CN103665135A - Sea cucumber tissue proteome extraction method - Google Patents
Sea cucumber tissue proteome extraction method Download PDFInfo
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- CN103665135A CN103665135A CN201310588498.3A CN201310588498A CN103665135A CN 103665135 A CN103665135 A CN 103665135A CN 201310588498 A CN201310588498 A CN 201310588498A CN 103665135 A CN103665135 A CN 103665135A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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Abstract
The invention discloses a sea cucumber tissue proteome extraction method. A series of steps such as liquid nitrogen grinding, ultrasonic treatment, ultra high-speed low-temperature centrifugation and proteome sample purification are performed to obtain proteomes with complete variety, higher concentration and purity from sea cucumber tissues, and the end product is particularly suitable for proteomics researches such as protein dimensional electrophoresis, high performance liquid chromatography (HPLC) and proteome mass spectrum identification, so that the great significance in carrying out related researches on sea cucumber proteomics is realized; with adoption of the extraction method, theoretical basis and practical guidance can be provided for offspring seed optimization, cultivation technique improvement and disease prevention and control of sea cucumbers, the settlement of the actual production problems about genetical characterization decline and frequent diseases of sea cucumbers is facilitated, and the economic benefit and the social benefit of the sea cucumber industry can be increased.
Description
Technical field
The present invention relates to a kind of extracting method of material, particularly a kind of extracting method of sea cucumber tissue protein group.
Background technology
Along with to the deepening continuously of genome research, people find gradually, can not disclose merely the rule of vital movement from genomic level completely, only have the genomic expression product of further investigation, could more directly understand the essence of life.Protein is the main executive of gene function, in vital movement, plays a significant role, and organism protein group is carried out to qualitative, quantitative, location and interactional research and can be the essence of illustrating vital movement theoretical foundation is provided.And the extraction of protein group sample is the key link in protein science research, the quality of protein group sample extraction will directly affect follow-up protein science correlative study and analysis.
Sea cucumber has higher edible pharmaceutical use and economic worth, the high value precious marine product with multi-efficiencies such as special nourishing and nutritive health-caries of extensively being admitted.But along with sea cucumber is propagated the continuous expansion of scale artificially, sea cucumber germplasm is degenerated and the farming disease harms problem is on the rise, and has restricted health and the Sustainable development of sea cucumber industry.Want sea cucumber proteomics to carry out deep research, first need to the protein group of sea cucumber carry out effectively, high-quality extraction, and also there is no now a kind of extracting method for sea cucumber tissue protein group.
Summary of the invention
The present invention is in order to solve the existing above-mentioned deficiency of prior art, proposes a kind ofly to obtain comparatively comprehensively kinds of protein, and purity and concentration extracting method of higher sea cucumber tissue protein group all.
Technical solution of the present invention is: a kind of extracting method of sea cucumber tissue protein group, is characterized in that: described method is carried out according to following steps:
A, get sea cucumber tissue and clean 3-5 time with 4 ℃, the 50mM Tris-HCL of pH=6.8, then, by putting into mortar after the pulverizing of sea cucumber tissue, while grind in mortar, add liquid nitrogen, until sea cucumber organizes fragment to be solid-state Powdered,
B, organize powder to be placed in ultraphonic pipe sea cucumber, and according to liquid ratio, be that the ratio of 200 μ l:0.1g is to the damping fluid that adds the 50mM Tris-HCL of pH=6.8 in ultraphonic pipe, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulates 30 times, then centrifugal treating 20min under 8000g, the condition of 4 ℃, get supernatant liquor and throw out, wherein supernatant liquor is sea cucumber and organizes solubility whole protein solution
C, the throw out obtaining in b step is put into ultraphonic pipe, and add 50mM Tris-HCL in ultraphonic pipe, 1mM EGTA, 1mM PMSF, the damping fluid of pH=7.4, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulate 30 times, then at 23000g, centrifugal treating 1h under the condition of 4 ℃, get supernatant liquor, by supernatant liquor at 150000g, centrifugal treating 1h under the condition of 4 ℃, abandon supernatant liquor, to adding final concentration in remaining solution, be 2% Triton X 114, stir process 1h under the condition of 4 ℃, again at 150000g, centrifugal treating 1h under the condition of 4 ℃, remove insolubles, at the Water Unders of 37 ℃, bathe 20min, afterwards at 1000g, centrifugal treating 20min under the condition of 4 ℃, take off a layer solution, this lower floor's solution is the extracting solution of sea cucumber histocyte membranin,
D, organize the extracting solution of the sea cucumber histocyte membranin obtaining in solubility whole protein solution and c step to merge the sea cucumber obtaining in b step after, mix according to the ratio of 1:5 with the acetone of-20 ℃, standing 12-20h under the condition of-20 ℃, then by mixing solutions centrifugal treating 30min under the condition of 8000g, remove supernatant liquor, precipitation is taken out under the environment of 4 ℃ and is dried, and dried product is and contains the sea cucumber tissue protein group that sea cucumber is organized solubility whole protein and sea cucumber histocyte membranin.
