CN103816562B - A rapid hemostasis product for war wounds and its preparation method - Google Patents

Abstract

The invention discloses a kind of scars of war quick-acting haemostatic powder product and its preparation method and application, this product comprises the matrix that with the addition of described Microbial transglutaminase (mTG), does the aminoacid sequence of wherein said Transglutaminase EC2.3.2.13 have as SEQ? ID? the protein sequence of the genes encoding described in NO:3, or have as SEQ? ID? protein sequence described in NO:4.More specifically, this hemostasia products is selected from hemostatic agent, hemostasis device, first-aid hemostatic bag or common hemostatic article, and described matrix is the absorbable fluid of human body or semi-solid form, or plastic glue or colloid form.It contains Microbial transglutaminase and gelatin.The present invention has quick-acting haemostatic powder, easy to use, anti-organ is adhered, promote wound healing, toxic side effect is low, absorb the advantage such as completely, except for except war hemostasis, also can be widely used in the wound quick-acting haemostatic powder of the field environment such as fire-fighting, security incident.

Description

A rapid hemostasis product for war wounds and its preparation method

technical field

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

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 blood loss within 5 to 10 minutes of being injured. . According to statistics, peripheral trunk vascular injuries account for more than 95% of all vascular injuries in wars. Hemostasis technology has become the most important link in the treatment of war wounds. Therefore, timely hemostasis in wars plays a key role in reducing the death rate. In addition, heavy hemorrhage also often causes death in safety accidents such as traffic accidents. In my country, at least 100,000 people die each year due to traffic accidents. %) and lead to death. Therefore, it is particularly important to develop rapid hemostasis products that are adapted to actual needs such as my country's war or safety accident wounds in time.

At present, there are 8 standards for emergency hemostatic products that are considered ideal in wars or accidents internationally, namely: (1) rapid hemostasis; (2) ready to use, no need to prepare or mix in advance; (3) easy to use, Does not require the participation of professional medical personnel; (4) light weight and durable; (5) storage period of at least 2 years and wide temperature range storage capacity (-10 ° C to 55 ° C); (6) no toxic side effects, no harm or the risk of spreading viral diseases; (7) low cost; (8) less side effects on wounds, which facilitates rapid wound healing. Products currently on the market include the following:

(1) Zeolites

Zeolite is a mineral in nature, and it is mostly distributed in mineral deposits or volcanic ash deposits at the bottom of lakes. Using aluminate polymers and silicates as raw materials, artificial zeolites similar to natural zeolites can be produced by controlling the reaction type and crystallization temperature. After dehydration and disinfection, natural or artificial zeolite crystals become a strong absorbent, which can selectively absorb various gases and liquids, and also absorb water in blood. According to this principle, the Military Medical College of the Chinese People's Liberation Army has developed a quick-acting hemostatic powder, which is a hemostatic product mainly composed of zeolite, which has the advantages of good hemostatic effect and low cost. However, zeolite-based hemostatic materials have some shortcomings: first, heat is released during the hemostasis process, which increases the local temperature, and the instantaneous temperature is close to 60-70 ° C, which is likely to cause burns at the wound; second, it is difficult to remove after hemostasis, causing physical and mental harm to the patient Pain may cause secondary bleeding and aggravate physical and mental pain; third, zeolite has a high density and is inconvenient to carry. However, since there is no better substitute at present, it is still a hemostatic product currently used for self-rescue in wars or field accidents.

(2) Quickclot

The U.S. military used Quickclot to stop the bleeding of battle wounds in the Afghanistan War and the Iraq War, and the effect was good, which significantly improved the survival rate of the wounded. The material is an artificially synthesized inert granular mineral composed of silicon oxide, aluminum, sodium, magnesium and a small amount of quartz. It has the effect of molecular sieve and moisture absorption. When used, it is covered on the bleeding wound and quickly absorbed into the blood clot The water can accelerate the blood coagulation process and make the blood scab form early. However, similar to zeolite, this material also has disadvantages such as heat generation and difficulty in clearing.

(3) Fibrin glue

In 2000, Rorbert (Rorbert LW. Tissue adhesive for battle field hemorrhage control [C] ADB209674, 2000: 1? 14.) developed a hemostatic dressing for war wounds, which consists of three layers: the innermost layer is a silicone film sprinkled with fibrin glue dry powder, the middle One layer is polyurethane foam, and the outer layer is medical pressure-sensitive adhesive, which can be used to stop bleeding from ruptured arteries. However, the disadvantages of this kind of materials are that they may cause infection of human or animal blood-borne diseases, complex structure, high cost, inconvenient use, slow hemostasis speed, and poor hemostatic effect on large blood vessels.

In addition, some fibrin glues require temporary addition of aprotinin during use (for example: Tisseel and Beriplast are equipped with bovine aprotinin, while Crosseal is equipped with tranexamic acid) to slow down the dissolution of fibrin during the coagulation process. In 2008, it was reported that aprotinin increased the mortality rate of cardiac surgery (FergussonDA, HebertPC, MazerCD, FremesS, MacAdamsC, MurkinJM, eta1. Acomparison of aprotinin and lysine analogues in high risk cardiac surgery [J]. NEnglJMed, 2008, 358: 23192331.), Bayer Medicine The company recalled its aprotinin preparation (trade name: Trasylo1) in various hospital pharmacies.

(4) Oxidized cellulose

Oxidized cellulose (trade name: Oxycel) is a kind of cellulose derivative, which is produced by oxidizing cellulose with nitric oxide. And oxidized regenerated cellulose (also known as instant yarn, Chinese name: soluble hemostatic gauze, trade name: Surgicel), is a kind of regenerated oxidized fiber woven yarn block, which belongs to carboxymethyl cellulose hemostatic material. A gauze-like or cotton-like absorbable hemostatic material processed into cellulose acid. Its hemostatic mechanism is that the acidic carboxyl group combines with Fe3+ in hemoglobin to form a brown glue block, which seals the end of the capillary and stops bleeding. Physiological blood coagulation mechanism (JamborovaG. PospisilovaN, SemeckyV, HysplerR, TichaA, PospechovaK, eta1. Microdispersedoxidizedcellulose as novel potential substance with hypolipidemic properties [J]. Nutrition, 2008, 24: 11741181.). Soluble hemostatic gauze can make blood coagulation components gather around it after close contact with the wound, and complete hemostasis in 2-8 minutes. Currently, it is commonly used in surgical wound bleeding and parts that are difficult to stop bleeding, such as bone surface bleeding. There is evidence that Surgicel begins to be absorbed by the body within one day of contact with the wound, and the absorption rate depends on the amount used, the local blood supply and the nature of the local tissue, and generally takes 4 to 8 weeks. The product has the advantages of good tissue compatibility, softness and thinness, and is easy to pack, apply and stuff. The disadvantage is that the hemostasis time is longer, and the effect on the rapid bleeding is poor, so it is not suitable as a wound hemostatic material alone (Zhou Ji. Introducing a new type of rectangular first aid kit [J]. People's Military Doctor, 2002, 45(1): 61. ). In addition, Surgicel can reduce the pH value around the damaged tissue. This highly acidic environment has a certain antibacterial ability, but the high acidic environment can also enhance the inflammatory response around the damaged tissue and delay wound healing (KrízovP, MsovL, SuttnarJ, SalajP, DyrJE, Homola J, eta1. The influence of intrinsic coagulation pathway on blood platelets activation by oxidized cellulose [J]. Biomed Mater Res A, 2007, 82: 274280.).

(5) Hemostatic gelatin

Hemostatic gelatin is extracted and purified from animal skins such as pigs or cows. Its porous structure can absorb blood 45 times heavier than itself. After absorbing blood, the volume expands to seal blood vessel cracks or wounds, activates platelets, and promotes the formation of blood clots. To achieve the purpose of hemostasis. Although hemostatic gelatin is derived from animal tissues, it has no antigenicity and no tissue reaction, and it can be digested and absorbed by the human body after 4 to 6 weeks of enzyme action in the body. Unlike the oxidized regenerated cellulose mentioned above, hemostatic gelatin does not affect the pH value of the tissue around the wound, so it is more suitable for use in combination with other hemostatic materials (IgaiH, YamamotoY, ChangSS, YamamotoM, TabataY, YokomiseH. Tracheal cartilage regeneration by slow release of basic fibroblast growth factor from gelatinsponge[J]. , 2007, 134: 170175.). However, gelatin hemostasis requires the participation of coagulation factors in the body, and the effect is not good for patients with coagulation disorders (Martinowite U, Spotnite WD. Fibrintissue adnesives [J]. Thromb Haemost, 1997, 78: 661.), and because it expands after absorbing blood, it may compress the surrounding area of the wound tissue, so it is not suitable for surgical sites close to nerves or with limited space. FloSeal, developed by Baxter, combines bovine gelatin matrix with thrombin preparations. The gelatin particles pack and compress the wound to stop bleeding through volume expansion, and thrombin accelerates the formation of blood clots. However, as mentioned above, the hemostatic effect of these two components depends on the body's coagulation mechanism. In addition, the thrombin in FloSeal comes from human plasma, and it has been confirmed that the two-step steam heating method can effectively reduce the viral load in plasma, but the manufacturer admits that there is no processing that can completely eliminate the virus infectivity in human plasma products Technology (ErethMH, SchaffM, EricsonEF, WetjenNM, NuttallGA, OliverWCJrComparativesafetyandefficacyoftopicalhemostaticagentsinaratneurosurgicalmodel[J]. Neurosurgery, 2008, 63: 369372.), indicating that this type of product has the risk of spreading diseases.

