title: Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.
abstract: Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression.
authors: School of Environmental Science and Engineering, Kochi University of Technology, Kochi, Japan.
authors: Vinh Van
authors: VV
authors: Hoang
authors: School of Environmental Science and Engineering, Kochi University of Technology, Kochi, Japan.
authors: Yanshuang
authors: Y
authors: Zou
authors: School of Environmental Science and Engineering, Kochi University of Technology, Kochi, Japan.
authors: Kentaro
authors: K
authors: Kurata
authors: School of Environmental Science and Engineering, Kochi University of Technology, Kochi, Japan. Electronic address: enomoto.keiichi@kochi-tech.ac.jp.
authors: Keiichi
authors: K
authors: Enomoto
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001671:Biochemistry / Q000379:methods*; D015153:Blotting, Western; D002772:Cholera Toxin / Q000378:metabolism*; D032485:Cryptomeria / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011058:Pollen / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D018984:Epitopes, T-Lymphocyte; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004247:DNA; D002772:Cholera Toxin
keywords: Cholera toxin B subunit; Cry j 1; Cry j 2; Japanese cedar pollen; Pollinosis; T-cell epitope