CN103788173A - Method for extracting ubiquitin and ubiquitin-like from animals - Google Patents
Method for extracting ubiquitin and ubiquitin-like from animals Download PDFInfo
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- CN103788173A CN103788173A CN201410071665.1A CN201410071665A CN103788173A CN 103788173 A CN103788173 A CN 103788173A CN 201410071665 A CN201410071665 A CN 201410071665A CN 103788173 A CN103788173 A CN 103788173A
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Abstract
The invention discloses a method for extracting ubiquitin and ubiquitin-like from animals. The method comprises the following steps: cleaning tissues, skins and meat of arthropods, annulatas, cnidarias and edible fish or poultry and tissues, skins and meat of livestock to obtain raw materials; mincing the raw materials, performing wet grinding to obtain slurry; uniformly mixing the slurry, adding sodium bicarbonate or sodium hydroxide, stirring, centrifuging and collecting the supernatant; concentrating the supernatant to 1/10-1/25 of the original volume to obtain a protein concentrated solution; adding ethanol, standing, centrifuging and collecting the precipitate; dissolving the precipitate to prepare a solution by using a Tris-Gly buffer solution, and filtering the solution twice, and collecting the trapped fluid with the molecular weight of 6000-14000, namely the ubiquitin and ubiquitin-like. The method is simple in production process and low in cost, and the ubiquitin and ubiquitin-like products with high safety and high nutritive value can be prepared.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method of extracting ubiquitin and ubiquitin-like from animal.
Background technology
Ubiquitin (Ubiquitin, Ub) is a peptide species, by 76 Amino acid profiles, the about 8.5kDa of its molecular weight, within 1975, separate first from the pancreas of calf, be found subsequently in many different organisms and tissue, the aminoacid sequence of ubiquitin has high conservative.In eukaryotic cell, ubiquitin, to be covalently bound on the lysine residue of substrate protein white matter, will be sent to " destructor plant " by the protein of more than 4 ubiquitin mark, by the identification of 26S proteasome degraded rapidly.In recent years, along with deepening continuously to ubiquitin research, find that ubiquitin is not only the mark of protein degradation, or cell maintains the basic regulative mode that those is subject to the protein level of composing type adjusting and environmental stimulus generation, intracellular protein ubiquitination participates in the various physiological processes of cell, such as: cell cycle progression, the generation of organoid, apoptosis, hyperplasia and differentiation regulate, the Quality Control of endoplasmic reticulum protein, albumen transhipment, inflammatory reaction, angtigen presentation and DNA repair, and cellular stress etc., in each vital movement, play an important role.
Along with increasing ubiquitin protein is in the news, the various types of modified protein like ubiquitin is also constantly found, as: SUMO, Rubl, Apgs and ApglZ etc.Ubiquitin-like albumen (Ubls) various kinds of cell function as signal envoy and ubiquitin-like co-controlling, repairs such as cell proliferation, apoptosis, cell cycle and DNA, is playing the part of important role with ubiquitin in human body self-regeneration function system.
Ubiquitin and ubiquitin-like albumen have biological function widely, the development and application values of ubiquitin will come into one's own gradually, the new way of finding the various diseases such as treatment tumor disease, heart disease, sacred disease from the research of ubiquitin and ubiquitin-like will be one of trend of future medicine, and ubiquitin and ubiquitin-like will provide new clue and direction in exploration and the application of the aspects such as plant gene engineering technology and raising stress resistance of plant for following agricultural.
Separate in little Pancreas Bovis seu Bubali from ubiquitin in 1975, at present the research of ubiquitin and ubiquitin-like is mainly concentrated on to mechanism of action aspect, along with the development of scientific and technical and modern analysis means, ubiquitin in increasing various animals and the mechanism of action of ubiquitin-like is just studied, but utilizes at present animal to extract and the method for purifying ubiquitin exists extraction process complexity, high in cost of production shortcoming for raw material.
Summary of the invention
The invention provides a kind of method of extracting ubiquitin and ubiquitin-like from animal, the method production technique is simple, cost is low, can make ubiquitin safe, that nutritive value is good and ubiquitin-like product.
