ISOLATION, DETERMINATION AND CLONING OF TRANSLOCATION DOMAIN OF EXOTOXIN A FROM PSEUDOMONAS AERUGINOSA

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BAYAT E.; KAMALI MAHDI; ZAREI MAHMOUDABADI A.; MORTAZAVI YOUSEF; EBRAHIM HABIBI A.; AMINI BAHRAM; JAVADI HAMID REZA; FARHADI N.; HAJI OJAGH FAGHIHI M.

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Journal: KOWSAR MEDICAL JOURNAL Year:2010 | Volume:15 | Issue:3 Page(s): 149-154

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Title

ISOLATION, DETERMINATION AND CLONING OF TRANSLOCATION DOMAIN OF EXOTOXIN A FROM PSEUDOMONAS AERUGINOSA

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Keywords

PSEUDOMONAS AERUGINOSAQ2

CLONINGQ3

EXOTOXIN AQ3

TRANSLOCATION DOMAINQ3

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 37oC 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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APA: Copy

BAYAT, E., KAMALI, MAHDI, ZAREI MAHMOUDABADI, A., MORTAZAVI, YOUSEF, EBRAHIM HABIBI, A., AMINI, BAHRAM, JAVADI, HAMID REZA, FARHADI, N., & HAJI OJAGH FAGHIHI, M.. (2010). ISOLATION, DETERMINATION AND CLONING OF TRANSLOCATION DOMAIN OF EXOTOXIN A FROM PSEUDOMONAS AERUGINOSA. KOWSAR MEDICAL JOURNAL, 15(3), 149-154. SID. https://sid.ir/paper/32743/en

Vancouver: Copy

BAYAT E., KAMALI MAHDI, ZAREI MAHMOUDABADI A., MORTAZAVI YOUSEF, EBRAHIM HABIBI A., AMINI BAHRAM, JAVADI HAMID REZA, FARHADI N., HAJI OJAGH FAGHIHI M.. ISOLATION, DETERMINATION AND CLONING OF TRANSLOCATION DOMAIN OF EXOTOXIN A FROM PSEUDOMONAS AERUGINOSA. KOWSAR MEDICAL JOURNAL[Internet]. 2010;15(3):149-154. Available from: https://sid.ir/paper/32743/en

IEEE: Copy

E. BAYAT, MAHDI KAMALI, A. ZAREI MAHMOUDABADI, YOUSEF MORTAZAVI, A. EBRAHIM HABIBI, BAHRAM AMINI, HAMID REZA JAVADI, N. FARHADI, and M. HAJI OJAGH FAGHIHI, “ISOLATION, DETERMINATION AND CLONING OF TRANSLOCATION DOMAIN OF EXOTOXIN A FROM PSEUDOMONAS AERUGINOSA,” KOWSAR MEDICAL JOURNAL, vol. 15, no. 3, pp. 149–154, 2010, [Online]. Available: https://sid.ir/paper/32743/en

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