Reptilase and its production process and application

A technology of hemocoagulase and snake venom, which is applied in the field of snake venom hemocoagulase and its production method and application, to achieve the effects of safe clinical use, shortening coagulation time, and shortening bleeding time

Inactive Publication Date: 2010-06-30
JINZHOU AHON PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Crotalase only breaks down fibrinopeptide A from the fibrinogen molecule, and prolonged incubation will lead to degradation of the fibrin chains

Method used

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  • Reptilase and its production process and application
  • Reptilase and its production process and application
  • Reptilase and its production process and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the production of hemocoagulase and hemocoagulase pharmaceutical preparation of white-browed snake venom of the present invention

[0037] Take 20 g of white-browed snake venom crystals and dissolve them in 1000 ml of 0.9% NaCl injection, centrifuge at 3000 rpm for 10 minutes, and discard the precipitate. Adjust the pH of the supernatant to 6.5, and then dilute to 1000 ml with 0.9% NaCl injection. To this solution, 670 ml of saturated ammonium sulfate solution was added, and the mixture was left at room temperature for 1 hour, then centrifuged at 3000 rpm for 10 minutes, and the precipitate was discarded. Add 552 ml of saturated ammonium sulfate solution to the supernatant, let it stand at room temperature for 1 hour, centrifuge the mixture at 3000 rpm for 10 minutes, discard the supernatant precipitate and dissolve it in 1000 ml of distilled water, and adjust the pH to 5.0. 7.7 ml of 90% phenol was added to this solution, and the mixture was left at room...

Embodiment 2

[0043] Embodiment 2, Determination of hemagglutinase-like activity of the white-browed snake venom of the present invention

[0044] The hemagglutinase of the white-browed snake venom of the present invention has a hemagglutinase-like effect and a thromboplastin-like effect, and the hemagglutinase-like effect of the white-browed snake venom on human fibrinogen is observed by adding EDTA to inhibit its thromboplastin-like effect effect.

[0045] Add Hemocoagulase sample 1ml (every sample is dissolved with 1ml 5 × 10-3mol / L EDTA) of the present invention in test tube, be divided into 4 groups, I, II, III are respectively Hemocoagulase high, middle , low-dose group, the doses are 2Ku / ml, 1Ku / ml, 0.5Ku / ml respectively, IV group is the blank group (add physiological saline 1ml), each tube acts on 37 ℃ water bath for 5 minutes after 5 hours, and then adds to each tube 0.1% human fibrinogen 1ml, observe and record the coagulation time of human fibrinogen. The results are shown in T...

Embodiment 3

[0050] Example 3, Determination of Thromboplastin-Like Activity of Hemocoagulase from Snake Venom of the Present Invention

[0051] The hemagglutinase-like effect of the white-browed snake venom of the present invention has a hemagglutinase-like effect and a thromboplastin-like effect, and the hemagglutase-like effect of the white-browed snake venom is observed by adding DFP (diisopropylfluorophosphoric acid) to inhibit the hemagglutase-like effect of the white-browed snake venom. Thromboplastin-like effect.

[0052] Add Hemocoagulase sample 1ml (every sample is dissolved with 2 * 10-3mol / L DFP solution) of the present invention in test tube, be divided into 4 groups, I, II, III group are respectively Hemocoagulase high, Hemocoagulase of Hemodon venom of the present invention, In the middle and low dose groups, the doses were 2Ku / ml, 1Ku / ml, and 0.5Ku / ml respectively. The IV group was the blank control group (with normal saline). Add 1ml of factor X-deficient standard plasma ...

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Abstract

The reptilase is extracted from snake poison of pallas pit viper, and its SDS-polyacrylamide gel electrophoresis exhibits three bands of molecular weight 54000 D, 34000 D and 15000 D separately. The reptilase producing process of the present invention includes the following steps: taking snake poison of pallas pit viper, dissolving in buffering liquid, centrifugal deposition, elution in saturatedphenol solution to obtain the grade-I reptilase product; dissolving the grade-I reptilase product in buffering liquid, ion exchange column chromatography and elution with buffering liquid to obtain refined reptilase liquid. The reptilase medicine of the present invention has very low toxicity, safe clinical application and wide application foreground.

Description

technical field [0001] The invention relates to a hemocoagulase and its production method and application, in particular to a snake venom hemocoagulase and its production method and application. Background technique [0002] Hemagglutinin uses fibrin I as a substrate, and quickly transfers to the injured part of the blood vessel to form a fibrin clot, preventing blood from flowing out from the injured part. The blood coagulation process is roughly divided into three stages, namely, the activation of coagulation factor X; the activation of procoagulation; and the transformation of fibrinogen into fibrin. The effect that can make fibrinogen into fibrin is called hemocoagulase-like effect; the effect that can be exerted by activating coagulation factor X and procoagulin is called thromboplastin-like effect. Snake venom is rich in proteases, among which a class of serine proteases is closely related to toxicological effects such as blood coagulation disorders caused by snake bi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/74A61K38/48A61P7/04
Inventor 于洪儒
Owner JINZHOU AHON PHARM CO LTD
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