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A kind of preparation method of thrombin

A thrombin and blood technology, applied in the field of bioengineering, can solve the problems of limiting the clinical application of thrombin products, low thrombin yield, high product impurities, etc., to achieve the effect of improving activity and purity, simple process flow and high specific activity

Active Publication Date: 2019-03-15
黄耀江
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The existing preparation methods are various combinations of the above-mentioned prothrombin extraction and thrombin purification methods, but no matter whether the thrombin product is prepared from animal blood or human blood, the existing preparation methods will have the following problems: thrombin produces The rate is low, the potency is unstable; the product impurities are high; these will limit the clinical application of thrombin products and cause safety hazards. In the preparation process, multiple purifications are often required to achieve high-purity thrombin

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preparation example Construction

[0034] (2) Preparation of chitosan-arginine anion resin

[0035] Add the porous chitosan microspheres prepared in step (1) into a solution of 2.0% arginine by mass fraction, shake for 20-30min, and prepare chitosan-arginine with a mass fraction of chitosan of 1.5%. Acid solution, then according to the molar ratio of arginine in the chitosan-arginine solution: dicyclohexylcarbodiimide is 1:0.5, in the chitosan-arginine solution, add DCC peptide Condensing agent, carry out condensation reaction for 20-40min, and finally filter, discard the filtrate, and collect filter residue to obtain chitosan-arginine anion resin.

[0036]Heparin-chitosan affinity magnetic microspheres of the present invention can be prepared according to the following method:

[0037] (1) The concentration of 0.3mol / L Fe 2+ , Fe 3+ The solution is mixed uniformly at a molar ratio of 1:2, and the volume mass ratio of the mixed solution to PEG4000 is 100 (mL):1, then add PEG4000, fully dissolve and quickly d...

Embodiment 1

[0041] (1) Collect 3500 mL of pig blood, add 350 mL of 3.8% sodium citrate solution to the blood, centrifuge, and take supernatant anticoagulated plasma, 1300 mL.

[0042] (2) Chitosan-arginine anion resin is added into water and soaked for 30min, fully balanced washing with 0.05mol / L of 0.08mol / L NaCl, Tris-HCl buffer solution of pH 7.2, adding anticoagulant to the anticoagulant plasma The chitosan-arginine anion resin after the equilibrium of plasma quality 0.5%, stirring and adsorption 60min, collects chitosan-arginine anion resin with filter cloth filtration; Wash chitosan-arginine anion resin with washing liquid 3 times; the washing liquid is 0.05 mol / L of 0.1 mol / L NaCl, Tris-HCl buffer solution with pH 7.2.

[0043] (3) The chitosan-arginine anion resin after above-mentioned washing is eluted 3 times with eluent, collects eluent, obtains prothrombin solution, 450mL; The eluent is 0.05mol of 1mol / L NaCl / L, Tris-HCl buffer at pH 7.2.

[0044] (4) Add 1mol / LCaCl to the ...

Embodiment 2

[0048] (1) Collect 4500mL of bovine blood, add 225mL of 4.5% sodium citrate solution to the blood, centrifuge, and take supernatant anticoagulated plasma, 1700mL;

[0049] (2) Chitosan-arginine anion resin is added into water and soaked for 30min, fully balanced washing with 0.05mol / L of 0.08mol / L NaCl, Tris-HCl buffer solution of pH 7.2, adding anticoagulant to the anticoagulant plasma The chitosan-arginine anion resin after the balance of plasma mass 1% is stirred and adsorbed for 30min, and the chitosan-arginine anion resin is collected by filter cloth filtration; the chitosan-arginine anion resin is washed with washing liquid 3 times; the washing liquid is 0.05 mol / L of 0.1 mol / L NaCl, Tris-HCl buffer solution with pH 7.2.

[0050] (3) The chitosan-arginine anion resin after above-mentioned washing is eluted 3 times with eluent, collects eluent, obtains prothrombin solution, 570mL; The eluent is 0.05mol of 1mol / L NaCl / L, Tris-HCl buffer at pH 7.2.

[0051] (4) Add 2mol / ...

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Abstract

The invention discloses a thrombin preparation method, and belongs to the technical field of bioengineering. The method comprises the following steps: adding chitosan-arginine anion resin to anticoagulant plasma in order to adsorb prothrombin, activating the prothrombin, and carrying out affinity chromatography separation by using heparin-chitosan affinity magnetic microspheres. The method adopts the chitosan-arginine anion resin to adsorb prothrombin, so the adsorption effect is good; and the heparin-chitosan affinity magnetic microspheres are adopted to carry out specific purification of thrombin, so the purification speed is fast, the enzyme activity is high, the highest specific activity of purified thrombin can reach 1928.7IU / mg, and the heparin-chitosan affinity magnetic microspheres can be reused after being processed. The method has the advantages of simple process flow, implementation at normal temperature under normal pressure, application in large-scale production, short production cycle, and simple operation, and only needs 2d to complete the whole process.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a preparation method of thrombin. Background technique [0002] Thrombin is a highly specific serine proteolytic enzyme, which can directly promote the conversion of fibrinogen in the blood into fibrin, and promote platelet aggregation to achieve the purpose of rapid hemostasis. Thrombin is activated by the hydrolysis of the prothrombin (prothrombin) precursor in the blood, and the thrombin product is activated by the extracted prothrombin. In the middle of the 20th century, people began to apply thrombin clinically. Due to its remarkable curative effect, its application is becoming more and more extensive. Later, it was proved that animal thrombin was applied to human body without thrombin antigenicity, and there was no allergic reaction and other adverse reactions when it was taken orally or externally. Thrombin extracted from plasma can be used clinically ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74
CPCC12N9/6429C12Y304/21005
Inventor 黄耀江蒋丹刘宇闫强商宇
Owner 黄耀江