摘要
目的构建含EB病毒(EBV)潜伏膜蛋白基因2A(LMP2A)的逆转录病毒表达载体,筛选建立携带该基因的高滴度产毒细胞系。方法逆转录-聚合酶链反应(RT-PCR)扩增获取目的基因LMP2A,定向插入逆转录病毒表达载体pMSCVpuro,形成重组质粒pMSCVpuro-2A,脂质体法将pMSCVpuro-2A转染逆转录病毒包装细胞PT67。嘌呤霉素筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒进行滴度测定,RT-PCR检测产毒PT67细胞目的基因的转录表达。结果限制性酶切、PCR及测序鉴定证实LMP2A正确插入逆转录病毒表达载体;筛选获得稳定产毒的抗性细胞克隆;收获病毒的滴度为3.2×107CFU/L,且重组腺病毒在PT67细胞能有效转录。结论携带LMP2A基因的重组逆转录病毒表达载体pMSCVpuro-2A构建成功,转染PT67细胞后包装出重组逆转录病毒,进而筛选获得了能转录表达LMP2A的产毒细胞系PT67-LMP2A。
Objective To establish a retroviral vector encoding Epstein-Barr virus （EBV） latent membrane protein 2A （LMP2A） and a stable virus producing cell line. Methods The target gene LMP2A amplified by RT-PCR was subeloned into retroviral vector pMSCVpuro. The recombinant plasmid pMSCVpuro-2A was transfeeted into packaging cell PT67 by lipofeetamine 2000.The transfeetants were selected by puromyein and viral titer tested. The expression of LMP2A in PT67 cell line was identified by RT-PCR. Results The recombinant retroviral vector pMSCVpuro-2A was identified by polymerase chain reaction （PCR）, restrictive analysis, and DNA sequencing. A stable virus producing cell line was selected and the viral titer was 3.2×10^7CFu/L. The expected 1 500 bp fragment was amplified from PT67-LMP2A cells,this result indicated that LMP2A could effectively express in packaging cell PT67. Conclusion The recombinant retroviral vector pMSCVpuro-2A was constructed successfully. A stable viral producing cell line PT67-LMP2A was selected and established. This study established a foundation for further study of LMP2A.
出处
《青岛大学医学院学报》
CAS
2007年第4期289-291,295,共4页
Acta Academiae Medicinae Qingdao Universitatis
