【摘要】 目的:构建含Ubc9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,深入研究SUMO化修饰的作用。方法:聚合酶链反应(PCR)扩增获取目的基因Ubc9,定向插入逆转录病毒表达载体pMSCVneo,形成重组质粒pMSCV-Ubc9;脂质体法将pMSCV-Ubc9转染逆转录病毒包装细胞PT67;G418筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒感染NIH3T3细胞。结果:限制性酶切和测序鉴定证实Ubc9正确插入逆转录病毒表达载体。G418筛选获得稳定产毒的抗性细胞克隆,收获病毒能有效感染NIH3T3细胞。结论:携带Ubc9基因的重组逆转录病毒表达载体pMSCV-Ubc9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Ubc9的产毒细胞系PT67-Ubc9。更多还原

【Abstract】 Objective: To establish a recombinant retrovirus vector containing Ubc9 gene and to establish a stable virus packaging cell line expressing Ubc9 effectively and stably.Methods: Ubc9 gene was amplified from the plasmid pcmv6-xl6-ubc9 by PCR technique and subcloned to the retroviral vector pMSCVneo to construct the recombined retrovirus vector.The recombinant plasmid was transfected into packaging cell line PT67 with PolyJetTM and the efficient virus-producing cell line PT67-Ubc9 was screened out following G418 selection and collected virus infected NIH/3T3 cells.Results: The recombinant retroviral vector pMSCV-Ubc9 was identified by restrictive analysis and DNA sequencing.A stable virus producing cell line was selected and the retrovirus was effectively transfected into NIH3T3 cells.Conclusion: The recombinant retroviral vector pMSCV-Ubc9 was constructed successfully.A stable viral producing cell line PT67-Ubc9 was selected and established.更多还原