高纯度纤溶酶-α2-纤溶酶抑制剂和凝血酶-抗凝血酶复合物的制备

Preparation of high-purity plasmin-α2-plasmin inhibitor and thrombin-antithrombin complex

为获得纤溶酶-α2-纤溶酶抑制剂(plasmin-α2-plasmin inhibitor complex,PIC)与凝血酶-抗凝血酶(thrombinantithrombin,TAT)单克隆抗体及建立相应复合物的检测方法,需要制备高纯度的PIC与TAT复合物。以脂血血浆为研究材料,进行血浆前处理,通过尿激酶激活血浆,经Lysine-sepharose亲和层柱,最后获得高纯度的PIC;处理过的血浆经过氯化钡吸附、饱和硫酸铵沉淀后,获得抗凝血酶(antithrombin,AT)与凝血酶原(prothrombin,Pro-T);采用Ecarin蛇毒激活Pro-T获得T,将T与AT分别通过Heparin-sepharose亲和柱以提高其纯度,使其在体外混合反应得到TAT混合物,再经Heparin-sepharose亲和柱除去未反应的T与AT,最终获得高纯度TAT。通过SDS-PAGE、紫外分光光度法与HISCL自动分析仪对产物进行鉴定与定量。结果表明:PIC最佳激活条件为2 mg尿激酶/100 mL血浆,37℃激活30 min,最终获得2.5 mg PIC,经SDS-PAGE鉴定纯度在90%以上;Pro-T的激活条件为0.5 mL蛇毒/100 mL血浆,37℃激活20 min,从100 mL血浆中获得2.1 mg T与6.2 mg AT;T与AT体外最佳反应质量比例为0.7∶1,经SDS-PAGE鉴定TAT的纯度在80%以上。

In order to obtain plasmin-α2-plasmin inhibitor complex(PIC)and thrombin-antithrombin(TAT)monoclonal antibodies and establish a detection method of the corresponding complex,it is necessary to prepare high-purity PIC and TAT complexes.With lipemia plasma as the research material,the plasma pretreatment was first performed.Plasma was activated by urokinase and passed through Lysine-sepharose affinity column to finally obtain high-purity PIC.After the treated plasma is adsorbed by barium chloride and precipitated with saturated ammonium sulfate,antithrombin and prothrombin are obtained.Ecarin snake venom is used to activate prothrombin to obtain thrombin.The thrombin and antithrombin were passed through Heparin-sepharose,respectively.Affinity column was used to improve its purity,and it was mixed and reacted in vitro to obtain a TAT mixture,and then through a Heparin-sepharose affinity column to remove unreacted thrombin and antithrombin,and finally obtain high-purity TAT.The product was identified and quantified by SDS-PAGE,ultraviolet spectrophotometry and HISCL automatic analyzer.The results showed that the best activation condition for PIC was 2 mg urokinase/100 mL plasma,activated at 37℃for 30 min;2.5 mg PIC was obtained,with a purity of more than 90%identified by SDS-PAGE.The activation condition of prothrombin was 0.5 mL snake venom/100 mL plasma,activated at 37℃for 20 min,and finally 2.1 mg thrombin and 6.2 mg antithrombin were obtained from 100 mL plasma.The optimal reaction mass ratio of thrombin and antithrombin in vitro was 0.7∶1,and the purity of TAT was more than 80%identified by SDS-PAGE.