含小鼠分泌型内皮抑素基因重组质粒的构建及表达

目的:克隆小鼠分泌型内皮抑素(sEndostatin)cDNA,构建pCMV-分泌型内皮抑素和pEgr-分泌型内皮抑素重组质粒,检测重组质粒在B16细胞中的表达. 方法:用RT-PCR方法从鼠肝脏中扩增出内皮抑素cDNA,经测序证实后,连入信号肽,构建pCMV-分泌型内皮抑素和pEgr-分泌型内皮抑素重组质粒,以脂质体转染小鼠B16细胞,用Western blot方法检测B16细胞上清中内皮抑素的表达.结果:测序表明扩增的分泌型内皮抑素序列与报道完全一致, Egr-1启动子和分泌型内皮抑素cDNA正确插入表达载体pcDNA3.1,Western blot证实转染B16细胞上清中有目的蛋白表达.结论:小鼠内皮抑素基因成功地被克隆和表达,为今后检测pEgr-分泌型内皮抑素的辐射诱导表达和恶性肿瘤的基因-放射治疗奠定基础.

Construction and expression of recombination plasmid containing mouse secretable endostatin gene

Objective:To clone mouse secretable endostatin (sEndostatin) cDNA,construct pCMV sEndostatin and pEgr sEndostatin recombinant plasmids and detect the expression of recombinant plasmids in B16 cells. Methods:Mouse endostatin cDNA was amplified from the murine liver by RT PCR. After the cDNA identified by sequencing, signal peptide was ligated, the pCMV sEndostatin and pEgr sEndostatin recombinant plasmids were constructed, plasmids were transfected into B16 cells with liposome and the expression of endostatin in B16 cells was detected by Western blot. Results:The sequence of mouse endostatin cDNA was identical with reported and to which a signal peptide was ligated. Egr 1 promoter and Endostatin cDNA was inserted into expression vector exactly. There was the expression of endostatin in B16 cells proved by Western blot. Conclusion:Mouse endostatin cDNA was cloned and expressed successfully in the study. The result provides a basis for examing expression of pEgr sEndostatin induced by radiation and gene radiotherapy of tumor.