title: Immunogenic characterization and epitope mapping of transmissible gastroenteritis virus RNA dependent RNA polymerase.
abstract: Coronavirus RNA synthesis is a sophisticated process performed by a viral multienzymatic replicase complex, together with cellular factors. A key enzyme of this replication complex is the RNA dependent RNA polymerase (RdRp). To study the replication of coronavirus genome, six monoclonal antibodies (mAbs) specific for transmissible gastroenteritis virus (TGEV) RdRp were generated and characterized. His-tagged RdRp was expressed in baculovirus, purified and used as immunogen to produce mAbs. The TGEV RdRp was recognized by these mAbs in the context of virus infection by immunofluorescence analysis and Western blot. Epitope mapping by Pepscan indicated that RdRp mAbs recognized four non-overlapping linear epitopes located in a 62-amino acid region of the N-terminal domain, suggesting that this region may constitute an immunodominant domain. The availability of TGEV RdRp mAbs will be instrumental to study coronavirus replication and to analyze the function of RdRp in pathogenesis.
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), C/ Darwin 3, Cantoblanco, 28049 Madrid, Spain. anogales@cnb.csic.es
authors.forename: Aitor
authors.initials: A
authors.lastname: Nogales
authors.forename: Carmen
authors.initials: C
authors.lastname: Galán
authors.forename: Silvia
authors.initials: S
authors.lastname: Márquez-Jurado
authors.forename: Mónica
authors.initials: M
authors.lastname: García-Gallo
authors.forename: Leonor
authors.initials: L
authors.lastname: Kremer
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.forename: Fernando
authors.initials: F
authors.lastname: Almazán
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000956:Antigens, Viral / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D017934:Coronavirus / Q000235:genetics / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005455:Fluorescent Antibody Technique; D012324:RNA-Dependent RNA Polymerase / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D012324:RNA-Dependent RNA Polymerase