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Agkistrodon halys venom thrombin and preparation method and application thereof

A technology of thrombin and snake venom, which is applied in the field of snake venom thrombin and its preparation, can solve the problems of softness and instability, and achieve high stability and safety, high safety, and good hemostatic performance

Active Publication Date: 2010-11-10
BEIJING SAISHENG PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The polymer clot formed by the connection of soluble fibrin monomers through secondary bonds such as hydrogen bonds can form a blood clot, but it is soft and unstable, and needs to be stored in Ca 2+ Participation. Only by the action of XIIIa can it be further transformed into a stable fibrin polymer

Method used

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  • Agkistrodon halys venom thrombin and preparation method and application thereof
  • Agkistrodon halys venom thrombin and preparation method and application thereof
  • Agkistrodon halys venom thrombin and preparation method and application thereof

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Effect test

Embodiment 1

[0054] (1) DEAE-Sepharose Fast Flow chromatography

[0055] Weighed 20 grams of snake venom purchased from Agkistrodon blomhoffii brevicaudus in Liaoning, dissolved it in 0.05 mol / L pH 8.0 Tris-HCl buffer, and centrifuged at 6000 rpm / min for 15 minutes to obtain a supernatant. Put the supernatant on a well-balanced DEAE-Sepharose Fast Flow chromatography column, first use 0.05mol / L Tris-HCl buffer to elute unadsorbed impurities, and then use a buffer solution containing 0-0.6mol / L NaCl for gradient Elution, collect each peak solution, take 0.1ml of each solution and add bovine plasma diluted twice with normal saline, if the plasma coagulates, it is the peak of thrombin activity. See figure 2 .

[0056] (2) Affinity chromatography

[0057] Put the active component collection solution of the above-mentioned thrombin activity peak on a well-balanced affinity chromatography column, first elute unadsorbed impurities with 0.05mol / L Tris-HCl pH8.0 buffer containing 0.5mol / L NaCl;...

Embodiment 2

[0064] The measurement result of the Agkistrodon halys venom thrombin made by embodiment 1 is as follows:

[0065] 1) Measured by SDS-polyacrylamide gel electrophoresis, a single band can be seen in the electropherogram, and the molecular weight is 32.3Kda. See Figure 5 .

[0066] 2) Determination of chromatographic conditions and system suitability test by high performance liquid chromatography: gel chromatography column: TSK GEL2000SWxl7.8mmX300mm, 5μm; mobile phase: 0.2mol / L sodium phosphate buffer (pH6.8); detection wavelength: 280nm ;; Injection volume: 20 μL; Column temperature: room temperature; Flow rate: 1.0ml / min. As a result, the purity was 98.56%. See Image 6 .

[0067] 3) As determined by the Procise 491 sequencer of the American ABI company, the enzyme consists of 236 amino acids, and the amino acid sequence is shown in SEQ ID NO: 1 (see figure 1 ).

[0068] 4) Activity measurement: the amount of enzyme that can coagulate 1.0ml of oxalated bovine whole b...

Embodiment 3

[0071] The thrombin of the invention can coagulate human plasma and bovine fibrinogen in vitro, and the coagulation specific activity of plasma is greater than 600 units / mg. The clot was dispersed in 5M urea solution, indicating that Agkistrodon venom thrombin did not activate coagulation factor XIII, and produced cross-linked fibrin, which could be rapidly degraded and cleared by plasmin.

[0072] Effect of intraperitoneal injection of Agkistrodon venom thrombin on the bleeding time of mice: Mice were intraperitoneally injected with normal saline, Agkistrodon venom thrombin, and Helizhizhi. 30 minutes after the administration, the tail of the mouse was quickly cut at 0.5 cm from the tip of the tail, and the outflowing blood was sucked up with filter paper regularly. The bleeding time was from the beginning of cutting the tail to the end of no bleeding. The results are shown in Table 2.

[0073] Table 2 Effect of intraperitoneal injection of Agkistrodon venom thrombin on blee...

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Abstract

The invention provides an agkistrodon halys venom thrombin with a single component and a preparation method and application thereof. The thrombin is the protein of (a) or (b), wherein (a) is the protein consisting of amino acid sequence displayed by SEQ ID NO:1, and (b) is the protein which is derived from (a), provided with agkistrodon halys venom thrombin activity and formed by replacing, deleting or adding one or a plurality of amino acids in the amino acid sequence of (a). The agkistrodon halys venom thrombin is clinically used for preventing bleeding and stopping bleeding. In the invention, the agkistrodon halys venom thrombin is obtained from agkistrodon halys venom via ion-exchange chromatography, affinity chromatography and gel chromatography. The invention also provides a drug composite containing the agkistrodon halys venom thrombin.

Description

technical field [0001] The invention relates to a snake venom thrombin and a preparation method thereof, in particular to a Agkistrodon halys venom thrombin and a preparation method thereof. [0002] The present invention also relates to the application of the snake venom thrombin. Background technique [0003] Blood coagulation is the process of changing blood from liquid to gel, which is an important part of hemostasis in mammals. Macfarlane et al. proposed the cascade reaction hypothesis of the coagulation process in 1964, and believed that coagulation is a series of coagulation factors activated by their prefactors to finally generate thrombin, and thrombin converts fibrinogen into fibrin clot a series of enzymatic reactions. This process includes the formation of fibrin monomers, polymerization and cross-linking of fibrin. Fibrinogen is synthesized by the liver and has two α-chains (Aa): two β-chains (Bβ), and two γ-chains (γ2), namely three pairs of different polype...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/74A61K38/48A61K47/18A61P7/04C12N15/57
Inventor 马骉孔双泉王靖宋梦薇孙爽
Owner BEIJING SAISHENG PHARMA
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