CN107674868B - Method for extracting thrombin from pig blood - Google Patents
Method for extracting thrombin from pig blood Download PDFInfo
- Publication number
- CN107674868B CN107674868B CN201711147689.0A CN201711147689A CN107674868B CN 107674868 B CN107674868 B CN 107674868B CN 201711147689 A CN201711147689 A CN 201711147689A CN 107674868 B CN107674868 B CN 107674868B
- Authority
- CN
- China
- Prior art keywords
- thrombin
- sodium chloride
- solution
- concentration
- supernatant
- 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
- 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
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21005—Thrombin (3.4.21.5)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for extracting thrombin from pig blood, which relates to the technical field of thrombin production and comprises the following steps: the pig blood is centrifuged to collect supernatant anticoagulated plasma, DEAE sepharose A-50 is used for adsorbing prothrombin in the supernatant anticoagulated plasma, sodium chloride solution I is used for washing the prothrombin, sodium chloride-calcium chloride mixed solution is used for eluting and activating the prothrombin, the obtained effluent is thrombin crude extract, 724 ion exchange resin is used for purifying, thrombin refined extract at the thrombin active peak is collected, viruses are removed from the thrombin refined extract, desalting and concentrating are carried out, and finally, a thrombin finished product is obtained by freeze-drying.
Description
Technical Field
The invention relates to the technical field of thrombin production, and particularly relates to a method for extracting thrombin from pig blood.
Background
Thrombin is a natural component of the body's blood coagulation system and is formed by activation of the precursor prothrombin by a prothrombin activator. Thrombin consists of two peptide chains, which are linked by disulfide bonds, and is a serine protease with high specificity. Thrombin hydrolyzes 4 Arg-Gly peptide bonds on fibrinogen to render fibrinogen insoluble, thereby coagulating blood. At present, the thrombin is mainly prepared from animal plasma and human plasma at home and abroad, the domestic pig industry has large scale and wide area, the pig blood resource is rich, the thrombin is widely applied clinically, and the thrombin is extracted from the pig blood, so that the pig blood resource is effectively utilized, and the problem of environmental pollution caused by pig blood waste is solved.
Chinese patent application publication No. CN105624134A discloses a method for preparing limited thrombin, which comprises adsorbing prothrombin from porcine plasma by using anion exchange chromatography medium, eluting anion chromatography column, activating prothrombin to obtain crude thrombin solution, performing ultrafiltration concentration to obtain thrombin concentrated solution, adsorbing thrombin by using cation exchange chromatography medium, eluting cation chromatography column to obtain thrombin solution, performing ultrafiltration desalination concentration, and lyophilizing to obtain the final product of limited thrombin.
In the actual operation process of the preparation method, trisodium citrate is used as an eluent for extracting thrombin from pig blood, and the trisodium citrate can be chelated with calcium ions to form a soluble citrate complex which is difficult to dissociate, so that the calcium ions are reduced, the prothrombin is prevented from being converted into thrombin, and then sufficient calcium ions need to be supplemented in the later period, the calcium ions added in the later period are usually chelated with the trisodium citrate, and the calcium ions left after the trisodium citrate is completely chelated promote the conversion of prothrombin into thrombin, so that the activation efficiency of prothrombin is reduced.
Disclosure of Invention
The invention aims to provide a method for extracting thrombin from pig blood, so that elution and activation of prothrombin are synchronously performed, and the activation efficiency of prothrombin is accelerated.
