中文摘要：

目的构建PhoP和PhoR基因过表达结核分枝杆菌国际标准强毒株H37Rv（以下简称H37Rv）,检测不同电压条件下电转化构建的PhoP/PhoR基因过表达H37Rv菌株PhoP/PhoR基因的表达水平。方法提取并鉴定结核分枝杆菌重组穿梭质粒pMV361-PhoP和pMV361-PhoR,利用电转化技术将结核分枝杆菌重组穿梭质粒pMV361-PhoP和pMV361-PhoR导入H37Rv的感受态细胞中,构建PhoP/PhoR基因过表达H37Rv,应用实时荧光定量PCR检测不同电压条件下电转化构建的PhoP/PhoR基因过表达H37Rv的PhoP/PhoR基因表达水平。结果成功构建PhoP/PhoR基因过表达H37Rv。应用实时荧光定量PCR检测不同电压条件下电转化构建的PhoP/PhoR基因过表达H37Rv的PhoP/PhoR基因表达水平,其中在电压为2 300～2 500V的范围内,电压越高,电转化菌的PhoP/PhoR基因表达量越高。结论成功构建了PhoP/PhoR基因过表达H37Rv菌株,以电压为2 500V电转化构建的PhoP/PhoR基因过表达H37Rv的PhoP/PhoR基因表达量较高。

英文摘要：

Objectives To construct a standard virulent strain of Mycobacterium tuberculosis（H37Rv）overexpressing the genes PhoP and PhoR and to detect levels of PhoP and PhoR expression by an H37 Rv strain of M.tuberculosis that had been constructed via electroporation at different voltages. Methods The M.tuberculosis recombinant shuttle plasmids pMV361 PhoP and pMV361 PhoR were extracted and identified.Electroporation was used to transfect the recombinant shuttle plasmids pMV361 PhoP and pMV361 PhoR into cells sensitized to H37 Rv in order to construct an H37 Rv strain overexpressing PhoP and PhoR.Real-time fluorescent quantitative PCR was used to detect levels of PhoP and PhoR overexpression by an H37 Rv strain constructed via electroporation at different voltages. Results An H37 Rv strain overexpressing PhoP and PhoR was successfully constructed.Real-time fluorescent quantitative PCR was used to detect levels of PhoP and PhoR overexpression by an H37 Rv strain constructed via electroporation at different voltages.In a voltage range of 2 300-2 500 V,the electroporated bacterium had higher levels of PhoP and PhoR expression at higher voltages. Conclusion A standard virulent strain of M.tuberculosis（H37Rv）overexpressing the genes PhoP and PhoR was successfully constructed.An H37 Rv strain of M.tuberculosis constructed via electroporation at a voltage of 2 500 V had high levels of PhoP and PhoR expression.