蚓激酶中纤溶酶谱鉴别方法的研究

Identification Method for Lumbrokinase by Fibrin Zymography

目的:建立蚓激酶的纤溶酶谱鉴别方法,同时考察5个企业产品及同一企业不同批次的一致性。方法:纤溶酶谱法,纤维蛋白原终浓度为5×10^(-4)g·ml^(-1),2.5%Triton-x-100溶液中复性30 min,在PBS缓冲液(p H=7.4)中37℃孵育30 min,供试品蛋白质浓度为5.0~37.5μg。结果:该方法灵敏度高,5家企业的活性蛋白条带均分布在15~40KD间,有6个相同特征活性蛋白条带;5个企业产品之间酶谱图略有差异,同一企业不同批次产品批间无差异。结论:该方法专属性较强,能够反映蚓激酶活性成分的分子量大小分布及种类,也能反映产品的批间一致性,生产工艺稳定性,且易于标准化,可作为蚓激酶的鉴别方法,同时为评价上市后产品质量一致性奠定了基础。

Objective：To establish a fibrin zymography method for identifying the active proteins in lumbrokinase,and investigate the production difference and batch consistency of 5 different manufacturers.Methods：Fibrin zymography was used with the final concentration of fibrinogen of 5 × 10^-4g·ml^-1,renature time of 30 min in 2.5% Triton-x-100,incubation time of 30 min in PBS buffer（p H = 7.4） at 37℃ and the protein concentration in the sample of 5.0-37.5 μg.Results：The sensitivity of the method was high,and the molecular weight distribution of active protein bands for the samples from five manufacturers was between 15 KD and 40 KD with 6common active protein bands.The zymography of the samples from the five manufacturers had slight difference,while various batches of the samples from the same manufacturer showed no difference.Conclusion：The method is special.It can reflect molecular weight distribution and species of active protein,batch consistency and production process stability.It is easy to be standardized and suitable for the identification of lumbrokinase,which can lay foundation for the quality consistency evaluation of marketed products.