The technical purpose of the invention is realized by the following technical scheme:
a method for extracting thrombin from pig blood comprises the following steps:
(1) adding trisodium citrate solution into pig blood, centrifuging the mixed solution until layering, collecting supernatant, and filtering the supernatant with double-layer absorbent gauze to obtain supernatant anticoagulant plasma;
(2) performing chromatography on the supernatant anticoagulant plasma obtained in the step (1) by using an A-50 chromatographic column, adsorbing prothrombin in the supernatant anticoagulant plasma on the A-50 chromatographic column, washing the A-50 chromatographic column by using a sodium chloride solution I, eluting by using a sodium chloride-calcium chloride mixed solution, and activating thrombin adsorbed on the A-50 chromatographic column to obtain a thrombin crude extract;
(3) performing chromatography on the thrombin crude extract obtained in the step (2) by using a resin chromatographic column, adsorbing thrombin in the thrombin crude extract on the resin chromatographic column, and eluting the thrombin adsorbed on the resin chromatographic column by using a sodium chloride solution II to obtain a thrombin refined extract;
(4) removing viruses from the thrombin refined extract obtained in the step (3), and then desalting and concentrating to obtain a thrombin concentrated solution;
(5) and (5) freeze-drying the thrombin concentrated solution obtained in the step (4) to obtain a white powdery thrombin finished product.
By adopting the scheme, the trisodium citrate solution is added into the pig blood to prevent the blood from coagulating, the pig blood leads blood cells and blood plasma to be layered after centrifugation, the collection of supernatant anticoagulated blood serum is facilitated through double-layer absorbent gauze, an A-50 chromatographic column adsorbs prothrombin in the supernatant anticoagulated blood plasma, the sodium chloride solution I cleans blood plasma of other components remained on the A-50 chromatographic column, and meanwhile, the residual trisodium citrate solution is washed away; the sodium chloride in the sodium chloride-calcium chloride mixed solution is used as an ion enhancer to promote the elution of the prothrombin adsorbed in the A-50 chromatographic column, and the calcium chloride is used as an activator to activate the prothrombin while eluting so as to realize the synchronous elution and activation and directly obtain the crude thrombin extract; and then purifying the thrombin crude extract by using an A-50 chromatographic column to obtain high-purity thrombin refined extract, and performing virus removal, desalination concentration and freeze-drying on the thrombin refined extract so as to prevent the influence of trisodium citrate on prothrombin activation, accelerate the prothrombin activation efficiency, simplify the thrombin preparation process and increase the storage time of thrombin.
More preferably:
the dosage of the trisodium citrate solution added into the pig blood in the step (1) is 7.0-8.0mL of the trisodium citrate solution added into 1L of the pig blood;
the concentration of the sodium chloride solution I for washing the A-50 chromatographic column in the step (2) is 0.1-0.3M/L;
the concentration of sodium chloride in the sodium chloride-calcium chloride mixed solution for eluting and activating the prothrombin in the step (2) is 1.5-2.5M/L, and the concentration of calcium chloride is 1.0-2.0M/L;
the concentration of the sodium chloride solution II for eluting the thrombin in the step (3) is 2.0-3.0 g/L.
By adopting the scheme, researches (experiments) show that the thrombin prepared by the used reagent within the parameter range has higher titer and protein concentration, and is convenient to be applied to the actual production process.
More preferably:
the dosage of the trisodium citrate solution added into the pig blood in the step (1) is 7.5mL of the trisodium citrate solution added into 1L of the pig blood; the concentration of the sodium chloride solution I for washing the A-50 chromatographic column in the step (2) is 0.2M/L;
the concentration of sodium chloride in the sodium chloride-calcium chloride mixed solution for eluting and activating the prothrombin in the step (2) is 2.0M/L, and the concentration of calcium chloride is 1.5M/L;
the concentration of the sodium chloride solution II for eluting the thrombin in the step (3) is 2.5 g/L.
By adopting the scheme, the titer and the protein concentration of the thrombin prepared by the used reagent according to the parameters are the highest through research (experiment).
More preferably: the washing process in the step (2) is carried out twice, and the dosage of the sodium chloride solution I in each washing is 24-26mL of the sodium chloride solution I added in each 1L of the supernatant anticoagulated plasma.
