Expressão da proteína E2 recombinante do vírus chikungunya em células eucariotas para utilização em plataformas diagnósticas

• Ano de defesa: 2023
• Autor(a) principal: Cid Oliveira de Queiroz
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; Brasil; ICB - DEPARTAMENTO DE MICROBIOLOGIA; Programa de Pós-Graduação em Microbiologia; UFMG
• Palavras-chave em Português: Chikungunya virus; diagnóstico; expressão; eucarioto; Microbiologia; Vírus Chikungunya; Diagnóstico
• Link de acesso: http://hdl.handle.net/1843/65885

The Chikungunya virus (CHIKV - family Togaviridae, genus Alphavirus) is an arbovirus that causes Chikungunya fever, whose symptoms include fever, arthralgia, myalgia, and headache. About 40% of patients develop a chronic arthralgia for months or years, resulting in a significant social and economic impact in endemic regions. The symptomatology and epidemiological profile of CHIKV may be similar to other arboviruses, such as Zika and Dengue, therefore, clinical diagnosis in Brazil becomes more challenging, where there is co-circulation of arboviruses. In the current scenario, national, cheap, easy-to-apply, and high-performance diagnostic tests are crucial for the control and treatment of CHIKV. This study aimed at producing the E2 protein of CHIKV in eukaryotic systems and evaluate its antigenicity, with the prospect of developing serological tests, ELISA, and IFL. The E2 envelope glycoprotein is associated with virus adsorption, being one of the main targets of the humoral response. The E2 gene was constructed for expression in eukaryotic systems to confer antigenicity and immunogenicity-associated post-translational modifications. Transient expression of CHIKV-E2 was observed in HEK293T and EXPI293 cells. The lentiviral system was used for stable expression in HEK293T, EXPI293, and CHO2353 cell lines. After optimizing stable expression, transient expression rates remained higher. Despite the expected size of 40 kDa, CHIKV-E2 was expressed at around 46 kDa. The divergence between the expected and identified size may be related to MPTs. The CHIKV-E2 protein expressed in EXPI293 was purified and evaluated in ELISA against sera from CHIKV-positive patients. The preliminary ELISA assay indicated 98.2% sensitivity and 100% specificity for IgG, as well as 77% sensitivity and 100% specificity for IgM. The preliminary ELISA assay using recombinant protein as the solid phase indicated 98.2% sensitivity and 100% specificity for IgG, as well as 77% sensitivity and 100% specificity for IgM.