【摘要】 克隆人血管内皮生长因子16 5 (Vascularendothelailgrowthfactor16 5 ,VEGF16 5 )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VEGF16 5DNA片段,将该片段插入质粒pUCmT中并测序鉴定。将测序正确的pUCmT VEGFl6 5与PET2 0b(+)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VEGF16 5的原核表达载体PET2 0b(+) -VEGF16 5 ,转化大肠杆菌BL2 1(DE3)pLysS ,IPTG诱导表达,表达产物用Ni NTAResin纯化,SDS PAGE及WesternBlot鉴定重组蛋白。结果表示:1.测序结果示扩增的VEGF16 5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2 3kd的重组蛋白条带;3.WesternBlot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VEGF16 5 ,为研究其功能奠定基础。更多还原

【Abstract】 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.更多还原