Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine
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Antibiotic resistant features of Acinetobacter baumannii is partly due to the decreased outer membrane proteins (OMPs) permeability. The OmpA is one of the most conserved proteins among A. baumannii with a considerable antigenic potential to stimulate the multidimensional immune system responses. The present study was aimed to clone the ompA gene into the eukaryotic expression vector with potential as DNA vaccine. The ompA gene of A. baumannii was amplified using polymerase chain reaction (PCR). The target DNA was cloned and sub-cloned into the pTZ57R/T and pBudCE4.1 vectors, respectively. The recombinant vectors containing ompA were then validated using colony PCR, vector sequencing and double-digestion strategies. The pBudCE4.1–ompA recombinant plasmid was transfected into the human dermal fibroblast cells (HDF) and presence of ompA transcript and protein was evaluated using reverse transcribed-PCR (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Our finding from colony PCR, sequencing and enzyme double digestion result confirmed that target gene has been successfully inserted into the pTZ57RT and pBudCE4.1. The presence of an expected band (1112 bp) in RT-PCR as wells as a ~ 38 kDa band during SDS-PAGE showed that the recombinant pBudCE4.1–ompA construct was efficiently transfected into the HDF cells and expressed. Altogether, our observation demonstrated that the recombinant pBudCE4.1–ompA construct was successfully produced although further experiments are needed.
KeywordsDNA vaccine Acinetobacter baumannii Outer membrane protein ompA gene
- A. baumanni
Dulbecco’s modified Eagle’s medium
Fetal calf serum
Granulocyte-macrophage colony-stimulating factor
Human dermal fibroblast
Infectious Diseases Society of America
Outer membrane protein
Outer membrane protein A
Outer membrane vesicles
Polymerase chain reaction
Reverse transcription polymerase chain reaction
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
This study is part of Ph.D. thesis which was granted by Fars Science and Research Branch, Islamic Azad University, Shiraz, I. R. Iran. The Authors are thankful from Farzam Latifi and all the colleagues in Biotechnology Research Center, Islamic Azad University Shahrekord for their gorgeous assistant in this project.
HA; literature review, manuscript writing and data collection. AD; literature review and manuscript revision, MK; manuscript revision, MB; manuscript revision and data collection and MJ; literature review.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
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