小鼠B7-H3-Fc融合蛋白的表达及其生物学活性的研究

Expression of mouse B7-H3-Fc fusion protein and characterization of its bioactivity

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。

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 LipfectAMINETM 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.