Patent Application: US-66054100-A

Abstract:
a determination of the nucleotide sequence for the genome of equine rhinovirus 1 , a horse - respiratory pathogen of heretofor uncertain taxonomic status , reveals a structure and predicted amino acid sequence that are more similar to those of foot - and - mouth disease viruses , the only members of the aphthovirus genus , than of other picornaviruses . these insights inform an understanding of erhv1 virion proteins and virus - like particles , as well as the design of probes , primers , antigens , vectors , diagnostics and tests of relevance to erhv1 .

Description:
the sequence of specific oligonucleotide primers used for the construction of expression plasmids are : erhv1 strain 393 / 76 was isolated from a nasal swab taken from a thoroughbred horse in south australia while it was being held in quarantine following importation from the united kingdom . the mare had an acute , systemic febrile illness . the virus was passaged 14 times in quine fetal kidney ( efk ) monolayer cell cultures and then once in vero cells . erhv1 virions were purified by a modification of the procedure described by abraham and colonno . cells were harvested 48 hours after infection . the infected cells and supernatant fluid were frozen and thawed three times and clarified by centrifuging at 2 , 000 × g for 20 min at 4 c . polyethylene glycol 6000 and nacl were added to the supernatant to final concentrations of 7 % and 380 mm , respectively , and the mixture was stirred overnight at 4 c . the precipitated virions were recovered by centrifuging at 10 , 000 × g for 15 min at 4 c and resuspended in 200 - 400 μl tne buffer ( 10 mm tris - hcl ph 8 . 0 , 100 mm nacl , 1 mm edta ) containing 1 % np40 . the suspension was clarified by centrifuging at 12 , 000 × g for 3 min before layering onto 15 % to 45 % ( wt / vol ) linear sucrose gradients ( 35 ml ) in tne buffer and centrifuging at 100 , 000 × g for 4 h at 4 c . gradients were fractioned and the fractions analyzed by sds - page . viral fractions were pooled , centrifuged at 200 , 000 × g for 2 h at 4 c , and the viral pellet was resuspended in a small volume of tne buffer , cdna synthesis and cloning . viral rna was reverse transcribed using an oligo - dt primer ( amersham ) or erhv1 specific primers p1 ( seq id no : 18 ) ( 5 ′- atccagcaagccgctgtccggttac - 3 ′) and p5 ( seq id no : 19 ) ( 5 ′- cgaagagacacctgcttc - 3 ′). viral rna was prepared as described in ( 1987 ) anal - biochem . 162 , 156 - 159 . viral rna and 100 pmol of primer were mixed , boiled for 2 min and cooled at room temperature . fist strand cdna was synthesized using 200 u of maloney murine leukemia virus reverse transcriptase ( promega ) in the presence of 0 . 8 mm dntps and 30 u of human placental rnase inhibitor ( pharmacia ) in a reaction volume of 25 μl . second strand cdna was synthesized using a cdna synthesis kit ( amersham ). the cdna fragments were ligated into puc18 , either as blunt ended fragments or after ligating bamh i adaptors ( pharmacia ), and the lighted products used to transform e . coli strain dh5α ( stratagene ). colonies were selected by hybridization , initially with an [ 32p ]- dctp - labelled cdna probe derived from reverse transcribed viral rna , and subsequently with [ 32p ]- dctp - labelled cloned viral cdna ( 16 ). the sequence between two cdna clones was obtained using the oligonucleotide primers p6 ( seq id no : 20 ) ( 5 ′- ttctggtggagaagtgacagc - 3 ′) and p7 ( seq id no : 21 ) ( 5 ′- gtgagccagcaacaattgc - 3 ′) in a polymerase chain reaction ( pcr ; 17 ) using the polymerase , vent exo + ( new england biolabs ). double - stranded dna was prepared using the alkaline lysis method and sequenced by dideoxy chain termination using modified t7 dna polymerase ( pharmacia ) and [ 35s ]- datp ( amersham ). sequence was read and analyzed using the geneworks software package ( intelligenetics , mountain view , calif .). the genbank database was searched using the fasta searching and comparison program . the protein alignment shown in fig3 was performed using the genetics computer group , inc . ( madison , wis ., usa , 1994 ) gap program with a gap creation penalty ( gcp ) of 3 . 0 and a gap extension penalty ( gep ) of 0 . 1 . the multiple alignments of nucleotide sequences were performed using clustalw . for pairwise alignments the slow method was used with a gcp of 10 and a gep of 0 . 1 . for multiple alignments a gcp of 10 and a gep of 0 . 05 was used with alignment of sequences which were more than 60 % divergent delayed and using weighted transitions . phylogenetic relationships were examined using the maximum likelihood method with the dnaml program of the phylogeny inference package ( phylip ) version 3 . 5c ( 1993 , j . felsenstein , department of genetics , university of washington , seattle ). the model used allowed for unequal expected frequencies of the four nucleotides , with the frequencies determined empirically from those present in the sequences analysed , and unequal rates of transitions and transversions . a single rate of change was assumed for all sites . the program was allowed to perform global rearrangements to optimize the tree . initial analyses were performed on polymerase sequences using a range of transition / transversion ratios to determine that which gave the maximal log likelihood . a ratio of 2 . 