Expression And Characterization Of Human CD40-IgG1Fc Fragment Fusion Protein And Its Effects On Apoptosis Of EBV-transformed B Cell Line

Aim: To construct eukaryotic expression vector CD40-IgG1Fc /pcDNA3.1(+) of human CD40-IgG1Fc fragment fusion protein, and to discriminate the stable expression of CD40-IgG1Fc fusion protein in CHO cells,and to investigate the influence of human CD40-IgG1Fc fragment fusion protein on apoptosis of EBV-transformed B lymphocyte.Methods: The gene encoding the CD40-IgG1Fc fusion protein were constructed in eukaryotic expression vector pcDNA3.1(+) by means of T-A cloning and subcloning techniques, then was transfected into CHO cells for stable expression. The expression of the fusion gene was detected by RT-PCR,Western blot and ELISA.The apoptosis was analysed using AO and EB double-staining methods and DNA ladder.Results: DNA sequencing and restriction endonuclease digestion analysis indicated that the Eukaryotic expression vector CD40-IgG1Fc/pcDNA3.1(+)was successfully constructed. After the recombinant plasmid was transfected into CHO cells, the stable expression of the fusion protein was demonstrated by western blot and ELISA.The obvious increasing of the apoptosis of B lymphocyte incubated with the CD40-IgG1Fc fusion protein was manifested by AO and EB double-staining methods and DNA ladder.Conclusion: The Eukaryotic expression vector of CD40-IgG1Fc/pcDNA3.1(+) was successfully constructed and stably expressed in CHO cells.The fusion protein could obviously promote the apoptosis of EBV-transformed B lymphocyte,and the apoptotic rate of B cells was dose- and time-dependent in experiment range.The purified human CD40-IgG1Fc fusion protein can be served as an important agent for further study in etiopathogenesis and immunotherapy of autoimmune diseases.