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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 recombinant human hsRAGE His fusion protein expression vector and to express and purify it in prokaryotic cells. Methods The cytoplasmic extracellular domain of hRAGE gene was amplified by PCR and cloned into pET-14b vector by molecular cloning technique. The correctness of the clones was confirmed by restriction analysis and sequence analysis. Escherichia coli BL21 (DE) 3 was transformed and the fusion protein was induced with isopropyl b-D thiogalactose (IPTG). The obtained inclusion bodies were treated with urea, and the fusion protein was purified by metal chelate affinity chromatography and refolded. The immunogenicity of the fusion protein and its binding activity with AGE were identified by Western blotting and ELISA. Results The cloned hsRAGE was completely correct. After the expression and purification, the fusion protein with relative molecular mass of about 45000 was obtained. After purification, the denatured protein can be recognized by the corresponding antibody after refolding. The resulting hsRAGE has the activity of binding to AGE and can inhibit the AGE-induced NF-kB expression in endothelial cells. Conclusion The prokaryotic expression vector of hsRAGE fusion protein was successfully constructed and highly expressed. A large number of renatured proteins were obtained. The protein was specific for immunogenicity, retained the binding activity with AGE and effectively blocked the AGE-RAGE interaction.