CN100465270C - Nereides protease, separating and purifying method and application thereof - Google Patents

Nereides protease, separating and purifying method and application thereof Download PDF

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CN100465270C
CN100465270C CNB021448280A CN02144828A CN100465270C CN 100465270 C CN100465270 C CN 100465270C CN B021448280 A CNB021448280 A CN B021448280A CN 02144828 A CN02144828 A CN 02144828A CN 100465270 C CN100465270 C CN 100465270C
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proteolytic enzyme
balance
concentrated
damping fluid
proteinase
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CN1500873A (en
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洪敏�
程熠
白若伦
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Abstract

The present invention provides one kind of N-V proteinase and its separation and purification process. The N-V proteinase has log-in number in Swiss prot of P83433, molecular weight of 27000-35000, isoelectric point of 5-7.5, optimal temperature of 45 deg.c and optimal pH value of 7.8. Inside its molecule, the 10 peptide section amino acid residue sequence features (1) AVYLAGMK (2) NFPNYYINLY (3) VYLAANPTASS (4) QTFNSDTL and (5) VYILDTGI. The N-V proteinase is one kind of proteinase capable of hydrolyzing fibrinogen and fibrillarin powerfully both inside and outside body, and may be used as thrombolytic medicine and blood coagulation resisting medicine for preventing and treating myocardial infarction, cerebral artery thrombosis and other diseases.

Description

N-V proteinase and separation and Extraction purification process and application
Technical field:
The present invention relates to a kind of thrombus dissolving plasmin, a kind of N-V proteinase and separation and Extraction purification process and application particularly are provided, relate to the technological field of biochemistry of the separation and Extraction purification process of plasmin.
Background technology:
Thromboembolic states be clinical modal also be the most serious a kind of disturbance of blood circulation disease.Treating above-mentioned disease, particularly use thrombolytics at the initial stage of a disease, reduce scleroproein and fibrinogen content, minimizing platelet aggregation, thrombus, recovery revascularization, is most important methods of treatment, can reduce mortality ratio, reduce sequela.At present, many clinically both at home and abroad with thrombolysis medicines such as urokinase and gram bolt enzyme, snake venom fiber eliminating enzymes.These drug prices are all very expensive, and different relative merits are also arranged, and as poor selectivity, half life is long, and antigenicity is strong, and toxicity is big, easily cause the secondary hemorrhage disease.Therefore, find that development novel thrombolytic class medicine is still the very significant scientific research project of pharmaceutical industry, has very big social benefit and economic benefit.
Summary of the invention:
The invention provides a kind of N-V proteinase, the separation and Extraction purification process and the application of this enzyme are provided simultaneously, overcome conventional thrombolytics drug price costliness, poor selectivity, shortcoming such as half life is short, antigenicity is strong, and can realize suitability for industrialized production.
N-V proteinase, English name: (hereinafter subtract title: N-V proteolytic enzyme): the login of this enzyme in Swiss prot is called N-V proteinase to N-V proteinase, and accession number is P83433.Its molecular weight is between 27000-35000, and iso-electric point is between 5-7.5, and optimum temperuture is 45 ℃, and optimal pH is 8-9.
The sequence of the amino-acid residue of 5 peptide sections of this enzyme intramolecule is characterized in that
(1)AVYLAGMK
(2)NFPNYYINLY
(3)VYLAANPTASS
(4)QTFNSDTL
(5)VYILDTGI
N-V proteinase is a kind of proteolytic ferment, and the former and scleroproein of external in vivo hydrolysis of fibrin consumingly can be as thrombolytic drug and anticoagulation medicine, the application of diseases such as prevention and treatment myocardial infarction, cerebral artery thrombosis formation.
N-V proteinase be direct hydrolysis in scleroproein, the proteolytic ferment that do not have kinases character, be from Annelita Phylum Annelida caterpillar steel Class Chaetopoda, crinosity order polychaeta, Nereidae Nereidae, separation and purification obtains.
The invention provides the separating and purifying method of five kinds of N-V proteinases:
(1) purifying process 1, may further comprise the steps:
1) get clam worm, clean the back and rub, add the pH4.0-9.0 of 1-10 times of volume, damping fluids such as 0.01-0.4mol/L phosphoric acid salt, acetate, Citrate trianion, Tris are made homogenate; 0-10 ℃, 3000-9000 rev/min centrifugal 15-60 minute, getting supernatant liquor is N-V proteolytic enzyme crude extract;
2) get N-V proteolytic enzyme crude extract, put and be cooled to 0-10 ℃ in the ice bath, under agitation condition, add the dehydrated alcohol of-20～0 ℃ of precooling, make concentration of ethanol reach 5～15%, remain on and make its generation precipitation in the ice bath;-10～10 ℃, 3000～9000 rev/mins centrifugal 15～60 minutes, abandon precipitation; Supernatant liquor adds the dehydrated alcohol of precooling while stirring, and final concentration is 25-45%, and it is fully precipitated;-10～0 ℃, 3000～9000 rev/mins centrifugal 15～60 minutes, abandon supernatant liquor, the precipitation drain as far as possible.Store method, precipitation is dissolved in an amount of pH4.0～9.0,0.01-0.4mol/L in the damping fluids such as phosphoric acid salt, acetate, Citrate trianion, Tris, packing ,-20 ℃ of preservations, maybe will precipitate lyophilize,-20 ℃ of preservations, the liquid storage of going bail for are with damping fluids such as pH4.0～9.0,0.01～0.4mol/L phosphoric acid salt, acetate, Citrate trianion, Tris dilution 10-50 doubly, ultrafiltration between cutoff value 100000～10000 obtains ultrafiltrated;
3) respectively with containing damping fluid abundant balance N-V proteolytic enzyme ultrafiltrated and DEAESepharose Fast Flow chromatography medias such as 5-10% alcoholic acid pH6.5-8.0,0.02～0.1mol/L phosphoric acid salt, acetate, Citrate trianion, Tris.The balance liquid upper prop, and employing linear gradient elution method wash-out (NaCl, neutral salt concentration such as KCl are collected active peak and freeze-drying from 0～0.5mol/L);
4) respectively with containing freeze drying activity sample and the CM Sepharose FastFlow chromatography media that the abundant balances of damping fluid such as 10-15% alcoholic acid pH4.0-6.5,0.02-0.1mol/L phosphoric acid salt, acetate, Citrate trianion, Tris obtain through previous step DEAE SepharoseFast Flow chromatography column purifying.Sample on the balance liquid adopts linear gradient elution method wash-out (NaCl, neutral salt concentration concentration such as KCl are from 0-0.5mol/L), collects active peak and freeze-drying, and identified activity and purity are carried out protein quantification, and obtaining N-V proteolytic enzyme productive rate is 0.2-0.7 ‰.
