人血管内皮生长因子DNA克隆和在大肠杆菌中的表达

克隆人血管内皮生长因子165(Vascular endothelail growth factor165，VEGF165)并在大肠杆菌中诱导表达。用RT-PCR法从人白血病细胞株TF1扩增VEGF165 DNA片段，将该片段插入质粒pUCmT中并测序鉴定。将测序正确的pUCmT-VEGF165与PET20b( )均用EcoRI和SalI双酶切，回收纯化目的基因片段与表达载体，构建VEGF165的原核表达载体PET20b( )-VEGF165，转化大肠杆菌BL21(DE3)pLysS，IPTG诱导表达，表达产物用Ni-NTA Resin纯化，SDS-PAGE及Western Blot鉴定重组蛋白。结果表示：1．测序结果示扩增的VEGF165序列与文献报道相符；2．SDS-PAGE电泳示IPTG诱导后出现分子质量约23kd的重组蛋白条带；3．Western Blot分析表明重组蛋白可与兔抗人VEGF单克隆抗体特异性结合。成功克隆、表达及纯化VEGF165，为研究其功能奠定基础。

Cloning and Expression of Vascular Endothelial Growth Factor 165 in E.coli

Purpose To clone and express vascular endothelial growth factor165(VEGF165)in E.coli.Methods The VEGF165 fragment was amplified with RT-PGR from human leukemic cell line TF1,the amplified fragment was inserted into pUCm-T and sequenced,then procaryotic expression vector PET20b( )-VEGF165 was constructed.After induced by IPTG,the recombinant VEGF165 was purified using Ni-NTA Resin column.SDS-PAGE analysed the expression and purification of VEGF165, and Western Blot identified recombinant VEGF165.Results The VEGF165 fragment was 505bp and its sequence was the same as the reported VEGF165.SDS-PAGE showed that VEGF165 was expressed and purified successfully,and Western Blot showed that recombinant VEGF165 could combine with rabbit anti-human VEGF MoAb specially. Conclusions VEGF165 was cloned,expressed and purified successfully,which laid the foundation for further studying of function of VEGF165.