人纤溶酶原K5基因在毕赤酵母中的表达及其抗肿瘤活性

Expression of Kringle 5 Gene of Human Plasminogen in Pichia pastoris and Antitumor Activity of Expressed Product

目的在毕赤酵母中表达人纤溶酶原K5基因,并检测hK5蛋白的抗肿瘤活性。方法根据酵母遗传密码的偏爱性优化设计hK5基因,构建重组表达质粒p819-8α-hK5,经G418加压筛选和甲醇诱导表达,并对摇瓶发酵条件进行优化。表达的hK5蛋白经纯化后,通过荷H22肿瘤昆明小鼠模型检测其抗肿瘤活性。结果筛选到2株高表达hK5蛋白的转化子,摇瓶表达量超过150mg/L,纯化后的蛋白纯度大于95.0%,并能够显著抑制昆明小鼠H22肿瘤的生长。结论hK5基因能够在毕赤酵母中高效分泌表达,且表达的hK5蛋白具有良好的抗肿瘤活性。

Objective To express the kringle 5 gene of human plasminogen （hKS） in Pichia pastoris and determine the antitumor activity of expressed hK5 protein. Methods Design and synthesize hK5 gene according to the codon bias of Pichia pastoris and insert into plasmid p819. Transform the constructed recombinant plasmid p819-8α-hK5 to Pichia pastoris GS115, screen transformants by G418 pressure screening for expression under induction of methanol. The condition for fermentation culture of recombinant Pichia pastoris in shaking flask was optimized. The expressed hK5 protein was purified by DEAE-Sepharose chromatography and G75 gel filtration, and evaluated for inhibitory activity to H22 tumor in KM mouse model. Results Two transformants for high expression of hK5 protein was screened. The expression level of hK5 protein in Pichia pastoris cultured in shaking flask under the optimized condition was more than 150 mg/L. The expressed hK5 protein reached a purity of more than 95. 0% after purification and inhibited the growth of H22 tumor in KM mice. Conclusion hK5 protein was highly expressed in Pichia pastoris and showed good antitumor activity.