生物学杂志

生物学杂志 ›› 2022, Vol. 39 ›› Issue (1): 110-.doi: 10. 3969/ j. issn. 2095-1736. 2022. 01. 110

• 技术方法 • 上一篇    下一篇

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

  

  1. 1. 江南大学 粮食发酵工艺与技术国家工程实验室,无锡 214122;
    2. 江苏拜明生物技术有限公司,盐城 224005
  • 出版日期:2022-02-18 发布日期:2022-02-15
  • 通讯作者: 白仲虎,博士,教授,主要从事发酵工程与体外诊断研究工作,E-mail:baizhonghu@jiangnan.edu.cn
  • 作者简介:周帅辉,硕士研究生,从事发酵工程与体外诊断试剂研究,E-mail:925397480@qq.com
  • 基金资助:
    江苏省重点研发计划项目(BE2018621)

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

  1. 1. National Engineering Laboratory of Grain Fermentation Technology, Jiangnan University, Wuxi 214122, China;
    2. Jiangsu Baiming Biotechnology Co. , Ltd. , Yancheng 224005, China
  • Online:2022-02-18 Published:2022-02-15

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摘要：

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Lysinesepharose 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。


关键词: 凝血酶, 抗凝血酶, 纤溶酶-α2-纤溶酶抑制剂, 亲和层析, 纯化

Abstract:

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.


Key words: thrombin, antithrombin, plasmin-α2-plasmin inhibition, affinity chromatography, purification

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