重组人IL-8在大肠杆菌中的表达及纯化工艺的优化

Improvement of the Method of the Expression of rhIL-8 in Escherichia Coli and the Purification

目的优化重组人IL-8(rhIL-8)大肠杆菌表达及纯化工艺,以便获得高纯度的重组蛋白。方法将已构建好的表达载体转化大肠杆菌HB101,经3-吲哚丙烯酸诱导,在发酵罐中进行表达,比较不同诱导剂浓度、温度、pH值及时间,以获得最佳表达工艺参数。离心收集菌体,超声破壁,收集包涵体和胞浆,其中包涵体经变性、复性处理,与包浆部分合并,经肝素亲和层析柱、阳离子交换柱和凝胶层析柱分离纯化,比较不同的洗脱条件,得到纯品,并对重组蛋白的纯度和生物学活性进行鉴定。结果用大肠杆菌HB101在发酵罐中表达rhIL-8,在30℃、pH为7.0的条件下加入50 mmol/L 3-吲哚丙烯酸,诱导28 h,得到约为100 mg/ml的高表达,其分泌的重组蛋白以包涵体和胞浆的形式存在,经三步层析纯化后,得到高纯度的rhIL-8(纯度>95%),经中性粒细胞趋化实验证实,所获IL-8具有良好的趋化白细胞迁移的作用。结论建立高效表达rhIL-8的原核表达和纯化工艺,所获的纯化IL-8具有良好的生物学活性。

Objective To improve the method of the expression of recombinant human interleukin- 8 （rhIL-8） in Escherichia Coli and It＇s purification to obtain high-purified rhIL-8. Method E. Coli was transfected with the constructed rhIL-8 plasmid and the engineering bacterium was induced to express the rhIL-8 by 3-indoleacryl acid. We establish the optimized method of the rhIL-8＇s expression based on the different concentration of the inducer, temperature, pH and time. Then bacterium was collected by centrifugation and lysed by sonication. Recombinant human IL-8 in the part of inclusion body was recovered by denaturation and renaturation, pooled it with the part of cytoplasm. The pooled fraction was further purified by three steps of chromatography, heparin affinity, ion exchange and gel filtration. We establish the optimized method of purification based on the different condition of chromatography. The purified samples were assessed by SDS PAGE, the quantity of IL-8 was determined by ELISA. Result There were 100 mg/ml rhIL-8 totally when expressing by E. Coli in the condition of 30℃, pH 7.0, 50 mmol/L 3-indolecrylic acid, 28 hours. The secreting protein, which formed in inclusion body and cytoplasm, was purified to 95 % by three steps of chromatography. The IL-8 has good biological activity approved by the test of neutrophil chemotactic. Conclusion High-efficient method of the expression of rhIL-8 in Escherichia Coli and purification method have been established, and the purified IL-8 has good biological activity.