Patent Publication Number: US-2003229910-A1

Title: Short fragment homologous replacement to provide BSE resistant cattle

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     [0001] This application claims priority from U.S. Provisional Application No. 60/373,149, filed Apr. 17, 2002, the disclosure of which is incorporated by reference herein. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] The modification of the genome of a cell can, in principle, be accomplished either by introducing a complete gene into the genome at a random position or by making a specific alteration in an existing, naturally occurring gene. In mammalian cells, endogenous enzymes that effect homologous recombination have been used to introduce disruptions in specific genes for more than a decade. The technique is termed homologous-recombination dependent gene targeting (hrdGT). Doetschman, T., et al., 1987, Nature 330, 576-78; Thomas K. R. &amp; Capecchi, M. R., 1987, Cell 51, 503-12. These efforts involve the introduction of large pieces (several kilobases (kb)) of duplex DNA into the cell in the presence of a genetic selection system that distinguishes between homologous recombination and random insertion.  
       [0003] The use of an alternative has been described in mammalian cells. The technique is termed single-stranded short fragment homologous replacement (ssSFHR). A DNA fragment of intermediate size, typically 400 to 800 bp, is manufactured by excision from a plasmid vector or, alternatively, synthesized by PCR from a template. The short fragment is denatured by heat and the complementary strands can be optionally purified from each other. The technology is described in U.S. Pat. No. 6,010,908 by D.C. Gruenert, and in the scientific literature. Kapsa, R., et al., 2001, Human Gene Therapy 12, 629-42 (repair of murine dystrophin, unseparated strands); Colosima, A., et al., 2001, Mol. Therapy Vol. 3, No. 3 (episomal DNA in mammalian cells, unseparated strands); Goncz, K. K., et al., 1998, Hum. Mol. Genetics 7, 1913-19 (human cystic fibrosis transmembrane conductance regulator (CFTR), unseparated strands); Kunzelman, K., et al., 1996, Gene Therapy 3, 859-867 (murine CFTR, unseparated strands). The ssSFHR technique differs from hrdGT in several respects. The nucleic acid is shorter (400-800 nt) compared to several kb for hrdGT; in ssSFHR, the exogenous polynucleotide is denatured, i.e., single stranded, but is homologous with the target gene except for a few mutator nucleotides; in hrdGT, foreign genes are embedded in the exogenous nucleic acid; and, in hrdGT, a selection system is employed that distinguishes between homologous and illegitimate recombination, where in ssSFHR no such selection is required because illegitimate recombination does not occur at rates comparable to that of homologous recombination.  
       [0004] The present invention concerns the use of ssSFHR to modify the genome of cattle so that they are resistant to Bovine Spongioform Encephalopathy (BSE). BSE is a type of the so-called transmissible spongioform encephalopathies (TSE), which include ovine scrapie and human Creutzfeldt-Jakob Disease (CJD), as well as other diseases. A recent epidemic of over 170,000 cases of BSE occurred in the United Kingdom, which resulted in transmission of at least 130 cases to humans. The epidemic is believed to have been caused by the use of scrapie-infected sheep in the preparation of processed animal feed for the cattle, a process that was discontinued in 1988 and resulted in the reduction of the numbers of cases. Pattison, J., 1998, Emerg Infect. Dis. 4, 390-4; Nathanson, N., et al., Am. J Epidemiol. 45, 959-69.  
       [0005] TSE are the unique infectious diseases that are not transmitted by a nucleic acid-based disease organism. Rather, TSE result from the abnormal conformation of a brain protein, the prion protein (PrP). Prusiner, S. B., 1991, Science 252, 1515-22. The pathologic conformation consists of a 142-amino acid fragment of the PrP that adopts a predominantly β-pleated sheet conformation, which form catalyzes the conversion of other PrP to assume the pathological conformation. Peretz, D., 2001, Protein Science 10, 854-63; Wadsworth, J. D., et al., 1999, Curr Opin Genet Dev 9, 338-45. Though the hypothesis that TSE results from an infectious conformational change in the PrP is not universally accepted outside of the English-speaking world (see, e.g., Lasmezas, C.I., et al., 1997, Science 275, 402-5), it is widely accepted and has been confirmed by examples of inherited protein conformation in yeast. Lindquist, S., 1996, Mol. Psychiatry 1, 376-9; Lindquist, S., 1997, Cell 89, 495-8.  
       [0006] Whatever uncertainty may remain about the etiology of TSE, the ablation of the host PrP gene results in an animal that is resistant to the disease. Prusiner, S. B., et al., 1993, PNAS 90, 10608-12; Weissmann, C., &amp; Aguzzi, A., 1999, Science 286, 914-15. In addition, dominant disease-resistant alleles of PrP having amino acid substitution can confer resistance to the disease as heterozygotes. Perrier, V. et al., 2002, PNAS 99, 13079-84. While certain biochemical and morphological abnormalities are associated with the PrP-ablated condition, the animals develop normally and appear healthy. Miele, G., et al., 2002, BBRC 291, 372-77; White, A. R., et al., 1999, Am J. Path. 155, 1723-30.  
       SUMMARY OF THE INVENTION  
       [0007] The invention provides a method of rendering cattle resistant to BSE by ablation of the bovine PrP gene. Fragments of bovine PrP gene are cloned into bacteria and mutated by known techniques of site directed mutagenesis. The mutated cloned gene is used as a template to generate a short fragment (henceforth “SF”) of between 200-1000 bp, preferably between 400 and 800 bp using conventional oligonucleotide primed polymerase chain reaction amplification. There can be more than one genetic alteration encoded in an SF, but the alterations should be limited in size and extent, so that not more than four consecutive nucleotides of the SF will not be homologous to the target gene. A second alteration without physiological effects may be introduced to facilitate the subsequent isolation of mutant cells that have homologously recombined the SF. The differences between the sequence of the SF and that of the target gene (the “heterologies”) can either be mismatches, insertions or deletions. Ablation is caused by insertion of a frame shift mutation or multiple stop codons.  
       [0008] The SF is converted to single strand SF (“ssSF”), and then into a strand separated form (“s 4 SF”). The sequence of the SF will preferably be examined to determine self-complementary sequences that will cause extensive self-complementary secondary structure.  
       [0009] Once formed, the s 4 SF can be introduced into a somatic cell, typically a fibroblast, so as to induce ablation of the PrP gene. Selection of mutated clones is performed by cloning and PCR screening. To facilitate PCR screening, it is preferred that the ablating mutation create a readily observable restriction site, so that mutant clones can be identified without sequencing. Cattle incorporating the mutated PrP gene can be recovered by nuclear transfer to oocytes, using known techniques. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTIONS  
     [0010] Preparation of the s 4 SF and generation of a mutant PrP gene. In one embodiment the invention consists of the use of a short fragment (SF) of single stranded DNA of between 200 and 1000 nt and, more preferably between 400 and 800 nt that is homologous (identical) with a fragment of the bovine PrP gene, except at a limited number of positions, typically fewer than 10, which are designed to introduce ablating mutations into the PrP gene and, optionally generate an additional alteration to facilitate identification of the modified PrP locus by a combination of allele-specific PCR and a secondary detection. The sequence of the bovine PrP gene is found at GENBANK accession No. AJ298878, the sequence of the PrP cDNA can be found as accession AB001468, which are hereby incorporated by reference.  
     [0011] Construction of the desired, mutated sequence can be most readily accomplished by in vitro site-directed mutagenesis. The techniques involved are well known in the art. Perrin, S., &amp; Gilliland, G., 1990, Nucleic Acid Research 18, 7433; Landt, O., et al., 1990, Gene 96, 125-8; Nassal M., &amp; Rieger, A., 1989, Nucleic Acids Research 18, 3077-8; Hemsley, A., et al., 1989, Nucleic Acids Research 17, 6545-51. Implementation of these techniques require that the target gene or a fragment of the gene that encompasses the sequenced to be modified is available in recombinant clones. Having constructed the appropriate desired sequence, the SF itself can be synthesized by routine polymerase chain reaction (“PCR”). When s 4 SF are to be used, the synthesis employs one 5′-biotinylated primer and one underivitized primer. The strands are separated as described below. The synthesis of 5′-biotinylated primers is well known. Cook, A. F., et al., 1988, Nucleic Acids Research 16, 4077-95; Connolly, B. A., 1988, Nucleic Acids Research 15, 3131-9.  
     [0012] After the SF is synthesized in a duplex form, i.e., the form in which the fragment is Watson-Crick bound to its complement, a single stranded SF can be prepared. The preparation is most simply accomplished by heat denaturation (heating to 95° C.) followed by rapid cooling to 4° C. This process results in a mixture of strands of both polarity having no or essentially no intermolecular Watson-Crick base pairings. However, continued incubation of the mixture at elevated temperatures can result in the formation of inter-molecular Watson-Crick pairings.  
     [0013] The separation of the complementary strands can be readily accomplished when one of the two primers used in the PCR synthesis of the SF is biotinylated. Separation of the product can be effected by binding the biotinylated strand to immobilized avidin as follows:  
