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目的对人血浆凝血因子Ⅷ(coagulation factorⅧ,FⅧ)制备过程中4种柱纯化前处理方法进行比较并分析其优缺点,为血源FⅧ的纯化制备提供借鉴与参考。方法冷沉淀经Tris溶解后的上清,分别用甘氨酸沉淀法、PEG沉淀法、酸沉淀法和氢氧化铝凝胶吸附法处理;用活化部分凝血活酶(APTT)法测定样品上清中FⅧ活性,计算总活性回收率和样品的比活性,用非还原性SDS-PAGE电泳比较去除杂蛋白的效果。结果 4种处理方法中,甘氨酸沉淀法总活性回收率最高[(94.00±7.60)%],其次是酸性沉淀法[(89.47±2.60)%]、PEG沉淀法[(80.92±9.67)%],氢氧化铝吸附法的总活性回收率最低[(78.65±7.52)%];甘氨酸沉淀法与PEG沉淀法去除杂蛋白的效果优于酸性沉淀法和氢氧化铝吸附法;经4种不同方法处理后,甘氨酸沉淀法所得样品中FⅧ比活最高[(0.6856±0.1258)IU/mg],其次是PEG沉淀法[(0.5773±0.0787)IU/mg]、酸性沉淀法[(0.3885±0.0301)IU/mg],氢氧化铝吸附法所得样品比活最低[(0.2879±0.0472)IU/mg]。结论在上述方法中,甘氨酸沉淀法和PEG沉淀法的总体效果较佳,且因后者操作更加简便、快捷,在实际生产过程中采用PEG沉淀法较为合适。
OBJECTIVE To compare and analyze the pretreatment methods of four kinds of columns during the preparation of human plasma coagulation factor VIII (FⅧ), and to analyze the advantages and disadvantages of these methods, so as to provide reference and reference for the purification and preparation of blood FⅧ. Methods The supernatant was precipitated by Tris, and then precipitated by glycine, PEG precipitation, acid precipitation and aluminum hydroxide gel adsorption respectively. FⅧ in the supernatant of the sample was determined by activated partial thromboplastin (APTT) Activity, calculate the total activity recovery and the specific activity of the sample, non-reducing SDS-PAGE electrophoresis to compare the removal of hybrid protein. Results The recovery of total activity of glycine precipitation was the highest among the four treatments [(94.00 ± 7.60)%], followed by acid precipitation [(89.47 ± 2.60)%] and PEG precipitation [(80.92 ± 9.67)%] The total activity recovery rate of aluminum hydroxide adsorption was the lowest [(78.65 ± 7.52)%]; the effect of glycine precipitation and PEG precipitation was better than acid precipitation and aluminum hydroxide adsorption by four different methods (0.6856 ± 0.1258) IU / mg], followed by PEG precipitation [(0.5773 ± 0.0787) IU / mg], acid precipitation [(0.3885 ± 0.0301) IU / mg]. The specific activity of the sample obtained by aluminum hydroxide adsorption was the lowest [(0.2879 ± 0.0472) IU / mg]. Conclusion In the above method, glycine precipitation method and PEG precipitation method overall effect is better, and because the latter operation easier and faster, in the actual production process using PEG precipitation method is more appropriate.