WO2014075374A1 - Agkistrodon acutus hemocoagulase-b - Google Patents
Agkistrodon acutus hemocoagulase-b Download PDFInfo
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- WO2014075374A1 WO2014075374A1 PCT/CN2013/000375 CN2013000375W WO2014075374A1 WO 2014075374 A1 WO2014075374 A1 WO 2014075374A1 CN 2013000375 W CN2013000375 W CN 2013000375W WO 2014075374 A1 WO2014075374 A1 WO 2014075374A1
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- hemocoagulase
- snake venom
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- 241001505404 Deinagkistrodon acutus Species 0.000 title abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000000502 dialysis Methods 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 15
- 239000002435 venom Substances 0.000 claims abstract description 14
- 210000001048 venom Anatomy 0.000 claims abstract description 14
- 231100000611 venom Toxicity 0.000 claims abstract description 14
- 150000001413 amino acids Chemical class 0.000 claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 11
- 238000000746 purification Methods 0.000 claims abstract description 10
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- 239000000243 solution Substances 0.000 claims description 43
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 34
- 239000003998 snake venom Substances 0.000 claims description 33
- 230000002439 hemostatic effect Effects 0.000 claims description 18
- 239000011780 sodium chloride Substances 0.000 claims description 17
- 239000003814 drug Substances 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 6
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- 238000003756 stirring Methods 0.000 claims description 5
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 238000005571 anion exchange chromatography Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 3
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- 241000271039 Agkistrodon Species 0.000 claims 1
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- 210000002966 serum Anatomy 0.000 claims 1
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- 108010027612 Batroxobin Proteins 0.000 abstract description 9
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 5
- 108010022999 Serine Proteases Proteins 0.000 abstract description 4
- 102000012479 Serine Proteases Human genes 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 4
- 102000003886 Glycoproteins Human genes 0.000 abstract description 3
- 108090000288 Glycoproteins Proteins 0.000 abstract description 3
- 238000004440 column chromatography Methods 0.000 abstract description 3
- 239000002808 molecular sieve Substances 0.000 abstract description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 abstract description 2
- 238000005349 anion exchange Methods 0.000 abstract 1
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- 238000004108 freeze drying Methods 0.000 abstract 1
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- 239000000126 substance Substances 0.000 abstract 1
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- 229940088598 enzyme Drugs 0.000 description 29
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 11
- 108010049003 Fibrinogen Proteins 0.000 description 9
- 102000008946 Fibrinogen Human genes 0.000 description 9
- 229940012952 fibrinogen Drugs 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 6
- 230000000740 bleeding effect Effects 0.000 description 6
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- 241000283690 Bos taurus Species 0.000 description 5
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- 101000772006 Bombus ignitus Venom serine protease Bi-VSP Proteins 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 3
- 108010073385 Fibrin Proteins 0.000 description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 3
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- 230000015271 coagulation Effects 0.000 description 3
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- 238000003028 enzyme activity measurement method Methods 0.000 description 3
- 229950003499 fibrin Drugs 0.000 description 3
- 230000023597 hemostasis Effects 0.000 description 3
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- 241000894007 species Species 0.000 description 3
- 206010036790 Productive cough Diseases 0.000 description 2
- 241000239226 Scorpiones Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
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- 210000003802 sputum Anatomy 0.000 description 2
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- 241000271037 Crotalinae Species 0.000 description 1
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- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 108010046562 botropase Proteins 0.000 description 1
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- 239000002574 poison Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
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Classifications
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- 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
Definitions
- the present invention relates to a serine protease, in particular to a scorpion snake venom hemagglutinase-B, and to a method for its isolation and purification.
- TLC thrombin-like enzyme
- the clotting enzyme is similar to the apparent action of thrombin (thrombin), which can convert fibrinogen in plasma into fibrin and "coagulate".
- the mechanism of action of the two enzymes is different.
- the thrombin-like enzyme does not activate the sputum factor in the coagulation system. Its interaction with fibrinogen produces only non-crosslinked fibrin, which does not cause thrombosis.
- the clot formed can be dissolved by 5M urea.
- Thrombin activates the sputum factor in the coagulation system, which interacts with fibrinogen to form cross-linked fibrin, which can lead to thrombosis, and the clot formed can not be dissolved by 5M urea.
- thrombin-like components are contained in more than 40 species of snake venom, and more than 30 species have been isolated and purified, and all or part of the amino acid sequence of more than 10 species of thrombin has been elucidated.
- the molecular weight of TLC has been found to be between 29 and 45 kD, most of which are acidic glycoproteins.
- TLC thrombin-like enzyme
- Another object of the present invention is to provide a method for isolating and purifying the above hemagglutinase-B.
- the hemagglutinase-B of the present invention is a highly active hemagglutinase (designated "hemagglutinase-B") isolated from the snake venom of Agkistrodon acutus, which has the SEQ ID No. Amino acid sequence.
- the enzyme has the following characteristics: 1 a single chain consisting of 236 amino acids, SDS-PAGE molecular weight is about 35kD, isoelectric point pi is 6.0, 2 contains 6 intrachain disulfide bonds (site: 7-141, 28- 44, 78-234, 120-188, 152-167, 178-203).
- heteropolysaccharide modifications consisting of 5 different monosaccharides at Asn 77 , 100 ' 229 .
- the cDNA library is screened, and the selected positive clones are separately subjected to sequencing and cloning analysis to obtain a gene encoding the protein of the present invention.
- the gene encoding the protein of the present invention can also be directly designed and synthesized according to the amino acid sequence of hemagglutinase-B of the present invention.
- the present invention also provides a method for purifying the above hemagglutinase-B, which comprises the following steps:
- step 3 the elution peak collected in step 2) is concentrated by ultrafiltration to a small volume, and then dialyzed to remove NaCl;
- the dialysis solution was loaded again onto a pre-equilibrated DEAE-Sephrose FF column, washed with 0.01 M PBS pH 7.0 ⁇ 7.5, and then 0.01 M with 0.02, 0.04, 0.06 M NaCl
- the PBS was fractionally eluted with pH 7.0 - 7.5, and the third elution peak in the eluate of 0.06 M NaCl solution was collected;
- the eluted peak collected in the above step 4) was concentrated by ultrafiltration to a small volume, precipitated with 80% ammonium sulfate, and centrifuged at 12000 g for 30 minutes to separate the precipitate.
- the pretreatment of the snake venom can be carried out by the following steps: weighing a few grams of snake venom, using a venom weight of 5 to 10 times the volume of 0.01 M pH 7.0 ⁇ 7.5 PBS in a 4 to 8 ° C chromatographic cabinet Stir for 30 ⁇ 60 minutes, centrifuge at 4 ⁇ 8°C, 5,000-10,000g for 10 ⁇ 20 minutes, pour the supernatant into the dialysis bag, and add the venom weight 5 ⁇ 10 times the volume of pre-cooled PBS. Stir in suspension and centrifuge again.
- step 2) and step 4) pre-equilibrated DEAE-Sephrose FF column with 0.01M pH 7.0 ⁇ 7.5 PBS, and then loaded.
- step 3) the purpose of dialysis is to remove the NaCl present in the solution.
- step 3) and step 5) The large volume eluent is concentrated by ultrafiltration (membrane cutoff value 5000-10000Da)
- the purpose of using Sephade X- G75 molecular sieve in step 6) is to further separate and purify the protein in the two anion column chromatography collections by molecular weight. Specifically, the precipitate was dissolved in PBS, and the column was eluted with a 0.01 M pH 7.4 eluate to collect the first peak of the eluate.
- the specific activity of hemagglutinase-B purified by the method of the present invention is not less than 2000 U/mg, and both reduction and non-reduction SDS-PAGE are one band; HPLC analysis has a purity of over 95%.
- the enzyme has good thermal stability and can maintain 90% enzyme activity at 40 ⁇ constant temperature for 60 days. Based on the weight of the venom of the snake venom, the purification yield of the method is 0.25% - 0.3%.
