[Efficient generation of recombinant adenoviruses expressing antiangiogenic fragment of human thrombospondin1].
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.
Liu
Peng
P
State Key Laboratory of Experimental Hematology, Institute of Hematology, Blood Diseases Hospital, CAMS, PUMC, Tianjin 300020, China.
Wang
Yi
Y
Yang
Ren-chi
RC
Gu
Jie
J
Cai
Ying-lin
YL
Han
Zhong-chao
ZC
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000096:biosynthesis / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000098:blood supply; D009389:Neovascularization, Pathologic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D019700:Thrombospondin 1 / Q000096:biosynthesis / Q000235:genetics*
D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D019700:Thrombospondin 1