嗜肺军团菌lvgA基因的克隆及其在大肠杆菌中的表达  

Cloning the lvgA Gene of Legionella Pneumophila and Detecting Its Expression in Escherichia coli

作　　者：刘明杰[1] 陈建平[1] 廖涛[1] 芦殿香[1] 陈宪[1] 

机构地区：[1]四川大学华西基础医学与法医学院寄生虫学教研室形态学实验室,成都610041

出　　处：《生物医学工程学杂志》2006年第3期605-608,共4页Journal of Biomedical Engineering

基　　金：国家自然科学基金资助课题(39870656)

摘　　要：本实验采用聚合酶链反应(PCR)从嗜肺军团菌基因组DNA中扩增得到军团菌毒力基因(L eg ionellav iru lence gene,lvg A gene),并将其定向连接入克隆载体pUC 18,构建重组质粒pU lvgA,重组子经限制性内切酶分析、聚合酶链式反应及测序鉴定后,再将lvgA基因亚克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pG lvgA,转化宿主菌JM 109,IPTG诱导表达,产物进行SDS-PAGE电泳、免疫印记分析鉴定。实验结果表明,成功扩增出627 bp的lvgA基因,构建了重组质粒pU lvgA及原核表达重组质粒pG lvgA,并使53.7 KD a的G ST-lvgA融合蛋白质在原核系统中得到了有效的表达。In order to clone lvgA gene（Legionella virulence gene） of Legionella pneumophila and detect its expression in prokaryotic cell, we amplified the lvgA gene from the total cell DNA of Legionella pneumophila with PCR,and then inserted it into the coloning vector pUC18. The recombinant plasmid pUlvgA was obtained. After the recombinant plasmid pUlvgA was identified with restriction enzyme analysis, polymerase chain reaction and nucleotide sequencing analysis, the lvgA gene was subcloned into the prokaryotic expression vector pGEX-4T-1. The prokaryotic expression recombinant plasmid pGlvgA was constructed. After IPTG induction ,the E. coli JM109 containing the recombinant plasmid pGlvgA expressed fusion protein. The expression of lvgA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicated that the lvgA gene of 627 bp long was amplified, the recombinant plasmids pUlvgA and pGlvgA were constructed successfully, and the GST-LvgA fusion protein of approximately 36 KDa in size was expressed in prokaryotic cell efficiently as expected.

关 键 词：军团菌 lvgA基因 聚合酶链式反应 基因表达 

分 类 号：R346[医药卫生—基础医学]