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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。
Objective: To construct an eukaryotic expression vector of Rv3586 gene of Mycobacterium tuberculosis (Mtb) cyclic diadenosine monophosphate (c-di-AMP) synthetase and express it in COS-7 cells. Methods: The three domains of Rv3586 were amplified by PCR using the Mtb genome as a template. The eukaryotic expression plasmids were respectively cloned into pEGFP-N3 and identified by colony PCR, plasmid digestion and sequencing. The recombinant plasmid was transfected into COS-7 cells by lipofectamine and the expression of the target gene in COS-7 cells was detected by indirect immunofluorescence. Results: Three Rv3586 domain genes were successfully amplified by PCR. The results of colony PCR, plasmid digestion and plasmid sequencing showed that the target fragment was inserted successfully. The eukaryotic expression vector containing the three domain genes of Rv3586 was successfully constructed. Indirect immunofluorescence assay showed that the three domain proteins of Rv3586 were successfully expressed in COS-7 cells. CONCLUSION: The eukaryotic expression vector of the three domain genes of Rv3586 has been successfully constructed and successfully expressed in COS-7 cells, which lays the foundation for the study of function and application of the subsequent Mtb Rv3586 domain.