By adopting the scheme, researches (experiments) show that the prothrombin on the column is cleaned in batches, and when 24-26mL of sodium chloride solution I is added into every 1L of supernatant anticoagulated plasma, the sodium chloride solution I has a good cleaning effect on the prothrombin, so that the protein concentration of thrombin is improved conveniently.
More preferably: the washing process in the step (2) is carried out in two times, and the amount of the sodium chloride solution I is 25mL per 1L of the supernatant anticoagulated plasma.
By adopting the scheme, researches (experiments) show that the prothrombin on the column is cleaned in batches, and when 25mL of sodium chloride solution I is added into every 1L of supernatant anticoagulated plasma, the sodium chloride solution I has the best cleaning effect on the prothrombin, so that the protein concentration of the thrombin is conveniently improved.
More preferably: the dosage of the sodium chloride-calcium chloride mixed solution for eluting and activating the prothrombin in the step (2) is 18-20mL of the sodium chloride-calcium chloride mixed solution added into every 100mL of the supernatant anticoagulated plasma.
By adopting the scheme, researches (experiments) show that when 18mL-20mL of sodium chloride-calcium chloride mixed solution is added into every 100mL of supernatant anticoagulant plasma, the protein concentration of thrombin is higher.
More preferably: the amount of the sodium chloride-calcium chloride mixed solution for eluting and activating prothrombin in the step (2) is 19mL of the sodium chloride-calcium chloride mixed solution added to every 100mL of the supernatant anticoagulated plasma.
By adopting the scheme, the research (experiment) shows that the protein concentration of the thrombin is the highest when 19mL of the sodium chloride-calcium chloride mixed solution is added into every 100mL of supernatant anticoagulated plasma.
More preferably: and (3) carrying out elution and activation operation in the step (2) in two times, wherein corresponding effluent is obtained by each elution and activation operation, and the sum of the two effluents is the crude thrombin extracting solution.
By adopting the above scheme, the data obtained from the multiple preparation of thrombin shows that the protein concentration of thrombin is the highest when elution and activation of prothrombin are performed in batches.
More preferably: and (4) removing viruses from the thrombin refined extract in the step (4) by using a filter membrane with the pore diameter of 20 nm.
By adopting the scheme, the filter membrane with the aperture of 20nm is used for removing the virus, and compared with the pasteur virus inactivation process, the activity loss to the thrombin is reduced.
In conclusion, the invention has the following beneficial effects:
1. by synchronously carrying out elution and activation, the process steps for preparing thrombin are reduced, the seizure of calcium ions by an anticoagulant during the activation of prothrombin is avoided, the process period is shortened, and the yield of thrombin is improved;
2. the sodium chloride is used as an ion enhancer, has good elution effect, does not generate toxicity, and reduces the production cost;
3. the filter membrane with the pore diameter of 20nm is used for removing viruses, so that the activity loss of thrombin is reduced, and the specific activity of thrombin is increased.
Detailed Description
Pretreatment:
1. preparing a trisodium citrate solution:
weighing 38g of trisodium citrate particles, dissolving the trisodium citrate particles in distilled water to enable the concentration of the trisodium citrate particles to be 38g/L, and adjusting the pH of the trisodium citrate particles to be 7.0-7.4 by using 1M/L HCl solution or 1M/L NaOH solution;
2. equilibration of DEAE Sepharose A-50 (abbreviated to A-50):
suspending A-50 in distilled water until dissolvedAfter 1 hour, pouring the supernatant and the A-50 floating in the supernatant, soaking the rest A-50 in 0.5mol/L NaOH solution, stirring, standing for 30min, washing A-50 with distilled water until the pH of the effluent from A-50 is 7, and adding 0.1mol/L NaH2PO4Treating the solution in the same operation process; finally, 0.01mol/L, pH of Na which is 6.5 is used2HPO4-NaH2PO4Balancing with buffer solution (PB) to make pH of effluent from A-50 reach stable value, vacuum pumping, and storing;
3. 724 ion exchange resin (resin for short) balance:
washing the resin with distilled water for 5-6 times, and gently stirring (not vigorously stirring to prevent resin from being damaged); soaking the soaked resin in 5% HCl solution (the amount of HCl solution is twice of the volume of the resin at this time) for 10min, and washing the resin with distilled water until the pH of effluent flowing out of the resin is 6; the resin was then soaked with 5% NaOH solution (twice the volume of the resin) for 10min, and the resin was rinsed with distilled water until the effluent from the resin had a pH of 9; soaking the resin in 5% HCl solution (the amount of HCl solution is three times of the volume of the resin) for more than 1H, washing with distilled water after the resin is converted into H-type, until the pH of effluent liquid flowing out of the resin is 7, and packing the resin into a column for later use.