- the hemagglutinase-B of the present invention has good blood coagulation activity and can be used as a raw material medicine for preparing various hemostatic drugs, that is, the present invention also includes a hemostatic drug containing the scorpion snake blood coagulase-B.
- the medicament may be a lyophilized powder, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostatic spray for various medical conditions requiring hemostasis to reduce the amount of bleeding. Can also be used to prevent bleeding, avoid or reduce the surgical site and surgery After bleeding.
- Figure 1 shows the SDS-PAGE-band of hemagglutinase-B after purification.
- Figure 2 shows the results of HPLC analysis of hemagglutinase-B after purification.
- FIG. 3 Schematic diagram of hemagglutinase-B heterozygous glycosylation structure. detailed description
- the percent sign "%" referred to in the present invention means mass percentage unless otherwise specified; however, the percentage of the solution, unless otherwise specified, means that the solution contains several grams of solute in 100 ml; the percentage between the liquids, It refers to the ratio of capacity at 20 °C. In the present invention, it is similar to the expression "pre-cooled with a venom weight of 10 times by volume", wherein the units of weight and volume are g and ml, respectively.
- the two supernatants were combined and centrifuged in a dialysis bag (molecular weight cutoff of 10,000 D), and dialyzed against 0.01 M pH 7.4 PBS for 24 hours in a 4 ° C chromatography cabinet, during which time the solution was changed three times.
- the pretreated snake venom solution was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column pre-equilibrated with 0.01 M pH 7.4 PBS, first washed with 0.01 M pH 7.4 PBS, and then with 0.02 M, 0.06 M NaCl, respectively. 0.01M pH 7.4
- the PBS was eluted in sections, and the elution peak of 0.06 M NaCl solution was collected to obtain a total of 1960 ml.
- the dialyzed enzyme solution was applied to a pre-equilibrated DEAE-Sepharose Fast Flow anion exchange chromatography column, washed with 0.01 M pH 7.4 PBS, and then 0.02 M, 0.04 M, 0.06 M NaCl 0.01 M, respectively.
- the PBS of pH 7.4 was eluted in sections, and the third eluting peak of the eluate of 0.06 M NaCl solution was collected, and a total of 660 ml was obtained.
- the mixture was concentrated to 120 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane), and the 120 ml was precipitated by 80% ammonium sulfate at 4 ° C overnight, and centrifuged at 12,000 g for 4 minutes at 4 ° C for 30 minutes. The pellet was suspended and dissolved in 10 ml of M pH 7.4 PBS. Immediately applied to a Sephdex-G75 column and eluted with 0.01 M pH 7.4 eluate. The first peak of the eluate was collected in 65 ml. The measurement confirmed the presence of the objective product.
- the 65 ml concentrate was placed in a dialysis bag, and dialyzed against ion-free water for 24 hours, during which time the solution was changed three times, 2000 ml each time. After dialysis, the volume was 73 ml and directly freeze-dried. 81 mg of the lyophilizate was obtained, and the specific activity of the enzyme was determined to be 2500 U/mg, and the final yield was 0.27%. Both reduced and non-reduced SDS-PAGE are banded (see Figure 1) and the molecular weight of the electrophoresis is approximately 35kD. The HPLC purity was 96.2% (see Figure 2). The isoelectric point pi of the enzyme was determined by isoelectric focusing electrophoresis to be 6.0.
- the amino acid and glycosylation assays were carried out by Edman digestion and peptide mapping mass spectrometry, and the amino acid sequence thereof is shown in SEQ ID No. 1.
- the hemagglutinase contains 236 amino acids, and the molecular weight is only 26116.7Dalton based on amino acid; the chain contains 6 disulfide bonds, and the sites are: Cys 7 -Cys 141 , Cys 28 -Cys 44 , Cys 78 -Cys 234 , Cys 120- Cys' 88 , Cys , 52 -Cys 167 , Cys 178 -Cys 203 o glycosylation sites are Asn 77 - G2F4S, Asn 100 - Hybrid, Asn 229 - G2F4S, glycosylation structure consists of 5 different monosaccharides The heterozygous polysaccharide is composed (see Figure 3).
- Example 2 30 g of the lyophilized powder of Agkistrodon acutus venom (batch number: 20090701, Guangxi Snake Venom Research Institute) was pretreated in the same manner as in Example 1.
- the pretreated snake venom solution was subjected to the first DEAE-Sephrose FF column chromatography in the same manner as in Example 1.
- the target substance appeared in the elution peak of 0.06 M NaCl by enzyme activity measurement and electrophoresis analysis, and the eluted collection solution was combined.
- a total of 2000ml was prepared and concentrated to 210ml by Millipore Pellicon 2 tangential flow ultrafilter (0.1M 2 cut off 10k membrane).
- the 210ml ultrafiltration concentrate was poured into a dialysis bag (10,000D) with 2000ml 0.01 M pH 7.4 PBS was dialyzed against 4'C for 24 hours, during which time the solution was changed 3 times.
- the dialyzed enzyme solution was applied to a DEAE-Sephrose FF column, and a second chromatography was carried out in the same manner as in Example 1. Hemagglutinase appeared in the third elution peak of the 0.06 M NaCl solution eluate, and the peak eluate was collected to obtain 648 ml.
- the mixture was concentrated to 130 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane), and the 130 ml was passed through 80 °/. Ammonium sulfate was precipitated at 4 ° C overnight, 12,000 g of 4 . After centrifugation for 30 minutes, the pellet was suspended and dissolved in 10 ml of 0.01 M pH 7.4 PBS, and immediately applied to a Sephdex-G75 column to collect 68 ml of the first peak of the eluate, which was confirmed by enzyme activity measurement. The 68 ml was dialyzed in the same manner as in Example 1 to a volume of 78 ml, which was directly freeze-dried.
- Example 1 The hemagglutinase-B isolated in Example 1 was diluted with physiological saline to an enzyme activity of 1 U/ml. A 1% solution of bovine fibrinogen (Sigma) was prepared in physiological saline.
- the agglutination test was separately performed in the order of the test tube numbers. Add a constant temperature of 1% bovine fibrinogen solution 200 ⁇ 1 to the test tube, immediately time the mixture, gently shake and mix, and let stand in 37 C water, observe the agglutination reaction in the tube. The timing is terminated when the solution is completely solidified.
- the freeze-dried pure enzyme (without adding any lyoprotectant) obtained in column 1 and column 2 was dispensed into a plurality of ampoules and sealed in a plurality of ampoules, and placed in a 40 ° C incubator for 30 days respectively.
- the enzyme activity was measured and taken at 60 days.
- Bovine fibrinogen assay 1.0% bovine fibrinogen (Sigma) solution prepared in physiological saline was placed in a small test tube, kept in a 37 ⁇ 0.5°C water bath for 3 minutes, and preheated at 37 ⁇ 0.5°C. The enzyme solution is 1 ml, immediately timed, and the fibrinogen solution shakes white flocs in 120 ⁇ 30 seconds, and the enzyme solution is 1 U/ml.
- Standard human plasma assay Take standard human plasma 1ml in a small test tube, preheat for 3 minutes in a 37 ° C ⁇ 0.5 ° water bath, add 37 ⁇ 0.5 ° C preheated enzyme solution 1 ml, immediately timed, human plasma When the white floc appeared in shaking within 60 ⁇ 20 seconds, the enzyme solution was 1 U/ml.
- the dilution factor is the number of enzyme activity units per ml of the original enzyme solution.
- the blood cobase-B obtained by the purification method of the invention has good coagulation activity, high enzyme specific activity and good thermal stability, and the enzyme has good thermal stability. . Therefore, the hemagglutinase-B of the present invention can be used as a raw material medicine for preparing various hemostatic drugs, and the medicine can be a lyophilized powder original medicine, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostasis. Liquid spray for various medical conditions where hemostasis is required to reduce the amount of bleeding. It can also be used to prevent bleeding and to avoid or reduce bleeding at the surgical site and after surgery.
