利用PCR技术扩增出牛结核杆菌mpb64基因片段，克隆到真核载体pcDNA3．1(+)上，构建重组质粒pCM64，将该重组质粒转染SP2/0细胞，间接免疫荧光试验和Western blotting检测目的基因的表达情况。结果表明，在转染了重组质粒pCM64的SP2/0细胞中出现绿色荧光，转染的SP2/0细胞泳道23 kDa处出现特异条带，说明mpb64基因在SP2/0细胞中成功进行了瞬时表达。
The mpb64 gene fragment amplified by PCR from Mycobacterium bovis was cloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pCM64 was obtained.Then the recombinant plasmid was transfected into SP2/0 cells in vitro.The expression of target gene was detected by indirect immunofluorescence and Western blotting.The result showed that the green fluorescence appeared in the SP2/0 cells transfected by pCM64 and the specific band about 23 kDa appeared in the electophoretogram of SP2/0 cells,indicaing that the target gene mpb64 had been successfully expressed.
[1] 张秀华,刘思国.牛分枝杆菌MPB64基因的克隆、鉴定及表达[J].中国生物工程杂志,2005,25(4):43-46.
[2] Buddle B M,S kinner M A,W edlock D N,et al.New generation vaccines and delivery systems for control of bovine tuberculosis in cattle and wildlife[J].Vet Immunol Immunopathol,2002,87:177-185.
[3] Wilkins M J,Meyerson J,Bartlett P C,et al.Human Mycobacterium bovisInfection and bovine tuberculosis outbreak,Michigan,1994-2007[J].Emerg Infect Dis,2008,14(4):657-660.
[4] Ju W,Liu J Y,Yu Y L.Expression of HSP65 from Mycobacterial tuberculosis in eukaryotic cell system[J].Immunol,2005,21:216-217.
[5] 萨姆布鲁克 J,拉塞尔D W.分子克隆实验指南[M].黄培堂,等译.北京:科学出版社,2002:888-897.
[6] Singh H B,Singh P,Jadaun G P,et al.Simultaneous use of two PCR systems targeting IS6110 and MPB64 for confirmation of diagnosis of tuberculous lymphadenitis[J].Commun Dis,2006,38(3):274-279.
