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目的 研究人血管内皮生长因子 (hVEGF16 5 )基因在Pichia .pastoris酵母中的表达 ,获得高效表达的具有生物学活性的重组VEGF16 5。方法 采用聚合酶链反应 (PCR)扩增hVEGF16 5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酵母载体中 ,构建重组表达质粒 pPIC9K hVEGF16 5 ,转化酵母宿主菌KM 71,筛选多拷贝整合转化子 ,摇瓶培养 ,1%甲醇诱导表达。结果 SDS PAGE分析显示 ,诱导 4d的培养上清中hVEGF16 5表达量达上清总蛋白的 30 %以上。ELISA及Westernblot实验表明 ,表达产物具有良好的抗原性和特异性。生物学活性检测证实其具有刺激HUVEC增殖的功能。结论 在Pichia .pastoris表达系统中实现了hVEGF16 5的高效分泌性表达
Objective To study the expression of human vascular endothelial growth factor (hVEGF16 5) gene in Pichia pastoris yeast and to obtain highly expressed recombinant VEGF16 5 with high biological activity. Methods hVEGF16 5 gene was amplified by polymerase chain reaction (PCR). After DNA sequence analysis, the recombinant plasmid pPIC9K hVEGF16 5 was inserted into Pichia pastoris yeast vector containing AOX1 promoter and α secretion signal peptide sequence. Host strain KM 71, screening multiple copies of integrated transformants, shake flask culture, 1% methanol induced expression. Results SDS PAGE analysis showed that the expression of hVEGF16 5 in the culture supernatant induced 4 days reached more than 30% of the total supernatant protein. ELISA and Western blot showed that the expressed product had good antigenicity and specificity. Biological activity test confirmed that it has the function of stimulating HUVEC proliferation. Conclusion Highly efficient secreted expression of hVEGF16 5 was achieved in the Pichia pastoris expression system