For this reason, technical scheme of the present invention is:
A method of extracting ubiquitin and ubiquitin-like from animal, comprises following steps:
1) tissue of tissue, skin, meat and the domestic animal of arthropods, annelid, cnidarian, edible fish or poultry, skin, meat water or mass concentration is clean lower than 0.3% organic acid sodium salt aqueous cleaning, obtain raw material; Described organic acid sodium salt is sodium lauryl sulphate, sodium polyacrylate, sodium formiate or sodium stearate;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 5～7 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=7.6～8.0; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are placed in to constant-temperature table under 4～10 ℃ of conditions and process 5～10h, or stir 1～2h under 4～10 ℃ of conditions, obtain homogeneous slurry;
4) be 0.2～1.0% sodium bicarbonate or 0.1～0.5% sodium hydroxide to adding mass concentration in described homogeneous slurry, fully stir, then supernatant liquor is collected in centrifugation;
5) by described supernatant concentration to 1/10～1/25 of original volume, obtain protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding centrifugal and collecting precipitation after leaving standstill when wherein ethanol mass concentration is 75%;
7) to adding in described precipitation Tris-Gly damping fluid that pH=7.6～8.0, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 14000 with molecular weight cut-off is also collected filtered solution; Then the ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
The method of step 5) concentrated supernatant is that to utilize aperture be the dialysis membrane circulating filtration of 0.0001～0.001 μ m.
Described arthropods is honeybee, ant, moth, family cicada, cricket, katydid, centipede, scorpio or locust; Described annelid is earthworm or leech; Described cnidarian is jellyfish; Described domestic animal is pig, ox or sheep; Described poultry is chicken or duck.
Described honeybee is wasp, wasp or honeybee; Described ant is termite or ant; Described moth is hawkmoth, noctuid or snout moth.
The present invention utilizes animal for raw material, obtains ubiquitin and ubiquitin-like albumen by steps such as defibrination, degreasing, concentrated, precipitation, ultrafiltration, and the advantage applies of the inventive method is at following 2 points:
1) from production technique, first the present invention concentrated crude protein liquid before utilizing alcohol precipitation, improve the concentration of crude protein liquid before precipitation, improve alcohol precipitation efficiency, then improved the extraction efficiency of ubiquitin and ubiquitin-like albumen, the reagent cost using in degreasing, alcohol precipitation step is cheap, and in leaching process, does not introduce the toxic substances such as organic solvent, has guaranteed to extract the security of ubiquitin and ubiquitin-like albumen.
2) product from obtaining, the propagation of the ubiquitin of producing and ubiquitin-like albumen more effective increase immune effect, Promote immunity cell compared with common protein powder, has improved when prepared using is worth and has obtained and had higher practical value and economic worth product.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described.
Embodiment 1
Extract ubiquitin and ubiquitin-like take leech as raw material, comprise the steps:
1) leech water is cleaned, not only remove the dirt of its body surface, also remove the dirt in digestive tube, obtain raw material;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 7 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=7.6; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are placed in to constant-temperature table under 10 ℃ of conditions and process 5h;
4) be that 1.0% sodium bicarbonate fully stirs to adding mass concentration in described homogeneous slurry, then utilize disk centrifugal separator to carry out centrifugation, collect supernatant liquor;
5) the dialysis membrane circulating filtration with 0.0001 μ m by described supernatant concentration, 1/20 of simmer down to original volume, obtains protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding when wherein ethanol mass concentration is 75%, leave standstill 2h, centrifugal 15min under l0000/min condition, collecting precipitation;
7) to adding in described precipitation Tris-Gly damping fluid that pH=7.6, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 1.4 ten thousand with molecular weight cut-off is also collected filtered solution; Then the neutral ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
The ubiquitin making and ubiquitin-like can be made capsule or tablet through following steps:
By above-mentioned ubiquitin and ubiquitin-like, add Zulkovsky starch or the medical starch of its weight 8～10%, stir evenly, in spray-drier, regulate optimal temperature spray to become fine powder, powder is carried out to capsulation or tabletting for bottling packing, sterilization.
