Construction Of Recombinant Eukaryotic Expression Vectors Of Hypoxia-induciable Factor-1α And Analysis Of Their Expression

Methods such as PCI, CABG, thrombolysis, and so on, play important role in coronary artery reconstruction. The methods have their indications, contraindications, and limitation. Scientists resorted to methods to promote angiogenesis. They used to place great expectations on vascular endothelial growth factor (VEGF), which in some recent studies had been proved to induce distorting and high-permeability blood vessels when used alone. Furthermore, it might promote growth of tumors. A transcription activator, the new found hypoxia inducible factor-la (HIF-1 a ) had been proved to be able to induce not only transcription of some genes that play key roles in angiogenesis, such as glucose and energy metabolism, erythropoiesis, etc. in vitro, but also hypervascularity without leakage or inflammation in skin of transgenic mice overexpressing recombinant HIF-1 a. Since HIF-1 a is apt to be degraded under normoxia, we intended to make site-directed mutagenesis on recombinant eukaryotic expression vector pcDNA3.1V5-HisA-HIF-la to obtain two mutant of HIF-1 a (pcDNAS.l V5-HisA -HIF-1 a -564Ala and pcDNA3.1 V5-HisA -HIF-1 a -564Ala-803Ala ) in our study. After transforming these three vectors into human lung microvascular cells, we investigated their expressions and tried to elucidate the influence of the mutations on their expressions and protein functions. We hope the jobs could pave the way for studying the mutants at levels of organization and animal and therefore help to select a kind of perfect angiogenesis stimulator in the future.MethodsSite-directed mutagenesis was performed on recombinant eukaryotic expression vector pcDNA3.1V5-HisA-HIF-1a to obtain mutated vector pcDNA3.1V5-HisA-HIF-la-564Ala, which was confirmed by DNA sequencing and then changed intopcDNA3.1V5-HisA-HIF-1a-564Ala-803Ala by the same method. The three kinds of recombinant plasmids were transformed to human lung microvascular endothelial cells transiently by means of Lipofectin. Immunofiuorescence, Western blotting and RT-PCR were used to examine the expression of HIF-la or its target gene VEGF in the transformed cells.ResultsDNA sequencings showed that two-stage mutagenesis were carried out successfully and we got the two constructs of pcDNA3.1 V5-HisA-HIF-la-564Ala and pcDNA3.1V5-His-HIF-1aA-564Ala-803Ala. The transformation of the mutated vectors resulted in accumulation of HIF-la protein and increase of mRNA levels of VEGF. The increase of VEGF mRNA by pcDNA3.1 V5-His-HIF-1a A-564Ala-803Ala are more intensive than that by pcDNA3.1 V5-His-HIF-l a A-564Ala. Angiotensin II administration enhanced expression of non-mutated vector instead of the other two mutated ones.ConclusionUnder normoxia, non-mutated HIF-la protein is subject to rapid degradation that can be inhibited by angiotensin II administration while mutated ones accumulate in the same condition. The above results illustrate 564 proline residue on HIF-1a is targeted for the degradation of the protein. Double mutated HIF-la protein is the most energic in transcription activation among the three kinds of proteins, which proves that 803 asparagine residue on HIF-la protein is related to inactivation of HIF-la in normoxic cells. We think that mutated HIF-la proteins have potent ability ofbecoming good angiogenesis factors and, are worth further studying and developing.