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目的分离、克隆十二指肠钩虫抗凝肽AduNAP1基因,在大肠埃希菌中重组表达AduNAP1,并检测其抗凝活性。方法以十二指肠钩虫成虫cDNA为模板,PCR扩增AduNAP1成熟肽编码序列,并克隆、连接到表达质粒pET32a-sumo,构建原核表达载体pET32a-sumo/AduNAP1。重组载体转入到大肠埃希菌BL21(DE3)中,用IPTG诱导表达。重组产物经Ni亲和层析后用SUMO蛋白酶酶切融合伴侣,纯化获得重组目的多肽。用SDS-PAGE分析蛋白表达及纯化情况,用凝血时间法(PT及aPTT)检测重组多肽的抗凝活性。结果扩增并克隆AduNAP1成熟肽编码序列,构建的原核表达载体pET32a-sumo/AduNAP-1转化大肠埃希菌表达rAduNAP1。纯化的rAduNAP1能延长PT及aPTT,但延长PT作用更为显著,其延长2倍PT及aPTT时间所需的浓度分别约为142nmol/L及406nmol/L。结论构建rAduNAP1表达载体,其表达产物具有较强抗凝活性,为进一步了解AduNAP1的生物学功能及作为抗凝新药开发应用奠定了基础。
Objective To isolate and clone the AduNAP1 gene of the hookworm, and to express AduNAP1 recombinantly in Escherichia coli. The anticoagulant activity of AduNAP1 was tested. Methods The coding sequence of the AduNAP1 mature peptide was amplified by PCR from the cDNA of adult B. hookerworm and cloned into the expression vector pET32a-sumo to construct the prokaryotic expression vector pET32a-sumo / AduNAP1. The recombinant vector was transformed into Escherichia coli BL21 (DE3) and induced with IPTG. The recombinant product was purified by Ni affinity chromatography and digested with SUMO protease to obtain the recombinant polypeptide of interest. The protein expression and purification were analyzed by SDS-PAGE, and the anticoagulant activity of the recombinant polypeptide was detected by the clotting time method (PT and aPTT). Results The coding sequence of AduNAP1 mature peptide was amplified and cloned. The prokaryotic expression vector pET32a-sumo / AduNAP-1 was constructed and transformed into Escherichia coli to express rAduNAP1. Purified rAduNAP1 can prolong the PT and aPTT, but to extend the role of PT more significantly, and its required time to extend the 2-fold PT and aPTT concentrations were about 142nmol / L and 406nmol / L. Conclusion The rAduNAP1 expression vector is constructed and its expression product has strong anticoagulant activity, which lays the foundation for further understanding of the biological function of AduNAP1 and its application as a new anticoagulant drug.