CN108395475B - Hirudin separation and purification method based on affinity chromatography - Google Patents
Hirudin separation and purification method based on affinity chromatography Download PDFInfo
- Publication number
- CN108395475B CN108395475B CN201810268931.8A CN201810268931A CN108395475B CN 108395475 B CN108395475 B CN 108395475B CN 201810268931 A CN201810268931 A CN 201810268931A CN 108395475 B CN108395475 B CN 108395475B
- Authority
- CN
- China
- Prior art keywords
- hirudin
- separating
- column
- affinity chromatography
- purifying
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a hirudin separation and purification method based on affinity chromatography, which comprises the steps of feeding a saturated leech living body with physiological saline, physically extruding to enable the leech living body to secrete saliva and collect the saliva, adding a precooled trichloroacetic acid extracting solution into an obtained leech saliva sample, uniformly mixing, leaching, centrifuging to obtain a supernatant, adjusting the pH to be 3.0-5.0, adding precooled acetone, uniformly mixing, standing overnight, recovering acetone in the supernatant, centrifuging, and taking a precipitate for later use; adding distilled water into the precipitate, dissolving completely, adjusting pH to 6.0-8.0, separating and purifying with thrombin Sepharose 4FF affinity column, detecting hirudin content by high performance liquid chromatography, collecting purified solution at peak of hirudin content, dialyzing the purified solution, adding water for 2-4 times, maintaining for 10-15 hr, rotary evaporating the ultrafiltered supernatant, removing water 70-80%, and freeze-drying the concentrated solution with freeze-drying machine to obtain hirudin product. The natural hirudin purified by the method has high purity, high activity, low cost, simple operation and easy repetition.
Description
Technical Field
The invention relates to a method for separating and purifying hirudin, belonging to the field of separation and purification of biomedicine.
Background
Researches show that the leech salivary gland secretion contains various bioactive substances such as hirudin and the like and has wide application prospect in medicine. The hirudin is an acidic polypeptide secreted by salivary glands of leeches, contains 65-66 amino acid residues, mainly comprises 3 isomers of HV1, HV2 and HV3, and has high structural homology. Hirudin is the thrombin-specific inhibitor found to date which has the strongest effect, and the activity of the hirudin (HV3) protein expressing engineering bacteria species depends on the strength of the antithrombin activity, which forms stable complexes with thrombin in equimolar ratios, in tight non-covalent bonds. In 1984, Haycraft discovered that leeches extract had anticoagulant effect. Jocoby isolated this active fraction from medical leeches for the first time in 1904 and named hirudin. In 1955, Markwardt succeeded in isolating and purifying pure hirudin from the heads of medical hirudo, and started basic biochemical and pharmacological studies, and in 1970 it was determined that hirudin is a specific inhibitor of thrombin. In 1986, Harvey et al obtained cDNA for hirudin, which was subsequently successfully expressed in a variety of hosts.
Hirudin is a very stable protein, extremely stable in the dry state, and its activity is not affected by a simple temperature increase (100 ℃ water bath) or by a change in pH (l.47-12.9), and is also very stable in the presence of certain denaturing agents, and its activity begins to decrease only when the temperature and pH are simultaneously increased. Generally, hirudin can stably exist in water at room temperature for 6 months, and can not be damaged when heated at 80 ℃ for 15 min. The stability of the solution is reduced by increasing the pH of the solution. The product can be stabilized at 20 deg.C in 0.lmol/LHCI or 0.1mol/LNaOH for 15min, and completely inactivated by treatment at 80 deg.C under pH = 13.
In 1955 Markwardt in germany isolated relatively pure hirudin from hirudo medicinalis for the first time, after which the purification and standardization techniques for hirudin became more and more complete. There are a number of research reports and patents available to date, and most of the work has been to use the head or even the whole body of Whitmania pigra Whitman as a starting material, to dehydrate it with ethanol and then precipitate it with acetone to obtain a crude hirudin product. In 1970, Markwardt adopted the steps of ethanol fractional precipitation, cation exchange, gel filtration, anion exchange and the like to obtain a pure hirudin product. In 1985, Wdsmam and Marhyardt improved the purification process by using ion exchange in combination with affinity chromatography to obtain hirudin of high purity. In 1986 Johnnes et al reported a five-step purification method in which a new high performance liquid chromatography technique was used.
