抗凝蛋白EH体外活性检测条件的建立  

The Antithrombin Activity Detection of EH in Vitro

作　　者：秦晓永[1] 于爱平[2] 王文文[3] 毕建进[2] 郭晓军[1] 袁洪水[1] 吴祖泽[2] 靳继德[2] 朱宝成[1] 

机构地区：[1]河北农业大学生命科学学院保定071001 [2]军事医学科学院放射与辐射医学研究所北京100850 [3]天津大学化工学院天津300072

出　　处：《中国生物工程杂志》2011年第5期108-112,共5页China Biotechnology

基　　金：国家“重大新药创制”科技重大专项资助项目(2009ZX09103-630)

摘　　要：EH蛋白是一种水蛭素衍生物,它是在水蛭素的N端引入了一小段寡肽,该寡肽可被凝血因子XIa(factor XIa,FXIa)和Xa(factor Xa,FXa)裂解释放水蛭素的抗凝血酶活性。比较在不同条件下FXa裂解EH蛋白的效果,包括裂解时间(2h,4h,6h,8h,10h,20h)、裂解温度(25℃,37℃,40℃),FXa与EH蛋白摩尔比(1∶300,1∶180,1∶60),反应溶液(水、生理盐水、裂解缓冲液)。分别采用凝块法和TH生色底物法检测EH蛋白经FXa裂解后的体外抗凝血酶活性。FXa对EH的裂解活性在一定条件下与反应时间和温度成正比,裂解活性随FXa的摩尔数增加而增强。反应溶液对其裂解活性影响较弱,其中生理盐水为最适裂解溶剂。最终确定EH体外活性检测时最适裂解条件为:用生理盐水稀释EH样品,FXa与EH的摩尔比为1∶180,在37℃裂解6h。此法裂解EH蛋白,操作方便,所测活性重现性好,适合作为EH蛋白质量控制中活性测定的方法。Recombinant protein EH originates from hirudin by linking three amino acids to its N-terminal.The three amino acids are recognized and cut by coagulate factor Xa（FXa） and XIa（FXIa）,that will release the antithrombin activity of hirudin.The cleavage efficiency of FXa to EH was compared under different conditions,incuding emzymolytic time and temperature,the molar ratio of FXa and EH,and reaction solution.Results showed that the antithrombin activity of EH increased proportionally,under certain conditions,as the reaction time and temperature increased,Moreover,the FXa cleavage activity is enhanced when the molar ratio of FXa and EH was increased.It seems that saline is more suitable reaction solutions for FXa to cut EH.So the optimal cleavage conditions for EH with FXa in vitro is that EH was reacted with FXa at 37℃ for 6 hours in saline solution with the molar ratio（1∶180） of FXa and EH.A practical and reproducible method for the activity determination and quality control of EH in vitro was developed.

关 键 词：水蛭素 EH蛋白 抗凝活性 FXa 

分 类 号：R973.2[医药卫生—药品]