人TACI-linker-BR3双受体融合基因真核表达载体构建及其在COS-7细胞中的表达

目的:构建人双受体融合基因真核表达载体--pcDNA3.1( )-TACI-linker-BR3-IgGFc,观察其在COS-7细胞中的表达.方法:应用基因工程技术构建重组载体,脂质体转染COS-7细胞,通过Western印迹、ELISA以及免疫组化方法,检测目的蛋白的表达及其与BAFF、APRIL蛋白相互作用情况.结果:融合基因在瞬时转染的真核细胞中获得表达,并分泌至上清,转染效率能达到48%,融合蛋白能与BAFF、APRIL蛋白有效结合而且融合蛋白中分子标签IgGFc未影响双受体融合蛋白的结构及其生物学活性.结论:人TACI-linker-BR3双受体基因与IgGFc融合基因载体构建成功,表达的融合蛋白具有生物学活性,为进一步探讨SLE等自身免疫疾病的发病机制和生物学治疗方法奠定了基础.

Construction of a eukaryotic vector containing human TACI-linker-BR3-IgGFc double receptor fusion gene and its expression in COS-7 cells

Objective:To construct a eukaryotic vector containing human TACI-linker-BR3-IgGFc double receptor fusion gene and to investigate its expression in COS-7 cells.Methods: The standard cloning technology was employed to construct the(title) vector,which was then used to transfect COS-7 cells by lipofectamine 2000.The expression of target protein and their interaction with BAFF and APRIL protein were examined by Western blot,ELISA,and immunohistochemistry methods.Results: The expression of the fusion protein was observed in transiently transfected COS-7 cells, with the transfection efficiency being 48%.The fusion protein was detected in the supernatant.It suggested that the fusion protein interacted well with BAFF and(APRIL) proteins;the IgGFc tag did not influence the natural assembly and the biological activity of fusion proteins.Conclusion: A vector containing TACI-linker-BR3 double receptor and IgGFc fusion gene has been successfully constructed and the(subsequent) fusion protein is bioactive,which paves a way for further research on the pathogenesis of autoimmune diseases and biotherapy strategy.