B7-H3Ig重组蛋白的真核表达及其对T细胞的作用

发布时间：2018-01-11 09:03

本文关键词：B7-H3Ig重组蛋白的真核表达及其对T细胞的作用　出处：《细胞与分子免疫学杂志》2013年01期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:Objective to express human B7-H3Ig recombinant protein and investigate the role of B7-H3 signal in T cells. Methods B7-H3Ig recombinant gene was constructed by overlapping PCR method. Then the recombinant retrovirus vector pGEZ-Term / B7-H3 IgA was obtained, and the L929 / B7-H3Ig gene was transfected into L929 cells to establish the L929 / B7-H3Ig gene transfected cell line. B7-H3Ig recombinant protein was purified, and B7-H3Ig was added to activated T cells stimulated by CD3 monoclonal antibody (mAb) in vitro. The effects of B7-H3Ig on the proliferation of T cells and the secretion of IL-10 and IFN- 纬 were observed. Results the purity of B7-H3Ig was 90% after being purified by Protein G column. The concentration of recombinant protein was 0.559 mg / mL. The recombinant protein binding test showed that CD3 mAb stimulated 48. Peak expression of B7-H3 receptor on T cells after h. B7-H3Ig enhanced CD3 in a dose-dependent manner. MAb induced T cell proliferation and IL-10 and IFN- 纬 secretion in vitro. Conclusion Eukaryotic expression of human B7-H3Ig gene transfected cell line was successfully established. B7-H3Ig co-stimulates T cell proliferation and IL-10 and IFN- 纬 secretion.
【作者单位】：安徽医科大学第二附属医院检验科;苏州大学医学生物技术研究所;
【基金】：国家自然科学基金(30901789) 安徽省高校省级自然科学基金(KJ2011B072)
【分类号】：R392.1
【正文快照】： B7-H3是CD28-B7家族成员,小鼠B7-H3仅有2IgB7-H3一种形式[1],人B7-H3有2种不同形式的剪切体:2IgB7-H3和4IgB7-H3,以后者为主要存在形式[2]。B7-H3蛋白表达于体外诱导培养的树突状细胞(dendritic cells,DC)、组织巨噬细胞以及活化的外周血单核细胞[2-4],在多种肿瘤组织包括肺

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