At present, the method for extracting hirudin is to grind leech or dry the leech and then extract the hirudin by water, ethanol, ultrafiltration or chromatography. However, the method has many defects, mainly including that the active ingredients are not high after extraction, the activity is generally about 100-4000 ATU/g, the yield is 0.27-4.47%, the activity is different according to different extracts, the impurity content is high, the method is not suitable for injection, the extraction method is complicated, the process of the method is slow, and the method is not suitable for industrial production, so that the existing hirudin extraction method is researched by many people, and the extraction method with high efficiency, low cost and high active ingredients is hoped to be found.
Disclosure of Invention
Aiming at the problems, the invention provides a method for separating and purifying hirudin, which is simple to operate and high in efficiency, and can obtain the hirudin with high purity and high activity.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a hirudin separation and purification method based on affinity chromatography comprises the following steps:
(1) coarse extraction of hirudin: feeding saturated leech living bodies with physiological saline, physically extruding to enable the leech living bodies to secrete saliva and collect, adding 2-4 times (V: V) of precooled trichloroacetic acid extract into the obtained leech saliva sample, uniformly mixing, leaching for 2-6 h, centrifuging for 10-20 min at 8000-;
(2) separation and purification: adding 4-6 times of distilled water into the precipitate, dissolving completely, adjusting pH to 6.0-8.0 with HCl, separating and purifying with thrombin Sepharose 4FF affinity column, eluting, detecting hirudin content with high performance liquid chromatography, and collecting purified solution at hirudin peak for use;
(3) desalting and concentrating: dialyzing the purified solution, repeatedly adding water for 2-4 times for 10-15 hr, performing rotary evaporation operation (30-60 deg.C, 0-180 r/min) on the ultrafiltered supernatant to remove 70-80% of water, and collecting concentrated solution;
(4) freeze-drying the concentrated solution by a freeze dryer, collecting the product, and sealing and storing to obtain the hirudin product.
And (3) detecting the activity of hirudin: the anticoagulant activity of the Markwardt thrombin is detected by a direct titration method.
And (3) detecting the content of a crude product: adding 2 times of distilled water into the saliva, shaking and mixing uniformly, and detecting the content of hirudin by HPLC.
The carrier activating agent in the thrombin Sepharose 4FF affinity column in the step (2) is carbonyl diimidazole, and the carrier activating solvent comprises but is not limited to DMSO and dioxane, wherein DMSO is preferred.
The preparation method of the thrombin Sepharose 4FF medium comprises the following steps: taking Sepharose 4FF with wet weight of 10.0 g in a Buchner funnel, repeatedly washing with deionized water, sequentially adding 25%, 50% and 75% DMSO aqueous solutions to the medium by 50 mL respectively, placing Sepharose 4FF in an anhydrous DMSO (dimethyl sulfoxide) triangular flask containing 20mL, adding 0.5-1.0 g CDI (carbonyl diimidazole) at 30 deg.C, and rotating at 100 r/min with a constant temperature oscillatorShaking at high speed for 15min, and repeatedly washing with deionized water to obtain activated Sepharose 4 FF; another 50 mg-100 mg of thrombin was dissolved in 50 mL of phosphate buffer (0.2 mol/L K)2HPO4-0.01 mol/L KCL, PH 8.0), adding the activated Sepharose 4FF into a thrombin-containing phosphate buffer solution, stirring and reacting for 10-15h at the temperature of 4-8 ℃ and the rotating speed of 50-80 r/min, performing suction filtration and washing according to deionized water, 1.0 mol/L KCL, deionized water, 0.1 mol/LHCL and deionized water (the volume of the whole solution is 4-6 times of that of the solution), and finally storing the Sepharose 4FF medium in the phosphate buffer solution at the temperature of 4 ℃ to obtain the thrombin-containing Sepharose 4FF medium.
