CN101280296A - Trimeresurus albolabris defibrase, preparation thereof and application thereof to pharmacy - Google Patents
Trimeresurus albolabris defibrase, preparation thereof and application thereof to pharmacy Download PDFInfo
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- CN101280296A CN101280296A CN 200810069758 CN200810069758A CN101280296A CN 101280296 A CN101280296 A CN 101280296A CN 200810069758 CN200810069758 CN 200810069758 CN 200810069758 A CN200810069758 A CN 200810069758A CN 101280296 A CN101280296 A CN 101280296A
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Abstract
The invention relates to fibrinolytic enzyme of trimeresurus albolabris venom, and the preparation method and the application thereof in pharmaceutical industry, and belongs to the biomedical field. Trimeresurus albolabris venom fibrinolytic enzyme is metal protease obtained in the venom of the domestic trimeresurus albolabris through separation and purification, has the activity of simultaneously degrading fibrinogen Aalpha chain and fibrinogen Bbeta chain, and is 66300 dalton under the reducing and non-reducing conditions of the apparent molecular weight on polyacrylamide gel electrophoresis. The preparation method comprises the following steps: firstly, pretreatment of trimeresurus albolabris venom; secondly, DEAE-Sephadex A-25 ion-exchange chromatography; thirdly, DEAE-Sephadex G-100 gel chromatography; fourthly, CM-Sephadex C-25 cation-exchange chromatography. The fibrinolytic enzyme of the trimeresurus albolabris venom has higher activity of simultaneously degrading fibrinogen Aalpha chain and fibrinogen Bbeta chain, and can be applied for curing thromboembolic disease.
Description
Technical field
The invention provides a kind of trimeresurus albolabris defibrase and preparation method thereof and the application in pharmacy, belong to biomedical sector.
Background technology
Existing the directly enzyme of solution fibrin (former) of a class in snake venom such as Viperinae, Crotalinae and glasses section, be called for short fiber eliminating enzyme, is a kind of can solution fibrin former, again can fibrinolytic fibrinoclase.There is not the zymogen forms of non-activity in the snake venom fiber eliminating enzyme, and has active proteolytic ferment, does not need activator to activate.Therefore can directly act on thrombosed scleroproein, thrombus is dissolved fast, improve patient's clinical symptom; By the dissolving plasma fibrinogen, reduce blood viscosity, suppress thrombosis, prevent to block once more.
According to its constructional feature, the snake venom fiber eliminating enzyme can be divided into serine protease (Serine proteinase) and metalloprotease (metalloproteinase) again.The falling fine activity and can be suppressed of serine protease, but do not suppressed, and the falling fine activity and can be suppressed of metalloprotease by EDTA by EDTA by common serpin such as diisopropylphosphofluoridate (DFP) and phenylmethylsulfonyl fluoride (PMSF).Fibrinogenic B β 2 chains of the preferential cracking of fibril propylhomoserin proteolytic enzyme fall in great majority, and A α 2 chains are then had only very low lytic activity; And most of fiber eliminating enzymes are metalloproteases, and preferential fibrinogenic A α 2 chains of cracking then have only cracking than low degree to B β 2 chains.
The thrombolysis medicine of present clinical use mainly is streptokinase, urokinase, tissue plasminogen activator, and the common feature of this class medicine is an indirect action, and onset is slow.We first from the venom of my home-made Trimeresurus albolabris (Gray) snake thrombolysis system such as the t-PA (tissue plasminogen activator) of the fiber eliminating enzyme of separation and purification and present clinical use, urokinase, streptokinase compared many advantages: trimeresurus albolabris defibrase is a kind of metalloprotease, and its activity is not suppressed by serpin in the blood; Because it does not activate the physiology plasmin and can avoid, as forming relevant side effects such as platelet activation with the physiology plasmin; Substrate is single-minded, long half time.These characteristics have all shown the potential value of trimeresurus albolabris defibrase as a kind of strong thrombus-dissolving agent.But, also there is not a cover from the white Trimeresurus stejnegeri venom of China, to extract purifying trimeresurus albolabris defibrase efficient ways and to the affirmation of its drug effect at present.
