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A kind of separation and purification method of hirudin based on affinity chromatography

A technology for separation and purification and hirudin, which is applied in the field of biomedical separation and purification, can solve the problems of low effective components, unsuitable for injection medicine, unsuitable for industrial production, etc.

Active Publication Date: 2021-09-10
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 embodiment, preparation of thrombin Sepharose 4FF affinity column of thrombin adsorbent: taking a wet weight of 10.0 g of Sepharose 4FF (Rong Junsheng was purchased from Shanghai Pharmaceutical Co., Ltd.) in the Buchner funnel, repeatedly washed with deionized water, were added to the medium, 25%, 50%, 75% DMSO aqueous solution of each of 50 mL, then placed Sepharose4FF containing dry DMSO (dimethyl sulfoxide) 20mL flask was added 0.5-1.0 g CDI (carbonyl diimidazole) 30 deg.] C thermostatic oscillator 100 r / min at 15 min with shaking speed, and washed repeatedly with deionized water, to give both an activated Sepharose4FF; another 50 mg-100 mg of thrombin (purchased Lanzhou Institute of biological products Co., Ltd.) was dissolved in 50 mL phosphate buffer (0.2mol / LK 2 HPO 4 -0.01 mol / L KCL, PH8.0), and the above obtained activated Sepharose 4FF was added to the phosphate buffer solution containing thrombin, 4-8 ℃, 50-80 r / min speed, the reaction was stirred ...

Embodiment 2

[0046] Effects of the present invention provides isolated and purified vectors activators hirudin, the following steps:

[0047] Using different carriers activator (the CDI, cyanogen bromide, epichlorohydrin) treated activated Sepharose 4FF in Example 1 based on the embodiment.

[0048] This method obtained: activity for further experiments hirudin obtained after Sepharose 4FF 0.8 g CDI activation of 6937ATU / g, a yield of 7.37%; the next step after 4FF 1.0 g of cyanogen bromide (the optimum amount) activated Sepharose hirudin activity was obtained experimental 4219ATU / g, 2.19% yield; for further experiments hirudin obtained after 4FF 130μmol / L activated by epichlorohydrin (optimum concentration) was Sepharose active 5231ATU / g, yield rate of 3.51%. Therefore, the support activator, CDI used in this experiment is a good effect of activating agent, can provide accurate scientific basis for activated Sepharose.

Embodiment 3

[0050] Effects of the present invention provides a carrier solvent activators Purification of hirudin, the following steps:

[0051] Using different carriers activating solvent (DMSO, dioxane, acetone, ethanol, isopropanol), treated activated Sepharose 4FF in Example 1 based on the embodiment.

[0052] Obtained in this example: the use of DMSO for further experiments as obtained after the solvent hirudin activity 7524ATU / g, 7.28% yield; using dioxane as an activity for further experiments hirudin obtained after solvent 6424 ATU / g, 3.18% yield; acetone for further experiments as obtained after the solvent hirudin activity was 3018 ATU / g, 2.31% yield; using ethanol and isopropanol after the next experiment obtained hirudin activity were 4359 ATU / g and 4893 ATU / g, a yield of 2.09% and 1.68%, respectively. Therefore, in this experiment the support activator, was dissolved with DMSO agent a good effect activator, can provide accurate scientific basis for activated Sepharose. ...

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PUM

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Abstract

The invention discloses a method for separating and purifying hirudin based on affinity chromatography. After feeding a living leech with physiological saline, physically extruding it to make it secrete saliva and collecting it, adding pre-cooled trichloroacetic acid to the obtained leech saliva sample Mix the extract, leaching, centrifuge to take the supernatant, adjust the pH to 3.0‑5.0, add pre-cooled acetone, mix and let it stand overnight, recover the acetone in the supernatant, centrifuge, take the precipitate, and set aside; add the precipitate to distilled water , fully dissolved, adjusted to pH 6.0‑8.0, separated and purified with thrombin Sepharose 4FF affinity column, detected the hirudin content by high performance liquid chromatography, took the purified solution at the peak of the hirudin content, dialyzed the above purified solution, and in the process Repeatedly add water 2-4 times for 10-15 hours, perform rotary evaporation on the ultrafiltered supernatant, remove 70-80% of water, take the concentrated liquid and freeze-dry it to obtain the hirudin product. The purified natural hirudin obtained by the method of the invention has high purity, high activity, low cost, simple operation and easy repetition.

Description

Technical field [0001] The present invention relates to a process for the separation and purification of hirudin, purified belonging biomedical separation. Background technique [0002] It found, hirudin and other biologically active substances contained in leech salivary gland secretion and has broad application prospect in medicine. Hirudin is an acidic polypeptide leech salivary gland secretion of animals, comprising 65 to 66 amino acid residues, mainly HV1, HV2, HV3 these three isomers, having high structural homology between them. Hirudin is the strongest action ever found specific inhibitors of thrombin, hirudin protein (the HV3) engineering bacteria species activity is dependent antithrombin activity intensity, the molar ratio of thrombin to it like non-covalent cohesive bond is stable complex. 1984, Haycraft found leech extract anticoagulant effect. 1904 Jocoby such active ingredients were isolated from the medical leech, and designated hirudin. 1955, Markwardt successful...

Claims

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Application Information

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Patent Type & Authority Patents(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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