Construction of murine phage antibody library and selection of ricin-specific single-chain antibodies

A murine phage antibody library containing 1.7 x 10(8) independent clones w as constructed and then screened by affinity panning of anti-ricin antibody fragments. First, mRNA was purified from the total RNA extracted from fres h spleens of nonimmunized mice of Kunming, and then the total antibody vari able region cDNA was amplified via reverse transcription-polymerase chain r eaction. Gene fragments encoding VH and VL were amplified and assembled int o a single gene by using a DNA linker encoding a pentadecapeptide (Gly(4)Se r)(3) through primary PCR, Finally the recombinant DNA fragments were clone d into the phagemid pCANTAB 5E vector and electroporated into Escherichia c oli TG1, The recombinant phage particles displaying functional single-chain fragment variable regions (scFvs) were rescued by reinfection of helper ph age M13KO7, thus constructing a murine antibody library. Two ricin-specific scFvs strains were selected from the phage antibody library by using affin ity panning, The target antigen, ricin, was biotinylated with photobiotin a nd the biotin-labeled ricin interacted with the phage antibody library. Sub sequent addition of streptavidin-coated paramagnetic beads isolated the bio tinylated ricin-binding phage particle complex, Washing, elution, and reinf ection of the isolated complex then proceeded sequentially, After six round s of panning, 2 strains of the 34 clones were verified to show greater spec ific affinity to ricin.