To sum up, first-aid hemostatic products used in wars or safety accidents in the world are divided into three categories (the first two are chemical materials):

Table 1

As can be seen from Table 1, there is still a lack of emergency hemostatic products that can fully meet the above standards.

To sum up, ideal hemostatic products for war or first aid should at least have the following common characteristics, namely: quick hemostasis, non-adhesive wounds, easy healing, low cost, easy absorption in the body, difficult to spread diseases and few sequelae, etc. However, there is currently no hemostatic agent that meets or approaches the above-mentioned criteria. In this regard, in recent years, people continue to research and develop new hemostatic materials.

Transglutaminase (protein-glutamic acid-γ-glutamine transaminase, Transglutaminase, referred to as TGase), also known as transglutaminase or γ-glutamyl transferase, consists of 331 Amino composition, catalyzing acyl transfer reaction, can catalyze intramolecular or intermolecular crosslinking of proteins, connection between proteins and amino acids, and hydrolysis of glutamine groups in protein molecules, thereby improving the structural and functional properties of proteins. Due to its excellent cross-linking properties, it is known as "the super adhesive of the 21st century". It has shown broad prospects in the fields of food, medicine, textiles, cosmetics, etc.

TGase widely exists in animal, plant and microbial tissues. Recent studies have found that TGase from different sources has differences in amino acid sequence, molecular weight, and physical and chemical properties of the enzyme. TGase derived from animals has different molecular weights, requires calcium ions to activate, and the active center of the enzyme is a cysteine residue site. TGase from animal sources is the most in-depth study of TGase (GTG) from guinea pig liver. The molecular weight of this enzyme is 90kD, and its enzymatic reaction requires calcium ion activation. It has strong specificity to the substrate, and because the enzyme contains 17 cysteine residues, it has poor thermal stability. At 50°C, after 10 minutes, only 40% of the enzyme activity remained. The effect of calcium ions on plant-derived TGase activity varies from species to species. TGase extracted from animal tissues has few sources, high cost, complicated separation procedures, and is not easy to popularize and apply.

What role TGase plays in plant tissues is still not very clear. Kang et al. extracted TGase from soybean leaves for research. However, the separation and purification process is complicated, and the yield of the enzyme is low. At present, there is no plant-derived TGase for commercial production.

However, microbial-derived TGase (mTG) has different enzymatic properties from animal-derived TGase. The molecular weight of mTG is between 23 and 45 kD, most of which are about 40 kD, which is about half of the molecular weight of animal-derived TGase. Importantly, mTG has strong thermal stability and high activity in a relatively wide pH range. At the same time, the enzymatic activity of mTG is not dependent on calcium ions, so it has a wide range of substrate-specific acyl donors, which is completely different from TGase from animal and plant sources. At the same time, compared with GTG, mTG has more remarkable stability under high pressure conditions. The above characteristics enable mTG to have a wider range of applications.

TGase derived from microorganisms is an extracellular enzyme that can be directly secreted into the medium. It is easier to separate and purify than TGase derived from animals and plants, and the raw materials for microbial fermentation are cheap and the enzyme production cycle is short. It is most suitable for large-scale industrial production, so it has been researched favored by those.

Studies have found that TGase can catalyze the formation of ε-(Y-glutamine)-lysine covalent bonds between glutamine and lysine between adjacent fibrin molecules, thereby forming cross-links between adjacent fibrin molecules. Transglutaminase and blood coagulation factor XIIIa (FXIIIa) both belong to the superfamily of transglutaminase, and their function is very similar to the latter, which can catalyze the acyl transfer reaction, so that the protein molecule or intermolecular cross-linking can be formed, thereby changing The structure and function of proteins, this reaction exists in many biological processes, including blood coagulation, wound healing, epidermal keratinization, and hardening of red blood cell membranes. Chinese patent "use of transglutaminase" (CN2008100475880) reports that transglutaminase can be used as a skin wound healing accelerator or its active ingredient, which contains 0.1-100U/ml or 0.1-100% of dry weight Transglutaminase as an active ingredient of a skin wound healing accelerator. However, this research is only on the use of transglutaminase as an adjuvant (i.e. accelerator) for wound healing, it neither involves the use of rapid hemostasis, nor does it study how the enzyme can achieve the effect of wound non-adhesion, while The stated scheme of 0.1-100U/ml or 0.1-100% dry weight is too broad and lacks specific and effective data, so it was not authorized in the end. Therefore, its related research still stays on how TG enzyme reduces the aspect of wound healing time (Talk Dan, Microbial transglutaminase to rat wound healing research, " Chinese Journal of Bioengineering ", the 27th volume 9th of 2007 period; Zhang Nianrong, research progress on the characteristics and application of glutaminase transaminase, "Western Leather", Volume 34, Issue 14, 2012).

Chinese patent application CN2007800512154 and its equivalent patent application WO2008076407 disclose a gelatin-transglutaminase hemostatic adjuvant and sealing material, which contains gelatin and non-toxic transglutaminase cross-linked material, which can be used to treat wound tissue. However, this invention uses conventional microbial source transglutaminase (USA, CHICAGO, lot number L-04207). This enzyme is not only expensive, but also the optimum reaction temperature is generally around 50°C-55°C. At normal temperature (25° C.), the enzyme activity is only about 20% of the optimal reaction temperature, which seriously affects the activity related to transglutaminase (see Example 1).

For this phenomenon, domestic researcher Cui Yanhua et al. (Progress in research on transglutaminase, "Biotechnology Bulletin", No. 1, 2009) showed that "mTG derived from different microorganisms has differences in enzymology, Even if there are large or small differences in isoelectric point, optimum pH, optimum temperature, thermal stability, etc. between TG enzymes from different strains or even from different strains of the same strain, these differences will directly It affects the application of enzymes." "For example, TG enzymes of Bacillus and Streptomyces have low gene homology, and there are also differences in enzymatic activity. The optimum temperature for the enzyme activity of S. mobaraensism TG is 50 ° C, The optimum temperature for mTG from B. subtilis is 60°C."

Because for war hemostatic products, the stability of enzymes is very important, especially for products stored at room temperature. However, the optimum reaction temperature of mTG in the above research is generally around 50°C-55°C, and the enzyme activity is only about 20% of the optimum reaction temperature at room temperature (25°C). Even at about body temperature (37°C), the enzyme The activity is only about half of the best enzyme activity (BuettnerK, HertelTC, PietzschM, AminoAcids.2012Feb; 42(2-3): 987-96.; MarxCK, HertelTC, PietzschM, JBiotechno1.2008Sep10; 136(3-4): 156-62.). In addition, the thermostability of these enzymes is relatively poor, and 10 minutes at 60° C. can cause about 80% loss of mTG enzyme activity (MarxCK, HertelTC, PietzschM, JBiotechno1. 2008 Sep10; 136(3-4): 156-62.). In view of the above-mentioned characteristics, German scientists have carried out research on this, and achieved certain results, and found enzymes with good thermal stability at high temperatures (such as the above-mentioned documents, and European patents WO2008020075A1, WO2010101256A1; US patent US20120021459A1), but these enzymes The optimal enzyme reaction temperature is still around 50°C, which has far restricted the application of transglutaminase as a medical product.

To solve this problem, the inventor's prior Chinese patent application CN2012101922407 proposed for the first time to use the non-enzymatic microbial transglutaminase zymogen, combined with an appropriate amount of activating enzyme and gelatin, to achieve a good wound hemostatic effect. However, the activating enzyme used in this research is dispase, which is a non-specific metalloprotease. Although it can disperse tissues and classify cells, some dispase can damage cells, and it is unstable when cultured, and may even introduce Mycoplasma pollution, and it is expensive, and it is necessary to mix dispase and transglutaminase in aqueous solution before use, and the two cannot be pre-mixed and made into related medical products, which greatly affects glutamine. Medical uses of the protransaminase enzyme.

