牛血凝血酶的制备工艺

目的研究1种高纯度牛凝血酶的制备工艺。方法采用不同终浓度的Ba2+吸附、硫酸铵分段盐析从牛血浆中提取凝血酶原,用Ca2+激活,活化的凝血酶用CM-Sepharose Fast Flow离子交换柱纯化。结果用冷冻1周血浆制备的凝血酶粗品产量、比活明显高于来自新鲜血浆的粗品;Ba2+终浓度在0.107 mol/L以上时凝血酶的产量较高;凝血酶原的最佳激活条件为:Ca2+终浓度0.05 mol/L,37℃,30~60 min;CM-Sepharose Fast Flow柱制备的凝血酶比活达到2 000 U/mg以上。结论该工艺适用于高纯度凝血酶的制备。

Preparation process of bovine thrombin

Purpose To establish a preparation process of bovine thrombin with high purity.Methods Barium Chloride and Ammonium sulfate were used to absorb and precipitate the prothrombin.The product was activated by Ca2+ and the activated product was purified by CM-Sepharose Fast Flow ion exchange chromatography.Results The specific activity and recovery of thrombin from bovine plasma frozen at-20℃ for a week were more than those from fresh plasma.The concentration of BaCl2 significantly affected the recovery of thrombin.The recovery was low when the was less than 0.107 mol/L.When Ca2+ was used to activate prothrombin at 37℃,the optimal final concentration of Ca2+ and activating time were 0.05 mol/L and 30-60 minutes and the specific activity of the highly purified thrombin obtained by CM-Sepharose Fast Flow after activating prothrombin with CaCl2 was more than 2 000 U/mg.Conclusion The preparation process is suitable for the manufacture of thrombin preparation.