Rescue and Amplification of Recombinant Human Adenovirus Type 41 in 293 Cells / 病毒学报
Chinese Journal of Virology
;
(6): 515-523, 2015.
Artigo
em Chinês
| WPRIM
| ID: wpr-296254
ABSTRACT
Human adenovirus type 41 (HAdV-41) is considered to be a "fastidious adenovirus". E1-deleted HAdV-41 cannot be rescued or amplified in 293 cells. To propagate recombinant HAdV-41 in 293 cells, the backbone plasmid pAdbone41 was reconstructed. That is, the E3 coding sequence of HAdV-41 was deleted and replaced with the HAdV-5 E4orf6 gene; and the E1A enhancer of HAdV-5 was inserted upstream of the E4 promoter of HAdV-41. Novel adenoviral plasmid pAd41E4EE-GFP was generated by homologous recombination of the shuttle plasmid pSh41-GFP with the modified backbone plasmid in the Escherichia coli BJ5183 strain. Adenovirus HAdV-41-E4EE-GFP was rescued by transfecting 293 cells with linearized pAd41E4EE-GFP. After seven rounds of propagation, viruses were purified by the CsCl ultracentrifugation method. HAdV-41-E4EE-GFP in 1.0 ml with a particle titer of 8 x 10(10) vp/mL was obtained which had a particle-to-infectious ratio of 50 1. The genome of HAdV-41-E4EE-GFP was confirmed by restriction analyses and polymerase chain reaction. These results showed that a novel HAdV-41 vector system was established in which recombinant HAdV-41 could be constructed and packaged in 293 cells.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Fisiologia
/
Plasmídeos
/
Recombinação Genética
/
Transfecção
/
Linhagem Celular
/
Adenovírus Humanos
/
Montagem de Vírus
/
Proteínas de Fluorescência Verde
/
Vetores Genéticos
/
Genética
Limite:
Humanos
Idioma:
Chinês
Revista:
Chinese Journal of Virology
Ano de publicação:
2015
Tipo de documento:
Artigo
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