CN103641910A - Separation and purification method and identification method of chicken apolipoprotein AI - Google Patents
Separation and purification method and identification method of chicken apolipoprotein AI Download PDFInfo
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- CN103641910A CN103641910A CN201310708396.0A CN201310708396A CN103641910A CN 103641910 A CN103641910 A CN 103641910A CN 201310708396 A CN201310708396 A CN 201310708396A CN 103641910 A CN103641910 A CN 103641910A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
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Abstract
The invention relates to a separation and purification method and an identification method of chicken apolipoprotein AI, and relates to the technical field of biology. The separation and purification method comprises the following steps: collecting chicken serums; preparing serum high-density lipoprotein; defatting HDL (High Density Lipoprotein); dissolving the defatted HDL; dialyzing and concentrating a defatted HDL solution; dialyzing the HDL solution by using a urea-10mmol/LTris-HCl-1mmol/LEDTA mixed solution of 6 mol/L to remove an organic solvent contained in the HDL solution, and then concentrating polyethylene glycol 6000 of 500 g/L to 2-3 mL to enhance the concentration of the HDL solution. The separation and purification method disclosed by the invention is simple and lays the foundation for the measurement and further research of the apo AI.
Description
Technical field:
The present invention relates to biological technical field, be specifically related to a kind of separation and purification and authentication method of chicken ApoA1.
Background technology:
A large amount of epidemiological studies show, cholesterol in serum high-density LP (HDL-C) and ApoA1 (apoA I) are to prevent the arteriosclerotic protection factor, are negative correlation with atherosclerotic cardiovascular systemic disease.Although apoA I and HDL-C have the dependency of highly significant, along with going deep into of research, many scholars think the two physiological significance and the clinical value running that exists lipoprotein in much difference; And apoA I represents the protein ingredient in HDL, account for 70% of HDL protein ingredient, the balance between reflection HDL synthesizes and decomposes.ApoA I can directly act on oxidation and the gathering that arterial wall prevents low-density lipoprotein (LDL), promotes that cholesterol flows out from arterial wall, atherosclerosis cardiovascular system diseases.In addition, the distinctive secondary structure block of apoA I can promote the specific protein polymeric immunoglobulin receptor on special fat surface on apoA I and cytolemma.HDL-C represents cholesterol level in HDL, and the effect of reflection serum, makes cholesterol and phosphatide on cytolemma dissolve and shift out simultaneously.Have document to point out, apoA I, when differentiating cardiovascular systems pathology, is obviously better than the indexs such as serum total cholesterol, triglyceride level and high density lipoprotein cholesterol.ApoA I, except outside the Pass having with adjusting HypercholesterolemicRats, also has close relationship with immunity and nervous function.But, also do not have special method to carry out separation, purifying and evaluation to chicken ApoA1 at present.
Summary of the invention:
The separation and purification and the authentication method that the object of this invention is to provide a kind of chicken ApoA1, the method for its separation and purification is simple, gives the measurement of apoA I and further studies and lay the foundation.
In order to solve the existing problem of background technology, the present invention by the following technical solutions: its separation purification method is:
1, the collection of chicken serum: venous blood collection under wing, after blood sampling, the syringe needle of taking away, gently in injecting tube.For preventing breaking of hemocyte, the syringe that cleans up and disinfect and test tube infiltrate with 0.85% physiological saline in advance, prevent blood clot and glass tube walls adhesion, cause serum to separate out bad.Standing 37 ℃ of incubator 1 h of test tube 45° angle, then put standing 3～4 h in 4 ℃ of refrigerators draw serum with capillary burette after blood coagulation blood clot retraction, then with centrifugal 10 min of 1000 * g, get in rearmounted-20 ℃ of refrigerators of supernatant serum packing, to add NaN3 and save backup.
2, the preparation of serum high-density LP: get serum 1.0 ml and add 1.0 ml precipitation agent I to mix, standing 10 min, centrifugal 15 min of 1000 * g, in sucking-off, honest and upright and thrifty 1.8 ml add 0.42 ml precipitation agent II to mix, standing 5 min, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is dissolved in 0.1 ml 12%NaCl and 1.0 ml 0.02 mol/L Tris-HCL liquid, PH=8.6, add standing 5 min of 0.26 ml precipitation agent II, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is carried out to redissolve, precipitation and again dissolution process are once, finally obtain the solution of crude product that contains HDL.
