               
               
                 98.1 
                 unknown 
               
               
                 98.2 
                 Unknown 
               
               
                 99.1 
                 Cadherin metallothionin 
               
               
                 99.2 
                 Unknown 
               
               
                 100.1 
                 Protein disulphide isomerase 
               
               
                 103.1 
                 Ribosomal protein 
               
               
                 106.2 
                 Cytochrome b 
               
               
                 109.1 
                 unknown 
               
               
                 109.2 
                 Metalloprotease ] 
               
               
                 112.1 
                 Cytochrome c oxidase 
               
               
                 113.1 
                 Ribosomal protein 
               
               
                 116.1 
                 Elongation factor 
               
               
                 117.2 
                 Unknown 
               
               
                 118.1 
                 unknown 
               
               
                 120.1 
                 
                   O. ostertagia 
                 
               
               
                 120.2 
                 Unknown 
               
               
                 125.2 
                 Rab3 GTPase-activating protein 
               
               
                 126.1 
                 ASP3 precursor (ES protein) 
               
               
                 126.2 
                 C-type single domain activation associated secreted protein ASP3 
               
               
                   
                 precursor 
               
               
                 127.2 
                 Alpha-ketoglutarate-dependent sulfonate dioxygenase 
               
               
                 128.1 
                 Troponin 
               
               
                 128.2 
                 Troponin family protein 
               
               
                 129.1 
                 Aspartyl protease 
               
               
                 134.2 
                 NADH dehydrogenase subunit 4 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   O. oslerlagi  L4 cDNA library screen (with antiserum from protected 
               
               
                 calves immunised with pool 3 fraction from Trial 3 - see FIG. 1) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Clone 1.1 
               
               
                 CGGCTTACGGCCGGGGACGTGAACAGGCAGCCAAGGCCGCAAAAGAGGCGAACAAAGCAGCTCGAGCAG 
               
               
                 CCAAAGCTGCCATGAACAAGGAAAAGAAGCCCGCTGCACAAAAAATGAAACCACCGAAGCCAGTGAAGA 
               
               
                 CTGCTGCACCCCGAGTCGGAGGAAAACGTTAAGCTTGGATAGCATGTTGTTTGTTATTGCAAATAAATA 
               
               
                 TTGTTATCGT 
               
               
                   
               
               
                 Clone2.1 
               
               
                 CATCGGCCNTAGGCGGGGGTATGAAGAATATCGACAAAGACGATACTTGCGTTATGTATTCTGTGCTGG 
               
               
                 CGTATGACGCAACCAGTGAAATTCACGAAACTATTGTGATGGTTCTCATAAAGAATGAGACGGGAAAAG 
               
               
                 TCAGATCTCACTACTTCAAGTATCAGGTGATAACTGATAAGACAACAAAGAAACAAAGCACTTGGATTG 
               
               
                 ACGACATGGACGCGCTTAATTTCATGTTAACGATAAGAAAGTGTAAGCTCGTCCCTTCTAGAGGTTAAA 
               
               
                 ATCCGTCTTGAATGAATGGACATGGAAATAAATTTTCGCAGCTGTAAGAAGG 
               
               
                   
               
               
                 Clone 4.1 
               
               
                 ACGGATATCAAGGTCGATTACAAGTCACATAACAAAGCTGGTGAATACCAGTTAGTTAGC 
               
               
                 TGTAGAAGGCCAATAAGTGACGAGGAATTGGAGGATCCAGACGTTGCTATGAAGCAACTGGAACTGCAA 
               
               
                 ATCAAAAAGGAAATGCTGATCTCGGATTTAATGAAGTCTAAGCGAAAGCTTACAAAAGAAGAGCTGAGT 
               
               
                 ATTCTCAATGAGGAACTGCCTGTTGGACAAGCGAAAAAGTCATGACCGATCACTCAGTTGTAGTATAGC 
               
               
                 TAGGTTTTCAATTAAC 
               
               
                   
               
               
                 Clone 5.1 
               
               
                 CGGCTTACGGCCGGGGTGGAACAAGATTTTCCGCATCCTTAAGGCCAAGGGCATGGCTCCGGAAATCCC 
               
               
                 TGAGGATCTCTACCATCTGATCAAGAAGGCGGTATCCATCCGAAAGCACCTTGAGCACTCGCGCAAGGA 
               
               
                 CATTGACAGCAAATACAGATTGATTCTTGTTGAGTCTCGAATCCATCGTTTGGCTCGCTACTACAAAAC 
               
               
                 CAGCCGTCAACTCCCAGCGACCTGGAAGTATGAGTCGGCGACCGCTGCCTCACTCATCTCATAAAGTTG 
               
               
                 TTTTGTGATTATTTGTTATAAATTGTTG 
               
               
                   
               
               
                 Clone 6.1 
               
               
                 GGCATTACGGCCGGGAGAGGAGGGAAGTTTACAAACCATACTACTTTGAATACATAGAGGAGGAAGACA 
               
               
                 AGATAACAAAGAAGTCCACTCGCAAAATAGGTCCGTTGACCGAAGAGGAATTCACAACTAAATATAACC 
               
               
                 ATTGCAAAAGGTGGTGATAATGGTCGTCTTGAACAAAATTTTGTCCACTTCGATGAAAATAAATTTCGC 
               
               
                 AATTGAC 
               
               
                   
               
               
                 Clone 7.1 
               
               
                 CGGCATTACGGCCGGGGCATAGAACAATTGTATTGAATGATACCATATCGCATCAAGCTT 
               
               
                 ATACAAGATTAAAGAAAGGACTTGGGACCTTGAAGTATCCCTGCATCGAGCGCGCTGTCATACAGGAAT 
               
               
                 TTCTGTACTGGCTCAAGATGGAAATGTCGCTAAAAATATATTACACCCTCCGCGGTCTCGAGATGTACA 
               
               
                 GGACTGATATCGAGGAGCTTCCCGAAGGAGCGCAATATGGATGCCACCATCTCCTTGCTCGAGGTTATA 
               
               
                 GCTTTCAGCTCATGCGTATTTTTTGCTTCTTCCGAACAACCCTAAAAAATGATTGGATTATCCATTAAT 
               
               
                 GACCTTTATAAGGTCTGTAAATTTCGCTACATTATGTGCTCGTTTGATAAAATCTGCATAAAACGATTC 
               
               
                 CGTACG 
               
               
                   
               
               
                 Clone 8.1 
               
               
                 CGGCATTACGGCCGGGGAGTCAAATGTTGTTCGTCCTCACAATCCTGTCCTTCCTCTTGGTGAATCTTG 
               
               
                 GCGCCTCGGATCAGCTTAAATATAAACAGTGCTTGGACCCCATAAGCATGGCTGACGTGTTTGTGTGGT 
               
               
                 TTTTCTTCCGGGAGGCTAGACGAGATATGGAATGGGATTGGATAGCTGCAACTGCGGCTGAGAAGGCAT 
               
               
                 TGGCTGACCCTTCTCTCGAAATGCGGGACTTCTGGAAAGCCAGCAATGGAGAGACACACGTCCGTTTAT 
               
               
                 GGGGGCGTCCACTCAATATGGTGATGAAGTTGACAAAAACCCTTCAAGGGTACAGGAAACGTTTTCCAG 
               
               
                 ATATCATGAAGATGAAGAGTAAAATATATGGATGCTGGTGCAGGTGCGACCTTAGTGAAACCGTCCTAG 
               
               
                 AAACGGTTTGCTTTTTCC 
               
               
                   
               
               
                 Clone 9.1 
               
               
                 GGGCATTACGGCCGGGGACACTCGAAATGTTGCCCATCCTTGGACTGTTCCTGCTGTTCCTTGGAAATG 
               
               
                 TCAACGCTCAGGGGAAAGAACCTTTGCCGAAACGGTGCGAAAAGATCTATGAGAGGTTCATTAAGGAGC 
               
               
                 ACACCAGAGGTTTGACTTGGAATGACGAACTGGCGTCCGAAGCTTTGGATATGCTGATGCGAGGATATT 
               
               
                 CTATGGACTTTTCATATGACTTGAAGCTTCTAGTGTCAGGGACGTTTCCGAATAGTGATAATTCGTCAT 
               
               
                 TGGAGGACAAGGTTTCTCTCACCTTGGAAAGTGCCATCTTTACACCAGAA 
               
               
                   