Example 1:
1. collection of supernatant anticoagulated plasma
1.1, collecting fresh pig blood with the separation time of less than 30min by using a clean glass container, adding a prepared trisodium citrate solution of 38g/L into the pig blood, adding 6.5mL of the trisodium citrate solution into every 1L of the pig blood, stirring for 30min, and refrigerating at the constant temperature at the temperature of 4 ℃ to obtain a pig blood treatment solution;
1.2, adjusting parameters of a centrifuge to 4 ℃ and 4020rpm, placing the pig blood treatment fluid in the centrifuge for centrifugation until layering, and collecting supernatant;
and 1.3, filtering the supernatant by using double-layer absorbent gauze, and collecting the supernatant to obtain anticoagulant plasma.
2. Collection of crude Thrombin extract
2.1, sampling the collected supernatant anticoagulated plasma into an A-50 chromatographic column which is balanced in advance, and adsorbing prothrombin in the supernatant anticoagulated plasma on the A-50 chromatographic column;
2.2, washing the prothrombin attached to the A-50 chromatographic column twice by using a sodium chloride solution I with the concentration of 0.2M/L, wherein the using amount of the sodium chloride solution I in each washing is that 25mL of the sodium chloride solution I is added into 1L of supernatant anticoagulated plasma;
and 2.3, eluting and activating prothrombin on the A-50 chromatographic column by using a sodium chloride-calcium chloride mixed solution twice, obtaining corresponding effluent liquid by each elution and activation operation, and collecting the effluent liquid twice to obtain a thrombin crude extract, wherein the concentration of sodium chloride in the sodium chloride-calcium chloride mixed solution is 2.0M/L, the concentration of calcium chloride is 1.5M/L, and the total dosage of the sodium chloride-calcium chloride mixed solution twice is that 19mL of the sodium chloride-calcium chloride mixed solution is added into each 100mL of supernatant anticoagulant plasma.
3. Collection of Thrombin extract
3.1, injecting the thrombin crude extract into resin chromatography which is balanced in advance, and adsorbing thrombin in the thrombin crude extract on a resin chromatography column;
and 3.2, eluting the resin by using a sodium chloride solution II with the concentration of 2.5g/L, simultaneously obtaining a chromatogram by using a high performance liquid chromatograph, and collecting an effluent liquid flowing out of the resin when a thrombin activity peak is reached, namely the thrombin refined extract.
4. Preparation of Thrombin concentrates
4.1, filtering the thrombin refined extract by using a filter membrane with the aperture of 20nm to remove viruses;
4.2, desalting and concentrating by using an ultrafiltration membrane with the aperture of 10kD, and adding glycine as a protective agent to obtain a thrombin concentrated solution.
