嗜肺军团菌mip基因重组质粒GFP-mip的构建及表达

The Construction and Expression of Recombinant Plasmid GFP-mip of Legionella Pneumophila Macrophage Infectivity Potentiator Gene

目的构建嗜肺军团菌mip基因的真核重组质粒GFP-mip,并观察其在NIH3T3细胞中的表达。方法以嗜肺军团菌DNA为模版,通过PCR扩增获得mip基因,将其定向克隆到绿色荧光质粒pEGFP-C1中,构建真核重组质粒GFP-mip。经限制性核酸内切酶XhoI和BamHI酶切鉴定、PCR和核酸序列分析后,通过脂质体法转染到NIH3T3细胞中,利用荧光显微镜观察重组质粒的稳定表达。结果扩增出了702bpmip基因,在细胞质和细胞膜观察到较强绿色荧光。结论成功构建了真核重组质粒GFP-mip,并在NIH3T3细胞中得到了表达。

Objective To construct recombinant plasmid GFP-mip of Legionella pneumophila macrophage infectivity potentiator gene and observe its expression in the NIH3T3 cells.Methods The macrophage infectivity potentiator gene was amplified from DNA of Legionella pneumophila by polymerase chain reation（PCR）,then cloned into pEGFP-C1 vector.The recombinant plasmid was named as GFP-mip and was analyzed with restriction endonuclease XhoI and BamHI digestion,PCR and DNA sequencing techniques.The NIH3T3 cell was transfected by recombinant plasmid GFP-mip with lipofection strategy.The stable expression products of macrophage infectivity potentiator gene were observed by the fluorescent microscope.Results 702bp mip gene was amplified.Under the fluorescent microscope,green fluorescent was observed in the cell cytoplasm and on the cell membrane.Conclusion The recombinant plasmid GFP-mip was constructed successfully and expressed in the NIH3T3 cells.