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Rapid hemostatic product for war wounds and preparation method thereof

A product, rapid technology, applied in the field of rapid hemostasis products, can solve problems such as insufficient activity

Active Publication Date: 2014-05-28
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0033] Therefore, the idea of ​​the present invention is to overcome the shortcomings of the existing transglutaminase, which is insufficient in activity at normal temperature, or requires auxiliary agent activation (i.e. activating the enzyme), and provides a novel transglutaminase as the main active ingredient, Rapid hemostatic materials and composite hemostatic products for hemostasis in war or other wounds

Method used

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  • Rapid hemostatic product for war wounds and preparation method thereof
  • Rapid hemostatic product for war wounds and preparation method thereof
  • Rapid hemostatic product for war wounds and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1 Enzyme Activity Determination

[0090] 1) Preparation of reagents for measuring enzyme activity

[0091] Solution A (0.5L): Add 0.015mol (5.06g) of substrate Na-CBZ-Gln-Gly, 0.05mol (3.475g) of hydroxylamine hydrochloride, 0.005mol (1.536g) of reduced glutathione to 400ml of distilled water in a beaker After stirring with a magnetic stirrer for 20 minutes, add 0.1mol (12.11g) Tris, adjust the pH to 6.0 with 6mol / L (or 1mol / L) hydrochloric acid, transfer the solution to a 500ml volumetric flask, and wash the beaker with distilled water for 3 Pour it into a volumetric flask once, and set the volume to 500ml.

[0092] Solution B: 3mol / L HCl, 12% trichloroacetic acid (W / V), 5% ferric chloride (W / V, dissolved in 0.1mol / L hydrochloric acid, then filtered) according to the volume ratio of 1:1:1 mix.

[0093] 2) Determination of enzyme activity

[0094] Experimental group: take 0.2ml of the sample to be tested, add 2ml of solution A and incubate at 37°C for 10 min...

Embodiment 2

[0095] The mutagenesis of embodiment 2 wild strains

[0096] The wild-type mTG strain is Streptomyces mobaraensis (such as the strain ATCC No. 29032 of the American ATCC or the strain ATCC No. 27441 of the American ATCC, or the strains of the China Microorganism Conservation Center such as CGMCC No. 4.1719 and CGMCC No. 4.5591).

[0097] Medium configuration: Gaoshi No. 1 medium: soluble starch 20g / L, KNO 3 1g / L, MgSO 4 ·7H 2 O0.5g / L, K 2 HPO 4 ·3H 2 O0.5g / L, NaCl0.5g / L, FeSO 4 ·7H 2 O0.01g / L, agar 20g / L, pH7.2-7.4. Fermentation medium: glycerin 20g / L, yeast extract 6g / L, fish meal peptone 25g / L, MgSO 4 ·7H 2 O2g / L, K 2 HPO 4 ·3H 2 O2g / L, pH7.4.

[0098] Add 10ml of cold sterile water to Gao's No. 1 medium, use an inoculation needle to fully scrape the surface hyphae, break up the spores, and filter with sterile filter paper.

[0099] The operation is carried out under red light or dark conditions, and the ultraviolet light is turned on 0.5 hours in advance to st...

Embodiment 3

[0100] The acquisition of embodiment 3 mutant mTG gene

[0101]Genome extraction: Inoculate the fresh mycelium of the mutant strain with the highest enzyme activity in liquid culture in Example 2 into fresh medium, and cultivate for about 24 hours; collect 10 ml of thalline by centrifugation; fresh thalline in a mortar (-20 ℃ (pre-cooled), grind repeatedly with liquid nitrogen until fine powder, quickly and evenly distribute into two 1.5mL centrifuge tubes; add 550μL of TE buffer, add 30μL of 20% SDS solution preheated at 65℃, and vortex for 5 seconds , add 20 μL of 20 mg / mL proteinase K, mix gently, and incubate at 37°C for 1 hour; add an equal volume of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1) for extraction, slightly invert to mix, and centrifuge at 10,000 rpm for 10 minutes ; Carefully draw the supernatant into a new centrifuge tube, add an equal volume of chloroform / isoamyl alcohol (24:1) for extraction, centrifuge at 10,000 rpm for 5 minutes; absorb the...

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Abstract

The invention discloses a rapid hemostatic product for war wounds, as well as a preparation method and application thereof. The rapid hemostatic product comprises a substrate with added microbial transglutaminase (mTG), wherein the amino acid sequence of the transglutaminase is the protein sequence coded by the gene shown in SEQ ID NO.: 3, or a protein sequence shown in SEQ ID NO.: 4. More specifically, the rapid hemostatic product is selected from a hemostatic agent, a hemostatic device, a hemostatic first-aid kit or a common hemostatic product, the substrate is in a fluid or semi-solid form, which can be absorbed by a human body, or in a malleable gel or colloid form, and the rapid hemostatic product contains the microbial transglutaminase and gelatin. The rapid hemostatic product has the advantages of rapid hemostasis, convenience for use, organ adhesion prevention, wound healing promotion, low toxic and side effects, complete absorption and the like; except for hemostasis in a war, the rapid hemostatic product can also be widely applied to rapid hemostasis for wounds in fire control, accidents and other wild environments.

Description

technical field [0001] The invention belongs to a rapid hemostatic product for field wounds, in particular to a hemostatic composition containing microbial transglutaminase (mTG) and its preparation method and application. The product can be used in fields such as wars, firefighting and safety accidents Environmental wounds quickly stop bleeding. Background technique [0002] Excessive blood loss is one of the leading causes of trauma-related death among civilians in peacetime, and controlling hemorrhage (or bleeding) is an important step in first aid and trauma care. In field environments such as combat, such as wars, firefighting, safety accidents, etc., due to the limited on-site rescue measures, time urgency, and poor conditions, excessive bleeding and death occur from time to time. According to the latest statistics, 90% of the officers and soldiers killed in the U.S. military’s anti-terrorist operations died on the way to the hospital, and nearly 1 / 4 died of excessive...

Claims

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Application Information

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IPC IPC(8): A61L15/38A61L15/42A61L26/00C12N9/10C12N15/54
CPCA61K38/00A61L15/44A61L29/048C12N9/1044C12Y203/02013
Inventor 易正芳吕方丛晓楠金明飞裴正培刘明耀
Owner EAST CHINA NORMAL UNIVERSITY
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