NSPc1-siRNA重组慢病毒的构建及其对U87细胞NSPc1基因的沉默效应  

Construction of NSPc1-si RNA recombinant lentivirus and its silencing effects in U87 cells

作　　者：樊嵘[1] 李辉[2] 沈静[3] 龚燕华[1] 

机构地区：[1]武警后勤学院生物化学与分子生物学教研室,天津300309 [2]武警后勤学院人体解剖与组织胚胎学教研室,天津300309 [3]武警后勤学院附属医院脑科医院神经内科,天津300162

出　　处：《武警后勤学院学报（医学版）》2014年第12期981-984,F0002,共5页Journal of Logistics University of PAP（Medical Sciences）

基　　金：国家自然科学基金青年项目(81201757);天津市自然科学基金青年项目(13JCQNJC09600);武警后勤部项目(WJHQ2012-13)

摘　　要：【目的】构建包装NSPc1-si RNA慢病毒颗粒,并在U87胶质瘤细胞中鉴定其感染及基因沉默效果。【方法】根据Gen Bank中基因信息,采用干扰序列设计软件设计靶点,制备合成GV118-si RNA目的质粒,转化感受态细胞,对于长出的克隆应用菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和对比分析,重组病毒质粒与另外2种辅助包装载体质粒通过Lipofectamine TM2 000共转染293T细胞,培养48 h后,收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度;并检测病毒颗粒在目的细胞U87胶质瘤细胞中的感染效率,实时荧光定量PCR(RT-PCR)和Western blotting方法检测NSPc1 si RNA慢病毒对U87中NSPc1的干扰作用。【结果】成功构建NSPc1 si RNA慢病毒载体GV118-si RNA,重组病毒滴度为4×108TU/ml;用该病毒体外感染U87胶质瘤细胞,当感染复数(MOI)为10时,感染效率大于90%;RT-PCR和Western blotting方法检测NSPc1基因沉默的效率分别为66.4%、60.0%。【结论】成功构建了NSPc1 si RNA慢病毒载体GV118-si RNA,该重组病毒包装后在体外感染U87胶质瘤细胞的效率较高,且具有显著的基因沉默效果。[Objective]To construct the lentivims vector that encode NSPcl-siRNA and examine the efficiency of its infection and gene silencing in the U87 cells. [ Methods ] According to genetic information in GenBank, target was designed by the interference sequence design software. The target plasmid GV118-siRNA was prepared by connecting the linearized vector GV118 and the DNA segment by using T4 DNA Ligase. The target plasmids were transformed into E.coli competent cells. The grown colonies were identified by colony PCR, and then the positive colonies were sequenced and comparative analyzed. Recombinant lentivirus vector plasmids and the other two auxiliary plasmids were coransfected into 293T cells by using Lipofectamine TM2 000, and the cell culture supematant was collected 48 h later. The virus supernatant was concentrated and virus titer was determinated in 293T cells. The infection efficiency of the constructed virus was examined in U87 cells and the gene silencing efficiency was determined by real time quantitative PCR and Western blotting. [ Results ] NSPcl siRNA was successfully cloned into lentivirus vector GV118, and the titer of constructed virus was 4 ×10^8 TU/ml. The infection efficiency of the virus in U87 cells was more than 90% at a multiplicity of infection （MOI） of 10. NSPcl was significantly knocked down by the recombinant lentivirus vector. The mRNA and protein expression levels of NSPcl were decreased by 66.4% and 56% respectively （P 〈 0.05）. [ Conclusion ] Lentivirus vector encoding NSPcl siRNA were successfully constructed and the recombinant virus can effectively infect U87 glioma cells and knock down NSPcl expression.

关 键 词：NSPC1 SIRNA 慢病毒载体 基因沉默 U87细胞 

分 类 号：R393[医药卫生—基础医学]