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Bionic affinity purification method of plasminogen activator

A technology of plasminogen and purification method, applied in the biological field, can solve the problems of unstable nature, high cost, inability to withstand online cleaning, etc., and achieve the effect of reducing large-scale purification steps and reducing production costs

Inactive Publication Date: 2006-06-14
上海荣君生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the D-Phe-D-Phe-Argal material used in this method needs to be synthesized by solid-phase peptide synthesis, and the preparation steps are cumbersome and costly; Stable and cannot withstand the harsh conditions of in-line cleaning
Although the complex purification process can be simplified, the above shortcomings limit large-scale applications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Preparation of separation materials

[0018] Take NH 2 -Sepharose (250ml), washed with 5 times the volume of 1M NaCl, then fully washed with deionized water, drained of water, poured into a reaction vessel, added 200ml of deionized water, and placed in an ice-salt bath for precooling. When the temperature dropped to 5°C, start stirring. Triazoxide (45 g) was dissolved in pre-cooled acetone and added to the reaction vessel. with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 3 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) and washed successively to obtain 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (190ml).

[0019] Take 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (200ml), weigh aminobenzamidine (20g) and dissolve it with deionized water (150ml), and dissolve it with 1-amino- Sepharose-3,5-dichloro-2,4,6-triazoxide was mixed evenly, placed at a temperature of 50° C. and stirred for ...

Embodiment 2

[0023] 1. Preparation of separation materials

[0024] Take NH 2 -Sepharose (250ml), washed with 5 times the volume of 1M NaCl, then fully washed with deionized water, drained of water, poured into a reaction vessel, added 200ml of deionized water, and placed in an ice-salt bath for precooling. When the temperature dropped to 5°C, start stirring. Triazoxide (45 g) was dissolved in pre-cooled acetone and added to the reaction vessel. with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 3 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) and washed successively to obtain 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (190ml).

[0025] Take 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (200ml), weigh aminobenzamidine (20g) and dissolve it with deionized water (150ml), and dissolve it with 1-amino- Sepharose-3,5-dichloro-2,4,6-triazoxide was mixed evenly, placed at a temperature of 50° C. and stirred for ...

Embodiment 3

[0029] 1. Preparation of separation materials

[0030] Take NH 2 -Sepharose (250ml), washed with 5 times the volume of 1M NaCl, then fully washed with deionized water, drained of water, poured into a reaction vessel, added 200ml of deionized water, and placed in an ice-salt bath for precooling. When the temperature dropped to 5°C, start stirring. Triazoxide (45 g) was dissolved in pre-cooled acetone and added to the reaction vessel. with saturated NaHCO 3 Keep the pH of the solution between 6 and 7, react at 5°C for 3 hours, take out, 3×10 times the volume of water / acetone (1:1, 1:3, 0:1, 1:1, 3:1, 1:0) and washed successively to obtain 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (190ml).

[0031] Take 1-amino-Sepharose-3,5-dichloro-2,4,6-triazoxide (200ml), weigh aminobenzamidine (20g) and dissolve it with deionized water (150ml), and dissolve it with 1-amino- Sepharose-3,5-dichloro-2,4,6-triazoxide was mixed evenly, placed at a temperature of 50° C. and stirred for ...

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PUM

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Abstract

The invention relates to a plasminogen activator bionics and purifying method. It belongs to biotechnology field. It includes the following steps: basal chromatography medium reacts with tri-chlorine tri-nitrogen zin; they react with pair amino benzene formamidine to compound bionics and separating material; the upper material is made into affinity column; the sample of the plasminogen activator is flowed through the affinity column; then the former is absorbed in the latter; cleaning the latter; cleaning it again after changing buffer solution condition; purging the plasminogen activator to gain a purified one. The advantages of the invention are less purifying steps, low cost, high efficiency, and can realize volume produce.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for biomimetic affinity purification of plasminogen activator. Background technique [0002] Tissue plasminogen activator is a serine protease that has a high affinity with fibrin and exhibits activity after binding to fibrin in the circulatory system. It can activate plasminogen to become plasmin, dissolve thrombus. Tissue plasminogen activator has a natural form, and there is also a form of artificially designed part of the domain deletion, such as the modified form of the deletion of the fibrin ring region, the epidermal growth factor (E) region and / or the Kringle-1 region, and Prolongs half-life in vivo, speeds up diffusion into and dissolves blood clots. Tissue plasminogen activator is a high-efficiency thrombolytic drug, and it is one of the six major gene recombination drugs on the US market. Commonly used purification methods for plasminogen activators...

Claims

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Application Information

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IPC IPC(8): C12N9/50
Inventor 李荣秀吴方庞艳萍
Owner 上海荣君生物医药科技有限公司
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