In the step (2), the column packing method comprises the following steps: packing 1/2-3/4 times of thrombin Sepharose 4FF medium in the volume of the separation column, washing the column bed with 15 times of phosphate buffer solution with pH 7.4-8.4 at 3-5mL/min, and balancing.
The eluent used in the elution in the step (2) is 0.01-0.03 mol/L KCL-0.1-0.3 mol/L HCL, and the elution speed is 1 ml/3 min per tube.
The detection conditions of the high performance liquid chromatography in the step (2) are as follows: a chromatographic column: c1810 × 200mm, detector: UV 210nm, mobile phase: methanol: water =3:1, column temperature: 25 ℃ and sample introduction: 10 μ L, flow rate: 1 mL/min.
The principle of the anticoagulant activity of the Markwardt thrombin direct titration method is as follows:
the component of the leech saliva with the highest anticoagulant activity is generally considered to be hirudin; the hirudin can be specifically combined with thrombin, the combination ratio is 1:1, the principle of inactivating thrombin is adopted, and the activity of natural hirudin can be detected by a Matkwardt thrombin direct titration method; the measurement of hirudin is in international units, expressed as ATU, and the thrombin activity is in international units NIH, i.e. 1ATU equals the amount of hirudin which neutralizes 1 NIH thrombin.
The specific operation for detecting the anticoagulant activity by the Markwardt thrombin direct titration method is as follows:
taking about 0.1 g of the powder of the product, precisely weighing, precisely adding 500 mu L of 0.9% physiological saline solution, fully stirring and dissolving to prepare 0.2 g/mL of test solution, precisely weighing 100 mu L of supernatant, placing in a 1.5 mL test tube, adding 200 mu L of 0.5% porcine fibrinogen tris (hydroxymethyl) aminomethane hydrochloric acid buffer solution (25 mL of 0.2m0L/L tris (hydroxymethyl) aminomethane solution and about 40mL of 0.1m0L/L hydrochloric acid solution, adding water to 100mL, adjusting the pH value to 7.4), shaking uniformly, placing in a water bath (37 ℃ +/-0.5 ℃) to slowly dropwise add each 1mL of 40U (unit) thrombin solution (5 mu L/min) to coagulate while gently shaking uniformly), recording the volume of consumed thrombin solution, and calculating according to the following formula:
U＝C1V1/C2V2W
in the formula: u- - -containing thrombin activity units per 1g, i.e. hirudin content, U/g;
c1- -concentration of thrombin solution, μ/ml;
c2- -concentration of test solution, g/ml;
v1 — volume of thrombin solution consumed, μ l;
v2- -amount of test solution added, μ l;
the method for detecting the hirudin content is also suitable for: quality monitoring of various preparations prepared from blood sucking Hirudo as main raw material is provided.
Has the advantages that:
the natural hirudin purified by the method has high purity, high activity, low cost, simple operation and easy repetition, and can be industrially produced. The natural hirudin product obtained by the process can be used as raw materials of foods, health products, medicines or cosmetics, has the effects of resisting coagulation, dissolving thrombus, improving blood circulation, promoting metabolism, etc., and can be clinically used for preventing and treating cardiovascular and cerebrovascular diseases, such as apoplexy, coronary heart disease, hyperlipemia, etc.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results described in the examples are merely illustrative of the invention and should not be construed as limiting the invention.
Example 1
In this example, the preparation method of thrombin adsorbent in thrombin Sepharose 4FF affinity column is:taking Sepharose 4FF (purchased from Shanghai Rongjun biomedical science and technology Co., Ltd.) with wet weight of 10.0 g, repeatedly washing with deionized water in a Buchner funnel, sequentially adding 50 mL of 25%, 50% and 75% DMSO aqueous solutions into a medium, then putting the Sepharose 4FF into a 20 mL-containing anhydrous DMSO (dimethyl sulfoxide) triangular flask, adding 0.5-1.0 g of CDI (carbonyl diimidazole) and shaking for 15min at the rotating speed of 100 r/min of a 30 ℃ constant temperature oscillator, and repeatedly washing with deionized water to obtain activated Sepharose 4 FF; another 50 mg-100 mg thrombin (from Lanzhou Biometrics institute, LLC) was dissolved in 50 mL phosphate buffer (0.2 mol/L K)2HPO4-0.01 mol/L KCL, PH 8.0), adding the activated Sepharose 4FF into a thrombin-containing phosphate buffer solution, stirring and reacting for 10-15h at the temperature of 4-8 ℃ and the rotating speed of 50-80 r/min, performing suction filtration and washing according to deionized water, 1.0 mol/L KCL, deionized water, 0.1 mol/LHCL and deionized water (the volume of the whole solution is 4-6 times of that of the solution), and finally storing the Sepharose 4FF medium in the phosphate buffer solution at the temperature of 4 ℃ to obtain the thrombin-containing Sepharose 4FF medium.
