小鼠内皮抑素基因真核表达载体的构建和表达

Construction and expression of mouse endostatin in eukaryotic expression vector

目的 构建小鼠内皮抑素 (endostatin) c DNA的真核表达载体并将其在体外培养的脑胶质瘤细胞内表达 .方法 将带有分泌信号的小鼠 endostatin c DNA克隆入真核表达载体 pc DNA3,构建 CMV启动子控制的载体 pc DNA- s Endo,并将上述载体转染体外培养的脑胶质瘤细胞 (SHG44 ) ,采用Western Blot鉴定其表达 ,应用牛微血管内皮细胞抑制实验检测其活性 .结果 成功地构建了真核表达载体 pc DNA-s Endo并转染入脑胶质瘤细胞 ,获得的分泌型 endostatin可以明显抑制牛毛细血管内皮细胞增殖 (ED5 0 =2 0 0μg· L- 1 ) .结论 获得有表达功能活性的 endostatin真核表达载体 。

AIM To construct eukaryotic expression vector carrying mouse endostatin cDNA and to express it in cultured gliomas cells. METHODS Endostatin cDNA with secretive signal was cloned into polylinker site of eukaryotic expression vector pcDNA3 to construct pcDNA sEndo, in which the transcription of endostatin cDNA was driven by CMV promoter; and endostatin cDNA was identified by restriction enzyme digestion. The vector was transfected into glioma cells (SHG44). The expressed endostatin protein was identified byWestern blot. Its activity was examined by the fauro micrangium endotheliocyte inhibition test. RESULTS The eukaryotic expression vector pcDNA sEndo was successfully constructed and transfected into glioma cells. The endostatin protein, which can inhibit endothelial cells proliferation (ED 50 =200 μg·L -1 ), was obtained. CONCLUSION The acquisition of the eukaryotic expression vector of functional endostatin gene is of significance for mouse glimas specific gene therapy.