(2) purifying process 2, may further comprise the steps:
1) ultra-filtration and separation and concentrated: get clam worm, clean the back and rub, add the pH4.0-9.0 of 1-10 times of volume, damping fluids such as 0.01-0.4mol/L phosphoric acid salt, acetate, Citrate trianion, Tris are made homogenate; 0-10 ℃, 3000-9000 rev/min centrifugal 15-60 minute, getting supernatant liquor is N-V proteolytic enzyme crude extract; N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution, measures A respectively 260And A 280Value, PAGE identifies purity;
2) CM-52 cellulose chromatography: use pH3-4.6 0.01-0.05mol/L acetate, the abundant balance concentrated solution of citrate buffer and CM-52 Mierocrystalline cellulose chromatography medium respectively; Balance liquid concentrates upper prop, adopts isocratic elution method wash-out; Use the 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, measure A respectively 260And A 280Value, PAGE identifies purity;
3) QAE Sephadex A50 column chromatography: with pH3-4.6 0.01-0.05mol/L acetate, the abundant balance QAE Sephadex of citrate buffer A50 chromatography media; With the concentrated solution abundant dialysis equilibrium of sort buffer liquid, the balance liquid upper prop adopts isocratic elution method wash-out, collects active peak, measures A respectively 260And A 280Value is used the 300000MW membrane ultrafiltration, and filtrate is held back concentrated by the 10000MW film, and PAGE identifies purity;
4) Phenyl-Sepharose CL-4B column chromatography: with abundant balances of damping fluid such as active peak concentrated solution pH5.0-8.0 0.01-0.025mol/L phosphoric acid salt, Tris; With the abundant balance Phenyl-Sepharose CL-4B of damping fluid chromatography medias such as pH5.0-8.0 0.01-0.025mol/L phosphoric acid salt, Tris; Balance liquid concentrates goes up sample, and (NaCl concentration is from 1.5～0.05mol/L) to adopt linear gradient elution method wash-out; Collect active peak, measure A respectively 260And A 280Value is used the 300000MW membrane ultrafiltration, and filtrate is held back concentrated by the 10000MW film, and PAGE identifies purity, and obtaining N-V proteolytic enzyme productive rate is 0.2-0.7 ‰.
(3) purifying process 3, may further comprise the steps:
1) ultra-filtration and separation and concentrated: get clam worm, clean the back and rub, add the pH4.0-9.0 of 1-10 times of volume, damping fluids such as 0.01-0.4mol/L phosphoric acid salt, acetate, Citrate trianion, Tris are made homogenate; 0-10 ℃, 3000-9000 rev/min centrifugal 15-60 minute, getting supernatant liquor is N-V proteolytic enzyme crude extract; N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution, measures A respectively 260And A 280Value, PAGE identifies purity;
2) adopt pH7.0-8.0, damping fluids such as 0.02-0.04mol/L phosphoric acid salt, Tris with the isocratic elution method, carry out DEAE Sepharose Fast Flow chromatography, collect active peak and ultrafiltration and concentration, measure A respectively 260And A 280Value, PAGE identifies purity;
3) adopt pH gradient elution (pH 4.0-6.5,0.01-0.02mol/L acetate, citrate buffer) and salt gradient elution process (pH 5.5-6.5,0.01-0.02mol/L acetate, citrate buffer, NaCl concentration is 0-0.5mol/L) combination, carry out CMSepharose Fast Flow chromatography, ultrafiltration and concentration is measured A respectively 260And A 280Value, PAGE identifies purity, obtaining N-V proteolytic enzyme productive rate is 0.2-0.7 ‰.
(4) purifying process 4, may further comprise the steps:
1) gets clam worm and clean the back rubbing, add pH6.0～8.0 of 1～5 times of volume, 0.1mol/LNa 2HPO 4-NaH 2PO 4Damping fluid is made homogenate; Centrifugal, getting supernatant liquor is the thrombolysin crude extract; Crude extract is through 15-60% (NH 4) 2SO 4Salt precipitation;
2) dissolution precipitation again through Sephacryl S-100 chromatography column chromatographic separation, is collected active peak; Active peak freeze-drying concentrates;
3) Phenyl-Sepharose CL-4B column chromatography: with pH6.0～8.0,0.025mol/LNa 2HPO 4-NaH 2PO 4+ 1mol/L (NH 4) 2SO 4The above-mentioned sample of the abundant balance of damping fluid, Phenyl-Sepharose CL-4B chromatography media; Sample on the balance liquid adopts linear gradient elution method wash-out ((NH 4) 2SO 4Concentration is from 1-0.05mol/L), collect active peak, ultrafiltration and concentration is measured A respectively 260And A 280Value, PAGE identifies purity.
