CN105861476B - Thrombolytic Drugs and its application based on cobra venom PIII type metalloproteinases - Google Patents
Thrombolytic Drugs and its application based on cobra venom PIII type metalloproteinases Download PDFInfo
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Abstract
Based on cobra venom PIII type metalloproteinases, the protease is that Naja kaouthia is also known as from Chinese cobra, and acquisition systematic name: is isolated and purified in the snake venom of Naja atra；The protease is single chain glycoprotein, and structure elucidation shows there is 3 structural domains: metalloproteinase structure domain, is rich in Cysteine domains at disintegrating element for poisonous spline structure domain, belongs to PIII type snake venom metalloproteinase.Its title is respectively Atrase A and Atrase B, sequence are as follows: AtraseA:SEQ ID No.1；AtraseB:SEQ ID No.2.Target protein and its Thrombolytic Drugs can induce endogenous plasminogen activator tPA release to have thrombolytic effect, can be used for the thromboembolism treatment of thrombotic diseases.
Description
Technical field
The present invention relates to the application fields of cobra venom PIII type metalloproteinases, in particular to a kind of to be based on cobra venom
The Thrombolytic Drugs of PIII type metalloproteinases and its application.
Background technique
Cardiovascular and cerebrovascular disease is the high-incidence critical illness in modern society, and the death rate is high, seriously threatens human life
Health.Wherein, the harm of thrombotic diseases is the most serious, clinically with acute myocardial infarction AMI, cerebral arterial thrombosis, acute lung bolt
Plug is main performance, and disease incidence, disability rate, lethality are higher.In the past 30 years, thrombolytic therapy is widely used in various blood vessel bolts
The treatment of plug property disease, achieves significant effect, it has also become treat the effective means of thrombotic diseases.China is that population is big
State, with socio-economic development, cerebrovascular disease incidence of disease increases increasingly, it has also become threatens the main disease of people's life health
Disease.The especially high-end drug of thrombolytic drug of country's clinical use is foreign study exploitation at present, and the country is mostly with follow-up study
Based on imitated.In western developed countries such as America and Europes, the Thrombolytic Drugs of clinical use are fine with the second generation drug recombination sense of organization at present
Based on dissolved preferment activator rt-PA (recombinant tissue type plasminogen activator, rt-PA),
Expensive, the country is difficult to popularize, and causes main force's thrombolytic drug that the country clinically uses at present still with first generation Thrombolytic Drugs urine
Based on kinases (urokinase, UK).There is an urgent need to research and develop the novel thrombolytic medicine of autonomous innovation to break external monopolization for the country.Base
There are biggish social reality demand and vast market prospect in thrombolytic drug, and current existing thrombolytic drug is generally still
The disadvantages of in the presence of specificity not high, half-life short, hemorrhage risk, find, find and research and develop thrombolysis with independent intellectual property rights
Effect is good, Small side effects novel thrombolytic drugs meet Strategic Demand of the country in health field, with important society's warp
Meaning of helping and value.
The research and development of thrombolytic drug are always the hot spot of drug research in the world.Thrombolytic drug is generally divided into three at present
Generation.The first generation is with streptokinase (streptokinase, SK) and UK for representative.The major defect of this kind of medicines is to fiber egg
It is white lower without selectivity or selectivity, systemic activation of fibrinolysis and hemorrhage risk can be caused.UK is still the domestic main force at present
Thrombolytic Drugs.The second generation is with sense of organization plasminogen activator (tissue type plasminogen activator, t-PA)
It represents.It can get the rt-PA of recombination using technique for gene engineering.The medicine is preferable to fibrinous selectivity, but is clinically
Obtain preferable thrombolytic effect and large dosage causes fibrinolytic too strong, causes in use, can lose to fibrinous selectivity
Blood.The medicine half-life short, only 3-4 minutes.In developed countries such as America and Europes, rt-PA has become mainstream Thrombolytic Drugs.Its price is high
Expensive, the country is not possible to popularize at present.Currently, developing third generation Thrombolytic Drugs both at home and abroad, including it is acylated plasminogen-chain and swashs
It enzyme activation compound (APSAC), recombinant single chain urokinase (rscu-PA), stqphylokinase (Sak) and sucks blood bat from South America
The plasminogen activator (Bat) extracted in bat, part have entered clinical test.
