可诱导共刺激分子和小鼠免疫球蛋白融合基因的分泌表达及生物学活性  

Expression and bioactivity of the fusion gene between extracellular portion of human ICOS and Fc portion of mouse IgG 2a

作　　者：王健[1] 张军[1] 沈茜[1] 

机构地区：[1]第二军医大学长海医院中心实验室,上海200433

出　　处：《中国免疫学杂志》2006年第5期411-415,共5页Chinese Journal of Immunology

基　　金：国家科技部"863"计划资助项目(2002AA214091)

摘　　要：目的：为获得重组可诱导共刺激分子胞外段与小鼠免疫球蛋白的融合蛋白（ICOS-Ig）并研究其生物学活性，以探讨它作为免疫拮抗剂阻断ICOS／B7RP-1共刺激通路的作用。方法：克隆编码人可诱导共刺激分子（ICOS）胞外片段，与编码小鼠免疫球蛋白IgG恒定片段（Ig）的基因融合，构建ICOS-Ig融合基因及其分泌型真核表达载体pSecTag2／Hygro A-ICOS-Ig；构建融合蛋白的稳定真核表达细胞株，用Western blot和ELISA法检测其表达、流式细胞仪（FACS）检测其配体结合活性、混合淋巴细胞反应（MLR）及细胞因子检测测定其生物学活性。结果：核苷酸序列测定显示克隆基因的核苷酸与Genebank序列一致：ELISA和Western blot检测表明转染细胞有分泌型重组蛋白的表达，其含量可达5～25μg／ml；该重组蛋白有配体结合活性，可体外抑制淋巴细胞增殖、减少IL-2、IFN-γ的分泌。结论：重组ICOS-Ig融合基因及其真核表达载体的构建完成并获得高效表达，该重组蛋白具有配体结合活性，可在体外通过抑制ICOS／B7RP-1共刺激通路抑制淋巴细胞增殖及细胞因子分泌。Objective： To produce a fusion protein （ICOS-Ig）between extraceilular portion of human ICOS and Fc portion of mouse IgG 2a and study on its biological activity. Methods：cDNA encoding the extraceilular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripheriai blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector that contains the sequence-encoding Fc portion of mouse IgG2a. Two resulting amplified PCR products were ligated into a secrection mammalian expression vector, pSecTag2/Hygro A. The recomhined vector was transfected into CHO ceils by lipofectamine2000. Results ：RT-PCR demonstrated the integration and mRNA synthesis of fusion gene. ELISA and Western blot analysis showed the expression of ICOS-Ig and its molecular weight was between 43-66 kD, its concentration was 5-25 μg/ml. FACS analysis assured that ICOS-Ig had ligand specific binding activity. Addition of ICOS-Ig to MLR resulted in inhibition of T-ceil proliferation and IL-2, IFN-γ secretion. Conclusion：The fused gene ICOS-Ig was constructed and expressed successfully. It had the bioactivity of inhibition of T cell proliferation and eytokine secretion.

关 键 词：可诱导共刺激分子 pSecTag2/Hygro A 融合蛋白 真核表达 

分 类 号：Q789[生物学—分子生物学]