3, the degreasing of HDL: treat that by prepared degreasing HDL solution is in the situation that constantly stirring, with liquid-transfering gun, be dropwise incorporated in 60 mL acetone-ethanol of-20 ℃ of precoolings, volume ratio is in 1:1 mixed solution, and degreasing 2.5 h abandon supernatant after centrifugal 15 min of 1000 * g, under similarity condition again after degreasing 1 time, centrifugal 15 min of 1000 * g, abandon supernatant, finally add 50 mL ether defatting 2.5 h, the centrifugal supernatant of abandoning, gained white precipitate is just degreasing HDL goods.
4, the dissolving of degreasing HDL: the HDL after degreasing is dissolved in 6 mol/L urea, 10mmol/L Tris-HCl 10 mmol/L dithiothreitol (DTT) 100 mL mixing solutionss, and pH 8.0, are put in 4 ℃ of refrigerator overnight.
5, the dialysis of degreasing HDL solution and concentrated: above-mentioned HDL solution is dialysed with 6 mol/L urea-10 mmol/L Tris-HCl-1 mmol/L EDTA mixing solutionss, after removing organic solvent wherein, with the polyethylene glycol 6000 of 500 g/L, be concentrated into 2-3mL, to improve the concentration of HDL solution.
6, the extraction of chicken ApoA1: column volume 1.5 * 40.0 cm, after DEAE Sepharose CL-6B dress post, with 0.03 mol/L Tris-6 mol/L urea, regulating pH is 8.0, and mixing solutions balance, gets after approximately 10 mL sample upper props, first with the 0.03 mol/L Tris-6 mol/L urea mixed solution that is equivalent to 2 times of bed volumes, wash post, then with 0～0.5 mol/L NaCl, carry out gradient elution, wash fast 1 mL/min, every pipe is collected 6 mL; Gradient liquid composition: A liquid: 0.03 mol/L Tris-HC-6 mol/L urea, pH is 8.0, mixed solution 500 mL; B liquid: 0.03 mol/L Tris-HC-6 mol/L urea-0.5 mol/L NaCl, pH is 8.0, mixed solution 500 mL.280 nm Ultraviolet Detectors detect, and merge near several pipes of each summit, after concentrating with sucrose, at-20 ℃ of freezer storages.
7, the evaluation of chicken ApoA1: adopt separation gel T=12.5%, C=2.6%, pH 8.8; Concentrated glue: T=5%, C=2.6%, pH 6.8; In 1 L electrode buffer, contain 3 g Tris, 14.4 g glycine, 1 g SDS, pH 8.3.While starting electrophoresis, concentrated glue is used 120 V voltage stabilizings, uses 240 V voltage stabilizings after entering separation gel.Use 1 g/L coomassie brilliant blue R_250 3 h that dye after electrophoresis completes, with 500 mL/L methyl alcohol and the decolouring of 100 mL/L glacial acetic acids, urea-SDS-PAGE electrophoresis result ApoA1 only presents single band subsequently, and the ApoA1 that preparation be described has been that electrophoresis is pure.
Described precipitation agent I is by phospho-wolframic acid 0.44 g, MgCl
26H
2o 1.1 g and 1mol/L NaOH 1.3 ml add distilled water to 100 ml, regulate PH=6.2.
Described precipitation agent II is by phospho-wolframic acid 4.4 g, MgCl
26H
2o 11.0 g and 1 mol/L NaOH 13.0 ml add distilled water to 100 ml, regulate PH=6.2.
The present invention has following beneficial effect: the method for its separation and purification is simple, gives the measurement of apoA I and further studies and lay the foundation.
Embodiment:
This embodiment is taked following technical scheme: the separation purification method of its chicken ApoA1:
1, the collection of chicken serum: venous blood collection under wing, after blood sampling, the syringe needle of taking away, gently in injecting tube.For preventing breaking of hemocyte, the syringe that cleans up and disinfect and test tube infiltrate with 0.85% physiological saline in advance, prevent blood clot and glass tube walls adhesion, cause serum to separate out bad.Standing 37 ℃ of incubator 1 h of test tube 45° angle, then put standing 3～4 h in 4 ℃ of refrigerators draw serum with capillary burette after blood coagulation blood clot retraction, then with centrifugal 10 min of 1000 * g, get in rearmounted-20 ℃ of refrigerators of supernatant serum packing, to add NaN3 and save backup.
