CN1537938A - New earthoworm plasmin and its preparation method - Google Patents
New earthoworm plasmin and its preparation method Download PDFInfo
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- CN1537938A CN1537938A CNA031219500A CN03121950A CN1537938A CN 1537938 A CN1537938 A CN 1537938A CN A031219500 A CNA031219500 A CN A031219500A CN 03121950 A CN03121950 A CN 03121950A CN 1537938 A CN1537938 A CN 1537938A
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Abstract
A novel plasmin extracted from earthworm and having 21 KD for its molecular weight, its preparing process, the composition containing it, and its application in preparing the medicine for treating thrombus are disclosed.
Description
The present invention relates to extract a kind of new plasmin, and relate to the preparation method of this plasmin, contain the composition of this plasmin from earthworm, and the purposes of this plasmin in the medicine of preparation treatment blood embolism disease.
At present, because the blood embolism disease that a variety of causes causes, as cerebral thrombosis, myocardial infarction, coronary heart disease, the sickness rate of extremity vascular embolism etc. is in rising trend; Only China is used to prevent and treat expense with aspect such as rehabilitation every year up to hundreds billion of yuans, this type of disease serious harm patient's life quality, increased the burden of patient family and society.Thereby, also make progress rapidly at the research and development of the clinical application of this type of disease; Methods of treatment comparatively commonly used clinically is the surprise thrombolytic therapy, and common medicine is: urokinase, fiber eliminating enzyme etc.This type of medicine must be at patient's back six hours innerlich anwendens of falling ill, and by quick thrombolysis, avoid half blanking bar death of the non-reversibility that causes because of ischemic, thereby do not stay sequela to the patient.This just requires thrombolytic drug to have thrombolytic efficiently effect, has no side effect simultaneously.
At present, domestic listing or at the thrombolytic drug that grinds much from raw material sources, can be divided into four classes:
(1) gene recombination product: as recombined streptokinase, lepirudin 023 ludon, t-PA etc.
(2) Bio-engineering Products: as staphylokinase, Nattokinase etc.
(3) biochemical drug: as urokinase, fiber eliminating enzyme, nevin fibrinolytic enzyme, Lumbrukinase etc.
Wherein, the gene recombination product mostly is intracellular protein, and the secretion type expression system is very few, thereby processing apparatus cost such as its thalline separation, broken wall, renaturation is higher.In addition, by the requirement of new biological product rules, the workload that material was prepared before it was clinical is big, consuming time long.
Microorganism sends out the thrombolysis enzyme that enzyme is produced, and the many genus of this kind new medicine grind the stage again, and fund input is limited, and development lags behind biochemical drug, though more higher as Nattokinase than living, the activity conformation instability, easy inactivation under the normal temperature is not suitable as medicament and uses.The plasmin research and development of other Bacillus subtilus, head mold are carried out, and quality waits checking.
The thrombolysis class biochemical drug of China listing is mainly snake venom fiber eliminating enzyme, urokinase etc., all exists deficiency on the quality, and fiber eliminating enzyme is human activin fibrinolytic system in vivo only, direct thrombolysis.There is dimer in the urokinase preparation, easily causes anaphylaxis.
In recent years, very active about the research and development of earthworm fibrinolysin.The isozyme of earthworm fibrinolysin is kind surplus in the of ten nearly, and molecular weight, iso-electric point difference are little, and the batch preparations single component formulations is difficulty comparatively.
The separation method that the purpose of this invention is to provide a kind of special isozyme of a kind of earthworm fibrinolysin.Utilize this method can be from the Eisenia foetida (place of production: China, Eisenia felida) is separated to a kind of new pure plasmin of a kind of molecular weight about 21 kilodaltons (KD), it and other component molecular weight difference are bigger, be easy to purifying and batch preparations, and active good, ratio high, nontoxicity alive; Molecular weight is little, antigen-reactive is low, is ideal earthworm fibrinolysin single component formulations raw material comparatively, is the ideal thrombolytic drug.
Therefore, another object of the present invention plasmin that to provide a kind of molecular weight from Eisenia foetida be the one-component of 21KD.
A further object of the present invention provides the thrombolytic drug composition that contains described plasmin.
