CN103816562A

CN103816562A - Rapid hemostatic product for war wounds and preparation method thereof

Info

Publication number: CN103816562A
Application number: CN201410035698.0A
Authority: CN
Inventors: 易正芳; 吕方; 丛晓楠; 金明飞; 裴正培; 刘明耀
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2013-12-02
Filing date: 2014-01-24
Publication date: 2014-05-28
Anticipated expiration: 2034-01-24
Also published as: CN103820412B; CN103820411B; CN103820412A; CN103816562B; CN103820411A

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

A kind of war wound quick-acting haemostatic powder product and preparation method thereof

Technical field

The invention belongs to the quick-acting haemostatic powder product for field wound, be specifically related to hemostatic composition that one contains glutamine of microbe transaminase (mTG) and its preparation method and application, this product can be used for the wound quick-acting haemostatic powder of the wild environments such as war, fire-fighting, security incident.

Background technology

Severe loss of blood be peacetime the common people because of one of dead principal element of wound, Bleeding control (or bleeding) is the important step of first aid and trauma care.In the wild environments such as operation, as war, fire-fighting, security incident etc., because salvage at scene measure is limited, it is pressed for time, condition is simple and crude, making to bleed too much causes that dead event happens occasionally.Show according to a up-to-date statistical data, U.S. army falls in battle in officers and men fight-terrorism warfare, have 90% dead in the way of being sent to hospital, and nearly 1/4 within injured 5～10 minutes just because severe loss of blood is died.According to statistics, in war, around major blood vessel wound accounts for the more than 95% of whole blood vessel injury, and hemostatic technique has become most important link in current treatment of war wound, so hemostasis in time plays a crucial role for reducing percentage of killed in action in war.In addition, in the security incidents such as vehicle accident is large, massive hemorrhage also usually causes death, often be only because vehicle accident causes at least 10 ten thousand people's death in China, in security incident, have at least 20% personnel to cause death because of massive blood loss (exceeding self-blood total amount more than 20%).Therefore the quick-acting haemostatic powder product that, timely development is adapted to the actual needs such as China's war or security incident wound is particularly important.

At present, think that in the world the desirable first aid hemostasia products for war or contingency has 8 standards, that is: (1) quick-acting haemostatic powder; (2) can use at any time, not need prepare in advance or mix; (3) be simple and easy to use, do not need professional health care personnel's participation; (4) lightweight and durable; The storage period of (5) at least 2 years and wide temperature range storage capability (10 ℃ to 55 ℃); (6) have no side effect, do not injure or the risk of the toxicity disease that spreads disease; (7) with low cost; (8) little to wound side effect, be convenient to wound healing.Commercially available prod comprises following several at present:

(1) zeolites

Zeolite is a kind of mineral of occurring in nature, is distributed in mineral precipitation or lakebed volcanic ash deposition area more.Take aluminate polymer and silicate as raw material, by controlling response type and crystallization temperature, can produce and the similar artificial zeolite of natural zeolite.Natural or artificial zeolite's crystalline solid, after dehydration, sterilization, becomes a kind of powerful absorbent, alternative multiple gases and the liquid of absorbing, the moisture in also can absorbing blood.According to this principle, Chinese People's Liberation Army's Military Medical Academy has been developed a kind of Quick-acting styptic powder, it be develop be mainly the hemostasia products of composition with zeolite, there is haemostatic effect better, the advantage such as cost is low.But zeolites hemostatic material has some shortcomings part: the one, release heat in hemostasis, raises local temperature, and instantaneous temperature approaches 60～70 ℃, easily causes site of injury burn; After two hemostasis, remove difficulty, cause body and mind misery to patient, likely cause secondary hemorrhage, increase the weight of body and mind misery; The 3rd, zeolite density is large, carries inconvenience.But due to current not succedaneum better, be applied at present therefore still become the hemostasia products that war or field accident are saved oneself

(2) Quickclot

U.S. army has used Quickclot to stop blooding to war wound wound in Afghan War and the war in Iraq, and effect is better, has significantly improved the wounded's survival rate.A kind of synthetic inert particulate mineral that this material is made up of Si oxide, aluminum, sodium, magnesium and a small amount of quartz, there is effect of molecular sieve and adsorption moisture, when use, covered in bleeding wounds, absorb rapidly the moisture in clot, accelerate coagulation process, blood crusts is formed ahead of time.But, and zeolites is seemingly, also there is the deficiency such as heating, clear difficulty in this material.

(3) Fibrin Glue class

Rorbert in 2000 (Rorbert LW.Tissue adhesive for battle field hemorrhage control[C] ADB209674,2000:1 14.) develop a kind of war wound bleeding-stopping dressing, it forms by three layers: innermost layer is the organosilicon membrane sprinkled with Fibrin Glue dry powder, middle one deck is polyurethane foamed material, skin is medical pressure-sensitive adhesive, and object is to be used for arteriorrhexis to stop blooding.But such material weak point is possible bring that human body or animal blood borne disease infect, complex structure, and cost is high, use inconvenience, anthemorrhagic speed slow, and poor etc. to trunk haemostatic effect.

In addition, some Fibrin Glue needs to add aprotinin (for example: Tisseel and Beriplast are all furnished with aprotinin bovine, and Crosseal is furnished with tranexamic acid) to slow down fibrinous dissolving in coagulation process in use temporarily.Within 2008, once there is report (the Fergusson D A that operation on heart mortality rate is increased about aprotinin, Hebert P C, Mazer C D, Fremes S, MacAdams C, Murkin J M, et a1.A comparison of aprotinin and lysine analogues in high risk cardiac surgery[J] .N Engl J Med, 2008,358:23192331.), Bayer pharmaceuticals has reclaimed its aprotinin preparations at each hospital pharmacy (trade name: Trasylo1) thereafter.

(4) oxidized cellulose class

Oxidized cellulose (trade name: Oxycel) is the one of cellulose derivative, is formed through oxidation of nitric oxide by cellulose.And oxidized regenerated cellulose (claims that speed is yarn, China claims: soluble stanching gauze, trade name: Surgicel), it is a kind of regenerating oxidation fibrage yarn piece, belonging to carboxymethyl cellulose class hemostatic material, is cellulose becomes cellulosic acid tulle shape or cotton shape absorbable hemostatic material through oxidation processes.Its hemostatic mechanism is to have acid carboxyl Fe3+ in hemoglobin to be combined, form brown blob of viscose, seal blood capillary end and stop blooding, simultaneously after close contact wound, can make the hemostatic composition in blood be gathered on mesh gauze, but do not rely on physiological clotting mechanism (Jamborova G.Pospisilova N, Semecky V, Hyspler R, Ticha A, Pospechova K, et a1.Microdispersed oxidized cellulose as a novel potential substance with hypolipidemic properties[J] .Nutrition, 2008, 24:11741181.).Soluble stanching gauze, after close contact wound, can make blood coagulation composition be gathered in around it, completes hemostasis at 2～8min, is usually used at present the position that surgical wound surface is hemorrhage and oozing of blood is difficult for stopping, as surface of bone oozing of blood etc.Evidence suggests that Surgicel just starts to be absorbed by body in one day in contact wound surface, absorption rate with consumption and local blood for and the character of local organization determine, one is 4～8 weeks.It is good that this product has histocompatibility, soft and belittle, is easy to the advantages such as bag, deposited, filling.Weak point is that bleeding stopping period is longer, poor to hemorrhage swift and violent person's effect, be not suitable for separately as wound hemostasis material (week swashs. introduce a kind of novel rectangle first-aid kit [J]. the medical officer people, 2002,45 (1): 61.).In addition, Surgicel can reduce damaged tissue pH value around, this peracidity environment has certain antibacterial ability, but peracidity environment can strengthen damaged tissue inflammatory reaction around simultaneously, delay (Kr í zov P cicatrizes a wound, M sov L, Suttnar J, Salaj P, Dyr J E, Homola J, et a1.The influence of intrinsic coagulation pathway on blood platelets activation by oxidized cellulose[J] .Biomed Mater Res A, 2007,82:274280.).