0 gave the maximal log likelihood and thus this ratio was used for all subsequent analyses of other sequences . sixty seven overlapping cdna clones and one pcr product clone were obtained and sequenced from both ends . the nucleotide in each position was determine at least twice , and 95 % of the sequence was obtained by sequencing in both directions . the predicted genomic structure of erhv1 was characteristic of picornaviruses , possessing one long open reading frame ( orf ) flanked by 5 ′- and 3 ′- ntr &# 39 ; s ( fig1 ). the nucleotide and predicted amino acid sequences of the erhv1 polyprotein are shown in fig2 a . partial sequence of the 5 ′- ntr ( 433 bases ) was also obtained fig2 b . there was a tract of 9 cs at position − 550 to − 542 . polyc tracts of various lengths have been observed in similar locations in fmdv and emcv . the actual length of the erhv1 polyc tract is uncertain as these sequences are known to be unstable when propagated in e . coli . a 14 nucleotide polypyrimidine tract , which possessed the tttc motif common to all picornaviruses , was present near the potential translation initiation codons . a region of 450 nucleotides upstream of the most likely initiation codon is predicted to contain an internal ribosome entry site ( ires ). this region showed most sequence identity ( 48 - 50 %) with corresponding sequences in fmdv and emcv . the 3 ′- ntr of erhv1 was 102 nucleotides excluding the polya tail ( data not shown ). in picornaviruses , there are two factors that influence which atg codon initiates translation , a requirement for the atg to be located at the 3 ′- end of the ires , and that this atg occurs in a sequence optimal for initiating translation , that is , a purine at position − 3 and a g in position + 4 . two pairs of in - frame atg codons were identified in the erhv1 genome . the second atg of the first pair is separated by 25 nucleotides from the beginning of the polypyrimidine tract ( fig2 b ), similar to the distance ( 25 to 27 nucleotides ) found in the corresponding regions in fmdv and emcv ( 24 ). the second atg of each pair occurs in an optimal context . therefore , the second atg of the first pair is most likely to be the translation initiation codon but it is possible that translation is also initiated from the second optimal atg , by a process of leaky scanning , or even from the other two , non - optimal atg codons . the predicted erhv1 coding sequence , beginning at the most likely initiation atg , extended for 6 , 741 bases and would encode a polyprotein of 2 , 247 amino acids . alignment of the erhv1 amino acid sequence with those of other picornaviruses showed that it was most similar to aphthoviruses and , to a lesser extent , to cardioviruses in all regions of the genome ( data not shown ). fig3 shows a comparison of the predicted amino acid sequence of erhv1 with that of fmdv . o1k . the two sequences were 40 % identical . the more conserved regions include : the 3d / polymerase ( 50 % identity ), vp4 ( 49 % identity ) and some regions of the 2c protein . erhv1 encodes a 2a protein of 16 amino acids , 14 of which were identical with those of fmdv 2a . erhv1 possessed only one copy of the vpg sequence . this is in contrast to fmdv which has 3 tandemly repeated , non - identical vpg sequences ( 27 - 29 ). table 1 shows the proteolytic cleavage sites of erhv1 predicted from the amino acid alignment ( fig3 ), and compares these with those of fmdv , emcv and theiler &# 39 ; s murine encephalomyelitis virus ( tmev ). most of the erhv1 cleavage sites could be assigned with reasonable confidence because of significant amino acid similarity with fmdv in the regions flanking the predicted cleavage site ; an exception was the 3a / 3b cleavage site where there was less sequence similarity . as is the case with fmdv , the predicted erhv1 3c protease cleavage sites were more variable than those of the cardioviruses . emcv and tmev . a phylogenetic tree was derived from the nucleotide sequences of complete picornavirus polyproteins ( fig4 a ). each branch of this tree was statistically , highly significant ( p & lt ; 0 . 01 ), with the 95 % confidence limits ranging from ± 7 % to ± 15 % of branch lengths . erhv1 was found to be most closely related to the aphthoviruses , although it was clear that erhv1 was considerably more distant from individual members of this genus than the aphthoviruses were from each other . a phylogenetic tree was also derived from the nucleotide sequences of picornavirus polymerase genes ( fig4 b ). each branch of this tree was statistically , highly significant ( p & lt ; 0 . 01 ) with 95 % confidence limits ranging from ± 14 % to ± 38 % of the branch lengths . again , erhv1 grouped with the aphthoviruses and the topology of the tree was the same as that obtained using data of the entire polyprotein ( fig4 a ). the vp1 nucleotide sequences were also similarly analyzed ( fig4 c ). most branches were statistically , highly significant ( p & lt ; 0 . 01 ), although , that between the erhv1 branch point and the branch point for the echovirus 22 - hepatovirus cluster was less so ( p & lt ; 0 . 05 ). the 95 % confidence limits of the branch lengths of this tree were considerable greater than for the other two trees , ranging from ± 18 % to ± 69 %. this tree did not group erhv1 with the aphthoviruses . with the exception of bovine enterovirus ( bev ), the tree had the same topology as those derived from the complete polyprotein and the polymerase sequences . it was also apparent that picornaviruses formed three clusters : enteroviruses - rhinoviruses , echovirus 22 - hepatovirus and cardioviruses - aphthoviruses - erhv1 . oligonucleotide primers : we have designed short oligonucleotide primers and used them in polymerase chain reactions ( pcr ) for the diagnosis of erhv infected horses . any of the erhv nucleotide sequences may be used for the design primer sets for use as diagnostic reagents . they may be highly specific for erhv1 or they may be designed to be more cross reactive so as to amplify single strand rna template from other erhv types e . g ., erhv 2 , 3 and 4 . as a specific example we have used the primer set shown in fig5 to diagnose erhv disease in several groups of seriously ill horses in circumstances in which , despite exhaustive efforts , we could not isolate the virus using conventional cell culture procedures . we no consider erhv a very under reported disease simply because , most of the time , nasal samples collected from horses experiencing severe , systemic clinical disease