Cruzipaína (Czp1) como bioindicador no diagnóstico de Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) em suco de açaí (Euterpe oleraceae) por ensaio Imunoenzimático
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Data
2023-03-15
Autores
Garcia, Alef Henrique
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Universidade Estadual Paulista (Unesp)
Resumo
A doença de Chagas é uma enfermidade infecciosa que afeta principalmente populações carentes em países menos desenvolvidos. É endêmica em 21 países das Américas, atingindo cerca de 6 a 7 milhões de pessoas e causando uma média de 14.000 mortes anualmente. O agente causador é o protozoário flagelado Trypanosoma cruzi, que é veiculado por triatomíneos, insetos hematófagos conhecidos como "barbeiros". Este estudo teve como objetivo avaliar as proteínas secretadas pela cepa Y (TcII) de T. cruzi em sua forma tripomastigota em solução salina tamponada com fosfato (PBS). Para tanto, técnicas de biologia molecular e bioquímica foram utilizadas para identificar a cisteína protease cruzipaína por anticorpos e analisar a quantidade secretada em suco de açaí por meio de Western blot. A solução de formas tripomastigotas em PBS foi utilizada como padrão para verificar se a quantidade secretada de cruzipaína era suficiente para um teste imunoenzimático. Utilizou-se o plasmídeo pET21a contendo o gene da cisteína protease natural cruzipaína Wild Type (W.T), com uma cauda His6-tag, transformado para expressão em E. coli resistente à ampicilina como controle positivo. Para a metaciclogênese induzida in vitro, utilizou-se a cepa Y de T. cruzi e camundongos heterogênicos machos da linhagem Swiss. Os animais com parasitemia alta foram eutanasiados para coleta de sangue, que foi cultivado em meio LIT. A cruzipaína recombinante expressa em Bl21 foi confirmada por Western blot com banda de 37 kDa. Os dados apresentados indicam que o método utilizado para expressar a cruzipaína em Bl21 foi eficiente, produzindo uma quantidade satisfatória de secretomas. As etapas foram consideradas efetivas para alcançar esse resultado. Os métodos são considerados preliminares e requerem validação e testes adicionais para determinar sua eficácia na detecção de cruzipaína em suco de açaí. No entanto, os resultados obtidos são promissores e ajudam a entender as propriedades da cruzipaína recombinante em ensaios imunoenzimáticos e em amostras de suco de açaí. É importante enfatizar que medidas devem ser tomadas para preservar a integridade da proteína durante o processo de quantificação de proteínas. A degradação do anticorpo usado afeta a eficiência do método dot blot.
Chagas disease is an infectious disease that primarily affects disadvantaged populations in less developed countries. It is endemic in 21 countries in the Americas, affecting around 6 to 7 million people and causing an average of 14,000 deaths annually. The causative agent is the flagellated protozoan Trypanosoma cruzi, which is transmitted by triatomine bugs, blood-sucking insects known as "kissing bugs". This study aimed to evaluate the proteins secreted by the Y strain (TcII) of T. cruzi in its trypomastigote form in phosphate-buffered saline (PBS) solution. Molecular biology and biochemistry techniques were used to identify the cysteine protease cruzipain by antibodies and to analyze the secreted quantity in açaí juice by Western blot. The trypomastigote forms in PBS solution were used as a standard to verify whether the secreted quantity of cruzipain was sufficient for an immunoassay test. The pET21a plasmid containing the natural cysteine protease gene cruzipain Wild Type (W.T), with a His6-tag, transformed for expression in ampicillin-resistant E. coli, was used as a positive control. For the in vitro induced metacyclogenesis, the Y strain of T. cruzi and male heterozygous Swiss mice were used. Animals with high parasitemia were euthanized for blood collection, which was cultured in LIT medium. The recombinant cruzipain expressed in Bl21 was confirmed by Western blot with a band of 37 kDa. The presented data indicate that the method used to express cruzipain in Bl21 was efficient, producing a satisfactory quantity of secretomes. The steps were considered effective to achieve this result. The methods are considered preliminary and require validation and additional tests to determine their effectiveness in detecting cruzipain in açaí juice. However, the results obtained are promising and help to understand the properties of recombinant cruzipain in immunoassays and açaí juice samples. It is important to emphasize that measures should be taken to preserve the protein integrity during the protein quantification process. The degradation of the used antibody affects the efficiency of the dot blot method.
Chagas disease is an infectious disease that primarily affects disadvantaged populations in less developed countries. It is endemic in 21 countries in the Americas, affecting around 6 to 7 million people and causing an average of 14,000 deaths annually. The causative agent is the flagellated protozoan Trypanosoma cruzi, which is transmitted by triatomine bugs, blood-sucking insects known as "kissing bugs". This study aimed to evaluate the proteins secreted by the Y strain (TcII) of T. cruzi in its trypomastigote form in phosphate-buffered saline (PBS) solution. Molecular biology and biochemistry techniques were used to identify the cysteine protease cruzipain by antibodies and to analyze the secreted quantity in açaí juice by Western blot. The trypomastigote forms in PBS solution were used as a standard to verify whether the secreted quantity of cruzipain was sufficient for an immunoassay test. The pET21a plasmid containing the natural cysteine protease gene cruzipain Wild Type (W.T), with a His6-tag, transformed for expression in ampicillin-resistant E. coli, was used as a positive control. For the in vitro induced metacyclogenesis, the Y strain of T. cruzi and male heterozygous Swiss mice were used. Animals with high parasitemia were euthanized for blood collection, which was cultured in LIT medium. The recombinant cruzipain expressed in Bl21 was confirmed by Western blot with a band of 37 kDa. The presented data indicate that the method used to express cruzipain in Bl21 was efficient, producing a satisfactory quantity of secretomes. The steps were considered effective to achieve this result. The methods are considered preliminary and require validation and additional tests to determine their effectiveness in detecting cruzipain in açaí juice. However, the results obtained are promising and help to understand the properties of recombinant cruzipain in immunoassays and açaí juice samples. It is important to emphasize that measures should be taken to preserve the protein integrity during the protein quantification process. The degradation of the used antibody affects the efficiency of the dot blot method.
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Palavras-chave
Doença de Chagas, Trypanosoma cruzi, Técnicas Imunoenzimáticas, Cisteína Proteases, Euterpe