Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.
Vet Immunol Immunopathol
; 269: 110729, 2024 Mar.
Article
em En
| MEDLINE
| ID: mdl-38377627
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 8â¯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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Tuberculose Bovina
/
Doenças dos Bovinos
Limite:
Animals
Idioma:
En
Revista:
Vet Immunol Immunopathol
Ano de publicação:
2024
Tipo de documento:
Article
País de publicação:
Holanda