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Abstract
The present invention provides agkistrodon acutus hemocoagulase-B. The agkistrodon acutus hemocoagulase-B is high-activity hemocoagulase separated from agkistrodon acutus venom. The hemocoagulase is a single-stranded glycoprotein comprising 236 amino acids, and has an amino acid sequence shown as SEQ ID No. 1. The strand comprises 6 pairs of disulfide bonds; the molecular weight of SDS-PAGE is approximately 35kD; the molecular weight after deglycosylation 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 site. The hemocoagulase is serine proteinase. The present invention further provides a separation and purification method of the hemocoagulase. The method comprise: removing undissolved substances by using pretreatment; performing anion-exchange column chromatography for two times and Sephdex-G75 molecular sieve chromatography for one time; obtaining an active elution peak; performing dialysis and lyophilization, so as to obtain the high-purity agkistrodon acutus venom hemocoagulase. The activity of the agkistrodon acutus venom hemocoagulase is not less than 2000U/mg; the purity in the HPLC analysis can exceed 95%; and the yield is 0.25% to 0.30% calculated according to the weight of an agkistrodon acutus venom raw material.
Description
尖吻蝮蛇血凝酶 -B 技术领域 Agkistrodon acutus hemagglutinase-B
本发明涉及一种丝氨酸蛋白酶,具体地说是一种尖吻蝮蛇蛇毒血 凝酶 -B, 本发明还涉及其分离纯化方法。 背景技术 The present invention relates to a serine protease, in particular to a scorpion snake venom hemagglutinase-B, and to a method for its isolation and purification. Background technique
蝮亚科(Crotalinae )蛇毒中存在一类与血液凝固相关的蛋白酶, 通常称之为 "类凝血酶" (thrombin-like enzyme, 简称: TLC )。 类凝 血酶与凝血酶(thrombin ) 的表观作用相似, 均可以使血浆中纤维蛋 白原转化为纤维蛋白而 "凝固"。 然而, 两种酶作用机理不同, 类凝 血酶不激活凝血系统中 ΧΠΙ因子,其与纤维蛋白原作用仅产生非交联 纤维蛋白, 不会导致血栓形成, 其形成的凝血块可被 5M尿素溶解; 而凝血酶能够激活凝血系统中 ΧΠΙ因子,其与纤维蛋白原作用形成交 联纤维蛋白,可导致血栓形成,其形成的凝血块不能被 5M尿素溶解。 There is a class of proteases associated with blood coagulation in Crotalinae venom, commonly referred to as "thrombin-like enzyme" (TLC). The clotting enzyme is similar to the apparent action of thrombin (thrombin), which can convert fibrinogen in plasma into fibrin and "coagulate". However, the mechanism of action of the two enzymes is different. The thrombin-like enzyme does not activate the sputum factor in the coagulation system. Its interaction with fibrinogen produces only non-crosslinked fibrin, which does not cause thrombosis. The clot formed can be dissolved by 5M urea. Thrombin activates the sputum factor in the coagulation system, which interacts with fibrinogen to form cross-linked fibrin, which can lead to thrombosis, and the clot formed can not be dissolved by 5M urea.
近 50年来的科学研究发现在 40多种蛇毒中含有类凝血酶成分, 有 30余种得到分离纯化,其中有 10余种类凝血酶的全部或部分氨基 酸序列得到阐明。 已发现的 TLC分子量多在 29 ~ 45kD之间, 大多数 为酸性糖蛋白。 Scientific research in the past 50 years has found that thrombin-like components are contained in more than 40 species of snake venom, and more than 30 species have been isolated and purified, and all or part of the amino acid sequence of more than 10 species of thrombin has been elucidated. The molecular weight of TLC has been found to be between 29 and 45 kD, most of which are acidic glycoproteins.
在以往已经发现的蛇毒 TLC 中, 蛋白质一级结构多为单链。 其 商品化典型代表产品 "立芷雪" (Reptilase ) 系由瑞士 Solco Basle公 司生产, 是由巴西矛头蝮蛇 i Bothmps. atrox )蛇毒中分离出的一种 凝血酶, 该酶前体由 255个氨基酸构成, N端有 24个氨基酸形成的 引导肽, 活性酶含 231氨基酸, SDS-PAGE分子量 34kD。 含 6个链 内二硫键(位点: 7-139, 26-42, 74-230, 118-184, 150-163, 174-199 ) , 为单链糖蛋白 (糖基化位点为 Asn 146,225 ), 脱去糖基后的分子量为 25517Da。 另一个已经商品化的产品是意大利 RAVIZZ公司生产的 "Botropase" , 该凝血酶是从美洲矛头蝮蛇 ( BothrOpsJara ca )蛇
毒中分离得到的。 活性酶含 231氨基酸, 在氨基酸序列上与 Reptilase 比较在 14个位点上有差别。 In the snake venom TLC that has been found in the past, the primary structure of the protein is mostly single-stranded. Its commercial representative product, "Reptilase", produced by Swiss company Solco Basle, is a thrombin isolated from the venom of the Brazilian snake, i Bothmps. atrox, which consists of 255 precursors. Amino acid composition, a guide peptide formed by 24 amino acids at the N-terminus, an active enzyme containing 231 amino acids, and a SDS-PAGE molecular weight of 34 kD. Contains 6 intrachain disulfide bonds (site: 7-139, 26-42, 74-230, 118-184, 150-163, 174-199), a single-chain glycoprotein (glycosylation site is Asn) 146 , 225 ), the molecular weight after removal of the glycosyl group is 25517 Da. Another product that has been commercialized is the "Botropase" produced by RAVIZZ, Italy, which is a snake from the American spear python (Bothr. Separated from the poison. The active enzyme contained 231 amino acids and differed in amino acid sequence from 14 sites in comparison with Reptilase.
此外, 国内学者沈居仁等 2006年从中国尖吻蝮蛇 Ugkistrodo" acMi )蛇毒中分离得到一种高活性血凝酶,该酶为单链, SDS- PAGE 分子量 36 ± 2kD, 等电点为 6.59, 比活为 2000U-3000U/mg, N-末端 15 个氨基酸序列为 VIGGNECDTNEEHFL。 以蛇毒原料重量计收得 率为 0.03%。 In addition, domestic scholar Shen Juren et al. isolated a high-activity hemagglutinase from the Chinese snake venom Ugkistrodo " acMi ) snake venom in 2006. The enzyme is single-stranded, the molecular weight of SDS-PAGE is 36 ± 2kD, and the isoelectric point is 6.59. The specific activity is 2000U-3000U/mg, and the N-terminal 15 amino acid sequence is VIGGNECDTNEEHFL. The yield of the snake venom raw material is 0.03%.
到目前为止, 商品化的蛇毒血凝酶种类较少, 因此寻找适合产业 化的高活性高收率的蛇毒血凝酶新品种尤显必要。 So far, there are few varieties of commercial snake venom hemagglutinase, so it is particularly necessary to find a new high-activity and high-yield snake venom hemagglutinase suitable for industrialization.
本发明人从我
acutus, 俗称五步蛇) 的蛇毒中分离纯化得到一种高活性血凝酶 -B。 发明内容 The inventor from me Acutus, commonly known as the five-step snake, is isolated and purified to obtain a highly active hemagglutinase-B. Summary of the invention
本发明的目的在于提供一种蛇毒血凝酶 -B,其是从尖吻蝮蛇蛇毒 中分离得到的一种类凝血酶(TLC )。 It is an object of the present invention to provide a snake venom hemagglutinase-B which is a thrombin-like enzyme (TLC) isolated from the snake venom of Agkistrodon acutus.
本发明的另一个目的在于提供了一种分离纯化上述血凝酶 -B 的 方法。 Another object of the present invention is to provide a method for isolating and purifying the above hemagglutinase-B.
本发明的目的还在于提供上述尖吻蝮蛇血凝酶 -B 在制备止血药 物中的用途。 It is also an object of the present invention to provide the use of the above-mentioned Agkistrodon acutus hemagglutinase-B in the preparation of a hemostatic drug.
