Rescue and Amplification of Recombinant Human Adenovirus Type 41 in 293 Cells.
- Author:
Xiaohui ZOU
;
Xiaojuan GUO
;
Rong XIAO
;
Min WANG
;
Zhuozhuang LU
;
Tao HONG
- Publication Type:Journal Article
- MeSH:
Adenoviruses, Human;
genetics;
growth & development;
physiology;
Cell Line;
Genetic Vectors;
genetics;
physiology;
Green Fluorescent Proteins;
genetics;
metabolism;
Humans;
Plasmids;
genetics;
metabolism;
Recombination, Genetic;
Transfection;
Virus Assembly
- From:
Chinese Journal of Virology
2015;31(5):515-523
- CountryChina
- Language:Chinese
-
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.
