人CD28-Fc融合蛋白在真核细胞中的表达与纯化

目的建立稳定表达CD28-Ig融合蛋白系统,提供用于研究的CD28-Ig融合蛋白。方法构建高表达的真核细胞表达载体,利用FAD批准的、可用于生产人用重组蛋白药物的CHO细胞进行表达,MTX加压筛选出稳定表达的细胞株。蛋白A纯化获得电泳纯融合蛋白。结果经RT-PCR,SDS-PAGE及Western印迹鉴定,CHO细胞上清中纯化的蛋白是CD28与Ⅰg的融合蛋白,并获得纯度较高的融合蛋白。结论成功建立CD28-Ig融合蛋白表达体系,并获得稳定表达融合蛋白的细胞株,为T细胞活化第二信号系统的基础研究奠定了基础。

Expression and Purification of Human CD28-Ig Fusion Potein in Eukaryotic Cells

Objective To establish a stable expression system of CD28-Ig fusion protein in mammalian cell line and to provide CD28-Ig fusion protein in the study of costimulatory signal sytem. Methods A stable mammalian expression vector pCI/CD28Ig was constructed which contained the CD28 extracellular region and genomatic human IgG1 Fc (including hinge, CH2, and CH3 regions) gene. The CD28-Ig fusion proteins expressed in CHO cell culture supernatant were identified by sandwich EL1SA. By using MTX and two times cloning with limiting dilution method, individual CHO engineering cell lines, which secret CD28-Ig fusion protein in the culture supernatant, were obtained. The fusion protein was obtained by purifying protein A. Results The proteins purified in culture supernatant was confirmed to be CD28-Ig fusion protein by RT-PCR, SDS-PAGE and Western blot assay. Conclusion A stable protein expressing CHO cell line which expresses CD28-Ig fusion protein was established and purified fusion protein was obtained.