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目的:构建结核分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物活性。方法:采用PCR技术克隆结核分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMVe-is,经酶切和测序鉴定其正确性,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155中,采用SDS-PAGE和Western blot检测eis基因在耻垢分枝杆菌中的表达。结果:成功构建结核杆菌eis基因穿梭表达载体pMV-eis;生长曲线说明重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE和Western blot检测证实eis在耻垢分枝杆菌中可表达出相对分子量约42kDa的Eis蛋白。结论:成功构建了eis基因穿梭表达质粒pMV-eis,且该重组质粒在耻垢分枝杆菌中具有生物活性,为下一步研究表达产物Eis的功能奠定了一定基础。
Objective: To construct a shuttle expression vector for eis gene of Mycobacterium tuberculosis and identify its biological activity in recombinant Mycobacterium smegmatis. Methods: The eis gene of Mycobacterium tuberculosis was cloned by PCR and the shuttle vector pMVe-is constructed. The correctness of the eis gene was confirmed by enzyme digestion and sequencing. The recombinant plasmid was transformed into the smegmatis branch by electroporation Bacillus mc2155, SDS-PAGE and Western blot eis gene expression in M. smegmatis. Results: The eis gene shuttle vector pMV-eis was constructed successfully. The growth curve indicated that the recombinant plasmid did not affect the growth of Mycobacterium smegmatis in vitro. SDS-PAGE and Western blot showed that eis was expressed in M. smegmatis The relative molecular weight of about 42kDa Eis protein. CONCLUSION: The eis gene shuttle plasmid pMV-eis was successfully constructed and the recombinant plasmid has biological activity in Mycobacterium smegmatis, which lays the foundation for further study on the function of Eis gene.