CN104357430A - Production method of acutase - Google Patents
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- 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
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Abstract
The invention relates to a production method of acutase. The production method comprises the following steps: (1) dissolving long-noded pit viper venom freeze-dried powder in excess 0.05mol/L Tris-HCL buffer solution with the pH value of 8.5; (2) putting the long-noded pit viper venom solution on a DEAT-Sepharose Fast Flow anion-exchange column to perform elution separation; (3) putting the solution obtained in the step (2) on a SOURCE 30Q gel column to perform chromatography, and then performing separation by virtue of phenyl sepharose high performance column chromatography; and (4) putting the solution obtained in the step (3) on a Hiload 26/60Superdex75PG column, and collecting a second protein peak with thrombin-like enzyme activity, thereby obtaining an acutase solution. The acutase solution prepared by the production method disclosed by the invention is a single component and has the advantage of high purity.
Description
Technical field
The present invention relates to field of biological pharmacy, particularly a kind of production method of Effect of Agkistrodon acutus Enzyme.
Background technology
Effect of Agkistrodon acutus Enzyme is a kind of thrombin-like enzyme of separation and purification from ahylysantinfarctase, is serine-type proteolytic enzyme mixt.Effect of Agkistrodon acutus Enzyme has thrombin-like enzyme activity, and its molecular weight is 24000 ~ 30000 dalton, and iso-electric point is 4.5 ~ 5.0.Long-nosed pit viper zymin Acutobin Injection can be used for treatment acute cerebral infarction and various thrombotic diseases, adopts intravenous drip administration clinically.
Document is had about Effect of Agkistrodon acutus Enzyme production technique, Chinese patent application publication number is the open Effect of Agkistrodon acutus Enzyme of patent document and the production technique thereof of CN1229136A, with dextrane gel G on known buffer solution 75 posts, collect A, B peak with thrombin-like enzyme activity, with DEAE dextrane gel A 50 column chromatography, respectively collect have thrombin-like enzyme activity V, VI, VII peak.By the V with thrombin-like enzyme activity collected, VI, VII peak uses CM dextrane gel C 50 column chromatography respectively, collects CM1, CM2, CM3 peak with thrombin-like enzyme activity respectively.CM1, CM2, CM3 peak of thrombin-like enzyme activity that has collected is filtered with superdex75PG post respectively, each get a, b two peak, collects the b peak with high thrombin-like enzyme activity, is mixed into the Effect of Agkistrodon acutus Enzyme of molecular weight 24000 ~ 30000 dalton and iso-electric point 4.5 ~ 5.0.The purity of Effect of Agkistrodon acutus Enzyme prepared by this production technique needs to be improved further, and production process is complicated, the production cycle is long, is not easy to Industry Promotion.
Also exist in prior art and adopt tentatively through gel filtration chromatography chromatography, again through the object component of ion-exchange chromatography chromatography gained, this component detected through gel electrophoresis is a protein ingredient, it is actual is the mixture of protein that molecular weight and iso-electric point are close or polypeptide, need be further purified, namely there is the problem that the purity of the Effect of Agkistrodon acutus Enzyme of acquisition is not high enough.In addition, the problem that the existing Effect of Agkistrodon acutus Enzyme production technique ubiquity production technique cycle is long.
Summary of the invention
Technical problem to be solved by this invention is: the production method of the Effect of Agkistrodon acutus Enzyme providing a kind of purity high, with short production cycle.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
A production method for Effect of Agkistrodon acutus Enzyme, comprises the following steps:
(1) under temperature is 4 ~ 10 DEG C of conditions, agkistrodon acutus venom lyophilized powder is dissolved in 0.04 ~ 0.06mol/L, pH value be 8 ~ 9 Tris in HCl damping fluid, leave standstill 60min, centrifugal acquisition supernatant liquor under 0 ~ 10 DEG C of condition, by supernatant liquor 0.01mol/L, pH value be 8 ~ 9 Tris HCl damping fluid dialyse, obtain agkistrodon acutus venom solution;
(2) by DEAE on described agkistrodon acutus venom solution Sepharose Fast Flow anion-exchange column, carry out wash-out separation, collect the protein peak in protein peak with thrombin-like enzyme activity, by collect protein peak merge, then with 0.01mol/L, pH value be 7 ~ 8 Tris HCl damping fluid dialyse.
