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https://www.arca.fiocruz.br/handle/icict/62993
DESIGN AND DEVELOPMENT OF MULTIEPITOPE CHIMERIC ANTIGENS BY BIOINFORMATIC AND BACTERIAL BASED RECOMBINANT EXPRESSION METHODS, WITH POTENTIAL APPLICATION FOR BOVINE TUBERCULOSIS SERODIAGNOSIS
Diagnosis
Multiepitope Chimeric Antigens
Immunoassay
Enzyme-Linked Immunosorbent Assay
Diagnóstico
Inmunoensayo
Ensayo de Inmunoadsorción Enzimática
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Empresa Brasileira de Pesquisa Agropecuária (Embrapa Pecuária Sudeste). São Carlos, SP, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Semiárido). Petrolina, PE, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Gado de Corte). Mato Grosso do Sul, MS, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Pecuária Sudeste). São Carlos, SP, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Semiárido). Petrolina, PE, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Gado de Corte). Mato Grosso do Sul, MS, Brasil.
Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.
Empresa Brasileira de Pesquisa Agropecuária (Embrapa Pecuária Sudeste). São Carlos, SP, Brasil.
Abstract
Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.
Keywords
Tuberculosis BovineDiagnosis
Multiepitope Chimeric Antigens
Immunoassay
Enzyme-Linked Immunosorbent Assay
Keywords in Spanish
Tuberculosis BovinaDiagnóstico
Inmunoensayo
Ensayo de Inmunoadsorción Enzimática
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