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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 construct a recombinant plasmid pEGFP-N1-CB with green fluorescent protein (GFP) as a reporter gene for transfection of COS-7 cells cultured in vitro to observe the expression of CB recombinant protein in eukaryotic cells And positioning. Methods: The sequence of CB fusion gene with the stop codon was amplified by PCR and cloned into eukaryotic expression vector pEGFP-N1 to construct recombinant expression vector pEGFP-N1-CB. After transfected COS-7 cells by liposome method, the mRNA and protein expression of COS-7 cells were verified by RT-PCR and Western blotting. The results of fluorescence microscopy and confocal laser scanning microscopy Observe the distribution and localization of CB-GFP fusion protein in cells. Results: The results of RT-PCR and western blot showed that the expression vector CB-GFP fusion gene pEGFP-N1-CB was expressed in COS-7 cells. Fluorescence microscopy showed that in the empty vector pEGFP-N1 transfected group, the fluorescence of COS-7 cells was diffusely distributed. In the pEGFP-N1-CB transfected group, the green fluorescence mainly accumulated in the cytoplasm. CONCLUSION: CB fusion gene can be highly expressed in eukaryotic cells COS-7, and the protein expression is mainly located in the cytoplasm. This study laid the foundation for the extraction of CB recombinant protein and further study of its function.