CN116478275A - Purification method of rat tail type I collagen - Google Patents
Purification method of rat tail type I collagen Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 40
- 102000012422 Collagen Type I Human genes 0.000 title claims abstract description 25
- 108010022452 Collagen Type I Proteins 0.000 title claims abstract description 25
- 238000000746 purification Methods 0.000 title claims abstract description 15
- 210000002435 tendon Anatomy 0.000 claims abstract description 58
- 239000012535 impurity Substances 0.000 claims abstract description 20
- 230000001954 sterilising effect Effects 0.000 claims abstract description 18
- 238000005185 salting out Methods 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract description 7
- 102000008186 Collagen Human genes 0.000 claims description 79
- 108010035532 Collagen Proteins 0.000 claims description 79
- 229920001436 collagen Polymers 0.000 claims description 79
- 239000000243 solution Substances 0.000 claims description 58
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 54
- 238000000605 extraction Methods 0.000 claims description 43
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 31
- 239000002244 precipitate Substances 0.000 claims description 28
- 239000000523 sample Substances 0.000 claims description 27
- 238000003756 stirring Methods 0.000 claims description 19
- 239000012528 membrane Substances 0.000 claims description 18
- 238000000502 dialysis Methods 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000000108 ultra-filtration Methods 0.000 claims description 16
- 239000007983 Tris buffer Substances 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 14
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 14
- 229940088598 enzyme Drugs 0.000 claims description 12
- 239000012488 sample solution Substances 0.000 claims description 12
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- 238000000703 high-speed centrifugation Methods 0.000 claims description 9
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- 239000005017 polysaccharide Substances 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 102000057297 Pepsin A Human genes 0.000 claims description 6
- 108090000284 Pepsin A Proteins 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 239000012510 hollow fiber Substances 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 229940055729 papain Drugs 0.000 claims description 4
- 239000004627 regenerated cellulose Substances 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
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- 102000004142 Trypsin Human genes 0.000 claims description 3
- 108090000631 Trypsin Proteins 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 3
- 235000019834 papain Nutrition 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 229960001322 trypsin Drugs 0.000 claims description 2
- 230000018044 dehydration Effects 0.000 claims 1
- 238000006297 dehydration reaction Methods 0.000 claims 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
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- OFHCOWSQAMBJIW-AVJTYSNKSA-N alfacalcidol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C OFHCOWSQAMBJIW-AVJTYSNKSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
- 229940096422 collagen type i Drugs 0.000 description 1
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- 238000004090 dissolution Methods 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
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- 230000006340 racemization Effects 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 210000000515 tooth Anatomy 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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- Gastroenterology & Hepatology (AREA)
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Abstract
The invention provides a purification method of rat tail type I collagen, which comprises the steps of sterilizing rat tail, peeling off tendons, mixing and extracting with acid enzyme, salting out and removing impurities, ultrafiltering and changing liquid, concentrating, sterilizing and filtering; the method can obtain the sterile rat tail type I collagen, has high sample purity and good effect of promoting cell adhesion and proliferation, and can be applied to the field of cell culture. The method for purifying the type I collagen provided by the invention has the advantages of simple method, simple and convenient operation and low production cost, and can be used for industrial mass production.
Description
The invention field:
the application belongs to the technical field of proteins, and particularly provides a method for purifying rat tail type I collagen.
The background technology is as follows:
collagen (collagen) is a natural protein which is widely used in animal skin, bones, cartilage, teeth, tendon and ligament and blood vessel, is a structural protein of great importance for connective tissue, plays a role in supporting organs and protecting organisms, and has strong biological activity and biological function. Up to now, 29 types of collagen have been found, and more studies have been made on type i-v collagen, which is a dominant protein of bone, skin, tendon and other fibrous connective tissue, accounting for 90% of the total amount of collagen in organisms. The type I collagen is fiber-forming collagen, can promote adhesion, growth, proliferation and differentiation of cells, and is widely applied to bone tissue repair materials, artificial skin materials, artificial blood vessels, heart valves, cell transplanting devices and the like. The type I collagen consists of two alpha 1 (I) chains and one alpha 2 (I) chain, and sometimes the two alpha chains are overlapped with each other to form a beta chain, or the three alpha chains are overlapped with each other to form a gamma chain, and the three alpha chains have a triple-helix structure.
