@2024 Afarand., IRAN
ISSN: 1735-7675 Kowsar Medical Journal 2010;15(3):149-154
ISSN: 1735-7675 Kowsar Medical Journal 2010;15(3):149-154
Isolation, determination and cloning of translocation domain of exotoxin A from Pseudomonas aeruginosa
ARTICLE INFO
Article Type
Original ResearchAuthors
Bayat E. (1 )Kamali M. (* )
Zare’ei Mahmoodabadi A. (2 )
Mortazavi Y. (3 )
Ebrahim Habibi A. (4 )
Amini B. (1 )
Javadi H. R. (5 )
Farhadi N. (5 )
Haj Ojagh Faghihi M. (6 )
(* ) Nano-Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
(1 ) Department of Biology, Faculty of Basic sciences, Sciences & Research Branch, Islamic Azad University, Tehran, Iran
(2 ) Department of Biochemistry, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran
(3 ) Department of Genetics & Molecular Medicine, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
(4 ) Endocrinology & Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran
(5 ) Nano-Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
(6 ) Department of Clinical Medicine, Vali-e-Asr Hospital, Zanjan University of Medical Sciences, Zanjan, Iran
Correspondence
Address:Phone:
Fax:
mehkamali@bmsu.ac.ir
Article History
Received:Accepted:
ePublished:
ABSTRACT
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 inpatients 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.
Materials & Methods Pseudomonas aeruginosa samples were isolated from burnt inpatients 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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