CN1234443A - Nereid kinase and its extraction method and use - Google Patents
Nereid kinase and its extraction method and use Download PDFInfo
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- CN1234443A CN1234443A CN98111280.3A CN98111280A CN1234443A CN 1234443 A CN1234443 A CN 1234443A CN 98111280 A CN98111280 A CN 98111280A CN 1234443 A CN1234443 A CN 1234443A
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Abstract
The present invention provides a low-cot, safe, nontoxic, and effective thrombolytic oral medicine-thrombolytic plasmin-nereid kinase extracted from nereid body. The molecular weight of the nereid kinase is 25000-55000, and its isoelectric point is 3-7. Its raw material source is rich,and said invented nereid kinase can be used for producing oral medicine for curing thrombotic diseases resulted from cerebral thrombosis and myocardiac infarction.
Description
The present invention relates to a kind of from perinereis aibuhitensis (Perinereis aibuhitensis) and the multiple tooth thrombus dissolving plasmin that can be used for the treating thrombotic diseases------Nereid kinase that extracts the energy fibrin degradation in clam worm (Perinereis nuntia) body that encloses.
Cardiovascular and cerebrovascular diseases such as cerebral thrombosis, myocardial infarction are only second to cancer at China's M ﹠ M.The treatment thrombolytic drugs such as urokinase, streptokinase that adopt are treated more clinically at present.But because medicine such as urokinase remains in some drawbacks as can only quietly annotating, can not be oral; Easily Fibrinogen causes bleeding in the degraded blood; Can not use continuously for a long time etc.And urokinase need extract from urine, and its raw-material collection is along with the quickening of urbanization speed difficulty further, and quality can not guarantee, pollutes easily in the transportation.Though the Lumbrukinase of developing recently has certain result of treatment, and oral effectively,, the used starting material earthworm of Lumbrukinase must be by propagating artificially, even must add some special nutritive ingredients, this has just improved the medicine production cost greatly.Thereby seek the thrombolytic-anticoagulant medicine that other are more effective, side effect is little, price is low and become the task of top priority.
The object of the present invention is to provide a kind of abundant raw materials, cheap, safety non-toxic, orally active thrombolytic drug, with remedy medicine such as urokinase produce and treatment aspect deficiency.
Clam worm is the Polychaeta annelid, and it is very wide to distribute in China coastal beach area, and resource is very abundant, and the overwhelming majority is underutilized, and is in the state that runs its course, and causes the serious waste of resource.We obtain in the clam worm body first can fibrin degradation plasmin, and the character of plasmin done preliminary study, find that the clam worm plasmin probably becomes antithrombotic reagent of new generation, and, be hopeful to develop into a kind of conventional treatment medicine because its raw material sources are abundant, cheap.Simultaneously, utilize clam worm to produce the natural resources that the thrombolysis medicine can also make full use of China, promote the Economic development of coastland.
We obtain plasmin from two kinds of clam worms of China's high yield, and the called after Nereid kinase.And confirm that by experiment its thrombolysis activity is not less than urokinase.The effect of its existing direct fibrin degradation again can plasminogen activation, fibrin degradation indirectly.Its molecular weight is between 25000-55000, and iso-electric point is between 3-7, and optimum reaction conditions is about 37 ℃, pH8.0.
Compare with urokinase, Nereid kinase has thermostability preferably, and urokinase is at 37 ℃ of very fast inactivations, so urokinase is not suitable for making oral preparations, and can only intravenous injection.Nereid kinase then might be developed into the novel thrombolytic drug of suitable for oral administration, and Nereid kinase still keeps its stability of activity more than 90% after one hour even surpasses the Lumbrukinase of development recently through 60 ℃.Lumbrukinase very fast loss of activity just in the time of 50 ℃.In addition, Nereid kinase is the highest 37 ℃ of its activity, and this explanation Nereid kinase is very suitable for making oral medicine.
The preparation of Nereid kinase can be divided into following step:
(1) fragmentation of pre-treatment and tissue
Clam worm is used with the state of living as far as possible, otherwise just should it is immediately freezing.Clam worm is cleaned with the clear water immersion, smashed to pieces with high-speed tissue mashing machine.
(2) extracting
Add 0.02mo1/L phosphate buffered saline buffer (containing 0.1mol/L NaCl) extract, 4 ℃ of extractings are more than 6 hours, and are centrifugal then, collect supernatant liquor.
