肉葡萄球菌tat-gfp融合基因的构建与表达

Construction of tat-gfp Fusion Gene and Its Expression in Staphylococcus carnosus

将PCR扩增的肉葡萄球菌铁离子过氧化物酶基因(fepB)的双精氨酸转运(Tat)信号肽DNA序列与绿色荧光蛋白基因(gfp)亚克隆到pCX9质粒中构建可表达的tat-gfp融合基因,再将形成的pCX19-Tat-GFP质粒电转化转入肉葡萄球菌宿主中。提取阳性克隆子质粒进行酶切验证,表明表达载体pCX19-Tat-GFP成功地构建并转化。用木糖诱导重组菌株后,分别使用荧光显微镜和SDS-PAGE检测外源GFP蛋白的表达分泌情况。结果显示,菌体能够发出绿色的荧光,且蛋白电泳能够在细胞质与细胞壁组分中检测到GFP蛋白条带,表明肉葡萄球菌宿主可以将表达载体pCX19-Tat-GFP表达的GFP在细胞质中正确折叠,并以活性形式转运到细胞壁部分。

PCR-amplified fepB-tat fragment of Staphylococcus carnosus TM300 and green fluorescent protein （gfp） gene were sub- cloned into plasmid pCX19 to generate the expressible tat-gfp fusion gene,and the resulting plasmid pCX19-Tat-GFP was transformed into S. carnosus TM300 host by electroporation. The positive clones were identified by double restriction enzyme digestion. By xylose induction, the target protein was observed active in vivo by fluorescence microscopy and also detectable in protoplast and cell wall fractions of S. carnosus TM300 host by SDS-PAGE,but not in the culture broth. The results suggested that Tat-GFP fusion protein produced by this recombinant was folded correctly in cytoplasm and actively translocated in cell wall,but not released extracellularly.