CN102925422A - Agkistrodon acutus hemocoagulase-B - Google Patents
Agkistrodon acutus hemocoagulase-B Download PDFInfo
- Publication number
- CN102925422A CN102925422A CN2012104589872A CN201210458987A CN102925422A CN 102925422 A CN102925422 A CN 102925422A CN 2012104589872 A CN2012104589872 A CN 2012104589872A CN 201210458987 A CN201210458987 A CN 201210458987A CN 102925422 A CN102925422 A CN 102925422A
- Authority
- CN
- China
- Prior art keywords
- hemocoagulase
- pbs
- snake venom
- agkistrodon acutus
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6418—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals from snakes
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Wood Science & Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biotechnology (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides agkistrodon acutus hemocoagulase-B which is high-activity hemocoagulase separated from agkistrodon acutus venom. The hemocoagulase is single-stranded glycoprotein comprising 236 amino acids, and has an amino acid sequence shown as SEQ ID No. (sequence identification number) 1. The strand comprises 6 pairs of disulfide bonds; molecular weight of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is approximately 35kD; the molecular weight of the hemocoagulase after being deglycosylated is 26116.7Da; and an isoelectric point (pI) is 6.0. A hemocoagulase molecule is modified by heterozygotic polysaccharides at an Asn<77, 100, 229> (asparagine) site. The hemocoagulase is serine proteinase. The invention also provides a separation and purification method of the hemocoagulase, which comprises the steps that undissolved substances are removed through pretreating; then two times of anion-exchange column chromatography and one time of Sephdex-G75 molecular sieve chromatography are conducted; an active elution peak is acquired; dialysis and lyophilization are conducted; and the high-purity agkistrodon acutus venom hemocoagulase is obtained. The specific activity of the agkistrodon acutus venom hemocoagulase is not less than 2000U/mg; the analysis purity of HPLC (high performance liquid chromatography) can reach above 95%; and the yield is 0.25-0.30% calculated according to the weight of an agkistrodon acutus venom raw material.
Description
Technical field
The present invention relates to a kind of serine protease, specifically a kind of Hemocoagulase agkistrodon-B the invention still further relates to its separation purification method.
Background technology
Have the class proteolytic enzyme relevant with blood coagulation in Crotalinae (Crotalinae) snake venom, (thrombin-like enzyme is called for short: TLC) usually to be referred to as " Thrombin-like enzyme ".Thrombin-like enzyme is similar to the apparent effect of zymoplasm (thrombin), can make all that Fibrinogen is converted into scleroproein and " solidifying " in the blood plasma.Yet, two kinds of enzyme mechanism differences, Thrombin-like enzyme does not activate the Χ III factor in the blood coagulation system, and itself and Fibrinogen effect only produce noncrosslinked fibrin, can not cause thrombosis, and the sludged blood of its formation can be dissolved by 5M urea; And zymoplasm can activate the Χ III factor in the blood coagulation system, and itself and Fibrinogen effect form crosslinked fibrin, can cause thrombosis, and the sludged blood of its formation can not be dissolved by 5M urea.
Scientific research over nearly 50 years is found to contain the Thrombin-like enzyme composition in more than 40 kinds of snake venom, has more than 30 kinds to obtain separation and purification, wherein has more than the 10 all or part of aminoacid sequences of planting Thrombin-like enzymes to be elucidated.The TLC molecular weight of having found is many between 29～45kD, and great majority are acid glycoprotein.
In the snake venom TLC that in the past had been found that, prlmary structure of protein mostly is strand.Its commercialization Typical Representative product " vertical root of Dahurian angelica snow " is to be produced by Switzerland Solco Basle company (Reptilase), by isolated a kind of zymoplasm in Brazilian spearhead pallas pit viper (Bothrops.atrox) snake venom, this enzyme precursor is by 255 Amino acid profiles, the N end has 24 amino acids formed guiding peptides, organized enzyme contains 231 amino acid, SDS-PAGE molecular weight 34kD.Contain 6 intrachain disulfide bonds (site: 7-139,26-42,74-230,1 18-184,150-163,174-199), (glycosylation site is Asn for strand glycoprotein
146,225), the molecular weight of sloughing behind the glycosyl is 255 17Da.Another commercial product be that Italian RAVIZZ company produces " Botropase ", this zymoplasm separates from America spearhead pallas pit viper (Bothrops.jararaca) snake venom and obtains.Organized enzyme contains 231 amino acid, relatively in 14 sites difference is arranged with Reptilase on aminoacid sequence.
In addition, domestic scholars Shen Ju benevolence etc. 2006 are separated from Chinese agkistrodon acutus (Agkistrodon acutus) snake venom and are obtained a kind of high reactivity hemocoagulase, this enzyme is strand, SDS-PAGE molecular weight 36 ± 2kD, iso-electric point is 6.59, specific activity is 2000U-3000U/mg, and terminal 15 aminoacid sequences of N-are VIGGNECDTNEEHFL.Collect yield as 0.03% take the snake venom raw material weight.
Up to the present, commercial snake venom blood coagulation enzyme kind is less, and the snake venom blood coagulation enzyme new variety of therefore seeking the high yield of high reactivity that is fit to industrialization are especially aobvious necessary.
Inventor's separation and purification from the snake venom of China's Guangxi agkistrodon acutus (Agkistrodon acutus is commonly called as Agkistrodon) obtains a kind of high reactivity hemocoagulase-B.
Summary of the invention
The object of the present invention is to provide a kind of snake venom blood coagulation enzyme-B, it is to separate a kind of Thrombin-like enzyme (TLC) that obtains from the agkistrodon acutus snake venom.
Another object of the present invention has been to provide the method for the above-mentioned hemocoagulase-B of a kind of separation and purification.
Hemocoagulase-B of the present invention separates the high reactivity hemocoagulase (called after " hemocoagulase-B ") that obtains from Chinese agkistrodon acutus (Agkistrodon acutus) snake venom, it has the aminoacid sequence shown in the SEQ ID No.1.This enzyme has following feature: 1. by the strand of 236 Amino acid profiles, the SDS-PAGE molecular weight is about 35kD, and iso-electric point pI is 6.0,2. contains 6 intrachain disulfide bonds (site: 7-141,28-44,78-234,120-188,152-167,178-203).3. at Asn
77,100,229Having the heterozygosis saccharan that is made of 5 kinds of different monose on the site modifies.4. enzymic activity can be suppressed fully by phenylmethylsulfonyl fluoride (PMSF), shows that it is a kind of serine protease.5. have preferably thermostability, 60 days enzymic activitys of 40 ℃ of constant temperature can keep 90%.
Should be appreciated that those skilled in the art can not affect under its active prerequisite according to aminoacid sequence disclosed by the invention, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.Therefore, albumen of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.1 is substituted, replaces and/or increases one or several amino acid, have with isoreactivity by the described protein derived protein that obtains.Those skilled in the art can be according to the present invention the N terminal amino acid sequence of hemocoagulase-B, the design synthetic oligonucleotide probe, extract mRNA from the agkistrodon acutus the poison gland, reverse transcription and construction cDNA library, by above-mentioned probe screening cDNA library, and to positive colony that the filters out clonal analysis that checks order respectively, thereby obtain the gene of code book invention albumen.Also can be according to the present invention the aminoacid sequence of hemocoagulase-B directly design the gene of composite coding albumen of the present invention.
The present invention also provides the purification process of above-mentioned hemocoagulase-B, and it comprises the steps:
1), snake venom pre-treatment;
2), with on the pretreated snake venom solution through the DEAE-Sephrose of pre-equilibration FF anion-exchange chromatography post, PBS with 0.01M pH7.0～7.5 washes post, with containing 0.02 and the PBS stepwise elution of the 0.01M pH7.0 of 0.06M NaCl～7.5, collect the elutriant of 0.06MNaCl solution again;
3), above-mentioned elutriant ultrafiltration and concentration to small volume is removed NaCl by dialysis;
4), the solution after will dialysing again loading to the DEAE-Sephrose FF chromatography column through pre-equilibration, PBS with 0.01M pH7.0～7.5 washes post, again with contain 0.02,0.04, the PBS stepwise elution of the 0.01M pH7.0 of 0.06M NaCl～7.5, collect the 3rd elution peak in the 0.06M NaCl eluant solution liquid;
5), with above-mentioned collection liquid ultrafiltration and concentration to small volume by 80% ammonium sulfate precipitation, centrifugal 30 minutes precipitation separations of 12000g.
6), with an amount of dissolution precipitation of PBS, be further purified through Sephadex-G75, more directly lyophilize behind the dialysis desalination.
Wherein, the pretreated method of step 1) snake venom be with snake venom with the 0.01MpH7.0 of an amount of precooling～7.5PBS dissolving, the centrifuging and taking supernatant liquor is dialysed, and (the dialysis tubing molecular weight cut-off is 7,000D～10,000D).Can remove insoluble impurity and micromolecule polypeptide by pre-treatment, and reduce solution ion strength.
Specifically can carry out as follows the pre-treatment of snake venom: take by weighing the some grams of snake venom, with the PBS of the 0.01M pH7.0 of 5 ~ 10 times of volume precoolings of snake venom weight～7.5 stirring and dissolving 30 ~ 60 minutes in 4 ~ 8 ℃ chromatography cabinet, in 4 ~ 8 ℃, 5,000 ~ 10, centrifugal 10 ~ 20 minutes of 000g, in centrifuged supernatant impouring dialysis tubing, centrifugation adds the PBS stirring suspension of 5 ~ 10 times of volume precoolings of snake venom weight, recentrifuge again.Merge the two times centrifugal supernatant liquor in dialysis tubing, in 4 ~ 8C to the PBS dialysis of 0.01M pH7.0～7.5 12 ~ 24 hours, during change liquid 2 ~ 4 times, to remove micromolecule polypeptide and to reduce solution ion strength.
Wherein, step 2) and step 4) adopt the PBS pre-equilibration DEAE-Sephrose FF chromatography column of 0.01M pH7.0～7.5, and then loading.
Wherein, the purpose of step 3) dialysis is to remove the NaCl that exists in the solution.The concentrated mode of large volume elutriant is ultrafiltration and concentration (film cutoff value 5000-10000Da) in step 3) and the step 5).
Wherein, adopting the purpose of Sephadex-G75 molecular sieve in the step 6) is further the protein in twice anion column chromatography gleanings to be carried out separation and purification by molecular size range.To precipitate particularly with upper prop after the PBS dissolving, with 0.01M pH7.4PBS elutriant wash-out, collect first peak of elutriant.
Hemocoagulase-B through the inventive method purifying is not less than 2000U/mg than vigor, and reduction and non-reduced SDS-PAGE are a band; The HPLC purity assay is more than 95%.This enzyme has preferably thermostability, and 60 days enzymic activitys of 40 ℃ of constant temperature can keep 90%.In the lyophilized venom raw material weight, this law purifying recovery rate is 0.25%-0.3%.
Hemocoagulase-B of the present invention has good blood coagulation activity, can be used as the bulk drug of making various haemostatic medicaments, i.e. the present invention also comprises the haemostatic medicament that contains described Agkistrodon acutus hemocoagulase atrox-B.This medicine can be the former medicine of lyophilized powder, lyophilized powder injection, hemostatic plaster, hemostasis pulvis, hemostasis tablet, hemostatic mastic or hemostasis liquid spray, needing to be used for hemostasis to reduce the various medical conditions of amount of bleeding.Also can be used to prevent hemorrhage, avoid or reduce operative site and postoperative hemorrhage.
Description of drawings
Fig. 1 shows SDS-PAGE one band of hemocoagulase-B behind the purifying.
Fig. 2 shows the HPLC analytical results of hemocoagulase-B behind the purifying.
Fig. 3 hemocoagulase-B heterozygosis glycosylation structural representation.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the routine techniques that the technique means that adopts among the embodiment is well known to those skilled in the art.
The percentage sign that relates among the present invention " % " if do not specify, refers to mass percent; But the per-cent of solution except as otherwise herein provided, refers to contain among the solution 100ml the some grams of solute; Per-cent between the liquid refers to the ratio of capacity in the time of 20 ℃.Similar among the present invention " 10 times of volume precoolings of usefulness snake venom weight " statement, the unit of weight and volume wherein is respectively g and ml.
The purifying of embodiment 1 snake venom blood coagulation enzyme-B
Get 30g agkistrodon acutus lyophilized venom (lot number: 20090701, Snake Venoms From Guangxi institute), with the PBS of the 0.01M pH7.4 of 10 times of volume precoolings of snake venom weight stirring and dissolving 30 minutes in 4 ℃ chromatography cabinet, in centrifugal 15 minutes of 4 ℃, 10000g, in centrifuged supernatant impouring dialysis tubing, centrifugation adds the PBS stirring suspension of 10 times of volume precoolings of snake venom weight, recentrifuge again.Merge the two times centrifugal supernatant liquor in dialysis tubing (molecular weight cut-off is 10,000D) in, in 4 ℃ of chromatography cabinets to the PBS dialysis of 0.01M pH7.4 24 hours, during change liquid 3 times.Pretreated snake venom solution is loaded to DEAE-Sepharose Fast Flow anion-exchange chromatography post through 0.01M pH7.4PBS pre-equilibration, wash post with 0.01M pH7.4PBS first, use respectively again the PBS stepwise elution of the 0.01M pH7.4 that contains 0.02M, 0.06M NaCl, collect the elution peak of 0.06M NaCl solution, be total to get 1960ml.
Through enzyme activity determination (with reference to 1. or 2. method of appendix) and electrophoretic analysis, object appears at the elution peak of 0.06M NaCl solution and collects in the liquid.Adopt Millipore Pellicon 2 tangential flow ultrafilter (0.1M
2Cut off 10k film) ultrafiltration and concentration is to 200ml, with this 200ml ultrafiltration and concentration liquid impouring dialysis tubing (10,000D) in, with the PBS of 2000ml 0.01M pH7.4 in 4 ℃ of dialysis 24 hours, during change solution 3 times.Enzyme solution after the dialysis is loaded to DEAE-Sepharose Fast Flow anion-exchange chromatography post through pre-equilibration, PBS with 0.01M pH7.4 washes post, use respectively again the PBS stepwise elution of the 0.01M pH7.4 of 0.02M, 0.04M, 0.06M NaCl, collect the 3rd elution peak of 0.06M NaCl eluant solution liquid, be total to get 660ml.With Millipore Pellicon 2 tangential flow ultrafilter (0.1M
2Cut off 5k film) ultrafiltration and concentration is to 120ml, this 120ml is spent the night through 4 ℃ of 80% ammonium sulfate precipitations, next day 12,4 ℃ of 000g are centrifugal 30 minutes, centrifugation is loaded to Sephdex-G75 series of strata post immediately with the 0.01M pH7.4PBS suspension dissolving of 10ml, with 0.01M pH7.4PBS elutriant wash-out, collect elutriant first peak 65ml, confirm to exist the purpose product through enzyme activity determination.This 65ml concentrated solution is packed in the dialysis tubing, dialysed 24 hours with deionized water, during change solution 3 times, each 2000ml.Volume is 73ml after the dialysis, directly lyophilize.Obtain lyophilized products 81mg, measuring the enzyme specific activity is 2500U/mg, ultimate yield 0.27%.Reduction and non-reduced SDS-PAGE are a band (see figure 1), and the electrophoresis molecular weight is roughly 35kD.HPLC purity 96.2%(sees Fig. 2).It is 6.0 that isoelectric focusing electrophoresis is measured this enzyme iso-electric point pI.
Carry out amino acid and glycosylation mensuration through Edman enzymolysis and peptide figure mass spectrum, its aminoacid sequence is shown in SEQ ID No.1.This hemocoagulase contains 236 amino acid, and only calculating molecular weight by amino acid is 26116.7Dalton; Chain includes 6 disulfide linkage, and the site is: Cys
7-Cys
141, Cys
28-Cys
44, Cys
78-Cys
234, Cys
120-Cys
188, Cys
152-Cys
167, Cys
178-Cys
203Glycosylation site is Asn
77G2F4S, Asn
100– Hybrid, Asn
229-G2F4S, the glycosylation structure consists of (see figure 3) by the heterozygosis saccharan of 5 kinds of different monose.
The purifying of embodiment 2 snake venom blood coagulation enzymes-B
Get 30g agkistrodon acutus lyophilized venom (lot number: 20090701, Snake Venoms From Guangxi institute), carry out pre-treatment by the method identical with embodiment 1.Pretreated snake venom solution is carried out the DEAE-Sephrose FF column chromatography first time by example 1 identical method, appear in the elution peak of 0.06MNaCl through enzyme activity determination and electrophoretic analysis object, merge wash-out and collect liquid, be total to get 2000ml, adopt Millipore Pellicon 2 tangential flow ultrafilter (0.1M
2Cut off10k film) ultrafiltration and concentration is to 210ml, with this 210ml ultrafiltration and concentration liquid impouring dialysis tubing (10,000D) in, with 2000ml 0.01M pH7.4PBS in 4 ℃ of dialysis 24 hours, during change solution 3 times.Enzyme liquid loading after the dialysis is carried out the chromatography second time by example 1 identical method to DEAE-Sephrose FF post.Hemocoagulase appears in the 3rd elution peak of 0.06MNaCl eluant solution liquid, collects this peak elutriant and gets 648ml.With Millipore Pellicon 2 tangential flow ultrafilter (0.1M
2Cut off 5k film) ultrafiltration and concentration is to 130ml, this 130ml is spent the night through 4 ℃ of 80% ammonium sulfate precipitations, 12,4 ℃ of 000g are centrifugal 30 minutes, precipitation is dissolved with the 0.01M pH7.4PBS suspension of 10ml, be loaded to immediately Sephdex-G75 series of strata post, collect elutriant first peak 68ml, confirm through enzyme activity determination.Be 78ml with this 68ml by the example 1 identical method rear volume of dialysing, directly lyophilize.Obtain lyophilized products 85mg, the mensuration specific activity of enzyme is 2460U/mg, ultimate yield 0.28%.SDS-PAGE is a band, and its electrophoresis molecular weight is roughly 35kD.HPLC purity 96.0%.
The serine stretch protein attribute experiment of embodiment 3 Agkistrodon acutus hemocoagulase atroxes-B
Bovine fibrinogen (Sigma company) solution with physiological saline preparation 1%.
Dissolve phenylmethylsulfonyl fluoride (PMSF, Merck company) with Virahol, strength of solution is 4mg/ml.
The experimental implementation step is as follows:
(1) gets 1% bovine fibrinogen solution 2ml, 37 ℃ of lower constant temperature 5 minutes.
(2) get three small test tubes, mark respectively 1#, 2#, 3#, every pipe adds the 1U/ml hemocoagulase of 200 μ l-C solution.
(3) add 10 μ l distilled water to the 1# test tube respectively, the 2# test tube added 10 μ l Virahols, and the 3# test tube adds 10 μ l PMSF, in 37 ° of C water bath heat preservations 5 minutes.
(4) carrying out separately respectively agglutination test by the test tube number order observes.Add good 1% bovine fibrinogen solution, the 200 μ l of constant temperature in test tube, immediately timing is shaken mixing simultaneously gently, leaves standstill in 37 ℃ of water-baths, observes the situation of agglutination reaction in the test tube.Stop timing when presenting complete curdled appearance with solution.
The results are shown in Table one
Table one, PMSF are on the impact of aggegation time
According to the experimental result in the table one, draw to draw a conclusion: 1. the PMSF of 100ppm concentration has suppressed this hemocoagulase-B activity fully, proves that this Agkistrodon acutus hemocoagulase atrox-B is serine protease.2. micro-Virahol on this agglutination reaction without impact.
The thermostability of embodiment 4 Agkistrodon acutus hemocoagulase atroxes-B is investigated experiment
The pure enzyme of lyophilize (the not adding any lyophilized vaccine) lyophilized powder that obtains in implementation column 1 and the implementation column 2 is sub-packed in a plurality of amperes of bottles and seals, be positioned in 40 ℃ of thermostat containers, take out mensuration enzyme specific activity during respectively at 30 days and 60 days.
The result shows: the level that still can keep original enzyme activity 90% in 95%, 60 day that the specific activity of two batches of enzymes can keep protoenzyme to live in 30 days.This result shows that the hemocoagulase that is separated to has preferably thermostability.The results are shown in Table two.
Table two, thermostability are investigated enzyme specific activity measurement result (40 ℃)
| |
0 day specific activity (u/mg) | 30 days specific activity (u/mg) | 60 days specific activity (u/mg) |
| |
2500 | 2380 | 2260 |
| |
2460 | 2360 | 2250 |
Appendix
Agkistrodon acutus hemocoagulase atrox unit definition and activity determination method
1. bovine fibrinogen assay method 1.0% bovine fibrinogen (Sigma company) the solution 1ml that gets physiological saline preparation puts in the small test tube, 37 ± 0.5 ℃ of water bath heat preservations 3 minutes, the enzyme solution 1ml to be measured that adds 37 ± 0.5 ℃ of preheatings, immediately timing, white floc sedimentation appears in fibrinogen solution jolting in 120 ± 30 seconds, and then this enzyme solution is 1U/ml.
2. the accurate human plasma 1ml of normal man's determination of plasma method label taking puts in the small test tube, put in 37 ℃ ± 0.5 ℃ water-bath preheating 3 minutes, and added the enzyme solution 1ml to be measured of 37 ± 0.5 ° of C preheatings, immediately timing, white floc sedimentation appears in human plasma jolting in 60 ± 20 seconds, and then this enzyme solution is 1U/ml.
Annotate: need when measuring unknown enzymatic activity high solution to dilute with deionized water, be used for measuring until reach 1U/ml; Its extension rate is every milliliter of units alive of the enzyme in the former enzyme solution.
Sequence table
＜110〉Beijing Kangchen Medicine Co., Ltd
＜120〉Agkistrodon acutus hemocoagulase atrox B
<130>
<160>2
<170>PatentIn version 3.5
<210>1
<211>236
<212>PRT
<213>Agkistrodon acutus
<220>
<221>CARBOHYD
<222>(77),(100),(229)
<400>1
Val Ile Gly Gly Asn Glu Cys Asp Thr Asn Glu His Arg Phe Leu
1 5 10 15
Val Ala Leu Asn Glu Leu Thr Ser Met Thr Phe Leu Cys Gly Gly
20 25 30
Thr Leu Ile Asn Gln Glu Trp Val Val Thr Ala Ala His Cys Asp
35 40 45
Arg Leu Gln Leu Tyr Leu Tyr Ile Gly Met His Asp Lys Tyr Val
50 55 60
Lys Phe Asp Asp Glu Gln Gly Arg Glu Pro Ile Glu Lys Tyr Phe
65 70 75
Tyr Asn Cys Ser Asn Asn Leu Thr Thr Arg Asp Lys Asp Ile Met
80 85 90
Leu Ile Arg Leu Asp Arg Pro Val Asp Asn Ser Thr His Ile Ala
95 100 105
Pro Leu Ser Leu Pro Ser Arg Pro Pro Ser Val Gly Ser Val Cys
110 115 120
Arg Val Met Gly Trp Gly Ala Ile Ser Pro Ser Arg Asp Val Leu
125 130 135
Pro Asp Val Pro His Cys Val Asn Ile Asn Leu Val Asn Asn Ala
140 145 150
Glu Cys Arg Arg Ala Tyr Pro Arg Leu Pro Ala Thr Ser Arg Thr
155 160 165
Leu Cys Ala Gly Val Met Gln Gly Gly Ile Asp Ser Cys Asn Arg
170 175 180
Asp Ser Gly Gly Pro Leu Ile Cys Asp Gly Gln Phe Gln Gly Val
185 190 195
Val Asn Trp Gly Gly Asn Pro Cys Ala Gln Pro Asn Met Pro Ala
200 205 210
Leu Tyr Thr Lys Val Tyr Asp Tyr Asn Asp Trp Ile Arg Ser Ile
215 220 225
Thr Ala Gly Asn Thr Thr Ala Ala Cys Pro Pro
230 235
<210>2
<211>15
<212>PRT
<213>Agkistrodon acutus
<400>2
Val Ile Gly Gly Asn Glu Cys Asp Thr Asn Glu Glu His Phe Leu
1 5 10 15
Claims (10)
1. Agkistrodon acutus hemocoagulase atrox-B, it has the aminoacid sequence shown in the SEQ ID No.1 or this sequence through replacing, lack and/or adding the aminoacid sequence with same function that one or several amino acid obtains.
2. Agkistrodon acutus hemocoagulase atrox-B as claimed in claim 1 is characterized in that, chain contains 6 pairs of disulfide linkage, at Asn
77,100,229Having the heterozygosis saccharan on the site modifies.
3. the medicine that contains claim 1 or 2 described Agkistrodon acutus hemocoagulase atrox-B.
4. medicine as claimed in claim 3, it is the former medicine of lyophilized powder, lyophilized powder injection, hemostatic plaster, hemostasis pulvis, hemostasis tablet, hemostatic mastic or hemostasis liquid spray.
5. the purification process of claim 1 or 2 described hemocoagulase-B, it comprises the steps:
1), snake venom pre-treatment;
2), with on the pretreated snake venom solution through the DEAE-Sepharose of pre-equilibration Fast Flow anion-exchange chromatography post, PBS with 0.01M pH7.0～7.5 washes post, with containing 0.2 and the PBS stepwise elution of the 0.01M pH7.0 of 0.6M NaCl～7.5, collect the elution peak of 0.06MNaCl solution again;
3), NaCl is removed in dialysis behind the above-mentioned elutriant ultrafiltration and concentration;
4), the solution after will dialysing is gone up the chromatography column through the DEAE-Sepharose of pre-equilibration Fast Flow again, PBS with 0.01M pH7.0～7.5 washes post, again with contain 0.02,0.04, the PBS stepwise elution of the 0.01M pH7.0 of 0.06M NaCl～7.5, collect the 3rd elution peak of 0.06M NaCl solution;
5), above-mentioned wash-out is collected liquid 80% ammonium sulfate precipitation, centrifugal collecting precipitation;
6), with an amount of dissolution precipitation of PBS, be further purified through Sephadex-G75, again through the dialysis desalination, lyophilize.
6. method as claimed in claim 5, wherein, the pretreated method of step 1) snake venom is that the centrifuging and taking supernatant liquor is dialysed with the PBS dissolving of snake venom with the 0.01M pH7.0 of an amount of precooling～7.5.
