CN1920017A - Separating and purifying method of agkistrodonacutus thrombin - Google Patents
Separating and purifying method of agkistrodonacutus thrombin Download PDFInfo
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- CN1920017A CN1920017A CN 200610122176 CN200610122176A CN1920017A CN 1920017 A CN1920017 A CN 1920017A CN 200610122176 CN200610122176 CN 200610122176 CN 200610122176 A CN200610122176 A CN 200610122176A CN 1920017 A CN1920017 A CN 1920017A
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- phosphoric acid
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Abstract
The invention relates the method for separating and purifying pallas-pit viper thrombosin, comprising the following steps: dissolving venom with phosphate buffered saline, removing pyrogen with chromatography column, second purifying, dialyzing, purifying again, third purifying, desalting, and certifying. The purity of pallas-pit viper thrombosin is above 95%, and the productivity is up to 6-8%.
Description
Technical field
The present invention relates to the echidnotoxin field, refer in particular to a kind of separation purification method of Halase.
Background technology
Utilization zymoplasm of separation and purification from snake venom is used for haemostatic medicament, and the clinical use of real a large amount of input is at present produced " Botropase " (Reptilase) by the plain high pharmaceutical factory of Basel, SUI; These product are zymoplasms of separation and purification from Brazilian spearhead pallas pit viper (Bothropejararaca) poison, all have been widely used in clinically in many countries, prove a kind of efficient, safe haemostatic medicament, and China also has a large amount of imports to be used for clinical use every year.This situation for a change, Chinese patent ZL0115567.1 " Ahylysantinfarctase thrombase and production method thereof ", disclose and utilized the Halase I that separation and purification obtains in special product of China agkistrodon acutus (Agkisrodon acutus) poison, its molecular weight is 30000, can be used as the monomer hemostatic agent clinically and uses.The present inventor finds also to contain another kind of zymoplasm by the research to ahylysantinfarctase in ahylysantinfarctase, can pass through processing method comparatively easily, and the separation and purification from the thick liquid of snake venom of this zymoplasm is come out.
Summary of the invention
The novel method that the purpose of this invention is to provide a kind of Halase separation and purification, have two molecular weight by the Halase of the inventive method separation and purification from the thick poison of agkistrodon acutus and be respectively 13875Da and 14581Da subunit, the ingredient amount is 29099Da, relative content greater than 95% single component protein, can be used for haemostatic medicament.
The object of the present invention is achieved like this: a kind of separation purification method of Halase is characterized in that comprising the steps: that the thick poison of (1) agkistrodon acutus adds the phosphoric acid buffer dissolving and makes thick malicious solution; (2) chromatography column is used the phosphoric acid buffer balance with NaOH solution removal pyrogen; (3) sample on the thick malicious solution, Xian takes off with 0 → 0.1M NaCl phosphoric acid buffer gradient, carries out purifying one time, collects with short human plasma coagulation activity, the Halase target component that HPLC and SDS-PAGE identify; (4) the target component of a purifying is dialysed; (5) the target component after the dialysis carries out chromatography once more, with 0 → 0.1MNaCl phosphoric acid buffer gradient elution, collects the high purity Halase target component of identifying with short human plasma coagulation activity, HPLC and SDS-PAGE; (6) set by step the method for (5) is carried out purifying for the third time to the component of collecting of purification of target once more; (7) to the Halase behind the chromatography for the third time with the desalination of Sephadex G-25 chromatography column; (8) to the short human plasma coagulation activity of the warp of the Halase behind the purifying, HPLC, the SDS-PAGE electrophoresis is identified.
Phosphoric acid buffer described in the separation purification method of above-mentioned a kind of Halase is the mixing solutions of Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, concentration 0.01M, pH7.6.
The described dialysis condition of step in the separation purification method of above-mentioned a kind of Halase (4) is: at 4 ℃ of phosphoric acid buffer dialysis 15h with 3～5 times of volumes, change liquid 4～5 times.
Method of the present invention utilizes phosphoric acid buffer that the thick poison of agkistrodon acutus is carried out separation and purification, the Halase purity height (greater than 95%) of separation and purification, and yield higher (can reach 6～8%) can provide a kind of new way for effective utilization of ahylysantinfarctase.
Description of drawings
Below in conjunction with drawings and Examples the present invention is described in further detail, but does not constitute any limitation of the invention.
Fig. 1 is a process flow diagram of the present invention.
Embodiment
Consult shown in Figure 1, the separation purification method of a kind of Halase of the present invention, its technical process is: phosphoric acid buffer dissolving snake venom → chromatography column is removed a pyrogen back balance → purifying → dialysis → purifying → three time purifying → desalination once more → evaluation, and details are as follows below by specific embodiment:
Embodiment 1
1. take by weighing ahylysantinfarctase 50g (supporting She Chang by Guangxi snake venom institute provides), add the phosphoric acid buffer dissolving of 400m10.01MpH7.6, refrigerate standby.
2.DEAE-Sepharose Fast Flow chromatography column is removed pyrogen and is handled
Chromatography column (3.6 * 4.0cm) and tubing system with the flushing of 200ml 1M NaOH solution, the about 500ml/h of flow velocity washes with water for injection again, the about 1500ml/h of flow velocity is until the spout alkali-free liquid.
3.DEAE-Sepharose Fast Flow chromatography column balance
The chromatography column of pyrogen will be removed, with the phosphoric acid buffer flushing of 0.01M pH7.6, the about 1500ml/h of flow velocity.Approximately the 3500ml phosphoric acid buffer can make spout pH consistent with the pH of phosphoric acid buffer, promptly reaches balance.
