摘要： 目的·制备以内源性凝血途径中关键的凝血因子Ⅸa（coagulation factor Ⅸa，FⅨa）为靶点的单克隆抗体，并研究其抗栓功能及作用机制。方法·通过免疫小鼠、杂交瘤技术、单克隆抗体细胞表达纯化技术，制备高纯度的抗FⅨa单克隆抗体；通过酶联免疫吸附测定筛选与FⅨa具有高亲和力的单抗，利用活化部分凝血活酶时间（activated partial thromboplastin time，APTT）和凝血酶原时间（prothrombin time，PT）评估单抗的抗栓效果；然后利用发色底物法检测其对FⅨa酶活性的影响；采用计算机模拟蛋白-蛋白对接的方法预测抗体与FⅨa相互作用可能的结合位点，并通过竞争实验（间接通过发色底物法）对这一结合位点进行验证。结果·制备得到1种高亲和力抗FⅨa的单克隆抗体FⅨa-4；虽然它不影响PT和FⅨa的酶活性，但可显著延长APTT至88.8 s，是对照组（25.5 s）的3.5倍，并且具有浓度依赖性。蛋白-蛋白对接预测结果发现，FⅨa-4不直接与FⅨa的底物催化位点结合，而是占据了FⅨa和FⅧa的结合区域。竞争实验进一步验证了上述结果，FⅨa-4以剂量依赖的方式抑制FⅩa的生成，FⅨa-4的浓度达到400 pmol/L即可使FⅩa的生成几乎完全被抑制，而FⅧa可纠正抗体的抑制作用达到近50%。结论·获得抗FⅨa的单抗FⅨa-4；FⅨa-4与FⅧa竞争性结合FⅨa阻碍了FⅧa-FⅨa复合物形成，阻断FⅩ向FⅩa的转化，该抗体主要通过抑制内源性凝血途径发挥抗栓作用。
关键词: 凝血因子Ⅸa, 单克隆抗体, 活化部分凝血活酶时间, 酶活性, 凝血因子Ⅷa, 抗栓作用 Abstract: Objective·To prepare a monoclonal antibody targeting coagulation factor Ⅸa (FⅨa), a key factor in endogenous coagulation pathways, and study its antithrombotic roles and mechanisms.
Methods·Immunization of mice, hybridoma technology, cell expression and purification were used to prepare the anti-FⅨa monoclonal antibodies of high purity. The monoclonal antibodies with high affinity for FⅨa were screened by enzyme-linked immunosorbent assay. The activated partial thromboplastin time (APTT) and prothrombin time (PT) were used to evaluate the antithrombotic effects of the monoclonal antibody. And then chromogenic substrate method was used to detect the effect of monoclonal antibody on the enzyme activity of FⅨa. The method of computer simulation of protein-protein docking was adopted to predict the possible binding sites between FⅨa and the antibody, and the binding site, was verified by competitive experiments (indirectly through the chromatic substrate method).
Results·FⅨa-4, an anti-FⅨa monoclonal antibody with high affinity, was generated. Although FⅨa-4 did not affect PT and the enzyme activity of FⅨa, it significantly prolonged APTT to 88.8 s, which was 3.5 times of the control group (25.5 s), in a concentration-gradient dependence. The protein-protein docking prediction results revealed that FⅨa-4 did not directly bind to substrate catalytic sites of FⅨa, but occupied the binding region of FⅨa and FⅧa. Competitive experiments further verified the above results. FⅨa-4 inhibited FⅩa production in a dose-dependent manner, almost completely inhibiting FⅩa production at the concentrations of 400 pmol/L, and FⅧa could correct the inhibition effect of the antibody up to nearly 50%.
Conclusion·The monoclonal antibody against FⅨa-4 is obtained. FⅨa-4 competes with FⅧa to bind FⅨa, and inhibits the conversion of FⅩ to FⅩa which is catalyzed by FⅧa-FⅨa complex; it plays an antithrombotic role mainly by inhibiting endogenous coagulation pathway.
Key words: coagulation factor Ⅸa (FⅨa), monoclonal antibody, activated partial thromboplastin time (APTT), enzyme activity, coagulation factor Ⅷa (FⅧa), antithrombosis PDF全文下载地址:
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