本发明血凝酶 -B是从中国尖吻蝮蛇( Agkistrodon acutus )蛇毒中 分离得到的高活性血凝酶(命名为 "血凝酶 -B" ),其具有 SEQ ID No. l 所示的氨基酸序列。该酶具有如下特征: ①由 236个氨基酸构成的单 链, SDS-PAGE分子量约为 35kD, 等电点 pi为 6.0, ②含 6个链内 二硫键(位点: 7-141 , 28-44, 78-234, 120-188, 152-167, 178-203 )。 The hemagglutinase-B of the present invention is a highly active hemagglutinase (designated "hemagglutinase-B") isolated from the snake venom of Agkistrodon acutus, which has the SEQ ID No. Amino acid sequence. The enzyme has the following characteristics: 1 a single chain consisting of 236 amino acids, SDS-PAGE molecular weight is about 35kD, isoelectric 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在 Asn 77, 100' 229位点上具有由 5种不同单糖构成的杂合多聚糖修饰。3 There are heteropolysaccharide modifications consisting of 5 different monosaccharides at Asn 77 , 100 ' 229 .
④酶活性可被苯甲基磺酰氟(PMSF)完全抑制, 表明其是一种丝氨酸 蛋白酶。 ⑤具有较好的热稳定性, 40°C恒温 60天酶活性可保持 90%。 4 Enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), indicating that it is a serine protease. 5 has good thermal stability, 60 ° C constant temperature 60 days of enzyme activity can be maintained at 90%.
应当理解,本领域技术人员可根据本发明公开的氨基酸序列, 在
不影响其活性的前提下, 取代、 缺失和 /或增加一个或几个氨基酸, 得到所述蛋白的突变序列。 因此, 本发明蛋白还包括 SEQ ID No.l所 示氨基酸序列经取代、 替换和 /或增加一个或几个氨基酸, 具有同等 活性的由所述蛋白衍生得到的蛋白质。本领域技术人员可以根据本发 明血凝酶 -B的 N端氨基酸序列, 设计合成寡核苷酸探针, 从尖吻蝮 蛇毒腺组织提取 mRNA, 反转录并构建 cDNA文库, 通过上述探针 筛选 cDNA文库, 并对筛选出的阳性克隆子分别进行测序克隆分析, 从而得到编码本发明蛋白的基因。 也可以根据本发明血凝酶 -B 的氨 基酸序列直接设计合成编码本发明蛋白的基因。 It will be understood that one skilled in the art can according to the amino acid sequences disclosed herein, The mutated sequence of the protein is obtained by substituting, deleting and/or adding one or several amino acids without affecting its activity. Accordingly, the protein of the present invention further comprises a protein derived from the protein having the amino acid sequence of SEQ ID No. 1 substituted, substituted and/or increased by one or several amino acids and having an equivalent activity. A person skilled in the art can design a synthetic oligonucleotide probe according to the N-terminal amino acid sequence of hemagglutinase-B of the present invention, extract mRNA from the tissue of Agkistrodon acutus, reverse transcription and construct a cDNA library through the above probe. The cDNA library is screened, and the selected positive clones are separately subjected to sequencing and cloning analysis to obtain a gene encoding the protein of the present invention. The gene encoding the protein of the present invention can also be directly designed and synthesized according to the amino acid sequence of hemagglutinase-B of the present invention.
本发明还提供上述血凝酶 -B的纯化方法, 其包括如下步骤: The present invention also provides a method for purifying the above hemagglutinase-B, which comprises the following steps:
1 )、 蛇毒预处理; 1), snake venom pretreatment;
2 )、 将预处理后的蛇毒溶液上经预平衡的 DEAE - Sephrose FF 阴离子交换层析柱, 用 0.01M pH7.0 ~ 7.5的 PBS洗柱, 再用含 0.02 和 0.06M NaCl的 0.01M pH7.0 - 7.5的 PBS分段洗脱, 收集 0.06M NaCr溶液的洗脱峰; 2), pre-treated the snake venom solution on a pre-equilibrated DEAE-Sephrose FF anion exchange chromatography column, wash the column with 0.01M PBS pH 7.0 ~ 7.5, and then use 0.01M pH7 containing 0.02 and 0.06M NaCl .0 - 7.5 PBS was eluted in sections, and the elution peak of 0.06 M NaCr solution was collected;
3 )、 将步骤 2 )得到的洗脱峰收集液超滤浓缩至小体积后经透析 去除 NaCl; 3), the elution peak collected in step 2) is concentrated by ultrafiltration to a small volume, and then dialyzed to remove NaCl;
4 )、 将透析后的溶液再次上样至经过预平衡的 DEAE - Sephrose FF层析柱, 用 0.01M pH7.0 ~ 7.5 的 PBS洗柱, 再用含 0.02、 0.04、 0.06M NaCl的 0.01M pH7.0 - 7.5 的 PBS分段洗脱,收集 0.06M NaCl 溶液洗脱液中的第三个洗脱峰; 4), the dialysis solution was loaded again onto a pre-equilibrated DEAE-Sephrose FF column, washed with 0.01 M PBS pH 7.0 ~ 7.5, and then 0.01 M with 0.02, 0.04, 0.06 M NaCl The PBS was fractionally eluted with pH 7.0 - 7.5, and the third elution peak in the eluate of 0.06 M NaCl solution was collected;
5 )、 将上述步骤 4 )得到的洗脱峰收集液超滤浓缩至小体积后经 80 %硫酸铵沉淀, 12000g离心 30分钟分离沉淀。 5), the eluted peak collected in the above step 4) was concentrated by ultrafiltration to a small volume, precipitated with 80% ammonium sulfate, and centrifuged at 12000 g for 30 minutes to separate the precipitate.
6)、 用 PBS适量溶解沉淀, 经 Sephadex-G75进一步纯化, 再经 透析除盐后直接冷冻干燥。 6) Dissolve the pellet in an appropriate amount with PBS, further purify it with Sephadex-G75, and then freeze-dry by dialysis and desalting.
其中, 步骤 1 )蛇毒预处理的方法是将蛇毒用适量预冷的 0.01M pH7.0 - 7.5 PBS溶解, 离心取上清液进行透析 (透析袋截留分子量为
7,000D~10,000D)。 通过预处理可以去除不溶的杂质以及小分子多肽, 并降低溶液离子强度。 Among them, step 1) snake venom pretreatment method is to dissolve the snake venom with an appropriate amount of pre-cooled 0.01M pH7.0 - 7.5 PBS, centrifuge to take the supernatant for dialysis (dialysis bag molecular weight cut-off is 7,000D~10,000D). Pretreatment can remove insoluble impurities as well as small molecule polypeptides and reduce solution ionic strength.
具体地说可通过如下步骤进行蛇毒的预处理: 称取蛇毒若干克, 用蛇毒重量 5~10倍体积预冷的 0.01M pH7.0 ~ 7.5 的 PBS于 4~8°C的 层析柜中搅拌溶解 30~60分钟, 于 4~8°C、 5,000-10,000g离心 10〜20 分钟,将离心上清液倾入透析袋中,离心沉淀再次加入蛇毒重量 5~10 倍体积预冷的 PBS搅拌悬浮, 再次离心。 合并两次离心上清液于透 析袋中, 于 4〜8°C对 0.01M pH7.0 ~ 7.5的 PBS透析 12〜24小时, 期 间换液 2~4次, 以去除小分子多肽和降低溶液离子强度。 Specifically, the pretreatment of the snake venom can be carried out by the following steps: weighing a few grams of snake venom, using a venom weight of 5 to 10 times the volume of 0.01 M pH 7.0 ~ 7.5 PBS in a 4 to 8 ° C chromatographic cabinet Stir for 30~60 minutes, centrifuge at 4~8°C, 5,000-10,000g for 10~20 minutes, pour the supernatant into the dialysis bag, and add the venom weight 5~10 times the volume of pre-cooled PBS. Stir in suspension and centrifuge again. Combine the two centrifugation supernatants into a dialysis bag and dialyze on 0.01 M pH 7.0 ~ 7.5 PBS for 12 to 24 hours at 4~8 °C, and change the solution 2~4 times to remove small peptides and reduce the solution. Ionic strength.
