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目的在毕赤酵母中表达人血管内皮生长因子189(vascular endothelial growth factor 189,VEGF189),并进行纯化,同时检测其在体外的生物活性。方法以人VEGF183为模板,经重叠PCR法获得VEGF189基因,亚克隆至pPIC9K中,获得重组表达质粒pPIC9K-VEGF189。将其转化至E.coli DH5α中进行扩增,提取质粒,用SacⅠ酶线性化,电转至毕赤酵母GS115中,经4 mg/ml G418-YPD平板筛选高拷贝克隆。采用甲醇进行诱导表达,并通过肝素琼脂糖树脂亲和层析进行纯化。血管通透性试验检测其生物活性。结果经PCR鉴定证明重组表达质粒pPIC9K-VEGF189构建正确。VEGF189蛋白的表达及纯化产物均可与兔抗人VEGF183多克隆抗体发生特异性结合,纯化蛋白纯度达95%以上,浓度为0.291 1 mg/ml,可增加血管的通透性。结论本实验通过酵母表达系统获得了具有活性的VEGF189蛋白,为在体外探讨该蛋白在血管生成中的作用奠定了基础。
Objective To express and purify vascular endothelial growth factor 189 (VEGF189) in Pichia pastoris and test its biological activity in vitro. Methods Human VEGF183 was used as template to obtain VEGF189 gene by overlap PCR and subcloned into pPIC9K to obtain recombinant plasmid pPIC9K-VEGF189. This was transformed into E. coli DH5α for amplification. Plasmids were extracted, linearized with SacI and electroporated into Pichia pastoris GS115. High-copy clones were selected by 4 mg / ml G418-YPD plates. Methanol was used for induction of expression and purified by heparin sepharose resin affinity chromatography. Vascular permeability test to test its biological activity. Results PCR identification proved that recombinant plasmid pPIC9K-VEGF189 was constructed correctly. The expression and purification of VEGF189 protein could specifically bind with rabbit anti-human VEGF183 polyclonal antibody. The purity of the purified protein reached above 95% with a concentration of 0.291 1 mg / ml, which could increase the permeability of blood vessel. Conclusion In this study, we obtained the active VEGF189 protein by yeast expression system, which lays the foundation for exploring the function of this protein in angiogenesis in vitro.