【摘要】 目的:用大肠杆菌表达获得重组线虫抗凝血肽5(rNAP 5),为研究开发NAP5的功能与应用提供原料来源。方法:将扩增的NAP5基因经BamHⅠ和HindⅢ双酶切后与表达载体pET-32a连接。构建好的重组表达质粒转化至大肠杆菌BL21(DE3)后,分别经IPTG和乳糖诱导表达,并探讨诱导表达条件,分析表达产物的可溶性情况。表达产物经镍亲和纯化后,用凝血酶原时间(PT)和活化部分凝血活酶时间(aPTT)检测体外抗凝活性。结果:成功构建了pET-32a/NAP5表达载体,IPTG和乳糖均能诱导目的蛋白在大肠杆菌BL21(DE3)中高效地可溶性表达。优化条件下每升LB培养基可获可溶性目的融合蛋白量达65.3mg。纯化的蛋白能明显延长PT及aPTT,7.0mg/L的蛋白平均约延长5.09倍aPTT,2.55倍PT。结论:在大肠杆菌中成功表达了具有很好生物活性的Trx-NAP5融合蛋白,为研究开发NAP5的功能与应用奠定了基础。更多还原

【Abstract】 Objective:To express nematode anticoagulant peptide 5(NAP 5) in Escherichia coli,and obtain enough recombinant protein for investigating its application and function.Methods: NAP 5 gene was amplified and inserted into BamHⅠand Hind Ⅲ digested pET32a vector.The construced recombinant plasmid of pET32a/ NAP5 was transferred into E.coli BL21(DE3),and the fusion protein was expressed induced by IPTG and lactose.After purifying by His affinity chromatography,the anticoagulant activity of recombinant fusion protein was detected by activated partial thromboblastin time(aPTT) and prothrombin time(PT) assay.Results:The prokaryotic expression plasmid pET-32a/NAP 5 was constructed successfully.The recombinant was transformed into E.coli BL21(DE3) and expressed by inducing with IPTG and lactose with high efficiency.The fusion protein was mostly expressed in soluble form and obtained 65.3 mg from 1000 ml LB culture medium under optimized condition.The puried fusion protein obviously prolonged the PT and aPTT of human plasma in vitro,7.0mg/L prolonged 5.09 times aPTT and 2.55 times PT. Conclusion:The Trx-NAP 5 fusion protein was expressed successfully in E.coli BL21(DE3) and it had obviously anticoagulant activity.The result of our study laid a basis for further research the function and application to NAP 5.更多还原