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Production of a polyclonal antiserum against the coat protein of Cucurbit yellow stunting disorder crinivirus expressed in Escherichia coli
Author(s) -
Cotillon A. C.,
Desbiez C.,
Bouyer S.,
WipfScheibel C.,
Gros C.,
Delécolle B.,
Lecoq H.
Publication year - 2005
Publication title -
eppo bulletin
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.327
H-Index - 36
eISSN - 1365-2338
pISSN - 0250-8052
DOI - 10.1111/j.1365-2338.2005.00804.x
Subject(s) - antiserum , polyclonal antibodies , escherichia coli , fusion protein , coat protein , biology , recombinant dna , virology , antigen , gene , biochemistry , rna , genetics
Cucurbit yellow stunting disorder crinivirus infects cucurbit crops in the Middle East and the Mediterranean area (Egypt, Morocco, Spain, Portugal, South of France since 2001). Transmitted by Bemisia tabaci in a semipersistent manner, this virus provokes interveinal yellowing on older leaves of cucurbits, and is reported to cause significant economic losses in cucumber crops in Lebanon. Since it is very difficult to purify CYSDV, the main technique used for detection is RT‐PCR, which is not appropriate for routine tests. Therefore, the coat protein gene of a French isolate of CYSDV was amplified by RT‐PCR, cloned into bacterial expression vector pET16b allowing fusion of the protein with a N‐terminal 10xHis‐tag for easier purification, and transformed into Escherichia coli , strain BL21(DE3). The recombinant coat protein was produced at a high level, purified from cell lysates on a Ni‐NTA resin column, and used as an antigen to immunize a rabbit (three intradermal then six subcutaneous injections every 3 or 4 weeks). The resulting antiserum can be successfully used in DAS‐ELISA tests.