Abstract:

BACKGROUND: Hypoxia inducing factor 1 is able to regulate co-expression of various gene and induce new vascular generation in bone defects areas. It can supply nutritional support and metabolism promotion for osteogenesis differentiation and osteogenesis activity in cells and accelerate bone healing. However, studies regarding the construction and expression of hypoxia inducing factor 1 are few. 
OBJECTIVE: To construct a new adenovirus eukaryotic expression vector which can express mutant hypoxia inducible factor 1 α (HIF1-α) interest protein and reporter molecule of human renilla reniformis green fluorescent protein (hrGFP) and to transfect it into HEK293 cells to observe its expression.
METHODS: Three aminoacid including the 402 location, the 564 location and the 803 location in gene coding region in donor vector pCMV6-XL5-HIF1α carrying HIF-1α were selected to complete site-directed mutagenesis and add new enzyme sites including Not Ⅰ and Pvu Ⅰ after removing stop codon pre and post gene sequence by polymerase chain reaction. The mutation was monitored by restriction enzyme and sequencing. The correct HIF-1α gene mutation HIF-1αmu was linked into adenovirus shuttle vector pShuttle-CMV-IRES- hrGFP-1 directionally. The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cell after sequencing identification and Pme Ⅰ restriction enzyme linearization. The transfection status was determined by hrGFP fluorescent expression.
RESULTS AND CONCLUSION: Aminoacid including the 402 location, the 564 location and the 803 location in gene coding region in HIF-1α turned to alanine after rite-directed mutagenesis and removing stop codon was successful. Enzyme restriction and sequencing confirmed that the recombinant adenoviral expressing vector was successful constructed. Results of fluorescence microscope showed there was a large number of green fluorescence expression in HEK293A cells, which confirmed that the recombinant adenovirus vector was successful transfected into HEK293A cells by Lipofectamine 2000.