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Hirudin separation and purification method based on affinity chromatography

A technology for separation and purification of hirudin, applied in the field of biomedical separation and purification, can solve the problems of being unsuitable for injection medicine, unsuitable for industrial production, and low active ingredients, and achieves the effects of low cost, simple operation, and high activity

Active Publication Date: 2018-08-14
长握生物科技(江苏)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned method has many shortcomings, mainly because the active ingredient is not high after extraction, the activity is generally about 100-4000ATU / g, the yield is between 0.27-4.47%, and the activity varies according to different extracts, impurities The content of hirudin is high, so it is not suitable for injection medicine. Some extraction methods are complicated, and some methods are relatively slow, so they are not suitable for industrial production. Therefore, many people are still studying the extraction methods of hirudin, hoping to find high efficiency, low cost, Extraction method with high active ingredient

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035]In this example, the preparation method of the thrombin adsorbent in the thrombin Sepharose 4FF affinity column is as follows: take Sepharose 4FF (purchased from Shanghai Rongjun Biomedical Technology Co., Ltd.) with a wet weight of 10.0 g in a Buchner funnel, Wash repeatedly with deionized water, add 25%, 50%, and 75% DMSO aqueous solutions to the medium in turn, each 50 mL, then put Sepharose4FF into a 20 mL anhydrous DMSO (dimethyl sulfoxide) Erlenmeyer flask, add Shake 0.5-1.0 g CDI (carbonyldiimidazole) at 30°C with a constant temperature oscillator at 100 r / min for 15 min, and wash repeatedly with deionized water to obtain activated Sepharose4FF; another 50 mg-100 mg thrombin (purchased (Lanzhou Institute of Biological Products Co., Ltd.) dissolved in 50 mL phosphate buffer (0.2mol / L K 2 HPO 4 -0.01 mol / L KCL, PH8.0), and the above-mentioned activated Sepharose 4FF was added to the phosphate buffer containing thrombin, 4-8°C, 50-80 r / min, stirring for 10-15 h , a...

Embodiment 2

[0046] The present invention provides the influence of different carrier activators on the separation and purification of hirudin, and the specific steps are as follows:

[0047] On the basis of Example 1, different carrier activators (CDI, cyanogen bromide, epichlorohydrin) were used to treat and activate Sepharose 4FF.

[0048] This method gains: 0.8 g CDI activates Sepharose 4FF and carries out the activity of the hirudin obtained in the next step experiment is 6937ATU / g, and the yield is 7.37%; 1.0 g cyanogen bromide (optimum addition amount) carries out the next step after activating Sepharose 4FF The activity of the hirudin obtained in the experiment was 4219ATU / g, and the yield was 2.19%; the activity of the hirudin obtained in the next experiment after 130 μmol / L epichlorohydrin activation (optimum concentration) Sepharose 4FF was 5231ATU / g, yielding The rate is 3.51%. Therefore, the carrier activator CDI used in this experiment is an activator with good effect, which...

Embodiment 3

[0050] The present invention provides the influence of different carrier activator solvents on the separation and purification of hirudin, and the specific steps are as follows:

[0051] On the basis of Example 1, different carrier activation solvents (DMSO, dioxane, acetone, ethanol, isopropanol) were used to treat and activate Sepharose 4FF.

[0052] The result of this example: the activity of the hirudin obtained in the next experiment after using DMSO as the solvent is 7524 ATU / g, and the yield is 7.28%; the activity of the hirudin obtained in the next experiment after using dioxane as the solvent is 6424 ATU / g, the productive rate is 3.18%; the activity of the hirudin obtained in the next step experiment after using acetone as the solvent is 3018 ATU / g, and the productive rate is 2.31%; the next step experiment obtained after using ethanol and isopropanol The activities of hirudin were 4359 ATU / g and 4893 ATU / g, and the yields were 2.09% and 1.68%, respectively. Therefor...

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PUM

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Abstract

The invention discloses a hirudin separation and purification method based on affinity chromatography. The hirudin separation and purification method comprises the following steps: after a leech living body is fed with normal saline, carrying out physical extrusion to enable the leach living body to secrete saliva, collecting the saliva, adding an obtained leech saliva sample into precooled trichloroacetic extraction liquid to be evenly mixed, carrying out extraction, carrying out centrifugation to take supernate, regulating pH (Potential of Hydrogen) to 3.0-5.0, adding precooled acetone to beevenly mixed and stand overnight, recovering the acetone in the supernate, carrying out centrifugation, and taking sediments for standby use; adding the sediments into distilled water to be fully dissolved, regulating pH to 6.0-8.0, adopting a thrombin Sepharose 4FF affinity column to carry out separation and purification, detecting the content of hirudin by an efficient liquid chromatogram, taking purified liquid on the content peak value of the hirudin, carrying out dialysis on the purified liquid, repeatedly adding water for 2-4 times in the process for 10-15h, carrying out rotary evaporation on the ultrafiltration supernate, and carrying out freeze-drying on concentrated solution by a freeze dryer after 70-80% of moisture is removed to obtain a hirudin product. The natural hirudin purified with the method has the advantages of high purity, high activity, low cost, simpleness in operation and easiness in repeating.

Description

technical field [0001] The invention relates to a method for separating and purifying hirudin, which belongs to the field of biomedical separation and purification. Background technique [0002] Studies have found that the secretion of leech salivary glands contains hirudin and other biologically active substances and has broad application prospects in medicine. Hirudin is an acidic polypeptide secreted by the salivary glands of leeches. It contains 65-66 amino acid residues and mainly has three isomers, HV1, HV2, and HV3. They have high structural homology. Hirudin is the strongest thrombin-specific inhibitor found so far. The activity of engineered strains expressing hirudin (HV3) protein depends on the intensity of antithrombin activity. It forms a non-covalent A stable complex with tightly bound bonds. In 1984, Haycraft found that leech extract had anticoagulant effect. In 1904, Jocoby isolated the active ingredient from medical leech for the first time and named it h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/815C07K1/22
CPCC07K14/815
Inventor 虞龙
Owner 长握生物科技(江苏)有限公司
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