美洲钩虫纤溶酶抑制剂NaKuI10的表达与活性鉴定

Prokaryotic expression and identification of the activity of NaKuI10,aplasmin inhibitor from Necator americanus

目的从美洲钩虫(Necator americanus)cDNA中扩增编码NaKuI10基因片段,构建原核表达系统,重组表达并纯化rNaKuI10,采用化学发色法和平板溶解试验等检测其对有关丝氨酸蛋白酶的抑制作用及纤溶活性。方法根据GenBank MH218867序列,从美洲钩虫cDNA中扩增出编码成熟肽NaKuI10基因片段并连接入表达载体,构建原核表达重组质粒pET32a-sumo/NaKuI10,鉴定正确的重组质粒转化至大肠埃希菌(Escherichia coli)BL21(DE3)中,用IPTG诱导表达,超声破碎细菌后用镍亲和层析纯化表达产物,在纯化柱上用SUMO蛋白酶切割融合标签获得rNaKuI10,用发色底物法检测rNaKuI10对各丝氨酸蛋白酶的抑制活性,通过纤维蛋白平板法和纤维蛋白原降解试验观察rNaKuI10对纤溶酶纤溶活性的抑制作用。结果成功构建了重组质粒pET32a-sumo/NaKuI10,表达产物纯化后获得可溶性rNaKuI10。2倍及等摩尔比的rNaKuI10分别能抑制100%及90.4%的人纤溶酶活性,22.4%及10.3%的人胰蛋白酶活性。但1、2、5倍摩尔比的rNaKuI10对人中性粒细胞弹性蛋白酶,组织蛋白酶G,蛋白酶3,胰糜蛋白酶,胰弹性蛋白酶,凝血酶,凝血因子Xa(FXa)、FXIa、FXIIa、FIXa,活化蛋白C,血浆激肽释放酶及FVIIa/TF均无明显抑制作用。rNaKuI10对人纤溶酶的抑制常数(ki)为(0.83±0.10)nmol/L。2 000nmol/L的rNaKuI10接近完全抑制1 000nmol/L的人纤溶酶对纤维蛋白平板中纤维蛋白的溶解作用,等摩尔比的rNaKuI10可完全抑制人纤溶酶对纤维蛋白原的降解作用。结论 NaKuI10是一种抑制活性强且特异性较好的纤溶酶抑制剂,其生物学功能还需进一步研究。

Objectives To isolate and express the cDNA of mature NakuI10,a novel Kunitz-type serine protease inhibitor from the human hookworm Necator americanus,and to identify its inhibitory activity against serine proteases and its fibrinolytic activity. Methods Based on the nucleotide sequence previously registered in GenBank（NO.MH218867）,cDNA encoding mature NaKuI10 was amplified from an N.americanus cDNA library and then ligated into an expression vector to construct the prokaryotic expression recombinant plasmid pET32 a-sumo/NaKuI10.The recombinant plasmid was verified as correct and then transformed into Escherichia coli BL21（DE3）.Protein expression was induced with IPTG.Cells were collected using centrifugation and lysed using sonication.The expressed product was purified using nickel affinity chromatography and the fusion tag was cleaved with SUMO protease to obtain rNaKuI10.Inhibitory activity against serine protease was detected using the chromogenic substrate method,and the inhibitory effect on plasmin hydrolysis was observed with the fibrin plate method and fibrin degradation test. Results rNaKuI10 was successfully expressed and purified from the supernatant in E.coli.Two-fold and equimolar ratios of rNaKuI10 inhibited 100% and 90.4% of human plasmin activity and 22.4% and 10.3% of human trypsin activity,respectively.rNaKuI10 at a molar ratio of one,two and five-fold had no obvious inhibitory effect on human neutrophil elastase,cathepsin G,protease 3,trypsin,pancreatic elastase,thrombin,coagulation factor Xa（FXa）,FXIa,FXIIa,FIXa,activation protein C,plasma kallikrein,and FVIIa/TF.The inhibitory constant（ki）for inhibition of human plasmin by rNaKuI10 was 0.83 ± 0.10 nmol/L.The fibrinolytic activity of human plasmin（1 000 nmol/L）on the fibrin plate was completely inhibited by 2 000 nmol/L rNaKuI10.Hydrolysis of fibrinogen by plasmin was completely inhibited by equimolar rNaKuI10. Conclusion NaKuI10 is a plasmin inhibitor with strong inhibitory activity and specificity,and its biological function still needs to be studied further.