关键词: 噬菌体展示技术, 抗体库, 单链抗体, 神经肽, 亲和淘选

Abstract: A murine phage antibody library containing 1.7×10⁸ independent clones was constructed and affinity panning of anti-N-peptide antibody fragments was carried out. First, mRNA was purified from the total RNA extracted from fresh spleens of nonimmunized mice of Kunming, then the cDNA library was achieved via reverse transcription PCR. Gene fragments encoding V_H and V_L were amplified and assembled into a single gene using a DNA linker encoding a polypeptide of 15 amino acid residues (Gly₄Ser)₃ through primary PCR. And finally the recombinant DNA fragments were cloned into the phagemid pCANTAB 5E vector and introduced into E.coli TG1 by means of electroporation. The phagemid particles displaying functional ScFvs were rescued by reinfection of helper phage M13KO7, thus a murine antibody library was obtained. One N-peptide-binding ScFv clone was selected from the phage antibody library using affinity panning. The target antigen, N-peptide was biotinylated using photobiotin first. Then the biotin-labelled N-peptide interacted with the phage antibody library. The streptavidin paramagnetic beads were added subsequently to bind the biotinylated N-peptide-specific phage particle complex. Washing, elution and reinfection were ensued sequentially. After 4 rounds panning, one clone was verified to show higher affinity to N-peptide by dot blot.

Key words: phage display, antibody library, single-chain F_V, N-peptide, affinity panning