               
               
                 Clone 10.1 
               
               
                 CGGCATTACGGCCGGGGTCCACTTCGATGAAATAAATTTCGCAGTTGT 
               
               
                   
               
               
                 Clone 12.1 
               
               
                 TCGGCTTACGGCCGGGGTGCGCAAGTATTTCAAAGGTGCTGAAAGCTTCACTGCCGATGACGTCCAAAA 
               
               
                 AAGCGATAGGTTTGCCGTCCAAGGTATGGCTCTGCTCACATCCGTGCACATTCTTGCCGACACCTATGA 
               
               
                 CAATGAGATGATCTTCCGTGCCTTCGTCCGTGATCTCATGAACCGACATAAGGAGCGAGGACTTGACCC 
               
               
                 TAAACTCTGGAAGGACTTCTGGGATATCTTCGAGAAGTTCCTGGAGAACCGCAAGCCACTAACTGCTGA 
               
               
                 CCAGAAGACTGCGCTTGATGCGATGGGCACAAGATTCAACGATGAAGCTCAGAAGCAACTGGCCGTCCT 
               
               
                 TGGACTTCCACACACATAAGAAACTCTCTTGGGAAATGCCTAGGTCTTGATGCGTCAGTGAATAAAGTG 
               
               
                 TTGTCAGCG 
               
               
                   
               
               
                 Clone 13.1 
               
               
                 TCGGCTTACGGCCGGGGGATGCGCGGTGCATTGGTGCCCTGACATGACATTCGCCGCGTGCGAGTATAA 
               
               
                 TCCCGCTGGAAATCTTCTTGGTTCTGTTGTTTACGAAAAAGGAGATCCATGTACAACTGACGCCGACTG 
               
               
                 CCAGTGCGAAGGTTGCGTTTGCAGCAGAGATGAGGCGCTATGCATTGCCCCAGCACATTGATTTAGCTG 
               
               
                 TCATTCTCAACCCACTTTTTCAAAGTTTGTGTGCTTTGACAGTTTCAAAGGATTCATCAACAGTCAAAT 
               
               
                 AAAAGGTTTTAC 
               
               
                   
               
               
                 Clone 14.1 
               
               
                 CGGCATTACGGCCGGGAGGGAGAGACGAACAGGATGATCACCGAATTGATATTATAAGTGCAGAGAAAG 
               
               
                 GAACAGCATCAACAGACCGTGTGACGACACCATTCATGACGAAGTATGAACGCGCTCGAGTTTTAGGTA 
               
               
                 CACGTGCTCTTCAGATTGCTATGGGCGCACCGGTGATGGTCGAGTTGGAAGGAGAAACGGATCCACTGG 
               
               
                 AAATTGCTCGCAAGGAGCTAAAACATCGACGTATTCCAATCATTGTTCGACGATACCTACCAGATGGCT 
               
               
                 CATTTGAAGATTGGTCCGTCGATCAGCTGCATGTGACCGACTGGTGATATGGCTGACCATGTAACTTCT 
               
               
                 GTTTCCTGTTACCTTTCTTCTCATACCTGTTGATCTTCATGAGCCTTTTTTTTTACATATTTGGTTATA 
               
               
                 TGTTATGTCATATTTGGAGCTATACACACATCCTCCCAGCAATGAAGTGATTGAC 
               
               
                   
               
               
                 Clone 19.1 
               
               
                 GCATACGGCCGGGGAGGGCTCTGATGTGGTTTGAGCAGTTTTATTCCGGGGTCATTACAATCGCGTTTG 
               
               
                 TTGCTGGTGCATGTTATATGAGTTATCCTTTCAATAAATGGGATGTTGGACGAGCCTATCGAAGGGATT 
               
               
                 ACTGCACTCCTGCAAGAATTGAGCTGTCGAAGCGTGATCATCGTTTAACTGGGAACCAATATGTCATCT 
               
               
                 CCGGTCTGGAATCGATCATCAAATAGATTGTCGGAACTTTTGATGCTCTGCTAGGTGTTGGAAGATTGG 
               
               
                 AGTTTTCTCACTGTCATTGTAGAAGTACATTTGATAACTTTGATTGTGTCGTGTAGCTATAGATTGATG 
               
               
                 AATATTAGAATATTT 
               
               
                 TTTGGTTTCATCATCGGAATGAAATTCGAACCCT 
               
               
                   
               
               
                 Clone 20.1 
               
               
                 AGAGCGATGAACGCACGTTATGTGAAGAGCCCAAGACATACTATTCAAGTCGATTACATT 
               
               
                 GAATACATGGATGAATTAGCCAGCTTAGTTGGATGCAAGCCGAACATCGCGCAGATTTTC 
               
               
                 AAATCAGATCCGATTTTAGCATTACAGCTCTACTTTGGTCCATGCGTTCCGTACGCGTAC 
               
               
                 AGGCTGCAGGGGCCTCATCCTTGGTCAGGAGCTCGAGACGCAATAATGACAGTTGATGAAAGAGTGTTC 
               
               
                 AAGGCGACAAATTCGAACAGGTACAAGGCCTCCACTGGGTATGGATACATTATCATTGCATCGATACTT 
               
               
                 CTTGTTTTGTTGCTTATCCTACTCTTTTAATTATGTACCACA 
               
               
                 ACCTTAAAAAGAATATTTTTGC 
               
               
                   
               
               
                 Clone 21.1 
               
               
                 CGGCATTACGGCCGGGGTTCAGATGACACTGAAAAAGAGCTCGATCTTCATCGGAACTGTCTGCGATAA 
               
               
                 TGGAGTCGCAAAGAAAGCTCAAGTACCACCAGAGGCATGCCATCACAAGATCTACCCAGAGATTGGTGA 
               
               
                 CAAGTTCTTGGAGATGCTCAGCACCCCCGGCAGCTACGACATGGAAGTGATTGAGAAGGAGGCTCATCA 
               
               
                 GTCGAACATCATCAAACTGCCAGCGATCAGCAGCGCTTTGAACAACTTTGTCGTTAAGGGTGACTGGCA 
               
               
                 GGCACAGATCGCACTCGTTCTTGGAGGTCAGACAATCGCACATATCAAGGCTCCATCAAATACTGATTG 
               
               
                 GCTCTATGTCAACTAGGCCTGGCATTTCATTGGAGTAACACCTAGGCAGTGATTCACTGAAATTCCGCA 
               
               
                 ATAAAAAATGAAATATGCAGGAAC 
               
               
                   
               
               
                 Clone 23.1 
               
               
                 CGGCATTACGGCCGGGTACACAAATCGTGTGCCAGTGATTACGTACAGCAGCTCTCTTTATAATTTCAT 
               
               
                 AACCACTGAACTCGAATATCGATACGTTCCTGGACGTCCATCTGCCTAAACGATTTCTGCATATGGACA 
               
               
                 CTATTATGGCCATCAGAGTGATAAAGTGCTGAAGTGACTTTTCTGTTGCTAAACTTCGCGGTTTAATAA 
               
               
                 AGTTTTTAGTTC 
               
               
                   
               
               
                 Clone 24.1 
               
               
                 CGGCATTACGGCCGGGGAATGGAATGATACTATCTCTGAACTAGCTAGGCAAGATGTCACGCAACCAGC 
               
               
                 AAGCTTATCTTCGGCTATATTGAGAGGTTATGAAGAAGCAAGTGATTGGGTCGATTTTCCACCGAAGGA 
               
               
                 TGAACAACCAATGGAGGATAAGGTGAACTCGACAATGCACACAAAGACCTTCAATCAACGTCTGTCAAA 
               
               
                 AGTGATAAGTGGTCTAACATGTGACGAAGCTATGTTTGGATGCTACTGCGATTACGGAAGTGGACCGTT 
               
               
                 TTCTGACGACATGCAGATCAAGTGTTTCTTCCAATGAGTTTTTCAAATCAGTAAATTTGTGAAATTTTC 
               
               
                 ACTGCATGAATGAACTAGAGACAC 
               
               
                   
               
               