     [0014] Double stranded SF (ds-SF) products can be prepared by PCR using two primers, one of which contained a biotin at the 5′ end.  
     [0015] Single strand preparation:  
     [0016] Single strands were generated by binding the biotinylated PCR product to avidin-magnetic beads (Dyonex).  
     [0017] The displaced strand (D-ssSF not containing biotin) was isolated by denaturing the bound dsPCR fragment under high pH (0.5 M NaOH) 1-2 minutes.  
     [0018] The “displaced strand” (supernatant) was removed from the beads using a magnet or centrifugation, neutralized with acid (27 ul cHCL per 500 :1 0.5 M NaOH) and dialyzed (1000×volume 0.1 M Tris pH 7.0, then 2 Times 1000×volume of water). The displaced strand was then concentrated by ethanol precipitation or spin concentrators.  
     [0019] The immobilized strand (B-SF) attached to the beads was neutralized with 2 Tris 0.1 M pH 7.0 washes followed by 2 water washes. The immobilized strand was removed from the magnetic beads in water following heat treatment (95° C.).  
     [0020] Both displaced and immobilized strands individually have activity.  
     [0021] Typically the displaced strand was more active. Either the coding or non-coding strand may be used to introduce the modification into the targeted gene.  
     [0022] The s 4 SF can be introduced into a bovine cell, such as a bovine fibroblast, by any method that can be used to introduce duplex DNA into the cell. The preferred method is by microinjection, which allows for individual inoculation of pre-selected, adherent cells in an controlled manner.  
     [0023] A cell containing the modified target gene can be isolated by cloning and PCR testing of the cloned cells prior to the regeneration of whole animals. Several methods can be employed to identify a clone of cells in which the SF has altered the PrP locus.  
     [0024] A particular method, which is suitable when the frequency of altered cells is low is termed “coupled detection” (CD) and was described in commonly owned U.S. patent application Ser. No. 10/298,859, filed Nov. 18, 2002, by R. Metz which is hereby incorporated by reference in its entirety. In CD, two alternations, typically between 50 and 100 nucleotides apart, are introduced using a single SF. After a population containing a putatively modified cell is obtained; the population is divided into replicate subgroups. A PCR reaction is performed on genomic DNA from a replicate of each subgroup using a PCR primers that will amplify the target sequence but not the SF. The products of this reaction are diluted and used as template for the second PCR reaction. The second PCR reaction is performed using a PCR primer that preferentially anneals to the sequence of one of the alterations compared to the wild-type sequence and a non-selective second primer. The PCR reaction is designed so that the product includes the site of the second alteration. Suitable selection of annealing temperature results in the preferential amplification of the altered fragment relative to the wild-type. The preferential amplification permits the ready detection of a single copy of the altered genotype in a subgroup of several thousand by detection of the second alteration in the PCR product. A second alteration that creates or deletes a restriction enzyme recognition such that the presence of a mutant locus can be detected by a restriction enzyme digest is particularly preferred because of the ease in detection.  
     [0025] Using CD, a large population of cells can be readily screened to detect a rare cell that contains the linked alterations. The screening is performed by subdividing the populations into subgroups or about 5,000. The replicates from subgroups that contain a single copy of the rare modified cell are further subdivided and cultured. Successive cycles of subdivision, replica formation and detection can be used to isolate the rare modified cell from the population.  
     [0026] In a less preferred method, a PCR primer that preferentially anneals to the mutant sequence compared to the wild-type sequence is used to PCR-amplify a genomic fragment from a population of cells under such temperature conditions that only the mutant sequence, if present, yields a product.  
     [0027] In an alternative embodiment, the alteration in the bovine PrP gene can be made to increase the relative stability of the soluble form of the PrP protein. Mutations that increase the stability have been identified by comparison of the susceptibility of different strains of sheep to scrapie with polymorphism in the ovine PrP gene. Mutations at positions 136, 154 and 171 were found to be protective. Drogenmuller, C., et al., 2001, Vet. Res. 149, 349-52. The same mutations can be introduced into the bovine PrP gene in an alternative embodiment of the invention.  
     [0028] In yet another embodiment, an alteration of the bovine PrP gene by mutations that confer a dominant disease-resistant phenotype can be introduced into the bovine PrP gene, so that animals would not need to be homozygous for the altered Prp gene to be resistant to the disease.  
     [0029] Generation of BSE resistant cattle: The generation of domestic animals containing site-specific mutations has been made possible by recent advances in nuclear transplantation from somatic cells into a competent germ-line cell, i.e., oocytes or cells of a blastocyst (blastomers). These techniques are referred to in general as “cloning” or “animal cloning” because they enable the practitioner to make a genetically identical individual from an explanted somatic cell. The techniques are described in detail in U.S. Pat. No. 6,147,276 and No. 6,252,133.  
     [0030] Scientific publications describing the technology teach that with some species-specific adaptations the techniques have proved successful in sheep, cattle and swine. Schnieke, A. E., et al., 1997, Science 278, 2130-33; Wilmut, I., et al., 1997, Nature 385, 810-3; Polejaeva, I. A., et al., 2000, Nature 407, 29-30. A current review of the field can be found in Kuhholtzer, B., &amp; Prather, R. S., 2000, Proc. Soc. Exp. Med. 224, 240-45.  
     [0031] It is expected that the originally mutated somatic nucleus will be heterozygous for the PrP mutation. Accordingly, the generation of BSE resistant stock will require interbreeding the founder stock in order to isolate the mutation in homozygous form when the alteration of the PrP gene is designed to prevent its translation. The presence of the modified PrP in offspring of parents carrying a PrP disease-resistant allele can be determined through a DNA-based assay which may include techniques commonly known in the art such as RFLP mapping, SNP detection, southern blots, PCR amplification and direct sequencing. As an alternative to generating an animal homozygous for alterations in the PrP gene, cell lines prepared from embryos derived in the first round of nuclear transfer cloning can be retargeted by SFHR to alter the second PrP allele. The alteration of the second allele can be the same as that of the first allele or alternatively it can be different to aid in the identification of cells having both PrP alleles modified. These mutant cell lines homozygous for altered PrP alleles heterozygous can be used to redone an animal homozygous for the desired PrP gene mutation.  
     [0032] DNA fragments for SFHR are synthesized by PCR in a two step process using a commercially available vector into which exon 3 of bovine PrP has been inserted. Two types of primers are used. A mutational primer is used to alter the PiP sequence in the vector.  
     [0033] After the mutation is introduced production primers are used to make the SFHR duplex DNA by PCR using the mutated vector as template.  
     [0034] Listed below of Forward and Reverse production primers (FP and RP) and mutational primers which are labeled according to the position of the mutation in the amino acid sequence.  
                                                          SFHR PCR Primers                   LOCATION   NUCLEOTIDE                                                     FP5   5′ GTGGCCATGTGGAGTGA   (SEQ ID NO:1)   281                           RP5   5′ CCCAACCTGGTAAAGATTAAG   (SEQ ID NO:2)   1061                           (rc-cttaatctttaccaggttggg)   (SEQ ID NO:3)                       FP4   5′ CTGTTTATAGCTGATGCCACT   (SEQ ID NO:4)   130                       RP4   5′ ACGGTTGCCTCCAGGAC   (SEQ ID NO:5)   375                           (rc-gtcctggaggcaaccgt)   (SEQ ID NO:6)                       RP3   5′ GGCTTACTGGGTTTGTTCC   (SEQ ID NO:7)   567                           (rc-ggaacaaacccagtaagcc)   (SEQ ID NO:8)                       RP2   5′ GGCCTGTAGTACACTTGGTTG   (SEQ ID NO:9)   745                           (rc-caaccaagtgtactacaggcc)   (SEQ ID NO: 10)                                 Mutagenic primers and SFHR combinations                                         Prp-mutant                        Primer set   Name   Sequence   SFHR                                             Prp-0-1   W9stop 5′ CACATAGGCAGTTAGATCCTGGTTCTC3′   (SEQ ID NO:11)   FP4/Rp2,                           FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-0-2   W18stop   5′ TTTGTGGCCATGTAGAGTGACGTGGGC3′   (SEQ ID NO:12)   FP4/Rp2,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-0-3   C24stop   5′ GACGTGGGCCTCTGAAAGAAGCGACCA3′   (SEQ ID NO:13)   FP4/Rp2,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-0-4   K3stop   5′ GTCATCATGGTGTAAAGCCACATAGGC3′   (SEQ ID NO:14)   FP4/Rp2,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-0-d5   V2del   5′ GTCATCATGGT:AAAAGCCACATAGGC3′   (SEQ ID NO:15)   FP4/Rp2,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-0-d6   H5del   5′ GGTGAAAAGCCA:ATAGGCAGTTGGAT3′   (SEQ ID NO:16)   FP4/Rp2,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5               Prp-ARR   Q178R   5′ AGGCCAGTGGATCGGTATAGTAACCAG3′   (SEQ ID NO:17)   FP4/RP5,                       FP4/RP3,                       FP4/RP4,                       FP4/RP5                  
 
