RESUMEN
The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the promoter for T7 RNA polymerase. Recombinant NP was synthesized in Escherichia coli and in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coli and of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.
Asunto(s)
Marburgvirus/metabolismo , Nucleoproteínas/metabolismo , Proteínas de Unión al ARN , Ribonucleoproteínas , Proteínas Virales , Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Recombinante , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Escherichia coli/genética , Humanos , Microscopía Electrónica/métodos , Proteínas de la Nucleocápside , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
Examining the specific activity has showed that recombinant vaccinia virus growth factor binds to appropriate receptors on the A-431 cell surface and prompts the healing acceleration of degree III burns in rats. This recombinant factor did not demonstrate pyrogenicity or toxicogenicity in tests on rabbits, guinea-pits, noninbred albino mice.
Asunto(s)
Quemaduras/tratamiento farmacológico , Epidermis/fisiología , Sustancias de Crecimiento/uso terapéutico , Virus Vaccinia/metabolismo , Cicatrización de Heridas/fisiología , Animales , Quemaduras/metabolismo , Quemaduras/patología , Células Cultivadas , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Femenino , Estudios de Seguimiento , Cobayas , Ratones , Microscopía Inmunoelectrónica , Conejos , Ratas , Proteínas Recombinantes , Resultado del Tratamiento , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Several detergents and chemical compounds--Tweens 40, 60, 80, Triton X-100, Triton WR-1340, polyvinylpyrrolidone, dimethylsulfoxide, urea, n-butyl, MESK, and combinations thereof were used for the isolation of surface proteins of vaccinia virus. Optimal conditions for the treatment of the virus with detergents were selected, permitting isolation of vaccinia virus surface proteins p35 and p61. Mouse experiments yielded data on the protective properties of the isolated proteins. Protein p35 may turn to be one of the major proteins responsible for the formation of protective immunity in vaccination with vaccinia virus.
Asunto(s)
Detergentes , Inmunidad/fisiología , Virus Vaccinia/fisiología , Proteínas del Envoltorio Viral/fisiología , Animales , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Microscopía Electrónica , Virus Vaccinia/ultraestructura , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
Comparative structural analysis of the 36K proteins of vaccinia, ectromelia, cowpox and variola viruses revealed that it is conservative among Orthopoxviruses. The possible role of this protein was suggested. A study of the synthesis kinetics of vaccinia and ectromelia virus 36K proteins established that they belong to late proteins. Electron microscopy of infected cells using protein A labelled with colloidal gold showed that the 36K protein is located in the viroplast and not coupled to virions or any cell organelles.
Asunto(s)
Secuencia Conservada , Genes Virales , Virus Vaccinia/genética , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Chlorocebus aethiops , Microscopía Electrónica , Datos de Secuencia Molecular , Virus Vaccinia/ultraestructuraRESUMEN
Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of beta-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.