In addition, the present inventor proposed in another previous Chinese patent application CN2012102021709 to obtain a highly active mutant enzyme by transforming an existing wild enzyme, which involves a recombinase obtained by mutating three sites (hereinafter referred to as 3 mutant enzyme). Although the mutant enzyme has a certain hemostatic effect, the overall effect, especially the anti-adhesion effect, needs to be improved, and the hemostatic speed (i.e., the activity of the enzyme) needs to be improved, thus affecting the war against the transglutaminase zymogen to a certain extent It is used for first aid, so it is only suitable for micro and small amount of hemostasis such as daily trauma.

Based on the good hemostatic effect and no side effects of microbial transglutaminase, it is currently necessary to provide a rapid hemostatic material and compound hemostatic product with microbial transglutaminase as the main active ingredient, which should have rapid hemostasis, avoid Organ or tissue adhesion, easy to absorb, not easy to spread diseases, low-cost medical hemostatic materials, to fill the blank needs of similar products for quick hemostasis at the scene of domestic wars or accidents.

Contents of the invention

In summary, the problem of the prior art is how to provide a rapid hemostatic product containing a new microbial transglutaminase for war or other wounds, wherein the enzyme should have a structure different from that of the prior art and have better performance Excellent, and at the same time, the rapid hemostasis product for war or other wounds should also have a product structure different from that of the prior art.

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 compound hemostatic products for hemostasis in war or other wounds. The structure of this new microbial transglutaminase is different from any mutants with new properties that have been reported. At the same time, it can satisfy the deviation of the optimal activity temperature to a lower temperature and has better thermal stability. . High temperature treatment showed that the mTG still showed high residual enzyme activity after treatment at 70°C. The reaction temperature shows that the optimal activity temperature of the enzyme is 40°C (closer to human body temperature), which is lower than that of wild-type mTG, and the specific enzyme activity at room temperature and 37°C is higher than that of wild-type mTG.

The main mutation sites of mTG with thermal stability reported in the previous literature are the mutation of serine at position 2 to tyrosine, or the mutation of serine at position 23 to valine or tyrosine, or the mutation of tyrosine at position 24 Acid mutation to asparagine, or lysine at position 294 to leucine or isoleucine; or serine at position 101 to proline, or glycine at position 250 to arginine, or mutation at position 157 Glycine is mutated to serine. These mutated transglutaminases do not have more than 3 amino acid mutation combinations, and do not have good thermal stability.

Therefore, the first object of the present invention is to provide a new microbial transglutaminase mTG, which has four amino acid mutations compared with wild-type mTG, and the mutations are respectively that the 23rd serine becomes Alanine, glycine at position 73 is changed to tyrosine, lysine at position 317 is changed to glutamine, and lysine at position 325 is changed to glutamine (ie S23A, G73Y, K317Q, K325Q). The points (hereinafter referred to as the four mutations) are different from the mutation points reported in the existing literature. In one embodiment, the transglutaminase mTG has a sequence as described in SEQ ID NO:4. In another embodiment, the transglutaminase mTG has the protein sequence encoded by the gene described in SEQ ID NO:3.

In a specific embodiment, the transglutaminase mTG, wherein the enzyme has an electrophoretic purity of at least 90% and a specific activity greater than 25 U/mg, preferably, a purity of more than 95% and a specific activity greater than 25 U / mg; where the optimal reaction temperature of the enzyme is between 25-45 ℃.

The second object of the present invention is to provide a gene sequence encoding the above mutant enzyme. In one embodiment, the sequence is selected from the gene as described in SEQ ID NO:3.

The third object of the present invention is to overcome the lack of activity of existing transglutaminase at normal temperature, or the shortcoming of needing auxiliary agent activation (i.e. activating enzyme), and to provide a kind of transglutaminase comprising the transglutaminase as the main active substrate Rapid hemostatic products at the scene of war or accidents (for example, used for hemostasis of organs or tissues of wounded in other traumatic environments of war), the hemostatic products comprise a matrix added with microbial transglutaminase (mTG), wherein the glutamine The amino acid sequence of the transaminase has the protein sequence encoded by the gene as described in SEQ ID NO:3, or has the protein sequence as described in SEQ ID NO:4.

In one embodiment, the matrix is a solid, semi-solid, powder or fluid that can be absorbed by the human body, or a plastic gel-like or colloidal form. In a specific embodiment, the electrophoretic purity of the microbial transglutaminase is at least 90%, and the specific activity is greater than 25 U/mg. Preferably, the purity is over 95%, and the specific activity is over 25 U/mg.

In one embodiment, the form of the microbial transglutaminase includes but is not limited to solid, semi-solid or fluid, plastic jelly or colloid, powder and other forms.

In one embodiment, in addition to microbial transglutaminase, the hemostatic product also contains other auxiliary materials, including but not limited to mineral powder, protein powder and the like. In a specific embodiment, said hemostatic product is a military hemostatic product.

In another embodiment, the hemostatic product is a hemostatic agent (including but not limited to powdered hemostatic product, solid hemostatic product, fluid or semi-fluid hemostatic product, liquid hemostatic product), hemostatic device, hemostatic first aid kit or first aid kit or common hemostatic products.

In a specific embodiment, the matrix or material in fluid or semi-solid form comprises a liquid fibrin sealant or glue, such as keratin, collagen, fluid gelatin, and the like. Among them, liquid fibrin sealant or glue has been used as an operating room aid for bleeding control for many years (J.L.Garza et al. (1990). J.Trauma.30:512-513; H.B.Kram et al. (1990 ). J. Trauma. 30:884-887), and single-donor fibrin seal materials have been widely used clinically in various surgical situations. In another specific embodiment, the liquid fibrin sealing material or glue comprises a dissolving solution, and the dissolving solution is a phosphate buffer solution with a pH ranging from 7.0 to 9.0.

In another specific embodiment, said plastic gel-like or colloidal forms include oxidized cellulose, oxidized regenerated cellulose, chitosan, keratin, collagen, gelatin sponge, and in a more specific embodiment, plastic The gelatinous or colloidal form of material can be a gelatin sponge, which acts as an adhesive material, mimics the fibrin-thrombin hemostatic cascade reaction with microbial transglutaminase to achieve hemostasis, and can be removed without difficulty as needed (T. Chen, Biomacromolecules. 2003 Nov-Dec; 4(6): 1558-63;).

In one embodiment, the matrix is selected from proteinaceous substances such as absorbable fibrin glue, keratin, collagen or gelatin, or carbohydrate substances such as oxidized cellulose, oxidized regenerated cellulose, chitosan, proteoglycans (e.g. poly-N-acetylglucosamine), glycolic acid polymers, lactic acid polymers. In a specific embodiment, the matrix comprises medical gelatin of various origins as well as liquid fibrin sealants or glues, such as keratin, collagen, generally produced from animal sources, recombinant sources or combinations thereof. In a more specific embodiment, the absorbable matrix is gelatin. Preferably, gelatin of animal origin, preferably fish and mammalian. More preferably, the mammal is selected from porcine and bovine gelatins. Mammalian type A high molecular weight gelatin, or 20% to 60% proline hydroxylated gelatin is preferred for this experiment.

In a preferred embodiment, powdered microbial transglutaminase (mT6) is dissolved in phosphate buffer to form a microbial transglutaminase solution; hydroxylated gelatin powder is dissolved in phosphate buffer , form a hydroxylated gelatin solution; the microbial transglutaminase (mT6) solution is mixed with the hydroxylated gelatin solution, and the gelatin containing the above-mentioned transaminase and 20%～60% proline hydroxylation can be prepared fluid composition. In a more preferred embodiment, the concentration of the microbial transglutaminase in the composition is in the range of 1 U/g to 180 U/g; the concentration of the hydroxylated gelatin solution is in the range of 15% w/v to 40% w/v range. In another more preferred embodiment, the volume ratio of the hydroxylated gelatin solution to the microbial transglutaminase solution is 10:1-1:10. The raw material shape of the hemostatic product described here is: (i) gelatin in powder, granule or other solid form; (ii) transglutaminase in powder, granule or other solid form.

The fourth object of the present invention is to provide a hemostatic device for hemostasis of war wounds, including the enzyme, composition or hemostatic product described in any one of the above embodiments. The form of the enzyme, composition or hemostatic product includes but is not limited to solid, semi-solid, fluid, liquid, gas (such as spray) and other forms, which can be made into gauze-like, sponge-like and other forms that can be produced according to needs .

In one embodiment, the hemostatic device is a tourniquet or a hemostatic gauze, comprising an outer layer of a tourniquet or a hemostatic gauze based on cotton yarn or soluble hemostatic gauze, and an inner layer for contacting and dressing a wound, wherein the inner layer A matrix powder or drug core containing said transglutaminase in the layer. In a particular embodiment, said inner layer is exposed to a protective film or foil.

