专利名称：黄鳍鲷肌动蛋白的基因序列的制作方法
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、细胞分裂以及伤口愈合有关；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)atggatgatgaaatcgccgcactggttgttgacaacggatccggtatgtgcaaagccggtttcgccggagacgacgcccctcgtgctgtcttcccctccatcgtcggtcgccccaggcatcagggtgtgatggtgggtatgggccagaaggacagctacgttggtgatgaggcccagagcaagagaggtatcctgaccctgaagtaccccatcgagcacggtattgtgaccaactgggatgacatggagaagatctggcatcacaccttctacaacgagctgagagttgcccctgaggagcaccctgtcctgctcacagaggcccccctgaaccccaaagccaacagggagaagatgacccagatcatgttcgagaccttcaacacccctgccatgtacgttgccatccaggctgtgctgtccctgtatgcctctggtcgtaccactggtatcgtcatggactccggtgatggtgtgacccacacagtgcccatctatgagggctatgccctgccccacgccatcctgcgtctggacttggccggccgcgacctcacagactacctcatgaagatcctgacagagcgtggctactccttcaccaccacagccgagagggaaatcgtgcgtgacatcaaggagaagctgtgctatgtcgcc
ctggacttcgagcaggagatgggcactgctgcctcctcctcctccctggagaagagctatgagctgcctgacggacaggtcatcaccatcggcaatgagaggttccgttgcccagaggccctcttccagccatccttcctcggtatggagtcctgcggaatccatgagaccacctacaacagcatcatgaagtgtgatgtcgacatccgtaaggacctgtatgccaacacagtgctgtctggaggtaccaccatgtaccctggcatcgctgacaggatgcagaaggagatcacagccctggccccatccaccatgaagattaagatcattgccccacctgagcgtaaatactctgtctggatcggaggctccatcctggcctccctgtccaccttccagcagatgtggatcagcaagcaggagtacgatgagtccggcccctccatcgtccaccgcaaatgcttc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 MetCys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe ProSer Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met GlyGln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly IleLeu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp AspAsp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg ValAla Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn ProLys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe AsnThr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr AlaSer Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val ThrHis Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile LeuArg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys IleLeu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu IleVal Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe GluGln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser TyrGlu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe ArgCys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser CysGly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val AspIle Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr ThrMet Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala LeuAla Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg LysTyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr PheGln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro SerIle 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F5’ATg gA(A/T)g(A/g)(T/C)gAA ATC gCC gC 3’R5’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，将具有插入片段的质粒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 ESequences producing significant alignments (Bits)Valuegi|56554009|gb|AAV97945.1|beta actin 2[Rivulus marmoratus]...7550.0gi|52547951|gb|AAR97600.2|beta actin[Epinepheius coioides]...7530.0gi|40362701|gb|AAR84618.1|beta actin[Acanthopagrus schlegelii]7530.0gi|57977261|dbj|BAD88412.1|beta cytoplasmic actin[Pagrus major7520.0gi|19309743|emb|CAD27237.1|beta-actin[oncorhynchus mykiss]...7520.0gi|45709360|gb|AAH67566.1|Bactin2[Danio rerio]＞gi|2822456|...7520.0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。
具体实施例方式
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F5’ATg gA(A/T)g(A/g)(T/C)gAA ATC gCC gC 3’R5’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，将具有插入片段的质粒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 ESequences producing significant alignments (Bits)Valuegi|56554009|gb|AAV97945.1|beta actin 2[Rivulus marmoratus]...7550.0gi|52547951|gb|AAR97600.2|beta actin[Epinephelus coioides]...7530.0gi|40362701|gb|AAR84618.1|beta actin[Acanthopagrus schlegelii]7530.0gi|57977261|dbj|BAD88412.1|beta cytoplasmic actin[Pagrus major7520.0gi|19309743|emb|CAD27237.1|beta-actin[Oncorhynchus mykiss]...7520.0gi|45709360|gb|AAH67566.1|Bactin2[Danio rerio]＞gi|2822456|....7520.0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>1atg gat gat gaa atc gcc gca ctg gtt gtt gac aac gga tcc ggt atg48Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met1617 13tgc aaa gcc ggt ttc gcc gga gac gac gcc cct cgt gct gtc ttc ccc96Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro3217 23 29tcc atc gtc ggt cgc ccc agg cat cag ggt gtg atg gtg ggt atg ggc144Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly4833 39 45cag aag gac agc tac gtt ggt gat gag gcc cag agc aag aga ggt atc192Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile6449 55 61
