title: Design and characterization of plasmids encoding antigenic peptides of Aha1 from Aeromonas hydrophila as prospective fish vaccines.
abstract: The current investigation aimed at designing DNA vaccines against Aeromonas hydrophila infections. The DNA vaccine candidates were designed to express two antigenic outer membrane protein (Aha1) peptides and to be delivered by a nanoparticle-based delivery system. Gene sequences of conserved regions of antigenic Aha1 [aha1(211-381), aha1(211-381)opt, aha1(703-999) and aha1(703-999)opt] were cloned into pVAX-GFP expression vector. The selected DNA vaccine candidates were purified from E. coli DH5α and transfected into Chinese hamster ovary cells. The expression of the antigenic peptides was measured in cells along post-transfection time, through the fluorescence intensity of the reporter GFP. The lipofection efficiency of aha-pVAX-GFP was highest after 24h incubation. Formulated PLGA-chitosan nanoparticle/plasmid DNA complexes were characterized in terms of size, size distribution and zeta potential. Nanocomplexes with average diameters in the range of 150-170nm transfected in a similar fashion into CHO cells confirmed transfection efficiency comparable to that of lipofection. DNA entrapment and further DNase digestion assays demonstrated ability for pDNA protection by the nanoparticles against enzymatic digestion.
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology Rourkela, Odisha, 769008 India.
authors.forename: Pradipta R
authors.initials: PR
authors.lastname: Rauta
authors.affiliation: Immunology and Molecular Medicine Laboratory, Department of Life Science, National Institute of Technology Rourkela, Odisha, 769008 India.
authors.forename: Bismita
authors.initials: B
authors.lastname: Nayak
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Monteiro
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address: marilia.mateus@tecnico.ulisboa.pt.
authors.forename: Marília
authors.initials: M
authors.lastname: Mateus
mesh_terms: D016980:Aeromonas hydrophila / Q000235:genetics*; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005393:Fish Diseases / Q000517:prevention & control*; D053758:Nanoparticles / Q000737:chemistry*; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D014612:Vaccines / Q000737:chemistry / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D014612:Vaccines
keywords: Aeromonas hydrophila; Aha1; Cell transfection; Nanocomplex; pDNA vaccine