人源化鼠抗人纤维蛋白单链抗体-尿激酶融合蛋白在大肠杆菌中的功能性表达

尿激酶是目前临床上广泛使用的一种有效的溶栓制剂 ,但由于尿激酶缺乏对血栓的特异亲和性 ,导致临床上尿激酶的使用剂量较大 ,容易出现全身性出血等副作用。因而开发对血栓特异的导向溶栓制剂是目前溶栓药研制的一个重要方向。本实验室在以前的工作中 ,利用噬菌体表面呈现技术 ,筛选到一种对人纤维蛋白特异的鼠单链抗体[1 ] ,在对该鼠抗体可变区进行结构模建的基础上[2 ] ,对其进行了人源化改造和体外亲和力成熟 ,得到亲和力和特异性较鼠抗体更好的人源化单链抗体[3 ] 。为研制导向溶栓制剂 ,本实验室构建了人源化单链抗体 低分子量尿激酶的…

The Functional Expression of Humanized ScFv-urokinase Fusion Protein in Escherichia coli

The fusion protein of Humanized mouse anti-human fibrin ScFv and the low molecuar weight urokinase (IIn-UK) contained seven disulfide bonds and formed inclusion body while expressing in normal E.coli strain.By coexpressing DsbC and using the special E.coli strain Origami(DE3) which was trxB/gor double mutant,the fusion protein IIn-UK was functionally expressed in the cytoplasm of E.coli. The expressed fusion protein in the soluble fraction was purified by using affinity chromatography specific against urokinase. The purified fusion protein could combine the thrombus in vitro ,and the specific activity of urokinase reached 80 000IU/mg fusion protein.The result showed that the fusion protein retained the activity of two moieties,and this study laid a foundation for further research of targeting thrombolytic agent.