【摘要】：目的分離美洲鉤蟲(Necator americanus)Kunitz型絲氨酸蛋白酶抑制劑1(Na Ku I1)c DNA,并進(jìn)行原核表達(dá),研究其抑制蛋白酶的效果。方法根據(jù)Gen Bank上預(yù)測的不完整的Na Ku I基因序列(XM_013449790)設(shè)計(jì)引物,運(yùn)用快速擴(kuò)增c DNA末端技術(shù)(SMART-RACE)分別從美洲鉤蟲成蟲c DNA中擴(kuò)增Na Ku I1 c DNA的5′和3′末端序列,拼接獲得全長Na Ku I1 c DNA。將Na Ku I1成熟肽編碼基因連接入原核表達(dá)載體,構(gòu)建重組質(zhì)粒p ET32a-sumo/Na Ku I1,轉(zhuǎn)化至大腸埃希菌(Escherichia coli)BL21(DE3)中,用異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)Na Ku I1融合蛋白表達(dá)。表達(dá)產(chǎn)物包涵體經(jīng)變性、復(fù)性、Ni-NTA親和層析純化后,用SUMO蛋白酶切割融合蛋白標(biāo)簽后獲得重組蛋白r Na Ku I1。用凝血時(shí)間法檢測r Na Ku I1的抗凝活性,發(fā)色底物法檢測其對人纖溶酶、胰蛋白酶、中性粒細(xì)胞彈性蛋白酶、組織蛋白酶G和蛋白酶3、豬胰蛋白酶和胰彈性蛋白酶及牛α-胰糜蛋白酶的抑制作用。結(jié)果獲得Na Ku I1全長c DNA,其編碼的多肽由84個(gè)氨基酸殘基組成,其中含16個(gè)氨基酸殘基組成的信號肽,68個(gè)氨基酸殘基組成的成熟肽。成熟肽原核表達(dá)產(chǎn)物為不可溶的包涵體。經(jīng)變性、復(fù)性后純化的r Na Ku I1無抗凝血活性。在100倍摩爾濃度比下,r Na Ku I1對人纖溶酶(5 nmol/L)、人胰蛋白酶(1 nmol/L)和豬胰蛋白酶(5 nmol/L)活性的抑制率近100%,對牛α-胰糜蛋白酶(1 nmol/L)和人中性粒細(xì)胞彈性蛋白酶(5 nmol/L)活性的抑制率分別約31.45%和25.18%,對人組織蛋白酶G、蛋白酶3和豬胰彈性蛋白酶均無抑制作用。r Na Ku I1抑制人胰蛋白酶及纖溶酶的抑制常數(shù)(ki)分別為(21.17±7.22)和(21.72±3.95)nmol/L。結(jié)論成功分離獲得Na Ku I1全長c DNA序列,其原核表達(dá)產(chǎn)物r Na Ku I1具有較強(qiáng)抑制胰蛋白酶和纖溶酶活性的特點(diǎn)。
[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 偽-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 偽 -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 鹵7.22) nmol/L. and (21.72 鹵3.95) nmol/L., respectively. The inhibitory constants of protease 3 and porcine pancreatic elastase were (21.17 鹵7.22) and (21.72 鹵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.
【作者單位】： 廣東醫(yī)科大學(xué)寄生蟲學(xué)暨臨床寄生蟲檢驗(yàn)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(yàn)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(xiàng)目-特色創(chuàng)新類(No.2015KTSCX050) 廣東省高等學(xué)校人才引進(jìn)專項(xiàng)基金(No.2050205)~~
【分類號】：R383.13

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