Study of expression and purification of CD40-Ig fusion protein in CHO cells
Objective To obtain CD40 Ig fusion protein, and investigate the therapeutic potentials of blocking the CD40/CD40L signal pathway in the prevention and treatment of graft versus host disease (GVHD). Methods The CD40 Fc fusion gene, derived from mammalian transient expressing vector pIG/40Ig, was inserted into constitutive expressing vector pcDNA3.1. Then the recombinant expression vector was transfected into CHO cells with Lipofectamine reagent. The CHO cells, secreting CD40 Ig fusion protein in the culture supernatant, were obtained after selecting by G418. That was confirmed by Sandwich ELISA and Western bolt assay. The transfected CHO cells were cloned two times by limited dilution method. After big batch serum free culture using roller bottle, the concentrated supernatant was purified by rProtein A affinity chromatograph method. The purity of the purified recombinant was confirmed by SDS PAGE. The binding of the CD40 Ig fusion protein to Jurkat cell was detected by FCAS. Results Mammalian constitutive expression vector p3.1/40Ig was constructed as described above. Then the mixed CHO clones, expressing CD40 Ig fusion protein, were selected by G418 0.8g/L for two weeks after transfection. Through twice limited dilution cloning, five single clones were obtained, and the best one named as B2. The big batch culture supernatant of B2 was harvested, concentrated, and purified through rProtein A affinity chromatograph. The purity of recombinant CD40 Ig protein is above 95%. FACS results shown that CD40 Ig can bind to the CD40L expressing on the Jurkat cells, which were stimulated by PHA P. Conclusion The CD40 Ig could mimic the native CD40 molecule and bind to its ligand. It provided a useful tool to investigate the role of blockade CD40/CD40L costimulation signal pathway in the prevention and therapy of GVHD.