RESUMEN
The E2 envelope protein is the main protective antigen of classical swine fever virus (CSFV). Importantly, gram-positive enhancer matrix (GEM) particles can work as an immunostimulant and/or carrier system to improve the immune effect of antigens. In this study, the artificially designed E2-Spy was expressed and glycosylated in Pichia pastoris, and subsequently conjugated with SpyCatcher-PA which was expressed in Escherichia coli. The conjugated E2-Spy-PA was displayed on the surface of GEM particles, generating the E2-Spy-PA-GEM complex. Blocking ELISA analysis and neutralization assays showed that both E2-Spy and E2-Spy-PA-GEM complexes induced high levels of anti-CSFV antibodies in mice. Furthermore, statistical analyses indicated that the E2-Spy-PA-GEM complex exhibited enhanced immunogenicity compared with E2-Spy alone.
Asunto(s)
Anticuerpos Antivirales/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Proteínas del Envoltorio Viral , Inmunidad Adaptativa , Animales , Virus de la Fiebre Porcina Clásica/metabolismo , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Saccharomycetales/genética , Porcinos , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.