(5) preparation property separation and Extraction purification process may further comprise the steps:
With Epoxy-activated Sepharose 6B is medium, makes the medium suspension; The rabbit desertification cocconase antibody of purifying is dissolved in the coupling buffer, mixes, dress post after effect is spent the night in shaking table with the gel particle suspension; Wash excessive antibody successively with coupling buffer, distilled water etc.; With the thanomin treatment gel of spending the night, with sealing residual activity group; With coupling buffer cleaning down affinity column, use sample buffer solution elution affinity media; Collect elutriant and carry out protein quantification, calculate the coupling rate; With the affinity media dress post for preparing, be last all product with the N-V proteinase ultrafiltrated, with the stepwise elution method, collect active peak and freeze-drying.
N-V proteinase of the present invention is at the prevention and the medicine that are used to prepare thrombotic diseases.
Pharmaceutical composition preferred weight ratio of the present invention is 0.1%～99.5% activeconstituents.
Medicine of the present invention preferably contains 1%～99% N-V proteinase and 99%～1% vehicle.
Medicine of the present invention preferably contains 10%～90% N-V proteinase and 90%～10% pharmaceutical excipient.
Medicine of the present invention preferably contains 30%～80% N-V proteinase and 70%～20% pharmaceutical excipient.
Medicine of the present invention preferably contains 60%～70% N-V proteinase and 40%～30% vehicle.
It is activeconstituents that medicine of the present invention contains the extract for the treatment of significant quantity, and contains one or more pharmaceutically acceptable carriers.
The present invention can composition form be applied to the patient of this treatment by administering modes such as vein, intramuscular injection, oral, rectum.Prepare various formulations as injection, tablet, granule, electuary, capsule, suppository, sprays, sustained release dosage, liquid oral formulation according to the conventional production method of pharmaceutical field.Also can make its activeconstituents and one or more carriers or medicament mixed, make required formulation.
Carrier above is meant the pharmaceutical carrier of pharmaceutical field routine, comprises as sanitas, pigment, thinner, vehicle, weighting agent, tackiness agent, correctives, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier.
The drug effect of N-V proteinase:
Pharmacodynamic experiment:
1. thrombus in vivo formation time experiment:
Collect according to national new drug (Western medicine) preclinical study governing principle, thrombolytic thrombus in vivo dissolution experiment---carotid artery thrombosis method, with the positive contrast medicine of urokinase, with the negative contrast of physiological saline, behind the intravenously administrable, to form thrombus, observe thrombus formation time with electricity irritation injury rats carotid artery, measure the clotting time simultaneously.
The thrombus in vivo experimental study of N-V proteolytic enzyme shows: N-V proteolytic enzyme and urokinase are under the prerequisite of Isodose, and the thrombus in vivo formation time of N-V proteolytic enzyme is longer than urokinase.
2. external thrombus forms
● external thrombus forms experiment: collect according to national new drug (Western medicine) preclinical study governing principle, with the positive contrast medicine of urokinase, with the negative contrast of physiological saline, after the rat vein administration, the abdominal cavity aorta is got blood, place extracorporeal thrombosis forming device to rotate, measure thrombus length, weight in wet base, dry weight.The result shows: antithrombotic formation effect is better than urokinase in the body of N-V proteolytic enzyme.
3. the relevant biochemical investigation of blood coagulation
With reference to N-V proteolytic enzyme medium lethal dose, select large, medium and small three dosage group intravenously administrables, time, method are with step 2.
● Fibrinogen mg/dL control group 266, low dose 207, middle dosage 142, heavy dose 132, urokinase 153, significant difference;
● PT control group 19.6, low dose 19.2, middle dosage 18.1, heavy dose 20.1, urokinase 19.3, difference is not remarkable;
● APTT control group 20.7, low dose 16.8, middle dosage 16.8, heavy dose 19.0, urokinase 19.9, difference is not remarkable;
4. clotting time experiment: extract eyeball behind the mouse tail vein injection N-V proteolytic enzyme, get blood.
● slide method unit second: control group 131, low dose of 194, middle dosage 233, heavy dose of 183 seconds;
● capillary tube technique unit second: control group 124, low dose of 180, middle dosage 329, heavy dose of 211 seconds;
5. hemolytic experiment in the body:
● the rat method: in the body injection little, in, the N-V proteolytic enzyme of big three dosage, get aorta abdominalis blood behind the different time, the Trisodium Citrate anti-freezing, in test tube, centrifugal 3000 revolutions per seconds, 5 minutes, blood divided two-layer up and down, upward is limpid blood plasma, no haemolysis;
● rabbit method: no haemolysis
The thermal source experiment of preparation
Detect no thermal source with tachypleus amebocyte lysate.
Qualitative identification experiment
N-V proteolytic enzyme is the antigen immune rabbit, separates antiserum(antisera).On agar plate, do antigen antibody reaction.N-V protease-producing immunoprecipitation line, other protein are all reactionless.
Active determination in vitro method---the fibrin plate of N-V proteolytic enzyme
1. material
Cryodesiccant Human Fibrinogen (Chinese biological goods calibrating institute)
Human blood zymoplasm (Chinese biological goods calibrating institute)
Human urokinase (Chinese biological goods calibrating institute)
Other medicine is homemade analytical pure
2. method:
(1) fibrinogen solution of preparation 0.5%: the 100mg fibrinogen powder is dissolved in 0.1MpH7.4Na 2HPO 4-NaH 2PO 4Damping fluid.