(5) hemostasis gelatin class

Hemostasis gelatin extracts and purification forms through the animal skins such as pig or cattle, and its loose structure can absorb and overweight self blood of 45 times, volumetric expansion sealing blood vessels breach or wound surface after absorbing blood, and activate platelet, and promote blood clot to form, reach hemostasis object.Although hemostasis gelatin derives from animal tissue, there is no antigenicity, inorganization reaction, staying in the body can be digested after 4～6 weeks through enzyme effect.Different from above-mentioned oxidized regenerated cellulose is, hemostasis gelatin can not affect the pH value of wound surrounding tissue, therefore be more suitable for combining with other hemostatic materials use (Igai H, Yamamoto Y, Chang S S, Yamamoto M, Tabata Y, Yokomise H.Tracheal cartilage regeneration by slow release of basic fibroblast growth factor from a gelatin sponge[J] .J Thorac Cardiovasc Surg, 2007,134:170175.).But gelatin hemostasis needs the participation of body thrombin, for not good enough (the Martinowite U of the patient's effect that has blood coagulation disorders, Spotnite W D.Fibrin tissue adnesives[J] .Thromb Haemost, 1997,78:661.), simultaneously because expanding and may oppress wound surrounding tissue after its absorbing blood, therefore be not suitable for the operative site near nerve or narrow space.The FloSeal being researched and developed by Baxter company combines Bos taurus domesticus Gmelin substrate and thrombin preparation, and gelatin particle is clogged hemostasis by compression by volumetric expansion to wound, and thrombin accelerates blood clot and forms.But as mentioned before, these two kinds of composition performance anastalsises all rely on body clotting mechanism.In addition, thrombin in FloSeal is from human plasma, at present verifiedly pass through two step steam heating processing and can effectively reduce the virus load in blood plasma, but processing technique (the Ereth M H of viral infectivity in human plasma goods admits still not have thoroughly to remove at present in manufacturer, Schaff M, Ericson E F, Wetjen N M, Nuttall G A, Oliver W C Jr Comparative safety and efficacy of topical hemostatic agents in a rat neurosurgical model[J] .Neurosurgery, 2008, 63:369372.), illustrate that this series products exists the risk of relaying disease.

In sum, be divided into three major types (first two is chemical material class) for the first aid hemostasia products of war or security incident in the world:

Table 1

As shown in Table 1, also lack the first aid hemostasia products that can meet above standard completely at present.

In sum, desirable war or first aid hemostasia products, it should at least have following general character, that is: hemostasis fast, wound is not adhered, easily in healing, with low cost, easy body, absorb, not fast spreading disease and sequela few etc.But still do not reach at present or the approaching hemorrhage that reaches above-mentioned standard.To this, in recent years, people constantly carry out the research and development of novel hemostatic material.

Glutamine transaminage (protein-glutamic acid-gamma-glutamyl amine transaminase; Transglutaminase; be called for short TGase); claim again transglutamin-ase 9 transaminase or gamma-glutamyl amine acyltransferase; formed by 331 amino; catalyzing acyl transfer reaction, can catalytic proteins the hydrolysis of glutamy amido in connection in molecule or between intermolecular crosslinked, protein and aminoacid and protein molecule, thereby improve the 26S Proteasome Structure and Function characteristic of protein.Due to its remarkable crosslinking feature, be described as " super binding agent of 21 century ".Show extensive prospect (Cui Yanhua etc., " biotechnology circular ", 2009 year first phase) in fields such as food, medicine, weaving, cosmetics

TGase is extensively present in animal and plant and microorganism body tissue.Nearest research discovery, the TGase of separate sources has difference in the physicochemical property of aminoacid sequence, molecular size range, enzyme.The TGase molecular weight of animal origin not etc., does not need calcium ion activatedly, and enzyme active center is cysteine residues site.The TGase of animal origin is the most deep with TGase (GTG) research of Guinea Pig Liver, and the molecular weight of this enzyme is 90kD, and its enzymatic reaction needs calcium ion activated.Strong to substrate specificity, because containing 17 cysteine residues, this enzyme causes its poor heat stability simultaneously.At 50 ℃, after 10 minutes, enzymatic activity is only residual 40%.Calcium ion to the TGase activity influence of plant origin because of species different.The TGase source of extracting from animal tissue is few, and cost is high, and separable programming complexity is difficult for applying.

It is still not fully aware of which kind of role TGase plays the part of in plant tissue, and Kang etc. extract TGase and are studied from soybean leaves.But separation purifying technique complexity, the yield of enzyme is lower.At present, not yet there is plant origin TGase for commercially producing.

But microbe-derived TGase (mTG) has different enzyme characteristics from the TGase of animal origin, mTG molecular weight is between 23～45kD, and majority is 40kD left and right, is the half left and right of animal origin TGase molecular weight.Importantly, mTG has stronger heat stability, and all has greater activity within the scope of relatively wide pH.Meanwhile, the enzymatic activity of mTG does not rely on calcium ion, so there is the acry radical donor of substrate specificity widely, this point is completely different from the TGase of plant and animal material.Meanwhile, mTG compares compared with GTG, has more significant stability under condition of high voltage.Above-mentioned characteristic makes mTG have more wide range of application.

Microbe-derived TGase belongs to exoenzyme, can direct secretion in culture medium. separation and purification is easy compared with the TGase of plant and animal material, and microorganism fermentation raw material cheapness, produce the enzyme cycle short, the most applicable large-scale industrial production that carries out, therefore enjoys the favor of researcher.

Research finds that glutamine and the lysine of TGase between can catalysis adjacent fiber protein molecular forms ε-(Y glutamine)-lysine covalent bond, thereby forms crosslinked between adjacent fiber protein molecular.Glutamine transaminage and factor XIII a (FXIIIa) belong to glutamine transaminage superfamily; function is very similar to the latter; can catalyzing acyl transfer reaction; make in protein molecule or intermolecular formation crosslinked; thereby change the 26S Proteasome Structure and Function of protein; this reaction is present in a lot of biological processes, comprises the sclerosis of blood clotting process, wound healing, epidermis keratinization, erythrocyte membrane.Chinese patent " purposes of glutamine transaminage " (CN2008100475880) has reported that glutamine transaminage can be used as skin wound healing promoters or its active component, wherein comprises the glutamine transaminage of 0.1-100U/ml or 0.1-100% dry weight as the active component of skin wound healing promoters.But, this research is only that using glutamine transaminage as adjuvant (being promoter) is for wound healing, it had not both related to the purposes of quick-acting haemostatic powder, do not study this enzyme and how to realize the effect that wound is not adhered yet, simultaneously the scheme of described 0.1-100U/ml or 0.1-100% dry weight is too wide in range, lacks concrete active data and finally authorized.Therefore, its correlational study still rests on the aspect how TG enzyme to reduce the wound healing time (talk redly, glutamine of microbe transaminase is to the research of rat Wound Healing, " Chinese biological engineering magazine ", the 27th the 9th phase of volume in 2007; Zhang Nianrong, glutaminase transaminase's characteristic and applied research progress thereof, " western leather ", the 34th the 14th phase of volume in 2012).

Chinese patent application CN2007800512154 and patent family application WO2008076407 thereof disclose a kind of gelatin-Transglutaminases hemostasis adjuvant and encapsulant, and the cross-linked material that it comprises gelatin and avirulence Transglutaminases, can be used for treating wound tissue.But, it is conventional microbial source transglutamin-ase 9 transaminase (USA that this invention is used, CHICAGO, lot number L-04207), this enzyme is not only expensive, and optimal reaction temperature one 50 ℃ of-55 ℃ of left and right, when room temperature (25 ℃), enzyme work only has 20% left and right of optimal reaction temperature, thereby has had a strong impact on the activity (referring to embodiment 1) that relates to glutamine transaminage.

For this phenomenon, magnificent (the glutamine transaminage progress that waits of domestic researcher Cui Yan, " biotechnology circular ", the 1st phase in 2009) paper show, " mTG that derives from different microorganisms there are differences in zymetology; even even there is this either large or small difference at aspects such as isoelectric point, IP, optimal pH, optimum temperature, heat stability between the TG enzyme from same strain different strains from different strains, these difference can directly affect the application of enzyme." " for example, the TG enzyme DNA homolog of bacillus TG enzyme and streptomyces is lower, and the enzyme activity also there are differences.The enzyme optimum temperature alive of S.mobaraensis mTG is 50 ℃, and is 60 ℃ from the mTG optimum temperature of B.subtilis.”

Due to the series products that stops blooding for war, the stability of enzyme is very important, particularly all the more so for the product of room temperature preservation.But the optimal reaction temperature of the mTG of above-mentioned research one 50 ℃ of-55 ℃ of left and right, when room temperature (25 ℃), enzyme work only has 20% left and right of optimal reaction temperature, even in body temperature (37 ℃) left and right, enzyme is lived and is also only had best enzyme half left and right (Buettner K alive, Hertel TC, Pietzsch M, Amino Acids.2012Feb; 42 (2-3): 987-96.; Marx CK, Hertel TC, Pietzsch M, J Biotechno1.2008Sep10; 136 (3-4): 156-62.).In addition the heat stability of these enzymes is poor, and 60 ℃ can cause mTG enzyme loss alive 80% left and right (Marx CK, Hertel TC, Pietzsch M, J Biotechno1.2008Sep10 for 10 minutes; 136 (3-4): 156-62.).For These characteristics, the scientist of Germany is studied this, and has obtained certain achievement, has found that the enzyme of Heat stability is good under high temperature is (as above-mentioned document, and European patent WO2008020075A1, WO2010101256A1; U.S. Pat 20120021459A1), but the best enzyme reaction temperature of these enzymes still 50 ℃ of left and right, this has restricted the application of glutamine transaminage as curable product far away.

