title: Prokaryotic High-Level Expression System in Producing Adhesin Recombinant Protein E of Nontypeable Haemophilus influenzae.
abstract: BACKGROUND
  Adhesion protein E (PE) of Haemophilus influenzae is a 16 - 18 kDa protein with 160 amino acids which causes adhesion to epithelial cells and acts as a major factor in pathogenesis.
  
  
  OBJECTIVE
  In this study, we performed cloning, expression and purification of PE as a candidate antigen for vaccine design upon further study.
  
  
  METHODS
  At first, the pe gene of NTHi ATCC 49766 strain (483 bp) was amplified by PCR. Then, to sequence the resulted amplicon, it was cloned into TA vector (pTZ57R/T). In the next step, the sequenced gene was sub-cloned in pBAD/gIII A vector and transformed into competent Escherichia coli TOP10. For overexpression, the recombinant bacteria were grown in broth medium containing arabinose and the recombinant protein was purified using metal affinity chromatography (Ni-nitrilotriacetic acid) (Ni-NTA agarose). Finally, the protein was detected using sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAG) and confirmed by western blotting.
  
  
  RESULTS
  The cloned gene was confirmed by PCR, restriction digestion and sequencing. The sequenced gene was searched for homology in GenBank and 99% similarity was found to the already deposited genes in GenBank. Then we obtained PE using Ni-NTA agarose with up to 7 mg/mL concentration.
  
  
  CONCLUSIONS
  The pe gene was successfully cloned and confirmed by sequencing. Finally, PE was obtained with high concentration. Due to high homology and similarity among the pe gene from NTHi ATCC 49766 and other NTHi strains in GenBank, we believe that the protein is a universal antigen to be used as a vaccine design candidate and further studies to evaluate its immunogenicity is underway.
authors.affiliation: Fars Science and Research Branch, Islamic Azad University, Marvdasht, IR Iran.
authors.forename: Minoo
authors.initials: M
authors.lastname: Tavakoli
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Saeed
authors.initials: S
authors.lastname: Bouzari
authors.affiliation: Department of Lung Diseases, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Seyed Davar
authors.initials: SD
authors.lastname: Siadat
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Shahin
authors.initials: S
authors.lastname: Najar Peerayeh
authors.affiliation: Department of Molecular Biology, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Anis
authors.initials: A
authors.lastname: Jafari
keywords: Ni-NTA agarose; Nontypeable Haemophilus influenzae; pBAD/gIII A