Patent Application: US-72669203-A

Abstract:
recombinant polypeptides are disclosed that are useful for diagnosing american trypanosomiasis , or chagas disease , a disease caused by the infectious agent trypanosoma cruzi . preferably , dna sequences encoding the recombinant proteins are placed in plasmid vectors to be expressed in an organism .

Description:
[ 0020 ] fig1 a - 1 h represent the recombinant proteins of the invention , with the various letters indicating known protein sequences , as follows . the figs . are schematic diagrams of the recombinant t . cruzi proteins , comprised of segments a through l . solid segments ( a , c , d , f , h , i , and k ) represent nonrepetitive proteins having amino acid sequences that are unrelated to each other . saw - tooth segments ( b , e , g , j , and l ) represent repetitive proteins having amino acid sequences that are unrelated to each other and unrelated to those of the nonrepetitive proteins . the relative sizes and numbers of repeats in the repetitive proteins are roughly represented in the figs . the sizes and shapes of the nonrepetitive segments bear no relation to the actual proteins . the following information refers to fig1 and 1 a - 1 h in which the recombinant proteins ag15 , fp3 , fp4 , fp5 , fp6 , fp7 , fp8 , fp9 and fp10 are depicted schematically . these proteins are derived from t . cruzi , the protozoan parasite that causes chagas disease , and are formed from of proteins a through l as indicated , and defined herein . there are no substantive amino acid similarities among proteins a through l . similarly there are no substantive dna sequence similarities among the segments that encode proteins a through l . the t . cruzi dna sequences that encode proteins a through l were cloned in combination into pgex and pet plasmid vectors , such as pet - 32a . strains of escherichia coli were transfected with the recombinant vectors bearing the t . cruzi dna sequences , and the bacteria were incubated in liquid culture under conditions favoring synthesis of the recombinant proteins . the latter proteins were subsequently affinity - purified and then used as target antigens in elisas . elisas in which proteins ag15 , fp3 , fp4 , fp5 , fp6 , fp7 , fp8 , fp9 , and fp10 , alone or in combination are employed as target antigens are useful as sensitive and specific detectors of anti - t . cruzi antibodies in blood specimens obtained from persons who are chronically infected with this parasite . the detection of such antibodies is the primary means of identifying persons who are chronically infected with t . cruzi . the following paragraphs contain information relating to the naming , localization , and function of proteins a through l , as well as the corresponding genbank accession numbers of the sequences to which they are related and relevant publications . it should be noted that the t . cruzi gene segments that encode protein segments a through l generally are shortened versions of the native coding regions . in this context , the constructs that encode single segments ( i . e ., fp5 and fp9 ), as well as all the others that encode more than one segment , are all unique , because , even if the individual components from which the various recombinant proteins of this invention are known , the segments of the invention have not been combined previously as described herein . protein ab . this hybrid recombinant protein , also designated ag15 [ seq id no . 2 ] in fig1 is derived from the tcr27 gene of t . cruzi [ seq id no . 1 ]. protein a is the amino terminal nonrepetitive portion of the tcr27 protein , and protein b is comprised of approximately 18 of the 14 amino acid repeats that make up the central portion of the tcr27 protein . the two native tcr27 genes sequenced contained approximately 69 and 105 of the 14 - amino acid repeats . nucleotide sequence data that include the ag15 dna sequence were deposited with genbank and embl databases by keiko otsu , john e . donelson , and louis v . kirchhoff with the accession number l04603 and are described in u . s . pat . no . 5 , 876 , 734 and no . 