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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。
OBJECTIVE: To construct retroviral expression vector containing Ubc9, screen and screen high-titer virus-producing cell lines carrying this gene and further study the effect of SUMOylation. Methods: The target gene Ubc9 was amplified by polymerase chain reaction (PCR) and inserted into retrovirus expression vector pMSCVneo to form recombinant plasmid pMSCV-Ubc9. The recombinant plasmid pMSCV-Ubc9 was transfected into retroviral packaging cell PT67 and G418 The toxin-producing cell clones were screened, the toxin-producing cell clones were expanded and virus-infected NIH3T3 cells were harvested. RESULTS: Restriction analysis and sequencing confirmed that Ubc9 was correctly inserted into the retroviral expression vector. G418 screening stable resistant to drug-producing cell clones harvested virus can effectively infected NIH3T3 cells. CONCLUSION: The recombinant retrovirus vector pMSCV-Ubc9 carrying Ubc9 gene was successfully constructed. The recombinant retrovirus was packaged after transfection into PT67 cells, and then the antiviral cell line PT67-Ubc9 transcribing Ubc9 was screened.