人胰岛新生相关蛋白基因的克隆及在毕赤酵母中的表达  

Cloning and secretory expression of islet neogenesis-associated protein in Pichia pastoris

作　　者：沙建平[1] 薛耀明[1] 陈炫[2] 曾展军[1] 魏民[1] 罗祥蓉[1] 何飞英[1] 王玲[1] 卓凤婷[1] 

机构地区：[1]南方医科大学南方医院内分泌科,广东广州510515 [2]暨南大学第一附属医院临床实验中心,广东广州510630

出　　处：《南方医科大学学报》2008年第7期1203-1206,共4页Journal of Southern Medical University

基　　金："十五"863生物领域高技术首批重点课题(N02001AA215161)~~

摘　　要：目的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试验。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XhoⅠ和EcoRⅠ酶切位点处,再将融合基因αINGAP重组到表达质粒pPIC9K的BamHⅠ和EcoRⅠ之间,SalⅠ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Westernblotting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。Objective To clone the recombinant human islet neogenesis-associated protein （rhINGAP） gene for its secretory expression in Pichia pastoris. Methods INGAP gene was amplified with PCR and inserted between Xho Ⅰ and EcoR Ⅰ downstream sites of the α factor of the recombinant plasmid α/pUC18. The fusion gene of α factor and INGAP was subsequently inserted between BamH Ⅰ and EcoR Ⅰ sites of the plasmid pPIC9K of P. pastor＆. After confirmation with restriction enzyme digestion and sequencing, the positive recombinant plasmid that integrated INGAP gene was linearized with SolⅠ digestion and transformed into the yeast host strain GS 115 through electroporation. The yeast transformants that harbored the INGAP gene with high copies were selected with the auxotroph medium and G418, followed then by PCR verification of the positive transformants, from which the expression of recombinant human INGAP was induced with methanol as the only carbone source. The antigenic activity of the desired protein was then detected using Western blotting and enzyme-linked immunosorbent assay （ELISA）. Results and Conclusion The recombinant expression plasmid INGAP/pPIC9K was successfully constructed, and 3 positive transformants were obtained. The expressed protein showed good antigenic activity as confirmed by Western blotting and ELISA.

关 键 词：胰岛新生相关蛋白 毕赤酵母 分泌表达 

分 类 号：Q78[生物学—分子生物学]