ISHS Acta Horticulturae 472: XVII International Symposium Virus and Virus-Like Diseases of Temperate Fruit Crops
CHARACTERIZATION OF PLUM POX VIRUS COAT PROTEIN EPITOPES USING FUSION PROTEINS EXPRESSED IN E. COLI |
| Authors: | T. Candresse, F. Rafia, J. Dunez, M. Navratil, D. Boscia, M. Cambra, M. Asensio, J.A. García, G. Pasquini, M. Barba |
| Keywords: | sharka, monoclonal antibodies, western blot, serotype, infectious clones, mutations |
| Abstract:
In order to gain an insight into the nature and identify of the epitopes recognized on PPV coat protein (CP) by monoclonal antibodies, fragments of the PPV CP were expressed as fusion proteins in E. coli. A PCR fragment cloning for the 43 C-terminal amino acids of the NIb and the N-terminal part of the CP (complete variable region plus 19 amino acids of the conserved core) was cloned into the pMal-c2 expression vector. Upon induction, the viral protein was expressed as a C-terminal fusion to the E. coli maltose binding protein (MBP). A series of truncated proteins, containing increasing deletions from the C-terminus of the viral CP were also expressed. These fusion proteins were confronted to 24 monoclonal antibodies directed against PPV, using western blot analysis. All but four of the Mabs tested reacted with the large fusion protein, demonstrating that the vast majority of the epitopes recognized on PPV CP are located in the expressed region. Comparison of the reactivity of the various Mabs towards the series of truncated proteins allowed the delineation of a minimum number of nine epitopes. Sequence comparison between PPV variants showing differential reactivity towards some of the Mabs confirmed the location of the cognate epitopes. These results provide for the first time an epitope map of plum pox virus. | |
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