人ICOS-Ig融合蛋白的真核表达及其生物学活性

目的:真核表达人ICOS胞外区与IgG Fc融合蛋白,并对其生物学活性进行初步分析。方法:以RT-PCR方法克隆人ICOS胞外区片段和人IgG Fc片段,定向插入真核表达载体pSecTag2,构建重组质粒pSecTag2-ICOS-Ig,脂质体转染CHO细胞,可溶性表达并纯化ICOS-Ig融合蛋白。采用SDS-PAGE、蛋白质印迹、ELISA对融合蛋白进行鉴定,配体结合活性实验和混合淋巴细胞增殖反应实验检测融合蛋白的活性。结果:构建的ICOS-Ig融合蛋白表达载体在CHO细胞中表达ICOS-Ig蛋白。纯化获得的ICOS-Ig可以与配体ICOSL特异性结合,并抑制同种T淋巴细胞增殖反应。结论:成功构建了人ICOS胞外区与人IgG Fc融合蛋白真核表达系统,获得有生物学活性的ICOS-Ig融合蛋白。

Eukaryotic expression of human ICOS-Ig fusion protein and analysis of its biological activity

Objective:To express human ICOS extracellular region and IgG Fc fusion protein using euckaryotic vector and to analyze its biological activity.Methods: Human ICOS extracellular region and IgG Fc fragment were cloned by RT-PCR and inserted into eukaryotic expression vector pSecTag2.The constructed plasmid pSecTag2-ICOS-Ig was stably transfected into CHO cells.Soluble ICOS-Ig fusion protein was collected from serum-free medium and purified with protein A affinity chromatography.The purified product was analyzed by SDS-PAGE,Western blotting assay, and ELISA.Fluorescence-activated cell sorting (FACS) and mixed lymphocyte reaction (MLR) were used to study the activity of the fusion protein.Results: ICOS extracellular region and IgG Fc fragment were successfully cloned into expression vector; ICOS-Ig fusion protein was expressed and purified in mammal cells.The purified fusion protein specifically bound to ICOSL and inhibited mixed lymphocyte reaction.Conclusion: A ICOS-Ig fusion protein expression system has been successfully constructed,and bioactive ICOS-Ig fusion protein has been obtained.