RESUMEN
Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.
Asunto(s)
Alérgenos/inmunología , Epítopos de Linfocito T/metabolismo , Lactococcus lactis/efectos de los fármacos , Nisina/farmacología , Rinitis Alérgica Estacional/terapia , Adyuvantes Inmunológicos/administración & dosificación , Alérgenos/genética , Vacunas Bacterianas/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/genética , Cryptomeria/inmunología , Epítopos de Linfocito T/efectos de los fármacos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Escherichia coli/genética , Humanos , Inmunoglobulina E/inmunología , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Nisina/administración & dosificación , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Plásmidos , Polen/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/prevención & controlRESUMEN
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.