摘要
目的筛选对人凝血因子Ⅷ吸附效果较好的新型离子交换树脂,研究其吸附性能。方法将初始浓度为(20±1)IU/ml的冷沉淀提取液,恒温摇床振荡吸附2 h,测定吸附后上清液中蛋白浓度及FⅧ活性,通过蛋白吸附量及FⅧ活性吸附率,获得人FⅧ吸附效果较好的树脂;P-15与A-15树脂相同条件下吸附FⅧ,计算FⅧ的活性吸附率以及蛋白吸附量,考察树脂骨架对吸附的影响,同种方法考察树脂孔径大小对吸附的影响;测定吸附过程中吸附液中蛋白浓度的变化,得到吸附动力学曲线,从而确定吸附速率。分别改变A-15树脂用量、吸附温度、冷沉淀粗提液的pH值以及NaCl浓度,计算FⅧ的活性吸附率以及蛋白吸附量,考察树脂用量、温度、pH值、NaCl浓度对吸附的影响。结果 A-15树脂对FⅧ的活性吸附率为(78.76±4.2)%,蛋白吸附量(77.32±3.8)mg/g;A-15树脂未接基团时对FⅧ的活性吸附率为(12.34±5.6)%,蛋白吸附量为(18.68±5.3)mg/g;随着孔径的增大,树脂对FⅧ的活性吸附率和蛋白吸附量均为先增大后减小;树脂为1.25 g时,即可满足对FⅧ的吸附要求;吸附速率常速为0.003 56 g/mg.min;摇床温度为25～30℃、pH值为6.5～7.0、NaCl浓度为(0.1～0.2)mol/L时为较佳吸附条件。结论 A-15树脂对人凝血因子Ⅷ的吸附效果较好,在较佳吸附条件下,树脂对FⅧ的活性吸附率为78±4%,蛋白吸附量为(77±4)mg/g。
Objective To find a new ion-exchange resin, for a better separation and purification of human coagulation factor Ⅷ and to study its adsorption properties. Methods The crude extracts of cold precipitation, which has an initial concentration of FⅧ was adsorbed for ：2 hours on a constant temperature oscillation shaker. The activity of FⅧ and protein concentration of the supernatant, in order to calculated the activity adsorption rate of FⅧ and protein adsorption. Comparing the activity adsorption rate of FⅧ and the protein concentration between the resins, we can get a resin, with a better adsorption capacity for FⅧ . The resin P-15, which is not combined with groups, and the resin A-15 adsorb the FⅧ ,under the same conditions. Calculating the activity adsorption rate of FⅧ and protein adsorption, in order to study the influence of skeleton of the resin on the adsorption capacity of FⅧ . The effects of pore size of the resin on the adsorption capacity of FⅧ were studied in the same way. To get the adsorption kinetics and determine the adsorption rate constant, measuring the diversification of the protein concentration in the adsorption process. Changing the amount of the resin A-15 ,the temperature of the shaker, the pH and the concentration of NaCl of the crude extracts of cryo-precipitate, respectively, and calculating the activity adsorption rate of FⅧ and protein adsorption. Results For the resin A-15, the activity adsorption rate of FV~ was 78. 76%, and the protein adsorption is 77. 32mg/g. For the resin P-15, which is not combined with chemical groups, the activity adsorption rate of FⅧ is 12. 34%, and the protein adsorption is 18.68mg/g; As the aperture of the resin increases, the activity adsorption rate of FⅧ and the protein adsorption are first increases and then decreases. And the speed constant of the adsorption is 0. 003 56g/mg·min. When the quality of the resin is 1.25g,they can meet the requirements of the adsorption of FⅧ . The better adsorption conditions are
出处
《中国输血杂志》
CAS
CSCD
北大核心
2012年第3期231-236,共6页
Chinese Journal of Blood Transfusion
基金
2009年国家卫生行业科研专项(200902008-2)
关键词
人凝血因子Ⅷ
离子交换树脂
分离纯化
Human coagulation factor Ⅷ
Ion exchange resins
Purification