(2) thrombin solution of preparation 20U/ml:, zymoplasm is dissolved in 0.1MpH7.4Na by demarcating unit 2HPO 4-NaH 2PO 4Damping fluid.
(3) preparation fibrin plate: with the plate of placing diameter 9cm on the water level gauge corrected plane, add 0.5% the fibrinogen solution of 9ml and the thrombin solution of 1.0ml 20u/ml, left standstill behind the mixing 15 minutes.
(4) measure activity: get negative control medicine (physiological saline), positive control drug (urokinase) and each 10ul of N-V proteolytic enzyme, point sample on fibrin plate was placed 15 minutes down for 37 ℃.With the line of apsides of the molten spot of kind of calliper, calculate molten spot area.
The result:
According to the size of molten spot area, judge the height of N-V protease activity.Molten spot area is big more, and the N-V protease activities is high more.
The protein quantification method of N-V proteolytic enzyme
1. method:
(1) preparation of standard reagent:
The smart Xylene Brilliant Cyanine G G-250 that claims 100mg is dissolved in 95% the ethanol, mixes with the phosphoric acid of 100mlw/v again, and the phosphate buffered saline buffer constant volume of 0.01M pH7.0 is in the volumetric flask of 1000ml.
(2) analytical procedure:
Traditional analysis method: get the 5ml standard reagent, add in the phosphate buffered saline buffer dissolved protein sample liquid of 0.1ml 0.01M pH7.0, thorough mixing was placed after 5 minutes, measured absorbancy at the 595nm place.
Microanalysis: get the 0.2ml standard reagent, add in the phosphoric acid salt dissolved protein sample liquid of 0.8ml 0.01M pH7.0, thorough mixing was placed after 5 minutes, measured absorbancy at the 595nm place.
(3) remarks:
The protein content linearity range of traditional analysis method: 0.2～1.4mg/ml
The protein content linearity range of microanalysis: 5～100ug/ml
(4) standard quantitative curve
Evenly get concentration point in traditional analysis method and microanalysis protein content linearity range, the absorbancy that with the protein concentration is X-coordinate, 595nm place is an ordinate zou, draws conventional and the quantitative curve of microstandard respectively, and does the straight line correlation check.
2 results:
Protein concentration and linearly positive correlation of A595 (P＜0.001) in regulation protein concentration scope.
The experiment of N-V proteolytic enzyme temperature effect
1. the pure product of 2mgN-V proteolytic enzyme electrophoresis are dissolved in 1ml physiological saline.
2. be placed on respectively in 20,30,37,45,50,60,70 ℃ of water baths and be incubated 1 hour.
3. fibrin plate is measured active (37 ℃, 15 minutes).
Kinase activity assay and Profibrinolysin activation analysis
(1) kinase activity assay
1. with the ultra-filtration centrifuge tube of 10000 cutoff value, use 10mM Tris pH7.4,150mM NaCl, 10mM MgCl 2The pure product of electrophoresis of damping fluid thorough washing N-V proteolytic enzyme wash three times.
2. absorption supernatant, under 37 ℃ of conditions, add ATP, [ 32P] substrate such as ATP and azo-casein, Fibrinogen, hatched jointly 15 minutes.
3. add SDS-polyacrylamide gel electrophoresis sample solution, boil termination reaction, the centrifuging and taking supernatant.
4. carry out SDS-polyacrylamide gel electrophoresis, fixing with 0.25%Coomassie blue (45% methyl alcohol, the preparation of 10% acetate), in the exposure of X-ray sheet, check the substrate band of phosphorylation.The result shows that N-V proteolytic enzyme does not have kinase activity.
Profibrinolysin activation analysis method
With physiological saline preparation Profibrinolysin solution, with pH7.4, the phosphate buffered saline buffer of 0.1mol/L preparation N-V protein enzyme solution.With 5 μ l, 1U/mL Profibrinolysin solution and 5 μ l N-V protein enzyme solutions (1. liquid), with 5 μ l physiological saline and 5 μ l N-V protein enzyme solutions (2. liquid), with 5 μ l, 10U/mL Profibrinolysin solution and 5 μ l N-V protein enzyme solutions (3. liquid), the difference mixed preparing, and be divided into three parts.
2. all dosings still at 1. liquid and 3. liquid adding ATP solution, 2. do not add in the liquid with (two), 1.The difference mixed preparing, and be divided into three parts.
3. each three parts of dosing that above-mentioned two kinds of methods are prepared, first part at direct point sample on fibrin plate after the mixing, placed 10 minutes down in 37 ℃ after second part of mixing, point sample on fibrin plate is placed 20 minutes point samples on fibrin plate down in 37 ℃ after the 3rd part of mixing.After point sample is finished, placed 15 minutes down in 37 ℃, the back is measured active.The result shows: N-V proteolytic enzyme does not have the Profibrinolysin activation.
N-V proteolytic enzyme clot dissolution experiment
A) get the Wister rat, anesthesia back abdominal aortic blood is positioned over plate, and room temperature forms thrombus, and cutting and weighing clot are in the test tube of packing into.
B) urokinase and N-V proteolytic enzyme are added in the clot test tube, 37 ℃ of constant temperature are in different time observation dissolving situation.The result shows: dissolving fully in 10 hours, and the phosphate buffered saline buffer control group does not have dissolving.