In another specific embodiment, the preparation method of the tourniquet or the hemostatic gauze is as follows:

The content of this paragraph is changed to the content of preparing enzyme powder, saying that 1-6g of microbial transglutaminase is dissolved in 240ml of 50mM HEPES buffer (PH=7), and the mT6 enzyme solution with an enzyme activity of 50-300u/ml is prepared. In addition, conventional excipients, such as gelatin, can also be selected to help maintain the activity of transglutaminase and exert a hemostatic effect.

Coated with medical adhesive on the inner side of cotton gauze or soluble hemostatic gauze,

Spray the matrix powder containing the above enzyme on the viscous inner layer to form a tourniquet or a core layer of hemostatic gauze;

A protective film or mucous membrane is arranged outside the drug core layer to make the hemostatic device.

In another embodiment, the hemostatic device is a rapid hemostasis spray device comprising a pressurized spray or a mixture of foam, gelatin and transglutaminase components. In a specific embodiment, the aforementioned hemostatic device comprises a mixture of a transglutaminase component, gelatin, a foam, or a portion of a spray. Administration of the hemostatic adjuvant ingredient mixture using these methods is optionally accomplished, for example, by storing the mixture components separately and mixing them immediately prior to administration; for example, alternatively by storing the components together in an inactive form and activating them immediately prior to administration. . The inactive form of the hemostatic adjuvant component is optionally provided as a frozen solution, a lyophilized powder for reconstitution, a spray-dried powder for reconstitution, and/or any other suitable form of inactive sealant mixture. one or more.

In other embodiments, the hemostatic device may also be a hemostatic first aid kit, a first aid kit, etc. containing the aforementioned gelatin and transglutaminase components. In other embodiments, the hemostatic device is a military hemostatic device.

The fifth object of the present invention is to provide a method for preparing a rapid hemostatic product for war or accident site wounds, the product comprises a matrix added with microbial transglutaminase, wherein the transglutaminase The amino acid sequence is shown in SEQ ID NO: 4.

Steps include:

(1) preparing a recombinant microorganism capable of expressing the microorganism transglutaminase, and carrying out recombinant expression;

(2) collecting and purifying the transglutaminase with said activity, and configuring it into an active solution;

(3) adding the active solution of the transglutaminase to the human body absorbable matrix by conventional methods;

(4) The matrix is subjected to conventional processing to obtain the above-mentioned rapid hemostatic product for war or accident site wounds.

In one embodiment, powdered microbial transglutaminase (mTG) is dissolved in phosphate buffer to form a microbial transglutaminase solution; powdered hydroxylated gelatin is dissolved in phosphate buffer , forming a hydroxylated gelatin solution; the microbial transglutaminase (mTG) solution is mixed with the hydroxylated gelatin solution to prepare a fluid comprising the above-mentioned transaminase and 20% to 60% proline hydroxylated gelatin combination. In a more preferred embodiment, the concentration of the microbial transglutaminase in the composition is in the range of 1 U/g to 180 U/g; the concentration of the hydroxylated gelatin solution is in the range of 15% w/v to 40% w/v range. In another more preferred embodiment, the volume ratio of the hydroxylated gelatin solution to the microbial transglutaminase solution is 10:1-1:10.

Optionally, the composition of the present invention may further include the formation of bionic blood clots for the purpose of hemostasis.

technical terms

"War trauma" or "accident trauma" as used herein refers to any damage to any tissue of the wounded by bullets, accidents at the scene of war or accident, which results in massive blood loss in the circulatory system, or even loss of limbs, etc. The tissue may be in vivo (eg, an organ or blood vessel), or in vitro (eg, head, limb, etc.). Loss of blood can be internal (eg, from a ruptured organ), or external (eg, from a laceration, scratch, cut, etc.). Wounds can be in soft tissue, such as an organ, or in hard tissue, such as bone. The trauma, if not treated promptly, should or could be life-threatening. Therefore, the hemostatic product used for war or accident site trauma of the present invention is not a hemostatic product for daily wounds (micro-trauma), such as patches such as Band-Aids, nor a hemostatic product for clinical surgical rescue, such as medical hemostasis Bags, hemostatic strips, hemostatic forceps, etc.

The term "field trauma" as used in this article refers to field trauma such as war trauma and safety accidents, such as traffic accidents, mine accidents, production accidents, etc. Unless otherwise specified, war-related trauma also includes traffic accidents, mine accidents, production accidents, etc., but war-related trauma or field trauma does not include daily life-related minor trauma or clinical surgical trauma.

As used herein, "hemostatic product" and hemostatic agent refer to rapid hemostatic products that contain transglutaminase (mTG) of the present invention and can be used for "war wounds" or "accident wounds", such as: hemostatic agent (including powder Shaped hemostatic products, solid hemostatic products, fluid or semi-fluid hemostatic products, liquid hemostatic products), hemostatic devices, hemostatic first aid kits or first aid kits, and existing hemostatic products. In common battlefield or on-site use, the non-device hemostatic product may also be called a hemostatic agent.

As used herein, "absorbable matrix or material" means a material that spontaneously and/or via the mammalian body breaks down into components that are consumed or eliminated in a manner that does not significantly interfere with wound healing and/or tissue regeneration, And without causing any significant metabolic disturbances. For example, the absorbable material may be a proteinaceous substance such as fibrin, keratin, collagen, or a carbohydrate substance such as alginate, chitin, cellulose, proteoglycans (e.g. poly-N-acetylglucosamine), ethanol Acid polymers, lactic acid polymers, or glycolic/lactic acid copolymers. For example the absorbable material may be a carbohydrate substance. Among them, the hemostatic dressing based on the above improved absorbable material, which includes multiple layers containing absorbable substances, see for example PCT/US03/28100, US Patent Application No. 0060155234.

As used herein, "non-absorbable material" means a material that cannot be broken down by the patient's body to be non-toxic, or to interfere with wound healing and/or tissue regeneration, or to cause any significant metabolic disturbance, such as sterile bandages, sterile Bacterial gauze, mineral hemostatic powder (silicon oxide powder, potassium iron oxo salt powder, etc.), etc.

Beneficial effect

The present invention uses microbial transglutaminase and gelatin as effective substances. Compared with the prior art, the beneficial effects of the present invention include that the hemostatic product containing microbial transglutaminase and gelatin can enhance the wound recovery effect of war wounds, and hemostasis The effect is obvious. Secondly, the raw material microbial transglutaminase used in the present invention is to improve the microbial transglutaminase through a certain technology without changing the function of traditional microbial transglutaminase and the original low toxicity. The heat resistance of the enzyme has obtained the microbial transglutaminase with better stability at normal temperature. In addition, the hemostatic product has the effects of rapid hemostasis, anti-wound adhesion, etc., and has the advantages of low side effects, good healing effect on wounds, quick onset of action, and good absorption. Especially, in war or accident trauma environment, the product of the present invention does not generate heat during hemostasis, does not need to be removed after application, has good tissue compatibility and absorbability, and overcomes the aforementioned disadvantages of generating heat, being difficult to remove, and being unable to absorb. During the use of this product, the exothermic reaction is not obvious, it will not cause burns to the human body, and it can be absorbed into the body without negative effects, avoiding the secondary damage caused by the later removal of other hemostatic products. In addition, the invention can complete the mixing operation of the hemostatic agent by one person, is convenient to use, and meets the needs of special conditions in wars and accident wounds.

Description of drawings

Fig. 1 represents the gel electrophoresis detection figure of mutant mTG gene in embodiment 3;

Fig. 2 represents the mass spectrometry analysis result of mTG protein in embodiment 5;

Fig. 3 shows the electropherogram of the finely purified mutant mTG protein of the present invention in Example 6, wherein the 4th lane is the purified protein;

Fig. 4 represents the determination of the specific activity of the wild-type mTG protein, the three-site mutant mTG protein and the mutant mTG protein of the present invention at different temperatures in Example 7;

Figure 5 shows the hemostatic effects of the novel hemostatic product of the present invention and the blank control group on femoral artery hemorrhage in rats. Wherein: Figure 5A represents the hemostatic effect of the control group on rat femoral artery bleeding, wherein Figure 5A-1 represents the exposure of the rat femoral artery, and 5A-2 represents severe bleeding after cutting the femoral artery; Figure B represents the new hemostatic product of the present invention Hemostatic effect on femoral artery bleeding in rats, wherein Figure 5B-1 represents the exposure of the femoral artery of rats, 5B-2 represents severe bleeding after cutting the femoral artery, and 5B-3 represents the bleeding stopped after applying the hemostatic product of the present invention for 3 minutes , 5B-4 means to detect the adhesiveness of the hemostatic product and the wound with tweezers after applying the present invention for 5 minutes;

Fig. 6 represents the statistics of the hemostatic amount of the femoral artery of rats respectively by the novel hemostatic product of the present invention and the blank control group;

Figure 7-A shows that the new hemostatic product of the present invention is applied immediately after the rupture of the femoral artery in rats, and Figure 7-B shows the recovery of the wound suture appearance of rat legs after 21 days of application, and Figure 7-C shows that the rat legs are dissected The recovery of rat leg muscles in the future;

Fig. 8-A (enlarged Fig. B) is a staining diagram of superficial subcutaneous normal tissue. Panel C (enlarged panel D) shows the tissue staining diagram of the composition and muscle histocompatibility tissue staining in the superficial subcutaneous layer, after the experimental group applied the hemostatic composition on the 7th day.

