CN102242102A - Preparation method of X factor activator from bothrops atrox venom - Google Patents
Preparation method of X factor activator from bothrops atrox venom Download PDFInfo
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Abstract
The invention provides a coagulation X factor activator from bothrops atrox venom, and an extraction method thereof. The activator is a protein with the activity of a coagulation X factor activating enzyme, and is collected by sequentially performing DEAE-Sepharose fast flow ion-exchange chromatography, dialysis, cellulose DEAE-52 ion-exchange chromatography and Sephadex-G75 gel filtration chromatography on the bothrops atrox venom. The coagulation X factor activating enzyme provided by the invention can be used as an active component for preparing medicaments for treating clinical hemorrhage such as surgical hemorrhage, medical hemorrhage, gynecological hemorrhage, facial hemorrhage, pediatric hemorrhage, hemorrhage after tissue biopsy and the like, and treating hemorrhagic disease, so that the coagulation X factor activating enzyme can play an important role in the field of biological pharmacy and has wide application prospects.
Description
Technical field
The present invention relates to enzyme and extracting method thereof, a kind of extracting method that derives from spearhead agkistrodon halyx pallas venom X factor activator thing particularly is provided.
Background technology
X factor activator enzyme is a strongest component of Blood clotting, factor X (FX) is a kind of plasma glycoprotein, play an important role in coagulation process, its activated form Fxa can be zymoplasm to the thrombogen activation under the booster action of factor V, Caz+, phosphatide.
The FX activating enzyme mainly is distributed in round spot frog snake (Rusesilviper) snake venom, in frog Ophiinae and Crotalinae and some elapid snake venom discovery is arranged also.FX activating enzyme (CRVV) is the X factor activating enzymes that extract from circle spot locust snake (Russelv coin er) snake venom in China's frog calmy poison, can specificity activate factor X and make the clotting of plasma, with foreign like product Stypven reagent roughly the same.The FX activating enzyme can activate the factor X (FX) in the blood specifically, promotes blood coagulation, and in clinical diagnosis, the various fields such as scientific research of the exploitation of novel drugs and protein structure and function all have huge applications to be worth.
Summary of the invention
The object of the present invention is to provide a kind of blood coagulation X factor activating enzyme that derives from the spearhead viper venom.
The present invention is for solving the problems of the technologies described above, take following technical scheme: a kind of X factor activator enzyme is to have the active albumen of blood coagulation X factor activating enzyme with what the spearhead viper venom carried out collecting behind DEAE-Sepharose fast flow ion exchange chromatography, dialysis, Mierocrystalline cellulose DEAE-52 ion exchange chromatography and the Sephadex-G75 gel permeation chromatography successively.
The blood coagulation X factor activating enzyme that derives from the spearhead viper venom of the present invention has following characteristics: be made up of metalloprotease (calcium ion dependency phosphatide dependent/non-dependent) and serine protease (calcium ion dependent/non-dependent) two big classes.
X factor activator enzyme extraction method of the present invention may further comprise the steps:
(1), the spearhead viper venom is dissolved in the Tris-HCl damping fluid of pH7-8,0.05mol/L, obtaining concentration is the spearhead viper venom liquid of 100-120g/L;
(2), the spearhead viper venom liquid that step (1) is obtained carries out ion exchange chromatography, the solid phase weighting material is DEAE-Sepharose fast flow, and elutriant is the Tris-HCl damping fluid straight line gradient elution of the pH7-8, the 0.05mol/L that contain 0-0.2mol/L NaCl;
(3), collect elutriant, under the wavelength of 280nm, elutriant is carried out the ultraviolet spectrometry range and detects, the collection of illustrative plates that is absorbed, Jiang Gefeng carries out the SDS-PAGE gel electrophoresis, collects the part that contains X factor activator enzyme;
(4), concentrate desalination, with the Tris-HCl damping fluid ultrafiltration and concentration of pH6.2,0.05mol/L and remove NaCl;
(5), ion exchange chromatography once more, the concentrated solution of step 4) carries out ion exchange chromatography, the solid phase weighting material is Mierocrystalline cellulose DEAE-52; Elutriant is the Tris-HCl damping fluid straight line gradient elution of the pH6.2, the 0.05mol/L that contain 0-0.5mol/LNaCl;
(6), each peak of elutriant that step (5) is obtained carries out the SDS-PAGE gel electrophoresis, collects the part that contains X factor activator enzyme;
(7), once more concentrate desalination, with the Tris-HCl damping fluid ultrafiltration and concentration of pH7.5,0.01mol/L and remove NaCl;
(8), step (7) concentrated solution is carried out gel permeation chromatography, the solid phase weighting material is Sephadex-G75, and elutriant is the Tris-HCl damping fluid of pH7.5,0.01mol/L, collects elutriant, therefrom collect and contain X factor activator enzyme part, obtain spearhead pallas pit viper X factor activator proenzyme liquid.
