Angiogenesis of triple mutant low-oxygen inducible factor 1 alpha induced by auxiliary activating factors and application thereof

A mutant and normoxic technology, applied in the field of recombinant adenovirus vector construction, can solve the problems of insufficient expression, low transcription activity, and inability to increase stability, and achieve the effect of avoiding damage and safely transferring genes

Inactive Publication Date: 2011-07-13
吴平生 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chun et al. successively confirmed by experiments (Chun, Choi et al.2000; Chun, Choi et al.2001; Chun, Choi et al.2002): HIF-1α 736 , HIF-1α 557 and HIF-1α 516 The three deficient types lack the C-TAD domain. Although the mutant HIF-1α can combine with HIF-1β and translocate from the cytoplasm to the nucleus, its transcripti...

Method used

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  • Angiogenesis of triple mutant low-oxygen inducible factor 1 alpha induced by auxiliary activating factors and application thereof
  • Angiogenesis of triple mutant low-oxygen inducible factor 1 alpha induced by auxiliary activating factors and application thereof
  • Angiogenesis of triple mutant low-oxygen inducible factor 1 alpha induced by auxiliary activating factors and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0091] Example 1 Construction of triple mutant HIF-1α recombinant adenoviral vector

[0092] 1. Recombinant pcDNA3.1(+)-HIF-1α native build

[0093] (1) Digest pcDNA3.1 / V5-HisA-HIF-1α with Kpn I and Xba I native , use the Omega gel recovery kit (refer to the kit instructions for specific steps) to recover the target gene fragment (HIF-1α nativec DNA fragment), about 2500bp in size, take appropriate amount after recovery and carry out agarose gel (1%) electrophoresis, the results are shown in Figure 6 .

[0094] The enzyme digestion reaction system is as follows:

[0095] pcDNA3.1 / V5-HisA-HIF-1α native About 1μg

[0096] KpnI 1 μL

[0097] Xba I 1 μL

[0098] 10×M Digestion Buffer 2μL

[0099] Add sterile deionized water to a total volume of 20 μL

[0100] (2), the same method as above with Kpn I, Xba I double digestion pcDNA3.1 (+), gel recovery linearized pcDNA3.1 (+) fragments (method is the same as HIF-1α native The recovery of cDNA fragment), about 5400bp ...

Embodiment 2

[0214] Example 2 Packaging, in vitro amplification and titer determination of recombinant adenovirus carrying triple mutant HIF-1α gene

[0215] 1. HEK293 cell preparation

[0216] The day before transfection, HEK293 cells were treated with 3-5×10 5 Cell density per well was seeded in a 6-well plate with DMEM medium containing 10% FBS at 37°C containing 5% CO 2 Continue culturing in an incubator until the cell confluence rate is around 60-70%, then transfection can begin.

[0217] 2. Preparation of recombinant adenovirus plasmid pAdeno-HIF-1α-Ala402-Ala564-Ala803

[0218] ①. The recombinant adenovirus plasmid pAdeno-HIF-1α-Ala402-Ala564-Ala803 was digested with Pac I to expose its inverted repeat sequence. The enzyme digestion reaction system is as follows:

[0219] PI-Sce I digestion reaction system:

[0220] pAdeno-HIF-1α-Ala402-Ala564-Ala803 about 3.0μg

[0221] PI-Sce I (1U / μL) 1.0μL

[0222] 10×BSA 3.0 μL

[0223] 10×NEB3 digestion buffer 3.0μL

[0224] Add steri...

Embodiment 3 3

[0252] Example 3 Expression and transcriptional activity of triple mutant HIF-1α under normoxic conditions in vitro

[0253] To evaluate the expression level of triple mutant HIF-1α under normoxic conditions in vitro.

[0254] Human lung-type microvascular endothelial cells (HMVEC-L) were prepared into a single cell suspension with EBM-2 medium, and 8.0×10 3 The cell density per well was inoculated in a 96-well plate and placed in a normoxic incubator (37°C 20% O 2 5%CO 2) after 10-12 hours of culture equilibrium, replaced with EBM-2 medium containing 2% FBS, placed under different conditions and continued to culture for 24 hours, using QuantiGene 2.0 Reagent System (Panomics, Inc.) to detect HIF-1α and downstream Expression levels of pro-angiogenic genes (VEGF, PLGF, PAI-1 and PDGF) under normoxic conditions.

[0255] Human pulmonary microvascular endothelial cells (HMVEC-L) were prepared into a single cell suspension with EBM-2 medium, and 75×10 4 / cm 2 The cell density...

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Abstract

The invention relates to angiogenesis of a triple mutant low-oxygen inducible factor 1 alpha induced by auxiliary activating factors and application thereof. The invention relates to the triple mutant low-oxygen inducible factor 1 alpha and a vector containing the triple mutant low-oxygen inducible factor 1 alpha for encoding nucleic acid, in particular a recombinant adenovirus vector and a medicinal composition containing the factor and the vector. Triple mutant HIF-1 alpha protein can be bound with the auxiliary activating factors such as response element binding protein (CREB)/adenovirus E1A associated protein P300 (CBP/p300), histone deacetylase (HDAC) and the like to promote expression of HIF-1 alpha downstream target genes such as a vascular endothelial growth factor (VEGF) and the like. The triple mutant protein, the nucleic acid, the vector and the medicinal composition are used for promoting angiogenesis and treating ischemic diseases such as coronary disease, peripheral artery ischemic vascular disease, intermittent claudication and the like.

Description

field of invention [0001] The invention relates to the field of triple mutant hypoxia-inducible factor 1α gene therapy. More specifically, the present invention provides a method for promoting angiogenesis by using the triple-mutant hypoxia-inducible factor-1α gene, including the construction of the triple-mutant hypoxia-inducible factor-1α gene, and the carrier containing the triple-mutant hypoxia-inducible factor-1α gene The construction of recombinant adenovirus vectors, especially the construction of recombinant adenovirus vectors; the triple mutant HIF-1α protein can co-activate with CREB binding protein / adenovirus E1A-related protein P300 (CBP / p300) and histone deacetylase (HDAC) Factor binding promotes the expression of HIF-1α downstream target genes such as vascular endothelial growth factor (VEGF), and they are used as therapeutic agents for coronary heart disease, peripheral arterial ischemic vascular disease, and intermittent claudication. Background technique ...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/63C12N15/86C12N15/861C12N5/10C12N1/21C12N1/15A61K48/00A61K38/17A61P9/10A61P9/14
Inventor 吴平生刘城宾建平王月刚赖艳娴谢宜军郭寿贵童锴胡英芳傅锐斌
Owner 吴平生
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