导向溶栓分子scFv-UK32中连接肽的引入和在昆虫细胞中的表达

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

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DNABacPAK6 Bsu36Idigest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 10 7IU mL ,比未引入连接肽的scFv UK32的表达活性 (2 5IU 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。WesternBlotting实验表明共转染上清可与Pro UK的单抗特异结合。

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.