     [0035] Example: The approach for generating animals resistant to Transmissible Spongiform Encephalopathy (TSE) or Bovine Spongiform Encephalopathy (BSE) will proceed in two parallel tracks. A non-functional PrP allele will be generated in a bovine primary cell line using SFHR (GenEdit) molecules (PrP0-1 and/or PrP0-2, other molecules disrupting the open reading frame may also be attempted such as PrP-0-3, -4, -5, -6 etc). In addition, mutagenic PCR primers will be used to insert a point mutation in a restriction enzyme recognition sequence within 100 nucleotides of any of the above mutations. Mutant cells will be generated which have incorporated the mutant sequence by homologous recombination, and clones of these cells will be screened for the presence of the mutant sequences. Replicate subcultures will be generated and DNA prepared for PCR-amplification. In order to increase our sensitivity and selectivity two rounds of PCR amplification will be performed. The first reaction will use a primer set flanking the PrP targeted region. The products from the first round reaction will be diluted 10,000 fold and used as a template for a an allele-enrichment PCR reaction, where one of the primers is designed to preferentially bind the mutant sequence to selectively enrich for sequences containing the PrP mutations. The allele-enriched PCR product will then be digested with the restriction enzyme whose recognition site was mutated. Uncut PCR products are those that contain the mutant sequences, whereas the presence of two fragments will represent the presence of the wildtype PrP. Subcultures containing the mutant form of PrP will be further subdivided and the process of screening for the mutant PrP will be reiterated until a pure subculture containing modified mutant cells is isolated.  
     [0036] From the modified cell line animals will be generated using nuclear transfer technology. The reduction of a functional PrP allele may have protective properties based on reduced gene product. A homozygous PrP-0 animal (PrP-0/PrP-0) can be generated by back crossing PrP-0 heterozygotes. In a similar fashion, a BSE resistant allele will be introduced into a breeding stock using SFHR molecules (bPrPAAR and/or other molecules affecting resistant phenotype) to introduce a polymorphism barrier to TSE (BSE). The current evidence for polymorphism barriers to TSE (Scrapie) has been described for scrapie resistant herds of sheep containing Alanine (A), Arginine (R) and Arginine (R) at codons 136, 154, and 171, respectively. The bovine sequence contains the resistance-associated amino acids at positions homologous to 136 and 154 and only the amino acid at 171 need be modified. Homozygote PrPARR/PrPAAR or PrPARR/PrP-0 animals can be generated using standard back crossing and cross breeding strategies with the appropriate homozygote/heterozygote animals.  
     [0037] A number of mutations that can be generated including several null alleles. Below are examples of three nonsense and two frame shift null mutations. Also given are the base substitutions that generate a bovine PrP-ARR. SFHR molecules will be single stranded coding or non-coding, or denatured double stranded. All null generating SFHR molecules will extend into intron 2 and terminate in the exon 3 (Coding region of PRP). The PrP-ARR alelle will need an SFHR molecule whose sequences are contained in exon 3.  
                                      bovine ex.   #-237   cta gga aac aga gcc agg aat tat ttt aag gtc                       bovine ex.   #-204   aac ttt gtc ctt aga gaa gga aga gtt gtg tta               bovine ex.   #-171   aca ctt tac cta taa tta ctt tcg tga gat gta               bovine ex.   #-138   tgg aat gtg aag aat att tat gac cta gac tgt               bovine ex.   #-105   tta tag ctg atg cca ctg cta tgc agt cat tat               bovine ex.   #-72   gct aca gac ttt aag tga ttt tta cat ggg cat               bovine ex.   #-39   atg atg ctg aca ccc tct tta ttt tgc agA TAA               bovine ex.   #-6   GTC ATC ATG GTG AAA AGC CAC ATA GGC AGT TGG   (SEQ ID NOS:18-19)                        M   V   K   S   H   I   G   S   W               PrP-0-1   #-6   GTC ATC ATG GTG AAA AGC CAC ATA GGC AGT TAG   (SEQ ID NOS:20-21)                        M   V   K   S   H   I   G   S   *               PrP-0-2   #-6   GTC ATC ATG GTG AAA AGC CAC ATA GGC AGT TGG   (SEQ ID NOS:22-23)                        M   V   K   S   H   I   C   S   W               PrP-0-3   #-6   GTC ATC ATG GTG AAA AGC CAC ATA GGC AGT TGG   (SEQ ID NOS:24-25)                        M   V   K   S   H   I   G   S   W               Prp-0-4   #-6   GTC ATC ATG GTG TAA AGC CAC ATA GGC AGT TGG   (SEQ ID NO:26)                    M    V  *               PrP-0-d5   #-6   GTC ATC ATG GT: AAA AGC CAC ATA GGC AGT TGG   (SEQ ID NOS:27- 28)                    M  V    K   A   T   *               PrP-0-d6   #-6   GTC ATC ATG GTG AAA AGC CA: ATA GGC AGT TGG   (SEQ ID NOS:29-30)                    M   V   K   S   Q   *               PrP-ARR   #-6   GTC ATC ATG GTG AAA AGC CAC ATA GGC AGT TGG   (SEQ ID NOS:31-32)                    M   V   K   S   H   I   G   S   W               bovine ex.   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               I   L   V   L   F   V   A   M   W   S   D               PrP-0-1   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               PrP-0-2   #28   ATC CTG GTT CTC TTT GTG GCC ATG TAG AGT GAC               I   L   V   L   F   V   A   M   *               PrP-0-3   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               I   L   V   L   F   V   A   M   W   S   D               Prp-0-4   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               PrP-0-d5   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               PrP-0-d6   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               PrP-ARR   #28   ATC CTG GTT CTC TTT GTG GCC ATG TGG AGT GAC               I   L   V   L   F   V   A   M   W   S   D               bovine ex.   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               V   G   L   C   K   K   R   P   K   P   G               PrP-0-1   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               PrP-0-2   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               PrP-0-3   #61   GTG GGC CTC TG A  AAG AAG CGA CCA AAA CCT GGA               V   G   L   *               Prp-0-4   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               PrP-0-d5   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               PrP-0-d6   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               PrP-ARR   #61   GTG GGC CTC TGC AAG AAG CGA CCA AAA CCT GGA               V   G   L   C   K   K   R   P   K   P   G               bovine ex.   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               G   G   W   N   T   G   G   S   R   Y   P               PrP-0-1   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               PrP-0-2   #94   GGA GGA TGC AAC ACT GGG GGG AGC CGA TAC CCA               PrP-0-3   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               Prp-0-4   #94   GGA GGA TGG AAC ACT GGG CGG AGC CGA TAC CCA               PrP-0-d5   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               PrP-0-d6   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               PrP-ARR   #94   GGA GGA TGG AAC ACT GGG GGG AGC CGA TAC CCA               G   C   W   N   T   C   G   S   R   Y   P               bovine ex.   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               G   Q   G   S   P   C   G   N   R   Y   P               PrP-0-1   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               PrP-0-2   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               PrP-0-3   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               Prp-0-4   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               PrP-0-d5   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               PrP-0-d6   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               PrP-ARR   #127   GGA CAG GGC AGT CCT GGA GGC AAC CGT TAT CCA               G   Q   G   S   P   G   G   N   R   Y   P               bovine ex.   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               P   Q   G   G   G   G   W   G   Q   P   H               PrP-0-1   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               PrP-0-2   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               PrP-0-3   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               Prp-0-4   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               PrP-0-d5   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               PrP-0-d6   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               PrP-ARR   #160   CCT CAG GGA GGG GGT GGC TGG GGT CAG CCC CAT               P   Q   G   G   G   G   W   G   Q   P   H               bovine ex.   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               G   G   G   W   G   Q   P   H   G   G   G               PrP-0-1   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               PrP-0-2   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               PrP-0-3   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               Prp-0-4   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               PrP-0-d5   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               PrP-0-d6   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               PrP-ARR   #193   GGA GGT GGC TGG GGC CAG CCT CAT GGA GGT GGC               G   G   G   W   G   Q   P   H   G   G   G               bovine ex.   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               W   G   Q   P   H   G   G   G   W   G   Q               PrP-0-1   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               PrP-0-2   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               PrP-0-3   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               Prp-0-4   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               PrP-0-d5   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               PrP-0-d6   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG               PrP-ARR   #226   TGG GGC CAG CCT CAT GGA GGT GGC TGG GGT CAG                 W   C   Q   P   H   G   G   G   W   G   Q               bovine ex.   #259   CCC CAT GGT GGT GGC GGC GGA CAC CCA CAT GGT               P   H   G   G   G   W   G   Q   P   H   G               PrP-0-1   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               PrP-0-2   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               PrP-0-3   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               Prp-0-4   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               PrP-0-d5   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               PrP-0-d6   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               PrP-ARR   #259   CCC CAT GGT GGT GGC TGG GGA CAG CCA CAT GGT               P   H   G   G   G   W   G   Q   P   H   C               bovine ex.   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               G   G   G   W   G   Q   G   G   T   H   G               PrP-0-1   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               PrP-0-2   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               PrP-0-3   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               Prp-0-4   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               PrP-0-d5   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               PrP-0-d6   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               PrP-ARR   #292   GGT GGA GGC TGG GGT CAA GGT GGT ACC CAC GGT               G   G   G   W   G   Q   G   G   T   H   G               bovine ex.   