Summary of the invention
The objective of the invention is based on above-mentioned prior art basis, a kind of have stronger former active trimeresurus albolabris defibrase of hydrolysis of fibrin and preparation method thereof and the application in pharmacy are provided.
For the purposes of the present invention, the invention provides following technical scheme:
Lip trimeresurus stejnegeri snake venom fiber eliminating enzyme, we therefrom separate a kind of metalloprotease that obtains in the venom of home-made Trimeresurus albolabris (Gray) snake, it has the former α chain of while hydrolysis of fibrin and the activity of β chain, inhibitors of metalloproteinase EDTA can suppress its activity, but mercaptoethanol and serine protease specific inhibitor PMSF can not suppress its activity, and it is 66300 dalton under reduction of the apparent molecular weight on the polyacrylamide gel electrophoresis and non-reduced condition.
The preparation method of lip trimeresurus stejnegeri snake venom fiber eliminating enzyme, collect the Trimeresurus albolabris (Gray) snake venom, concentrated postlyophilization makes thick malicious lyophilized powder, get the thick malicious lyophilized powder 1～5g of Trimeresurus albolabris (Gray) snake venom, be dissolved in 4～6ml pH 7.5～8.5,0.04 in～0.06mol Tris-HCl the damping fluid, with whizzer with the centrifugal 8～12min of 3000～5000r/min, get supernatant liquor, through DEAE-Sephadex A-25 ion exchange chromatography, 0.04～0.06mol Tris-HCl damping fluid linear gradient elution with concentration range 0.01～0.6mol/L NaCl1000ml, elution flow rate is 0.20～0.30ml/min, collect gained I peak elutriant lyophilize, (this I peak be DEAE-Sephadex A-25 ion exchange chromatography collection of illustrative plates left side number come first peak) is dissolved in 3ml0.01～0.6mol/L Tris-HCl damping fluid; Through DEAE-Sephadex G-100 gel chromatography, with pH7.5～8.5,0.01～0.02mol/L Tris-HCl buffer solution elution, speed is 0.11～0.17ml/min, collect isolated I peak elutriant (the isolated I of G-100 gel chromatography column peak be Sephadex G-100 chromatography collection of illustrative plates left side number come first peak), after freeze-drying is dry, again through CM-Sephadex C-25 cation-exchange chromatography, with pH 5～6.5, concentration is the NaAc buffer solution elution of 0.009～0.011mol/L, elution flow rate is 0.25ml/min, gets main peak and gets final product.Use high performance liquid chromatography (HPLC) and SDS-amine gel disk electrophoresis (SDS-PAGE) to identify its purity then, molecular weight determination takes SDS-amine gel disk electrophoresis to measure.
Trimeresurus albolabris defibrase is as the application of preparation treatment thrombus disease.
The pharmacological results with the trimeresurus albolabris defibrase of inventing illustrates drug action of the present invention and beneficial effect below:
1. fibrinogen plate assay is measured the fine effect of falling of trimeresurus albolabris defibrase and is got 2% agar-agar soln (with pH 7.4,0.15M/L NaCl, 50mM/L Tris-HCl buffer preparation) 5ml, add 1ml before the bed board and contain 10 unit thrombin solutions, in 37 ℃ of-45 ℃ of water-baths, (use pH7.4 with 0.4% fibrinogen solution, contain 0.15M/L NaCl, 50M/L Tris-HCl buffer preparation) 5ml, pour into after the mixing in the culture dish (diameter 90mm) of sterilizing, put after room temperature forms gel, " dressing plate ".With dressing plate in 85 ℃ of incubators, heat take out behind the 30min " heated plate ".Beat the aperture of diameter 3mm as required on flat board, every hole application of sample 10ul puts 37 ℃ of insulation 18h, takes out observations.The result shows that trimeresurus albolabris defibrase is can both solution fibrin on the dull and stereotyped and heated plate at general fibre albumen former, illustrates that it falls fine mechanism is based on direct fibrinolytic; Fibrinolytic activity obviously is better than the thick poison of Isodose;
And with the solusphere diameter amount double-log that fine component falls in Trimeresurus albolabris (Gray) is mapped, there is certain dose-effect relationship in the two.