Embodiment:
Embodiment 1
Get the 200g clam worm, clean the back and rub, add the pH7.0 of 4.5 times of volumes, 0.01mol/LNa 2HPO 4-NaH 2PO 4Damping fluid is made homogenate; 4 ℃, 9000 rev/mins centrifugal 30 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.Get N-V proteolytic enzyme crude extract 1000ml, put and be cooled to 0 ℃ in the ice bath, under agitation condition, add the dehydrated alcohol of-20 ℃ of precoolings, make concentration of ethanol reach 5%, remain on and make its generation precipitation in the ice bath;-10 ℃, 9000 rev/mins centrifugal 30 minutes, abandon precipitation; Supernatant liquor adds the dehydrated alcohol of-20 ℃ of precoolings while stirring, and final concentration is 30%, and it is fully precipitated;-10 ℃, 9000 rev/mins centrifugal 30 minutes, abandon supernatant liquor, the precipitation drain as far as possible.Precipitation is dissolved in an amount of pH5.8,0.01mol/LNa 2HPO 4-NaH 2PO 4In the damping fluid, packing.With pH8.0,0.02mol/L Tris-HCl damping fluid dilutes 5 times with preceding, and ultrafiltration between cutoff value 100000-10000 obtains ultrafiltrated.Respectively with containing 5% alcoholic acid pH8.0,0.02mol/L Tris-HCl damping fluid abundant balance N-V proteolytic enzyme ultrafiltrated and DEAE Sepharose Fast Flow chromatography media.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (NaCl concentration is from 0-0.5mol/L), collects active peak and freeze-drying, identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Respectively with containing freeze drying activity sample and the CM Sepharose Fast Flow chromatography media that 15% alcoholic acid pH4.0, the abundant balance of 0.02mol/L acetate-sodium acetate buffer obtain through previous step DEAE Sepharose Fast Flow chromatography column purifying.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (NaCl concentration is from 0-0.5mol/L), identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Obtaining N-V proteolytic enzyme productive rate is 0.23 ‰.
Embodiment 2
Get the 200g clam worm, clean the back and rub, add the pH8.0 of 8 times of volumes, the 0.08mol/LTris damping fluid is made homogenate; 4 ℃, 6000 rev/mins centrifugal 60 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.Get N-V proteolytic enzyme crude extract 1000ml, put and be cooled to 0 ℃ in the ice bath, under agitation condition, add the dehydrated alcohol of-20 ℃ of precoolings, make concentration of ethanol reach 10%, remain on and make its generation precipitation in the ice bath;-10 ℃, 6000 rev/mins centrifugal 60 minutes, abandon precipitation; Supernatant liquor adds the dehydrated alcohol of-20 ℃ of precoolings while stirring, and final concentration is 25%, and it is fully precipitated;-10 ℃, 8000 rev/mins centrifugal 30 minutes, abandon supernatant liquor, the precipitation drain as far as possible.Precipitation is dissolved in an amount of pH5.8, in the 0.01mol/L HAC-NaAC damping fluid, packing.With preceding with pH7.8,0.04mol/L Na 2HPO 4-NaH 2PO 45 times of damping fluid dilutions, ultrafiltration between cutoff value 100000-10000 obtains ultrafiltrated.Respectively with containing 5% alcoholic acid pH7.8,0.04mol/L Na 2HPO 4-NaH 2PO 4Damping fluid abundant balance N-V proteolytic enzyme ultrafiltrated and DEAE Sepharose Fast Flow chromatography media.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (NaCl concentration is from 0-0.5mol/L), collects active peak and freeze-drying, identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Respectively with containing freeze drying activity sample and the CM Sepharose Fast Flow chromatography media that 25% alcoholic acid pH6.0, the abundant balance of 0.02mol/L citrate buffer obtain through previous step DEAE Sepharose Fast Flow chromatography column purifying.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (KCl concentration is from 0-0.5mol/L), identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Obtaining N-V proteolytic enzyme productive rate is 0.21 ‰.
Embodiment 3
Get the 200g clam worm, clean the back and rub, add the pH5.0 of 8 times of volumes, the 0.04mol/LHAC-NaAC. damping fluid is made homogenate; 4 ℃, 4000 rev/mins centrifugal 60 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.Get N-V proteolytic enzyme crude extract 1000ml, put and be cooled to 0 ℃ in the ice bath, under agitation condition, add the dehydrated alcohol of-20 ℃ of precoolings, make concentration of ethanol reach 10%, remain on and make its generation precipitation in the ice bath;-10 ℃, 4000 rev/mins centrifugal 60 minutes, abandon precipitation; Supernatant liquor adds the dehydrated alcohol of-20 ℃ of precoolings while stirring, and final concentration is 25%, and it is fully precipitated;-10 ℃, 6000 rev/mins centrifugal 45 minutes, abandon supernatant liquor, the precipitation drain as far as possible.Precipitation is dissolved in an amount of pH5.8, in the 0.01mol/LHAC-NaAC damping fluid, packing.With preceding with pH7.8,0.04mol/L Na 2HPO 4-NaH 2PO 45 times of damping fluid dilutions, ultrafiltration between cutoff value 100000-10000 obtains ultrafiltrated.Respectively with containing 20% alcoholic acid pH6.0,0.03mol/L citrate buffer abundant balance N-V proteolytic enzyme ultrafiltrated and DEAE Sepharose Fast Flow chromatography media.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (NaCl concentration is from 0-0.5mol/L), collects active peak and freeze-drying, identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Respectively with containing freeze drying activity sample and the CM Sepharose Fast Flow chromatography media that 25% alcoholic acid pH4.0, the abundant balance of 0.02mol/L citrate buffer obtain through previous step DEAE Sepharose Fast Flow chromatography column purifying.The balance liquid upper prop adopts straight line linear gradient elution method wash-out (KCl concentration is from 0-0.5mol/L), identifies purity with fibrin plate method identified activity with polyacrylamide gel electrophoresis, carries out protein quantification.Obtaining N-V proteolytic enzyme productive rate is 0.26 ‰.
