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Thrombin extracting and separating process

A separation method, thrombin technology, applied in the field of bioengineering, can solve problems such as not easy to be widely used, high cost, complicated preparation process, etc., and achieve the effect of easy industrial production, saving cost and extraction time, and simple and easy extraction method

Inactive Publication Date: 2009-07-15
马忠仁 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They prepared it into a crude product of thrombin or prothrombin, and then purified it by ion exchange chromatography, gel chromatography and affinity chromatography, and freeze-dried to obtain thrombin with high purity. Its preparation process is complicated, the technical requirements are high, the cost is expensive, and it is not easy to be widely used

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] The present invention has the following sequential steps:

[0014] a. First, take 1000 mL of bovine blood as a raw material, add 3.8% W / V sodium citrate as an anticoagulant to the blood, stir evenly, and centrifuge at 4000 rpm for 20 minutes below 4°C to obtain plasma.

[0015] b. Dilute the obtained plasma 9 times by volume with deionized water, adjust the pH value to 5.30 with 2mol / L acetic acid, let it stand at 4°C for 10 hours, then centrifuge at 4000 rpm for 20 minutes, and settle The supernatant was then discarded to obtain a prothrombin precipitate.

[0016] c. Precipitate the obtained prothrombin with 1.5% W / V CaCl 2 Dissolve in 300 ml of deionized water, place at 4°C for 1.5 hours, then centrifuge at 4,000 rpm for 20 minutes, discard the precipitate after settling, and obtain the supernatant.

[0017] d. Place the obtained supernatant in a 37°C water bath and stir, while stirring, slowly add 10% W / V (NH 4 ) 2 SO 4 , making it with Ca ++ A chemical reactio...

Embodiment 2

[0022] The present invention has the following sequential steps:

[0023] a. First, take 1000 mL of bovine blood as a raw material, add 3.8% W / V sodium citrate as an anticoagulant to the blood, stir evenly, and centrifuge at 4000 rpm for 20 minutes below 5°C to obtain plasma.

[0024] b. Dilute the obtained plasma 10 times by volume with deionized water, adjust the pH value to 5.30 with 2mol / L acetic acid, let it stand at 5°C for 12 hours, then centrifuge at 4000 rpm for 20 minutes, and settle The supernatant was then discarded to obtain a prothrombin precipitate.

[0025] c. Precipitate the obtained prothrombin with 2.0% W / V CaCl 2 Dissolve in 300 ml of deionized water, place at 5°C for 1.5 hours, then centrifuge at 4,000 rpm for 20 minutes, discard the precipitate after settling, and obtain the supernatant.

[0026] d. Place the obtained supernatant in a 37°C water bath and stir, while stirring, slowly add 15% W / V (NH 4 ) 2 SO 4 , making it with Ca ++ A chemical reacti...

Embodiment 3

[0030] The present invention has the following sequential steps:

[0031] a. First, take 1000 mL of bovine blood as a raw material, add 3.8% W / V sodium citrate as an anticoagulant to the blood, stir evenly, and centrifuge at 4000 rpm for 20 minutes below 4°C to obtain plasma.

[0032] b. Dilute the obtained plasma 9 times by volume with deionized water, adjust the pH value to 5.30 with 2mol / L acetic acid, let it stand at 4°C for 11 hours, then centrifuge at 4000 rpm for 20 minutes, and settle The supernatant was then discarded to obtain a prothrombin precipitate.

[0033] c. Precipitate the obtained prothrombin with 1.8% W / V CaCl 2 Dissolve in 300 ml of deionized water, place at 4°C for 1.5 hours, then centrifuge at 4,000 rpm for 20 minutes, discard the precipitate after settling, and obtain the supernatant.

[0034] d. Place the obtained supernatant in a 37°C water bath and stir, while stirring, slowly add 13% W / V (NH 4 ) 2 SO 4 , making it with Ca ++ A chemical reactio...

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Abstract

The invention relates to the technical field of bioengineering, in particular to a method for extracting and separating thrombin. The present invention uses CaCl2 to activate prothrombin to become thrombin while adding (NH4)2SO4 to generate CaSO4 to precipitate and adsorb thrombin, so as to simplify its production process, shorten the subsequent purification cycle and reduce costs. The extraction method of the present invention is simple and easy to implement, and is easy to industrialized production. It does not go through complex steps such as ion exchange chromatography and affinity chromatography, but directly goes through the steps of elution, ultrafiltration and freeze-drying, which saves cost and extraction time. The required 7 days is shortened to 2 to 3 days. About 0.7g of freeze-dried thrombin powder can be obtained per liter of blood, and its specific activity is about 100U / mg (protein).

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for extracting and separating thrombin. Background technique [0002] Thrombin is a serine proteolytic enzyme that catalyzes the cleavage of fibrinopeptides A and B in fibrinogen, making it into insoluble fibrin, thereby promoting blood coagulation. A lot of research has been done on it at home and abroad, and it has been used clinically as a new type of quick-acting local hemostatic drug. The general extraction and separation methods are organic solvent methods such as ethanol, acetone and ether, barium citrate adsorption method and magnesium hydroxide adsorption method. They prepared it into a crude product of thrombin or prothrombin, and then purified it by ion exchange chromatography, gel chromatography and affinity chromatography, and freeze-dried to obtain thrombin with high purity. The preparation process is complicated, the technical requirements are hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74
Inventor 马忠仁印明善冯玉萍李明生
Owner 马忠仁
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