B7-H3Ig重组蛋白的真核表达及其对T细胞的作用  

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

作　　者：陈礼文[1,2] 张光波[2] 朱一蓓[2] 张学光[2] 

机构地区：[1]安徽医科大学第二附属医院检验科,安徽合肥230601 [2]苏州大学医学生物技术研究所,江苏苏州215007

出　　处：《细胞与分子免疫学杂志》2013年第1期27-30,共4页Chinese Journal of Cellular and Molecular Immunology

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)

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。Objective To construct a recombinant eukaryotic expression vector containing the human B7-H31g gene and investigate the effects of B7-H3 signal on T cells. Methods Being constructed by overlap extension PCR, the recombinant gene fragment of B7-H31g was inserted into pGEZ-Term vector. The recombinant pGEZ-Term/B7-H31g vector was used to transfect L929 cells to purify B7-H31g from conditioned medium harvested from cultured L929/B7-H31g cells. T cells were then stimulated with agonistic anti-CD3 mAb in the presence or absence of purified B7-H31g, and cell proliferation and the secretion of IL-IO and IFN-γ were analyzed 72 h later. Results The concentration of B7-H31g was 0.5.59 mg/mL with a purity of more than 90% after protein G affinity chromatography. B7-H31g binding test showed that the putative B7-H3 receptor was presen- ted in activated T cells and the maximum binding capacity was observed 48 h later. B7-H31g enhanced, in a dose-dependent manner, the proliferation and IL-10 and IFN-γ secretion of T cells stimulated with agonistic anti-CD3 mAb. Conclusion L929/ B7-H31g transfectants have been successfully constructed. B7-H3 is a co-stimulator for T cell proliferation and IL-IO and IFN-γ expressions.

关 键 词：共信号分子 基因转染 基因表达 增殖 细胞因子 

分 类 号：R392.1[医药卫生—免疫学]