| 李薇1 刘晓萍1△ 徐祥2 谭艳2 于业军3 王苗苗3 任书亭3.Pmscv-Ubc9 逆转录病毒表达载体的构建及产毒细胞系的建立[J].现代生物医学进展英文版,2011,11(14):2628-2631. |
| Pmscv-Ubc9 逆转录病毒表达载体的构建及产毒细胞系的建立 |
| Construction of Retrovirus Vector with Ubc9 Gene and RecombinantVirus Producing Cell Line |
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| DOI： |
| 中文关键词: Ubc9 基因 逆转录病毒载体pMSCVneo PT67 细胞 |
| 英文关键词: Ubc9 gene Retroviral Vector pMSCVneo PT67 |
| 基金项目:国家自然科学基金项目资助（No. 30970651） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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