Isolation, expression and characterization of two single-chain variable fragment antibodies against an endo-polygalacturonase secreted by Sclerotinia sclerotiorum.
Canola is a very important economic crop in the world and canola stem rot caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive and non-host-specific fungus, can reduce yield significantly. This fungus secretes numerous cell wall degrading enzymes including an endo-polygalacturonase, SSPG1d, which has been detected at early stages of infection. In this report we describe the isolation of two recombinant antibodies of the single-chain variable fragment (ScFv) format from RNA of mice immunized with recombinant SSPG1d (rSSPG1d) or a peptide derived from SSPG1d (peptide 3796) that was predicted to be antigenic. The ScFvs were isolated using the established phage display technology. These recombinant antibodies were expressed, purified and refolded to functional antibodies with a yield of 120-500mug per liter of cell culture. Recombinant antibodies were characterized using various techniques including enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Of the two ScFvs, it appears that only ScFv-rSSPG1d is able to detect whole SSPG1d produced by the fungus. Thus our results indicate that this ScFv may have utility in the detection of the SSPG1d enzyme in an antibody-based diagnostic test for S. sclerotiorum infection.
Yang
Bo
B
Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G2P5, Canada.
Yajima
William
W
Das
Dipankar
D
Suresh
Mavanur R
MR
Kav
Nat N V
NN
D000818:Animals; D000908:Antibodies, Fungal / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D001203:Ascomycota / Q000201:enzymology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D019151:Peptide Library; D010935:Plant Diseases / Q000382:microbiology; D011096:Polygalacturonase / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D020349:Surface Plasmon Resonance
D000908:Antibodies, Fungal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D011096:Polygalacturonase