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Updated: Aug 7, 2026
Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5
Published on: January 28, 2016
[Efficient generation of recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1]
Peng Liu1, Yi Wang, Ren-chi Yang
1State Key Laboratory of Experimental Hematology, Institute of Hematology, Blood Diseases Hospital, CAMS, PUMC, Tianjin 300020, China.
Objective:
To construct recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1 (TSP1f).
Methods:
TSP1f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was subcloned into a shuttle vector pShuttle-CMV. After sequence confirmation, the resultant plasmid was linearized by the restriction endonuclease Pme I and cotransformed with the supercoiled adenoviral vector pAdEasy-1 into Escherichia coli strain BJ5183. Recombinants were selected by Kanamycin resistance and screened by restriction endonuclease digestion. Then, the recombinant adenoviral construct was cleaved with Pac I and transfected into the packaging cell line 293. The adenoviral vector ADV-TSP1f was propagated in 293 cells and purified by cesium chloride (CsCl) density centrifugation. PCR and Western blot analysis were performed to confirm TSP1f expression.
Results:
Of 43 Kanamycin-resistant colonies obtained from cotransformation, all of the 10 smallest ones were the correct recombinants. TSP-1f was expressed efficiently by ADV-TSP1f. The virus stock titer after CsCl banding was 1.0 x 10(11) pfu/mL.
Conclusions:
Generating recombinant adenoviruses using AdEasy System results in highly efficient viral production and significantly decrease the time required to construct usable viruses. ADV-TSP1f can be further used in in vivo gene therapy studies.
