The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.
Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.
Jørgensen
Trond Ø
TØ
Department of Marine Biotechnology, NFH/University of Tromsø, 9037 Tromsø, Norway. trondj@nfh.uit.no
Solem
Stein Tore
ST
Espelid
Sigrun
S
Warr
Greg W
GW
Brandsdal
Bjørn Olav
BO
Smalås
Arne
A
D000818:Animals; D017104:Bacteriophage M13 / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D006241:Haptens / Q000276:immunology / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008863:Microspheres; D008958:Models, Molecular; D019151:Peptide Library; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D020317:Salmo salar / Q000235:genetics / Q000276:immunology*; D014301:Trinitrobenzenes / Q000276:immunology / Q000378:metabolism
D022801:Complementarity Determining Regions; D000939:Epitopes; D006241:Haptens; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D014301:Trinitrobenzenes; D016650:Fluorescein-5-isothiocyanate