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Preparation method and use of heparin oligosaccharide of specific length

A technology of heparin oligosaccharides and heparinase, which is applied in the field of medicine and can solve problems such as no practical application

Inactive Publication Date: 2011-12-14
黄欣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, low-molecular-weight heparins with anticoagulant activity are prepared by chemical methods both at home and abroad. Conrad H.E. (1995) published a patent for preparing heparin oligosaccharides with anti-proliferative activity of smooth muscle cells by chemical methods, but it has not been practically applied so far.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Corynebacterium preparation: each liter contains 1g of NaCl, K 2 HPO 4 2.5g, MgSO 4 0.5g, 2g heparin, pH to 6.5 culture medium in 1000ml Erlenmeyer flask, incubate at 30-33°C, 200 rpm shaker for 36 hours.

Embodiment 2

[0020]Preparation of heparinase: The cells are disrupted by ultrasound, and the obtained cell-free crude enzyme solution is first adsorbed with DEAE-cellulose. The DEAE-cellulose was fully balanced with 0.2mol / L, pH6.8 phosphate buffer, added to the crude enzyme solution to absorb foreign proteins, and the supernatant was collected by centrifugation. Then use hydroxyapatite to adsorb, fully balance the hydroxyapatite with 0.2mol / L, pH6.8 phosphate buffer, add it to the enzyme solution treated with DEAE-cellulose, centrifuge to collect the precipitate, and be adsorbed The enzyme was eluted once with the same buffer solution containing 1mol / L NaCl, the eluate was concentrated by ultrafiltration and desalted, and the desalted heparin enzyme solution was fully balanced with SP- Sepharose FF column chromatography was further purified, and the same buffer salt containing 0-0.5mol / L NaCl linear gradient was used for gradient elution, and three heparinases were separated and purified,...

Embodiment 3

[0022] Preparation and separation and purification of anti-smooth muscle cell proliferative active heparin oligosaccharides: a method for producing heparin oligosaccharides with heparinase according to the present invention refers to: using commercial heparin as raw material, using partially purified heparinase I, in a certain Heparin is degraded under conditions to prepare heparin oligosaccharides. Prepare 2% heparin solution with 0.05mol / L, pH7.0 phosphate buffer, use 0.1 unit of enzyme per gram of heparin, react at 20-30°C, stop the reaction when A232 is 0.55-0.65. The obtained reaction mixture is ultrafiltered through a filter membrane with a cut-off molecular weight of less than 10KD to remove macromolecular heparin and enzyme protein, concentrated and desalted to obtain a heparin-oligosaccharide mixture. The mixed heparin oligosaccharides were fractionated by Sephadex G-50 column, and then a series of partially purified heparin oligosaccharides were extracted by concentr...

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PUM

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Abstract

The invention relates to a preparation method and application of heparin oligosaccharides of a specific length. In the invention, heparin is degraded by heparinase, and heparin with a specific sugar chain length is prepared by ultrafiltration, gel chromatography, high performance liquid chromatography and the like. oligosaccharides.

Description

technical field [0001] The invention belongs to the technical field of medicine, and more precisely relates to a method for producing heparin oligosaccharides by using heparinase. Background technique [0002] Cardiovascular and cerebrovascular diseases are common and frequently-occurring diseases among middle-aged and elderly people, ranking first in the disease spectrum and death spectrum in many countries and regions. Atherosclerosis (AS) is the main pathological basis of cardiovascular and cerebrovascular diseases, so the prevention and treatment of AS is the fundamental measure to prevent and treat cardiovascular and cerebrovascular diseases. The pathogenesis of AS is long, and the etiology and pathogenesis are still not very clear. In recent years, with the development and interpenetration of cell biology, molecular biology, immunology and other disciplines, there has been a new understanding and development of the etiology of AS. The occurrence of AS is multifactori...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/00C12N9/88C12R1/15
Inventor 黄欣
Owner 黄欣
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