CAJ | 학술논문

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Western blotting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.
목적 장재필적효모중분비표체구유생물학활성적중조인이도신생상관단백(rhINGAP),용우INGAP적생리공능연구화동물실험.방법 통과PCR확증INGAP기인,삽입도중조질립α/pUC18적α인자신호서렬적XhoⅠ화EcoRⅠ매절위점처,재장융합기인αINGAP중조도표체질립pPIC9K적BamHⅠ화EcoRⅠ지간,SalⅠ매절선성화중조질립INGAP/pPIC9K,전천공전화필적효모,영양결함형배양기MD화G418사선함고고패표체합적효모전화자,PCR감정시부함유목적 기인INGAP,갑순유도rhINGAP적표체,SDS-PAGE화Western blotting감정목적단백,용ELISA정량측정생물학활성.결과 성공구건료중조표체질립INGAP/pPIC9K, 사선득도3개양성전화자,표체산물구유량호적항원활성.결론 실현료rhINGAP재필적효모중적고효분비성표체급순화.
Objective To express and purify the human islet neogenesisassociated protein(rhINGAP))gene in Pichia pastoris. Methods INGAP gene was amplified with PCR and inserted into the sites between XhoⅠand EcoRⅠin the downstream of the α factor of the recombinant plasmid α/pUC18. Then, the fusion gene of αINGAP was digested and inserted into the sites between BamHⅠand EcoRⅠin 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 SalⅠ 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. ResultsExpression plasmid recombinant INGAP /pPIC9K 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 rhINGAP in Pichia pastoris have been satisfactorily achieved.