Identificação de proteínas imunoreativas de Staphylococcus aureus isolado de mastite bovina

• Ano de defesa: 2010
• Autor(a) principal: Fabres, Mary Hellen Araújo
• Orientador(a): Ribon, Andréa de Oliveira Barros
• Banca de defesa: Fietto, Luciano Gomes , Brito, Maria Aparecida Vasconcelos Paiva e , Oliveira, Leandro Licursi de
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Mestrado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Proteínas imunoreativas; Staphylococcus aureus; Mastite bovina
• Palavras-chave em Inglês: Immunoreactive proteins; Staphylococcus aureus; Bovine mastitis
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR
• Link de acesso: http://locus.ufv.br/handle/123456789/2462

Staphylococcal mastitis is a highly prevalent pathogen of the mammary gland that may progresses to a chronic state. The earlier diagnosis is very important due to the high cost of mastitis and risks to human health. This work aimed the identification of immunogenic proteins as target antigens for specific diagnosis of mastitis caused by Staphylococcus aureus. Within this purpose we used the Western Blotting method for the selection of specific immunogens expressed during infection. Protein extracts were prepared from cultures of the mastitis isolates Staph. aureus 3993, 4124, 4163, Staph. epidermidis, Staph. intermedius, Staph. chromogens, Staph. hyicus coagulase positive, and Streptococcus agalactiae. Proteins were resolved by SDS-PAGE 14% and electrotransferred on nitrocellulose membrane. The detection was performed with antisera raised against total protein extracts obtained from cultures grown until exponential or stationary phase. Two and five bands, respectively, were immunorevealed with extracts produced from secreted and cell-wall proteins obtained from stationary cultures. These bands, found only in Staph aureus strains, were excised from the gel, digested with trypsin and analyzed by mass spectrometry. Another approach for selection immunogens was constructed a genomic library of Staph. aureus was constructed in vector pUC18 using DNA fragments between 0.5 and 8 kb. Antiserum produced using bacterial protein extract was employed for library screening. This methodology enabled the identification of 74 positive clones and 20 were chosen for sequencing. Nucleotide sequences were aligned to the genome of Staph. aureus strain RF122, a causative agent of contagious bovine mastitis, and identified 18 different loci that included proteins related to metabolism, toxins, adhesion, bacterial cell-wall, as well as regulatory and hypothetical proteins. Analysis revealed that the fibrinogen-binding protein (Efb) encoded by the fib gene is unique to Staph. aureus and thus a promising candidate for the development of specific serodiagnostic assays. Further work is needed to sequence the eluted proteins and to express those identified from the genomic library.