because of erhv infection do not yield the virus in cell culture . in one particular group of horses , we detected the presence of erhv by pcr and confirmed that the horses were both actively infected and seriously ill with erhv by use of paired serum samples which showed that there was a concomitant rise in erhv1 serum neutralising antibody . vigorous attempts to isolate the virus in cell cultures yielded negative results . oligonucleotide probes : virus specific oligonucleotides are used as probes to detect the presence of the virus in infected samples from diseased horses and other animals . this may be especially important given the systemic nature of the illness , i . e ., it is a foot - and - mouth - like , generalized disease with virus distributed throughout the body in many organs and tissues ; it is not just a simple “ common cold - like ” illness as the name rhinovirus implies . the significance of the sequence in moving the virus out of the rhinovirus genus and into a new genus proposed to be called “ equirhinovirus ” in the picornaviridae family does not represent merely a taxonomic change but represents a paradigm shift in how erhv1 and related viruses must now be regarded as pathogens for the horse and other animal species . diagnostic antigens : individual virion proteins , in particular vp1 , vp2 and vp3 , can be expressed in any one of a number of heterologous expression systems to provide antigens to detect specific antibody to erhv1 present in blood . such expression systems , which are well established for e . coli , yeast , vaccinia virus and baculovirus , allow for the production of large quantities of protein to a high degree of purity . the expressed virion proteins may be used in simple immunoassays , such as elisa , to detect erhv1 specific antibody . virion proteins expressed in this way also serve as effective vaccines against erhv1 disease . production of virus like particles ( vlps ): we have used the sequence information to construct recombinant plasmids containing the p1 - 2a - 3c region of the genome ( see fig1 a and fig6 ). these plasmid constructions are of course critically dependent on the erhv1 sequence that has been determined although the strategy that we are adopting , in general , is similar to that described in j . virol 66 , 4557 - 4564 . some early plasmid constructions have been inserted into e . coli and baculovirus expression systems based on prior art with similar viruses such as poliomyelitis of humans and foot - and - mouth disease virus of cattle and other cloven hoofed animals . the rt pcr double stranded dna of the p1 - 2a - 3c region of the erhv1 genome is transcribed , within the transformed e . coli or insect cell for baculovirus , into messenger rna as a single transcript which is then translated into a mini polyprotein . the 3c protease activity results in the cleavage of the mini polyprotein into its constituent parts namely 1a ( vp4 ), 1b ( vp2 ), 1c ( vp3 ) and 1d ( vp1 ), 2a and 3c ( see fig1 a and fig6 ) and that the vp component parts then self assemble into vlps i . e ., virus particles that lack nucleic acid and are therefore non infectious i . e ., are unstable to cause disease . two important applications of erhv vlps are as follows : ( a ) the vlps are very useful as highly effective , safe , high antigen - mass vaccines for the control erhv1 disease . if erhv1 disease is confirmed , as we believe to be the case , as significant and responsible for much hither to undiagnosed illness that results in many lost training days , many expensive treatments , much serious illness because of secondary infections following on the primary erhv1 infection , and much poor performance , then the utility of the vaccine based on the vlps that are the subject of this invention will be very great and likely to have world - wide application . with improved methods for the diagnosis of erhv1 infection such as by pcr and elisa as described herein , it is likely that other members of the proposed new equirhinovirus genus within the family picornaviridae including for example erhv2 , erhv3 , may be similarly diagnosed . indeed suitably selected pcr primer sets based on the erhv1 sequence could be used to detect these other equine rhinoviruses . the sequencing of these genomes could provide a basis for their specific diagnosis . it is also evident that the construction of vlp &# 39 ; s based on expression plasmids similar to those described herein for erhv1 , could be readily adapted to these other equine rhinoviruses leading for example to production of combined erhv vaccines to cover all antigenic types as may be extant or as may emerge by antigenic variation , as is very much a part of the biology of fmdv , in the future . polyvalent vlp vaccines incorporating a range of erhv antigenic types are obvious extensions based on the work described herein . ( b ) erhv vlps can be used as a delivery vector that will provide not only protection against erhv disease but will be used to deliver other therapeutic and useful substances to the horses following administration by parenteral or other routes . such delivery vectors can be produced by inserting into , for example the p1 region at some appropriate site , double stranded dna coding for antigenic epitopes of other virus and infections agents of horse as well as epitopes derived from other non infectious sources for example reproductive hormones . for the detection of erhv1 antibodies in infected or vaccinated horses various stranded tests can be used . vlp &# 39 ; s may be used in such test for example in an elisa test for antibody . other diagnostic tests based on recombinant antigens derived from the erhv1 sequence can be devised along similar lines to those reported for fmdv in which the absence of protein 2c from clarified inactivated whole virus fmd , fmdv or fmdv vlp vaccines may be used as the basis for distinguishing infected from vaccinated animals where the vaccine is