RESUMO
We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.
Assuntos
Antígenos de Protozoários/genética , Citoesqueleto/química , Genes de Protozoários/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/análise , RNA Mensageiro/análise , RNA de Protozoário/análise , Sequências Repetitivas de Ácido Nucleico/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Transcrição Gênica , Trypanosoma cruzi/imunologiaRESUMO
A Trypanosoma cruzi DNA fragment encoding an immunodominant repetitive antigen (H49) was subcloned into a protein purification and expressed system. Purified H49 peptide reacted specifically in an enzyme-linked immunosorbent assay (ELISA) with sera from T. cruzi-infected patients, but not with sera from patients with other parasitic diseases such as leishmaniasis and T. rangeli-infection. The H49 recombinant ELISA was able to detect specific antibodies in 84% of chronic chagasic serum samples tested. One of the major advantage of the recombinant ELISA for serodiagnosis of chronic Chagas' disease resides in its high specificity (100%). Our data suggest that recombinant peptides could provide a practical basis for specific diagnosis tests for Chagas' disease.