For addressing this problem, the inventor's formerly Chinese patent application CN2012101922407 proposes to utilize first does not have the glutamine of microbe transaminase proenzyme that enzyme is lived, and in conjunction with appropriate kinase and gelatin, obtains good wound hemostasis effect.But, the kinase that this institute uses is Bacillus polymyxa Neutral proteinase, this is a kind of nonspecific metalloproteases, although it can dispersion tissue, classification cell, but part Bacillus polymyxa Neutral proteinase can injure cell, and unstable in the time cultivating, even may introduce mycoplasma contamination, and it is expensive, need before use Bacillus polymyxa Neutral proteinase and glutamine transaminage proenzyme to carry out aqueous solution simultaneously, the two can not be pre-mixed and to make relevant curable product, this has greatly affected the medical application of glutamine transaminage proenzyme.

In addition, the inventor proposes to utilize the existing wild enzyme of transformation to obtain high activity mutant enzyme at previous another Chinese patent application CN2012102021709, and it has related to 3 recombinases (hereinafter to be referred as 3 mutant enzymes) that site mutation obtains.Although this mutant enzyme has certain haemostatic effect, but general effect particularly preventing adhesiving effect haves much room for improvement, and anthemorrhagic speed (being the activity of enzyme) need to improve, therefore affect to a certain extent the war first aid purposes of glutamine transaminage proenzyme, caused it to be only applicable to the purposes micro-, that stop blooding in a small amount such as daily wound.

Based on the good haemostatic effect of glutamine of microbe transaminase and the feature having no side effect, a kind of rapid hemostatic material and compound hemostatic product take glutamine of microbe transaminase as main active need to be provided at present, it should have quick-acting haemostatic powder, avoid organ or tissue's adhesion, easily absorb, not fast spreading disease, hemostatic material in medical use with low cost, to fill up the blank needs of civil war or scene of the accident quick-acting haemostatic powder similar products.

Summary of the invention

In sum, prior art problem is how to provide a kind of new glutamine of microbe transaminase, quick-acting haemostatic powder product for war or other wounds of containing, wherein this enzyme should there is the prior art of being different from structure and performance more excellent, the quick-acting haemostatic powder product of simultaneously described war or other wounds also should have the product structure that is different from prior art.

Therefore, thinking of the present invention is to overcome under existing glutamine transaminage room temperature active not enough, or need adjuvant to activate the shortcoming of (being kinase), provide a kind of take Novel transglutaminase as main active, for rapid hemostatic material and the compound hemostatic product of war or other wound hemostasis.This new glutamine of microbe transaminase, its structure is different from any mutant with new capability of having reported, can meet in optimum activity temperature simultaneously and departs from and have a good heat stability to lower temperature.High-temperature process demonstration, this mTG has still embodied very high remnant enzyme activity after 70 ℃ of processing.Reaction temperature shows, the optimum activity temperature of this enzyme is 40 ℃ (more approaching human body temperature), lower than wild type mTG, simultaneously higher than wild type mTG than enzyme work when room temperature and 37 ℃.

The main mutational site of the mTG with heat stability that comprises foregoing bibliographical information is that the mutant serine of 2 is tyrosine, or the mutant serine of 23 is valine or tyrosine, or the tyrosine of 24 sports agedoite, or the lysine mutation of 294 is leucine or isoleucine; Or the mutant serine of 101 is proline, or the glycine mutation of 250 is arginine, or the glycine mutation of 157 is serine.The glutamine transaminage of these sudden changes does not have the combination of more than 3 amino acid mutation, and does not have good heat stability.

Therefore, first object of the present invention is to provide a kind of new glutamine of microbe transaminase mTG, there is the amino acid mutation in four sites in this mTG compared with wild type mTG, sudden change is respectively the 23rd serine and becomes that alanine, 73 glycine become tyrosine, 317 lysine becomes glutamine and 325 lysines become glutamine (being S23A, G73Y, K317Q, K325Q), and these four mutational sites (hereinafter to be referred as four sudden changes) are all different from the mutational site of existing bibliographical information.In one embodiment, described glutamine transaminage mTG has the sequence as described in SEQ ID NO:4.In another embodiment, described glutamine transaminage mTG has the protein sequence of the gene code as described in SEQ IDNO:3.

In a specific embodiments, described glutamine transaminage mTG, wherein this enzyme electrophoresis purity is for more than at least 90%, and specific activity is greater than 25U/ milligram, and preferably, purity is more than 95%, and specific activity is greater than 25U/ milligram; Wherein the optimal reactive temperature of this enzyme is between 25-45 ℃.

Second object of the present invention is to provide a kind of gene order of the said mutation enzyme of encoding.In one embodiment, described sequence is selected from the gene as described in SEQ ID NO:3.

The 3rd object of the present invention is to overcome under existing glutamine transaminage room temperature active not enough, or need adjuvant to activate the shortcoming of (being kinase), a kind of war or scene of the accident quick-acting haemostatic powder product (for example stopping blooding for the organ or tissue of the wounded under other wound environment of war) that this glutamine transaminage is main active matrix that comprise is provided, this hemostasia products comprises the substrate of having added glutamine of microbe transaminase (mTG), the aminoacid sequence of wherein said glutamine transaminage has the protein sequence of the gene code as described in SEQ ID NO:3, or there is the protein sequence as described in SEQ ID NO:4.

In one embodiment, described substrate is the absorbable solid of human body, semisolid, powder or fluid, or plastic glue or colloid form.In a specific embodiments, the electrophoresis purity of described glutamine of microbe transaminase is for more than at least 90%, and specific activity is greater than 25U/ milligram.Preferably, purity is more than 95%, and specific activity is greater than 25U/ milligram.

In one embodiment, the form of described glutamine of microbe transaminase includes but not limited to the forms such as solid, semisolid or fluid, plastic glue or colloid, powder.

In one embodiment, in described hemostasia products, except glutamine of microbe transaminase, also contain other adjuvants, described adjuvant includes but not limited to mineral nitrogen powder, egg albumen powder etc.In a specific embodiments, described hemostasia products is military hemostasia products.

In another embodiment, described hemostasia products is hemorrhage (including but not limited to powder hemostatic article, solid, shaped hemostatic article, fluid or semifluid hemostatic article, liquid hemostatic article), hemostasis device, first-aid hemostatic bag or emergency case or common hemostatic article.

In a specific embodiments, the substrate of described fluid or semi-solid form or material comprise liquid fiber albumen encapsulant or glue class, such as keratin, collagen protein, fluid gelatin etc.Wherein, liquid fiber albumen encapsulant or glue class (people (1990) .J.Trauma.30:512-513 such as J.L.Garza that used many years as the operating room auxiliary material of hemorrhage control; The people such as H.B.Kram (1990) .J.Trauma.30:884-887), and single donor fibrin encapsulant is widely used in various surgery situation clinically.In another embodiment, described liquid fiber albumen encapsulant or glue class comprise lysate, and described lysate is phosphate buffer, phosphate buffer PH scope from 7.0 to 9.0.

In another embodiment, described plastic glue or colloid form comprise oxidized cellulose, oxidized regenerated cellulose, chitosan, keratin, collagen protein, gelfoam, in a more particular embodiment, the material of plastic glue or colloid form can be gelfoam, it can be used as jointing material, realize hemostasis with glutamine of microbe transaminase simulation fibrin-thrombin hemostasis cascade reaction, and can be had no problem on demand and be removed (T.Chen, Biomacromolecules.2003 November-December; 4 (6): 1558-63; ).

In one embodiment, described substrate is selected from protein material, such as absorbable fibre albumin glue, keratin, collagen protein or gelatin, or glucide, for example, such as oxidized cellulose, oxidized regenerated cellulose, chitosan, proteoglycan (poly-n-acetyl glucosamine amine), glycolic acid polymer, lactic acid polymer.In a specific embodiments, described substrate comprises medical gelatin and liquid fiber albumen encapsulant or the glue in various sources, such as keratin, collagen protein, one from animal origin, recombinant sources or its combinations produce source.In a more particular embodiment, can absorption base be gelatin.Preferably, the preferred fish of animal origin and mammiferous gelatin.More preferably, mammal is selected from the gelatin of pig and cattle.The A type high molecular gelatin of this experiment preferred mammal, or the gelatin of 20%～60% proline hydroxylation.

