CN1478793A - Method of extracting soluble substrate protein from pearl material - Google Patents
Method of extracting soluble substrate protein from pearl material Download PDFInfo
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- CN1478793A CN1478793A CNA031459560A CN03145956A CN1478793A CN 1478793 A CN1478793 A CN 1478793A CN A031459560 A CNA031459560 A CN A031459560A CN 03145956 A CN03145956 A CN 03145956A CN 1478793 A CN1478793 A CN 1478793A
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- nacre
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- concentration
- phosphate buffer
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- 238000000034 method Methods 0.000 title claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 title claims description 12
- 108090000623 proteins and genes Proteins 0.000 title claims description 12
- 239000000463 material Substances 0.000 title abstract description 5
- 239000000758 substrate Substances 0.000 title description 4
- 238000000502 dialysis Methods 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 5
- 238000010521 absorption reaction Methods 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 229940111685 dibasic potassium phosphate Drugs 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 9
- 239000001488 sodium phosphate Substances 0.000 claims description 9
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 9
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 9
- 239000011159 matrix material Substances 0.000 claims description 8
- 238000000108 ultra-filtration Methods 0.000 claims description 6
- 238000004440 column chromatography Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 5
- 238000010612 desalination reaction Methods 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 2
- 239000012507 Sephadex™ Substances 0.000 claims description 2
- 239000012141 concentrate Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000010298 pulverizing process Methods 0.000 claims description 2
- 150000003384 small molecules Chemical class 0.000 claims description 2
- 208000001132 Osteoporosis Diseases 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract description 2
- 238000001035 drying Methods 0.000 abstract 2
- 108060003393 Granulin Proteins 0.000 abstract 1
- 229910019142 PO4 Inorganic materials 0.000 abstract 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 abstract 1
- 238000013375 chromatographic separation Methods 0.000 abstract 1
- 238000004587 chromatography analysis Methods 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 230000008021 deposition Effects 0.000 abstract 1
- 238000011033 desalting Methods 0.000 abstract 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 239000007791 liquid phase Substances 0.000 abstract 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 1
- 239000010452 phosphate Substances 0.000 abstract 1
- 229910052700 potassium Inorganic materials 0.000 abstract 1
- 239000011591 potassium Substances 0.000 abstract 1
- 235000004252 protein component Nutrition 0.000 abstract 1
- 238000003756 stirring Methods 0.000 abstract 1
- 239000011049 pearl Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241000490568 Pinctada fucata Species 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 229910021642 ultra pure water Inorganic materials 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000011164 ossification Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 235000005956 Cosmos caudatus Nutrition 0.000 description 1
- 244000293323 Cosmos caudatus Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000012010 growth Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 210000004409 osteocyte Anatomy 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
A process for extracting soluble matrix protein from nacre includes such steps as adding pearl powder to buffer liquid of disodium hydrogen phosphate and potassium bihydrogen phosphate, stirring, centrifugal deposition, collecting supernatant, superfilter, dialysis, desalting, concentrating, drying, chromatography, collecting micro-molecular components, liquid-phase chromatographic separation, collecting the protein component with absorptive peak, and drying. It can be used to prepare the medicine for preventing and treating osteoporosis and the biomedical materials, and for culturing pearl.
Description
Technical field
The present invention relates to a kind of method of from nacre, extracting the solubility nacre substrate protein, belong to the medical tissue engineering field.
Background technology
Pearl is the product of biomineralization in the mollusk Margarita body, is exactly the treasure of decorating and keeping healthy since ancient times.Pinctada fucata (Pinctada fucata) is to be used to one of main shellfish that produces sea water pearls in the world, and China's sea water pearls is nearly all produced by pinctada fucata.The nacre of black lip nacreous layer is the same with pearl, and composition and pearl are very nearly the same by its mantle secretion, and be main by the aragonite crystal (CaCO more than 95%
3) form with the organic substrate less than 5%.Only account for the organic substrate of nacre under weight 5%, in control nacre forming process, play an important role.Nacre has good biological fitness and osteogenic activity, Growth and Differentiation that can the obvious stimulation osteocyte, and can not cause serious immune response.Domestic and international research shows that the material that has the induced osteogenesis effect in the nacre is present in its water-soluble base in the recent period.But because the restriction of protein separation method has hindered progress of research, also limited this discovery in medically application.
