血管生成素与绿色荧光蛋白融合基因的构建、表达及生物活性

Construction and Expression of Human Angiogenin/Gfp Fusion Gene and Its Biological Function

通过RT PCR的方法从人外周血白细胞扩增血管生成素 (Ang)cDNA .在计算机分子结构模建的基础上 ,通过柔性连接臂构建了Ang与Gfp融合基因 ,并在大肠杆菌DH5α中实现了高效表达 .重组蛋白占菌体总蛋白的 32 %.融合蛋白经初步纯化后 ,在紫外线激发下可见明显的绿色荧光 ,同时能够显著地促进鸡胚尿囊膜毛细血管的新生 ,而且所获融合蛋白在体外具有促进人脐静脉血管内皮细胞增殖的作用 .这种双功能融合蛋白的表达为阐明Ang的核转位过程奠定了基础 。

Angiogenin(Ang) is the first angiogenic protein factor purified from serum free supernatants of an established human adenocarcinoma cell line (HT 29).The cDNA encoding angiogenin was isolated by RT PCR from human peripheral white blood cells. On the basis of computer modelling, the fusion gene of angiogenin and Gfp was contructed by DNA recombination in vitro with a flexible linker. The fusion gene was expressed in E.coli , and the target protein amounted to 32% of total bacteria protein . After the purification of the fusion protein, the green fluorescence was observed under the ultraviolet light, and the angiogenesis of chicken chorioallantoic membrane was improved by the fusion protein. The proliferation of endothelial cell of umbilical vein ( ECV304 )was also enhanced by the recombinant protein. These results made a solid foundation to clarity the procedure of nuclear translocation of Ang.