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Hemostat, preparation method thereof, and application thereof

A technology of hemostatic agent and activator, applied in the field of hemostatic agent and its preparation, can solve the problem of not being able to use mature MTG industrial application, etc., and achieve a technology that is conducive to preservation and maintenance of activity, increases safety and reliability, and has good stability. Effect

Active Publication Date: 2012-10-17
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above reaction cannot be used for the industrial application of mature MTG

Method used

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  • Hemostat, preparation method thereof, and application thereof
  • Hemostat, preparation method thereof, and application thereof
  • Hemostat, preparation method thereof, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Fermentation of microbial transglutaminase zymogen

[0027] In this example, the strain producing the progenase of transglutaminase is Streptomyces mobaraensis. In the present invention, the microbial transglutaminase proenzyme can also be produced by fermentation from Bacillus subtilis, Streptomyces, Corynebacterium glutamicum and the like.

[0028] Slant medium (g / L): starch 20, KNO3 1, MgSO4 7H2O 0.5, K2HPO4 3H2O 0.5, NaCl 0.5, FeSO4 7H2O 0.01, agar 20. Fermentation medium (g / L): glycerin 20, yeast extract 6, peptone 25, MgSO4 7H2O 2, K2HPO4 3H2O 2 10.

[0029] Colonies were first inoculated on slant medium and cultured for 7 days. All microorganisms were collected, inoculated into the fermentation medium, samples were taken at 8, 12, 16, 20, and 24 hours respectively, and the expression level of pro-MTG was determined by electrophoresis.

[0030] After 8, 12, 16, 20, and 24 hours of fermentation of the enzyme-producing strains, electrophoresis was pe...

Embodiment 2

[0031] Example 2 Separation and purification of microbial transglutaminase zymogen

[0032] The fermentation broth fermented for 20 hours in the above-mentioned Example 1 was centrifuged to remove bacteria. Collect the supernatant, add 2 times the volume of absolute ethanol, and stir for 30 minutes. The precipitate was collected by centrifugation. Protein separation and purification using ion exchange columns. The filler is DEAE-825, and the balance liquid is 0.05M tris buffer, pH7.4. The mobile phase was 0.1 mol / L sodium dihydrogen phosphate buffer at pH 6.0. Each fraction was collected and detected by electrophoresis to obtain pro-MTG protein with better purity. The fraction was desalted and stored lyophilized. Such as figure 2 The results of electrophoresis showed that the target components were collected after ion exchange, and the purity of the purified target protein detected by electrophoresis was above 99%.

[0033] The protein obtained under the above experime...

Embodiment 3

[0034] Embodiment 3 microbial transglutaminase zymogen and the stability comparison of glutamine transaminase

[0035] The pro-MTG and MTG were placed and treated at different temperatures for 1 hour respectively, wherein the zymogen pro-MTG was treated with dispase, and MTG was directly treated with buffer. Then adjust it to 37°C, add an equal volume of sterile water containing 50% (w / w) lipase or lipase-free, respectively, and measure the enzyme activity of MTG after 30 minutes, compare different treatment points with the initial enzyme activity, and calculate the relative residual enzyme activity. Compared with the untreated enzyme activity, 1.0 represents 100% enzyme activity, that is, there is no loss of enzyme activity.

[0036] Experimental results such as image 3 As shown, the enzymatic activity of pro-MTG only slightly decreased after being treated at 70 degrees Celsius for 1 hour, while the enzymatic activity of MTG had been reduced by 50%. Pro-MTG still retains ...

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Abstract

The invention discloses a hemostat, comprising gelatin, microbial transglutaminase zymogen, and an activating agent of the microbial transglutaminase zymogen. The ratio of gelatin to microbial transglutaminase zymogen to the activating agent is 100000:1-200:1-100. The invention also discloses a preparation method and an application of the hemostat. The hemostat provided by the invention has better stability and hemostatic effect, and enhanced safety and reliability.

Description

technical field [0001] The invention relates to the related technical field of a hemostatic material, in particular to a hemostatic agent and its preparation method and application. Background technique [0002] In the prior art, gelatin has been widely used for hemostasis, such as Surgiflo® fluid gelatin of Johnson & Johnson Medical Equipment Co., Ltd., chitosan gelatin (Chinese patent application number: 200310121182.X), modified gelatin (Chinese patent application number: 200910244834.6) And other gelatin for hemostasis, such as Chinese patents 02121201.5, 98117763.8, 03126852.8, 02104346.9, 01142070.7, etc.; Gelatin has a hemostatic effect and can be used to stop bleeding in trauma or surgery. [0003] Microbial transglutaminase (abbreviated MTG) belongs to the same family of enzymes as coagulation factor FACTOR XIII, and many studies have shown its role in coagulation reactions. MTG can be used to cross-link gelatin to form a good hollow or network, for example, it ca...

Claims

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Application Information

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IPC IPC(8): A61L15/44A61L15/32A61K38/48A61P7/04A61K38/39
Inventor 金明飞刘明耀高红亮易正芳
Owner EAST CHINA NORMAL UNIVERSITY
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