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

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sTNFRII与IgG Fc,然后利用重叠延伸PCR得到嵌合体基因sTNFRII-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2mg/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5×104U/LTNF-α的EC50为170μg/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。

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

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 IgGFc, 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-alpha 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-alpha to L929 cells, and the EC(50) of the fusion protein to inhibit 5 x 10(4) U/L of TNF-alpha was 170 microg/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.