(3) purification of enzyme
1. salting-out process
The ammonium sulfate solids of porphyrize added in the above-mentioned supernatant liquor saltout, get the 20-70% precipitation.Again with 0.02mol/L phosphate buffered saline buffer dissolution precipitation.
2. gel-filtration
With Sephadex G-25 column chromatography or dialysis method desalination, column chromatographies such as G-75 or G-100 carry out chromatographic separation, and chromatography column volume size is 2.6 * 100cm, and elutriant is all 0.02mol/L phosphoric acid salt (PBS).Survey the 280nm light absorption value with ultraviolet spectrophotometer.Collect active peak, must purer product.
3. ion-exchange chromatography
Select for use CM-Mierocrystalline cellulose and DEAE-cellulose column that purer product are carried out chromatography again, with the 0-1mol/LNaCl/PBS gradient elution, the 280nm ultraviolet detection.Collect active peak, the dialysis postlyophilization.
4. ultrafiltration
Can will directly collect protein between the 10000-60000 behind the salting-out process gained resolution of precipitate, and then carry out next step separation in the ultrafiltration mode.
5. electrophoresis
Be prepared with the 10-12% polyacrylamide gel electrophoresis, can obtain pure plasmin.
6. affinity chromatography
Carry out affinity chromatography with Benzamidine-Sepharose 4B.
7. through the sample of column chromatography, use the HPLC purifying, can get pure enzyme.
Through above-mentioned the whole bag of tricks separation and purification, the Nereid kinase yield is between 0.4-0.6 ‰ (it is heavy to account for clam worm alive), and 500 gram clam worms can obtain about 4000-50000U Nereid kinase.
Nereid kinase can be used for the oral medicine of the thrombotic diseases that production for treating cerebral thrombosis, myocardial infarction etc. cause.The active mensuration of Nereid kinase adopts flat band method and external clot dissolution method, and specific practice is as follows:
(1) flat band method
Higher and not only can be qualitative but also can be quantitative because of flat band method sensitivity, so this research flat band methods that adopt more.
Reagent:
Parenogen 0.25% (W/V) solution: lysate is 17 parts of physiological saline, 1 part of 0.03mol/L, pH7.8 phosphoric acid buffer.Fibrinogen is a Sigma company product.
Thrombin of beef: Nat'l Pharmaceutical ﹠ Biological Products Control Institute produces.Be made into every m160NIH unit (2.2NIH unit=1BP unit) with physiological saline.
Phosphate buffered saline buffer: 0.03mol/L, pH7.8.
Nereid kinase sample: be made into finite concentration with phosphate buffered saline buffer.
Operation:
At diameter is in the 10.5cm plate, add the 17ml fibrinogen solution, add the 0.4ml thrombin solution again, shake up fast, leave standstill 30min, the rear plate substantially transparent that condenses is colourless, with microsyringe point sample 20ul (during detection by quantitative, with the urokinase standard substance that are diluted to different concns on the time point), cover with glass again, insulation is taken out behind the 18h in 37 ℃ of casees, measures vertical two diameters of solusphere with slide calliper rule, multiplies each other one long-pendingly.Make ordinate zou with solusphere diameter product, the concentration of urokinase standard substance is the X-coordinate mapping, can find the unit of activity of sample on figure.
(2) external clot dissolution method
Get people's fresh blood, treat that it is frozen into piece and inhales serum deprivation with paper, make blood clot (the diameter 8mm of identical size, the garden column of high 5mm), puts into the test tube of numbering, add the plasmin extracting solution, compare with the damping fluid that does not add plasmin simultaneously, put 37 ℃ and observe the clot dissolution situation.The generally dissolving substantially about 18 hours of clot in the test tube of adding Nereid kinase, and control tube does not have any variation.This method can be used to do qualitative experiment.
The property research of plasmin:
(1) temperature is to the active influence of enzyme
Use 0.02mol/L, the pH7.8 phosphate buffered saline buffer is made into the solution of 2mg/ml with Nereid kinase, is divided into 6 parts, places 1 hour at 10 ℃, 20 ℃, 37 ℃, 50 ℃, 60 ℃, 70 ℃ respectively, measures remaining activity with flat band method then.Measurement result after 18 hours, (see accompanying drawing 1, temperature is to the active influence of Nereid kinase) has maximum vigor about 37 ℃.