5. Thrombin freeze drying
5.1, subpackaging the thrombin concentrated solution and putting the thrombin concentrated solution into a cabinet at normal temperature;
5.2, controlling the temperature of the clapboard to be reduced to-40 ℃ within 40min, and keeping the temperature for 3 h;
5.3, starting a vacuum pump, and controlling the vacuum degree to be 7-15 Pa;
after 5.4 h and 2h, heating the partition plate to-30 ℃, and keeping for 3 h;
after 5.5 h and 4h, heating the partition plate to-20 ℃, and keeping for 6 h;
after 5.6 h and 8h, heating the partition plate to 0 ℃, and keeping for 20 h;
after 5.7 hours and 10 hours, heating the partition plate to 30 ℃, and keeping for 6 hours;
5.8, keeping the vacuum degree below 5Pa for 1 h;
5.9, vacuum pressing and taking out of the cabinet to obtain a thrombin finished product.
Example 2: the difference from example 1 is that: 7.0mL of trisodium citrate solution was added per 1L of pig blood.
Example 3: the difference from example 1 is that: 7.5mL of trisodium citrate solution was added per 1L of pig blood.
Example 4: the difference from example 1 is that: 8.0mL of trisodium citrate solution was added per 1L of pig blood.
Example 5: the difference from example 1 is that: 8.5mL of trisodium citrate solution was added per 1L of pig blood.
Example 6: the difference from example 3 is that: the concentration of the sodium chloride solution I is 0.05M/L.
Example 7: the difference from example 3 is that: the concentration of the sodium chloride solution I is 0.1M/L.
Example 8: the difference from example 3 is that: the concentration of the sodium chloride solution I is 0.3M/L.
Example 9: the difference from example 3 is that: the concentration of the sodium chloride solution I is 0.4M/L.
Example 10: the difference from example 3 is that: 23mL of sodium chloride solution I was added to 1L of the supernatant anticoagulated plasma.
Example 11: the difference from example 3 is that: 24mL of sodium chloride solution I was added to 1L of the supernatant anticoagulated plasma.
Example 12: the difference from example 3 is that: 26mL of sodium chloride solution I was added to 1L of the supernatant anticoagulated plasma.
Example 13: the difference from example 3 is that: 27mL of sodium chloride solution I was added to 1L of the supernatant anticoagulated plasma.
Example 14: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.0M/L, and the concentration of calcium chloride is 0.5M/L.
Example 15: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.0M/L, and the concentration of calcium chloride is 1.0M/L.
Example 16: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.0M/L, and the concentration of calcium chloride is 1.5M/L.
Example 17: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.0M/L, and the concentration of calcium chloride is 2.0M/L.
Example 18: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.0M/L, and the concentration of calcium chloride is 2.5M/L.
Example 19: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.5M/L, and the concentration of calcium chloride is 0.5M/L.
Example 20: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.5M/L, and the concentration of calcium chloride is 1.0M/L.
Example 21: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.5M/L, and the concentration of calcium chloride is 1.5M/L.
Example 22: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.5M/L, and the concentration of calcium chloride is 2.0M/L.
Example 23: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 1.5M/L, and the concentration of calcium chloride is 2.5M/L.
Example 24: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.0M/L, and the concentration of calcium chloride is 0.5M/L.
Example 25: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.0M/L, and the concentration of calcium chloride is 1.0M/L.
Example 26: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.0M/L, and the concentration of calcium chloride is 2.0M/L.
Example 27: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.0M/L, and the concentration of calcium chloride is 2.5M/L.
Example 28: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.5M/L, and the concentration of calcium chloride is 0.5M/L.
Example 29: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.5M/L, and the concentration of calcium chloride is 1.0M/L.
Example 30: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.5M/L, and the concentration of calcium chloride is 1.5M/L.
Example 31: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.5M/L, and the concentration of calcium chloride is 2.0M/L.
Example 32: the difference from example 3 is that: the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride is 2.5M/L, and the concentration of calcium chloride is 2.5M/L.
Example 33: the difference from example 3 is that: 17mL of a sodium chloride-calcium chloride mixed solution was added to 100mL of the supernatant anticoagulated plasma.
Example 34: the difference from example 3 is that: 18mL of a sodium chloride-calcium chloride mixed solution was added to every 100mL of the supernatant anticoagulated plasma.