Summary of the invention
The purpose of this invention is to provide a kind of from nacre the proteic method of separation and purification dissolvable matrix.
Present method comprises the following steps:
1) add in Sodium phosphate dibasic-potassium phosphate buffer in the nacre after will pulverizing, stirred 72 hours in 4 ℃, centrifugal, collect supernatant;
2) supernatant liquor is after ultrafiltration, dialysis desalination concentrate, dry nacre dissolvable matrix;
3) dissolvable matrix is carried out column chromatography, collect the small molecule component that absorption peak is arranged under the 280nm;
4) above component is carried out high performance liquid chromatography and separate, collect the protein ingredient that absorption peak is arranged under the 280nm, the dry finished product that gets.
In said process, described used when pearl powder is stirred under 4 ℃ of conditions be 2～5 times of pearl powder quality, pH is 7.0, Sodium phosphate dibasic-potassium phosphate buffer of concentration 1～2mM.
During mode desalinations such as described employing ultrafiltration, dialysis, super rate is leant on molecular weight cut-off with dialysis tubing less than 3KD; The cryodesiccated mode of dry employing.
Filler in the described column chromatography is a Sephadex G-75 dextrane gel, moving phase is that pH is 7.0, Sodium phosphate dibasic-potassium phosphate buffer of concentration 1～2mM, collection for having the molecular weight of absorption less under the 280nm, the later relatively component that elutes.
Filler in the described high performance liquid chromatography is the C18 particle, and moving phase is the concentration acetonitrile solution that gradient increases progressively between 0-100%, collection the protein ingredient that elutes by the 3rd peak in three major protein peaks that absorption is arranged under the 280nm of correspondence time.
With the nacre dissolvable matrix albumen of present method separation and purification, its molecular weight is about 10kD, is rich in glycine (37.2%), leucine (15.7%) and L-Ala (13.3%).Experiment in vitro shows that this albumen has good induced osteogenesis effect is arranged, and can independently impel calcium carbonate crystal to form orderly aragonite crystal, illustrate that its vitro culture and fields such as raising pearl production and quality at preparation prevention and treatment medicine for treating osteoporosis, preparation medical bio novel material, pearl or class pearl material have broad application prospects.
Embodiment
Embodiment 1,
1) get nacres after 100 grams are pulverized, adds 250 milliliters of pH7.0, concentration is Sodium phosphate dibasic-potassium phosphate buffer of 2mM, stirred 72 hours in 4 ℃, 8000 rev/mins centrifugal 30 minutes, the collection supernatant;
2) supernatant liquor is the ultrafiltration post ultrafiltration final vacuum lyophilize of 3KD through molecular weight cut-off, dissolves again with 10 ml distilled waters to be placed in the dialysis tubing, spends the night with 1 liter of distill water dialysis, once more vacuum lyophilization;
3) above-mentioned product dissolves with Sodium phosphate dibasic-potassium phosphate buffer of 1 milliliter of 1mM, carry out SephadexG-75 dextrane gel column chromatography, Sodium phosphate dibasic-potassium phosphate buffer wash-out with 1mM, being collected in has the molecular weight of absorption less under the 280nm, the later relatively component that elutes, place dialysis tubing to spend the night vacuum lyophilization with 1 liter of distill water dialysis;
4) said components is with 5 milliliters of ultrapure water (ultrapure water, HPLC analyzes dedicated water, pure degree is higher than distilled water, prepare with Milli-Q) dissolving, 0.45 carrying out C18 post high performance liquid chromatography behind the micron membrane filtration separates, with the concentration acetonitrile solution wash-out that gradient increases progressively between 0-100%, be collected in the 3rd peak in three major protein peaks that absorption is arranged under the 280nm the protein ingredient that elutes of corresponding time, vacuum lyophilization;
5) component of above-mentioned collection (is named dihomocinchonine acid again with two quinic acids after dissolving again with ultrapure water, bicinchoninicacid) the reagent method is measured protein concentration, with the bovine serum albumin is contrast, records the albumen of collecting altogether and is equivalent to 1.6 milligrams of bovine serum albumins approximately;
6) sodium laurylsulfonate-poly-about 10kD of propionic acid amide gel electrophoresis (SDS-PAGE) analysis revealed gained molecular weight of albumen, amino acid composition analysis shows that this albumen is rich in glycine (37.2%), leucine (15.7%) and L-Ala (13.3%).
