CN103468771B - Method for extracting collagens from bovine achilles tendon - Google Patents
Method for extracting collagens from bovine achilles tendon Download PDFInfo
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- CN103468771B CN103468771B CN201310200479.9A CN201310200479A CN103468771B CN 103468771 B CN103468771 B CN 103468771B CN 201310200479 A CN201310200479 A CN 201310200479A CN 103468771 B CN103468771 B CN 103468771B
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 241000283690 Bos taurus Species 0.000 title abstract description 5
- 210000001361 achilles tendon Anatomy 0.000 title abstract 4
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 25
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 15
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 11
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for extracting collagens from bovine achilles tendon. The method comprises the following steps: refrigerating the bovine achilles tendon, cutting into blocks, refrigerating, crushing, filling crushed bovine achilles tendon into a temperature-controllable chromatography column, continuously injecting an extract liquid into the temperature-controllable chromatography column, controlling the temperature of circulating water in a jacket of the chromatography column consistent to the temperature of the extract liquid, and collecting the extract liquid from an outlet of the chromatography column; carrying out enzymatic hydrolysis, carrying out chromatographic separation in an affinity chromatography mode, wherein a ligand of an affinity chromatography media is a polypeptide; and leaching out uncombined proteins, and eluting to obtain proteins which can be combined with the chromatography media, that is, highly pure collagens. The method is simple to operate, and the prepared collagens have the advantages of high purity, low random degradation and the like.
Description
Technical field
The invention belongs to technical field of biological material, particularly, the present invention relates to a kind of method extracting collagen from ox heel string.
Technical background
Collagen accounts for 33% of total protein in mammalian body as the main component of extracellular matrix, be almost present in a organized way and to the form of biological cells and tissues, structure and function, there is material impact.Tropocollagen molecule is slender rod shaped, is about 300nm, and diameter is about 1.5nm, and molecular weight is about 300kDa.Tropocollagen molecule is made up of 3 polypeptide chains, every bar peptide chain about 1000 amino-acid residues, and tropocollagen molecule has triple-helix structure, and the form that every bar polypeptide chain of composition collagen rotates with left hand exists, and three polypeptide chains are wound collagen with the form of right-handed helix.According to the difference forming collagen polypeptide chain amino acid sequence, collagen can be divided into broad variety, and in mammalian body, found 27 kinds of dissimilar collagens at present, wherein the content of type i collagen is the highest.There is some difference for the performances such as the physical strength of different collagen and thermostability, and also there is some difference for the interaction between other biomacromolecule, cell or material interface, prepare different biomaterial and certain requirement is existed to collagen-type and ratio etc.
Collagen has good biological characteristics, comprise biocompatibility, poor antigen, biodegradable, Bioabsorbable and good water-absorbent etc., collagen is very extensive in field application such as organizational project, regenerative medicine, medicine equipment and medicine controlled releasings as a kind of desirable biomaterial, if collagen is the critical material preparing the artificial organ such as styptic sponge, microbial film, operating sutures, cell culturing bracket, tissue bulking material and artificial skin, artificial cornea, artificial liver.Collagen has good adhesiving effect to cell, therefore in organizational project and regenerative medicine field, the critical material of collagen or a kind of desirable improvement material surface performance and raising Biocompatibility, as in tissue engineering material, the three-dimensional rack prepared of collagen is used to can be used for inoculation chondrocyte and for cartilaginous tissue reparation and regeneration, as in tissue regeneration material, collagen for the bladder wall acellular matrix transplant and regeneration effect good.In recent years, the medicine equipment printed based on 3D and the development of organizational project technology of preparing rapidly, cause growing with each passing day to the demand of collagen.
Much collagen is insoluble in water under native state, and direct preparation of high-purity collagen exists certain difficulty, is the focus of collagen research in technical field of biological material in recent years.Adopt engineered method can prepare recombinant collagen (recombined collagen, number of patent application CN102351954.A; A kind of recombination human source collagen protein and preparation method thereof, application number 102443057.A), but this technology can only to prepare the molecular weight of collagen low and be difficult to prepare the collagen with triple-helix structure.Duan Rui etc. adopt (a kind of process for refining of macromolecular collagen protein, publication number 200910184481.5) adopt acidic solution facture to collect collagen suspended layer after carry out collagen and refine, the method operation steps is simple, but what collagen preparation process adopted is precipitation process, the co-precipitation that non-specific adsorption causes often causes in the collagen prepared containing a certain amount of foreign protein.Preparation patent (the application number: 200980101280.2) of high-purity collagen protein of the application such as Huang Linghui, grind after raw materials pretreatment, obtain collagen crude product, and adopt further the method for Acid precipitation to carry out collagen to refine, the method removing foreign protein still belongs to the precipitator method, and the collagen treating process based on the precipitator method is difficult to prepare high-purity collagen.Li Jie etc. take animal cartilage as raw material, after pulverizing, adopt stomach en-to degrade, adopt and saltout and centrifugal method acquisition non denatured collagen (non denatured II collagen type production method, application number CN102154425.A), this process need saltout process and centrifugal process repeatedly, process is comparatively loaded down with trivial details.
