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目的 建立快速分离纯化降纤酶工艺方法适合于规模化生产。 方法 以五步蛇毒为原料 ,采用 EDTA络合—硫氰酸钾沉淀初步分离与快速离子交换和凝胶层析纯化相结合 ,分离纯化出符合新部颁标准的降纤酶成分。 结果 经初步分离五步蛇粗毒可使出血毒去除 92 % ,非类凝血酶 ( TLE)成分去除65.1 7% ,TLE活性回收保持 92 .68%以上。层析组份中至少含有 4种 TLE,其中 1种为降纤酶成分。初步化学分离 ,离子交换柱层析和凝胶过滤制得的降纤酶 ,其比活性大于 1 2 0 0 Bu/mg蛋白 ,达 1 50 3Bu/mg蛋白 ;每克五步蛇毒干粉可收获降纤酶 2 998.1 1 6Bu;纯度达到 SDS-PAGE电泳只在标准蛋白质分子量360 0 0 Da附近呈现单一电泳区带 ;HPLC单一峰。 结论 建立了快速分离纯化降纤酶工艺方法 ,快速高效 ,适合规模生产
Objective To establish a rapid separation and purification defibrase method suitable for large-scale production. Methods Five-step snake venom was used as raw material, and EDTA complexation-potassium thiocyanate precipitation was preliminarily separated and combined with rapid ion exchange and gel chromatographic purification to separate and purify the defibrase composition in line with the new standard. Results After preliminary separation of the five-step snake crude poison, 92% of the hemorrhagic poisons were removed, 65.17% of the non-thrombolytic (TLE) components were removed, and TLE activity recovery was maintained above 92.68%. There are at least four TLEs in the chromatographic component, one of which is a defibrase component. The initial chemical separation, ion exchange column chromatography and gel filtration defibrase, its specific activity is greater than 1 200 Bu/mg protein, up to 1 50 3 Bu / mg protein; per gram of five-step snake venom powder can be harvested Fibrilase 2 998.1 1 6Bu; purity reached SDS-PAGE electrophoresis only showed a single electrophoresis band around the standard protein molecular weight of 360 0 Da; HPLC single peak. Conclusion A rapid and efficient method for the separation and purification of defibrase was established. It is suitable for large-scale production.