CN1332241A - Agkistrodon acutus reptilase and its production method - Google Patents
Agkistrodon acutus reptilase and its production method Download PDFInfo
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- CN1332241A CN1332241A CN 01115567 CN01115567A CN1332241A CN 1332241 A CN1332241 A CN 1332241A CN 01115567 CN01115567 CN 01115567 CN 01115567 A CN01115567 A CN 01115567A CN 1332241 A CN1332241 A CN 1332241A
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- reptilase
- ahylysantinfarctase
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Abstract
本发明提供一种具有止血功能的尖吻蝮蛇毒凝血酶Ⅰ及其生产方法。经阴离子交换剂柱层析法分离纯化和快速蛋白纯化工作站程序再纯化,本发明从我国特产尖吻蝮(Agkistrodon acutus)的蛇毒中得到纯度在97%以上的尖吻蝮蛇毒凝血酶Ⅰ;经测定,该酶Ⅰ由A、B两个亚基(链)组成,其中,A亚基含有135个氨基酸,B亚基含有126个氨基酸。与现有蛇毒凝血酶(如Reptilase)相比,它是一个全新的新型酶止血剂;经药理、毒理、药代等研究表明,本发明的尖吻蝮蛇毒凝血酶Ⅰ是一个见效快、副作用小、安全有效的新型止血剂。The invention provides a Agkistrodon venom thrombin I with hemostatic function and a production method thereof. After separation and purification by anion exchanger column chromatography and re-purification by a fast protein purification workstation program, the present invention obtains Agkistrodon acutus venom thrombin I with a purity of more than 97% from the snake venom of Agkistrodon acutus, a special product in my country; It was determined that the enzyme Ⅰ was composed of two subunits (chains), A and B, among which, the A subunit contained 135 amino acids, and the B subunit contained 126 amino acids. Compared with the existing snake venom thrombin (such as Reptilase), it is a brand-new new enzyme hemostatic agent; pharmacology, toxicology, pharmacokinetics and other studies have shown that the Agkistrodon venom thrombin I of the present invention is a quick-acting, A new type of hemostatic agent with few side effects, safe and effective.
Description
Technical field under the present invention:
The invention belongs to biochemical field of medicaments.Further, the present invention relates to Ahylysantinfarctase thrombase I, this reptilase I has hemostatic function clinically.Research background of the present invention:
The present invention system single component that separation and purification obtains from point kiss Pallas pit viper (Agkistrodon acutus is commonly called as Agkistrodon, the Hundred-pace pit viper) snake venom of China's special product, it has two isozyme, measure through physics and chemistry, Ahylysantinfarctase thrombase I is made up of two subunits, and molecular weight is 30000.Be made up of 135 amino acid through determined amino acid sequence A chain, the B chain is made up of 126 amino acid.Have significantly or short blood coagulation effect of highly significant and anastalsis through the test of pesticide effectiveness, be applicable to the prevention and the treatment of various hemorrhagic diseases.
Abroad, (Holleman, W H. etc., 1976 J..Biol.Chem. such as Holleman, 251:1663.) obtained reptilase (Hemocoagulase, Reptilase, Batroxbin) with affinity chromatography separation and purification in Brazilian spearhead Pallas pit viper (Bothrope jararaca) snake venom, through (Itoh, N etc., J.Biol-Chem.262:3132-3135 such as Itoh.N., 1987) measure, Batroxobin is a strand, is made up of 230 amino acid, and molecular weight is 3100.V.Klobusitzky (1936) at first finds and this zymin (containing at least two above components) is developed into hemostatic agent-Reptilase, and applies for a patent (referring to the Reptilase working instructions).The medicine of China's import " reptilase " promptly is to be produced by the plain tall and big pharmaceutical factory of Basel, SUI (Reptilase).Annual millions of of the imports " reptilase " of China, annual annual sales amount in China can reach more than 3-4 hundred million Renminbi according to estimates.
