Isolation, determination and cloning of translocation domain of exotoxin A from Pseudomonas aeruginosa

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Kowsar Medical Journal Pages: 49-54 - : * PhD PhD PhD PhD MSc MSc MSc ()... MSc MSc () *... ()... A. A : EF-2.... A... : A. PCR A DNA.. PCR. pet28a PCR PCR. SDS-PAGE. A PCR : IPTG.. A :. A : Isolation, determination and cloning of translocation domain of exotoxin A from Pseudomonas aeruginosa Bayat E. MSc, Kamali M.* PhD, Zare ei Mahmoodabadi A. 2 PhD, Mortazavi Y. 3 PhD, Ebrahim Habibi A. 4 PhD, Amini B. MSc, Javadi H. R. 5 MSc, Farhadi N. 5 MSc, Haj Ojagh Faghihi M. 6 MSc Nano-Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 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 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 mm concentration incubated at 37 C for 2 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. Keywords: Pseudomonas aeruginosa, Cloning, Exotoxin A, Translocation Domain نويسنده مسي ول: مهدي كمالي. تمام درخواستها بايد به نشاني mehkamali@bmsu.ac.ir فرستاده شوند. دريافت مقاله: 89//24 پذيرش مقاله: 89/4/8

TSI MR VP (% ) LB. - A : DNA.( ) : A PBF: 5 - TGC TGC ACT ACT CCA TGG TC - 3 PBR: 5 - ATC GGT ACC AGC CAG TTC AG -3 A EcoRI : HindIII PTF:5 - ATT TC GAATTC GCG GCA GCC TGG CCG -3 PTR:5 - ACC AC AAGCTT GCT GGC CGC GCC GG -3 : DNA. LB High Pure PCR ( Template Preparation, Roche). A DNA :A PCR. PCR /. DNA / / dttp dgtp dctp datp MgSo 4 * PCR PWO DNA. DNA / PCR % PCR.. PCR :PCR ( ) TaqI ( ) HindIII EcoRI. A..[ ] A A A.[ ].[] CD9 []. ( -) A A.[ ]. (eef-2).[] Ia Ib I. A Ia. II.[] Ib III [].[ ] (eef-2) A %.[ ] A A.[] A.[].[] A. A......

PCR HindIII EcoRI. TaqI T7. : LB - D-B / OD (IPTG).... % SDS-PAGE His-. Tag DNA A % PCR. % PCR A. ). DNA.( PCR. TaqI PCR DNASIS. TaqI. HindIII EcoRI. % * PCR. High Pure PCR Product ( Purification Kit,Roche). % (pet28a) :. LB %. HindIII PCR EcoRI. High Pure PCR Cleanup ) % ( Micro kit, Roche. PCR : : * T 4 - High pure. ( PCR cleanup micro kit, Roche). :. BL2(DE3) E. coli -. E.. SOC coli BL2(DE3). LB ( ). PCR :. ) LB (.. PCR

T7. % SDS-PAGE. 90 bp 4 3 2 bpp ١٠٠٠ ٩٠٠ ٨٠٠ ٧٠٠ ٦٠٠ ٥٠٠ ٤٠٠ ٣٠٠ ٢٠٠ ١٠٠ bp bp A PCR ( PCRP - bp DNA -.% - ( bp ) A - ( bp ) A PCR bpp ) A PCRP ( PCR ( A PCRR ( DNAA (bp) (.% ( bp A HindIII EcoRI A.bp 3 2 SDS-PAGE (. % A ( ( (. IPTG IPTG. IPTG %/. KDa Vol.. 5, No. 3, Fall 200 bp A PCR ( ( bp DNA (.% PWO DNA PCR bpp % PCR (.TaqI PCR ( DNAD (. PCR bpp PCR. % 2 bpp bp bp p p

].[.. A % G+C A. (%/).[ ] HindIII. PCR EcoRI.[] A.[] PCR pet28a.. pet32a.[] toxc. A.[ ] A.[] A (). A. A. IPTG A..[] C A ( ( (. His-Tag ( ) A.. His-. Tag. A.... A A.[]... eef-2.[] A A. A KDa

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