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               Q   W   N   K   P   S   K   P   K   T   N               PrP-0-1   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               PrP-0-2   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               PrP-0-3   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               Prp-0-4   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               PrP-0-d5   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               PrP-0-d6   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               PrP-ARR   #325   CAA TGG AAC AAA CCC AGT AAG CCA AAA ACC AAC               Q   W   N   K   P   S   K   P   K   T   N               bovine ex.   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               M   K   H   V   A   G   A   A   A   A   G               PrP-0-1   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               PrP-0-2   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               PrP-0-3   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               Prp-0-4   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               PrP-0-d5   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               PrP-O-d6   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               PrP-ARR   #358   ATG AAG CAT GTG GCA GGA GCT GCT GCA GCT GGA               M   K   H   V   A   G   A   A   A   A   G               bovine ex.   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               A   V   V   G   G   L   G   G   Y   M   L               PrP-0-1   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               PrP-0-2   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               PrP-0-3   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               Prp-0-4   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               PrP-0-d5   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               PrP-0-d6   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               PrP-ARR   #391   GCA GTG GTA GGG GGC CTT GGT GGC TAC ATG CTG               A   V   V   G   G   L   G   G   Y   M   L               bovine ex.   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               G   S   A   M   S   R   P   L   I   H   F               PrP-0-1   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               PrP-0-2   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               PrP-0-3   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               Prp-0-4   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               PrP-0-d5   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               PrP-0-d6   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT               PrP-ARR   #424   GGA AGT GCC ATG AGC AGG CCT CTT ATA CAT TTT                 G   S   A   M   S   R   P   L   I   H   F               bovine ex.   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               G   S   D   Y   E   D   R   Y   Y   R   E               PrP-0-1   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               PrP-0-2   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               PrP-0-3   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               Prp-0-4   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               PrP-0-d5   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               PrP-0-d6   #457   GGC AGT GAC TAT GAG GAC CGT TAC TAT CGT GAA               PrP-ARR   #457   GGC AGT GAC TAT GAG GAC CGT TAG TAT CGT GAA                 G   S   D   Y   E   D   R   Y   Y   R   E               bovine ex.   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               N   M   H   R   Y   P   N   Q   V   Y   Y               PrP-0-1   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               PrP-0-2   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               PrP-0-3   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               Prp-0-4   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               PrP-0-d5   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               PrP-0-d6   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               PrP-ARR   #490   AAC ATG CAC CGT TAC CCC AAC CAA GTG TAC TAC               N   M   H   R   Y   P   N   Q   V   Y   Y               bovine ex.   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               R   P   V   D   Q   Y   S   N   Q   N   N               PrP-0-1   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               PrP-0-2   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               PrP-0-3   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               Prp-0-4   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               PrP-0-d5   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               PrP-0-d6   #523   AGG CCA GTG GAT CAG TAT AGT AAC CAG AAC AAC               PrP-ARR   #523   AGG CCA GTG GAT CGG TAT AGT AAC CAG AAC AAC               R   P   V   D   R   Y   S   N   Q   N   N                                * AA171               bovine ex.   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               F   V   H   D   C   V   N   I   T   V   K               PrP-0-1   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               PrP-0-2   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               PrP-0-3   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               Prp-0-4   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               PrP-0-d5   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               PrP-0-d6   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG               PrP-ARR   #556   TTT GTG CAT GAC TGT GTC AAC ATC ACA GTC AAG                 F   V   H   D   C   V   N   I   T   V   K               bovine ex.   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG                 E   H   T   V   T   T   T   T   K   G   E               PrP-0-1   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               PrP-0-2   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               PrP-0-3   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               Prp-0-4   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               PrP-0-d5   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               PrP-0-d6   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG               PrP-ARR   #589   GAA CAC ACA GTC ACC ACC ACC ACC AAG GGG GAG                 E   H   T   V   T   T   T   T   K   G   E               bovine ex.   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               N   F   T   E   T   D   I   K   M   M   H               PrP-0-1   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               PrP-O-2   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               PrP-0-3   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               Prp-0-4   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               PrP-0-d5   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               PrP-0-d6   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               PrP-ARR   #622   AAC TTC ACC GAA ACT GAC ATC AAG ATG ATG GAG               N   F   T   E   T   D   I   K   M   M   H               bovine ex.   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-0-1   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-0-2   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-0-3   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               Prp-0-4   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-0-d5   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-0-d6   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               PrP-ARR   #655   CGA GTG GTG GAG CAA ATG TGC ATT ACC CAG TAC               R   V   V   E   Q   M   C   I   T   Q   Y               bovine ex.   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               Q   R   E   S   Q   A   Y   Y   Q   R   G               PrP-0-1   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               PrP-0-2   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               PrP-0-3   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               Prp-0-4   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               PrP-0-d5   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               PrP-0-d6   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG               PrP-ARR   #688   CAG AGA GAA TCC CAG GCT TAT TAC CAA CGA GGG                 Q   R   E   S   Q   A   Y   Y   Q   R   G               bovine ex.   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               A   S   V   I   L   F   S   S   P   P   V               PrP-0-1   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               PrP-0-2   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               PrP-0-3   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               Prp-0-4   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               PrP-0-d5   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               PrP-G-d6   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               PrP-ARR   #721   GCA AGT GTG ATC CTC TTC TCT TCC CCT CCT GTG               A   S   V   I   L   F   S   S   P   P   V               bovine ex.   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA                 I   L   L   I   S   F   L   I   F   L   I               PrP-0-1   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               I   L   L   I   S   F   L   I   F   L   I               PrP-0-2   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               PrP-0-3   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               Prp-0-4   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               PrP-0-d5   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               PrP-0-d6   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               PrP-ARR   #754   ATC CTC CTC ATC TCT TTC CTC ATT TTT CTC ATA               I   L   L   I   S   F   L   I   F   L   I               bovine ex.   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT                 V   G  *               PrP-0-1   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               PrP-0-2   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               PrP-0-3   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               Prp-0-4   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               PrP-0-d5   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               PrP-0-d6   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT               PrP-ARR   #787   GTA GGA TAG GGG CAA CCT TCC TGT TTT CAT TAT                 V   G  *               bovine ex.   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               PrP-0-1   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               PrP-0-2   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               PrP-0-3   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               prp-0-4   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               PrP-0-d5   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT               PrP-0-d6   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGC AGT               PrP-ARR   #820   CTT CTT AAT CTT TAC CAG GTT GGG GGA GGG AGT          
 
