AnÃlise da antigenicidade e expressÃo de quatro novas proteÃnas hipotÃticas de Leishmania.(Leishmania) Chagasi

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Leishmaniasis are parasitary infectious-diseases endemic in many countries caused by protozoan parasites of the genus Leishmania. Current strategies to combat visceral leishmaniasis, the most lethal and often fatal form of the disease unless treated, have not so far been successful. For this reason identification of recombinant antigens that can be used in diagnosis and development of vaccines against visceral leishmaniasis is crucial. In previous studies, a serological screening, using sera from infected humans and dogs against expression libraries, genomic and cDNA, of L. chagasi permitted the identification of many clones coding for hypothetical proteins of this parasite. To perform this work we selected four clones, many of them coding repetitive motifs- containing proteins: Lc131 (from cDNA library) and Lcg27, Lcg36 and Lcg53 (from genomic library). The complete or partial inserts of selected clones, coding or not for repetitive motifs, were obtained by PCR and/or digestion with restriction enzymes and were cloned/sub-cloned in plasmidial expression vectors. The resulting recombinant proteins, fused to a poly-histidines tag at the N-terminal region, were expressed in Escherichia coli and purified by affinity chromatography. Western-blot assays using polyclonal sera from rabbits raised against proteins Lc131, Lcg53 and Lcg36 showed that these proteins are expressed in promastigotes of L. chagasi. To evaluate their antigenic potential, the complete and truncated proteins containing repetitive motifs, were analyzed by ELISA using sera from three distinct groups of dogs (1- infected with L. chagasi; 2- dogs with other parasitoses; and 3- control group with healthy dogs). The full length proteins presented the higher sensibility: Lc131 (80.43%), Lcg27 (84.78%), Lcg36 (80.43%) e Lcg53 (82.60%) showing that they are naturally immunogenic. Analysis of truncated forms showed that the repetitive regions are responsible for almost all of the antigenicity of these proteins. Using a combination of the proteins Lcg27 and Lcg36 we obtained 97.82% of sensibility with the available sera. Indeed, this combination showed low cross-reactivity with sera from dogs with other parasitoses (25%), demonstrating their potential to be used in serological tests.

ASSUNTO(S)

antÃgeno leishmania genetica antigens leishmania diagnÃstico diagnose

ACESSO AO ARTIGO

Documentos Relacionados