Expression of Recombinant Zonula Occludens Toxin (Zot) ofVibrio Cholerae and Biological Activity on Rabbit Ileal Loops

Introduction: Vibrio cholerae causes potentially lethal disease, cholera, through the elaboration of the intestinal secretion of cholera toxin .The cholera enterotoxin (CT) has been considered as the major virulence factor of V. cholerae. This microorganism also produces other putative toxins such as Zonula Occludens toxin (Zot) and Accessory cholera enterotoxin (Ace). Zot has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. The Zot toxin alters ion transport and causes fluid accumulation in ligated rabbit ileal loops which results in mild diarrhea. Materials and Methods: In this study, gene coding for the Zot toxin was amplified from V. cholera isolate 62013. The PCR product containing the Zot gene was cloned in pET28a expression vector .The recombinant Zot gene was transformed into E. coli (DH5 ?) and then retransformed into E. coli Tuner for expression. Expression of recombinant protein induced by isopropythio-B-D-galctoside (IPTG) at different concentration and was examined by SDS-PAGE and Western blotting. Rabbit ileal loops experiment was conducted. Results: Cloning of Zot was confirmed by colony-PCR and enzymatic digestion. The recombinant Zot with molecular weights of 45KDa and 22kDa was expressed and reacted with rabbit anti-Vibrio cholerae polyclonal antibody in western-blot analysis. Zot protein significantly causes fluid accumulation in ligated rabbit ileal loops test. Conclusion: Our findings indicated that a prokaryotic expression system for Zot protein was successfully constructed. This expression system can be useful as a tool for production of Zot protein for vaccine purposes.