     [0038] 
    
     
       
         1 
         
           
             32  
           
           
             1  
             17  
             DNA  
             Artificial Sequence  
             
               forward primer  
             
           
            1 

gtggccatgt ggagtga                                                    17 

 
           
             2  
             21  
             DNA  
             Artificial Sequence  
             
               reverse primer  
             
           
            2 

cccaacctgg taaagattaa g                                               21 

 
           
             3  
             21  
             DNA  
             Artificial Sequence  
             
               reverse complement of SEQ ID NO2  
             
           
            3 

cttaatcttt accaggttgg g                                               21 

 
           
             4  
             21  
             DNA  
             Artificial Sequence  
             
               forward primer  
             
           
            4 

ctgtttatag ctgatgccac t                                               21 

 
           
             5  
             17  
             DNA  
             Artificial Sequence  
             
               reverse primer  
             
           
            5 

acggttgcct ccaggac                                                    17 

 
           
             6  
             17  
             DNA  
             Artificial Sequence  
             
               reverse complement of SEQ ID NO5  
             
           
            6 

gtcctggagg caaccgt                                                    17 

 
           
             7  
             19  
             DNA  
             Artificial Sequence  
             
               reverse primer  
             
           
            7 

ggcttactgg gtttgttcc                                                  19 

 
           
             8  
             19  
             DNA  
             Artificial Sequence  
             
               reverse complement of SEQ ID NO7  
             
           
            8 

ggaacaaacc cagtaagcc                                                  19 

 
           
             9  
             21  
             DNA  
             Artificial Sequence  
             
               reverse primer  
             
           
            9 

ggcctgtagt acacttggtt g                                               21 

 
           
             10  
             21  
             DNA  
             Artificial Sequence  
             
               reverse complement of SEQ ID NO9  
             
           
            10 

caaccaagtg tactacaggc c                                               21 

 
           