A further object of the present invention provides the purposes of described plasmin in the medicine of preparation treatment blood embolism disease.
Earthworm fibrinolysin of the present invention is more higher than living, and activity stabilized, 4 ℃ preserve down 2 years constant, aqueous solution normal temperature is placed 2 years activity down and is only reduced by 5%.Many components preparation is through several years up to ten thousand cases, and no anaphylaxis takes place.Above-mentioned checking explanation, earthworm fibrinolysin is at the quality requirements that can reach than aspects such as work, vigor stability and low antigenicities as clinical medicine for treating thrombus thing, and in the technical problem that has solved the batch extracting single component, this enzyme single-component preparation utmost point is hopeful to obtain clinical official written reply.
The object of the present invention is achieved like this:
Clean earthworm with the preferred sodium-chlor of non-toxic salt, the earthworm of cleaning is ground, centrifugal, get supernatant liquor and carry out carrying out sieve chromatography after the affinity chromatography, carry out ion exchange chromatography at last, collect the high-abundance proteins peak.
Preferably, the Affi-Gel of the used affinity chromatography of the present invention is with trypsin inhibitor link coupled CNBr activated agarose.
Preferably, the used molecular sieve chromatography of the present invention is Sephacryl S-100.
Preferably, the used ion exchange column of the present invention is a Sepharose Fast Flow ion exchange column.
The invention has the advantages that the particular combinations by different purification process, the molecular weight of 21KD plasmin component of Eisenia foetida and the molecular weight difference of other component are widened, abundance is concentrated, and be easy to separate, and this method is fit to commercially produce.The present invention be advantageous in that pre-treatment takes water-ethanol-extracted, segmentation alcohol precipitation.Exempted the required high speed centrifugation operation of water extraction, reduction equipment drops into significantly.Separation and purification adopts the high aglucon of specificity to carry out affinity chromatography.Adopt hydrophobic chromatography to exempt dialysis, desalination operation.
Embodiment
Describe the present invention in detail below by embodiment, where face limits the scope of the invention but these embodiment are not in office.
Embodiment 1: the preparation of Eisenia foetida 21KD plasminogen single component
Material: the Eisenia foetida (place of production: China)
Preparation:
1, pre-treatment
100 gram earthworms are cleaned, added 10% heavy NaCl of earthworm, placed 10 minutes, clean again.
2, grind centrifugal
The earthworm of handling such as adds at heavy 45% ethanol, grind with colloidal mill, on reset and add dehydrated alcohol to alcohol concn and reach 80%, the room temperature standing over night, supernatant discarded, precipitation pH7.6,0.02MPBS dissolves, and places 24 hours, the centrifugal 30min of 3000rpm gets supernatant liquor.
3, affinity chromatography:
Affinity glue: with trypsin inhibitor link coupled CNBr activated agarose (commercially available).
Chromatography: above-mentioned centrifugal supernatant liquor, last affinity chromatographic column is with the NH of pH11
4OH dissociates, and the thing that dissociates is poured in the dialysis tubing normal saline dialysis 24 hours.
4, sieve chromatography:
Liquid after the dialysis is concentrated into a bag interior liquid volume 15-20ml with polyoxyethylene glycol 20,000, last with physiological saline equilibrated Sephacryl S-100 (high 100cm φ 2.5cm) post (commercially available), collect the higher protein peak of abundance, to pH8.2 0.02MTris-HCl damping fluid dialysis desalination.
5, ion exchange chromatography:
Through above-mentioned damping fluid equilibrated Sepharose Fast Flow ion exchange column (commercially available),, collect the higher protein peak of abundance on the liquid after the dialysis with the buffer solution for gradient elution (0-0.2mol/L) that contains 0.7MNaCl.
6, electrophoresis:
Dialysis back liquid is done electrophoresis, uses SDS-PAGE, and gum concentration is 15%.The result is a protein band, and molecular weight is 21KD, has fibrinolytic.This is a plasmin of the present invention.
7, iso-electric point:
Prove that by the isoelectric focusing electrophoresis test-results pI of the iso-electric point of this plasmin is about 3.8.