(2) pH is to the influence of enzymic activity
Be made into the damping fluid of a series of different pH with phosphoric acid salt, Nereid kinase is dissolved in wherein (2mg/ml), under different pH, measure the vigor of enzyme then, found that its optimal pH is about 8.0 with flat band method.(see accompanying drawing 2, PH is to the active influence of Nereid kinase).
Again enzyme is dissolved in the phosphate buffered saline buffer of different pH, 37 ℃ are incubated 1 hour, transfer pH to 8.0 then, measure the residue vigor, result's (see accompanying drawing 3, different PH are to the influence of Nereid kinase stability).Nereid kinase stability about pH8.0 is best.(3) proplasmin activates determination of activity and takes out 15ul respectively from several parts of different Nereid kinase samples, add the proplasmin that 15ul concentration is 10 units/ml more respectively, make ultimate density for containing the 5mg/ml sample, 5 units/ml Profibrinolysin, contrast replaces Profibrinolysin with the 15ul damping fluid, measures active then with flat band method.Found that the solusphere of sample on flat board that adds Profibrinolysin is obviously greater than contrast.The existing direct fibrinolytic effect of Nereid kinase is described thus, the indirect activation effect is arranged again.
Description of drawings:
Fig. 1, temperature is to the active influence of Nereid kinase.
Fig. 2, PH is to the active influence of Nereid kinase.
Fig. 3, different PH are to the influence of Nereid kinase stability.
Embodiment 11. gets 500 grams clam worm alive, soaks with clear water, and flush away silt dirt drains water then, smashs to pieces with tissue mashing machine.2. add 1000ml 0.02mol/L phosphate buffered saline buffer (containing 0.1mol/L NaCl) extract, 4 ℃ of extractings are more than 6 hours, and the pH value is controlled at 7-8.8000rpm then, 4 ℃ of centrifugal 15min collect supernatant liquor.3. with the abundant porphyrize of ammonium sulfate, take by weighing ammonium sulfate and add and make into 20% saturation ratio in the above-mentioned supernatant liquor.Leave standstill about 2 hours centrifugal collection supernatant liquor.Add ammonium sulfate to 70% saturation ratio again, leave standstill, centrifugal collecting precipitation.Use the 0.02mol/LPBS dissolution precipitation again.4. (the column volume size is 2.6 * 100cm), and the application of sample amount is about 100ml, and elutriant is 0.02mol/L phosphoric acid salt (PBS) with the desalination of Sephadex G-25 column chromatography.With the crude extract lyophilize after desalting ,-20 ℃ of preservations are standby.5. take by weighing the above-mentioned dried frozen aquatic products of 50mg, with 0.02mol/L PBS dissolving, (the post size is 2.6 * 100cm) to carry out initial gross separation, with ultraviolet spectrophotometer survey 280nm light absorption value, collects active peak, must purer product with Sephadex G-100 column chromatography.6. (2.6 * 30cm) further separate, and with 0-1M NaCl/PBS gradient elution, the 280nm ultraviolet detection is collected active peak with DEAE-Sephadex A post with above-mentioned purer product.The dialysis postlyophilization.7. be prepared with the 10-12% polyacrylamide gel electrophoresis at last, obtain pure plasmin.Yield is 0.4 ‰, and per 500 gram clam worms can get the 8000-40000U Nereid kinase.Embodiment 21. is with embodiment 1.2. with embodiment 1.3. with embodiment 1.4. dialysis desalting, lyophilize ,-20 ℃ of preservations are standby.5. get above-mentioned dried frozen aquatic products 50mg, with 0.02mol/L PBS dissolving, (2.6 * 100cm) carry out initial gross separation, collect active peak with Sephadex G-75 column chromatography.6. (2.6 * 30cm), gradient elution is collected active peak above-mentioned active ingredient to be carried out column chromatography with the CM-Mierocrystalline cellulose.Dialysis is desalted.7. (2.6 * 30cm) further separate above-mentioned active ingredient, and 0-1M NaCl/PBS gradient elution is collected active peak, ultrafiltration and concentration to use the DEAE-cellulose chromatography again.8. obtain the pure product of plasmin with HPLC (FPLC) chromatography at last.Yield is 0.5 ‰, and per 500 gram clam worms can get the 4000-35000U Nereid kinase.Embodiment 31. is with embodiment 1.2. with embodiment 1.3. with embodiment 1.4. with the ultra-filtration membrane ultrafiltration of PSPP, collect the protein of molecular weight between 10000-60000.5. said components is added Benzamidine-Sepharose 4B affinity column, with the PBS wash-out of 0.02mol/L pH7.8, non-specific heteroproteins is washed, earlier again with containing the arginic PBS wash-out of 1mol/L urea and 0.5mol/L, collect active peak, ultrafiltration and concentration.6. obtain pure plasmin with HPLC (FPLC) chromatography again.Yield is 0.6 ‰, and per 500 gram clam worms can get the 30000-50000U Nereid kinase.
