Beginning Steps in Construction of a Plasmid Free from Antibiotic Resistant Genes for DNA Vaccine


Objectives: Bacterial cell cultures containing antibiotic resistant genes represent a problem of growing concern. Spreading of bacterial genes encoding antibiotics resistance provides an environmental hazard, which is worsened by the release of antibiotics from the fermentation facility as well. This study has been undertaken to construct ,a new plasmid free from antibiotics to be used for DNA vaccine in the next future.Subject and Methods: DNA vaccine has initiated a new era of vaccine research. Restriction enzyme and T4 DNA ligation from NEB were used for Restriction/Ligation of DNA and two PCR methods were used in this study long and short PCR. All plasmid and PCR products were analysed on 1% agarose gel. Results: A plasmid free from antibiotic resistant genes was constructed from pBR322 by delete the bla gene by long PCR and tetr gene was deleted by direct repeat homologous recombination the final constructed plasmid is confirmed by gel electrophoresis in compare with two control, origin pBR322 and pBR322-∆amp in addition to specific lambda DNA marker.Conclusion: It is concluded that the vector used as DNA vaccine which be free from antibiotic can play the same role as those contain antibiotic and eventually prevent horizontal separation of antibiotic resistant gene