The column packing method comprises the following steps: the thrombin Sepharose 4FF medium in 2/3 column volumes was loaded onto a 20X 100 mm column, and the column was washed at 3-5mL/min with 15 bed volumes of phosphate buffer pH8.0 and washed to equilibrium.
A method for separating and purifying hirudin specifically comprises the following steps:
(1) coarse extraction of hirudin: feeding saturated leech living bodies with physiological saline, physically extruding to enable the leech living bodies to secrete saliva and collect, adding 3 times (V: V) of precooled trichloroacetic acid extract into the obtained leech saliva sample, uniformly mixing, leaching for 2 hours, centrifuging at 10000 r/min for 20 min, taking supernatant, adding 4 times of precooled acetone into the leech saliva sample after adjusting the pH value of the mixture to be 4.0, uniformly mixing, standing overnight, recovering the acetone in the supernatant, centrifuging at 10000 r/min for 20 min, and taking precipitate for later use;
(2) separation and purification: adding 5 times of distilled water into the precipitate, fully dissolving, adjusting pH to 7.0 by using HCL, separating and purifying by using a thrombin Sepharose 4FF affinity column, eluting by using 0.025 mol/L KCL-0.2 mol/L HCL, wherein the concentration can maintain the activity and the stability of an affinity medium, and can effectively separate and elute hirudin at the same time, the elution speed is 1 ml/3 min per tube, detecting the content of hirudin by using a high performance liquid chromatography of a collected solution, and taking a purified solution at the peak value of the content of hirudin for later use;
(3) desalting and concentrating: dialyzing the purified solution, wherein the dialysis membrane is a dialysis bag with cut-off molecular weight of 7000 or less, adding water repeatedly for 10 hr for 4 times, subjecting the ultrafiltered supernatant to rotary evaporation (30-60 deg.C, 0-180 r/min), removing water 70%, and collecting the concentrated solution;
(4) freeze-drying the concentrated solution by a freeze dryer, collecting the product, and sealing and storing;
(5) and (3) activity detection: the anticoagulant activity of the Markwardt thrombin is detected by a direct titration method.
(6) And (3) detecting the content of a crude product: adding 2 times of distilled water into the saliva, shaking and mixing uniformly, and detecting the content of hirudin by HPLC.
The hirudin purified product obtained by the method has the activity of 7350ATU/g and the yield of 7.51 percent by using a Markwardt thrombin direct titration method to detect the hirudin, so that the hirudin with high purity and high activity is obtained.
Example 2
The invention provides the influence of different carrier activators on the separation and purification of hirudin, which comprises the following steps:
activation of Sepharose 4FF was carried out on the basis of example 1 by treatment with different support activators (CDI, cyanogen bromide, epichlorohydrin).
The method comprises the following steps: 0.8 g of hirudin obtained by next experiment after CDI activates Sepharose 4FF has the activity of 6937ATU/g and the yield of 7.37%; 1.0 g cyanogen bromide (the best adding amount) activates Sepharose 4FF, and then the activity of hirudin obtained by the next experiment is 4219ATU/g, and the yield is 2.19%; the activity of hirudin obtained in the next experiment after activating Sepharose 4FF (optimum concentration) by 130. mu. mol/L epichlorohydrin was 5231ATU/g, and the yield was 3.51%. Therefore, the carrier activator CDI used in the experiment is an activator with good effect, and can provide accurate scientific basis for activating the agarose gel.
