Isolation, determination and cloning of translocation domain of exotoxin A from Pseudomonas aeruginosa
Trauma Monthly: 15 (3); 149-154 Article Type: Research Article
M, Zareei Mahmoodabadi
Y, Ebrahim Habibi
A, et al. Isolation, determination and cloning of translocation domain of exotoxin A from Pseudomonas aeruginosa,
Online ahead of Print
Aims: Pseudomonas aeruginosa Exotoxin A is an important virulence factor of this bacterium. Exotoxin A is made of three domains: binding domain, translocation domain and catalytic domain. Exotoxin A inhibits protein synthesis by ADP-ribosylating EF-2 factor in eukaryote cells. Translocation domain has an important function in translocation of toxin into the cell. Purpose of this study was to product recombinant domain of translocation for antibody production against it.
Materials & Methods: Pseudomonas aeruginosa samples were isolated from burnt inp atien ts of Moosavi Hospital in Zanjan and were identified by biochemistry tests. Bacteria genomic DNA was extracted and Exotoxin A presence was approved by PCR. Translocation domain of Exotoxin A was reproduced by PCR and PCR products were cloned in a pET28a plasmid. Clones were sequenced, screened and enzyme-digested by PCR. Recombinant protein was approved by SDS-PAGE and western blotting with its specific antibody.
Results: PCR and enzyme digestion results approved the cloning of translocation domain of Exotoxin A and results of Exotoxin A translocation domain sequencing was the same as the Gene Bank database. Expression of recombinant translocation domain protein was determined in IPTG 1mM concentration incubated at 37°C for 12 hours.
Conclusion: Expression of recombinant protein is higher than Pseudomonas aeruginosa. The whole toxin is not necessary for vaccine production and this recombinant protein can be used instead.
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