             11  
             27  
             DNA  
             Artificial Sequence  
             
               W9stop primer  
             
           
            11 

cacataggca gttagatcct ggttctc                                         27 

 
           
             12  
             27  
             DNA  
             Artificial Sequence  
             
               W18stop primer  
             
           
            12 

tttgtggcca tgtagagtga cgtgggc                                         27 

 
           
             13  
             27  
             DNA  
             Artificial Sequence  
             
               C24stop primer  
             
           
            13 

gacgtgggcc tctgaaagaa gcgacca                                         27 

 
           
             14  
             27  
             DNA  
             Artificial Sequence  
             
               K3stop  
             
           
            14 

gtcatcatgg tgtaaagcca cataggc                                         27 

 
           
             15  
             26  
             DNA  
             Artificial Sequence  
             
               V2del primer  
             
           
            15 

gtcatcatgg taaaagccac ataggc                                          26 

 
           
             16  
             26  
             DNA  
             Artificial Sequence  
             
               H5del primer  
             
           
            16 

ggtgaaaagc caataggcag ttggat                                          26 

 
           
             17  
             27  
             DNA  
             Artificial Sequence  
             
               Q178R primer  
             
           
            17 

aggccagtgg atcggtatag taaccag                                         27 

 
           
             18  
             1089  
             DNA  
             Bos taurus  
             
               CDS  
               (238)..(1032)  
             
           
            18 

ctaggaaaca gagccaggaa ttattttaag gtcaactttg tccttagaga aggaagagtt     60 

gtgttaacac tttacctata attactttcg tgagatgtat ggaatgtgaa gaatatttat    120 

gacctagact gtttatagct gatgccactg ctatgcagtc attatgctac agactttaag    180 

tgatttttac atgggcatat gatgctgaca ccctctttat tttgcagata agtcatc       237 

atg gtg aaa agc cac ata ggc agt tgg atc ctg gtt ctc ttt gtg gcc      285 
Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 
1               5                   10                  15 

atg tgg agt gac gtg ggc ctc tgc aag aag cga cca aaa cct gga gga      333 
Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 
            20                  25                  30 

gga tgg aac act ggg ggg agc cga tac cca gga cag ggc agt cct gga      381 
Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 
        35                  40                  45 

ggc aac cgt tat cca cct cag gga ggg ggt ggc tgg ggt cag ccc cat      429 
Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 
    50                  55                  60 

gga ggt ggc tgg ggc cag cct cat gga ggt ggc tgg ggc cag cct cat      477 
Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
65                  70                  75                  80 

gga ggt ggc tgg ggt cag ccc cat ggt ggt ggc tgg gga cag cca cat      525 
Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
                85                  90                  95 

ggt ggt gga ggc tgg ggt caa ggt ggt acc cac ggt caa tgg aac aaa      573 
Gly Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys 
            100                 105                 110 

ccc agt aag cca aaa acc aac atg aag cat gtg gca gga gct gct gca      621 
Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 
        115                 120                 125 

gct gga gca gtg gta ggg ggc ctt ggt ggc tac atg ctg gga agt gcc      669 
Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala 
    130                 135                 140 

atg agc agg cct ctt ata cat ttt ggc agt gac tat gag gac cgt tac      717 
Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr 
145                 150                 155                 160 

tat cgt gaa aac atg cac cgt tac ccc aac caa gtg tac tac agg cca      765 
Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro 
                165                 170                 175 

gtg gat cag tat agt aac cag aac aac ttt gtg cat gac tgt gtc aat      813 
Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn 
            180                 185                 190 

atc aca gtc aag gaa cac aca gtc acc acc acc acc aag ggg gag aac      861 
Ile Thr Val Lys Glu His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn 
        195                 200                 205 

ttc acc gaa act gac atc aag atg atg aag cga gtg gtg gag caa atg      909 
Phe Thr Glu Thr Asp Ile Lys Met Met Lys Arg Val Val Glu Gln Met 
    210                 215                 220 

tgc att acc cag tac cag aga gaa tcc cag gct tat tac caa cga ggg      957 
Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly 
225                 230                 235                 240 

gca agt gtg atc ctc ttc tct tcc cct cct gtg atc ctc ctc atc tct     1005 
Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser 
                245                 250                 255 

ttc ctc att ttt ctc ata gta gga tag gggcaacctt cctgttttca           1052 
Phe Leu Ile Phe Leu Ile Val Gly 
            260 

ttatcttctt aatctttacc aggttggggg agggagt                            1089 

 
           
             19  
             264  
             PRT  
             Bos taurus  
           
            19 

Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 
1               5                   10                  15 

Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 
            20                  25                  30 

Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 
        35                  40                  45 

Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 
    50                  55                  60 

Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
65                  70                  75                  80 

Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
                85                  90                  95 

Gly Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys 
            100                 105                 110 

Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 
        115                 120                 125 

Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala 
    130                 135                 140 

Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr 
145                 150                 155                 160 

Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro 
                165                 170                 175 

Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn 
            180                 185                 190 

Ile Thr Val Lys Glu His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn 
        195                 200                 205 

Phe Thr Glu Thr Asp Ile Lys Met Met Lys Arg Val Val Glu Gln Met 
    210                 215                 220 

Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly 
225                 230                 235                 240 

Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser 
                245                 250                 255 

Phe Leu Ile Phe Leu Ile Val Gly 
            260 

 
           
             20  
             858  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(33)  
             
           
            20 

gtcatc atg gtg aaa agc cac ata ggc agt tag atcctggttc tctttgtggc      53 
       Met Val Lys Ser His Ile Gly Ser 
       1               5 

catgtggagt gacgtgggcc tctgcaagaa gcgaccaaaa cctggaggag gatggaacac    113 

tggggggagc cgatacccag gacagggcag tcctggaggc aaccgttatc cacctcaggg    173 

agggggtggc tggggtcagc cccatggagg tggctggggc cagcctcatg gaggtggctg    233 

gggccagcct catggaggtg gctggggtca gccccatggt ggtggctggg gacagccaca    293 

tggtggtgga ggctggggtc aaggtggtac ccacggtcaa tggaacaaac ccagtaagcc    353 

aaaaaccaac atgaagcatg tggcaggagc tgctgcagct ggagcagtgg tagggggcct    413 

tggtggctac atgctgggaa gtgccatgag caggcctctt atacattttg gcagtgacta    473 

tgaggaccgt tactatcgtg aaaacatgca ccgttacccc aaccaagtgt actacaggcc    533 

agtggatcag tatagtaacc agaacaactt tgtgcatgac tgtgtcaata tcacagtcaa    593 

ggaacacaca gtcaccacca ccaccaaggg ggagaacttc accgaaactg acatcaagat    653 

gatgaagcga gtggtggagc aaatgtgcat tacccagtac cagagagaat cccaggctta    713 

ttaccaacga ggggcaagtg tgatcctctt ctcttcccct cctgtgatcc tcctcatctc    773 

tttcctcatt tttctcatag taggataggg gcaaccttcc tgttttcatt atcttcttaa    833 

tctttaccag gttgggggag ggagt                                          858 

 
           