                 Clone 25.1 
               
               
                 TCGGCATTACGGCCGGGGACGCTGAGGGTATTGAACCAGACGATTTGGAGGAGATGTACAAGAAAGCTC 
               
               
                 ACGAACGCATTCGAAGTCAACCAGATCATGTTGCTCCAGCTCCTAAGAAAGTCGAGAAAAAGAGCTATC 
               
               
                 GCATCCATAAGATCAGCTTGGAGGAGAGAAAGAAACGCATTGAGGAGAAGAAAGCGCTGCTCCTGCTAC 
               
               
                 TGAAGAAACAGCAAGACGCCGCAATGTCGTGAATGCACAAGCTGTTGATTTACAGCAATAAAGTTGTTG 
               
               
                 AAGTC 
               
               
                   
               
               
                 Clone 26.1 
               
               
                 TCGGCATTACGGCCGGGGGATGAATGGCGAAAAGGCCCTGACGGCTTACCAGGCTCTCCTGGTCAAGCT 
               
               
                 GGAACCCCTGGTGAACCCGGCGAGCGTGGAGTTTGTCCAAAATACTGCGCCATTGATGGAGGAGTTTTC 
               
               
                 TTTGAAGATGGAACAAGGCGTTAAATTACATCAGATCTTGACATATCAGCAGTTGCTTCTAGGAAATTG 
               
               
                 TACCCACCACAAAATAAATGTATTCAAACG 
               
               
                   
               
               
                 Clone 28.1 
               
               
                 CGGCATTACGGCCGGGGTGAGTGAAAATGTACTGAACACAACAATAAGTGTGTGAAGTACGTGAATCTA 
               
               
                 TACTATATATTTTGCGCCTTATTCATAATGTTAGGTGGATTTCGAACGGGA 
               
               
                 TTATGAGAGTTGCCTTAACATTTTATCGACCTCATTTGTGGTCGAAATATGAAATTTGTG 
               
               
                 TTTCAAATGTCAAATGTTCGTTCAAATTATGCGTAGATATGTCATGTAAATAAATTTCAT 
               
               
                 GAACTTCT 
               
               
                   
               
               
                 Clone 30.1 
               
               
                 GAATCATTGTTTGACGTTCAAGTTCATATAACTAGTGTTTGCTGATGTTTTTTAAACGGT 
               
               
                 CATTTATTCATTAGGATGATGATTATTCATTGCTTTGTGAATAAACCTAATAAATAATAT 
               
               
                 CAGGTTCCCGCTATTTCTAGCCAATTATTCACCGATATAGGAAGTTGTTGTTACGTTACT 
               
               
                 AGCTTTGTAATCTGCTTTGTATGTCATTAAAGAGACTTCCGTATAGTGGCCGTTTTTCAA 
               
               
                 AGTTCTCGTGTTATTTTCAGAACAAGTAAAGAATTCTT 
               
               
                   
               
               
                 Clone 32.1 
               
               
                 TCGGCATTACGGCCGGGGACGCTTTCAAAGAAGGCGCGCGTGGAGACGCAGCGACTGATACAACCACAG 
               
               
                 CTTCTCGACCTCGGCGCAGTTCAAGCATCAGTTCTCAAAGACAATCTGAACGTTCTAGAGGCGAACGGC 
               
               
                 TTCGGATTCGAGTTTAAAGATGGCGATGACGGCTGTACAATCCCTCTGCTAGTCTCCGCACCCGTTCTT 
               
               
                 CATAGTTGGCAATTTGATAAAAGCGATATTGAAGAGATCCTAACTGTGGTTTCCGAATTTCCTGGCGTA 
               
               
                 ATGTACCGTCCAGCTAAGCTTCGCCGAATATTCGCTTCTAGAGCCCGTCGAAAATCCGTGATGATTGGC 
               
               
                 ACAACACTTACCACGGCACAAATGCAAACTATTGTTCATCACCTCGGTACACTGGATCAACCTTGGAAC 
               
               
                 TGTCCACATGGTCGCCCAACTCTTCGACATCTCGTTGACTTGCAAAACATAACCTTGTAATGTCTAATC 
               
               
                 ACTATTTCACTTTCTTAAGTTGCTATTGGAAGTACTAATGAACGATATGTTTACTCATGATTTTCAGTT 
               
               
                 CAACAAAGCAAGATTTTGAATATTTATTATGTTCTATAAAAGTTTCAG 
               
               
                 TTG 
               
               
                   
               
               
                 Clone 33.1 
               
               
                 CGGCATTACGGCCGGGTCAGAAGACGAGGATTTATTTACGGAATGGTTGAAGGATACTGCTGGAGTGTC 
               
               
                 CTCCAATCATGCTAAGAGTGCGTACAATTGTCTCAATGCGTGGGCGGAGCAATATATCTGATATTGATC 
               
               
                 TGCTAATTGAAATGTTTAGTGTGAAAATTGTATGAATTACTA 
               
               
                 CTTTTGATTTCATTTGTTCGTATTACGGTTCGTGGAATTCGGTGTGTACGACTTAACTGA 
               
               
                 CTTGTTCTAAATTGTGTCAATTTGTCATTTCGATAGGGATGCTCGAATAAACGACTATTT 
               
               
                 TC 
               
               
                   
               
               
                 Clone 37.1 
               
               
                 TATAGAAAATATTGTATTTATTTATACCTTTTATGTTAATTATATTAATTTAATATTTTTGTTGTTTTA 
               
               
                 TTTAAGTTAATTATAAAAATATTATATAAAAAATAAGTAGAGCT 
               
               
                   
               
               
                 Clone 38.1 
               
               
                 TCGGCATTACGGCCGGGATCGCAGATCAAGACTTGCGCAAGGATTGGTGAGAAAAAATACCGGCAGGTA 
               
               
                 TCTTCCAAAAGCAGCGAACATGTTCAAACTTGTTTATAACTGTTCACTTGAAACGTCAGCTAGAGAAGC 
               
               
                 GGCTGACAGATGTACGACCGCCCCATCAACATCACTTCCAAGCGGTGTCAAAGAAAATATCCACAGCGT 
               
               
                 TGCGAAGTCGATGGCCCGGTATCGTGTCGATGCTATGAAAGAGGCGGCCAGGTATTGGTGGAAGCAAGT 
               
               
                 GAGACTCGTCGATGGAATTGGAATGAAAGTCATATTCAGAGCGAAGCATGAAAGCAGCCCGATCAGATA 
               
               
                 TTTCACTCTAATGGCATGGGCAACCACCAAAACGATAGGGTGTGCTGTTTCTGAAAATTGTGGAAGCGC 
               
               
                 ATGGTTCGTGGCGTGCCACTACTATGGGGGAGGAAGTATGGTCGACGATGCCGTTTACGAGAGAGGCAC 
               
               
                 GCCATGCTCAGCTTGTCCTACTGGCTACTTCTGCAATGAGATGAAGTTATGTCAATCGGCGAATTGATT 
               
               
                 GAATTCGTGCACATTTCTTGTGGTAAATGAAGTTCTTCAGCTCG 
               
               
                   
               
               
                 Clone 39.1 
               
               
                 CGGCATTACGGCCGGGTGATGAAGGCAGAAATGGAGCGATTAGGTTTCAATCCATATGGCGAACATGCT 
               
               
                 GAAAAGCGATTGAAACCCGACTATTCTAATTCGCCTAAGGATAATCCTGCTGCTAAGGCATTTGAGGGT 
               
               
                 TTAGAGCAATAAGAATGTTATCAAATATTAAATATGCTTTTT 
               
               
                 TGCTTTTATTCATGAGTTGTTAAAATAGGTAGTGAACATTGTAGCATTGTTAGTTTTGTT 
               
               
                 CCAGATTGTTATTATTTTTTGTTTTGCACACATGACCTGTATAAAGAGTTGTTGAT 
               
               
                   
               
               
                 Clone 41.1 
               
               
                 GGATCCAAAGCCGACTGTCCTCACATATCGACTGTCCTCTAATCTTCAGTCATGAAGCTG 
               
               
                 GTTGTTCTGGCTATTCTGTTGTGCGCGGCTTATTCTGTGTATGCACAGAACTGTTTCTTA 
               
               
                 ATCACTAATCTGAGTGGTGCCACATGGGGAAAGACAGAATTTTATCGTGACGAAGAACTT 
               
               
                 CGTGGAAAGATTGAAAGGAAGCTGGAGAAGAAAATCGGCATAGACGATACTTTCGTTATGTATTCTGTG 
               
               
                 CTGGCGTTTGAGGCAAATAATGAAATTCACGAATCTATTGTGATGGTTCTCATAAAGGATGAGAAGGGA 
               
               
                 AAAGTCAGATCTCACTACTTCAAGTATCAGGTGATAACTGATAGGACAACAAAGGAACGAAGAACCTGG 
               
               
                 ATTGACGACATGGACGCGCTTAATTTCATGTTAACGATAAGAAAGTGTAAGCTCGTCCCTTCTAGAGGT 
               
               
                 TAAAATCCGTTTTGAATGAATGGACATGGAAATAAATTTTCGCAGTC 
               
               
                   
