马尔他布氏杆菌KdtA基因的克隆及原核表达  

Cloning and prokaryotic expression of KdtA gene from Brucella melitensis

作　　者：彭冬梅[1] 李国华[1] 李亚颖 贾晓晓[1] 徐开莲 朱华培[1] 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969-972,共4页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31460670)

摘　　要：为了研究马尔他布氏杆菌KdtA蛋白的功能,根据GenBank中公布的马尔他布氏杆菌M5-90株KdtA基因序列,利用DNAMAN软件设计上、下游引物,以M5-90株的基因组DNA为模板,采用PCR对其进行扩增,凝胶回收纯化后得到1 341bp的目的片段;然后构建克隆重组质粒pMD20-T-KdtA,将其转化到大肠杆菌DH5α;待测序正确后,构建原核表达质粒pET-28a(+)-KdtA,再将该质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导表达,用SDS-PAGE和Western-blot对基因的表达情况进行分析检测。结果表明,成功克隆了KdtA基因,并在大肠杆菌BL21(DE3)中表达了KdtA基因;经诱导后得到了预期的目的蛋白,证明KdtA基因得到了成功表达。To study the function of KdtA protein,according to Brucella melitensis M5-90 strain KdtA gene＇s sequence from GenBank,the upstream and downstream primers were designed by using the DNAMAN software and KdtA gene was amplified with whole genomic DNA as the template.The KdtA gene was obtained in 1 341 bp.The constructed recombinant plasmid pMD20-T-KdtA was transformed into Escherichia coli DH5α,and the recombinant prokaryotic expression plasmid pET-28a（＋）-KdtA was successfully constructed,and transformed into E.coli BL21（DE3）.The expression of fusion protein His-KdtA was induced with IPGT,and identified by SDS-PAGE and Western-blot.The results showed that KdtA gene was cloned successfully from B.melitensis M5-90 strain,and under induction with IPTG the target protein was obtained with a molecular weight as same as the expected size,which suggested that the prokaryotic expression vector was successfully constructed and KdtA gene was successfully expressed in E.coli BL21（DE3）.

关 键 词：马尔他布氏杆菌 KdtA基因 克隆 原核表达 

分 类 号：S852.614[农业科学—基础兽医学]