牛FADD基因过表达和RNAi载体构建及在牛胎儿成纤维细胞中的表达

Over-expression and RNAi Vector Construction of Cattle FADD Gene and Its Expression in Bovine Fetal Fibroblasts

FADD(Fas-associated death domain protein)存在于Fas/FasL系统中,是信号传导通路的一个信号连接蛋白,FADD通过传递凋亡信号,从而介导细胞凋亡。为进一步验证FADD基因抑制细胞增殖和促进细胞凋亡的作用,从牛卵巢组织中扩增FADD基因,将FADD基因连接到带有绿色荧光蛋白报告基因的真核表达载体pAcGFP-N1中,构建过表达FADD基因载体,并构建FADD基因的RNAi载体;用脂质体介导法将FADD基因RNAi载体、真核表达载体转染到牛胎儿成纤维细胞中,观察有无荧光的表达,并使用Real-Time qPCR和Western blot方法检测FADD基因mRNA、蛋白水平的表达情况。结果表明:成功构建出FADD基因RNAi载体和高表达载体,重组质粒转染牛胎儿成纤维细胞24 h后在荧光显微镜下可观察到绿色荧光,转染效率可达50%。

Fas-associated death domain protein（ FADD） is connected to a signal protein signaling pathway in Fas / FasL system which mediates apoptosis guide by passing apoptotic signals. To further verify the function of FADD gene in inhibiting cell proliferation and promoting apoptosis,we cloned FADD gene in bovine ovary tissue with molecular cloning technique,directionally cloned the amplified FADD gene into eukaryotic expression vector pAcGFP-Nl including AcGFP and constructed the fusion protein recombinant plasmid. Using gene-silencing technology,we constructed RNA interference（ RNAi） vector. And then we transfected pAcGFP-N1-FADD and RNAi into bovine fetal fibroblasts（ BEF） cell mediated by Lipofectamine 2000,observed the expression of AcGFP and detected the mRNA and protein level of FADD by Real-Time qPCR and Western blot. The results showed that cattle FADD gene was successfully cloned,and RNAi vectors and pAcGFP-N1-FADD fusion protein eukaryotic expression vector were successfully constructed. Recombinant plasmid bovine fetal fibroblasts（ BEF） under a fluorescence microscope after 24 h green fluorescence were observed,and the transfection efficiency was up to 50%. In this study,we used Real-Time qPCR and Western blot methods to verify the changes of FADD in gene expression level after transfection,and provided an experimental basis for the subsequent induction of apoptosis gene and further study of FADD materials.