               
               
                 Clone 42.1 
               
               
                 TTACGGCCGGGCAGCAAGTTCAACGAAATATCGTCGAAGCACCAGCACCATGCAAAGCATGCTC 
               
               
                   
               
               
                 Clone 44.1 
               
               
                 TCGGCATTACGGCCGGGATGGAGCAGAAGGTAAACGCGACAATGCACGGCACTCTTCACCATCAGCTTA 
               
               
                 TAACAGCGGTCAAAAATCAACACGCCACTTTTGGATGCTATTGCGAGCTCGTCACTGATAGAAGTGGAT 
               
               
                 GGAGCGATATGTTAATCAATTGTTTCTTCAAATGACTCCTTTTTACTTCGGCGACTTTGTCAAATTTAC 
               
               
                 TGCATCTAAAATGAAAAAAAAAATTTCGCATAA 
               
               
                 ACGTTTTTTG 
               
               
                   
               
               
                 Clone 45.1 
               
               
                 GGCATTACGGCCGGGTGGCCATTGGATATCAAAGTTGCTCTGATTGGTTCCTGCACAAATTCTTCATAT 
               
               
                 GAGGATATGACTCGAGCTGCATCCATCGCTAAGCAGGCACTTGACAAAGGGTCGAAAGCTAAAACCCTG 
               
               
                 TTCACTATCACACCTGGATCGGAACAAGTTCGCGCTACAATTGAAAGGGATGGAATTTCTAAAATTTTC 
               
               
                 AGCGACTTCGGAGGAATGGTGCTGGCAAATGCGTGCGGTCCTTGCATTGGACAATGGGATCGCCAAGAT 
               
               
                 GTGAAGAAAGGAGAGAAAAATACCATTGTCACATCATATAACCGAAATTTCACTGGAAGAAACGATGCG 
               
               
                 AATCCCGCTACACACGGTTTTGTCACATCTCCTGACATTGTTACTGCACTCTCCATTACTGGAAGGCTC 
               
               
                 GACTTCGATCCCACAAAAGATCCGATTACTGCACCGGATGGCTCGAAATTCGTGCTCAAGCCTCCAACG 
               
               
                 GGAGATGATCTGCCACAGAAGGGGTACGATCCTGGTGAGGATACTTTCCAGTCGCCATCACAATCTGGA 
               
               
                 GAGGTTGTGGTCGACCCTAAATCGGATCGTCTGCAACTTCTTCAACCCTTCGATAAGTGGGATGGCAAA 
               
               
                 GACCTAGAGGACATGATCATTCTGATCAAAGTCAAAGGAAAGTGCACAACTGATCACATTTCGGCTGCC 
               
               
                 GGACCATGGCTGAAATACCGAGGTCATCTTGACAACTTTTCCAACAACTTATTCCTACCAG 
               
               
                   
               
               
                 Clone 46.1 
               
               
                 GGGCATTACGGCCGGGAAGGAGATCGTGACAGAGACAGGCGAGATCGACGCTACTGACTCATGTGGCTG 
               
               
                 TTCGGTGGAATATTCTCCGTGACATTTTGTTATGGTGTTATGGTTGTTCGTAATACTCTGCTCGATTAA 
               
               
                 TTATTAAACTCATATTTTTGTTCATGTAGTATTCTTTCGGAA 
               
               
                 TTCTAATGATGGTTCTAAGATTTGTTGTGAAGGTTTTCCGTCTGTACGTTCCAAATGGTA 
               
               
                 TTCTTTTTTTTCGCGCTGAGAATCTTGTCTTTTCTGTTGCCCTATTATTTATAGAGATTG 
               
               
                 CAAAGGTCAGTAGCTTTCTTTACAGTTTTCGTTGCAATCATGTCAATAAAAACTTCCTCT 
               
               
                 GCTC 
               
               
                   
               
             
          
         
       
     
         [0039]    In a further embodiment, the antigens and/or vaccines/vaccine compositions provided by this invention may comprise proteins and/or peptides comprising sequences encoded by any one of the nucleic acid sequences shown in Table 3. 
         [0040]    Table 4, below, identifies some specific proteins/peptides encoded by cDNA sequences comprising the nucleic acid sequences detailed in Table 3. 
         [0000]    
       
         
               
             
               
               
             
               
               
             
           
               
                   
               
               
                   O. ostertagi  L4 cDNA library screen 
               
             
          
           
               
                 Clone 
                 I.D. 
               
               
                   
               
             
          
           
               
                 1.1 
                 Chain A, Cekdm7a From  C.Elegans , Complex With H3k4me3 
               
               
                   
                 Peptide And Nog 
               
               
                 2.1 
                 Serpentine Receptor, class H family member (srh-214) 
               
               
                 4.1 
                 Unknown 
               
               
                 5.1 
                 Ribosomal Protein, Small subunit family member (rps-13) 
               
               
                 6.1 
                 17 kDa ES antigen protein 
               
               
                 7.1 
                 putative L3 ES protein 
               
               
                 8.1 
                 AIDA-1b, putative 
               
               
                 9.1 
                 Putative L3 ES protein 
               
               
                 12.1 
                 Globin-like ES protein F6 
               
               
                 13.1 
                 C-type single domain activation associated secreted protein ASP3 
               
               
                   
                 precursor 
               
               
                 14.1 
                 DNA-directed RNA polymerases I, II, and III 14.4 kDa  
               
               
                   
                 polypeptide 
               
               
                 19.1 
                 Unknown 
               
               
                 20.1 
                 Flavin-containing MonoOxygenase family member (fmo-1) 
               
               
                 21.1 
                 Unknown 
               
               
                 23.1 
                 Metalloprotease I 
               
               
                 24.1 
                 Putative L3 ES protein 
               
               
                 25.1 
                 60S ribosomal protein L5 
               
               
                 26.1 
                 Collagen col-34 
               
               
                 28.1 
                 Nuclear Hormone Receptor family member (nhr-9) 
               
               
                 30.1 
                 Seven TM Receptor family member (str-66) 
               
               
                 32.1 
                   C. briggsae  CBR-PMS-2 protein 
               
               
                 33.1 
                 Barrier to Autointegration Factor family member (baf-1) 
               
               
                 38.1 
                 Secreted protein 4 precursor 
               
               
                 39.1 
                 Elongation factor Tu homologue precursor 
               
               
                 41.1 
                 17 kDa ES antigen protein 
               
               
                 44.1 
                   C. briggsae  CBR-CDH-4 protein 
               
               
                 45.1 
                 ACOnitase family member (aco-2) 
               
               
                 46.1 
                 INneXin family member (inx-14) 
               
               
                   
               
             
          
         
       
     
         [0041]    In a further embodiment, the invention relates to one or more of the  Ostertagia ostertagi  antigens identified in  FIGS. 6 and 14 . These antigens have been identified by mass spectrometry analysis of gel slices obtained from PAGE analysis of L4 antigen preparations obtained in accordance with this invention. 
         [0042]    In addition to providing L4 antigens for use in raising immune responses in animals, the present invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have affinity or specificity for) any of the L4 antigens described herein—including those comprising sequences encoded by the cDNA sequences of Table 1. Production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example “Basic methods in Antibody production and characterisation” Howard &amp; Bethell, 2000, Taylor &amp; Francis Ltd. Such antibodies may be used in diagnostic procedures, as well as for passive immunisation. 
         [0043]    The present invention further provides a vaccine for use in preventing or controlling disease in bovine hosts caused by non-blood feeding nematode parasites. The vaccine may be a polypeptide or polynucleotide vaccine. 
         [0044]    The invention further provides a method for immunising bovine animals against non-blood feeding nematode parasites, said method comprising the step of administering to the bovine a vaccine of the invention. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0045]    The present invention will now be described in detail with reference to the following figures which show: 
     
