LMP1基因重组慢病毒载体的构建及体外表达  

Construction and identification of a recombinant lentivirus vector of latent membrane protein 1

作　　者：陈小艳[1] 张弓[1] 刘芳[1] 李慧灵[1] 方唯意[2] 江庆萍[2] 赵彤[1] 

机构地区：[1]南方医科大学南方医院病理科,广东广州510515 [2]南方医科大学肿瘤研究所,广东广州510515

出　　处：《南方医科大学学报》2009年第5期837-840,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30670812,30770908)

摘　　要：目的构建EB病毒潜伏膜蛋白I(LMP1)基因重组慢病毒载体,并检测其在体外的表达。方法采用DNA重组技术,将LMP1基因克隆到带绿色荧光蛋白的慢病毒表达载体质粒pCDF中,筛选阳性克隆,经限制性酶切、PCR扩增和DNA测序鉴定重组载体。以脂质体介导法将慢病毒包装系统的包装质粒pFIV-34N、包膜质粒pVSV-G和带目的基因的质粒pCDF-LMP1共同转染到包装细胞293FT细胞内包装病毒,荧光显微镜观察包装细胞报告基因的表达情况,收集病毒上清,浓缩鉴定,测定重组病毒的滴度。将重组慢病毒转染小鼠B淋巴瘤细胞系A20,RT-PCR和Western blotting检测LMP1的表达。结果重组慢病毒表达载体质粒经限制性酶切和DNA测序分析证实LMP1基因准确克隆入pCDF的多克隆位点,LMP1序列与GenBank中的数据完全一致。荧光显微镜下观察可见293FT细胞的细胞浆与细胞膜有大量的绿色荧光,重组慢病毒浓缩后滴度为107Tu/ml,慢病毒转染A20细胞的效率>90%。RT-PCR和Western blotting检测A20细胞内有LMP1的表达。结论成功构建了LMP1基因重组慢病毒表达载体,慢病毒可高效转染A20细胞,为后期研究EBV致瘤基因LMP1在淋巴瘤发病机制中的作用奠定基础。Objective To construct a recombinant lentivirus vector of latent membrane protein 1 （LMP1） and detect the expression of LMP1 in vitro. Methods The LMP1 fragment including all the exons was amplified by PCR and inserted to the downstream of CMV promoter in the lentivirus vector pCDF. The three plasmids （packaging plasmid pFIV-34N, envelope plasmid pVSV-G and target plasmid pCDF-LMP1） were packaged into 293FT cells via liposome. The virus supematant was harvested, concentrated and titrated. Mouse B lymphoma cell line A20 was transfected with the recombinant lentivirus vector of LMP 1, and the expression of LMP 1 in A20 cells was detected by RT-PCR and Western blotting. Results DNA sequencing confirmed that the sequence ofPCR-amplified LMP1 was consistent with the GenBank data. The LMP1 gene fragment was cloned into pCDF in the right direction, and the open reading frame of LMP1 was maintained. The 3 plasmids were effectively transferred into 293FT cells, which emitted green fluorescence in the cytoplasm and on the cell membrane under fluorescence microscope. The titer of the lentivirus vector reached 107 Tu/ml with a transfection efficiency 〉90% in A20 cells. LMP1 expression was detected by RT-PCR and Western blotting in transfected A20 ceils. Conclusion The recombinant lentivirus vector of LMP1 constructed can be effectively transfected into A20 ceils, which provides a basis for exploring the role of LMP1 in the pathogenenisis oflymphoma.

关 键 词：潜伏膜蛋白1 基因重组 慢病毒载体 A20细胞 

分 类 号：R346[医药卫生—基础医学]