论文部分内容阅读
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 secrete and express biologically active recombinant human islet neogenesis-related protein (rhINGAP) in Pichia pastoris for physiological function studies and animal experiments of INGAP. Methods The INGAP gene was amplified by PCR and inserted into the Xho Ⅰ and EcoR Ⅰ restriction sites of the α-factor signal sequence of recombinant plasmid α / pUC18. The fusion gene αINGAP was recombined into BamH Ⅰ and EcoR Ⅰ of the expression plasmid pPIC9K , Sal Ⅰ digested linearized recombinant plasmid INGAP / pPIC9K, electroporated into Pichia pastoris, auxotrophic medium MD and G418 screening yeast transformants containing high copy expression cassette, PCR identified whether the target gene INGAP, methanol-induced rhINGAP The target protein was identified by SDS-PAGE and Western blottmg, and the biological activity was quantified by ELISA. Results The recombinant expression plasmid INGAP / pPIC9K was successfully constructed. Three positive transformants were screened out and the expressed product had good antigenic activity. Conclusion The rhINGAP expression and purification in Pichia pastoris was achieved efficiently.