Embodiment 2: the bioactive mensuration of plasmin of the present invention
The bioactive measuring method of plasmin is well known to a person skilled in the art.Issue standard with reference to Ministry of Health of the People's Republic of China, " agarose-human fibrin flat band method " in " lumbrukinase capsule quality standard ", heating respectively (60 ℃, 30 minutes; 80 ℃, 30 minutes) and heat treated not, make heated plate and ordinary flat, get plasmin 30 micrograms of the present invention with micropipet, add in the dull and stereotyped hole, the solusphere area is surveyed in 37 ℃ of insulations 6 hours down.Recording plasmin of the present invention is 160000 milligrams/centimetre than work
2, show that plasmin of the present invention has higher thrombolysis activity.
Embodiment 3: the mensuration of plasmin thermostability of the present invention
Plasmin of the present invention is prepared into the solution and the aqueous solution of pH4 0.02MTris-HCl respectively, respectively at 4 ℃, 15 ℃, 25 ℃, 35 ℃, 45 ℃, 55 ℃, 60 ℃, 70 ℃ of down insulations 1 hour, and respectively wherein two samples 4 ℃ and the placement for a long time down of normal temperature (25 ℃) normal pressure.Adjust temperature to 35 ℃, measure enzymic activity.The result shows that plasmin of the present invention is stable below 60 ℃.4 ℃ preserve down 2 years constant, aqueous solution normal temperature is placed 2 years activity down and is only reduced by 5%.
Embodiment 4: the preparation of drug combination that contains plasmin of the present invention
Get plasmin 50 micrograms of the present invention, be dissolved in 5 ml distilled waters.Sealing, normal temperature is placed down.
Claims (7)
1. plasmin that the molecular weight from Eisenia foetida is the one-component of 21KD.
2. the preparation method of the plasmin of claim 1 is characterised in that with non-toxic salt and cleans earthworm, and the earthworm of cleaning is ground, and is centrifugal, gets supernatant liquor and carries out carrying out sieve chromatography after the affinity chromatography, carries out ion exchange chromatography at last, collects the high-abundance proteins peak.
3. the preparation method of the plasmin of claim 2, the Affi-Gel that is characterised in that described affinity chromatography is with trypsin inhibitor link coupled CNBr activated agarose.
4. the preparation method of the plasmin of claim 2 is characterised in that described molecular sieve chromatography is Sephacryl S-100.
5. the preparation method of the plasmin of claim 2 is characterised in that described ion exchange column is a Sepharose Fast Flow ion exchange column.
6. the thrombolytic drug composition that contains the plasmin of claim 1.
7. the purposes of the plasmin of claim 1 in the medicine of preparation treatment blood embolism disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031219500A CN1537938A (en) | 2003-04-18 | 2003-04-18 | New earthoworm plasmin and its preparation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031219500A CN1537938A (en) | 2003-04-18 | 2003-04-18 | New earthoworm plasmin and its preparation method |
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| CN1537938A true CN1537938A (en) | 2004-10-20 |
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| CNA031219500A Pending CN1537938A (en) | 2003-04-18 | 2003-04-18 | New earthoworm plasmin and its preparation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010029453A1 (en) * | 2008-09-10 | 2010-03-18 | Pt.Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| CN102119928A (en) * | 2011-02-21 | 2011-07-13 | 江中药业股份有限公司 | Preparation method of lumbrokinase enteric-coated pellets |
| CN105400761B (en) * | 2015-10-21 | 2019-02-15 | 山东睿鹰先锋制药有限公司 | A kind of low molecular weight fibrinolysin and its preparation method and application |
-
2003
- 2003-04-18 CN CNA031219500A patent/CN1537938A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010029453A1 (en) * | 2008-09-10 | 2010-03-18 | Pt.Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| AU2009290466B2 (en) * | 2008-09-10 | 2014-06-12 | Pt. Dexa Medica | Composition of thrombolytic agent and anti thrombosis and also its production method |
| CN102119928A (en) * | 2011-02-21 | 2011-07-13 | 江中药业股份有限公司 | Preparation method of lumbrokinase enteric-coated pellets |
| CN105400761B (en) * | 2015-10-21 | 2019-02-15 | 山东睿鹰先锋制药有限公司 | A kind of low molecular weight fibrinolysin and its preparation method and application |
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