Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

Authors

  • Fakhri Haghi Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
  • Shahin Najar Peerayeh Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
  • Seyed Davar Siadat Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran
  • Mehran Montajabiniat Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

DOI:

https://doi.org/10.3855/jidc.1557

Keywords:

Neisseria meningitidis, PorA, Ni-NTA agarose, pET-32a

Abstract

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA.

Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porAplasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification.

Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle.

Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

Author Biography

Fakhri Haghi, Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

Department of Bacteriology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran

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Published

2011-11-14

How to Cite

1.
Haghi F, Najar Peerayeh S, Siadat SD, Montajabiniat M (2011) Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B. J Infect Dev Ctries 5:856–862. doi: 10.3855/jidc.1557

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Section

Original Articles