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目的真核表达人B7-H3Ig重组蛋白,探讨B7-H3信号在T细胞中的作用。方法重叠PCR方法构建B7-H3Ig重组基因,进而获得重组逆转录病毒载体pGEZ-Term/B7-H3Ig,转染L929细胞建立L929/B7-H3Ig基因转染细胞株,纯化获得B7-H3Ig重组蛋白。体外激发型CD3单克隆抗体(mAb)刺激活化T细胞过程中加入B7-H3Ig,观察其对T细胞增殖以及IL-10与IFN-γ分泌的作用。结果 B7-H3Ig经Protein G柱纯化后纯度>90%,浓度达0.559 mg/mL。重组蛋白结合实验表明CD3 mAb刺激48 h后T细胞表面B7-H3受体表达达到高峰。B7-H3Ig以剂量依赖方式促进CD3 mAb诱导的T细胞体外增殖与IL-10和IFN-γ分泌。结论成功建立真核表达人B7-H3Ig基因转染细胞株,B7-H3Ig协同刺激T细胞增殖与IL-10和IFN-γ分泌。
Objective To eukaryotic expression of human B7-H3Ig recombinant protein to explore the role of B7-H3 signaling in T cells. Methods Recombinant B7-H3Ig gene was constructed by overlapping PCR. The recombinant retroviral vector pGEZ-Term / B7-H3Ig was constructed and transfected into L929 cells to construct the recombinant cell line L929 / B7-H3Ig. The B7-H3Ig recombinant protein was purified. B7-H3Ig was added during the activation of T cells stimulated by CD3 monoclonal antibody (mAb) in vitro, and its effect on T cell proliferation and secretion of IL-10 and IFN-γ was observed. Results The purity of B7-H3Ig was> 90% purified by Protein G column and the concentration was 0.559 mg / mL. Recombinant protein binding experiments showed that the expression of B7-H3 on the surface of T cells reached a peak at 48 h after CD3 mAb stimulation. B7-H3Ig promoted CD3 mAb-induced T cell proliferation and IL-10 and IFN-γ secretion in vitro in a dose-dependent manner. Conclusion B7-H3Ig cells transfected with eukaryotic expressing human B7-H3Ig were successfully established and synergistically stimulate the proliferation of T cells and the secretion of IL-10 and IFN-γ.