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目的构建结核杆菌泛素样蛋白-蛋白酶体系统中Pup、Dop、PafA和Mpa基因的重组大肠埃希菌-分枝杆菌穿梭表达质粒pMV361/Pup、pMV361/Dop、pMV361/PafA和pMV361/Mpa,并对其进行鉴定。方法提取结核杆菌国际标准强毒株H37Rv基因组DNA,以此为模板PCR扩增Pup、Dop、PafA和Mpa基因编码序列并测序。扩增产物克隆至大肠埃希菌-分枝杆菌穿梭表达质粒pMV361,并转化E.coli DH5α感受态细胞,利用PCR技术、双酶切技术及基因测序技术对构建的重组穿梭表达质粒进行鉴定。结果 PCR扩增的Pup、Dop、PafA和Mpa基因片段分别为213、1 683、1 377、和1 848bp,与预期长度一致;双酶切鉴定Pup、Dop、PafA和Mpa基因均成功插入穿梭表达质粒pMV361,测序显示插入穿梭表达质粒pMV361的Pup、Dop、PafA和Mpa基因序列与GenBank公布的序列一致。结论成功构建了重组大肠埃希菌-分枝杆菌穿梭表达质粒pMV361/Pup、pMV361/Dop、pMV361/PafA和pMV361/Mpa,为进一步研究结核分枝杆菌泛素样蛋白-蛋白酶体系统奠定了基础。
Objective To construct the recombinant E. coli-Mycobacterium shuttle plasmid pMV361 / Pup, pMV361 / Dop, pMV361 / PafA and pMV361 / Mpa for Pup, Dop, PafA and Mpa genes in the ubiquitin-proteasome system of Mycobacterium tuberculosis, And its identification. Methods The genomic DNA of H37Rv strain was extracted from Mycobacterium tuberculosis isolates. The coding sequences of Pup, Dop, PafA and Mpa were amplified by PCR and sequenced. The amplified product was cloned into Escherichia coli - mycobacterium shuttle plasmid pMV361 and transformed into E. coli DH5α competent cells. The recombinant shuttle expression plasmid was identified by PCR, double enzyme digestion and gene sequencing. Results The Pup, Dop, PafA and Mpa gene fragments were 213,1 683,1 377 and 1 848 bp, respectively, which were consistent with the expected length. The double-digestion of Pup, Dop, PafA and Mpa genes was successfully inserted into shuttle expression Plasmid pMV361, sequencing showed that the sequence of Pup, Dop, PafA and Mpa inserted into shuttle expression plasmid pMV361 was consistent with that published in GenBank. Conclusion The recombinant E. coli-mycobacterium shuttle plasmid pMV361 / Pup, pMV361 / Dop, pMV361 / PafA and pMV361 / Mpa were constructed successfully, which laid the foundation for further study on the ubiquitin-proteasome system of Mycobacterium tuberculosis .