4.DEAE-Sepharose purifying of Fast Flow chromatography column
Snake venom solution is pumped into last sample in the post, with above-mentioned phosphoric acid buffer wash-out, the about 1500ml/h of flow velocity, the protein peak of absorbed component does not significantly descend, behind the lowest point, begin to carry out straight line gradient Xian and take off, collect elution fraction, determine target components with short human plasma coagulation activity, HPLC and SDS-PAGE with the phosphoric acid buffer that 6000ml contains 0 → 0.1M NaCl.
5. dialysis
The target components of collecting is poured in the dialysis tubing, at 4 ℃ of about 15h of above-mentioned phosphoric acid buffer dialysis with 5 times of volumes, changes liquid 5 times, to remove small-molecule substance.
6.DEAE-Sepharose Fast Flow chromatography column is purifying once more
The target component that dialysis is collected, after the last sample, phosphoric acid buffer gradient Xian who contains 0 → 0.1M NaCl with 3000ml takes off, and collects elution fraction, determines target components with short human plasma coagulation activity, HPLC and SDS-PAGE equally.
7.DEAE-Sepharose three purifying of Fsat Flow
Experimental implementation is consistent with the purifying once more of program and DEAE-Sepharose Fast Flow.
8.Sephadex G25 desalination
Sephadex G25 chromatography column is earlier washed post with 2000ml1M NaOH, the removal pyrogen, and washing to spout with water for injection does not have alkalescence.Sample on the target components of purifying is used the water for injection wash-out, and the about 1800ml/h of flow velocity collects elution peak, the i.e. about 400mg of the purified product of Halase.
The Halase qualification result of the inventive method separation and purification is as follows:
1. detect through the Waters high performance liquid chromatograph, Halase is the protein of one-component, the relative percentage composition of principal constituent＞95%.
2. show the protein that Halase is made of 15.3KD and 14.2KD subunit through SDS-polyacrylamide gel electrophoresis detected result.Measure through time-of-flight mass spectrometer, the molecular weight that Halase is made up of two subunits (molecular weight is respectively 13875Da and 14581Da) is the protein of 29099Da.
3. the isoelectric point determination result shows, the iso-electric point of Halase (pI) is 5.73.
4. the sugar determination result shows, the sugared content of the neutrality of Halase is 2.15%, shows that this product is a kind of glycoprotein.
5. human plasma setting time: 60 ± 20 seconds.
6. amino acid sequence analysis
Detecting instrument: sequenator ABI Procise 491.
The terminal amino acid sequence measurement result is as follows:
| Sequence 1 NO. | Residue | Sequence 1 NO. | Residue |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | Asp Cys Ser Ser Gly Trp Ser Ser Tyr Glu Gly His Cys Tyr Lys | 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 | Asp Cys Pro Ser Asp Trp Ser Ser Tyr Glu Gly His Cys Tyr Lys |
7. uv-spectrophotometric scanning
Halase is dissolved in water, and shows that in the result of 200nm-400nm wavelength region interscan this product has maximum absorption band at the 280nm place.
In sum, the Halase of method of the present invention institute separation and purification is a kind of protein-based haemostatic medicament.
Claims (3)
1. the separation purification method of a Halase is characterized in that comprising the steps: that the thick poison of (1) agkistrodon acutus adds the phosphoric acid buffer dissolving and makes thick malicious solution; (2) chromatography column is used the phosphoric acid buffer balance with NaOH solution removal pyrogen; (3) sample on the thick malicious solution, Xian takes off with 0 → 0.1M NaCl phosphoric acid buffer gradient, carries out purifying one time, collects with short human plasma coagulation activity, the Halase target component that HPLC and SDS-PAGE identify; (4) the target component of a purifying is dialysed; (5) the target component after the dialysis carries out chromatography once more, with 0 → 0.1MNaCl phosphoric acid buffer gradient elution, collects the high purity Halase target component of identifying with short human plasma coagulation activity, HPLC and SDS-PAGE; (6) set by step the method for (5) is carried out purifying for the third time to the component of collecting of purification of target once more; (7) to the Halase behind the purifying for the third time with the desalination of Sephadex G-25 chromatography column; (8) to the short human plasma coagulation activity of the warp of the Halase behind the purifying, HPLC, the SDS-PAGE electrophoresis is identified.
2. method according to claim 1 is characterized in that described phosphoric acid buffer is the mixing solutions of Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, concentration 0.01M, pH7.6.
3. method according to claim 1 is characterized in that the described dialysis condition of step (4) is: at 4 ℃ of phosphoric acid buffer dialysis 15h with 3～5 times of volumes, change liquid 4～5 times.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610122176 CN1920017A (en) | 2006-09-18 | 2006-09-18 | Separating and purifying method of agkistrodonacutus thrombin |
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| CN 200610122176 CN1920017A (en) | 2006-09-18 | 2006-09-18 | Separating and purifying method of agkistrodonacutus thrombin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN105586330A (en) * | 2016-03-25 | 2016-05-18 | 中山大学 | Haemocoagulase acutus thrombin and preparation method thereof |
-
2006
- 2006-09-18 CN CN 200610122176 patent/CN1920017A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN102757948B (en) * | 2012-05-28 | 2014-02-05 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN105586330A (en) * | 2016-03-25 | 2016-05-18 | 中山大学 | Haemocoagulase acutus thrombin and preparation method thereof |
| CN105586330B (en) * | 2016-03-25 | 2019-04-02 | 中山大学 | A kind of Halase and preparation method thereof |
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Open date: 20070228 |