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Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning.
- Source :
-
Journal of Biotechnology . Jan2020, Vol. 307, p193-201. 9p. - Publication Year :
- 2020
-
Abstract
- • Easy-to-use approaches are lacking for site-directed modification of adenovector (Ad). • Construct an intermediate plasmid for site-directed mutagenesis by DNA assembly. • Restore modified intermediate plasmid to adenoviral plasmid by restriction-ligation. • Combined DNA assembly and restriction-ligation can be used for Ad modification. Commonly used and well accepted approaches are lacking for site-directed modification of adenoviral vectors. Here, we attempt to introduce an easy-to-implement strategy for such purpose with an example of establishing a replication competent adenoviral vector system from pKAd5 plasmid, an infectious clone of human adenovirus 5 (HAdV-5). PCR products of GFP expression cassette and plasmid backbone were fused with the EcoRI/NdeI-digested fragment of pKAd5 to generate a modified intermediate plasmid pMDXE3GA by DNA assembly. NdeI-digested fragment of pMDXE3GA was brought back to pKAd5 to form the adenoviral plasmid pKAd5XE3GA by restriction-ligation cloning. Recombinant adenovirus HAdV5-XE3GA was rescued, amplified and purified. The expression of GFP and the propagation of virus in adherent HEp-2 and suspension K562 cells were investigated. Expression of target gene was significantly enhanced in both cell lines infected with HAdV5-XE3GA due to virus replication. However, propagation of virus could not sustain in culture of K562 cells. Shuttle plasmid pSh5RC-GFP was constructed to facilitate exchange of transgene. In summary, the strategy of combined DNA assembly and restriction-ligation cloning is functional, cost-effective and suitable for genetic modification of adenovirus. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 01681656
- Volume :
- 307
- Database :
- Academic Search Index
- Journal :
- Journal of Biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 140316357
- Full Text :
- https://doi.org/10.1016/j.jbiotec.2019.11.009