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Hemocoagulase extracted from bothrops atrox and preparation method and application thereof

A technology of hemagglutinin and snake venom, applied in the field of biomedicine, can solve the problems of difficult separation and preparation requirements, poor heat source removal effect, inconvenient large-scale production, etc., to achieve clinical drug safety, reduce process environmental exposure risks, short cycle effect

Pending Publication Date: 2018-10-02
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the hemocoagulase purification chromatography steps disclosed in this method are 3 steps, gradient elution is used, and the yield in the purification process is low or the heat source removal effect is not good on the basis of ensuring purity.
[0011] In summary, the extraction methods of snake venom hemagglutinin disclosed in literature or patent applications in the prior art are difficult to meet the ideal separation and preparation requirements, and are not convenient for large-scale production

Method used

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  • Hemocoagulase extracted from bothrops atrox and preparation method and application thereof
  • Hemocoagulase extracted from bothrops atrox and preparation method and application thereof
  • Hemocoagulase extracted from bothrops atrox and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 extracts hemocoagulase from the venom of Agkistrodon lancehead

[0069] extraction steps

[0070] 1. Snake venom pretreatment

[0071] Accurately weigh 80 g of Agkistrodon halys venom (purchased from Liaoning Yuanda Nuokang Biopharmaceutical Co., Ltd.), dissolve it in pH7.4, and have a concentration of 0.02M Tris-HCl buffer solution to obtain a concentration of 100mg / ml solution. Centrifuge at 4°C for 10 min, and take the supernatant.

[0072] 2. DEAE Sephadex A-25 ion exchange chromatography (column I, purchased from GE Company)

[0073] The chromatographic column (4.5×50cm, Shanghai Jinhua glass chromatographic column) was equilibrated with pH 7.4, 0.02M Tris-HCl buffer solution for 3 column volumes before sample loading, flow rate: 2ml / min. Check the bacterial endotoxin in the subcolumn fluid, if the bacterial endotoxin does not exceed 0.25EU / ml, it can be ready for sample loading.

[0074] Take the supernatant in step 1 and load it, and after el...

Embodiment 2

[0093] Example 2 Extracting hemocoagulase from the venom of Agkistrodon lancehead

[0094] extraction steps

[0095] 1. Snake venom pretreatment

[0096] Accurately weigh 80 g of Agkistrodon lanceolata venom, dissolve it in pH 7.0, 0.04M Tris-HCl buffer to obtain a solution with a concentration of 200 mg / ml, centrifuge at 3000 rpm at 4°C for 20 min, and take the supernatant.

[0097] 2. DEAE Sephadex A-25 ion exchange chromatography (column Ⅰ)

[0098] The chromatographic column (4.5×50cm, Shanghai Jinhua glass chromatographic column) was equilibrated with pH 7.0, 0.04M Tris-HCl buffer solution for 3 column volumes before sample loading, flow rate: 2ml / min. Check the bacterial endotoxin in the subcolumn fluid, if the bacterial endotoxin does not exceed 0.25EU / ml, it can be ready for sample loading.

[0099] Take the supernatant in step 1 and load it with pH 7.0 and 0.01M Tris-HCl buffer to elute for 2 column volumes, then use the above-mentioned Tris-HCl buffer with pH ...

Embodiment 3

[0111] Example 3 Research on coagulation function of hemagglutinin

[0112] This study investigated the effect of Agkistrodon haemocoagulase on coagulation function and fibrin formation in patients with bleeding disorders in vitro

[0113] experiment one

[0114] 1. Purpose of the experiment

[0115] To investigate the effect of haemocoagulase of Agkistrodon lancehead on plasma activated partial thromboplastin time (APTT), prothrombin time (PT), activation of coagulation factor Ⅹ (FX) and generation of thrombin in patients with bleeding disorders.

[0116] 2. Experimental objects and methods

[0117] Specimen preparation and reagents: According to the principle of informed consent, 3.8% sodium citrate anticoagulated plasma was collected from 20 normal people, aged 20-45 years, half male and half male. 25 patients with hemophilia A, 7 patients with vitamin K-dependent coagulation factor deficiency, 3 patients with von Willebrand's disease (VWD), 3 patients with FX deficien...

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Abstract

The invention provides a hemocoagulase extracted from bothrops atrox and also provides a preparation method of the hemocoagulase. The method comprises the following steps of: pretreating the bothropsatrox; sequentially passing the diethylaminoethyl cross-linked dextran gel A-25 ion exchange chromatography, benzamidine agarose gel 4-FF (HS) affinity chromatography, and cross-linked dextran gel G-75 gel filtration chromatography; and obtaining hemocoagulase. The invention also provides the application of the bothrops atrox hemocoagulase. in the preparation of a medicament for treating bleedingdisorder. Compared with the prior art, the preparation process steps are less, the period is short, and the exposure risk of the process environment is reduced; the product yield is high, the processhas strong ability to remove impurities, the yield of the product can be obtained more than 35% and the purity is over 98%; the process stability is high and the product quality is good, and the process is beneficial to clinical medication safety.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for extracting hemocoagulase from Agkistrodon venom, and also relates to a method for extracting hemocoagulase from Agkistrodon venom and the use of the hemagglutinase. Background technique [0002] Blood coagulation is a complex physiological process, which can be roughly divided into three stages: the first stage, the activation of coagulation factor X (FX); the second stage, the activation of prothrombin; the third stage, the conversion of fibrinogen to fibrin. [0003] Extracting hemostatic medicine from snake venom has a long history. At present, the more successful clinical application has the hemostatic drug Reptilase produced in Switzerland, which stops bleeding immediately. Its main component is snake venom thrombin-like enzyme (Snake Venom Thrombin-like Enzyme, extracted from the venom of Brazilian snakes (Bothrops Jrarace, Lachesisatrox), SVTLE), a seri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64A61K38/48A61P7/04
CPCA61K38/00C12N9/6418
Inventor 崔亮亮石皎李秀琳李秀娜丁忠福葛琳孙东薛百忠王宏英薛雁
Owner GRAND LIFE SCI (LIAONING) CO LTD
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