Clonagem, expressão e purificação de antígeno recombinante no imunodiagnóstico da angiostrongilíase abdominal

• Ano de defesa: 2007
• Autor(a) principal: Silva, Ana Cristina Arámburu da
• Orientador(a): Graeff-Teixeira, Carlos
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
• Palavras-chave em Português: ZOOLOGIA; PARASITOS; NEMATÓDEOS; DOENÇAS PARASITÁRIAS; PROTEÍNAS DO CHOQUE TÉRMICO; DIAGNÓSTICO
• Link de acesso: http://hdl.handle.net/10923/5299

Abdominal angiostrongyliasis is a zoonotic infection caused by Angiostrongylus costaricensis, a nematode with an intra-vascular location in the mesentery. The diagnosis is only achieved with a pathological examination of tissue fragments ressected during surgical treatment in complicated clinical courses (intestinal perforation and/or obstruction). Immunodiagnostic test show many difficulties with cross-reacting antibodies and diversity of the humoral reactivity. Therefore, efforts have been directed towards identification and purification of parasite specific antigens for development of serodiagnosis methods. In the present study we analized the use of recombinant A. costaricensis heat shock protein (HSP) 20 in ELISA for the serodiagnosis. One of the cDNA clones, obtained and expressed a small heat shock protein HSP 20 with 147 amino acids, which had high sequence identity with other nematode small heat shock proteins. The gene enconding the HSP 20 was sub-cloned using PCR technique. The sequence corresponding was subcloned in the pET-23a-d (+) expression vector, to construct a recombinant HSP 20 protein containing six histidine residues at the Nterminal. High level expression of the HSP 20 by Escherichia coli BL21 (DE3) harbouring the HSP 20 gene and containing its expression vector was observed upon induction with 1 mM IPTG at 37 °C. One-step purification of the recombinant HSP 20 was achieved with Ni-NTA resin. Western blot analysis, using sera from patients with confirmed angiostrongyliasis abdominal, indicated that the purified HSP 20 expression product was immunogenic. Sensitivity of ELISA-HSP 20 was 33%. However, 100% specificity of this recombinant protein suggests its usefullness in combination with other antigen molecules, in an approach of multiple screening tests for the most accurate serodiagnosis of abdominal angiostrongyliasis.