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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。结论 重组逆转录病毒成功地将EB病毒LMP2基因整合到细胞中并得以表达
Objective To search for an effective way to express Epstein Barr (EB) Latent Membrane Protein 2 (LMP2) in eukaryotic cells and investigate the function of LMP2 protein and to explore its role in inducing cellular immune response. Methods The gene of LMP2 protein of Epstein-Barr virus (EBV) was recombined into the retroviral vector LXSN. The recombinant plasmid was introduced into PT6 7 cells by lipofectamine. The resistant clone was screened by G418. The supernatant containing recombinant virus was collected and infected into fibroblasts Cell NIH 3T3, the virus titer was determined, the DNA of the transfected cells was extracted for PCR identification, and the expression of foreign genes in NIH 3T3 infected with virus was detected by indirect immunofluorescence. Results The virus titer in the culture supernatant was 5. 8 × 10 5 PFU ml. The result of polymerase chain reaction (PCR) confirmed that the DNA of transfected cells contained the specific fragment of the target gene. Immunofluorescence results showed that the EBV LMP2 gene was expressed in mouse fibroblasts. Conclusion The recombinant retrovirus successfully integrated the Epstein-Barr virus LMP2 gene into cells and expressed