The Expression Of CTLA-4/IgG4 Fusion Protein At High Level In CHO Cells

Objective: To establish the basis of the function study and the clinical application of the CTLA-4/IgG4 by studying the expression of the CTLA-4/IgG4 fusion protein in the eukaryotic (Chinese hamster ovary cell, CHO) CHO cells and purifying CTLA-4/IgG4 fusion protein expressed in the CHO cells and selecting the CHO cells that can express CTLA-4/IgG4 fusion protein at high level through transfecting the plasmids of pMMGR and pMM-CTLA4-IgG4/WG into the CHO cells.Methods: The plasmids of pMMGR and pMM-CTLA4-IgG4/WG were linearized by digesting the plasmid pMMGR with Nde I and the plasmid pMM-CTLA4-IgG4/WG with Not I . The CHO/dhfr-(dihydrofolate reductase,dhfr) cells were transfected with linearized plasmids pMM-CTLA4-IgG4/WG and pMMGR. The CH0/pCTLA-4+/pMMGR+ clons were selected by MTX and G418.The CH0/pCTLA-4+/pMMGR+ clones were cultured in suspension culture with serum-free media. The expression level of the CTLA-4/IgG4 fusion protein in the different density MTX media was detected through SDS-PAGE. The biologic identification of the expressed CTLA-4/IgG4 fusion pritein molecular weight and purity were detected by SDS-PAGE.The antigen specificity of the expressed CTLA-4/IgG4 fusion protein that have been purified with protein A affinity chromatography is studied respectively through Western blot.Results: The CHO cells that can express CTLA-4/IgG4 fusion protein at high level is selected by SDS-PAGE for the CTLA-4/IgG4 fusion protein that have been purified with protein A affinity chromatography , a protein band that match well with the predicted molecular weight is found .Conclusion: The CHO cells that can express CTLA-4/IgG4 fusion protein were obtained. Those cells can express CTLA-4/IgG4 fusion protein that is of antigen-specific at high level are successfully selected in the pressure of MTX.