California Department of Food and Agriculture Pierce ’ s Disease
نویسندگان
چکیده
The purpose of this project was to identify peptides that can bind and inactivate Xylella fastidiosa (Xf) polygalturonase (PG), an essential virulence factor that Xf needs to cause PD. With the year of funding we received for this project we have completed parts of objectives one and two. For objective one we were able to obtain a small amount of active recombinant Xf PG that we were able to detect using reducing sugar assays. Since most of the protein that we were able to produce was in the form of insoluble inactive inclusion bodies, we also tried a number of different expression constructs to increase the amount of active Xf PG we could generate. While we were working on increasing the amount of active Xf PG we could produce we went ahead with objective two. We synthesized two 14mer peptides derived from the Xf PG sequence, one which targeted the active site directly and a second that targeted an area providing entry into the active site. Polyclonal antibodies raised against each of these peptides were used in a western blot analysis that confirmed that the antibodies created against each 14-mer peptide could also recognize full length recombinant Xf PG. Peptide 2 was immobilized on a polystyrene surface and used as a “target” in a phage panning experiment using a scFv antibody library attached to the pIII protein of M13 phage. At the end of the third round of selection a polyclonal ELISA with BSA conjugated peptide 2 as the target was run which showed that each library (I and J) of scFvs showed a higher binding affinity to BSA conjugated peptide 2 than to BSA alone, or to the wells of the plate. We then sequenced the heavy chain variable portion of the scFvs giving the highest absorbance reading and although none of the eight clones selected from each of libraries shared the same sequence they did have similarities to each other.
منابع مشابه
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Abbreviations CDC US Centers for Disease Control FDA US Food and Drug Administration FR Federal Register FSMA Food Safety Modernization Act GAPs Good Agricultural Practices GMPs Good manufacturing practices HACCP Hazard Analysis and Critical Control Points HARPC Hazard Analysis and Risk-based Preventive Controls LGMA The California Leafy Greens Marketing Agreement USDA US Department of Agriculture
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