Summary of the invention
The problems such as the collagen purity existed for traditional collagen preparation process is low, sepn process is loaded down with trivial details, the object of the invention is to provide a kind of method extracting collagen from ox heel string, and the bovine collagen purity of preparation is high and keep the natural structure of triple helical.A kind of method extracting collagen from ox heel string of the present invention, the method comprises the following steps:
(1) weigh: take fresh ox heel string, WATER-WASHING METHOD removes appurtenant.
(2) freezing: freezen protective 12 ~ 24 hours under-40 DEG C of conditions.
(3) stripping and slicing: freezing ox heel string is cut into bulk (0.5cm × 0.5cm).
(4) pulverize: within freezing 6 ~ 12 hours under-40 DEG C of conditions, be placed on crusher for crushing by being cut into block ox heel string.
(5) extract: the ox heel string after pulverizing is placed in the empty chromatography column of temperature controllable, the aperture of its bottom screen is 100 ~ 300 orders, be continuously that the deionized water of 38-45 DEG C is injected into chromatography column from chromatography column top by temperature, the circulating water temperature controlling chromatography column chuck is consistent with the temperature injecting water in chromatography column; Collecting the solution flowed out bottom chromatography column, is collagen crude extract.
(6) enzymolysis: add proteolytic enzyme in collagen crude extract, the ratio control of albumen and enzyme, at 50:1 to 200:1, stirs 24 ~ 36 hours at a certain temperature.
(7) refining: the solution after enzymolysis is crossed loading to affinity column, the aglucon of chromatography media is a peptide species, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTCVAYSELRD, and applied sample amount is 5% ~ 16% of chromatography media volume.Use the aqueous solution albumen do not adsorbed or polypeptide drip washing to be gone out, adopt the acetonitrile-aqueous solution of 10% ~ 30% to be eluted by the albumen be adsorbed on chromatography media, collect elutriant.
(8) freeze-drying: the collagen lyophilize refining by affinity chromatography, obtains high-purity collagen.
Preferably, in described step (4) leaching process, be temperature controllable chromatography column for extracting the device of collagen.
Preferably, in described step (4) leaching process, the circulating water temperature of chromatography column chuck is identical with the temperature injecting water in chromatography column.
Preferably, in described step (7) treating process, the chromatography scheme that collagen treating process adopts is affinity chromatography.
Preferably, in described step (7) treating process, the aglucon of affinity chromatography medium is a peptide species, and the aminoacid sequence of this polypeptide can be ICTTNEGVMYRIGDEWDKEHDMGH MMRCTCVGNGRGEWTCVAYSELRD, may also be the polypeptide containing this aminoacid sequence.
Compared with prior art, its advantage comprises in the present invention:
(1) be mechanical valve by the preprocessing process of ox heel string, not with an organic solvent.
(2) adopt the chromatography column of temperature controllable to carry out collagen extraction, the collagen extracted shifts out in time, avoids thermal destruction, is conducive to the triple-helix structure keeping collagen.
(3) adopt the method for affinity chromatography to carry out the refining of collagen, purification efficiency is high.
(4) collagen adopting the present invention to prepare has the advantages such as purity is high, random degenerate rate is low.
Specific embodiment
Below by specific embodiment, the present invention is specifically described; what be worth proposition is; the present embodiment is just for a kind of implementation method of the present invention; the whole of patent of the present invention can not be represented; more limiting the scope of the invention can not be interpreted as; under the precondition of design not departing from essence of the present invention, can also make some adjustment or improvement, these all belong to protection scope of the present invention.
Embodiment 1:
(1) weigh: get fresh ox heel string 50g, residue is removed in washing.
(2) freezing: freezen protective 12 hours under-40 DEG C of conditions.
(3) stripping and slicing: the bulk ox heel string after freezing being cut into 0.5cm × 0.5cm.
(4) pulverize: within freezing 8 hours under-40 DEG C of conditions, be placed on crusher for crushing pulp by being cut into block ox heel string.
(5) extract: the ox heel string after pulverizing is placed in (diameter 5cm, height 20cm) in temperature controllable chromatography column, bottom extraction vessel, establishes 300 object nylon wires.Be continuously that the deionized water of 42 DEG C is injected in warm chromatography by temperature, the circulating water temperature controlling chromatography column chuck is 42 DEG C; Collect the solution flowed out from chromatography column outlet.
(6) enzymolysis: add stomach en-in the collagen crude extract that step (5) obtains, the ratio control of albumen and enzyme, at 100:1, stirs 24 hours at 37 DEG C.
(7) refining: the enzymolysis solution pH that step (6) obtains is adjusted to 7.0, loading is to affinity column (diameter 5cm, height 60cm), the matrix of chromatography media is agarose, aglucon is polypeptide, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTCVAYSELRD, and applied sample amount is 60ml.Use deionized water the albumen do not adsorbed or polypeptide drip washing to be gone out, adopt the 30% acetonitrile solution wash-out of 80ml, collect elutriant.
