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目的:制备B7-H3-Fc融合蛋白,研究其对T细胞的共刺激作用。方法:首先采用PCR技术分别从pMD19-T/小鼠B7-H3和pMD19-T/hIgG1(Fc)重组载体中扩增出小鼠B7-H3胞外段基因和人IgG1重链Fc恒定区基因。通过重叠PCR技术将2段基因连接成B7-H3-Fc,经EcoR I和BglII双酶切后插入真核表达载体pIRES2-EGFP构建成pIRES2-EG-FP/B7-H3-Fc重组载体。脂质体法转染CHO细胞,经G418加压筛选能稳定分泌表达小鼠B7-H3-Fc融合蛋白的基因转染细胞,并经Western blot鉴定。该转基因细胞无血清培养后,收集细胞上清、超滤浓缩后行经Protein G柱纯化,获得纯品B7-H3-Fc融合蛋白。通过CCK-8以及ELISA方法检测小鼠B7-H3-Fc融合蛋白对T细胞体外增殖及细胞因子分泌的影响。结果:成功地构建了能稳定表达B7-H3-Fc融合蛋白基因的CHO转基因细胞株,该融合蛋白能够剂量依赖性地促进T细胞体外增殖及IL-2和IFN-γ等细胞因子分泌。结论:本研究提示B7-H3作为重要的共刺激分子,在调节T细胞免疫应答中发挥了正性共刺激作用。
Objective: To prepare B7-H3-Fc fusion protein and study its costimulatory effect on T cells. Methods: The mouse B7-H3 extracellular domain gene and the human IgG1 heavy chain Fc constant region gene were amplified from pMD19-T / mouse B7-H3 and pMD19-T / hIgG1 (Fc) . The recombinant plasmid pIRES2-EG-FP / B7-H3-Fc was inserted into the eukaryotic expression vector pIRES2-EGFP by EcoR I and BglII digestion. CHO cells were transfected by lipofectamine. The transfected cells stably secreting mouse B7-H3-Fc fusion protein were screened by G418 and identified by Western blot. After the serum-free culture of the transgenic cells, the supernatant of the cells was collected, concentrated by ultrafiltration and purified by Protein G column to obtain pure B7-H3-Fc fusion protein. The effects of B7-H3-Fc fusion protein on proliferation and cytokine secretion of T cells were examined by CCK-8 and ELISA. Results: The transgenic CHO cell line stably expressing the B7-H3-Fc fusion protein gene was successfully constructed. The fusion protein can promote the proliferation of T cells in vitro and the secretion of cytokines such as IL-2 and IFN-γ in a dose-dependent manner. Conclusion: This study suggests that B7-H3, as an important costimulatory molecule, plays a positive co-stimulatory role in the regulation of T cell immune responses.