Example 3
The invention provides the influence of different carrier activator solvents on the separation and purification of hirudin, which comprises the following steps:
sepharose 4FF was activated by treatment with different carrier-activating solvents (DMSO, dioxane, acetone, ethanol, isopropanol) on the basis of example 1.
The examples obtained: the activity of hirudin obtained in the next experiment after DMSO was used as a solvent was 7524 ATU/g, and the yield was 7.28%; the activity of hirudin obtained by the next experiment after using dioxane as a solvent is 6424 ATU/g, and the yield is 3.18%; the activity of hirudin obtained by the next experiment after using acetone as a solvent is 3018 ATU/g, and the yield is 2.31%; the activity of hirudin obtained in the next experiment after using ethanol and isopropanol was 4359 ATU/g and 4893 ATU/g, respectively, and the yield was 2.09% and 1.68%, respectively. Therefore, the carrier activator dissolving agent DMSO used in the experiment is an activator with a good action effect, and can provide accurate scientific basis for activating the agarose gel.
Example 4
The method for separating and purifying hirudin provided by the invention comprises the following specific steps
(1) Coarse extraction of hirudin: feeding saturated leech living bodies with physiological saline, physically extruding to enable the leech living bodies to secrete saliva and collect, adding 3 times (V: V) of precooled trichloroacetic acid extract into the obtained leech saliva sample, uniformly mixing, leaching for 2 hours, centrifuging at 10000 r/min for 20 min, taking supernatant, adding 4 times of precooled acetone into the leech saliva sample after HCL (hydrogen chloride) adjusts the PH to be 4.0, uniformly mixing, standing overnight, recovering acetone in the supernatant, centrifuging at 10000 r/min for 20 min, and taking precipitate for later use;
(2) separation and purification: adding 5 times of distilled water into the precipitate, fully dissolving, adjusting pH to 6.5 with HCl, separating and purifying with thrombin Sepharose 4FF affinity column, eluting with 0.025 mol/L KCL-0.2 mol/L HCl at an elution speed of 1 ml/3 min per tube, detecting hirudin content with high performance liquid chromatography, and collecting purified solution at the peak of hirudin content for use;
(3) desalting and concentrating: dialyzing the purified solution, adding water for 4 times, maintaining for 10 hr, performing rotary evaporation (30-60 deg.C, 0-180 r/min) on the ultrafiltered supernatant to remove water 75%, and collecting the concentrated solution;
(4) freeze-drying the concentrated solution by a freeze dryer, collecting the product, and sealing and storing;
(5) and (3) activity detection: the anticoagulant activity of the Markwardt thrombin is detected by a direct titration method.
(6) And (3) detecting the content of a crude product: adding 2 times of distilled water into the saliva, shaking and mixing uniformly, and detecting the content of hirudin by HPLC.
The activity of the hirudin purified product obtained by the method is 7518ATU/g and the yield is 6.79% by using Markwardt thrombin direct titration method, compared with the prior art, the product yield and the final activity of the hirudin purified product are greatly improved.
Claims (7)
1. A hirudin separation and purification method based on affinity chromatography is characterized by comprising the following steps:
1) coarse extraction of hirudin: feeding saturated leech living bodies with physiological saline, physically squeezing to enable the leech living bodies to secrete saliva and collect, leaching and centrifuging the obtained leech saliva samples, and taking precipitates for later use;
2) separation and purification: adding distilled water into the precipitate obtained in the step 1), fully dissolving, adjusting the pH value to 6.0-8.0, separating and purifying by using a thrombin Sepharose 4FF affinity column, detecting the hirudin content by using a high performance liquid chromatography, and taking a purified solution at the peak value of the hirudin content for later use;
3) desalting and concentrating: dialyzing the purified solution, repeatedly adding water for 2-4 times for 10-15 hr, rotary evaporating the ultrafiltered supernatant to remove water 70-80%, and collecting the concentrated solution;
4) freeze-drying the concentrated solution by a freeze dryer to obtain a hirudin product;
step 2) the carrier activator in the thrombin Sepharose 4FF affinity column is carbonyl diimidazole; the carrier activating solvent is DMSO.
