title: Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.
abstract: Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.
authors.affiliation: Department of Natural Resources and Environmental Sciences, University of Illinois, Urbana, IL 61821, USA.
authors.forename: Ruth Elena
authors.initials: RE
authors.lastname: Soria-Guerra
authors.forename: Angel G
authors.initials: AG
authors.lastname: Alpuche-Solís
authors.forename: Sergio
authors.initials: S
authors.lastname: Rosales-Mendoza
authors.forename: Leticia
authors.initials: L
authors.lastname: Moreno-Fierros
authors.forename: Elise M
authors.initials: EM
authors.lastname: Bendik
authors.forename: Luzmila
authors.initials: L
authors.lastname: Martínez-González
authors.forename: Schuyler S
authors.initials: SS
authors.lastname: Korban
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000097:blood; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015139:Blotting, Southern; D015153:Blotting, Western; D002736:Chloroplasts / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007114:Immunization / Q000379:methods; D007422:Intestines / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D037342:Pertussis Toxin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013744:Tetanus Toxin / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D004167:Diphtheria Toxin; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D037342:Pertussis Toxin