Production of single chain antibodies against mycotoxin aflatoxin B1 in recombinant Escherichia coli

Thesis (master`s)--서울대학교 대학원 :농생명공학부,2004.Aflatoxins B1 (AFB1) is a secondary metabolite produced mostly by Aspergillus flavus and A. parasiticus strains and the most toxic to human and several animal species as food contaminant. The single chain variable region (scFv) genes of a monoclonal antibody against AFB1 were cloned and expressed in recombinant Escherichia coli. A fusion of myeloma cells (Sp2/0-Ag14) and spleen cells from the immunized mice, and hybridoma cells were cultured to obtain anti-AFB1 monoclonal antibody. The antibody was found to be very specific to AFB1 with 1.3 ng/ml IC50 and 0.1 ng/ml detection limit. The complementary DNA (cDNA) for the anti-AFB1 antibody by reverse transcription-polymerase chin reaction (RT-PCR). Amino acid sequence analysis of the cDNA identified that the variable region was composed of heavy chain (VH) as a type of IgG1 and light chain (VL) as a type of λ. Overlap-extension PCR (OE-PCR) using flexible DNA linker encoding polypeptide (Gly4Ser)3 led to combination of VH and VL genes and their stable expression in recombinant E. coli. Co-expression of molecular chaperones or ubiquitin fusion system increased an expression level of anti AFB1 scFv. In vitro refolding of anti AFB1 scFv inclusion bodies was optimized by changing environmental condition of refolding and its biological activity was determined by competitive direct enzyme linked immunosorbent assay (cdELISA). The optimized condition was obtained when using 50 mM tris, 10 mM GSH, 1 mM GSSG and 0.02% tween 20.Maste