CN101585874B - Method for separating and purifying sea-mussel mucin by using salting out and dialyzing - Google Patents
Method for separating and purifying sea-mussel mucin by using salting out and dialyzing Download PDFInfo
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- CN101585874B CN101585874B CN2009100875676A CN200910087567A CN101585874B CN 101585874 B CN101585874 B CN 101585874B CN 2009100875676 A CN2009100875676 A CN 2009100875676A CN 200910087567 A CN200910087567 A CN 200910087567A CN 101585874 B CN101585874 B CN 101585874B
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 238000005185 salting out Methods 0.000 title abstract 4
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims abstract description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 15
- 241000237536 Mytilus edulis Species 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 235000020638 mussel Nutrition 0.000 claims description 9
- 238000004043 dyeing Methods 0.000 claims description 8
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 8
- 238000010186 staining Methods 0.000 claims description 8
- 150000003536 tetrazoles Chemical class 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 5
- 241001062009 Indigofera Species 0.000 claims description 4
- 235000000177 Indigofera tinctoria Nutrition 0.000 claims description 4
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 229940097275 indigo Drugs 0.000 claims description 4
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 4
- 239000002253 acid Substances 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- MUUHXGOJWVMBDY-UHFFFAOYSA-L tetrazolium blue Chemical compound [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC=CC=2)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 MUUHXGOJWVMBDY-UHFFFAOYSA-L 0.000 abstract 1
- WFAJBYGUONQMCI-UHFFFAOYSA-N acetic acid urea Chemical compound NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.NC(=O)N.C(C)(=O)O WFAJBYGUONQMCI-UHFFFAOYSA-N 0.000 description 6
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 6
- 238000000605 extraction Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000001426 native polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 208000002177 Cataract Diseases 0.000 description 2
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 206010025482 malaise Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000004375 Angiodysplasia Diseases 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000237524 Mytilus Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 230000035587 bioadhesion Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003653 coastal water Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000004410 intraocular pressure Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108010004563 mussel adhesive protein Proteins 0.000 description 1
- 239000003988 mussel adhesive protein Substances 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
Abstract
The invention relates to a method for separating and purifying the sea-mussel mucin by using salting out and dialyzing, the salting out and the dialyzing utilizes the proteinaceous physicochemical character to purify by a non- chromatograph method, and solves the problem of high apparatus and material cost in the chromatograph method for purifying the sea-mussel mucin, a quick, low-cost and high-concentration method for separating and purifying the sea-mussel mucin is provided. The strong acid is used for extracting, the salting out and the dialyzing are used for purifying, the sea-mussel mucin is determined by adopting the acetic acid-carbamide- polyacrylamide gel electrophoresis via the blue tetrazolium specificity colour development, and the purity is determined by the inversed phase chromatography quantitatively.
Description
Technical field
The present invention relates to a kind of separation and purification method of protein, relate to a kind of use method with the dialysis method separating and purifying sea-mussel mucin of saltouing specifically.
Background technology
(Mussel adhesive protein MAP), also is Mytilus edulis byssus albumen (Mytilus edulisfoot protein to sea-mussel mucin; Mefp) from the seashells Mytilus edulis, Mytilus edulis, it has the ability of the tolerance wave effect in the coastal waters; It generates and stores a kind of albumin glue in special body of gland; Be discharged into through byssus on the solid surface of one type on rock, form water resisting combination, thus oneself is fixing.Show that in research on glass albumin glue has formed spot, extend from spot and open that very strong tensile strength (10 is arranged
6-10
7Newtonmeter
-2), and material has comprised sea-mussel mucin MAP in the spot.
Sea-mussel mucin contains L-3,4-dopa (L-DOPA), and it is formed by the effect of tyrosine oxidase to tyrosine residues.The L-DOPA residue is owing to oxidizing reaction is cross-linked with each other, and crosslinking reaction has caused strong and persistent splicing in mussel and the special surface fiber, and the mechanism of its bioadhesion is very interesting.Sea-mussel mucin adheres to does not have toxicity and immunogenicity to the people, and binding ability and life-span do not receive the influence of water, arouse attention as medical operation especially ophthalmologic operation glue.
