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Protein Bach1 for inhibiting angiogenesis and application thereof

An angiogenesis and protein technology, applied in the field of biotechnology and medicine, can solve the problem of unclear role of Bach1 in angiogenesis

Inactive Publication Date: 2016-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that human Bach1 protein contains 736 amino acids and has a molecular weight of 92kDa. Bach1 inhibits the expression of heme oxygenase-1 (heme oxygenase-1, HO-1) and participates in the regulation of cellular oxidative stress response and cell cycle, but The role of Bach1 in angiogenesis is still unclear, and no related reports have been seen so far

Method used

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  • Protein Bach1 for inhibiting angiogenesis and application thereof
  • Protein Bach1 for inhibiting angiogenesis and application thereof
  • Protein Bach1 for inhibiting angiogenesis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Construction of Bach1 expression vector

[0026] The following primers were synthesized based on the sequences at both ends of the human Bach1 gene. The upstream primer (5'-GGATCCATGTCTCTGAGTGAGAACTC-3') contained a BamHI cleavage site, and the downstream primer (5'-GAATTCCTCATCAGTAGTACATTTAT-3') contained a XhoI cleavage site for the construction of Bach1 Prokaryotic or eukaryotic expression vector; adopt Trizol reagent (U.S. Invitrogen Company) to extract the total RNA of human vascular endothelial cells, adopt SuperScript cDNA Synthesis Kit (U.S. Fermentas Company) to synthesize a strand of cDNA, use this synthetic cDNA as a template, and use the above Primers were used for PCR amplification. The Taq enzyme amplified by PCR was purchased from Japan Toyobo Co., Ltd. The reaction program was pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 2 minutes and 30 seconds, and 35 c...

Embodiment 2

[0027] Embodiment 2: Expression of Bach1 fusion protein

[0028] Transform the Bach1 prokaryotic expression vector PGEX-Bach1 constructed above into Escherichia coli BL21, pick a single colony and inoculate it in 2×YT medium containing 100ng / ml ammonium ammonia, shake and culture overnight at 37°C to activate the strains, according to the ratio of 1:100 Inoculate 500ml of 2×YT liquid medium at a volume ratio of 500ml, expand the culture at 37°C until the OD600 is about 0.9, add IPTG to a final concentration of 1mM, and collect the cells by centrifugation after induction at 18°C ​​for 24 hours, and buffer the total cells with Tris solution (50mM Tris, 150mM NaCl, pH7.5), and then suspended with an appropriate amount of Tris buffer, after sonication, the lysed supernatant and the precipitate after sonication were analyzed by SDS-PAGE electrophoresis, which showed that Bach1 fusion The expression of the protein accounted for more than 80% of the total protein of the bacteria, mos...

Embodiment 3

[0029] Example 3: Establishment of mouse lower limb ischemia model

[0030]An 8-week-old normal C57BL / 6J mouse was anesthetized with 4.5% chloral hydrate, the lower limbs were depilated and fixed, the mice were treated routinely, the fascia and adipose tissue were separated, the femoral artery and femoral vein were found, and after the common femoral artery branch , separate the arteries and veins supplying the inner muscle group, ligate the arteries, and then 5 cm down, separate the arteries and veins, and ligate the arteries, cut off the blood vessels between the two ligation points, strip and remove the section of blood vessels, suture the incision, and Inject normal saline or adenovirus (2×10 8 number of viral particles); the experiment was divided into three groups: normal saline control group, control adenovirus group, and Bach1 adenovirus group. Bach1 adenovirus or control virus (Ad-GFP) was provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., and the blood f...

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Abstract

The invention belongs to the field of biological and medicine, and relates to protein for inhibiting angiogenesis and an application thereof. The protein Bach1 is taken as a drug target for intervention of relative disease to angiogenesis, animal experiment and clinic experiment are carried out, the result displays that the protein Bach1 can obviously inhibit the recovering of blood flow of mice ischemic lower limb and angiogenesis; after gene Bach1 is specifically knockout, angiogenesis of the mice ischemic lower limb is obviously promoted; overexpression of Bach1 can inhibit angiogenesis in a growth process of zebra fish and inhibit the propagation, migration and tubular formation of human vascular endothelial cell; conversely, the Bach1 gene deletion can promote the above function of human vascular endothelial cell; the protein can inhibit expression of VEGF and IL-8, and the angiogenesis of relative disease can be effectively inhibited. The protein Bach1 can be used for preparing a preparation for treating tumor due to angiogenesis or eye angiogenesis disease.

Description

technical field [0001] The invention belongs to the fields of biotechnology and medicine, and relates to a protein for inhibiting angiogenesis and its application. Background technique [0002] The prior art discloses that angiogenesis refers to the growth of a new vascular network in the original capillary network by sprouting or non-sprouting, including the degradation of the perivascular matrix, the proliferation, migration and formation of tubular structures of endothelial cells; A primary capillary network is wrapped by vascular support cells such as smooth muscle cells and pericytes, gradually forming a mature blood vessel with a complete structure. Studies have shown that angiogenesis plays an important role in physiological processes such as embryonic development and injury repair in vivo. The main pathological changes of diseases such as rheumatoid arthritis. At present, macromolecular anti-angiogenesis drugs such as Avastin have been approved by the US FDA and ha...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12A61K38/17A61P35/00A61P27/02A61P9/00
Inventor 孟丹蒋丽陈思锋王新红刘俊许魏香香牛琮徐洁赵凤娣
Owner FUDAN UNIV
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