Fas相关死亡结构域蛋白荧光真核表达载体的构建及其鉴定

Construction and identifcation of pEGFP-N1-FADD eukaryotic fluorenscent expression vector

目的:构建真核表达载体pEGFP-N1-FADD并检测其在结肠癌细胞株SW480中的表达.方法:设计人FADD特异性引物,从人结肠癌细胞SW480细胞提取总RNA,通过RT-PCR方法获取人FADD全长cDNA,定向克隆至真核表达载体pEGFP-N1.应用PCR、酶切和DNA测序进行鉴定,确认后转染人结肠癌细胞SW480.G418抗性筛选获得FADD稳定表达细胞克隆,应用Westernblot检测FADD的表达水平.结果:测序及酶切鉴定证明获得人全长FADD基因,FADD基因正确插入pEGFP-N1中,在荧光显微镜下观察到绿色荧光蛋白在SW480细胞中的稳定表达,Westernblot检测结果显示稳定转染pEGFP-N1-FADD的细胞FADD表达水平增高,是未转染细胞的2.34倍.结论:成功构建真核表达载体pEGFP-N1-FADD,并且在SW480细胞中稳定表达.

AIM： To construct the recombinant plasmid pEGFP-N1-FADD with gene recombinant technique and detect its expression in SW480 cells. METHODS： FADD full length cDNA was amplified by RT-PCR with total RNA extracted from human colon carcinoma SW480 cells as template, and cloned into eukaryotic expression vector pEGFP-N1. Recombinant plasmid of pEGFP-N1-FADD was identified by restriction endonuclease analysis and DNA sequencing. Then the pEGFP-N1- FADD plasmid was transfected into SW480 cells. SW480 cell clones with FADD over-expression was screened by G418 selection. FADD expression was determined by Western blot analysis. RESULTS： The full-length human FADD cDNA was obtained and identified correct through sequencing and enzyme digestion. FADD cDNA was correctly inserted into pEGFP-N1. The ex- pression of EGFP in SW480 cells transfected with pEGFP-N1-FADD was observed by fluorescence microscopy. Western blotting analysis showed that FADD expression significantly increased af- ter pEGFP-N1-FADD transfection in SW480 cells in comparison with that in the controls. CONCLUSION： The eukaryotic expression vector pEGFP-N1-FADD is successfully constructed, which can stably express FADD in SW480 cells.