Cloning and Expression of binding Subunit of Shiga-Like Toxin Type 2 Gene and it's Immunization Study in Animal Model

Enterohemorrhagic Escherichia coli (EHEC) which produce shiga like toxin type 2 (Stx2) is the major cause of bloody diarrhea. This pathogen could lead to hemolytic uremic syndrome (HUS) and renal failure with high mortality rate. Stx is the major virulence factor of EHEC and the neutralization of toxin by specific antibodies is known as the best way to prevention and even cure of HUS. In this study, cloning, expression, purification and immunization of Stx2 B subunit which is responsible for toxin binding to the target cell surface, was described.
The stx2B gene was amplified by PCR and subcloned into pET28a expression vector and transformed into E. coli BL21-DE3. The expression of recombinant protein was evaluated and rSTX2B was purified by Ni-NTA column. The purified rSTX2B was administered subcutaneously to Balb/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX2B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune responce was verified by an in vitro assay on Hela cells and an in vivo assay on mice by challenging of them with lethal dose of Stx2.
The IgG titration was verified the inducing of humeral response in immunized mice. The Hela cell assay indicate that Stx2 toxin was neutralized by immune mice sera. In the challenge assay 70% of immunized mice were survived.
These results indicate that recombinant rSTX2B can induce neutralizing immune response in mice and can be used as a major component in EHEC vaccines development.