The animal collagen can be extracted by acid method, alkali method, neutral salt method and hot water method. The acid extraction method has short extraction time, can keep the structural integrity of collagen molecules to the greatest extent, keeps better biological characteristics, and has low production cost. However, the acid extraction method has low yield and low efficiency, and collagen end peptide is reserved, so that the risk of immunogenicity is high. The alkaline method has simple extraction operation and high extraction speed, but the alkaline method has low extraction rate, can cause hydrolysis of peptide bonds, and can damage all amino acids containing hydroxyl and sulfhydryl in collagen molecules to generate racemization and damage the triple helix structure of collagen. Therefore, practical applications are relatively few. The salt method refers to neutral salt solution with a certain concentration such as NaCl, tris (Tris-HCl) and citrate, which can dissolve collagen. NaCl is used in the extraction process to increase the surface charge of collagen, so that acting force between the collagen and water molecules is enhanced, and the dissolution of the collagen is accelerated. However, when the salt concentration is too high, the salting-out reaction may decrease the yield. The hot water extraction method is a method for obtaining collagen by carrying out decalcification pretreatment on raw materials, immersing the raw materials in water with a certain temperature and carrying out a series of purification processes, and the hot water extraction method is simple and convenient. However, the extraction temperature of the hot water method is too high, the activity is lost, the extraction is incomplete when the temperature is too low, and the physiological function of the original collagen is not achieved. The enzymatic extraction is the most commonly used method at present, and has the characteristics of short extraction time, high yield, high purity, stable physicochemical properties, less environmental pollution and the like; meanwhile, the collagen with good biological activity can be obtained due to mild extraction conditions of the enzyme method. However, the maximum problem of the enzymatic extraction is that the hydrolysis is not thorough, the extraction efficiency is low, and the industrial production is not facilitated.
Patent CN113201569a describes a method for extracting and purifying bovine tendon, niu Jijian is digested with enzyme, then each subunit of bovine type I collagen is purified with alcohol concentration of 15% -95%, and finally dialyzed with semipermeable membrane for 3 days, and this method can produce a large amount of alcohol waste liquid, and the semipermeable membrane is long in dialysis time, and protein is easy to denature. Patent CN112760351a describes a method for extracting and purifying collagen from fish skin, degreasing fish skin with NaOH and isopropanol, dialyzing for 5 days with dialysis bag after enzymatic extraction, and also dialyzing for too long, and degreasing with NaOH to generate a large amount of alkali waste liquid. Patent CN109602943a describes a preparation method of bovine tendon collagen, which comprises the steps of removing fat Niu Jijian, swelling with acetic acid, extracting with pepsin, salting out to obtain crude and pure collagen, dialyzing to purify, and changing the liquid to obtain collagen, wherein the same dialysis takes 6 days, the total extraction and purification time is 15 days, the operation is complex, and the production period is long.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for extracting and purifying rat tail collagen in a large scale, which aims to solve the technical problems of more purification process steps, high cost, long time consumption, large amount of organic waste liquid and alkali waste liquid production and the like in the prior art. The method combines acid method and enzyme method extraction and purification, and can obtain high-purity sterile collagen solution for cell culture.
In one aspect, the present application provides a method for purifying rat tail type I collagen, the method comprising: (1) tendon pretreatment: sterilizing the tail, peeling and taking tendons;
(2) Tendon impurity removal: removing soluble polysaccharide, and dehydrating and degreasing;
(3) Collagen extraction: adding acid-proteolytic enzyme extract into tendon, and extracting;
(4) Collagen purification: adding neutral salt to separate out collagen;
(5) Collagen dialysis and concentration.
Further, the step (3) is carried out extraction under low temperature condition, and supernatant is collected after high-speed centrifugation; preferably, the low temperature condition is 2-8 ℃ and the extraction time is 48-72h.
Further, the acid in step (3) may be any one selected from acetic acid, citric acid, hydrochloric acid.
Further, the proteolytic enzyme in the step (3) is preferably any one of pepsin, trypsin, papain.
Further, the concentration of the proteolytic enzyme in step (3) is 0.1% -1.0%.
Further, the step (4) is to slowly add neutral salt into the collagen extraction supernatant, stir while adding to promote collagen precipitation, add alkaline solution to adjust the pH of the sample solution to 7.2-7.4, then place in a low temperature environment, and wait for precipitation of white flocculent precipitate. The white precipitate is collected by high-speed centrifugation, acetic acid solution is added into the precipitate, the white precipitate is redissolved by shaking, a sample is filtered by a depth filter, and insoluble substances in the solution are removed.
