NF-κB受体激活因子配体在大肠杆菌中的表达、纯化及其活性  

Expression of Receptor Activator of NF-κB Ligand in E.coli and Purification and Activity of Expressed Product

作　　者：杨忠东[1] 何成[1] 

机构地区：[1]国药集团上海生物制品研究所有限责任公司,上海200052

出　　处：《中国生物制品学杂志》2011年第12期1385-1390,共6页Chinese Journal of Biologicals

摘　　要：目的在大肠杆菌中表达并纯化NF-κB受体激活因子配体(Receptor activator of NF-κB ligand,RANKL),并检测其生物学活性。方法 PCR扩增RANKL基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-RANKL,转化E.coli BL21(DE3),IPTG诱导表达,经SDS-PAGE分析目的蛋白的表达和表达形式。表达产物经镍离子柱亲和层析、阴离子交换层析及阳离子交换层析进行纯化,纯化产物经SDS-PAGE分析纯度,Western blot检测其反应原性,BCA试剂盒测定蛋白浓度,并经MTT法及RAW264.7细胞分化试验测定其生物学活性。结果重组表达质粒pET-28a-RANKL经双酶切及测序证实构建正确;表达的重组RANKL蛋白相对分子质量约为20 000,部分以包涵体形式存在;经纯化后纯度可达98.8%,可与相应抗体反应形成特异条带,蛋白浓度为56.72μg/ml,体外活性试验显示,RANKL具有良好的生物学活性。结论成功原核表达了RANKL,纯化的重组RANKL具有生物学活性,为骨代谢调控机制的研究及相关药物的开发提供了材料。Objective To express receptor activator of NF-κB ligand（RANKL） in E.coli,purify the expressed product and determine its biological activity.Methods RANKL gene was amplified by PCR and cloned into prokaryotic expression vector pET-28a（＋）.The constructed recombinant plasmid pET-28a-RANKL was transformed to E.coli BL21（DE3） and induced with IPTG.The expression level and form of target protein were analyzed by SDS-PAGE.The expressed product was purified by nickel ion affinity column chromatography,anion-exchange chromatography and cation-exchange chromatography,and determined for purity by SDS-PAGE,for reactogenicity by Western blot,for protein concentration by BCA kit,and for biological activity by MTT method and RAW264.7 cell differentiation test.Results Restriction and sequencing proved that recombinant plasmid pET-28a-RANKL was constructed correctly.The expressed recombinant RANKL protein,with a relative molecular mass of about 20 000,partially existed in a form of inclusion body,and reached a purity of 98.8% after purification.The purified RANKL,with a protein concentration of 56.72 μg / ml,formed a specific reaction band with the corresponding antibody,and showed high biological activity as proved by activity test in vitro.Conclusion RANKL with biological activity was successfully expressed in prokaryotic cells and purified,which provided a material for study on bone metabolism regulation and development of the related drugs.

关 键 词：NF-κB受体激活因子配体 大肠杆菌 原核细胞 基因表达 生物活性 

分 类 号：Q78[生物学—分子生物学]