CTLA-4/Ig在CHO/DG44细胞中的高效表达

The High Level Expression of CTLA-4/Ig Fusion Protein in CHO Cell

目的研究CTLA-4/Ig融合蛋白在CHO真核表达系统中的表达,并纯化所表达的CTLA-4/Ig融合蛋白,为其功能研究和临床应用奠定基础。方法将线性化的pMM-CTLA-4-IgG4/WG和pMMGR转染CHO/DG44细胞,对阳性克隆进行无血清悬浮培养,Western blot检测细胞培养液中CTLA-4/Ig融合蛋白的表达,筛选出高效稳定表达CTLA-4/Ig融合蛋白的CHO细胞;Protein A亲和层析纯化所表达的CTLA-4/Ig融合蛋白;SDS-PAGE电泳、Western印迹等对纯化的CTLA-4/Ig融合蛋白的相对分子质量、纯度、抗原特异性进行生物学鉴定。结果筛选出了能高效稳定表达CTLA-4/Ig融合蛋白的CHO细胞;纯化后的CTLA-4/Ig融合蛋白在SDS-PAGE电泳后出现一条与预期相对分子质量大小相符的蛋白条带;该蛋白能与羊抗人IgG-HRP抗体特异结合。结论获得了能够高效稳定表达具有生物学活性的CTLA-4/Ig融合蛋白的CHO细胞。

Objective To establish the basis in the function study and clinical application of the fusion protein of cytotoxic T lymphocyte antigen 4 CTLA-4 and IgG Crl （CTLA-4/Ig）, by studying the expression of CTLA-4/Ig fusion protein in eukaryotic CHO cells and purifying CTLA-4/Ig fusion protein expressing in CHO cells. Methods The CHO/dhfr- cells were transfected with linearized plasmlds of pMM-CTLA-4-IgG4/WG and pMMGR. The clones of CHO/pCTLA-4＋/pMMGR＋ were in suspension cultured in serum-free culture media. The expression level of CTLA-4/Ig fusion protein in cell culture media was detected through Western blot. The biological identification of relative molecular mass, purity and antigen specificity of the expressing CTLA-4/Ig fusion protein that have been purified with protein A affinity chromatography was studied by SDS-PAGE or Western blot respectively. Results The CHO cells that could steadily express at high level the CTLA-4/Ig fusion protein was selected for cloning. By SDS-PAGE for the CTLA-4/Ig fusion protein that have been purified with protein A affinity chromatography, a protein band was found to match well with the predicted relative molecular mass. This protein could specifically bind with human CTLA-4 monoclonal antibody. Conclusion The CHO cells that can express steadily CTLA-4/Ig fusion protein that is of the bioactivity at high level are successfully obtained.