CTLA-4/Ig融合蛋白在COS-7中的表达、纯化及鉴定

Expression, purification and identification of CTLA-4/Ig fusion protein in COS-7 cells

目的 探讨pCTLA 4/Ig融合蛋白表达载体在哺乳动物细胞中的表达及表达产物的鉴定、纯化 ,为其功能研究和临床应用奠定基础。方法 将pCTLA 4/Ig表达质粒转染COS 7细胞 ,双抗体夹心ELISA检测细胞培养上清中融合蛋白表达 ;ProteinA亲和层析纯化 ,SDS PAGE、Western印迹、3H TdR掺入等进行分子量、纯度、抗原特异性及生物学活性鉴定。结果 在pCTLA 4/Ig质粒转染后的 48h和 96h上清中 ,均能检测到CTLA 4/Ig融合蛋白表达 ;经亲和层析纯化后 ,在SDS PAGE胶上出现一条分子量约 5 0× 10 3 的与预期分子量大小相符的蛋白条带 ,该蛋白能与抗人CTLA 4单抗特异结合 ;该纯化融合蛋白能抑制人MLR ,抑制率为6 0 %～ 70 %。结论 在哺乳动物细胞中成功地表达并纯化了有生物学活性的重组人CTLA 4/Ig融合蛋白。

Objective To study the expression of pCTLA 4/Ig fusion protein expression vector in mammalian cells and the purification and identification of its expressive product in vitro for the further study of its function and clinical application. Methods Expression vector pCTLA 4/Ig was transfected into COS 7 with lipid reagent. The expression of fusion protein in the supernatant of the cell culture media was detected with double antibody sandwich ELISA. After purified with protein A affinity chromatography, the product was studied with SDS PAGE, Western blot and 3H TdR incorporation to determine its molecular weight, purity, antigen specificity and bioactivity. Results The fusion protein could be detected in the supernatant of cell culture media 48 and 96 h after the transfection of pCTLA 4/Ig. A protein band with molecular weight of 50×10 3 was found in the SDS PAGE gel. This protein specifically bound with human CTLA 4 monoclonal antibody and inhibited 60% to 70% proliferation of peripheral blood mononuclear cell (PBMC) in mixed lymphocyte reaction (MLR). Conclusion Recombined bioactive human CTLA 4/Ig fusion protein can be successfully expressed and purified in mammalian cells.