Compared with the existing technology, tool has the following advantages in the present invention:
The extracting method of sea cucumber tissue protein group disclosed in this invention, utilize the series of steps such as liquid nitrogen grinding, supersound process, ultra-high speed low-temperature centrifugation and protein group Sample Purification on Single, can from sea cucumber tissue, obtain kind comprehensively, the equal higher protein group of concentration and purity, its final product is particularly suitable for carrying out the proteomics researches such as Two-Dimensional Gel Electrophoresis, high performance liquid chromatography (HPLC) and protein group Mass Spectrometric Identification, to carrying out the correlative study of sea cucumber proteomics, has comparatively great meaning; It can for the seed of sea cucumber preferably, cultural technique improves and disease prevention and control are provided fundamental basis and practical advice, contributes to solve the actual production problems such as sea cucumber germplasm is degenerated, disease takes place frequently, and is conducive to improve economic benefit and the social benefit of sea cucumber industry.
Embodiment
An extracting method for sea cucumber tissue protein group, carries out according to following steps:
A, first get appropriate sea cucumber tissue, with 4 ℃, the 50mM Tris-HCL of pH=6.8, clean 3-5 time, then sea cucumber tissue is shredded with the clean scissors of sterilizing, put into mortar, while grinding in mortar, add liquid nitrogen, until sea cucumber organizes fragment to be solid-state Powdered; Under the condition of liquid nitrogen flash freezer, grind the degraded of protein in effectively preventing process of lapping, to guarantee activity and the structural integrity of tissue protein.
B, then sea cucumber is organized powder be placed in ultraphonic pipe, and according to liquid ratio, be that the ratio of 200 μ l:0.1g is to the damping fluid that adds the 50mM Tris-HCL of pH=6.8 in ultraphonic pipe, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulates 30 times, then centrifugal treating 20min under 8000g, the condition of 4 ℃, get supernatant liquor and throw out, wherein supernatant liquor is sea cucumber and organizes solubility whole protein solution; This step can effectively avoid pollution of nucleic acid and outer source ion to introduce, to guarantee the purity of protein example.
C, the throw out obtaining in b step is put into ultraphonic pipe, and add 50mM Tris-HCL in ultraphonic pipe, 1mM EGTA, 1mM PMSF, the damping fluid of pH=7.4, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulate 30 times, then at 23000g, centrifugal treating 1h under the condition of 4 ℃, get supernatant liquor, by supernatant liquor at 150000g, centrifugal treating 1h under the condition of 4 ℃, abandon supernatant liquor, to adding final concentration in remaining solution, be 2% Triton X 114, stir process 1h under the condition of 4 ℃, again at 150000g, centrifugal treating 1h under the condition of 4 ℃, remove insolubles, at the Water Unders of 37 ℃, bathe 20min, afterwards at 1000g, centrifugal treating 20min under the condition of 4 ℃, take off a layer solution, this lower floor's solution is the extracting solution of sea cucumber histocyte membranin, this step can effectively avoid pollution of nucleic acid and outer source ion to introduce equally, to guarantee the purity of protein example.
D, organize the extracting solution of the sea cucumber histocyte membranin obtaining in solubility whole protein solution and c step to merge the sea cucumber obtaining in b step after, mix according to the ratio of 1:5 with the acetone of-20 ℃, standing 12-20h under the condition of-20 ℃, then by mixing solutions centrifugal treating 30min under the condition of 8000g, remove supernatant liquor, precipitation is taken out under the environment of 4 ℃ and is dried, and dried product is and contains the sea cucumber tissue protein group that sea cucumber is organized solubility whole protein and sea cucumber histocyte membranin; In this step, adopt acetone as except salt solvent, can effectively remove the various salt ions in sea cucumber tissue protein group extracting solution, further guaranteed purity and the concentration of gained protein group.
