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Method for efficiently extracting and purifying blood coagulation factor IX and blood coagulation factor X

A high-efficiency technology for coagulation factors, applied in the field of purification of blood products, can solve the problems of limited specificity, achieve good stability, mild elution conditions, and less loss

Inactive Publication Date: 2011-08-10
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

FIX / FX-bp has high binding activity to IX and X, and its specificity is limited to FIX and FX

Method used

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  • Method for efficiently extracting and purifying blood coagulation factor IX and blood coagulation factor X
  • Method for efficiently extracting and purifying blood coagulation factor IX and blood coagulation factor X
  • Method for efficiently extracting and purifying blood coagulation factor IX and blood coagulation factor X

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0030] (1) Preparation of FIX / FX-bp-Sepharose 4B

[0031] Weigh the required amount of CNBr-activated Sepharose-4B powder, then slowly pour it into 10 times the volume of 1mmol / L HCl solution, stir gently to disperse the gel particles. After the gel was swollen, the gel was washed with coupling buffer (0.2M boric acid-borax buffer pH=8.0). Add 1 volume of FIX / FX-bp solution to 1 volume of well-balanced activated agarose, stir and couple at low speed for 16-20 hours. Then add excess ethanolamine to react, then use high and low pH buffer solution (pH=4.0, 0.1mol / L sodium acetate buffer containing 0.5mol / L NaCl; pH=8.3, 0.1mol / L NaCl containing 0.5mol / L NaCl) L boric acid-borax buffer) to wash the gel three times alternately, and then use affinity buffer (0.02mol / L Tris-HCl, pH 7.4, 5mmol / L CaCl 2 ) and elution buffer (0.02mol / L Tris-HCl, pH 7.4, 5mmol / L EDTA) were washed alternately, and finally soaked in the equilibrium buffer containing 0.1% sodium azide, and stored at 4°C. ...

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Abstract

The invention provides a method for efficiently extracting and purifying a blood coagulation factor IX and a blood coagulation factor X. The method provided by the invention comprises the following steps: collecting blood, treating, and collecting blood plasma; and extracting an IX crude extract and an X crude extract from the blood plasma, and purifying. The method is characterized in that the purifying step is to use FIX / FX-bp-Sepharose 4B to purify the blood coagulation factor IX crude extract and the blood coagulation factor X crude extract respectively through the affinity chromatography; and the affinity ligand FIX / FX-bp is selected from ACF I, ACF II or AHP. The purified blood coagulation factor IX can be directly used as a medicament and the purified blood coagulation factor X canbe directly used as a reagent.

Description

1. Technical field [0001] The invention relates to a method for purifying blood products in the field of biotechnology, in particular to a method for efficiently extracting and purifying coagulation factor IX and coagulation factor X. 2. Background technology [0002] Coagulation factor IX (FIX for short) and factor X (Coagulation factor X, FX for short) are both vitamin K-dependent factors, both of which are synthesized by liver cells and secreted into the blood, and in the coagulation reaction Play their respective activities [1]. Both FIX and FX are glycoproteins. The molecular weight of human FIX is 57,000, containing about 17% polysaccharide [2]; the molecular weight of human FX is 72,000, containing about 10% polysaccharide [3]. [0003] FIX and FX are located at the intersection of exogenous and exogenous pathways in the cascade reaction of coagulation, so they play a very important role in the coagulation process. The content of FIX in plasma is generally believed ...

Claims

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Application Information

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IPC IPC(8): C07K14/745C07K1/18C07K1/22
Inventor 徐小龙沈登科
Owner UNIV OF SCI & TECH OF CHINA
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