人血浆凝血因子Ⅷ柱纯化前处理方法的比较  

Comparison of four pre-column treatment methods for human plasma coagulation factor Ⅷ pre-purification

作　　者：王方[1,2] 吕茂民[2] 赵雄[2] 余蓉[1] 章金刚[2] 

机构地区：[1]四川大学华西药学院微生物与生化药学教研室,成都610041 [2]军事医学科学院野战输血研究所,北京100850

出　　处：《军事医学》2015年第3期199-202,共4页Military Medical Sciences

基　　金：国家863计划资助项目(2012AA021902)

摘　　要：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 four pretreatment methods of human plasma coagulation factor Ⅷ （FⅧ） before column purification. Methods Cryoprecipitate was dissolved in Tris, before the supernatant was treated with glycine precipitation, PEG precipitation, acid precipitation and aluminum hydroxide gel adsorption, respectively. Activated partial thromboplastin （APTT） was used to measure the activity of the supernatant clotting Fvm after treatment. The total activity recovery and specific activity of the final samples were used to weigh the efficacy of those methods. The purity of the intended protein was estimated by non-reducing SDS-PAGE electrophoresis. Results Total activity recovery of glycine precipitation was the highest （94.00%±7.60% ）, followed by that of acidic precipitation （89.47% ±2.60% ） and PEG precipitation （80.92% ± 9.67% ） methods. The lowest was aluminum hydroxide gel adsorption （78.65% ± 7.52% ）. Glyeine precipitation and PEG precipitation could more effectively remove contaminating protein than acid precipitation and aluminum hydroxide gel adsorption. Treated by four different methods, the specific activity of FⅧ of glycine precipitation sample was the highest （0. 6856 ± 0. 1258 IU/mg）, followed by PEG precipitation （0.5773 ± 0. 0787 IU/mg） and acidic precipitation （0. 3885± 0. 0301 IU/mg）. The specific activity of aluminum hydroxide gel adsorption was the lowest （0. 2879 ±0. 0472 IU/mg）. Conclusion PEG precipitation is more effective for the actual production process than the other three methods.

关 键 词：人凝血因子Ⅷ 冷沉淀 柱纯化前处理 

分 类 号：R392-33[医药卫生—免疫学]