HuR基因的慢病毒干扰载体构建及其病毒包装

摘要

目的 构建人HuR基因慢病毒干扰载体和基因敲低细胞株,为揭示C/EBPβ3'UTR RNA 与HuR蛋白相互作用抑制肝癌细胞增殖的分子机制奠定基础.方法 根据shRNA设计原则设计人HuR基因的shRNA序列,用化学合成法合成shDNA序列,以LV10(pGLVU6/RFP/Puro)为载体骨架,构建慢病毒干扰载体LV10-HuR,并与慢病毒包装辅助载体(pLP1、pLP2和pLP/VSVG)共转染HEK293T细胞,收集病毒液纯化后感染SMMC-7721细胞并用嘌呤霉素杀死未感染的肝癌细胞,荧光显微镜下挑取单克隆红色荧光细胞集落,扩大培养后通过Western blot验证肝癌细胞内源性HuR蛋白表达.结果 经DNA测序鉴定,LV10-HuR正是所需的慢病毒干扰载体.通过共转染获得的重组慢病毒颗粒感染的肝癌细胞在荧光显微镜下显示红色荧光.嘌呤霉素筛选和单克隆细胞集落挑取获得显示红色荧光细胞株,western blot证明稳转细胞内源性HuR基因表达下降,证明LV10-HuR慢病毒干扰载体可以抑制肝癌细胞内源性HuR基因表达.结论 成功构建慢病毒干扰载体LV10-HuR,包装出有活性的重组慢病毒颗粒,建立HuR基因敲低肝癌细胞株.%Objective To uncover the mechanism how to suppress hepatocellular carcinoma cell proliferation between C/EBPβ3'UTR RNA and HuR protein interaction,it is necessary to construct human HuR gene lentivirus interference vector and knockdown hepatocellular carcinoma cell line.Methods shRNA of human HuR gene was designed based on shRNA design principles and shDNA was chemically synthesized which was then inserted into lentvirus interference vector LV10(pGLVU6/RFP/Puro)to build LV10-HuR.Then,the LV10-HuR was co-transfected into HEK293T cell with lentivirus packaging auxiliary vectors including pLP1,pLP2 and pLP/VSVG.Reconstructed lentivirus particles were corrected and purified to infect hepatocellular carcinoma cell SMMC-7721 which then was observed under fluorescent microscope to detect the activity of reconstructed lentivirus particle.Puromycin was utilized to screen the infected cells and the formed monoclonal cell colony showing red fluorescence was picked and cultured continually,western blot was employed to identify expression of endogenous HuR protein.Results LV10-HuR was just the needed lentivirus interference vector identified by DNA sequencing.Lentivirus particles were harvested by co-transfection of HEK293T cells with LV10-HuR and lentivirus packaging auxiliary vectors,and then were used to infect hepatocellular carcinoma cells of SMMC-7721 which showed red fluorescence under fluorescenct microscope.Then,the infected cell was screened by puromycin to form monoclonal cell colony whose endogenous HuR gene expression was suppressed.Conclusion Human HuR lentivirus interference vector LV10-HuR is constructed and it could package activated reconstructed virus particle which could suppress the expression of endogenous HuR gene in hepatocellular carcinoma cell.