CB-GFP融合基因在COS-7细胞中的表达与定位

目的：构建以绿色荧光蛋白（green fluorescent protein，GFP）为报告基因的重组表达质粒pEGFP-N1-CB，转染体外培养的COS-7细胞，以观察CB重组蛋白在真核细胞中的表达及定位。方法：PCR方法扩增得到去除终止密码的cB融合基因序列，克隆入真核表达载体pEGFP-N1中，构建重组表达载体pEGFP-N1-CB。脂质体法转染体外培养的COST细胞后，以RT-PCR和Western印迹方法验证其mRNA及蛋白的表达，并在活细胞状态下用荧光显微镜、激光共聚焦显微成像技术直接观察CB-GFP融合蛋白在细胞中的分布和定位。结果：RT．PCR及Western印迹结果均证明CB—GFP融合基因表达载体pEGFP-N1-CB在COS．7细胞中获得了表达。荧光显微镜观察显示，在空载体pEGFP-N1转染组中，COST细胞内荧光呈弥散分布；重组质粒pEGFP-N1-CB转染组中，绿色荧光主要聚集在细胞浆中。结论：CB融合基因能在真核细胞COST中得到高效表达，且蛋白表达主要定位于细胞浆中，本试验为CB重组蛋白的提取及进一步功能研究奠定了基础。

Expression and localization of recombinant CB-GFP fusion plasmid in COS-7 cell line

Objective: To verify the expression of the recombinant CB gene in mammalian cells,CB-GFP(green fluorescent protein)fusion gene eukaryotic expression vector pEGFP-N1-CB was constructed,and transfected into COS-7 cells.The expression of foreign gene was detected by laser confocus microscope.Methods:The encoding region of CB without terminator was obtained by PCR,and cloned into pGEM-T vector.After the double enzyme cutting,the recombinant CB gene was inserted into the expression vector pEGFP-N1.Thus,the plasmid pEGFP-N1-CB with fusion gene CB-GFP was constructed,and then transfected into COS-7 cells by liposome.The mRNA expression of CB was detected by RT-PCR.The expression and subcellular localization of the fusion protein was detected by Western blot and laser confocus microimaging.Results: RT-PCR verified CB mRNA expression in COS-7 cells.The efficient expression of CB-GFP fusion protein was observed and the fluorescence was mainly located in the cytoplasm of COS-7 cells.Conclusion: The recombinant CB protein can be expressed efficiently in mammalian cells,and this laid a foundation for the further functional researches on recombinant CB protein.