美洲钩虫广谱丝氨酸蛋白酶抑制剂NaKuI3的鉴定及特性研究

发布时间：2017-12-31 09:26

本文关键词：美洲钩虫广谱丝氨酸蛋白酶抑制剂NaKuI3的鉴定及特性研究　出处：《中国病原生物学杂志》2017年06期 　论文类型：期刊论文

【摘要】：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225bp,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α-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。
[Abstract]:Objective to express NaKuI3, a serine protease inhibitor, in prokaryotic expression of Necator americanus. Methods Primer was designed according to GenBank No. XM013451492. Using the adult cDNA of hookworm as template, the 3 'and 5' cDNA terminal sequences of NaKuI3 were isolated by SMART-RACE method. The recombinant expression plasmid pET32a-sumo-Na KuI3 was constructed and transformed into Escherichia coli BL21 (DE3). RNaKuI3 was induced by IPTG and purified by Ni-NTA affinity and digested by SUMO. The inhibitory activity of rNaKuI3 on serine protease was detected by chromogenic substrate method. Results A novel Kunitz type serine protease inhibitor -NaKuI3 was successfully obtained from hookworm. NA. Its complete reading frame ORF is 225bp, encoding a peptide composed of 74 amino acid residues and a signal peptide with 19 amino acid residues. RNaKuI3 pair of porcine trypsin. Human fibrinolytic enzyme, human neutrophil elastase and human protease 3 had strong inhibitory effects, and the inhibition constant was 16.40 卤2.08 nmol, respectively. The activity of human cathepsin G was also inhibited by 24.39 卤4.25nmol (38.39 卤3.92nmol) and 89.90 卤13.91nmol (P < 0.05). But the inhibitory effect on bovine 伪 -chymotrypsin and porcine pancreatic elastase was weak. Conclusion the fusion protein NaKuI3 is successful, and the polypeptide can inhibit many serine proteases. It is a broad-spectrum serine protease inhibitor, which lays a foundation for the further study of the physiological function of NaKuI3.
【作者单位】：广东医科大学寄生虫学教研室;
【基金】：国家自然科学基金项目(No.81171599) 广东省教育厅重点科研项目-特色创新类(No.2015KTSCX050) 广东医科大学科研基金项目(No.M2014004)
【分类号】：R383.13
【正文快照】： 丝氨酸蛋白酶抑制剂广泛存在于植物、动物、病毒和微生物中,通过调节丝氨酸蛋白酶的活性参与许多重要生理反应,如消化、血液凝固、纤维蛋白溶解、炎症反应、免疫应答、信号传导等[1]。近年来研究较多的丝氨酸蛋白酶抑制剂是Kunitz型丝氨酸蛋白酶抑制剂(Kunitz type serine pro

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