a non - replicating form of erhv1 or a deletion mutant of erhv1 in which a particular non - structural protein gene has been deleted . precedent for this comes from studies of fmdv as reported in for example lubroth , grubman , burrage , newman & amp ; brown , 1996 , absence of protein 2c from clarified foot - and - mouth disease virus vaccines provides the basis for distinguishing convalescent from vaccinated animals , vaccine 14 ( 5 ), 419 - 427 . preparation and use of virus - like particles and other proteins based on erhv1 sequence from the sequence of erhv1 it is possible to clone certain segments of the viral genome into a variety of vectors for expression in a variety of different expression systems . there is a straight forward and storing literature for fmdv that provides a very clear precedent for what can be done for erhv1 . examples include the expression of fmdv p1 - 2a in a baculovirus ( abrams c c & amp ; belsham g j , 1994 , the antigenicity of foot - and - mouth disease virus p1 - 2a polyprotein and empty capsids produced in vaccinia virus and baculovirus expression systems . in viith meeting of the european study group on the molecular biology of picornaviruses , aug . 6 - 11 , 1994 , korpilampi , finland ) or vaccina virus systems ( abrams c c , king a m q & amp ; belsham g j , 1995 , assembly of foot - and - mouth disease virus empty capsids synthesized by a vaccinia virus expression system . journal of general virology 76 : 3089 - 3098 ) to obtain vlps or viral proteins . we have prepared similar plasmids in which p1 - 2a , p1 - 2a - 3c and these two sequences in a myristolated form have been inserted into p fastbac 1 baculovirus vector ( gibco / brl ) and into a pet vector ( novogene ) for expression in insect cells and e . coli respectively . these expressed products either as protein antigens or as vlps , have utility as the basis for diagnostic tests or vaccines . accordingly , such references are herein incorporated in support of the full description and enablement of the invention where the disclosed methods of preparing diagnostics , vaccines , vectors , host systems and kits are fully described and applicable to the like aspects of the current invention . erhv is also a human pathogen . we have unpublished data to confirm that humans have serum neutralising antibody to erhv1 that is indicative of infection . one of the laboratory workers connected with the conduct of the sequencing and who handled infections virus has specific antibody in high amounts ( serum neutralising antibody titre 1 : 640 to erhv1 ). we are currently extending these studies and anticipate finding a significant incidence of infection in humans world wide particularly among those humans who work with horses . the improved diagnostic methods outlined above , perhaps also the vaccine , are expected to have application in human medicine . ttttgtactg acatg atg gcg gcg tct aag gtg tat aga gtt tgc gag cag 471 act ctg ctg gca ggt gcc gtt cgc atg atg gac aaa ttc ttg caa aag 519 thr leu leu ala gly ala val arg met met asp lys phe leu gln lys aga act gtt ttt gtc ccc cat ctt gac aaa aca att cgt ttg act gga 567 arg thr val phe val pro his leu asp lys thr ile arg leu thr gly ctc cac aat tat gac aat act tgc tgg ttg aat gcc ttg aca caa ctg 615 aca cag att ctt gga att cgg ctt ttt gat gaa cac ttc ggc aat aga 663 thr gln ile leu gly ile arg leu phe asp glu his phe gly asn arg ggt ctg ttc act cgg aaa aca att gat tgg gtg agt gac cag act ggt 711 gly leu phe thr arg lys thr ile asp trp val ser asp gln thr gly ata aaa gat cta aaa tca gga gca ccg cca ctc gtg gtg gtg tac aaa 759 ctg tgg caa cat gga cac ttg gat gtc ggt acg atg gag aaa ccc cgg 807 leu trp gln his gly his leu asp val gly thr met glu lys pro arg tcg att act cta tgg tct ggc ccc aaa gtg tgt ctt tct gat ttc tgg 855 ser ile thr leu trp ser gly pro lys val cys leu ser asp phe trp gcc tgt gtt tcg gca aaa ccg gga cat gca gta ttc tac ctt ctc aca 903 ala cys val ser ala lys pro gly his ala val phe tyr leu leu thr agc gag ggt tgg atc tgt gtt gat gac aag aaa ata tac cca gaa aca 951 ser glu gly trp ile cys val asp asp lys lys ile tyr pro glu thr ccc aaa aca gag gat gta ctt gtt ttt gcg ccc tat gac ttt gag tca 999 pro lys thr glu asp val leu val phe ala pro tyr asp phe glu ser ctg ggc aag gac cca cca aag cta cac cag aga tat gaa aaa gca ttt 1047 leu gly lys asp pro pro lys leu his gln arg tyr glu lys ala phe gag ctc agt ggc gga ggt aca tcc act cca aca act ggc aac caa aac 1095 atg tcc gga aac agt ggt tca att gtt caa aat ttt tac atg caa cag 1143 tac cag aat tca att gac gca gac ctg gga gac aat gtg att agc cct 1191 tyr gln asn ser ile asp ala asp leu gly asp asn val ile ser pro gaa ggc cag ggc agc aac act agt agt tca acc tca tca agc caa tcc 1239 tct ggc ttg ggc ggg tgg ttc tct agt ttg ctg aac ctt gga aca aaa 1287 cta ctg gct gac aag aag aca gaa gag act aca aac att gaa gac aga 1335 att gaa aca aca gtg gtt gga gtc act att att aat tca caa gga tct 1383 gtt gga aca acc tac tgt tac tcc aaa ccg gat ggt aga cca cca tcc 1431 aca gtg tca gac cca gtt acc aga ctt gga ccc acg ctt tcc agg cac 1479 tac aca ttt aag gta ggt gag tgg ccc cat tct caa tca cat ggt cac 1527 tyr thr phe lys val gly glu trp pro his ser gln ser his gly his gca tgg atc tgt ccg ttg cca ggt gac aaa ctc aag aag atg ggc agt 1575 ala trp ile cys pro leu pro gly asp lys leu lys lys met gly ser ttt cat gag gtt gtc aaa gcc cac cac ctg gtc aag aac ggc tgg gat 1623 phe his glu val val lys ala his his leu val lys asn gly trp asp gtg gtt gtg cag gtg aat ccc tca ttt gct cac tcc ggg ccg ctg tgt 1671 val val val gln val asn pro ser phe ala his ser gly pro leu cys gta gca gca gtg ccg gag tac gaa cac aca cat gag aaa gca ctc aag 1719 tgg tct gag ctt gag gaa cca gct tac aca tac caa caa ctt tca gtt 1767 ttt ccc cac cag ttg cta aat ttg agg aca aat tca tca gtg cat ttg 1815 gtg atg ccc tac att ggg cca ggc caa cca aca aat ctg act ttg cac 1863 val met pro tyr ile gly pro gly gln pro thr asn leu thr leu his aac ccg tgg acc