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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。
OBJECTIVE: To construct a shuttle vector for green fluorescent protein (EGFP) reporter gene promoter driven by the promoter of membrane-bound proton-translocating ATPase (F-ATPase), and to observe its expression in Escherichia coli. Methods The promoter region of F-ATPase was amplified by using the genome of Streptococcus mutans (UA159) as a template. The green fluorescent expression vector pFgfp promoter, F-ATPase promoter and green fluorescent protein- Connected to the shuttle plasmid pDL276 to construct the recombinant vector pLFgfp. Results The recombinant plasmid pLFgfp digestion and gene sequence analysis confirmed that the target fragment was inserted successfully. The E. coli transformed with the recombinant vector had the expression of green fluorescent protein, and could continue to be expressed with the passage of bacteria. Conclusion The shuttle vector pLFgfp initiated by F-ATPase promoter was successfully constructed and laid the foundation for the study of the expression of F-ATPase-resistant virulence factors in biofilm environment.