Every year is carried out 1,100 ten thousand routine cataract operations in the whole world, and the cataractous sickness rate of China is very high, more than 50 years old among the crowd cataract incidence more than 29.64%, 70 years old crowd's sickness rate up to 60%.Along with the aggravation of social senilization, this numeral also can rise.At present the doctor can close with mode with two kinds and finish ophthalmologic operation: let wound heal voluntarily or use the nylon line suture otch, every kind of method of closing all has shortcoming: self-healing has to be infected and the danger of intraocular liquid leakage; Stitching has the danger of infection inflammation and angiodysplasia.And the sea-mussel mucin sea-mussel mucin can be crosslinked under lower concentration, and form the low viscosity liquid may be injected into irregularly shaped position, this solution solidifiable and be filled into designated space, but in several minutes closure of incisions, be less than the stitching required time.Sea-mussel mucin has also formed not destructible strip of paper used for sealing, under greater than the pressure of 12 times of normal people's intraocular pressures, intraocular liquid is revealed.Sea-mussel mucin has and the proximate specific refractory power of cornea, can disturbance not arrive retina.
In addition, the biological degradation character of sea-mussel mucin makes it very friendly to environment, also can be used for other field; Like the general bonding and sealing agent of biochemical bonding reagent, medical tissue, medical callus matrix, the bonding and sealing agent of dentist; Medical equipment top coat reagent, underwater operation equipment coating etc.
Saltout and dialysis is to utilize proteinic physicochemical property, carry out purifying with non-stratographic method, the target product concentration height that equipment cost is low, the experiment material cost is low, consuming time less, obtain need not concentrate.
Summary of the invention
The objective of the invention is to overcome chromatographic process purifying sea-mussel mucin equipment and the high problem of material cost, adopt the method saltout and dialyse, provide a kind of fast, the sea-mussel mucin separation purification method of low-cost, high density.
The objective of the invention is to realize through following technical scheme.
The method of saltouing and dialysing to the separating and purifying sea-mussel mucin of core of using provided by the invention comprises the steps:
1) with 0.5-2.5% perchloric acid or/and 1-10% acetate serves as to extract solvent, 100g mussel byssus adds 300-1000ml and extracts solvent, 10-25 ℃ of lixiviate 10-20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed and it evenly is suspended in extract in the solvent;
2) remove residue with the centrifugal 30-50min of 10000-16000r/min or with 45 μ m membrane filtrations, keep supernatant;
3) with the sodium-chlor of the 6-12M of the 1-6 times of volume 1-6h that under 15-25 ℃, saltouts;
4) 4 ℃, centrifugal 3-10min under the 10000-15000rpm, collecting precipitation is with the acetate dissolving of 1-3%;
5) 4 ℃, the wherein insoluble composition of the centrifugal 3-10min removal of 10000-15000rpm dissolves with the acetate of 1-3% once more;
6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 3-10h of 0.5-3%, 4 ℃ of preservations;
7) identify sea-mussel mucin from the angle of molecular weight: adopt 12-20% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 10-25 ℃ of dyeing 2-4h with HMW standard protein mark, confirms that the target protein molecular weight is 130kD;
8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 12-20% strength acetic acid-urea (Acetic acid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 10-25 ℃ of dyeing 1-3h, sea-mussel mucin will be developed the color by specificity;
9) analyze sea-mussel mucin purity with inverse bonded phase silica gel chromatographic column of C8 or Source polystyrene columns: with the acetonitrile-water is the moving phase isocratic elution, and 280nm detects.
The provided by the invention use to saltout and to dialyse is the method for the separating and purifying sea-mussel mucin of core, provide a kind of fast, the sea-mussel mucin separation purification method of low-cost, high density.
