用合成多肽免疫制备尿激酶原单克隆抗体

Preparation of monoclonal antibodies specific for prourokinase with synthetic peptide

根据尿激酶原与尿激酶一级结构的区别并结合计算机分子模拟 ,设计合成了包括尿激酶原Thr15 2～Glu16 3肽段的 13肽 ,然后与载体蛋白KLH偶联作为免疫原 ,用B淋巴细胞融合技术获得了 3种尿激酶原特异性单克隆抗体。这 3种抗体仅与尿激酶原和合成多肽反应 ,而不与尿激酶及其结构类似物组织型纤溶酶原激活剂、凝血酶、纤维蛋白原反应。琼脂双向免疫扩散实验及酶活性抑制实验表明 ,3种抗体均为IgG类的IgG1亚类 ,所有 3种抗体均不抑制酶活力。探讨了这组抗体用于尿激酶原结构与功能及其定量。

The sole structural difference between prourokinase and urokinase is whether or not cleavaged at the single peptide bond (Lys158 Ile159). To mimic the sequence spanning the primary site, we synthesized the peptide (Thr Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Cys), and the computer molecular modeling make it true that twelve amino acid sequence exposed to the surface of Prourokinase molecular. The Balb/c mice were immunized with the peptide linked to KLH (Keyhole limpet hemocyanin) , three Monoclonal Antibodies(McAbs) 1NFB3?1NFC1 and 2NFC2 were obtained by hybridoma technique. The reactivity of these McAbs against related protein like Urokinase , t PA, Thrombin(T),and Fibrinogen(F) was tested and no reactivity has been seen ; but the specific reactivity against Prourokinase has been seen. All these McAbs belong to IgG1 subclass and none of them inhibited the enzymatic activity of Prourokinase. The potential value of these McAbs in studying the structure and function as well as the measurement of Prourokinase levels in vivo from samples those with a low Prourokinase/Urokinase ratio was also dicussed.