RESUMEN
OBJECTIVE: To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6. METHODS: The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed. RESULTS: There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity. CONCLUSION: The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.
Asunto(s)
Virus de la Viruela de las Aves de Corral/genética , Productos del Gen gag/genética , Proteína gp120 de Envoltorio del VIH/genética , Interleucina-6/genética , Animales , Anticuerpos Antivirales/sangre , Western Blotting , Células Cultivadas , Embrión de Pollo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Viruela Aviar/sangre , Viruela Aviar/inmunología , Viruela Aviar/virología , Virus de la Viruela de las Aves de Corral/inmunología , Productos del Gen gag/metabolismo , Vectores Genéticos/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Inmunización/métodos , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Vacunas Virales/genética , Vacunas Virales/inmunología , Vacunas Virales/metabolismoRESUMEN
A fowl pox-based recombinant virus TROVAC-NDV (vFP96.5) was developed expressing the fusion and hemagglutinin-neuraminidase glycoproteins from a velogenic strain of Newcastle disease virus (NDV). Studies in specific-pathogen-free birds indicated that inoculation of a single dose of the recombinant led to the induction of significant levels of hemagglutination-inhibiting antibody that were maintained to 8 wk postinoculation. Further, the recombinant induced protective immunity against a combined intramuscular velogenic NDV challenge and respiratory NDV challenge. In commercial broiler chickens that were inoculated in the presence of maternally derived NDV immunity, the level of the NDV-specific humoral response was dampened, but significant levels of protection against both a lethal intramuscular NDV challenge and a fowl poxvirus challenge were obtained.