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 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。
Under these conditions the fibrinolytic activity of the supernatant can reach 820 urokinase units per milliliter broth . ba - dfe was purified from the supernatant of b . amyloiquefaciens dc - 4 culture broth by ammonium sulfate precipitation , ion - exchange chromatography on cm - and deae - sepharose fast flow , hydrophobic interaction chromatography on phenyl sepharose 6 fast flow and gel filtration on sephadex g - 50 . the purified enzyme displayed thermophilic , hydrophilic and strong fibrinolytic activity 通过硫酸铵分级沉淀、 cm - sepharosefastflow和deae - sepharosefastflow离子交换层析、 phenylsepharose6fastflow疏水层析和sephadexg - 50凝胶过滤等方法，从解淀粉芽孢杆菌dc - 4的发酵液中分离纯化出电泳纯的ba - dfe 。
