Construction of single-chain Fv with two possible CDR3H conformations but similar inter-molecular forces that neutralize bovine herpesvirus 1
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Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V?-VH orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V? and VH. Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V? and VH. Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle. © 2009 Elsevier Ltd. All rights reserved.
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