CN1928086A

CN1928086A - 黄鳍鲷肌动蛋白的基因序列

Info

Publication number: CN1928086A
Application number: CN 200610037560
Authority: CN
Inventors: 江世贵; 邱丽华; 张汉华; 刘振兴
Original assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Current assignee: South China Sea Fisheries Research Institute Chinese Academy Fishery Sciences
Priority date: 2006-09-07
Filing date: 2006-09-07
Publication date: 2007-03-14

Abstract

本发明从近源物种肌动蛋白的基因的CDS序列的保守区设计的兼并引物，从黄鳍鲷的脾脏中提取总RNA，并用PCR法扩增，克隆到黄鳍鲷肌动蛋白的基因的表达序列。该基因序列的获得不但使通过体外重组技术获得具有免疫活性的重组蛋白成为可能，而且为进一步探讨其功能提供了理论基础。

Description

黄鳍鲷肌动蛋白的基因序列

技术领域

本发明涉及分子生物学中基因克隆领域，尤其涉及黄鳍鲷肌动蛋白基因序列。

背景技术

肌动蛋白是一种看家蛋白，被定义成一种分子发动机，与细胞移动、细胞***以及伤口愈合有关；同时肌动蛋白还参与DNA转录，可以召集RNA聚合酶II，在RNA聚合酶II开始转录时充当一个分子发动机。如果肌动蛋白受到抑制，转录就无法开始。DNA转录这个过程对细胞的所有活动都非常重要。这些功能将使我们能够在遗传转录出错时进行有效干预；肌动蛋白可以作为辅助因子，具有降解细胞骨架的病毒酶的功能。

目前随着海水养殖业的发展，病害问题日趋严重，因此从鱼体自身入手，克隆相关的功能基因，获得纯化的具活性的体外重组蛋白，提高鱼体抗病能力是当今病害防治的趋势。至今对黄鳍鲷细胞方面的基因研究还较少，尤其对肌动蛋白基因的研究还属空白。

发明内容

本发明的目的是通过以近源物种(虹鳟、牙鲆、鲤鱼)肌动蛋白基因的CDS序列的保守区设计的兼并引物，并用PCR法扩增，结合RACE技术克隆到黄鳍鲷肌动蛋白基因的全表达序列。所得的基因序列为：

核苷酸序列(1125个碱基)：

atggatgatgaaatcgccgcactggttgttgacaacggatccggtatgtgcaaagccggt

ttcgccggagacgacgcccctcgtgctgtcttcccctccatcgtcggtcgccccaggcat

cagggtgtgatggtgggtatgggccagaaggacagctacgttggtgatgaggcccagagc

aagagaggtatcctgaccctgaagtaccccatcgagcacggtattgtgaccaactgggat

gacatggagaagatctggcatcacaccttctacaacgagctgagagttgcccctgaggag

caccctgtcctgctcacagaggcccccctgaaccccaaagccaacagggagaagatgacc

cagatcatgttcgagaccttcaacacccctgccatgtacgttgccatccaggctgtgctg

tccctgtatgcctctggtcgtaccactggtatcgtcatggactccggtgatggtgtgacc

cacacagtgcccatctatgagggctatgccctgccccacgccatcctgcgtctggacttg

gccggccgcgacctcacagactacctcatgaagatcctgacagagcgtggctactccttc

accaccacagccgagagggaaatcgtgcgtgacatcaaggagaagctgtgctatgtcgcc

ctggacttcgagcaggagatgggcactgctgcctcctcctcctccctggagaagagctat

gagctgcctgacggacaggtcatcaccatcggcaatgagaggttccgttgcccagaggcc

ctcttccagccatccttcctcggtatggagtcctgcggaatccatgagaccacctacaac

agcatcatgaagtgtgatgtcgacatccgtaaggacctgtatgccaacacagtgctgtct

ggaggtaccaccatgtaccctggcatcgctgacaggatgcagaaggagatcacagccctg

gccccatccaccatgaagattaagatcattgccccacctgagcgtaaatactctgtctgg

atcggaggctccatcctggcctccctgtccaccttccagcagatgtggatcagcaagcag

gagtacgatgagtccggcccctccatcgtccaccgcaaatgcttc

氨基酸序列(375个氨基酸)：

Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro

Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly

Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile

Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp

Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val

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

Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn

Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala

Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr

His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu

Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile

Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile

Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu

Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr

Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg

Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys

Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp

Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr

Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu

Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys

Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe

Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser

Ile Val His Arg Lys Cys Phe

上述目的是通过以下技术方案来实现的：

解剖黄鳍鲷取出脾脏，提取总RNA，使之与反转录引物(oligo-dT接头引物)混合，进行反转录，合成第一链cDNA。

从GenBank中下载近源物种(如虹鳟、牙鲆、鲤鱼等)肌动蛋白同源基因CDS序列，利用Clustal W软件进行多序列比对，确定保守区，根据保守区序列设计兼并引物，扩增片段大小约为1125bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成得到以下引物序列：

