BACKGROUND: Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness. METHODS: An Ad41 E1B55K-transduced 293 cell line (293E12) was established as the packaging cell line. A backbone plasmid (pAdbone41) and a shuttle plasmid (pSh41-CMV) were constructed based on the Ad41 genome. Replication-defective adenovirus (Ad41-GFP) was rescued in 293E12 after being transfected with the linearized adenoviral plasmid, which was generated by homologous recombination of pAdbone41 and the shuttle plasmid carrying the GFP gene in Escherichia coli strain BJ5183. The packaging ability of 293E12, the stability of the Ad41-GFP genome and the acid-resistant property of Ad41-GFP were all investigated. RESULTS: A 293E12 cell could produce approximately 9000 viral particles of Ad41-GFP, which is close to the amount in the control virus (Ad5-GFP) amplified in one 293 cell. Ad41-GFP contained a genetically stable genome after being passaged eight times in 293E12 cells. More significantly, Ad41-GFP was more resistant to acid exposure than Ad5-GFP. It retained almost complete viability when exposed to hydrochloric acid with a pH value of 2 for 30 min, whereas Ad5-GFP lost 99% of its viability under the same conditions. Ad41-GFP was also more tolerant to treatment with artificial digestive fluid. CONCLUSIONS: An Ad41 vector system was successfully constructed, which consisted of the backbone plasmid, shuttle plasmid and packaging cell line 293E12. This system can be utilized to generate genetically stable and acid-resistant recombinant Ad41 carrying any gene of interest. Copyright (c) 2008 John Wiley & Sons, Ltd.
BACKGROUND:Human adenovirus serotype 41 (Ad41) is a natural pathogen of the digestive tract and can cause gastroenteritis. There has been interest in reconstructing Ad41 as a gene delivery vector targeting the gastrointestinal tract, which is hampered by its fastidiousness. METHODS: An Ad41 E1B55K-transduced 293 cell line (293E12) was established as the packaging cell line. A backbone plasmid (pAdbone41) and a shuttle plasmid (pSh41-CMV) were constructed based on the Ad41 genome. Replication-defective adenovirus (Ad41-GFP) was rescued in 293E12 after being transfected with the linearized adenoviral plasmid, which was generated by homologous recombination of pAdbone41 and the shuttle plasmid carrying the GFP gene in Escherichia coli strain BJ5183. The packaging ability of 293E12, the stability of the Ad41-GFP genome and the acid-resistant property of Ad41-GFP were all investigated. RESULTS: A 293E12 cell could produce approximately 9000 viral particles of Ad41-GFP, which is close to the amount in the control virus (Ad5-GFP) amplified in one 293 cell. Ad41-GFP contained a genetically stable genome after being passaged eight times in 293E12 cells. More significantly, Ad41-GFP was more resistant to acid exposure than Ad5-GFP. It retained almost complete viability when exposed to hydrochloric acid with a pH value of 2 for 30 min, whereas Ad5-GFP lost 99% of its viability under the same conditions. Ad41-GFP was also more tolerant to treatment with artificial digestive fluid. CONCLUSIONS: An Ad41 vector system was successfully constructed, which consisted of the backbone plasmid, shuttle plasmid and packaging cell line 293E12. This system can be utilized to generate genetically stable and acid-resistant recombinant Ad41 carrying any gene of interest. Copyright (c) 2008 John Wiley & Sons, Ltd.
Authors: Hang Shi; Irene Xagoraraki; Kristin N Parent; Merlin L Bruening; Volodymyr V Tarabara Journal: Appl Environ Microbiol Date: 2016-07-29 Impact factor: 4.792
Authors: Marta Miralles; María Mercedes Segura; Meritxell Puig; Assumpció Bosch; Miguel Chillon Journal: PLoS One Date: 2012-07-31 Impact factor: 3.240