Production and use of polyclonal antibodies to the coat protein of apple stem grooving virus expressed in Escherichia coli

Production and use of polyclonal antibodies to the coat protein of apple stem grooving virus expressed in Escherichia coli

The coat protein (cp) gene of Apple stem grooving Capillovirus was amplified by RT-PCR, cloned, sequenced and subcloned in the expression plasmid pMal-c2. The ASGV cp gene was expressed in E. coli as a fusion protein (fp) containing a fragment of E. coli maltose binding protein (MBP). Bacterial cell...

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Bibliographic Details
Published in:Acta horticulturae no. 657; pp. 35 - 40
Main Authors: Nickel, O, Fajardo, T.V, Trivilin, A.P, Targon, M.L.N.P, Machado, M.A
Format: Journal Article
Language:English
Published: International Society for Horticultural Science 01-01-2004
Subjects:
affinity chromatography
amylose
antiserum
Apple stem grooving virus
apples
binding proteins
coat proteins
cultivars
enzyme-linked immunosorbent assay
Escherichia coli
genes
immunoblotting
maltose
plasmids
polyclonal antibodies
rabbits
reverse transcriptase polymerase chain reaction
virion
Brazil
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Summary:The coat protein (cp) gene of Apple stem grooving Capillovirus was amplified by RT-PCR, cloned, sequenced and subcloned in the expression plasmid pMal-c2. The ASGV cp gene was expressed in E. coli as a fusion protein (fp) containing a fragment of E. coli maltose binding protein (MBP). Bacterial cells were disrupted by sonication and the ASGVcp/MBP-fp was purified by amylose resin affinity chromatography. Polyclonal antibodies from rabbits immunized with the fp, gave specific reactions to ASGV from several infected apple cultivars at dilutions of up to 1:2000 in indirect ELISA. The ASGVcp/MBP-fp reacted to antisera raised against it as well as to commercial antisera against ASGV virions in immunoblotting. These results are a fundamental contribution to the development of an efficient ASGV-indexing of propagative and mother stock materials in Southern Brazil.
Bibliography:http://www.actahort.org/books/657/657_1.htm
ISSN:0567-7572
2406-6168
DOI:10.17660/ActaHortic.2004.657.1