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

发布时间：2018-11-15 12:28

【摘要】：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。
[Abstract]:Objective to isolate and express 1 (Na Ku I1) c DNA, of (Necator americanus) Kunitz serine protease inhibitor of hookworm, and to study its inhibitory effect on protease. Methods primers were designed based on incomplete Na Ku I gene sequence (XM_013449790) predicted by Gen Bank. The 5 'and 3' terminal sequences of Na Ku I1 c DNA were amplified from c DNA of hookworm by rapid amplification of c DNA terminal technique (SMART-RACE). The full-length Na Ku I1 c DNA. was obtained by splicing. Na Ku I1 mature peptide encoding gene was ligated into prokaryotic expression vector, and the recombinant plasmid p ET32a-sumo/Na Ku I1 was constructed and transformed into Escherichia coli (Escherichia coli) BL21 (DE3). The expression of Na Ku I 1 fusion protein was induced by isopropyl 尾-D-thiogalactoside (IPTG). After denaturation, renaturation and purification by Ni-NTA affinity chromatography, the recombinant protein r Na Ku I1 was obtained by cleavage the fusion protein label with SUMO protease. The anticoagulant activity of r Na Ku I1 was detected by clotting time method. The anticoagulant activity of r Na Ku I1 was detected by chromogenic substrate method. Inhibitory effects of porcine trypsin, pancreatic elastase and bovine 伪-chymotrypsin. Results the full-length c DNA, of Na Ku I1 was composed of 84 amino acid residues, including 16 amino acid residues signal peptides and 68 amino acid residues composed of mature peptides. The prokaryotic expression product of mature peptide is insoluble inclusion body. After denaturation and renaturation, the purified r Na Ku I1 had no anticoagulant activity. The inhibitory rate of, r Na Ku I1 on the activities of human plasminogen (5 nmol/L), human trypsin (1 nmol/L) and porcine trypsin (5 nmol/L) was 100% at a 100-fold molar ratio. The inhibitory rates of bovine 伪 -chymotrypsin (1 nmol/L) and human neutrophil elastase (5 nmol/L) were 31.45% and 25.18%, respectively. The inhibitory constants of. R Na Ku I1 on human trypsin and plasmin were (21.17 卤7.22) nmol/L. and (21.72 卤3.95) nmol/L., respectively. The inhibitory constants of protease 3 and porcine pancreatic elastase were (21.17 卤7.22) and (21.72 卤3.95) nmol/L., respectively. Conclusion the full-length c DNA sequence of Na Ku I1 was successfully isolated and its prokaryotic expression product r Na Ku I1 was characterized by strong inhibition of trypsin and plasminogen activity.
【作者单位】：广东医科大学寄生虫学暨临床寄生虫检验学教研室;广东医科大学病原生物学研究所;广东省医学分子诊断重点实验室;
【基金】：国家自然科学基金(No.81171599) 广东省教育厅重点科研项目-特色创新类(No.2015KTSCX050) 广东省高等学校人才引进专项基金(No.2050205)~~
【分类号】：R383.13

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