F：5’ATg gA(A/T)g(A/g)(T/C)gAA ATC gCC gC 3’

R：5’TTA gAA gCA TTT(g/A)Cg gTg gA 3’。

以合成的第一链cDNA作为模板，用兼并引物进行PCR扩增，所扩增的PCR产物用1.2％的琼脂糖凝胶电泳进行检测，从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，提取质粒DNA。经简并引物PCR检测后，将具有***片段的质粒DNA用M13通用引物进行双向测序。测序所得序列利用Clustal W软件进行拼接即可得到黄鳍鲷肌动蛋白基因。

所得到的PCR产物在1.2％的琼脂糖凝胶电泳上进行分离检测后，将PCR产物纯化后，克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，用3730测序仪对质粒DNA进行测序。测序结果用BLAST软件( http://www.ncbi.nim.nih.gov/)进行同源性测定，来确定为黄鳍鲷肌动蛋白基因同源序列。比对结果：

Score E

Sequences producing significant alignments： (Bits) Value

gi|56554009|gb|AAV97945.1|beta actin 2[Rivulus marmoratus]... 755 0.0

gi|52547951|gb|AAR97600.2|beta actin[Epinepheius coioides]... 753 0.0

gi|40362701|gb|AAR84618.1|beta actin[Acanthopagrus schlegelii] 753 0.0

gi|57977261|dbj|BAD88412.1|beta cytoplasmic actin[Pagrus major 752 0.0

gi|19309743|emb|CAD27237.1|beta-actin[oncorhynchus mykiss]... 752 0.0

gi|45709360|gb|AAH67566.1|Bactin2[Danio rerio]＞gi|2822456|... 752 0.0

根据Iron1995年的报道，只要两个序列间用BLAST软件比对后E-Value值小于0.005，则两个序列之间具有绝对的同源性，即证明序列为同一种基因序列。从对比结果看：目的基因序列BLAST比对后，与其他物种的肌动蛋白的E值绝对小于0.005，所以证明所克隆的目的基因是肌动蛋白的同源序列。

该基因序列的获得不但使通过体外重组技术获得具有免疫活性的重组蛋白成为可能，而且为进一步探讨其功能提供了理论基础。肌动蛋白可以作为研究其他功能基因的体内表达规律的内参照，来调节基因再体内的复制水平，因此获得黄鳍鲷肌动蛋白基因序列使进一步研究功能基因的体内表达调控规律成为了可能。

具体实施方式

下面通过以下具体实施方式对本发明作进一步阐述，但是本发明的内容完全不局限于此。

1.总RNA的提取

取鲜活健康黄鳍鲷(体重约400g)在实验室内暂养2d后(水温约24℃，气泵充气)，从胸腔注射脂多糖(LPS，10μg/mL)200μL。刺激6h后，解剖鱼体取出脾脏约100mg，分别放入1mL Trizol(Gibco，Japan)中进行匀浆，按照试剂盒使用说明提取总RNA。

2.cDNA第一链的合成

取黄鳍鲷总RNA 5μg与反转录引物(oligo-dT接头引物)1μL(10pmol/L)混合，70℃加热5min后，立即放置冰上，然后加入5×buffer，2.5mmol/L dNTP混合液，Ribonuclease Inhibitor，M-MLV反转录酶，反应体系为25μL。反应过程为42℃60min，70℃15min，最后放入-80℃保存备用。

3.兼并引物设计依据，引物合成的方法

从GenBank中下载近源物种(如虹鳟、牙鲆、鲤鱼等)肌动蛋白同源基因CDS序列，利用Clustal W软件进行多序列比对，确定保守区，根据保守区序列设计兼并引物，扩增片段大小约为1125bp。引物设计后采用β-乙睛亚磷酰胺化学法进行DNA合成。

引物序列为：

F：5’ATg gA(A/T)g(A/g)(T/C)gAA ATC gCC gC 3’

R：5’TTA gAA gCA TTT(g/A)Cg gTg gA 3’。

4.黄鳍鲷肌动蛋白基因cDNA全序列的克隆

以近源物种肌动蛋白基因CDS序列的保守区设计的兼并引物，扩增片段大小约为1125bp。

以上述合成的第一链cDNA作为模板，用兼并引物进行PCR扩增，反应体系为：10x PCR反应缓冲液5μL，25mmol/L MgCl₂3μL，2.5mmol/L dNTP 2μL，10nmol/L引物dTF和dTR各2μL，Taq酶1.25U，用PCR水将反应体系补充至50μL。反应条件为：1个循环，94℃变性5min；35个循环：94℃变性45s，54℃退火45s，72℃延伸45s；1个循环，72℃延伸10min；4℃保温。所扩增的PCR产物用1.2％的琼脂糖凝胶电泳进行检测，从凝胶中纯化回收目的产物。然后将纯化的PCR产物克隆到pMD-18T载体中，转化大肠杆菌JM-109感受态细胞，挑取阳性克隆，提取质粒DNA。经简并引物PCR检测后，将具有***片段的质粒DNA用M13通用引物进行双向测序。测序所得序列利用Clustal W软件进行拼接。

