Background and Objective: Antibody against Pseudomonas aeruginosa exotoxin A can be used in immunotherapy together with antibiotics to treat acute burn patients. Exotoxin A is one of the virulence factors in Pseudomonas aeruginosa that comprises of three domains, binding domain, translocation and catalytic domain. The purpose of this study was to produce recombinant domain of the catalytic part of this microorganism in order to produce antibody against it. Methods and Materials: Pseudomonas aeruginosa samples were isolated from burn patients hospitalized in Mousavi Hospital, Zanjan, Iran and Pseudomonas aeruginosa species were identified by Biochemical tests. Bacteria genomic DNA was extracted and exotoxin A gene determined by PCR. Catalytic domain of exotoxin A was amplified by PCR. Products and plasmid extracts was digested by restriction enzymes. Subsequently PCR products and plasmids transformed into E. coli BL21 (DE3). Clones containing gene of interest was determined by PCR, restriction enzyme and sequencing. Results: The sequence homology of the catalytic domain of exotoxin A was compared with that of the published gene data bank. The results showed a complete homology between our gene species and the published genome in data banks. Conclusion: The results of this study showed that about 90% of the isolated bacteria contained exotoxin A and there was a sequence homology between our species and published gene data banks.

Background and Objective: Pseudomonas aeruginosa is an opportunistic pathogen that causes severe and lethal infections in immunocompromised individuals. This bacterium possesses a single polar flagellum. Flagellum and its subunit Flagellin play important roles in the pathogenesis of P. aeruginosa. Flagellin induces immune responses by interaction of its N-terminal domain with TLR-5. Our main aims of this study were cloning and expression of N-terminal domains of flagellin and evaluation of antibodies raised against it on motility inhibition of P. aeruginosa. Material and Methods: The DNA sequence coding for the first 161 amino acids of flagellin was PCR amplified and cloned into a pET-28a expression vector. Recombinant protein was over expressed in BL-21(DE3), and purified by Ni-NTA resin. The immune reactivity of recombinant truncated flagellin was evaluated by Western blotting. The recombinant protein was injected into a rabbit and antibodies raised against it were evaluated for the cell motility inhibition of P. aeruginosa 8821M. Results: The N-terminal domain of Flagellin was successfully overexpressed in Escherichia coli BL-21(DE3) host strain. Anti-native and anti-N-terminal flagellin antibodies reacted with the recombinant protein. Motility inhibition assay demonstrated that polyclonal antiserum against N-teminal flagellin is able to inhibit the motility of P. aeruginosa 8821M. Conclusion: The N-terminal domain of flagellin may be used for development of a new recombinant vaccine against P. aeruginosa infections.

Background and Objective: As the main virulence factor of Helicobacter pylori, HP-NAP has an important role in immunoprotection against this pathogen. This antigen is a strong candidate as a part of multi-component vaccine in the clinical trial against this bacterium. Due to NAP importance, it was used in this study as a template for optimization of heterologous genes with a low A-T content and low expression in E. coli. Materials and Methods: A synthetic single gene that could reach the highest level of expression in the host was designed by using bioinformatics tools. Results: A number of factors that influence gene expression level were changed for HP-NAP gene optimizing: the codon usage bias in E. coli was changed the G+C content was upgraded from 38% to 45% and the stem-loop structure was broken. These could result to prolong of the half-life of the mRNA and overexpression of recombinant of HP-NAP protein up to 800 mg per liter. Conclusion: Applying of bioinformatics tools was appropriated to optimize of HP-NAP overexpression in E. coli. From our results, it appears that combination of In Silico and experimental approach is a logical approach for expression of heterologous genes in another host.