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目的 原核表达美洲钩虫(Necator americanus)丝氨酸蛋白酶抑制剂NaKuI3,检测其对丝氨酸蛋白酶的抑制活性. 方法 根据美洲钩虫序列(GenBank No.XM_013451492)设计引物,以美洲钩虫成虫cDNA为模板,通过SMART-RACE方法相继分离NaKuI3的3'和5'cDNA末端序列;构建重组表达质粒pET32a sumo/NaKuI3,将其转化至大肠埃希菌BL21(DE3),IPTG诱导表达并用Ni-NTA亲和纯化和SUMO酶酶切获得rNaKuI3,用发色底物法检测rNaKuI3对丝氨酸蛋白酶的抑制活性. 结果 成功从美洲钩虫中获得了新的Kunitz型丝氨酸蛋白酶抑制剂-NaKuI3全长cDNA,推测其完整阅读框(ORF)为225 bp,编码74个氨基酸残基组成的多肽,具有19个氨基酸残基组成的信号肽.rNaKuI3对猪胰蛋白酶、人纤溶酶、人中性粒细胞弹性蛋白酶和人蛋白酶3均有较强抑制作用,抑制常数(ki)分别为(16.40±2.08) nmol、(24.39±4.25) nmol、(38.39±3.92)nmol和(89.90±13.91)nmol,对人组织蛋白酶G也具有一定抑制活性,但对牛a-胰糜蛋白酶、猪胰弹性蛋白酶抑制作用较弱. 结论 融合蛋白美洲钩虫NaKuI3获得成功.该多肽对多种丝氨酸蛋白酶有抑制作用,是一种广谱的丝氨酸蛋白酶抑制剂,为进一步研究NaKuI3的生理学功能奠定了基础.“,”Objectives To express a new Kunitz-type serine protease inhibitor,named NaKuI3,from the hookworm Necator americanus in Escherichia coli and to characterize its inhibition of serine proteases.Methods Specific primers were designed based on a nucleotide sequence in GenBank (No.XM_013451492).The 3'and 5'cDNA ends of NaKuI3 were obtained from the adult N.americanus cDNA using SMART-RACE.The recombinant plasmid pET32a-sumo/NaKuI3 was constructed and expressed in E.coli BL21 (DE3),and protein expression was induced with IPTG.After the fusion protein was digested with SUMO protease,recombinant NaKuI3 was purified using Ni-NTA resin affinity chromatography.A single stage chromogenic assay was used to detect inhibition of serine proteases.Results The full-length cDNA of NaKuI3 was amplified successfully from N.americanus.The open reading frame consisted of 225 bp encoding a peptide of 74 amino acids with a signal peptide of 19 amino acid residues.rNaKuI3 had potent inhibitory activity against porcine trypsin with a ki of 16.40±2.08 nmol,against human plasmin with a ki of 24.39±4.25 nmol,against neutrophil elastase with a ki of 38.39±3.92 nmol,and against proteinase 3 with a ki of 89.90±13.91 nmol.rNaKuI3 also inhibited human cathepsin G and had weaker inhibitory activity against bovine a-pancreatic chymotrypsin and porcine pancreatic elastase.Conclusion NaKuI3 is a broad-spectrum Kunitz-type serine protease inhibitor from N.americanus that possesses inhibitory activity against several serine proteases.This study has laid the foundation for further study of the physiological functions of NaKuI3.