血管生成抑制因子arresten的真核表达体系的建立

Eukaryotic Expression of Human Arresten

目的构建血管生成抑制因子arresten的真核表达细胞克隆体系。方法用脂质体转染法,通过真核表达载体pSecTag2-arresten将arresten基因导入CHO细胞,经抗生素Zeocin筛选获得阳性细胞克隆,逆转录-聚合酶链反应(RT-PCR)检测arrestenmRNA表达,免疫蛋白印迹技术(Western-blot)测arresten蛋白的表达。结果经过筛选获得阳性克隆的CHO细胞,RT-PCR、Western-blot结果提示arresten基因成功导入CHO细胞并进行表达。结论获得了稳定表达人arresten因子的CHO细胞克隆,为深入研究arresten在抑制肿瘤血管生成中的作用机制奠定了良好的基础。

Objective To construct the eukaryotic expression of arresten in CHO cell. Methods By gene transfected way, pSecTag2-arresten was transfected into CHO cell line. The transfected cells were grown in DMEM medium comaining Zeocin at 72 hours after transfection, and the positive clone s were selected in Zeocin medium until the 15^th day. Expression of arresten mRNA in CHO cell was examined by RT- PCR, the expression of arresten protein was examined by Western-Blot. Results Arresten gene was expressed successfully in transfected CHO cell. Conclusion The positive CHO cell clones expressing human arresten gene stable obtained, which may be a promising cell model for studying the biological function of the arresten gene and role of arresten in the angiogenesis.