Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Detalhes bibliográficos
Autor(a) principal: Kurtenbach, Eleonora
Data de Publicação: 2006
Outros Autores: Favacho, Alexsandra R. M., Sardi, Silvia Inês, Gouvea, Vera S.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UFBA
Texto Completo: http://repositorio.ufba.br/ri/handle/ri/13803
Resumo: Texto completo: acesso restrito. p. 196–203
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spelling Kurtenbach, EleonoraFavacho, Alexsandra R. M.Sardi, Silvia InêsGouvea, Vera S.Kurtenbach, EleonoraFavacho, Alexsandra R. M.Sardi, Silvia InêsGouvea, Vera S.2013-11-19T20:49:41Z20061046-5928http://repositorio.ufba.br/ri/handle/ri/13803v. 46, n. 2Texto completo: acesso restrito. p. 196–203Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure–function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-08-27T15:03:28Z No. of bitstreams: 1 1-s2.0-S1046592805003359-main.pdf: 1116869 bytes, checksum: 9868a3eaec7c83a1a863781bd5c20e9f (MD5)Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-19T20:49:41Z (GMT) No. of bitstreams: 1 1-s2.0-S1046592805003359-main.pdf: 1116869 bytes, checksum: 9868a3eaec7c83a1a863781bd5c20e9f (MD5)Made available in DSpace on 2013-11-19T20:49:41Z (GMT). No. of bitstreams: 1 1-s2.0-S1046592805003359-main.pdf: 1116869 bytes, checksum: 9868a3eaec7c83a1a863781bd5c20e9f (MD5) Previous issue date: 2006http://dx.doi.org.ez10.periodicos.capes.gov.br/10.1016/j.pep.2005.09.014reponame:Repositório Institucional da UFBAinstname:Universidade Federal da Bahia (UFBA)instacron:UFBARotavirus spikeVP8* subunitExpression in Escherichia coliHis-Tag purificationCloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coliProtein Expression and Purificationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article10000-01-01info:eu-repo/semantics/openAccessengORIGINAL1-s2.0-S1046592805003359-main.pdf1-s2.0-S1046592805003359-main.pdfapplication/pdf1116869https://repositorio.ufba.br/bitstream/ri/13803/1/1-s2.0-S1046592805003359-main.pdf9868a3eaec7c83a1a863781bd5c20e9fMD51LICENSElicense.txtlicense.txttext/plain1762https://repositorio.ufba.br/bitstream/ri/13803/2/license.txt1b89a9a0548218172d7c829f87a0eab9MD52TEXT1-s2.0-S1046592805003359-main.pdf.txt1-s2.0-S1046592805003359-main.pdf.txtExtracted texttext/plain35775https://repositorio.ufba.br/bitstream/ri/13803/3/1-s2.0-S1046592805003359-main.pdf.txtd4a984e0ede7deaf2b5462d10f1891a8MD53ri/138032022-07-05 14:03:34.582oai:repositorio.ufba.br:ri/13803VGVybW8gZGUgTGljZW7vv71hLCBu77+9byBleGNsdXNpdm8sIHBhcmEgbyBkZXDvv71zaXRvIG5vIHJlcG9zaXTvv71yaW8gSW5zdGl0dWNpb25hbCBkYSBVRkJBCgogICAgUGVsbyBwcm9jZXNzbyBkZSBzdWJtaXNz77+9byBkZSBkb2N1bWVudG9zLCBvIGF1dG9yIG91IHNldQpyZXByZXNlbnRhbnRlIGxlZ2FsLCBhbyBhY2VpdGFyIGVzc2UgdGVybW8gZGUgbGljZW7vv71hLCBjb25jZWRlIGFvClJlcG9zaXTvv71yaW8gSW5zdGl0dWNpb25hbCBkYSBVbml2ZXJzaWRhZGUgRmVkZXJhbCBkYSBCYWhpYSBvIGRpcmVpdG8KZGUgbWFudGVyIHVtYSBj77+9cGlhIGVtIHNldSByZXBvc2l077+9cmlvIGNvbSBhIGZpbmFsaWRhZGUsIHByaW1laXJhLCAKZGUgcHJlc2VydmHvv73vv71vLiBFc3NlcyB0ZXJtb3MsIG7vv71vIGV4Y2x1c2l2b3MsIG1hbnTvv71tIG9zIGRpcmVpdG9zIGRlIAphdXRvci9jb3B5cmlnaHQsIG1hcyBlbnRlbmRlIG8gZG9jdW1lbnRvIGNvbW8gcGFydGUgZG8gYWNlcnZvIGludGVsZWN0dWFsIGRlc3NhIFVuaXZlcnNpZGFkZS4gCgogICAgUGFyYSBvcyBkb2N1bWVudG9zIHB1YmxpY2Fkb3MgY29tIHJlcGFzc2UgZGUgZGlyZWl0b3MgZGUgZGlzdHJpYnVp77+977+9bywgZXNzZSB0ZXJtbyBkZSBsaWNlbu+/vWEgZW50ZW5kZSBxdWU6IAoKICAgIE1hbnRlbmRvIG9zICBkaXJlaXRvcyBhdXRvcmFpcywgcmVwYXNzYWRvcyBhIHRlcmNlaXJvcywgZW0gY2FzbyAKZGUgcHVibGljYe+/ve+/vWVzLCBvIHJlcG9zaXTvv71yaW8gcG9kZSByZXN0cmluZ2lyIG8gYWNlc3NvIGFvIHRleHRvIAppbnRlZ3JhbCwgbWFzIGxpYmVyYSBhcyBpbmZvcm1h77+977+9ZXMgc29icmUgbyBkb2N1bWVudG8gKE1ldGFkYWRvcyBkZXNjcml0aXZvcykuCgogRGVzdGEgZm9ybWEsIGF0ZW5kZW5kbyBhb3MgYW5zZWlvcyBkZXNzYSB