嗜肺军团菌htpA基因的克隆及其原核表达

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。

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

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.