Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli

Detalhes bibliográficos
Autor(a) principal: Tokano,Dorismey Vieira
Data de Publicação: 2008
Outros Autores: Kawaichi,Marisa Emiko, Venâncio,Emerson José, Vidotto,Marilda Carlos
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Brazilian Archives of Biology and Technology
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132008000300006
Resumo: The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.
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spelling Cloning and characterization of the iron uptake gene iutA from avian Escherichia coliAvian Escherichia coliiron uptakeaerobactin receptorvirulence factoriutA geneThe aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.Instituto de Tecnologia do Paraná - Tecpar2008-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132008000300006Brazilian Archives of Biology and Technology v.51 n.3 2008reponame:Brazilian Archives of Biology and Technologyinstname:Instituto de Tecnologia do Paraná (Tecpar)instacron:TECPAR10.1590/S1516-89132008000300006info:eu-repo/semantics/openAccessTokano,Dorismey VieiraKawaichi,Marisa EmikoVenâncio,Emerson JoséVidotto,Marilda Carloseng2008-07-16T00:00:00Zoai:scielo:S1516-89132008000300006Revistahttps://www.scielo.br/j/babt/https://old.scielo.br/oai/scielo-oai.phpbabt@tecpar.br||babt@tecpar.br1678-43241516-8913opendoar:2008-07-16T00:00Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar)false
dc.title.none.fl_str_mv Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
title Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
spellingShingle Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
Tokano,Dorismey Vieira
Avian Escherichia coli
iron uptake
aerobactin receptor
virulence factor
iutA gene
title_short Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
title_full Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
title_fullStr Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
title_full_unstemmed Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
title_sort Cloning and characterization of the iron uptake gene iutA from avian Escherichia coli
author Tokano,Dorismey Vieira
author_facet Tokano,Dorismey Vieira
Kawaichi,Marisa Emiko
Venâncio,Emerson José
Vidotto,Marilda Carlos
author_role author
author2 Kawaichi,Marisa Emiko
Venâncio,Emerson José
Vidotto,Marilda Carlos
author2_role author
author
author
dc.contributor.author.fl_str_mv Tokano,Dorismey Vieira
Kawaichi,Marisa Emiko
Venâncio,Emerson José
Vidotto,Marilda Carlos
dc.subject.por.fl_str_mv Avian Escherichia coli
iron uptake
aerobactin receptor
virulence factor
iutA gene
topic Avian Escherichia coli
iron uptake
aerobactin receptor
virulence factor
iutA gene
description The aim of this work was to isolate, clone and characterize the iron uptake gene iutA from avian pathogenic E. coli (APEC). The iutA gene was isolated from the strain APEC 9, serotype O2:H9, which was cloned in the expression vector pET101/D-TOPO. The gene of 2.2 Kb was sequenced (AY602767, which showed high similarity to the iutA gene from three plasmids, two from APEC, pAPEC-02-ColV (AY545598.4) and pTJ100 (AY553855.1), and one from a human invasive E. coli strain, the pColV K30. The recombinant protein IutA was over expressed in E. coli BL21(DE-3) and was solubilized with urea and purified by Ni-NTA column. This method produced a relatively high yield of r-IutA of approximately 74kDa, which was used to produce the antibody anti-IutA. This anti-IutA reacted with the protein r-IutA and native IutA of APEC 9, as demonstrated by Western blot, showing that the r-IutA conserved epitopes and its antigenicity was preserved. The anti-IutA IgY was able to inhibit the IutA biological activity, inhibiting the sensitivity to cloacin DF13 of APEC9. However, it did not inhibit the growth of APEC9 in M9 and did not protect the chickens inoculated with the APEC, suggesting that the APEC possessed another iron acquisition mechanism distinct of aerobactin.
publishDate 2008
dc.date.none.fl_str_mv 2008-06-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132008000300006
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132008000300006
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S1516-89132008000300006
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Instituto de Tecnologia do Paraná - Tecpar
publisher.none.fl_str_mv Instituto de Tecnologia do Paraná - Tecpar
dc.source.none.fl_str_mv Brazilian Archives of Biology and Technology v.51 n.3 2008
reponame:Brazilian Archives of Biology and Technology
instname:Instituto de Tecnologia do Paraná (Tecpar)
instacron:TECPAR
instname_str Instituto de Tecnologia do Paraná (Tecpar)
instacron_str TECPAR
institution TECPAR
reponame_str Brazilian Archives of Biology and Technology
collection Brazilian Archives of Biology and Technology
repository.name.fl_str_mv Brazilian Archives of Biology and Technology - Instituto de Tecnologia do Paraná (Tecpar)
repository.mail.fl_str_mv babt@tecpar.br||babt@tecpar.br
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