运用绿色荧光蛋白探讨肉葡萄球菌双精氨酸分泌途径

GFP Translocation of Twin-arginine Secretion Pathway in Staphylococcus carnosus

根据肉葡萄球菌(Staphylococcus carnosus)TM300的基因组序列设计引物,经PCR扩增得到其tufA基因的启动子Peftu片段与大肠杆菌–葡萄球菌穿梭载体pBT2-Tat-GFP连接,构建了穿梭质粒pBT2-ETG.结果表明,穿梭质粒pBT2-ETG成功地转入大肠杆菌与肉葡萄球菌宿主中稳定表达具有活性的绿色荧光蛋白GFP,并被转运到肉葡萄球菌宿主的细胞壁,为进一步研究肉葡萄球菌双精氨酸(Tat)转运系统正确分泌其他外源蛋白奠定了实验基础.

According to the genome sequence of Staphylococcus carnosus TM300 genome,a pair of primers was designed for PCR amplification of promoter Peftu of tufA gene. The PCR-amplified promoter Peftu fragment was cloned into plasmid pBT2-Tat-GFP and chemically transformed into E. coli host. A shuttle vector pBT2-ETG was thereby constructed and succ-essfully transformed into E. coli and S. carnosus TM300 hosts,respectively. The experimental results reveal that GFP was expressed by the E. coli-Staphylococcus shuttle vector pBT2-ETG in both E. coli and S. carnosus hosts. The expressed GFP was translocated to the cell walls of S. carnosus host in fluorescent active form. Thereby,a preliminary experimental basis was laid for the further study of other exogenous protein secretion via twin-arginine translocation pathway in S. carnosus.