Figure 9 shows the tissue staining of the composition and muscle tissue compatibility after the hemostatic composition was applied in the experimental group on the 21st day, Figure A (enlarged figure B) is the superficial subcutaneous layer, and Figure C (enlarged figure D) is the deep subcutaneous layer, indicated by the arrow for the composition.

Fig. 10 represents the statistics of the hemostatic amount of the rat liver by the novel hemostatic product of the present invention (containing microbial transglutaminase and gelatin) and the blank control group in Example 9;

Figure 11 shows the hemostatic composition of the present invention (containing microbial transglutaminase and gelatin) and the blank control group respectively on the hemostatic and anti-exudation effect schematic diagrams of the rat liver hemorrhage model, wherein:

Figures 11A and 11C show the livers of the control group and the experimental group exposed before treatment, respectively;

Figures 11B and 11D respectively show the hemostasis of natural bleeding for 10 seconds after making liver wounds in the control group and the experimental group;

Figures 11E and 11F respectively show the hemostasis of the control group at 2 minutes and 30 seconds and 5 minutes after applying the composition of the present invention;

Figures 11G and 11H show that the adhesiveness of the composition of the present invention is checked manually;

Figure 12 represents the absorption situation of the composition of the present invention in the rat liver hemorrhage model, and sub-graphs 1-5 represent respectively in experimental group B (applying novel hemostatic product of the present invention) and control group A (Johnson & Johnson: absorbable hemostatic Gelatin sponge (trade name: Surgiflo ^TM ) before treatment, after application for 2 minutes and 30 seconds, 5 days, and 20 days, the recovery of rat liver injury.

detailed description

The present invention will be described in further detail in conjunction with the following specific examples and accompanying drawings, and the protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope. The process, conditions, reagents, experimental methods, etc. for implementing the present invention are general knowledge and common knowledge in the art except for the content specifically mentioned below, and the present invention has no special limitation content.

Example 1 Enzyme Activity Determination

1) Preparation of reagents for measuring enzyme activity

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.

Solution B: 3mol/L HCl, 12% trichloroacetic acid (W/V), 5% ferric chloride (W/V, dissolved in 0.1mol/L hydrochloric acid, then filtered) mixed in a volume ratio of 1:1:1 .

2) Determination of enzyme activity

Experimental group: Take 0.2ml of the sample to be tested, add 2ml of solution A and incubate at 37°C for 10 minutes, then add 2ml of solution B. Control group: take 0.2ml of the sample to be tested, add 2ml of solution B and incubate at 37°C for 10 minutes, then add 2ml of solution A. Absorbance was measured at 525 nm.

The mutagenesis of embodiment 2 wild strains

The wild-type mTG strain is Streptomyces mobaraensis (such as the strain ATCC number 29032 of the US ATCC or the strain ATCC number 27441 of the US ATCC, or the strains of the China Microorganism Conservation Center such as CGMCC number 4.1719 and CGMCC number 4.5591).

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 O 0.01g/L, agar 20g/L, pH7.2-7.4. Fermentation medium: glycerol 20g/L, yeast extract 6g/L, fish powder peptone 25g/L, MgSO ₄ ·7H ₂ O2g/L, K ₂ HPO ₄ ·3H ₂ O2g/L, pH7.4.

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.

The operation is carried out under red light or dark conditions, and the ultraviolet light is turned on 0.5 hours in advance to stabilize the light source. Take 3 mL of spore suspension with a concentration of about 10 ⁷ spores/mL and place it in a sterilized plate with a diameter of 6 cm, stir it with a magnetic stirrer, and irradiate different irradiation distances and different ultraviolet rays under the condition of a UV lamp power of 15W. time for mutagenesis. After being protected from light for 1 hour, the spore suspensions of different dilutions were spread on Gaussian medium in a dark environment, and cultured at 30°C for 7 days. Pick a single colony, culture it in the fermentation medium for 24 hours, and measure the enzyme activities of the selected strain and the wild strain at 37°C.

The acquisition of embodiment 3 mutant mTG gene

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 supernatant and mix with an equal volume of isopropanol (pre-cooled at 4°C) Mix well, then centrifuge at 10,000rpm for 5 minutes, and discard the supernatant. The precipitate was washed (inverted and centrifuged for 1 minute) with 1 mL of 70% ethanol (pre-cooled at 4°C), air-dried, dissolved by adding 40 uLTE, and stored at -20°C for future use.

Primer design: A pair of primers were designed to obtain the full sequence of the mTG gene by polymerase chain reaction, the sequence is selected from: SEQNO: 1: ctcaacgaaagcgctccggccgcttc

SEQ NO: 2: cgctcacatcacggccagccctgc

PCR reaction system and reaction conditions: Mix the genomic DNA and primers obtained above, and carry out PCR reaction according to the following conditions: Taq enzyme reaction solution (2 times concentration) 25 μL, primer Pf (2.5 mmol/mL) 2 μL, primer Pr (2.5 mmol/mL) /mL) 2μL, DNA 2μL and sterile water. Denaturation at 94°C for 1 minute, annealing at 55°C for 30 seconds, and 2 minutes at 72°C, a total of 40 cycles.

The PCR purity was detected by electrophoresis, and the results are shown in Figure 1. The left lane is the molecular weight standard DNA, and the right lane is the electrophoresis result of the PCR fragment. The PCR fragment size was between 1000bp and 2000bp, consistent with the theoretical value of 1058bp.

The sequencing of embodiment 4 mutation mTG gene

Add 2 times the volume of absolute ethanol to the PCR product in Example 3, mix well, and let stand at room temperature for 5 minutes. Centrifuge at 12000rmp for 10 minutes, discard the supernatant; wash the pellet once with 70% ethanol. Dry and dissolve the pellet with 50 µL sterile water. React according to the following system: 1 μL of PCR product dissolved in sterile water, 0.5 μL of pUC19-T vector (Takara company), 0.5 μL of ligase, 5 μL of buffer solution (10 times concentration) and 43 μL of sterile water, mix well, and react at 16 °C 20 minutes. Take 5 μL of the product after the above reaction, add it to 100 μL Escherichia coli DH5α competent cells (Takara Company), react at 4°C for 30 minutes, and react at 42°C for 90 seconds, add LB medium, incubate at 37°C for 1 hour, and spread the bacterial solution Cloth with LB plate on. After the colony is formed, send it to the sequencing company for sequencing. Mutation sites were detected compared to the wild-type mTG gene.

After sequencing by conventional methods, it was found that the sequence of the mutant gene was shown as SEQ ID NO: 3, and thus its corresponding protein amino acid sequence was deduced as shown in SEQ ID NO 4.

The general purification of embodiment 5mTG albumen

Fermentation medium and bacterial strain are the same as embodiment 2, the Streptomyces spores of Maoyuan are inoculated into 30mL fermentation medium, cultivated for 28 hours. Centrifuge at 12,000 rpm in a centrifuge at 4°C for 30 minutes to remove bacteria and solid impurities, and obtain 20 mL of culture supernatant. Add 40 mL of absolute ethanol and place at 4°C for 1 hour. Centrifuge at 12,000 rpm in a centrifuge at 4°C for 30 minutes, discard the supernatant, collect the precipitate, place the precipitate in an ampoule, and freeze-dry it in a freeze dryer for 2 days to obtain the initially purified wild-type mTG protein.

The spores of the mutated Streptomyces Maoyuan were inoculated into a 250ml Erlenmeyer flask containing 30mL fermentation medium, and cultivated for 24 hours. Other operating steps are the same as above, and the mutant mTG protein is purified.

The purified protein was analyzed by mass spectrometry to see if its structure was consistent with theoretical deduction. The final mass spectrometric analysis results are shown in Figure 2: the mass spectrometric molecular weight is 37934.9 Da, which is consistent with the molecular structure deduced from gene sequencing. It shows that the protein sequence is indeed SEQNO4. After comparison, compared with the wild-type sequence, there are 4 mutations in the SEQ ID NO; 4, respectively, the 23rd serine is changed to alanine, the 73rd glycine is changed to tyrosine, and the 317th lysine is changed to Glutamine and lysine at position 325 were changed to glutamine (ie S23A, G73Y, K317Q, K325Q).