2. The method for separating and purifying hirudin based on affinity chromatography according to claim 1, wherein the pH is adjusted to 3.0-5.0 with HCl in step 1) to inactivate part of the impurity proteins without affecting the activity of the objective product.
3. The method for separating and purifying hirudin based on affinity chromatography according to claim 1, wherein the leaching step in step 1) is: adding pre-cooled trichloroacetic acid extract, mixing, leaching, centrifuging to obtain supernatant, adjusting pH to 3.0-5.0, adding pre-cooled acetone, mixing, standing overnight, recovering acetone from supernatant, centrifuging, and collecting precipitate.
4. The method for separating and purifying hirudin based on affinity chromatography according to claim 1, wherein the column packing method of step 2) comprises: packing 1/2-3/4 times column volume of thrombin Sepharose 4FF medium in a separation column, washing the column bed with 15 times column volume of p H7.4.4-8.4 phosphate buffer solution at 3-5mL/min flow rate, and washing to balance.
5. The method for separating and purifying hirudin based on affinity chromatography of claim 1, wherein the eluent in step 2) is (0.01-0.03) mol/L KCl- (0.1-0.3) mol/L HCl, and the elution rate is 1 ml/3 min per tube.
6. The method for separating and purifying hirudin based on affinity chromatography according to claim 2, wherein the conditions of the HPLC detection in step (2) are as follows: a chromatographic column: c1810 × 200mm, detector: UV 210nm, mobile phase: methanol: water =3:1, column temperature: 25 ℃ and sample introduction: 10 μ L, flow rate: 1 mL/min.
7. The method for separating and purifying hirudin based on affinity chromatography of claim 1, wherein the dialysis membrane used in the dialysis of step 3) is a dialysis bag with a cut-off of 7000.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810268931.8A CN108395475B (en) | 2018-03-29 | 2018-03-29 | Hirudin separation and purification method based on affinity chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810268931.8A CN108395475B (en) | 2018-03-29 | 2018-03-29 | Hirudin separation and purification method based on affinity chromatography |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108395475A CN108395475A (en) | 2018-08-14 |
| CN108395475B true CN108395475B (en) | 2021-09-10 |
Family
ID=63096860
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810268931.8A Active CN108395475B (en) | 2018-03-29 | 2018-03-29 | Hirudin separation and purification method based on affinity chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108395475B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734489B (en) * | 2019-12-05 | 2021-07-23 | 昆明学院 | Device and method for extracting hirudin from living leeches in laboratory |
| CN112480243B (en) * | 2021-01-06 | 2023-05-05 | 广西科康科技集团有限公司 | Process and equipment for large-scale hirudin separation and purification production |
| CN113072638B (en) * | 2021-03-16 | 2022-12-23 | 宁波博睿瀚达生物科技有限公司 | Method for removing endotoxin in recombinant hirudin protein solution |
| CN115386445A (en) * | 2022-09-23 | 2022-11-25 | 文泽沂 | Preparation method of natural hirudin wine |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5164304A (en) * | 1989-05-04 | 1992-11-17 | Sri International | Method and vectors for stabilizing hirudin and human laminin b1 expression |
| CN1423929A (en) * | 2003-01-15 | 2003-06-18 | 周维海 | Culture of hiruto and extraction of hirutin |
| CN102191264A (en) * | 2011-04-02 | 2011-09-21 | 江苏省中医药研究院 | Production method of recombined hirudin |
| CN103509105A (en) * | 2012-06-29 | 2014-01-15 | 郑州英诺生物科技有限公司 | Production technology capable of efficiently separating high-activity hirudin based on anion exchange column |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2687681B1 (en) * | 1992-02-20 | 1995-10-13 | Transgene Sa | POLYETHYLENEGLYCOL-HIRUDINE CONJUGATES, THEIR PREPARATION PROCESS AND THEIR USE FOR THE TREATMENT OF THROMBOSES. |