7. method as claimed in claim 5, wherein, the pretreated method of step 1) snake venom is: take by weighing the some grams of snake venom, with the PBS of the 0.01MpH7.0 of 5 ~ 10 times of volume precoolings of snake venom weight～7.5 stirring and dissolving 30 ~ 60 minutes in 4 ~ 8 ℃ chromatography cabinet, in 4 ℃, 5,000 ~ 10, centrifugal 10 ~ 20 minutes of 000g, with centrifuged supernatant impouring dialysis tubing, the PBS stirring suspension of 5 ~ 10 times of volume precoolings of snake venom weight is used in centrifugation again, and recentrifuge, merging two times centrifugal supernatant liquor are packed in the dialysis tubing, in 4 ~ 8 ℃ to the PBS of 0.01M pH7.0～7.5 dialysis 12 ~ 24 hours, during change solution 2 ~ 4 times.
8. method as claimed in claim 5, wherein, step 2) and step 4) adopt the PBS pre-equilibration DEAE-Sepharose Fast Flow chromatography column of 0.01M pH7.0～7.5, then loading.
9. such as each described method of claim 5 ~ 8, wherein, step 3) employing molecular retention value is that the ultra-filtration membrane of 5,000 ~ 10,000D carries out ultrafiltration and concentration.
10. such as each described method of claim 5 ~ 8, wherein, the method for step 6) purifying is that upper Sephadex-G75 chromatography column with 0.01M pH7.4PBS elutriant wash-out, was collected first peak of elutriant after the precipitation that step 5) is collected was dissolved with PBS.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210458987.2A CN102925422B (en) | 2012-11-14 | 2012-11-14 | Agkistrodon acutus hemocoagulase-B |
| PCT/CN2013/000375 WO2014075374A1 (en) | 2012-11-14 | 2013-04-01 | Agkistrodon acutus hemocoagulase-b |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210458987.2A CN102925422B (en) | 2012-11-14 | 2012-11-14 | Agkistrodon acutus hemocoagulase-B |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102925422A true CN102925422A (en) | 2013-02-13 |
| CN102925422B CN102925422B (en) | 2014-08-06 |
Family
ID=47640353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210458987.2A Active CN102925422B (en) | 2012-11-14 | 2012-11-14 | Agkistrodon acutus hemocoagulase-B |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN102925422B (en) |
| WO (1) | WO2014075374A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075374A1 (en) * | 2012-11-14 | 2014-05-22 | 北京康辰药业有限公司 | Agkistrodon acutus hemocoagulase-b |
| CN105586330A (en) * | 2016-03-25 | 2016-05-18 | 中山大学 | Haemocoagulase acutus thrombin and preparation method thereof |
| CN106243224A (en) * | 2016-08-05 | 2016-12-21 | 安徽威尔试剂盒科技有限责任公司 | One utilizes Agkistrodon acutus venom differential protein to prepare sero-fast method and application thereof |
| CN116121279A (en) * | 2021-10-08 | 2023-05-16 | 北京康辰药业股份有限公司 | Hemagglutinin and screening method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766100A (en) * | 2005-07-25 | 2006-05-03 | 康辰医药发展有限公司 | Agkistrodonacutus thrombin preparation method and uses |
| CN1944642A (en) * | 2006-09-12 | 2007-04-11 | 沈居仁 | Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method |
| CN101358184A (en) * | 2008-09-27 | 2009-02-04 | 康辰医药股份有限公司 | Hemocoagulase agkistrodon |
| CN102242103A (en) * | 2010-05-10 | 2011-11-16 | 辽宁诺康医药有限公司 | Preparation method of agkistrodon acutus venom thrombin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925422B (en) * | 2012-11-14 | 2014-08-06 | 北京康辰药业有限公司 | Agkistrodon acutus hemocoagulase-B |
-
2012
- 2012-11-14 CN CN201210458987.2A patent/CN102925422B/en active Active
-
2013
- 2013-04-01 WO PCT/CN2013/000375 patent/WO2014075374A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766100A (en) * | 2005-07-25 | 2006-05-03 | 康辰医药发展有限公司 | Agkistrodonacutus thrombin preparation method and uses |
| CN1944642A (en) * | 2006-09-12 | 2007-04-11 | 沈居仁 | Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method |
| CN101358184A (en) * | 2008-09-27 | 2009-02-04 | 康辰医药股份有限公司 | Hemocoagulase agkistrodon |
| CN101560510A (en) * | 2008-09-27 | 2009-10-21 | 北京方策方程医药科技有限公司 | Agkistrodon acutus hemocoagulase atrox |
| CN102242103A (en) * | 2010-05-10 | 2011-11-16 | 辽宁诺康医药有限公司 | Preparation method of agkistrodon acutus venom thrombin |
