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

X Gao, Y Huang, S Zhu
IUBMB Life 1999, 48 (5): 513-7
A murine phage antibody library containing 1.7 x 10(8) independent clones was constructed and then screened by affinity panning of anti-ricin antibody fragments. First, mRNA was purified from the total RNA extracted from fresh spleens of nonimmunized mice of Kunming, and then the total antibody variable region cDNA was amplified via reverse transcription-polymerase chain reaction. Gene fragments encoding VH and VL were amplified and assembled into a single gene by using a DNA linker encoding a pentadecapeptide (Gly4Ser)3 through primary PCR. Finally the recombinant DNA fragments were cloned into the phagemid pCANTAB 5E vector and electroporated into Escherichia coli TG1. The recombinant phage particles displaying functional single-chain fragment variable regions (scFvs) were rescued by reinfection of helper phage M13KO7, thus constructing a murine antibody library. Two ricin-specific scFvs strains were selected from the phage antibody library by using affinity panning. The target antigen, ricin, was biotinylated with photobiotin and the biotin-labeled ricin interacted with the phage antibody library. Subsequent addition of streptavidincoated paramagnetic beads isolated the biotinylated ricin-binding phage particle complex. Washing, elution, and reinfection of the isolated complex then proceeded sequentially. After six rounds of panning, 2 strains of the 34 clones were verified to show greater specific affinity to ricin.

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