title: Expression of grape class IV chitinase in Spodoptera frugiperda (Sf9) insect cells.
abstract: BACKGROUND
  Most of pathogenesis related (PR) proteins possess complicated structures; hence their active recombinant forms are usually produced in eukaryotic systems. In this study, we employed an insect cell line to express a recombinant form of a previously identified grape PR3 allergen categorised as class IV chitinase.
  
  
  METHODS
  Grape chitinase cDNA was amplified by RT-PCR and inserted into pFastBacHTA using restriction enzymes. The recombinant pFastBacHTA was applied for the transformation of Escherichia coli DH10Bac cells. The purified recombinant bacmid was used for transfection of Sf9 cells. Finally, the IgE-immunoreactivity of purified recombinant protein was evaluated using grape allergic patient's sera. Moreover, polyclonal anti-6His-tag and monoclonal anti-chitinase antibodies were used for further assessment of recombinant protein.
  
  
  RESULTS
  SDS-PAGE analysis of the transfected Sf9 cells showed expression of a monomeric 25kDa and a dimeric 50 kDa recombinant protein. Western blotting revealed considerable IgE reactivity of the recombinant protein with grape allergic patients' sera. Furthermore, confirmatory assays showed specific reactivity of the recombinant protein with anti-His tag and anti-chitinase antibodies.
  
  
  CONCLUSIONS
  This study showed that, in contrast to E. coli, insect cells are suitable hosts for the production of a soluble and IgE-reactive recombinant form of grape class IV chitinase. This recombinant allergen could be used for component resolved diagnosis of grape allergy or other immunodiagnostic purposes.
authors.affiliation: Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: R
authors.initials: R
authors.lastname: Falak
authors.affiliation: Allergy Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
authors.forename: A R
authors.initials: AR
authors.lastname: Varasteh
authors.affiliation: Department of Biology, Faculty of Science, Islamic Azad University-Mashhad Branch, Mashhad, Iran.
authors.forename: H
authors.initials: H
authors.lastname: Ketabdar
authors.affiliation: Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Electronic address: SankianM@mums.ac.ir.
authors.forename: M
authors.initials: M
authors.lastname: Sankian
mesh_terms: D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000818:Animals; D016367:Baculoviridae; D015153:Blotting, Western; D002688:Chitinases / Q000235:genetics* / Q000276:immunology*; D018076:DNA, Complementary; D005512:Food Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D061987:Sf9 Cells; D018411:Spodoptera; D014162:Transfection; D027843:Vitis / Q000276:immunology*
chemical_list: D000485:Allergens; D018076:DNA, Complementary; D011994:Recombinant Proteins; D002688:Chitinases
keywords: Baculovirus; Chitinase; Component resolved diagnosis; Food allergy; Pathogenesis related proteins; Vitis vinifera