其中, 步骤 2 )和步骤 4 )采用 0.01M pH7.0 ~ 7.5 的 PBS预平衡 DEAE - Sephrose FF层析柱, 然后再上样。 Among them, step 2) and step 4) pre-equilibrated DEAE-Sephrose FF column with 0.01M pH 7.0 ~ 7.5 PBS, and then loaded.
其中, 步骤 3 )透析的目的是去除溶液中存在的 NaCl。 步骤 3 ) 和步骤 5 ) 中大体积洗脱液浓缩方式为超滤浓缩 (膜截留值 5000-10000Da )„ Among them, step 3) the purpose of dialysis is to remove the NaCl present in the solution. Step 3) and step 5) The large volume eluent is concentrated by ultrafiltration (membrane cutoff value 5000-10000Da)
其中, 步骤 6 ) 中采用 SephadeX-G75分子筛的目的是进一步将 两次阴离子柱层析收集物中的蛋白质按分子量大小进行分离纯化。具 体地将沉淀用 PBS溶解后上柱, 用 0.01M pH7.4 PBS洗脱液洗脱, 收集洗脱液第一个峰。 The purpose of using Sephade X- G75 molecular sieve in step 6) is to further separate and purify the protein in the two anion column chromatography collections by molecular weight. Specifically, the precipitate was dissolved in PBS, and the column was eluted with a 0.01 M pH 7.4 eluate to collect the first peak of the eluate.
经本发明方法纯化的血凝酶 -B比活力不低于 2000U/mg, 还原和 非还原 SDS-PAGE均为一条带; HPLC分析纯度 95%以上。该酶具有 较好的热稳定性, 40Ό恒温 60天酶活性可保持 90%。 以蛇毒冻干粉 原料重量计, 本法纯化收得率为 0.25% - 0.3%。 The specific activity of hemagglutinase-B purified by the method of the present invention is not less than 2000 U/mg, and both reduction and non-reduction SDS-PAGE are one band; HPLC analysis has a purity of over 95%. The enzyme has good thermal stability and can maintain 90% enzyme activity at 40 Ό constant temperature for 60 days. Based on the weight of the venom of the snake venom, the purification yield of the method is 0.25% - 0.3%.
本发明血凝酶 -B 具有良好的凝血活性, 可作为制成各种止血药 物的原料药, 即本发明还包括含有所述尖吻蝮蛇血凝酶 -B 的止血药 物。该药物可以是冻干粉原药、冻干粉注射剂、止血贴剂、止血粉剂、 止血片剂、止血膏剂或者止血液体喷雾剂, 用于需要止血以减少出血 量的各种医疗情况。 也可用来预防出血,避免或减少手术部位及手术
后出血。 附图说明 The hemagglutinase-B of the present invention has good blood coagulation activity and can be used as a raw material medicine for preparing various hemostatic drugs, that is, the present invention also includes a hemostatic drug containing the scorpion snake blood coagulase-B. The medicament may be a lyophilized powder, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostatic spray for various medical conditions requiring hemostasis to reduce the amount of bleeding. Can also be used to prevent bleeding, avoid or reduce the surgical site and surgery After bleeding. DRAWINGS
图 1 显示纯化后血凝酶 -B的 SDS-PAGE—条带。 Figure 1 shows the SDS-PAGE-band of hemagglutinase-B after purification.
图 2 显示纯化后血凝酶 -B的 HPLC分析结果。 Figure 2 shows the results of HPLC analysis of hemagglutinase-B after purification.
图 3 血凝酶 -B杂合糖基化结构示意图。 具体实施方式 Figure 3 Schematic diagram of hemagglutinase-B heterozygous glycosylation structure. detailed description
以下实施例进一步说明本发明的内容,但不应理解为对本发明的 限制。 在不背离本发明精神和实质的情况下, 对本发明方法、 步骤或 条件所作的修改或替换, 均属于本发明的范围。 The following examples are intended to further illustrate the invention, but are not to be construed as limiting the invention. Modifications or substitutions of the methods, steps or conditions of the invention are intended to be within the scope of the invention.
若未特别指明,实施例中所采用的技术手段为本领域技术人员所 熟知的常规技术。 The technical means employed in the examples are conventional techniques well known to those skilled in the art unless otherwise specified.
本发明中涉及到的百分号 "%", 若未特别说明, 是指质量百分 比; 但溶液的百分比, 除另有规定外, 是指溶液 100ml中含有溶质若 干克; 液体之间的百分比, 是指在 20°C时容量的比例。 本发明中类 似 "用蛇毒重量 10倍体积预冷的" 表述, 其中的重量和体积的单位 分别是 g和 ml。 实施例 1 蛇毒血凝酶 -B的纯化 The percent sign "%" referred to in the present invention means mass percentage unless otherwise specified; however, the percentage of the solution, unless otherwise specified, means that the solution contains several grams of solute in 100 ml; the percentage between the liquids, It refers to the ratio of capacity at 20 °C. In the present invention, it is similar to the expression "pre-cooled with a venom weight of 10 times by volume", wherein the units of weight and volume are g and ml, respectively. Example 1 Purification of snake venom hemagglutinase-B
取 30g尖吻蝮蛇蛇毒冻干粉(批号: 20090701,广西蛇毒研究所), 用蛇毒重量 10倍体积预冷的 0.01M pH7.4 的 PBS于 4°C的层析柜中 搅拌溶解 30分钟, 于 4°C、 lOOOOg离心 15分钟, 将离心上清液倾入 透析袋中, 离心沉淀再次加入蛇毒重量 10倍体积预冷的 PBS搅拌悬 浮,再次离心。合并两次离心上清液于透析袋 (截留分子量为 10,000D) 中,在 4°C层析柜中对 0.01M pH7.4的 PBS透析 24小时,期间换液 3 次。 将预处理后的蛇毒溶液上样到经过 0.01M pH7.4PBS 预平衡的 DEAE - Sepharose Fast Flow 阴离子交换层析柱, 先用 0.01M pH7.4PBS洗柱, 再分别用含 0.02M、 0.06M NaCl的 0.01M pH7.4的
PBS分段洗脱, 收集 0.06M NaCl溶液的洗脱峰, 共得 1960ml。 Take 30g of Agkistrodon acutus venom lyophilized powder (batch number: 20090701, Guangxi Snake Venom Research Institute), stir and dissolve in a 4°C chromatography cabinet with 10 times volume of venom weighted 0.01M pH 7.4 in a chromatography cabinet for 30 minutes. After centrifugation at 1000 °C for 15 minutes at 4 ° C, the centrifugation supernatant was poured into a dialysis bag, and the pellet was centrifuged again to add 10 volumes of pre-cooled PBS with a snake venom weight, and the mixture was again centrifuged. The two supernatants were combined and centrifuged in a dialysis bag (molecular weight cutoff of 10,000 D), and dialyzed against 0.01 M pH 7.4 PBS for 24 hours in a 4 ° C chromatography cabinet, during which time the solution was changed three times. The pretreated snake venom solution was loaded onto a DEAE-Sepharose Fast Flow anion exchange chromatography column pre-equilibrated with 0.01 M pH 7.4 PBS, first washed with 0.01 M pH 7.4 PBS, and then with 0.02 M, 0.06 M NaCl, respectively. 0.01M pH 7.4 The PBS was eluted in sections, and the elution peak of 0.06 M NaCl solution was collected to obtain a total of 1960 ml.
