一种蛇毒类凝血酶纯化新方法

A new purification process for snake venom thrombin⁃like enzyme

目的提供一种蛇毒类凝血酶的新纯化方法。方法蛇毒经预处理后,依次经过苯甲脒琼脂糖凝胶亲和色谱、阳离子交换色谱和疏水色谱后获得蛇毒类凝血酶,分别对活性、分子量及纯度进行检测,并与使用现有专利方法制备的蛇毒类凝血酶进行质量比较。结果新方法制备的蛇毒类凝血酶与现有专利方法相比,分子量无明显差异,但新方法的比活力更高,且纯度更高,同时新方法的工艺稳定性更佳。结论新方法与现有专利方法相比,简化了工艺步骤,缩短了纯化周期,减少了过程环境暴露风险,可获得高纯度类凝血酶,工艺稳定性高,产品质量一致性好,利于临床安全用药。

Objective To provide a new purification process for snake venom thrombin⁃like enzyme.Methods The snake venom was pretreated and purified by Benzamidine Sepharose 6B affinity chromatography,SP Sepharose High Performance cation exchange chromatography and Phenyl Sepharose 6 Fast Flow hydrophobic chromatography,and finally the venom thrombin⁃like enzyme was obtained.The enzyme was detected by coagulometer for activity,reduced SDS⁃PAGE for molecular weight,non⁃reduced SDS⁃PAGE,capillary electrophoresis and high⁃performance liquid chromatography for purity.And the quality comparison was studied between the enzyme purified by the new process and the existing patented process.Results There was no significant difference in molecular weight between the two kinds of purification process,but the enzyme purified by the new process had the higher specific activity,purity and better process stability than that by the existing patented process.Conclusion Compared with the existing patented process,the new process simplifies the process steps,shortens the purification cycle,reduces the risk of exposure to the process environment.The high⁃purity thrombin⁃like enzymes with good process stability and quality consistency could be obtained,which is conducive to clinical drug safety.