Identificação proteômica, seqüência de nucleotídeos, expressão heteróloga e reatividade imunológica da triose fosfato isomerase de Paracoccidioides brasiliensis
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Data
2004-05-03
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Universidade Federal de Goiás
Resumo
An 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.
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PEREIRA, 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.