Example 35: the difference from example 3 is that: 20mL of a sodium chloride-calcium chloride mixed solution was added to 100mL of the supernatant anticoagulated plasma.
Example 36: the difference from example 3 is that: every 100mL of supernatant anticoagulated plasma was added with 21mL of a sodium chloride-calcium chloride mixed solution.
Example 37: the difference from example 3 is that: the concentration of the sodium chloride solution II is 1.5 g/L.
Example 38: the difference from example 3 is that: the concentration of the sodium chloride solution II is 2.0 g/L.
Example 39: the difference from example 3 is that: the concentration of the sodium chloride solution II is 3.0 g/L.
Example 40: the difference from example 3 is that: the concentration of the sodium chloride solution II is 3.5 g/L.
The thrombin in the above examples was subjected to titer and protein concentration measurements:
the thrombin titer standard was determined according to the "second method for determining the amount of thrombin in accordance with the" Lowry method "in the determination of the protein content in 0731, the fourth rule, on pages 1561 and 1562 in the second pharmacopoeia of the people's republic of China 2015, respectively.
The test results are given in the following table:
example 3 was selected as the preferred example and comparative testing was performed.
Comparative example 1: example 1, example 2, example 4 and example 5
In comparison with example 3, in comparative example 1, the thrombin prepared in example 3 is prepared by changing the dosage of the trisodium citrate solution, and the titer and the protein concentration of the thrombin are higher when the dosage of the trisodium citrate is 7.0-8.0mL/L of pig blood.
Comparative example 2: example 6, example 7, example 8 and example 9
In comparison with example 3, in comparative example 2, the thrombin prepared in example 3 was prepared by changing the concentration of the sodium chloride solution I, and the titer and protein concentration of thrombin were higher at the concentration of the sodium chloride solution I of 0.1-0.3M/L.
Comparative example 3: example 10, example 11, example 12 and example 13
In comparison with example 3, in comparative example 3, the thrombin prepared in example 3 is prepared by changing the amount of the sodium chloride solution I, and the titer and the protein concentration of the thrombin are higher when the amount of the sodium chloride solution I is 24-26mL/L of the supernatant anticoagulated serum.
Comparative example 4: example 14 to example 32
In comparison with example 3, in comparative example 4, the thrombin prepared in example 3 was prepared by changing the concentration of the mixed solution of sodium chloride and calcium chloride, and the titer and protein concentration of thrombin were higher when the mixed solution of 1.5-2.5M/L sodium chloride-1.0-2.0M/L calcium chloride was used.
Comparative example 5: example 33, example 34, example 35 and example 36
In comparative example 5, compared with example 3, the thrombin prepared in example 3 with the elution amount of the mixed solution of sodium chloride and calcium chloride changed has higher titer and protein concentration when the total amount of the mixed solution of sodium chloride and calcium chloride is 18-20mL/100mL of the supernatant anticoagulant serum.
Comparative example 6: example 37, example 38, example 39 and example 40
In comparison with example 3, in comparative example 7, the thrombin prepared in example 3 was prepared by changing the concentration of the sodium chloride solution II, and the titer and protein concentration of thrombin were higher at the concentration of the sodium chloride solution II of 2.0 to 3.0 g/L.
The experiments prove that the titer and the protein concentration of the thrombin can be influenced by the dosage of the trisodium citrate, the concentration of the sodium chloride I, the dosage of the sodium chloride solution I, the concentration of the sodium chloride-calcium chloride mixed solution, the elution amount of the sodium chloride-calcium chloride mixed solution and the concentration of the sodium chloride solution II. Wherein, when 7.5mL/L of trisodium citrate of pig blood, 25mL/L of sodium chloride solution I of supernatant anticoagulated serum with concentration of 0.2M/L, 2.0M/L-1.5M/of sodium chloride-calcium chloride mixed solution of supernatant anticoagulated plasma with concentration of 19mL/100mL and 2.5g/L of sodium chloride solution II are used, the titer of protein and the concentration of protein are higher.