In a preferred embodiment, Powdered glutamine of microbe transaminase (mT6) is dissolved in phosphate buffer, forms glutamine of microbe transaminase solution; Hydroxylation gelatin powder is dissolved in phosphate buffer, forms hydroxylation gelatin solution; Described glutamine of microbe transaminase (mT6) solution is mixed with hydroxylation gelatin solution, can prepare the fluid composition of the gelatin that comprises above-mentioned transaminase and 20%～60% proline hydroxylation.In a preferred embodiment, described glutamine of microbe transaminase concentration is within the scope of 1U/g～180U/g in described compositions; Described hydroxylation gelatin solution concentration is within the scope of 15%w/v～40%w/v.In another preferred embodiment, the volume ratio of described hydroxylation gelatin solution and glutamine of microbe transaminase solution is 10:1～1:10.The stock material shapes of hemostasia products described here is: (i) gelatin of powder, granule or other solid forms; (ii) the transglutamin-ase 9 transaminase of powder, granule or other solid forms.

The 4th object of the present invention is to provide the hemostasis device that a kind of war wound hemostasis is used, and comprises the enzyme described in any one, compositions or hemostasia products in above-mentioned embodiment.The form of described enzyme, compositions or hemostasia products includes but not limited to the forms such as solid, semisolid, fluid, liquid, gas (as spray), can make gauze shape, the as required various and form that produces such as spongy.

In one embodiment, described hemostasis device is tourniquet or hemostatic gauze, comprise tourniquet or hemostatic gauze skin take cotton yarn or soluble stanching gauze as body, with the internal layer for contacting and binding up a wound, in wherein said internal layer, contain substrate powder or the medicated core of described glutamine transaminage.In a specific embodiments, described internal layer shows to be rich in protective film or pad pasting.

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

This section of content prepared enzyme powder content instead and claimed 1-6g glutamine of microbe transaminase to be dissolved in 240ml50mM HEPES buffer (PH=7), is mixed with enzyme and lives as the mT6 enzymatic solution of 50-300u/ml.In addition, also can select the conventional adjuvant that contributes to keep transglutaminase activity and be conducive to bring into play haemostatic effect, as gelatin etc.

At the interior laminated medical adhesive that scribbles of cotton yarn or soluble stanching gauze,

The substrate powder that contains above-mentioned enzyme is sprayed on the sticking internal layer of tool, forms tourniquet or hemostatic gauze drug core layer;

In drug core layer outside, protective film or mucosa are set, make described hemostasis device.

In another embodiment, described hemostasis device is the sprayer unit of quick-acting haemostatic powder, comprises the spraying of pressurization or the mixture of foam, gelatin and glutamine transaminage component.In a specific embodiments, above-mentioned hemostasis device comprises the mixture of a part for transglutamin-ase 9 transaminase composition, gelatin, foam or spraying.Use for example the using selectively by storing mixture component separately and immediately before using mix them of hemostasis adjunct ingredient mixture of these methods; For example selectively complete by immediately activating them with inactive form together storage component with before using.One or more in the non-activity encapsulant mixture of powder, the spray-dired powder that need to reconstitute and/or any other applicable form of the lyophilizing that the hemostasis adjuvant component of inactive form is selectively provided as frozen soln, need to reconstitute.

In other embodiment, described hemostasis device can also be first-aid hemostatic bag, the emergency case etc. that contains above-mentioned gelatin and glutamine transaminage component.In other embodiments, described hemostasis device is military hemostasis device.

The 5th object of the present invention is to provide a kind of method of the quick-acting haemostatic powder product for the preparation of war or scene of the accident wound, this product comprises the substrate of having added glutamine of microbe transaminase, and the aminoacid sequence of wherein said glutamine transaminage is as shown in SEQ ID N0:4.

Step comprises:

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

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

(3) by conventional method, by the living solution of described glutamine transaminage be added on human body can absorption base in;

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

In one embodiment, Powdered glutamine of microbe transaminase (mTG) is dissolved in phosphate buffer, forms glutamine of microbe transaminase solution; Powdered hydroxylation gelatin is dissolved in phosphate buffer, forms hydroxylation gelatin solution; Described glutamine of microbe transaminase (mTG) solution is mixed with hydroxylation gelatin solution, can prepare the fluid composition of the gelatin that comprises above-mentioned transaminase and 20%～60% proline hydroxylation.In a preferred embodiment, described glutamine of microbe transaminase concentration is within the scope of 1U/g～180U/g in described compositions; Described hydroxylation gelatin solution concentration is within the scope of 15%w/v～40%w/v.In another preferred embodiment, the volume ratio of described hydroxylation gelatin solution and glutamine of microbe transaminase solution is 10:1～1:10.

Selectively, the present composition may further include and forms bionical clot and reach the object of hemostasis.

technical term

" war wound " used herein or " accident wound " refer in war or the scene of the accident, is subject to any infringement that bullet, contingency carry out any tissue of the wounded, and it causes a large amount of blood loss of blood circulation, deficiency of skeletal limb etc. even.Tissue can be in-vivo tissue (for example organ or blood vessel), or vitro tissue (such as head, extremity etc.).The loss of blood can be (for example organ from breaking) in body, or external (for example, from laceration, scuffing, incised wound etc.).Wound can for example, in soft tissue (organ), or for example, in sclerous tissues's (bone).This wound is not processed in time, should maybe may cause life threatening.Therefore, of the present invention is not the hemostasia products for daily wound (Wicresoft's wound) for war or scene of the accident wound quick-acting haemostatic powder product, as patches such as adhesive bandages, neither be used for clinical operation and rescue the hemostasia products of class, as medical hemostatic bag, hemostasis adhesive tape, mosquito forceps etc.

" field wound " used herein refers to the field condition wounds such as war wound, security incident, as vehicle accident, mine accident, production accident etc.If not otherwise specified, war class wound has also comprised the wounds such as vehicle accident, mine accident, production accident, but war class wound or field wound do not comprise daily life category microtrauma or clinical operation wound class.

" hemostasia products " used herein and hemorrhage refer to contain quick-acting haemostatic powder goods glutamine transaminage of the present invention (mTG) and that can be used for " war wound " or " accident wound ", for example: hemorrhage (comprising powdery hemostatic article, solid, shaped hemostatic article, fluid or semifluid hemostatic article, liquid hemostatic article), hemostasis device, first-aid hemostatic bag or emergency case and existing hemostatic article.In common battlefield or on-the-spot use, the hemostasia products of described non-device class also can be described as hemorrhage.

" absorbability substrate or material " used herein refers to spontaneous and/or is decomposed into the material of component via body of mammals, described component is consumed or eliminates in the mode of significantly not disturbing wound healing and/or tissue regeneration, and can not cause any significant metabolism disorder.For example, but resorbable material protein material, such as fibrin, keratin, collagen protein, or glucide, for example, such as alginate, chitin, cellulose, proteoglycan (poly-n-acetyl glucosamine amine), glycolic acid polymer, lactic acid polymer or glycolic/lactic acid copolymer.But for example resorbable material glucide.Wherein, based on the bleeding-stopping dressing of above improved resorbable material, it comprises the multiple layers that comprise absorbability material, for example, referring to PCT/US03/28100, and No. 0060155234th, U.S. Patent application.

" non-resorbable material " used herein refers to and can not be decomposed into avirulence by patient body or not disturb wound healing and/or tissue regeneration or can not cause the material of any significant metabolism disorder, as aseptic binder, sterile gauze, minerals styptic powder (Si oxide powder, potassium ferrum oxyacid salt powder etc.) etc.

Beneficial effect

The present invention is take glutamine of microbe transaminase and gelatin as active substance, compared with the prior art, beneficial effect of the present invention comprises, the hemostasia products that contains glutamine of microbe transaminase and gelatin can strengthen war traumatic wounds recovery effects, and haemostatic effect is obvious.Secondly, the raw material glutamine of microbe transaminase that the present invention uses, under the function and original hypotoxic situation that do not change traditional glutamine of microbe transaminase, by the thermostability of certain technique improvement glutamine of microbe transaminase, obtain the glutamine of microbe transaminase at normal temperatures with better stability.In addition, this hemostasia products has quick-acting haemostatic powder, anti-wound and the effect such as is adhered, and low side effect, wound is had good healing effect, acts on rapid-actionly, absorbs the advantages such as good.Especially, in war or accident wound environment, after product of the present invention does not generate heat, applies in hemostasis, without removing, histocompatibility and absorbability are good, the shortcoming such as overcome heating noted earlier, be difficult to remove, can not absorb.In the use procedure of this product, exothermic reaction is not obvious, can human body do not caused and be burnt, and can be absorbed to enter in body and do not produce negative interaction, avoided other class hemorrhage product later stages to remove and the secondary injury that brings.In addition, the present invention can the single married operation that can complete hemorrhage, easy to use, has adapted to the needs of specific condition in war and accident wound.

