人可溶性晚期糖基化终产物受体的原核表达及活性鉴定

Prokaryotic expression and activity identification of recombinant human soluble receptor for advanced glycation end products

目的构建人可溶性晚期糖基化终产物受体(hsRAGE)His融合蛋白表达载体并在原核细胞表达与纯化。方法PCR扩增hRAGE基因编码区的胞质外段,利用分子克隆技术将其重组于pET-14b载体中。利用酶切、序列分析鉴定克隆正确性。转化大肠杆菌BL21(DE)3,用异丙基b-D硫代半乳糖(IPTG)诱导融合蛋白表达。以尿素对获得的包涵体进行处理,用金属螯合亲和层析的方法纯化融合蛋白并进行复性。以Western印迹和ELISA法对融合蛋白的免疫原性及与AGE的结合活性进行鉴定。结果克隆的hsRAGE完全正确,经过表达与纯化,获得了相对分子质量约45000的融合蛋白。纯化得到的变性蛋白经复性后可被相应抗体识别。所得到的hsRAGE具有与AGE相互结合的活性,并能够抑制AGE诱导的内皮细胞NF-kB的表达。结论成功地构建了hsRAGE融合蛋白原核表达载体且获得高效表达,得到大量的复性蛋白;该蛋白具有特异的免疫原性,保持与AGE的结合活性,并能够有效阻断AGE-RAGE相互作用。

Objective To construct a eukaryotic expression vector for His-tagged human soluble receptor for advanced glycation end products (hsRAGE),and study the function and biological activity of the fusion protein.Methods The coding sequence of hsRAGE was amplified from the recombinant pCI-neo/RAGE by PCR method.The fragment was cloned into pET-14b vector and identified by digestion with restriction enzymes and sequence analysis.After transformation of the vector into E.coli BL21(DE3),the fusion protein was expressed successfully in response to IPTG induction in the form of inclusion body.After extraction from the bactrerial cells and washing,the fusion protein was dissolved in 8 mol/L urea and purified using immobilized metal ion affinity chromatography.Renaturing of the fusion protein occurred after gradually removal of the denaturants by dialysis.The protein was subsequently identified by Western blotting,enzyme-linked immunosorbent assay (ELISA) and blockade experiments in the endothelial cells.Results The recombinant hsRAGE/pET-14b was verified by enzyme digestion and sequencing.A fusion protein with a molecular weight of about 45 kD was purified,which could interact with anti-RAGE antibody and AGE and block AGE-induced nuclear factor (NF)-kB activation in the endothelial cells.Conclusions His-hsRAGE fusion protein has been cloned,expressed and purified successfully and identified to possess immunogenicity,binding activity to AGE and blocking effect on AGE-RAGE.