2.SDS-amine gel disk electrophoresis (SDS-PAGE) method is measured the fine effect of falling of trimeresurus albolabris defibrase
Get 2.5mg/ml Fibrinogen and trimeresurus albolabris defibrase at pH7.4, contain 0.15mol/LNaCl, mix among the 50mmol/L Tris-HCI buffer, 37 ℃ of water are educated insulation, got 50ul solution in 5 minutes, 30 minutes, 2 hours in insulation respectively, with 50ul electrophoresis sample preparation liquid (2%SDS, 5% β-thin basic ethanol) mixing. boiling water boiled 5 minutes.The cooling back is gone up sample and is carried out SDS-PAGE.The result shows the Trimeresurus albolabris (Gray) snake venom fibrinolysis component former A α chain of hydrolysis of fibrin rapidly, slow cracking Fibrinogen B β chain, the γ chain there is not influence (as shown in Figure 1) wherein, Marker is a standard protein, 1 swimming lane is a Fibrinogen, 2 swimming lanes are the Fibrinogen of trimeresurus albolabris defibrase hydrolysis after 5 minutes, 3 swimming lanes are the Fibrinogen of trimeresurus albolabris defibrase hydrolysis after 0.5 hour, and 4 swimming lanes are the Fibrinogen of trimeresurus albolabris defibrase hydrolysis after 2 hours.
The beneficial effect that can draw trimeresurus albolabris defibrase of the present invention from above-mentioned experimental result is:
Trimeresurus albolabris defibrase has the fibrinogenic effect of direct hydrolysis, and former A α chain and the B β chain of hydrolysis of fibrin simultaneously, and generally can only the former a kind of chain of hydrolysis of fibrin from other venin-derived fiber eliminating enzyme, hydrolysis A α chain, hydrolysis B β chain, the snake venom fiber eliminating enzyme of two chains of hydrolysis simultaneously seldom.Therefore, trimeresurus albolabris defibrase of the present invention is that a kind of having that oneself is developed from the distinctive medicinal organism resource of China can while hydrolysis of fibrin former A α chain and the active metalloprotease of B β chain.
Description of drawings
Fig. 1 is the SDS-PAGE collection of illustrative plates of trimeresurus albolabris defibrase hydrolysis of fibrin former A α chain and B β chain;
Fig. 2 is a DEAE-Sephadex A-25 ion exchange chromatography collection of illustrative plates;
Fig. 3 is a Sephadex G-100 chromatography collection of illustrative plates;
Fig. 4 is a CM-Sephadex C-50 ion exchange chromatography collection of illustrative plates;
Embodiment
(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom:
Employing is stung the ware method of gathering venom and is gathered snake venom from the Trimeresurus albolabris (Gray) snake, the Trimeresurus albolabris (Gray) snake venom of collecting concentrates postlyophilization to make the thick malicious lyophilized powder of poison, get the thick malicious lyophilized powder 1g of Trimeresurus albolabris (Gray) snake venom, be dissolved in 5ml pH8.0,0.05mol in the Tris-HCl damping fluid, with the centrifugal 10min of 3000r/min, it is stand-by to get supernatant liquor with whizzer;
(2) DEAE-Sephadex A-25 ion exchange chromatography
Use concentration 0.05mol/L, the Tris-HCl damping fluid balance SephadexA-25 post of pH8.0, chromatography column: 2cmx60cm; With sample on the supernatant liquor that makes in the step (1), with the linear tonsure wash-out of the 0.05mol Tris-HCl damping fluid of concentration range 0.6mol/L NaCl1000ml, elution flow rate is 0.25ml/min, (referring to Fig. 2) is dissolved in the I peak elutriant lyophilize that the SephadexA-25 column chromatography for separation goes out in the 3ml 0.6mol/L Tris-HCl damping fluid and makes stand-by solution;
(3) DEAE-Sephadex G-100 gel chromatography
With pH 7.6,0.01mol/L Tris-HCl damping fluid balance DEAE-Sephadex G-100 gel chromatography column, with sample on the stand-by solution that makes at last in the step (2), elution flow rate is 0.14ml/min, (referring to figure
3) the isolated I of DEAE-Sephadex G-100 gel chromatography column peak elutriant lyophilize isolate is stand-by;
(4) CM-Sephadex C-25 cation-exchange chromatography
With pH 5.8, the NaAc damping fluid balance CM-Sephadex C-50 chromatography column of concentration 0.01mol/L, sample on the I peak lyophilize isolate that step (3) is obtained is at last used above-mentioned buffer solution elution, and elution flow rate is 0.25ml/min.CM-Sephadex C-50 ion exchange chromatography collection of illustrative plates as shown in Figure 4.