Further, the neutral salt in the step (4) is selected from any one of ammonium sulfate and sodium chloride; the alkaline solution is selected from 1M Tris pH8.5 solution; the low temperature condition is 2-8 ℃ and the salting-out time is 1-16h.
Further, the step (5) is to dialyze the collagen solution prepared in the step (4) with an ultrafiltration membrane under a certain pressure, replace the collagen solution with 6mM acetic acid with 5 times of the sample volume for 3-9 hours until the pH of the purified collagen solution is 3-7, and then filter the purified collagen solution with a 0.45+0.22um sterilization filter to obtain a sterile rat tail type I collagen solution; optionally, wherein the pressure is 0.05MPa to 0.3MPa; the aperture of the ultrafiltration membrane is 30kDa; the ultrafiltration membrane is made of PES and regenerated cellulose; the ultrafiltration membrane is hollow fiber or ultrafiltration membrane bag.
Further, the step (2) is to put the peeled tendon into an impurity removing solution, stir overnight to remove soluble polysaccharide and other impurities, finally submerge the tendon in absolute ethyl alcohol for 20-40min, dehydrate and degrease, and weigh the tendon; optionally, wherein the impurity removal solution is 50mM Tris+1M NaCl pH7.4.
Further, the step (1) is to immerse the rat tail in 75% alcohol for sterilization for 30-60min, take out the rat tail for peeling, and strip tendons for temporary storage in precooled PBS.
The rat tail described herein may be the tail of a variety of murine animals, including but not limited to the rat tail, the mouse tail of the kind commonly found in production and experimentation; the rat tail can be either fresh rat tail or cryopreserved rat tail.
Drawings
FIG. 1 is an SDS-PAEG gel electrophoresis of collagen type I according to the present invention. Wherein 1 is sigma type I collagen, and 2 is type I collagen.
FIG. 2 shows the results of detection of the absorption peaks of type I collagen according to the present invention.
FIG. 3 shows the results of the detection of Mycoplasma collagen I according to the present invention.
FIG. 4 shows the results of the detection of the cell activity of type I collagen according to the present invention.
Detailed Description
The present invention is described in detail below with reference to specific examples. The following examples are given for illustration only, and the scope of the invention is defined by the claims and is not limited to the following examples.
Example 1
(1) Tendon pretreatment: immersing the frozen and preserved mouse tail in 75% alcohol for sterilizing for 30min, taking out the mouse tail, peeling, and temporarily storing the peeled tendon in precooled PBS.
(2) Tendon impurity removal: putting the peeled tendon into 50mM Tris+1M NaCl pH7.4 solution, stirring overnight to remove soluble polysaccharide and other impurities, immersing the tendon in absolute ethanol for 30min, dehydrating and degreasing, and weighing tendon mass.
(3) Collagen extraction: tendon-pressing: acetic acid-enzyme extract=1:150, 0.05mol/L acetic acid-pepsin solution was added to the tendon, pepsin concentration was 0.1%, and extraction was performed at 4 ℃ with stirring for 72h. The extract was centrifuged at high speed under conditions of 11000 Xg for 20min, and the supernatant was collected after centrifugation.
(4) Collagen purification: slowly adding ammonium sulfate solid into the collagen extraction supernatant, stirring while adding, and finally keeping the concentration of ammonium sulfate at 30%. Adding 1mol/L Tris solution to regulate the pH value of the sample solution to 7.2-7.4, and then placing the sample solution in a refrigerator at the temperature of 4 ℃ for standing for 1h, and waiting for precipitation of white flocculent precipitate. The white precipitate was collected by high-speed centrifugation, 50mM acetic acid was added to the precipitate, the white precipitate was sufficiently dissolved by shaking, and the sample was filtered with a depth filter to remove insoluble matters in the solution.
(5) Collagen dialysis and concentration: the solution is dialyzed and desalted by a 30KD ultrafiltration membrane bag, the ultrafiltration membrane bag is made of PES, the pressure at the reflux end is controlled to be 0.2MPa, the solution is replaced by 6mM acetic acid with the volume of 5 times of the sample, the dialysis time is about 4 hours, and then the solution is filtered by a 0.45+0.22um sterilization filter, so that a sterile rat tail I type collagen sample is obtained.
Example 2
(1) Tendon pretreatment: immersing fresh mouse tail in 75% alcohol for sterilizing for 30min, taking out the mouse tail, peeling, and temporarily storing tendon in precooled PBS.