6 , 228 , 601 , issued to louis v . kirchhoff and keiko otsu ( each of which is herein incorporated by reference in its entirety ). these references also present dna and inferred protein sequences that include the ag15 dna and inferred protein sequences . the ag15 dna and inferred protein sequences are additionally presented in otsu k , donelson j e , kirchhoff l v . “ interruption of a trypanosoma cruzi gene encoding a protein containing 14 - amino acid repeats by targeted insertion of the neomycin phosphotransferase gene .” mol biochem parasitol 1993 ; 57 : 317 - 330 , herein incorporated by reference in its entirety . protein c . this is a calcium binding protein of t . cruzi , initially called 1f8 and later designated the flagellar calcium binding protein ( fcabp ) [ seq id no 4 ]. the accession number of the original 1 f8 dna sequence [ seq id no 3 ] deposited in genbank is k03278 . the protein c dna and inferred protein sequences are presented in gonzalez a , lemer t j , huecas m , sosa - pineda b , nogueira n , lizardi p m . “ apparent generation of a segmented mrna from two separate tandem gene families in trypanosoma cruzi .” nucleic acids res 1985 ; 13 ( 16 ): 5789 - 804 , herein incorporated by reference in its entirety . [ 0027 ] fig1 a shows a first protein ( fp3 ) [ seq id no . 22 ] in accordance with the invention . specifically , fp3 corresponds essentially to the combination of ag15 ( fig1 ), and by protein c . the dna sequence encoding fp3 [ seq id no 21 ], also essentially corresponds to the sequences coding for ag15 and protein c . protein d . this is the protein core of a surface glycoprotein of t . cruzi that is referred to as gp72 [ seq id no 6 ]. the accession number of the original gp72 dna sequence [ seq id no 5 ] deposited in genbank is m65021 . the protein d dna and inferred protein sequences are presented in cooper r , inverso j a , espinosa m , nogueira n , cross g a . “ characterization of a candidate gene for gp72 , an insect stage - specific antigen of trypanosoma cruzi .” mol biochem parasitol 1991 ; 49 ( 1 ): 45 - 59 , herein incorporated by reference in its entirety . [ 0029 ] fig1 b shows a second protein ( fp4 ) [ seq id no 8 ] in accordance with the invention . the dna sequence [ seq id no 7 ] that encodes protein dabc which is a single continuous coding region , essentially corresponds to the dna sequences from which it was constructed . protein e . this is a segment of the flagellar repetitive protein ( fra ) [ seq id no 10 ] of t . cruzi comprised of approximately nine repeats consisting of 68 amino acids each , shown as fig1 c ( fp5 ). the accession number of the original protein e dna sequence [ seq id no 9 ] deposited in genbank is j04015 . the protein e dna and inferred protein sequences are presented in lafaille j j , linss j , krieger m a , souto - padron t , de souza w , goldenberg s . “ structure and expression of two trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes .” mol biochem parasitol 1989 ; 35 ( 2 ): 127 - 136 , herein incorporated by reference in its entirety . protein fgh . this is a protein [ seq id no 12 ] encoded by a modified version of the t . cruzi tcr39 gene that was artificially constructed [ seq id no 11 ], shown as fig1 e ( fp7 ). the modification entailed reducing the length of the central portion of the tcr39 gene that encodes the 12 - amino acid repeats . protein f is the amino terminal nonrepetitive segment of the tcr39 protein . protein g is comprised of approximately 13 of the 12 - amino acid repeats that make up the central portion of the tcr39 protein . protein h is the carboxy terminal nonrepetitive segment of the tcr39 protein . the accession number of the original , i . e ., the unmodified , protein fgh dna sequence deposited in genbank is u15616 . the tcr39 dna and inferred protein sequences , which include the entire protein fgh sequences , are presented in gruber a , zingales b . “ trypanosoma cruzi : characterization of two recombinant antigens with potential application in the diagnosis of chagas &# 39 ; disease .” exp parasitol 1993 ; 76 ( 1 ): 1 - 12 , herein incorporated by reference in its entirety . [ 0032 ] fig1 d shows another hybrid recombinant protein ( fp6 , protein fghe ) [ seq id no 14 ] in accordance with the invention . the dna sequence that encodes protein fghe [ seq id no 13 ], which is a single continuous coding region , essentially corresponds to the dna sequences from which it was constructed . protein ijk . this is a protein [ seq id no 16 ] encoded by a