人可溶性血管内皮细胞生长因子受体Flt-1基因在链霉菌中的克隆表达

Cloning and Expression of VEGF Receptor Flt-1 Gene in S.lividans TK24

应用RT PCR技术 ,从人脐静脉内皮细胞中扩增出编码人可溶性血管内皮细胞生长因子 (VEGF)受体Flt 1胞外区前四个结构域的基因片段 ,亚克隆至pUCl8质粒进行测序 ,将目的基因片段连接至链霉菌表达载体pSGLgpp ,获得重组质粒pSGLgpp F ,将其转化至Streptomy ceslividansTK2 4 ,获得基因工程菌株Sreptomyceslividans (pSGLgpp F) ,对其培养上清液进行SDS PAGE及Westernblot分析 ,结果显示 ,在 6 3 6kD处有特异性条带出现 ,表明sFLT 1在链霉菌中获得了成功表达 ,受体配基结合实验显示表达产物与VEGF可特异性结合 。

Using RNA extracted from human umbilical vein endothelium cell as a template,the gene VEGF receptor Flt\|1 was amplified by RT\|PCR.Recombinant plasmid pSGLgpp\|F was constructed and was transformed into S.lividans TK24.With the detection of SDS\|PAGE and Western blot,a specific band being same to the reports near 63.6kd was found.The results showed sFlt\|1 was successfully expressed in S.lividans .The result of the binding assay of receptor\|ligand for sFlt\|1 showed sFlt\|1 has the biological activity of binding with its ligand VEGF.