人Notch-1基因RNA干扰慢病毒载体的构建及鉴定  

Construction and identification of a lentiviral vector of RNA interference containing human Notch-1 gene

作　　者：张庆庆[1,2] 张森林[1,2] 朱迎兰 董震[2] 曹罡[2] 陈伟 

机构地区：[1]南方医科大学南京临床医学院口腔科 [2]南京军区南京总医院口腔科,南京210002

出　　处：《华西口腔医学杂志》2014年第3期267-272,共6页West China Journal of Stomatology

基　　金：国家自然科学青年基金资助项目(81102051);江苏省自然科学基金面上资助项目(BK2011659)

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、T4DNA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Westernblot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5.8×108TU·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Westernblot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-1RNAi慢病毒载体。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 BamHⅠres-triction, EcoRⅠdouble digestion, and T4 DNA-ligase ligation. After the transformation into competent DH5αbacteria, the candidate clones were identified by KpnⅠand EcoRⅠdouble 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 trans-fection efficiency was assessed using a fluorescent microscope. The lentiviral vector particles were also transfected into ACC-M cells. The Notch-1 expression in the transfected cells was assayed by quantitative reverse transcription polymerase chain reaction （QRT-PCR） and Western blot analysis. Results The lentiviral RNAi vector pLenOR-THM-Notch1 for Notch-1 gene was constructed successfully. Strong green fluorescence was observed in the 293T cells under fluorescent microscope after co-transfection of the cells with the four plasmids of lentiviral vector. The virus in the supernatant reached a titer of 5.8× 108 TU·mL-1. The transfection efficiency of the collected virus exceeded 90% in 293T cells with 1 as a multiplicity of in-fection. The third lentiviral vector was found to significantly inhibit the Notch-1 expression at the mRNA and protein levels. Conclusion The lentiviral RNAi vector of Notch-1 has been successfully constructed and identified.

关 键 词：Notch-1基因 慢病毒载体 RNA干扰 

分 类 号：R739.8[医药卫生—肿瘤]