毛首鞭形线虫丝氨酸蛋白酶抑制剂TtSerpin1对蛋白酶的抑制作用

发布时间：2018-04-10 16:29

  本文选题：毛首鞭形线虫 + 丝氨酸蛋白酶抑制剂　； 参考：《中国寄生虫学与寄生虫病杂志》2017年04期

【摘要】：目的原核表达、分离纯化毛首鞭形线虫(Trichuris trichiura)丝氨酸蛋白酶抑制剂1(TtSerpin1),并观察其对蛋白酶的抑制作用。方法从毛首鞭形线虫成虫cDNA中扩增TtSerpin1编码序列(Gen Bank登录号为MF401634),将其连接入原核表达载体,构建重组质粒pET32a-sumo/TtSerpin1。将重组质粒转化入大肠埃希菌(Escherichia coli)BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷诱导TtSerpin1融合蛋白表达。表达的包涵体蛋白经变性、复性、镍亲和层析纯化、SUMO蛋白酶酶切融合标签后获得rTtSerpin1。用发色底物法检测其对人组织蛋白酶G、中性粒细胞弹性蛋白酶、蛋白酶3、纤溶酶和胰蛋白酶,猪胰蛋白酶、胰弹性蛋白酶及牛α-胰糜蛋白酶的抑制作用。结果成功构建了重组质粒pET32a-sumo/TtSerpin1,并在E.coli中成功表达。表达产物主要为包涵体,复性、纯化后的rTtSerpin1具有较好的蛋白酶抑制活性。1 000 nmol/L的rTtSerpin1对人组织蛋白酶G(100 nmol/L)、中性粒细胞弹性蛋白酶(10 nmol/L)、蛋白酶3(200 nmol/L),猪胰弹性蛋白酶(10 nmol/L),牛α-胰糜蛋白酶(1 nmol/L)的蛋白酶活性抑制率分别为60.89%、82.84%、21.21%、58.32%、96.98%,但对人纤溶酶、胰蛋白酶及猪胰蛋白酶抑制活性较弱。rTtSerpin1对人组织蛋白酶G及中性粒细胞弹性蛋白酶的抑制常数(K_i)分别为(949.80±91.51)、(242.70±53.41)nmol/L。结论 rTtSerpin1对多种丝氨酸蛋白酶具有较强抑制作用。
[Abstract]:Objective to express and purify Trichuris trichiura, a serine protease inhibitor of Trichuris trichiura, in prokaryotic expression, and to observe its inhibitory effect on protease.Methods TtSerpin1 encoding sequence Gen Bank was amplified from adult nematodes cDNA and ligated into prokaryotic expression vector to construct recombinant plasmid pET32a-sumo-TtSerpin1.The recombinant plasmid was transformed into Escherichia coli BL21DE3) and the expression of TtSerpin1 fusion protein was induced by isopropyl- 尾 -D- thiogalactoside.The expressed inclusion body protein was denatured, renatured, purified by nickel affinity chromatography and digested with Sumo protease to obtain rTtSerpin1.The inhibition of human cathepsin G, neutrophil elastase, protease 3, fibrinolytic enzyme and trypsin, porcine trypsin, trypsin and bovine 伪 -chymotrypsin were detected by chromogenic substrate method.Results the recombinant plasmid pET32a-sumo-TtSerpin1 was successfully constructed and expressed in E.coli.The expression product is mainly inclusion body, renaturation,Purified rTtSerpin1 has a relatively good protease inhibitory activity of 1.000 nmol/L rTtSerpin1 to human cathepsin Gon 100nmol / L, neutrophil elastase 10nmol / L, protease 3200nmol / L, porcine trypsin 10nmol / L, bovine 伪 -chymotrypsin 1 nmol / L) protease.The inhibitory constants of trypsin and porcine trypsin. RTtSerpin1 on human cathepsin G and neutrophil elastase were 949.80 卤91.51 and 242.70 卤53.41 nmol / L, respectively. The inhibitory constants of rTtSerpin1 on human cathepsin G and neutrophil elastase were 242.70 卤53.41 nmol / L, respectively.Conclusion rTtSerpin1 can inhibit many serine proteases.
【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)~~
【分类号】：R382.2
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