Claims (3)
1. thrombus dissolving plasmin, it is characterized in that the molecular weight of Nereid kinase is between 25000-55000 from perinereis aibuhitensis and multiple tooth thrombus dissolving plasmin one Nereid kinase that extracts in the clam worm body that encloses, iso-electric point is between 3-7, optimum reaction conditions is 37 ℃, about pH8.0.
2. the production method of a thrombus dissolving Nereid kinase, it is characterized in that smashing into clam worm to pieces homogenate with high-speed tissue mashing machine, and add the extract extracting, with ammonium sulfate precipitation, get the 20-70% precipitation, with Sephadex G-25, Sephadex G-75 or dialysis or ultrafiltration process desalination, be further purified with methods such as dextrane gel, ion-exchange cellulose or affinity chromatography and electrophoresis or HPLC then again.
3. Nereid kinase according to claim 1 is characterized in that Nereid kinase can be used for the medicine of the thrombotic diseases that production for treating cerebral thrombosis, myocardial infarction etc. cause.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN98111280A CN1088472C (en) | 1998-05-05 | 1998-05-05 | Nereid kinase and its extraction method and use |
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| Application Number | Priority Date | Filing Date | Title |
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| CN98111280A CN1088472C (en) | 1998-05-05 | 1998-05-05 | Nereid kinase and its extraction method and use |
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| CN1234443A true CN1234443A (en) | 1999-11-10 |
| CN1088472C CN1088472C (en) | 2002-07-31 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892210A (en) * | 2010-06-28 | 2010-11-24 | 广西医科大学 | Sipunculus nudus plasmin and preparation method thereof |
| CN102002488A (en) * | 2010-10-29 | 2011-04-06 | 广西医科大学 | Phascolosoma esculenta plasmin and preparation method thereof |
| CN106916803A (en) * | 2015-12-24 | 2017-07-04 | 上海中医药大学 | A kind of perinereis aibihitensis Grube fibrinolytic protein enzyme and its production and use |
| CN110093335A (en) * | 2019-05-05 | 2019-08-06 | 北京颢美细胞基因生物技术有限公司 | A kind of extracting method of Nereid kinase |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1080956A (en) * | 1993-04-24 | 1994-01-19 | 北京市首都生物技术研究所 | Process for preparing earthworm-suishuan enzyme |
| CN1120070A (en) * | 1994-12-26 | 1996-04-10 | 合肥兆峰科大药业有限公司 | Point pallas pit viper poisonous fibre-dissolving enzyme and its purifying method |
| CN1174889A (en) * | 1997-06-16 | 1998-03-04 | 中国医学科学院医药生物技术研究所 | Fibrinolysin generated from streptomycin |
-
1998
- 1998-05-05 CN CN98111280A patent/CN1088472C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892210A (en) * | 2010-06-28 | 2010-11-24 | 广西医科大学 | Sipunculus nudus plasmin and preparation method thereof |
| CN101892210B (en) * | 2010-06-28 | 2012-09-05 | 广西医科大学 | Sipunculus nudus plasmin and preparation method thereof |
| CN102002488A (en) * | 2010-10-29 | 2011-04-06 | 广西医科大学 | Phascolosoma esculenta plasmin and preparation method thereof |
| CN102002488B (en) * | 2010-10-29 | 2013-06-26 | 广西医科大学 | Phascolosoma esculenta plasmin and preparation method thereof |
| CN106916803A (en) * | 2015-12-24 | 2017-07-04 | 上海中医药大学 | A kind of perinereis aibihitensis Grube fibrinolytic protein enzyme and its production and use |
| CN110093335A (en) * | 2019-05-05 | 2019-08-06 | 北京颢美细胞基因生物技术有限公司 | A kind of extracting method of Nereid kinase |
| CN110093335B (en) * | 2019-05-05 | 2022-10-18 | 北京颢美细胞基因生物技术有限公司 | Extraction method of nereis kinase |
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| CN1088472C (en) | 2002-07-31 |
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