modified version of the t . cruzi shed acute phase antigen ( sapa ) gene that was artificially constructed [ seq id no 15 ], as shown in fig1 f ( fp8 ). the modification entailed reducing the length of the central portion of the sapa gene that consists of 12 - amino acid repeats . protein i is the amino terminal nonrepetitive segment of the sapa protein . protein j is comprised of approximately nine of the 12 - amino acid repeats that make up the central portion of the sapa protein . protein k is the carboxy terminal nonrepetitive segment of the sapa protein . the accession number of the original , i . e ., the unmodified , protein ijk dna sequence deposited in gen bank is j03985 . the sapa dna and protein sequences , which include the entire protein ijk sequences , are presented in affranchino j l , pollevick g d , frasch a c c . “ the expression of the major shed trypanosoma cruzi antigen results from the developmentally - regulated transcription of a small gene family .” febs lett 1991 ; 280 : 316 - 320 , herein incorporated by reference in its entirety . protein l . this is a microtubule - associated repetitive protein ( map ) [ seq id no 18 ] of t . cruzi that is comprised of approximately five repeats consisting of 38 amino acids each , as depicted in fig1 g ( fp9 ). the accession number of the original protein l dna sequence [ seq id no 17 ] deposited in genbank is s68286 . the protein l dna and inferred protein sequences are presented in kemer n , liegeard p , levin m j , hontebeyrie - joskowicz m . “ trypanosoma cruzi : antibodies to a map - like protein in chronic chagas &# 39 ; disease cross - react with mammalian cytoskeleton .” experimental parasitology 1991 ; 73 ( 4 ): 451 - 459 , herein incorporated by reference in its entirety . [ 0035 ] fig1 h shows another hybrid recombinant protein ( fp10 , protein ijkl ) [ seq id no 20 ] in accordance with the invention . the dna sequence that encodes protein ijkl [ seq id no 19 ], which is a single continuous coding region , essentially corresponds to the dna sequences from which it was constructed . additionally , combinations of the various recombinant proteins depicted in the figs . may be used . while it is possible to combine one or more of the recombinant proteins to form longer recombinant proteins , typically more than one recombinant protein is used simultaneously . for example , simultaneous uses of fp4 and fp5 , fp5 and fp6 , as well as fp4 and fp6 , and combinations using more than two recombinant proteins ( e . g ., fp4 , fp6 and fp10 ) are considered within the scope of the present invention . it is believed that the sensitivity and specificity of the assays according to the invention are sufficient to meet fda standards for screening the blood supply of the united states . additionally , as described in u . s . pat . no . 6 , 228 , 601 ( herein incorporated by reference in its entirety ), polypeptides need not correspond exactly over their entire lengths to be considered within the scope of the invention . for example , a wide variety of polypeptides which contain at least one epitope embodied in the polypeptides of the invention can be used in accordance with the present invention . based on the nucleotide sequences , polypeptide molecules also can be produced ( 1 ) that include sequence variations , relative to the naturally - occurring sequences , ( 2 ) that have one or more amino acids truncated from the naturally - occurring sequences and variations thereof , or ( 3 ) that contain the naturally - occurring sequences and variations thereof as part of a longer sequence . in this description , polypeptide molecules in categories ( 1 ), ( 2 ) and ( 3 ) are said to “ correspond ” to the amino acid sequences of the recombinant proteins of the invention . such polypeptides also are referred to as “ variants .” the category of variants within the present invention includes , for example , fragments and muteins of proteins a though l , as well as larger molecules that consist essentially at least one protein sequence a through l , alone or in combination with other proteins a to l . in this regard , a molecule that “ consists essentially of ” protein a to l , alone or in combination with any other proteins a to l , is one that is immunoreactive with samples from persons infected with t . cruzi , but that does not react with samples from patients with leishmaniasis , schistosomiasis , and other parasitic and infectious diseases , with samples from patients with autoimmune disorders and other illnesses , and with specimens from normal persons . a “ mutein ” is a polypeptide that is homologous to the protein to which it corresponds , and that retains the basic functional attribute — the ability to react selectively with samples from persons infected with t . cruzi — of the corresponding region . for purposes of this description , “ homology ” between two sequences connotes a likeness short of identity indicative of a derivation of the first sequence from the second . in particular , a polypeptide is “ homologous ” to the corresponding protein if a comparison of amino acid sequences between the polypeptide and the corresponding region reveals an identity of greater than 40 %, preferably greater than 50 % and more preferably 70 %. such sequence comparisons can be performed via known algorithms , such as those described in pearson w r , lipman d j . “ improved tools for biological sequence comparison .” proc natl acad sci usa 1988 ; 85 ( 8 ): 2444 - 2448 , herein incorporated by reference in its entirety , which are readily implemented by computer . a fragment of a protein of the invention is a molecule in which one or more amino acids are truncated from that protein . muteins and fragments can be produced , in accordance with the present invention , by known de novo synthesis techniques . also exemplary of variants within the present invention are molecules that are longer than a protein of the invention , but that contain the region or a mutein thereof within the longer sequence . for example , a variant may include a futher fusion partner in addition to the protein of the invention . such a fusion partner may allow easier purification of recombinantly - produced polypeptides . for example , use of a glutathione - s - transferase ( 26 kilodaltons , gst ) fusion partner allows purification of recombinant polypeptides on glutathione agarose beads . the portion of the sequence of a such molecule other than that portion of the sequence corresponding to the region may or may not be homologous to the sequence of a protein of the invention . it will be appreciated that polypeptides shorter than the corresponding protein of the invention but that retain the ability to react selectively with samples from persons infected with t . cruzi are suitable for use in the present invention . thus , variants may be of the same length , longer than or shorter than the protein of the invention , and also include sequences in which there are amino acid substitutions of the parent sequence . these variants must retain the ability to react selectively with samples from persons infected with t . cruzi . in one embodiment , the assay of the invention uses fp4 as target antigen . table ii compares the results obtained by testing 45 pre - screened argentinean specimens in an table ii ripa + − fp4 elisa + 9 0 − 0 36 the data in table ii show that in this group of specimens , the sensitivity and specificity of the fp4 elisa were both 100 % similarly , the performance of an fp4 + fp6 elisa in comparison to ripa was table iii ripa + − fp4 + fp6 elisa + 10 1 − 0 78 the data shown in table iii indicate that in this group of samples , the sensitivity of the fp4 + fp6 elisa was 100 % and the specificity was 98 . 7 %. as shown in fig2 in a fp4 + fp6 elisa , performed using standard procedures , a group of previously characterized ripa - positive samples from several chagas - endemic countries gave a mean reactivity ( absorbance ) of 2 . 99 . thus fp4 + fp6 is the preferred embodiment among the recombinant proteins tested alone and in combination in that experiment . it should be apparent that embodiments other than those specifically described above may come within the spirit and scope of the present invention , such as recombinant proteins comprised of different combinations and / or spatial arrangements of proteins a to l . hence , the present invention is not limited by the above description . tat ggc ccg agc tgt ggt gct tga gga tgg agc gct tta cgt ggc gga 48 caa tgc caa caa cct cgt tcg aga aat ctc caa tgg cgt tgt cac ttc 96 gtt tat tac gga agg act gct ggg ccc atc gta cat caa acc