The raw material of " reptilase " is Brazilian spearhead agkistrodon halyx pallas venom, in the not distribution of this snake of China, and, this product is that (specification sheets comprises two components to the composite components composition, according to our practical measurement component more than three is arranged then) (Mo Weinan etc., " the big pharmacy of China ", 1996,7 (3): 137).According to the general requirement of pharmaceutical preparation, for well, and purity is high more good more with monomer for the composition of medicine.Inventor's separation and purification from point kiss Pallas pit viper (do not belong to together, plant with the spearhead Pallas pit viper genus) snake venom of China's special product obtains the single component Ahylysantinfarctase thrombase I of high purity (more than 97%).
Through pharmacological testing, the inventor finds that this reptilase I has short coagulating and haemostatic effect, and this effect is better than " reptilase " (Reptilase).Toxicity and pharmacokinetic to this reptilase I show that this reptilase I can be applied to clinical fully.
The purpose of this invention is to provide a kind of highly purified monomer hemostatic agent Ahylysantinfarctase thrombase I; This reptilase I has anastalsis clinically.Technical scheme of the present invention:
Technical process of the present invention is as follows:
Injection Ahylysantinfarctase thrombase I ... his research of Xingqi of the going forward side by side of → feature of identifying reptilase I.
Particularly, technical process of the present invention comprises the steps:
1. snake venom dissolves with the Tris-HCl damping fluid, and centrifugation is got supernatant liquor through anion exchange chromatography, with Tris-Hcl damping fluid NaCl straight line gradient elution, can obtain four above snake venom simultaneously and coagulate enzyme sample enzyme (Thrombin like enyemy) component;
2. wherein two reptilase I components must be made with extra care reptilase I solution through twice above purifying of anion exchange chromatography or with quick protein purification workstation program purifying again, through capillary zone electrophoresis, HPLC (anti-phase) chromatogram, nucleus magnetic resonance, record purity 97-99%, form by two subunits.Its yield is 5-8%, and 2000/g snake venom can manufacture a finished product.
When producing Ahylysantinfarctase thrombase I, can also produce fiber eliminating enzyme with treatment thrombus function and Ahylysantinfarctase thrombase II with hemostatic function, this can make valuable snake venom raw material be fully utilized.
3. purified reptilase I is got reptilase I stoste through desalination again;
4. the reptilase I stoste of gained is filled a prescription (adding vehicle) respectively again, filtration, packing, lyophilize, capping promptly get venin for injection zymoplasm I finished product.
5. identify the feature of reptilase I:
(1) with the dissolving of Tris-HCl pH7.2-8.8 damping fluid,, can obtain 4 above rough segmentation venomous snake thrombin sample enzyme components simultaneously with above-mentioned pH of buffer 7.2-8.8 damping fluid sodium-chlor straight line gradient elutions through the anionite column chromatography;
(2) with the thick component of reptilase I of gained again through twice above column chromatography purification of anionite, with pH7.2-8.8 damping fluid, twice above wash-out purifying of sodium-chlor straight line gradient or must make with extra care reptilase I solution with quick protein purification instrument program purifying;
(3) characterized of this enzyme:
A. through capillary zone electrophoresis, can get its relative content is more than 97%;
The visible two protein subunit bands of B.SDS-polyacrylamide gel electrophoresis, molecular weight are respectively
Be 16000,14000;
C. use its enzyme activity of 4mg/ml people (or ox) pure blood fibrinogen assay, its than vigor＞120U/mg through protein sequencing, be two subunits, A subunit (chain) is formed (its sequence such as SEQ ID NO1) by 135 amino acid, and B subunit (chain) is formed (its sequence such as SEQ ID NO2) by 126 amino acid.The pairing of A subunit and B subunit as shown in Figure 1.
By comparison, Reptilase is a strand, is made up of 230 amino acid.
Therefore, Ahylysantinfarctase thrombase I is a brand-new hemostatic agent fully.From present skill
It also can not produce art with the clone.
6. with " reptilase " comparative studies (Reptilase):
The above-mentioned reptilase I that is obtained by the present invention and commercial " reptilase " that can get (Reptilase) are compared research.
