Construction and expression of adeno-associated virus vector harboring human hypoxia inducible factor-1α gene
- Author:
Xi-Qing CHAI
1
Author Information
1. Graduate School of Hebei Medical University
- Publication Type:Journal Article
- Keywords:
Adeno-associated virus;
Hippocampus;
Hypoxia-inducible factor-1α;
Primary culture
- From:
Academic Journal of Second Military Medical University
2010;28(6):633-636
- CountryChina
- Language:Chinese
-
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.
