2014-12-152004-05-03PEREIRA, Luiz Augusto. Identificação proteômica, seqüência de nucleotídeos, expressão heteróloga e reatividade imunológica da triose fosfato isomerase de Paracoccidioides brasiliensis. 2004. 65 f. Dissertação (Mestrado em Medicina Tropical e Saúde Publica) - Universidade Federal de Goiás, Goiânia, 2004.http://repositorio.bc.ufg.br/tede/handle/tede/3778An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C digested peptides of the purified protein which presented, molecular mass of 29-kDa and pI of 5.8, were subjected to sequence analysis of its amino acids. Search at databases comparing the sequence of amino acids from the three peptides of the native protein, revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249 amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained 6 exons interrupted by 5 introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidenced the correlation between the phylogeny provided by the deduced proteins and introns positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was over expressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that has been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed Paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation in assays for serodiagnosis of the disease.application/pdfAcesso AbertoParacoccidioides brasiliensisTriose fosfato isomeraseClonagem do gene e análise estruturalProteína recombinanteReatividade imunológicaGene cloning and structural analysisRecombinant proteinImmunological reactivityCIENCIAS BIOLOGICAS::MICROBIOLOGIADissertação