att gtt att tta att ttg tct gaa ttg aca gga cct 1911 ggc caa act gtg cct gtg acc atg tcg gtg gct ccc atc gat gca atg 1959 gtt aat ggg cct ctt cca aat cca gag gca ccg att aga gtg gtg tct 2007 gtg cct gaa tca gat tct ttt atg tct tca gta cct gat aat tcg act 2055 cca cta tac ccc aag gtt gtg gtc cca ccg cgc caa gtt cct ggc cgg 2103 ttt aca aat ttc att gat gtg gca aaa cag aca tat tca ttt tgt tcc 2151 phe thr asn phe ile asp val ala lys gln thr tyr ser phe cys ser att tct gga aaa cct tat ttt gag gtt acc aac acc tct ggg gac gag 2199 ile ser gly lys pro tyr phe glu val thr asn thr ser gly asp glu cca ctg ttt cag atg gat gtg tcg ctc agt gcg gca gag cta cat ggc 2247 pro leu phe gln met asp val ser leu ser ala ala glu leu his gly act tac gta gct agt ttg tca tca ttt ttt gca cag tac aga ggc tca 2295 ctt aat ttc aac ttt att ttc act ggt gca gca gcc act aag gca aag 2343 ttt ctg gtt gct ttt gtg cct ccc cac agt gca gcg ccc aaa acg cgc 2391 gat gaa gca atg gcg tgc atc cat gcc gtg tgg gat gtt ggc ttg aac 2439 asp glu ala met ala cys ile his ala val trp asp val gly leu asn tca gct ttt tct ttt aat gta cct tat ccc tcc cct gct gac ttc atg 2487 gcc gtt tat tct gcg gaa cgg acg gtt gtg aat gtc tct gga tgg ctt 2535 ala val tyr ser ala glu arg thr val val asn val ser gly trp leu caa gtt tat gca cta aca gct cta act tca act gac att gcc gtg aac 2583 agt aaa ggc cgt gtg ctg gtt gct gtt tcc gcc ggc cca gac ttc tcc 2631 ctt cgt cac ccg gcg gac ctg ccc gac aag cag gtt acc aat gtg gga 2679 leu arg his pro ala asp leu pro asp lys gln val thr asn val gly gag gat ggt gaa ccc ggt gag aca gag cct cgt cat gct ttg tca ccc 2727 gtg gac atg cac gtg cac aca gat gtc agt ttc ttg ctt gac cgg ttc 2775 ttt gat gtt gag aca ctt gag ctt tca aat ttg aca ggt tct cct gcc 2823 phe asp val glu thr leu glu leu ser asn leu thr gly ser pro ala aca cat gtt ctg gat ccg ttt ggc tcg act gcc caa ctg gct tgg gca 2871 thr his val leu asp pro phe gly ser thr ala gln leu ala trp ala cgt ctg cta aac act tgc acc tac ttc ttt tct gat ttg gaa ttg tca 2919 atc cag ttt aaa ttt acc acc act ccg tcc tct gtt gga gag ggc ttt 2967 gtg tgg gtg aag tgg ctc cct gtt gga gca cca acc aag acc aca gat 3015 gct tgg cag tta gaa gga ggt gga aat tca gtt aga att caa aaa ttg 3063 ala trp gln leu glu gly gly gly asn ser val arg ile gln lys leu gcc gtt gca ggg atg tgc ccc act gtt gtg ttc aag att gca ggc tcc 3111 ala val ala gly met cys pro thr val val phe lys ile ala gly ser cgt tca caa gcc tgt gct tca gcg ttg cca tat aca tca atg tgg cgt 3159 arg ser gln ala cys ala ser ala leu pro tyr thr ser met trp arg gtt gtg cca gtc ttt tac aat ggc tgg ggt gca cct acc aaa gaa aag 3207 val val pro val phe tyr asn gly trp gly ala pro thr lys glu lys gca acc tac aat tgg ctt cct ggt gca cac ttt ggt tcc atc ttg ctg 3255 ala thr tyr asn trp leu pro gly ala his phe gly ser ile leu leu act tct gat gcg cat gat aaa gga ggg tgc tac ttg cgg tat gct ttc 3303 thr ser asp ala his asp lys gly gly cys tyr leu arg tyr ala phe cgc gcg cca gcg atg tat tgc cct cga ccc att ccg ccg gct ttt acg 3351 cgt cca gcg gac aaa acc aga cat aaa ttt ccc act aac atc aac aaa 3399 cag tgt act aat tac tct ctc ctc aaa ttg gct gga gat gtt gag agc 3447 gln cys thr asn tyr ser leu leu lys leu ala gly asp val glu ser aac cct ggc ccc act att ttt tcc aaa gca tca gca gac ctg aat gcc 3495 asn pro gly pro thr ile phe ser lys ala ser ala asp leu asn ala ttg tca acg tcg cta ggt gaa ttg act ggc atg cta aaa gat ctt aaa 3543 gcc aag gca gaa act tat tcc ccg ttt tac aaa atg gcc aaa atg ctt 3591 ttc aaa ctt gca aca cta gct gtg gca gct atg agg aca aag gac cca 3639 gta gtg gtg gtt atg ttg att gct gat ttc gga ttg gag gtc ttt gac 3687 act ggg ttt ttc ttt tcc tac ttt caa gag aag ttg cag cct tat atg 3735 thr gly phe phe phe ser tyr phe gln glu lys leu gln pro tyr met aaa act att cct ggt aag att tct gat ttg gtc act gat gcg gct acg 3783 gct gcc gcc caa att cca aag gga gtg tat tct ttt gtg tcg tca ttt 3831 ttc gaa acg cct gaa gga gtg gtt gag aag cag gtg tct ctt cgg aca 3879 phe glu thr pro glu gly val val glu lys gln val ser leu arg thr gtg aat gac ata ttt gct ttg ctt aaa aat tct gat tgg ttc ata aag 3927 act ctt gtt gcc ctc aag aaa tgg ctg aca tcc tgg ttt gct caa gaa 3975 caa cag gca gat gat gcg ctc tat tca gaa ttg gaa aaa tat ccc ttg 4023 tac aag tta aaa ttg aag gaa cct gat act caa gag gaa gcg cgc cag 4071 tgg ttt aaa gac atg cag cag cgt gct ctc gct gtg aag gac aaa ggt 4119 trp phe lys asp met gln gln arg ala leu ala val lys asp lys gly ctc ttt tcc ctc ctg caa att cca tta gtt aac ttg ccc cag agc cgt 4167 cca gag ccc gtt gta tgc gtc ctt cgg ggc gca tca ggg caa ggc aaa 4215 pro glu pro val val cys val leu arg gly ala ser gly gln gly lys tct tat ttg gca aat ctg atg gct caa gca att tcg ctt ctc ttg gtt 4263 ggc aag cag gac agt gtg tgg agt tgt cct cct gac ccc aca tat ttt 4311 gly lys gln asp ser val trp ser cys pro pro asp pro thr tyr phe gat ggc tat aac gga cag gct gtg gtg att atg gat gca ttg ggc cag 4359 gat ccg aat ggt gct gac ttt aaa tat ttt tgc cag atg gtc tct aca 4407 asp pro asn gly ala asp phe lys tyr phe cys gln met val ser thr aca gct ttt gta cca cct atg gcc cat ttg gat gat aaa ggc att cca 4455 thr ala phe val pro pro met ala his leu asp asp lys gly ile pro ttt act tct cct gtt gtt att tgt act aca aat ttg cat tca tct ttt 4503 acc cct att act gtt tct tgt cct gaa gct ctt aag agg agg ttt cgg 4551 thr pro ile thr val ser cys pro glu ala leu lys arg arg phe arg ttt gat gtg acg gtg tcc gct aaa ccg ggc ttt gtg cgc act gtt ggt 4599 tca aac cag ctt ttg aat ctc cca ctt gct ctt aag cca gct ggt ctt 4647 ccc cca cac cct atc ttt gaa aat gac atg ccc att ata aat ggg cag 4695 gct gtt aaa ttg gct ctt tct ggt gga gaa gtg aca gct ttt gag ctt 4743 att gag atg ata ctg tca gaa gtt caa aac aga caa gac aca cac aaa 4791 ile glu met ile leu ser glu val gln asn arg gln asp thr his lys atg ccc att ttt aaa caa tca tgg tct gat ttg ttc aga aag tgt aca 4839 met pro ile phe lys gln ser trp ser asp leu phe arg lys cys thr act gat gag gaa cag aaa atg ttg cag ttt tta att gac aat aaa