嗜肺军团菌mip基因真核重组质粒的构建与表达
[Construction and expression of eucaryotic recombinant plasmid of Legionella pneumophila mip gene].
作者信息
Wang Tao, Chen Jian-Ping, Li Hong, Lei Zhang, Tao Da-Chang, Yang Chun-Lei
机构信息
Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, China.
出版信息
Sichuan Da Xue Xue Bao Yi Xue Ban. 2005 Nov;36(6):773-5.
OBJECTIVE
To construct recombinant plasmid of Legionella 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 X 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 X 10(3) Mip protein in NIH3T3 cells.
目的
构建嗜肺军团菌mip基因重组质粒并检测其在NIH3T3细胞中的表达。
方法
采用PCR扩增嗜肺军团菌mip基因。将扩增的DNA连接到pcDNA3.1(+)载体上。重组质粒命名为pcDNA3.1-mip。采用脂质体转染法将重组质粒pcDNA3.1-mip转染NIH3T3细胞。通过免疫荧光和Western-blot检测mip基因的瞬时和稳定表达产物。
结果
发现细胞膜和细胞内有高绿色荧光。表明pcDNA3.1-mip成功转染NIH3T3细胞。嗜肺军团菌兔血清抗体检测到用pcDNA3.1-mip转染的NIH3T3细胞。有相对分子质量为24×10(3)的蛋白，而在用pcDNA3.1(+)转染的NIH3T3细胞中未见到该蛋白。显示了mip基因的蛋白表达。
结论
成功构建了嗜肺军团菌mip基因重组质粒，并在NIH3T3细胞中检测到相对分子质量为24×10(3)的Mip蛋白。
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