Title : Human Single-Chain Antibodies to ETA: Potential Therapeutic Agent against Pseudomonal Infection
Pseudomonas exotoxin A (ETA) is the most potent virulence determinant produced by Pseudomonas aeruginosa which is one of the foremost causes of life-threatening nosocomial infections. The ETA catalyzes the ADP-ribosylation of eukaryotic elongation factor-2 halting protein synthesis and leading to mammalian cell death. Engineered single-chain antibodies (HuscFvs) against exotoxin A should raise hope for treatment of the fatal entity. In this study, ETA antigens were used as antigens in the phage bio-panning for selecting phage clones that bound to the respective antigens from a previously constructed HuscFv phage display library. The selected phage clones were transfected to E. coli, grown and induced for HuscFvs expression. HuscFvs in the E. coli lysates were tested for target binding ability. Genes coding for ETA-bound-HuscFvs were sub-cloned for large scale production and tested further for neutralizing activities. Furthermore, the homology modeling and molecular docking of ETA to HuscFvs were also conducted. The ETA bound-HuscFvs were successfully produced. From phage bio-panning, 241 E. coli clones were selected. The 155 clones were carried huscfv gene. The expressed HuscFvs of 37 clones bound to the native ETA. Based on the deduced amino acid sequences and numbering according to the Kabat and Chothia scheme, the HuscFv clones were characterized into 7 different types. The three candidates of the transformed E. coli clones containing genes coding for the target-bound HuscFvs that gave high binding ability were sub-cloned for large scale HuscFv production and the inhibitory activities of HuscFv were successfully investigated for their activities (apoptosis assays) and molecular docking studies.