摘要
目的：实现鸡α干扰素（ChIFNα）在毕赤酵母GS115中的表达,并考察糖基化对其表达的影响。方法：人工合成按照毕赤酵母偏好密码子优化的ChIFNα基因,克隆至分泌表达载体pPIC9,电转到毕赤酵母GS115中进行表达。利用定点突变对ChIFNα基因序列中4个糖基化位点进行缺失,SDS-PAGE分析N-糖基化对毕赤酵母表达ChIFNα的影响。结果：ChIFNα在GS115中获得了表达,在摇瓶发酵条件下,表达量为35.2 mg/L;构建了缺失1~4个糖基化位点的4个突变体,在GS115中实现了表达,分析表达结果显示,与未突变的ChIFNα对照相比,突变体的糖基化程度和表达量都有大幅度下降。结论：N-糖基化对于毕赤酵母表达ChIFNα具有重要影响,无糖基化的ChIFNα在毕赤酵母中表达量极低。
Objective： To express chicken interferon-α（ChIFNα） in Pichia pastoris,and investigate the effect of glycosylation on the secretion of ChIFNα.Methods： The ChIFNα gene,optimized for the preferred condons of the yeast and synthesised,was cloned into the P.pastoris expression vector pPIC9 and expressed in GS115 strains.In addition,four glycosylation minus ChIFNα mutants,constructed by site-directed mutagenesis were also expressed in P.pastoris.The effect of glycosylation on the secretion was assayed by SDS-PAGE.Results： Expression system produced ChIFNα into the broth medium under shaking flask growth conditions with yield of about 35.2 mg/L.The mutant proteins were assayed by SDS-PAGE,compared with the wild-type,glycosylation and expression of the mutants fell sharply.Conclusion： N-glycosylation plays a vital effect on the secretion of ChIFNα from P.pasto ris,and the yield of ChIFNα without glycosylation minus is very low.
出处
《生物技术通讯》
CAS
2012年第5期690-693,共4页
Letters in Biotechnology
关键词
鸡α干扰素
定点突变
N-糖基化
毕赤酵母
chicken interferon-α
site-directed mutagenesis
N-linked glycosylation
Pichia pastoris