gta cag 144 ccg tac aaa tgg cgc tca tga ctt gtt tgt gtc gga cac ggg caa atc 192 pro tyr lys trp arg ser leu val cys val gly his gly gln ile acg cat cat ttt tgc ccc acc tca gaa aaa aac gtt cat cac agt gtt 240 tat aac agg att cca gcc gga tgt tct tca aat tag cga gaa gag tcg 288 tyr asn arg ile pro ala gly cys ser ser asn arg glu glu ser ttt gat gtt tgc cat ctg caa ttc cac gaa aat tct tgc gat taa tat 336 phe asp val cys his leu gln phe his glu asn ser cys asp tyr gca ggg agc cac aac ccc gaa gga gta ctg gca agt tgg aaa tgc gga 384 ala gly ser his asn pro glu gly val leu ala ser trp lys cys gly ctg cat ggg cta tca gag ttc cct cat gct cac gac cga gga gga taa 432 act cct cta cta cgg cat att aaa tgg aac ccc atc cat cat gtc ttt 480 thr pro leu leu arg his ile lys trp asn pro ile his his val phe acc cgc cac caa aac gaa gac gga agc acc cag aat ttg ccc gga tgt 528 thr arg his gln asn glu asp gly ser thr gln asn leu pro gly cys gtt gtt gca gtg gcc aca tgg gcc cat tgt ttc gct tgt gaa tat taa 576 caa aca tgc att tta cgt tgt tac cgc ctc caa tgt ata cat tgt aca 624 tga tgg ctc gta tca tcc gac tgg atc cat ggc cca gct cca aca ggc 672 trp leu val ser ser asp trp ile his gly pro ala pro thr gly aga aaa taa tat cac taa ttc caa aaa aga aat gac aaa gct acg aga 720 arg lys tyr his phe gln lys arg asn asp lys ala thr arg aaa agt gaa aaa ggc cga gaa aga aaa att gga cgc cat taa ccg ggc 768 aac caa gct gga aga gga acg aaa cca agc gta caa agc agc aca caa 816 ggc aga gga gga aaa ggc taa aac att tca acg cct tat aac att tga 864 gtc gga aaa tat taa ctt aaa gaa aag gcc aaa tga cgc agt ttc aaa 912 val gly lys tyr leu lys glu lys ala lys arg ser phe lys tcg gga taa gaa aaa aaa ttc tga aac cgc aaa aac tga cga agt aga 960 gaa aca gag ggc ggc tga ggc tgc caa ggc cgt gga gac gga gaa gca 1008 gag ggc agc tga ggc cac gaa ggt tgc cga agc gga gaa gcg gaa ggc 1056 agc tga ggc cgc caa ggc cgt gga gac gga gaa gca gag ggc agc tga 1104 agc cac gaa ggt tgc cga agc gga gaa gca gaa ggc agc tga ggc cgc 1152 caa ggc cgt gga gac gga gaa gca gag ggc agc tga agc cac gaa ggt 1200 tgc cga agc gga gaa gca gag ggc agc tga agc cat gaa ggt tgc cga 1248 agc gga gaa gca gaa ggc agc tga ggc cgc caa ggc cgt gga gac gga 1296 gaa gca gag ggc agc tga agc cac gaa ggt tgc cga agc gga gaa gca 1344 gaa ggc agc tga ggc cgc caa ggc cgt gga gac gga gaa gca gag ggc 1392 agc tga agc cac gaa ggt tgc cga agc gga gaa gca gaa ggc agc tga 1440 ggc cgc caa ggc cgt gga gac gga gaa gca gag ggc agc tga agc cac 1488 asn leu gln trp arg cys his phe val tyr tyr gly arg thr ala gly pro ile val his gln thr val gln pro tyr lys trp arg ser ser glu lys asn val his his ser val tyr asn arg ile pro ala gly arg glu glu ser phe asp val cys his leu gln phe his glu asn ser tyr ala gly ser his asn pro glu gly val leu ala ser trp lys cys thr pro leu leu arg his ile lys trp asn pro ile his his val phe thr arg his gln asn glu asp gly ser thr gln asn leu pro gly cys trp leu val ser ser asp trp ile his gly pro ala pro thr gly arg phe gln lys arg asn asp lys ala thr arg lys ser glu lys gly arg tca aaa agg acg tcg cca gca ccc ttc cgt gcg ctc ctg ctg ccg gtc 886 gcg cag ttt gat tta agg cag cag cag ctg gtt ata cag gat ttc ttc 982 atc agt cgc tcc tgc gca gga tgt tca cag ggg caa acc gat ggc cca 1030 agc ggt gcc ggc aca ctc ttc act gcc gcc ggt ggt tcg ctt ggc aaa 1078 gat gct tcc acg ctg ctg ttg tgt gac caa ggt ggt ggt ggc tcc agc 1126 gtg cgt ttg gtg aac aaa tcc ggc att ttc acc ctt gcc ggt agt aaa 1174 val arg leu val asn lys ser gly ile phe thr leu ala gly ser lys acg acg cgt ggc aat caa aat ggt ccg gcg gcg acg gca ctc ttc aac 1222 atg ccc cga gct gtg gtg ctt gag gat gga gcg ctt tac gtg gcg gac 1270 agt gcc aac aac ctc gtt cga gaa atc tcc aat ggc att gtc act tcg 1318 ttt att acg gag gga ctg ctg ggc cca tcg tac atc aaa ccg tac agc 1366 cgt cca aat ggc gcc cat gac ttg ttt gtg tcg gac acg ggc aaa tct 1414 arg pro asn gly ala his asp leu phe val ser asp thr gly lys ser cgc atc att ttt gcc cca ctt cag aaa caa acg ttc atc aca gtg ttt 1462 ata aca gga ttc cag ccg gat gtt ctt caa att agc gag aag agt cgt 1510 ile thr gly phe gln pro asp val leu gln ile ser glu lys ser arg ttg atg ttt gcc atc tgc aat tcc acg aaa att ctt tcg att aat atg 1558 cag gga gcc aca acc ccg aag gat tac tgg caa gtt gga aat gcg gac 1606 gln gly ala thr thr pro lys asp tyr trp gln val gly asn ala asp tgc atg ggc