嗜肺军团菌htpA基因的克隆及其原核表达  

Cloning of the htpA gene of Legionella pneumophila and detection of its expression in prokaryotic cell

作　　者：刘明杰[1] 陈建平[1] 廖涛[2] 王涛[1] 陈宪[1] 田玉[1] 张雷[1] 张莉[1] 

机构地区：[1]四川大学华西医学中心基础医学与法医学院寄生虫学教研室暨形态学实验室四川大学时间生物学卫生部重点实验室,成都610041 [2]川北医学院附属医院检验科,南充637000

出　　处：《中国人兽共患病学报》2006年第2期114-117,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目(30300302)

摘　　要：目的克隆并检测嗜肺军团菌htpA基因在原核系统中表达情况,为进一步研究HtpA蛋白的免疫性能作必要的准备。方法采用聚合酶链反应(PCR)从嗜肺军团菌基因组DNA中扩得军团菌热休克蛋白A基因htpA,并将其定向克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pGhtpA,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,转化宿主菌大肠杆菌JM109,IPTG诱导表达,产物进行SDS-PAGE电泳、免疫印迹分析鉴定。结果扩增出了291bp完整的htpA基因,构建了原核表达重组质粒pGhtpA,并检测到约36kDa的GST-htpA融合蛋白质表达条带。结论成功克隆了嗜肺军团菌htpA基因并使HtpA蛋白在原核表达系统中得到了有效的表达,为进一步研究其免疫学特性奠定了基础。To clone the htpA gene of Legionella pneurnophila and to detect its expression in prokaryotic cell, the htpA gene was amplified from the genomic DNA of Legionella pneumophila with PCR, and then was inserted into the prokaryotic expression vector pGEX-4T-1.To construct the prokaryotic expression recombinant plasmid pGhtpA. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, the E. coli JM109 containing the recombinant plasmid pGhtpA was induced with IPTG, The expression of htpA was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. The results indicate that the htpA gene of 291bp long was amplified and the prokaryotic expression recombinant plasmid pGhtpA was constructed successfully, and the GST-HtpA fusion protein of approximately 36kDa in size was expressed efficiently as expected, which make it possible to further study its role in immune responses.

关 键 词：嗜肺军团菌 htpA基因 基因克隆 基因表达 

分 类 号：R378[医药卫生—病原生物学]