高等学校化学学报

高等学校化学学报 ›› 2004, Vol. 25 ›› Issue (6): 1046.

• 研究论文 • 上一篇    下一篇

溶栓与抗栓双功能尿激酶原突变体的模拟、构建与表达

张磊亮, 焦建伟, 邵开峰, 俞梅敏, 茹炳根   

  1. 北京大学生命科学学院, 蛋白质工程国家重点实验室, 北京 100871
  • 收稿日期:2003-06-17 出版日期:2004-06-24 发布日期:2004-06-24
  • 通讯作者: 茹炳根(1936年出生),男,教授,博士生导师,从事蛋白质化学与蛋白质功能研究.E-mail:rulab@pku.edu.cn E-mail:rulab@pku.edu.cn
  • 基金资助:

    国家自然科学基金(批准号:30200057)资助

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

ZHANG Lei-Liang, JIAO Jian-Wei, SHAO Kai-Feng, YU Mei-Min, RU Bing-Gen   

  1. College of Life Sciences, National Lab of Protein Engineering, Peking University, Beijing 100871, China
  • Received:2003-06-17 Online:2004-06-24 Published:2004-06-24

PDF (PC)

被引次数

7

摘要： 将抗栓肽(Decorsin)嫁接到低分子量尿激酶原(scuPA-32k)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scuPA-32k基因融合在一起,构建新的嵌合体基因dscuPA,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92000IU/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dscuPA不但具有较强的溶栓功能,而且具有抗栓功能.

关键词: 低分子量尿激酶原, 抗栓肽, 血小板聚集

Abstract: 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 Km=1.36 μmol/L and kcat=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.

Key words: ScuPA-32k, Decorsin, Platelet aggregation

中图分类号: 

TrendMD: