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Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse
BACKGROUND: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Avicenna Research Institute
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838764/ https://www.ncbi.nlm.nih.gov/pubmed/24285994 |
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author | Haghighat, Setareh Siadat, Seyed Davar Sorkhabadi, Seyed Mehdi Rezayat Sepahi, Abbas Akhavan Mahdavi, Mehdi |
author_facet | Haghighat, Setareh Siadat, Seyed Davar Sorkhabadi, Seyed Mehdi Rezayat Sepahi, Abbas Akhavan Mahdavi, Mehdi |
author_sort | Haghighat, Setareh |
collection | PubMed |
description | BACKGROUND: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. METHODS: A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-β-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. RESULTS: Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. CONCLUSION: Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies. |
format | Online Article Text |
id | pubmed-3838764 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Avicenna Research Institute |
record_format | MEDLINE/PubMed |
spelling | pubmed-38387642013-11-27 Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse Haghighat, Setareh Siadat, Seyed Davar Sorkhabadi, Seyed Mehdi Rezayat Sepahi, Abbas Akhavan Mahdavi, Mehdi Avicenna J Med Biotechnol Original Article BACKGROUND: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. METHODS: A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-β-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. RESULTS: Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. CONCLUSION: Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies. Avicenna Research Institute 2013 /pmc/articles/PMC3838764/ /pubmed/24285994 Text en Copyright © 2013 Avicenna Research Institute http://creativecommons.org/licenses/by-nc/3.0/ This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly. |
spellingShingle | Original Article Haghighat, Setareh Siadat, Seyed Davar Sorkhabadi, Seyed Mehdi Rezayat Sepahi, Abbas Akhavan Mahdavi, Mehdi Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title | Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title_full | Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title_fullStr | Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title_full_unstemmed | Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title_short | Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse |
title_sort | cloning, expression and purification of penicillin binding protein2a (pbp2a) from methicillin resistant staphylococcus aureus: a study on immunoreactivity in balb/c mouse |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838764/ https://www.ncbi.nlm.nih.gov/pubmed/24285994 |
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