摘要
探索共表达蛋白质折叠辅助因子Ero1、PDI和Bi P对毕赤酵母GS115分泌表达融合蛋白质IFNβ-HSA的影响。将构建的蛋白质折叠辅助因子表达载体p GAP-Ero1、p GAP-PDI、p GAPBi P和空载对照线性化后,电击转化重组毕赤酵母GS115/IFNβ-HSA细胞,用含有400μg/m L zeocin的YPD平板筛选阳性转化子,并采用PCR和Western blot法进一步鉴定。阳性转化子进行摇瓶发酵后,采用SDS-PAGE及Western blot法分析共表达Ero1、PDI和Bi P对IFNβ-HSA表达水平的影响。结果表明,Ero1、PDI和Bi P成功地在胞内过量表达,且不影响宿主细胞的正常生长;共表达Ero1和PDI分别使IFNβ-HSA的表达量提高了80%和90%,而共表达Bi P则对IFNβ-HSA的表达水平无明显影响。
To explore the expression level of co-expression of folding factors Ero1,PDI and BiP on production of IFNβ-HSA by Pichia pastoris GS115.The pGAP-Ero1,pGAP-PDI,pGAP-BiP expression plasmids and the empty plasmid pGAPZ B （control） were linearized and integrated into the genome of P.pastoris GS115-IFNβ-HSA by electroporation.The recombinant yeasts were screened with 400 μg/mL zeocin.Positive recombinants were identified by genomic PCR and Western blot.The transformants were fermented in flask and the expression products were confirmed by SDS-PAGE and Western blot.The expression levels of IFNβ-HSA co-expressed with Ero1,PDI and BiP were analysed.The results indicated that Ero1,PDI and BiP were successfully expressed in recombinants with no effect on growth.Compared with Bip,co-expressed with Ero1 and PDI significantly improved the expression level of IFNβ-HSA,the expression level was 80％（Ero1） and 90％（PDI） higher than control.
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2014年第12期1246-1250,共5页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(30970029)
江苏省优势学科建设工程资助项目
