Site-directed modification of adenoviral vector with combined DNA assembly and restriction-ligation cloning.
J Biotechnol
; 307: 193-201, 2020 Jan 10.
Article
in En
| MEDLINE
| ID: mdl-31751597
ABSTRACT
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Adenoviridae
/
Genetic Vectors
Limits:
Humans
Language:
En
Journal:
J Biotechnol
Journal subject:
BIOTECNOLOGIA
Year:
2020
Document type:
Article
Affiliation country:
China