Embodiment 4
Get clam worm 300g, clean the back and rub, add the pH7.0 of 2 times of volumes, the 0.025mol/L phosphate buffered saline buffer is made homogenate; 4 ℃, 9000 rev/mins centrifugal 15 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution.Use abundant balance concentrated solution of pH4.60.01mol/L citric acid-Trisodium Citrate+0.01mol/L NaCl damping fluid and CM-52 Mierocrystalline cellulose chromatography medium respectively.Balance liquid concentrates upper prop, adopts isocratic elution method wash-out, ultrafiltration and concentration.With the pH4.60.01mol/L citric acid-Trisodium Citrate+abundant balance QAE Sephadex of 0.01mol/L NaCl damping fluid A50 chromatography media.With the concentrated solution abundant dialysis equilibrium of sort buffer liquid, the balance liquid upper prop adopts isocratic elution method wash-out, collects active peak, ultrafiltration and concentration.With active peak concentrated solution pH7.0 0.025mol/L Na 2HPO 4-NaH 2PO 4The abundant balance of+1.5mol/LNaCl damping fluid.With pH7.0 0.025mol/L Na2HPO 4-NaH 2PO 4The abundant balance Phenyl-Sepharose CL-4B of+1.5mol/L NaCl damping fluid chromatography media.Balance liquid concentrates upper prop, and (NaCl concentration is collected active peak from 1.5～0.05mol/L), measures A respectively to adopt straight line linear gradient elution method wash-out 260And A 280Value, ultrafiltration and concentration, PAGE identifies purity, obtaining N-V proteolytic enzyme productive rate is 0.3 ‰.
Embodiment 5
Get clam worm 300g, clean the back and rub, add the pH5.0 of 5 times of volumes, the 0.02mol/L acetate buffer is made homogenate; 4 ℃, 6000 rev/mins centrifugal 30 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution.Use pH5.0 respectively, abundant balance concentrated solution of 0.02mol/L acetate buffer and CM-52 Mierocrystalline cellulose chromatography medium.Balance liquid concentrates upper prop, adopts isocratic elution method wash-out, ultrafiltration and concentration.Use pH5.0, the abundant balance QAE Sephadex of 0.02mol/L acetate buffer A50 chromatography media.With the concentrated solution abundant dialysis equilibrium of sort buffer liquid, the balance liquid upper prop adopts isocratic elution method wash-out, collects active peak, ultrafiltration and concentration.With active peak concentrated solution pH7.5 0.025mol/L Na 2HPO 4-NaH 2PO 4The abundant balance of+1mol/LNaCl damping fluid.With pH7.5 0.025mol/L Na 2HPO 4-NaH 2PO 4The abundant balance Phenyl-Sepharose CL-4B of+1mol/L NaCl damping fluid chromatography media.Balance liquid concentrates upper prop, and (NaCl concentration is collected active peak from 1～0.05mol/L), measures A respectively to adopt straight line linear gradient elution method wash-out 260And A 280Value, ultrafiltration and concentration, PAGE identifies purity, obtaining N-V proteolytic enzyme productive rate is 0.35 ‰.
Embodiment 6
Get clam worm 300g, clean the back and rub, add the pH6.0 of 10 times of volumes, the 0.02mol/L acetate buffer is made homogenate; 4 ℃, 4000 rev/mins centrifugal 60 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution.Use pH6.0 respectively, abundant balance concentrated solution of 0.02mol/L acetate buffer and CM-52 Mierocrystalline cellulose chromatography medium.Balance liquid concentrates upper prop, adopts isocratic elution method wash-out, ultrafiltration and concentration.Use pH6.0, the abundant balance QAE Sephadex of 0.02mol/L acetate buffer A50 chromatography media.With the concentrated solution abundant dialysis equilibrium of sort buffer liquid, the balance liquid upper prop adopts isocratic elution method wash-out, collects active peak, ultrafiltration and concentration.With active peak concentrated solution pH7.5 0.01mol/L Na 2HPO 4-NaH 2PO 4The abundant balance of+0.5mol/L NaCl damping fluid.Use pH7.5 0.01mol/LNa 2HPO 4-NaH 2PO 4The abundant balance Phenyl-Sepharose CL-4B of+0.5mol/L NaCl damping fluid chromatography media.Balance liquid concentrates upper prop, and (NaCl concentration is collected active peak from 0.5～0.05mol/L), measures A respectively to adopt straight line linear gradient elution method wash-out 260And A 280Value, ultrafiltration and concentration, PAGE identifies purity, obtaining N-V proteolytic enzyme productive rate is 0.33 ‰.
Embodiment 7
1) antigenic acquisition: adopt preparative electrophoresis to obtain target protein-N-V proteinase.Continuous system polyacrylamide gel electrophoresis, gel strength are 10%.Electrophoresis finishes back stripping glue, downcuts a fillet blob of viscose, puts into staining fluid dyeing 10 minutes, promptly can be observed active zone; Remaining gel piece is wrapped with preservative film, is kept under 4 ℃ of conditions.Painted blob of viscose is alignd on sheet glass with undyed blob of viscose, downcut on the undyed big blob of viscose and the corresponding adhesive tape of activated protein band, the adhesive tape of cutting-out is enclosed in the dialysis tubing, in adorn a small amount of buffer A, put into horizontal strip electrophoresis groove electrophoresis, 80mA, electrophoresis 4 hours.Liquid in the collecting bag is used the distill water dialysis desalination, and is centrifugal, crosses the millipore filtration degerming, and freeze-drying then promptly obtains the pure antigen of electrophoresis.
