Construction, Identification And Expression Of Human Hypoxia Inducible Factor-Ala564-Ala803 Recombinant Adenovirus Vector

IntroductionCoronary heart disease (CHD) is one of the gravest diseases threatening human's health. The treatments for CHD now mainly include the conventional medical treatment, percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). However, a significant proportion of patients has symptoms refractory to medical treatment, yet are unsuitable for conventional revascularization therapies. So it is necessary to find an alternative strategy. In recent years, with the progress in molecular biology, gene therapy for angiogenesis in CHD has emerged as a novel promising approach in the coronary revascularization.Among the numerous vascular growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been studied widely. But there were evidences indicated that VEGF gene therapy could result in vascular permeability, tissue edema and etc, while FGF therapy was associated with proteinuria. Until now, the clinical studies about angiogenesis induced by VEGF and FGF have been almost stopped due to their side effects. Researchers are searching for a new gene, which can induce mature vascular bed without adverse effects. Hypoxia inducible factor 1(HIF-1) is a DNA-binging factor, regulating transcription of many Hypoxia responsive genes (HRG) and angiogenesis. It is composed of termed HIF-1βand an oxygen-sensitive a-subunit. HIF-1αdetermines HIF-1's activity. Preclinical phase study shows that HIF with constitutive expression activity can induce angiogenesis with complete physiological function. Under normoxic conditions, with prolyl hydroxylation of the two proline residues Pro564 and Pro402, HIF-1αprotein is easy to be degraded by ubiquitin-proteasome systerm and the half-life is less than 5min. Under hypoxic conditions, the process of hydroxylation is inhibited and activity HIF-1 protein is formed which can induce its physiological function such as angiogenesis and etc. The activity of transcription is regulated by residues Asn803 in the COOH-terminal transactivation domain (CAD) of HIF-1αand can be enhanced with the interception of hydroxylation. Now provasoformation about HIF-1αis major in the condition of native gene transfection with the hypoxia model, but there is few report about mutation of HIF-1αpromoting angiogenesis. Consequently, having finished the mutation of Pro564 and Pro564/Asn803 in HIF-1αgene and construction of recombinant adenovirus vectors termed as Adeno-HIF-1αand Adeno - HIF - 1αAla564, we are to construct the recombinant adenovirus vectors termed as Adeno-HIF-1α-Ala564-Ala803, in order to get high levels of gene expression.ObjectiveTo construct the recombinant adenovirus vectors termed as Adeno-HIF-1α- Ala564-AlaS03, in order to get high levels of gene expression. It will provide the basis for the further study of the effects of HIF-1αand its mutant to therapeutic angiogenesis in CHD and the clinical use in the future.MethodsThe expression cassette containing HIF- 1αcDNA was obtained from the recombinant pShuttle2- HIF-1α-Ala564-AlaS03 with double digestion of PI- SceⅠandⅠ- CeuⅠ, then ligated to Adeno-ⅩViral DNA with in vitro ligation. The recombinant adenoviral plasmid was identified and transfected into the adenoviral packaging cell HEK293A by lipofectamine 2000 mediated gene transfer method to pack the virus. The recombinant adenovirus was confirmed by polymerase chain reaction (PCR) and the titer was determined. The protein of HIF were detected by Western blot. ResultsThe recombinant pAdeno- HIF1α-Ala564-Ala803 was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing analysis. The transfected HEK293A cells were lysed by freeze thawing to obtain the recombinant adenovirus in the lysate. The PCR product of the lysate was confirmed the presence of recombinant adenovirus. The viral titer was 6.3×10~8 pfu/mL. The HEK293A transfected by recombinant Adeno-HIF-1α-Ala564-Ala803 expressed HIF-1αprotein stabely.ConclusionsThe recombinant adenovirus containing the mutant HIF-1αgene was successfully constructed. It provided the foundation of HIF—1α-Ala564-Ala803 gene therapy for CHD.