title: Generation and characterization of a recombinant antibody fragment that binds to the coat protein of grapevine leafroll-associated virus 3.
abstract: Pathogen-specific recombinant antibodies have been used to characterize pathogen infections and to engineer resistance in crops. We selected a single-chain antibody fragment (scFvLR3cp-1) specific for the coat protein of grapevine leafroll-associated virus 3 (GLRaV-3), one of the agents of grapevine leafroll (GLR) disease, from a phage display library. The antibody binds specifically to the entire length of GLRaV-3 particles and has a high binding affinity value (K(D)) of 42 nM. The amino acid motif AQEPPRQ located at the N terminus of the GLRaV-3 coat protein was identified as the antibody-binding epitope by PEPSCAN analysis. To evaluate scFv stability in the reducing environment of the plant cell cytosol, transient expression assays were performed using Nicotiana benthamiana as a model plant. Capture ELISA demonstrated that the scFv fragment was produced and retained its antigen-binding capacity in the plant cytosol. Further functional assays showed that scFvLR3cp-1 binds with high specificity to at least four members of the family Closteroviridae. Therefore, the GLRaV-3-specific scFv fragment could be an ideal candidate for mediating broad-spectrum virus resistance if produced in transgenic grapevine plants.
authors.affiliation: Institute for Molecular Biotechnology (Biology VII), RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Orecchia
authors.forename: Greta
authors.initials: G
authors.lastname: Nölke
authors.forename: Pasquale
authors.initials: P
authors.lastname: Saldarelli
authors.forename: Mariangela
authors.initials: M
authors.lastname: Dell'Orco
authors.forename: Kerstin
authors.initials: K
authors.lastname: Uhde-Holzem
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.forename: Giovanni
authors.initials: G
authors.lastname: Martelli
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D000914:Antibodies, Viral / Q000276:immunology*; D000915:Antibody Affinity; D036022:Capsid Proteins / Q000737:chemistry / Q000276:immunology*; D029381:Closteroviridae / Q000276:immunology*; D003429:Cross Reactions; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000276:immunology*; D019151:Peptide Library; D010935:Plant Diseases / Q000821:virology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014026:Nicotiana / Q000378:metabolism*; D027843:Vitis / Q000821:virology*
chemical_list: D000914:Antibodies, Viral; D036022:Capsid Proteins; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv