Characterization of the biological activities of lyophilized recombinant adenovirus expressing the triple mutant of hypoxia inducible factor-1α.
- Author:
Yu TAO
1
;
Yue-gang WANG
;
Xuan WEI
;
Cheng LIU
;
Dong-dong CHEN
;
Ping-sheng WU
Author Information
- Publication Type:Journal Article
- MeSH: Adenoviridae; genetics; metabolism; Antibodies, Monoclonal; genetics; Freeze Drying; Genetic Vectors; HEK293 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; biosynthesis; genetics; Mutant Proteins; genetics; metabolism
- From: Journal of Southern Medical University 2011;31(3):469-472
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α (Ad-HIF-1α-564/402/803).
METHODSAd-HIF-1α-564/402/803 was amplified from HEK293A cells and purified by ultracentrifugation in CsCl gradient solutions. The infection efficiency was observed by X-gal staining. The lyophilized adenovirus was prepared under appropriate conditions. Before and after lyophilization, the effect of Ad-HIF-1α-564/402/803 on hMVEC proliferation was evaluated by MTS assay. The recombinant adenovirus was confirmed by PCR and DNA sequence analysis before and 1 day, 6 months and 12 months after lyophilization, and hMVECs infected with Ad-HIF-1α-564/402/803 at these time points were examined for HIF-1α protein expression using Western blotting.
RESULTSNo significant changes were observed in the effect of lyophilized Ad-HIF-1α-564/402/803 on hMVECs proliferation at the optimal multiplicity of infection of 100 pfu/cell (P>0.05). At the 4 time points, the recombinant adenovirus HIF-1α showed no structural alterations or significant changes in the expression level of HIF-1α protein in the transfected hMVECs (P>0.05).
CONCLUSIONLyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.
