布鲁氏菌外膜蛋白16基因的克隆及原核表达  

Cloning and Prokaryotic Expression of Outer Membrane Proteins 16 Gene of Brucella melitensis

作　　者：贾晓晓[1] 焦寒伟[1] 郭莳雨[1] 史巧芸[1] 荣辉[1] 张珈宁[1] 朱华培[1] 杜丽[1] 成鹰[1] 王凤阳[1] 

机构地区：[1]海南大学农学院,海南省热带动物繁育与疫病研究重点实验室,海口市动物基因工程重点实验室,海南海口570228

出　　处：《中国畜牧兽医》2013年第9期51-54,共4页China Animal Husbandry & Veterinary Medicine

基　　金："十二五"农村领域国家科技计划课题(2011AA100302);"863"项目(2013AA102524)

摘　　要：为了成功克隆外膜蛋白16(outer membrane proteins 16,Omp16)基因并对其进行原核表达,试验根据GenBank中羊布鲁氏菌M5-90株外膜蛋白Omp16基因序列(登录号:JF918760.1)设计1对引物,从布鲁氏菌基因组中扩增出大小约为507bp的目的基因片段,凝胶回收纯化目的片段,连接入pMD20-T质粒,转化E.coli DH5α并测序,测序正确后再亚克隆入pET-28a(+)表达载体,构建重组质粒pET-Omp16,转化入E.coli BL21(DE3),经IPTG诱导其表达,最后用Western blotting分析方法鉴定诱导得到的蛋白。结果表明,成功构建了pET-Omp16原核表达载体,并在E.coli BL21中表达了Omp16基因,诱导得到的蛋白经鉴定与目的蛋白大小一致,证明成功表达了目的基因。To successfully clone the outer membrane proteins 16 （Omp16） gene and make prokaryotic expression in E.coli, one pair of primers was designed according to B. melitensis M5-90 strain Omp16 gene sequence in GenBank, and then obtained Omp16 gene which was about 507 bp by PCR from the Brucella genome. After purifying, Omp16 gene was inserted into pMD20-T vector to construct recombinant plasmid pMD-Omp16. pMD-Omp16 transformed into E.coli DH5α and identified it by sequencing, then subcloned to vector pET-28a（＋）. The constructed recombinant plasmid pET-Omp16 was transformed into E.coli BL21（DE3） for expression under induction of IPTG. Lastly, the expression products of recombinant protein His-Omp16 was identified by Western blotting. The results showed that the prokaryotic expression vector was successfully constructed and expressed Omp16 gene in E.coli BL21.

关 键 词：布鲁氏菌 外膜蛋白16基因 克隆 原核表达 

分 类 号：Q78[生物学—分子生物学]