gat 4887 tca gaa att ctc agg gcg ttt gtt tca gaa cgc tcc att tta cta cat 4935 gaa gag tat ctt aaa tgg gag tca tat atg acc agg aga gcc aag ttt 4983 glu glu tyr leu lys trp glu ser tyr met thr arg arg ala lys phe cac cgc ctg gct gct gat ttt gct atg ttt cta tcc att ctt act tca 5031 ctg att gtt att ttt tgt tta gtt tat tct atg tat caa ctt ttt aag 5079 acc cct gac gag caa tca gct tat gat cct tca act aag cca aaa cca 5127 aag acc cag gaa gtg aaa aca ctg aag att agg act gag act ggt gta 5175 cca gca act gac ttg caa caa tcc atc atg aaa aat gtt cag cca att 5223 pro ala thr asp leu gln gln ser ile met lys asn val gln pro ile gag ctt tac ctt gac aat gaa ttg gtt act gac tgc tct gcc ttg ggt 5271 glu leu tyr leu asp asn glu leu val thr asp cys ser ala leu gly gtt tat gac aat tca tat ttg gtg ccc ctt cat ttg ttt gaa ttt gat 5319 ttt gat acc att gtg ctt ggt gga cgt cat tac aag aaa gct gag tgt 5367 phe asp thr ile val leu gly gly arg his tyr lys lys ala glu cys gag aag gta gag ttt gag ctt gaa gtg aat gga gac gtg gtg tca tca 5415 gat gcg tgt cta ctt cga gtg tca tcg ggg cct aaa gtt aga aat att 5463 asp ala cys leu leu arg val ser ser gly pro lys val arg asn ile gtt cat ctt ttt aca aat gaa att gaa ttg aag aaa atg acc caa gtg 5511 val his leu phe thr asn glu ile glu leu lys lys met thr gln val aca gga atc atg aat tca cca cac cag gca cgc act gtg ttt ttt ggc 5559 thr gly ile met asn ser pro his gln ala arg thr val phe phe gly agt ttt ttg aca gtg agg aag tcc atc tta aca tcg gat ggg act gta 5607 atg ccc aat gtt ttg tcc tat gcc gct cag acc tcg cgt ggg tat tgt 5655 met pro asn val leu ser tyr ala ala gln thr ser arg gly tyr cys ggc gct gca att gtt gct ggc tca cct gcc cgc ata att ggt atc cat 5703 tca gct ggc act gga tct gtt gca ttt tgc tcc ctg gtg tcc aga gac 5751 gcg ctg gag caa ctc tgg ccc cag aaa cag ggc aac gtt agt cgc ctt 5799 ala leu glu gln leu trp pro gln lys gln gly asn val ser arg leu gat gac gat gtg agg gtg tct gtt ccg cgc cgc tcc aaa ttg gtg aaa 5847 tca ttg gct tac ccc att ttc aaa cct gac tat ggc cca gcg cca ctc 5895 tct caa ttt gac aag cgc ctg tca gac ggc gtg aag ctg gat gaa gtg 5943 gtt ttt gct aaa cat act gga gac aag gag att tcc gca cag gac cag 5991 val phe ala lys his thr gly asp lys glu ile ser ala gln asp gln aaa tgg ctc ttg cgt gcg gcg cat gta tac gcc cag aag gtt ttc tcc 6039 lys trp leu leu arg ala ala his val tyr ala gln lys val phe ser cgg att gga ttt gac aac cag gct ttg act gaa aaa gag gcc att tgt 6087 arg ile gly phe asp asn gln ala leu thr glu lys glu ala ile cys ggc att cct ggc ctt gac aag atg gag cag gac acc gct ccc ggg ctg 6135 gly ile pro gly leu asp lys met glu gln asp thr ala pro gly leu ccc tat gct cag caa aat aag aga agg aaa gac atc tgt gat ttt gaa 6183 pro tyr ala gln gln asn lys arg arg lys asp ile cys asp phe glu gag ggc cgg ctg aag ggc gcc gaa ctc caa aag gac aga ttt atg gct 6231 ggt gac tac tct aat ttg gtc tat caa tca ttt ttg aaa gat gag atc 6279 gly asp tyr ser asn leu val tyr gln ser phe leu lys asp glu ile cgc cca ctt gag aaa gtt agg gct gga aag acc cgc ctg att gac gtg 6327 arg pro leu glu lys val arg ala gly lys thr arg leu ile asp val ccg ccg atg ccc cat gtg gtg gtt ggt agg cag ctc ttg ggc cgg ttt 6375 gtg gca aaa ttt cat gaa gca aat gga ttt gac att ggc tca gcc att 6423 val ala lys phe his glu ala asn gly phe asp ile gly ser ala ile gga tgt gac cca gat gtg gac tgg act cgg ttt ggc ctc gag ttg gag 6471 gly cys asp pro asp val asp trp thr arg phe gly leu glu leu glu cgt ttc agg tat gta tat gcc tgt gac tac tca cgg ttc gat gcc aac 6519 cat gca gct gat gca atg aga gtt gtg ctt aac tac ttt ttc tct gag 6567 his ala ala asp ala met arg val val leu asn tyr phe phe ser glu gac cac ggt ttc gac cct ggt gtg cct gct ttt att gag tca ctg gtt 6615 asp his gly phe asp pro gly val pro ala phe ile glu ser leu val gat tca gtg cat gcc tat gaa gag aaa agg tat aac atc tac ggt ggc 6663 asp ser val his ala tyr glu glu lys arg tyr asn ile tyr gly gly ttg cca tcc ggg tgt tcc tgc aca tca att ttg aat acc atc ttg aac 6711 aat gtt tac att ctt gca gct atg atg aag gct tat gag aat ttt gag 6759 cca gat gac att cag gtc att tgc tat ggg gac gac tgc ctc att gct 6807 tct gat ttt gaa att gat ttc caa caa ctg gtg cct gtc ttt tct agt 6855 ttt gga cag gta ata act aca gct gac aag act gat ttt ttt aaa ctg 6903 aca acg ctt tcg gag gtg acc ttc ctt aag cgc gct ttt gtt ctg acg 6951 gcc ttt tac aag cca gtg atg gat gtg aag acc ctt gaa gca atc tta 6999 ala phe tyr lys pro val met asp val lys thr leu glu ala ile leu agc ttt gtt cgc cca ggc aca cag gct gaa aag ctc ctg tcc gtg gcg 7047 ser phe val arg pro gly thr gln ala glu lys leu leu ser val ala cag ttg gca ggc cac tgc gaa ccg gag cag tat gag cgc ctg ttt gag 7095 gln leu ala gly his cys glu pro glu gln tyr glu arg leu phe glu ccc ttt gct ggg atg tat ttc gtc cct act tgg cga ctt gcg cct gca 7143 pro phe ala gly met tyr phe val pro thr trp arg leu ala pro ala gtg gtt gat gaa gct tgg atg cta aat tct ttt tgactttgtt tttctttgtt 7196 met ala ala ser lys val tyr arg val cys glu gln thr leu leu ala gly ala val arg met met asp lys phe leu gln lys arg thr val phe val pro his leu asp lys thr ile arg leu thr gly leu his asn tyr arg lys thr ile asp trp val ser asp gln thr gly ile lys asp leu lys ser gly ala pro pro leu val val val tyr lys leu trp gln his gly his leu asp val gly thr met glu lys pro arg ser ile thr leu trp ser gly pro lys val cys leu ser asp phe trp ala cys val ser ala lys pro gly his ala val phe tyr leu leu thr ser glu gly trp asp val leu val phe ala pro tyr asp phe glu ser leu gly lys asp pro pro lys leu his gln arg tyr glu lys ala phe glu leu ser gly ile asp ala asp leu gly asp asn val ile ser pro glu gly gln gly pro val thr arg leu gly pro thr leu ser arg his tyr thr phe lys val gly glu trp pro his ser gln ser his gly his ala trp ile cys pro leu pro gly asp lys leu lys lys met gly ser phe his glu val glu glu pro ala tyr thr tyr gln gln leu ser val phe pro his gln ile asp val ala lys gln thr tyr ser phe cys ser ile ser gly lys pro tyr phe glu val thr asn thr ser gly