嗜肺军团菌mip基因的克隆与表达

Cloning and expression of the mip gene of Legionella pneumophila

目的 扩增嗜肺军团菌mip基因 ,导入载体 pUC18,构建重组质粒 pLpmip ,并在原核系统中表达。 方法 采用聚合酶链式反应 (PCR)从嗜肺军团菌扩增得到巨噬细胞感染增强蛋白基因 (mip基因 ) ,导入载体pUC18,构建重组质粒 pLp mip并转化大肠杆菌JM 10 9,并用限制性酶切分析、聚合酶链式反应、序列分析、硫酸十二烷酸钠 -聚丙烯酰胺凝胶电泳 (SDS-PAGE)、Western印迹进行鉴定。结果 扩增出 82 8bp的mip基因 ;构建重组质粒 pLpmip ;表达出 2 4kD的抗原区带。结论成功扩增 82 8bp的嗜肺军团菌mip基因 ,构建重组质粒 pLpmip ,并在原核系统中表达出 2 4KD的抗原区带。

To construct the recombinant plasmid pLpmip and to detect its expression in the prokaryotic cells JM109,the mip gene of Legionella pneumophila was amplified by PCR from a template of sero group I of this organism.The amplified DNA was digested with BamH 1 and Sal 1 and ligated into vector pUC18. The recombinant plasmid was identified by restriction analysis and PCR with further confirmation by sequencing analysis.SDS PAGE and Western blotting were used to detect the expression of mip gene products.It was found that mip gene of 828 bp in length was amplified,and the recombinant plasmid could be constructed. The protein expressed in the prokaryotic expression system had a size of 24 kDa. It concludes that the mip gene of L pneumophila is successfully cloned and expressed in the present study.