A method for affinity purification of blood coagulation factor VIII using peptide ligand

A blood coagulation factor and peptide ligand technology, which is applied to the preparation method of peptides, blood coagulation/fibrinolysis factors, factor VII, etc., can solve the problems of poor stability and increased cost, and achieve easy preparation, increased safety, and high-efficiency purification. Effect

Active Publication Date: 2019-08-23
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the end of the 1970s, the affinity chromatography process using coagulation factor VIII antibody as a ligand began to be applied to the purification of coagulation factor VIII products. However, due to the poor stability of this type of coagulation factor VIII products, it is necessary to add human albumin As a protective agent, the specific activity can only reach 15IU / mg protein, and it also increases the cost of the product

Method used

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  • A method for affinity purification of blood coagulation factor VIII using peptide ligand
  • A method for affinity purification of blood coagulation factor VIII using peptide ligand
  • A method for affinity purification of blood coagulation factor VIII using peptide ligand

Examples

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Embodiment 1

[0030] A method for affinity purification of blood coagulation factor VIII using peptide ligands, comprising the following steps:

[0031] (1) Preparation of affinity chromatography resin for purification:

[0032] a. Preparation of the peptide ligand solution: Weigh 2.8 μmol of the peptide ligand and dissolve it in 0.8 ml double distilled water, stir until the peptide ligand is completely dissolved to obtain the peptide ligand solution; A polypeptide with an affinity reaction for factor VIII; the polypeptide is GCVSGCLCWEYC;

[0033] b. Agarose gel pretreatment: Take 8ml of Sepharose CL-4B stored in 20% ethanol aqueous solution, that is, Sepharose CL-4B, and use excess distilled water to repeatedly wash the Sepharose CL-4B to remove the agar Ethanol on the surface of agarose gel CL-4B to obtain wet agarose gel CL-4B;

[0034] c. Put the wet agarose gel CL-4B obtained after the treatment in step b into a centrifuge tube, add 20ml of pre-cooled 1mM HCl into the centrifuge tube,...

Embodiment 2

[0043] A method for affinity purification of blood coagulation factor VIII using peptide ligands, comprising the following steps:

[0044] (1) Preparation of affinity chromatography resin for purification:

[0045] a. Preparation of the peptide ligand solution: Weigh 3.0 μmol of the peptide ligand and dissolve it in 1 ml of double distilled water, stir until the peptide ligand is completely dissolved to obtain a peptide ligand solution; VIII A polypeptide with an affinity reaction; the polypeptide is RKWNCTDHEYC;

[0046] b. Agarose gel pretreatment: Take 10ml of Sepharose CL-4B stored in 20% ethanol aqueous solution, that is, Sepharose CL-4B, and use excess distilled water to repeatedly wash the Sepharose CL-4B to remove the agar Ethanol on the surface of agarose gel CL-4B to obtain wet agarose gel CL-4B;

[0047] c. Put the wet agarose gel CL-4B obtained after the treatment in step b into a centrifuge tube, add 25ml of pre-cooled 1mM HCl into the centrifuge tube, vortex an...

Embodiment 3

[0056] A method for affinity purification of blood coagulation factor VIII using peptide ligands, comprising the following steps:

[0057] (1) Preparation of affinity chromatography resin for purification:

[0058] a. Preparation of the peptide ligand solution: Weigh 3.2 μmol of the peptide ligand and dissolve it in 2 ml of double distilled water, stir until the peptide ligand is completely dissolved to obtain the peptide ligand solution; VIII A polypeptide with an affinity reaction; the polypeptide is EYCPC;

[0059] b. Agarose gel pretreatment: Take 12ml of Sepharose CL-4B stored in 20% ethanol aqueous solution, that is, Sepharose CL-4B, and use excess distilled water to repeatedly wash the Sepharose CL-4B to remove the agar Ethanol on the surface of agarose gel CL-4B to obtain wet agarose gel CL-4B;

[0060] c. Put the wet agarose gel CL-4B obtained after the treatment in step b into a centrifuge tube, add 25ml of pre-cooled 1mM HCl into the centrifuge tube, vortex and mi...

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Abstract

The invention discloses a method for implementing affinity purification on a coagulation factor VIII by using a peptide ligand. According to the method, the human-coagulation-factor-VIII-specific peptide ligand is used as an affinitive substrate, and the specific peptide ligand is fixed to a sepharose gel carrier to implement quick, specific and efficient purification on the coagulation factor VIII. The peptide ligand is a polypeptide capable of performing affinity reaction with the coagulation factor VIII. The polypeptide is any one of EYCPC, GCVSGCLCWEYC and RKWNCTDHEYC, or the mixture of EYCPC, GCVSGCLCWEYC and RKWNCTDHEYC in any ratio. The coagulation factor VIII obtained by the method has the advantages of high yield and high specific activity; and the method can implement large-scale purification and preparation on the coagulation factor VIII.

Description

technical field [0001] The invention relates to the field of biological separation and purification, in particular to a method for affinity purification of blood coagulation factor VIII by using peptide ligands. Background technique [0002] Coagulation factor VIII (coagulation factor VIII, FVIII) is an important coagulation factor in the intrinsic coagulation pathway. As a cofactor of coagulation factor IXa, it participates in the activation of coagulation factor X, and its content in plasma is about 0.1mg / L . Inherited deficiency of FVIII will lead to hemophilia A (or hemophilia A), so it is also called antihemophilic globulin or antihemophilic factor. Intravenous injection of FVIII products to replace hemophilia A is the main treatment at present. [0003] As early as the 19th century, European and American countries began to treat the bleeding symptoms of hemophilia A patients through blood transfusion, but the effect of this treatment was not obvious; in 1956, a crude...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/755C07K1/36C07K1/34C07K1/22C07K1/16
CPCC07K14/755
Inventor 陈正跃许建文陈美光王建刚段素芳高夏欢
Owner XINXIANG MEDICAL UNIV
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