Be divided into 4 groups with 32 of rabbit (male and female half and half), every group 8,1,3 groups is Ahylysantinfarctase thrombase I, 1,3U/kg body weight dosage, 2,4 groups with the test of making comparisons of " reptilase " 1,3U/kg body weight dosage, and the hematometry whole blood coagulation time is got by ear vein in 10 minutes, 20 minutes, 1 hour, 3 hours, 7 hours, 12 hours and 24 hours after medication respectively.
Studies show that, the reptilase I instant effect that the present invention obtains (significantly short Blood clotting was promptly arranged in 10 minutes, Reptilase then needs just to have remarkable blood coagulation enhancing effect in the time of 20 minutes), its blood coagulation enhancing effect significantly is better than Reptilase, and non-evident effect.
Make rat, rabbit hemostasis trial through modeling again, experimental result shows that 0.8U/mg body weight dosage promptly has remarkable anastalsis.Show that reptilase I that the present invention obtains has and " reptilase " (Reptilase) identical clinical function.
It should be noted that in operating process of the present invention solution with water when rough segmentation gained reptilase I and repurity is a ultrapure water; The used damping fluid of dissolving snake venom is 0.05-0.10M Tris-HCl damping fluid or glycine-NaOH damping fluid; The used anionite of rough segmentation is DEAE-SephadexA-50 or QAE-Sephadex A-25, and the used sodium-chlor gradient of rough segmentation is 0-1.0M; Purification process is characterized in that twice used anionite of above column chromatography purification is Q-Sepharose, DEAE-Sephadex A-50; Twice used damping fluid of above column chromatography purification wash-out is 0.3-0.9M.With AKTA or the quick protein purification workstation of Backman, used post is Q-post, and its elution process carries out by setup program.And full process operations should be carried out in 10 ± 2 ℃ GMP environment.Beneficial effect of the present invention:
The invention provides a kind of Ahylysantinfarctase thrombase I; This zymoplasm is that separation and purification obtains from ahylysantinfarctase, has adaptable clinically hemostatic function, is single component, has that anthemorrhagic performance is good, the effect of alternative similar medicine.
Embodiments of the invention:
Below narrate embodiments of the invention.Need to prove that embodiments of the invention have only illustration for the present invention and effect without limits.
The production of embodiment one, 990901 crowdes of Ahylysantinfarctase thrombase I:
DEAE-Sephadex A-50 (SIGMA St.Louis, MO 63178 U.S.A.) 80g is soaked more than 12 hours dress post (4 * 110cm with 0.05M pH7.8Tris-Hcl damping fluid
2), reequilibrate.Simultaneously ahylysantinfarctase 3g is dissolved among the above-mentioned damping fluid 10ml dissolving after 2000 rev/mins centrifugal 10 minutes, get the supernatant liquor upper prop.
In storage bottle and each 1500ml of gradient bottle, storage bottle NaCl concentration is 1M with above-mentioned damping fluid, and gradient bottle NaCl concentration is 0, carries out straight line gradient elution of NaCl0-1.0M, and elutriant is collected with automatic Fraction Collector, every pipe 8-10ml, per hour 7-8 pipes.Collect liquid and under 280nm, measure absorbance value, establish its albumen peak number with absorbance value and the mapping of pipe number, the collection liquid at each peak merge the rough segmentation component.
Then each rough segmentation component is measured its thrombin-like enzyme activity, general 5-8 peaks have this enzyme activity, and 5,6 peaks are defibrase (fiber eliminating enzyme), and 7,8 peaks are reptilase.
(about 80-100ml) through the about 100mg of G-25 desalination freeze-drying, goes up Q-Sepharose post (3 * 110cm again to get 8 peaks
2), with NaCl (0-0.6M) straight line gradient elution, collect liquid and measure its absorbance value by last method, and press the peak and collect elutriant in wide-necked bottle, measure enzyme activity and electrophoretogram, establish its desired component, generally be divided into 3 peaks, peak 2 is reptilase I (about 60ml), gets the about 60-80mg of lyophilized powder (purity about 90-94%) through G-25 desalination again.
Lyophilized powder is made into 10mg/ml with 0.05M pH8.0 Tris-HCl damping fluid, with AKTA or the quick protein purification workstation of Backman purifying, Q6-post (6ml) is gone up sample 1ml at every turn, and elution program is 0-30-60% wash-out, is drawn and is received required component by computer program.
