人sTNFR II-IgG Fc融合蛋白在毕赤酵母菌中的表达及其产物分析

Expression and characterization of human sTNFR II-IgG Fc fusion protein in Pichia pastoris

目的： 在毕赤酵母菌中表达sTNFR Ⅱ-IgG Fc融合蛋白.检测融合蛋白是否形成二聚体,并对其N-糖链进行分析.方法： 从人淋巴细胞中提取总RNA, 经RT-PCR扩增目的基因sTNFRⅡ与IgG Fc, 然后利用重叠延伸PCR得到嵌合体基因sTNFRⅡ-IgG Fc, 并将其插入pPIC9载体中.以重组载体转化巴斯德毕赤酵母菌中进行克隆与表达.采用ELISA筛选高表达sTNFRⅡ-IgG Fc融合蛋白的重组毕赤酵母菌株, 对融合蛋白通过还原和非还原SDS-PAGE分析其二聚体结构, 采用L929细胞检测融合蛋白抗TNF-α的生物学活性, 最后利用荧光辅助糖电泳（FACE）分析融合蛋白的N-糖链.结果： 成功地建立了可分泌表达sTNFR Ⅱ-IgG Fc融合蛋白的重组酵母菌株, 摇瓶培养后融合蛋白的表达量为2 mg/L.SDS-PAGE和Western blot表明, 该融合蛋白能自然形成二聚体结构; 可抑制TNF-α对L929细胞的杀伤作用, 其中和5×10^4 U/L TNF-α的EC50为170 μg/L.FACE分析融合蛋白N-糖链的大小为11 ～13个糖残基.结论： 在毕赤酵母菌中成功地表达了sTNFR Ⅱ-IgG Fc融合蛋白, 为在毕赤酵母菌中表达其他的Fc融合蛋白和抗体提供了参考.

AIM： To find if human soluble tumor necrosis factor receptor Ⅱ （p75） fused IgG Fc protein （sTNFR Ⅱ- IgG Fc） could be expressed in Pichia pastoris with an active dimmer form and characterize its N-linked oligosaccharides. METHODS： Two gene fragment, human sTNFR Ⅱ and IgG. Fc, were got by RT-PCR from leucocytes stimulated with LPS. And the chimeric gene sTNFR Ⅱ-IgG Fc achieved through gene splicing by over lap extension （SOE） method was cloned into pPIC9 and transformed into methanotropic yeast Pichia pastoris. The fusion protein purified by Protein A affinity column was analyzed with SDS-PAGE electrophoresis under reducing or non-reducing conditions and immunological methods. The anti-TNF-α biological activity assay of fusion protein was performed with L929 cells and detected with MTT colorimetry. The N-linked oligosaccharides hydrolyzed from fusion protein were labeled with 8-amino-1, 3, 6- naphthalene trisulfonic acid （ANTS） were analyzed with fluorophore-assisted carbohydrate eletrophoresis （FACE） as well. RESULTS： The recombinant P. pastoris strain that expressed human sTNFR Ⅱ-IgG Fc fusion protein was constructed. The expression level of fusion protein in 2 L flask reached 2 mg/L. SDS-PAGE and Western blot showed the expressed fusion protein purified by protein was a dimer linked with inter-molecular disulfide linkage. The fusion protein neutralized cytotoxic activity of TNF-α to L929 cells, and the EC50 of the fusion protein to inhibit 5 × 10^4 U/L of TNF-α was 170 μg/L. The FACE analysis showed there are 11 to 13 hexoses on each N-linked oligosaccharide. CONCLUSION： The human sTNFR Ⅱ-IgG Fc fusion protein is expressed successfully in P. pastoris and it could be a reference for the future expression of other Fc fusion proteins or immunoglobulins in Pichia pastoris.