RESUMEN
A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.
Asunto(s)
Pepsina A/metabolismo , Pepsinógeno C/metabolismo , Agrobacterium tumefaciens/genética , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Activación Enzimática , Vectores Genéticos , Hemoglobinas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Oryza/genética , Oryza/metabolismo , Pepsina A/química , Pepsina A/genética , Pepsinógeno C/química , Pepsinógeno C/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TemperaturaRESUMEN
The pentameric B subunit of Escherichia coli heat-labile enterotoxin (LTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic tobacco plants to express a fusion protein consisting of the synthetic LTB and a synthetic neutralizing epitope of porcine epidemic diarrhea virus (PEDV), causing an enteric disease that is especially severe in piglets. Both components of the fusion proteins were detected in Western blot analysis, and binding assay confirmed that plant-synthesized pentameric LTB-PEDV fusion bound to the intestinal membrane GM1-ganglioside receptor. This suggested that the fusion protein retained both its native antigenicity and the ability to form pentamers.
Asunto(s)
Toxinas Bacterianas/genética , Coronavirus/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Nicotiana/genética , Plantas Modificadas Genéticamente , Animales , Secuencia de Bases , Clonación Molecular , Coronavirus/inmunología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Cartilla de ADN , Epítopos/genética , Epítopos/metabolismo , Escherichia coli , Gangliósido G(M1)/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/metabolismo , Enfermedades de los Porcinos/virologíaRESUMEN
Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.
Asunto(s)
Toxinas Bacterianas/genética , Cloroplastos/metabolismo , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Nicotiana/genética , Toxinas Bacterianas/metabolismo , Enterotoxinas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Vectores Genéticos , Immunoblotting , Mapeo Físico de Cromosoma , Plantas Modificadas Genéticamente , Plásmidos , Unión Proteica , Receptores de Superficie Celular/metabolismo , Transformación GenéticaRESUMEN
Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.