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2050-12-31
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EXPRESSÃO DE TETRASPANINA CO029 COMO BIOMARCADOR TUMORAL PARA PREPARAÇÃO DE ANTICORPOS MONOCLONAIS: AVALIAÇÃO ANTIGÊNICA EM CÉLULAS DE CÂNCER COLORRETAL
Proteínas recombinantes
Anticorpos monoclonais
Biomarcadores
Biomarcadores tumorais
Alternative title
Tetraspanin co029 expression as a tumor biomarker for monoclonal antibodies preparation: antigenic assessment in colorectal cancer cellsAuthor
Affilliation
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil/School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil/School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
School of Pharmacy. 0Federal University of Ouro Preto. Ouro Preto, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil/Infectious Diseases. University of Georgia. Athens, GA, United States
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil/School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
School of Pharmacy. 0Federal University of Ouro Preto. Ouro Preto, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
School of Medicine. Federal University of Minas Gerais. Belo Horizonte, MG, Brazil
Diagnostic and Therapy of Infectious Diseases and Cancer. Rene Rachou Institute. Oswaldo Cruz Foundation. Belo Horizonte, MG, Brazil/Infectious Diseases. University of Georgia. Athens, GA, United States
Abstract in Portuguese
Introdução: A identificação de biomarcadores de câncer inovadores é uma busca contínua muito relevante. Além disso, seu papel no diagnóstico do câncer e no manejo clínico mudou radicalmente nos últimos anos, com grande ênfase na classificação molecular do câncer, na identificação do alvo t Western blotting erapêutico e na responsividade do protocolo terapêutico. As tetraspaninas são uma família de proteínas transmembrana correlacionadas com o estágio do tumor, tipo de tumor e evolução do paciente, que afetam o crescimento celular, morfologia, invasão e metástase. Métodos: Expressamos o antígeno de tetraspanina co029 humana transmembrana de 31kDa em células hospedeiras de expressão de Escherichia coli usando a plataforma Gateway®. As técnicas de Western blotting e ELISA, juntamente com o sequenciamento de genes, confirmaram a identidade da proteína recombinante TSP co029. Obtiveram-se quarenta clones produtores de anticorpos contra TSP co029. Esses anticorpos foram incubados com células de câncer colorretal humano em diferentes condições. ELISA e imunohistoquímica também foram realizados. Resultados: A tetraspanina expressa tinha uma conformação adequada e integridade antigênica para produzir anticorpos com afinidade para o biomarcador TSP co029 nativo. A afinidade da proteína recombinante purificada e dos anticorpos foram confirmados por western blotting, coloração fluorescente de células de câncer colorretal humano em microscopias fluorescentes e confocais, e por ELISA e imunohistoquímica. Conclusão: Nossos dados mostraram que a proteína recombinante e os anticorpos produzidos neste estudo permitiram a confirmação da proteína tetraspanina co029 apresentada na superfície de células de câncer colorretal humano.
Abstract
Introduction: The identification of innovative cancer biomarkers is a very relevant ongoing quest. Moreover, their role in cancer diagnosis and clinical management has been radically changed in the last few years with the major emphasis on cancer molecular classification, therapeutic target identification, and therapeutic protocol responsivity. tetraspanins are a family of transmembrane proteins correlated with tumor stage, tumor type and patient outcome affecting cell growth, morphology, invasion, and metastasis. Methods: We expressed the 31kDa transmembrane human tetraspanin co029 antigen in Escherichia coli expression host cells using Gateway® platform. Western blotting and ELISA techniques, together with gene sequencing, confirmed the identity of TSP co029 recombinant protein. Forty clones producing antibodies against TSP co029 were obtained. These antibodies were incubated with human colorectal cancer cells in different conditions. ELISA and immunohistochemistry were also performed. Results: The expressed tetraspanin had an appropriate conformation and antigenic integrity to produce antibodies with affinity to the native TSP co029 biomarker. The affinity of the purified recombinant protein and antibodies were confirmed by western blotting, florescent staining of human colorectal cancer cells in fluorescence and confocal microscopies and by ELISA and immunohistochemistry. Conclusion: Our data showed that the recombinant protein and antibodies produced in this study allowed the confirmation of tetraspanin co029 protein presented on the surface of human colorectal cancer cells.
Keywords in Portuguese
TetraspaninasProteínas recombinantes
Anticorpos monoclonais
Biomarcadores
Biomarcadores tumorais
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