title: Molecular Cloning, Expression and Purification of Truncated hpd Fragment of Haemophilus influenzae in Escherichia coli.
abstract: BACKGROUND
  Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in children, causing otitis media, sinusitis, conjunctivitis, pneumonia, and occasionally invasive infections. Protein D (PD) belongs to the minor outer-membrane proteins of H. influenza. Moreover, it has been shown that this protein is one of the most potent vaccine candidates against the NTHi strain.
  
  
  OBJECTIVE
  In the present study, a new truncated form of PD was designed based on conserved areas, and recombinant truncated PD was expressed.
  
  
  METHODS
  Truncated PD was designed using bioinformatics tools, and a 345 bp fragment of the truncated hpd gene was amplified by polymerase chain reaction (PCR) from H. influenzae and subsequently cloned into the prokaryotic expression vector pBAD-gIIIA. In addition, for the expression of the recombinant protein, the pBAD-truncated PD plasmid was transformed into competent TOP10 cells. The recombinant protein was expressed with Arabinose. The expressed protein was purified by affinity chromatography using Ni-NTA resin.
  
  
  RESULTS
  The cloning of PD was confirmed by colony-PCR and enzymatic digestion. Arabinose 0.2% was able to efficiently induce protein expression. The SDS-PAGE analysis showed that our constructed pBAD-PD-TOP10 efficiently produced a target recombinant protein with a molecular weight of 16 kDa. A high concentration of the recombinant protein was obtained via the purification process by affinity chromatography. The recombinant PD was reacted with peroxidase-conjugated rabbit anti-mouse immunoglobulins.
  
  
  CONCLUSIONS
  Our results showed that the recombinant protein produced by the pBAD vector in the Escherichia coli system was very efficient.
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Ava
authors.initials: A
authors.lastname: Behrouzi
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Saeid
authors.initials: S
authors.lastname: Bouzari
authors.affiliation: Department of Tuberculosis and Pulmonary Research, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Anis
authors.initials: A
authors.lastname: Jafari
authors.affiliation: Department of Biology, School of Basic Science, Science and Research Branch, Islamic Azad University, Tehran, IR Iran.
authors.forename: Shiva
authors.initials: S
authors.lastname: Irani
keywords: Expression Vector; Haemophilus influenzae; Ni-NTA; Recombinant Protein