嗜肺军团菌pilE基因的克隆及其原核表达

目的克隆嗜肺军团菌Ⅳ型菌毛蛋白pilE基因,构建重组质粒pET-pilE,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Ⅳ型菌毛蛋白pilE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-pilE,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429bp完整的pilE基因,构建的原核表达重组质粒pET-pilE表达出35.7 kDa的融合蛋白质。结论成功构建了军团菌pilE基因的原核表达载体,并在大肠杆菌中得到了高效表达,为以后深入研究奠定了基础。

Cloning of pilE Gene of Legionella pneumophila and Detection of Its Expression in Prokaryotic Cell

Objective To clone the pilE gene of Legionella pneumophila,to construct recombinant plasmid and to detect its expression in prokaryotic cell.Method The pilE gene was amplified from the genomic DNA of Legionella pneumophila with PCR,and then was inserted into the prokaryotic expression vector pET-32a( ).The prokaryotic expression recombinant plasmid pET-pilE was constructed.After identification with restriction enzyme analysis,PCR and nucleotide sequencing analysis,the E.coli BL21 containing the recombinant plasmid pET-pilE was induced with IPTG.The expression of pilE was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis.Result The pilE gene of 429 bp long was amplified,the recombinant plasmid pET-pilE was constructed,and the Trx-PilE fusion protein of approximately 35.7kDa in size was expressed.Conclusion The prokaryotic expression recombinant plasmid pET-pilE was constructed successfully and its expressed efficiently in E.coli,which lays a basis for further study of PiLE protein.