嗜肺军团菌ip基因的克隆及其原核表达  

Cloning of the ip Gene of Legionella Pneumophila and Detection of It's Expression in Prokaryotic Cell

作　　者：杨志伟[1] 曹秀琴[1] 

机构地区：[1]宁夏医学院基础学院微生物学与免疫学教研室,银川750004

出　　处：《宁夏医学院学报》2008年第6期696-699,共4页Journal of Ningxia Medical College

基　　金：宁夏高等学校科学技术研究项目(2007年)

摘　　要：目的克隆嗜肺军团菌主要免疫原蛋白ip基因,构建重组质粒pET-ip,并在原核系统中表达。方法采用聚合酶链反应(PCR),从嗜肺军团菌基因组DNA中扩增军团菌主要免疫原蛋白ip基因,并将其定向克隆至原核表达载体pET-32a(+),构建原核表达重组质粒pET-ip,经限制性内切酶鉴定、PCR及测序分析后,转化宿主菌大肠杆菌BL21,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792 bp完整的ip基因,构建的原核表达重组质粒pET-ip表达出49 kDa Trx-IP的融合蛋白质,Western-blot证实获得蛋白为所需要的目的蛋白。结论成功构建了军团菌ip基因的原核表达载体,并在大肠杆菌中得到了高效表达。Objective To clone the ip gene of Legionella pneumophila, to construct recombinant plasmid and to detect its expression in prokaryotic cell. Methods The ip gene was amplified from the genomie DNA of Legionella pneumophila with PCR, and then was inserted into the prokaryotic expression vector pET-32a（ ＋ ）. The prokaryotic expression recombinant plasmid pET-ip was constructed. After identification with restriction enzyme analysis, PCR and nucleotide sequencing analysis, the E. coli BL21 containing the recombinant plasmid pET-ip was induced with IPTG. The expression of ip was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis. Results The ip gene of 792 bp long was amplified and the recombinant plasmid pET- ip was constructed. SDS-PAGE analysis showed that the Trx- IP fusion protein of approximately 49 kDa in size was expressed. The expressed proteins could be confirmed by western blot. Conclusion The prokaryotic expression recombinant plasmid pET-ip was constructed successfully and its expressed efficiently in E. coli.

关 键 词：军团菌 ip基因 克隆 基因表达 

分 类 号：R378[医药卫生—病原生物学]