A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification
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| Autor principal: | |
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| Data de publicació: | 2013 |
| Altres autors: | , , , , , |
| Format: | Article |
| Idioma: | eng |
| Font: | Repositório Institucional da UnB |
| Download full: | http://repositorio.unb.br/handle/10482/23836 https://dx.doi.org/10.1186/1743-422X-10-262 |
Sumari: | Background: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts. |
| _version_ | 1871442488644861952 |
|---|---|
| author | Araújo, Daniel Mendes Pereira Ardisson de |
| author2 | Rocha, Juliana Ribeiro Costa, Márcio Hedil Oliveira da Bocca, Anamélia Lorenzetti Dusi, André Nepomuceno Resende, Renato de Oliveira Ribeiro, Bergmann Morais |
| author2_role | author author author author author author |
| author_browse | Araújo, Daniel Mendes Pereira Ardisson de Bocca, Anamélia Lorenzetti Costa, Márcio Hedil Oliveira da Dusi, André Nepomuceno Resende, Renato de Oliveira Ribeiro, Bergmann Morais Rocha, Juliana Ribeiro |
| author_facet | Araújo, Daniel Mendes Pereira Ardisson de Rocha, Juliana Ribeiro Costa, Márcio Hedil Oliveira da Bocca, Anamélia Lorenzetti Dusi, André Nepomuceno Resende, Renato de Oliveira Ribeiro, Bergmann Morais |
| author_role | author |
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| bitstream.url.fl_str_mv | http://repositorio2.unb.br/jspui/bitstream/10482/23836/1/ARTIGO_%20BaculovirusMediatedStrategy.pdf http://repositorio2.unb.br/jspui/bitstream/10482/23836/2/license.txt |
| collection | Repositório Institucional da UnB |
| dc.contributor.author.fl_str_mv | Araújo, Daniel Mendes Pereira Ardisson de Rocha, Juliana Ribeiro Costa, Márcio Hedil Oliveira da Bocca, Anamélia Lorenzetti Dusi, André Nepomuceno Resende, Renato de Oliveira Ribeiro, Bergmann Morais |
| dc.date.accessioned.fl_str_mv | 2017-07-17T12:35:28Z |
| dc.date.available.fl_str_mv | 2017-07-17T12:35:28Z |
| dc.date.issued.fl_str_mv | 2013-08-15 |
| dc.identifier.citation.fl_str_mv | ARAÚJO, Daniel Mendes Pereira Ardisson de et al. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification. Virology Journal, v. 10, Article 262, 15 ago. 2013. Disponível em: <https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262>. Acesso em: 9 jun. 2017. doi: https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262 |
| dc.identifier.doi.pt_BR.fl_str_mv | https://dx.doi.org/10.1186/1743-422X-10-262 |
| dc.identifier.uri.fl_str_mv | http://repositorio.unb.br/handle/10482/23836 |
| dc.language.iso.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | BioMed Central |
| dc.rights.driver.fl_str_mv | info:eu-repo/semantics/openAccess |
| dc.source.none.fl_str_mv | reponame:Repositório Institucional da UnB instname:Universidade de Brasília (UnB) instacron:UNB |
| dc.subject.keyword.pt_BR.fl_str_mv | Alho - doenças e pragas Vírus Proteínas |
| dc.title.pt_BR.fl_str_mv | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| dc.type.driver.fl_str_mv | info:eu-repo/semantics/article |
| dc.type.status.fl_str_mv | info:eu-repo/semantics/publishedVersion |
| description | Background: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | UNB_c4e800cf6331d12972e5092db8971ec1 |
| identifier_str_mv | ARAÚJO, Daniel Mendes Pereira Ardisson de et al. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification. Virology Journal, v. 10, Article 262, 15 ago. 2013. Disponível em: <https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262>. Acesso em: 9 jun. 2017. doi: https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262 |
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| institution | UNB |
| instname_str | Universidade de Brasília (UnB) |
| language | eng |
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| network_name_str | Repositório Institucional da UnB |
| oai_identifier_str | oai:repositorio.unb.br:10482/23836 |
| publishDate | 2013 |
| publishDateSort | 2013 |
| publisher.none.fl_str_mv | BioMed Central |
| reponame_str | Repositório Institucional da UnB |
| repository.mail.fl_str_mv | repositorio@unb.br |
| repository.name.fl_str_mv | Repositório Institucional da UnB - Universidade de Brasília (UnB) |
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| spelling | Araújo, Daniel Mendes Pereira Ardisson deRocha, Juliana RibeiroCosta, Márcio Hedil Oliveira daBocca, Anamélia LorenzettiDusi, André NepomucenoResende, Renato de OliveiraRibeiro, Bergmann Morais2017-07-17T12:35:28Z2017-07-17T12:35:28Z2013-08-15ARAÚJO, Daniel Mendes Pereira Ardisson de et al. A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification. Virology Journal, v. 10, Article 262, 15 ago. 2013. Disponível em: <https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262>. Acesso em: 9 jun. 2017. doi: https://virologyj.biomedcentral.com/articles/10.1186/1743-422X-10-262http://repositorio.unb.br/handle/10482/23836https://dx.doi.org/10.1186/1743-422X-10-262BioMed Central© 2013 Ardisson-Araújo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.info:eu-repo/semantics/openAccessA baculovirus-mediated strategy for full-length plant virus coat protein expression and purificationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleAlho - doenças e pragasVírusProteínasBackground: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. Results: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3′-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. Conclusions: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.engreponame:Repositório Institucional da UnBinstname:Universidade de Brasília (UnB)instacron:UNBORIGINALARTIGO_ BaculovirusMediatedStrategy.pdfARTIGO_ BaculovirusMediatedStrategy.pdfapplication/pdf1282316http://repositorio2.unb.br/jspui/bitstream/10482/23836/1/ARTIGO_%20BaculovirusMediatedStrategy.pdfa03b269e8b6a3b8df10bfc9cd399d5dbMD51open accessLICENSElicense.txtlicense.txttext/plain160http://repositorio2.unb.br/jspui/bitstream/10482/23836/2/license.txtcd7f2de909e07c008880b5201378c22eMD52open access10482/238362023-05-24 20:34:36.798open accessoai:repositorio.unb.br:10482/23836U3VibWlzcz9vIGVmZXRpdmFkYSBwb3IgaW50ZWdyYW50ZSBkYSBlcXVpcGUgZG8gUmVwb3NpdD9yaW8gSW5zdGl0dWNpb25hbCBkYSBVbkIgZGUgYWNvcmRvIGNvbSBsaWNlbj9hIGNvbmNlZGlkYSBwZWxvIGF1dG9yIGUvb3UgZGV0ZW50b3IgZG9zIGRpcmVpdG9zIGF1dG9yYWlzLg==Repositório InstitucionalPUBhttps://repositorio.unb.br/oai/requestrepositorio@unb.bropendoar:2023-05-24T23:34:36Repositório Institucional da UnB - Universidade de Brasília (UnB) |
| spellingShingle | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification Araújo, Daniel Mendes Pereira Ardisson de Alho - doenças e pragas Vírus Proteínas |
| status_str | publishedVersion |
| title | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| title_full | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| title_fullStr | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| title_full_unstemmed | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| title_short | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| title_sort | A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification |
| topic | Alho - doenças e pragas Vírus Proteínas |
| url | http://repositorio.unb.br/handle/10482/23836 https://dx.doi.org/10.1186/1743-422X-10-262 |