Embodiment 2
Extract ubiquitin and ubiquitin-like with earthworm raw material, comprise the steps:
1) the polyacrylic acid sodium water solution that is 0.29% by earthworm by concentration cleans, and not only removes the dirt of its body surface, also removes the dirt in digestive tube, obtains raw material;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 6 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=7.6; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are placed in to constant-temperature table under 4 ℃ of conditions and process 7h;
4) be that 0.8% sodium hydroxide fully stirs to adding mass concentration in described homogeneous slurry, then utilize disk centrifugal separator to carry out centrifugation, collect supernatant liquor;
5) the dialysis membrane circulating filtration with 0.001 μ m by described supernatant concentration, 1/20 of simmer down to original volume, obtains protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding when wherein ethanol mass concentration is 75%, leave standstill 2h, centrifugal 20min under 8000/min condition, collecting precipitation;
7) to adding in described precipitation Tris-Gly damping fluid that pH=7.6, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 1.4 ten thousand with molecular weight cut-off is also collected filtered solution; Then the neutral ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
The ubiquitin making and ubiquitin-like can be made capsule or tablet through following steps:
By above-mentioned ubiquitin and ubiquitin-like, add Zulkovsky starch or the medical starch of its weight 8～10%, stir evenly, in spray-drier, regulate optimal temperature spray to become fine powder, powder is carried out to capsulation or tabletting for bottling packing, sterilization.
Embodiment 3
Extract ubiquitin and ubiquitin-like take honeybee as raw material, comprise the steps:
1) honeybee is cleaned by the aqueous sodium formate solution that concentration is 0.1%, not only removed the dirt of its body surface, also remove the dirt in digestive tube, obtain raw material;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 6 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=8; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are placed in to constant-temperature table under 6 ℃ of conditions and process 10h;
4) be that 0.2% sodium hydroxide fully stirs to adding mass concentration in described homogeneous slurry, then utilize disk centrifugal separator to carry out centrifugation, collect supernatant liquor;
5) the dialysis membrane circulating filtration with 0.0001 μ m by described supernatant concentration, 1/15 of simmer down to original volume, obtains protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding when wherein ethanol mass concentration is 75%, leave standstill 2h, centrifugal 20min under 6000/min condition, collecting precipitation;
7) to adding in described precipitation Tris-Gly damping fluid that pH=8.0, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 1.4 ten thousand with molecular weight cut-off is also collected filtered solution; Then the neutral ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
The ubiquitin making and ubiquitin-like can be made capsule or tablet through following steps:
By above-mentioned ubiquitin and ubiquitin-like, add Zulkovsky starch or the medical starch of its weight 8～10%, stir evenly, in spray-drier, regulate optimal temperature spray to become fine powder, powder is carried out to capsulation or tabletting for bottling packing, sterilization.
Embodiment 4
Extract ubiquitin and ubiquitin-like take scorpio as raw material, comprise the steps:
1) scorpio water is cleaned, not only remove the dirt of its body surface, also remove the dirt in digestive tube, obtain raw material;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 5 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=8.0; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are stirred to 1h under 6 ℃ of conditions, obtain homogeneous slurry;
4) be that 0.2% sodium bicarbonate fully stirs to adding mass concentration in described homogeneous slurry, then utilize disk centrifugal separator to carry out centrifugation, collect supernatant liquor;
5) the dialysis membrane circulating filtration with 0.0001 μ m by described supernatant concentration, 1/20 of simmer down to original volume, obtains protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding when wherein ethanol mass concentration is 75%, leave standstill 2h, centrifugal 10min under l2000/min condition, collecting precipitation;
7) to adding in described precipitation Tris-Gly damping fluid that pH=8.0, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 1.4 ten thousand with molecular weight cut-off is also collected filtered solution; Then the neutral ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
The ubiquitin making and ubiquitin-like can be made capsule or tablet through following steps:
By above-mentioned ubiquitin and ubiquitin-like, add Zulkovsky starch or the medical starch of its weight 8～10%, stir evenly, in spray-drier, regulate optimal temperature spray to become fine powder, powder is carried out to capsulation or tabletting for bottling packing, sterilization.