    
     
       FIGURE LEGENDS 
         [0046]      FIG. 1 . Flow chart of the methods used to prepare the antigen fractions for the calf vaccination trials. 
           [0047]      FIG. 2 . SDS-PAGE and immunoblot analysis of ConA binding membrane proteins from  H. contortus  and  O. ostertagi.  a, coomassie blue stained. b, Immunoblot probed with sera from calves immunised with ConA binding membrane proteins from adult  O. ostertagi  (Smith et al, 2000). M, molecular weight markers. Lanes 1 and 4, adult  H. contortus;  lanes 2 and 5, adult  O. ostertagi;  lanes 3 and 6, L4  O. ostertagi.  Lanes 1-3, non reducing SDS-PAGE. Lanes 4-6 reducing SDS-PAGE. 
           [0048]      FIG. 3 . Kinetics of the antibody response following vaccination of calves with  O. ostertagi  L4 antigens or adjuvant alone in Experiment 1. Closed squares=vaccinates; open triangles=controls. 
           [0049]      FIG. 4 . Group mean faecal egg counts of vaccinated and control calves in Experiment 1. Closed squares=vaccinated animals; open triangles=controls. 
           [0050]      FIG. 5 . Group mean worm counts of vaccinated and control calves in Experiment 1. 
           [0051]      FIG. 6 . Mass spectrometry fingerprint analysis of the protective ConA binding fraction used in Trial 1. 
           [0052]      FIG. 7 . SDS-PAGE of the preparations used as antigens in Trial 2. Lane 1=ConA binding fraction; lane 2=Pool 1; lanes 3 and 4 were combined to provide Pool 2 and lane 5=Pool 3. The remaining lanes contain molecular weight markers. 
           [0053]      FIG. 8 . Kinetics of the antibody response following vaccination of calves with  O. ostertagi  L4 fractions or adjuvant alone in Trial 2. Closed squares=group 2.1. Open squares=group 2.2. Closed circles=group 2.3. Open circles=group 2.4. Open triangles=group 2.5 (controls). 
           [0054]      FIG. 9 . Group mean faecal egg counts of vaccinated and control calves in Trial 2. Closed squares=group 2.1. Open squares=group 2.2. Closed circles=group 2.3. Open circles=group 2.4. Open triangles=group 2.5 (controls). 
           [0055]      FIG. 10 . Group mean worm counts of the calves in Trial 2. *, P&lt;0.05. 
           [0056]      FIG. 11 . Chromatography and gel profiles of the preparations used as antigens in Trial 3. 
           [0057]      FIG. 12 . Group mean faecal egg counts of vaccinated and control calves in Trial 3. 
           [0058]      FIG. 13 . Group mean cumulative faecal egg counts of vaccinated and control calves in Trial 3. 
           [0059]      FIG. 14 . Mass spectrometry fingerprint analysis of the protective Peak 3 fraction used in Trial 3. 
           [0060]      FIG. 15 . Chromatography and gel profiles of the preparations used as antigens in Trial 4. 
           [0061]      FIG. 16 . Group mean faecal egg counts of vaccinated and control calves in Trial 4. 
       
    
    
