Construction and expression of adeno-associated virus vector harboring human hypoxia inducible factor-1α gene / 第二军医大学学报
Academic Journal of Second Military Medical University
;
(12): 633-636, 2010.
Article
in Chinese
| WPRIM
| ID: wpr-841113
ABSTRACT
Objective:
To construct a recombinant adeno-associated virus (AAV) vector harboring human hypoxia inducible factor-1α (HIF-1α) gene and to express it in primarily cultured neurons in the hippocampus.Methods:
Human HIF-1α gene was inserted into AAV vector plasmid pSNAV 2.0 to generate a recombinant plasmid pSNAV-HIF-1α, then pSNAV-HIF-1α was transfected into BHK-21 cells and selected by G418. The construction of rAAV-HIF-1α was achieved by infecting G418-resistant BHK-21 cells with recombinant herpes simplex virus. The recombinant rAAV-HIF-1α was confirmed by polymerase chain reaction(PCR). The titer of rAAV-HIF-1α was detected by dot-blot with digoxin labelled cytomegalovirus probe. The purity of rAAV-HIF-1α was detected by SDS-PAGE. The expression of HIF-1α protein was determined by Western blot.Results:
PCR demonstrated that human HIF-1α gene was successfully inserted into rAAV-HIF-1α. The Virus titer was 1 × 1012/ml and the virus purity exceeded 98%. Western nblot analysis demonstrated that rAAV-HIF1α infected hippocampal neurons expressed HIF-1α protein.Conclusion:
rAAV-HIF-1α has been successfully constructed and it is capable of expressing HIF-1α in cultured hippocampal neurons.
Full text:
Available
Index:
WPRIM (Western Pacific)
Language:
Chinese
Journal:
Academic Journal of Second Military Medical University
Year:
2010
Type:
Article
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