通俗环毛蚓抗血栓活性蛋白成分的快速富集及活性考察

Rapid Enrichment and Activity Research of Antithrombotic Proteins from Pheretima vulgaris

目的:以地龙品种中的通俗环毛蚓为研究对象,对其蛋白总提物进行纯化富集以获得抗血栓活性蛋白成分(PvQ),对PvQ进行抗血栓活性研究,以期为地龙的抗血栓物质基础研究提供参考。方法:采用AKTA-Pure型蛋白纯化系统,以体外活性为导向,富集通俗环毛蚓PvQ;采用纤维蛋白平板法、纤维蛋白原-凝血酶时间法(Fibg-TT法)对PvQ的纤溶及抗凝活性进行测定;利用体外血栓溶解试验评价PvQ对体外血栓的溶解能力,同时,考察了不同温度及pH对PvQ活性的影响。结果:成功富集得到通俗环毛蚓PvQ,活性测定表明其具有显著的纤溶及抗凝活性。体外血栓试验结果显示,37℃孵育5 h后PvQ可水解超过80%的血栓块。此外,温度和pH对于PvQ活性影响显著,在≥60℃或酸性条件(pH<7)下PvQ活性显著降低或失活,而在≤50℃和碱性条件下PvQ活性则不受影响或受较少影响。结论:建立了可行的通俗环毛蚓PvQ制备方法,PvQ可同时作用于纤维蛋白和纤维蛋白原,从而发挥双重纤溶作用,表现出良好的纤溶及抗凝活性,为其用于治疗血栓性疾病提供了科学阐释,并可为地龙抗血栓蛋白类产品的开发提供参考。

Objective: A strong antithrombotic protein component,named PvQ,was purified and enriched from total protein of Pheretima vulgaris,a traditional Chinese medicine. Moreover,we evaluated its fibrinolytic and anticoagulant activity,and expected to provide reference for the research on antithrombotic substances of Pheretima. Method: A rapid in vitro activity-oriented separation combined with the AKTA-Pure protein purification system conducted on P. vulgaris. Meanwhile,the fibrinolytic and anticoagulant activities of PvQ were measured by fibrin plate method and fibrinogen-thrombin time(Fibg-TT)method. And the in vitro thrombolysis assay was used for evaluating the lysis ability of PvQ to thrombus. Then the stability of PvQ was also analyzed for its anticoagulant activity at different pH and temperature. Result: The PvQ was successfully enriched and its activity was determined to have significant fibrinolytic and anticoagulant activities. And the result of in vitro thrombolysis assay revealed that PvQ could hydrolyze more than 80% of thrombus after 5 h of incubation at 37 ℃. In addition,the changes of temperature and pH had significant effects on antithrombotic activity,and this study showed that PvQ was rapidly inactivated at ≥60 ℃ or in acidic conditions(pH<7).While,the activity of PvQ was unaffected or less affected at ≤50 ℃ and under alkaline conditions. Conclusion:A feasible preparation method of PvQ is established,and it can affect fibrin and fibrinogen at the same time,thus exerting a dual fibrinolytic effect and possessing significant fibrinolytic and anticoagulant activities. It provides a scientific interpretation for the treatment of thrombotic diseases by PvQ and a reference for the development of antithrombotic protein products of Pheretima.