(3) solution step (2) obtained is after 0.22 μm of filtering with microporous membrane, upper SOURCE 30Q gel column, carry out chromatography, collect the protein peak with thrombin-like enzyme activity, upper phenyl sepharose high performance chromatography column after the protein peak solution collected is merged, carry out chromatography, to there is the protein peak of high thrombin-like enzyme activity after chromatography, Zhen Kong Du below 0.08MPa, concentrate under 5 ~ 10 DEG C of conditions, then 0.01mol/L is used, pH value be 7 ~ 8 Tris HCl damping fluid dialyse, by the solution that obtains after dialysis Zhen Kong Du below 0.08MPa, concentrating under reduced pressure under 5 ~ 10 DEG C of conditions, concentrated solution is through 0.22um filtering with microporous membrane,
(4) Hiload 26/60Superdex75PG post on solution step (3) obtained, collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
Beneficial effect of the present invention is:
(1) obtained Effect of Agkistrodon acutus Enzyme solution is one-component, purity in gel electrophoresis for single band and through high performance liquid phase be detected as one unimodal, purity is greater than 98%;
(2) with short production cycle, production technique favorable reproducibility.
Accompanying drawing explanation
Fig. 1 be the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention agkistrodon acutus venom DEAE the color atlas of Sepharose Fast Flow column chromatography;
Fig. 2 is the color atlas with VIII and Ⅸ protein peak SOURCE 30Q column chromatography of thrombin-like enzyme activity that the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention is collected;
Fig. 3 is the color atlas of the S2 peak with thrombin-like enzyme activity collected of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention and S3 peak phenyl sepharose high performance column chromatography;
Fig. 4 is the color atlas with the P3 peak Hiload 26/60Superdex75PG column chromatography of thrombin-like enzyme activity that the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention is collected;
Fig. 5 is the thin layer scanning figure of Effect of Agkistrodon acutus Enzyme solution (lot number 01) molecular weight subunit of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention;
Fig. 6 is the thin layer scanning figure of Effect of Agkistrodon acutus Enzyme solution (lot number 01) the electrophoretic method purity test of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention;
Fig. 7 is the color atlas of Effect of Agkistrodon acutus Enzyme solution (lot number 01) the high performance liquid chromatography purity test of the production method of the Effect of Agkistrodon acutus Enzyme of the embodiment of the present invention.
Embodiment
By describing technology contents of the present invention in detail, realized object and effect, accompanying drawing is coordinated to be explained below in conjunction with embodiment.
The design of most critical of the present invention is: by the dissolving of agkistrodon acutus venom lyophilized powder, centrifugal, dialysis, through the compounding practice of column chromatography, dialysis etc., prepare highly purified Effect of Agkistrodon acutus Enzyme solution.
The production method of a kind of Effect of Agkistrodon acutus Enzyme of the present invention, comprises the following steps:
(1) under temperature is 4 ~ 10 DEG C of conditions, agkistrodon acutus venom lyophilized powder is dissolved in 0.04 ~ 0.06mol/L, pH value be 8 ~ 9 Tris in HCl damping fluid, leave standstill 60min, centrifugal acquisition supernatant liquor under 0 ~ 10 DEG C of condition, by supernatant liquor 0.01mol/L, pH value be 8 ~ 9 Tris HCl damping fluid dialyse, obtain agkistrodon acutus venom solution;
(2) by DEAE on described agkistrodon acutus venom solution Sepharose Fast Flow anion-exchange column, collect the protein peak in protein peak with thrombin-like enzyme activity, by collect protein peak merge, then with 0.01mol/L, pH value be 7 ~ 8 Tris HCl damping fluid dialyse.
