大鼠SOCS3腺病毒的构建及鉴定

Construction and identification adenovirus of SOCS3

目的:构建大鼠细胞因子信号途径抑制物3(SOCS3)腺病毒载体,并对腺病毒感染后的HEK293细胞的SOCS3表达情况进行观察。方法:提取大鼠腹腔巨噬细胞中总RNA,克隆出SOCS3基因,亚克隆于含有GFP报告基因的穿梭质粒pAdtrack-CMV,通过pAdeasy-1腺病毒系统将含有目的基因的穿梭质粒和骨架质粒共转入BJ5183感受态细胞。线性化重组的病毒转染入HEK293细胞,并在其中对重组的腺病毒进行包装及扩增。结果:构建的大鼠SOCS3腺病毒能够高效率的感染HEK293细胞,mRNA和蛋白水平均证实SOCS3表达显著增加。结论:大鼠SOCS3腺病毒载体构建成功,可用于体内转染或细胞试验的进一步研究。

Objective：To construct and identify the recombinant adenovirus containing SOCS3 and detect expression in HEK293 cells.Methods： The SOCS3 gene was cloned by RT-PCR after total RNA extracted from peritoneal macrophage of rat,then subcloned with shuttle plasmid pAdtrack-CMV containing GFP reporter gene.pAdeasy-1 adenovirus system was used for co-transfecting shuttle plasmid and frame plasmid to BJ5183 cells.Reconstructed adenovirus was transfected to HEK293 cell after linearization and mRNA and protein level of SOCS3 in the cell were detected.Results： SOCS3 gene was cloned and recombinant adenovirus pAd-SOCS3-GFP was constructed successfully.Recombinant adenovirus was transfected to HEK293 cell and the mRNA and protein level of SOCS3 in the cell were both increased significantly.Conclusion： Recombinant SOCS3 adenovirus has been constructed successfully and could be used for transfection in vivo or in vitro.