经酶活测定(参照附录①或⑦方法)和电泳分析, 目的物出现在 0.06M NaCl溶液的洗脱峰收集液中。采用 Millipore Pellicon 2 切向流 超滤器( 0.1M2cut off 10k膜)超滤浓缩至 200ml, 将该 200ml超滤浓 缩液倾入透析袋(10,000D)中 , 用 2000ml 0.01M pH7.4的 PBS于 4°C 透析 24小时, 期间更换溶液 3次。 将透析后的酶溶液上样到经过预 平衡的 DEAE - Sepharose Fast Flow 阴离子交换层析柱, 用 0.01M pH7.4的 PBS洗柱, 再分别用 0.02M、 0.04M, 0.06M NaCl的 0.01M pH7.4的 PBS分段洗脱, 收集 0.06M NaCl溶液洗脱液的第三个洗脱 峰, 共得 660ml。 用 Millipore Pellicon 2 切向流超滤器( 0.1M2cut off 5k膜)超滤浓缩至 120ml, 将该 120ml经 80%硫酸铵沉淀 4°C过夜, 次日 12,000g 4°C离心 30分钟, 离心沉淀用 10ml的謹 M pH7.4PBS 悬浮溶解, 立即上样到 Sephdex-G75层析柱, 用 0.01M pH7.4PBS洗 脱液洗脱, 收集洗脱液第一个峰 65ml, 经酶活测定确认存在目的产 物。 将该 65ml浓缩液装入透析袋中, 用无离子水进行透析 24小时, 期间更换溶液 3次, 每次 2000ml。 透析后体积为 73ml, 直接冷冻干 燥。 获得冻干物 81mg, 测定酶比活为 2500U/mg, 最终收率 0.27%。 还原和非还原 SDS-PAGE均为一条带 (见图 1 ), 电泳分子量大致为 35kD。 HPLC纯度 96.2 % (见图 2 )。 等电聚焦电泳测定该酶等电点 pi为 6.0。 After the enzyme activity assay (refer to the method of Appendix 1 or 7) and electrophoresis analysis, the target appeared in the elution peak collection solution of 0.06 M NaCl solution. The mixture was concentrated to 200 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 10 k membrane), and the 200 ml ultrafiltration concentrate was poured into a dialysis bag (10,000 D) using 2000 ml of 0.01 M pH 7.4. The PBS was dialyzed at 4 ° C for 24 hours, during which time the solution was changed 3 times. The dialyzed enzyme solution was applied to a pre-equilibrated DEAE-Sepharose Fast Flow anion exchange chromatography column, washed with 0.01 M pH 7.4 PBS, and then 0.02 M, 0.04 M, 0.06 M NaCl 0.01 M, respectively. The PBS of pH 7.4 was eluted in sections, and the third eluting peak of the eluate of 0.06 M NaCl solution was collected, and a total of 660 ml was obtained. The mixture was concentrated to 120 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane), and the 120 ml was precipitated by 80% ammonium sulfate at 4 ° C overnight, and centrifuged at 12,000 g for 4 minutes at 4 ° C for 30 minutes. The pellet was suspended and dissolved in 10 ml of M pH 7.4 PBS. Immediately applied to a Sephdex-G75 column and eluted with 0.01 M pH 7.4 eluate. The first peak of the eluate was collected in 65 ml. The measurement confirmed the presence of the objective product. The 65 ml concentrate was placed in a dialysis bag, and dialyzed against ion-free water for 24 hours, during which time the solution was changed three times, 2000 ml each time. After dialysis, the volume was 73 ml and directly freeze-dried. 81 mg of the lyophilizate was obtained, and the specific activity of the enzyme was determined to be 2500 U/mg, and the final yield was 0.27%. Both reduced and non-reduced SDS-PAGE are banded (see Figure 1) and the molecular weight of the electrophoresis is approximately 35kD. The HPLC purity was 96.2% (see Figure 2). The isoelectric point pi of the enzyme was determined by isoelectric focusing electrophoresis to be 6.0.
经 Edman酶解和肽图质谱进行氨基酸和糖基化测定, 其氨基酸 序列如 SEQ ID No.l所示。 该血凝酶含 236个氨基酸, 仅按氨基酸计 算分子量为 26116.7Dalton; 链内含 6个二硫键,位点为: Cys7-Cys141, Cys28-Cys44, Cys78-Cys234, Cys120-Cys'88, Cys,52-Cys167, Cys178-Cys203 o 糖 基化位点为 Asn77 - G2F4S, Asn100 - Hybrid, Asn229 - G2F4S, 糖基化结 构由 5种不同单糖的杂合多聚糖构成 (见图 3 )。
实施例 2 蛇毒血凝酶 -B的纯化 The amino acid and glycosylation assays were carried out by Edman digestion and peptide mapping mass spectrometry, and the amino acid sequence thereof is shown in SEQ ID No. 1. The hemagglutinase contains 236 amino acids, and the molecular weight is only 26116.7Dalton based on amino acid; the chain contains 6 disulfide bonds, and the sites are: Cys 7 -Cys 141 , Cys 28 -Cys 44 , Cys 78 -Cys 234 , Cys 120- Cys' 88 , Cys , 52 -Cys 167 , Cys 178 -Cys 203 o glycosylation sites are Asn 77 - G2F4S, Asn 100 - Hybrid, Asn 229 - G2F4S, glycosylation structure consists of 5 different monosaccharides The heterozygous polysaccharide is composed (see Figure 3). Example 2 Purification of snake venom hemagglutinase-B
取 30g尖吻蝮蛇蛇毒冻干粉(批号: 20090701,广西蛇毒研究所), 按与实施例 1相同的方法进行预处理。将经预处理的蛇毒溶液按实例 1相同的方法进行第一次 DEAE-Sephrose FF柱层析, 经酶活测定和 电泳分析目的物出现在 0.06MNaCl 的洗脱峰中, 合并洗脱收集液, 共得 2000ml, 釆用 Millipore Pellicon 2 切向流超滤器( 0.1M2 cut off 10k膜)超滤浓缩至 210ml, 将该 210ml 超滤浓缩液倾入透析袋 (10,000D)中, 用 2000ml 0.01M pH7.4PBS于 4'C透析 24小时, 期间 更换溶液 3次。 透析后的酶液上样至 DEAE- Sephrose FF柱中, 按实 例 1相同的方法进行第二次层析。 血凝酶出现在 0.06MNaCl溶液洗 脱液的第三个洗脱峰中, 收集该峰洗脱液得 648ml。 用 Millipore Pellicon 2 切向流超滤器( 0.1M2 cut off 5k膜)超滤浓缩至 130ml, 将 该 130ml经 80°/。硫酸铵沉淀 4°C过夜, 12,000g 4。C离心 30分钟, 沉 淀用 10ml的 0.01M pH7.4PBS悬浮溶解, 立即上样到 Sephdex-G75 层析柱, 收集洗脱液第一个峰 68ml, 经酶活测定确认。 将该 68ml按 实例 1相同的方法进行透析后体积为 78ml, 直接冷冻干燥。 获得冻 干物 85mg,测定酶比活力为 2460U/mg,最终收率 0.28%。 SDS-PAGE 为一条带, 其电泳分子量大致为 35kD。 HPLC纯度 96.0 %。 实施例 3 尖吻蝮蛇血凝蒔 -B的丝氨酸蛋白属性实验 30 g of the lyophilized powder of Agkistrodon acutus venom (batch number: 20090701, Guangxi Snake Venom Research Institute) was pretreated in the same manner as in Example 1. The pretreated snake venom solution was subjected to the first DEAE-Sephrose FF column chromatography in the same manner as in Example 1. The target substance appeared in the elution peak of 0.06 M NaCl by enzyme activity measurement and electrophoresis analysis, and the eluted collection solution was combined. A total of 2000ml was prepared and concentrated to 210ml by Millipore Pellicon 2 tangential flow ultrafilter (0.1M 2 cut off 10k membrane). The 210ml ultrafiltration concentrate was poured into a dialysis bag (10,000D) with 2000ml 0.01 M pH 7.4 PBS was dialyzed against 4'C for 24 hours, during which time the solution was changed 3 times. The dialyzed enzyme solution was applied to a DEAE-Sephrose FF column, and a second chromatography was carried out in the same manner as in Example 1. Hemagglutinase appeared in the third elution peak of the 0.06 M NaCl solution eluate, and the peak eluate was collected to obtain 648 ml. The mixture was concentrated to 130 ml by ultrafiltration using a Millipore Pellicon 2 tangential flow ultrafilter (0.1 M 2 cut off 5k membrane), and the 130 ml was passed through 80 °/. Ammonium sulfate was precipitated at 4 ° C overnight, 12,000 g of 4 . After centrifugation for 30 minutes, the pellet was suspended and dissolved in 10 ml of 0.01 M pH 7.4 PBS, and immediately applied to a Sephdex-G75 column to collect 68 ml of the first peak of the eluate, which was confirmed by enzyme activity measurement. The 68 ml was dialyzed in the same manner as in Example 1 to a volume of 78 ml, which was directly freeze-dried. 85 mg of the lyophilizate was obtained, and the specific activity of the enzyme was determined to be 2460 U/mg, and the final yield was 0.28%. SDS-PAGE is a band with an electrophoretic molecular weight of approximately 35 kD. The HPLC purity was 96.0%. Example 3 Serine protein property test of blood clot-B in Agkistrodon acutus
将实施例 1分离得到的血凝酶 -B用生理盐水稀释至酶活 lU/ml。 用生理盐水配制 1 %的牛纤维蛋白原 (Sigma公司 )溶液。 The hemagglutinase-B isolated in Example 1 was diluted with physiological saline to an enzyme activity of 1 U/ml. A 1% solution of bovine fibrinogen (Sigma) was prepared in physiological saline.
