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  

Construction of human GRP94 lentivirus vector and its application in screening human cervical cancer cell line HeLa for stable expression

作　　者：杨娅楠[1] 王媛[1] 左志宇[1] 王鑫廷[1] 杨洁[1] YANG Ya-nan WANG Yuan ZUO Zhi-yu WANG Xin-ting YANG Jie(Department of Immunology,Tianjin Medical University, Tianjin 300070, Chin)

机构地区：[1]天津医科大学免疫学系,天津300070

出　　处：《天津医科大学学报》2017年第4期290-294,319,共6页Journal of Tianjin Medical University

基　　金：国家青年科学基金资助项目(31300709)

摘　　要：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pLV_X-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。Objective： To construct human GRP94 gene lentiviral vector and screen the GRP94 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-Hyg to obtain recombinant lentiviralvector pLVX-FLAG-SND1. After transfection of 293T cells with pLVX-FLAG-GRP94 for 48h, the expression of GRP94 protein was detected by Western blotting. PLVX-FLAG-GRP94 recombinant plasmid was co-transfected with 293T cells to obtain recombinant lentivirus carrying FLAG-GRP94. The expression of GRP94 protein was detected by Western Blotting method. 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 cells 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.

关 键 词：葡萄糖调节蛋白94 慢病毒载体 He LA SND1 

分 类 号：Q7[生物学—分子生物学]