tat cag agt tct ctc atg ctc acg acc gag gag gat aaa 1654 cys met gly tyr gln ser ser leu met leu thr thr glu glu asp lys ctc ctc tac tac ggc ata tta aat gga acc cca tcc atc atg tct tta 1702 ccc gcc acc aaa acg aag acg gaa gca ccc aga att tgc ccg gat gtg 1750 ttg ttg cgg tgg cca cat ggg ccc att gtt tcg ctt gtg aat att aac 1798 aaa cat gca ttt tac gtt gtt acc gcc tcc aat gta tac att gta cat 1846 gat ggc tct tat cat ccg act gtg acg ccg aca cct cct ctg aca ccg 1894 acg cct aca cca gaa gtg aca ccc aca cct act gtg acc ccg acg cct 1942 aca ccg gaa gtg aca ccg aca ccg cca gtg act ccg agc ccc acc atc 1990 aca atc cac cgg ggt ttt gct gtg gca gcc ttt cct gcc caa agt ctt 2038 thr ile his arg gly phe ala val ala ala phe pro ala gln ser leu cca atc gaa gac ccg cgg ctt atg cat gaa ctg ctt tct tgg tta atg 2086 aag gat gta ggg att gcg ttc gaa tcc acg gac ttt ttt gcc gta ttt 2134 cct cca gat aga gag gtt ttg gtg ccc ggt tat gta aat gtc tcc acc 2182 pro pro asp arg glu val leu val pro gly tyr val asn val ser thr tgg aat aac ttg acg gtg cta ttc aac ttt gac cgc acc att gtc atc 2230 acg gaa tat ttc act cca gag ggc atg tct tca gag gag gga cag gcc 2278 cga ctc ttc gct tcg ccg tgg tac tgg acg aga aat ttc ctt gat tca 2326 arg leu phe ala ser pro trp tyr trp thr arg asn phe leu asp ser tta aag aaa aca gta gct tgg aag gac ttg gag gcg ttt tgc atg gtc 2374 leu lys lys thr val ala trp lys asp leu glu ala phe cys met val aac tgt gtt gaa cac tgt gag aca atg aca ttc cat aag tca gaa tgt 2422 gta ggc tac gtc cgg ccc cca gta tgc aac gac gtc tgt gtg ggg gcg 2470 gta gtg tcc tcc gtg gtg ctt ggc gcc aca ggt atc gca ctc att gca 2518 ctg atg gtt gga agt tcg gcg aac tta cgg agc gct gtg att ctt gtt 2566 ser ser val arg leu val asn lys ser gly ile phe thr leu ala gly phe asn met pro arg ala val val leu glu asp gly ala leu tyr val tyr ser arg pro asn gly ala his asp leu phe val ser asp thr gly val phe ile thr gly phe gln pro asp val leu gln ile ser glu lys ser arg leu met phe ala ile cys asn ser thr lys ile leu ser ile asn met gln gly ala thr thr pro lys asp tyr trp gln val gly asn ala asp cys met gly tyr gln ser ser leu met leu thr thr glu glu asp lys leu leu tyr tyr gly ile leu asn gly thr pro ser ile met asp val leu leu arg trp pro his gly pro ile val ser leu val asn leu met lys asp val gly ile ala phe glu ser thr asp phe phe ala gln ala arg leu phe ala ser pro trp tyr trp thr arg asn phe leu asp ser leu lys lys thr val ala trp lys asp leu glu ala phe cys atg gcc cga gct gtg gtg ctt gag gat gga gcg ctt tac gtg gcg gac 48 aat gcc aac aac ctc gtt cga gaa atc tcc aat ggc gtt gtc act tcg 96 ttt att acg gaa gga ctg ctg ggc cca tcg tac atc aaa ccg tac agc 144 cgt aca aat ggc gct cat gac ttg ttt gtg tcg gac acg ggc aaa tca 192 arg thr asn gly ala his asp leu phe val ser asp thr gly lys ser cgc atc att ttt gcc cca cct cag aaa aaa acg ttc atc aca gtg ttt 240 ata aca gga ttc cag ccg gat gtt ctt caa att agc gag aag agt cgt 288 ile thr gly phe gln pro asp val leu gln ile ser glu lys ser arg ttg atg ttt gcc atc tgc aat tcc acg aaa att ctt gcg att aat atg 336 cag gga gcc aca acc ccg aag gag tac tgg caa gtt gga aat gcg gac 384 gln gly ala thr thr pro lys glu tyr trp gln val gly asn ala asp tgc atg ggc tat cag agt tcc ctc atg ctc acg acc gag gag gat aaa 432 cys met gly tyr gln ser ser leu met leu thr thr glu glu asp lys ctc ctc tac tac ggc ata tta aat gga acc cca tcc atc atg tct tta 480 ccc gcc acc aaa acg aag acg gaa gca ccc aga att tgc ccg gat gtg 528 ttg ttg cag tgg cca cat ggg ccc att gtt tcg ctt gtg aat att aac 576 aaa cat gca ttt tac gtt gtt acc gcc tcc aat gta tac att gta cat 624 gat ggc tcg tat cat ccg act gga tcc atg gcc cag ctc caa cag gca 672 asp gly ser tyr his pro thr gly ser met ala gln leu gln gln ala gaa aat aat atc act aat tcc aaa aaa gaa atg aca aag cta cga gaa 720 aaa gtg aaa aag gcc gag aaa gaa aaa ttg gac gcc att aac cgg gca 768 acc aag ctg gaa gag gaa cga aac caa gcg tac aaa gca gca cac aag 816 gca gag gag gaa aag gct aaa aca ttt caa cgc ctt ata aca ttt gag 864 tcg gaa aat att aac tta aag aaa agg cca aat gac gca gtt tca aat 912 ser glu asn ile asn leu lys lys arg pro asn asp ala val ser asn cgg gat aag aaa aaa aat tct gaa acc gca aaa act gac gaa gta gag 960 aaa cag agg gcg gct gag gct gcc aag gcc gtg gag acg gag aag cag 1008 agg gca gct gag gcc acg aag gtt gcc gaa gcg gag aag cgg aag gca 1056 