(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom:
Employing is stung the ware method of gathering venom and is gathered snake venom from the Trimeresurus albolabris (Gray) snake, the Trimeresurus albolabris (Gray) snake venom of collecting concentrates postlyophilization to make the thick malicious lyophilized powder of poison, get the thick malicious lyophilized powder 5g of Trimeresurus albolabris (Gray) snake venom, be dissolved in 5ml pH8.5,0.06mol in the Tris-HCl damping fluid, with the centrifugal 12min of 5000r/min, it is stand-by to get supernatant liquor with whizzer;
(2) DEAE-Sephadex A-25 ion exchange chromatography
Use concentration 0.06mol/L, the Tris-HCl damping fluid balance SephadexA-25 post of pH 7.0, chromatography column: 2cmx60cm; With sample on the supernatant liquor that makes in the step (1), with the linear tonsure wash-out of the 0.06mol Tris-HCl damping fluid of concentration range 0.01mol/L NaCl1000ml, elution flow rate is 0.30ml/min, I peak elutriant lyophilize with the SephadexA-25 column chromatography for separation goes out is dissolved in the 3ml 0.01mol/LTris-HCl damping fluid and makes stand-by solution;
(3) DEAE-Sephadex G-100 gel chromatography
With pH 8.5,0.02mol/L Tris-HCl damping fluid balance DEAE-Sephadex G-100 gel chromatography column, with sample on the stand-by solution that makes at last in the step (2), elution flow rate is 0.17ml/min, and the isolated I of DEAE-Sephadex G-100 gel chromatography column peak elutriant lyophilize isolate is stand-by;
(4) CM-Sephadex C-25 cation-exchange chromatography
With pH 6.5, the NaAc damping fluid balance CM-Sephadex C-50 chromatography column of concentration 0.011mol/L, sample on the I peak lyophilize isolate that step (3) is obtained is at last used above-mentioned buffer solution elution, and elution flow rate is 0.20ml/min.The collection main peak gets final product.
(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom:
Employing is stung the ware method of gathering venom and is gathered snake venom from the Trimeresurus albolabris (Gray) snake, the Trimeresurus albolabris (Gray) snake venom of collecting concentrates postlyophilization to make the thick malicious lyophilized powder of poison, get the thick malicious lyophilized powder 2.5g of Trimeresurus albolabris (Gray) snake venom, be dissolved in 5mlpH 7.5,0.04mol in the Tris-HCl damping fluid, with the centrifugal 8min of 4500r/min, it is stand-by to get supernatant liquor with whizzer;
(2) DEAE-Sephadex A-25 ion exchange chromatography
Use concentration 0.04mol/L, the Tris-HCl damping fluid balance SephadexA-25 post of pH 8.0, chromatography column: 2cmx60cm; With sample on the supernatant liquor that makes in the step (1), 0.04mol Tris-HCl damping fluid linear gradient elution with concentration range 0.6mol/LNaCl1000ml, elution flow rate is 0.20ml/min, I peak elutriant lyophilize with the SephadexA-25 column chromatography for separation goes out is dissolved in the 3ml 0.6mol/LTris-HCl damping fluid and makes stand-by solution;
(3) DEAE-Sephadex G-100 gel chromatography
With pH 7.5,0.01mol/L Tris-HCl damping fluid balance DEAE-Sephadex G-100 gel chromatography column, with sample on the stand-by solution that makes at last in the step (2), elution flow rate is 0.11ml/min, and the isolated I of DEAE-Sephadex G-100 gel chromatography column peak elutriant lyophilize isolate is stand-by;
(4) CM-Sephadex C-25 cation-exchange chromatography
With pH 5, the NaAc damping fluid balance CM-Sephadex C-50 chromatography column of concentration 0.009mol/L, sample on the I peak lyophilize isolate that step (3) is obtained is at last used above-mentioned buffer solution elution, and elution flow rate is 0.30ml/min.The collection main peak gets final product.
