变形链球菌F-ATPase启动子荧光蛋白载体的构建及表达

目的构建由质子移位膜ATP酶(membrane-bound proton-translocating ATPase,F-ATPase)启动子启动的绿色荧光蛋白报告基因穿梭表达载体,观察其在大肠埃希菌中的表达同时鉴定表达产物。方法以变形链球菌(UA159)基因组为模板,扩增F-ATPase启动子片段,构建由F-ATPase启动子启动的绿色荧光表达载体pFgfp,酶切F-ATPase启动子及绿色荧光蛋白编码基因,连接到穿梭质粒pDL276,构建重组载体pLFgfp。结果重组质粒pLFgfp酶切及基因序列分析证实目的片段成功插入,重组载体转化后的大肠埃希菌有绿色荧光蛋白的表达,并能随着细菌传代继续表达。结论 F-ATPase启动子启动的绿色荧光蛋白穿梭表达载体pLFgfp构建成功,为研究生物膜环境中耐酸菌F-ATPase毒力因子的表达奠定基础。

Construction of a green fluorescent protein vector driven by F-ATPase promoter of Streptococcus mutans and its expression in Escherichia coli

Objective To construct a green fluorescent protein expression vector driven by F-ATPase operon promoter, and characterize its expression in Escherichia coll. Methods The promoter of F-ATPase was amplified from Streptococcus mutans UA159, and inserted into plasmid pEgfp-N1 to construct pFgfp with a green fluorescent coding gene, then connected to the shuttle vector pDL276 to construct pLFgfp. Results The successful construction of pLFgfp plasmid was confirmed by enzyme digestion and gene sequencing. The E. coli transformed with the pLFgfp plasmid expressed reporter gene GFP, and continued to express as the bacteria passaged. Conclusion This study successfully constructed the recombinant plasmid pLFgfp. The recombination vector constructed can be used to study F-ATPase gene expression by green fluorescence detection.