pEgr-ssEndostatin的构建及其在体外的辐射诱导表达

Construction of pEgr-ssEndostatin and mouse endostatin expression in vitro induced by ionizing radiation

为探讨 pEgr-ssEndostatin 重组质粒转染 B16 细胞后接受不同辐射剂量以及接受同一剂量不同时程endostatin 表达的规律。采用 RT-PCR 方法从鼠肝脏中扩增出 Endostatin cDNA,连入信号肽,构建了pEgr?ssEndostatin 重组质粒,用脂质体介导的转染法转染小鼠 B16 细胞,用 ELISA 方法检测 B16 细胞上清中endostatin 的表达水平。结果表明的分泌型 Endostatin 序列与报道完全一致,Egr-1 启动子和 Endostatin cDNA正确插入表达载体 pcDNA3.1;pEfr-ssEndostatin 具有辐射诱导表达特性。提示小鼠 Endostatin 基因成功地被克隆和表达,Egr-1 启动子具有辐射激活和诱导下游基因表达增强的功能。

To study the regularity of mouse endostatin expression in B16 cells transfected by pEgr?ssEndostatin after different doses of X-ray irradiation and different time after the irradiation, mouse endostatin cDNA was ampli- fied by RT?PCR and signal peptide was ligated to it. The pEgr?ssEndostatin recombinant plasmid was constructed and transfected into B16 cells with liposome and cells were irradiated. The expression of endostatin in the supernatant medium of B16 cells was detected by ELISA. The results showed that the cDNA sequence of mouse secretable en- dostatin is identical to the literatures. Egr?1 promoter and secretable endostatin cDNA were inserted into expression vectors correctly. pEgr?ssEndostatin had the expression property induced by radiation. It suggests that the Egr?1 promoter can be activated by ionizing irradiation and expression of downstream gene can be regulated.