Accompanying drawing explanation

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

Fig. 2 represents the mass spectrometry results of mTG albumen in embodiment 5;

Fig. 3 represents the electrophoretogram that the present invention in embodiment 6 suddenlys change after the polishing purification of mTG albumen, the albumen that wherein 4 swimming lanes are purification;

Fig. 4 represents in embodiment 7 that the suddenly change specific activity of mTG albumen of under different temperatures wild type mTG albumen, three site mutation mTG albumen and the present invention measures;

Fig. 5 represents that novel hemostasia products of the present invention and blank group are respectively to the hemorrhage haemostatic effect of rat femoral.Wherein: Fig. 5 A represents that matched group is to the hemorrhage haemostatic effect of rat femoral, and wherein Fig. 5 A-1 represents exposed Rats femoral artery, after 5A-2 represents to cut off femoral artery, severe is bled; Figure B represents that novel hemostasia products of the present invention is to the hemorrhage haemostatic effect of rat femoral, wherein Fig. 5 B-1 represents exposed Rats femoral artery, after 5B-2 represents to cut off femoral artery, severe is bled, 5B-3 represents to apply hemostasia products of the present invention and bleeds and stop later for 3 minutes, and 5B-4 represents to apply 5 minutes later cohesives that detect hemostasia products and wound with tweezers of the present invention;

Fig. 6 represents the hemostasis statistics of variables to rat femoral respectively of novel hemostasia products of the present invention and blank group;

Fig. 7-A is illustrated in rat femoral and breaks and be applied with novel hemostasia products of the present invention, and Fig. 7-B applies the wound suture outward appearance recovery situation of rat shank after 21 days, and Fig. 7-C represents that solution cuts the recovery situation of rat leg muscle after rat shank open;

Fig. 8-A (enlarged drawing B) is subcutaneous shallow-layer normal structure colored graph.Figure C (enlarged drawing D) is illustrated in subcutaneous shallow-layer, and experimental group applied hemostatic composition after the 7th day, the tissue staining figure of compositions and muscular tissue compatibility tissue staining.

Fig. 9 represents that experimental group applied hemostatic composition after the 21st day, compositions and muscular tissue compatibility tissue staining, and figure A (enlarged drawing B) is subcutaneous shallow-layer, and figure C (enlarged drawing D) is subcutaneous deep, and arrow indication is compositions.

Figure 10 represents novel hemostasia products of the present invention (containing glutamine of microbe transaminase and gelatin) and the hemostasis statistics of variables to rat liver respectively of blank group in embodiment 9;

Figure 11 represents the hemostasis to rat liver Hemorrhage Model and anti-ooze out effect schematic diagram respectively of hemostatic composition of the present invention (containing glutamine of microbe transaminase and gelatin) and blank group, wherein:

The matched group exposing before Figure 11 A and 11C difference display process and the liver of experimental group;

Figure 11 B and 11D show respectively the hemostasis situation of naturally bleeding for 10 seconds after the liver wound of manufacturing matched group and experimental group;

Figure 11 E and 11F show that respectively matched group applies 2 points of hemostasis situations of 30 seconds, 5 minutes after the present composition;

Figure 11 G and 11H represent the cohesive with the manual mode check present composition;

Figure 12 represents the absorbing state of compositions of the present invention in rat liver Hemorrhage Model, component 1-5 is illustrated respectively in experimental group B (applying novel hemostasia products of the present invention) and control group A (Johson & Johnson: absorbable hemostatic gelfoam, trade name Surgiflo ^tM) before processing, apply the recovery situation of 2 points of injury of livers after 30 seconds, 5 days, 20 days.

The specific embodiment

In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Do not deviating under the spirit and scope of inventive concept, variation and advantage that those skilled in the art can expect are all included in the present invention, and take appending claims as protection domain.Implement process of the present invention, condition, reagent, experimental technique etc., except the content of mentioning specially below, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.

Embodiment 1 enzyme activity determination

1) survey enzyme reagent preparation alive

A liquid (0.5L): add 400ml distilled water in beaker 0.015mol (5.06g) substrate Na-CBZ-Gln-Gly, 0.05mol (3.475g) oxammonium hydrochloride., 0.005mol (1.536g) reduced glutathion, by magnetic stirrer after 20 minutes, add 0.1mol (12.11g) Tris, with 6mol/L (or lmol/L) hydrochloric acid tune pH to 6.0, solution is moved in 500ml volumetric flask, pour in volumetric flask for 3 times with distilled water wash beaker, be settled to 500ml.

B liquid: 3mol/L HCl, 12% trichloroacetic acid (W/V), 5% ferric chloride (W/V, is dissolved in 0.1mol/L hydrochloric acid, refilters) mix by the volume ratio of 1:1:1.

2) enzyme activity determination

Experimental group: get 0.2ml testing sample, add 37 ℃ of incubations of 2ml A liquid 10 minutes, then add 2ml B liquid.Matched group: get 0.2ml testing sample, add 37 ℃ of incubations of 2ml B liquid 10 minutes, then add 2mlA liquid.Measure light absorption value in 525nm.

The mutation of embodiment 2 wild strains

Wild type mTG bacterial strain is luxuriant source streptomycete (Streptomyces mobaraensis) (as the strains A TCC numbering 27441 of the strains A TCC numbering 29032 of U.S. ATCC or U.S. ATCC, or the bacterial strain that center is preserved in Chinese microorganism is as CGMCC numbering 4.1719 and CGMCC numbering 4.5591).

Culture medium configuration: Gause I culture medium: soluble starch 20g/L, KNO ₃1g/L, MgSO ₄7H ₂o0.5g/L, K ₂hPO ₄3H ₂o0.5g/L, NaCl0.5g/L, FeSO ₄7H ₂o0.01g/L, agar 20g/L, pH7.2-7.4.Fermentation medium: glycerol 20g/L, yeast extract 6g/L, fish meal protein peptone 25g/L, MgSO ₄7H ₂o2g/L, K ₂hPO ₄3H ₂o2g/L, pH7.4.

In Gause I culture medium, add the cold sterilized water of 10ml, with the abundant scraping of Inoculating needle surface mycelia, break up spore, aseptic filter paper filters.

Operate under red light or lucifuge condition and carry out, within 0.5 hour, open uviol lamp in advance, make light stability.Get concentration and be about 10 ⁷it is 6 centimetres of sterilizing plates that the spore suspension 3mL of individual spore/mL is placed in diameter, utilizes magnetic stir bar to stir, under uviol lamp power 15W condition, and different irradiation distance, the different time of ultraviolet irradiation is carried out mutation.After lucifuge 1 hour, in dark situation, dilution difference spore suspension is coated in Gao Shi culture medium, cultivated 7 days for 30 ℃.Picking list bacterium colony is cultivated 24 hours in fermentation medium, and while measuring respectively 37 ℃, the enzyme of candidate strain and wild strain is lived.

Obtaining of embodiment 3 mutant mTG genes

Genome extracting: the fresh mycelia of the mutant strain the highest enzymatic activity of liquid culture in embodiment 2 is inoculated in to fresh culture, cultivates about 24 hours; Centrifugal collection 10ml thalline; New fresh thalli is in mortar (20 ℃ of pre-coolings), and liquid nitrogen grinding is to fine-powdered repeatedly, and average mark is filled in 2 1.5mL centrifuge tubes rapidly; Add TE buffer 550 μ L, add the 20%SDS solution 30 μ L of 65 ℃ of preheatings, in 5 seconds of vortex oscillation, add 20mg/mL E.C. 3.4.21.64 20 μ L, mix gently, 37 ℃ of insulations 1 hour; Add the saturated phenol/chloroform/isoamyl alcohol of equal-volume Tris (25:24:1) extracting, slightly put upside down and mix, centrifugal 10 minutes of 10000rpm; The careful supernatant of drawing, to new centrifuge tube, adds equal-volume chloroform/isoamyl alcohol (24:1) extracting, 10000rpm, centrifugal 5 minutes; Draw supernatant and equal-volume isopropyl alcohol (4 ℃ of pre-coolings) and mix, then centrifugal 5 minutes of 10000rpm, abandons supernatant.1mL70% ethanol (4 ℃ of pre-coolings) washing for precipitation (put upside down, centrifugal 1 minute), natural air drying, adds 40uL TE dissolving, and-20 ℃ save backup.

Design of primers: designed pair of primers and can obtain by polymerase chain reaction the complete sequence of mTG gene, this sequence is selected from: SEQ NO:1:ctcaacgaaa gcgctccggc cgcttc

SEQ?NO：2：cgctcacatc?acggccagcc?ctgc

PCR reaction system and reaction condition: the genomic DNA of above-mentioned acquisition and primer are mixed, carry out PCR reaction by following condition: Taq enzyme reaction solution (2 times of concentration) 25 μ L, primer Pf (2.5mmol/mL) 2 μ L, primer Pr (2.5mmol/mL) 2 μ L, DNA2 μ L and sterilized water.30 seconds, 72 ℃ of 1 minute, 55 ℃ annealing of 94 ℃ of degeneration 2 minutes, 40 circulations altogether.

