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A Monoclonal Antibody That Specifically Binds Chitosan In Vitro and In Situ on Fungal Cell Walls
( Schubert Max ),( Siham Agdour ),( Rainer Fischer ),( Yvonne Olbrich ),( Helga Schinkel ),( Stefan Schillberg ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.8
We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders, resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody could bind to chitosans of varying composition, but demonstrated the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.
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Janina Kirchhoff,Andreas Schiermeyer,Katja Schneider,Rainer Fischer,W. Michael Ainley,Steven R. Webb,Helga Schinkel,Stefan Schillberg 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.4
Genome editing tools such as zinc-finger nucleases provide novel strategies for genetic manipulation in plants. Unlike agrobacterium- mediated or direct gene transfer, which introduce genes randomly into the genome and thereby potentially resulting in high variation of gene expression, the targeted gene addition provides predictable integration of DNA sequences into a specified location of the plant genome. We investigated whether various independent cell lines that all contain a transgene placed in the same genomic locus by zinc-finger nuclease-mediated homologous recombination (HR) would yield a more reproducible and homogeneous level of expression compared to integration events generated via agrobacterium-mediated transformation at random sites. The variance of gene expression of targeted HR events and random integration events was analyzed in Nicotiana tabacum L cv. Bright Yellow 2 (BY-2) suspension cells by measuring protein amount produced from the transgene by flow cytometry, thus providing the first report on positional effects of marker gene expression in a quickly proliferating plant suspension cell line. Marker protein levels of targeted HR and single-copy random events covered a similar range; however, the uniformity of protein expression in a given cell line was significantly higher in targeted events than in lines with randomly inserted transgene; the same is true for the overall viability of protoplasts from HR lines. In conclusion, using targeted insertion into a qualified locus of a well-characterized line leads to more reliable results than random insertion into the genome.
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