大鼠组织因子基因克隆及其在C6细胞系中的表达

目的:克隆大鼠组织因子(tissue factor,TF)基因,构建真核表达载体pEGFP-N1-TF,转染C6大鼠神经胶质瘤细胞并检测转染细胞内TF表达水平.方法:采用RT-PCR法从Wistar大鼠肺组织中扩增TF基因,克隆到真核表达载体pEGFP-N1上.通过测序鉴定重组质粒中插入TF的完整性和可靠性.应用脂质体法将鉴定正确的重组质粒转入C6细胞中,荧光显微镜下观察EGFP报告基因的表达强度和转染效率,并对转染细胞的TF-eGFP融合蛋白进行Western blot检测.结果:成功构建pEGFP-N1-TF真核表达载体,转染C6细胞24 h后,在荧光显微镜下可以观测到荧光,并通过Western blot技术检测到TF-eGFP融合蛋白的表达.结论:重组真核表达载体pEGFP-N1-TF构建成功,转染C6细胞后获得了良好的瞬时表达.

Cloning of rat tissue factor gene and its expression in C6 cell line

Objective To clone the rat tissue factor gene(tissue factor,TF) and construct eukaryotic expression vector of pEGFP-N1-TF,transfect the C6 glima cells with the vector and detect the expression of TF gene in the transfected cells.Methods Amplified TF gene from wistar rat by RT-PCR was cloned into the eukaryotic expression vector pEGFP-N1.The integrity and fidelity of TF gene contained in the recombinant plasmids pEGFP-N1-TF were identified by sequencing.Correct recombinant plasmids were transfected into C6 cells by liposome,The expression intensity and transfection efficiency of EGFP reporter gene were observed with fluorescence microscope.Western blot assays were used to analyze the expression of TF-eGFP fusion protein in transfected C6 cells.Results pEGFP-N1-TF eukaryotic expression vector was constructed and transfected into C6 cells successfully.Fluorescence was observed after 24 h through fluorescence microscope.TF-eGFP fusion protein was detected in the transfected C6 cells by Western blot.Conclusions The recombinant eukaryotic vector pEGFP-N1-TF was constructed successfully with transient expression in transfected C6 cells.