Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests
Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiologic...
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Published in | Revista brasileira de parasitologia veterinaria Vol. 29; no. 3; p. e005820 |
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01.01.2020
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Abstract | Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.
Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil. |
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AbstractList | Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.
Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil. Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil. |
Author | Labruna, Marcelo Bahia Ferreira, Eliane de Oliveira Machado, Sérgio Lisboa Soares, João Fábio Toma, Helena Keiko Almosny, Nádia Regina Pereira Medeiros, Miguel Ângelo da Silva Matta, Maria Adelaide do Valle Xavier, Márcia de Souza Meirelles, Maria de Nazareth Silveira Leal de Silva, Maria Helena da |
AuthorAffiliation | Universidade Castelo Branco Universidade Federal Fluminense Fundação Oswaldo Cruz Universidade de São Paulo Universidade Federal do Rio de Janeiro Universidade Federal do Rio Grande do Sul |
AuthorAffiliation_xml | – name: Fundação Oswaldo Cruz – name: Universidade Federal do Rio Grande do Sul – name: Universidade de São Paulo – name: Universidade Federal do Rio de Janeiro – name: Universidade Castelo Branco – name: Universidade Federal Fluminense |
Author_xml | – sequence: 1 givenname: Miguel Ângelo da Silva surname: Medeiros fullname: Medeiros, Miguel Ângelo da Silva organization: Universidade Castelo Branco, Brasil; Universidade Federal Fluminense, Brasil – sequence: 2 givenname: Maria Helena da surname: Silva fullname: Silva, Maria Helena da organization: Universidade Federal do Rio de Janeiro, Brasil – sequence: 3 givenname: Maria Adelaide do Valle surname: Matta fullname: Matta, Maria Adelaide do Valle organization: Fundação Oswaldo Cruz, Brasil – sequence: 4 givenname: Eliane de Oliveira orcidid: 0000-0002-9977-6460 surname: Ferreira fullname: Ferreira, Eliane de Oliveira organization: Universidade Federal do Rio de Janeiro, Brasil – sequence: 5 givenname: Sérgio Lisboa surname: Machado fullname: Machado, Sérgio Lisboa organization: Universidade Federal do Rio de Janeiro, Brasil – sequence: 6 givenname: João Fábio surname: Soares fullname: Soares, João Fábio organization: Universidade Federal do Rio Grande do Sul, Brasil – sequence: 7 givenname: Marcelo Bahia surname: Labruna fullname: Labruna, Marcelo Bahia organization: Universidade de São Paulo, Brasil – sequence: 8 givenname: Helena Keiko surname: Toma fullname: Toma, Helena Keiko organization: Universidade Federal do Rio de Janeiro, Brasil – sequence: 9 givenname: Márcia de Souza surname: Xavier fullname: Xavier, Márcia de Souza organization: Universidade Federal Fluminense, Brasil – sequence: 10 givenname: Maria de Nazareth Silveira Leal de surname: Meirelles fullname: Meirelles, Maria de Nazareth Silveira Leal de organization: Universidade Castelo Branco, Brasil; Fundação Oswaldo Cruz, Brasil – sequence: 11 givenname: Nádia Regina Pereira surname: Almosny fullname: Almosny, Nádia Regina Pereira organization: Universidade Federal Fluminense, Brasil |
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Cites_doi | 10.1590/S1984-29612011000100002 10.1016/j.tvjl.2008.07.013 10.1016/j.vetmic.2013.02.015 10.1128/CDLI.6.3.392-399.1999 10.1038/227680a0 10.1093/jmedent/41.5.126 10.1128/CVI.00361-06 10.4142/jvs.2014.15.2.241 10.1016/S0195-5616(03)00031-7 10.1128/IAI.01494-06 10.1007/BF02623551 10.1128/JCM.29.9.2024-2029.1991 10.1590/S1517-83822008000300014 10.1093/nar/7.6.1513 10.1128/JCM.32.9.2107-2112.1994 10.1016/j.ttbdis.2015.10.003 10.1016/j.ttbdis.2014.01.011 10.1016/j.tvjl.2010.02.001 10.1177/104063879600800406 10.1128/IAI.71.5.2516-2524.2003 10.1128/IAI.74.1.711-720.2006 10.1590/S1984-29612014055 10.2460/ajvr.67.2.206 10.1196/annals.1355.079 10.1128/JCM.39.1.315-322.2001 10.1128/IAI.01347-10 10.1128/JCM.36.9.2671-2680.1998 |
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DocumentTitleAlternate | Expressão e análise antigênica da proteína RTP36 recombinante da amostra São Paulo de Ehrlichia canis para testes sorológicos |
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Keywords | TRP36 protein epidemiology diagnóstico diagnostic proteína TRP36 epidemiologia Ehrlichia canis serologic tests testes sorológicos |
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Snippet | Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is... |
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SubjectTerms | diagnostic Ehrlichia canis epidemiology PARASITOLOGY serologic tests TRP36 protein VETERINARY SCIENCES |
Title | Expression and antigenic analysis of the recombinant TRP36 protein from Ehrlichia canis São Paulo strain for serologic tests |
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