Obtenção de gp43 de Paracoccidioides brasiliensis recombinante em Escherichia coli e produção de anticorpos policlonais para gp43 em galinhas poedeiras imunizadas com vacina de DNA

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in Latin America, caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis (Pb). It is considered the 8th cause of death by parasitic and infectious diseases in Brazil. Immunological assays are used for diagnosis, follow-up of patients and epidemiological studies. 43 kDa glycoprotein (gp43), the main antigen used in immunoassays, has carbohydrates epitopes that contribute to the cross reaction with sera of patients with histoplasmosis and Jorge Lobo´s disease. The production of a recombinant gp43 in E. coli could minimize the cross reaction, because it is unglycosylated. In the present work, the gene of gp43 was cloned in the expression vector pET30a+ (Novagen) and transformed in E. coli BL21 (DE3) Star. The peptide of approximately 42 kDa present in the extract protein of the bacterial culture induced with IPTG was recognized in Western blot by serum of a dog positive for gp43. However, this protein could not be purified in affinity column of nickel. Alternatively, it was proposed the production of a DNA vaccine containing the gene of gp43 in order to immunize laying hens and obtain polyclonal antibodies anti-gp43 that could be used in immunoassays. The gp43 gene was cloned in the vaccine plasmid (pcDNA3.1), sequenced and used for immunization of three laying hens. The production of anti-gp43 IgY antibodies was evaluated through ELISA using native gp43 as antigen; and it was observed that just one chicken showed increase in the levels of antibodies. The present work showed that DNA vaccine induced the production of antibodies anti-gp43.

ASSUNTO(S)

patologia experimental paracoccidioidomicose doenças transmissíveis doenças parasitárias experimental pathology paracoccidioidomycosis

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