Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155)

摘要

Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding V"H and V"L regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of V"H and V"L fragments was 402bp and 363bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly"4Ser)"3 as a linker between V"H and V"L regions and subcloned into pPICZ@aA, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of V"H and V"L were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.