Staff View: Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycop...

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Main Authors:	Dambros, Régia Maria Feltrin, Ribeiro, Bergman Moraes, Aguiar, Raimundo Wagner de Souza, Schaefer, Rejane, Esteves, Paulo Augusto, Perecmanis, Simone, Simon, Neide Lisiane, Silva, Nayara Cavalcante, Coldebella, Michele, Ciacci-Zanella, Janice Reis
Format:	Artigo
Language:	English
Published:	Sociedade Brasileira de Microbiologia 2017
Subjects:	Aujeszky Glycoprotein E Baculovírus Recombinant Glicoproteína E Recombinante
Online Access:	http://repositorio.unb.br/handle/10482/27099
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spelling	ir-10482-270992019-03-28T14:08:23Z Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system Clonagem e expressão da glicoproteina E (gE) do vírus da doença de Aujeszky em sistema de baculovirus Dambros, Régia Maria Feltrin Ribeiro, Bergman Moraes Aguiar, Raimundo Wagner de Souza Schaefer, Rejane Esteves, Paulo Augusto Perecmanis, Simone Simon, Neide Lisiane Silva, Nayara Cavalcante Coldebella, Michele Ciacci-Zanella, Janice Reis Aujeszky Glycoprotein E Baculovírus Recombinant Aujeszky Glicoproteína E Baculovírus Recombinante Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively. A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA), um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE) é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da amplificação foi clonado no vetor pGem®-T Easy e subclonado no plasmídeo de expressão pFastBac™1. O DNA recombinante pFastBac-gE-VDA foi usado para a transposição sítio-específica no baculovírus recombinante (bacmid). Após seleção por antibióticos e cor, as colônias com o recombinante bacmid-pFastBac-gE-VDA foram selecionadas e a presença do gene da gE foi confirmada por PCR. O DNA recombinante viral, bacmid-pFastBac-gE-VDA, foi usado para cotransfecção de células de inseto Trichoplusia ni e a presença do recombinante e a proteína gE foi determinada por PCR, por SDS-PAGE e Western blotting, respectivamente. 2017-12-07T04:48:34Z 2017-12-07T04:48:34Z 2007 Artigo Braz. J. Microbiol.,v.38,n.3,p.494-499,2007 1517-8382 http://repositorio.unb.br/handle/10482/27099 10.1590/S1517-83822007000300021 en Acesso Aberto application/pdf Sociedade Brasileira de Microbiologia http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822007000300021&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_abstract&pid=S1517-83822007000300021&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_pdf&pid=S1517-83822007000300021&lng=en&nrm=iso
institution	REPOSITORIO UNB
collection	REPOSITORIO UNB
language	English
topic	Aujeszky Glycoprotein E Baculovírus Recombinant Aujeszky Glicoproteína E Baculovírus Recombinante
spellingShingle	Aujeszky Glycoprotein E Baculovírus Recombinant Aujeszky Glicoproteína E Baculovírus Recombinante Dambros, Régia Maria Feltrin Ribeiro, Bergman Moraes Aguiar, Raimundo Wagner de Souza Schaefer, Rejane Esteves, Paulo Augusto Perecmanis, Simone Simon, Neide Lisiane Silva, Nayara Cavalcante Coldebella, Michele Ciacci-Zanella, Janice Reis Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
description	Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.
format	Artigo
author	Dambros, Régia Maria Feltrin Ribeiro, Bergman Moraes Aguiar, Raimundo Wagner de Souza Schaefer, Rejane Esteves, Paulo Augusto Perecmanis, Simone Simon, Neide Lisiane Silva, Nayara Cavalcante Coldebella, Michele Ciacci-Zanella, Janice Reis
author_sort	Dambros, Régia Maria Feltrin
title	Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
title_short	Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
title_full	Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
title_fullStr	Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
title_full_unstemmed	Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system
title_sort	cloning and expression of aujeszky's disease virus glycoprotein e (ge) in a baculovirus system
publisher	Sociedade Brasileira de Microbiologia
publishDate	2017
url	http://repositorio.unb.br/handle/10482/27099
_version_	1641988400599793664
score	13.657419

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

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