A blood coagulation factor X activator and its preparation method

A technology for coagulation factor and activator, applied in the field of biomedicine, can solve the problems of increased production cost, complicated preparation method, and inability to use large-scale industrial production, and achieves production cost saving, high purity, and is conducive to large-scale industrialization. production effect

Active Publication Date: 2021-09-28
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In summary, the current purified raw materials for coagulation factor X activators are mainly separated and purified from viper venom, and the preparation method is cumbersome, which greatly increases the production cost and cannot be used in large-scale industrial production.

Method used

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  • A blood coagulation factor X activator and its preparation method
  • A blood coagulation factor X activator and its preparation method
  • A blood coagulation factor X activator and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1 Separation and purification of coagulation factor X activator from Agkistrodon lancehead venom

[0117] 1. Dissolving snake venom

[0118] Get 10g Agkistrodon lanceolata venom (snake venom purchased from Liaoning Yuanda Nuokang Biopharmaceutical Co., Ltd.), dissolve it in a chromatographic cabinet at 4-8°C with 100ml pH8.0, 0.05mol / L Tris-HCl buffer solution, and centrifuge at 3000g After 15 minutes, take the supernatant for later use.

[0119] 2. DEAE-Sepharose Fast Flow column chromatography: 2.6×20cm (filler purchased from GE Company)

[0120] 2-1) DEAE-Sepharose Fast Flow column packing: Take about 90ml of DEAE-Sepharose Fast Flow filler stored in 20% ethanol, first replace the alcohol with pure water and then fill a 2.6×20cm chromatographic column, and then use pH8.0, 0.05mol / L Tris-HCl buffer equilibrated over 10 column volumes, linear flow rate 100cm / h, 280nm wavelength ultraviolet absorption detection.

[0121] 2-2) Sample loading and elution: pum...

Embodiment 2

[0152] Example 2 Separation and purification of coagulation factor X activator from Agkistrodon lancehead venom

[0153] 1. Dissolving snake venom

[0154] Take 10g Agkistrodon lanceolata venom (purchased from Liaoning Yuanda Nuokang Biopharmaceutical Co., Ltd.), dissolve it in a chromatographic cabinet at 4-8°C with 100ml pH7.4, 0.01mol / L Tris-HCl buffer solution, and centrifuge at 3000g After 15 minutes, take the supernatant for later use.

[0155] 2. DEAE-Sepharose Fast Flow column chromatography: 2.6×20cm (purchased from GE Company)

[0156] 2-1) Packing with DEAE-Sepharose Fast Flow: Take about 90ml of DEAE-Sepharose Fast Flow filler stored in 20% ethanol, first replace the alcohol with pure water and then fill a 2.6×20cm column, then use pH7.4, 0.01mol / L Tris-HCl buffer equilibrated over 10 column volumes, linear flow rate 100cm / h, 280nm wavelength UV absorption detection.

[0157] 2-2) Sample loading and elution: pump the centrifuged snake venom supernatant into th...

Embodiment 3

[0179] Example 3 Separation and purification of Agkistrodon venom coagulation factor X activator

[0180] 1. Dissolving snake venom:

[0181] Take 10g Agkistrodon lanceolata venom, dissolve it in 100ml pH8.5, 0.1mol / L Tris-HCl buffer solution in a chromatographic cabinet at 4-8°C, centrifuge at 3000g for 15min, and take the supernatant for later use.

[0182] 2. DEAE-Sepharose Fast Flow column chromatography: 2.6×20cm (filler purchased from GE Company)

[0183] 2-1) Packing with DEAE-Sepharose Fast Flow: Take about 90ml of DEAE-Sepharose Fast Flow filler stored in 20% ethanol, first replace the alcohol with pure water and then fill a 2.6×20cm column, then use pH8.5, 0.1mol / L Tris-HCl buffer equilibrated over 10 column volumes, linear flow rate 100cm / h, 280nm wavelength ultraviolet absorption detection.

[0184] 2-2) Sample loading and elution: pump the centrifuged snake venom supernatant in step 2-1) into the balanced chromatographic column at a linear flow rate of 60 cm / h...

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Abstract

The invention provides a blood coagulation factor X activator, which is derived from bothrops atrox snake venom, and the invention also provides a preparation method of the blood coagulation factor X activator. The present invention also provides the application of the blood coagulation factor X activator in the preparation of medicine for treating bleeding disorders and a pharmaceutical composition for treating bleeding. Compared with the prior art, the blood coagulation factor X activator provided by the present invention has high purity, and the purity is 100% when detected by the size exclusion chromatography SEC column, and the purity is as high as 96% when detected by the C4 reverse-phase column. The method provided by the present invention first uses anion-cation exchange packing chromatography, and then performs affinity chromatography or composite packing chromatography, which not only can make the purity of coagulation factor X activator close to electrophoretic pure after affinity column chromatography, but also greatly extend the The service life of the affinity filler is extended, thereby saving the production cost and being conducive to large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a coagulation factor X activator derived from the venom of Bothrops atrox, and also relates to a preparation method and application of the activator. Background technique [0002] Snake venom is rich in various proteases and polypeptides that act on many links of the blood coagulation system of mammals. These components are specific to blood coagulation factors and are widely used in anticoagulation, antithrombosis and hemostasis. Coagulation factor X activator (FXA) specifically acts on coagulation factor X (FX), which rapidly converts FX into activated coagulation factor X (FXa), and prothrombin is activated under the action of FXa to form active thrombin , thrombin acts on plasma fibrinogen to convert it into fibrin, and then plays a hemostatic role. The coagulation factor X activator obtained from snake venom is more effective than the existing hemostatic drugs, and ha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/04
CPCA61K38/00C12N9/6418C12Y304/21006
Inventor 丁忠福李秀琳李秀娜崔亮亮李香南李萍薛雁王宏英薛百忠
Owner GRAND LIFE SCI (LIAONING) CO LTD
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