             21  
             8  
             PRT  
             Bos taurus  
           
            21 

Met Val Lys Ser His Ile Gly Ser 
1               5 

 
           
             22  
             858  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(60)  
             
           
            22 

gtcatc atg gtg aaa agc cac ata ggc agt tgg atc ctg gtt ctc ttt        48 
       Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe 
       1               5                   10 

gtg gcc atg tag agtgacgtgg gcctctgcaa gaagcgacca aaacctggag          100 
Val Ala Met 
15 

gaggatggaa cactgggggg agccgatacc caggacaggg cagtcctgga ggcaaccgtt    160 

atccacctca gggagggggt ggctggggtc agccccatgg aggtggctgg ggccagcctc    220 

atggaggtgg ctggggccag cctcatggag gtggctgggg tcagccccat ggtggtggct    280 

ggggacagcc acatggtggt ggaggctggg gtcaaggtgg tacccacggt caatggaaca    340 

aacccagtaa gccaaaaacc aacatgaagc atgtggcagg agctgctgca gctggagcag    400 

tggtaggggg ccttggtggc tacatgctgg gaagtgccat gagcaggcct cttatacatt    460 

ttggcagtga ctatgaggac cgttactatc gtgaaaacat gcaccgttac cccaaccaag    520 

tgtactacag gccagtggat cagtatagta accagaacaa ctttgtgcat gactgtgtca    580 

atatcacagt caaggaacac acagtcacca ccaccaccaa gggggagaac ttcaccgaaa    640 

ctgacatcaa gatgatgaag cgagtggtgg agcaaatgtg cattacccag taccagagag    700 

aatcccaggc ttattaccaa cgaggggcaa gtgtgatcct cttctcttcc cctcctgtga    760 

tcctcctcat ctctttcctc atttttctca tagtaggata ggggcaacct tcctgttttc    820 

attatcttct taatctttac caggttgggg gagggagt                            858 

 
           
             23  
             17  
             PRT  
             Bos taurus  
           
            23 

Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 
1               5                   10                  15 

Met 

 
           
             24  
             858  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(78)  
             
           
            24 

gtcatc atg gtg aaa agc cac ata ggc agt tgg atc ctg gtt ctc ttt        48 
       Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe 
       1               5                   10 

gtg gcc atg tgg agt gac gtg ggc ctc tga aagaagcgac caaaacctgg         98 
Val Ala Met Trp Ser Asp Val Gly Leu 
15                  20 

aggaggatgg aacactgggg ggagccgata cccaggacag ggcagtcctg gaggcaaccg    158 

ttatccacct cagggagggg gtggctgggg tcagccccat ggaggtggct ggggccagcc    218 

tcatggaggt ggctggggcc agcctcatgg aggtggctgg ggtcagcccc atggtggtgg    278 

ctggggacag ccacatggtg gtggaggctg gggtcaaggt ggtacccacg gtcaatggaa    338 

caaacccagt aagccaaaaa ccaacatgaa gcatgtggca ggagctgctg cagctggagc    398 

agtggtaggg ggccttggtg gctacatgct gggaagtgcc atgagcaggc ctcttataca    458 

ttttggcagt gactatgagg accgttacta tcgtgaaaac atgcaccgtt accccaacca    518 

agtgtactac aggccagtgg atcagtatag taaccagaac aactttgtgc atgactgtgt    578 

caatatcaca gtcaaggaac acacagtcac caccaccacc aagggggaga acttcaccga    638 

aactgacatc aagatgatga agcgagtggt ggagcaaatg tgcattaccc agtaccagag    698 

agaatcccag gcttattacc aacgaggggc aagtgtgatc ctcttctctt cccctcctgt    758 

gatcctcctc atctctttcc tcatttttct catagtagga taggggcaac cttcctgttt    818 

tcattatctt cttaatcttt accaggttgg gggagggagt                          858 

 
           
             25  
             23  
             PRT  
             Bos taurus  
           
            25 

Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 
1               5                   10                  15 

Met Trp Ser Asp Val Gly Leu 
            20 

 
           
             26  
             858  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(15)  
             
           
            26 

gtcatc atg gtg taa agccacatag gcagttggat cctggttctc tttgtggcca        55 
       Met Val 
       1 

tgtggagtga cgtgggcctc tgcaagaagc gaccaaaacc tggaggagga tggaacactg    115 

gggggagccg atacccagga cagggcagtc ctggaggcaa ccgttatcca cctcagggag    175 

ggggtggctg gggtcagccc catggaggtg gctggggcca gcctcatgga ggtggctggg    235 

gccagcctca tggaggtggc tggggtcagc cccatggtgg tggctgggga cagccacatg    295 

gtggtggagg ctggggtcaa ggtggtaccc acggtcaatg gaacaaaccc agtaagccaa    355 

aaaccaacat gaagcatgtg gcaggagctg ctgcagctgg agcagtggta gggggccttg    415 

gtggctacat gctgggaagt gccatgagca ggcctcttat acattttggc agtgactatg    475 

aggaccgtta ctatcgtgaa aacatgcacc gttaccccaa ccaagtgtac tacaggccag    535 

tggatcagta tagtaaccag aacaactttg tgcatgactg tgtcaatatc acagtcaagg    595 

aacacacagt caccaccacc accaaggggg agaacttcac cgaaactgac atcaagatga    655 

tgaagcgagt ggtggagcaa atgtgcatta cccagtacca gagagaatcc caggcttatt    715 

accaacgagg ggcaagtgtg atcctcttct cttcccctcc tgtgatcctc ctcatctctt    775 

tcctcatttt tctcatagta ggataggggc aaccttcctg ttttcattat cttcttaatc    835 

tttaccaggt tgggggaggg agt                                            858 

 
           
             27  
             857  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(24)  
             
           
            27 

gtcatc atg gta aaa gcc aca tag gcagttggat cctggttctc tttgtggcca       54 
       Met Val Lys Ala Thr 
       1               5 

tgtggagtga cgtgggcctc tgcaagaagc gaccaaaacc tggaggagga tggaacactg    114 

gggggagccg atacccagga cagggcagtc ctggaggcaa ccgttatcca cctcagggag    174 

ggggtggctg gggtcagccc catggaggtg gctggggcca gcctcatgga ggtggctggg    234 

gccagcctca tggaggtggc tggggtcagc cccatggtgg tggctgggga cagccacatg    294 

gtggtggagg ctggggtcaa ggtggtaccc acggtcaatg gaacaaaccc agtaagccaa    354 

aaaccaacat gaagcatgtg gcaggagctg ctgcagctgg agcagtggta gggggccttg    414 

gtggctacat gctgggaagt gccatgagca ggcctcttat acattttggc agtgactatg    474 

aggaccgtta ctatcgtgaa aacatgcacc gttaccccaa ccaagtgtac tacaggccag    534 

tggatcagta tagtaaccag aacaactttg tgcatgactg tgtcaatatc acagtcaagg    594 

aacacacagt caccaccacc accaaggggg agaacttcac cgaaactgac atcaagatga    654 

tgaagcgagt ggtggagcaa atgtgcatta cccagtacca gagagaatcc caggcttatt    714 

accaacgagg ggcaagtgtg atcctcttct cttcccctcc tgtgatcctc ctcatctctt    774 

tcctcatttt tctcatagta ggataggggc aaccttcctg ttttcattat cttcttaatc    834 

tttaccaggt tgggggaggg agt                                            857 

 
           