Electrophoresis detection PCR purity, as shown in Figure 1, left side swimming lane is molecular weight standard DNA to result, the right swimming lane is PCR fragment electrophoresis result.PCR clip size is between 1000bp and 2000bp, and 1058bp is consistent with theoretical value.

The suddenly change order-checking of mTG gene of embodiment 4

To the dehydrated alcohol that adds 2 times of volumes in the PCR product in embodiment 3, mix, room temperature is placed 5 minutes.Centrifugal 10 minutes of 12000rmp, abandons most supernatant; Wash precipitation once with 70% ethanol.Dry, with 50 μ L sterilized water dissolution precipitations.React by following system: aseptic water-soluble PCR product 1 μ L, pUC19-T carrier (Takara company) 0.5 μ L, ligase 0.5 μ L, buffer (10 times of concentration) 5 μ L and sterilized water 43 μ L, mix, 16 ℃ are reacted 20 minutes.Above-mentioned reacted product is got to 5 μ L, join 100 μ L bacillus coli DH 5 alpha competent cells (Takara company), 4 ℃ are reacted 30 minutes, and 42 ℃ are reacted 90 seconds, add LB culture medium, cultivate 1 hour for 37 ℃, by the coating of bacterium liquid and LB flat board.After bacterium colony forms, send the order-checking of order-checking company.Compare with wild type mTG gene, detect mutational site.

Check order through conventional method, find that the sequence of this mutant gene is as shown in SEQ ID NO:3, infer that thus its corresponding protein amino acid sequence is as shown in SEQ ID NO4.

The general purification of embodiment 5mTG albumen

Fermentation medium and bacterial strain be with embodiment 2, and luxuriant source streptomycete spore inoculating, to 30mL fermentation medium is housed, is cultivated 28 hours.Centrifugal 30 minutes of 12000rmp in 4 ℃ of centrifuges, removes thalline and solid impurity, obtains 20mL culture medium supernatant.Add 40mL dehydrated alcohol, place 1 hour for 4 ℃.In 4 ℃ of centrifuges, centrifugal 30 minutes of 12000rmp, abandons supernatant, and collecting precipitation, is placed in ampoule bottle by precipitation, and lyophilizing 2 days in freezer dryer obtains the wild type mTG albumen of preliminary purification.

The luxuriant source streptomycete spore inoculating of sudden change, to being equipped with in the 250ml triangular flask of 30mL fermentation medium, is cultivated 24 hours.Other operating procedures are the same, the purification mTG albumen that obtains suddenling change.

Whether its structure of protein utilization mass spectral analysis of purification is inferred consistent with theory.Final mass spectrometry results shows as shown in Figure 2: its mass spectrum molecular weight is shown as 37934.9Da, meets with the molecular structure of the deduction of gene sequencing.Illustrate that protein sequence is really SEQ NO4.Through comparison, with respect to wild-type sequence, this SEQ ID NO; 4 have 4 sites to undergo mutation, and are respectively the 23rd serine and become that alanine, 73 glycine become tyrosine, 317 lysine becomes glutamine and 325 lysines become glutamine (being S23A, G73Y, K317Q, K325Q).

The suddenly change polishing purification of mTG albumen of embodiment 6 the present invention

Utilize ion exchange column to carry out albumen polishing purification in the albumen obtaining in embodiment 5.Filler is DEAE-Sephrose fast flow (GE healthcare), the tris/HCl buffer that balance liquid is 0.05M, pH9.0, the tris/HCl of the NaCl50mM that eluent is 0-1mol/L, pH9.0.16 column volumes of linear elution, collect each component, and electrophoresis detection obtains the good mTG albumen of purity.By lyophilizing after this component desalination.Experimental result shows that this albumen size is consistent with the molecular size range of expection 37.9kDa, and purity is very high, and electrophoresis only has single band, and purity was greater than for 99% (as shown in Figure 3).The albumen that the 4th swimming lane is purification.Thus, also further to have confirmed the protein sequence analyzed in embodiment 5 be really SEQ NO4 to the result of embodiment 6.

Under embodiment 7 different temperatures, suddenly change mTG albumen specific activity measure

The wild type mTG albumen that purification is obtained and the present invention suddenly change mTG albumen at 25 ℃, 37 ℃, 45 ℃, 55 ℃, 75 ℃, measure respectively enzyme live.Measure enzyme and live method therefor with described in embodiment 1.The solution that surveying lives uses preheating 2 minutes at corresponding temperature respectively before reaction.Experimental result shows (as shown in Figure 4), and at differential responses temperature, what the present invention suddenlyd change mTG has shown the diverse tendency with wild type mTG than enzyme work.The present invention suddenlys change mTG optimal reaction temperature 45 ℃ of left and right, and wild type mTG optimal reaction temperature is 55 ℃ of left and right, and mTG optimal reaction temperature of the present invention is starkly lower than wild type mTG.And at 25 ℃-75 ℃, mTG of the present invention is than the enzyme wild type that is all significantly higher than alive.In the time of 37 ℃, mTG of the present invention has reached 80U/ milligram than enzyme work, has lived high more than 100% than wild type mTG enzyme, has also improved 50% than 3 site mutations of patent CN2012102021709 formerly.In addition, under the hot conditions more than 55 ℃ wild type mTG activity sharply decline and mTG of the present invention still retain high enzyme live.65 ℃-75 ℃ time, mTG of the present invention still retains high enzyme and lives, and almost inactivation of wild type mTG now.Visible, compare than wild type mTG and 3 site mutation enzymes, the present invention mTG that suddenlys change has significantly good enzyme activity and heat-resistant stability, enzyme activity and haemostatic effect positive correlation, point out 4 sudden change mTG haemostatic effects of the present invention to be better than wild type mTG and 3 site mutation enzymes, and its environment for use is more wide in range, there is better adaptability for war or accident wound site environment.

Embodiment 8 gene engineering method are prepared mTG of the present invention

In order to guarantee also to obtain mutant gene under the prerequisite that there is no luxuriant source streptomycete mutant strain, the present embodiment utilizes the method for synthetic to obtain mTG mutant.

The gene order of said mutation is synthesized to complete genome sequence in business-like biotech firm (as above marine growth engineering company, Ying Jun biotech company etc.), and be cloned into commercialization coli expression carrier pET-32a (as Merck biotech firm or other biotech firm; Any other expression vector as pET-32b or other suitable carrier all can) EcoRV site in, form the expression vector that can express genes of interest.In e. coli bl21, obtain expressing the genetic engineering bacterium of this albumen according to conventional technical transform.This genetic engineering bacterium is inoculated in 20ml LB culture medium, and after 16 hours, adding solubility according to 1/1000 of culture volume 37 ℃ 200 rpms cultivations is 1mol/L lactose solution, continues to cultivate 4 hours, collects thalline.

Under the power of 200w, use the broken thalline of Ultrasonic Cell Disruptor; Centrifugal 5 minutes supernatant discarded of 10000rmp, are resuspended in precipitation in the solution containing 8mol/L carbamide, blow and beat 3 times; Centrifugal 5 minutes of 10000rmp, collects supernatant purification as described below.

The Ni-NTA Spin test kit that utilizes QIAGEN company, carries out purification according to wherein said method.Concrete grammar carries out according to its description completely, has finally obtained the fusion rotein of the mTG that contains sudden change.

To the Bacillus polymyxa Neutral proteinase that adds 1/10 quality in fusion rotein solution according to the quality of fusion rotein.37 ℃ effect 30 minutes after,

According to the method purification of embodiment 6, obtain mTG albumen of the present invention.

Embodiment 9: rat femoral hemostasis experiment

This case study on implementation provides the case study on implementation to external massive hemorrhage quick-acting haemostatic powder under product simulation battlefield surroundings of the present invention.

Indication of the present invention containing the hemostatic material lyophilized powder proportioning of glutamine transaminage is: glutamine transaminage 5400U; Hydroxylation gelatin 25g; Sorbitol 5g.Preparation method is: claim the 25g hydroxylation gelatin heated and stirred that adds water that it is dissolved completely; Claim 5g sorbitol, add in above-mentioned solution, stir it is dissolved completely; Use again 0.22 μ tm membrane filtration degerming, be placed to room temperature and inject water and be settled to 100ml.Aseptic filtration adds 5400U glutamine transaminage, after mix homogeneously, is placed in freeze dryer and carries out lyophilization.After dry end, shatter powdered, this hemostatic composition packing is made to finished product.

14 of SD rats are selected in experiment, are divided at random blank group and experimental group (mTG-gelatin), 7 every group.

