EB病毒潜伏膜蛋白2重组逆转录病毒的构建及表达

Expression of Epstein Barr virus latent membrane protein 2 in murine fibroblasts by retroviral mediated gene transfer

目的 寻找在真核细胞中表达Epstein Barr(EB)病毒潜伏膜蛋白 2 (Latentmembraneprotein 2 ,LMP2 )的有效途径 ,研究LMP2蛋白功能及深入探讨其在诱导细胞免疫应答中的作用。方法 将EB病毒LMP2蛋白的基因重组至逆转录病毒载体LXSN上 ,通过脂质体将重组质粒导入PT6 7细胞 ,G418筛选抗性克隆 ,收集含重组病毒的上清 ,将其感染小鼠成纤维细胞NIH 3T3 ,测定病毒滴度 ,提取转染细胞DNA进行PCR鉴定 ,间接免疫荧光法检测外源基因在感染病毒的NIH 3T3中的表达。结果 培养上清的病毒滴度为 5 .8× 10 5 PFU ml,聚合酶链反应 (PCR)结果证实 ,转染细胞的DNA中含有目的基因的特异性片段。免疫荧光结果表明EBV LMP2基因在小鼠成纤维细胞中获得表达。

Objective This study is to find an efficient way to express EB virus latent membrane protein 2 in mammalian cells, and provide for further investigate on the function of LMP2 protein and its role in cellular immunity. Methods The LMP2 gene was cloned into the EcoR Ⅰ site of a retroviral vector LXSN and the recombinant LXSN LMP2 was tranfected into PT67 by lipofect TAMINE.After screened by G418,the supernatant of G418 resistant cells was used to infect murine fibroblasts NIH 3T3 to determine the virus titer. DNA was extracted from transfected cells and tested by PCR. Indirect immunofluorescence assay was used to detect the expression of the inserted gene. Results The virus titer was 5.8 ×10 5 PFU/ml. The result of PCR showed that the LMP2 gene had been integrated into the DNA of transfected cells. Indirect immunofluoresecence showed that the LMP2 gene had been expressed in the murine fibroblasts. Conclusion EB virus LMP2 gene had been integrated into the genome of cells by retroviral mediated transfer and the target gene had been expressed.