RESUMO
For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.
Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Epitopos/genética , Epitopos/metabolismo , Escherichia coli/genética , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/química , Epitopos/química , Epitopos/imunologia , Escherichia coli/metabolismo , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Febre Aftosa/virologia , Vírus da Febre Aftosa/química , Vírus da Febre Aftosa/classificação , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Glutationa Transferase/metabolismo , Imunização , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Reprodutibilidade dos Testes , Sorotipagem , Vacinas Virais/biossíntese , Vacinas Virais/química , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
Foot-and-mouth disease (FMD) is caused by the FMD virus (FMDV) and results in severe economic losses in livestock farming. For rapid FMD diagnostic and therapeutic purposes, an effective antibody against FMDV is needed. Here, we developed a high-affinity antibody against FMDV by FACS-based high throughput screening of a random library. With the FITC-conjugated VP1 epitope of FMDV and high-speed FACS sorting, we screened the synthetic antibody (scFv) library in which antibody variants are displayed in the periplasm of Escherichia coli. After three rounds of sorting, we isolated one antibody fragment (#138-scFv) against the VP1 epitope of FMDV. Next, to improve its affinity, a mutation library of #138-scFV was constructed by error-prone PCR and screened by FACS. After three rounds of sorting, we isolated one antibody (AM-32 scFv), which has a higher binding affinity (KD=42.7nM) than that of the original #138-scFv. We also confirmed that it specifically binds to whole inactivated FMDV.