Acta Scientiarum Naturalium Universitatis Pekinensis

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Construction and Expression of a Recombinant Antibody-targeted Plasminogen Activator

YANG Jiashu, JIANG Pengchen, RU Binggen   

  1. National Laboratory of Protein Engineering and plant genetic engineering, College of Life Sciences, Peking University, Beijing, 100871
  • Received:1998-05-14 Online:1999-07-20 Published:1999-07-20



  1. 北京大学蛋白质工程和植物基因工程国家重点实验室,北京,100871

Abstract: A novel plasminogen activator was constructed using scFv of SZ51 as targeted molecule, and scu-PA-32k as effect molecule. SZ51 was a monoclonal antibody of GMP140 on activated human platelets. Polymerase chain reaction (PCR) was used to amplify the region of VK and VH from Fab of SZ51, and scu-PA-32k(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via Nde I site. After transforming in E.coli BL21 (DE3) plyS and inducing with IPTG, the recombinant protein was expressed in inclusion bodies. It was shown in Western-Blotting that the protein could interact weakly with the multiple clonal antibody of urokinase in 8 mol/L of Urea. After renaturation and partial purification, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate, the specific activity was about 17500 IU/mg, which showed the recombinant protein restored the activity of u-PA quiet well. The yield was almost 1.5mg/100g wet bacteria.

Key words: GMP140, monoclonal antibody, urokinase, single-chain of fragments of variable region of antibody (scFv), DNA recombination, fibrinolytic activity

摘要: 以针对人活化血小板表面糖蛋白GMP140的单克隆抗体SZ51的单链抗体作为导向分子,以单链尿激酶32kd变体(scu-PA-32k)作为效应分子,构建了一种新型的导向性溶栓剂。通过聚合酶链式反应(PCR),分别从SZ51的Fab片段基因中扩增出VK和VH区;从尿激酶原基因中扩增出scu-PA-32k基因。通过合适的linker及酶切位点,将VK, VH及scu-PA-32k基因相连接并装入表达载体pET-5a中的Nde I位点,在大肠杆菌中经IPTG诱导表达了重组蛋白。Western-Blotting检测到在8mol/L尿素存在下,该重组蛋白与抗尿激酶的多克隆抗体之间仍有弱的结合反应。表达产物经变复性处理和部分分离纯化后,在纤维蛋白平板上表现有较好的纤溶活性,且这种活性是通过激活纤维蛋白溶酶原为纤溶酶而实现的。重组蛋白纤溶活性的比活达到17500IU/mg,较好地保留了尿激酶的纤溶活性,重组蛋白的产率约每100g湿菌为1.5mg。

关键词: 人活化血小板表面糖蛋白GMP140, 单克隆抗体, 尿激酶, 单链抗体, DNA重组, 纤溶活性

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