2) immune animal
2.1) bacille Calmette-Guerin vaccine sensitization: give every rabbit two metapedes intradermal injections bacille Calmette-Guerin vaccine alive, every 1mg.
2.2) fundamental immunity: carry out fundamental immunity behind the fortnight.In autoclaved mortar, add Freund's incomplete adjuvant, drip the bacille Calmette-Guerin vaccine of deactivation again, grind, make Freund's complete adjuvant (2mg bacille Calmette-Guerin vaccine/ml Freund's complete adjuvant) while dripping; Freeze dried antigen is dissolved in an amount of autoclaved buffer A, is added dropwise in the Freund's complete adjuvant, grind while dripping, add again benzylpenicillin sodium solution, Vetstrep solution a little, grind evenly, make water-in-oil emulsion, antigen concentration is 100 μ g/ml emulsions.Give every subcutaneous multi-point injection in rabbit back, every 2ml.
2.3) booster immunization: per 3 all booster immunizations once, method is basic identical with fundamental immunity, different is to replace Freund's complete adjuvant with Freund's incomplete adjuvant, the dosage of booster shots can suitably reduce.Behind each booster immunization 7～10 days, auricular vein was got blood, uses two-way agar diffusion test [31]Measure antiserum titre, when tire 〉=8 the time, only from auricular vein bloodletting 20～30ml/.Continue after the bloodletting to raise, continue immunity.Tire stable after, can 1 week of every interval from auricular vein bloodletting 20～30ml/ only, descend until serum titer, get whole blood from rabbit aorta at last.
2.4) in four rabbits of pure thrombolysin immunity, with other two rabbits of clam worm homogenate supernatant liquor immunity, in contrast with electrophoresis.
2.5) get antiserum(antisera)
Each blood sample collection that obtains makes it to solidify in the room temperature placement in the centrifuge tube of sterilization (not adding antithrombotics).Unscrew grumeleuse with a glass rod along tube wall, room temperature is placed a few hours again, and antiserum(antisera) is separated out fully; 5000 rev/mins centrifugal 15 minutes, supernatant is antiserum(antisera).Packing ,-20 ℃ of storages.
3) purification of IgG
3.1) get an amount of antiserum(antisera) freeze thawing, add the buffer B mixing of isopyknic precooling.Drip saturated (NH under the condition of ice bath 4) 2SO 4Solution, the limit edged stirs, and makes (the NH of solution 4) 2SO 4Saturation ratio reaches 50%, 4 ℃ and left standstill 2 hours, 4000 rev/mins centrifugal 20 minutes, abandon supernatant.
3.2) the buffer B dissolving of precipitation with volume, dropwise add saturated (NH under the condition of ice bath 4) 2SO 4Solution, the limit edged stirs, and makes (the NH of solution 4) 2SO 4Saturation ratio reaches 33%, 4 ℃ and left standstill 2 hours, 4000 rev/mins centrifugal 20 minutes, abandon supernatant; Repeat twice of 4.2 operation.
3.3) precipitation is dissolved in a small amount of buffer B, the dialysis tubing of packing into, 4 ℃ to damping fluid C dialysis desalination balance, and centrifugal, supernatant is purer IgG solution.
3.4) go up the step and obtain purer IgG and cross DEAE Sepharose Fast Flow column chromatography, level pad is damping fluid C, and elutriant is damping fluid D, and isocratic elution is collected first peak, concentrates with PEG20000.Foreign protein damping fluid E wash-out.The IgG that purifies identifies with SDS-PAGE.
4) preparation of affinity chromatography medium and affinity chromatography: with Epoxy-activated Sepharose6B is medium, makes the medium suspension; The anti-thrombolysin antibody of the rabbit of purifying is dissolved in the coupling buffer, mixes with the gel particle suspension, effect was adorned post after 16 hours in 37 ℃ of shaking tables; Wash excessive antibody successively with coupling buffer, distilled water etc.; Use the 1M thanomin, 37 ℃ of treatment gel of spending the night are with sealing residual activity group; With coupling buffer cleaning down affinity column, use sample buffer solution elution affinity media; Collect elutriant and carry out protein quantification, calculate the coupling rate.With the affinity media dress post for preparing, be last all product with the thrombolysin ultrafiltrated, with the stepwise elution method, collect active peak and freeze-drying.
Embodiment 8
Get clam worm 300g, clean the back and rub, add the pH8.0 of 2 times of volumes, 0.03mol/L Tris damping fluid is made homogenate; 4 ℃, 6000 rev/mins centrifugal 30 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution.With pH8.0, abundant balance concentrated solution of 0.03mol/L Tris damping fluid and DEAE Sepharose Fast Flow chromatography media, and carry out isocratic elution, collect active peak and ultrafiltration and concentration.With abundant balance concentrated solution of acetate buffer and CM Sepharose Fast Flow chromatography media, (pH4.5-pH6.5) carries out the pH gradient elution with the 0.02mol/L acetate buffer, (NaCl 0-0.5mol/L) carries out salt gradient wash-out, ultrafiltration and concentration with pH6.5 0.02mol/L acetate buffer.Adopt pH7.0, the 0.02mol/L phosphate buffered saline buffer carries out Sephadex G-50 chromatography, measures A respectively 260And A 280Value, PAGE identifies purity.Obtaining N-V proteolytic enzyme productive rate is 0.32 ‰.