The thrombin prepared by the method improves the activation efficiency of prothrombin and simplifies the preparation process under the condition of ensuring the titer and the protein concentration of the thrombin.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the present invention.
Claims (5)
1. A method for extracting thrombin from pig blood is characterized by comprising the following steps:
(1) adding a trisodium citrate solution into the pig blood, wherein the dosage of the trisodium citrate solution in the pig blood is 7.0-8.0mL of the trisodium citrate solution added into every 1L of the pig blood, centrifuging the mixed solution until the mixed solution is layered, collecting supernatant, and filtering the supernatant by using double-layer absorbent gauze to obtain supernatant anticoagulant plasma;
(2) performing chromatography on the supernatant anticoagulant plasma obtained in the step (1) by using an A-50 chromatographic column, adsorbing prothrombin in the supernatant anticoagulant plasma on the A-50 chromatographic column, washing the A-50 chromatographic column twice by using a sodium chloride solution I with the concentration of 0.1-0.3M/L, eluting by using a sodium chloride-calcium chloride mixed solution, and activating the prothrombin adsorbed on the A-50 chromatographic column to obtain a thrombin crude extract;
the washing process is carried out twice, and the dosage of the sodium chloride solution I is 24-26mL of the sodium chloride solution I added into every 1L of the supernatant anticoagulated plasma during each washing;
the concentration of sodium chloride in the mixed solution of sodium chloride and calcium chloride for eluting and activating prothrombin is 1.5-2.5M/L, and the concentration of calcium chloride is 1.0-2.0M/L;
the dosage of the sodium chloride-calcium chloride mixed solution for eluting and activating the prothrombin is 18-20mL of the sodium chloride-calcium chloride mixed solution added into every 100mL of the supernatant anticoagulated plasma;
the elution and activation operation is carried out in two times, corresponding effluent liquid is obtained by each elution and activation operation, and the sum of the two effluent liquid is the thrombin crude extract;
(3) performing chromatography on the thrombin crude extract obtained in the step (2) by using a resin chromatographic column, adsorbing thrombin in the thrombin crude extract on the resin chromatographic column, and eluting the thrombin adsorbed on the resin chromatographic column by using a sodium chloride solution II with the concentration of 2.0-3.0g/L to obtain a thrombin refined extract;
(4) removing viruses from the thrombin refined extract obtained in the step (3), and then desalting and concentrating to obtain a thrombin concentrated solution;
(5) and (5) freeze-drying the thrombin concentrated solution obtained in the step (4) to obtain a white powdery thrombin finished product.
2. The method of claim 1, wherein the thrombin is extracted from pig blood by:
the dosage of the trisodium citrate solution added into the pig blood in the step (1) is 7.5mL of the trisodium citrate solution added into 1L of the pig blood;
the concentration of the sodium chloride solution I for washing the A-50 chromatographic column in the step (2) is 0.2M/L;
the concentration of the sodium chloride in the sodium chloride-calcium chloride mixed solution for eluting and activating the prothrombin in the step (2) is 2.0M/L,
the concentration of calcium chloride is 1.5M/L;
the concentration of the sodium chloride solution II for eluting the thrombin in the step (3) is 2.5 g/L.
3. The method of claim 1, wherein the thrombin is extracted from pig blood by: the washing process in the step (2) is carried out in two times, and the amount of the sodium chloride solution I is 25mL per 1L of the supernatant anticoagulated plasma.
4. The method of claim 1, wherein the thrombin is extracted from pig blood by: the amount of the sodium chloride-calcium chloride mixed solution for eluting and activating prothrombin in the step (2) is 19mL of the sodium chloride-calcium chloride mixed solution added to every 100mL of the supernatant anticoagulated plasma.
