Identificação, caracterização e síntese peptídica associada à enolase de Sporothrix schenckii: aplicação em plataforma imunossensora eletroquímica portátil para o diagnóstico da Esporotricose Felina.
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Data
2022-03-22
Autores
Castilho, Bruna Mateus de
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Universidade Estadual Paulista (Unesp)
Resumo
A esporotricose é uma infecção causada por fungos de espécies pertencentes ao gênero Sporothrix, afetando humanos e animais. Esta infecção apresenta ampla distribuição mundial, mas no Brasil é considerada emergente e representa importante agravo à saúde humana e animal. O diagnóstico laboratorial precoce e com maior acurácia torna-se imprescindível como medida epidemiológica de controle de novas infecções e disseminação da doença nas populações humana e animal. Para medidas de controle eficazes é fundamental métodos de diagnóstico acurados e precoces. Nesse contexto, com grande potencial para aplicação em diagnósticos, os imunossensores eletroquímicos são vistos como ferramentas inovadoras. Assim, o objetivo desta tese foi desenvolver uma plataforma imunodiagnóstica a partir da proteína enolase do Sporothrix schenckii (EnoSs), usando sensor eletroquímico, para o diagnóstico da esporotricose felina. Com base em estudos prévios sobre o potencial imunogênico da proteína recombinante enolase do Sporotrhix schenckii (rSsEno), determinamos a imunorreatividade da rSsEno frente a amostras de soros de gatos positivos (n=20) e negativos (n=6) para esporotricose, aplicando o ensaio imunoenzimático indireto (ELISA). A SsEno demonstrou atividade com concentrações de 1µg. A partir desses resultados com a rSsEno, propomos o emprego deste antígeno em um novo método sorológico para esporotricose. A predição de epítopos mais imunogênicos e antigênicos da EnoSs em célula B foi realizada com o propósito de diminuir possíveis reações inespecíficas da proteína recombinante em plataforma mais sensível baseada em sensor eletroquímico. Os servidores web de predição utilizados foram: BEPIpred 2.0, DLBEtope, IBCEL-EL e Lbtope. O melhor epítopo da EnoSs obtido foi sintetizado por Síntese Química em Fase Sólida usando a estratégica Fmoc/tBut, posteriormente caracterizado por Espectrometria de Massas ms/ms e analisado e purificado por HPLC. A plataforma diagnóstica com sensor eletroquímico foi desenvolvida empregando o sensor DRP-220AT da Metrohm/DropSens, pela imobilização do peptídeo em monocamada organizada, sobre o eletrodo de trabalho, com cistamina (20 mM) e glutaraldeído (5 mM). As reações imunoquímicas ocorreram com os soros positivos e negativos previamente analisados pelo método de ELISA, observando redução da corrente entre 0 e -0,5 mA e com o Potencial/V entre 0,0 e 0,15V da transdução do sinal eletroquímico. A plataforma eletroquímica desenvolvida é promissora como novo método imunodiagnóstico para a esporotricose felina.
Sporotrichosis is an infection caused by fungi of species belonging to the genus Sporothrix, affecting humans and animals. This infection has a worldwide distribution, but in Brazil it’s considered emerging and represents an important threat to human and animal health. Early and more accurate laboratory diagnosis is essential as an epidemiological measure to control new infections and the spread of the disease in humans and animals. For effective control measures we need accurate and early diagnostic methods. In this context, with great potential for application in diagnostics, electrochemical immunosensors are seen as innovative tools. The aim of this thesis was to develop an immunodiagnostic platform from the Sporothrix schenckii protein enolase (EnoSs), using an electrochemical sensor, for the diagnosis of feline sporotrichosis. Based on previous studies on the immunogenic potential of the recombinant protein enolase from Sporotrhix schenckii (rSsEno), we determined the immunoreactivity of rSsEno against sera samples from positive (n=20) and negative (n=6) sporotrichosis cats, applying the indirect enzyme immunoassay (ELISA). SsEno demonstrated activity at concentrations of 1µg. From these results with rSsEno, we propose the use of this antigen in a new serological method for sporotrichosis. The prediction of more immunogenic and antigenic epitopes of EnoSs in B cells was performed with the purpose of reducing possible nonspecific reactions of the recombinant protein in a more sensitive platform based on an electrochemical sensor. The prediction web servers used were: BEPIpred 2.0, DLBEtope, IBCEL-EL and Lbtope. The best epitope of EnoSs obtained was synthesized by Solid Phase Chemical Synthesis using the Fmoc/tBut strategy, later characterized by Mass Spectrometry ms/ms and analyzed and purified by HPLC. The diagnostic platform with an electrochemical sensor was developed using the Metrohm/DropSens DRP-220AT sensor, by immobilizing the peptide in an organized monolayer, on the working electrode, with cystamine (20 mM) and glutaraldehyde (5 mM). The immunochemical reactions occurred with the positive and negative sera previously analyzed by the ELISA method, observing a reduction in the current between 0 and - 0.5 mA and with the Potential/V between 0.0 and 0.15V of the electrochemical signal transduction. The electrochemical platform developed is promising as a new immunodiagnostic method for feline sporotrichosis.
Sporotrichosis is an infection caused by fungi of species belonging to the genus Sporothrix, affecting humans and animals. This infection has a worldwide distribution, but in Brazil it’s considered emerging and represents an important threat to human and animal health. Early and more accurate laboratory diagnosis is essential as an epidemiological measure to control new infections and the spread of the disease in humans and animals. For effective control measures we need accurate and early diagnostic methods. In this context, with great potential for application in diagnostics, electrochemical immunosensors are seen as innovative tools. The aim of this thesis was to develop an immunodiagnostic platform from the Sporothrix schenckii protein enolase (EnoSs), using an electrochemical sensor, for the diagnosis of feline sporotrichosis. Based on previous studies on the immunogenic potential of the recombinant protein enolase from Sporotrhix schenckii (rSsEno), we determined the immunoreactivity of rSsEno against sera samples from positive (n=20) and negative (n=6) sporotrichosis cats, applying the indirect enzyme immunoassay (ELISA). SsEno demonstrated activity at concentrations of 1µg. From these results with rSsEno, we propose the use of this antigen in a new serological method for sporotrichosis. The prediction of more immunogenic and antigenic epitopes of EnoSs in B cells was performed with the purpose of reducing possible nonspecific reactions of the recombinant protein in a more sensitive platform based on an electrochemical sensor. The prediction web servers used were: BEPIpred 2.0, DLBEtope, IBCEL-EL and Lbtope. The best epitope of EnoSs obtained was synthesized by Solid Phase Chemical Synthesis using the Fmoc/tBut strategy, later characterized by Mass Spectrometry ms/ms and analyzed and purified by HPLC. The diagnostic platform with an electrochemical sensor was developed using the Metrohm/DropSens DRP-220AT sensor, by immobilizing the peptide in an organized monolayer, on the working electrode, with cystamine (20 mM) and glutaraldehyde (5 mM). The immunochemical reactions occurred with the positive and negative sera previously analyzed by the ELISA method, observing a reduction in the current between 0 and - 0.5 mA and with the Potential/V between 0.0 and 0.15V of the electrochemical signal transduction. The electrochemical platform developed is promising as a new immunodiagnostic method for feline sporotrichosis.
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Palavras-chave
Sporothrix brasiliensis, Felinos, Bioinformática, Biossensor, Diagnóstico, Felines, Bioinformatics, Biosensor, Diagnosis