In the X factor activator enzyme extraction method of the present invention, be acquisition better implement effect in the step (1), can be earlier remove impurity in the spearhead viper venom liquid with centrifugation method, centrifugal condition can be selected according to practical situation, as centrifugal 15min under at 4500rpm.
In the X factor activator enzyme extraction method of the present invention, the DEAE-Sepharosefast flow ion exchange column in the step (2) before use, earlier with the Tris-HCl damping fluid balance of pH7-8,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, Tris-HCl damping fluid with the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent carries out the straight line gradient elution then, and flow velocity is 3.0ml/min; Described step 2) the Tris-HCl damping fluid in is preferably the Tris-HCl damping fluid of pH7.5,0.05mol/L..
In the X factor activator enzyme extraction method of the present invention, the collection method in the step (3) can be: detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm; Detection method with X factor activator enzyme part is: the SDS-PAGE electrophoresis.
In the X factor activator enzyme extraction method of the present invention, step (4) concentrates desalination can select cutoff 10000 molecular weight film cross-flow ultrafiltrations for use, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH6.2,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength to remove micromolecule polypeptide and desalination.
In the X factor activator enzyme extraction method of the present invention, in the step (5) Mierocrystalline cellulose DEAE-52 chromatography column before use, with the Tris-HCl damping fluid balance of pH6.2,0.05mol/L 5-6 hour, flow velocity was 3.0ml/min earlier; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH6.2,0.05mol/L and equivalent then, flow velocity is 3.0ml/min.
In the X factor activator enzyme extraction method of the present invention, before the Sephadex-G75 chromatography column uses in the step (8), the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, treat supernatant liquor move to fully the glue face following after, above the glue face, add the buffer solution elution identical with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step (3) to collect the part that contains X factor activator enzyme in the elutriant.
The invention provides a kind of X factor activator enzyme that from the spearhead pit viper venom, extracts.Described X factor activator enzyme has following characteristics: (1) is made up of metalloprotease (calcium ion dependency phosphatide dependent/non-dependent) and serine protease (calcium ion dependent/non-dependent) two big classes; (2) extracting method of this enzyme is based on the chromatographic technique of routine, realize the purpose of clean cut separation by conversion chromatography condition and regulation technology parameter, and can operate at normal temperatures, and have that condition is simple amplifies easily, cost low (the chromatography glue of selection can be recycled), the high and high advantage of product purity of yield.Based on These characteristics, can X factor activator enzyme of the present invention be that activeconstituents preparation treatment surgery is hemorrhage, internal medicine is hemorrhage, Obstetric and Gynecologic Department are hemorrhage, department of eye is hemorrhage, paediatrics is hemorrhage and biopsy after the medicine of clinical hemorrhage and hemorrhagic diseases such as hemorrhage, to play a significant role in field of biological pharmacy, have a extensive future.
Embodiment
Below in conjunction with specific embodiment the present invention is described in further detail.
Method therefor is ordinary method if no special instructions among the following embodiment.
Embodiment 1
The extracting method of the described X factor activator of present embodiment enzyme is: get 10g spearhead viper venom dry powder (lot number 080709), Tris-HCl damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH7, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with DEAE-Sepharose fast flow chromatography column with the Tris-HCl damping fluid balance of pH7,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, Tris-HCl damping fluid with the pH7 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7,0.05mol/L and equivalent carries out the straight line gradient elution then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has X factor activator enzyme part through electrophoretic analysis, merge elutriant, altogether 232ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, by the dilution of Tris-HCl damping fluid, the ultrafiltration and concentration with pH6.2,0.05mol/L, the redilution mode of ultrafiltration and concentration again reduces solution ion strength, concentrating sample 40ml to remove micromolecule polypeptide and desalination; Last sample is to Mierocrystalline cellulose DEAE-52 chromatography column, and the Tris-HCl damping fluid balance of usefulness pH6.2,0.05mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH6.2,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.01mol/L concentrates desalination once more, go up sample Sephadex-G75 chromatography column then, the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 45ml, results albumen 34mg, the HPLC purity assay is 97.5%, SDS-PAGE is a band.
Embodiment 2
The extracting method of the described X factor activator of present embodiment enzyme is: get 10g spearhead viper venom dry powder (lot number 080709), Tris-HCl damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH7.5, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with DEAE-Sepharose fast flow chromatography column with the Tris-HCl damping fluid balance of pH7.5,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, Tris-HCl damping fluid with the pH7.5 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7.5,0.05mol/L and equivalent carries out the straight line gradient elution then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has X factor activator enzyme part through electrophoretic analysis, merge elutriant, altogether 285ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, concentrate desalination, concentrating sample 40ml by Tris-HCl damping fluid with pH6.2,0.05mol/L; Last sample is to Mierocrystalline cellulose DEAE-52 chromatography column, and the Tris-HCl damping fluid balance of usefulness pH6.2,0.05mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH6.2,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.01mol/L concentrates desalination once more, go up sample Sephadex-G75 chromatography column then, the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 58ml, results protein 36 .2mg, the HPLC purity assay is 98.3%, SDS-PAGE is a band.
Embodiment 3
The extracting method of the described X factor activator of present embodiment enzyme is: get 10g spearhead viper venom dry powder (lot number 080709), Tris-HCl damping fluid stirring and dissolving 30min in 4-8 ℃ chromatography cabinet with pH8.0, the 0.05mol/L of 100ml precooling, the centrifugal 15min of 4500rpm gets supernatant liquor.Before the chromatography earlier with DEAE-Sepharose fast flow chromatography column with the Tris-HCl damping fluid balance of pH8.0,0.05mol/L 5-6 hour, flow velocity is 3ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, Tris-HCl damping fluid with the pH8.0 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH8.0,0.05mol/L and equivalent carries out the straight line gradient elution then, and flow velocity is 3.0ml/min; Detect the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm, has X factor activator enzyme part through electrophoretic analysis, merge elutriant, altogether 242ml, with cutoff 10000 molecular weight film cross-flow ultrafiltrations, concentrate desalination, concentrating sample 40ml by Tris-HCl damping fluid with pH6.2,0.05mol/L; Last sample is to Mierocrystalline cellulose DEAE-52 chromatography column, and the Tris-HCl damping fluid balance of usefulness pH6.2,0.05mol/L is 5-6 hour earlier, and flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical with balance liquid, the Tris-HCl damping fluid straight line gradient elution of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH-6.2,0.05mol/L and equivalent then, flow velocity is 3.0ml/min; Tris-HCl damping fluid with pH7.5,0.0lmol/L concentrates desalination once more, go up sample Sephadex-G75 chromatography column then, the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, add the buffer solution elution identical with balance liquid above the glue face, flow velocity is 3.0ml/min.Collect sample 39ml, results albumen 33.4mg, the HPLC purity assay is 97.9%, SDS-PAGE is a band.
Claims (10)
1. preparation method who derives from spearhead agkistrodon halyx pallas venom X factor activator thing, it is characterized in that: described spearhead agkistrodon halyx pallas venom blood coagulation X factor activating enzyme is to have the active albumen of blood coagulation X factor activating enzyme with what the spearhead viper venom carried out collecting behind DEAE-Sepharose fast flow ion exchange chromatography, dialysis, Mierocrystalline cellulose DEAE-52 ion exchange chromatography and the Sephadex-G75 gel permeation chromatography successively; Described first time, DEAE-Sepharose fast flow ion exchange chromatography carried out the straight line gradient elution with the Tris-HCl damping fluid of the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent; Described Mierocrystalline cellulose DEAE-52 ion exchange chromatography carries out the straight line gradient elution with the Tris-HCl damping fluid of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH6.2,0.05mol/L and equivalent; The described Sephadex-G75 gel permeation chromatography Tris-HCl buffer solution elution of pH7.5,0.01mol/L.
2. according to the described preparation method who derives from spearhead agkistrodon halyx pallas venom X factor activator thing of claim 1, it is characterized in that: described spearhead agkistrodon halyx pallas venom blood coagulation X factor activating enzyme can be divided into metalloprotease and serine protease two big classes, metalloprotease is by two light chains and the glycoprotein that heavy chain forms through interchain disulfide bond, and its activity is a calcium ion dependency phosphatide dependent/non-dependent; The serine stretch protein enzyme molecular weight that activates FX is less, and its activity is the calcium ion dependent/non-dependent.
3. according to the described preparation method who derives from spearhead agkistrodon halyx pallas venom X factor activator thing of claim 1, it is characterized in that: the preparation method of described spearhead agkistrodon halyx pallas venom X factor activator enzyme may further comprise the steps:
(1) snake venom pre-treatment;
(2) with on the pretreated snake venom solution through the DEAE-Sepharose of pre-equilibration fast flow ion exchange chromatography, carry out the straight line gradient elution with the Tris-HCl damping fluid of the pH7-8 that contains 0.2mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH7-8,0.05mol/L and equivalent;
(3) collect elutriant, under the wavelength of 280nm, elutriant is carried out the ultraviolet spectrometry range and detect, the collection of illustrative plates that is absorbed, Jiang Gefeng carries out the SDS-PAGE gel electrophoresis, collects the part that contains X factor activator enzyme;
(4) above-mentioned elutriant is carried out ultrafiltration and concentration and remove NaCl;
(5) ion exchange chromatography once more, the concentrated solution of step (4) carries out ion exchange chromatography, and the solid phase weighting material carries out the straight line gradient elution for Mierocrystalline cellulose DEAE-52 with the Tris-HCl damping fluid of the pH6.2 that contains 0.5mol/LNaCl, the 0.05mol/L of the Tris-HCl damping fluid of pH6.2,0.05mol/L and equivalent;
(6) each peak of elutriant that step (5) is obtained carries out the SDS-PAGE gel electrophoresis, collects the part that contains X factor activator enzyme;
(7) ultrafiltration and concentration is removed NaCl;
(8) step (7) concentrated solution is carried out gel permeation chromatography, the solid phase weighting material is Sephadex-G75, and elutriant is the Tris-HCl damping fluid of pH7.5,0.01mol/L.
4. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: the pretreated method of described step (1) snake venom is with the Tris-HCl damping fluid dissolving of snake venom with pH7-8, the 0.05mol/L of an amount of precooling; Step (1) also comprises the step of removing the impurity in the spearhead viper venom liquid with centrifugation method.
5. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: DEAE-Sepharose fast flow ion exchange column before use in the described step (2), the Tris-HCl damping fluid balance of first usefulness pH7-8,0.05mol/L 5-6 hour, flow velocity is 3.0ml/min; During chromatography, treat the spearhead pit viper venom move to fully the glue face following after, above the glue face, add the damping fluid identical with balance liquid, carry out the straight line gradient elution then, flow velocity is 3.0ml/min.
6. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: the collection method in the described step (3) is for detecting the collection of illustrative plates manual collection according to ultraviolet spectrometry range under the 280nm; Detection method with X factor activator enzyme part is: the SDS-PAGE gel electrophoresis.
7. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, its feature in: the concentration method in the described step (4) for the Tris-HCl damping fluid ultrafiltration and concentration of pH6.2,0.05mol/L and remove NaCl.
8. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: Mierocrystalline cellulose DEAE-52 chromatography column before use in the described step (5), the Tris-HCl damping fluid balance of first usefulness pH6.2,0.05mol/L 5-6 hour, flow velocity is 3.0ml/min; During chromatography, after treating that the spearhead pit viper venom moves to below the glue face fully, above the glue face, add the damping fluid identical, carry out the straight line gradient elution then with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step (3) to collect the part that contains X factor activator enzyme in the elutriant.
9. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: the concentration method in the described step (7) is with the Tris-HCl damping fluid ultrafiltration and concentration of pH7.5,0.01mol/L and removes NaCl.
10. according to the preparation method of the described spearhead agkistrodon halyx pallas venom of claim 3 X factor activator enzyme, it is characterized in that: before the Sephadex-G75 chromatography column uses in the described step (8), the Tris-HCl damping fluid balance of first usefulness pH7.5,0.01mol/L 5-6 hour, flow velocity is 3.0ml/min, during chromatography, after treating that supernatant liquor moves to below the glue face fully, above the glue face, add the buffer solution elution identical with balance liquid, flow velocity is 3.0ml/min, uses the method identical with step (3) to collect the part that contains X factor activator enzyme in the elutriant.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643791A (en) * | 2012-04-18 | 2012-08-22 | 暨南大学 | Purification method for snake venom serine protease Harobin |
| CN103376329A (en) * | 2012-04-19 | 2013-10-30 | 蓬莱诺康药业有限公司 | Characterization method of stypticity of hemocoagulase atrox for injection and effective components thereof |
| CN104531631A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for preparing venin antithrombotic enzyme |
| CN109207461A (en) * | 2017-07-07 | 2019-01-15 | 辽宁远大诺康生物制药有限公司 | A kind of factor X activator and preparation method thereof |
-
2010
- 2010-05-10 CN CN2010101666897A patent/CN102242102A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643791A (en) * | 2012-04-18 | 2012-08-22 | 暨南大学 | Purification method for snake venom serine protease Harobin |
| CN102643791B (en) * | 2012-04-18 | 2013-06-12 | 暨南大学 | Purification method for snake venom serine protease Harobin |
| CN103376329A (en) * | 2012-04-19 | 2013-10-30 | 蓬莱诺康药业有限公司 | Characterization method of stypticity of hemocoagulase atrox for injection and effective components thereof |
| CN104531631A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for preparing venin antithrombotic enzyme |
| CN109207461A (en) * | 2017-07-07 | 2019-01-15 | 辽宁远大诺康生物制药有限公司 | A kind of factor X activator and preparation method thereof |
| CN109207461B (en) * | 2017-07-07 | 2021-09-28 | 远大生命科学(辽宁)有限公司 | Blood coagulation factor X activator and preparation method thereof |
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Application publication date: 20111116 |