ctg acc ctg aag tac ccc atc gag cac ggt att gtg acc aac tgg gat240Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp8065 71 77gac atg gag aag atc tgg cat cac acc ttc tac aac gag ctg aga gtt288Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val9681 87 93gcc cct gag gag cac cct gtc ctg ctc aca gag gcc ccc ctg aac ccc336Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro11297 103 109aaa gcc aac agg gag aag atg acc cag atc atg ttc gag acc ttc aac384Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn128113 119 125acc cct gcc atg tac gtt gcc atc cag gct gtg ctg tcc ctg tat gcc432Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala144129 135 141tct ggt cgt acc act ggt atc gtc atg gac tcc ggt gat ggt gtg acc480Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr160145 151 157cac aca gtg ccc atc tat gag ggc tat gcc ctg ccc cac gcc atc ctg528His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu176161 167 173cgt ctg gac ttg gcc ggc cgc gac ctc aca gac tac ctc atg aag atc576Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile192177 183 189
ctg aca gag cgt ggc tac tcc ttc acc acc aca gcc gag agg gaa atc624Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile208193 199 205gtg cgt gac atc aag gag aag ctg tgc tat gtc gcc ctg gac ttc gag672Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu224209 215 221cag gag atg ggc act gct gcc tcc tcc tcc tcc ctg gag aag agc tat720Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr240225 231 237gag ctg cct gac gga cag gtc atc acc atc ggc aat gag agg ttc cgt768Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg256241 247 253tgc cca gag gcc ctc ttc cag cca tcc ttc ctc ggt atg gag tcc tgc816Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys272257 263 269gga atc cat gag acc acc tac aac agc atc atg aag tgt gat gtc gac864Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp288273 279 285atc cgt aag gac ctg tat gcc aac aca gtg ctg tct gga ggt acc acc912Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr304289 295 301atg tac cct ggc atc gct gac agg atg cag aag gag atc aca gcc ctg960Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu320305 311 317
gcc ccg tcc acc atg aag att aag atc att gcc cca cct gag cgt aaa1008Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys336321 327 333tac tct gtc tgg atc gga ggc tcc atc ctg gcc tcc ctg tcc acc ttc1056Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe352337 343 349cag cag atg tgg atc agc aag cag gag tac gat gag tcc ggc ccc tcc1104Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser368353 359 365atc gtc cac cgc aaa tgc ttc1125Ile Val His Arg Lys Cys Phe375369 375<210>2<2ll>375<212>PRT<213>黄鳍鲷(Sparus latus)<400>2Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met1617 13Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro3217 23 29Ser Ile Val Gly Arg Pro Arg hIs Gln Gly Val Met Val Gly Met Gly4833 39 45Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile64
49 55 61Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp8065 71 77Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val9681 87 93Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro11297 103 109Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn128113 119 125Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala144129 135 141Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr160145 151 157His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu176161 167 173Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile192177 183 189Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile208193 199 205Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu224209 215 221Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr240225 231 237Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg256