(2) Tendon impurity removal: putting the peeled tendon into 50mM Tris+1M NaCl pH7.4 solution, stirring overnight to remove soluble polysaccharide and other impurities, immersing the tendon in absolute ethanol for 30min, dehydrating and degreasing, and weighing tendon mass.
(3) Collagen extraction: tendon-pressing: hydrochloric acid-enzyme extract=1:100 ratio, 0.1mol/L hydrochloric acid-trypsin solution was added to the tendon, trypsin concentration was 0.5%, and extraction was performed at 2 ℃ with stirring for 48h. The extract was centrifuged at high speed under conditions of 11000 Xg for 20min, and the supernatant was collected after centrifugation.
(4) Collagen purification: slowly adding sodium chloride solid into the collagen extraction supernatant, and stirring while adding, wherein the final concentration of sodium chloride is 30%. And adding 1mol/L Tris solution to adjust the pH of the sample solution to 7.2-7.4, and then placing the sample solution in a refrigerator at the temperature of 2 ℃ for standing for 4 hours, and waiting for precipitation of white flocculent precipitate. The white precipitate was collected by high-speed centrifugation, 50mM acetic acid was added to the precipitate, the white precipitate was sufficiently dissolved by shaking, and the sample was filtered with a depth filter to remove insoluble matters in the solution.
(5) Collagen dialysis and concentration: and (3) dialyzing and desalting the solution by using 30KD hollow fiber, wherein the hollow fiber is made of regenerated cellulose, the pressure at the reflux end is controlled to be 0.1MPa, the solution is replaced by 6mM acetic acid with 5 times of the sample volume, the dialysis time is about 6 hours, and then filtering is carried out by using a 0.45+0.22um sterilizing filter, so that a sterile rat tail type I collagen sample is obtained.
Example 3
(1) Tendon pretreatment: immersing fresh rat tail in 75% alcohol for sterilizing for 30min, taking out the rat tail, peeling, and temporarily storing tendon in precooled PBS.
(2) Tendon impurity removal: putting the peeled tendon into 50mM Tris+1M NaCl pH7.4 solution, stirring overnight to remove soluble polysaccharide and other impurities, immersing the tendon in absolute ethanol for 30min, dehydrating and degreasing, and weighing tendon mass.
(3) Collagen extraction: tendon-pressing: citrate-enzyme extract = 1:150 ratio, 0.2mol/L citrate-papain solution was added to the tendon, papain concentration was 1.0%, and extraction was performed at 8 ℃ with stirring for 72h. The extract was centrifuged at high speed under conditions of 11000 Xg for 20min, and the supernatant was collected after centrifugation.
(4) Collagen purification: slowly adding sodium chloride solid into the collagen extraction supernatant, and stirring while adding, wherein the final concentration of sodium chloride is 40%. And adding 1mol/L Tris solution to adjust the pH of the sample solution to 7.2-7.4, and then placing the sample solution in an 8 ℃ refrigerator for standing for 8 hours to wait for precipitation of white flocculent precipitate. The white precipitate was collected by high-speed centrifugation, 50mM acetic acid was added to the precipitate, the white precipitate was sufficiently dissolved by shaking, and the sample was filtered with a depth filter to remove insoluble matters in the solution.
(5) Collagen dialysis and concentration: the solution was desalted by dialysis with 30KD hollow fiber, PES as the material of the hollow fiber, the reflux pressure was controlled to 0.3MPa, the solution was replaced with 5 times the sample volume of 6mM acetic acid for about 3 hours, and then the solution was filtered with 0.45+0.22um sterilizing filter to obtain a sterile rat tail type I collagen sample.
Example 4
(1) Tendon pretreatment: immersing the frozen rat tail in 75% alcohol for sterilizing for 30min, taking out the rat tail, peeling, and temporarily storing the peeled tendon in precooled PBS.
(2) Tendon impurity removal: putting the peeled tendon into 50mM Tris+1M NaCl pH7.4 solution, stirring overnight to remove soluble polysaccharide and other impurities, immersing the tendon in absolute ethanol for 30min, dehydrating and degreasing, and weighing tendon mass.
(3) Collagen extraction: tendon-pressing: acetic acid-enzyme extract=1:100 ratio, 0.5mol/L acetic acid-pepsin solution was added to the tendon, pepsin concentration was 0.5%, and extraction was performed at 4 ℃ with stirring for 48h. The extract was centrifuged at high speed under conditions of 11000 Xg for 20min, and the supernatant was collected after centrifugation.
(4) Collagen purification: slowly adding ammonium sulfate solid into the collagen extraction supernatant, stirring while adding, and finally obtaining ammonium sulfate with a concentration of 40%. Adding 1mol/L Tris solution to regulate the pH value of the sample solution to 7.2-7.4, and then placing the sample solution in a refrigerator at the temperature of 4 ℃ for standing for 16 hours to wait for precipitation of white flocculent precipitate. The white precipitate was collected by high-speed centrifugation, 50mM acetic acid was added to the precipitate, the white precipitate was sufficiently dissolved by shaking, and the sample was filtered with a depth filter to remove insoluble matters in the solution.
(5) Collagen dialysis and concentration: and (3) carrying out dialysis and desalination on the solution by using a 30KD ultrafiltration membrane bag, wherein the ultrafiltration membrane bag is made of regenerated cellulose, the pressure of a reflux end is controlled to be 0.2MPa, the solution is replaced by 6mM acetic acid with the volume of 5 times of the sample, the dialysis time is about 4 hours, and then a 0.45+0.22um sterilization filter is used for filtering, so that a sterile rat tail I collagen sample is obtained.
Example 5
(1) Tendon pretreatment: immersing the frozen and preserved mouse tail in 75% alcohol for sterilizing for 30min, taking out the mouse tail, peeling, and temporarily storing the peeled tendon in precooled PBS.
(2) Tendon impurity removal: putting the peeled tendon into 50mM Tris+1M NaCl pH7.4 solution, stirring overnight to remove soluble polysaccharide and other impurities, immersing the tendon in absolute ethanol for 30min, dehydrating and degreasing, and weighing tendon mass.
(3) Collagen extraction: tendon-pressing: acetic acid-enzyme extract=1:50 ratio, 0.2mol/L acetic acid-pepsin solution was added to the tendon, pepsin concentration was 1.0%, and extraction was performed at 8 ℃ with stirring for 48h. The extract was centrifuged at high speed under conditions of 11000 Xg for 20min, and the supernatant was collected after centrifugation.
(4) Collagen purification: slowly adding ammonium sulfate solid into the collagen extraction supernatant, stirring while adding, and finally obtaining ammonium sulfate with concentration of 20%. Adding 1mol/L Tris solution to adjust the pH value of the sample solution to 7.2-7.4, and then placing the sample solution in a refrigerator at 8 ℃ for standing for 4 hours to wait for precipitation of white flocculent precipitate. The white precipitate was collected by high-speed centrifugation, 50mM acetic acid was added to the precipitate, the white precipitate was sufficiently dissolved by shaking, and the sample was filtered with a depth filter to remove insoluble matters in the solution.
(5) Collagen dialysis and concentration: the solution is dialyzed and desalted by a 30KD ultrafiltration membrane bag, the ultrafiltration membrane bag is made of PES, the pressure at the reflux end is controlled to be 0.05MPa, the solution is replaced by 6mM acetic acid with the volume of 5 times of the sample, the dialysis time is about 9 hours, and then the solution is filtered by a 0.45+0.22um sterilization filter, so that a sterile rat tail type I collagen sample is obtained.
Example 6
Collagen promoting 3T3 cell proliferation in vitro
(1) Sigma rat tail type I collagen (acetic acid extraction, naCl precipitation) and example 1 type I collagen were diluted to appropriate concentrations with cell culture medium, diluted collagen solution was added to cell culture plates, and the plates were placed in a 37℃incubator for 1-3h incubation.
(2) The plates were washed 2-3 times with PBS to wash away unbound collagen.
(3) NIH-3T3 cell suspension was added to the cell culture plate and transferred to a 5% CO2 incubator at 37℃for 40-48h.
(4) The cell growth status of the blank and sigma, type I collagen group of the invention was observed under a microscope.
Collagen sample detection:
sample recovery rates are compared, sigma standard products with different concentrations are prepared, absorbance values at 205nm are measured, and a standard curve is drawn. The sample concentrations at the different extraction and purification stages were determined and the recovery of the samples was calculated. As a result, it was found that the extraction ratio of tendon and enzyme solution and the salting-out concentration had the greatest influence on the recovery rate of the sample. In example 5, tendon: the ratio of acetate-enzyme extract was 1:50, and the mass of the extracted sample was only 35% of that of the other examples, and the salting-out recovery rate was 70% of that of the other examples when the salting-out neutral salt concentration was 20%. The purity of the samples obtained for all examples remained consistent, being higher than 95%.
