α-2b干扰素重组质粒的建立及在毕赤酵母中的表达

Construction of Recombinant pPIC9K-IFNα-2b and Expression in Pichia pastoris

目的:采用基因工程手段构建基因工程菌表达α-2b干扰素。方法:根据毕赤酵母密码子嗜好性原理设计并合成α-2b干扰素基因序列,将其插入到毕赤酵母Pichia pastoris的分泌型表达质粒pPIC9K中,得到重组分泌表达质粒pPIC9K-IFNα-2b,并用电转化法转化P.pastoris GS115。筛选出整合型His+Muts菌株,进一步用G418筛选获得高拷贝转化子,经5d诱导表达后SDS-PAGE检测。结果:菌落PCR和序列测定结果显示重组表达质粒已成功构建,SDS-PAGE结果显示目的蛋白已成功表达。结论:在毕赤酵母中成功表达α-2b干扰素。

Objective： A recombinanted DNA Pichia pastroris was constructed to express mature IFNα- 2b with Pichia pastroris expression system. Methods： According to the synonymous codon bias of Pichia pastoris, The cDNA fragment coding for mature IFNα- 2b was designed and synthesized. Then the gene was inserted into the plasmid pPIC9K of Pichia pastoris to obtain secretory plasmid pPICgK - IFNα - 2b. After electroporation of Pichia pastoris GS115 （His^- Mut^＋ ）,some high- copy transformants（His^＋ Mut^s ） were selected by G418. After 5 days induction, the result was tested by SDS - PAGE. Results： PCR and DNA sequencing results showed that the recombined plasmid was constructed successfully. SDS - PAGE result showed that IFNα - 2b was expressed successfully. Conclusion： IFNα - 2b was successfully expressed in Pichia pastroros.