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A blood coagulation factor X activator and a preparation method thereof

A technology for coagulation factors and activators, applied in the field of biomedicine, can solve the problems of complicated preparation methods, increased production costs, and inability to use large-scale industrial production.

Active Publication Date: 2019-01-15
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 a preparation method thereof
  • A blood coagulation factor X activator and a preparation method thereof
  • A blood coagulation factor X activator and a preparation method thereof

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 coagulation factor X activator derived from the venom of Bothrops Atrox, and also provides a preparation method of the coagulation factor X activator. The present invention also provides the use of the coagulation factor X activator in the preparation of a drug for the treatment of hemorrhagic disorders and a pharmaceutical composition for the treatment of hemorrhage. Compared with the prior art, the coagulation factor X activator provided by the invention has high purity, and is detected by using a volume exclusion chromatography SEC column, and the purity is 100%, and is detected by using a C4 reversed-phase column, and the purity is as high as 96%. The method of the present invention first uses anion and cation exchange carrier chromatography, then affinity chromatography or composite packing chromatography can not only make the purity of coagulation factor X activator close to electrophoretic purity, but also greatly prolong the service life of affinity packing, so as to save production cost and be 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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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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