吉林农业大学学报Issue(2)：199-204,212,7.DOI:10.13327/j.jjlau.2014.1907
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
摘要
Abstract
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 Lipofec-tamine 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 .关键词
FADD/RNAi载体/pAcGFP-N1/牛胎儿成纤维细胞Key words
FADD/RNA interference(RNAi)vector/pAcGFP-N1/bovine fetal fibroblasts(BEF)分类
农业科技引用本文复制引用
赵茂鑫,于海滨,李傲楠,赵志辉,杨润军,芦春艳..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[J].吉林农业大学学报,2014,(2):199-204,212,7.基金项目
国家“863”计划项目（2013AA102505），国家自然科学基金项目（31000991），教育部新教师基金项目 （）
