Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats.
Rats are widely used as a model in the study of many important diseases, yet their utility in research is limited by the lack of robust technology for the production of rat recombinant antibodies. Here we have identified and compiled putative rat immunoglobulin sequences by bioinformatic analysis of recently available genomic and EST databases, and used this information to design PCR primers to amplify rat V(H) and V(L) domain coding DNA representing the expressed repertoire of the animal. These primer sets were successfully tested and used to produce a scFv phage display library from rats that had been infected by the blood fluke, Schistosoma mansoni. The library was selected for scFvs that bind to extracellular loop epitopes on either of two known immunogenic S. mansoni membrane proteins, Sm23 and SmTsp2, both of the tetraspanin protein family. Two unique scFvs were identified that bind to each tetraspanin and shown to have excellent specificity for the target, including recognition of the native tetraspanins produced by the fluke. The primers and methods developed for rat scFvs should be of use to others seeking to characterize the antibody repertoire and/or prepare recombinant antibodies from this model.
Sepulveda
Jorge
J
Tufts Cummings School of Veterinary Medicine, North Grafton, MA 01536, United States.
Shoemaker
Charles B
CB
D000595:Amino Acid Sequence; D000818:Animals; D000937:Antigen-Antibody Reactions; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology; D017931:DNA Primers / Q000235:genetics* / Q000276:immunology; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors / Q000235:genetics; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D011916:Rats, Inbred F344; D012550:Schistosoma mansoni / Q000276:immunology; D012555:Schistosomiasis mansoni / Q000097:blood / Q000276:immunology*
D000947:Antigens, Helminth; D017931:DNA Primers; D000939:Epitopes; D015801:Helminth Proteins; D007128:Immunoglobulin Fragments; D019151:Peptide Library; C055966:immunoglobulin Fv; C067614:antigen Sm23, Schistosoma