十二指肠钩虫抗凝肽AduNAP1的原核表达及其抗凝活性研究

Cloning and expression of nematode anticoagulant peptide 1(AduNAP1)fromAncylostoma duodenale

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。

Objectives To isolate,clone,and express the gene coding for nematode anticoagulant peptide 1（AduNAP1,GenBank No.ACD80354）and to analyze the anticoagulant activity of recombinant AduNAP1（rAduNAP1）. MethodsThe nucleotide sequence encoding mature AduNAP1 was amplified from the cDNA of adult Ancylostoma duodenale and then ligated into pET32a-sumo to construct the recombinant pET32a-sumo/AduNAP1.Expression of pET32a-sumo/AduNAP1 in E.coli BL21（DE3）was induced with IPTG.The recombinant fusion protein was purified using Ni-NTA affinity chromatography,and its fusion partner was cleaved with SUMO protease on resin.Prothrombin time（PT）and activated partial thromboplastin time（aPTT）assays were used to analyze the anticoagulant activity of rAduNAP1.The expression and purification of recombinant protein was analyzed using SDS-PAGE. Results The gene encoding mature AduNAP1 was amplified from the cDNA of adult A.duodenale,and rAduNAP1 was successfully obtained in prokaryotic expression system.Purified rAduNAP1 substantially prolonged the PT and aPTT.A concentration of 142nmol/L doubled the normal human plasma PT and a concentration of 406nmol/L doubled the aPTT. Conclusions AduNAP1 was successfully overexpressed in E.coli and had potent anticoagulant activity.This study has laid the foundation for the further investigation of the biological function and use of AduNAP1.