Cloning and Expression of the Binding Subunit of Shiga-Like Toxin Type ۲ Gene and Immunization Study in an Animal Model

Objective: Enterohemorrhagic Escherichia coli (EHEC) which produces shiga-like toxin type ۲ (Stx۲) is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome (HUS) and renal failure with a high mortality rate. Stx۲ is the major virulence factor of EHEC. Neutralization of toxin by specific antibodies is known to be the best way to prevent and cure HUS. In this study, we describe the cloning, expression, purification, and immunization of the Stx۲B subunit which is responsible for toxin binding to the target cell surface. Methods: The Stx۲B gene was amplified by PCR and subcloned into a pET۲۸a expression vector and transformed into E. coli BL۲۱-DE۳. We evaluated recombinant protein expression and rSTX۲B was purified by the Ni-NTA column. The purified rSTX۲B was administered subcutaneously to BALB/c mice in three separate doses as an immunogenic candidate. The raising of anti-rSTX۲B antibodies in immunized mice sera was evaluated by Elisa assay. The neutralizing immune response was verified by an in vitro assay on HeLa cells and an in vivo assay on mice by challenging them with a lethal dose of Stx۲. Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx۲ toxin was neutralized by immune mice sera. In the challenge assay, ۷۰% of immunized mice survived. Conclusion: Recombinant rSTX۲B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines.

کلیدواژه ها:

Enterohemorrhagic Escherichia coli ، Immunization ، Vaccine candidate ، Shiga-like toxin ، سم شبه شیگا ، ایمنی زایی ، کاندید واکسن ، باکتری اشریشیا کلی انتروهموراژیک