gct gag gcc gcc aag gcc gtg gag acg gag aag cag agg gca gct gaa 1104 gcc acg aag gtt gcc gaa gcg gag aag cag aag gca gct gag gcc gcc 1152 aag gcc gtg gag acg gag aag cag agg gca gct gaa gcc acg aag gtt 1200 gcc gaa gcg gag aag cag agg gca gct gaa gcc atg aag gtt gcc gaa 1248 gcg gag aag cag aag gca gct gag gcc gcc aag gcc gtg gag acg gag 1296 aag cag agg gca gct gaa gcc acg aag gtt gcc gaa gcg gag aag cag 1344 aag gca gct gag gcc gcc aag gcc gtg gag acg gag aag cag agg gca 1392 gct gaa gcc acg aag gtt gcc gaa gcg gag aag cag aag gca gct gag 1440 gcc gcc aag gcc gtg gag acg gag aag cag agg gca gct gaa gcc acg 1488 aag gtt gcc gaa gcg gag aag gat atc gat ccc atg ggt gct tgt ggg 1536 tcg aag gac tcg acg agc gac aag ggg ttg gcg agc gat aag gac ggc 1584 aag aac gcc aag gac cgc aag gaa gcg tgg gag cgc att cgc cag gcg 1632 att cct cgt gag aag acc gcc gag gca aaa cag cgc cgc atc gag ctc 1680 ttc aag aag ttc gac aag aac gag acc ggg aag ctg tgc tac gat gag 1728 gtg cac agc ggc tgc ctc gag gtg ctg aag ttg gac gag ttc acg ccg 1776 val his ser gly cys leu glu val leu lys leu asp glu phe thr pro cga gtg cgc gac atc acg aag cgt gca ttc gac aag gcg agg gcc ctg 1824 ggc agc aag ctg gag aac aag ggc tcc gag gac ttt gtt gaa ttt ctg 1872 gag ttc cgt ctg atg ctg tgc tac atc tac gac ttc ttc gag ctg acg 1920 gtg atg ttc gac gag att gac gcc tcc ggc aac atg ctg gtt gac gag 1968 val met phe asp glu 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aaa 144 cca tca cca ttt gga cag gcc gca gca ggt gac aaa cca cca cca ttt 192 gga cag gcc gca gca ggt gac aaa cca cca cca ttt gga cag gcc gca 240 gca ggt gac aaa cca tca cta ttt gga cag gcc gca gca ggt gac aaa 288 cca tca cca ttt gga cag gcc gca gca ggt gac aaa cca cca cca ttt 336 gga cag gcc gca gca ggt gac aaa cca tca cta ttt gga cag gcc gca 384 gca ggt gac aaa cca tca cca ttt gga cag gcc gca gca ggt gac aaa 432 cca cca cca ttt gga cag gcc gca gca ggt gac aaa cca cca cca ttt 480 gga cag gcc gca gca ggt gac aaa cca tca cta ttt gga cag gcc gca 528 gca ggt gac aaa cca tca cca ttt gga cag gga act gcg ttt gat gcc 576 tct cga agc act gtg ttt gcg aat gcg cct ggt gtt gcc cag gtg 621 ttt aat cct tct acg gac aaa ttg aag cta aac caa caa aat aag cct 48 at att gca aat aat aaa caa aaa aca aca ctc gaa aaa act caa aca 96 gaa caa aaa aca gcg cca ttt gga cag ggc gca gca ggg tgg aca aaa 144 cca tca cca ttt gga cag gcc gca gca ggt gac aaa cca cca cca ttt 192 gga cag gcc gca gca ggt gac aaa cca cca cca ttt 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ctt ctt tac aac cgt cag ctg aat gcc gag 432 gag atc agg acc ttg ttc ttg agc cag gac ctg att ggc acg gaa gca 480 glu ile arg thr leu phe leu ser gln asp leu ile gly thr glu ala cac atg ggc agc agc agc ggc agc agt gcc cac ggt acg ccc tcg att 528 ccc gtt gac agc agt gcc cac ggt aca ccc tcg act ccc gtt gac agc 576 agt gcc cac ggt acg ccc tcg act ccc gtt gac agc agt gcc cac ggt 624 aca ccc tcg act ccc gtt gac agc agt gcc cac ggt aca ccc tcg act 672 ccc gtt gac agc agt gcc cac ggt aag ccc tcg act ccc gct gac agc 720 agt gcc cac agt acg ccc tcg act ccc gct gac agc agt gcc cac agt 768 acg ccc tca att ccc gct gac agc agt gcc cac agt acg ccc tca gct 816 ccc gct gac aac ggc gcc aat ggt acg gtt ttg att ttg tcg 858 asp pro thr tyr arg phe ala asn his ala phe thr leu val ala ser glu lys his gln trp gln pro ile tyr gly ser thr pro val thr pro thr gly ser trp glu met gly lys arg tyr his val val leu thr met ala asn lys ile gly ser val tyr ile asp gly glu pro leu glu gly ser gly gln thr val val pro asp 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ggg ccg cgc cac gtt gat ccc gac cac 576 ttc cgc tcg acg act cat gac gcg tac agg ccc gtt gat ccc tcg gcg 624 tac aag cgc gcc ttg ccg cag gaa gag caa gag gat gtg ggg ccg cgc 672 tyr lys arg ala leu pro gln glu glu gln glu asp val gly pro arg cac gtt gat ccc gac cac ttc cgc tcg ac 701 ser ala tyr lys arg ala leu pro gln glu glu gln glu asp val gly tyr lys arg ala leu pro gln glu glu gln glu asp val gly pro arg gat cca acg tat cgt ttt gca aac cac gcg ttc acg ctg gtg gcg tcg 48 asp pro thr tyr arg phe ala asn his ala phe thr leu val ala ser gtg acg att cac gag gtt ccg agc gtc gcg agt cct ttg ctg ggt gcg 96 agc ctg gac tct tct ggt ggc aaa aaa ctc ctg ggg ctc tcg tac gac 144 gag aag cac cag tgg cag cca ata tac gga tca acg ccg gtg acg ccg 192 glu lys his gln trp gln pro ile tyr