RESUMEN
A nondefective recombinant human adenovirus 5 (Ad5) carrying the gene encoding for the porcine rotavirus outer capsid protein VP7 in the E3 region of the Ad5 genome has been obtained. mRNA for VP7 in recombinant Ad5KV14/VP7 infected cells was demonstrated by means of RT-PCR. Radioimmunoprecipitation of cell extracts infected with Ad5KV14/VP7 on early (12 h p.i.) and late (24 h p.i.) stages of adenovirus infection with rabbit polyclonal antiserum raised to purified rotavirus virions revealed a recombinant protein possessing the same electrophoretic mobility, 37 kDa, as the native rotavirus K VP7. One-dimensional peptide mapping also demonstrated the identity of the recombinant and native VP7.
Asunto(s)
Adenoviridae/genética , Antígenos Virales , Proteínas de la Cápside , Cápside/genética , Regulación Viral de la Expresión Génica , Rotavirus/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Ensayo de Radioinmunoprecipitación , Recombinación GenéticaRESUMEN
A ribozyme was constructed of the catalytic domain of tobacco ringspot virus satellite RNA and flanking sequences complementary to the target, secreted alkaline phosphatase (SEAP) mRNA. The ribozyme specifically cleaved the substrate in vitro; compared with that constant temperature. The relationship between specific endoribonuclease activity of the ribozyme and Mg2+ concentration was shown. The ribozyme was active in 293 cell line which was cotransfected with plasmids carrying the SEAP gene under control of RSV LTR promoter and the ribozyme gene under early HCMV promoter: SEAP activity reduced by half.
Asunto(s)
Fosfatasa Alcalina/genética , ARN Catalítico/genética , ARN Mensajero/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Bases , Línea Celular , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Nepovirus/genética , Oligodesoxirribonucleótidos , Plásmidos , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , ARN Viral/genéticaRESUMEN
The plasmid pH5KV14 containing the right end of the Ad5 viral genome (45-100 map units) with deleted nonessential E3 region (78.5-84 map units) has been constructed. A helper-independent recombinant virus Ad5KV14 has been obtained as a result of in vivo transformation of the line 293 human cells by the pH5KV14 plasmid DNA and an EcoRI-A DNA fragment of the virus Ad5 and subsequent recombination. A nondefective recombinant virus Ad5KV17 containing the Ad5 genome with the E3 region substituted for the plasmid DNA fragment coding for the kanamycin-resistance has been constructed by a similar procedure.
Asunto(s)
Adenoviridae/genética , ADN Recombinante/genética , Vectores Genéticos , Genoma Viral , Resistencia a la Kanamicina/genética , Células Cultivadas , Humanos , Plásmidos , Eliminación de SecuenciaRESUMEN
The terminal fragment of avian adenovirus CELO has been cloned in a plasmid vector. The obtained recombinant plasmid pCBE1 carries the terminal BamHI-E fragment of CELO DNA. Transfection of a nonpermissive culture of Rat2 cell line by the plasmid DNA results in formation of transformation focuses. The cloned BamHI-E fragment of CELO DNA is concluded to contain the viral oncogene. Thus, the CELO genome region deriving the BamHI-E fragment is "left".