Current clinically common Thrombolytic Drugs are plasminogen activator, and have been listed and whole in the thrombolytic drug ground
For the pharmaceutical grade protein for belonging to serine protease.
Snake venom is natural most complicated toxalbumin mixture.Containing structure function multiplicity with cardiovascular system in snake venom
For the activated protein of target, it has also become the valuable source library of modern new drug and reagent for clinical diagnosis research and development.Foreign countries are in 20 generation
Discipline has researched and developed the protease preparation for acting on hematological system since the sixties successively, such as from the red mouth cobra-venom point of Malaysia
From protease ancrod (Ancrod), separate from Brazilian spearhead cobra-venom preparation Batroxobin (batroxobin) and
Japanese Tung Ling ketai enzyme (batroxobin).The research work of this respect has been actively developed since the end of the seventies in last century in the country
Make.Relevant fibrinolysin is successively isolated and purified out from cobra-venom and Ahylysantinfarctase.Currently, the country has approved 2 kinds of snake venom systems
Agent enters clinical use: first is that Defibrase, is to produce to divide in agkistrodon halys ussuriensis snake venom and Jiangsu and Zhejiang Provinces production cobra-venom from China, Changbai Mountain, northeast
The thrombin-like enzyme obtained from purifying；Another kind is Effect of Agkistrodon acutus Enzyme, is to isolate and purify acquisition from Ahylysantinfarctase (long-noded pit viper)
Thrombin-like enzyme.This kind of enzyme preparation is that they can, improvement former with fibrin degradation for the mechanism of thrombotic diseases prevention and treatment
The effect of hemorheology and fibrinolysis activity.In addition, the product of this kind of Defibrase degradation in vivo fibrinogen can excite
Vascular endothelial cell discharges t-PA, plasminogen is changed into fibrinolysin, to promote fibrinolysis activity.It is above-mentioned both at home and abroad to develop
Defibrase belongs to Snake Venom Serine Proteases.
The present invention relates to based on cobra venom PIII type metalloproteinases Thrombolytic Drugs and its application.Snake venom metalloproteinase
(snake venom metalloproteinase, SVMP) belongs to metalloproteinases M12 family Reprolysin member, mainly
It is present in Viperidae and Crotalidae snake venom, the distinguishing feature shared in structure is to contain one section in the activated centre of enzyme
The conserved sequence of " HEXGHXXGXXHD ", zinc ion is in conjunction with the conserved region.The significant toxic characteristic of SVMP is that have blood poison
Property, there is hydrolyzed cellular basilar memebrane and extracellular matrix, anticoagulant, rush to coagulate, platelet aggregation-against isoreactivity, in blood circulation poison class snake more
Key player is played in the toxic effect of poison.It can be by snake venom metalloprotease according to molecular size range and contained structural domain number
Enzyme is divided into 4 classes: PI type snake venom metalloproteinase only contains a metalloproteinase structure domain；PII type contains a metalloprotein
Enzyme domains and a disintegrating element for poisonous or class disintegrin domain；One is rich in cysteine structure more than PIII type ratio PII type
Domain, and PIV type is in C-terminal that there is also the 4th structural domain-c-type lectin-like domains.
So far, isolate and purify out few SVMP in cobra venom never of the same race, as atrahagin,
Mocarhagin, kaouthiagin, NN-PF3 etc..Generally, since cobra venom does not show apparent proteolytic enzyme
Activity, therefore, in cobra venom SVMP carry out research it is few.These fibrinolysins obtained from cobra venom or
SVMP majority can hydrolysis of fibrin original α chain, and all show the activity research of platelet aggregation-against, antiplatelet is poly-
For the main mechanism integrated as digestion platelet membrane glycoprotein GPIb or vWF, it is poly- that these protein molecules are likely to become antiplatelet
The potential drug molecule of collection or fibrinogen reducing, and all do not studied and reported in terms of for thrombolysis.From Viperidae and
The SVMP prepared is separated in Crotalidae snake venom due to its intrinsic bleeding toxicity, it is made to be not used to the system of related drugs
It is standby.