gly ser thr pro val thr pro acc gga tcg tgg gag atg ggt aag agg tac cac gtg gtt ctt acg atg 240 thr gly ser trp glu met gly lys arg tyr his val val leu thr met gcg aat aaa att ggc tcc gtg tac att gat gga gaa cct ctg gag ggt 288 ala asn lys ile gly ser val tyr ile asp gly glu pro leu glu gly tca ggg cag acc gtt gtg cca gac gag agg acg cct gac atc tcc cac 336 ser gly gln thr val val pro asp glu arg thr pro asp ile ser his ttc tac gtt ggc ggg tat gga agg agt gat atg cca acc ata agc cac 384 phe tyr val gly gly tyr gly arg ser asp met pro thr ile ser his gtg acg gtg aat aat gtt ctt ctt tac aac cgt cag ctg aat gcc gag 432 gag atc agg acc ttg ttc ttg agc cag gac ctg att ggc acg gaa gca 480 glu ile arg thr leu phe leu ser gln asp leu ile gly thr glu ala cac atg ggc agc agc agc ggc agc agt gcc cac ggt acg ccc tcg att 528 ccc gtt gac agc agt gcc cac ggt aca ccc tcg act ccc gtt gac agc 576 agt gcc cac ggt acg ccc tcg act ccc gtt gac agc agt gcc cac ggt 624 aca ccc tcg act ccc gtt gac agc agt gcc cac ggt aca ccc tcg act 672 ccc gtt gac agc agt gcc cac ggt aag ccc tcg act ccc gct gac agc 720 agt gcc cac agt acg ccc tcg act ccc gct gac agc agt gcc cac agt 768 acg ccc tca att ccc gct gac agc agt gcc cac agt acg ccc 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glu tyr lys arg ala leu pro gln glu glu gln glu asp val gly pro arg tyr arg pro val asp pro ser ala tyr lys arg ala leu pro gln glu atg gcc cga gct gtg gtg ctt gag gat gga gcg ctt tac gtg gcg gac 48 aat gcc aac aac ctc gtt cga gaa atc tcc aat ggc gtt gtc act tcg 96 ttt att acg gaa gga ctg ctg ggc cca tcg tac atc aaa ccg tac agc 144 cgt aca aat ggc gct cat gac ttg ttt gtg tcg gac acg ggc aaa tca 192 arg thr asn gly ala his asp leu phe val ser asp thr gly lys ser cgc atc att ttt gcc cca cct cag aaa aaa acg ttc atc aca gtg ttt 240 ata aca gga ttc cag ccg gat gtt ctt caa att agc gag aag agt cgt 288 ile thr gly phe gln pro asp val leu gln ile ser glu lys ser arg ttg atg ttt gcc atc tgc aat tcc acg aaa att ctt gcg att aat atg 336 cag gga gcc aca acc ccg aag gag tac tgg caa gtt gga aat gcg gac 384 gln gly ala thr thr pro lys glu tyr trp gln val gly asn ala asp tgc atg ggc tat cag agt tcc ctc atg ctc acg acc gag gag gat aaa 432 cys met gly tyr gln ser ser leu met leu thr thr glu glu asp lys ctc ctc tac tac ggc ata tta aat gga acc cca tcc atc atg tct tta 480 ccc gcc acc aaa acg aag acg gaa gca ccc aga att tgc ccg gat gtg 528 ttg ttg cag tgg cca cat ggg ccc att gtt tcg ctt gtg aat att aac 576 aaa cat gca ttt tac gtt gtt acc gcc tcc aat gta tac att gta cat 624 gat ggc tcg tat cat ccg act gga tcc atg gcc cag ctc caa cag gca 672 asp gly ser tyr his pro thr gly ser met ala gln leu gln gln ala gaa aat aat atc act aat tcc aaa aaa gaa atg aca aag cta cga gaa 720 aaa gtg aaa aag gcc gag aaa gaa aaa ttg gac gcc att aac cgg gca 768 acc aag ctg gaa gag gaa cga aac caa gcg tac aaa gca gca cac aag 816 gca gag gag gaa aag gct aaa aca ttt caa cgc ctt ata aca ttt gag 864 tcg gaa aat att aac tta aag aaa agg cca aat gac gca gtt tca aat 912 ser glu asn ile asn leu lys lys arg pro asn asp ala val ser asn cgg gat aag aaa aaa aat tct gaa acc gca aaa act gac gaa gta gag 960 aaa cag agg gcg gct gag gct gcc aag gcc gtg gag acg gag aag cag 1008 agg gca gct gag gcc acg aag gtt gcc gaa gcg gag aag cgg aag gca 1056 gct gag gcc gcc aag gcc gtg gag acg gag aag cag agg gca gct gaa 1104 gcc acg aag gtt gcc gaa gcg gag aag cag aag gca gct gag gcc gcc 1152 aag gcc gtg gag acg gag aag cag agg gca gct gaa gcc acg aag gtt 1200 gcc gaa gcg gag aag cag agg gca gct gaa gcc atg aag gtt gcc gaa 1248 gcg gag aag cag aag gca gct gag gcc gcc aag gcc gtg gag acg gag 1296 aag cag agg gca gct gaa gcc acg aag gtt gcc gaa gcg gag aag cag 1344 aag gca gct gag gcc gcc aag gcc gtg gag acg gag aag cag agg gca 1392 gct gaa gcc acg aag gtt gcc gaa gcg gag aag cag aag gca gct gag 1440 gcc gcc aag gcc gtg gag acg gag aag cag agg gca gct gaa gcc acg 1488 aag gtt gcc gaa gcg gag aag gat atc gat ccc 1521 arg thr asn gly ala his asp leu phe val ser asp thr gly lys ser ile thr gly phe gln pro asp val leu gln ile ser glu lys ser arg gln gly ala thr thr pro lys glu tyr trp gln val gly asn ala asp cys met gly tyr gln ser ser leu met leu thr thr glu glu asp lys asp gly ser tyr his pro thr gly ser met ala gln leu gln gln ala ser glu asn ile asn leu lys lys arg pro asn asp ala val ser asn