重组激活基因1shRNA慢病毒载体的构建与鉴定  

Construction and Identification of RNAi Lentiviral Vector Targeting Rat Recombination Activating Gene 1

作　　者：陈浩浩[1] 周星娟[1] 周婧[1] 李一乔[1] 韩曙[1] 凌树才[1] 

机构地区：[1]浙江大学医学院人体解剖与细胞生物学系,杭州310058

出　　处：《细胞生物学杂志》2009年第5期671-676,共6页Chinese Journal of Cell Biology

基　　金：国家自然科学基金资助项目(No.30570586)~~

摘　　要：构建3对针对大鼠海马神经元重组激活基因(Rag1)的RNA干扰重组表达载体及1对无关序列的载体,分别命名为shRNA1、shRNA2、shRNA3及negative。经基因测序确认后,采用慢病毒载体系统分别包装4个载体,而后分别感染体外培养的大鼠原代海马神经细胞,收集感染后96h的细胞,通过免疫荧光观察细胞感染情况,并且利用RT-PCR和Western印迹检测Rag1mRNA及蛋白质的表达。经测序鉴定合成的shRNA序列正确,并且慢病毒感染细胞效果良好。LV-shRNA1组、LV-shRNA2组、LV-shRNA3组与正常对照组相比,Rag1mRNA表达都明显减少(P<0.01),其中LV-shRNA3组的抑制效率最高为(76.4±3.5)%,Western印迹结果与其基本一致,而LV-negative组对Rag1mRNA及其表达的蛋白质都无明显影响。实验结果表明,我们成功构建了能特异性抑制大鼠海马神经元Rag1表达的shRNA慢病毒表达载体。Three-pair shRNA （named as shRNA1, shRNA2 and shRNA3） targeting rat recombination activating gene 1 （Ragl） and one-pair independent sequence （negative） were chemically synthesized and confirmed by sequencing analysis. The four different pRNAT-U6.2/Lenti vectors were packaged with lentiviral vector system, and were transferred into the cultured rat hippocampal neuron cells in vitro respectively. After being harvested for 96 h, cells were observed by immunofluorescence, then assayed for expression of Rag 1 by RT-PCR and Western blot. The results indicated that four lentiviral vectors were correct by sequencing analysis and could be infected into most hippocampal neuron cells in vitro. The expression of Ragl mRNA could be down-regulated by LV-shRNA1, LV-shRNA2 and LV-shRNA3 groups respectively compared with the control group （P〈0.01）, and LV-shRNA3 group got the highest efficiency, with the inhibition rate of （76.4±3.5）%. The results of Western blot were similar with that of RT-PCR. The negative lentiviral vector had no such inhibitory effects. In conclusion, the lentiviral vectors that can specifically inhibit the expression of Ragl have been constructed successfully.

关 键 词：重组激活基因1 神经元 SHRNA RT-PCR WESTERN印迹 

分 类 号：Q78[生物学—分子生物学]