RESUMO
Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 µg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Mycobacterium bovis/imunologia , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Bovina/prevenção & controle , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Bovinos , Clonagem Molecular , Códon , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Imunogenicidade da Vacina , Interferon gama/biossíntese , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Vacinas contra a Tuberculose/genética , Vacinas contra a Tuberculose/imunologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia , Vacinação/métodosRESUMO
Epithelioid sarcoma is a rare, aggressive soft tissue tumor of unknown histogenesis showing predominantly epithelioid cytomorphology. We conducted a conventional and molecular cytogenetic study of a 27-year-old male with epithelioid sarcoma with angiomatoid features. Cytogenetic analysis of epithelioid sarcoma metaphase spreads by GTG-banding revealed a diploid chromosome complement with structural and numerical aberrations. Comparative genomic hybridization analysis demonstrated the amplification of 3p24-pter, 4p15.2-p16 and 18q23, while chromosome losses involved 3p13-p14, 3q24-q26.1, 9q21, and 11q21. Fluorescence in situ hybridization assessment showed normal hybridization patterns for the C-MYC and CCND1 loci; CCND1 RNA overexpression was detected by real-time polymerase chain reaction analysis. Genetic evaluation of this rare condition may be useful in determining if epithelioid sarcoma is associated with a distinct genetic background.