RNA干扰EGFR基因家族真核表达载体的构建.pdfVIP

重庆医科大学学报2008年第33卷第4期 (Journal of Chongqing Medical University 2008．Vo1．33 No．4) 一 399一 论 著 文章编号：0253—3626(2008)04—0399—04 RNA干扰EGFR基因家族真核表达载体的构建 黄 环 ，吴永忠 ，李少林，，郭启帅。，林远洪。，彭志平 (1．重庆医科大学附属第一医院肿瘤科，重庆 400016；2．重庆市肿瘤研究所，重庆 400030； 3．重庆医科大学基础医学院放射医学教研室，重庆 400016) 【摘 要】目的：构建针对表皮生长因子受体(Epidermal growth factor recepter，EGFR)家族的小分子发夹状RNA(Small hairpin RNA，shRNA)干扰质粒表达载体，为本课题下一步研究提供有力工具。方法：选择放疗不敏感的卵巢癌细胞SKOV3中高表达 的EGFR家族同源性基因为靶基因，根据shRNA的设计原则，设计实验组及对照组shRNA。体外合成2段编码短发夹RNA序 列的DNA单链，退火，克隆到相应载体pGensil一1的polⅢ启动子下游。结果：经酶切和DNA测序鉴定成功构建了EGFR家族 同源性基因的RNA干扰表达载体，分别命名为pGensil一1一E及pGensil一1一C。为下一步体外、体内实验奠定了基础。 【关键词】EGFR家族；RNA干扰；真核细胞表达载体 【中国图书分类法分类号】Q784 【文献标识码】A 【收稿日期】2007—09—10 Construction of eukaryotic expression plasmid experssing shRNA targeting EGFR gene family HUANG Huan．et (Department of Ontology，the First Affiliated Hospital，Chongqing Medical University) 【Abstract】Objective：To construct the eukaryotic expression plasmid expressing shRNA targeting EGFR gene family as a tool for following experiments in vitro and in vivo．Methods：EFGR family highly expressed in low radiosensitive ovarian cancer cells SKOV3，chosen highly expressed homologous genes in EFGR family aft targeted gene．According to guidelines for shRNA design，designed experimental shRNA and negative control shRNA．In vitro synthesized 2 of the coding sequence of short hairpin RNA DNA single-strands。annealing，and cloned into the corresponding vector pGensil一1 of pol Ⅲ promoter downstream，named pGensil一1一E and pGensil一1一C． Confirm ed by restrict endonuclease digestion and DNA sequencing． Conclusions：Eukaryotic expression plasmid experssing shRNA targeting homologous genes in EFGR family was constructed successfully． 【Key words】EFGR family；RNA interference；Eukarvotic expression vector 妇科恶性肿瘤是危及妇女生命的顽症。三大妇 R