SOCS－3基因真核载体的构建及瞬时表达鉴定

Construction and identification of eukaryotic expression plasmid carrying mouse SOCS-3 gene

目的构建含小鼠细胞因子信号抑制蛋白-3（SOCS-3）基因的重组pcDNA3.1质粒，同时瞬时转染后，鉴定其在非洲绿猴肾细胞株COS-7中的表达。方法自小鼠肝脏标本中提取RNA进行RT—PCR，经克隆后获得pUC19-SOCS-3质粒，从中切出目的基因片段克隆至质粒pcDNA3．1中，以构建重组质粒pcDNA3.1-SOCS-3。后将构建完成的质粒瞬时转染COS-7细胞株，并随机分为未转染质粒组（对照组）、转染空质粒组和转染重组SOCS-3基因质粒组，同时采用免疫印迹法测定SOCS-3的表达。结果经测序鉴定证实，SOCS-3与美国国立生物技术信息中心核苷酸序列数据库提供的原始序列完全一致；同时经瞬时转染COS-7细胞后，转染重组质粒组的SOCS-3蛋白表达水平（光密度比值）显著高于对照组和转空质粒组（P〈0．01）。结论成功构建含小鼠SOCS-3基因的真核表达质粒，为进一步研究该基因在脂肪代谢中的抑制调节作用提供了技术平台。

Objective To construct and identify eukaryotic expression plasmid carrying mouse suppressors of cytokine signaling-3 （SOCS-3） gene. Methods Total RNA was extracted from mouse liver, the plasmid of pUC19-SOCS-3 was successfully constructed after RT-PCR and TA cloned. The SOCS-3 fragment was cloned into peDNA3.1 to construct the recombinant pcDNA3.1-SOCS-3. Plasmid pcDNA3.1-SOCS-3 was verified by restriction digestion and gene sequencing. The recombinant plasmid was transfected into COS-7 cell line and the express products were identified by western blot analysis. The non-transfected COS-7 cells and COS-7 cells transfected with empty vector were served as controls. Results The sequence of target gene was identical with that of NCBI gene bank. The OD value of SOCS-3 expression in recombinant pcDNA3.1-SOCS-3 group was significantly higher than that of un-transfected control group and empty vector group （110.0 vs 38.9, 110.0 vs 46.5; P〈0.01）. Conclusion Plasmid pcDNA3.1-SOCS-3 was constructed and expressed in COS-7 cells successfully.