结核分支杆菌耐利福平相关基因rpoB真核表达载体的构建及鉴定

Construction and identification of the eukaryotic expression vector of rifampin-resistant mycobacterimn tuberculosis related gene rpoB

目的构建结核分支杆菌耐利福平相关基因rpoB真核表达载体。方法 PCR技术扩增获得rpoB基因,连接入pB luescriptⅡSK克隆载体上,将重组质粒转化入大肠杆菌DH5α内并提取质粒,酶切与DNA测序鉴定准确,再用双酶切方法将rpoB基因定向连接到真核表达载体pQE-Trisystem中,构成pQE-Tri-rpoB,将pQE-Tri-rpoB转化入大肠杆菌DH5α内并提取质粒,双酶切鉴定、DNA测序鉴定准确,脂质体转染P815细胞后,以RT-PCR方法检测mRNA表达。结果构建了重组质粒pQE-Tri-rpoB,RT-PCR结果证明rpoB可在P815细胞中转录。结论成功构建了结核分支杆菌耐利福平相关基因rpoB真核表达载体,rpoB基因可以在P815细胞中表达。

Objective To analyze the effect of rpoB eukaryotic expression vector transfection.Methods The rpoB was amplified by PCR.Then it was cloned to the cloning vector：pBluescript Ⅱ SK vector,identified corrected by digestion with restriction endonucleases and sequencing,Then it was cloned to the expression vector：pQE-Trisystem vector,The recombinant pQE-Tri-rpoB was transformed into E.coli.DH5α,identified by double digestion with restriction endonucleases and sequencing,The recombinant plasmids were transfected into P815 cells by lipofectamine method.The expression of mRNA was detected with RT-PCR.Results It was demonstrated that the genes of rpoB strain could be transcripted within P815 cells.Conclusion indicating the successful construction of the eukaryotic recombinant vector for genes of rpoB strain and the expression of the target protein.