Abstract:

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) can regulate the co-expression of various genes, and can induce angiogenesis with integrated physiological function.
OBJECTIVE: To construct a novel adenoviral eukaryotic expression vector that can co-express mutant hypoxia-inducible factor-1 alpha (HIF-1α) target protein and humanized Renilla reniformis green fluorescent protein (hrGFP) reporter molecule under normoxic conditions.
METHODS: The human HIF-1α gene carried by target gene donor plasmid pCMV6-XL5-HIF1α was sequenced and the site of restriction enzyme in above gene was analyzed. Site-directed mutagenesis of three amino acids including the 402 location, the 564 location, and the 803 location in gene coding region in HIF-1α were performed by polymerase chain reaction and sequencing was also done for monitoring mutation. The HIF-1α gene mutated correctly (HIF-1αmu) was coupled to adenoviral shuttle vector pShuttle-CMV-IRES- hrGFP-1. The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cells after sequencing identification and Pme Ⅰ restriction enzyme linearization. HIF-1αmu and hrGFP gene as well as hemeo-expression elements of hrGFP gene were reconstructed into adenoviral genome plasmids using homologous recombination mechanism in bacterium. Recombinants were obtained by Pac Ⅰ restriction enzyme digestion and sequencing identification.
RESULTS AND CONCLUSION: Amino acids including the 402 location, the 564 location and the 803 location in gene coding region in HIF-1α had become alanine after site-directed mutagenesis. Recombinant adenoviral expressing vector was successful as confirmed by restriction enzyme digestion and sequencing. These findings demonstrate that a novel recombinant adenoviral mutant eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was successfully constructed.