Expressão heteróloga em sistema procarioto da NTPDase-1 do Trypanosoma cruzi e avaliação do potencial uso no imunodiagnóstico na doença de Chagas

AUTOR(ES)

Tatiana Dias Araújo

DATA DE PUBLICAÇÃO

2008

RESUMO

In this work were optimized the time of induction and the concentration of IPTG to get an expression of large quantities NTPDase-1 from the T. cruzi recombinant. Moreover, it was evaluate the concentrations of sodium chloride needed for a greter recovery of NTPDase-1 T. cruzi recombinant full purest, because it showed lower purity when eluted with salt concentrations recommended by manufacturers of resins used. The difference in purification of proteins recombinates study in full and without missing signal peptide was assessed used resins containing the metal ion cobalt or nickel. It is possible to have more pure protein with just a purification with resin containing nickel, and requiring two consecutive purificações to take a similar purity when using resin with cobalt. The soluble and insoluble fractions NTPDase-1 T. cruzi recombinant full and without signal peptide was purified and analyzed by gel electrophoresis 10% SDSPAGE and Western blotting. As the purification of inclusion bodies with very low protein concentration or non-existent, was the only viable purify sobrendante to have a higher concentration of protein to be used in tests of immunodiagnosis. Tests western blotting were performed to confirm the presence of the protein recombinate. Besides being possible, with these tests, assess the possibility of a recognition and signal peptide cleavage of the bacterium E. coli. Serum of dogs experimentally positive for T. cruzi strains Berenice-78, Y and AAS in acute and chronic stages of Chagas disease, were submitted to immunoreactivity by ELISA taking as NTPDase-1 antigen of T. cruzi recombinant full or without the signal peptide, being evaluated by anti-IgG conjugated to biotin. It was possible to see that there was a separation of sera infected and uninfected with acute and chronic disease using the recombinant protein without the signal peptide. These results of immunodiareactivity may have been influenced by the purity of the antigen and the conformation of the protein purified.

ASSUNTO(S)

biologia molecular 1. tripanossomo - teses. 2. imunodiagnóstico - teses. 3. proteínas - teses. i. universidade federal de ouro preto. ii. título.

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