溶栓与抗栓双功能尿激酶原突变体的模拟、构建与表达

Simulation,Construction and Expression of the Recombinant dscuPA with Thrombolytic and Antithrombus Bifunction

将抗栓肽 ( Decorsin)嫁接到低分子量尿激酶原 ( scu PA-3 2 k)上 ,可以期望获得既具有抗血小板聚集活性 ,又具有溶栓活性的新型基因工程蛋白质分子 .利用计算机辅助分子设计手段模拟了该融合蛋白的分子结构 ,证明其活性区可以正常发挥功能 .根据大肠杆菌偏好密码子合成 Decorsin的基因 ,与 scu PA-3 2 k基因融合在一起 ,构建新的嵌合体基因 dscu PA,并在大肠杆菌中通过 IPTG进行诱导表达 ,该重组蛋白在大肠杆菌中以包涵体的形式存在 .对包涵体进行变性和复性并通过层析纯化得到目的蛋白质 .用纤维蛋白平板法测得重组蛋白的比活为 92 0 0 0 IU/mg.激活纤溶酶原的酶促动力学性质与天然低分子量尿激酶相似 ,且有较强的抑制血小板聚集的功能 .重组蛋白 dscu PA不但具有较强的溶栓功能 ,而且具有抗栓功能 .

A recombinant chimeric plasminogen activator(dscuPA) was constructed, consisting of the decorsin(platelet aggregation inhibitor) and a low molecular mass(32 000) single-chain urokinase(scuPA-32k,comprising Leu144 through Leu 411). The structure of the designed protein was predicted and simulated. The recombinant protein was produced in \%E. Coli \%after IPTG induction and exited in inclusion body. After refolded in vitro,the chimeric protein was purified by chromatography. The special activity of the chimera was 92 000 IU/mg detected by fibrin plate determination. The chimera could activate plasminogen following Michaelis-Menten kinetics with \%K\%\-m=1.36 μmol/L and \%k\%\-\{cat\}=\{0.002 8 s\+\{-1\}\},corresponding to that of scuPA. It was also shown that chimera inhibited ADP-induced platelet aggregation in a concentration dependent manner. These results showed that the chimeric protein not only had high thrombolytic activity but also had anti-thrombus function. [WT5HZ]