RESUMEN
Baculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10B(AC)Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was useful for HA purification.
Asunto(s)
Baculoviridae/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H5N2 del Virus de la Influenza A/genética , Animales , Western Blotting , Línea Celular , Cromatografía de Afinidad , Clonación Molecular , Concanavalina A , Fluorescencia , Proteínas Fluorescentes Verdes/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/biosíntesis , Glicoproteínas Hemaglutininas del Virus de la Influenza/aislamiento & purificación , Lectinas de Plantas , Proteínas Recombinantes de Fusión/genética , SpodopteraRESUMEN
An enzyme-linked immunosorbent assay (ELISA) using the expressed sigmaC and sigmaB proteins which induce neutralizing antibodies as the coating antigen (sigmaC-sigmaB-ELISA) for the detection of antibodies to avian reovirus in chickens was developed and compared with serum neutralization and conventional ELISA tests. These assays were used to examine the sera from chickens vaccinated experimentally and farm chickens. The correlation rate between serum neutralization and a sigmaC-sigmaB-ELISA was 100% (156/156), and that between serum neutralization and conventional ELISA was 89.1% (139/156). The results revealed that preparation of an ELISA by using sigmaC and sigmaB of ARV as the coating antigen in detecting the field chicken sera in comparison with the conventional ELISA gave a titer more correlated to the serum neutralization test. The sigmaC-sigmaB-ELISA showed a higher correlation with the serum neutralization-positive and -negative sera than that obtained with conventional ELISA. This combination antigen may thus be the best suited for preparing an ELISA for improving the determination of the immune status of chicken flocks or for detection of chicken infections with avian reovirus.
Asunto(s)
Anticuerpos Antivirales/sangre , Proteínas de la Cápside , Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Orthoreovirus Aviar/aislamiento & purificación , Enfermedades de las Aves de Corral/virología , Proteínas de Unión al ARN , Infecciones por Reoviridae/veterinaria , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Cápside/genética , Pollos , Chlorocebus aethiops , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática/normas , Expresión Génica , Pruebas de Neutralización , Orthoreovirus Aviar/genética , Orthoreovirus Aviar/inmunología , Enfermedades de las Aves de Corral/sangre , Enfermedades de las Aves de Corral/inmunología , Infecciones por Reoviridae/sangre , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Vero , Proteínas Virales/genéticaRESUMEN
Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The sigmaC and sigmaB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express sigmaC and sigmaB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB have been successfully constructed. After infection, both His6-tagged recombinant sigmaC (rsigmaC) and sigmaB (rsigmaB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rsigmaC and rsigmaB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rsigmaC and rsigmaB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-sigmaC and BacSC-sigmaB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB can be a potential vaccine against ARV infections.