猪流行性腹泻病毒纳米抗体酵母表达载体的构建及其表达分析

Construction and expression analysis of Pichia pastoralis expression vector nanoantibody against Porcine Epidemic Diarrhea Virus

[目的]探究猪流行性腹泻病毒纳米抗体SH-Fc蛋白在毕赤酵母中的分泌表达情况,以期获得毕赤酵母表达系统,从而进一步研究PEDV的治疗方法。[方法]利用相关生物学分析软件对目的蛋白进行理化性质,磷酸化位点及二、三级结构的分析。将p UC57-SH-Fc质粒与酵母表达载体p PIC9K双酶切,构建真核表达载体p PIC9K-SH-Fc,经SalⅠ酶线性化后电转入毕赤酵母GS115中,利用G418抗性筛选以及菌落PCR的方法鉴定阳性菌株,阳性菌株经过30℃、0.8%甲醇诱导72 h,用SDS-PAGE和Western Blotting鉴定诱导表达后的上清液中蛋白表达情况。[结果]成功构建了真核分泌表达载体p PIC9K-SH-Fc,成功在毕赤酵母中表达目的蛋白,表达产物分子量与理论值55.1k Da相符合。[结论]成功诱导SH-Fc蛋白的分泌表达,获得了分泌表达目的蛋白的毕赤酵母表达系统,为后续研究PEDV的防治提供参考和奠定基础。

[Objective] The secretion and expression of porcine epidemic diarrhea virus nano-antibody SH-Fc protein in Pichia pastorais was investigated in order to obtain the expression system of Pichia pastorais,so as to further study the treatment of PEDV.[Method] The physicochemical properties,phosphorylation sites and secondary and tertiary structures of the target protein were analyzed by using relevant biological analysis software. The eukaryotic expression vector p PIC9 K-SH-Fc was constructed by digesting p UC57-SH-Fc with yeast expression vector p PIC9 K. After being linearized by SalⅠ,the eukaryotic expression vector p PIC9 K-SH-Fc was electroporated into Pichia pastoris GS115. The positive strains were identified by G418 resistance screening and colony PCR. The positive strains were induced by 0. 8% methyl alcohol at 30 ℃ for 72 hours. The protein expression in the supernatant was identified by SDS-PAGE and Western Blotting.[Result] The eukaryotic secretory expression vector p PIC9 K-SH-Fc was successfully constructed and the target protein was successfully expressed in Pichia pastoris. The molecular weight of the expressed product was consistent with the theoretical value of 55. 1 k Da. [Conclusion] The secretion expression of SH-FC protein was successfully induced,and the Pichia pastoris expression system for secretory expression of the target protein was obtained,providing reference and foundation for subsequent PEDV prevention and control studies.