Embodiment 6 The fine purification of mutant mTG protein of the present invention

The protein obtained in Example 5 was finely purified using an ion exchange column. The filler is DEAE-Sephrosefastflow (GE healthcare), the balance solution is 0.05M tris/HCl buffer, pH9.0, the eluent is 0-1mol/L NaCl50mM tris/HCl, pH9.0. After 16 column volumes were linearly eluted, each fraction was collected and detected by electrophoresis to obtain mTG protein with better purity. The fraction was desalted and lyophilized. Experimental results showed that the size of the protein was consistent with the expected molecular weight of 37.9 kDa, and the purity was very high. There was only a single band in electrophoresis, and the purity was greater than 99% (as shown in FIG. 3 ). Lane 4 is the purified protein. Thus, the results of Example 6 further confirmed that the protein sequence analyzed in Example 5 is indeed SEQNO4.

The specific activity determination of mutant mTG protein under the different temperature of embodiment 7

The purified wild-type mTG protein and the mutant mTG protein of the present invention were tested for enzyme activity at 25°C, 37°C, 45°C, 55°C, and 75°C, respectively. The method used for measuring the enzyme activity is the same as that described in Example 1. The solution used in the bioassay was preheated at the corresponding temperature for 2 minutes before the reaction. The experimental results show (as shown in FIG. 4 ), at different reaction temperatures, the specific enzyme activity of the mutant mTG of the present invention shows a trend completely different from that of the wild-type mTG. The optimal reaction temperature of the mutant mTG of the present invention is about 45°C, while the optimal reaction temperature of the wild-type mTG is about 55°C, and the optimal reaction temperature of the mTG of the present invention is obviously lower than that of the wild-type mTG. Moreover, at 25°C-75°C, the mTG specific enzyme activity of the present invention is significantly higher than that of the wild type. At 37°C, the mTG specific enzyme activity of the present invention reaches 80U/mg, which is more than 100% higher than the wild-type mTG enzyme activity, and 50% higher than the 3-site mutation of the prior patent CN2012102021709. In addition, the activity of the wild-type mTG drops sharply under high temperature conditions above 55°C, while the mTG of the present invention still retains a relatively high enzyme activity. At 65°C-75°C, the mTG of the present invention still retains high enzyme activity, while the wild-type mTG is almost inactivated at this time. It can be seen that compared with the wild-type mTG and the 3-site mutant enzyme, the mutant mTG of the present invention has significantly better enzyme activity and heat resistance stability, and the enzyme activity is positively correlated with the hemostatic effect, suggesting that the 4-mutant mTG of the present invention has a hemostatic effect It is superior to wild-type mTG and 3-site mutant enzyme, and its use environment is wider, and it has better adaptability to the scene environment of war or accident trauma.

Embodiment 8 Genetic engineering method prepares mTG of the present invention

In order to ensure that the mutant gene can be obtained without the mutant strain of Streptomyces maoyuan, the mTG mutant was obtained by artificial synthesis in this example.

Synthesize the whole gene sequence of the above-mentioned mutated gene sequence in a commercial biological company (such as Shanghai Bioengineering Company, Yingjun Biotechnology Company, etc.), and clone it into a commercial E. company or other bio-companies; any other expression vector such as pET-32b or other suitable vectors) in the EcoRV site to form an expression vector that can express the target gene. Transformed into Escherichia coli BL21 according to conventional techniques to obtain genetically engineered bacteria capable of expressing the protein. Inoculate the genetically engineered bacteria into 20ml of LB medium, culture at 37°C and 200 rpm for 16 hours, then add lactose solution with a solubility of 1mol/L according to 1/1000 of the volume of the medium, continue to cultivate for 4 hours, and collect the bacteria body.

Use an ultrasonic breaker at a power of 200w to crush the bacteria; centrifuge at 10,000rmp for 5 minutes, discard the supernatant, resuspend the pellet in a solution containing 8mol/L urea, and pipette 3 times; centrifuge at 10,000rmp for 5 minutes, and collect the supernatant as follows The purification.

Utilize the Ni-NTASpin kit of QIAGEN Company, and perform purification according to the method described therein. The specific method was carried out completely according to the instructions thereof, and finally the fusion protein containing the mutated mTG was obtained.

Add 1/10 mass of dispase to the fusion protein solution according to the mass of the fusion protein. After 30 minutes at 37°C,

Purify according to the method in Example 6 to obtain the mTG protein of the present invention.

Embodiment 9: Rat femoral artery hemostasis experiment

This implementation case provides an implementation example of the product of the present invention for rapid hemostasis of extracorporeal hemorrhage under a simulated battlefield environment.

The ratio of the hemostatic material freeze-dried powder containing transglutaminase referred to in the present invention is: transglutaminase 5400U; hydroxylated gelatin 25g; sorbitol 5g. The preparation method is: weigh 25g of hydroxylated gelatin, add water, heat and stir to dissolve completely; weigh 5g of sorbitol, add it to the above solution, stir to dissolve completely; then use a 0.22μtm filter membrane to filter and sterilize, place it at room temperature and add water for injection to set Make up to 100ml. Add 5400U transglutaminase by sterile filtration, mix evenly, and place in a lyophilizer for freeze-drying. After drying, it is pulverized into powder, and the hemostatic composition is packaged to make a finished product.

In the experiment, 14 SD rats were selected and randomly divided into blank control group and experimental group (mTG-gelatin), with 7 rats in each group.

The rat's femoral artery was cut using a surgical scalpel. After about 10 seconds of heavy bleeding, use gauze to remove accumulated blood immediately prior to application. The hemostatic composition is prepared when the hemostatic composition is administered. After application of the composition, the femoral artery still completely stopped bleeding in less than 3 minutes. After 5 min, the hemostatic product was manually detected using a camera. Observe the experimental effect of gauze hemostasis and new hemostatic product hemostasis respectively, and take pictures for records. And count the amount of bleeding.

The experimental results are shown in Fig. 5A (control group) and Fig. 5B (experimental group), wherein Fig. 5A-1 represents the exposed rat femoral artery, and 5A-2 represents severe bleeding after cutting the femoral artery; Fig. B represents the novel hemostatic product of the present invention Hemostatic effect on femoral artery bleeding in rats, wherein Figure 5B-1 represents the exposure of the femoral artery of rats, 5B-2 represents severe bleeding after cutting the femoral artery, and 5B-3 represents the bleeding stopped after applying the hemostatic product of the present invention for 3 minutes , 5B-4 indicates that the adhesion between the hemostatic product and the wound was detected with tweezers after 5 minutes of applying the present invention.

Therefore, Fig. 5A shows that the femoral artery of rats bled severely after the femoral artery was cut open, and the bleeding could not be stopped by pressing with gauze for 3 minutes. It shows that the hemostasis with gauze is unsuccessful for femoral artery bleeding.

On the contrary, Fig. 5B shows: After the rat femoral artery was cut open for 10 seconds to naturally bleed, a hemostatic product was applied, and it could be observed that the bleeding of the femoral artery had been stopped by the hemostatic product in less than 3 minutes, and with forceps in 5 minutes Detect the adhesion of the hemostatic product to the rat leg muscles and the viscosity of the hemostatic product, and find that the hemostatic product has good viscosity, and the hemostatic product and the rat leg muscle have good adhesion and are not easy to fall off.

While implementing the above scheme, the blood flow of each experimental group was collected, and an electronic balance was used to weigh and count the blood flow of the rat femoral artery within 3 minutes. The results are shown in Figure 6: the new hemostatic product successfully stopped bleeding in the femoral artery of rats within three minutes. The above experimental results show that the hemostatic product of the present invention has good rapid hemostatic performance.

Embodiment 10: Histocompatibility research of novel hemostatic product

The ratio of the hemostatic material freeze-dried powder containing transglutaminase referred to in the present invention is: transglutaminase 5400U; hydroxylated gelatin 25g; sorbitol 5g. The preparation method is: weigh 25g of hydroxylated gelatin, add water, heat and stir to dissolve completely; weigh 5g of sorbitol, add it to the above solution, stir to dissolve completely; then use a 0.22μtm filter membrane to filter and sterilize, place it at room temperature and add water for injection to set Make up to 100ml. Add 5400U transglutaminase by sterile filtration, mix evenly, and place in a lyophilizer for freeze-drying. After drying, it is pulverized into powder, and the hemostatic composition is packaged to make a finished product.

15 rats were used in the experiment. Randomly divided into 5 as the blank control group, 10 as the experimental group (mTG-gelatin).

For 10 rats in the experimental group, the femoral artery of the rats was cut with a scalpel. After about 10 seconds of heavy bleeding, use gauze to remove accumulated blood immediately prior to application. The hemostatic composition is prepared when the hemostatic composition is administered. After application of the composition, the femoral artery still completely stopped bleeding in less than 3 minutes. After 5 min, the hemostatic product was manually detected using a camera. Afterwards, the rats were sutured and housed in separate cages. Tissue sections were taken from the thigh muscles at the experimental site on the 7th day and 21st day respectively. Take 5 rats each time. The experimental results are shown in FIG. 7 , indicating that the wound recovery and muscle changes of the rat legs were just applied and 21 days after the application of the composition.

