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目的探讨不同吸附体系及其柠檬酸钠、氯化钠、pH、电导4因素对凝血酶原复合物(PCC)中蛋白C、蛋白S、蛋白Z、抗凝血酶收率及比活的影响。方法选择柠檬酸钠、氯化钠、pH3因素3水平设计正交试验,配制9种PCC平衡吸附体系;凝胶平衡后,吸附血浆,洗涤、洗脱,制备PCC浓缩物;测定浓缩物中蛋白C和S、抗凝血酶活性、蛋白Z含量、总体积及蛋白浓度,获得四类抗凝蛋白收率及比活,比较不同吸附体系收率、比活总趋势;利用正交试验直观分析法分析柠檬酸钠、氯化钠、pH对蛋白C、S和Z收率及比活的影响;测定吸附体系电导率,分析电导率与4类抗凝蛋白收率及比活的关系。结果不同吸附体系4类抗凝蛋白收率及比活有较大差异,抗凝血酶二者均极低。柠檬酸钠对蛋白C收率及比活、蛋白S收率影响较大;pH对蛋白S比活、蛋白Z收率及比活影响极差分别为:0.120、6.847、0.716,高于其他因素;氯化钠对其影响均最小。柠檬酸钠浓度越低,蛋白C收率及比活越高,蛋白S收率则越低,0.015 mol/L水平蛋白Z收率为49.11%,高于其他水平,而蛋白S和Z比活最低;pH越低,蛋白Z收率则越高,pH7.0水平蛋白C和S收率及比活、蛋白Z比活最高;氯化钠浓度0.075 mol/L水平,蛋白C收率及比活、蛋白S和Z比活分别为:29.37%、0.533 IU/mg、0.401 IU/mg、2.362μg/mg,均高于其他水平,而蛋白S、Z收率最低。不同电导率条件,蛋白C收率及蛋白Z比活变化较大,其中蛋白Z与S收率、蛋白Z与S比活、蛋白C收率与比活变化趋势基本一致;(10.00±0.20)ms/cm条件,蛋白C、S和Z比活较高。结论不同吸附体系抗凝血酶收率及比活均极低,蛋白C、S和Z收率及比活间无规律性。氯化钠对蛋白C、S和Z收率及比活影响较小,柠檬酸钠、氯化钠、pH不同水平对蛋白C、S和Z收率影响效应无共性,0.01 mol/L柠檬酸钠,0.075 mol/L氯化钠、pH7.0水平蛋白C、S和Z比活最佳。电导率与四类抗凝蛋白收率及比活间无线性关系。
Objective To investigate the effects of different adsorption systems and sodium citrate, sodium chloride, pH and conductance on the protein C, protein S, protein Z, antithrombin yield and specific activity of prothrombin complex (PCC) . Methods Sodium citrate, sodium chloride and pH3 factor 3 were chosen to design orthogonal experiment. Nine kinds of PCC equilibrium adsorption systems were prepared. After the gel was equilibrated, the plasma was absorbed, washed and eluted to prepare PCC concentrate. The protein in the concentrate C and S, antithrombin activity, protein Z content, total volume and protein concentration, the yield and specific activity of four types of anticoagulant protein were obtained. The yield and specific activity of different adsorption systems were compared. The orthogonal experiment The effects of sodium citrate, sodium chloride and pH on the yield and specific activity of proteins C, S and Z were analyzed. The conductivity of the adsorption system was determined. The relationship between the conductivity and the yield and specific activity of the four types of anticoagulant protein was analyzed. Results The yield and specific activity of four types of anti-coagulation proteins in different adsorption systems were quite different. Antithrombin both were extremely low. Sodium citrate has a great effect on the yield and specific activity of protein C and the yield of protein S. The effect of pH on protein S specific activity, protein Z yield and specific activity are 0.120, 6.847 and 0.716 respectively, which is higher than other factors ; Sodium chloride has the least impact on it. The lower the concentration of sodium citrate, the higher the yield and specific activity of protein C, the lower the yield of protein S, and the yield of protein Z at 0.015 mol / L was 49.11%, higher than other levels, while the protein S and Z specific activities The lowest, the lower the pH, the higher the yield of protein Z, the protein C and S yield and specific activity of protein at pH7.0, the highest specific activity of protein Z; the sodium chloride concentration of 0.075 mol / L, protein C yield and ratio The specific activities of live, protein S and Z were 29.37%, 0.533 IU / mg, 0.401 IU / mg and 2.362μg / mg respectively, all higher than other levels, while the protein S and Z yields were the lowest. The yield of protein C and the specific activity of protein Z varied greatly under different conductivity conditions, among which the yield of protein Z and S, the ratio of protein Z to S, the yield of protein C and the specific activity were basically the same (10.00 ± 0.20) ms / cm conditions, protein C, S and Z higher than the specific activity. Conclusion The antithrombin yield and specific activity of the different adsorption systems are extremely low, and the yield and specific activity of protein C, S and Z are not regular. Sodium chloride had little effect on the yield and specific activity of protein C, S and Z. The effects of different concentrations of sodium citrate, sodium chloride and pH on the yield of protein C, S and Z were not common. The concentration of 0.01 mol / L citric acid Sodium, 0.075 mol / L sodium chloride, pH7.0 level of protein C, S and Z specific activity best. The relationship between conductivity and four types of anticoagulant yield and specific activity.