CN1159437C - Ahylysantinfarctase thrombase and its production process - Google Patents
Ahylysantinfarctase thrombase and its production process Download PDFInfo
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- CN1159437C CN1159437C CNB011155671A CN01115567A CN1159437C CN 1159437 C CN1159437 C CN 1159437C CN B011155671 A CNB011155671 A CN B011155671A CN 01115567 A CN01115567 A CN 01115567A CN 1159437 C CN1159437 C CN 1159437C
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- Prior art keywords
- reptilase
- ahylysantinfarctase
- thrombase
- subunit
- thrombin
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- Expired - Lifetime
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- 108090000190 Thrombin Proteins 0.000 title claims abstract description 25
- 238000004519 manufacturing process Methods 0.000 title abstract description 7
- 108010027612 Batroxobin Proteins 0.000 claims abstract description 53
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- 230000002439 hemostatic effect Effects 0.000 claims abstract description 8
- 238000004440 column chromatography Methods 0.000 claims abstract description 7
- 238000001742 protein purification Methods 0.000 claims abstract description 7
- 238000013016 damping Methods 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 18
- 230000011218 segmentation Effects 0.000 claims description 9
- 235000001014 amino acid Nutrition 0.000 claims description 8
- 238000010612 desalination reaction Methods 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 238000007670 refining Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 16
- 239000003998 snake venom Substances 0.000 abstract description 14
- 229940088598 enzyme Drugs 0.000 abstract description 13
- 238000000746 purification Methods 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 7
- 241001505404 Deinagkistrodon acutus Species 0.000 abstract description 5
- 238000000926 separation method Methods 0.000 abstract description 5
- 229960004072 thrombin Drugs 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 4
- 238000005259 measurement Methods 0.000 abstract description 2
- 150000001450 anions Chemical class 0.000 abstract 1
- 239000003053 toxin Substances 0.000 abstract 1
- 231100000765 toxin Toxicity 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 239000011780 sodium chloride Substances 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000008176 lyophilized powder Substances 0.000 description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 241001474977 Palla Species 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 5
- 239000012507 Sephadex™ Substances 0.000 description 5
- 241000271897 Viperidae Species 0.000 description 5
- 230000023555 blood coagulation Effects 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 101000772006 Bombus ignitus Venom serine protease Bi-VSP Proteins 0.000 description 3
- 241001449342 Chlorocrambe hastata Species 0.000 description 3
- 239000012614 Q-Sepharose Substances 0.000 description 3
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000005515 capillary zone electrophoresis Methods 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000271039 Agkistrodon Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241000270295 Serpentes Species 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 229940030225 antihemorrhagics Drugs 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 239000002874 hemostatic agent Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 231100000611 venom Toxicity 0.000 description 2
- 241000271517 Bothrops jararaca Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 102000002067 Protein Subunits Human genes 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960002210 batroxobin Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 230000000025 haemostatic effect Effects 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention provides agkistrodon acutus venin thrombin I having the hemostatic function and a production method thereof, which has the technical scheme that anion exchanger column chromatography is used for separation and purification, and a fast protein purification workstation program is used for repurification, and agkistrodon acutus venin thrombin I with the purity of more than 97% is obtained from agkistrodon acutus venin of a local product in China; the measurement shows that the thrombin is composed of two subunits (chains) of A and B, wherein the subunit A contains 135 amino acids, the subunit B contains 126 amino acids. Compared with the existing reptilase (such as Reptilase), the present invention is totally new enzyme hemostatic; the research of the medicine theory, toxin theory, medicine generation, etc. shows that the agkistrodon acutus venin thrombin I is new hemostatic having the advantages of instant effect, small side effect, high efficiency and high safety.
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 132 amino acid through determined amino acid sequence A chain, the B chain is made up of 123 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.
Summary of the invention
Technical scheme of the present invention:
Technical scheme of the present invention is as shown below:
Injection Ahylysantinfarctase thrombase I ... his research of Xingqi of the going forward side by side of → feature of identifying reptilase I.