     MATERIALS AND METHODS 
     Animals 
       [0062]    All calves were reared and housed indoors in conditions designed to exclude accidental infection with nematode parasites. Those used as donors for  O. ostertagi  eggs or fourth stage larvae were of various breeds and aged between 3 and 12 months at the time of infection. Those used in the vaccine trials were castrated Holstein-Fresian crosses aged between 6 and 12 months at the start of each trial. 
       Parasites 
       [0063]    Infective larvae were from strains of  O. ostertagi  which have been maintained at Moredun Research Institute for several years. 
       Parasitological Techniques 
       [0064]    The methods for faecal egg counting and enumeration of worm burdens have been described before (31,32) . Fourth stage  O. ostertagi  larvae were harvested from donor calves which had been infected with a single dose of approximately 200,000 L3 seven days earlier. Soon after the animals had been killed by captive bolt and pithing, the abomasums were removed and the contents discarded. After a brief rinse in warm saline, each abomasum was pinned mucosal surface uppermost to a block of polystyrene which was then inverted and floated in a large Baermann funnel containing warm saline. Following four hours at 37° C., fourth stage larvae were drained from the base of the funnel. The funnels were then incubated at 4° C. overnight by which time any larvae still in suspension had settled out and could be drawn off. All larvae were frozen at −70° C. until required for antigen extraction. 
       SDS-PAGE 
       [0065]    Prior to SDS-PAGE samples were heated at 100° C. for 3 min in an equal volume of 63 mM Tris-HCl pH6.8 containing 5% (w/v) SDS, ±10 mM DTT under non-reducing or reducing (10 mM DTT) conditions and separated on 4-12% gradient acrylamide gels (BIORAD, Hercules, Calif., USA). Molecular weight markers (Fermentas, Burlington, Ontario, Canada) were run on each gel and the gels were either stained with coomassie blue R250 (SIGMA, St. Louis, Mo., USA) (0.025% in 40% methanol/10% acetic acid) and destained in 20% methanol/10% acetic acid, or silver stained as follows. After SDS-PAGE the gels were washed 3 times in distilled water, and then fixed overnight in 40% methanol/10% glacial acetic acid. This was followed by incubation for 20 min in 20% methanol/5% acetic acid then 4×15 min washes in distilled water. Gels were then incubated in 50 ml 5 mg/L DTT for 45 min, then for 40 min in 50 ml 0.1% w/v AgNO 3 , followed by 2 rapid washes in water and 2 washes in 25 ml 3% Na 2 CO 3 . The gels were then developed in 50 ml 3% Na 2 CO 3  with the addition of 25 μl formalin, and the development stopped after 15 min by adding 20 ml 2.3 M citric acid. 
       Immunoblotting 
       [0066]    SDS-PAGE separated proteins were transferred to PVDF membrane (Millipore, Billerica, Mass., USA) using a semi-dry apparatus. Membranes were blocked in 10% Marvel (Premier Foods International, Spalding, Lincs., UK) in 10 mM Tris, 0.5M NaCl, 0.05% (v/v) Tween-20, 0.02% (w/v) thimerosal (TNTT), the assay diluent and wash buffer, overnight at 4° C. Periodate treatment was carried out by washing the membrane twice, for 20 minutes, in 50 mM NaAc pH 4.5, then incubating for 1 h in 50 mM NalO 4 /50 mM NaAc, in the dark at room temperature. After further washes of 2×10 minutes in 50 mM NaAc, then 2×10 minutes in TNTT, the membrane was incubated for 30 minutes in 50 mM NaBH 4 , after which it was washed for 3×10 minutes in TNTT. Membrane strips were incubated with pooled serum samples from each group, diluted 1/300 in TNTT, for 2 h at room temperature. They were then washed 3×5 minutes in TNTT, then incubated with rabbit anti-bovine immunoglobulin horseradish peroxidise conjugated antibody diluted 1/1000 in TNTT (P0159, DAKOcytomation, Glostrup, Denmark). 
       Protein Concentrations 
       [0067]    These were estimated by the bicinchoninic protein assay reagent according to the manufacturer&#39;s instructions (Pierce, Thermo Fisher Scientific Inc., Waltham, Mass., USA). 
       Preparation of Immunogens 
       [0068]    Triton X-100 extracts of  Ostertagia  L4 membranes were prepared as detailed for  Haemonchus    
         [0069]    (33), and diluted four-fold with 10 mM Tris-HCl, 0.5M NaCl, 0.05% NaN 3 , 10 μM MnCl 2 , 100 μM CaCl 2 , pH 7.4 (Lectin Wash Buffer, LWB). The solution was pumped (8 ml/h) at 4° C. through ConcanavalinA (ConA) lectin cross linked to agarose beads (Vector Laboratories, Burlingame, Calif., USA) contained in a column. After thorough washing in LWB/0.5% reduced Triton X-100 the column was eluted with LWB/0.25% CHAPS/0.2M methylmannopyranoside/0.2M methylglucopyranoside ( FIG. 1  flow chart). For elution, sufficient sugar solution was pumped onto each column to cover the beads, then the flow was stopped for approximately one hour. The pump was re-started and the peak monitored at OD 280  was retained as the “1 hour eluate”. The elution process was then repeated exactly, except that the flow was stopped overnight to produce an “overnight eluate”. The eluates were pooled and passed through a column of Sephadex G-25 to remove the sugar and exchange the buffer to 10 mM Tris-HCl, 0.1% CHAPS , pH 7.4 and stored at −70° C. before use as immunogens. 
         [0070]    The ConA eluate was fractionated on a MonoQ anion exchange column, 1 ml bed volume (Pharmacia, Pfizer, Kent, UK) equilibrated in 10 mM Tris/0.1% CHAPS pH7.4. The ConA eluate was applied to the column (1 ml/min), and unbound proteins were collected. The bound proteins were eluted by a linear gradient increase in NaCl from 0 M to 1 M over 20 ml, with 10×2 ml fractions being collected. The fractions were then pooled as follows:—Pool 1=unbound material and proteins eluted with up to 0.1M NaCl; Pool 2=fractions eluted between 0.1 and 0.5M NaCl and Pool 3=fractions eluted between 0.5 and 1.0M NaCl (Fig flow chart). Further batches of Pool 2 and 3 material were prepared and fractionated by gel filtration using a Superose 12 column equilibrated with 10 mM Tris, 0.1% CHAPS , 0.5M NaCl pH 7.4 and flowing at 0.5 ml/min. Two hundred ul of Pool 2 containing 0.75 μg of protein was separated in 2 runs was fractionated on a single 30 cm column. Pool 3 was fractionated under identical conditions except two 30 cm columns were coupled in series to improve the resolution. 
       ELISA for Antibodies to Immunogens 
       [0071]    Microtitre plates were coated overnight at 4° C. with 50 μl coating protein per well (ConA eluate), at 0.5 μg/ml in 50 mM sodium bicarbonate buffer, pH 9.6. The plates were washed six times with wash buffer (PBS, 0.05% v/v Tween-20), then incubated with 200 μl 10% (w/v) infasoy (Cow and Gate, Trowbridge, Wiltshire, UK) in TNTT overnight at 4° C. After washing, 50 μl serum per well, diluted 1:2000 in TNTT, were added for 1 h at room temperature. The wells were re-washed and 50 μl peroxidase conjugated rabbit anti-bovine immunoglobulin diluted 1:1000 in TNTT added for 1 h at room temperature. After a final wash, 50 μl o-phenylenediamine dihydrochloride substrate (Sigma) were added to each well. After 10 min in the dark, the colour reaction was stopped by addition of 25 μl 2.5 M sulphuric acid per well and OD values read at 490 nm. Each test sample was assayed in triplicate. Pooled serum taken at the time of challenge from the group of calves in Experiment 2 immunised with the unfractionated ConA eluate was included on each plate as a reference sample, and OD values expressed relative to this value. 
       Mass Spectrometry Analysis of Protein Fractions 
       [0072]    The ConA binding fraction used in Trial 1 and Peak 3 employed in. Trial 3 were fractionated by 1-dimensional SDS-PAGE under reducing conditions. Each sample (approximately 10 μg) was mixed with 10 μL SDS-PAGE sample buffer (0.05 m Tris, pH 6.8, containing 5% (w/v) SDS, 20% (v/v) glycerol, 0.01% (w/v) bromophenol blue and 10 mm DTT), boiled for 5 min before loading onto 10% gels with a 3% stacking gel. After protein separation, gels were stained with colloidal Coomassie Blue (SimplyBlue™ SafeStain, Invitrogen), destained in water and the image of each track captured. Mass spectrometry analysis was performed at the Moredun Research Institute&#39;s Proteomics Facility &lt;http://www.mri.sari.ac.uk/fgu-functional-genomics-services.asp&gt;. Each gel track was sliced horizontally into about 27 equal gel slices of approximately 2-5 mm each and individual slices were finely chopped (approximately 1 mm3), transferred to clean 0.5 mL Eppendorf tubes and processed using standard in-gel reduction, alkylation and trypsinolysis steps (15). Digest supernatants of 20 μL final volume were transferred to HPLC sample vials and stored at 4° C. until required for liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. Liquid chromatography was performed using an Ultimate 3000 nano-HPLC system (Dionex) comprising a WPS-3000 well-plate micro auto-sampler, a FLM-3000 flow manager and column compartment, a UVD-3000 UV detector, an LPG-3600 dual-gradient micropump and an SRD-3600 solvent rack controlled by Chromeleon chromatography software (Dionex: http://www1.dionex.com). Samples of 4 μL were applied to the column by direct injection. Peptides were eluted by the application of a 15-min linear gradient from 8% to 45% solvent B (80% acetonitrile, 0-1% formic acid) and directed through a 3-nL UV detector flow cell. LC was interfaced directly with a 3-D high capacity ion trap mass spectrometer (Esquire HCTpIus™, Bruker Daltonics) utilizing a low-volume (50 μL/min maximum) stainless steel nebuliser (Agilent, catalogue number G1946-20260) and ESI. MS/MS analysis was performed as previously described (16). A peak list file was generated from the resultant data and submitted to a local database server using the MASCOT search engine for protein database searching against NCBInr &lt;http://www.ncbi.nlm.nih.gov/&gt; and Nembase &lt;http://www.nematodes.org/nematodeESTs/nembase.html&gt; databases. The modifications used in these searches were a global modification of carbamidomethyl (C) and a variable modification of oxidation (M). The tolerances used were; for MS data, 1.5 Da, and for MS/MS data, 0.5 Da. Matches achieving a significant molecular weight search (MOWSE) score were considered significant if two peptides matched for each protein, each of which had to contain an unbroken b or y ion series of a minimum of four amino acid residues. The other criterion considered in assigning a positive identification for each protein was a concordance between the calculated theoretical molecular mass value of the protein and the observed position of the peptide on 1-D gel electrophoresis. 
         [0000]      Ostertagia Ostertagi  L4 cDNA Library Construction and Validation 
         [0073]    This was made in Lambda TriplEX (Clontech) and amplified ×1 according to the manufacturer&#39;s instructions as described previously (Skuce et al, 1999). Ten-fold dilutions of the unamplified primary library were made in SM buffer over the dilution range 10 −1 -10 −5 . A 10 μl aliquot of each dilution was mixed with 200 μl of XL 1-Blue plating cells (OD. 600 =0.5) and incubated for 30 min at 37° C. to allow the phagemids to bind to the cells. After incubation, 4 ml of NZY top agarose at 48° C. was added and the mixture was plated onto pre-warmed 100 mm diameter LB-agar plates. After the top agarose solidified the plates were incubated overnight at 37° C. The 10 −1  dilution plate had 293 plaques, therefore the primary library contained 2.93×10 5  pfu/ml. 
         [0074]    The amplified cDNA library was titrated as above except that the top NZY agarose was supplemented with 100 μl of 100 mM IPTG and 80 μl of 50 mg/ml X-gal to allow the selection of a blue “wild type” phagemid plaque. Several blue plaques were identified and agar plugs containing individual plaques taken into 0.5 ml SM buffer containing 20 μl CHCl 3 , to prevent bacterial growth, and stored at +4° C. 
         [0075]    The SM buffer supernatant, prepared as above, containing a wild type Lambda TriplEx phagemid was titrated and a dilution that gave near confluent plaques was selected. This clone was plated as above and grown overnight at 37° C. After overnight incubation the plates were flooded with 5 ml of SM buffer and agitated gently on an orbital rocker for 5 h. The resulting suspension of  E. coli/ phagemid was divided into 1 ml aliquots and subjected 3 rounds of freezing and thawing (−80° C. for 30 min followed by 37° C. for 5 min) to lyse both the  E. coli  and phagemid. The resulting lysate was stored at −80° C. until required. 
         [0000]    Immunoscreening of cDNA Library 
         [0076]    The unamplified primary library was diluted 10 −1  in SM buffer and plated in NZY top agarose onto LB agar plates as above and incubated at 42° C. for 6 h. A nitrocellulose filter, pre-treated with 10 mM IPTG, was placed on top of the top agarose of each plate and incubated at 37° C. overnight. After overnight incubation the plates were transferred to +4° C. for 1 h. The filters were marked to ensure correct orientation later and carefully lifted off the plates. The filters were washed extensively (several changes of TNTT buffer over ˜6 h) and blocked overnight in a solution of 1% w/v gelatine in TNTT at +4° C. When immunoscreening using sera from Trial 3 the filters were blocked in TNTT alone, as this was shown to give a lower level of background staining when the blots were developed. 
         [0077]    Filters were probed for 1 h at room temperature with pooled serum from the best protected groups (Groups 2.4 and 3.3 see Table 5 and  FIG. 1 ) in trial2 and 3 (diluted to 1/400 in TNTT buffer). The serum had been pre-absorbed as follows:—500 μl was mixed with 500 μl of  E. coli/ lambda TriplEx freeze/thaw lysate at +4° C. overnight, centrifuged and the supernatant retained for subsequent use. Bovine IgG was detected with a biotin-labelled monoclonal antibody to bovine IgG (Dako) diluted to 1/4000 in TNTT for 1 h at room temperature. In Trial 3 Bovine IgG was detected with a similar antibody (Sigma) diluted to 1/2000 
         [0078]    Biotin was detected with Streptavidin-HRPO conjugate (Sigma) diluted 1/2000 or 1/5000 in TNTT for 1 h at room temperature. The filters were washed between each step with 3×5 min washes in TNTT 
         [0079]    Finally, HRPO activity was revealed with 3,3-diaminobenzidine (SigmaFast, Sigma) prepared as per the manufacturer&#39;s instructions. 
         [0080]    Immuno-positive plaques were picked into 0.5 ml SM buffer with 20 μl CHCl 3  and subjected to a second round of screening to obtain clones of each positive plaque. 
         [0000]    PCR of Insert DNA from Immuno-Positive Clones 
         [0081]    The  O. ostertagi  DNA encoded in the immuno positive clones was amplified by per using primers directed at the pTriplEx vector sequence flanking the cloning site. The primer sequences were; 
         [0000]                            TriplEx Forward:           5′-CTC GGG AAG CGC GCC ATT GTG-3′                       TriplEx Reverse:           5′-TGC GGC CGC ATG CAT AAG CTT G-3′            
The PCR reaction mixture contained 2 μl of a freeze/thaw lysate prepared from individual immuno-positive clones, as template and 23 μl of a reaction mix containing 1× reaction buffer (Bioline), 5 mM MgCl2, 200 μM dNTPs, 1 μM of each primer and 1 U Taq polymerase per reaction.
 
