Design and testing of PCR primers for the construction of scFv libraries representing the immunoglobulin repertoire of rats

Abstract

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.

Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

“…Several months later and prior to tissue harvest, the sheep received another 250-g boost of A-HC and two additional weekly doses of 2 g BoNT/A1 holotoxin. Peripheral blood lymphocytes (PBLs) were obtained from blood, and cDNA was produced from PBL mRNA by reverse transcriptase, using random hexamer and oligo(dT) primers as previously described (23).…”

“…The V L and V H domains were sequentially cloned into the JSC phage display vector (23) to produce a library with about 7 ϫ 10 6 phage, with Ͼ80% containing inserts with both V H and V L domains. This scFv display library, representing the antibody repertoire of the BoNT/A-immunized sheep, was panned on Immunotubes (Nunc) coated with 1 g/ml BoNT/A holotoxin, using standard procedures.…”

“…DNAs encoding unique scFv clones were transferred to the JSC-his periplasmic expression vector (23) such that each scFv was produced with an epitope tag, or E tag (GAPVPYPDPLEPR), and six histidines at the carboxyl end. In one instance, a second copy of the E tag coding DNA was engineered at the amino-terminal coding end of scFv#7 (scFv#7-E2).…”

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

“…The preparation and characterization of the scFv phage-display library used in this study has been described previously by Sepulveda and Shoemaker (2008). Briefly, the scFv-display library was prepared using antibody V H and V L coding DNA amplified by PCR from the B cells of rats that were twice infected by S. mansoni cercariae.…”

“…The library has a complexity of approximately 10 7 with >90% displaying full-length scFvs. For panning, an aliquot of the library containing 10 9 colony forming units (CFU) of phage was brought to log phase growth and infected with helper phage to produce an amplified phage preparation displaying scFvs (Sepulveda and Shoemaker, 2008). …”