Exendin-4-Tβ4融合蛋白在毕赤酵母中的表达研究

Studies on the Expression of Exendin-4-Tβ4 Fusion Protein in Pichia pastoris

为实现Exendin-4-Tβ4(以下简称Ex4-Tβ4)融合蛋白在毕赤酵母中的表达,采用重叠PCR法扩增出Ex4-Tβ4融合基因,将其克隆入表达质粒pPIC9KH,采用毕赤酵母GS115作为宿主菌,利用电转化法将重组载体pPIC9KH-Ex4-Tβ4转入GS115中,利用甲醇作为诱导物,对重组载体进行诱导表达,成功构建重组质粒pPIC9KH-Ex4-Tβ4,SDS-PAGE证明Ex4-Tβ4在毕赤酵母中能高效表达,成功地在毕赤酵母中表达了Ex4-Tβ4融合蛋白.

Objective： To produce the Exendin-4-Tβ4 （hereinafter referred to as Ex4-Tβ4） fusion protein in Pichia pastoris.Methods： The whole sequence of the fusion protein was amplified by overlapping PCR and cloned into expression vector pPIC9KH.After verification by restriction digestion and sequencing,the vector was transferred into P.pastoris strain GS115 with electronic pulse.The recombinant was obtained through culture on a nutrition-deficient medium,and then the strains with multiple copy foreign gene were induced by methanol.Result： The expressive vectors were successfully constructed,and SDS-PAGE indicated that the protein had been expressed in P.pastoris.Conclusion： Ex4-Tβ4 fusion protein can be expressed in P.pastoris,which lays the foundation for large scale production of Ex4-Tβ4 and may play a significant role in the therapy of diabetes and diabetes ulcer.