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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2011, Vol. 31 Issue (5): 108-112    
技术与方法     
抗凝蛋白EH体外活性检测条件的建立
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
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
The Antithrombin Activity Detection of EH in Vitro
QIN Xiao-yong1, YU Ai-ping2, WANG Wen-wen3, BI Jian-jin2, GUO Xiao-jun1, YUAN Hong-shui1, WU Zu-ze2, JIN Ji-de2, ZHU Bao-cheng1
1. College of Life Science, Agricultural University of Hebei, Baoding 071001, China;
2. Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing 100850, China;
3. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
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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。

关键词: 水蛭素EH蛋白抗凝活性FXa    
Abstract:

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.

Key words: Hirudin    EH    Antithrombin activity    Coagulate factor Xa
收稿日期: 2011-01-20 出版日期: 2011-05-27
ZTFLH:  R973+.2  
基金资助:

国家"重大新药创制"科技重大专项资助项目(2009ZX09103-630)

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引用本文:

秦晓永, 于爱平, 王文文, 毕建进, 郭晓军, 袁洪水, 吴祖泽, 靳继德, 朱宝成. 抗凝蛋白EH体外活性检测条件的建立[J]. 中国生物工程杂志, 2011, 31(5): 108-112.

QIN Xiao-yong, YU Ai-ping, WANG Wen-wen, BI Jian-jin, GUO Xiao-jun, YUAN Hong-shui, WU Zu-ze, JIN Ji-de, ZHU Bao-cheng. The Antithrombin Activity Detection of EH in Vitro. China Biotechnology, 2011, 31(5): 108-112.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I5/108


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