(3) solution step (2) obtained is after 0.22 μm of filtering with microporous membrane, upper SOURCE 30Q gel column, carry out chromatography, collect the protein peak with thrombin-like enzyme activity, upper phenyl sepharose high performance chromatography column after the protein peak collected is merged, carry out chromatography, to there is the protein peak of high thrombin-like enzyme activity after chromatography, Zhen Kong Du concentrated under below 0.08MPa 5 ~ 10 DEG C of conditions, then 0.01mol/L is used, pH value be 7 ~ 8 Tris HCl damping fluid dialyse, by the solution that obtains after dialysis Zhen Kong Du below 0.08MPa, concentrating under reduced pressure under 5 ~ 10 DEG C of conditions, concentrated solution is through 0.22um filtering with microporous membrane,
(4) Hiload 26/60Superdex75PG post on solution step (3) obtained, collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
Technical conceive of the present invention: protein ingredient contained by agkistrodon acutus venom is complicated, the original production technique of Effect of Agkistrodon acutus Enzyme adopt successively SephadexG 75, DEAE SephadexA 50 and SephadexG 75 carry out separation and purification, obtain three isozyme.Adopt the production technique of this patent can effectively remove non-targeted protein ingredient and bacterial endotoxin, obtain single protein ingredient, the purity of product is high, and with short production cycle, technological process is easy to control, favorable reproducibility.
From foregoing description, beneficial effect of the present invention is:
(1) obtained Effect of Agkistrodon acutus Enzyme solution is one-component, purity in gel electrophoresis for single band and through high performance liquid chromatography be detected as one unimodal, purity is greater than 98%;
(2) with short production cycle, production technique favorable reproducibility.
Further, agkistrodon acutus venom described in step (1) is dissolved in Tris carry out under 0 ~ 10 DEG C of condition after HCl damping fluid centrifugal, the supernatant liquor of gained again with Tris the dialysis of HCl damping fluid.
Seen from the above description, the described centrifugal effect with further removal of impurities.
Further, described dialysis adopts dialysis tubing to carry out, and the molecular weight cut-off of described dialysis tubing is 7000 dalton.
Further, the liquid after dialysis treatment in step (1) is filtered with 0.22 μm of millipore filtration.
Seen from the above description, the preliminary filtration treatment of the liquid after to dialysis is carried out being filtered into 0.22 μm of millipore filtration, can the carrying out of convenient subsequent operations.
Further, by agkistrodon acutus venom solubilize described in step (1) in Tris HCl damping fluid, then carry out centrifugal under 0 ~ 10 DEG C of condition, the supernatant liquor Tris of gained HCl damping fluid dialyse.
Further, by the protein solution with thrombin-like enzyme activity of collection in step (3) prior to concentrating under very empty degree below 0.08M Pa, 5 ~ 10 DEG C of conditions, then dialyse with Tris HCl damping fluid.
Further, by the solution after dialysis in step (3) in Zhen Kong Du below 0.08MPa, concentrate under 5 ~ 10 DEG C of conditions.
Further, Hiload 26/60Superdex75PG post on the concentrated solution obtain step (3), collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
Embodiments of the invention one are:
Step 1: at temperature is 4 ~ 10 DEG C, agkistrodon acutus venom lyophilized powder is dissolved in 0.04 excessive ~ 0.06mol/L, pH value be 8 ~ 9 Tris in HCl damping fluid, leave standstill more than 60min, 0 centrifugal acquisition supernatant liquor at 10 DEG C, get supernatant liquor and put (molecular weight cut-off 7000 dalton) in dialysis tubing, with the Tris HCl damping fluid dialysis that 0.01mol/L, pH value are 8 ~ 9, with except desalting and biological micromolecule impurity; By the 0.22 μm of filtering with microporous membrane of the agkistrodon acutus venom solution through dialysing.
Step 2: the solution DEAE that step 1 is obtained Sepharose Fast Flow anion-exchange column wash-out be separated, to remove containing hemorrhage poisonous substance matter.Fig. 1 be DEAE the color atlas of Sepharose Fast Flow column chromatography, its X-coordinate is time (min), and ordinate zou is response value (mAU).As shown in Figure 1, obtain 11 protein peaks, collect respectively and there is VIII peak of thrombin-like enzyme activity and the first half peak of Ⅸ, merge the first half peak collection liquid of VIII peak and Ⅸ, again with concentration be 0.01mol/L, pH value be 7 ~ 8 Tris HCl damping fluid dialyse, the molecular weight cut-off of dialysis tubing used is 7000 dalton, with except desalting and amino acid.This step DEAE Sepharose Fast Flow post disengaging time only use 6 ~ 7 hours, there is the advantage that efficiency is high.