Embodiment
Embodiment 1, with perchloric acid extraction, saltout and separate sea-mussel mucin with dialysing
1) serve as to extract solvent with 0.5% perchloric acid, 100g mussel byssus adds 300ml and extracts solvent, and 25 ℃ of lixiviate 10min use whisking appliance that refrigerated mussel byssus is smashed at a high speed it evenly is suspended in the extraction solvent;
2) remove residue with the centrifugal 30min of 10000r/min, keep supernatant;
3) with the sodium-chlor of the 6-12M of the 2 times of volumes 1-6h that under 15-25 ℃, saltouts;
4) 4 ℃, centrifugal 3min under the 10000rpm, collecting precipitation, acetate dissolving with 2%;
5) 4 ℃, the wherein insoluble composition of the centrifugal 3min removal of 10000rpm dissolves with 2% acetate once more;
6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 5h of 2%, 4 ℃ of preservations;
7) identify sea-mussel mucin from the angle of molecular weight: adopt 15% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 25 ℃ of dyeing 2h with HMW standard protein mark, confirm that the target protein molecular weight is 130kD;
8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 15% strength acetic acid-urea (Aceticacid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 25 ℃ of dyeing 2h, sea-mussel mucin will be developed the color by specificity;
9) analyze sea-mussel mucin purity with the inverse bonded phase silica gel chromatographic column of C8: C8 chromatographic column 4.6 * 250mm; 5 μ m;
is the moving phase isocratic elution with acetonitrile-water (10-90), and 280nm detects.
Embodiment 2, with acetic acid extraction, saltout and separate sea-mussel mucin with dialysing
1) serve as to extract solvent with 10% acetate, 100g mussel byssus adds 1000ml and extracts solvent, 25 ℃ of lixiviates, and 20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed it evenly is suspended in the extraction solvent;
2) remove residue with 45 μ m membrane filtrations, keep supernatant;
3) with the sodium-chlor of the 10M of the 3 times of volumes 6h that under 25 ℃, saltouts;
4) 4 ℃, centrifugal 3min under the 15000rpm, collecting precipitation, acetate dissolving with 3%;
5) 4 ℃, the wherein insoluble composition of the centrifugal 3min removal of 15000rpm dissolves with 3% acetate once more;
6) with 4 ℃ of dialysis of acetate (dialysis tubing molecular weight cut-off 12000) 10h of 3%, 4 ℃ of preservations;
7) identify sea-mussel mucin from the angle of molecular weight: adopt 12% concentration sodium laurylsulfonate (SDS)-polyacrylamide gel electrophoresis (PAGE); Cma staining; 25 ℃ of dyeing 2h with HMW standard protein mark, confirm that the target protein molecular weight is 130kD;
8) blue (NBT) specific stain of tetrazole is identified sea-mussel mucin: adopt 12% strength acetic acid-urea (Aceticacid-Urea) polyacrylamide gel electrophoresis (Native PAGE); Staining fluid adopts tetrazole indigo plant-Padil solution (pH10); 25 ℃ of dyeing 2h, sea-mussel mucin will be developed the color by specificity;
9) the Source polystyrene columns is analyzed sea-mussel mucin purity: with acetonitrile-water (15-85) is the moving phase isocratic elution, and 280nm detects.
Claims (1)
1. the use method of separating and purifying sea-mussel mucin of saltouing and dialyse comprises following step:
1) with 0.5-2.5% perchloric acid or/and 1-10% acetate serves as to extract solvent, 100g mussel byssus adds 300-1000ml and extracts solvent, 10-25 ℃ of lixiviate 10-20min uses whisking appliance that refrigerated mussel byssus is smashed at a high speed and it evenly is suspended in extract in the solvent;
2) remove residue with the centrifugal 30-50min of 10000-16000r/min or with 45 μ m membrane filtrations, keep supernatant;
3) with the sodium-chlor of the 6-12M of the 1-6 times of volume 1-6h that under 15-25 ℃, saltouts;
4) 4 ℃, centrifugal 3-10min under the 10000-15000rpm, collecting precipitation is with the acetate dissolving of 1-3%;
5) 4 ℃, the wherein insoluble composition of the centrifugal 3-10min removal of 10000-15000rpm dissolves with the acetate of 1-3% once more;
6) with 4 ℃ of dialysis of the acetate of 0.5-3% 3-10h,, wherein 12000,4 ℃ of preservations of dialysis tubing molecular weight cut-off;
7) identify sea-mussel mucin from the angle of molecular weight: adopt 12-20% concentration sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Cma staining; 10-25 ℃ of dyeing 2-4h with HMW standard protein mark, confirms that the target protein molecular weight is 130kD;
