人可诱导共刺激分子胞外片段和小鼠免疫球蛋白Ig融合基因的表达及生物学活性鉴定

目的:探讨用基因工程方法表达的人可诱导共刺激分子(ICOS)与小鼠免疫球蛋白的融合蛋白竞争抑制ICOSB7RP1共剌激通路的作用.方法:以RT-PCR方法克隆编码人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,构建携带融合基因的高效真核表达载体pcDNA4/HisMAX A-ICOS-Ig.采用脂质体法转染CHO细胞,用Western 印迹法检测稳定表达细胞中融合基因的表达,流式细胞术检测重组蛋白的配体结合活性.以适当浓度的重组融合蛋白作为拮抗剂与BALB/c和C57BL小鼠脾单个核细胞进行混合培养,观察重组蛋白在体外抑制淋巴细胞增殖的效果.结果:构建了携带融合基因的高效真核表达载体pcDNA4/HisMAX A-ICOS-Ig;Western印迹法检测表明转染细胞有重组蛋白的表达;流式细胞术分析显示重组蛋白有配体表达细胞的结合活性;该重组融合蛋白作为拮抗剂可体外抑制混合淋巴细胞增殖.结论:构建并成功表达了ICOS-Ig融合基因高效真核表达载体;该重组蛋白具有配体结合活性,可在体外通过抑制ICOS-B7RP1共刺激通路抑制淋巴细胞增殖.

Fusion protein expression of extracellular portion of human inducible costimulator and Fc portion of mouse IgG2a and its bioactivity in vitro

Objective:To study the bioactivity of inducible costimulator Ig(ICOS-Ig) as an inhibitor of ICOS-B7RP-1 costimulatory pathway in vitro. Methods: cDNA encoding the extracellular domain of human ICOS was prepared by RT-PCR from the RNA of the stimulated human peripheral blood mononuclear cells. The Fc portion of mouse IgG2a was cloned by PCR from the vector containing the sequence-encoding Fc portion of mouse IgG2a.The above 2 PCR products were ligated into a clone vector: pGL-3-Basic. The fusion gene was then cloned and ligated into a mammalian expression vector: pcDNA4/HisMAX A. The recombined vector was transfected into CHO cells by Lipofectamine2000 and the expression of the fusion protein was identified by Western blot. The mixture lymphoproliferation reaction(MLR) of the lymphocytes derived from BALB/c and C57BL mice was used to detect the fusion protein function in vitro. Results: Western blot analysis showed the expression of fusion protein, with the molecular weight being 43 000-66 000. FACS analysis assured that expression products had ligand specific binding activity. MLR was inhibited by the fusion protein. Conclusion: The constructed recombinant fusion protein has ligand specific binding activity and can inhibit the lymphoproliferation.