Step 3: there is VIII peak of thrombin-like enzyme activity and the first half peak of Ⅸ by what dialysed, use SOURCE30Q column chromatography, Fig. 2 is the color atlas of SOURCE 30Q column chromatography, and its X-coordinate is time (min), and ordinate zou is response value (mAU).As shown in Figure 2, obtain multiple protein peak, collect S2, S3 protein peak with thrombin-like enzyme activity respectively; S2, S3 protein peak with thrombin-like enzyme activity collected is mixed by phenyl sepharose high performance chromatographic separation, to remove the impurity in S2, S3.Fig. 3 is the color atlas of phenyl sepharose high performance column chromatography, and its X-coordinate is time (min), and ordinate zou is response value (mAU).As shown in Figure 3, collect after there is the P3 protein peak of thrombin-like enzyme activity, through rotatory evaporator, very empty degree below 0.08MPa, concentrating under reduced pressure under 5 ~ 10 DEG C of conditions.Concentrated solution loads in dialysis tubing, and the molecular weight cut-off of dialysis tubing is 7000 dalton.With 0.01mol/L, pH value be 7 8 Tris HCl damping fluid dialysis, desalination, then Zhen Kong Du below 0.08MPa, carry out concentrating under reduced pressure under 5 ~ 10 DEG C of conditions, concentrated solution is through 0.22um filtering with microporous membrane.
Step 4: the P3 protein peak Hiload 26/60Superdex75PG post after filtering is filtered, to remove pyrogen and macromole impurity.Fig. 4 is the color atlas of Hiload 26/60Superdex75PG column chromatography, and its X-coordinate is time (min), and ordinate zou is response value (mAU).As shown in Figure 4, collect highly purified there is second protein peak of thrombin-like enzyme activity after, through 0.22 μm of aseptic suction filtration of millipore filtration, be Effect of Agkistrodon acutus Enzyme solution.
The mass analysis of Effect of Agkistrodon acutus Enzyme:
1, Rate activity measures
1.1 titration
The preparation of reference material solution
Get Effect of Agkistrodon acutus Enzyme solution reference material, add 0.01mol/L tris buffer and (get Tutofusin tris 0.121g, sodium-chlor 0.878g, add water appropriate dissolving, regulate pH to 7.4 with 0.5mol/L hydrochloric acid soln, add water to 100ml and obtain) to make concentration be respectively containing the solution of 0.4,0.6,0.8,1.0 units in every 1ml.
Assay method
Get test tube (10mm × 100mm, internal diameter should be 8.0mm ± 0.2mm) 4, respectively add human fibrinogen solution 0.1ml, put in 37 DEG C ± 0.5 DEG C water-bath and be incubated 2min, add the reference material solution 0.4ml of 4 kinds of concentration respectively successively, shake up timing immediately, in 37 DEG C ± 0.5 DEG C water-bath, observe the fibrinous presetting period.Often kind of concentration determination 5 times, averages (difference measuring maximal and minmal values for 5 times must not exceed 10% of mean value, otherwise resurveys).With the logarithm of reference material concentration for X-coordinate, the logarithm in presetting period is ordinate zou, drawing standard curve or calculating regression equation (correlation coefficient r >=0.990).Described human fibrinogen solution is: get human fibrinogen 1, adds above-mentioned damping fluid (pH7.4) and makes the solution containing human fibrinogen 4mg in every 1ml, put 37 DEG C of water-baths and make abundant dissolving in 30 ~ 60 minutes.Get this product to measure as stated above, try to achieve units by typical curve or regression equation.
1.2 protein contents: it is appropriate that precision measures this product, with 0.9% sodium chloride solution to make in every 1ml about containing the solution of 0.1mg albumen as need testing solution, precision measures need testing solution 1.0ml, measures protein content with Forint phenol method (Chinese Pharmacopoeia (version two in 2010) annex VII M protein determination second method).
