【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,并進行原核表達,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)計引物,運用快速擴增c DNA末端技術(shù)(SMART-RACE)分別從美洲鉤蟲成蟲c DNA中擴增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。表達產(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個氨基酸殘基組成,其中含16個氨基酸殘基組成的信號肽,68個氨基酸殘基組成的成熟肽。成熟肽原核表達產(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,其原核表達產(chǎn)物r Na Ku I1具有較強抑制胰蛋白酶和纖溶酶活性的特點。
[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)分子診斷重點實驗室;
【基金】：國家自然科學(xué)基金(No.81171599) 廣東省教育廳重點科研項目-特色創(chuàng)新類(No.2015KTSCX050) 廣東省高等學(xué)校人才引進專項基金(No.2050205)~~
【分類號】：R383.13

【相似文獻】

相關(guān)期刊論文 前10條

1 任海南,肖樹華,戴志強;美洲鉤蟲的掃描電鏡觀察[J];中國寄生蟲學(xué)與寄生蟲病雜志;1989年02期

2 楊元清,楊惠中,任海南,成寶珠,莊兆農(nóng),陸曉艷;美洲鉤蟲的組織化學(xué)觀察[J];中國寄生蟲學(xué)與寄生蟲病雜志;1989年03期

3 ;人體美洲鉤蟲在小白鼠體內(nèi)的部分發(fā)育[J];國外醫(yī)學(xué)參考資料(寄生蟲病分冊);1974年01期

4 陳祖澤;;美洲鉤蟲在乳兔體內(nèi)完成發(fā)育和感染后體液抗體、白細(xì)胞反應(yīng)及血清蛋白的改變[J];廣東衛(wèi)生防疫資料;1982年02期

5 任海南,成寶珠;美洲鉤蟲實驗感染金色倉鼠的研究[J];寄生蟲學(xué)與寄生蟲病雜志;1983年04期

6 黃先翔,韓家俊;體外培養(yǎng)美洲鉤蟲成蟲的研究[J];寄生蟲學(xué)與寄生蟲病雜志;1985年01期

7 周慧娟;人體美洲鉤蟲的抗止血作用[J];國外醫(yī)學(xué)(寄生蟲病分冊);1996年04期

8 蔣誠者，，俞仲清;十二指腸鉤蟲和美洲鉤蟲性別組成的比較觀察[J];中國寄生蟲病防治雜志;1995年01期

9 汪浩;十二指腸及美洲鉤蟲交合傘伸展?fàn)顟B(tài)標(biāo)本的制作技術(shù)[J];實用寄生蟲病雜志;1998年04期

10 武蘇平;人體美洲鉤蟲乙酰膽堿脂酶的免疫原性[J];國外醫(yī)學(xué)(寄生蟲病分冊);1994年01期

本文編號：2333299