241 247 253Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys272257 263 269Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp288273 279 285Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr304289 295 301Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu320305 311 317Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys336321 327 333Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe352337 343 349Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser368353 359 365Ile Val His Arg Lys Cys Phe375369 37权利要求
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 atg48Met Asp Asp Glu Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met1617 13tgc aaa gcc ggt ttc gcc gga gac gac gcc cct cgt gct gtc ttc ccc96Cys Lys Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg Ala Val Phe Pro3217 23 29tcc atc gtc ggt cgc ccc agg cat cag ggt gtg atg gtg ggt atg ggc144Ser Ile Val Gly Arg Pro Arg His Gln Gly Val Met Val Gly Met Gly4833 39 45cag aag gac agc tac gtt ggt gat gag gcc cag agc aag aga ggt atc192Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg Gly Ile6449 55 61ctg acc ctg aag tac ccc atc gag cac ggt att gtg acc aac tgg gat240Leu Thr Leu Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp8065 71 77gac atg gag aag atc tgg cat cac acc ttc tac aac gag ctg aga gtt288Asp Met Glu Lys Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg Val9681 87 93gcc cct gag gag cac cct gtc ctg ctc aca gag gcc ccc ctg aac ccc336Ala Pro Lys Lys His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn Pro11297 103 109aaa gcc aac agg gag aag atg acc cag atc atg ttc gag acc ttc aac384Lys Ala Asn Arg Glu Lys Met Thr Gln Ile Met Phe Glu Thr Phe Asn128113 119 125acc cct gcc atg tac gtt gcc atc cag gct gtg ctg tcc ctg tat gcc432Thr Pro Ala Met Tyr Val Ala Ile Gln Ala Val Leu Ser Leu Tyr Ala144129 135 141tct ggt cgt acc act ggt atc gtc atg gac tcc ggt gat ggt gtg acc480Ser Gly Arg Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr160145 151 157cac aca gtg ccc atc tat gag ggc tat gcc ctg ccc cac gcc atc ctg528His Thr Val Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu176161 167 173cgt ctg gac ttg gcc ggc cgc gac ctc aca gac tac ctc atg aag atc576Arg Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile192177 183 189ctg aca gag cgt ggc tac tcc ttc acc acc aca gcc gag agg gaa atc624Leu Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg Glu Ile208193 199 205gtg cgt gac atc aag gag aag ctg tgc tat gtc gcc ctg gac ttc gag672Val Arg Asp Ile Lys Glu Lys Leu Cys Tyr Val Ala Leu Asp Phe Glu224209 215 221cag gag atg ggc act gct gcc tcc tcc tcc tcc ctg gag aag agc tat720Gln Glu Met Gly Thr Ala Ala Ser Ser Ser Ser Lys Glu Lys Ser Tyr240225 231 237gag ctg cct gac gga cag gtc atc acc atc ggc aat gag agg ttc cgt768Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg Phe Arg256241 247 253tgc cca gag gcc ctc ttc cag cca tcc ttc ctc ggt atg gag tcc tgc816Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys272257 263 269gga atc cat gag acc acc tac aac agc atc atg aag tgt gat gtc gac864Gly Ile His Glu Thr Thr Tyr Asn Ser Ile Met Lys Cys Asp Val Asp288273 279 285atc cgt aag gac ctg tat gcc aac aca gtg ctg tct gga ggt acc acc912Ile Arg Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr304289 295 301atg tac cct ggc atc gct gac agg atg cag aag gag atc aca gcc ctg960Met Tyr Pro Gly Ile Ala Asp Arg Met Gln Lys Glu Ile Thr Ala Leu320305 311 317gcc cca tcc acc atg aag att aag atc att gcc cca cct gag cgt aaa1008Ala Pro Ser Thr Met Lys Ile Lys Ile Ile Ala Pro Pro Glu Arg Lys336321 327 333tac tct gtc tgg atc gga ggc tcc atc ctg gcc tcc ctg tcc acc ttc1056Tyr Ser Val Trp Ile Gly Gly Ser Ile Leu Ala Ser Leu Ser Thr Phe352337 343 349cag cag atg tgg atc agc aag cag gag tac gat gag tcc ggc ccc tcc1104Gln Gln Met Trp Ile Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser368353 359 365atc gtc cac cgc aaa tgc ttc1125Ile Val His Arg Lys Cys Phe375369 37全文摘要
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。
文档编号C07K14/435GK1928086SQ200610037560
公开日2007年3月14日 申请日期2006年9月7日 优先权日2006年9月7日
发明者江世贵, 邱丽华, 张汉华, 刘振兴 申请人:中国水产科学研究院南海水产研究所