RESUMEN
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.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium bovis/inmunología , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis Bovina/prevención & control , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Bovinos , Clonación Molecular , Codón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Histidina/genética , Histidina/metabolismo , Inmunogenicidad Vacunal , Interferón gamma/biosíntesis , Mycobacterium bovis/química , Mycobacterium bovis/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Alineación de Secuencia , Vacunas contra la Tuberculosis/genética , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Bovina/inmunología , Tuberculosis Bovina/microbiología , Vacunación/métodosRESUMEN
This study examined the impact of emulsifier type on the physicochemical characteristics and antifungal capacity of oregano oil-in-water emulsions: Tween 80, hydroxylated soy lecithin, and gum arabic. GC/MS analysis showed that the major components of the Lippia graveolens essential oils were thymol (31.7%), p-cymene (18.7%), and carvacrol (14.6%). The oil-in-water emulsions were made using ultrasonic technology in which thymol and carvacrol quantities were 12.26-13.67 g/L and 5.6-6.2 g/L, respectively. The droplet size of the emulsions followed the next descendent order: gum arabic > lecithin > T80. The zeta potential of the emulsions favored the stability against coalescence. Finally, the antifungal activity of the emulsions was evaluated, in which, 30 µL/mL of gum arabic or hydroxylated soy lecithin emulsions inhibited the growth of Candida albicans. The result suggests that Mexican oregano essential oil emulsions can be used as an antifungal against of C. albicans.
