Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida.
To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used. DFR was overproduced in Escherichia coli, purified and used for immunization. From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification. Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing. They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot. These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run.
De Jaeger
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Departement Genetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, Belgium.
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Van Montagu
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Fischer
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D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase