CN101684151B - Protein matter with coagulation activity - Google Patents
Protein matter with coagulation activity Download PDFInfo
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- CN101684151B CN101684151B CN2008101965853A CN200810196585A CN101684151B CN 101684151 B CN101684151 B CN 101684151B CN 2008101965853 A CN2008101965853 A CN 2008101965853A CN 200810196585 A CN200810196585 A CN 200810196585A CN 101684151 B CN101684151 B CN 101684151B
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Abstract
The invention relates to single chain protein matter with coagulation activity. The gel electrophoresis of the reducing SDS-polyacrylamide of the protein matter is performed to form a single strip under the molecular weight of 31+/-2kDa, the molecular weight is measured to be 30500+/-1000Da by a MALDI-TOF method, the isoelectric point is 4.4-4.8, the area normalization method is utilized to measure the purity which is more than 98%, and fifteen-amino acid sequence of N terminal end measured by the Edman degradation method is VIGGNECDTNEHRFL. The protein matter has stronger coagulation activity on human fibrinogen in vitro, and the specific activity thereof is 500-1500U/mg. Verified by in vitro coagulation activity experiment tests, the single chain protein matter with coagulation activityprovided by the invention has better coagulation activity; the preparation method thereof utilizes frequently-used separation and purification measures in the extraction separation process of currentbiochemical filed, thereby having strong process maneuverability, simple and easy operation and stable volume of production.
Description
Technical field
The present invention relates to a kind of protein matter and preparation method thereof, relate in particular to a kind of method that has the protein matter of blood coagulation activity and from snake venom, extract this material with blood coagulation activity.
Background technology
Contain multiple protein and polypeptide in the snake venom, they are special proteolytic enzyme and active polypeptide mostly.These materials possibly participated in digestion as support substance, and also are hemorrhage poison of snake venom and Neurotoxic major cause.1767, Fontana narrated the Blood clotting of snake venom first, after this over more than 200 year, relevant snake venom is applied to the research of blood system and constantly scores achievements.Since the seventies in 20th century, many snake venom class medicines have developed into clinical medicine like Ancrod, Batroxobin, Crotalase and Reptilase, and they are playing the part of important role in the treatment of blood and cardiovascular disorder.The World Health Organization will separation and purification from spearhead pallas pit viper (Borheratrox) snake venom the thrombin-like enzyme called after: " Batroxobin ", it has the various clinical purposes, as is used for hemostasis and is called as " Retilase "; Be used to decompose Fibrinogen, suppress thrombosis and be called " Defibrase "; Be called " Reptilase-Reagent " as diagnostic reagent.Wherein the higher upright root of Dahurian angelica snow (Reptilase) of popularity is that the plain high pharmaceutical factory of Switzerland makes the medicine that is used to treat and prevent bleeding that from pallas pit viper, extracts.Wherein contain two kinds of compositions: the factor X fat dependency of batroxobin and trace activates agent (FXA); Because it has quick-acting; Efficiently, safe ready and do not receive plurality of advantages such as the blood plasma thrombin inhibitors influences is widely used in treatment and prevents various hemorrhagic diseasess.Batroxobin wherein is a Thrombin-like enzyme, is strand gp through identifying it, can obtain by separating in several snake venom; Along with the snake kind is different, there is some difference for its character, research comparatively be clear that spearhead pallas pit viper batroxobin; Relative molecular weight 43kDa, iso-electric point is 6-7, is strand gp.Factor X fat dependency in the upright root of Dahurian angelica snow activates agent (FXA) and also can from several pallas pit vipers, extract and obtain, and is not quite similar for the report of its molecular weight and iso-electric point, and it is molecular weight 83kDa that the America spearhead Pallas pit viper FXA of report is arranged, the acidic protein of iso-electric point 4.9; It is 104kDa or the like that the FXA molecular weight of report circle spot adder snake venom is also arranged, the FXA character of just reporting at present, and most FXA albumen have the bigger general character of molecular weight, and this possibly be to the requirement of structure because it brings into play specific function in vivo.
Nineteen fifty-seven Ouyang separates from the agkistrodon acutus snake venom and has obtained thrombin-like enzyme and its character has been carried out more deep research, this enzyme is named be Acutin.Domestic scholars is also studied the blood coagulation activity material of pallas pit viper, and obtains a series of progress.Kunming, Yunnan animal the Acutin that will from agkistrodon acutus, extract be called " IBGO ", be used for clinical treatment cerebral thrombosis, heart clot, eyeground vein thrombus and vasculitis, evident in efficacy.Owing to contain multiple albumen and peptide material in the snake venom with external blood coagulation activity; The separation and purification difficulty; It is a kind of launch sale of nation's booth (injection white-browed snake venom blood coagulation enzyme) of raw material production with Changbai Mountain, northeast agkistrodon halys ussuriensis snake venom that present domestic snake-poison hemostatic series products has only Jinzhou, Liaoning great medicine company difficult to understand; SDS-the polyacrylamide gel electrophoresis of the reptilase in this product contains 54kDa; 34kDa and 15kDa three bands are not seen the pharmacological action of the thrombin-like enzyme of these three electrophoretic bands at present to full coverage.Agkistrodon acutus has Anhui (south), Chongqing, Jiangxi, Zhejiang, Fujian (the north), Hunan, Hubei, Guangxi (the north), Guizhou, Guangdong (the north) and Taiwan Province etc. in the distribution area of China; Have a very wide distribution; Domestic how tame mechanism is studied the blood coagulation activity component in the agkistrodon acutus snake venom; Obtain certain achievement, but so far, do not had agkistrodon acutus snake poison blood coagulation medicine successfully to go on the market as yet.The inventor finds a kind of new protein matter with blood coagulation activity through test of long duration research, and accomplishes the present invention based on this.
Summary of the invention
The present invention provides a kind of substance A with blood coagulation activity, it is characterized in that: it is a single chain protein class material, and its reductibility SDS-polyacrylamide gel electrophoresis is single band at 31 ± 2kDa; It is 30500 ± 1000Da that the MALDI-TOF method is measured its molecular weight; Iso-electric point is 4.4-4.8; Adopt area normalization method to measure purity more than 98%; 15 aminoacid sequences of N-terminal that the Edman edman degradation Edman is measured are following: VIGGNECDTNEHRFL.It has stronger congealing activity external to the human fibrinogen, and specific activity is 500-1500U/mg.
Substance A with blood coagulation activity provided by the invention can be extracted from snake venom and obtain, and also can after the complete sequence of measuring substance A, adopt biochemical field engineered method commonly used to obtain.Preferably from snake venom, extract and obtain.
The present invention also provides a kind of said method with substance A of blood coagulation activity of from snake venom, extracting, and may further comprise the steps:
(1) take by weighing snake venom, after the Tris-HCl damping fluid dissolving with pH7.0, the centrifugal removal suspended substance of low-temperature and high-speed is got supernatant, gets said substance A bullion with blood coagulation activity;
(2) above-mentioned bullion repeatedly obtains the said pure article of substance A with blood coagulation activity behind the purifying through anion exchange chromatography, ultrafiltration and gel exclusion chromatography etc.
Said method specifically comprises the steps:
(1) take by weighing snake venom, with Tris-HCl damping fluid dissolving of pH7.0, the centrifugal removal suspended substance of low-temperature and high-speed is got supernatant, gets said substance A bullion with blood coagulation activity;
(2 said bullions carry out SOURCE30Q anion exchange chromatography purifying, and the concentration of salt solution gradient elution is removed most of small-molecule substance, lipid and part basic protein;
(3) adopt that ultrafiltration process concentrates, desalination, use Sephacryl S200 gel filtration chromatography purifying then, remove and target protein molecular weight different impurity, collect and contain said substance A component with blood coagulation activity;
(4) component with above-mentioned collection adopts the anion exchange chromatography purifying once more, obtains to contain said substance A component with blood coagulation activity behind the employing isocratic elution;
(5) above-mentioned (4) component that obtains of step carries out can obtaining the said pure article of substance A with blood coagulation activity after the desalination freeze-drying after concentrating again.
In the said extracted method, the preferred agkistrodon acutus snake venom of snake venom; Anion-exchange column is preferably SOURCE30Q or Hitrap Q XL; It is the filter membrane of 0.3～20kDa that ultra-filtration process adopts molecular weight cut-off, and preferably adopting molecular weight cut-off is the filter membrane of 5～10kDa, most preferably the filter membrane of 10kDa.In the said extracted method, each step is all being carried out below 16 ℃.
The present invention provides described substance A with blood coagulation activity to have the purposes in the medicine of coagulation function in preparation on the other hand.Said medicine with coagulation function can be used to prevent clinical operation hemorrhage, palpuses minimizings such as treatment traumatic hemorrhage and whole body and local hemostasis bleed or the various medical conditions of hematostatic under hemorrhage and hemorrhagic acute disease.
Further aspect of the present invention provides a kind of blood-clotting agent, and it contains described substance A and proper supplementary material or excipient with blood coagulation activity.
Substance A with blood coagulation activity provided by the invention is through external blood coagulation activity verification experimental verification, has a blood coagulation activity preferably external; Its preparation method adopts separation and means of purification commonly used in the extraction separation process of present biochemical field, and technology is workable, and is easy to operation, stable yield.
Embodiment
Further specify content of the present invention with specific embodiment below, but and do not mean that by any way and limit the invention.The snake venom that uses among the present invention is from the agkistrodon acutus snake venom.
The preparation of embodiment 1 substance A
Get the snake poison lyophilized crystal 6 g of agkistrodon acutus and be dissolved in concentration 20mmol/L; In the Tris-HCl damping fluid of pH7.0, centrifugal 30min under 4 ℃ of 1000rpm gets supernatant and carries out SOURCE30Q anion column chromatography purification; Adopt the method for NaCl solution linear gradient elution; At first with behind 5 column volumes of 20%NaCl solution washing again with 10 column volumes of 40%NaCl eluant solution, collect and also to merge the active constituent peak, carry out Sephacryl S200 gel filtration chromatography purifying after adopting the ultra-filtration membrane ultrafiltration and concentration of molecular weight cut-off 10kDa; Collect and merge the active constituent peak; Carry out SOURCE30Q anion column chromatography purification again, adopt the method for NaCl isocratic elution, at first with behind 3 column volumes of 20%NaCl eluant solution again with the 40%NaCl eluant solution; Collect the active constituent peak of 40%NaCl eluant solution, promptly get high purity substance A after the ultrafiltration desalination freeze-drying.The molecular weight that the MALDI-TOF method records extract A is 30522Da, and iso-electric point is 4.5, and 15 aminoacid sequences of N-terminal that the Edman edman degradation Edman is measured are following: VIGGNECDTNEHRFL.
The preparation of embodiment 2 substance A
Getting the snake poison lyophilized crystal 8g of agkistrodon acutus, to be dissolved in concentration be 20mmol/L; In the Tris-HCl damping fluid of pH7.0; Centrifugal 30min under 4 ℃ of 1000rpm gets supernatant and carries out SOURCE30Q anion column chromatography purification, adopts the method for NaCl solution linear gradient elution; At first with behind 3 column volumes of NaCl solution washing of 20% again with 12 column volumes of 30%NaCl eluant solution; Collect and merging active constituent peak, carry out Sephacryl S200 gel filtration chromatography purifying behind the ultra-filtration membrane ultrafiltration and concentration of employing molecular weight cut-off 5kDa, collect merging active constituent peak; Carry out Hitrap Q XL anion column chromatography purification again; Adopt the method for NaCl solution isocratic elution, at first with behind 3 column volumes of 10%NaCl eluant solution again with the 30%NaCl eluant solution, collect the active constituent peak that merges the 30%NaCl eluant solution after ultrafiltration desalination freeze-drying promptly get high purity substance A.The molecular weight that the MALDI-TOF method records extract A is 30530Da, and iso-electric point is 4.7, and 15 aminoacid sequences of N-terminal that the Edman edman degradation Edman is measured are following: VIGGNECDTNEHRFL.
The external blood coagulation specific activity of embodiment 3 substance A is measured
2.1 material
Substance A 1,2,3 lot sample article adopt the method for embodiment 1 or 2 to make
Ba Quting is available from Peng Lai Nuo Kang pharmaceutcal corporation, Ltd
2.2 method
Adopt unit protein mass blood coagulation activity thing that human plasma is investigated the external blood coagulation specific activity of substance A of the present invention index time of coagulation.Concrete operation method is: get one of normal man's blood plasma, add 1ml water for injection dissolving after, precision is measured 0.2ml and is put in the small test tube; Insulation is 3 minutes in 37 ℃ of water-baths, and precision adds the need testing solution 0.2ml of preheating, mixing immediately; Timing; Collude water intaking with clean minute hand and bathe the mixed solution under the keeping warm mode, stop timing when observing solution to the minute hand Pick Wire first time simultaneously, this time is the presetting period.Adopt 1U/ml crust Qu Ting as contrast, the need testing solution after the suitable dilution is carried out determination of activity.Wherein, the gross activity method of calculation are with 1U/ml crust Qu Tingwei contrast, and the multiple that diluted sample is diluted during to 1U/ml is the sample gross activity, and unit is that the protein content in the U/ml need testing solution adopts the lowry method to measure.Specific activity=gross activity/total protein content
2.3 experimental data
| Batch | Sample gross activity (U/ml) | Protein content (mg/ml) | Sample specific activity (U/mg) |
| 1 | 302 | 0.36 | 838.89 |
| 2 | 380 | 0.34 | 1117.65 |
| 3 | 312 | 0.38 | 821.05 |
2.4 experimental result
Experimental result explanation, substance A provided by the invention has a blood coagulation activity external.
Claims (3)
1. substance A of from the agkistrodon acutus snake venom, extracting with blood coagulation activity, it is characterized in that: it is a single chain protein class material, its reductibility SDS-polyacrylamide gel electrophoresis is single band at 31 ± 2kDa; It is 30500 ± 1000Da that the MALDI-TOF method is measured its molecular weight; Iso-electric point is 4.4-4.8; Adopt area normalization method to measure purity more than 98%; 15 aminoacid sequences of N-terminal that the Edman edman degradation Edman is measured are following: VIGGNECDTNEHRFL, its external specific activity that records is 500-1500U/mg.
2. the described substance A of from the agkistrodon acutus snake venom, extracting with blood coagulation activity of claim 1 has the purposes in the medicine of coagulation function in preparation.
3. blood-clotting agent, it contains the described substance A of from the agkistrodon acutus snake venom, extracting with blood coagulation activity of claim 1 and proper supplementary material or excipient.
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| CN2008101965853A CN101684151B (en) | 2008-09-26 | 2008-09-26 | Protein matter with coagulation activity |
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| CN2008101965853A CN101684151B (en) | 2008-09-26 | 2008-09-26 | Protein matter with coagulation activity |
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| CN101684151B true CN101684151B (en) | 2012-06-20 |
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Citations (6)
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| CN1181421A (en) * | 1997-11-12 | 1998-05-13 | 中国科学院上海生物化学研究所 | Gene sequence of fibrin ferment of Agkistrodon acutus snakes |
| CN1332242A (en) * | 2001-04-29 | 2002-01-23 | 中国科学院昆明动物研究所 | Ahylysantinfarctase thrombase and its production process |
| CN1332241A (en) * | 2001-04-29 | 2002-01-23 | 中国科学院昆明动物研究所 | Ahylysantinfarctase thrombase and its production process |
| CN1502693A (en) * | 2002-11-22 | 2004-06-09 | 唐松山 | Zymoplasmoid of venom of Deinagkistrodon acutus with hemostatic activity |
| CN1944642A (en) * | 2006-09-12 | 2007-04-11 | 沈居仁 | Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method |
| CN1958790A (en) * | 2006-07-27 | 2007-05-09 | 唐松山 | New thrombin in snake poison categories of hundred-paced pit viper, and styptic activity |
-
2008
- 2008-09-26 CN CN2008101965853A patent/CN101684151B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181421A (en) * | 1997-11-12 | 1998-05-13 | 中国科学院上海生物化学研究所 | Gene sequence of fibrin ferment of Agkistrodon acutus snakes |
| CN1332242A (en) * | 2001-04-29 | 2002-01-23 | 中国科学院昆明动物研究所 | Ahylysantinfarctase thrombase and its production process |
| CN1332241A (en) * | 2001-04-29 | 2002-01-23 | 中国科学院昆明动物研究所 | Ahylysantinfarctase thrombase and its production process |
| CN1502693A (en) * | 2002-11-22 | 2004-06-09 | 唐松山 | Zymoplasmoid of venom of Deinagkistrodon acutus with hemostatic activity |
| CN1958790A (en) * | 2006-07-27 | 2007-05-09 | 唐松山 | New thrombin in snake poison categories of hundred-paced pit viper, and styptic activity |
| CN1944642A (en) * | 2006-09-12 | 2007-04-11 | 沈居仁 | Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method |
Non-Patent Citations (1)
| Title |
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| 郑颖等.蛇毒血凝酶的比较.《蛇志》.2008,第20卷(第03期), * |
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