Epitopos imunodominantes da MSP1a de Anaplasma marginale e suas aplicações diagnósticas e vacinais

• Ano de defesa: 2011
• Autor(a) principal: Santos, Paula de Souza
• Orientador(a): Goulart Filho, Luiz Ricardo
• Banca de defesa: Szabó, Matias Pablo Juan , Azevedo, Vasco Ariston de Carvalho , Vidotto, Odilon , Mineo, Tiago Wilson Patriarca
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Uberlândia
• Programa de Pós-Graduação: Programa de Pós-graduação em Genética e Bioquímica
• Departamento: Ciências Biológicas
• País: BR
• Palavras-chave em Português: Anaplasma marginale; Phage Display; Epitopo; Bioeletrodo; Peptídeos; Anaplasmose - Vacina; Anaplasmose - Diagnóstico
• Palavras-chave em Inglês: Epitope; Bioelectrode
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::GENETICA
• Link de acesso: https://repositorio.ufu.br/handle/123456789/15730

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system.