人胰岛新生相关蛋白在毕赤酵母中的表达和纯化

Expression and purification of human islet neogenesis-associated protein in Pichia pastoris

目的将在毕赤酵母中分泌表达具有生物学活性的重组人胰岛新生相关蛋白(rhINGAP),用于INGAP的生理功能研究和动物实验。方法通过PCR扩增INGAP基因,插入到重组质粒α/pUC18的α因子信号序列的Xho Ⅰ和EcoR Ⅰ酶切位点处,再将融合基因αINGAP重组到表达质粒pPIC9K的BamH Ⅰ和EcoR Ⅰ之问,Sal Ⅰ酶切线性化重组质粒INGAP/pPIC9K,电穿孔转化毕赤酵母,营养缺陷型培养基MD和G418筛选含高拷贝表达盒的酵母转化子,PCR鉴定是否含有目的基因INGAP,甲醇诱导rhINGAP的表达,SDS-PAGE和Western blottmg鉴定目的蛋白,用ELISA定量测定生物学活性。结果成功构建了重组表达质粒INGAP/pPIC9K,筛选得到3个阳性转化子,表达产物具有良好的抗原活性。结论实现了rhINGAP在毕赤酵母中的高效分泌性表达及纯化。

Objective To express and purify the human islet neogenesis-associated protein （rhINGAP））gene in Piehia pastoris. Methods INGAP gene was amplified with PCR and inserted into the sites between Xbo [ and EcoR ] in the downstream of the a factor of the recombinant plasmid a/pUC18. Then, the fusion gene of aINGAP was digested and inserted into the sites between BamH Ⅰ and EcoR Ⅰ in the expression plasmid pPIC9K of P. pastoris. After the positive recombinant plasmid integrated into INGAP was confirmed by restriction enzyme digestion and sequencing, it was linearized with Sal digestion and transformed into the yeast host strain GS115 through electroporation. The yeast transformants that harbor the desired gene INGAP with high copy were selected by the auxotroph medium G418, and verified by PCR technique. The condition of hake-flask culture was optimized, and the recombinant human INGAP expression was induced with methanol as the only carbone soure. The antigen activity of the desired protein was detected by Western blot and ELISA method. Results Expression plasmid recombinant INGAP/pPICgK was successfully constructed, and three positive transformants were obtained. The expressed protein had satisfactory antigen activity, which was confirmed by the Western blot and ELISA method. Conclusions Cloning and secretion expression of rhlNGAP in Pichia pastoris have been satisfactorily achieved.