5.对黄鳍鲷肌动蛋白基因的测定

Score E

Sequences producing significant alignments： (Bits) Value

gi|56554009|gb|AAV97945.1|beta actin 2[Rivulus marmoratus]... 755 0.0

gi|52547951|gb|AAR97600.2|beta actin[Epinephelus coioides]... 753 0.0

gi|40362701|gb|AAR84618.1|beta actin[Acanthopagrus schlegelii] 753 0.0

gi|57977261|dbj|BAD88412.1|beta cytoplasmic actin[Pagrus major 752 0.0

gi|19309743|emb|CAD27237.1|beta-actin[Oncorhynchus mykiss]... 752 0.0

gi|45709360|gb|AAH67566.1|Bactin2[Danio rerio]＞gi|2822456|.... 752 0.0

根据Iron1995年的报道，只要两个序列间用BLAST软件比对后E-Value值小于0.005，则两个序列之间具有绝对的同源性，即证明序列为同一种基因序列。从对比结果看目的基因序列BLAST比对后，与其他物种的肌动蛋白的E值绝对小于0.005，所以证明所克隆的目的基因是肌动蛋白的同源序列。

序列表

<110>中国水产科学研究院南海水产研究所

<120>黄鳍鲷肌动蛋白的基因序列

<160>2

<210>1

<211>1125

<212>RNA

<213>黄鳍鲷(Sparus latus)

<220>

<221>CDS

<222>(1)...(1125)

<400>1

atg gat gat gaa atc gcc gca ctg gtt gtt gac aac gga tcc ggt atg 48

Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met 16

1 7 13

tgc aaa gcc ggt ttc gcc gga gac gac gcc cct cgt gct gtc ttc ccc 96

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro 32

17 23 29

tcc atc gtc ggt cgc ccc agg cat cag ggt gtg atg gtg ggt atg ggc 144

Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly 48

33 39 45

cag aag gac agc tac gtt ggt gat gag gcc cag agc aag aga ggt atc 192

Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile 64

49 55 61

ctg acc ctg aag tac ccc atc gag cac ggt att gtg acc aac tgg gat 240

Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp 80

65 71 77

gac atg gag aag atc tgg cat cac acc ttc tac aac gag ctg aga gtt 288

Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val 96

81 87 93

gcc cct gag gag cac cct gtc ctg ctc aca gag gcc ccc ctg aac ccc 336

Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro 112

97 103 109

aaa gcc aac agg gag aag atg acc cag atc atg ttc gag acc ttc aac 384

Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn 128

113 119 125

acc cct gcc atg tac gtt gcc atc cag gct gtg ctg tcc ctg tat gcc 432

Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala 144

129 135 141

tct ggt cgt acc act ggt atc gtc atg gac tcc ggt gat ggt gtg acc 480

Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr 160

145 151 157

cac aca gtg ccc atc tat gag ggc tat gcc ctg ccc cac gcc atc ctg 528

His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 176

161 167 173

cgt ctg gac ttg gcc ggc cgc gac ctc aca gac tac ctc atg aag atc 576

Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 192

177 183 189

ctg aca gag cgt ggc tac tcc ttc acc acc aca gcc gag agg gaa atc 624

Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile 208

193 199 205

gtg cgt gac atc aag gag aag ctg tgc tat gtc gcc ctg gac ttc gag 672

Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu 224

209 215 221

cag gag atg ggc act gct gcc tcc tcc tcc tcc ctg gag aag agc tat 720

Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr 240

225 231 237

gag ctg cct gac gga cag gtc atc acc atc ggc aat gag agg ttc cgt 768

Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg 256

241 247 253

tgc cca gag gcc ctc ttc cag cca tcc ttc ctc ggt atg gag tcc tgc 816

Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys 272

257 263 269

gga atc cat gag acc acc tac aac agc atc atg aag tgt gat gtc gac 864

Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp 288

273 279 285

atc cgt aag gac ctg tat gcc aac aca gtg ctg tct gga ggt acc acc 912

Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr 304

289 295 301

atg tac cct ggc atc gct gac agg atg cag aag gag atc aca gcc ctg 960

Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu 320

305 311 317

gcc ccg tcc acc atg aag att aag atc att gcc cca cct gag cgt aaa 1008

Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys 336

321 327 333

tac tct gtc tgg atc gga ggc tcc atc ctg gcc tcc ctg tcc acc ttc 1056

Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 352

337 343 349

cag cag atg tgg atc agc aag cag gag tac gat gag tcc ggc ccc tcc 1104

Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 368

353 359 365

atc gtc cac cgc aaa tgc ttc 1125

Ile Val His Arg Lys Cys Phe 375

369 375

<210>2

<2ll>375

<212>PRT

<213>黄鳍鲷(Sparus latus)