Claims (1)
1. one kind is extracted the proteic method of dissolvable matrix from nacre, it is characterized in that being made up of the following step:
1) add in Sodium phosphate dibasic-potassium phosphate buffer in the nacre after will pulverizing, stirred 72 hours in 4 ℃, centrifugal, collect supernatant;
2) supernatant liquor is after ultrafiltration, dialysis desalination concentrate, dry nacre dissolvable matrix;
3) dissolvable matrix is carried out column chromatography, collect the small molecule component that absorption peak is arranged under the 280nm;
4) above component is carried out high performance liquid chromatography and separate, collect the protein ingredient that absorption peak is arranged under the 280nm, the dry finished product that gets;
In said process, described used when pearl powder is stirred under 4 ℃ of conditions be 2～5 times of pearl powder quality, pH is 7.0, Sodium phosphate dibasic-potassium phosphate buffer of concentration 1～2mM;
During mode desalinations such as described employing ultrafiltration, dialysis, super rate is leant on molecular weight cut-off with dialysis tubing less than 3KD; The cryodesiccated mode of dry employing;
Filler in the described column chromatography is a Sephadex G-75 dextrane gel, moving phase is that pH is 7.0, Sodium phosphate dibasic-potassium phosphate buffer of concentration 1～2mM, collection for having the molecular weight of absorption less under the 280nm, the later relatively component that elutes;
Filler in the described high performance liquid chromatography is the C18 particle, and moving phase is the concentration acetonitrile solution that gradient increases progressively between 0-100%, collection the protein ingredient that elutes by the 3rd peak in three major protein peaks that absorption is arranged under the 280nm of correspondence time.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031459560A CN1478793A (en) | 2003-07-18 | 2003-07-18 | Method of extracting soluble substrate protein from pearl material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031459560A CN1478793A (en) | 2003-07-18 | 2003-07-18 | Method of extracting soluble substrate protein from pearl material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1478793A true CN1478793A (en) | 2004-03-03 |
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ID=34155940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA031459560A Pending CN1478793A (en) | 2003-07-18 | 2003-07-18 | Method of extracting soluble substrate protein from pearl material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1478793A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381400B (en) * | 2008-10-14 | 2011-06-15 | 晶旺生物科技股份有限公司 | Extraction of nano pearl organic matrix and molecular-weight gradation method |
| CN109553674A (en) * | 2017-09-26 | 2019-04-02 | 深圳先进技术研究院 | Shell pearl layer stromatin and its preparation method and application |
-
2003
- 2003-07-18 CN CNA031459560A patent/CN1478793A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101381400B (en) * | 2008-10-14 | 2011-06-15 | 晶旺生物科技股份有限公司 | Extraction of nano pearl organic matrix and molecular-weight gradation method |
| CN109553674A (en) * | 2017-09-26 | 2019-04-02 | 深圳先进技术研究院 | Shell pearl layer stromatin and its preparation method and application |
| WO2019061862A1 (en) * | 2017-09-26 | 2019-04-04 | 深圳先进技术研究院 | Shell nacre matrix protein, preparation method therefor, and use thereof |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |