重组活性小鼠组织因子及其阻断性单抗的制备与应用

组织因子是一种位于细胞膜上的糖蛋白，是外源性凝血过程的关键启动因子，近年来其在肿瘤细胞迁移等其他过程中的重要作用也已逐渐被揭示．构建了融合有His标签的小鼠组织因子胞外区段重组蛋白基因，利用昆虫杆状病毒蛋白表达系统成功表达并得到大量可溶性重组小鼠组织因子．利用血浆凝集实验和鼠尾流血时间实验对此重组小鼠组织因子进行的活性检测表明，此重组蛋白具有良好的生物活性，可以引起血浆凝血或缩短鼠尾流血时间．同时，利用此重组蛋白为抗原，制备了小鼠组织因子的小鼠源功能阻断性单克隆抗体，在血浆凝集实验中证明其对小鼠组织因子的活性有明显抑制作用．利用此阻断性单抗，成功地在小鼠深静脉血栓模型中减轻了血栓形成，证明组织因子在深静脉血栓的病程发展中起重要作用，这也是组织因子阻断性单抗在此类动物模型中的首次成功应用．通过此项工作，成功地建立了大量制备具有良好生物活性的重组小鼠组织因子蛋白的方法，并进而得到了小鼠组织因子功能阻断性单抗，为利用各种小鼠动物模型对组织因子在各项生命活动中的作用进行深入研究奠定了良好的基础．

Generation and Application of Active Recombinant Mouse Tissue Factor and Its Function-blocking Monoclonal Antibody

Tissue factor (TF) is a cell surface glycoprotein playing an important role in the initiation of the blood coagulation cascade. The functions of TF in other physiological or pathological activities, such as tumor cell migration, are also acknowledged gradually in recent years. A recombinant protein of mouse tissue factor (mTF) extracellular part fused with His tag was constructed, and it was expressed and purified successfully in high-level soluble form. This recombinant mTF can effectively initiate plasma clotting in vitro and enhance blood coagulation in vivo, which prove its biological activity. Using this recombinant mTF as immunogen, a murine monoclonal function-blocking antibody to mTF was also generated, which can inhibit mTF-initiated plasma clotting in vitro. And by treating with this function-blocking antibody, the thrombus formation in a murine deep vein thrombosis model was attenuated successfully, which suggests the important role of tissue factor in deep vein thrombosis. In all, with the active mTF recombinant protein and the mTF function-blocking antibody, the functional investigations of TF in murine models of various research areas become more convenient and feasible.