The experimental results showed that the wounds on the legs of rats had healed after 21 days of application of the new hemostatic product. After dissection, the hemostatic product disappeared compared with when it was just applied, and there was no significant difference between the leg muscles and normal tissues. It shows that the new hemostatic product can be absorbed well and has no adverse effects on rats.

Then, among the 5 control groups, the muscles of 5 rats were taken for slices on the first day as blank controls to obtain normal muscle tissue slices.

The experimental steps for tissue sectioning are as follows:

1. Material collection and fixation:

Tissue blocks (thickness of one strand not exceeding 0.5 cm) were removed from rat thighs and put into pre-prepared fixative solution 10% formalin.

2. Dehydration and transparency:

One uses alcohol from low concentration to high concentration as a dehydrating agent to gradually remove the water in the tissue block. Then place the tissue block in xylene, a clearing agent soluble in both alcohol and paraffin, to be transparent.

3. Embedding in wax:

Place the transparent tissue block in the melted paraffin and keep it warm in a paraffin box. Embedding was performed after the paraffin was completely immersed in the tissue block.

4. Slicing and patching:

Fix the embedded wax block on a microtome and cut into thin slices. The cut slices were ironed in heated water, then attached to glass slides, and dried in a constant temperature oven at 45°C.

5. Dewaxing dyeing:

stained with HE

The HE staining process is:

①Stain the sections that have been soaked in distilled water in the hematoxylin aqueous solution for several minutes.

②Color separation in acid water and ammonia water, each for a few seconds.

③ Rinse with running water for 1 hour and then pour into distilled water for a while.

④ Dehydrate in 70% and 90% alcohol for 10 minutes each.

⑤ Stain in alcohol eosin staining solution for 2-3 minutes.

6. Dehydration and transparency:

The stained sections were dehydrated with pure alcohol, and then transparentized with xylene.

7. Sealing:

Neutral color gum for mounting. After the gum is slightly dry, stick a label, and the sliced specimen can be used.

The experimental results are shown in Figures 8-9, in which Figure 8-A (enlarged Figure B) is a stained image of normal subcutaneous superficial tissue. Panel C, (enlarged panel D) represents the tissue staining diagram of the composition and muscle histocompatibility tissue staining in the superficial subcutaneous layer, after the experimental group applied the hemostatic composition on the 7th day. The results showed that the hemostatic composition (pointed by the arrow) had begun to be absorbed into the muscle tissue as observed in the tissue section on the 7th day.

Figure 9 shows the tissue staining of the composition and muscle tissue compatibility after the hemostatic composition was applied in the experimental group on the 21st day, Figure A (enlarged figure B) is the superficial subcutaneous layer, and Figure C (enlarged figure D) is the deep subcutaneous layer, indicated by the arrow For the composition, on the 21st day, the hemostatic product has entered the muscle tissue and degraded.

The results also showed that after 21 days, compared with the muscle tissue section of the normal control group in Figure 8A (enlarged Figure B), there was no significant difference in the subcutaneous superficial muscle tissue section in Figure 9A (enlarged Figure B). It shows that the histocompatibility and absorbability of the hemostatic product are good within 21 days after application of the hemostatic product.

The above experimental results show that the novel hemostatic product of the present invention has good tissue compatibility and absorbability.

Example 11 Comparison of the haemostatic product of the present invention on hemostatic effect and local temperature change of femoral artery traumatic bleeding in SD rats

This implementation case provides an implementation example of the comparison between the present invention and the traditional military hemostatic agent on the wounded hemostatic effect and local temperature change.

The ratio of the hemostatic material freeze-dried powder containing transglutaminase referred to in the present invention is: transglutaminase 5400U; hydroxylated gelatin 25g; sorbitol 5g. The preparation method is: weigh 25g of hydroxylated gelatin, add water, heat and stir to dissolve completely; weigh 5g of sorbitol, add it to the above solution, stir to dissolve completely; then use a 0.22μm filter membrane to filter and sterilize, place it at room temperature and add water for injection to set Make up to 100ml. Add 5400U transglutaminase by sterile filtration, mix evenly, and place in a lyophilizer for freeze-drying. After drying, it is pulverized into powder, and the hemostatic composition is packaged to make a finished product.

In the experiment, 21 SD rats were selected and randomly divided into blank control group, zeolite powder control group and experimental group (mTG-gelatin), 7 rats in each group. The purchased SD rats were acclimatized in the experimental animal room for 1 week, and the experiment was started to establish a model of vascular rupture trauma. Rats were anesthetized with respiratory anesthesia; fixed on the operating table in supine position, disinfected, made a 3 cm longitudinal incision on the anterior upper part of the right thigh, fully exposed and freed the femoral artery about 1.5 cm, and the femoral vein was about 1.5 cm, and placed a thermometer in the blood vessel , Muscle. Cut off the femoral artery and femoral vein with a scalpel, and immediately sprinkle hemostatic materials on the bleeding wound (each bleeding wound uses 25g of test materials: negative control starch, positive control zeolite particles, hemostatic powder of the present invention) to stop bleeding, and press for 2 minutes. , observe and record the hemostatic effect of the animal wounds in each group. Statistical processing: Chi-square test was used to compare the significance of the difference in hemostatic effect; the significance level a=0.05.

Table 2. Hemostatic effect and local temperature changes on femoral arteriovenous rupture bleeding in SD rats

group the rat Number of hemostasis Average hemostasis time (min) Effective rate of hemostasis local temperature negative control 7 0 0% 37°C Zeolite powder control 7 7 2 100% 60～70℃ this invention 7 7 2.5 80% 37～50℃

Compared with the negative control group, ^** P<0.01

The experimental results show that the hemostatic agent of the present invention and the positive control zeolite powder can effectively stop bleeding after being sprinkled on the femoral artery and vein rupture bleeding site and pressing the femoral artery wound for 3 minutes. Negative control starch sprinkled on the bleeding site and pressing the femoral artery wound for 3 minutes could not effectively stop the bleeding, and all SD rats that were not treated with other interventions died. The positive control zeolite particles raise the local temperature, but the effect of the hemostatic agent of the present invention on raising the local temperature is much weaker than that of the zeolite particles, and the local temperature does not exceed 50°C. Experimental results show that although the hemostatic agent of the present invention has a slightly lower hemostatic speed than zeolite powder, it still has a strong and quick hemostatic effect, which is sufficient to meet the requirements of rapid hemostasis in war. Moreover, the hemostatic agent of the present invention has a weaker effect on raising local temperature than zeolite, does not cause local tissue thermal damage, and its comprehensive effect is better than that of traditional zeolite hemostatic agents.

Example 12: Hemostasis and wound exudation prevention experiments of the composition of the present invention in a rat liver hemorrhage model

This embodiment provides the rapid hemostatic effect on human internal organs under simulated combat environment of the product (microbial transglutaminase (mTG)-gelatin composition) according to the present invention.

In the experiment, 14 SD rats with a weight of 300g±20g were selected and randomly divided into a control group and an experimental (mTG-gelatin) group, with 7 rats in each group.

The specific activity of microbial transglutaminase used in the present invention is: 1-50U/mg

The ratio of the hemostatic material freeze-dried powder containing transglutaminase referred to in the present invention is: transglutaminase 5400U; hydroxylated gelatin 25g; sorbitol 5g. The preparation method is: weigh 25g of hydroxylated gelatin, add water, heat and stir to dissolve completely; weigh 5g of sorbitol, add it to the above solution, stir to dissolve completely; then use a 0.22μm filter membrane to filter and sterilize, place it at room temperature and add water for injection to set Make up to 100ml. Add 5400U transglutaminase by sterile filtration, mix evenly, and place in a lyophilizer for freeze-drying. After drying, it is pulverized into powder, and the hemostatic composition is packaged to make a finished product.

For the control group, no hemostatic product (or hemostatic agent, the same below) was added, just to observe the natural hemostasis time.

Adult rats were placed under general anesthesia before the experiment, and the vital signs of the rats were monitored and maintained throughout the experiment.

For experimental and control administrations, the laparotomy was routinely performed, and after exposure of the liver, a sagittal incision 1 cm long and 0.5 cm deep was made in the left lobe of the liver using a scalpel. After approximately 10 seconds of active bleeding, the accumulated blood was removed with gauze just prior to application of the mTG-gelatin solution.

The test solution was applied to the incision on the left lobe of the liver. A gel formed about two minutes after application and bleeding was completely stopped in less than about 2.5 minutes after application. After 5 minutes, the tissue was shaken vigorously and forceps were used to apply pull across the wound site, but the gel was still intact and the wound closed.