         [0082]    The PCR protocol was as follows;
   94° 5 min, denaturation   
 
         [0084]    Then 30 cycles of:— 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
             
             
               
                   
                 94°  
                 1 min 
               
               
                   
                 64° 
                 1 min or 54° 2 min 
               
               
                   
                 72° 
                 1 min or 3 min 
               
               
                   
               
             
          
         
       
     
         [0085]    Followed by
   72° 7 min, sequence extension   
 
         [0087]    PCR products were purified, using a proprietary clean up kit (Qiagen) and sequenced using the Pyrosequencer or sent to Eurofins (MWG) for sequencing 
       Design of Protection Experiments 
       [0088]    Four immunisation-challenge trials were conducted with weight balanced groups of calves. The number of animals assigned to each group and the dose of antigen each group received is laid out in Table 5. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Age and weight of the vaccinated calves, the type and dose of antigen 
               
               
                 administered to each group and summary protection data 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                   
                   
                 mean % 
               
               
                   
                 Age 
                 Wt range 
                   
                   
                   
                 Dose/ 
                   
                 Protection 
               
             
          
           
               
                 Trial 
                 (m) 
                 (kg) 
                 Group 
                 N 
                 Antigen 
                 shot (ug) 
                   
                 eggs 
                 worms 
               
               
                   
               
             
          
           
               
                 1 
                 6 to 8 
                 nd 
                 1.1 
                 7 
                 ConA 
                 95 
                   
                 60 
                 47 
               
               
                   
                   
                   
                 1.2 
                 8 
                 Adjuvant only 
                 0 
               
               
                 2 
                  8 to 10 
                 155 to 240 
                 2.1 
                 7 
                 ConA 
                 50 
                   
                 70 
                 51 
               
               
                   
                   
                   
                 2.2 
                 7 
                 Pool 1 
                 16 
                   
                 85 
                 0 
               
               
                   
                   
                   
                 2.3 
                 6 
                 Pool 2 
                 19 
                   
                 83 
                 38 
               
               
                   
                   
                   
                 2.4 
                 6 
                 Pool 3 
                 14 
                   
                 78 
                 63 
               
               
                   
                   
                   
                 2.5 
                 7 
                 Adjuvant only 
                 0 
               
               
                 3 
                 10 to 12 
                 170 to 360 
                 3.1 
                 7 
                 Pool 2 - peak 1 
                 25 
                   
                 30 
                 0 
               
               
                   
                   
                   
                 3.2 
                 7 
                 Pool 2 - peak 2 
                 14 
                   
                 61 
                 0 
               
               
                   
                   
                   
                 3.3 
                 7 
                 Pool 2 - peak 3 
                 16 
                   
                 83 
                 0 
               
               
                   
                   
                   
                 3.4 
                 7 
                 Adjuvant only 
                 0 
               
               
                 4 
                 10 to 11 
                 175 to 290 
                 4.1 
                 7 
                 Pool 3 - peak 2 
                 10 
                   
                 54 
                 13 
               
               
                   
                   
                   
                 4.2 
                 7 
                 Pool 3 - peak 3 
                 1 
                   
                 35 
                 26 
               
               
                   
                   
                   
                 4.3 
                 7 
                 Pool 3 - peak 4 
                 2 
                   
                 47 
                 0 
               
               
                   
                   
                   
                 4.4 
                 7 
                 Adjuvant only 
                 0 
               
               
                   
                   
                   
                   
                   
                   
                   
                 mean 
                 62 
                 22 
               
               
                   
               
             
          
         
       
     
         [0089]      FIG. 1  shows a flow chart of how the different immunogen fractions were prepared. All groups were immunised three times at three week intervals and challenged with 50,000  O. ostertagi  L3 one week later. Immunogens were diluted with cold phosphate buffered saline, pH 7.4, (PBS) and mixed with QuilA (Superfos Biosector) so that each calf received either 20 mg (Trial 1) or 5 mg (Trials 2, 3 and 4) of adjuvant at each immunisation. Control immunogen was prepared identically, except that PBS was substituted for antigen, and administered to all challenge control animals. One ml of immunogen was injected intramuscularly into each side of the neck. All animals were bled at approximately weekly intervals to monitor the kinetics of the antibody response. Details of antigens, dose and numbers of animals are given in Table 5. 
         [0000]    Statistical methods 
         [0090]    Arithmetic group means are shown throughout with their standard errors. Significant differences between groups were calculated by the t test in Trial 1 and by analysis of variance followed by Tukey&#39;s test in Trials2, 3 and 4. To satisfy Bartlett&#39;s test for equal variances the egg data i was log transformed prior to analysis. 
       Results 
     Yield of Fourth Stage Larvae and ConA Binding Membrane Proteins 
       [0091]    Recovery of fourth stage  O. ostertagi  larvae from donor calves ranged from 5% to 20% of the dose given. The yield of ConA binding membrane proteins was approximately 0.3 mg per 100,000 fourth stage larvae. 
       Comparison of L4 and Adult ConA Binding Proteins 
       [0092]    ConA binding integral membrane proteins, prepared in the same way from adult  Haemonchus  conforms or fourth and adult stages of  O. ostertagi  were compared by gel analysis and western blotting. Coomassie stained gels indicated differences in the profiles of all three fractions ( FIG. 2   a ), although additional bands present in the L4 but not in the adult  O. ostertagi  preparations were of most interest in this case. When the three ConA binding fractions were probed with anti-sera from calves which had been immunised with material obtained in the same way from adult  Ostertagia,  additional bands were still detected in the L4 fraction ( FIG. 2   b ). 
         [0093]    Digesta from a worm free calf was treated in exactly the same way as the L4s, but no protein peak was detected when the ConA column was eluted with sugar (not shown). 
         [0000]    Protective Capacity of Glycoproteins from  O. ostertagi  L4s
 