8 secondary program separated and collected liquid are merged about 20ml, again through Sephadex G-25 desalination, lyophilize gets the about 40mg of lyophilized powder, be 160U/mg than vigor after testing, through the SDS-polyacrylamide gel electrophoresis is 14000,16000 daltonian two subunits, measuring purity through reverse-phase chromatography is 98%, it is 97% that capillary electrophoresis is measured its purity.
At last, get 6000 of venin for injection zymoplasm I finished products through prescription (adding vehicle), ultrafiltration, packing, freeze-drying, gland, promptly every gram is slightly malicious to get 2000.
The production of embodiment two, 990902 crowdes of Ahylysantinfarctase thrombase I:
To go up example and adorn post (4 * 110cm with DEAE-Sephadex A-50 regeneration back
2) with 0.08M pH8.8Tris-HCl damping fluid balance 8 hours.Simultaneously ahylysantinfarctase 3.0g is dissolved among the above-mentioned damping fluid 10ml dissolving after 2000 rev/mins centrifugal 10 minutes, get the supernatant liquor upper prop.
In storage bottle and each 1500ml of gradient bottle, storage bottle Nacl concentration is 1M with above-mentioned damping fluid, and gradient bottle Nacl concentration is 0, carries out straight line gradient elution of Nacl 0-1.0M, and elutriant is collected with automatic Fraction Collector, every pipe 8-10ml, per hour 7-8 pipes.Collect liquid and under 280nm, measure absorbance value, establish its albumen peak number with absorbance value and the mapping of pipe number, the collection liquid at each peak merge the rough segmentation component.
Then, each rough segmentation component is measured its thrombin-like enzyme activity, generally speaking, 5-8 peaks have this enzymic activity, and 5,6 peaks are fiber eliminating enzyme, and 7,8 peaks are reptilase.
Get 8 peaks (about 80ml) through G-25 post (3 * 110cm
2) the chromatography desalination, lyophilized powder is 90mg, again through Q-Sepharose post (3 * 110cm
2) chromatography, with Nacl (0-0.6M) straight line gradient elution, collect liquid and press its absorbance value of said determination, press the peak and collect elutriant in wide-necked bottle, measure enzyme activity and electrophoretogram, establish it and want component, generally be divided into three peaks, peak 2 is reptilase I (about 65ml), again through G-25 post (3 * 110cm
2) chromatography desalination freeze-drying, getting the about 65mg of lyophilized powder (purity 90-93%), specific activity of enzyme is 160U/mg.
With lyophilized powder 0.08M, pH8.2 Tris-Hcl damping fluid is made into 10mg/ml concentration, quick protein purification workstation purifying with AKTA (amershampharmacia biotech U.S.A.) or Backmam (U.S.A.) production, with Q6-post (6ml), each sample 1ml that goes up, elution program is 0-60% wash-out, is drawn and is collected required component by computer program.
Each secondary program separated and collected liquid is merged about 30ml, and through G-25 desalination, lyophilize gets lyophilized powder 45mg again, through the SDS-polyacrylamide gel electrophoresis is 14000,16000 daltonian two subunits, through capillary zone electrophoresis, rp-hplc analysis, purity is 97.2%.
Add the vehicle prescription at last, ultrafiltration, packing, freeze-drying, gland get 7600 of the every bottle 1 injection Ahylysantinfarctase thrombase I of unit finished products, thick malicious 2533 finished products of promptly every gram.
