Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies


Autoria(s): Fajardo,Thor V.M.; Barros,Danielle R.; Nickel,Osmar; Kuhn,Gilmar B.; Zerbini,F. Murilo
Data(s)

01/12/2007

Resumo

Grapevine leafroll-associated virus 3 (GLRaV-3), the main viral species of the grapevine leafroll complex, causes yield and quality reduction in grapes (Vitis spp.). The coat protein gene was RT-PCR-amplified from total RNA extracted from infected grapevine leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pRSET-C expression vector. The recombinant plasmid was used to transform Escherichia coli BL21:DE3 and express the capsid protein. The coat protein, fused to a 6 His-tag, was purified by affinity chromatography using an Ni-NTA resin. The identity of the purified protein was confirmed by SDS-PAGE and Western blot. The in vitro-expressed protein was quantified and used for rabbit immunizations. The antiserum was shown to be sensitive and specific for the detection of GLRaV-3 in grapevine extracts in Western blot and DAS-ELISA assays, with no unspecific or heterologous reactions against other non-serologically related viruses being observed.

Formato

text/html

Identificador

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-41582007000600007

Idioma(s)

en

Publicador

Sociedade Brasileira de Fitopatologia

Fonte

Fitopatologia Brasileira v.32 n.6 2007

Palavras-Chave #grapevine #GLRaV-3 #recombinant protein #serology #antibodies
Tipo

journal article