asp glu pro leu phe gln met asp val ser leu ser ala ala glu leu his gly thr tyr val ala phe val pro pro his ser ala ala pro lys thr arg asp glu ala met ala cys ile his ala val trp asp val gly leu asn ser ala phe ser ala glu arg thr val val asn val ser gly trp leu gln val tyr ala leu thr ala leu thr ser thr asp ile ala val asn ser lys gly arg ala asp leu pro asp lys gln val thr asn val gly glu asp gly glu pro gly glu thr glu pro arg his ala leu ser pro val asp met his asp pro phe gly ser thr ala gln leu ala trp ala arg leu leu asn thr cys thr tyr phe phe ser asp leu glu leu ser ile gln phe lys glu gly gly gly asn ser val arg ile gln lys leu ala val ala gly met cys pro thr val val phe lys ile ala gly ser arg ser gln ala cys ala ser ala leu pro tyr thr ser met trp arg val val pro val trp leu pro gly ala his phe gly ser ile leu leu thr ser asp ala his asp lys gly gly cys tyr leu arg tyr ala phe arg ala pro ala tyr ser leu leu lys leu ala gly asp val glu ser asn pro gly pro phe ser tyr phe gln glu lys leu gln pro tyr met lys thr ile pro glu gly val val glu lys gln val ser leu arg thr val asn asp ile phe ala leu leu lys asn ser asp trp phe ile lys thr leu val ala leu lys glu pro asp thr gln glu glu ala arg gln trp phe lys asp met gln gln arg ala leu ala val lys asp lys gly leu phe ser leu asn leu met ala gln ala ile ser leu leu leu val gly lys gln asp ser val trp ser cys pro pro asp pro thr tyr phe asp gly tyr asn ala asp phe lys tyr phe cys gln met val ser thr thr ala phe val pro pro met ala his leu asp asp lys gly ile pro phe thr ser pro val ser cys pro glu ala leu lys arg arg phe arg phe asp val thr val ser ala lys pro gly phe val arg thr val gly ser asn gln leu ile phe glu asn asp met pro ile ile asn gly gln ala val lys leu leu ser glu val gln asn arg gln asp thr his lys met pro ile phe lys gln ser trp ser asp leu phe arg lys cys thr thr asp glu glu lys trp glu ser tyr met thr arg arg ala lys phe his arg leu ala phe cys leu val tyr ser met tyr gln leu phe lys thr pro asp glu val lys thr leu lys ile arg thr glu thr gly val pro ala thr asp leu gln gln ser ile met lys asn val gln pro ile glu leu tyr leu asp asn glu leu val thr asp cys ser ala leu gly val tyr asp asn ser tyr leu val pro leu his leu phe glu phe asp phe asp thr ile leu arg val ser ser gly pro lys val arg asn ile val his leu phe asn ser pro his gln ala arg thr val phe phe gly ser phe leu thr val arg lys ser ile leu thr ser asp gly thr val met pro asn val gly ser val ala phe cys ser leu val ser arg asp ala leu glu gln leu trp pro gln lys gln gly asn val ser arg leu asp asp asp val pro ile phe lys pro asp tyr gly pro ala pro leu ser gln phe asp his thr gly asp lys glu ile ser ala gln asp gln lys trp leu leu arg ala ala his val tyr ala gln lys val phe ser arg ile gly phe asp asn gln ala leu thr glu lys glu ala ile cys gly ile pro gly leu asp lys met glu gln asp thr ala pro gly leu pro tyr ala gln gln asn lys arg arg lys asp ile cys asp phe glu glu gly arg leu lys gly ala glu leu gln lys asp arg phe met ala gly asp tyr ser asn leu val tyr gln ser phe leu lys asp glu ile arg pro leu glu lys val arg ala gly lys thr arg leu ile asp val pro pro met pro his glu ala asn gly phe asp ile gly ser ala ile gly cys asp pro ala met arg val val leu asn tyr phe phe ser glu asp his gly phe asp pro gly val pro ala phe ile glu ser leu val asp ser val his ala tyr glu glu lys arg tyr asn ile tyr gly gly leu pro ser gly leu ala ala met met lys ala tyr glu asn phe glu pro asp asp ile gln val ile cys tyr gly asp asp cys leu ile ala ser asp phe glu pro val met asp val lys thr leu glu ala ile leu ser phe val arg his cys glu pro glu gln tyr glu arg leu phe glu pro phe ala gly met tyr phe val pro thr trp arg leu ala pro ala val val asp glu gln leu ala trp ala arg leu leu asn thr cys thr tyr phe phe ser thr lys thr thr asp ala trp gln leu glu gly gly gly asn ser val arg ile gln lys leu ala val ala gly met cys pro thr val val phe lys ile ala gly ser arg ser gln ala cys ala ser ala leu pro tyr thr ser met trp arg val val pro val phe tyr asn gly trp gly ala pro thr lys glu lys ala thr tyr asn trp leu pro gly ala his phe gly ser ile leu leu thr ser asp ala his asp lys gly gly cys tyr asp pro val thr arg leu gly pro thr leu ser arg his tyr thr phe lys val gly glu trp pro his ser gln ser his gly his ala trp ile cys pro leu pro gly asp lys leu lys lys met gly ser phe his glu gln val asn pro ser phe ala his ser gly pro leu cys val ala ala val pro glu tyr glu his thr his glu lys ala leu lys trp ser glu leu glu glu pro ala tyr thr tyr gln gln leu ser val phe pro his pro arg gln val pro gly arg phe thr asn phe ile asp val ala lys gln thr tyr ser phe cys ser ile ser gly lys pro tyr phe glu val thr asn thr ser gly asp glu pro leu phe gln met asp val ser leu phe ala gln tyr arg gly ser leu asn phe asn phe ile phe thr gly ser ala ala pro lys thr arg asp glu ala met ala cys ile his ala val trp asp val gly leu asn ser ala phe ser phe asn val pro tyr pro ser pro ala asp phe met ala val tyr ser ala glu arg thr val ser thr asp ile ala val asn ser lys gly arg val leu val ala val ser ile asp ala asp leu gly asp asn val ile ser pro glu gly gln ttgtactgac atg atg gcg gcg tct aag gtg tat aga gtt tgc gag cag 829 act ctg ctg gca ggt gcc gtt cgc atg atg gac aaa 865 met ala ala ser lys val tyr arg val cys glu gln thr leu leu ala met asn thr thr asp cys phe ile ala leu val gln ala ile arg glu ile lys ala leu phe leu ser arg thr thr gly lys met glu leu thr leu tyr asn gly glu lys lys thr phe tyr ser arg pro asn asn his asp asn cys trp leu asn ala ile leu gln leu phe arg tyr val glu glu pro phe phe asp trp val tyr ser ser pro glu asn leu thr leu thr gly ile gly thr ala ser arg pro ser glu val cys met val asp gly thr asp met cys leu ala asp phe his ala gly ile phe leu lys gly gln glu his ala val phe ala cys val thr ser asn gln trp tyr val leu val phe val pro tyr asp gln glu pro leu asn gly glu trp gly tyr ala thr ala glu asp phe val ser gly pro asn thr ser gly phe asp trp val thr