LMP1基因重组慢病毒载体的构建及体外表达

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Westernblotting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Westernblotting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.

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

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 supernatant was harvested,concentrated and titrated. Mouse B lymphoma ce...