Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes

Bibliographic Details
Main Author: Miura,Valéria Chamas
Publication Date: 2017
Other Authors: Aoki,Sérgio Moraes, Peitl Junior,Paulo, Pires,Lilian Campos, Dalmagro,Priscila, Nakamura,Alex Akira, Meireles,Marcelo Vasconcelos
Format: Article
Language: eng
Source: Revista Brasileira de Parasitologia Veterinária (Online)
Download full: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S1984-29612017000200205
Summary: Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
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spelling Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposesCryptosporidiumglycoprotein GP60polyclonal antibodydiagnosisAbstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.Colégio Brasileiro de Parasitologia Veterinária2017-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S1984-29612017000200205Revista Brasileira de Parasitologia Veterinária v.26 n.2 2017reponame:Revista Brasileira de Parasitologia Veterinária (Online)instname:Colégio Brasileiro de Parasitologia Veterinária (CBPV)instacron:CBPV10.1590/s1984-29612017032info:eu-repo/semantics/openAccessMiura,Valéria ChamasAoki,Sérgio MoraesPeitl Junior,PauloPires,Lilian CamposDalmagro,PriscilaNakamura,Alex AkiraMeireles,Marcelo Vasconceloseng2017-07-12T00:00:00Zoai:scielo:S1984-29612017000200205Revistahttp://www.scielo.br/scielo.php?script=sci_serial&lng=pt&pid=1984-2961https://old.scielo.br/oai/scielo-oai.php||zacariascbpv@fcav.unesp.br1984-29610103-846Xopendoar:2017-07-12T00:00Revista Brasileira de Parasitologia Veterinária (Online) - Colégio Brasileiro de Parasitologia Veterinária (CBPV)false
dc.title.none.fl_str_mv Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
title Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
spellingShingle Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
Miura,Valéria Chamas
Cryptosporidium
glycoprotein GP60
polyclonal antibody
diagnosis
title_short Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
title_full Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
title_fullStr Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
title_full_unstemmed Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
title_sort Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes
author Miura,Valéria Chamas
author_facet Miura,Valéria Chamas
Aoki,Sérgio Moraes
Peitl Junior,Paulo
Pires,Lilian Campos
Dalmagro,Priscila
Nakamura,Alex Akira
Meireles,Marcelo Vasconcelos
author_role author
author2 Aoki,Sérgio Moraes
Peitl Junior,Paulo
Pires,Lilian Campos
Dalmagro,Priscila
Nakamura,Alex Akira
Meireles,Marcelo Vasconcelos
author2_role author
author
author
author
author
author
dc.contributor.author.fl_str_mv Miura,Valéria Chamas
Aoki,Sérgio Moraes
Peitl Junior,Paulo
Pires,Lilian Campos
Dalmagro,Priscila
Nakamura,Alex Akira
Meireles,Marcelo Vasconcelos
dc.subject.por.fl_str_mv Cryptosporidium
glycoprotein GP60
polyclonal antibody
diagnosis
topic Cryptosporidium
glycoprotein GP60
polyclonal antibody
diagnosis
description Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.
publishDate 2017
dc.date.none.fl_str_mv 2017-06-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
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dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/s1984-29612017032
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dc.publisher.none.fl_str_mv Colégio Brasileiro de Parasitologia Veterinária
publisher.none.fl_str_mv Colégio Brasileiro de Parasitologia Veterinária
dc.source.none.fl_str_mv Revista Brasileira de Parasitologia Veterinária v.26 n.2 2017
reponame:Revista Brasileira de Parasitologia Veterinária (Online)
instname:Colégio Brasileiro de Parasitologia Veterinária (CBPV)
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instname_str Colégio Brasileiro de Parasitologia Veterinária (CBPV)
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reponame_str Revista Brasileira de Parasitologia Veterinária (Online)
collection Revista Brasileira de Parasitologia Veterinária (Online)
repository.name.fl_str_mv Revista Brasileira de Parasitologia Veterinária (Online) - Colégio Brasileiro de Parasitologia Veterinária (CBPV)
repository.mail.fl_str_mv ||zacariascbpv@fcav.unesp.br
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