Aim: To obtain mouse B7-H3-Fc fusion protein and to investigate its biological function and effects on T lymphocyte activation.
Methods: The genes coding extracellular domain of mouse B7-H3 and the Fc fragment of human IgG1 were amplified from pMD19-T/mouse B7-H3 and pMD19-T/human IgG1 vectors by PCR. The two genes were combined with mouse B7-H3-Fc fragment by overlap PCR. Then the resulting gene fragment was inserted into eukaryotic vector pIRES2-EGFP after digested with EcoR I and Bgl II to construct the recombinant vector pIRES2-EGFP/B7-H3-Fc. The recombinant vector was transfected into CHO cells with LipfectAMINE™ 2000, and the cells were further selected with G418. The collected supernatant of the transfected cell line cultured in serum-free media was ultrafiltrated and concentrated, then purified by Protein G column. The expression of mouse B7-H3-Fc fusion protein was confirmed by Western blot. Effects of fusion protein on T cells proliferation and cytokine production in vitro was studied by methods of CCK8 and ELISA.
Results: The results showed that the transfected CHO cell line secreting mouse B7-H3-Fc fusion protein was constructed successfully. In vitro, mouse B7-H3-Fc fusion protein obviously promoted the proliferation of T cells in a dose-dependent manner.
Conclusion: A transfected CHO cell line stably expressing mouse B7-H3-Fc fusion protein has been obtained and the B7-H3-Fc fusion protein stimulates the proliferation of T cells and cytokines production in vitro.