hsRAGE-IgG Fc融合蛋白的表达及其生物学活性

Expression of hsRAGE-IgG Fc Fusion Protein and Its Bioactivity

将编码人可溶性晚期糖基化终产物受体-免疫球蛋白G Fc段(hsRAGE-IgG Fc)融合蛋白的DNA片段克隆到大肠杆菌表达载体pET-20b中,构建重组表达质粒,转化大肠杆菌Rosetta(DE3)并表达。SDS-PAGE分析表明其表达形式为包涵体,相对分子质量约为66kDa,表达量占菌体总蛋白的38.4%。经复性后,获得纯度为96.6%的融合蛋白,得率约为29.5mg/L。经Western印迹法鉴定,该融合蛋白可与sRAGE抗体产生阳性反应。同时,hsRAGE-IgG Fc融合蛋白可以显著抑制晚期糖基化终产物(AGE)引起的ECV-304细胞NF-κB p65表达的上调,其活性与hsRAGE相似。

The human soluble receptor for advanced glycation end products（hsRAGE）fused with the immunoglobulinin G Fc fragment（IgG Fc）was expressed in Rosetta（DE3）by IPTG induction.The result of SDS-PAGE analysis indicated the hsRAGE-IgG Fc fusion protein was expressed in the form of inclusion body.The expression level of the fusion protein was about 40% of the total cellular protein.After renaturation and purification,a purity of about 96% of the fusion protein was obtained.Result of Western blotting indicates the fusion protein can specifically combine with sRAGE antibody and significantly inhibit the over expression of NF-κB p65 of ECV-304 cells induced by AGEs.