             28  
             5  
             PRT  
             Bos taurus  
           
            28 

Met Val Lys Ala Thr 
1               5 

 
           
             29  
             857  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(24)  
             
           
            29 

gtcatc atg gtg aaa agc caa tag gcagttggat cctggttctc tttgtggcca       54 
       Met Val Lys Ser Gln 
       1               5 

tgtggagtga cgtgggcctc tgcaagaagc gaccaaaacc tggaggagga tggaacactg    114 

gggggagccg atacccagga cagggcagtc ctggaggcaa ccgttatcca cctcagggag    174 

ggggtggctg gggtcagccc catggaggtg gctggggcca gcctcatgga ggtggctggg    234 

gccagcctca tggaggtggc tggggtcagc cccatggtgg tggctgggga cagccacatg    294 

gtggtggagg ctggggtcaa ggtggtaccc acggtcaatg gaacaaaccc agtaagccaa    354 

aaaccaacat gaagcatgtg gcaggagctg ctgcagctgg agcagtggta gggggccttg    414 

gtggctacat gctgggaagt gccatgagca ggcctcttat acattttggc agtgactatg    474 

aggaccgtta ctatcgtgaa aacatgcacc gttaccccaa ccaagtgtac tacaggccag    534 

tggatcagta tagtaaccag aacaactttg tgcatgactg tgtcaatatc acagtcaagg    594 

aacacacagt caccaccacc accaaggggg agaacttcac cgaaactgac atcaagatga    654 

tgaagcgagt ggtggagcaa atgtgcatta cccagtacca gagagaatcc caggcttatt    714 

accaacgagg ggcaagtgtg atcctcttct cttcccctcc tgtgatcctc ctcatctctt    774 

tcctcatttt tctcatagta ggataggggc aaccttcctg ttttcattat cttcttaatc    834 

tttaccaggt tgggggaggg agt                                            857 

 
           
             30  
             5  
             PRT  
             Bos taurus  
           
            30 

Met Val Lys Ser Gln 
1               5 

 
           
             31  
             858  
             DNA  
             Bos taurus  
             
               CDS  
               (7)..(801)  
             
           
            31 

gtcatc atg gtg aaa agc cac ata ggc agt tgg atc ctg gtt ctc ttt        48 
       Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe 
       1               5                   10 

gtg gcc atg tgg agt gac gtg ggc ctc tgc aag aag cga cca aaa cct       96 
Val Ala Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro 
15                  20                  25                  30 

gga gga gga tgg aac act ggg ggg agc cga tac cca gga cag ggc agt      144 
Gly Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser 
                35                  40                  45 

cct gga ggc aac cgt tat cca cct cag gga ggg ggt ggc tgg ggt cag      192 
Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln 
            50                  55                  60 

ccc cat gga ggt ggc tgg ggc cag cct cat gga ggt ggc tgg ggc cag      240 
Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln 
        65                  70                  75 

cct cat gga ggt ggc tgg ggt cag ccc cat ggt ggt ggc tgg gga cag      288 
Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln 
    80                  85                  90 

cca cat ggt ggt gga ggc tgg ggt caa ggt ggt acc cac ggt caa tgg      336 
Pro His Gly Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp 
95                  100                 105                 110 

aac aaa ccc agt aag cca aaa acc aac atg aag cat gtg gca gga gct      384 
Asn Lys Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala 
                115                 120                 125 

gct gca gct gga gca gtg gta ggg ggc ctt ggt ggc tac atg ctg gga      432 
Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly 
            130                 135                 140 

agt gcc atg agc agg cct ctt ata cat ttt ggc agt gac tat gag gac      480 
Ser Ala Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp 
        145                 150                 155 

cgt tac tat cgt gaa aac atg cac cgt tac ccc aac caa gtg tac tac      528 
Arg Tyr Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr 
    160                 165                 170 

agg cca gtg gat cgg tat agt aac cag aac aac ttt gtg cat gac tgt      576 
Arg Pro Val Asp Arg Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys 
175                 180                 185                 190 

gtc aat atc aca gtc aag gaa cac aca gtc acc acc acc acc aag ggg      624 
Val Asn Ile Thr Val Lys Glu His Thr Val Thr Thr Thr Thr Lys Gly 
                195                 200                 205 

gag aac ttc acc gaa act gac atc aag atg atg aag cga gtg gtg gag      672 
Glu Asn Phe Thr Glu Thr Asp Ile Lys Met Met Lys Arg Val Val Glu 
            210                 215                 220 

caa atg tgc att acc cag tac cag aga gaa tcc cag gct tat tac caa      720 
Gln Met Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln 
        225                 230                 235 

cga ggg gca agt gtg atc ctc ttc tct tcc cct cct gtg atc ctc ctc      768 
Arg Gly Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu 
    240                 245                 250 

atc tct ttc ctc att ttt ctc ata gta gga tag gggcaacctt cctgttttca    821 
Ile Ser Phe Leu Ile Phe Leu Ile Val Gly 
255                 260 

ttatcttctt aatctttacc aggttggggg agggagt                             858 

 
           
             32  
             264  
             PRT  
             Bos taurus  
           
            32 

Met Val Lys Ser His Ile Gly Ser Trp Ile Leu Val Leu Phe Val Ala 
1               5                   10                  15 

Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys Pro Gly Gly 
            20                  25                  30 

Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly 
        35                  40                  45 

Gly Asn Arg Tyr Pro Pro Gln Gly Gly Gly Gly Trp Gly Gln Pro His 
    50                  55                  60 

Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
65                  70                  75                  80 

Gly Gly Gly Trp Gly Gln Pro His Gly Gly Gly Trp Gly Gln Pro His 
                85                  90                  95 

Gly Gly Gly Gly Trp Gly Gln Gly Gly Thr His Gly Gln Trp Asn Lys 
            100                 105                 110 

Pro Ser Lys Pro Lys Thr Asn Met Lys His Val Ala Gly Ala Ala Ala 
        115                 120                 125 

Ala Gly Ala Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala 
    130                 135                 140 

Met Ser Arg Pro Leu Ile His Phe Gly Ser Asp Tyr Glu Asp Arg Tyr 
145                 150                 155                 160 

Tyr Arg Glu Asn Met His Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro 
                165                 170                 175 

Val Asp Arg Tyr Ser Asn Gln Asn Asn Phe Val His Asp Cys Val Asn 
            180                 185                 190 

Ile Thr Val Lys Glu His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn 
        195                 200                 205 

Phe Thr Glu Thr Asp Ile Lys Met Met Lys Arg Val Val Glu Gln Met 
    210                 215                 220 

Cys Ile Thr Gln Tyr Gln Arg Glu Ser Gln Ala Tyr Tyr Gln Arg Gly 
225                 230                 235                 240 

Ala Ser Val Ile Leu Phe Ser Ser Pro Pro Val Ile Leu Leu Ile Ser 
                245                 250                 255 

Phe Leu Ile Phe Leu Ile Val Gly 
            260