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Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria
Published on: May 8, 2013
Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant
Setareh Haghighat1, Seyed Davar Siadat, Seyed Mehdi Rezayat Sorkhabadi
1Department of Biology, Faculty of Basic Sciences, Science and Research branch, Islamic Azad University, Tehran, Iran.
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.
Insights
This study developed a recombinant Penicillin-Binding Protein 2a (PBP2a) from Staphylococcus aureus as a vaccine candidate. Vaccinated mice showed a high antibody response, indicating PBP2a
Area of Science:
- Microbiology
- Immunology
- Vaccine Development
Background:
- Staphylococcus aureus is a growing threat due to antibiotic resistance.
- Penicillin-binding protein modifications contribute to S. aureus resistance.
- Vaccines are crucial for controlling S. aureus infections.
Purpose of the Study:
- To develop and produce recombinant PBP2a as a vaccine candidate.
- To assess the humoral immune response to PBP2a in mice.
Main Methods:
- Amplified and cloned the mecA gene encoding PBP2a.
- Expressed and purified recombinant PBP2a in E. coli.
- Immunized mice with PBP2a and measured antibody titers via ELISA.
Main Results:
- Confirmed successful cloning and expression of recombinant PBP2a (13 kDa).
- Demonstrated high titers of specific antibodies against PBP2a in vaccinated mice.
- Confirmed the immunogenicity of the PBP2a vaccine candidate.
Conclusions:
- Recombinant PBP2a successfully induced specific antibodies in mice.
- PBP2a shows potential as a vaccine candidate against Staphylococcus aureus infections.
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