LMP1与绿色荧光蛋白融合基因慢病毒的制备

Production of lentivirus of LMP1 and GFP recombinant gene

目的:制备EB病毒LMP1与绿色荧光蛋白(GFP)融合基因慢病毒.方法:采用RT-PCR方法获得LMP1全外显子片段,使之克隆到带绿色荧光蛋白慢病毒表达载体质粒FUGW中.经限制性酶切、PCR扩增和测序鉴定重组载体.在脂质体介导下将慢病毒包装系统的包装结构基因pCMV.△8.9、包膜基因VSVG、目的基因FUGW-LMP1导入病毒包装细胞293FT,荧光显微镜检测基因的表达,包装成病毒后,收集病毒上清,浓缩,PCR鉴定.结果:限制性内切酶酶切和DNA测序分析证实RT-PCR获得的LMP1 cDNA片段与Gen-Bank中的数据完全吻合;LMP1准确克隆入FUGW的多克隆位点.慢病毒的3种质粒可高效转染入293FT细胞,荧光显微镜下观察可见大量的绿色荧光.绿色荧光位于细胞的胞膜及胞质.结论:成功制备了LMP1与GFP融合基因慢病毒,为研究EBV瘤基因LMP1在多种疾病中的作用机制提供适合的稳定转染细胞的载体.

AIM： To produce lentivirus of latentmembrane protein 1 （ LMPI ） and green fluorescent protein （GFP） recombinant gene. METHODS： The fragment including all the exons of LMPI was amplified by RT-PCR and was recombined to the downstream of CMV promoter in the lentivirus vector FUGW. The three plasmids expressing lentivirus [ packaging plasmid pCMV.△8.9, envelope plasmid VSVG and target plasmid FUGW-LMP1 ] were packaged into virus packaging cell line 293FT using lipofectamine method. The virus-producing cell supernatant was harvested and concentrated. RESULTS： It was proved that the sequence of the RT-PCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The LMP1 cDNA fragment was cloned in the vector FUGW in the right direction and the open reading fragment of LMP1 was maintained. The three plasmids were effectively transferred into 293FT. Much green fluorescence was observed by fluorescence microscopy. CONCLUSION： The lentivirus of LMP1 and GFP recombinant gene are successfully produced.