锯缘青蟹精氨酸激酶基因的克隆与表达  

Cloning and Expression of Arginine Kinase from Mud Crab(Scylla serrata)

作　　者：阮韦伟[1,2] 沈苑[1,2] 曹敏杰[1,2] 苏文金[1,2] 蔡秋凤[1,2] 刘光明 

机构地区：[1]集美大学生物工程学院,福建厦门361021 [2]福建省高校水产科学技术与食品安全重点实验室,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2011年第5期346-351,共6页Journal of Jimei University：Natural Science

基　　金：国家自然科学基金资助项目(31071519);福建省自然科学基金资助项目(2010J06012;2008J0067);福建省科技计划重点资助项目(2010N0019);福建省高校新世纪优秀人才计划资助项目(NCETFJ-2007);集美大学创新团队基金资助项目(2010A005);李尚大集美大学学科建设基金资助项目(ZC2010008)

摘　　要：通过分子生物学方法,克隆得到锯缘青蟹(Scylla serrata)精氨酸激酶(Arginine Kinase,AK)开放阅读框基因序列.测序结果显示:该开放阅读框基因的序列长度为1 071 bp,编码356个氨基酸残基;该序列登录Genbank(GQ:851626),序列比对结果显示,锯缘青蟹精氨酸激酶与凡纳滨对虾(Litopenaeus vannamei)精氨酸激酶的氨基酸序列同源性高达94%,与岸蟹(Carcinus maenas)、中华绒鳌蟹(Eriocheir sinensis)等甲壳类动物的精氨酸激酶也有较高的同源性,均高于90%;将克隆得到的锯缘青蟹精氨酸激酶基因与pGEX-4T-3载体连接,转化大肠杆菌BL21,经IPTG诱导得到分子质量为65 ku的融合表达蛋白(GST-AK),经兔抗锯缘青蟹精氨酸激酶多克隆抗体的免疫印迹分析证实,GST-AK具有抗原性.The open reading frame gene sequences of Mud crab arginine kinase was cloned through the molecular biology technology method.The cloning of arginine kinase gene amplified with sequence analysis showed that the cloned DNA fragments had open reading frames of 1 071 bp,and predicted to encode proteins with 356 amino acid residues.The obtained gene was deposited in Genbank under accession number of GQ 851626.Sequence alignment revealed that the Mud crab arginine kinase shared 94 % of homology to Litopenaeus vannamei,and more than 90 % homology to other shellfishes,such as Carcinus maenas,Eriocheir sinensis.Mud crab arginine kinase gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL21.The expressed protein revealed a band of about 65 ku on SDS-PAGE.Western blot analysis using anti-Mud crab AK polyclonal antibody revealed positive reaction to the GST-AK fusion protein.

关 键 词：锯缘青蟹 精氨酸激酶 过敏原 CDNA克隆 重组表达 

分 类 号：Q816[生物学—生物工程]