Claims (3)
1, trimeresurus albolabris defibrase is characterized in that separating from the venom of China Trimeresurus albolabris (Gray) snake pure
Change a kind of metalloprotease that obtains, and have the activity of while fibrin degradation former A α chain and B β chain,
It is 66300 dalton under reduction of the apparent molecular weight on the polyacrylamide gel electrophoresis and non-reduced condition.
2, a kind of preparation method of trimeresurus albolabris defibrase is characterized in that may further comprise the steps:
(1) pre-treatment of Trimeresurus albolabris (Gray) snake venom: the Trimeresurus albolabris (Gray) snake venom of collection concentrates postlyophilization to make thick malicious lyophilized powder, get the thick malicious lyophilized powder 1～5g of Trimeresurus albolabris (Gray) snake venom, be dissolved in 5ml pH 7.5～8.5,0.04 in～0.06mol Tris-HCl the damping fluid, with the centrifugal 8～12min of 3000～5000r/min, it is stand-by to get supernatant liquor with whizzer;
(2) DEAE-Sephadex A-25 ion exchange chromatography
With concentration 0.04～0.06mol/L, the Tris-HCl damping fluid balance SephadexA-25 post of pH 7.0～8.0; With sample on the supernatant liquor that makes in the step (1), with concentration range 0.01～0.6mol/LNaCl 1000ml and the linear tonsure wash-out of 0.04～0.06mol Tris-HCl damping fluid, elution flow rate is 0.20～0.30ml/min, with the I peak elutriant lyophilize that the SephadexA-25 column chromatography for separation goes out, be dissolved in 3ml 0.01～0.6mol/L Tris-HCl damping fluid and make stand-by solution;
(3) DEAE-Sephadex G-100 gel chromatography
With pH 7.5～8.5,0.01～0.02mol/L Tris-HCl damping fluid balance DEAE-SephadexG-100 gel chromatography column, with sample on the stand-by solution that makes at last in the step (2), elution flow rate is 0.11～0.17ml/min, and the isolated I of DEAE-Sephadex G-100 gel chromatography column peak elutriant lyophilize isolate is stand-by;
(4) CM-Sephadex C-25 cation-exchange chromatography
With pH 5～6.5, the NaAc damping fluid balance CM-SephadexC-50 chromatography column of concentration 0.009～0.011mol/L, sample on the stand-by isolate of I lyophilize that step (3) is obtained is at last used above-mentioned buffer solution elution, and elution flow rate is 0.20～0.30ml/min.
3, trimeresurus albolabris defibrase is as the application of preparation treatment thrombus disease.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732700B (en) * | 2009-12-18 | 2012-07-25 | 刘天银 | Health-care wine and preparation method thereof |
| CN105567666A (en) * | 2016-01-07 | 2016-05-11 | 贵州省中国科学院天然产物化学重点实验室 | Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
| CN110791491A (en) * | 2019-12-11 | 2020-02-14 | 昆明龙津药业股份有限公司 | Method for extracting defibrase from snake venom |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1189559C (en) * | 2003-02-28 | 2005-02-16 | 边六交 | Method for separating and purifying batroxobin from snake venom |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101732700B (en) * | 2009-12-18 | 2012-07-25 | 刘天银 | Health-care wine and preparation method thereof |
| CN105567666A (en) * | 2016-01-07 | 2016-05-11 | 贵州省中国科学院天然产物化学重点实验室 | Anticoagulant drug based on cobra venom PIII type metalloprotease and applications thereof |
| CN105567666B (en) * | 2016-01-07 | 2020-06-02 | 贵州省中国科学院天然产物化学重点实验室 | Anticoagulant based on cobra venom PIII type metalloprotease and application thereof |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
| CN110791491A (en) * | 2019-12-11 | 2020-02-14 | 昆明龙津药业股份有限公司 | Method for extracting defibrase from snake venom |
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