Claims (1)
1. an extracting method for sea cucumber tissue protein group, is characterized in that: described method is carried out according to following steps:
A. get sea cucumber tissue and clean 3-5 time with 4 ℃, the 50mM Tris-HCL of pH=6.8, then, by putting into mortar after the pulverizing of sea cucumber tissue, while grind in mortar, add liquid nitrogen, until sea cucumber organizes fragment to be solid-state Powdered,
B. sea cucumber is organized powder to be placed in ultraphonic pipe, and according to liquid ratio, be that the ratio of 200 μ l:0.1g is to the damping fluid that adds the 50mM Tris-HCL of pH=6.8 in ultraphonic pipe, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulates 30 times, then centrifugal treating 20min under 8000g, the condition of 4 ℃, get supernatant liquor and throw out, wherein supernatant liquor is sea cucumber and organizes solubility whole protein solution
C. the throw out obtaining in b step is put into ultraphonic pipe, and add 50mM Tris-HCL in ultraphonic pipe, 1mM EGTA, 1mM PMSF, the damping fluid of pH=7.4, under the condition of 4 ℃, carry out supersound process, working hour 4s in supersound process process, intermittent time 10s, circulate 30 times, then at 23000g, centrifugal treating 1h under the condition of 4 ℃, get supernatant liquor, by supernatant liquor at 150000g, centrifugal treating 1h under the condition of 4 ℃, abandon supernatant liquor, to adding final concentration in remaining solution, be 2% Triton X 114, stir process 1h under the condition of 4 ℃, again at 150000g, centrifugal treating 1h under the condition of 4 ℃, remove insolubles, at the Water Unders of 37 ℃, bathe 20min, afterwards at 1000g, centrifugal treating 20min under the condition of 4 ℃, take off a layer solution, this lower floor's solution is the extracting solution of sea cucumber histocyte membranin,
D. the sea cucumber obtaining in b step is organized after the extracting solution merging of the sea cucumber histocyte membranin obtaining in solubility whole protein solution and c step, mix according to the ratio of 1:5 with the acetone of-20 ℃, standing 12-20h under the condition of-20 ℃, then by mixing solutions centrifugal treating 30min under the condition of 8000g, remove supernatant liquor, precipitation is taken out under the environment of 4 ℃ and is dried, and dried product is and contains the sea cucumber tissue protein group that sea cucumber is organized solubility whole protein and sea cucumber histocyte membranin.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880546A (en) * | 2015-04-28 | 2015-09-02 | 中国科学院近代物理研究所 | Sample preparation method for mice brain tissue proteome analysis |
| CN105116044A (en) * | 2015-08-14 | 2015-12-02 | 中国海洋大学 | Method for identifying thelenota ananas by means of special peptide fragment group |
| CN107299077A (en) * | 2017-06-18 | 2017-10-27 | 广东博溪生物科技有限公司 | A kind of extracting method of bovine brain pituitary extract |
| CN110308228A (en) * | 2019-08-09 | 2019-10-08 | 陈溪 | A kind of analysis method of sea cucumber protein group |
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| CN101906130A (en) * | 2010-07-07 | 2010-12-08 | 北华大学 | Wild ginseng protein extracting method suitable for dielectrophoresis |
| CN102288467A (en) * | 2011-07-20 | 2011-12-21 | 浙江工业大学 | Preparation method of animal protein sample for proteomic analysis |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880546A (en) * | 2015-04-28 | 2015-09-02 | 中国科学院近代物理研究所 | Sample preparation method for mice brain tissue proteome analysis |
| CN104880546B (en) * | 2015-04-28 | 2017-12-12 | 中国科学院近代物理研究所 | The sample preparation methods of Mice brain tissues Proteomic analysis |
| CN105116044A (en) * | 2015-08-14 | 2015-12-02 | 中国海洋大学 | Method for identifying thelenota ananas by means of special peptide fragment group |
| CN105116044B (en) * | 2015-08-14 | 2018-06-22 | 中国海洋大学 | A kind of method for differentiating plum blossom ginseng using specificity peptide fragment group |
| CN107299077A (en) * | 2017-06-18 | 2017-10-27 | 广东博溪生物科技有限公司 | A kind of extracting method of bovine brain pituitary extract |
| CN107299077B (en) * | 2017-06-18 | 2020-09-01 | 广东博溪生物科技有限公司 | Method for extracting bovine pituitary extract |
| CN110308228A (en) * | 2019-08-09 | 2019-10-08 | 陈溪 | A kind of analysis method of sea cucumber protein group |
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Application publication date: 20140326 |