Expressão e purificação da proteína recombinante NTPDase2 de Leishmania infantum e sua aplicação no imunodiagnóstico da leishmaniose canina

• Autor(a) principal: Souza, Ronny Francisco de
• Data de Publicação: 2012
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/327
• topic: Proteína recombinante; Leishmaniose canina; Imunodiagnóstico; Recombinant protein; Canine leishmaniasis; Immunodiagnostic; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR

In Brazil leishmaniasis are zoonotic diseases character, being transmitted by blood-sucking sand flies of the family Psychodidae. The development of clinical forms: visceral or cutaneous, is closely related to species involved in infection and immune status of the infected individual. The dog is currently the main reservoir of epidemiological importance. The elimination of infected dogs is now one of the epidemiological surveillance activities, being guided by serodiagnosis. In this study, NTPDase2 L. infantum is heterologously expressed in E. coli and purified. rNTPDase2 purified protein was used in ELISA assay of sera of two banks. A bank with samples mounted in the city of Caratinga-MG, an endemic area for Canine Visceral Leishmaniasis (CVL) and the other bank with samples of the city of Governador Valadares-MG, a region endemic for American Cutaneous leishmaniasis (ACL). Also included in this study serum samples from 30 dogs from non-endemic area for CVL experimentally infected with T. cruzi. For samples purified Caratinga the rNTPDase2 was used at a concentration of 0,5 μg. The sensitivity was 100% (95% CI: 92,60% to 100,0%), specificity 100% (95% CI: 86,77% to 100,0%) and the index of agreement was excellent (k = 1). For samples Valadares antigen was used in three concentrations: 0,5, 1 and 2 μg. For these concentrations the sensitivity ranged from 91,67 to 95,35% and specificity was 100%, the level of agreement was excellent (k> 0,8). Tissue samples from 48 dogs in the city of Governador Valadares were also used for direct parasitological test. In the technique was used to imprint the spleen and liver tissue stained with fast Panotic (sensitivity = 79,16%). Using lymph node cuts of 0.5 μM and stained with slow Giemsa, H & E, the sensitivity was 100% for both techniques. In immunohistochemistry, using anti rabbit rNTPDase2 hyperimmune serum, the sensitivity was 89,58%. The rNTPDase2 also been used in immunochromatographic assay pilot demonstrating the potential for use in this type of test. In this study it is shown that rNTPDase2 can be used as a new potential antigen for the diagnosis of CVL.