美洲钩虫蛋白酶抑制剂NaKuI1对蛋白酶的抑制作用的鉴定和特性研究

Identification and characterization of protease inhibition effect of Kunitz-type serine protease inhibitor 1 (NaKuI1) from Necator americanus

目的分离美洲钩虫(Necator americanus)Kunitz型丝氨酸蛋白酶抑制剂1(Na Ku I1)c DNA,并进行原核表达,研究其抑制蛋白酶的效果。方法根据Gen Bank上预测的不完整的Na Ku I基因序列(XM_013449790)设计引物,运用快速扩增c DNA末端技术(SMART-RACE)分别从美洲钩虫成虫c DNA中扩增Na Ku I1 c DNA的5′和3′末端序列,拼接获得全长Na Ku I1 c DNA。将Na Ku I1成熟肽编码基因连接入原核表达载体,构建重组质粒p ET32a-sumo/Na Ku I1,转化至大肠埃希菌(Escherichia coli)BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导Na Ku I1融合蛋白表达。表达产物包涵体经变性、复性、Ni-NTA亲和层析纯化后,用SUMO蛋白酶切割融合蛋白标签后获得重组蛋白r Na Ku I1。用凝血时间法检测r Na Ku I1的抗凝活性,发色底物法检测其对人纤溶酶、胰蛋白酶、中性粒细胞弹性蛋白酶、组织蛋白酶G和蛋白酶3、猪胰蛋白酶和胰弹性蛋白酶及牛α-胰糜蛋白酶的抑制作用。结果获得Na Ku I1全长c DNA,其编码的多肽由84个氨基酸残基组成,其中含16个氨基酸残基组成的信号肽,68个氨基酸残基组成的成熟肽。成熟肽原核表达产物为不可溶的包涵体。经变性、复性后纯化的r Na Ku I1无抗凝血活性。在100倍摩尔浓度比下,r Na Ku I1对人纤溶酶(5 nmol/L)、人胰蛋白酶(1 nmol/L)和猪胰蛋白酶(5 nmol/L)活性的抑制率近100%,对牛α-胰糜蛋白酶(1 nmol/L)和人中性粒细胞弹性蛋白酶(5 nmol/L)活性的抑制率分别约31.45%和25.18%,对人组织蛋白酶G、蛋白酶3和猪胰弹性蛋白酶均无抑制作用。r Na Ku I1抑制人胰蛋白酶及纤溶酶的抑制常数(ki)分别为(21.17±7.22)和(21.72±3.95)nmol/L。结论成功分离获得Na Ku I1全长c DNA序列,其原核表达产物r Na Ku I1具有较强抑制胰蛋白酶和纤溶酶活性的特点。

Objective To isolate and express a Kunitz-type serine protease inhibitor, named NaKuI1, from the human hookworm Necator americanus, and identify its protease inhibitory activity. Methods Primers were designed based on the predicted nucleotide sequence GenBank No.XM_013449790, which coded for a predicted Kunitz type serine protease inhibitor consisting of 63-amino acids. The 3′ and 5′ ends of NaKuI1 cDNA were amplified from the N. americanus adult worm cDNA with rapid amplification of cDNA ends （SMART-RACE） technique. The nucleotide sequence encoding mature NaKuI1 was cloned and ligated into pET32a-sumo vector to construct the recombinant plasmid pET32a-sumo/NaKuI1, which was then transferred into Escherichia coli BL21 （DE3）. Expression of NaKuI1 fusion protein was induced with IPTG. The expressed products in inclusion bodies were denatured, refolded, then purified by Ni-NTA resin affinity chromatography, and cleaved with SUMO protease to obtain the recombinant protein rNaKuI1. Activated partial thromboplastin time （aPTT） and prothrombin time （PT） assays were used to explore the anticoagulant activity of rNaKuI1, and a single-stage chromogenic assay was used to detect the inhibitory effect on serine proteases. Results The full-length cDNA of NaKuI1 was obtained, and it includes an open-reading frame of 255 nucleotides encoding a 84-amino-acid peptide, consisting of a signal peptide of 16 amino acids and a mature peptide of 68 amino acids. NaKuI1 fusion protein expressed in E. coli BL21 （DE3） was in a form of inclusion body, and purified rNaKuI1 had no anticoagulant activity. At a 100-fold molar ratio, rNaKuI1 exhibited nearly 100% inhibition on enzymatic activity of human fibrinolytic enzyme （5 nmol/L）, human trypsin （1 nmol/L） and porcine trypsin （5 nmol/L）, and 31.45% and 25.18% inhibition on bovine pancreatic α-chymotrypsin （1 nmol/L） and human neutrophil elastase （5 nmol/L）, respectively. However, it did not affect the enzymatic activity of human cathepsin G, proteinase 3 and porcine pancreatic elastase. rNaKuI1 inhibited human pancreatic trypsin and plasmin with ki values of（21.17 ± 7.22） nmol/L and（21.72 ± 3.95） nmol/L, respectively. Conclusion The full-length cDNA of NaKuI1 is obtained, which shows strong inhibitory activities against trypsin and plasmin.