title: Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse.
abstract: 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.
  
  
  CONCLUSIONS
  Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies.
authors.affiliation: Department of Biology, Faculty of Basic Sciences, Science and Research branch, Islamic Azad University, Tehran, Iran.
authors.forename: Setareh
authors.initials: S
authors.lastname: Haghighat
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.forename: Seyed Mehdi Rezayat
authors.initials: SM
authors.lastname: Sorkhabadi
authors.forename: Abbas Akhavan
authors.initials: AA
authors.lastname: Sepahi
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mahdavi
keywords: Methicillin-resistant Staphylococcus aureus; PBP2a; Recombinant vaccine