Cobra venom PIII type metalloproteinases Atrase A and Atrase B in this case is at present from Zhoushan kind glasses
Only 2 PIII by structure elucidation identification of acquisition are isolated and purified in snake (Naja atra, Chinese cobra) snake venom
Type metalloproteinases, to the measurement of its sequential structure and analysis shows, Atrase A and Atrase B by
Metalloproteinase domain, Disintegrin-like domain and Cysteine-rich domain tri- knots
Structure domain composition.Atrase A and Atrase B have significant molten in addition to significant platelet aggregation-against and anticoagulating active
Bolt characteristic is not available for other cobra venom metalloproteinases or nevin fibrinolytic enzyme.It can be effectively molten in low dosage
Bolt, effect are better than positive control drug UK and rt-PA.Its mechanism is by acting on complement releasing induction of endogenous t-PA
It puts, thus plasminogen activation, efficient thrombolysis.And in its Cysteine-rich domain and Disintegrin-like
Contain the RGD that can be identified with thrombi activated blood platelet GPIIb/IIIa receptor-specific and class RGD sequence in domain respectively,
Structure basis with targeting thrombolysis.Target protein inhibits platelet aggregation with the novel thrombolysis activity of mechanism and effectively and shows
Write the structure of anticoagulant multi-efficiency and targeting thrombolysis, direction and trend phase of these features with thrombolytic drug research and development of new generation
It coincide, so that it is had unique structure and effect advantage as thrombolytic drug, Thrombolytic Drugs are prepared with target protein, have other
Particular advantages not available for snake venom metalloproteinase or serine protease.
Summary of the invention
To solve the above-mentioned problems of the prior art, the purpose of the present invention is to provide based on cobra venom PIII type gold
The Thrombolytic Drugs of Proteases and its application.
In order to achieve the above objectives, the technical solution of the present invention is as follows:
Based on cobra venom PIII type metalloproteinases, the protease is that Zhoushan kind glasses are also known as from Chinese cobra
Systematic name: snake isolates and purifies acquisition in the snake venom of Naja atra；The protease is single chain glycoprotein, and structure elucidation shows have
Have 3 structural domains: metalloproteinase structure domain, is rich in Cysteine domains at disintegrating element for poisonous spline structure domain, belongs to PIII type snake
Noxious metals protease.
Further, it is described be based on cobra venom PIII type metalloproteinases, title be respectively Atrase A and
Atrase B, wherein Atrase A deduced molecular weight is 46717.14, isoelectric point 7.281；Atrase B deduced molecular weight is
44749.39, isoelectric point 7.238；The nucleotide similarity of two sequences is 85.9%, and amino acid similarity is 65.4%, sequence
It is classified as: AtraseA:SEQIDNo.1；AtraseB:SEQIDNo.2；
Further, the activity based on cobra venom PIII type metalloproteinases with hydrolysis of fibrin original α chain,
The activity can be completely inhibited by metal-chelator EDTA, EGTA, 1,10-phenanthroline and reducing agent DTT；Target protein
The accurate molecular quality of Atrase A and Atrase B are respectively 49.3kDa and 49.4kDa, but its apparent molecular weight is according to different
Measuring method it is different, range is between 45kDa-65kDa: Atrase A is in reproducibility and irreducibility SDS-PAGE
Respectively 64.6 and 57.1kDa, gel filtration method be measured as 56.5kDa；Atrase B is in reproducibility and irreducibility SDS-
It is respectively 62.2 and 55.8kDa in PAGE, gel filtration method is measured as 54.3kDa.Etc. point focusing measurement result show become
The isoelectric point of target protein Atrase A and Atrase B are respectively 9.6 and 9.7 under the conditions of property, and target protein does not hydrolyze junket egg
White activity, also without Activity of arginine estrase and fibrinolytic activity；Target protein contains glycosyl, and neutral sugar content is
5%.
Based on the Thrombolytic Drugs of cobra venom PIII type metalloproteinases, cobra venom PIII type metal egg is based on by above-mentioned
White enzyme adds freeze drying powder injection made of other auxiliary agents.
Further, the Thrombolytic Drugs are grouped as by following groups: cobra venom PIII type metalloproteinases, sodium phosphate, essence
Propylhomoserin, Tween 80.
