ICOS/Fc在CHO细胞中的稳定表达

目的:探讨Picos/Fc融合蛋白基因在中国仓鼠卵巢细胞(CHO)中的稳定表达及其表达产物的纯化和初步鉴定,为其功能研究和临床应用奠定基础.方法:将Picos/Fc表达质粒及含G418抗性基因neor的质粒Pegfp共转染CHO细胞,经G418抗性培养基筛选、克隆化培养、夹心ELISA检测,挑选100%强阳性且表达均一的细胞克隆,扩大培养取上清经Protein A亲和层析纯化,SDS-PAGE和Western印迹等进行分子质量、纯度、抗原特异性的初步鉴定.结果:获得稳定表达ICOS/Fc蛋白的CHO细胞单克隆,制备并经Protein A亲和层析柱获得纯化的ICOS/Fc融合蛋白,SDS-PAGE胶可见一条Mr约70×103的与预期分子质量大小相符的蛋白条带,Western印迹法可在同一位置检测到该蛋白与HRP酶标抗人Fc mAb特异结合.结论:在CHO细胞中成功地建立了稳定表达ICOS/Fc融合蛋白的细胞系,表达并纯化了有抗原特异性的重组ICOS/Fc融合蛋白.

Stable expression of ICOS/Fc in CHO cells

AIM: To study the stable expression of pICOS/Fc fusion protein expression vector in Chinese hamster ovary (CHO) cells and to purify and identify its expression product in vitro. METHODS: Expression vector pICOS/Fc and resistance plasmid pEGFP were cotransfected into CHO cells with electroporation. The selective medium containing G418 was then employed to select the positive colony. After repeating cloning culture and double antibody sandwich ELISA detection, the cell clones with stable expression were obtained. After purified with protein A affinity chromatography, the product was studied with SDS-PAGE and Western blotting to determine its molecular weight, purity and antigen specificity. RESULTS: CHO cell clones stably expressing pICOS/Fc fusion proteins were obtained and the fusion protein was available from the supernatant of cell culture media after purified with protein A affinity chromatography. A protein band with molecular weight of 70 KDa was found in the SDS-PAGE. This protein specifically bound with anti-human Fc monoclonal antibody labeled HRP. CONCLUSION: Recombined mouse ICOS/Fc fusion protein with antigen specificity is successfully and stably expressed in CHO cells.