人IL-37b在大肠埃希菌中的表达、纯化及活性鉴定

Expression,purification and activity assay of human IL-37b in E.coli

目的表达重组IL-37b蛋白并去除内毒素,鉴定其活性。方法构建原核表达载体p ET28/IL-37b,转化大肠杆菌感受态细胞;经IPTG诱导表达的重组蛋白由Ni2+-NTA凝胶进行变性条件下的亲和层析纯化;用SDS-PAGE分析、考马斯亮蓝染色鉴定重组蛋白是否为目的蛋白;去除蛋白中原核表达所产生的内毒素;将蛋白作用于受LPS刺激的RAW 264.7细胞,收集培养上清,通过ELISA方法检测IL-6的表达水平,鉴定蛋白的生物学活性。结果表达了纯度较好的重组IL-37b蛋白,降低了其中原核表达所产生的内毒素,经鉴定其具备良好的生物学活性。结论成功表达了具备良好生物学活性的IL-37b蛋白。

Objective To investigate the expression of recombinant IL-37 b protein and removal of the endotoxin,and identify its biological activity.Methods The prokaryotic expression vector p ET28/IL-37 b was constructed and to transform Escherichia coli（E.coli） Rosetta.After induction with IPTG,the recombinant protein was purified through Ni2 ＋-NTA gel column and identified by SDS-PAGE and Coomassie brilliant blue staining.Then,the endotoxin protein was removed and was treated with LPS-stimulated RAW 264.7 cells.The culture supernatant was collected.The expression of IL-6 was detected by ELISA and the biological activity of the protein was identified.Results The recombinant IL-37 b with high purity was expressed and the endotoxin produced by prokaryotic expression was reduced,and it was identified to have good biological activity.Conclusions In this study a recombinant IL-37 b protein with high biological activity is successfully obtained.