Use electronic balance to weigh and count the hemorrhage of rat liver in 2.5 minutes, the result is as shown in Figure 10, the experimental result data shows that the composition of the present invention can inhibit the hemorrhage of rat liver 93% in 2 minutes and 30 seconds , can quickly achieve hemostatic effect (**, P<0.01).

Figure 11 shows a schematic diagram of the hemostatic composition of the present invention and the blank control group on the hemostatic and anti-exudation effects of the rat liver hemorrhage model, wherein Figures 11A and 11C show the livers of the control group and the experimental group exposed before treatment, such as Figures 11B and 11D show the active bleeding for 10 seconds after making the liver wounds of the control group and the experimental group, respectively. Figure 11E shows the control group 2 minutes and 30 seconds after application of the composition of the invention, Figure 11F shows the situation after 5 minutes of application of the composition of the invention, and Figures 11G and 11H show the adhesion of the composition of the invention manually tested.

For the control group, the bleeding cannot be stopped naturally for 2 minutes and 30 seconds without taking hemostatic measures. For the experimental group, the liver wound would stop bleeding rapidly after adding the hemostatic composition for 2 minutes and 30 seconds. As shown in FIG. 11E , the composition of the present invention has very strong adhesiveness, which binds the wound and stops the bleeding. After 5 minutes, as shown in Figures 11G and 11H, it was detected with tweezers that the adhesive strength of the composition could completely block the wound, prevent blood or other body fluids from seeping out, and would not cause the wound to bleed again. Moreover, the tweezers can lift the hemostatic product and the liver together without bleeding at this time, showing the extremely strong hemostatic and anti-vibration effects of the hemostatic product of the present invention, which is very beneficial for the rehabilitation of patients. If the mTG solution is stored at 4°C and the experiment is performed immediately after taking it out, the wound will stop bleeding after 3 minutes after adding the composition, the wound will be glued, and the bleeding will stop.

Example 13: Absorption of the composition of the present invention in a rat liver hemorrhage model

This example provides the natural absorption of the product (microbial transglutaminase (mTG)-gelatin composition) according to the present invention after simulating hemostasis in the battlefield environment.

14 SD rats with a weight of 300g±20g were selected for the experiment and divided into control group (Johnson & Johnson: absorbable hemostatic gelatin sponge, trade name Surgiflo ^TM ) and experimental (mTG-gelatin) group, 7 rats in each group.

The ratio of the hemostatic material freeze-dried powder containing transglutaminase referred to in the present invention is: transglutaminase 5400U; hydroxylated gelatin 25g; sorbitol 5g. The preparation method is: weigh 25g of hydroxylated gelatin, add water, heat and stir to dissolve completely; weigh 5g of sorbitol, add it to the above solution, stir to dissolve completely; then use a 0.22μm filter membrane to filter and sterilize, place it at room temperature and add water for injection to set Make up to 100ml. Add 5400U transglutaminase by sterile filtration, mix evenly, and place in a lyophilizer for freeze-drying. After drying, it is pulverized into powder, and the hemostatic composition is packaged to make a finished product.

Control group A: The control group used an internationally commercialized hemostatic product (the product of Johnson & Johnson, absorbable hemostatic gelatin sponge, trade name Surgiflo ^TM ), and observed the absorption.

Animal state: Adult rats were under general anesthesia, and the vital signs of the rats were detected and maintained throughout the test period.

Experimental procedure: Use surgical scissors to open the abdominal cavity of the rat to expose the liver. Use a scalpel to make a sagittal incision 1 cm long and 0.5 cm deep in the left lobe of the liver. After active bleeding for about 10 seconds, use gauze to remove the blood accumulated on the surface.

The test solution was applied to the incision on the left lobe of the liver, a gel formed within about two minutes of application, and the bleeding stopped completely within about 2.5 minutes. After 5 minutes, the tissue was shaken vigorously and forceps were used to apply pull across the wound site, the gel was still intact and the wound site remained closed. After the purpose of hemostasis is achieved, the liver wound does not need to be sutured, and the abdominal cavity is routinely sutured. After 5 days, 10 days and 20 days respectively, the experimental animals were killed, and the absorption of the hemostatic composition at the liver wound was observed.

The experimental results are shown in Figure 12. The results of the experimental group B show that the hemostatic composition of the present invention has not been fully absorbed after 20 days, but as the wound heals, the hemostatic composition is gradually absorbed, and the absorption degree is not much different from that of the international commercial hemostatic product group .

Claims (18)

1. a glutamine of microbe transaminase mTG, is characterized in that this mTG there occurs the amino acid mutation in four sites compared with wild-type mTG, i.e. S23A, G73Y, K317Q, K325Q.

2. glutamine transaminage mTG according to claim 1, wherein this enzyme has the protein sequence of the genes encoding as described in SEQIDNO:3; Or, there is the protein sequence as described in SEQIDNO:4.

3. for the gene order of enzyme described in claim 1 or 2 of encoding.

4. the quick-acting haemostatic powder product for war or scene of the accident wound, it is characterized in that, comprise the matrix that with the addition of described glutamine of microbe transaminase mTG, the sequence of wherein said glutamine transaminage has the protein sequence of genes encoding as claimed in claim 3, or has protein sequence as claimed in claim 1 or 2.

5. hemostasia products as claimed in claim 4, it is characterized in that, wherein hemostasia products is selected from hemostatic agent, hemostasis device, first-aid hemostatic bag or common hemostatic article, and described matrix is the absorbable fluid of human body, semisolid, powder or plastic glue or colloid form.

6. hemostasia products as claimed in claim 5, it is characterized in that, the matrix of described fluid or semi-solid form or material comprise liquid fiber albumen sealing material or glue class.

7. hemostasia products as claimed in claim 6, wherein said liquid fiber albumen sealing material or glue class are selected from Keratin sulfate, collagen protein or fluid gelatin.

8. hemostasia products as claimed in claim 6, it is characterized in that, described liquid fiber albumen sealing material or glue class comprise lysate, and described lysate is phosphate buffered saline buffer, and phosphate buffered saline buffer PH scope is from 7.0 to 9.0.

9. hemostasia products as claimed in claim 5, it is characterized in that, described plastic glue or colloid form comprise SURGICEL, oxidized regenerated cellulose, chitosan, Keratin sulfate, collagen protein, gelfoam.

10. the hemostasia products as described in any one of claim 4-6, it is characterized in that, described matrix is selected from absorbable fibre albumin glue, Keratin sulfate, the medical gelatin in collagen protein or various source and liquid fiber albumen sealing material or glue, or is selected from SURGICEL, oxidized regenerated cellulose, chitosan, protein-polysaccharide, glycolic acid polymer, lactic acid polymer.

11. hemostasia products as claimed in claim 10, wherein said protein-polysaccharide is poly-n-acetyl glucosamine amine.

12. hemostasia products as claimed in claim 10, it is characterized in that, described matrix is gelatin.

13. hemostasia products as claimed in claim 12, is characterized in that, described matrix is selected from mammiferous A type high molecular gelatin, or containing 20% ~ 60% proline(Pro) by the gelatin of hydroxylation.

14. hemostasia products as claimed in claim 5, it is characterized in that, the stock material shapes of described hemostasia products is: the gelatin of (i) powder, particle or other solid forms; (ii) the transglutamin-ase 9 transaminase of powder, particle or other solid forms.

15. hemostasia products as claimed in claim 5, it is characterized in that, described hemostasia products is tourniquet or hemostatic gauze, comprise with cotton yarn or the soluble stanching gauze tourniquet that is body or hemostatic gauze outer, with for the internal layer contacted and bind up a wound, containing the matrix powder of described glutamine transaminage or medicine core in wherein said internal layer.

16. hemostasia products as claimed in claim 5, is characterized in that, described hemostasia products is the spraying plant of quick-acting haemostatic powder, comprise the spraying of pressurization or the mixture of foam, gelatin and transglutamin-ase 9 transaminase component.

17. hemostasia products as described in claim 12-14, it is characterized in that, described hemostasia products is containing above-mentioned gelatin and the first-aid hemostatic bag of glutamine transaminage component, the military hemostasia products of first-aid chest.

18. prepare the quick-acting haemostatic powder method for product for war or scene of the accident wound described in any one of claim 4-17, comprise the following steps:

(1) preparation can express the recombinant microorganism of described glutamine of microbe transaminase, and carries out recombinant expressed;

(2) collect also purifying and there is the glutamine transaminage of described activity, and be configured to living solution;

(3) by ordinary method, the living solution of described glutamine transaminage is added in human body Absorbable rod matrix;

(4) described matrix is carried out conventional processing, namely obtain the above-mentioned quick-acting haemostatic powder product for war or scene of the accident wound.