表皮生长因子受体基因RNA干扰慢病毒载体的构建和鉴定研究  

Construction and Identification of Lentiviral Vector-mediated RNA Interference of Epidermal Growth Factor Receptor

作　　者：金晶[1] 马跃辉[1] 詹仁雅[1] 周永庆[1] 俞建波[1] 

机构地区：[1]浙江大学医学院附属第一医院,浙江省杭州市310003

出　　处：《临床合理用药杂志》2013年第15期16-18,共3页Chinese Journal of Clinical Rational Drug Use

摘　　要：目的构建表皮生长因子受体(EGFR)基因RNA干扰(RNAi)慢病毒载体,为其体内外实验研究提供依据。方法合成EGFR基因的miRNA干扰序列,构建pcDNATM6.2-GW/EmGFP-miR-EGFR质粒,测序验证插入序列正确,连接到慢病毒载体pLenti6.3-MCS/V5 DEST中,获得pLenti6.3-EGFR-miRNA质粒,与慢病毒包装质粒Packaging MIX、POLOdelivererTM3000 Transfection Reagent共转染293T细胞株,细胞包装产生慢病毒颗粒,测定慢病毒滴度。结果 PCR和测序结果证实,EGFR miRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液滴度为1.8×106ifu/ml。结论成功构建EGFR基因RNAi慢病毒载体,为研究其在胶质瘤细胞中的生物学功能奠定基础。Objective To construct a lentiviral ventor-mediated RNA interference of EGFR gene and provide the basis for further experiments in vivo and in vitro. Methods A miRNA of EGFR gene was synthesized.A plasmid pcDNATM6.2-GW/EmGFP-miR-EGFR was constructed and confirmed by sequencing,then was cloned into lentiviral expression vector pLenti6.3-MCS/V5 DEST.The recombined vector pLenti6.3-EGFR-miRNA was cotransfected with the Packaging MIX and POLOdelivererTM 3000 Transfection Reagent into 293T cells.Virus was synthesized and the titer was measured. Results PCR and DNA sequencing demonstrated that the inserted EGFR miRNA sequence was corrected.The titer of lentiviral virus was 1.8×106 ifu/ml. Conclusion The lentivirus RNAi vector targeting EGFR has been successfully constructed,which will lay a foundation for the further study on biological function of EGFR in glioma cells.

关 键 词：受体 表皮生长因子 RNA干扰 慢病毒载体 

分 类 号：R739.4[医药卫生—肿瘤]