摘要
目的 建立重组人IL - 8(rhIL - 8)大肠杆菌表达后的高效分离纯化工艺 ,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HB10 1,经IPTG诱导进行高效表达。将离心收集到的菌体用超声破壁分离 ,收集包涵体和胞浆 ,其中包涵体部分经变性和复性处理后 ,与胞浆部分合并 ,进行肝素亲和层析柱分离 ,再经离子交换柱和凝胶层析柱过滤 ,得到最终纯品。蛋白纯度用SDS -PAGE鉴定 ,ELISA法检测重组IL - 8含量。检测纯化后重组蛋白的生物学活性和热源质。结果 用大肠杆菌HB10 1表达重组人IL - 8,具有较高的表达量 (2 5mg/ml) ,其分泌的重组蛋白以包涵体和胞浆的形式存在。经多步柱层析纯化后 ,可获得高纯度的重组IL- 8蛋白 (纯度 >95 % )。该蛋白在体外具有趋化中性粒细胞游走的作用 ,兔温法检测热源质含量符合要求。结论 大肠杆菌可高效表达rhIL - 8,该工艺流程可从大肠杆菌载体中获得具有生物学活性的高纯度rhIL - 8。
Objective To establish a method for obtained high purity of recombinant human interleukin-8 from Escherichia coli expressing system.Methods E.Coli HB101 was transfected with the constructed recombinant IL-8 plasmid and the clone was screened in LB medium by Amp +.Engineering bacterium was induced to expressing the recombinant IL-8 for 28 h by IPTG,then bacterium was collected by centrifugation and lysied by repeated freeze-thaw and sonication.Recombinant IL-8 in the part of inclusion body was recovering 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.The purity of samples was assessed by SDS-PAGE,and the quantity of IL-8 was determined by ELISA.Results There are 25mg/ml rhIL-8 in total when expres sing by E.coli,which forming in inclusion body and cytoplasmic protein.By three steps chromatography,rhIL-8 can be purified to 95%(SDS-PAGE),and the purified rhIL-8 aslo show chemota tic activity to neutrophil in virto.Conclusion The process design can be a scheme to obtain the high purity rhIL-8 from Escherichia coli.
出处
《苏州医学院学报》
2000年第4期340-343,共4页
Acta Academiae Medicinae Suzhou
基金
江苏省科委优秀青年教师基金!(BQ96016)
核工业总公司民品技术开发项目!(H7410262)资助课题
关键词
大肠杆菌
纯化
分离
rhIL-8
recombinant IL-8
chromotography
Escherichia coli
