Cloning and Expression of the Binding Subunit of Shiga-Like Toxin Type 2 Gene and Immunization Study in an Animal Model

Authors
R. Kazemi 1
A. Akahavian Tehrani 1
M. Jafari 1
J. Amani 2
A. Mousavi 1
A. H. Salmanian 1
1 Department of Plant Bioproducts, Institute of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
2 Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
Abstract
Objective: Enterohemorrhagic Escherichia coli (EHEC) which produces shiga-like toxin type 2 (Stx2) is a major cause of bloody diarrhea. This pathogen can lead to hemolytic uremic syndrome (HUS) and renal failure with a high mortality rate. Stx2 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 Stx2B subunit which is responsible for toxin binding to the target cell surface.
Methods: The Stx2B gene was amplified by PCR and subcloned into a pET28a expression vector and transformed into E. coli BL21-DE3. We evaluated recombinant protein expression and rSTX2B was purified by the 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 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 Stx2.
Results: The IgG titration verified the induction of a humeral response in immunized mice. The HeLa cell assay indicated that the Stx2 toxin was neutralized by immune mice sera. In the challenge assay, 70% of immunized mice survived.
Conclusion: Recombinant rSTX2B can induce a neutralizing immune response in mice. It can be used as a major component in development of EHEC vaccines.

Keywords

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Pathobiology Reserach
Volume 18, Issue 4
January 2016
Pages 45-60