表达绿色荧光蛋白的重组耻垢分枝杆菌的构建

目的 建立可表达绿色荧光蛋白的耻垢分枝杆菌，便于对耻垢分枝杆菌进行直观检测和快速定量。方法利用PCR技术从真核表达质粒pLVTH扩增获得绿色荧光蛋白的编码基因，克隆人大肠埃希菌一分枝杆菌穿梭载体pMV261，建立重组质粒pMVGFP，并经酶切鉴定证实。利用电穿孔技术将pMVGFP转化入耻垢分枝杆菌，利用卡那霉素抗性筛选重组耻垢分枝杆菌克隆，扩大培养后直接涂片，荧光显微镜镜检。结果重组质粒pMVGFP构建正确；将重组耻垢分枝杆菌在荧光显微镜下观察，证实绿色荧光蛋白在重组耻垢分枝杆菌中的表达。结论自发释放荧光的重组耻垢分枝杆菌的成功建立，为研究结核病致病机制和快速筛选化学药物等奠定了基础。

Construction of Recombinant Mycobacterium Smegmatis Expressing GFP Protein

Objective In order to directly detect and quickly quantify Mycobacterium smegmatis strain, the recombinant M. smegmatis expressing GFP protein was constructed. Methods GFP encoding fragment was amplified by PCR from eukaryotic expression plasmid pLVTH and cloned into E. coli-mycobacterium shuttle vector pMV261. The resultant plasmid pMVGFP was identified by digestion with enzymes, pMVGFP transformed M. smegmatis clones were selected by karnamycin resistance. Expression of GFP in transformed M. smegmatis clones was observed under fluorescent mi- croscope. Results Recombinant plasmid pMVGFP was constructed correctly. The pMVGFP transformed M. smegmatis sufficiently expressed GFP. Conclusion The established recombinant M. smegmatis expressing GFP may be usable in the study of pathogenesis of tuberculosis and rapid screening of new anti-TB drugs.