title: Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.
abstract: Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.
authors.affiliation: Université de Bretagne Occidentale, Quimper, 2, rue de l'Université
authors.forename: Vincent
authors.initials: V
authors.lastname: Le Moigne
authors.forename: Georges
authors.initials: G
authors.lastname: Robreau
authors.forename: Wahib
authors.initials: W
authors.lastname: Mahana
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D048014:Clostridium tyrobutyricum / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D018274:Electroporation; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D005260:Female; D005408:Flagellin / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D005816:Genetic Complementation Test; D007371:Interferon-gamma / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009068:Movement; D009841:Oligonucleotides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology; D011995:Recombination, Genetic; D013154:Spleen / Q000276:immunology; D018417:Th1 Cells / Q000276:immunology*; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000939:Epitopes; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D005408:Flagellin; D015847:Interleukin-4; D007371:Interferon-gamma