Uso de plantas como biorreatores para a produção heteróloga da proteína NS2B do Zika vírus como insumo para diagnóstico

• Autor(a) principal: Herazo, Mario Alberto Maestre
• Data de Publicação: 2020
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da Universidade Federal do Ceará (UFC)
• Texto Completo: http://www.repositorio.ufc.br/handle/riufc/53360
• topic: Vírus Zika; Proteínas recombinantes; Expressão transitória; Diagnóstico

Zika is an emerging infectious disease caused by the Zika virus (ZIKV), and it is related to neurological disorders such as microcephaly and Guillain-Barré syndrome. The detection and differentiation of ZIKV from other similar arboviviruses is based mainly on molecular methods (RT-PCR and serological tests). However, this has been a problem due to cross- reactions mainly in serum samples. Many proteins used in the serological diagnosis of the disease are produced recombinantly, mainly on a prokaryotic platform. However, the use of plants for the expression of recombinant proteins for diagnostic purposes has become a promising alternative because it is economical, safe and fast compared to other platforms. The objective of this work was to evaluate the possibility of expressing the non-structural protein from ZIKV, NS2B, on a plant platform to be used for diagnostic purposes. Therefore, the NS2B protein gene was fused with a Hydrophobin I (HFBI) tag and expressed transiently in Nicotiana benthamiana plants using Agrobacterium tumefaciens as a vector. The detection of NS2B::HFBI in extracts of infiltrated leaves was done using western blot technic and an anti-c-myc antibody, subsequently the protein was purified through aqueous two-phase separation (ATPS), followed by hydrophobic interaction chromatography. After purification, the recombinant protein had its antigenicity assessed by WB using a commercially specific antibody and sera from ZIKV + patient. ELISA-IgM tests for dengue and Zika of patients with positive sera were also performed, with a sensitivity of 95.0% and 100% specificity for ZIKV. In subcutaneous mouse immunization tests, a recombinant protein was able to induce an immune response, generating antibodies that, after purified was used for the detection of viral protein in the serum of ZIKV patients. Based on the obtained results, this work showed that the plant produced NS2B has high potential in the diagnostics kits for ZIKV.