Non-Patent Citations (2)
| Title |
|---|
| YING-MING WANG ET AL.: "Serine protease isoforms of Deinagkistrodon acutus venom cloning, sequencing and phylogenetic analysis", 《BICHEMICAL JOURNAL》 * |
| 欧光武等: "尖吻蝮蛇类凝血酶的研究现状", 《中国医药导报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075374A1 (en) * | 2012-11-14 | 2014-05-22 | 北京康辰药业有限公司 | Agkistrodon acutus hemocoagulase-b |
| CN105586330A (en) * | 2016-03-25 | 2016-05-18 | 中山大学 | Haemocoagulase acutus thrombin and preparation method thereof |
| CN105586330B (en) * | 2016-03-25 | 2019-04-02 | 中山大学 | A kind of Halase and preparation method thereof |
| CN106243224A (en) * | 2016-08-05 | 2016-12-21 | 安徽威尔试剂盒科技有限责任公司 | One utilizes Agkistrodon acutus venom differential protein to prepare sero-fast method and application thereof |
| CN116121279A (en) * | 2021-10-08 | 2023-05-16 | 北京康辰药业股份有限公司 | Hemagglutinin and screening method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014075374A1 (en) | 2014-05-22 |
| CN102925422B (en) | 2014-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Thaler et al. | A simple two‐step isolation procedure for human and bovine antithrombin II/III (heparin cofactor): a comparison of two methods | |
| CN101560510B (en) | Agkistrodon acutus hemocoagulase atrox | |
| US3931399A (en) | Process for isolating a fibrin-stabilizing factor | |
| CN102925422B (en) | Agkistrodon acutus hemocoagulase-B | |
| CN103539831A (en) | Prunus armeniaca alpha-glucosidase inhibiting peptide as well as preparation method and application of inhibiting peptide | |
| JPS59137417A (en) | Preparation of colonization stimulation factor and kallikrein originated from human urine | |
| CN103539833A (en) | High-activity alpha-glucosidase inhibitory peptide and preparation method and application thereof | |
| JP2003518513A (en) | Separation of fibrinogen from plasma protease | |
| CN100494365C (en) | Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method | |
| CN103160485B (en) | Agkistrodon acutus hemocoagulase atrox-C | |
| CN101880656B (en) | Agkistrodon halys venom thrombin and preparation method and application thereof | |
| Zhang et al. | Flammulin Purified from the Fruit Bodies of Flammulina velutipes (Curt.: Fr.) P. Karst. | |
| Smith et al. | A factor VIII concentrate of intermediate purity and higher potency | |
| CN1159438C (en) | Ahylysantinfarctase thrombase and its production process | |
| CN103184207A (en) | Agkistrodon acutus streptokinase and preparation method and application thereof | |
| CN1548534B (en) | Reptilase and its production process and application | |
| CN103145800A (en) | Centipede zymolyte anti-thrombus polypeptide | |
| US20100120120A1 (en) | Haemocoagulase | |
| CN101092612A (en) | New thrombin with hemostaitic activityin in category of snake poison of hundred-paced pit | |
| CN1159437C (en) | Ahylysantinfarctase thrombase and its production process | |
| CN101580826A (en) | Snake-poison hemostatic enzyme | |
| CN1920017A (en) | Separating and purifying method of agkistrodonacutus thrombin | |
| Jamieson et al. | Membrane glycoproteins of blood platelets | |
| CN102146134A (en) | Method for efficiently extracting and purifying blood coagulation factor IX and blood coagulation factor X | |
| CN105586330A (en) | Haemocoagulase acutus thrombin and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 100085, A, building 7, building 9, Wah Wah building, 707 3rd Street, Beijing, Haidian District Patentee after: BEIJING KONRUNS PHARMACEUTICAL CO., LTD. Address before: 100085, A, building 7, building 9, Wah Wah building, 707 3rd Street, Beijing, Haidian District Patentee before: Konruns Pharmaceutical Co., Ltd. |
|
| C56 | Change in the name or address of the patentee | ||
| CP02 | Change in the address of a patent holder |
Address after: 101500 Miyun Economic Development Zone, Beijing, No. 11 South Road Patentee after: BEIJING KONRUNS PHARMACEUTICAL CO., LTD. Address before: 100085, A, building 7, building 9, Wah Wah building, 707 3rd Street, Beijing, Haidian District Patentee before: BEIJING KONRUNS PHARMACEUTICAL CO., LTD. |