人Notch1基因RNA干扰慢病毒载体构建及鉴定.docVIP

人Notch-1基因RNA干扰慢病毒载体的构建及鉴定 　　[文章编号] 1000-1182(2014)03-0267-06 　　[摘要] 目的 构建及鉴定人Notch-1基因的RNA干扰（RNAi）慢病毒载体，寻找最佳RNAi慢病毒载体。方法 针对人Notch-1基因序列，按照RNAi序列设计原则，设计3段RNAi靶点序列（shRNA1~3），通过限制性内切酶BamHⅠ和EcoRⅠ双酶切、T4 DNA连接酶连接，将Notch-1基因序列插入慢病毒载体pLenOR-THM，构建pLenOR-THM-Notch-1重组载体。质粒转化感受态DH5α细菌，筛选阳性克隆，经KpnⅠ和EcoRⅠ双酶切及测序鉴定正确后通过脂质体将慢病毒四质粒系统共转染293T细胞，进行慢病毒包装并测定病毒滴度，观察感染效率。各组病毒载体转染ACC-M细胞后，运用定量逆转录聚合酶链反应和Western blot检测Notch-1基因mRNA和蛋白的表达水平。结果 成功构建慢病毒载体pLenOR-THM-Notch-1，四质粒共转染293T细胞后可见大量绿色荧光；浓缩后的病毒滴度为 　　5.8×108 TU?mL-1；以复感染系数为1时感染293T细胞，感染效率在90％以上。QRT-PCR和Western blot检测结果表明，pLenOR-Notch-1-shRNA3组受抑制程度最高。结论 成功构建了人Notch-1 RNAi慢病毒载体。 　　[关键词] Notch-1基因；慢病毒载体；RNA干扰 　　[中图分类号]R 782 [文献标志码] A [doi] 10.7518/hxkq.2014.03.014 　　 　　Construction and identification of a lentiviral vector of RNA interference containing human Notch-1 gene Zhang Qing-qing1,2, Zhang Senlin 1,2, Zhu Yinglan1,2, Dong Zhen2, Cao Gang2, Chen Wei1,2. (1. Dept. of Stomatology, Nanjing School of Clinical Medicine, Southern Medical University, Nanjing 210002, China; 2. Dept. of Stomatology, Nanjing General Hospital of Nanjing Military Command, Nanjing 210002, China) 　　[Abstract] Objective To construct and identify a lentiviral vector of RNA interference targeting human Notch-1 gene. Methods To determine the Notch-1 gene sequences, three RNAi target sequences (shRNA1-3) were designed in accordance with the RNAi sequence design principles and cloned into the lentiviral vector pLenOR-THM by endonuclease BamH#8198;Ⅰ#8198;res-triction, EcoR#8198;Ⅰ#8198;double digestion, and T4 DNA-ligase ligation. After the transformation into competent DH5α bacteria, the candidate clones were identified by Kpn#8198;Ⅰ#8198;and EcoR#8198;Ⅰ#8198;double digestion and DNA sequencing. The recombinant and three packaging plasmids were co-transfected into human embryonic kidney cell line 293T cells by lipofectamine to produce the lentiviral particles. The viral titer was determined. The 293T cells were infected by the lentiviral particles obtained, and