本文選題：毛首鞭形線蟲 + 絲氨酸蛋白酶抑制劑　； 參考：《中國寄生蟲學(xué)與寄生蟲病雜志》2017年04期

【摘要】：目的原核表達(dá)、分離純化毛首鞭形線蟲(Trichuris trichiura)絲氨酸蛋白酶抑制劑1(TtSerpin1),并觀察其對蛋白酶的抑制作用。方法從毛首鞭形線蟲成蟲cDNA中擴(kuò)增TtSerpin1編碼序列(Gen Bank登錄號為MF401634),將其連接入原核表達(dá)載體,構(gòu)建重組質(zhì)粒pET32a-sumo/TtSerpin1。將重組質(zhì)粒轉(zhuǎn)化入大腸埃希菌(Escherichia coli)BL21(DE3)中,用異丙基-β-D-硫代半乳糖苷誘導(dǎo)TtSerpin1融合蛋白表達(dá)。表達(dá)的包涵體蛋白經(jīng)變性、復(fù)性、鎳親和層析純化、SUMO蛋白酶酶切融合標(biāo)簽后獲得rTtSerpin1。用發(fā)色底物法檢測其對人組織蛋白酶G、中性粒細(xì)胞彈性蛋白酶、蛋白酶3、纖溶酶和胰蛋白酶,豬胰蛋白酶、胰彈性蛋白酶及牛α-胰糜蛋白酶的抑制作用。結(jié)果成功構(gòu)建了重組質(zhì)粒pET32a-sumo/TtSerpin1,并在E.coli中成功表達(dá)。表達(dá)產(chǎn)物主要為包涵體,復(fù)性、純化后的rTtSerpin1具有較好的蛋白酶抑制活性。1 000 nmol/L的rTtSerpin1對人組織蛋白酶G(100 nmol/L)、中性粒細(xì)胞彈性蛋白酶(10 nmol/L)、蛋白酶3(200 nmol/L),豬胰彈性蛋白酶(10 nmol/L),牛α-胰糜蛋白酶(1 nmol/L)的蛋白酶活性抑制率分別為60.89%、82.84%、21.21%、58.32%、96.98%,但對人纖溶酶、胰蛋白酶及豬胰蛋白酶抑制活性較弱。rTtSerpin1對人組織蛋白酶G及中性粒細(xì)胞彈性蛋白酶的抑制常數(shù)(K_i)分別為(949.80±91.51)、(242.70±53.41)nmol/L。結(jié)論 rTtSerpin1對多種絲氨酸蛋白酶具有較強(qiáng)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 偽 -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 偽 -chymotrypsin 1 nmol / L) protease.The inhibitory constants of trypsin and porcine trypsin. RTtSerpin1 on human cathepsin G and neutrophil elastase were 949.80 鹵91.51 and 242.70 鹵53.41 nmol / L, respectively. The inhibitory constants of rTtSerpin1 on human cathepsin G and neutrophil elastase were 242.70 鹵53.41 nmol / L, respectively.Conclusion rTtSerpin1 can inhibit many serine proteases.
【作者單位】： 廣東醫(yī)科大學(xué)寄生蟲學(xué)暨臨床寄生蟲檢驗學(xué)教研室;廣東醫(yī)科大學(xué)病原生物學(xué)研究所;廣東省醫(yī)學(xué)分子診斷重點(diǎn)實(shí)驗室;
【基金】：國家自然科學(xué)基金(No.81171599) 廣東省教育廳重點(diǎn)科研項目-特色創(chuàng)新類(No.2015KTSCX050)~~
【分類號】：R382.2
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本文編號：1732042