Performance test:
(1) ubiquitin and ubiquitin-like albumen are to mice spleen lymphocytes proliferation ability test
Get 1,2 ubiquitin and the ubiquitin-likes that extract in example, choose small white mouse and test.
Test method: mouse is put to death, and the aseptic spleen of getting, grinds spleen gently with tweezers, with Hank's liquid washed twice, centrifugal 10min collecting cell under 1000r/min condition, makes individual cells suspension with the RPMI-1640 nutrient solution containing serum.Adjusting cell concn is 4 × 10
6individual/mL.Cell suspension is added in 96 well culture plates, 150 μ L/ holes, white contrast adds the not RPMI-1640 nutrient solution 50 μ L containing serum, and other each group adds the protein soln 50 μ L with the RPMI-1640 nutrient solution containing serum is not configured to, each sample do three parallel, be placed in 37 ℃, contain 5%CO
2cO
2in incubator, cultivate 72h.
Cultivation finishes front 4h, adds XTT-PMS30 μ L/ hole, after cultivation finishes, surveys the absorbance value in each hole under 450nm wavelength condition, with this value representation cell proliferation degree in microplate reader.
Experimental result: following table is the direct impact of different proteins on mice spleen lymphocytes proliferation.Visible 2～5 groups of blank groups compare that there were significant differences, and the protein obtaining in example all can promote the propagation of splenic lymphocyte, obtain ubiquitin and ubiquitin-like albumen and more can promote compared with common albumen the propagation of splenic lymphocyte in example.There is significant difference (P < 0.05) compared with blank
| Group | Dosage (protein liquid concentration) | A 450（X±S） |
| 1. blank | 0μg/ml | 0.653±0.013 |
| 2. commercially available common protein powder | 200μg/ml | 0.731±0.019 |
| 3. example 1 makes albumen | 200μg/ml | 0.806±0.022* |
| 4. example 1 makes albumen | 250μg/ml | 0.813±0.024* |
| 5. example 2 makes albumen | 250μg/ml | 0.802±0.019* |
2～5 compare with blank 1: * P < 0.05
(2) ubiquitin and ubiquitin-like albumen generate and detect test mouse antibodies
40 mouse are divided into 4 groups at random, are respectively: blank 1 control group, and gavage shown in 2 groups, 3 groups, 4 groups accordings to the form below, gavage 25 days continuously,
| Group | Number of mice (only) | Dosage (protein liquid concentration) |
| 1. blank | 10 | Physiological saline |
| 2. commercially available common protein powder | 10 | 0.5g/kg |
| 3. example 1 makes albumen | 10 | 0.5g/kg |
| 4. example 2 makes albumen | 10 | 0.5g/kg |
The 25th day, every mouse peritoneal injection 2%SRBC0.2mL, continue gavage 5d, mouse is put to death, the aseptic spleen of getting, grinds spleen gently with tweezers, by Hank's liquid washed twice, centrifugal 10min collecting cell under 1000r/min condition, uses containing the RPMI-1640 nutrient solution of serum and makes individual cells suspension.Then adjusting cell concn is 4 × 10
6individual/mL cell suspension is for subsequent use.To containing 0.5mL(45～50 ℃) add 50 μ L10%SRBC and 20 μ L splenocyte suspensions in the small test tube of top layer substratum, mix rapidly, be added on and hang on slide, after solidifying, slide is put upside down, put CO
2in incubator, cultivate 1.5h, add the complement of SA damping fluid dilution, continue to cultivate 1.5h, calculate antibody-producting cell number in spleen.
Experimental result: successive administration, after 25 days, is measured antibody-producting cell number in spleen.From following table, 2,3,4 groups all can significantly improve spleen antibody cell generate number (P < 0.05), wherein 3,4 groups more remarkable than 2 groups of impacts.
| Group | Number of mice (only) | Antibody-producting cell number/10 6（X±S） |
| 1. blank | 10 | 183.90±17.03 |
| 2. commercially available common protein powder | 10 | 302.80±30.53* |
| 3. protein powder in example 3 | 10 | 378.30±23.42* |
| 4. protein powder in example 4 | 10 | 395.50±26.49* |
2～4 compared with control group 1, * P < 0.05.
