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人SUMO病毒载体构建及稳转神经母细胞瘤SHSY5Y细胞系的建立 被引量：1

Construction of a retroviral vector expressing human SUMO gene and establishment of its stable transfected SHSY5Y cell line
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摘要 目的构建表达人小泛素化相关修饰蛋白(SUMO)基因的逆转录病毒载体,并观察其稳定感染神经母细胞瘤SHSY5Y细胞后SUMO蛋白的表达情况。方法用EcoRⅠ和XhoⅠ双酶切pcDNA 3.0-HA-SUMO1、pcDNA3.0-HA-SUMO2、pcDNA 3.0-HA-SUMO3质粒,得到3×Flag-SUMOs基因的编码序列,定向插入到pBabe-puro载体中,经扩增、筛选阳性克隆、酶切和测序验证后,转染293T细胞进行病毒包装、扩增、纯化获取逆转录病毒颗粒。将逆转录病毒颗粒感染SHSY5Y细胞,经嘌呤霉素筛选后抗性克隆集落长出。Western blot检测稳定转染的pBabe-3×Flag-SUMOs-SHSY5Y单克隆细胞SUMO1～3的表达。结果 pBabe-3×Flag-SUMOs-puro质粒构建正确。稳定转染的SHSY5Y细胞系3×Flag-SUMOs表达明显增强。结论成功构建了稳定表达SUMO的pBabe-3×Flag-SUMOs-SHSY5Y细胞系。 Objective To construct a retroviral vector of SUMO gene in order to generate a SHSYSY cell line stably expressing exogenous SUMO and to detect SUMO protein in SHSY5Y cells. Methods pcDNA3.0-HA-SUMO1, pcDNA 3.0-HA-SUMO2, pcDNA3.0-HA-SUMO3 plasmids were double digested by EcoR I and Xho I to get the 3 x Flag-SUMOs coding sequence, which was then subcloned into pBabe-puro vector. Positive clone was sequencing verified and amplified. Cell 293T were transfected with pBabe-puro and viral packaging plasmid to produce purified retroviral particles. SHSYSY cell was then infected with retroviral particles. Puromycin resistant clones were selected. The expression of 3 × Flag-SUMOs was verified by method of Western blot. Results Restriction endonulease analysis and DNA sequencing showed that the pBabe-3 × Flag-SUMOs-puro plasmid was constructed successfully. The enhanced expression of 3 × Flag-SUMOs in stably infected SHSY5Y cells was proved by Flag antibody. Conclusion SHSY5Y cell lines stably expressing genes encoding 3 × Flag-SUMOs were established successfully.
作者 常晶 陆菡 薛庆生 于布为
机构地区 上海交通大学医学院附属瑞金医院
出处 《山东医药》 CAS 2013年第9期1-3,共3页 Shandong Medical Journal
基金 国家自然科学基金资助项目(81102513 81072703)
关键词 小泛素化相关修饰蛋白 逆转录病毒 神经母细胞瘤细胞 small ubiquitin-like modifier retrovirus neuroblastoma cell
分类号 R329.2 [医药卫生—人体解剖和组织胚胎学]
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山东医药
2013年 第9期

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