人GRP94基因慢病毒载体构建及稳定表达GRP94蛋白的人宫颈癌HeLa细胞株筛选及鉴定

摘要

Objective:To construct human GRP94 gene lentiviral vector and screen the Hela cell line stably expressing GRP94 protein,to provide a cell line model for the study of GRP94 gene regulation in cervical cancer.Methods:The GRP94 gene fragment was amplified from HeLa cells by RT-PCR and ligated into lentiviral vector expression plasmid pLVx-IRES-Puro to obtain recombinant lentiviralvector pLVx-FLAG-GRP94.After transfection of 293T cells with pLVx-FLAG-GRP94 for 48 h,the expression of FLAG-GRP94 fusion protein was detected by Western blot.PLVX-FLAG-GRP94 recombinant plasmid was co-transfected with 293T cells to obtain recombinant lentivirus carrying FLAG-GRP94.The expression of FLAG-GRP94 protein in 293T cells transtected with recombinant plasmid was detected by Western blot.The GRP94-binding protein lysate was obtained by FLAG-small peptide elution using HeLa cell line which could stably express FLAG-GRP94.SDS-PAGE electrophoresis and silver staining were performed to select the binding protein.Results:Recombinant lentiviral vector was identified by restriction enzyme digestion and gene sequencing.The expression of GRP94 protein in transfected 293T cells was higher than that in wild type HeLa cells (P ＜0.01).The expression of GRP94 protein in HeLa ceils was significantly higher than that in wild-type HeLa cells (P＜0.01).The SND1 protein,which was closely related to tumor development and progression,was successfully detected by mass spectrometry and verified by immnoprecipitation.Conclusion:The GRP94 gene lentiviral vector pLVx-FLAG-GRP94 could be successfully constructed,and the HeLa cell line stably expressing GRP94 protein is screened,which lays a foundation for further study of the mechanism of tumorigenesis and development.%目的:构建人葡萄糖调节蛋白94(GRP94)基因慢病毒载体,筛选稳定表达GRP94蛋白的人宫颈癌细胞株,筛选GRP94结合蛋白,为探讨GRP94对宫颈癌的调控作用提供细胞模型.方法:采用RT-PCR法从HeLa细胞中扩增GRP94基因片段,连接到慢病毒载体pLVX-IRES-Puro中,获得重组载体.瞬时转染293T细胞,采用Western blot法检测GRP94蛋白表达量.pLVX-FLAG-GRP94重组质粒通过与包装质粒共转染293T细胞,获得重组慢病毒.以慢病毒感染HeLa细胞,筛选并鉴定稳定表达GRP94蛋白的细胞株.用稳定表达FLAG-GRP94的HeLa细胞株,银染后利用质谱筛选结合蛋白,并经免疫共沉淀验证.结果:重组慢病毒载体经双酶切和基因测序比对鉴定正确.HeLa细胞经慢病毒感染、药物筛选后获得的稳定表达株中GRP94蛋白表达量高于野生型HeLa细胞(P＜0.01).成功钓取出一种与肿瘤发生发展密切相关的SND1蛋白.结论:成功构建了GRP94基因慢病毒载体pLVx-FLAG-GRP94,并筛选出稳定表达GRP94蛋白的HeLa细胞株,为进一步明确肿瘤的发生、发展机制奠定了基础.