结核杆菌活动期高表达基因Rv1776c的克隆及其在耻垢分枝杆菌中的表达

Cloning of high expression gene Rv1776c in active phase of Mycobacterium tuberculosis and its expression in Mycobacterium smegmatis

目的构建表达结核分枝杆菌Rv1776c基因的重组耻垢分支杆菌,并鉴定该基因在重组耻垢分支杆菌中的活性。方法采用PCR技术克隆结核分枝杆菌Rv1776c基因,构建大肠埃希菌-分支杆菌穿梭表达质粒pMV-Rv1776c,通过酶切和测序鉴定其正确性,用电穿孔法将重组质粒转染到耻垢分支杆菌mc^2155中。以SDS-PAGE及Western blot检测证实Rv1776c蛋白在重组耻垢分支杆菌内的表达。结果重组耻垢分支杆菌构建成功,生长曲线说明重组质粒不会影响耻垢分支杆菌的体外生长;SDSPAGE及Western blot检测证实Rv1776c在耻垢分枝杆菌内表达出相对分子量约56kD的Rv1776c蛋白。结论成功构建了Rv1776c基因的穿梭质粒pMV-Rv1776c,且该质粒在耻垢分枝杆菌内具有生物活性,为进一步研究其表达产物的功能提供基础。

Objective To construct a recombinant Mycobacterium tuberculosis Rv1776c gene,and identify its activity in recombinant Mycobacterium smegmatis.Methods PCR method was used to clone Mycobacterium tuberculosis Rv1776c gene.Escherichia coli-mycobacteria shuttle expression plasmid pMV-Rv1776c was constructed,and confirmed by enzyme digestion and sequencing.The recombinant plasmid was transfected into Mycobacterium smegmatis mc^2 155 with electroporation method.The expression of Rv1776c protein in recombinant Mycobacterium smegmatis was confirmed by using SDS-PAGE and Western blot.Results The recombinant Mycobacterium smegmatis was constructed successfully.The growth curve showed that the recombinant plasmid did not affect the growth of Mycobacterium smegmatis in vitro.SDS-PAGE and Western blot confirmed that the Rv1776c expressed in the Mycobacterium smegmatis had a relative molecular weight of about 42 kD.Conclusion The shuttle plasmid pMV-Rv1776c of Rv1776c gene was successfully constructed,and its biological activity was also expressed in Mycobacterium smegmatis,which provides the basis for further research on its function.