Acta Scientiarum Naturalium Universitatis Pekinensis

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Introduction of A Linking Peptide to the Thrombolysis-Targeted Protein and Its Expression in Insect Cell

JU Wujian,HU Meihao   

  1. College of Life Sciences, Peking University, Beijing, 100871
  • Received:2000-07-25 Online:2001-11-20 Published:2001-11-20



  1. 北京大学生命科学学院,北京,100871

Abstract: The recombinant PCR technic was used to introduce a linking peptide (KLGGGG) to the site between scFv (single chain form of the monoclonal antibody SZ-51 specific for the glycoprotein GMP140 on activated platelet membrane) and UK32 (low molecular weight form of pro-urokinase), to make the scFv-linker-UK32 chimeric gene. This gene was cloned into the transfer vector pBacPAK9, and cotransfected with BacPAK6/Bsu36I digest into Sf 9 cells. The fusion protein was secreted into the medium. In the fifth day after the cotransfection, the supernatant of the medium showed 107 IU/mL fibrinolytic activity, higher than 25 IU/mL fibrinolytic activity of scFv-UK32. ELISA showed that the supernatant had the binding activity to activated platelet. Wastern Blotting also indicated that the supernatant could bind to the monoclonal antibody of urokinase B chain.

Key words: targeted thrombolysis, linking peptide, insect cell, expression

摘要: 为了提高重组导向溶栓分子scFv-UK32的溶纤活性,通过重组PCR方法在编码scFv与UK32的碱基之间引入编码KLGGGG连接肽的碱基序列,并克隆到转移载体pBacPAK9上,通过与线性病毒DNA BacPAK6/Bsu36I digest共转染到昆虫细胞Sf 9内,进行表达。表达产物分泌到上清中,共转染后第5d(天)用纤维平板法测得Sf 9细胞上清溶纤活性达到107 IU/mL,比未引入连接肽的scFv-UK32的表达活性(25 IU/mL)高。ELISA实验表明共转染上清具有明显对活化血小板特异结合能力。Western Blotting实验表明共转染上清可与Pro-UK的单抗特异结合。

关键词: 导向溶栓, 连接肽, 昆虫细胞, 表达

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