Further, the Thrombolytic Drugs freeze drying powder injection is grouped as after redissolving by following groups: the 0.01M phosphoric acid that pH is 7.4
Containing based on cobra venom PIII type metalloproteinases purification of samples 0.5mg, mass fraction 0.15% in sodium buffer 0.5ml
Arginine, mass fraction be 0.01% Tween 80.
Further, the cobra venom PIII type metalloproteinases is preparing the application in Thrombolytic Drugs.
Compared with the existing technology, the invention has the benefit that
Target protein of the present invention and its Thrombolytic Drugs have apparent thrombolytic effect: passing through inducing endogenous Plasminogen activation
Agent tPA release is to generate apparent thrombolytic effect.Target protein by inducing endogenous plasminogen activator tPA discharge from
And the mechanism of thrombolytic effect is generated, and targeting thrombus is had based on its structure feature.Can be used for thrombotic disease or
The treatment that thrombus generates under related medical conditions, without bleeding risk, thrombolytic effect is significant.
Detailed description of the invention
The primary structure of Fig. 1 target protein (AtraseA and AtraseB)；
The SDS-PAGE electrophoresis of Fig. 2 target protein；
Wherein: swimming lane 1 and 4 is respectively the Atrase B under non-reduced and reducing condition, and swimming lane 2 and 5 is respectively non-reduced
With the Atrase A under reducing condition, swimming lane 3 is standard molecular weight albumen；
Target protein isoelectric focusing under Fig. 3 Denaturing；Wherein: swimming lane 1 is Atrase A, and swimming lane 2 is Atrase B；
It is prepared by the purifying of Fig. 4 target protein Atrase A；Wherein: A.SP Sephadex C-25 chromatography；B.Sephacryl
S-200 chromatography；C.HiTrap Heparin HP chromatography；
It is prepared by the purifying of Fig. 5 target protein Atrase B；Wherein, A.SP Sephadex C-25 is chromatographed；B.HiTrap
Heparin HP chromatography；C.HiTrap Q HP chromatography；D.HiTrap SP HP chromatography；
The influence diagram of Fig. 6 Thrombolytic Drugs Human Umbilical Vein Endothelial Cells fibrinolytic function molecule release；Wherein, A:*p<0.05,**P < 0.01, with
Blank is compared；#p<0.05,##P < 0.01, compared with 1h group；B:*P < 0.05,**P < 0.01, compared with Blank；#P < 0.05,##p
< 0.01, compared with 1h group；*P < 0.05,**P < 0.01, compared with Blank；#P < 0.05,##P < 0.01, compared with 1h group；
The influence of Fig. 7 Thrombolytic Drugs Human Umbilical Vein Endothelial Cells fibrinolytic；Wherein: A:*P < 0.05,**P < 0.01, with (Atrase A+
INHS) group is compared；#P < 0.05,##P < 0.01, compared with Blank；B:*P < 0.05,**P < 0.01, with (Atrase A+INHS) group
It compares；C:*P < 0.05,**P < 0.01, compared with Blank；
Fig. 8 low dosage Thrombolytic Drugs to the influence diagram of fibrinolytic in rat body, wherein*P < 0.05,**P < 0.01, with
Control is compared；
Fig. 9 high dose Thrombolytic Drugs to the influence diagram of fibrinolytic in rat body, wherein*P < 0.05,**P < 0.01, with
Control is compared；
Thrombolytic effect figure of Figure 10 Thrombolytic Drugs to rat carotid artery thrombus, wherein P < 0.001, model group and sham-operation
Group is compared；P < 0.001, low dose group is compared with model group；P < 0.05, high dose group is compared with low dose group；P < 0.01, low dose
Amount group is compared with Alteplase group；P < 0.001, high dose group is compared with Alteplase group；P < 0.001, Alteplase group and urine
Kinases group is compared.
Specific embodiment
Technical solution of the present invention is described in further detail with reference to the accompanying drawings and detailed description:
Embodiment 1:
Target protein is isolated and purified from Chinese cobra (Naja kaouthia) (systematic name: Naja atra) snake venom
It obtains.Target protein is single chain glycoprotein, and structure elucidation shows there is 3 structural domains: metalloproteinase structure domain, disintegrating element for poisonous
Spline structure domain is rich in Cysteine domains, belongs to PIII type snake venom metalloproteinase (Fig. 1).