Use surgical knife to cut the femoral artery of rat.Severe was bled after approximately 10 seconds, and before using, the instant gauze that uses is removed the blood of accumulating.Before using hemostatic composition, hemostatic composition is ready to.After applying compositions, femoral artery has still stopped bleeding being less than in 3 minutes completely.After 5 minutes, use and take the photograph manually detection hemostasia products of son.Observe respectively the experiment effect of gauze hemostasis and the hemostasis of novel hemostasia products, Taking Pictures recording.And statistic fluid blood volume.

Experimental result is shown in Fig. 5 A (matched group) and Fig. 5 B (experimental group), and wherein Fig. 5 A-1 represents exposed Rats femoral artery, and after 5A-2 represents to cut off femoral artery, severe is bled; Figure B represents that novel hemostasia products of the present invention is to the hemorrhage haemostatic effect of rat femoral, wherein Fig. 5 B-1 represents exposed Rats femoral artery, after 5B-2 represents to cut off femoral artery, severe is bled, 5B-3 represents to apply hemostasia products of the present invention and bleeds and stop later for 3 minutes, and 5B-4 represents to apply 5 minutes later cohesives that detect hemostasia products and wound with tweezers of the present invention.

Therefore, Fig. 5 A shows: rat femoral is cut off rear femoral artery and bled seriously, presses and still can not stop blooding for 3 minutes with gauze.Illustrate that femoral artery bleeds unsuccessful with gauze hemostasis.

On the contrary, Fig. 5 B shows: rat femoral is cut off nature and bled after 10 seconds, apply hemostasia products, can observe bleeding of femoral artery in being less than 3 minutes is stopped by hemostasia products, time standby tweezers at 5 minutes detect hemostasia products and the adhesion situation of rat leg muscle and the viscosity of hemostasia products, find that hemostasia products viscosity is good, and the adhesion of hemostasia products and rat leg muscle is all right, difficult drop-off.

Implementing in above scheme, to collect the blood flow of each experimental group, adopt electronic balance weighing and add up the amount of bleeding of rat femoral in 3 minutes.Result is as shown in Figure 6: rat femoral is bled with the successfully hemostasis in three minutes of novel hemostasia products, and the quick-acting haemostatic powder of above-mentioned experimental result explanation hemostasia products of the present invention is functional.

Embodiment 10: the tissue compatible Journal of Sex Research of novel hemostasia products

15 rats are selected in experiment.Be divided at random 5 for blank group, 10 is experimental group (mTG-gelatin).

Wherein 10 experimental grouies, use surgical knife to cut the femoral artery of rat.Severe was bled after approximately 10 seconds, and before using, the instant gauze that uses is removed the blood of accumulating.Before using hemostatic composition, hemostatic composition is ready to.After applying compositions, femoral artery has still stopped bleeding being less than in 3 minutes completely.After 5 minutes, use and take the photograph manually detection hemostasia products of son.Be rat stitching, sub-cage rearing afterwards.The huckle muscle that treating excess syndrome is tested position in the 7th day and 21 days respectively carries out tissue slice.Get 5 rats at every turn.Experimental result shows as shown in Figure 7: be just applied with compositions and applying the wound recovery situation of compositions rat shank after 21 days and the situation of change of muscle.

Experimental result shows, rat shank apply novel hemostasia products after 21 days wound recover, solution has disappeared when observing hemostasia products contrast and just applied after cutting open, and leg muscle does not have significant difference than normal structure.Illustrate that novel hemostasia products is can absorbing state good, does not cause harmful effect to rat.

Then, wherein 5 matched groups, first day is got 5 rat muscles and is cut into slices, and as blank, obtains normal muscular tissue section.

Wherein the experimental procedure of tissue slice is as follows:

1, draw materials with fixing:

Taking off piece of tissue (one thickness is no more than 0.5 centimetre) from rat thigh position drops into fixative 10% formalin preparing in advance.

2, dewater transparent:

One is used by low concentration and makes dehydrant to alcohol in high concentration, sloughs gradually the moisture content in piece of tissue.Again piece of tissue is placed in and is both dissolved in ethanol, be dissolved in again in the clarifier dimethylbenzene of paraffin transparent.

3, waxdip embedding:

Transparent piece of tissue is placed in to the paraffin having dissolved, puts into wax-dissolving box insulation.After immersing piece of tissue completely, paraffin carries out embedding.

4, section and paster:

Embedded wax stone is fixed on microtome, thinly slices.The thin slice cutting is put in the water of heating and plates, then is attached on microscope slide, puts in 45 ℃ of calorstats and dries.

5, dewaxing dyeing:

Dye with HE

HE dyeing course is:

1. put into hematoxylin aqueous solution and dye several minutes entering section after distilled water.

2. color separation in sour water and ammonia, each several seconds.

3. flowing water rinses after 1 hour and enters distilled water for a moment.

4. enter in 70% and 90% ethanol and dewater each 10 minutes.

5. enter ethanol Yihong dyeing liquor dyeing 2-3 minute.

6, dewater transparent:

Section after dyeing is dewatered through absolute alcohol, then makes to cut into slices transparent through dimethylbenzene.

7, sealing:

Muted color natural gum carries out mounting.After natural gum is slightly dry, stick mark writing paper, section preparation just can use.

As Figure 8-9, wherein Fig. 8-A (enlarged drawing B) is subcutaneous shallow-layer normal structure colored graph to experimental result.Figure C, (enlarged drawing D) is illustrated in subcutaneous shallow-layer, and experimental group applied hemostatic composition after the 7th day, the tissue staining figure of compositions and muscular tissue compatibility tissue staining.Result shows, observes hemostatic composition (arrow indication) started to be absorbed with and enter muscular tissue inside at the tissue slice of the 7th day.

Fig. 9 represents that experimental group applied hemostatic composition after the 21st day, compositions and muscular tissue compatibility tissue staining, figure A (enlarged drawing B) is subcutaneous shallow-layer, figure C (enlarged drawing D) is subcutaneous deep, arrow indication is compositions, and in the 21st day, hemostasia products has entered the inner degraded of muscular tissue.

Result also shows, after 21 days, compares with the muscular tissue section of Normal group Fig. 8 A (enlarged drawing B), and subcutaneous rat shallow-layer muscular tissue tissue slice Fig. 9 A (enlarged drawing B) does not have notable difference.Illustrate and apply after hemostasia products in the time of 21 days, histocompatibility and the absorbability of hemostasia products are good.

Above experimental result shows: histocompatibility and the absorbability of the novel hemostasia products of the present invention are good.

The contrast of embodiment 11 hemostasia products of the present invention to SD rat femoral wound hemorrhage haemostatic effect and local variations in temperature

This case study on implementation provides the present invention and the case study on implementation of traditional military hemorrhage to wounded's haemostatic effect and the contrast of local variations in temperature.

Indication of the present invention containing the hemostatic material lyophilized powder proportioning of glutamine transaminage is: glutamine transaminage 5400U; Hydroxylation gelatin 25g; Sorbitol 5g.Preparation method is: claim the 25g hydroxylation gelatin heated and stirred that adds water that it is dissolved completely; Claim 5g sorbitol, add in above-mentioned solution, stir it is dissolved completely; Use again 0.22 μ m membrane filtration degerming, be placed to room temperature and inject water and be settled to 100ml.Aseptic filtration adds 5400U glutamine transaminage, after mix homogeneously, is placed in freeze dryer and carries out lyophilization.After dry end, shatter powdered, this hemostatic composition packing is made to finished product.

21 of SD rats are selected in experiment, are divided at random blank group, zeolite powder matched group and experimental group (mTG-gelatin), 7 every group.The SD rat of buying starts to test after experimental animal room adapts to 1 week, sets up rupture of blood vessel trauma model.Breathe anesthetis anesthetized rat; Face upward position and be fixed on operating-table, sterilization, makes 3cm stringer otch at right lateral thigh front upper part, fully exposes and the about 1.5cm of free femoral artery, and the about 1.5cm of femoral vein, puts temperature measurer between blood vessel, muscle.Cut off femoral artery, femoral vein with scalpel, immediately hemorrhage wound surface being sprinkled to hemostatic material (each hemorrhage wound surface all uses the tested material of 25g: negative control starch, positive control zeolite granular, styptic powder of the present invention) stops blooding, press after 2min the haemostatic effect of the each treated animal wound surface of observed and recorded.Statistical procedures: adopt the relatively significance of haemostatic effect difference of X 2 test; Significance level a=0.05.

