Purificação e identificação de proteína de Babesia bovis com atividade lectínica

• Autor(a) principal: Santos, Eliziária Cardoso dos
• Data de Publicação: 2011
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/2333
• topic: Babesia; Lectina; Babesia; Lectin; CNPQ::CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

The present study aimed to purify and identify merozoite protein of B. bovis with lectin activity. For such, total soluble merozoite antigens (Bb-STAg) were subjected to affinity chromatography on immobilized fetuin agarose column. The resin adsorbed protein, eluted with NaCl, was named Fetuin Binding Protein (FBP-B.bovis). The homogeneity and characterization of molecular weight of FBP were assessed by SDS-PAGE, that revealed the protein band of 27 kilodaltons which wasn t glycosylated when combined with Pro-Q Emerald 300 glycoprotein Stain. To identify the constituent of the sample, the protein spot was cut from the gel, chemically treated and had their internal amino acids sequenced by mass spectrometry. The sequence analysis showed that the fragment was related to the bovine serum albumin of Bos taurus with mass/charge of 1567.726 in the most intense peak with score of 62 and 98,98% confidence. The fraction of the FBP-B. bovis was also subjected to sequencing of the NH2-terminal region by Automatic Edman degradation analysis which analysis in the BLAST, involving all the microorganisms, identified the following as part of a putative protein uncharacterized of Dappu pulex (flea d 'water), with E-value of 6.5, 90% identity, 90% of positivity and mass about 26.939 kilodaltons. Considering the same sequence, other BLAST analysis restricted to organisms of Apicomplexa phylum was made, and their amino acid sequence was identify as putative protein of B. bovis evolutionarily conserved with total score of 52,8, covering 71% and E-value 0.40. The isolated protein fragment was also used to immunize BALB/c mice to produce of specific antibodies that after being purified by affinity chromatography were titrated by Dot-ELISA that results in recognition of FBP-B. bovis (title 102,400). That away, was possible to obtain a protein fraction of merozoite of B. bovis with lectin activity, with the internal amino acid belonging to the protein bovine albumin serum and amino acids of the NH2-terminal region belonging to a putative protein of B. bovis that was recognized by specific anti-FBP B. bovis. According to the results presented, we believe this protein fraction of merozoite of B. bovis with lectin activity purified can be one important mediators involved in erythrocyte parasitemia. In future studies, it is proven hypothesis, it may open perspectives for new processes for therapeutic intervention against bovine babesioses and greater control of the disease.