The target protein correlation physical and chemical index that table 1 is derived according to primary structure
As shown in table 1: target protein has the activity of hydrolysis of fibrin original α chain, which can be by metal-chelator
EDTA, EGTA, 1,10-phenanthroline and reducing agent DTT complete inhibition.Target protein Atrase A and Atrase B's
Actual molecular weight respectively may be about 49.3kDa and 49.4kDa, sequence are as follows: AtraseA:SEQIDNo.1；AtraseB:
SEQIDNo.2；But according to different measuring methods, its apparent molecular weight is different, and range is between 45kDa-65kDa
(Atrase A is respectively 64.6 and 57.1kDa (Fig. 2) in reproducibility and irreducibility SDS-PAGE, and gel filtration method is surveyed
It is set to 56.5kDa；Atrase B is respectively 62.2 and 55.8kDa (Fig. 2), gel in reproducibility and irreducibility SDS-PAGE
Filter method is measured as 54.3kDa).Etc. point focusing measurement result show under Denaturing target protein Atrase A and
The isoelectric point of Atrase B is respectively 9.6 and 9.7 (Fig. 3).Target protein does not have caseinhydrolysate active, without arginine ester yet
Enzymatic activity and fibrinolytic activity.Target protein contains glycosyl, and neutral sugar content is about 5%.
Target protein Atrase A's isolates and purifies
The thick poison of cobra (Naja atra) is dissolved in the sodium acetate buffer of pH 5.8,20mmol/L, by this solution loading
In SP Sephadex C-25column (2.6 × 40cm) chromatographic column balanced with same buffer, with same buffering
Liquid is eluted with the flow velocity of 30ml/h, after the unadsorbed albumen on column is eluted completely, carries out the NaCl of same buffer
Linear gradient elution (0-1mol/L) (Fig. 4 A).
The above-mentioned part in peak with fibrinogen hydrolysing activity is collected, using Sephacryl S-
200column (2.6 × 110cm) carries out molecular sieve gel chromatography, with the 10mmol/L phosphorus of pH 7.4, the NaCl containing 0.1mol/L
Phthalate buffer carries out the balance and elution (Fig. 4 B) of chromatographic column under the flow velocity of 20ml/h.
The part in sieve chromatography with fibrinogen hydrolysing activity is collected, is merged, buffer is changed to by ultrafiltration
Then the phosphate buffer of pH 7.0,10mmol/L are loaded to the HiTrap Heparin balanced with same buffer
HP column (5ml) heparin affinity chromatography column carries out same buffer after eluting the unadsorbed albumen on column completely
NaCl linear gradient elution (0-0.5mol/L) collects the last one eluting peak, as target protein Atrase A (Fig. 4 C).System
Standby target protein is a band in reduction and non-reduced SDS-PAGE and isoelectric focusing.
The part in above-mentioned heparin affinity chromatography with fibrinogen hydrolysing activity is collected, is merged, ultrafiltration, by buffer
It is changed to the phosphate buffered saline of pH 7.4,10mmol/L, measures protein content, cryopreservation is spare.
Target protein Atrase B's isolates and purifies
The thick poison of cobra (Naja atra) is dissolved in the sodium acetate buffer of pH 5.8,20mmol/L, by this solution loading
In SP Sephadex C-25column (2.6 × 40cm) chromatographic column balanced with same buffer, with same buffering
Liquid is eluted with the flow velocity of 30ml/h, after the unadsorbed albumen on column is eluted completely, carries out the NaCl of same buffer
Linear gradient elution (0-1mol/L) (Fig. 5 A).
The component in SP Sephadex C-25 chromatographic grade elution fraction with fibrinogen hydrolysing activity is collected, is closed
And ultrafiltration, buffer is changed to the phosphate buffer of pH 7.0,10mmol/L, is then loaded to same buffer
HiTrap Heparin HP column (5ml) the heparin affinity chromatography column balanced, the unadsorbed albumen on column is complete
After elution, the NaCl linear gradient elution (0-0.6mol/L) of same buffer is carried out, collects the last one eluting peak (Fig. 5 B).