用异丙醇溶解苯甲基磺酰氟(PMSF, Merck公司), 溶液浓度为 mg/mL Dissolve phenylmethylsulfonyl fluoride (PMSF, Merck) with isopropanol at a solution concentration of mg/mL
实验搡作步骤如下: The experimental steps are as follows:
( 1 ) 取 1%牛纤维蛋白原溶液 2ml, 37°C下恒温 5分钟。 (1) Take 1 ml of 1% bovine fibrinogen solution and incubate at 37 °C for 5 minutes.
( 2 ) 取三支小试管, 分别标注 W、 2#、 3#,每管加入 200μ1的 lU/ml 血凝酶 -B溶液。
( 3 ) 分别向 1#试管加入 ΙΟμΙ蒸馏水, 2#试管加入 ΙΟμΙ异丙醇, 3# 试管加入 lC^l PMSF, 于 37°C水浴保温 5分钟。 (2) Take three small test tubes, respectively labeled W, 2#, 3#, and add 200 μl of lU/ml hemagglutinase-B solution to each tube. (3) Add ΙΟμΙ distilled water to the 1# tube, add ΙΟμΙ isopropanol to the 2# tube, add lC^l PMSF to the 3# tube, and incubate for 5 minutes at 37 °C in a water bath.
( 4 ) 按试管编号顺序分别单独进行凝集试验观察。 向试管中加入恒 温好的 1 %牛纤维蛋白原溶液 200μ1, 立即计时, 同时轻轻摇动 混匀, 于 37 C水洛中静置, 观察试管中凝集反应的情况。 以溶 液呈现完全凝固状态时终止计时。 (4) The agglutination test was separately performed in the order of the test tube numbers. Add a constant temperature of 1% bovine fibrinogen solution 200μ1 to the test tube, immediately time the mixture, gently shake and mix, and let stand in 37 C water, observe the agglutination reaction in the tube. The timing is terminated when the solution is completely solidified.
结果见表一 The results are shown in Table 1.
表一、 PMSF对凝集吋间的影响 Table 1. Effect of PMSF on agglutination
根据表一中的实验结果,得出以下结论:① lOOppm浓度的 PMSF 完全抑制了该血凝酶 -B活性, 证明该尖吻蝮蛇血凝酶 -B为丝氨酸蛋 白酶。 ②微量异丙醇对本凝集反应无影响。 实施例 4 尖吻蝮蛇血凝晦 -B的热稳定性考察实验 Based on the experimental results in Table 1, the following conclusions were drawn: PMSF at a concentration of 1 OO ppm completely inhibited the activity of hemagglutinase-B, demonstrating that the blood coagulase-B is a serine protease. 2 Trace isopropanol has no effect on the agglutination reaction. Example 4 Thermal stability test of blood clot 晦-B of Agkistrodon acutus
将实施列 1和实施列 2中获得的冷冻干燥纯酶(未添加任何冻干 保护剂)冻干粉分装于多个安培瓶中密封, 放置于 40°C恒温箱中, 分别于 30天和 60天时取出测定酶比活。 The freeze-dried pure enzyme (without adding any lyoprotectant) obtained in column 1 and column 2 was dispensed into a plurality of ampoules and sealed in a plurality of ampoules, and placed in a 40 ° C incubator for 30 days respectively. The enzyme activity was measured and taken at 60 days.
结果显示: 两批酶的比活 30天可保持原酶活的 95%, 60天仍能 保持原酶活性 90%的水平。这种结果表明所分离到的血凝酶具有较好 的热稳定性。 结果见表二。 The results showed that the specific activity of the two batches of enzymes maintained 95% of the original enzyme activity for 30 days, and remained at 90% of the original enzyme activity for 60 days. This result indicates that the isolated hemagglutinase has good thermal stability. The results are shown in Table 2.
尖吻蝮蛇血凝酶单位定义及活性测定方法 Definition of hemagglutinase unit of Agkistrodon acutus and method for measuring activity
①牛纤维蛋白原测定法 取生理盐水配制的 1.0 %牛纤维蛋白原 (Sigma公司) 溶液 lml置小试管中, 37±0.5°C水浴保温 3分钟, 加入 37±0.5°C预热的待测酶溶 液 lml, 立即计时, 纤维蛋白原溶液在 120±30秒内振摇出现白色絮团, 则该酶 溶液为 lU/ml。 1 Bovine fibrinogen assay 1.0% bovine fibrinogen (Sigma) solution prepared in physiological saline was placed in a small test tube, kept in a 37±0.5°C water bath for 3 minutes, and preheated at 37±0.5°C. The enzyme solution is 1 ml, immediately timed, and the fibrinogen solution shakes white flocs in 120±30 seconds, and the enzyme solution is 1 U/ml.
②标准人血浆测定法 取标准人血浆 lml置小试管中,置 37°C士 0.5Γ水浴中预热 3分钟, 加入 37±0.5°C预热的待测酶溶液 lml, 立即计时, 人血浆在 60±20秒内 振摇出现白色絮团, 则该酶溶液为 lU/ml。 2 Standard human plasma assay Take standard human plasma 1ml in a small test tube, preheat for 3 minutes in a 37 ° C ± 0.5 ° water bath, add 37 ± 0.5 ° C preheated enzyme solution 1 ml, immediately timed, human plasma When the white floc appeared in shaking within 60 ± 20 seconds, the enzyme solution was 1 U/ml.
注:测定未知高酶活性溶液时需用无离子水进行稀释,直至达到 lU/ml用于测定; 其稀释倍数即为每毫升原酶溶液中的酶活单位数。 工业实用性 Note: When measuring the unknown high enzyme activity solution, it needs to be diluted with ion-free water until it reaches lU/ml for determination; the dilution factor is the number of enzyme activity units per ml of the original enzyme solution. Industrial applicability
通过本发明所述的纯化方法所得到的尖吻蝮蛇血凝酶 -B,具有良 好的凝血活性、较高的酶比活性和较好的热稳定性, 且这种酶的热稳 定较好。 因此本发明血凝酶- B 可作为制成各种止血药物的原料药, 该药物可以是冻干粉原药、 冻干粉注射剂、 止血贴剂、 止血粉剂、 止 血片剂、止血膏剂或者止血液体喷雾剂, 用于需要止血以减少出血量 的各种医疗情况。 也可用来预防出血,避免或减少手术部位及手术后 出血。
The blood cobase-B obtained by the purification method of the invention has good coagulation activity, high enzyme specific activity and good thermal stability, and the enzyme has good thermal stability. . Therefore, the hemagglutinase-B of the present invention can be used as a raw material medicine for preparing various hemostatic drugs, and the medicine can be a lyophilized powder original medicine, a lyophilized powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic cream or a hemostasis. Liquid spray for various medical conditions where hemostasis is required to reduce the amount of bleeding. It can also be used to prevent bleeding and to avoid or reduce bleeding at the surgical site and after surgery.