SDS-PAGE detection is carried out on purified rat tail collagen, and the result is shown in figure 1, the extracted collagen electrophoresis band is the same as the sigma main band, and mainly consists of alpha 1 and alpha 2 bands, a dimer beta chain formed by 2 alpha chains and a trimer gamma chain formed by alpha chains, and the other bands are few, so that the extracted collagen has high purity, contains less impurities and has the sample purity higher than 95 percent.
The purified rat tail collagen solution is subjected to full-wave scanning by an ultraviolet spectrophotometer, the wavelength range is 190nm-900nm, the wavelength interval is 1nm, the result is shown in figure 2, the peak types of the extracted collagen and the sigma sample are consistent, and the maximum absorbance value is provided at the position of 205nm-215nm, so that the collagen meets the common ultraviolet absorption characteristic of the collagen.
The purified rat tail collagen solution was tested for mycoplasma residue by PCR, and the results are shown in fig. 3, and the test result was negative, indicating that the sample did not contain mycoplasma.
The activity of purified rat tail collagen rat tail cells is detected, and the detection result is shown in figure 4, compared with a blank control, sigma rat tail collagen and the rat tail collagen group provided by the invention, the cell number is obviously increased, and the effect of obviously promoting cell proliferation is achieved.
Claims (10)
1. A method for purifying rat tail type I collagen, the method comprising:
(1) Tendon pretreatment: sterilizing the tail, peeling and taking tendons;
(2) Tendon impurity removal: removing soluble polysaccharide, and dehydrating and degreasing;
(3) Collagen extraction: adding acid-proteolytic enzyme extract into tendon, and extracting;
(4) Collagen purification: adding neutral salt to separate out collagen;
(5) Collagen dialysis and concentration.
2. The method according to claim 1, wherein the extraction is performed under low temperature conditions in the step (3), and the supernatant is collected after high-speed centrifugation; preferably, the low temperature condition is 2-8 ℃ and the extraction time is 48-72h.
3. The method of claim 1, wherein the acid in step (3) is selected from any one of acetic acid, citric acid, hydrochloric acid.
4. The method according to claim 1, wherein the proteolytic enzyme in step (3) is preferably any one of pepsin, trypsin, papain.
5. The method of claim 4, wherein the concentration of proteolytic enzyme in step (3) is 0.1% -1.0%.
6. The method according to claim 1, wherein the step (4) is to slowly add neutral salt to the collagen extraction supernatant while stirring to precipitate collagen, and to adjust the pH of the sample solution to 7.2-7.4 by adding alkali solution, and then to stand in a low temperature environment to wait for precipitation of white flocculent precipitate. The white precipitate is collected by high-speed centrifugation, acetic acid solution is added into the precipitate, the white precipitate is redissolved by shaking, a sample is filtered by a depth filter, and insoluble substances in the solution are removed.
7. The method of claim 6, wherein the neutral salt in step (4) is selected from any one of ammonium sulfate, sodium chloride; the alkaline solution is 1M Tris pH8.5 solution; the low temperature condition is 2-8 ℃ and the salting-out time is 1-16h.
8. The method according to claim 1, wherein the step (5) is to subject the collagen solution prepared in (4) to dialysis with an ultrafiltration membrane under a certain pressure, to replace the solution with 5 times the sample volume of 6mM acetic acid for 3-9 hours until the pH of the purified collagen solution is 3-7, and then to filter with a 0.45+0.22um sterilization filter, to obtain a sterile rat tail type I collagen solution; optionally, wherein the pressure is 0.05MPa to 0.3MPa; the aperture of the ultrafiltration membrane is 30kDa; the ultrafiltration membrane is made of PES and regenerated cellulose; the ultrafiltration membrane is hollow fiber or ultrafiltration membrane bag.
9. The method according to claim 1, wherein the step (2) is to put the peeled tendon into an impurity removing solution, stir overnight to remove soluble polysaccharide and other impurities, and finally submerge the tendon in absolute ethanol for 20-40min for dehydration and degreasing, and weigh the tendon mass; optionally, wherein the impurity removal solution is 50mM Tris+1M NaCl pH7.4.
10. The method of claim 1, wherein step (1) is to immerse the rat tail in 75% alcohol for sterilization for 30-60min, remove the rat tail and peel off, and strip the tendon for temporary storage in pre-chilled PBS.
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| CN119306820B (en) * | 2024-12-18 | 2025-06-27 | 天津松果生物医疗科技有限公司 | Natural active collagen extraction and purification method |
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