1.3 Rate activity: be calculated as follows
Albumen mg number in Rate activity=every 1ml trial-product potency unit number/every 1ml trial-product
Table 1 is Effect of Agkistrodon acutus Enzyme solution Rate activity chart.
Table 1
As shown in Table 1, Effect of Agkistrodon acutus Enzyme solution lot number 01,02 and 03 3 batch of Rate activity is respectively 14.8u/mg, 13.4u/mg and 13.6u/mg.
2, subunit molecules flow measurement:
Get this product appropriate, with SDS polyacrylamide gel electrophoresis (Chinese Pharmacopoeia (version two in 2010) annex V F electrophoretic method the 5th method) measure its molecular weight subunit.Standard substance be SDS polyacrylamide gel electrophoresis lower molecular weight standard protein, 43000), BCA (molecular weight: 31000 dalton), trypsin inhibitor (molecular weight: 20100 dalton), hen's egg-white lysozyme (molecular weight: 14400 dalton) composition comprises: rabbit phosphorylase B (molecular weight: 97400 dalton), bovine serum albumin (molecular weight: 66200 dalton), rabbit Actin muscle (molecular weight:.By the gel slab after electrophoresis in the upper scanning of Shimadzu dual wavelength flying spot thin layer chromatography scanner (CS 9301PC type), reference wavelength 700nm, measure wavelength 580nm, obtain Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03) three batches of molecular weight subunits and be respectively 14327.166,14263.507,14184.903.The thin layer scanning figure of Effect of Agkistrodon acutus Enzyme solution lot number 01 molecular weight subunit is shown in Fig. 5, and the X-coordinate of Fig. 5 is for exhibition is apart from (mm), and ordinate zou is absorbancy.
3, purity test
3.1SDS polyacrylamide gel electrophoresis
Get this product, according to SDS polyacrylamide gel electrophoresis (Chinese Pharmacopoeia (version two in 2010) annex V F electrophoretic method the 5th method) check, point sample 20ul, by the gel slab after electrophoresis in the upper scanning of Shimadzu dual wavelength flying spot thin layer chromatography scanner (CS 9301PC type), reference wavelength 700nm, measure wavelength 580nm, calculate by areas of peak normalization method, obtain Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03) three batches of electrophoretic method master tape content and be respectively 99.276%, 99.576%, 98.294%, be all greater than 98%.Fig. 6 is the X-coordinate of Effect of Agkistrodon acutus Enzyme solution lot number 01 electrophoretic method purity test thin layer scanning figure, Fig. 6 is exhibition distance (mm), and ordinate zou is absorbancy.
3.2 high performance liquid chromatography
Be weighting agent (TSK gel G3000SW with gel, 7.5mm × 300mm), (Sodium phosphate dibasic 4.37g and SODIUM PHOSPHATE, MONOBASIC 1.22g is got with 0.02mol/L phosphate buffered saline buffer, be dissolved in 1000ml water, adjust ph is 7.0 to obtain) the 0.1mol/L metabisulfite solution made is moving phase; Flow velocity is per minute 1.0ml, and determined wavelength is 280nm.Effect of Agkistrodon acutus Enzyme solution (lot number 01,02 and 03) three batches detects through high performance liquid chromatography, and purity is respectively 98.908%, 99.341%, 98.623%, is all greater than 98%.The high-efficient liquid phase chromatogram of Effect of Agkistrodon acutus Enzyme solution lot number 01 batch is shown in Fig. 7, and X-coordinate is time (min), and ordinate zou is response value (uV);
In sum, the obtained Effect of Agkistrodon acutus Enzyme solution of the production method of Effect of Agkistrodon acutus Enzyme provided by the invention is one-component, purity in gel electrophoresis for single band and through high performance liquid chromatography be detected as one unimodal, purity is greater than 98%; This production method with short production cycle, production technique favorable reproducibility.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every equivalents utilizing specification sheets of the present invention and accompanying drawing content to do, or be directly or indirectly used in relevant technical field, be all in like manner included in scope of patent protection of the present invention.