| GB9309509D0 (en) * | 1993-05-07 | 1993-06-23 | Merck Patent Gmbh | Thrombin inhibitors |
| CN101279999B (en) * | 2008-05-21 | 2012-07-18 | 大连理工大学 | Method for modifying hirudin by polyethyleneglycol assisted by anion exchange column |
-
2018
- 2018-03-29 CN CN201810268931.8A patent/CN108395475B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5164304A (en) * | 1989-05-04 | 1992-11-17 | Sri International | Method and vectors for stabilizing hirudin and human laminin b1 expression |
| CN1423929A (en) * | 2003-01-15 | 2003-06-18 | 周维海 | Culture of hiruto and extraction of hirutin |
| CN102191264A (en) * | 2011-04-02 | 2011-09-21 | 江苏省中医药研究院 | Production method of recombined hirudin |
| CN103509105A (en) * | 2012-06-29 | 2014-01-15 | 郑州英诺生物科技有限公司 | Production technology capable of efficiently separating high-activity hirudin based on anion exchange column |
Non-Patent Citations (2)
| Title |
|---|
| On the isolation of the thrombin inhibitor hirudin;P.Walsmann,F.Markwardt;《Thrombosis Research》;19851115;第40卷(第4期);第563-569页 * |
| 重组水蛭素克隆的表达及其产物的分离纯化;郭娟 等;《中华血液学杂志》;19980331;第19卷(第3期);第146页摘要、第147页第2.7节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108395475A (en) | 2018-08-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108395475B (en) | Hirudin separation and purification method based on affinity chromatography | |
| FR2461500A1 (en) | PROCESS FOR PRODUCING A SUBSTANCE CAPABLE OF STIMULATING THE PROLIFERATION AND DIFFERENTIATION OF HUMAN GRANULOPOIETIC STEM CELLS | |
| CA2491716C (en) | Processes for the preparation of fibrinogen | |
| JP3110078B2 (en) | Mass production of high-purity antimicrobial peptides | |
| CN112661811B (en) | Antihypertensive peptide, long-acting antihypertensive peptide and preparation method thereof | |
| Bagdy et al. | Large scale preparation of hirudin | |
| KR0153774B1 (en) | Anti-thrombins | |
| NO138145B (en) | PROCEDURE FOR PREPARING AN ENZYME PREPARATION | |
| ES2373035T3 (en) | METHOD FOR ELIMINATING HUMAN SEROALBUMIN POLYMERS. | |
| CN109651502B (en) | Method for simultaneously separating and purifying blood coagulation factors IX, X and VII from human plasma | |
| JP2003518513A (en) | Separation of fibrinogen from plasma protease | |
| US3879369A (en) | Anti-coagulant isolation from malayan pit viper using affinity chromatography | |
| CN107056903B (en) | Method for extracting pharmaceutical-grade ultrahigh-purity nisin by agarose gel affinity chromatography | |
| CN106589068B (en) | Sea bream antioxidant polypeptide and preparation method thereof | |
| CN115947829A (en) | Method for separating and purifying hirudin based on thrombin affinity magnetic microspheres and application thereof | |
| US4617378A (en) | Purification of biologically active human immune interferon | |
| CN115873095A (en) | Purification method of recombinant human bone morphogenetic protein-2 dimer | |
| Surovtsev et al. | Purification of bacteriocins by chromatographic methods | |
| CN112457377B (en) | Periplaneta americana polypeptide and application thereof | |
| CN103910790B (en) | A kind of method preparing Human plasma protein C concentrate | |
| RU2256464C1 (en) | Method for preparing human c-1 esterase inhibitor amd product for using in medicine | |
| JP7375012B2 (en) | Extraction and purification method of hirudin mutant and its use | |
| CN109957008B (en) | Extraction and purification method of hirudin mutant and application thereof | |
| CN115975997B (en) | Secondary ultrafiltration dialysate for purifying human coagulation factor IX and purification method | |
| CN113045634B (en) | Preparation method of complement factor H |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211103 Address after: 215107 No. 1269, Xinzhou highway, Lili Town, Wujiang District, Suzhou City, Jiangsu Province Patentee after: Changzhou Biotechnology (Jiangsu) Co.,Ltd. Address before: 215000 Bamboo Garden Road, Suzhou high tech Zone, Jiangsu Province, No. 209 Patentee before: SUZHOU ZHIHUI BIOTECHNOLOGY CO.,LTD. |
|
| TR01 | Transfer of patent right |