Aim: To construct a recombinant eukaryotic expression vector containing pcDNA3.1-mOX40-Ig fusion gene and obtain mOX40-Ig fusion protein with bioactivity by transfecting CHO cells.
Methods: The gene fragment encoding the human IgG1Fc was amplified by RT-PCR and the eukaryotic expression vector pcDNA3.1-hIgG1Fc was constructed. After sequencing, mOX40 extracellular gene was cloned from pIRES2-EGFP-OX40 by PCR and then inserted into the recombinant vector pcDNA3.1-hIgG1Fc. The right recombinant was transfected into CHO cells with lipofectin Ragent and its expression was detected by RT-PCR and sandwich-ELISA. After purified by protein A affinity column chromatography, the mOX40-Ig fusion protein was identified by SDS-PAGE and its effect on the proliferation of B cells in vitro was studied by (3)H-TdR method.
Results: The hIgG1Fc, mOX40 extracellular gene and mOX40-Ig gene were consistent with DNA sequencing.The expression of mOX40-Ig fusion protein in CHO cells was confirmed by RT-PCR, sandwich-ELISA and SDS-PAGE. (3)H-TdR analysis showed the mOX40-Ig fusion protein stimulated the proliferation of B cells in vitro.
Conclusion: A eukaryotic expression vector containing pcDNA3.1-mOX40-Ig has been constructed successfully and the stable expression of mOX40-Ig fusion protein with bioactivity has been acquired, which lays a solid basis for further study of the application related to OX40.