The buffer for the eluting peak amalgamation liquid collected in above-mentioned heparin affinity chromatography is changed to the 25mM of pH8.9
Then Tris-HCl buffer is loaded to the HiTrap Q HP chromatographic column (1ml) balanced with same buffer, will not inhale
It is attached to after the albumen on column elutes completely, carries out the NaCl linear gradient elution (0-0.5mol/L) of same buffer, collect ladder
Spend the first big peak (Fig. 5 C) of elution.
The buffer for the eluting peak amalgamation liquid collected in above-mentioned HiTrap Q HP chromatography is changed to the 10mM phosphorus of pH 7.0
Then phthalate buffer is loaded to the HiTrap SP HP chromatographic column (1ml) balanced with same buffer, by it is unadsorbed
After albumen on column elutes completely, carries out the NaCl gradient elution (0-0.5mol/L) of same buffer: successively carrying out 0-
The horizontal elution of the gradient elution, 0.2mol/L of 0.2mol/L and the horizontal elution of 0.3mol/L, collect the last one eluting peak,
As target protein Atrase B (Fig. 5 D).The target protein of preparation is in reduction and non-reduced SDS-PAGE and isoelectric focusing
It is a band.
Target protein amalgamation liquid is concentrated by ultrafiltration, and after buffer is changed to the PBS of pH 7.4, measurement albumen contains
Amount, cryopreservation are spare.
Preparation
The sample of purifying is prepared into Thrombolytic Drugs freeze drying powder injection by the manufacturing requirements of powder ampoule agent for injection.It is described molten
Bolt medicine specification is 0.5mg/ branch, is grouped as by following groups: the Thrombolytic Drugs freeze drying powder injection is grouped as after redissolving by following groups: pH
For in 7.4 0.01M sodium phosphate buffer 0.5ml containing based on cobra venom PIII type metalloproteinases purification of samples 0.5mg,
The Tween 80 that arginine that mass fraction is 0.15%, mass fraction are 0.01%.
Target protein of the present invention and its Thrombolytic Drugs have apparent thrombolytic effect: passing through inducing endogenous Plasminogen activation
Agent tPA release is to generate apparent thrombolytic effect.Target protein by inducing endogenous plasminogen activator tPA discharge from
And the mechanism of thrombolytic effect is generated, and targeting thrombus is had based on its structure feature.Can be used for thrombotic disease or
The treatment that thrombus generates under related medical conditions, without bleeding risk, thrombolytic effect is significant.
Embodiment 2:
1. the effect of cobra venom PIII type metalloproteinases inducing endothelial cell fibrinolytic function up-regulation (is with Atrase A
Example)
1.1 materials and reagent
Human microvascular endothelial cell (mvec) (human microvascular endothelial cells, HMEC)；RPMI-
1640 culture mediums are purchased from Gibco；Fetal calf serum (fetal bovine serum, FBS) is that the ocean Tianjin Hao biological products science and technology has
Limit Products；t-PA(tissue plasminogen activator),PAI-1(plasminogen activator
Inhibitor) content detection ELISA kit and Activity Assay Kit are purchased from U.S. Assaypro company；Normal human serum
(normal human serum, NHS) being donated blood by several healthy volunteers is prepared, and is frozen after packing in -80 DEG C, through complement
Activity determination is normal, spare；Human serum (inactivated normal human serum, INHS) is inactivated by NHS in 56 DEG C
It is incubated for 30min and obtains；Atrase A's isolates and purifies preparation ditto；Remaining reagent be meet requirement of experiment analysis it is pure.
1.2 method
NHS, Atrase A and serum-free 1640 are mixed with the ratio of 3:2:10,37 DEG C of incubation 1.5h, preparation is incubated for mixed
Object is closed, it is spare.By HMEC with 1 × 104A/hole is inoculated in 96 porocyte culture plates, abandons supernatant afterwards for 24 hours, is washed with the 1640 of no FBS
One time, 150ul mixtures incubated is added, adds 50ul 1640 culture medium containing serum.It tests while being provided with INHS+Atrase
A is incubated for control group, NHS control group, INHS control group, Atrase A control group and blank control group.Each group after sample-adding 1h,
6h, 12h take cell conditioned medium, and 3000r/min is centrifuged 10min, takes supernatant, -80 DEG C freeze.T-PA, PAI-1 in cells and supernatant
Content and activity by kit specification operate.
