犬钩虫抗凝肽AcaNAP8的原核表达及其抗凝活性

目的在大肠杆菌中重组表达犬钩虫抗凝肽AcaNAP8基因，并检测表达产物抗凝活性。方法设计引物，将AcaNAP8成熟肽编码序列克隆、连接到原核表达载体pET32a，构建重纽表达质粒；成功构建的重组表达质粒转化到大肠杆菌BL21（DE3）中用IPTG诱导表达，SDS—PAGE分析表达情况；诱导表达的重组蛋白用Ni亲和层析进行纯化；用凝血酶原时间（PT）和活化的部分凝血活酶时间（aPTT）检测重组产物体外抗凝活性。结果成功构建了pET32a／AcaNAP8原核表达重组质粒，在大肠杆菌中成功表达并获得了重组AcaNAP8，表达产物能明显延长PT及aPTT。结论在大肠杆菌中成功表达了AcaNAP8融合蛋白，表达产物具有显著抗凝活性。该实验为进一步探讨AcaNAP8的作用机制及应用奠定了基础。

Cloning, expression and anticoagulant activities of anticoagulant peptide AcaNAP8 from Ancylostoma caninum

Aim To express nematode anticoagulant peptide 8 from Ancylostoma canium （AcaNAP8） in E. coli and analyse its anticoagulant activities in vitro. Methods The nucleotide sequence encoding mature AcaNAP8 was cloned and hgated into pET32a to construct the recombinant pET32a/AcaNAP8 plasmid. The recombinant AcaNAP8 were expressed in E. coli BL21 （DE3） by inducing with IPTG and analysed by SDS-PAGE. The recombinant protein was affinity - purified by the Protino Hi-IDA Resin and its anticoagulant activity was measured with activated partial thromboblastin time （aPTY） and prothromhin time （PT） assay in vitro. Results AcaNAP8 was successfully ligated into pET32a plasmid and expressed in E. eoli BI21 （DE3） after inducing by IPTG. The recombinant AcaNAP8 prolonged the PT and aPTT of human plasma in vitro. The prolongation of PT was more marked than that of aPTT. Conclusions AcaNAP8 had remarkable anticoagulant activities in vitro. The present study laid a basis for further research the mechanism of action and application of AcaNAP8.