EB病毒BHRF1基因慢病毒载体的构建及功能鉴定  

Construction and identification of a lentiviral vector expressing BHRF1 gene of Epstein-Barr virus

作　　者：孙静[1] 孙强明 李建芳 李彦涵 乌美妮 廖芸 王晶晶 胡云章 

机构地区：[1]中国医学科学院协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118

出　　处：《现代肿瘤医学》2013年第6期1197-1201,共5页Journal of Modern Oncology

基　　金：云南省应用技术基础研究计划项目

摘　　要：目的:构建并制备能够有效表达EB病毒BHRF1基因的重组慢病毒载体,观察BHRF1表达对人胚肺成纤维细胞(KMB17)凋亡的影响。方法:采用RT-PCR法,从B95-8细胞扩增EBV BHRF1基因,克隆至pWPI GW慢病毒载体上,与pVSVG及pSPAX质粒共转染人胚肾293T细胞,包装出重组慢病毒。将纯化后的重组慢病毒直接感染293T和KMB17细胞,荧光显微镜、实时定量RT-PCR、免疫印迹等方法检测BHRF1在细胞中的表达水平。流式细胞仪检测BHRF1表达对凋亡诱导剂或无血清培养诱发KMB17细胞凋亡的影响。结果:重组慢病毒介导BHRF1在293T和KMB17细胞内获得表达,能有效地抑制KMB17细胞的凋亡。结论:成功构建了表达BHRF1基因的重组慢病毒载体。Objective：To construct a lentiviral vector expressing BHRF1 gene of Epstein-Barr virus and observe the effect of BHRF1 expression on the apoptosis of human embryonic lung fibroblast（KMB17）.Methods： BHRF1 sequence was amplified from the B95-8 cell by RT-PCR and cloned into pWPI GW vector.The recombinant plasmid pWPI GW/BHRF1 was then co-transfected into human embryonic kidney 293T cells with pVSVG and pSPAX.The recombinant lentivirus was packaged and amplified,followed by infection of 293T and KBM17 cells.Then,the expression level of target protein was examined by fluorescence microscope,realtime RT-PCR and Western blot.The effect of BHRF1 expression on the apoptosis of KMB17 cell induced by apoptosis inducer or serum starvation was detected by flow cytometry.Results： The recombinant lentiviral vector expressing BHRF1 was successfully constructed.Fluorescence microscope,realtime RT-PCR and Western blot demonstrated that BHRF1 was successfully expressed in 293T and KBM17 cells,and flow cytometry assay showed that BHRF1 can effectively suppress KMB17 cell apoptosis.Conclusion： A lentiviral vector carrying BHRF1 gene is successfully constructed.

关 键 词：EB病毒 BHRF1 慢病毒载体 细胞凋亡 

分 类 号：R730.231.3[医药卫生—肿瘤]