r结核分枝杆菌PhoP／PhoR基因重组穿梭表达质粒的构建及鉴定

为构建结核杆菌PhoPR双组分系统PhoP、PhoR基因的重组穿梭质粒pMV361-PhoP、pMV361-PhoR并对其进行鉴定。采用结核分枝杆菌国际标准强毒株H37Rv菌株基因组DNA为模版，利用PCR技术分别扩增PhoP、PhoR基因编码序列，先克隆于T—Vector pMD19（Simple），再亚克隆至大肠杆菌-分枝杆菌穿梭表达载体pMV361，并转化E.coli DH5α感受态细胞，提取所得的阳性克隆子获得重组表达质粒pMV361-PhoP、pMV361-PhoR，通过PCR及双酶切鉴定。结果显示，结核分枝杆菌国际标准强毒株H37Rv菌株基因组DNA中扩增出的PhoP、PhoR基因与GenBank公布的序列一致，经过PCR及双酶切鉴定，表明PhoP、PhoR基因均成功插入了pMV361穿梭载体。由此可知，成功构建了重组穿梭质粒pMV361-PhoP、pMV361-PhoR，为进一步研究PhoPR双组分系统奠定了基础。

Construction and Identification of E. coli-Mycobacterium Recombinant Shuttle Expression Plasmid for PhoP/PhoR of Mycobacterium Tuberculosis

To construct and identify the recombinant shuttle plasmid pMV361-PhoP and pMV361-PhoR of Mycobacterium tuberculosis of PhoPR two-component system. PCR was used to amplify PhoP, PhoR from the genomic DNA of the Mycobacterium tuberculosis H37Rv. The gene was inserted initially into T-Vector pMD19 （Simple） ,and then was subcloned into pMV361 and was transformed into E. coli DHSa. We extracted the positive clones to obtain recombinant plasmid and identified them by restriction en-donuclease and PCR.The cloned PhoP and PhoR of Mycobacterium tuberculosis H37Rv were consistent with the sequence in GenBank. The recombinant plasmid pMV361-PhoP and pMV361-PhoR were successfully constructed after restriction endonuclease and PCR. The pMV361-PhoP and pMV361-PhoR were successfully constructed,which laid a foundation for further study.