牛源麦氏弧菌ASP基因的克隆与原核表达  

Cloning and prokaryotic expression of ASP gene from bovine Vibrio metschnikovii

作　　者：林永润[1] 李章程[1] 刀丽梅 程方俊[1,2] 曹立亭[1,2] 蒋梦娜[1] 孙莹莹[1] 张婷婷[1] LIN Yong-run LI Zhang-cheng DAO Li-mei CHENG Fang-jun CAO Li-ting JIANG Meng-na SUN Ying-ying ZHANG Ting-ting(Department of Veterinary Medicine, Rongchang Campus of Southwest University, Chongqing 402460, China Chongqing Engineering Research Center of Veterinary Science, Chongqing 402460, China)

机构地区：[1]西南大学荣昌校区动物医学系,重庆402460 [2]重庆市兽医科学工程研究中心,重庆402460

出　　处：《中国兽医科学》2017年第7期884-889,共6页Chinese Veterinary Science

基　　金："十二五"国家科技支撑计划项目(2011BAD36B00);重庆市重点科技攻关资助项目(cstc2012jcsfjfzhX0021);重庆高校创新团队建设计划(CXTDG201602003)

摘　　要：为获得牛源麦氏弧菌重组碱性丝氨酸蛋白酶(ASP),笔者扩增并构建了以麦氏弧菌FD39-1的ASP基因为插入片段的重组表达质粒p ET32a-ASP,将该质粒转化至大肠杆菌Rosetta中诱导表达并纯化,采用SDS-PAGE与Western-blot技术检测目的蛋白是否表达成功。结果显示,扩增的ASP基因与GenBank中相应基因序列(Kp126900.1)的同源性达100%。ASP基因在大肠杆菌中成功表达,表达蛋白相对分子质量大小约为58 ku,与预期一致。Western-blot显示在预期位置出现阳性条带。本研究为麦氏弧菌ASP的生物学特性与亚单位疫苗的制备奠定了基础。In order to study recombinant alkaline serine proteinase（ASP） from bovine Vibrio metschnikovii,the DNA sequence of ASP gene from Vibrio Metschnikovii（FD39-1 isolate） was amplified by PCR,purified,and linked into the pET32a（＋） vector.The recombinant plasmids（p ET32a-ASP） were trans formed into Escherichia coli Rosetta,and positive clone was selected to induce expression by IPTG.The results showed the ASP gene was successfully obtained,and sequence analysis revealed it had 100% sequence identity to the gene（Kp126900.1）from GenBank database.Western-blot confirmed ASP gene was successfully expressed in E.coli and the recombinant protein was about 58 ku.This research lay foundations for the further studies on Vibrio metschnikovii biological characteristics and subunit vaccines.

关 键 词：麦氏弧菌 丝氨酸蛋白酶 克隆 原核表达 

分 类 号：S852.651.5[农业科学—基础兽医学]