嗜肺军团菌m ip基因真核表达重组质粒构建与表达

Construction and Expression of Eucaryotic Recombinant Plasmid of Legionella pneumophila mip Gene

目的构建嗜肺军团菌m ip基因的真核表达重组质粒pcDNA 3.1-m ip,并研究其在细胞中的表达。方法PCR扩增嗜肺军团菌m ip基因,导入载体pcDNA 3.1(+),构建真核表达重组质粒pcDNA 3.1-m ip。用脂质体法将重组质粒pcDNA 3.1-m ip转染N IH 3T 3细胞,采用免疫荧光法和W estern-b lot分别鉴定pcDNA 3.1-m ip的瞬时表达产物和稳定表达产物。结果在细胞膜和细胞内观察到较强的绿色荧光,表明pcDNA 3.1-m ip成功转入N IH 3T 3细胞,并在细胞膜和细胞内获得短暂表达;用嗜肺军团菌兔血清抗体检测pcDNA 3.1-m ip稳定转染的N IH 3T 3细胞,在相对分子质量24×103处检测到阳性杂交信号,表明pcDNA 3.1-m ip在细胞内表达出M ip蛋白。空质粒pcDNA 3.1(+)转染的N IH 3T 3细胞未检测到绿色荧光和杂交信号。结论成功构建m ip基因真核表达重组质粒,并在N IH 3T 3细胞中表达出相对分子质量为24×103的M ip蛋白。

Objective To construct recombinant plasmid of LegioneUa pneumophila mip gene and detect its expression in NIH3T3 cells. Methods mip gene of Legionella pneumophila was amplified by PCR. The amplified DNA was ligated to pcDNA3.1 （＋） vector. The recombinant plasmid was named pcDNA3.1-mip. NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-mip with Lipofection strategy. Transient and stable products of mip gene were detected by immunofluorescence and Western-blot. Results It was found that there was high green fluorescence on the cell membrane and inside the cell. It showed that NIH3T3 cell was transfected by pcDNA3. 1-mip successfully. Rabbit serum antibody of Legionella pneumophila detected the NIH3T3 cell transfected with pcDNA3.1-mip. There was the protein in relative molecular weight 24×10^3, whereas no evidence for the protein in NIH3T3 cell transfected with pcDNA3.1 （＋） was seen. The protein expression of mip gene was shown, Conclusion We have successfully constructed the recombinant plasmid of Legionella pneumophila mip gene and detected the relative molecular weight 24×10^3 Mip protein in NIH3T3 cells.