CB-GFP融合基因在COS-7细胞中的表达与定位

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

目的:构建以绿色荧光蛋白(green fluorescent prote in,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COS-7细胞后,以RT-PCR和W estern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W estern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,COS-7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COS-7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。

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. Mter 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.