Particularly, technical scheme 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 enzyme) component;
2. wherein two reptilase I components get high purity 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 at 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 elution through the anionite column chromatography;
(2) with the thick component of reptilase I of gained again through twice above column chromatography purification of anionite, use the 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 is respectively 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 NO 1) by 132 amino acid, and B subunit (chain) is formed (its sequence such as SEQ ID NO 2) by 123 amino acid.By comparison, Reptilase is a strand, is made up of 230 amino acid.Therefore, Ahylysantinfarctase thrombase I is a brand-new hemostatic agent fully.It also can not be produced with the clone from present technology.
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-Sephadex A-50 or QAE-SephadexA-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 the 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.
Description of drawings:
Fig. 1 represents the pairing of A subunit and B subunit.
Embodiment
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 NaCl 0-1.0M, and elutriant is collected with automatic Fraction Collector, every pipe 8-10ml, per hour 7-8 pipe.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 peak has this enzyme activity, and 5,6 peaks are defibrase (fiber eliminating enzyme), and 7,8 peaks are reptilase.
Get 8 peaks (about 80-100ml) through the about 100mg of G-25 desalination freeze-drying, go up Q-Sepharose post (3 * 110cm again
2), with straight line gradient elution of NaCl (0-0.6M), 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 (the about 90-94% of purity) through the 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 the 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 pipe.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, the 5-8 peak has 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 straight line gradient elution of NaCl (0-0.6M), 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 the 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 again through the G-25 desalination, lyophilize gets lyophilized powder 45mg, 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 that the present invention relates to
SEQ ID NO 1 (aminoacid sequence of Ahylysantinfarctase thrombase I subunit A):
10 20 30 40
DFNCPPGWSAYDHCYIKEPKNWDDAEKFCTEQADGGHLVS
50 60 70 80
IESKGERDFLAQLVSQSIESIEDHVWTGLRLQNKEKQCST
90 100 110 120
EWSDGSSLSFENLLELYMRKCGALDRDTGFHKWINLGCIQ
130
LNPFVCKFPPQC
SEQ ID NO 2 (aminoacid sequence of Ahylysantinfarctase thrombase I subunit B):
10 20 30 40
GFCCPLRWSSYEHCYVKEKKTWDDAEKFCTEQRKGGHLVS
50 60 70 80
IHSKEEADFLVHLAYP----ILDLSLIWLGLSNMWNDCKREWSD
90 100 110
GSKLDFKSWAKTSDCLIGKTDGDN---QWINLD
120
CSKKHYFVCKFKL--
Claims (3)
1. an Ahylysantinfarctase thrombase I is characterized in that this enzyme I is made up of A and two subunits of B, wherein, the A subunit is made up of 132 amino acid, have the aminoacid sequence shown in the SEQ ID NO 1, the B subunit is made up of 123 amino acid, has the aminoacid sequence shown in the SEQ ID NO 2.
2. the pharmaceutical composition with clinical hemostatic function is characterized in that this pharmaceutical composition contains the Ahylysantinfarctase thrombase I and the necessary additional excipient of claim 1.
3. a method of producing Ahylysantinfarctase thrombase I as claimed in claim 1 is characterized in that this method comprises the steps:
(1) dissolve ahylysantinfarctase 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, purifying obtains refining 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011155671A CN1159437C (en) | 2001-04-29 | 2001-04-29 | Ahylysantinfarctase thrombase and its production process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011155671A CN1159437C (en) | 2001-04-29 | 2001-04-29 | Ahylysantinfarctase thrombase and its production process |
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| CN1332241A CN1332241A (en) | 2002-01-23 |
| CN1159437C true CN1159437C (en) | 2004-07-28 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1218747C (en) * | 2003-08-14 | 2005-09-14 | 吴忠 | Medicinal use of agkistrodon acutus thrombase for treating hemorrhagic disease |
| 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 |
| CN101358184A (en) * | 2008-09-27 | 2009-02-04 | 康辰医药股份有限公司 | Hemocoagulase agkistrodon |
| 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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2001
- 2001-04-29 CN CNB011155671A patent/CN1159437C/en not_active Expired - Lifetime
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| Publication number | Publication date |
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| CN1332241A (en) | 2002-01-23 |
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