Trial 1: Immunisation with the ConA Lectin Binding Fraction
 
       1a) Antigen Used for Immunisation. 
       [0094]    The gel profile of the preparation used to immunise the vaccinated calves in Trial 1 was very similar to that shown in  FIG. 2  lane 3. 
       1b) Antibody Response 
       [0095]    Serum antibody titres in the control group remained at background concentrations throughout ( FIG. 3 ). In contrast, a marked response was observed in the vaccinated group by week 5, two weeks after the second vaccination. This response reached a peak on week 8 two weeks after the third immunisation. 
       1c) Egg and Worm Counts 
       [0096]    Mean egg counts of the immunised calves were always lower than controls throughout the experiment, although the difference was not statistically significant on Days 28 and 30 ( FIG. 4 ). However the group means of the cumulative eggs per gram over Days 19 to 30 were significantly different (P=0.01), with the vaccinated animals shedding 60% fewer eggs. Significantly (P&lt;0.01) fewer worms were recovered from the vaccinates (1909±252) compared to the controls (3621±414 and  FIG. 4 ). Small numbers of early fourth stage larvae were found in some calves but no difference between vaccinates and controls was observed. 
       1d) Identity of Components in the Protective Fraction by Mass Spectrometry. 
       [0097]    This is shown in  FIG. 6   
         [0098]    Trial 2: Immunisation with Sub Fractions of the ConA Lectin Binding Glycoproteins. 
         [0099]    This trial was done partly to determine whether the level of protection detected in the first trial could be improved if fractions were prepared which were more enriched for the protective components but also to find out whether simpler fractions could be equally protective. 
         [0000]    2a Antigens used for Immunisation 
         [0100]    A flow chart depicting how these preparations were made is shown in  FIG. 1 , the details relating to which calves received which fraction and the dose of protein administered are presented in Table 5 whereas the SDS-PAGE profiles of the immunogens are shown in  FIG. 7 . 
         [0000]      2   b ) Antibody Response 
         [0101]    The kinetics of the antibody responses of each group is shown in  FIG. 8 . All vaccinated groups showed a similar antibody response to  O. ostertagi  L4 antigen, and had a significantly (P&lt;0.01) higher antibody titres compared to the control group from one week after the second immunisation until the end of the experiment. 
       2c) Egg and Worm Counts 
       [0102]    All four vaccinated groups showed significantly reduced egg counts compared to the adjuvant only control group from Day 20 to Day 29 ( FIG. 9 ). The group means of the cumulative egg counts over Days 19 to 34 were significantly lower in each of the vaccinated groups compared to the controls, with the corresponding percentage protection ranging from 70 to 85% as detailed in Table 5. Only Group 4 showed a significant reduction in worm burden at necropsy, with 64% fewer worms than the control group ( FIG. 10  and Table 5). 
         [0103]    2d) Identification of components in the best protected group by cDNA library screening. The sequences and, where possible, the corresponding identities of 135 immuno positive clones selected by the calves immunised with the pool 2 fraction are shown in Tables 1 and 2 
         [0000]    Trial 3: Immunisation with Sub-Fractions of the 0.1 to 0.5M MonoQ pool. 
         [0104]    The object of this trial was to separate the protective antigens identified by Group 2.2 by gel filtration in order to narrow the identity of the candidate protective polypeptides. (No attempt was made to do this for the unbound fraction as too little protein was available for the task) 
       3a) Antigens Used for Immunisation. 
       [0105]    The peaks separated by gel filtration together with an SDS PAGE analysis of the polypeptides present in each of the three antigen pools used to immunise the calves are shown in  FIG. 11 . Details of the groups and doses of protein administered are laid out in Table 5. 
       3b) Egg and Worm Counts 
       [0106]    The kinetics of the group mean egg counts of the calves in Trial 3 are shown in  FIG. 12  and the cumulative counts are presented in  FIG. 13 . The mean egg output of the calves immunised with Peak 3 was consistently lower than the control calves with an overall reduction of 83% (Table 5). However, there was a large variance in the egg output of the control calves in this trial so this difference just failed to be statistically significant (P=0.056). The number of worms recovered from any of the vaccinated groups and the control group was very similar (Table 5). 
       3c) Identity of the Components in Peak 3, the Most Protective of the Pool3 Sub-Fractions. 
       [0107]    Ten μg of the Peak 3 fraction was separated by SDS-PAGE and subjected to mass spectrometry as described in the Methods. About 16 polypeptide bands were visible  FIG. 14 . Twelve significant identities were obtained ranging from about 18 to 110 kDa in molecular weight and these are listed in  FIG. 14 . 
         [0000]    3 d ) Identification of Components in the Best Protected Group by cDNA Library Screening. 
         [0108]    The sequences and, where possible, the corresponding identities of 46 immuno positive clones selected by the calves immunised with the pool 3 fraction are shown in Tables 3 and 4. 
         [0000]    Trial 4: Immunisation with Sub Fractions of the 0.1 to 0.5M MonoQ pool 
         [0109]    As before the object of this trial was to determine whether the components responsible for the protection in Group 3.3 could be separated with a view to simplifying their identity. 
       4a) Antigens Used for Immunisation. 
       [0110]    The peaks separated by gel filtration together with an SDS PAGE analysis of the polypeptides present in each of the three antigen pools used to immunise the calves are shown in  FIG. 15 . Details of the groups and doses of protein administered are laid out in Table 5. 
       4b) Egg and Worm Counts 
       [0111]    The kinetics of the group mean egg counts of the calves in Trial 4 are shown in  FIG. 16  and the overall protective effects are summarised in Table 5. The mean egg outputs of all four groups of immunised calves were lower than the controls but there was little to choose between the efficacy of the different fractions. 
       Discussion 
       [0112]    There was little doubt that the ConA binding fraction of fourth stage  O. ostertagi  membrane extracts contained protective antigens, since all the groups vaccinated with this antigen or one of its derivatives had lower egg counts than their respective controls. As a crude measure, the mean percent reduction in egg output of all 11 groups immunised with this preparation or sub-fractions of it was 62%. More impressively, three of these fractions reduced cumulative egg counts by more than 80% (Table 5). The effect against worm numbers was more variable however, and did not necessarily correlate with the degree of egg reduction (Table 5). However, the two best fractions did reduce worm numbers by 50% or more (Table 5). 
         [0113]    These protection figures were better than those achieved with the same ConA binding extract of adult  O. ostertagi  where eggs were only reduced by between 30 and 50% and there was no measurable effect against worm numbers (Smith et al 2000). These results support the hypothesis developed in the Introduction that fourth stage  O. ostertagi  were likely to be more susceptible to the gut antigen approach to vaccination than their adult counterparts and that gut membrane antigens sourced from this developing stage are likely to be more efficacious. This idea does not seem to have been mooted before and could have general applicability to various other non-blood feeding nematode parasite genera across a range of hosts. 
         [0114]    Obtaining large numbers of fourth stage  O. ostertagi  is a laborious and expensive procedure. The trials reported here were made possibly by a regular supply of donor calves which were scheduled to be culled anyway after having been the subject of unrelated studies at the Institute. 
         [0115]    Because it was not possible to obtain the L4s without some contaminating digesta, the possibility existed that plant material was the source of some of the bands present in the antigen preparations. This possibility was discounted when attempts to make similar preparations from worm free abomasal digesta did not yield any protein. Presumably the cellulose cell walls of the plant cells which make up the bulk of the digesta are resistant to Triton extraction. 
         [0116]    Another possibility was that some of the L4 preparation polypeptides, which were additional to those observed in similar preparations from adult worms, were bovine in origin—perhaps from small pieces of abomasal tissue leaching from the mucosa when it was being incubated at 37 C to recover the larvae. However, an immunoblot developed with serum from calves immunised with  O. ostertagi  proteins revealed that several of these bands could not have been bovine proteins. 
         [0117]    This discovery of apparently novel bands in the L4 fraction prompted a protection trial. The encouragingly positive result from the first vaccine experiment lead onto 3 further “fractionate and vaccinate” trials where the overall objective was to determine whether simpler fractions containing fewer components would be just as if not more efficacious. It was striking how little native protein was actually required to achieve a good level of protective immunity (Table 5), but because of the difficulty and expense of obtaining large numbers of  Ostertagia  L4s, synthetic antigens, probably derived by recombinant DNA techniques, will be essential for a commercial vaccine. Obviously, the cDNAs of the protective polypeptides are required to do this and some progress was made in that direction through a combination of mass spectrometry and cDNA library immunoscreening. Much remains to be done however before a single protective antigen can be identified 
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