Construction and normoxic expression of mutant hypoxia inducible factor-1α adenovirus vector

Objective To study the angiogenesis promoting effect of hypoxia inducible tactor (HIF)-lα gene at bone detect and loss location under normoxic conditions, construct a new adenovims vector which can express mutant hypoxia HIF-lo and human renilla reniformis green fluorescent protein (hrGFP) simultaneously and detect the expression of the vector in rabbit marrow stromal cells (MSCs).Methods Site-directed mutagenesis of the 402,564 and 803 position amino acids in HIF-1 α gene coding sequence (CDS) was done by using polymerase chain reaction (PCR) technique. The mutant HIF-1αt and hrGFP gene were recombined into pAdEasy-1 adenovirus system, the viruses were packed and the titer was assayed. The recombinant adenovirus was transfected into MSCs following MOI =100, and the HIF-1omRNA and protein expression was detected by using reverse transcription (RT)-PCR and Western blotting.Results ( 1 ) The 402, 564 and 803 amino acids all had site-directed mutagenesis into 2-aminopropionic acid. (2) There was statistically significant difference in the HIF-1α mRNA expression between group A or B and group C or D (P ＜0. 01 ). The HIF-1α protein expression in group A was significantly higher than other three groups (P ＜ 0. 01 ). Conclusion ( 1 ) A novel adenovirus vector namely Ad-CMV-HIF-1α muIRES-hrGFP-1 was constructed successfully; (2) The expression of HIF-1α gene after mutation is abundant and efficient under the normoxic conditions. Key words: Hypoxia inducible factor 1 alpha; Gene mutation; Recombinant adenovirus vector; Marrow stromal cells