Establishment of A Ⅲa-Deleted Human Type-7 Adenoviral Vector System
We wished to obtain an adenoviral vector that can efficiently amplify exogenous genes without producing replicative progeny viruses.We developed a"single cycle"adenoviral vector with one deletion of theⅢ a gene.This study was conducted on the basis of pK-Ad7E3GFP,a previously constructed human adenovirus type-7 vector carrying green fluorescent protein(GFP)in the E3 region.First,primers were designed to amplify the AMP-ORI fragment containing the ampicillin-resistance gene and the plasmid origin of replication.pK-Ad7E3GFP was digested with restriction enzyme Asc I to generate two fragments of 29.1 kb and 7.7 kb in size.Then,the 7.7-kb fragment was assembled with the AMP-ORI fragment to produce the first intermediate plasmid pA-Ad7E3GAscI.Using overlap-extension polymerase chain reaction(PCR),enzyme digestion,and DNA assembly,the Ⅲ a gene on pA-Ad7E3GAscI was removed,and the second intermediate plasmid pA-AscD Ⅲ a was produced.The plasmid pA-AscD Ⅲ a was digested with Pme I,which was mixed with the 29.1-kb fragment by Gibson DNA assembly,to construct the recombinant adenoviral plasmid pK-Ad7D Ⅲ aE3GFP.Meanwhile,the eukaryotic expression vector pTPL-Ⅲ a carrying the Ⅲ a gene was constructed and transfected into 293 cells to obtain stably transfected 293-Ⅲ a cells by G418 screening.Pme I-linearized pK-Ad7D ⅢaE3GFP was used to transfect 293-Ⅲ a cells.GFP foci were observed on monolayer cells 7 days after transfection,suggesting rescue of the Ad7D Ⅲ aE3GFP recombinant virus.The viral genome was extracted for identification by PCR,and the results were as expected.The typical icosahedral morphology of adenovirus was seen under an electron microscope after negative staining of cell-culture supernatants.The virus was also seen in a lattice arrangement in cultured cells.All of these observations suggest rescue of Ad7D Ⅲ AE3GFP.We established a single-cycle human type-7 adenoviral vector with deletion of the Ⅲ a gene.Our data could lay the foundation for the research and application of this vector.
Ⅲ a geneHuman adenovirus type-7 vectorSingle-cycle vectorDNA assemblyVirus rescue
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