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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 express recombinant IL-37b protein and remove endotoxin and identify its activity. Methods The prokaryotic expression vector p ET28 / IL-37b was constructed and transformed into E.coli competent cells. The recombinant protein induced by IPTG was purified by affinity chromatography on denaturing Ni2 + -NTA gel. SDS-PAGE analysis Lysostaphin (LPS) -stimulated RAW 264.7 cells, the supernatant was collected and the expression of IL-6 was detected by ELISA. The expression of IL-6 was detected by ELISA. Identify the biological activity of the protein. The results showed that the recombinant IL-37b protein with good purity was expressed, and the endotoxin produced by prokaryotic expression was reduced. It was identified as having good biological activity. Conclusion The IL-37b protein with good biological activity was successfully expressed.