5. The method of claim 1, wherein the thrombin is extracted from pig blood by: and (4) removing viruses from the thrombin refined extract in the step (4) by using a filter membrane with the pore diameter of 20 nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147689.0A CN107674868B (en) | 2017-11-17 | 2017-11-17 | Method for extracting thrombin from pig blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711147689.0A CN107674868B (en) | 2017-11-17 | 2017-11-17 | Method for extracting thrombin from pig blood |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107674868A CN107674868A (en) | 2018-02-09 |
| CN107674868B true CN107674868B (en) | 2020-06-05 |
Family
ID=61150077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711147689.0A Active CN107674868B (en) | 2017-11-17 | 2017-11-17 | Method for extracting thrombin from pig blood |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107674868B (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104328101B (en) * | 2014-10-27 | 2017-05-10 | 长春雷允上药业有限公司 | Preparation method of thrombin |
| CN106474461B (en) * | 2015-08-24 | 2019-12-27 | 上海洲跃生物科技有限公司 | Method for preparing freeze-dried human thrombin |
| CN105624134B (en) * | 2016-02-26 | 2019-05-21 | 福建华灿制药有限公司 | The preparation method of restricted fibrin ferment |
| CN107058270A (en) * | 2017-05-08 | 2017-08-18 | 湖南格制药有限公司 | The preparation method and its production system of pig thrombiase |
-
2017
- 2017-11-17 CN CN201711147689.0A patent/CN107674868B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107674868A (en) | 2018-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3094167B2 (en) | Purification method of immune serum globulin | |
| CN104672328B (en) | A kind of production method of Human Antithrombin Ⅲ | |
| CA2024667C (en) | Process for preparing a concentrate of blood coagulation factor viii-von willebrand factor complex from total plasma | |
| RU2015141849A (en) | METHOD FOR CLEANING THE BLOOD COAGING FACTOR VIII | |
| CN104109202B (en) | Method for adsorbing human prothrombin complex from plasma | |
| CN101974070B (en) | Preparation process of human prothrombin compound | |
| CN108048433B (en) | Preparation method of human prothrombin complex | |
| CN109651502B (en) | Method for simultaneously separating and purifying blood coagulation factors IX, X and VII from human plasma | |
| CN205073379U (en) | Plasma exchange adsorbs filters clean system | |
| CN106676089B (en) | Method for preparing human prothrombin complex from blood plasma | |
| JP3471379B2 (en) | Method for preparing human antithrombin-III | |
| CN107674868B (en) | Method for extracting thrombin from pig blood | |
| CN103724428A (en) | Method for improving column efficiency purified ulinastatin through resin regeneration | |
| CN106928344A (en) | Method for being reduced from the solution containing clotting factor and/or remove FXI and FXIa | |
| CN116497009A (en) | Method for extracting defibrase from snake venom powder | |
| CN104513308A (en) | Method of resin regeneration for improving column efficiency and purifying ulinastatin, and drug composition containing ulinastatin | |
| CN107459572B (en) | Method for concentrating hirudin in fermentation liquor | |
| UA44261C2 (en) | METHOD OF OBTAINING VITAMIN K-DEPENDENT PROTEINS USING MEMBRANE CHROMATOGRAPHY METHODS | |
| CN101439047B (en) | Technological process for improving stable FVII yield of human prothrombin complexes | |
| CN105002153B (en) | A kind of preparation method of fibrin ferment | |
| CN106520737A (en) | Method for purifying urokinase through resin regeneration column efficiency improvement | |
| CN103910790B (en) | A kind of method preparing Human plasma protein C concentrate | |
| CN108441490B (en) | Process for preparing human prothrombin complex by flow adsorption method | |
| CN105753974A (en) | Ulinastatin purification method based on affinity chromatography column | |
| FI62103B (en) | REQUIREMENTS FOR THE REQUIREMENT OF HEPARIN MED HOEG SPECIFIC ACTIVITIES |
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 |