Construction of adenovirus-mediated eukaryotic expression vector co-expressing mutant hypoxia-inducible factor-1 alpha target protein and humanized Renilla reniformis green fluorescent protein reporter molecule under normoxic conditions
10.3969/j.issn.1673-8225.2010.20.041
- VernacularTitle:常氧条件下同时表达突变型低氧诱导因子1α目的蛋白和人源化绿色荧光蛋白报告分子腺病毒真核表达载体的构建
- Author:
Danping LIU
;
Guoxian WANG
;
Liang HU
;
Chen LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2010;14(20):3787-3792
- CountryChina
- Language:Chinese
-
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-1a) 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.