Embodiment 9
Get clam worm 300g, clean the back and rub, add the 10% alcoholic acid pH8.0 that contains of 2 times of volumes, 0.03mol/L Tris damping fluid is made homogenate; 4 ℃, 6000 rev/mins centrifugal 60 minutes, getting supernatant liquor is N-V proteolytic enzyme crude extract.N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution.With pH7.5, abundant balance concentrated solution of 0.025mol/LTris damping fluid and DEAE Sepharose Fast Flow chromatography media, and carry out isocratic elution, collect active peak and ultrafiltration and concentration.With abundant balance concentrated solution of citrate buffer and CM Sepharose Fast Flow chromatography media, (pH4.5-pH6.5) carries out the pH gradient elution with the 0.04mol/L acetate buffer, (NaCl 0-0.5mol/L) carries out salt gradient wash-out, ultrafiltration and concentration with pH6.5 0.04mol/L citrate buffer.Adopt pH7.0, the 0.04mol/L phosphate buffered saline buffer carries out Sephadex G-75 chromatography, measures A respectively 260And A 280Value, PAGE identifies purity.Obtaining N-V proteolytic enzyme productive rate is 0.39 ‰.
Embodiment 10
1) gets the 500g clam worm, clean the back and rub, add the pH7.0 of 4.5 times of volumes, 0.1mol/LNa 2HPO 4-NaH 2PO 4Damping fluid is made homogenate; 4 ℃, 9000 rev/mins centrifugal 30 minutes, getting supernatant liquor is the thrombolysin crude extract; Crude extract is through 15-60% (NH 4) 2SO 4Salt precipitation;
2) dissolution precipitation again through Sephacryl S-100 chromatography column chromatographic separation, is collected active peak; Active peak freeze-drying concentrates;
3) Phenyl-Sepharose CL-4B column chromatography: with pH7.0,0.025mol/LNa 2HPO 4-NaH 2PO 4+ 1mol/L (NH 4) 2SO 4The above-mentioned sample of the abundant balance of damping fluid, Phenyl-Sepharose CL-4B chromatography media; Sample on the balance liquid adopts linear gradient elution method wash-out ((NH 4) 2SO 4Concentration is from 1-0.05mol/L), collect active peak, ultrafiltration and concentration is measured A respectively 260And A 280Value, PAGE identifies purity.
Embodiment 11
N-V protease preparation technology
Type of preparation and every bottled amount: freeze-dried preparation, N-V proteolytic enzyme 1-3mg/ bottle;
Excipient kind and consumption: N.F,USP MANNITOL 10-40mg, dextran 10-40mg, L Methionin 5-15mg, human serum albumin 2-10mg, each moiety is with any proportioning of above-mentioned consumption, with folk prescription or compound form, freeze-drying excipient.
Freeze-drying operation: one-level freeze-drying :-30～-20 ℃ freeze-drying 16-48 hour; Secondary freeze-drying: O-10 ℃ freeze-drying 16-24 hour.

Claims (1)

1, N-V proteinase is in the purposes of preparation treatment thrombus disease medicine, and described N-V proteinase obtains by following separation and Extraction purification process:
1) ultra-filtration and separation and concentrated: get clam worm, clean the back and rub, add the pH4.0-9.0 of 1-10 times of volume, 0.01-0.4mol/L phosphoric acid salt or acetate or Citrate trianion or Tris damping fluid are made homogenate; 0-10 ℃, 3000-9000 rev/min centrifugal 15-60 minute, getting supernatant liquor is N-V proteolytic enzyme crude extract; N-V proteolytic enzyme crude extract 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, get concentrated solution, measures A respectively 260And A 280Value, PAGE identifies purity;
2) CM-52 cellulose chromatography: use pH3-4.6 respectively, abundant balance concentrated solution of 0.01-0.05mol/L acetate or citrate buffer and CM-52 Mierocrystalline cellulose chromatography medium; Balance liquid concentrates upper prop, adopts isocratic elution method wash-out; Use the 300000MW membrane ultrafiltration, filtrate is held back concentrated by the 10000MW film, measure A respectively 260And A 280Value, PAGE identifies purity;
3) concentrated solution that obtains QAE Sephadex A50 column chromatography: use the abundant balance QAE Sephadex of pH3-4.6 0.01-0.05mol/L acetate or citrate buffer A50 chromatography media and step 2 respectively), the balance liquid upper prop, adopt isocratic elution method wash-out, collect active peak, measure A respectively 260And A 280Value is used the 300000MW membrane ultrafiltration, and filtrate is held back concentrated by the 10000MW film, and PAGE identifies purity;
4) Phenyl-Sepharose CL-4B column chromatography: with the active peak concentrated solution pH5.0-8.0 that obtains in the step 3), 0.01-0.025mol/L phosphoric acid salt or the abundant balance of Tris damping fluid; Use pH5.0-8.0,0.01-0.025mol/L phosphoric acid salt or the abundant balance Phenyl-Sepharose CL-4B of Tris damping fluid chromatography media; Balance liquid concentrates upper prop, adopts 1.5～0.05mol/LNaCl neutral salt solution straight line gradient elution; Collect active peak, measure A respectively 260And A 280Value is used the 300000MW membrane ultrafiltration, and filtrate is held back concentrated by the 10000MW film, gets proteolytic enzyme.
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