摘要
目的制备重组美洲钩虫Ascris型蛋白酶抑制剂1(rNaTIL-1),并观察其抗凝血活性及抑制丝氨酸蛋白酶作用。方法从美洲钩虫成虫c DNA中扩增获得NaTIL-1编码基因,连接入原核表达质粒p ET32a-sumo后转入到大肠埃希菌BL21(DE3)中,用IPTG诱导表达,重组产物经Ni-NTA亲和层析纯化,纯化柱上用SUMO蛋白酶切割融合伴侣后获得rNaTIL-1。用凝血时间法检测rNaTIL-1的抗凝活性,用发色底物法检测rNaTIL-1对丝氨酸蛋白酶的抑制作用。结果成功构建了原核表达质粒p ET32a-sumo/NaTIL-1,在E.coli BL21(DE3)中表达产物大部分可溶,并纯化获得了rNaTIL-1。rNaTIL-1对血浆凝血酶原时间和活化部分凝血活酶时间均无明显影响。在100倍摩尔比的过量浓度下,rNaTIL-1对人胰凝乳蛋白酶、糜酶、纤溶酶、血浆激肽释放酶和猪胰蛋白酶、胰弹性蛋白酶均无明显抑制作用,但能抑制(91.6±0.9)%的人中性粒细胞弹性蛋白酶(HNE)活性。rNaTIL-1抑制HNE的IC50值约为(325.50±1.05)nmol/L,Ki值约为(0.34±0.04)μmol/L。结论NaTIL-1无抗凝血活性,但具有较强的抑制HNE活性,可能在美洲钩虫参与调控宿主炎症反应和逃避宿主免疫中发挥作用。
Objective To prepare recombinant Ascris-type protease inhibitor 1(rNaTIL-1)from Necator americanus,and observe its anticoagulant activity and inhibitory activity against serine protease.Methods The gene encoding NaTIL-1 was amplified from the cDNA of Necator americanus,and then ligated into the prokaryotic expression plasmid pET32 a-sumo and transferred into Escherichia coli BL21(DE3),and the recombinant product was expressed by inducing with IPTG.rNaTIL-1 was obtained after purification of fusion protein with Ni-NTA affinity chromatography,and then cleavage of the fusion partner on the chromatography column with SUMO protease.The clotting time was used to detect the anticoagulant activity of rNaTIL-1,and the chromogenic substrate method was used to detect the inhibitory activity of rNaTIL-1 on serine proteases.Results The recombinant plasmid pET32 a-sumo/NaTIL-1 was successfully constructed and transferred into E.coli BL21(DE3).The recombinant fusion protein was mainly expressed as soluble products and the purified rNaTIL-1 was obtained after cleavage of the fusion tag.rNaTIL-1 shows no effect on prothrombin time and activated partial thromboplastin time.At an excess concentration of 100-fold molar ratio,rNaTIL-1 had no obvious inhibitory activity against human pancreatic chymotrypsin,chymase,plasmin,plasma kallikrein and porcine pancreatic trypsin,pancreatic elastase,but had significant inhibitory activity against human neutrophil elastase(HNE).Its IC50 value for inhibiting HNE was(325.50±1.05)nmol/L,and the Ki value was(0.34±0.04)μmol/L.Conclusion This study demonstrated that NaTIL-1 had no anticoagulant activity,but shows potently inhibitory activity against HNE,and it might play a role in the regulating host inflammation and evading host immunity for Necator americanus.
作者
朱艳
高如秀
雷蕾
卢晓湧
崔咏诗
邓莉
邵正
彭礼飞
ZHU Yan;GAO Ru-xiu;LEI Lei;LU Xiao-yong;CUI Yong-shi;DENG Li;SHAO Zheng;PENG Li-fei(Department of Parasitology Parasitology,Guangdong Medical University,Zhanjiang,Guangdong 524023,China)
出处
《热带医学杂志》
CAS
2021年第5期531-534,579,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(81171599)。
