中文摘要：

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pcDNA3．0-HA—SUM01、pcDNA3．0．HA．SUM02、pcDNA3．0。HA．SUM03质粒，得到3XFlag—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。Westernblot检测稳定转染的pBabe-3×Flag．SUMOs．SI-ISY5Y单克隆细胞SUM01—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。

英文摘要：

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.