Claims
1、 尖吻蝮蛇血凝酶 -B, 其具有 SEQIDNo.l所示的氨基酸序列 或该序列经替换、 缺失和 /或添加一个或几个氨基酸得到的具有同等 功能的氨基酸序列。 1. Agkistrodon hemagglutinin-B, which has the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence with equivalent functions obtained by replacing, deleting and/or adding one or more amino acids to this sequence.
2、 如权利要求 1所述的尖吻蝮蛇血凝酶 -B, 其特征在于, 链内 含有 6对二硫键, 在 Asn 77' ,ο 229位点上具有杂合多聚糖修饰。 2. The adder adder hemocoagulase-B according to claim 1, characterized in that the chain contains 6 pairs of disulfide bonds and has a hybrid polysaccharide modification at the Asn 77 ' , o 229 position.
3、 含有权利要求 1或 2所述尖吻蝮蛇血凝酶 -Β的药物。 3. A medicine containing the adder adder hemagglutinin-B described in claim 1 or 2.
4、 如权利要求 3所述的药物, 其为冻干粉原药、 冻干粉注射剂、 止血贴剂、 止血粉剂、 止血片剂、 止血膏剂或止血液体喷雾剂。 4. The drug according to claim 3, which is a freeze-dried powder original drug, a freeze-dried powder injection, a hemostatic patch, a hemostatic powder, a hemostatic tablet, a hemostatic ointment or a hemostatic body spray.
5、权利要求 1或 2所述血凝酶 -Β的纯化方法,其包括如下步骤: 5. The purification method of hemagglutinin-B according to claim 1 or 2, which includes the following steps:
1)、 蛇毒预处理; 1), snake venom pretreatment;
2)、将预处理后的蛇毒溶液上经预平衡的 DEAE - Sepharose Fast Flow阴离子交换层析柱, 用 0.01MpH7.0- 7.5的 PBS洗柱, 再用含 0.2和 0.6MNaCl的 0.01MpH7.0 ~ 7.5的 PBS分段洗脱, 收集 0.06M NaCl溶液的洗脱峰; 2). Place the pretreated snake venom solution on the pre-equilibrated DEAE-Sepharose Fast Flow anion exchange chromatography column, wash the column with 0.01M pH7.0-7.5 PBS, and then use 0.01MpH7.0 containing 0.2 and 0.6M NaCl. Stepwise elution with PBS of ~7.5, collect the elution peak of 0.06M NaCl solution;
3)、 将上述步骤 2)得到的洗脱峰收集液超滤浓缩后透析去除 NaCl; 3). Concentrate the elution peak collection liquid obtained in step 2) above by ultrafiltration and then dialyze to remove NaCl;
4)、 将透析后的溶液再次上经预平衡的 DEAE - Sepharose Fast Flow层析柱, 用 0.01MpH7.0~7.5的 PBS洗柱, 再用含 0.02、 0.04、 0.06MNaCl的 0.01M pH7.0 ~ 7.5的 PBS分段洗脱, 收集 0.06MNaCl 溶液的第三个洗脱峰; 4). Put the dialyzed solution on the pre-equilibrated DEAE-Sepharose Fast Flow chromatography column again, wash the column with 0.01M PBS pH7.0~7.5, and then use 0.01M pH7.0 containing 0.02, 0.04, and 0.06M NaCl. Stepwise elution with PBS of ~7.5, collect the third elution peak of 0.06M NaCl solution;
5)、将上述步骤 4)得到的洗脱峰收集液超滤浓缩后用 80%硫酸 铵沉淀, 离心收集沉淀; 5). Concentrate the elution peak collection solution obtained in step 4) above by ultrafiltration and precipitate with 80% ammonium sulfate, and centrifuge to collect the precipitate;
6)、 用 PBS适量溶解沉淀, 经 Sephadex-G75进一步纯化, 再经 透析除盐, 冷冻干燥。 6) Dissolve the precipitate with an appropriate amount of PBS, further purify it with Sephadex-G75, remove salt through dialysis, and freeze-dry.
6、 如权利要求 5所述的方法, 其中, 步骤 1)蛇毒预处理的方 法是将蛇毒用适量预冷的 0.01M pH7.0 - 7.5的 PBS溶解, 离心取上
清液进行透析。 6. The method of claim 5, wherein step 1) snake venom pretreatment method is to dissolve the snake venom with an appropriate amount of pre-cooled 0.01M PBS pH 7.0-7.5, and centrifuge to take out the venom. The serum is dialyzed.
7、 如权利要求 5所述的方法, 其中, 步骤 1 )蛇毒预处理的方 法是: 称取蛇毒若干克, 用蛇毒重量 5~10 倍体积预冷的 0.01M pH7.0 ~ 7.5的 PBS于 4~8°C的层析柜中搅拌溶解 30〜60分钟,于 4°C、 5,000~10,000g离心 10〜20分钟, 将离心上清液倾入透析袋, 离心沉 再用蛇毒重量 5~10倍体积预冷的 PBS搅拌悬浮, 再次离心, 合并 两次离心上清液装入透析袋中,于 4〜8'C对 0.01M pH7.0 ~ 7.5的 PBS 透析 12〜24小时, 期间更换溶液 2〜4次。 7. The method of claim 5, wherein step 1) snake venom pretreatment method is: weigh several grams of snake venom, and add pre-cooled 0.01 M pH 7.0 to 7.5 PBS with a volume of 5 to 10 times the weight of the snake venom. Stir and dissolve in a chromatography cabinet at 4 to 8°C for 30 to 60 minutes, centrifuge at 4°C and 5,000 to 10,000g for 10 to 20 minutes, pour the centrifugal supernatant into a dialysis bag, centrifuge and weigh the snake venom. Stir and suspend 5 to 10 times the volume of pre-cooled PBS, centrifuge again, combine the two centrifugation supernatants and put them into a dialysis bag, and dialyze against 0.01M PBS of pH 7.0 to 7.5 for 12 to 24 hours at 4 to 8°C. The solution was replaced 2 to 4 times during this period.
8、 如权利要求 5所述的方法, 其中, 步骤 2 )和步骤 4 )釆用 0.01M pH7.0 ~ 7.5 的 PBS预平衡 DEAE - Sepharose Fast Flow层析 柱, 然后上样。 8. The method of claim 5, wherein step 2) and step 4) use 0.01M PBS of pH 7.0 ~ 7.5 to pre-equilibrate the DEAE-Sepharose Fast Flow chromatography column, and then load the sample.
9、 如权利要求 5〜8任一项所述的方法, 其中, 步骤 3 )采用分 子截留值为 5,000〜10,000D的超滤膜进行超滤浓缩。 9. The method according to any one of claims 5 to 8, wherein step 3) uses an ultrafiltration membrane with a molecular cutoff value of 5,000 to 10,000D for ultrafiltration concentration.
10、 如权利要求 5〜8任一项所述的方法, 其中, 步骤 6 )纯化的 方法是将步骤 5 ) 收集的沉淀用 PBS溶解后, 上 Sephadex-G75层析 柱, 用 0.01M pH7.4 PBS洗脱, 收集洗脱液第一个峰。
10. The method according to any one of claims 5 to 8, wherein the purification method in step 6) is to dissolve the precipitate collected in step 5) with PBS, then put it on a Sephadex-G75 chromatography column, and use 0.01M pH7. 4 PBS elution, collect the first peak of the eluate.
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