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

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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 pBudCE4.1–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.

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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

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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.

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Authors and Affiliations

  1. Department of Molecular Genetics, Fars Science and Research Branch, Islamic Azad University, Shiraz, Islamic Republic of Iran

    Hossein Ansari

  2. Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Islamic Republic of Iran

    Abbas Doosti

  3. Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Islamic Republic of Iran

    Mohammad Kargar

  4. Department of Medical Genetics, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Islamic Republic of Iran

    Mahdi Bijanzadeh

  5. Department of Molecular Genetics, Marvdasht Branch, Islamic Azad University, Marvdasht, Islamic Republic of Iran

    Hossein Ansari & Mojtaba Jaafarinia

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  1. Hossein Ansari
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  2. Abbas Doosti
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  4. Mahdi Bijanzadeh
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  5. Mojtaba Jaafarinia
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Contributions

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.

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Correspondence to Abbas Doosti.

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Ansari, H., Doosti, A., Kargar, M. et al. Cloning of ompA gene from Acinetobacter baumannii into the eukaryotic expression vector pBudCE4.1 as DNA vaccine. Indian J Microbiol 58, 174–181 (2018). https://doi.org/10.1007/s12088-017-0705-x

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