[Eukaryotic expression of recombinant B7-H3Ig protein and its effects on T cells]

To construct a recombinant eukaryotic expression vector containing the human B7-H3Ig gene and investigate the effects of B7-H3 signal on T cells.

Being constructed by overlap extension PCR, the recombinant gene fragment of B7-H3Ig was inserted into pGEZ-Term vector. The recombinant pGEZ-Term/B7-H3Ig vector was used to transfect L929 cells to purify B7-H3Ig from conditioned medium harvested from cultured L929/B7-H3Ig cells. T cells were then stimulated with agonistic anti-CD3 mAb in the presence or absence of purified B7-H3Ig, and cell proliferation and the secretion of IL-10 and IFN-γ were analyzed 72 h later.

The concentration of B7-H3Ig was 0.559 mg/mL with a purity of more than 90% after protein G affinity chromatography. B7-H3Ig binding test showed that the putative B7-H3 receptor was presented in activated T cells and the maximum binding capacity was observed 48 h later. B7-H3Ig enhanced, in a dose-dependent manner, the proliferation and IL-10 and IFN-γ secretion of T cells stimulated with agonistic anti-CD3 mAb.

L929/B7-H3Ig transfectants have been successfully constructed. B7-H3 is a co-stimulator for T cell proliferation and IL-10 and IFN-γ expressions.