Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom
Autor(a) principal: | |
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Data de Publicação: | 2017 |
Outros Autores: | , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.3390/toxins9090259 http://hdl.handle.net/11449/175114 |
Resumo: | Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy. |
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Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venomAllergyAntigen 5DiagnosisHeterologous expressionPolybia paulistaPolybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaLaboratório de Imunologia Translacional-LIT Departamento de Clínica Médica Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n◦ 126, Cidade Universitária “Zeferino Vaz”System Biology Department Biomedical Research Division Center for Genetic Engineering and Biotechnology, Ave. 31, e/158 and 190, P.O. Box 6162, CubanacanCentro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental LageadoLaboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela VistaCentro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental LageadoFAPESP: 2013/26451-9FAPESP: 2014/13936-7CNPq: 455422/2014-1Universidade Estadual Paulista (Unesp)Universidade Estadual de Campinas (UNICAMP)Center for Genetic Engineering and BiotechnologyBazon, Murilo Luiz [UNESP]Perez-Riverol, Amilcar [UNESP]Dos Santos-Pinto, José Roberto Aparecido [UNESP]Fernandes, Luis Gustavo RomaniLasa, Alexis MusacchioJusto-Jacomini, Débora Laís [UNESP]Palma, Mario Sergio [UNESP]De Lima Zollner, RicardoBrochetto-Braga, Márcia Regina [UNESP]2018-12-11T17:14:26Z2018-12-11T17:14:26Z2017-09-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.3390/toxins9090259Toxins, v. 9, n. 9, 2017.2072-6651http://hdl.handle.net/11449/17511410.3390/toxins90902592-s2.0-850285917462-s2.0-85028591746.pdf2901888624506535Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengToxins0,955info:eu-repo/semantics/openAccess2023-11-22T06:15:10Zoai:repositorio.unesp.br:11449/175114Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T18:25:36.212183Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
title |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
spellingShingle |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom Bazon, Murilo Luiz [UNESP] Allergy Antigen 5 Diagnosis Heterologous expression Polybia paulista |
title_short |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
title_full |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
title_fullStr |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
title_full_unstemmed |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
title_sort |
Heterologous expression, purification and immunoreactivity of the antigen 5 from polybia paulista wasp venom |
author |
Bazon, Murilo Luiz [UNESP] |
author_facet |
Bazon, Murilo Luiz [UNESP] Perez-Riverol, Amilcar [UNESP] Dos Santos-Pinto, José Roberto Aparecido [UNESP] Fernandes, Luis Gustavo Romani Lasa, Alexis Musacchio Justo-Jacomini, Débora Laís [UNESP] Palma, Mario Sergio [UNESP] De Lima Zollner, Ricardo Brochetto-Braga, Márcia Regina [UNESP] |
author_role |
author |
author2 |
Perez-Riverol, Amilcar [UNESP] Dos Santos-Pinto, José Roberto Aparecido [UNESP] Fernandes, Luis Gustavo Romani Lasa, Alexis Musacchio Justo-Jacomini, Débora Laís [UNESP] Palma, Mario Sergio [UNESP] De Lima Zollner, Ricardo Brochetto-Braga, Márcia Regina [UNESP] |
author2_role |
author author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade Estadual de Campinas (UNICAMP) Center for Genetic Engineering and Biotechnology |
dc.contributor.author.fl_str_mv |
Bazon, Murilo Luiz [UNESP] Perez-Riverol, Amilcar [UNESP] Dos Santos-Pinto, José Roberto Aparecido [UNESP] Fernandes, Luis Gustavo Romani Lasa, Alexis Musacchio Justo-Jacomini, Débora Laís [UNESP] Palma, Mario Sergio [UNESP] De Lima Zollner, Ricardo Brochetto-Braga, Márcia Regina [UNESP] |
dc.subject.por.fl_str_mv |
Allergy Antigen 5 Diagnosis Heterologous expression Polybia paulista |
topic |
Allergy Antigen 5 Diagnosis Heterologous expression Polybia paulista |
description |
Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy. |
publishDate |
2017 |
dc.date.none.fl_str_mv |
2017-09-01 2018-12-11T17:14:26Z 2018-12-11T17:14:26Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.3390/toxins9090259 Toxins, v. 9, n. 9, 2017. 2072-6651 http://hdl.handle.net/11449/175114 10.3390/toxins9090259 2-s2.0-85028591746 2-s2.0-85028591746.pdf 2901888624506535 |
url |
http://dx.doi.org/10.3390/toxins9090259 http://hdl.handle.net/11449/175114 |
identifier_str_mv |
Toxins, v. 9, n. 9, 2017. 2072-6651 10.3390/toxins9090259 2-s2.0-85028591746 2-s2.0-85028591746.pdf 2901888624506535 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
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Toxins 0,955 |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
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UNESP |
institution |
UNESP |
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Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1808128931294871552 |