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[The high level expression of CTLA-4/Ig fusion protein in CHO cell]
Li-heng Zheng1, Quan-sheng Liu
1Department of Cytobiology, College of Life Science, Sichuan University, Chengdu 610041, China.
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 plasmids 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.
