【作者】 黄爱民；

【导师】 廖共山；

【作者基本信息】 广西医科大学 ， 药理学， 2005， 硕士

【摘要】 目的:从广西菲牛蛭中分离纯化菲牛蛭素(Bufrudin),并对其物理、生化性质及体内外抗凝作用进行研究。方法:用30%硫酸铵分步沉淀、离子交换、凝胶过滤和高效液相色谱法分离纯化广西菲牛蛭消化液中的菲牛蛭素,用SDS-聚丙烯酰胺凝胶电泳测定其蛋白分子量,等电聚焦法测定等电点,并测定活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、凝血酶原时间(PT)以及对凝血因子的影响。结果:从广西菲牛蛭中分离出两种特异凝血酶抑制剂—菲牛蛭素A(Bufrudin A ,BDA)、菲牛蛭素B(Bufrudin B ,BDB)。BDA 分子量约为15.2KD,等电点为3.97;BDB 分子量为14.6KD,等电点为4.61。紫外扫描发现,BDA在230nm处有最大吸收,而BDB的最大吸收则在200-210nm之间。在体内外凝血实验中,BDA、BDB 均能明显延长活化部分凝血活酶时间(APTT)、凝血时间(PT)及凝血酶原时间(TT),但二者的三个指标有明显差异性。二者相比较,BDA 的凝血酶时间(TT)的作用较BDB 更为显著,而BDB 凝血酶原时间(PT)的作用强于BDA。对于凝血因子,BDA 对Ⅱ、Ⅷ、Ⅻ因子产生抑制作用,对Ⅴ、Ⅸ、Ⅹ、Ⅺ因子无影响;BDB 则对Ⅱ、Ⅷ、Ⅹ、Ⅻ因子产生抑制作用,对Ⅴ、Ⅸ、Ⅺ因子无影响。结果:广西菲牛蛭中的抗凝物质BDA、BDB 均有很强的抗凝活性。更多还原

【Abstract】 Objective :To purify bufrudin from the GuangxiHirudinaria manillensis and study the physical and biochemicalcharacterization and anticoagulation effect in vitro on human andrabbit plasma coagulateon. Methods :The anticoagulation proteinbufrudin was purified from the Saliva of Guangxi Hirudinariamanillensis by 30 % ammonium sulfate preparation and columnchromatography on DEAE cellulose ,Sephadex G-50 and Re-HPLC .UsingSDS-PAGE calculated their molecular weights and then estimatedisoelectric point by the isoelectric focusing electrophoresis , thenassayed anticoagulant activities in vitro by Activated partialthromboplastin time(APTT) , Prothrombin time(PT),Thrombin time (TT)on human and rabbit plasma and their affection on Blood clotting factor.Result :The purified Bufrudin A (BDA) showed a single band by SDS-PAGEwith a molecular weight of 15.2KD,and Bufrudin B(BDB) show the samecharacter but the molecular weight is 14.6 KD.The isoelectric pointof BDA was PH 3.97 and BDB was PH 4.61 by the isoelectric focusingelectrophoresis .This protein BDA or BDB all prolonged acitivatedpartial thromboplsatin time(APTT) ,thrombin time (TT)and prothrombintime(PT),but there were some different charater between them .Comparatively,BDA effected stronger on thrombin time (TT) but BDBeffected more on prothrombin time(PT). BDA didn’t affect other bloodclotting factor but inhibited the blood clotting factor Ⅱ、Ⅷ、Ⅻ.BDBonly inhibited the blood clotting factorⅡ、Ⅷ、Ⅹ、Ⅻ. Conclusion:Both of the anticoagulantion protein BDA and BDB have stronganticoagulantion activity ,BDA and BDB inhibited the coagulationprocess by effecting on the thrombin .更多还原