结核分枝杆菌Rv3586结构域基因真核表达载体的构建及鉴定

目的:构建结核分枝杆菌(Mycobacterium tuberculosis,Mtb)环二腺苷酸(cyclic diadenosine monophosphate,c-di-AMP)合成酶Rv3586结构域基因的真核表达载体,并在COS-7细胞中表达。方法:以Mtb基因组为模板PCR扩增Rv3586三个结构域基因,分别克隆入pEGFP-N3真核表达质粒,用菌落PCR、质粒酶切和测序方法对插入序列进行鉴定。通过脂质体将重组质粒转染COS-7细胞,间接免疫荧光法检测目的基因在COS-7细胞内的表达。结果:PCR成功扩增出Rv3586三个结构域基因,菌落PCR、质粒酶切和质粒测序鉴定结果表明成功插入目的片段,包含Rv3586的三个结构域基因的真核表达载体构建成功。间接免疫荧光法结果显示,Rv3586三个结构域蛋白在COS-7细胞中表达成功。结论:成功构建Rv3586三个结构域基因的真核表达载体,并在COS-7细胞中表达成功,为后续Mtb Rv3586结构域的功能和应用研究奠定了基础。

Construction and Identification of Eukaryotic Expression Vectors for Mycobacterium tuberculosis Rv3586 Domains

ABSTRACT Objective: To construct eukaryotic expression plasmids for three domains of Rv3586, a dinucleotide cyclase of cyclic diadenosine monophosphate (c-di-AMP) of Mycobacterium tuberculosis, and express in COS-7 cells. Methods: Three genes of Rv3586 domains were amplified by PCR and cloned into eukaryotic expression vector pEGFP-N3, which were identified by colony PCR, plasmid enzyme digestion and DNA sequencing. The recombinant plasmids were transfected into COS-7 cells by liposome. The expression of target genes in COS-7 cells was detected by indirect immunofluorescence. Results: Three genes of Rv3586 domains were amplified by PCR and cloned into eukaryotic expression plasmids respectively, which were confirmed by colony PCR, enzyme digestion and DNA sequencing. And the results of indirect immunofluorescence implied that target genes were expressed in COS-7 cells. Conclusion: The eukaryotic expression plasmids of Rv3586 domains were constructed and expressed in COS-7 cells successfully, which will be helpful for the further research on function and application of M. tuberculosis Rv3586.