<400>2

Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met 16

1 7 13

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro 32

17 23 29

Ser Ile Val Gly Arg Pro Arg hIs Gln Gly Val Met Val Gly Met Gly 48

33 39 45

Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile 64

49 55 61

Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp 80

65 71 77

Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val 96

81 87 93

Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro 112

97 103 109

Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn 128

113 119 125

Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala 144

129 135 141

Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr 160

145 151 157

His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 176

161 167 173

Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 192

177 183 189

Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile 208

193 199 205

Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu 224

209 215 221

Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr 240

225 231 237

Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg 256

241 247 253

Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys 272

257 263 269

Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp 288

273 279 285

Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr 304

289 295 301

Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu 320

305 311 317

Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys 336

321 327 333

Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 352

337 343 349

Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 368

353 359 365

Ile Val His Arg Lys Cys Phe 375

369 375

Claims

1、一种黄鳍鲷肌动蛋白基因，其特征在于具有以下RNA核苷酸及相应的氨基酸序列：

atg gat gat gaa atc gcc gca ctg gtt gtt gac aac gga tcc ggt atg 48

Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met 16

1 7 13

tgc aaa gcc ggt ttc gcc gga gac gac gcc cct cgt gct gtc ttc ccc 96

Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro 32

17 23 29

tcc atc gtc ggt cgc ccc agg cat cag ggt gtg atg gtg ggt atg ggc 144

Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly 48

33 39 45

cag aag gac agc tac gtt ggt gat gag gcc cag agc aag aga ggt atc 192

Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile 64

49 55 61

ctg acc ctg aag tac ccc atc gag cac ggt att gtg acc aac tgg gat 240

Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp 80

65 71 77

gac atg gag aag atc tgg cat cac acc ttc tac aac gag ctg aga gtt 288

Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val 96

81 87 93

gcc cct gag gag cac cct gtc ctg ctc aca gag gcc ccc ctg aac ccc 336

Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro 112

97 103 109

aaa gcc aac agg gag aag atg acc cag atc atg ttc gag acc ttc aac 384

Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn 128

113 119 125

acc cct gcc atg tac gtt gcc atc cag gct gtg ctg tcc ctg tat gcc 432

Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala 144

129 135 141

tct ggt cgt acc act ggt atc gtc atg gac tcc ggt gat ggt gtg acc 480

Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr 160

145 151 157

cac aca gtg ccc atc tat gag ggc tat gcc ctg ccc cac gcc atc ctg 528

His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 176

161 167 173

cgt ctg gac ttg gcc ggc cgc gac ctc aca gac tac ctc atg aag atc 576

Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile 192

177 183 189

ctg aca gag cgt ggc tac tcc ttc acc acc aca gcc gag agg gaa atc 624

Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile 208

193 199 205

gtg cgt gac atc aag gag aag ctg tgc tat gtc gcc ctg gac ttc gag 672

Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu 224

209 215 221

cag gag atg ggc act gct gcc tcc tcc tcc tcc ctg gag aag agc tat 720

Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr 240

225 231 237

gag ctg cct gac gga cag gtc atc acc atc ggc aat gag agg ttc cgt 768

Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg 256

241 247 253

tgc cca gag gcc ctc ttc cag cca tcc ttc ctc ggt atg gag tcc tgc 816

Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys 272

257 263 269

gga atc cat gag acc acc tac aac agc atc atg aag tgt gat gtc gac 864

Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp 288

273 279 285

atc cgt aag gac ctg tat gcc aac aca gtg ctg tct gga ggt acc acc 912

Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr 304

289 295 301

atg tac cct ggc atc gct gac agg atg cag aag gag atc aca gcc ctg 960

Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu 320

305 311 317

gcc cca tcc acc atg aag att aag atc att gcc cca cct gag cgt aaa 1008

Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys 336

321 327 333

tac tct gtc tgg atc gga ggc tcc atc ctg gcc tcc ctg tcc acc ttc 1056

Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe 352

337 343 349

cag cag atg tgg atc agc aag cag gag tac gat gag tcc ggc ccc tcc 1104

Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser 368

353 359 365

atc gtc cac cgc aaa tgc ttc 1125

Ile Val His Arg Lys Cys Phe 375

369 375