马尔他布氏杆菌LpxK基因的克隆及原核表达  

Cloning and prokaryotic expression of LpxK gene from Brucella melitensis

作　　者：李国华[1] 彭冬梅[1] 李亚颖 贾晓晓[1] 朱华培[1] 徐开莲 赵天靖 庞峰[1] 王凤阳[1] 杜丽[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：《中国兽医科学》2015年第9期959-962,共4页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31460670)

摘　　要：为了克隆马尔他布氏杆菌LpxK基因并对其进行原核表达,根据GenBank中马尔他布氏杆菌M5-90株LpxK基因序列信息设计引物,从M5-90株基因组扩增出1 026bp的目的基因;然后将其连接入pMD20-T载体,转化入大肠杆菌DH5α并测序;测序正确后将目的基因连接入pET-28a(+)质粒,构建重组质粒pET-28a(+)-LpxK,并将其转化入大肠杆菌BL21(DE3),用IPTG诱导其表达,用SDS-PAGE和Western-blot鉴定诱导表达的蛋白。结果表明,成功构建了pET-28a(+)-LpxK原核表达载体,并在大肠杆菌BL21(DE3)中表达了LpxK基因,表达的融合蛋白大小约41ku且主要以包涵体形式存在。To clone the LpxK gene and make prokaryotic expression in Escherichia coli,one pair of primers was designed according to Brucella melitensis M5-90 genome sequence,and then the LpxK gene was obtained,which was about 1 026 bp in size.The LpxK gene was inserted into pMD20-T vector.The pMD20-T-LpxK vector was transformed into E.coli DH5α.After sequencing,the LpxK gene was ligated into pET-28a（＋）to construct the recombinant expression plasmid pET-28a（＋）-LpxK,and then transformed into E.coli BL21（DE3）.After induction with IPTG,the protein was detected by SDS-PAGE and Western-blot.The results showed that the pET-28a（＋）-LpxKprokaryotic expression vector was successfully constructed and expressed in E.coli BL21（DE3）.The expression fusion protein was about 41 ku in molecular weight and it existed in the form of inclusion bodies.

关 键 词：马尔他布氏杆菌 LpxK基因 克隆 原核表达 

分 类 号：S852.614[农业科学—基础兽医学]