8) the blue specific stain of tetrazole is identified sea-mussel mucin: adopt 12-20% strength acetic acid-urea-polyacrylamide gel electrophoresis, staining fluid adopts tetrazole indigo plant-Padil solution of pH10,10-25 ℃ of dyeing 1-3h, and sea-mussel mucin will be developed the color by specificity;
9) analyze sea-mussel mucin purity with inverse bonded phase silica gel chromatographic column of C8 or Source polystyrene columns: with the acetonitrile-water is the moving phase isocratic elution, and 280nm detects.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100875676A CN101585874B (en) | 2009-06-30 | 2009-06-30 | Method for separating and purifying sea-mussel mucin by using salting out and dialyzing |
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|---|---|---|---|
| CN2009100875676A CN101585874B (en) | 2009-06-30 | 2009-06-30 | Method for separating and purifying sea-mussel mucin by using salting out and dialyzing |
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| CN101585874A CN101585874A (en) | 2009-11-25 |
| CN101585874B true CN101585874B (en) | 2012-08-22 |
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| CN103087976A (en) * | 2013-01-05 | 2013-05-08 | 高敏 | Material for promoting cell growth adhering to wall and preparation method of material |
| WO2014186937A1 (en) * | 2013-05-20 | 2014-11-27 | Gao Min | Preparation method of mussel adhesive protein gel, mussel adhesive protein gel and use thereof |
| DK3326640T3 (en) | 2015-07-20 | 2021-09-06 | Jiangyin Bengt I Samuelsson Inst Of Life Science Co Ltd | Applications of mussel adhesive protein product in treatment and prevention of diseases related to melanin |
| EP3326639B1 (en) | 2015-07-20 | 2023-11-01 | Jiangyin Bengt I. Samuelsson Institute of Life Science Co., Ltd. | Mussel adhesive protein product and applications thereof in suppression of skin inflammations |
| WO2017011986A1 (en) | 2015-07-20 | 2017-01-26 | 赵兵 | Air filter |
| WO2017028025A1 (en) | 2015-08-14 | 2017-02-23 | 江阴市本特塞缪森生命科学研究院有限公司 | Mussel adhesive protein product and use thereof for inhibiting mucosal inflammation |
| CN105949299A (en) * | 2016-06-08 | 2016-09-21 | 杨子中 | Extracting method of mussel mucin |
| CN112870761A (en) * | 2021-01-12 | 2021-06-01 | 上海建冶科技股份有限公司 | Preparation process and preparation device of mussel mucin for increasing base material adhesive force |
| CN113632876B (en) * | 2021-08-11 | 2022-12-30 | 山西大学 | Production process for adding artificial meat into colored wheat bran |
| CN114456250B (en) * | 2022-03-30 | 2023-12-26 | 成都英普博集生物科技有限公司 | Mussel-like mucin purification process |
| CN114853863B (en) * | 2022-04-28 | 2023-11-21 | 西安德诺海思医疗科技有限公司 | Recombinant mussel-like mucin purification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6335430B1 (en) * | 1998-09-28 | 2002-01-01 | Magnus Qvist | Process of producing polyphenolic adhesive proteins and proteins produced in accordance with the process |
| CN101348520A (en) * | 2007-12-14 | 2009-01-21 | 北京康铭优盛生化技术有限公司 | Method for separating and purifying sea-mussel mucin by using mixing adsorption chromatography |
-
2009
- 2009-06-30 CN CN2009100875676A patent/CN101585874B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6335430B1 (en) * | 1998-09-28 | 2002-01-01 | Magnus Qvist | Process of producing polyphenolic adhesive proteins and proteins produced in accordance with the process |
| CN101348520A (en) * | 2007-12-14 | 2009-01-21 | 北京康铭优盛生化技术有限公司 | Method for separating and purifying sea-mussel mucin by using mixing adsorption chromatography |
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Denomination of invention: Method for separating and purifying sea-mussel mucin by using salting out and dialyzing Effective date of registration: 20190111 Granted publication date: 20120822 Pledgee: Bank of China Limited Jiangyin Branch Pledgor: JIANGYIN USUN BIOCHEMICAL TECHNOLOGY Co.,Ltd. Registration number: 2019320000034 |
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Denomination of invention: A method for separating and purifying mussel mucin by salting out and dialysis Effective date of registration: 20210727 Granted publication date: 20120822 Pledgee: Jiangyin branch of Bank of China Ltd. Pledgor: JIANGYIN USUN BIOCHEMICAL TECHNOLOGY Co.,Ltd. Registration number: Y2021980006856 |
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