Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine

  • Hossein Ansari
  • Abbas Doosti
  • Mohammad Kargar
  • Mahdi Bijanzadeh
  • Mojtaba Jaafarinia
Original Research Article
  • 11 Downloads

Abstract

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 pBudCE4.1ompA 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 pBudCE4.1ompA construct was efficiently transfected into the HDF cells and expressed. Altogether, our observation demonstrated that the recombinant pBudCE4.1ompA construct was successfully produced although further experiments are needed.

Keywords

DNA vaccine Acinetobacter baumannii Outer membrane protein ompA gene 

Abbreviations

A. baumanni

Acinetobacter baumanni

cDNA

Complementary DNA

DMEM

Dulbecco’s modified Eagle’s medium

EDTA

Ethylenediaminetetraacetic acid

FCS

Fetal calf serum

g

Gram

GMSF

Granulocyte-macrophage colony-stimulating factor

h

Hour

HDF

Human dermal fibroblast

IDSA

Infectious Diseases Society of America

IPTG

Isopropyl-β-d-thiogalactoside

LB

Luria-Bertani

ml

Milliliter

min

Minute

MDR

Multidrug resistance

NTC

Negative control

OMP

Outer membrane protein

OmpA

Outer membrane protein A

OMVs

Outer membrane vesicles

PCR

Polymerase chain reaction

PNAG

Polysaccharide poly-N-acetyl-β-(1-6)-glucosamine

RT-PCR

Reverse transcription polymerase chain reaction

s

Second

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

U

Unit

Notes

Acknowledgements

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.

Authors Contribution

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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Copyright information

© Association of Microbiologists of India 2018

Authors and Affiliations

  • Hossein Ansari
    • 1
    • 5
  • Abbas Doosti
    • 2
  • Mohammad Kargar
    • 3
  • Mahdi Bijanzadeh
    • 4
  • Mojtaba Jaafarinia
    • 5
  1. 1.Department of Molecular Genetics, Fars Science and Research BranchIslamic Azad UniversityShirazIslamic Republic of Iran
  2. 2.Biotechnology Research Center, Shahrekord BranchIslamic Azad UniversityShahrekordIslamic Republic of Iran
  3. 3.Department of Microbiology, Jahrom BranchIslamic Azad UniversityJahromIslamic Republic of Iran
  4. 4.Department of Medical Genetics, School of MedicineAhvaz Jundishapur University of Medical SciencesAhvazIslamic Republic of Iran
  5. 5.Department of Molecular Genetics, Marvdasht BranchIslamic Azad UniversityMarvdashtIslamic Republic of Iran

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