1bml2ZXJzaWRhZGUgCmVtIG1hbnRlciBzdWEgcHJvZHXvv73vv71vIGNpZW5077+9ZmljYSBjb20gYXMgcmVzdHJp77+977+9ZXMgaW1wb3N0YXMgcGVsb3MgCmVkaXRvcmVzIGRlIHBlcmnvv71kaWNvcy4gCgogICAgUGFyYSBhcyBwdWJsaWNh77+977+9ZXMgZW0gaW5pY2lhdGl2YXMgcXVlIHNlZ3VlbSBhIHBvbO+/vXRpY2EgZGUgCkFjZXNzbyBBYmVydG8sIG9zIGRlcO+/vXNpdG9zIGNvbXB1bHPvv71yaW9zIG5lc3NlIHJlcG9zaXTvv71yaW8gbWFudO+/vW0gCm9zIGRpcmVpdG9zIGF1dG9yYWlzLCBtYXMgbWFudO+/vW0gbyBhY2Vzc28gaXJyZXN0cml0byBhbyBtZXRhZGFkb3MgCmUgdGV4dG8gY29tcGxldG8uIEFzc2ltLCBhIGFjZWl0Ye+/ve+/vW8gZGVzc2UgdGVybW8gbu+/vW8gbmVjZXNzaXRhIGRlIApjb25zZW50aW1lbnRvIHBvciBwYXJ0ZSBkZSBhdXRvcmVzL2RldGVudG9yZXMgZG9zIGRpcmVpdG9zLCBwb3IgCmVzdGFyZW0gZW0gaW5pY2lhdGl2YXMgZGUgYWNlc3NvIGFiZXJ0by4KCiAgICBFbSBhbWJvcyBvIGNhc28sIGVzc2UgdGVybW8gZGUgbGljZW7vv71hLCBwb2RlIHNlciBhY2VpdG8gcGVsbyAKYXV0b3IsIGRldGVudG9yZXMgZGUgZGlyZWl0b3MgZS9vdSB0ZXJjZWlyb3MgYW1wYXJhZG9zIHBlbGEgCnVuaXZlcnNpZGFkZS4gRGV2aWRvIGFvcyBkaWZlcmVudGVzIHByb2Nlc3NvcyBwZWxvIHF1YWwgYSBzdWJtaXNz77+9byAKcG9kZSBvY29ycmVyLCBvIHJlcG9zaXTvv71yaW8gcGVybWl0ZSBhIGFjZWl0Ye+/ve+/vW8gZGEgbGljZW7vv71hIHBvciAKdGVyY2Vpcm9zLCBzb21lbnRlIG5vcyBjYXNvcyBkZSBkb2N1bWVudG9zIHByb2R1emlkb3MgcG9yIGludGVncmFudGVzIApkYSBVRkJBIGUgc3VibWV0aWRvcyBwb3IgcGVzc29hcyBhbXBhcmFkYXMgcG9yIGVzdGEgaW5zdGl0dWnvv73vv71vLgo=Repositório InstitucionalPUBhttp://192.188.11.11:8080/oai/requestopendoar:19322022-07-05T17:03:34Repositório Institucional da UFBA - Universidade Federal da Bahia (UFBA)false
dc.title.pt_BR.fl_str_mv Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
dc.title.alternative.pt_BR.fl_str_mv Protein Expression and Purification
title Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
spellingShingle Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
Kurtenbach, Eleonora
Rotavirus spike
VP8* subunit
Expression in Escherichia coli
His-Tag purification
title_short Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
title_full Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
title_fullStr Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
title_full_unstemmed Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
title_sort Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli
author Kurtenbach, Eleonora
author_facet Kurtenbach, Eleonora
Favacho, Alexsandra R. M.
Sardi, Silvia Inês
Gouvea, Vera S.
author_role author
author2 Favacho, Alexsandra R. M.
Sardi, Silvia Inês
Gouvea, Vera S.
author2_role author
author
author
dc.contributor.author.fl_str_mv Kurtenbach, Eleonora
Favacho, Alexsandra R. M.
Sardi, Silvia Inês
Gouvea, Vera S.
Kurtenbach, Eleonora
Favacho, Alexsandra R. M.
Sardi, Silvia Inês
Gouvea, Vera S.
dc.subject.por.fl_str_mv Rotavirus spike
VP8* subunit
Expression in Escherichia coli
His-Tag purification
topic Rotavirus spike
VP8* subunit
Expression in Escherichia coli
His-Tag purification
description Texto completo: acesso restrito. p. 196–203
publishDate 2006
dc.date.issued.fl_str_mv 2006
dc.date.accessioned.fl_str_mv 2013-11-19T20:49:41Z
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://repositorio.ufba.br/ri/handle/ri/13803
dc.identifier.issn.none.fl_str_mv 1046-5928
dc.identifier.number.pt_BR.fl_str_mv v. 46, n. 2
identifier_str_mv 1046-5928
v. 46, n. 2
url http://repositorio.ufba.br/ri/handle/ri/13803
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.source.pt_BR.fl_str_mv http://dx.doi.org.ez10.periodicos.capes.gov.br/10.1016/j.pep.2005.09.014
dc.source.none.fl_str_mv reponame:Repositório Institucional da UFBA
instname:Universidade Federal da Bahia (UFBA)
instacron:UFBA
instname_str Universidade Federal da Bahia (UFBA)
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reponame_str Repositório Institucional da UFBA
collection Repositório Institucional da UFBA
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