Claims (5)
1. a method of extracting ubiquitin and ubiquitin-like from animal, is characterized in that comprising following steps:
1) tissue of tissue, skin, meat and the domestic animal of arthropods, annelid, cnidarian, edible fish or poultry, skin, meat water or mass concentration is clean lower than 0.3% organic acid sodium salt aqueous cleaning, obtain raw material;
2) will be described raw material carry out wet-milling after rubbing, in wet-milling process, adding quality is that the concentration of 5～7 times of described raw materials is the Tris-Gly damping fluid of 25mmol/L, pH=7.6～8.0; After wet-milling, carry out homogenate, obtaining granularity is 50～150 object slurries;
3) described slurries are placed in to constant-temperature table under 4～10 ℃ of conditions and process 5～10h, or stir 1～2h under 4～10 ℃ of conditions, obtain homogeneous slurry;
4) be 0.2～1.0% sodium bicarbonate or 0.1～0.5% sodium hydroxide to adding mass concentration in described homogeneous slurry, fully stir, then supernatant liquor is collected in centrifugation;
5) by described supernatant concentration to 1/10～1/25 of original volume, obtain protein concentrated solution;
6) to add mass concentration in described protein concentrated solution be 95% ethanol until stop adding centrifugal and collecting precipitation after leaving standstill when wherein ethanol mass concentration is 75%;
7) to adding in described precipitation Tris-Gly damping fluid that pH=7.6～8.0, concentration are 25mmol/L to resolution of precipitate, remove insolubles, collect solution;
8) ultrafiltration membrance filter that solution step 7) being obtained is 14000 with molecular weight cut-off is also collected filtered solution; Then the ultrafiltration membrance filter that is 6000 by described filtered solution with molecular weight cut-off is also collected trapped fluid, and described trapped fluid is described ubiquitin and ubiquitin-like.
2. the method for claim 1, is characterized in that: described organic acid sodium salt is sodium lauryl sulphate, sodium polyacrylate, sodium formiate or sodium stearate.
3. the method for claim 1, is characterized in that: in step 5), the method for concentrated supernatant is that to utilize aperture be the dialysis membrane circulating filtration of 0.0001～0.001 μ m.
4. the method for claim 1, is characterized in that: described arthropods is honeybee, ant, moth, family cicada, cricket, katydid, centipede, scorpio or locust; Described annelid is earthworm or leech; Described cnidarian is jellyfish; Described domestic animal is pig, ox or sheep; Described poultry is chicken or duck.
5. method as claimed in claim 4, is characterized in that: described honeybee is wasp, wasp or honeybee; Described ant is termite or ant; Described moth is hawkmoth, noctuid or snout moth.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105622739A (en) * | 2016-02-15 | 2016-06-01 | 天津替代医学科技股份有限公司 | Method for extracting ubiquitin and like ubiquitin from protein milk |
| CN114075544A (en) * | 2020-08-21 | 2022-02-22 | 复旦大学附属华山医院 | Method for preparing de-ubiquitin erythrocyte product based on ultrafiltration centrifugation |
-
2014
- 2014-02-28 CN CN201410071665.1A patent/CN103788173A/en active Pending
Non-Patent Citations (2)
| Title |
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| 张建社等: "《蛋白质分离与纯化技术》", 1 September 2009 * |
| 黄新敏等: "泛素蛋白的研究进展", 《广东农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105622739A (en) * | 2016-02-15 | 2016-06-01 | 天津替代医学科技股份有限公司 | Method for extracting ubiquitin and like ubiquitin from protein milk |
| CN114075544A (en) * | 2020-08-21 | 2022-02-22 | 复旦大学附属华山医院 | Method for preparing de-ubiquitin erythrocyte product based on ultrafiltration centrifugation |
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Application publication date: 20140514 |