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目的研究人 ＶＥＧＦ１６５基因在 Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ酵母中的表达，获得高效表达、具有生物学活性的重组ｈＶＥＧＦ１６５。方法采用ＰＣＲ扩增ｈＶＥＧＦ１６５基因，经ＤＮＡ序列分析后，插入含ＡＯＸ１启动子和α分泌信号肽序列的Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ酵母表达载体中，构建重组质粒ｐＰＩＣ９Ｋ／ｈＶＥＧＦ１６５。经转化酵母宿主菌ＫＭ７１，筛选多拷贝整合转化子，摇瓶培养，用 １０ ｍＬ／Ｌ甲醇诱导表达。表达产物经Ｈｅｐａｒｉｎ－Ｓｅｐｈａｒｏｓｅ ＣＬ６Ｂ亲和层析纯化后，以人脐静脉内皮细胞（ＨＵＶＥＣ）测定其生物学活性。结果ＳＤＳ－ＰＡＧＥ分析显示，表达产物以可溶性分子的形式存在于上清中，诱导４ｄ的表达量达上清中总蛋白的６０％以上。 Ｗｅｓｔｅｒｎ ｂｌｏｔ表明，表达产物具有良好的抗原性和特异性。经Ｈｅｐ－ａｒｉｎ－Ｓｅｐｈａｒｏｓｅ ＣＬ６Ｂ亲和层析纯化， ｒｈＶＥＧＦ１６５的纯度可达到９０％以上。生物学活性检测证实，其具有刺激脐静脉内皮细胞（ＨＵＶＥＣ）增殖的活性。结论在 Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ表达系统中，实现了ｈＶＥＧＦ１６５的高效分泌性表达。
Objective To study the expression of human VEGF165 gene in Pichia pastoris yeast and to obtain highly expressed recombinant hVEGF165 with biological activity. Methods The hVEGF165 gene was amplified by PCR. After DNA sequence analysis, the recombinant plasmid pPIC9K / hVEGF165 was constructed by inserting Pichia pastoris yeast expression vector containing AOX1 promoter and α secretion signal peptide sequence. The transformed yeast host strain KM71 was screened for multiple copies of the integrated transformants and cultured in shake flasks with 10 mL / L methanol to induce expression. The expressed product was purified by affinity chromatography with Heparin-Sepharose CL6B and its biological activity was measured by human umbilical vein endothelial cells (HUVECs). Results SDS-PAGE analysis showed that the expressed product was soluble in the supernatant in the form of soluble molecules, and the expression level reached more than 60% of the total protein in the supernatant 4 days after induction. Western blot showed that the expressed product has good antigenicity and specificity. Purified by Hep-arin-Sepharose CL6B affinity chromatography, the purity of rhVEGF165 can reach more than 90%. The biological activity test confirmed that it has the activity of stimulating the proliferation of umbilical vein endothelial cells (HUVECs). Conclusion In the Pichia pastoris expression system, efficient secretion of hVEGF165 was achieved.