ser asp ser phe gly arg cys his leu leu glu ala tyr met arg asn gly trp asp val glu val thr ala val gly asn gln phe asn gly gly cys leu leu val ala met val pro glu leu tyr ser ile gln lys arg glu leu tyr gln leu thr leu phe pro his gln val gly val asn arg tyr asp gln tyr lys val his lys pro trp thr phe thr asn leu leu asp val ala glu ala cys pro thr phe leu arg phe glu gly gly val pro tyr val thr thr lys thr asp ser asp arg ile asn leu his phe met phe thr gly pro thr asp ala lys ala arg glu ala ala ala his cys ile his ala glu trp asp thr gly leu asn cys leu phe gln ile thr his gly lys ala asp gly asp ala leu val val leu ala ser ala gly lys asp phe glu leu arg leu pro val asp his thr asp val ser phe ile met asp arg phe val lys val thr pro glu ile ala val lys his glu gly asp leu thr trp val pro asn gly arg thr leu pro thr ser phe asn tyr gly ala ile lys ala thr arg val thr glu leu leu tyr arg met lys arg ala glu thr tyr cys pro ile val ala pro val lys gln thr leu asn phe asp leu leu lys leu arg ser asn phe ser lys leu val glu thr ile asn gln met gln glu asp met ser thr lys his gly pro asp phe asn arg leu val ser ala phe glu glu leu ala ile gly val lys ala ile arg thr gly leu asp ile met leu ala asp thr gly leu glu ile leu asp ser thr phe val lys asn gly glu trp leu val lys leu ile leu ala ile arg asp trp ile lys ala trp ile ala ser glu glu lys phe val thr met thr asp leu val pro gly ile leu glu lys gln arg asp leu asn asp pro ser lys tyr lys glu ala lys glu trp leu asp asn ala arg gln ala cys ala ile ser thr his phe thr gly arg ile asp ser val trp tyr cys val met asp asp leu gly gln asn pro asp gly lys asp phe lys tyr leu glu asp lys gly lys pro phe asn ser lys val ile ile ala thr thr asn leu tyr ser gly phe thr pro arg thr met val cys pro asp ala leu asn arg arg phe his phe asp ile asp val ser ala lys asp thr his ala asn pro val ala met phe gln tyr asp cys ala leu leu asn gly met ala val glu met lys arg met gln gln asp met phe lys asp arg val glu leu his glu lys val ser ser his pro ile phe lys asp ser ile lys glu glu leu arg pro leu ile gln gln thr ser phe arg lys arg gln lys met val asp asp ala val asn glu tyr ile glu thr leu pro gly gln lys ala cys asp asp val asn ser glu pro ala glu gly pro tyr ala gly pro met glu arg gln lys pro leu lys val glu ser gly ala pro pro thr asp leu gln lys met val met gly asn thr lys pro val glu leu ile leu asp gly lys thr val ala ile cys cys ala thr gly val phe gly thr ala tyr leu val pro arg his leu phe ala glu lys tyr asp lys ile met val asp gly arg ala met thr asp ser asp tyr arg val phe glu phe glu ile lys val lys gly gln ile phe ser gly glu ala leu thr tyr lys asp ile val val cys met asp gly asp thr met pro gly leu phe ala tyr arg ala ala thr lys phe ile val gly thr his ser ala gly gly asn gly val gly tyr cys ser cys val ser arg ser met leu leu lys met lys ala his ile asp his gly val phe asn pro glu phe gly pro ala ala leu ser asn lys asp pro arg leu asn glu gly val val leu asp glu val ile phe ser lys his lys gly asp thr lys met ser glu glu asp lys ala leu phe thr ala asn ala pro leu ser ile tyr glu ala ile lys gly val asp leu gln gly lys arg arg gly ala leu ile asp phe glu asn gly thr tyr lys phe val cys gln thr phe leu lys asp glu ile arg pro leu glu lys val arg ala gly lys thr arg ile val asp val leu pro val glu his ile leu tyr thr arg met met ile gly arg phe cys ala gln met his ser asn asn gly pro gln ile gly ser ala val gly cys asn pro asp val asp trp gln arg phe gly thr his phe ala gln tyr arg asp ala met asn ile met phe glu glu val phe arg thr glu phe gly phe his pro asn ala glu trp ile leu lys thr leu val asn thr glu his ala tyr glu asn lys arg ile thr val gly gly gly met pro ser asp leu asp phe glu ala leu lys pro his phe lys ser leu gly gln thr ile thr pro ala asp lys ser asp lys gly phe val leu gly his ser ile thr asp val thr phe leu lys arg his phe his met asp tyr gly thr gly phe tyr lys pro val met ala ser lys thr leu glu ala ile leu ser phe ala arg arg gly thr ile gln glu lys leu ile ser val ala gly leu ala val his ser gly pro asp glu tyr arg arg leu met ala ala ser lys val tyr arg val cys glu gln thr leu leu ala gly ala val arg met met asp lys phe leu gln lys arg thr val phe val pro his leu asp lys thr ile arg leu thr gly leu his asn tyr arg lys thr ile asp trp val ser asp gln thr gly ile lys asp leu lys ser gly ala pro pro leu val val val tyr lys leu trp gln his gly his leu asp val gly thr met glu lys pro arg ser ile thr leu trp ser gly pro lys val cys leu ser asp phe trp ala cys val ser ala lys pro gly his ala val phe tyr leu leu thr ser glu gly trp asp val leu val phe ala pro tyr asp phe glu ser leu gly lys asp pro pro lys leu his gln arg tyr glu lys ala phe glu leu ser gly ile asp ala asp leu gly asp asn val ile ser pro glu gly gln gly pro val thr arg leu gly pro thr leu ser arg his tyr thr phe lys val gly glu trp pro his ser gln ser his gly his ala trp ile cys pro leu pro gly asp lys leu lys lys met gly ser phe his glu val glu glu pro ala tyr thr tyr gln gln leu ser val phe pro his gln ile asp val ala lys gln thr tyr ser phe cys ser ile ser gly lys pro tyr phe glu val thr asn thr ser gly asp glu pro leu phe gln met asp val ser leu ser ala ala glu leu his gly thr tyr val ala phe val pro pro his ser ala ala pro lys thr arg asp glu ala met ala cys ile his ala val trp asp val gly leu asn ser ala phe ser ala glu arg thr val val asn val ser gly trp leu gln val tyr ala leu thr ala leu thr ser thr asp ile ala val asn ser lys gly arg ala asp leu pro asp lys gln val thr asn val gly glu asp gly glu pro gly glu thr glu pro arg his ala leu ser pro val asp met his asp pro phe gly ser thr ala gln leu ala trp ala arg leu leu asn thr cys thr tyr phe phe ser asp leu glu leu ser ile gln phe lys glu gly gly gly asn ser val arg ile gln lys leu ala val ala gly met cys pro thr val val phe lys ile ala gly ser arg ser gln ala cys ala ser ala leu pro tyr thr ser met trp arg val val pro val 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