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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。
To construct and identify the recombinant shuttle plasmids pMV361-PhoP and pMV361-PhoR of PhtP and PhoR genes of Mycobacterium tuberculosis PhoPR two-component system. Using the genomic DNA of H37Rv strain of Mycobacterium tuberculosis as a template, the coding sequences of PhoP and PhoR genes were amplified respectively by PCR and cloned into T-Vector pMD19 (Simple) and subcloned into E.coli - The recombinant plasmid pMV361-PhoP and pMV361-PhoR were transformed into E.coli DH5α competent cells. The positive clones were extracted and identified by PCR and restriction enzyme digestion. The results showed that PhoP and PhoR genes amplified from genomic DNA of H37Rv strain of Mycobacterium tuberculosis were the same as those published in GenBank. PCR and double enzyme digestion showed that both PhoP and PhoR genes were successfully inserted into pMV361 Shuttle carrier. Thus, we successfully constructed recombinant shuttle plasmid pMV361-PhoP, pMV361-PhoR, which laid the foundation for further study of PhoPR two-component system.