中国生物工程杂志

The single-chain Fv cDNA of a monoclonal antibody specific for glycoprotein of rabies virus, was cloned into the PE40 (a truncated form of pseudomonas exotoxin) gene inserted fusion protein expression plasmid pET-PE40 to form the expression vector pET-ScFv-PE40, The recombinant plasmid was identified by restriction enzyme digestion and sequencing.After transfection of E.coli BL21 and induction by IPTG,the recombinant immunotoxin fusion protein was highly expressed in E.coli BL-21 in the form of inclusion bodies(up to 32.29% of total cellular proteins). The fusion protein was refolded by constant gradient dialysis and purified with anion exchange chromatography, hydrophobic interaction and gel filtration chromatography, the interest protein was about 96% pure. Indirect immunofluorescent staining showed that recombinant immunotoxin could specifically bind to rabies virus-infected cells. Cytotoxicity analysis by MTT showed that the fusion proteins could kill rabies virus-infected cells specifically,while having no toxicity to normal cells.