Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B

Detalhes bibliográficos
Autor(a) principal: Radaelli, Paula
Data de Publicação: 2008
Outros Autores: Fajardo, Thor Vinícius Martins, Nickel, Osmar, Eiras, Marcelo, Pio-Ribeiro, Gilvan
Tipo de documento: Artigo
Idioma: por
Título da fonte: Pesquisa Agropecuária Brasileira (Online)
Texto Completo: https://seer.sct.embrapa.br/index.php/pab/article/view/1039
Resumo: The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.
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spelling Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus BProdução de anti‑soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll‑associated virus 2 e Grapevine virus BVitis; GLRaV‑2; GVB; indirect ELISA; recombinant protein; Western blotVitis; GLRaV‑2; GVB; ELISA indireto; proteína recombinante; Western blotThe objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.O objetivo deste trabalho foi produzir e caracterizar anti-soros específicos contra isolados brasileiros do Vírus do enrolamento-da-folha da videira 2 (GLRaV-2) e do Vírus B da videira (GVB), desenvolvidos a partir das proteínas capsidiais expressas em Escherichia coli, e testar seu possível uso para a detecção destes dois vírus em videiras infectadas. Os genes da proteína capsidial (CP) foram amplificados via RT-PCR, clonados e seqüenciados. Foram, subseqüentemente, subclonados, e os plasmídeos recombinantes foram empregados na transformação das células de E. coli e na expressão das proteínas capsidiais. As proteínas capsidiais recombinantes foram purificadas, e suas identidades foram confirmadas em SDS-PAGE e "Western blot" e utilizadas para imunizar coelhos. Os anti-soros produzidos contra essas proteínas foram capazes de reconhecer as proteínas recombinantes correspondentes em "Western blot", de detectar GLRaV-2 e GVB em tecidos infectados de videiras pelo ELISA indireto, e de discriminar videiras sadias e infectadas, com absorbâncias (A405) de 0,08/1,15 e 0,12/1,30, respectivamente. A expressão dos genes CP pode produzir grandes quantidades de proteínas virais, com elevada antigenicidade, e os anti-soros de GLRaV-2 e GVB obtidos neste trabalho possibilitam a diagnose confiável desses vírus.Pesquisa Agropecuaria BrasileiraPesquisa Agropecuária BrasileiraCNPqRadaelli, PaulaFajardo, Thor Vinícius MartinsNickel, OsmarEiras, MarceloPio-Ribeiro, Gilvan2008-10-31info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://seer.sct.embrapa.br/index.php/pab/article/view/1039Pesquisa Agropecuaria Brasileira; v.43, n.10, out. 2008; 1405-1411Pesquisa Agropecuária Brasileira; v.43, n.10, out. 2008; 1405-14111678-39210100-104xreponame:Pesquisa Agropecuária Brasileira (Online)instname:Empresa Brasileira de Pesquisa Agropecuária (Embrapa)instacron:EMBRAPAporhttps://seer.sct.embrapa.br/index.php/pab/article/view/1039/5530https://seer.sct.embrapa.br/index.php/pab/article/downloadSuppFile/1039/680info:eu-repo/semantics/openAccess2012-06-17T11:44:06Zoai:ojs.seer.sct.embrapa.br:article/1039Revistahttp://seer.sct.embrapa.br/index.php/pabPRIhttps://old.scielo.br/oai/scielo-oai.phppab@sct.embrapa.br || sct.pab@embrapa.br1678-39210100-204Xopendoar:2012-06-17T11:44:06Pesquisa Agropecuária Brasileira (Online) - Empresa Brasileira de Pesquisa Agropecuária (Embrapa)false
dc.title.none.fl_str_mv Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
Produção de anti‑soros policlonais a partir de proteínas capsidiais recombinantes de Grapevine leafroll‑associated virus 2 e Grapevine virus B
title Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
spellingShingle Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
Radaelli, Paula
Vitis; GLRaV‑2; GVB; indirect ELISA; recombinant protein; Western blot
Vitis; GLRaV‑2; GVB; ELISA indireto; proteína recombinante; Western blot
title_short Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
title_full Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
title_fullStr Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
title_full_unstemmed Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
title_sort Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll‑associated virus 2 and Grapevine virus B
author Radaelli, Paula
author_facet Radaelli, Paula
Fajardo, Thor Vinícius Martins
Nickel, Osmar
Eiras, Marcelo
Pio-Ribeiro, Gilvan
author_role author
author2 Fajardo, Thor Vinícius Martins
Nickel, Osmar
Eiras, Marcelo
Pio-Ribeiro, Gilvan
author2_role author
author
author
author
dc.contributor.none.fl_str_mv
CNPq
dc.contributor.author.fl_str_mv Radaelli, Paula
Fajardo, Thor Vinícius Martins
Nickel, Osmar
Eiras, Marcelo
Pio-Ribeiro, Gilvan
dc.subject.por.fl_str_mv Vitis; GLRaV‑2; GVB; indirect ELISA; recombinant protein; Western blot
Vitis; GLRaV‑2; GVB; ELISA indireto; proteína recombinante; Western blot
topic Vitis; GLRaV‑2; GVB; indirect ELISA; recombinant protein; Western blot
Vitis; GLRaV‑2; GVB; ELISA indireto; proteína recombinante; Western blot
description The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.
publishDate 2008
dc.date.none.fl_str_mv 2008-10-31
dc.type.none.fl_str_mv
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://seer.sct.embrapa.br/index.php/pab/article/view/1039
url https://seer.sct.embrapa.br/index.php/pab/article/view/1039
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv https://seer.sct.embrapa.br/index.php/pab/article/view/1039/5530
https://seer.sct.embrapa.br/index.php/pab/article/downloadSuppFile/1039/680
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Pesquisa Agropecuaria Brasileira
Pesquisa Agropecuária Brasileira
publisher.none.fl_str_mv Pesquisa Agropecuaria Brasileira
Pesquisa Agropecuária Brasileira
dc.source.none.fl_str_mv Pesquisa Agropecuaria Brasileira; v.43, n.10, out. 2008; 1405-1411
Pesquisa Agropecuária Brasileira; v.43, n.10, out. 2008; 1405-1411
1678-3921
0100-104x
reponame:Pesquisa Agropecuária Brasileira (Online)
instname:Empresa Brasileira de Pesquisa Agropecuária (Embrapa)
instacron:EMBRAPA
instname_str Empresa Brasileira de Pesquisa Agropecuária (Embrapa)
instacron_str EMBRAPA
institution EMBRAPA
reponame_str Pesquisa Agropecuária Brasileira (Online)
collection Pesquisa Agropecuária Brasileira (Online)
repository.name.fl_str_mv Pesquisa Agropecuária Brasileira (Online) - Empresa Brasileira de Pesquisa Agropecuária (Embrapa)
repository.mail.fl_str_mv pab@sct.embrapa.br || sct.pab@embrapa.br
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