Claims (7)
1. a production method for Effect of Agkistrodon acutus Enzyme, is characterized in that, comprises the following steps:
(1) under temperature is 4 ~ 10 DEG C of conditions, agkistrodon acutus venom lyophilized powder is dissolved in 0.04 ~ 0.06mol/L, pH value be 8 ~ 9 Tris in HCl damping fluid, leave standstill 60min, centrifugal acquisition supernatant liquor under 0 ~ 10 DEG C of condition, by supernatant liquor 0.01mol/L, pH value be 8 ~ 9 Tris HCl damping fluid dialyse, obtain agkistrodon acutus venom solution;
(2) by DEAE on described agkistrodon acutus venom solution Sepharose Fast Flow anion-exchange column, carry out wash-out separation, collect the protein peak in protein peak with thrombin-like enzyme activity, by collect protein peak merge, then with 0.01mol/L, pH value be 7 ~ 8 Tris HCl damping fluid dialyse;
(3) solution step (2) obtained is after 0.22 μm of filtering with microporous membrane, upper SOURCE 30Q gel column, carry out chromatography, collect the protein peak with thrombin-like enzyme activity, upper phenyl sepharose high performance chromatography column after the protein peak collected is merged, by the protein solution with thrombin-like enzyme activity of collection prior to very empty degree below 0.08M Pa, concentrate under 5 ~ 10 DEG C of conditions, use 0.01mol/L again, pH value be 7 ~ 8Tris HCl damping fluid dialysis, by dialysis after solution in Zhen Kong Du below 0.08MPa, concentrate under 5 ~ 10 DEG C of conditions,
(4) upper Hiload 26/60Superdex75PG post after solution step (3) obtained carries out filtration with 0.22 μm of millipore filtration, collects second protein peak with thrombin-like enzyme activity, is Effect of Agkistrodon acutus Enzyme solution.
2. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, is characterized in that, described dialysis adopts dialysis tubing to carry out, and the molecular weight cut-off of described dialysis tubing is 7000 dalton.
3. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, is characterized in that, is filtered by the liquid after dialysis treatment in step (1) with 0.22 μm of millipore filtration.
4. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, it is characterized in that, by agkistrodon acutus venom solubilize described in step (1) in Tris HCl damping fluid, then carry out centrifugal under 0 ~ 10 DEG C of condition, the supernatant liquor Tris of gained HCl damping fluid dialyse.
5. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, it is characterized in that, by the protein solution with thrombin-like enzyme activity of collection in step (3) prior to concentrating under very empty degree below 0.08M Pa, 5 ~ 10 DEG C of conditions, then dialyse with Tris HCl damping fluid.
6. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, is characterized in that, by the solution after dialysis in step (3) in Zhen Kong Du below 0.08MPa, concentrate under 5 ~ 10 DEG C of conditions.
7. the production method of Effect of Agkistrodon acutus Enzyme according to claim 1, is characterized in that, is filtered by the solution after dialysis in step (3) with 0.22 μm of millipore filtration.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109444319A (en) * | 2018-10-25 | 2019-03-08 | 吕梁学院 | A kind of method of astragalin content in measurement BEIQI MUSHROOM |
| CN111019991A (en) * | 2019-12-09 | 2020-04-17 | 黄山市三祈生物医药科技有限公司 | Method for preparing Agkistrodon acutus polypeptide by enzymolysis |
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2014
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Non-Patent Citations (2)
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| 孔毅: "一种新的尖吻腹蛇毒类凝血酶的纯化及性质研究", 《第一届全国化学工程与生物化工年会论文摘要集(下)》 * |
| 郑颖等: "尖吻蝮蛇毒38 k凝血酶样酶的分离纯化工艺", 《药物生物技术》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109444319A (en) * | 2018-10-25 | 2019-03-08 | 吕梁学院 | A kind of method of astragalin content in measurement BEIQI MUSHROOM |
| CN111019991A (en) * | 2019-12-09 | 2020-04-17 | 黄山市三祈生物医药科技有限公司 | Method for preparing Agkistrodon acutus polypeptide by enzymolysis |
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