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Affinity chromatographic process of separating and purifying plasmin

A technology of separation and purification, plasmin, applied in biochemical equipment and methods, enzymes, ion exchange and other directions, can solve the problems of complex operation, long process route, low recovery rate, etc. The effect of improving recovery and purity

Inactive Publication Date: 2007-11-07
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These all have the deficiencies such as long operational route, low recovery rate (generally only 10%-40%), complicated operation, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Separating and purifying the sodium soybean kinase fermented liquid by affinity chromatography, comprising the following steps and process conditions:

[0026] (1) Preparation of affinity chromatography medium:

[0027] a Dissolve 100 mg of human fibrinogen in 2 mL of sterile water to prepare a fibrinogen solution with a concentration of 50 mg / mL, and incubate at 37°C for 15-30 minutes until completely dissolved;

[0028] b Dissolve 300mg of agarose in 20mL of 40mmol / L barbiturate-HCl buffer solution at pH 7.8 to prepare a solution with a mass concentration of 1.5%, sterilize and cool to 50°C;

[0029] c Mix the solutions prepared in step a and step b, then add 5IU / mL human thrombin, mix well, and keep warm for 30 minutes;

[0030] d Use a syringe to drop the mixture in step c into 50mL of tetrachlorethylene precooled to 4°C to make spherical particles;

[0031] e Wash the spherical particles with deionized water and keep warm at 37°C for 2h;

[0032] f. Add glutaral...

Embodiment 2

[0039] Using affinity chromatography to separate and purify fermented soybean fibrinolytic enzyme broth, the following steps and process conditions are adopted:

[0040] (1) Preparation of affinity medium:

[0041] a Dissolve 200 mg of human fibrinogen in 2 mL of sterile water to prepare a fibrinogen solution with a concentration of 100 mg / mL, and keep warm at 35°C for 15-30 minutes until completely dissolved;

[0042] b Dissolve 240mg of agarose in 20mL of 40mmol / L barbiturate-HCl buffer solution at pH 7.6 to prepare a solution with a mass concentration of 1.2%, sterilize and cool to 48°C;

[0043] c Mix the solutions prepared in step a and step b, then add 4IU / mL human thrombin, mix well, and keep warm for 20 minutes;

[0044] d Use a syringe to drop the mixture in step c into 50 mL of tetrachlorethylene precooled to 2°C to make spherical particles;

[0045] e Wash the spherical particles with deionized water and keep warm at 40°C for 1h;

[0046] f. Add glutaraldehyde wi...

Embodiment 3

[0050] The crude enzyme solution of nattokinase is separated and purified by affinity chromatography, and the following steps and process conditions are adopted:

[0051] (1) Preparation of affinity medium:

[0052] a Dissolve 160 mg of human fibrinogen in 2 mL of sterile water to prepare a fibrinogen solution with a concentration of 80 mg / mL, and keep warm at 40°C for 15-30 minutes until completely dissolved;

[0053]b Dissolve 360mg of agarose in 20mL of 40mmol / L barbiturate-HCl buffer solution at pH 8.0 to prepare a solution with a mass concentration of 1.8%, sterilize and cool to 52°C;

[0054] c Mix the solutions prepared in step a and step b, then add 10IU / mL human thrombin, mix well, and keep warm for 40 minutes;

[0055] d. Drop the mixed solution in step c into 50mL tetrachlorethylene precooled to 10°C with a syringe to make spherical particles;

[0056] e Wash the spherical particles with deionized water and keep warm at 35°C for 1.5h;

[0057] f. Add glutaraldehy...

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PUM

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Abstract

The present invention discloses affinity chromatographic process of separating and purifying plasmin. The process includes preparing affinity chromatographic medium and affinity chromatography. The preparation of affinity chromatographic medium includes the steps of: dissolving human fibrinogen in sterilized water at 35-40 deg.c, agarose in barbital sodium-HCl buffer solution, sterilizing and cooling; adding human thrombin and dropping into vinyl tetrachloride at 2-10 deg.c to form spherical grain; washing with ion-free water and maintaining at 35-40 deg.c for 1-2 hr; and adding glutaraldehyde to cross link. The affinity chromatography includes the steps of balancing the chromatographic column with the buffer liquid at 2-10 deg.c, adding the sample solution, rinsing with the buffer liquid and final eluting with the eluting solution. The present invention has high plasmin recovering rate and high plasmin purity.

Description

technical field [0001] The invention relates to a technology for separating and purifying plasmin for treating thrombotic diseases, in particular to a method for separating and purifying plasmin by using affinity chromatography. Background technique [0002] Embolism diseases seriously endanger human life and health, especially cardiovascular and cerebrovascular embolism diseases are one of the most serious and common diseases that endanger the health of middle-aged and elderly people. Thrombosis is becoming the disease with the highest mortality rate day by day, and dissolving thrombus is an important means to treat this type of disease. Therefore, the research on thrombolytic drugs has been widely valued, and the drugs used in clinical treatment today include streptokinase (Streptokinase; SK), urokinase (Urokinase; UK), tissue-type plasminogen activator (Tissue Plasminogen Activator; tPA) and so on. The fibrinolytic enzymes being researched and developed include nattokin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/68B01D15/08
Inventor 郭勇崔堂兵刘柳
Owner SOUTH CHINA UNIV OF TECH
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