Attached: the aminoacid sequence SEQ ID NO1 that the present invention relates to (aminoacid sequence of Ahylysantinfarctase thrombase I subunit A):
10 20 30 40DFNCPPGWSAYDHCYIKEPKNWDDAEKFCTEQADGG.H
50 70LVS?I?ESKGERDFLAQLVSQS?IES?IEDHVWTGLRLQN?K?E
88 100 110KQCSTEWSDGS?S?LSFEN?L?LELYMRKCGALDRDTGFHK
120 135WINLGC IQLNPFVCKFPPQCSEQ ID NO2 (aminoacid sequence of Ahylysantinfarctase thrombase I subunit B):
10 20 30 40GFCCPLRWSSYEHCYVKEKKTWDDAEKFCTEQRKGG.H
50 70LVSVHSREEADFLVHLAYP----ILDLS?LIWMGLSNMW
88 100 110NDCKREWSDGTKLDFKSWAKTS?D?CLIGKTDGDN---Q
120 135WINLDCSKKHYFVCKFKL--
Claims (7)
1. Ahylysantinfarctase thrombase I is characterized in that this enzyme I is that separation and purification obtains from the snake venom of the point kiss Pallas pit viper of China's special product, has hemostatic function, and is made up of A and two subunits of B;
2. the Ahylysantinfarctase thrombase I of claim 1, wherein said hemostatic function is meant that this enzyme I can be used for mammiferous clinically hemostasis;
3. the Ahylysantinfarctase thrombase I of claim 1, wherein said hemostatic function is meant that this enzyme I can be used for people's hemostasis clinically;
4. the Ahylysantinfarctase thrombase I of claim 1, two subunits of wherein said A and B (chain) are made up of 135 and 126 amino acid respectively, and have the aminoacid sequence shown in SEQ ID NO1 and SEQ ID NO2;
5. the pharmaceutical composition with clinical hemostatic function is characterized in that this pharmaceutical composition contains the Ahylysantinfarctase thrombase I with hemostatic function;
6. the pharmaceutical composition of claim 5, wherein said pharmaceutical composition also contains pharmaceutically acceptable carrier and/or vehicle;
7. a method of producing Ahylysantinfarctase thrombase I is characterized in that this method comprises the steps:
(1) ahylysantinfarctase dissolves with damping fluid, centrifugation, and, use buffer solution elution through the anionite column chromatography, can obtain defibrase (fiber eliminating enzyme) and reptilase rough segmentation component simultaneously;
(2) with the thick component of reptilase I of gained more respectively through anionite column chromatography and quick protein purification workstation program, purifying must be made with extra care reptilase I solution;
(3) smart reptilase I solution is got reptilase I stoste through desalination;
(4) gained reptilase I stoste is filled a prescription, filtration, packing and lyophilize.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB011155671A CN1159437C (en) | 2001-04-29 | 2001-04-29 | Agkistrodon venom thrombin and production method thereof |
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| CNB011155671A CN1159437C (en) | 2001-04-29 | 2001-04-29 | Agkistrodon venom thrombin and production method thereof |
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| CN1332241A true CN1332241A (en) | 2002-01-23 |
| CN1159437C CN1159437C (en) | 2004-07-28 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005017139A1 (en) * | 2003-08-14 | 2005-02-24 | Liu, Xianying | Thrombin from venom of agkistrodon acutus used as drugs for the treatment of haemorrhage |
| WO2010034176A1 (en) * | 2008-09-27 | 2010-04-01 | 康辰医药股份有限公司 | A throbin-like enzyme of agkistrodon acutus |
| CN101684151B (en) * | 2008-09-26 | 2012-06-20 | 江苏正大天晴药业股份有限公司 | Protein matter with coagulation activity |
| 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 |
-
2001
- 2001-04-29 CN CNB011155671A patent/CN1159437C/en not_active Expired - Lifetime
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005017139A1 (en) * | 2003-08-14 | 2005-02-24 | Liu, Xianying | Thrombin from venom of agkistrodon acutus used as drugs for the treatment of haemorrhage |
| CN101684151B (en) * | 2008-09-26 | 2012-06-20 | 江苏正大天晴药业股份有限公司 | Protein matter with coagulation activity |
| WO2010034176A1 (en) * | 2008-09-27 | 2010-04-01 | 康辰医药股份有限公司 | A throbin-like enzyme of agkistrodon acutus |
| US8476054B2 (en) | 2008-09-27 | 2013-07-02 | Konruns Pharmaceutical Co., Ltd. | Thrombin-like enzyme of Agkistrodon acutus |
| 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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| Publication number | Publication date |
|---|---|
| CN1159437C (en) | 2004-07-28 |
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