1.3 statistical analysis
Experimental data withIt indicates.One-way analysis of variance is carried out using 16.0 software of SPSS, Multiple range test is adopted between group
With LSD method.
1.4 result
The incubation object of Atrase A and NHS can obviously raise HMEC release t-PA and PAI-1, measure the t- of fibrinolytic
PA/PAI-1 obviously raises (Fig. 6).The result of Chromogenic assay measurement also indicates that the incubation object of Atrase A and NHS can make fibrinolytic
Active obvious up-regulation (Fig. 7).The experimental results showed that the up-regulation of HMEC fibrinolytic function is related with effect of the target protein to complement.
Embodiment 2:
1. influence of the cobra venom PIII type metalloproteinases to fibrinolytic function in rat body (by taking Atrase A as an example)
1.1 experimental material
Preparing for Thrombolytic Drugs Atrase A is the same；T-PA, PAI-1, PAP detection kit；Other reagents are to meet reality
Test the reagent or drug of requirement.
SPF grades of male SD rats are purchased from Chongqing Teng Xin experimental animal Co., Ltd, 250-280g of weight, quality certification number
SCXK (Chongqing) 2007-0005.Zoopery and animal welfare meet relevant animal experiment management regulations.Experimental animal feeding is in clear
Clean, constant temperature, constant humidity environment in, ad lib and water inlet before testing.
1.2 experimental group
Animal is grouped at random.Low dose group is divided into control group, 2h, 6h, 12h and group for 24 hours, middle dose group be divided into control group,
2h, 6h, 12h, for 24 hours, 48h, 72h, 96h group, high dose group be divided into control group, 2h, 6h, 12h, for 24 hours, 48h, 120h, 196h group.
Every group in low dose group and middle dose group is 10 rats, and every group is 6 rats in high dose group.
1.3 blood specimen collection
Sample is passed through into tail vein injections to rat according to the dosage of design.According to different groups of other collect specimen times
It is required that opening abdominal cavity in advance after 1% yellow Jackets anesthetized rat of 20min, blood sample is acquired by abdominal aorta, is used
0.109mol/L sodium citrate is carried out anticoagulant in 1:9 ratio.
The measurement of 1.4 fibrinolytic functions
The anticoagulation of acquirement is centrifuged 15min with 3000r/min, takes supernatant, obtains blood plasma, packing freezes in -80 DEG C, standby
With.T-PA, PAI-1, PAP of each plasma specimen are measured according to t-PA, PAI-1, PAP detection kit specification.
1.5 statistical analysis
Experimental data withIt indicates.One-way analysis of variance is carried out using 16.0 software of SPSS, Multiple range test is adopted between group
With LSD method.
1.6 result
Low dosage Thrombolytic Drugs (0.3mg/kg) can obviously raise the release of t-PA in rat body, and (figure is not influenced on PAI-1
8)；High dose Thrombolytic Drugs (3.0mg/kg) can obviously raise t-PA and PAI-1 (Fig. 9) in rat body.The up-regulation of PAP is shown greatly
The activation and up-regulation of fibrinolytic in mouse body.
Embodiment 3:
1. the drug action of thrombolysis in cobra venom PIII type metalloproteinases body (by taking Atrase A as an example)
1.1 experimental material
Preparing for Thrombolytic Drugs Atrase A is the same, and it is the single band of electrophoresis, the sample point of purifying that SDS-PAGE, which detects purity,
Dress freezes in -80 DEG C；Aspirin is purchased from Sigma Co., USA；Urokinase (urokinase, UK) is the beautiful pearl system of Li Zhu group
Pharmaceutical factory product；Recombinant Human Tissue Typceplasminogen Activator's Alteplase (rt-PA) is Germany Boehringer Ingelheim
Products；Other reagents are the reagent or drug for meeting requirement of experiment.
SPF grades of male SD rats are purchased from Chongqing Teng Xin experimental animal Co., Ltd, weight 210-240g, quality certification number SCXK
(Chongqing) 2007-0005.Zoopery and animal welfare meet relevant animal experiment management regulations.Experimental animal feeding in cleaning,
Constant temperature, constant humidity environment in, ad lib and water inlet before testing.
