VEGF_(165)cDNA在酵母中的表达、纯化及其生物学活性研究

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。

Expression, purification and biological activity analysis of human vascular endothelial growth factor (VEGF_(165)) in Pichia pastoris

Aim To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expressed recombinant human VEGF165 with good biological activity. Methods Amplifying human VEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptides, the recombinant expression plasmids pPIC9K/hVEGF165 was constructed and transformed into KM71. The multiple insert transformants were screened， fermented in flasks and induced by 10 mL/L methanol. Results After 4 days of methanol induction, the expressed hVEGF165 reached up to 60％ of total proteins in supernatant by SDS-PAGE. Western blot assay proved the expressed hVEGF165 having good antigenicity and high specificity. The recombinant protein was further purified with Heparin-Sepharose CL6B affinity chromatography, and was proved to have good biological activity in stimulating HUVEC proliferation. Conclusion High-level expression of secreted hVEGF165 has been successfully achieved in Pichia pastoris expression system.