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

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서울대학교 대학원
아플라톡신 B1Aflatoxin b1scFvScfv간접경합방식의 효소면역측정법Hybridoma하이브리도마Chaperoneoverlap-extension 중합효소연쇄반응Ubiquitin재접힘Inclusion body내포체Refolding샤페론Competitive direct enzyme linked immunosorbent aasay유비퀴틴Overlap-extension pcr
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.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Theses (Master's Degree_식품·동물생명공학부)
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