(8) freeze-drying: the elutriant that step (7) obtains is carried out lyophilize, obtains high-purity collagen.
Sequence table
Kao Lisen bio tech ltd, <110> Hebei
<120> mono-kind extracts the method for collagen from ox heel string
<160> 1
<210> 1
<211> 48
<212> PRT
<213> artificial sequence
<400> 1
Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile Gly Asp Glu
1 5 10 15
Trp Asp Lys Glu His Asp Met Gly His Met Met Arg Cys Thr Cys
20 25 30
Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Val Ala Tyr Ser Glu
35 40 45
Leu Arg Asp
Claims (1)
1. from ox heel string, extract a method for collagen, it is characterized in that extracting method comprises the following steps:
(1) weigh: take fresh ox heel string, WATER-WASHING METHOD removes appurtenant;
(2) freezing: freezen protective 12 ~ 24 hours under-40 DEG C of conditions;
(3) stripping and slicing: freezing ox heel string is cut into bulk;
(4) pulverize: be placed on crusher for crushing by being cut into block ox heel string freezing 6-12 hour under-40 DEG C of conditions;
(5) extract: the ox heel string after pulverizing is placed in the empty chromatography column of temperature controllable, the aperture of its bottom screen is 100 ~ 300 orders, be continuously that the deionized water of 38-45 DEG C is injected into chromatography column from chromatography column top by temperature, the circulating water temperature controlling chromatography column chuck is consistent with the temperature injecting water in chromatography column; Collecting the solution flowed out bottom chromatography column, is collagen crude extract;
(6) enzymolysis: add proteolytic enzyme in collagen crude extract, the ratio control of albumen and enzyme, at 50:1 to 200:1, stirs 24 ~ 36 hours;
(7) refining: by the solution loading after enzymolysis to affinity column, the aglucon of chromatography media is a peptide species, its aminoacid sequence is ICTTNEGVMYRIGDEWDKEHDMGHMMRCTCVGNGRGEWTCVAYSELRD, and applied sample amount is 5% ~ 16% of chromatography media volume; Use water the albumen do not adsorbed or polypeptide drip washing to be gone out, adopt the acetonitrile-aqueous solution of 10% ~ 30% to be eluted by the albumen be adsorbed on chromatography media, collect elutriant;
(8) freeze-drying: the collagen lyophilize refining by affinity chromatography, obtains high-purity collagen.
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| CN201310200479.9A CN103468771B (en) | 2013-05-27 | 2013-05-27 | Method for extracting collagens from bovine achilles tendon |
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| CN201310200479.9A CN103468771B (en) | 2013-05-27 | 2013-05-27 | Method for extracting collagens from bovine achilles tendon |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105601731B (en) * | 2014-09-03 | 2018-07-13 | 浙江崇山生物制品有限公司 | Purify the method for ox heel string collagen and its preparation of sponge |
| CN106148466A (en) * | 2016-08-30 | 2016-11-23 | 夏佳文 | A kind of preparation method of yak collagen protein |
| CN107913785A (en) * | 2016-10-11 | 2018-04-17 | 浙江崇山生物制品有限公司 | A kind of efficiently animal tendon breaking method |
| CN106589113A (en) * | 2016-11-17 | 2017-04-26 | 北京华信佳音医疗科技发展有限责任公司 | Method for extracting collagen from bovine achilles tendons |
| CN107227330B (en) * | 2017-07-10 | 2020-12-08 | 山东省千佛山医院 | A kind of extraction method of bovine Achilles tendon type I collagen |
| CN110467668A (en) * | 2019-09-10 | 2019-11-19 | 河北考力森生物科技有限公司 | A kind of extracting method of type III collagen |
| CN111363772A (en) * | 2020-04-08 | 2020-07-03 | 平凉市华科生物技术有限公司 | Method for preparing collagen peptide by hydrolyzing bovine bone and collagen peptide thereof |
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| CN101215733A (en) * | 2008-01-14 | 2008-07-09 | 四川大学 | Collagen-based PEG Composite Fiber and Its Spinning Process |
| CN101363040A (en) * | 2008-09-19 | 2009-02-11 | 无锡贝迪生物工程有限公司 | Method for preparing collagen protein |
| CN101747427A (en) * | 2010-03-04 | 2010-06-23 | 威海市宇王集团有限公司 | Method for separating functional polypeptide from fish collagen |
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2013
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|---|---|---|---|---|
| CN101215733A (en) * | 2008-01-14 | 2008-07-09 | 四川大学 | Collagen-based PEG Composite Fiber and Its Spinning Process |
| CN101363040A (en) * | 2008-09-19 | 2009-02-11 | 无锡贝迪生物工程有限公司 | Method for preparing collagen protein |
| CN101747427A (en) * | 2010-03-04 | 2010-06-23 | 威海市宇王集团有限公司 | Method for separating functional polypeptide from fish collagen |
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| Title |
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| 牛跟腱制造海绵状胶原蛋白纤维的技术研究;王秋卓;《中国医药生物技术》;20110831;第6卷(第4期);第1.2.1.1、1.2.1.2节 * |
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