Table 2, to SD rat thigh arteriovenous rupture hemorrhage haemostatic effect and local variations in temperature

Group	Rat	Hemostasis quantity	Average bleeding stopping period (min)	Hemostatic efficiency	Local temperature
						Negative control	7	0	?	0％	37℃
Zeolite powder contrast	7	7	2	100％	60～70℃
						The present invention	7	7	2.5	80％	37～50℃

With negative control group comparison, ^*p<0.01

Experimental result shows: hemorrhage of the present invention and positive control zeolite powder are sprinkling upon strand arteriovenous fracture bleeding part and press after femoral artery wound surface 3min, can effectively stop blooding.Negative control starch can not effectively stop blooding after being sprinkling upon bleeding part and pressing femoral artery wound surface 3min, and the SD rat of no longer applying other intervening measure treatments is all dead.Positive control zeolite granular raises local temperature, but the effect that hemorrhage of the present invention raises local temperature is far weaker than zeolite granular, and local temperature is no more than 50 ℃.Although experimental result shows that hemorrhage anthemorrhagic speed of the present invention is a little less than zeolite powder anthemorrhagic speed, but still has potent quick-acting anastalsis, is enough to meet the requirement of war quick-acting haemostatic powder.And hemorrhage of the present invention makes local temperature rising effect be weaker than zeolite, does not cause local organization hot injury, and its resultant effect is better than traditional zeolites hemorrhage.

Embodiment 12: the hemostasis of compositions of the present invention in rat liver Hemorrhage Model and prevent that wound from oozing out experiment

This embodiment provides according to the present invention the quick-acting haemostatic powder effect to inside of human body internal organs under product (glutamine of microbe transaminase (mTG)-gelatin composition) sham fight environment.

14 of the SD rats of 300g ± 20g are selected in experiment, are divided at random matched group and experiment (mTG-gelatin) group, 7 every group.

In the present invention, use glutamine of microbe transaminase specific activity to be: 1-50U/ milligram

For matched group, do not add any hemostasia products (or hemorrhage, lower with), just in order to observation of nature bleeding stopping period.

Start to make adult rat in generalized anesthetic state before experiment, at whole duration of test, detect and maintain the vital sign of rat.

For using of experiment and contrast, routine operation is opened abdominal cavity, exposes after liver, uses surgical knife to manufacture 1 centimeter length, 0.5 centimetre of dark sagittal otch at liver lobus sinister.After active approximately 10 seconds of bleeding, before using, mTG-gelatin solution use in time gauze to remove the blood of accumulating.

Experimental solutions is applied to the otch on liver lobus sinister.Use latter about two minutes gel formation and use after bleed and stopped completely being less than in approximately 2.5 minutes.After 5 minutes, firmly shake tissue and use tweezers cross wound site and apply pulling force, but still intact and wound closure of gel.

The amount of bleeding that uses electronic balance weighing and add up rat liver in 2.5 minutes, result as shown in figure 10, experimental result data shows, compositions of the present invention can suppress the amount of bleeding of rat liver 93% in the time of 2 points and 30 seconds, can reach rapidly haemostatic effect (* *, P<0.01).

Figure 11 represents hemostatic composition of the present invention and the blank group hemostasis to rat liver Hemorrhage Model and the anti-effect schematic diagram that oozes out respectively, the matched group that wherein Figure 11 A and 11C expose before display process respectively and the liver of experimental group, as Figure 11 B and 11D show respectively active situation of bleeding for 10 seconds after the liver wound of manufacturing matched group and experimental group.Figure 11 E represents that matched group applies 2 points of situations of 30 seconds after the present composition, and Figure 11 F represents to apply after the present composition situation of 5 minutes, and Figure 11 G and 11H represent the cohesive with the manual mode check present composition.

For matched group, lower 2 points of the measure situations of not taking to stop blooding can not be stopped blooding for 30 seconds naturally.For experimental group, liver wound is adding the hemostasis rapidly after 30 seconds of 2 points of hemostatic compositions.As shown in Figure 11 E, present composition cohesive is very strong, and wound is bonding, bleeds and stops.After 5 minutes, as shown in Figure 11 G and 11H, detect that with tweezers composition bond intensity can stop up wound completely, anti-Hemostatic Oral Liquid or other body fluid ooze out, and can not allow wound again hemorrhage.And tweezers can be mentioned hemostasia products together with liver, at this moment also do not bleed, show hemostasis and anti-vibration effect that hemostasia products of the present invention is extremely strong, this rehabilitation for patient is very favourable.If mTG solution is stored at 4 ℃, after taking out, to test, wound also can stop blooding adding after compositions after 3 minutes, and wound is bonding, bled and stopped.

Embodiment 13: the absorbing state of compositions of the present invention in rat liver Hemorrhage Model

This embodiment provides situation about naturally absorbing after hemostasis in product (glutamine of microbe transaminase (mTG)-gelatin composition) Synthetic Theatre of War environment lower body according to the present invention.

14 of the SD rats of 300g ± 20g are selected in experiment, are divided into matched group (Johson & Johnson: absorbable hemostatic gelfoam, trade name Surgiflo ^tM) and experiment (mTG-gelatin) group, 7 every group.

Control group A: matched group uses hemostasia products (Johson & Johnson's product, absorbable hemostatic gelfoam, trade name Surgiflo for international commodityization ^tM), and observe absorbing state.

Animal state: adult rat, in generalized anesthetic state, at whole duration of test, detects and maintain the vital sign of rat.

Experimental procedure: open rat abdominal cavity with operating scissors, expose liver, use scalpel to manufacture 1 centimeter length, 0.5 centimetre of dark sagittal otch at liver lobus sinister, after active approximately 10 seconds of bleeding, remove the blood that accumulate on surface with gauze.

Experimental solutions is applied to the otch on liver lobus sinister, uses about two minutes gel formation, in approximately 2.5 minutes, bleed and stop completely.After 5 minutes, firmly shake tissue and use tweezers cross wound site and apply pulling force, and gel is still complete, and wound site remains closed.Realize after hemostasis object, liver wound does not need to sew up, the conventional abdominal cavity of sewing up.After 5 days, 10 days, 20 days, put to death laboratory animal respectively, observe the absorbing state of liver wound hemostatic composition.

As shown in figure 12, the result of experimental group B shows to apply hemostatic composition of the present invention and absorbs not yet completely after 20 days experimental result, but with wound healing, hemostatic composition is absorbed gradually, and degree of absorption and international commodity hemostasia products group are more or less the same.

Claims

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

2. glutamine transaminage mTG claimed in claim 1, wherein this enzyme has the protein sequence of the gene code as described in SEQ ID NO:3; Or, there is the protein sequence as described in SEQ ID NO:4.

3. for the gene order of enzyme described in the claim 1-3 that encodes.

4. the quick-acting haemostatic powder product for war or scene of the accident wound, it is characterized in that, comprise the substrate of having added described glutamine of microbe transaminase mTG, the aminoacid sequence of wherein said glutamine transaminage has the protein sequence of gene code 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 hemorrhage, hemostasis device, first-aid hemostatic bag or common hemostatic article, and described substrate is the absorbable fluid of human body, semisolid, powder or plastic glue or colloid form.

6. hemostasia products as claimed in claim 5, is characterized in that, the substrate of described fluid or semi-solid form or material comprise liquid fiber albumen encapsulant or glue class, such as keratin, collagen protein, fluid gelatin.

7. hemostasia products as claimed in claim 6, is characterized in that, described liquid fiber albumen encapsulant or glue class comprise lysate, and described lysate is phosphate buffer, phosphate buffer PH scope from 7.0 to 9.0.

8. hemostasia products as claimed in claim 5, is characterized in that, described plastic glue or colloid form comprise oxidized cellulose, oxidized regenerated cellulose, chitosan, keratin, collagen protein, gelfoam.

9. the hemostasia products as described in any one of claim 4-8, it is characterized in that, described substrate is selected from protein material, comprise medical gelatin and liquid fiber albumen encapsulant or the glue in absorbable fibre albumin glue, keratin, collagen protein or various sources, or glucide, such as oxidized cellulose, oxidized regenerated cellulose, chitosan, proteoglycan, glycolic acid polymer, lactic acid polymer; Wherein, described proteoglycan is poly-n-acetyl glucosamine amine.

10. hemostasia products as claimed in claim 9, is characterized in that, described matrix optimization is gelatin.

11. hemostasia products as claimed in claim 10, is characterized in that, described substrate is selected from mammiferous A type high molecular gelatin, or the gelatin of 20%～60% proline hydroxylation.

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

13. hemostasia products as claimed in claim 5, it is characterized in that, described hemostasia products is tourniquet or hemostatic gauze, comprise tourniquet or hemostatic gauze skin take cotton yarn or soluble stanching gauze as body, with the internal layer for contacting and binding up a wound, in wherein said internal layer, contain substrate powder or the medicated core of described glutamine transaminage.

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

15. hemostasia products as claimed in claim 5, is characterized in that, described hemostasia products is the military hemostasis such as first-aid hemostatic bag, emergency case that contains above-mentioned gelatin and glutamine transaminage component.

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