[Cloning and expression of the binding subunit of shiga-like toxin type 2 gene and immunization study in an animal model]

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

[Production of chimeric tir-intimin protein from E. coli O157:H7 in the tobacco [Nicotiana tobbacum] plant and its immunological evaluation in an animal model]

Escherichia coli [E.coli] O157:H7 is one of the most important pathogenic causes of hemorrhagic colitis in humans. Cattle are the main reservoirs of this bacteria and vaccination is a key mechanism for its control. The intimin, translocated intimin receptor [tir], and EspA proteins are virulence factors expressed by the LEE locus of enterohemorrhagic E. coli. EspA protein is a member of the type III secretion system [TTSS] needle complexes that delivers the tir protein into the host cell. Surface arrayed intimin docks the bacterium to the translocated intimin receptor [Tir]. This intimate linkage is the starting point for attachment and effacing lesions. We hypothesize that the chimeric recombinant forms of two of these three effectors, as edible-based immunogens, would reduce colonization of E. coli O157:H7 in the mice model. We constructed a synthetic gene [it] composed of eae [i] and tir [t] attached together by a peptide linker. The synthetic gene [it] was codon optimized based on the tobacco [Nicotiana tobbacum] plant and cloned into plant expression vectors adjacent to CaMV35S promoters for expression in transgenic tobacco plants. The antigen produced in this plant was orally fed to mice. Immunization of the mice model by the transgenic plant that contained the divalent immunogen showed the presence of IgG antibodies against E. coli O157:H7. This method could be an effective tool for protecting against E. coli O157:H7 hemorrhagic colitis

Regeneration of glyphosate-tolerant Nicotiana tabacum after plastid transformation with a mutated variant of bacterial aroA gene

Presence of antibiotic resistance markers has always been considered as one of the main safety concerns in transgenic plants and their derived products. Elimination of antibiotic selectable markers from transgenics is a major hurdle for finding efficient and safe candidates. Herbicide tolerance genes might be attractive alternatives. In this study, a variant form of the 5-enoylpyruvyl shikimate-3-phosphate synthase [EPSPS] gene that harbors glycine at position 96 to alanine and alanine 183 to threonine substitutions and confers higher resistance to the broad-spectrum herbicide, glyphosate, was substituted against the spectinomycin resistant gene as a sole selectable marker for plastid transformation of Nicotiana tabacum. Plastid transformation was carried out using the biolistic delivery procedure while delivery parameters such as rupture disk pressure, bombardment distance, etc had been optimized first. A previous study showed that the glyphosate herbicide imposes lethal effects on the structure and integrity of the plastid membrane, even at low concentrations. In order to overcome this problem, a modified procedure for selection of transplastomic cells was used. A long preculture incubation period followed by a gradual increased in glyphosate concentration led to sufficient expression of the transgene. Tolerant calli were thus regenerated through direct selection of transformed plastids in the presence of the glyphosate

Site-directed mutagenesis, expression and biological activity of E. coli 5-enolpyruvylshikimate 3-phosphate synthase gene

Site-directed mutagenesis [SDM] as a powerful technique was used to change two important and conserved amino acids in 5-enolpyruvylshikimate 3- phosphate synthase [EPSPS] gene of E. coli. The mutations changed glycine 96 to alanine and alanine 183 to threonine. These two amino acids are very important for intraction of the wide spectrum herbicide, glyphosate, to EPSP synthase enzymes. By designing mutagen primers and overlapping extension method, three kinds of altered bacterial EPSPS enzymes with first, second and both mutations were produced. These modified enzymes are expected to show decreased affinity for herbicide, with least alteration in their enzymatic activity. These altered genes were cloned under the control of chemically inducible T7 promoter and over expressed in E. coli. Biological activity analyses in the presence of glyphosate show that the bacteria containing the mutated enzymes, especially the enzyme with two mutations, were more tolerant to glyphosate