结核分枝杆菌FurB基因真核表达质粒的构建及表达

目的构建结核分枝杆菌铁调控基因furB真核表达载体。方法以BamHⅠ和HindIII双酶切pQE-80L-furB获得furB基因,克隆入真核表达载体pcDNA3.1(-),重组质粒酶切鉴定后以阳离子聚合物转染COS-7细胞,分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达。结果构建了重组质粒pcDNA3.1(-)-furB,RT-PCR结果证明furB可在COS-7细胞中转录,用间接免疫荧光检测,显示COS-7细胞内有FurB蛋白的表达。结论成功构建了结核分枝杆菌furB基因的真核表达载体pcDNA3.1(-)-furB,furB基因可以在COS-7细胞中表达。

Construction and expression of the eukaryotic expression vector for furB gene of Mycobacterium tuberculosis

To construct and express the eukaryotic expression vector for furB gene of Mycobacterium tuberculosis,the furB gene was obtained from plasmid pQE-80L-furB digested by BamHI and HindIII,and then was inserted into the eukaryotic expression vector pcDNA3.1(-).The recombinant plasmid pcDNA3.1(-)-furB was transfected to COS-7 cells by use of liposome.By use of the RT-PCR and indirect immunofluorescent assay,the expressions of mRNA and the expressed proteins were determined.respectively.The experimental results showed that the gene furB was cloned into pcDNA3.1(-) correctly and its expression of proteins encoded by this gene could be detected in COS-7 cells.It is concluded that the furB gene of M.tuberculopsis can be expressed successfully in the COS-7 cells,thus to provide basis for the further study on the function of this gene..