HIF-1 plays an important role in promoting angiogenesis and microvascular system formation[7]. HIF-1 is a transcription factor which widely exists in the mammalian and human body under hypoxic conditions, consisting of α and β two subunits. Its physiological function is primarily regulated by α subunit[8-10]. HIF-1α is sensitive to oxygen concentration in the local microenvironment in vivo[11].
Hypoxia in local tissue can induce HIF-1 expression, and HIF-1 expression correlates to hypoxia in time-dependent manner[12-14]. Accumulative evidence[15-16] exists that under normoxic conditions, plasmid expressing exogenous HIF-1α can acquire the physiological function similar to hypoxia induction. But under normoxic conditions, HIF-1α produced by cells would be quickly degraded. The underlying mechanism is that HIF-1α CDS region encodes 826 amino acids, in which there is an oxygen-dependent degradation domain composed by more than 200 amino acids, under the catalysis of proline hydroxylase, the 402 and 564 location proline residues in the HIF-1α molecule ODD region are hydroxylated, and then HIF-1α would be degraded by ubiquitin-specific protease.
Lando et al[17] reported that the 803 location asparagine in the COOH-terminal transactivation domain determines the transcription activity of HIF-1α, and terminating the hydroxylation by chemical inhibitor or gene recombination would lead to strong transcription activity of HIF-1α.
Wan et al[18] reported that during the stage of bone formation in mouse models, HIF-1α is a necessary regulatory factor for mature angiogenesis and can accelerate bone tissue regeneration. Wang et al[19] confirmed that during bone formation in mice, HIF-1α was highly expressed, the concentration of vascular endothelial growth factors was increased, obvious vascularization with no adverse reactions was observed.These findings demonstrated that HIF-1α pathway during bone development was closely related to complete vascularization.
Elson et al[3] reported that HIF-1α without ODD can induce angiogenesis in newly formed skin with integreted function. Generally, large fragment of gene loss (such as ODD) can cause protein function loss which has not been found. HIF-1α with point mutation expresses protein structure similar to that expressed by normal HIF-1α. Changing some properties of protein, for example, easy to be degraded and inactivated, benefits for maintaining the physiological function of protein. Therefore, amino acid substitution is a sensible choice for HIF-1α gene recombination.
In 2001, Ivan et al[20] performed site-specific mutagenesis of HIF-1α Pro564 location into alanine and found that mutant HIF-1α was more stable and hard to be degraded than wide-type HIF-1α. Jaakkola et al[21] did identical experiments and acquired similar outcomes. Lando et al[17] reported that HIF-1α activity was lower when one amino acid was substituted by proline 564 than two animo acids were substituted by recombinant ODD proline 564 and CAD region asparagine 803, then theoretically, substitution of three amino acids (including proline 402, 564, and asparagine 803) would yield the maximum activity of HIF-1α under normoxic conditions.
Bacterial homologous recombination is a high-efficiency method to construct adenoviral vector[22]. This method first needs to construct adenoviral shuttle plasmid carrying target gene, then which was transfected with E coli BJ5183 together with adenoviral backbone plasmid. Target gene and hemeo-expression elements were inserted into adenoviral genome plasmids using homologous recombination mechanism in bacterium.
pShuttle-CMV-IRES-hrGFP-1, a novel adenoviral shuttle plasmid developed by Stratagene Company in USA, possesses hemeo-expression elements and homologous recombination sequence which are necessary in eukaryotic gene expression, including CMV promotor, multiple clone site, and PolyA signal region. FLAG epitope gene (FLAG is artificially synthesized epitope, and its amino acid residue sequence is DYKDDDDK), internal ribosome entry site (IRES) and hrGFP gene are designed, arranged accordingly after polyclonal site. Because of presence of IRES, target gene inserted into polyclonal sites and GFP gene are simutaneously expressed in the form of double cistrons. Double gene expression vector is a new development direction of vector reconstruction, which expresses hrGFP reporter gene and HIF-1α target gene in the form of double cistrons and at the same time, HIF-1α protein expression can be easily detected. Compared with enhanced green fluorescent protein (EGFP), the excitation spectrum and emission spectrum of hrGFP is narrower, for this reason, in various cell lines, hrGFP is brighter and is much easier to be detected, and its stability and toxicity are much lower than EGFP, so hrGFP is a more suitable one as reporter gene in the cell lines.
In the present study, three amino acid sites degraded under normoxic conditions from pMD19-T-HIF1α carried HIF-1α gene sequence were removed through site-directed mutagenes using PCR technique, thus, HIF-1α can be expressed under normoxic conditions; in addition, Not Ⅰ restriction enzyme digestion site was added before initiation codon and Pvu Ⅰ restriction enzyme digestion site was added before termination codon in target gene CDS region, and to ensure correct reading frame, single basic group “G” was added to remove the termination codon in target gene coding region. After Not Ⅰ/Pvu Ⅰ double enzyme digestion of pShuttle-CMV-IRES-hrGFP-1 shuttle plasmid, target gene fragment can be coupled between Not Ⅰ and Puv Ⅰ among multiple clone sites of shuttle plasmid; moreover, three termination codons of pShuttle-CMV- IRES-hrGFP-1 shuttle vectors were removed due to enzyme digestion, which contributes to the hemeo-expression of hrGFP reporter gene. Finally, pAd-HIF1αmu-IRES-hrGFP-1 expression vector was constructed using homologous recombination mechanism in bacterium. Macrorestriction map shows the same vector structure as expected. Sequencing and restriction enzyme identification results confirmed that a novel recombinant adenoviral mutant enkaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was successfully constructed. This will add evidence to future study of HIF-1α gene in promoting mature angiogenesis in bone defect regions under normoxic conditions.