1.2 experimental groups and method
Rat is randomly divided into sham-operation group (sham), model group (mode1), urokinase group and rt-PA group and Atrase
A high and low dose group, each group 8.It is commented using rat carotid artery thrombus model using urokinase and rt-PA as positive control drug
The thrombolytic effect of valence target protein.Urokinase group (100,000 U/kg) and rt-PA group (10mg/kg) and Atrase A high low dosage
Group (3.0mg/kg, 0.3mg/kg) 30min after modeling, passes through the disposable drug administration by injection of tail vein.1.5h after administration, passes through the right side
The blood sampling of side arteria carotis communis, sodium citrate is anticoagulant, and anticoagulation is centrifuged 15min with 3 000r/min, anticoagulant blood plasma is prepared, for measuring
The indexs such as t-PA, PAP.It takes blood to finish, cuts left common carotid thrombi blood vessel rapidly, be put on filter paper and blot residual blood,
Its weight is measured with electronic analytical balance.
1.3 statistical analysis
Experimental data withIt indicates.One-way analysis of variance, Multiple range test between group are carried out using 16.0 software of SPSS
Using LSD method.
1.4 result
Low dosage Thrombolytic Drugs can effective thrombolysis, high dose can further enhance the effect of thrombolysis, and effect is superior to the positive
Comparison medicine UK and rt-PA (Figure 10).
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
The change or replacement expected without creative work, should be covered by the protection scope of the present invention.Therefore, of the invention
Protection scope should be determined by the scope of protection defined in the claims.
Claims (1)
1. the Thrombolytic Drugs based on cobra venom PIII type metalloproteinases, which is characterized in that the protease is from Chinese glasses
Snake is also known as Naja kaouthia, systematic name: acquisition is isolated and purified in the snake venom of Naja atra；The protease is single-stranded sugared egg
White, structure elucidation shows there is 3 structural domains: metalloproteinase structure domain, is rich in cysteine knot at disintegrating element for poisonous spline structure domain
Structure domain belongs to PIII type snake venom metalloproteinase；
Described to be based on cobra venom PIII type metalloproteinases, title is respectively Atrase A and Atrase B, wherein
Atrase A deduced molecular weight is 46717.14, isoelectric point 7.281；Atrase B deduced molecular weight is 44749.39, waits electricity
Point is 7.238；The nucleotide similarity of two sequences is 85.9%, and amino acid similarity is 65.4%, sequence are as follows: AtraseA:
SEQ ID No.1；AtraseB:SEQ ID No.2；
The activity based on cobra venom PIII type metalloproteinases with hydrolysis of fibrin original α chain, which can be golden
Belong to chelating agent EDTA, EGTA, 1,10-phenanthroline and reducing agent DTT complete inhibition；Target protein Atrase A and
The accurate molecular quality of Atrase B is respectively 49.3kDa and 49.4kDa, but according to its apparent molecular weight of different measuring methods
Different, range is between 45kDa-65kDa: Atrase A is respectively 64.6 in reproducibility and irreducibility SDS-PAGE
And 57.1kDa, gel filtration method are measured as 56.5kDa；Atrase B distinguishes in reproducibility and irreducibility SDS-PAGE
For 62.2 and 55.8kDa, gel filtration method is measured as 54.3kDa, etc. the measurement results of point focusing show mesh under Denaturing
The isoelectric point of mark albumin A trase A and Atrase B is respectively 9.6 and 9.7, and target protein does not have caseinhydrolysate activity,
There is no Activity of arginine estrase and fibrinolytic activity；Target protein contains glycosyl, neutral sugar content 5%；
The Thrombolytic Drugs based on cobra venom PIII type metalloproteinases its by it is above-mentioned be based on cobra venom PIII type metal egg
White enzyme adds freeze drying powder injection made of other auxiliary agents；
The Thrombolytic Drugs are grouped as by following groups: being based on cobra venom PIII type metalloproteinases, sodium phosphate, arginine, tween
80；
Thrombolytic Drugs freeze drying powder injection is grouped as after redissolving by following groups: being contained in the 0.01M sodium phosphate buffer 0.5ml that pH is 7.4
Have based on cobra venom PIII type metalloproteinases purification of samples 0.5mg, the arginine that mass fraction is 0.15%, mass fraction
For 0.01% Tween 80.
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