摘要
目的:在乳酸克鲁维酵母中表达人可溶性肿瘤坏死因子受体Ⅱ(sTNFRⅡ)与IgG Fc的融合蛋白。方法:首先获得sTNFRⅡ-IgGFc融合基因片段,然后构建至乳酸克鲁维酵母表达载体pKLAC1中,获得sTNFRⅡ-IgGFc的表达载体,并将其电转化乳酸克鲁维酵母(Δura3),通过ELISA方法筛选高表达sTNFRⅡ-IgGFc融合蛋白的重组乳酸克鲁维酵母菌株,采用还原和非还原SDS-PAGE分析融合蛋白是否形成二聚体结构,Western印迹验证sTNFRⅡ-IgGFc融合蛋白在乳酸克鲁维酵母(Δura3)中的表达。结果:构建了sTNFRⅡ-IgGFc表达载体pKLAC1-sTNFRⅡ-IgGFc,获得了表达sTNFRⅡ-IgGFc的乳酸克鲁维酵母菌株,SDS-PAGE和Western印迹表明该融合蛋白能自发形成类似于抗体的二聚体结构。结论:实现了sTNFRⅡ-IgGFc融合蛋白在乳酸克鲁维酵母(Δura3)中的表达。
Objective：To express the human soluble tumor necrosis factor receptor Ⅱ and IgG Fc fusion protein（sTNFRⅡ-IgGFc） in Kluyveromyces lactis（Δura3）.Methods：The sTNFRⅡ-IgGFc gene was obtained from vector pPIC9-sTNFRⅡ-IgGFc by XhoⅠ/NotⅠ digestion,and cloned into pKLAC1 to construct vector pKLAC1-sTNFRⅡ-IgGFc.pKLAC1-sTNFRⅡ-IgGFc was linearized and transformed into K.lactis（Δura3）.The fusion protein was expressed and analyzed with SDS-PAGE and Western blot under reducing or non-reducing conditions.Results：The expression vector pKLAC1-sTNFRⅡ-IgGFc was constructed.The recombinant K.lactis strain expressing human sTNFRⅡ-IgGFc fusion protein was obtained.The results of SDS-PAGE and Western blot showed that the expressed fusion protein could naturally form a dimer through the inter-molecular disulfide linkage in the hinge region of IgGFc.Conclusion：The human sTNFRⅡ-IgGFc fusion protein was expressed successfully in K.lactis（Δura3）.
出处
《生物技术通讯》
CAS
2011年第1期11-14,共4页
Letters in Biotechnology
基金
国家高技术研究发展计划(2007AA02Z103)
国家自然科学基金(30870050)
国家科技重大新药创制(2009ZX09031-002)
国家科技支撑计划(2008BAI66B03)