2, the preparation of serum high-density LP: get serum 1.0 ml and add 1.0 ml precipitation agent I to mix, standing 10 min, centrifugal 15 min of 1000 * g, in sucking-off, honest and upright and thrifty 1.8 ml add 0.42 ml precipitation agent II to mix, standing 5 min, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is dissolved in 0.1 ml 12%NaCl and 1.0 ml 0.02 mol/L Tris-HCL liquid, PH=8.6, add standing 5 min of 0.26 ml precipitation agent II, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is carried out to redissolve, precipitation and again dissolution process are once, finally obtain the solution of crude product that contains HDL.
3, the degreasing of HDL: treat that by prepared degreasing HDL solution is in the situation that constantly stirring, with liquid-transfering gun, be dropwise incorporated in 60 mL acetone-ethanol of-20 ℃ of precoolings, volume ratio is in 1:1 mixed solution, and degreasing 2.5 h abandon supernatant after centrifugal 15 min of 1000 * g, under similarity condition again after degreasing 1 time, centrifugal 15 min of 1000 * g, abandon supernatant, finally add 50 mL ether defatting 2.5 h, the centrifugal supernatant of abandoning, gained white precipitate is just degreasing HDL goods.
4, the dissolving of degreasing HDL: the HDL after degreasing is dissolved in 6 mol/L urea, 10mmol/L Tris-HCl 10 mmol/L dithiothreitol (DTT) 100 mL mixing solutionss, and pH 8.0, are put in 4 ℃ of refrigerator overnight.
5, the dialysis of degreasing HDL solution and concentrated: above-mentioned HDL solution is dialysed with 6 mol/L urea-10 mmol/L Tris-HCl-1 mmol/L EDTA mixing solutionss, after removing organic solvent wherein, with the polyethylene glycol 6000 of 500 g/L, be concentrated into 2-3mL, to improve the concentration of HDL solution.
6, the extraction of chicken ApoA1: column volume 1.5 * 40.0 cm, after DEAE Sepharose CL-6B dress post, with 0.03 mol/L Tris-6 mol/L urea, regulating pH is 8.0, and mixing solutions balance, gets after approximately 10 mL sample upper props, first with the 0.03 mol/L Tris-6 mol/L urea mixed solution that is equivalent to 2 times of bed volumes, wash post, then with 0～0.5 mol/L NaCl, carry out gradient elution, wash fast 1 mL/min, every pipe is collected 6 mL; Gradient liquid composition: A liquid: 0.03 mol/L Tris-HC-6 mol/L urea, pH is 8.0, mixed solution 500 mL; B liquid: 0.03 mol/L Tris-HC-6 mol/L urea-0.5 mol/L NaCl, pH is 8.0, mixed solution 500 mL.280 nm Ultraviolet Detectors detect, and merge near several pipes of each summit, after concentrating with sucrose, at-20 ℃ of freezer storages.
7, the evaluation of chicken ApoA1: adopt separation gel T=12.5%, C=2.6%, pH 8.8; Concentrated glue: T=5%, C=2.6%, pH 6.8; In 1 L electrode buffer, contain 3 g Tris, 14.4 g glycine, 1 g SDS, pH 8.3.While starting electrophoresis, concentrated glue is used 120 V voltage stabilizings, uses 240 V voltage stabilizings after entering separation gel.Use 1 g/L coomassie brilliant blue R_250 3 h that dye after electrophoresis completes, with 500 mL/L methyl alcohol and the decolouring of 100 mL/L glacial acetic acids, urea-SDS-PAGE electrophoresis result ApoA1 only presents single band subsequently, and the ApoA1 that preparation be described has been that electrophoresis is pure.
Described precipitation agent I is by phospho-wolframic acid 0.44 g, MgCl
26H
2o 1.1 g and 1mol/L NaOH 1.3 ml add distilled water to 100 ml, regulate PH=6.2.
Described precipitation agent II is by phospho-wolframic acid 4.4 g, MgCl
26H
2o 11.0 g and 1 mol/L NaOH 13.0 ml add distilled water to 100 ml, regulate PH=6.2.
This embodiment has following beneficial effect: the method for its separation and purification is simple, gives the measurement of apoA I and further studies and lay the foundation.
Claims (4)
1. the separation and purification of chicken ApoA1 and an authentication method, is characterized in that its separation purification method is:
(1) collection of chicken serum: venous blood collection under wing, after blood sampling, the syringe needle of taking away, gently in injecting tube;
For preventing breaking of hemocyte, the syringe that cleans up and disinfect and test tube infiltrate with 0.85% physiological saline in advance, prevent blood clot and glass tube walls adhesion, cause serum to separate out bad;
Standing 37 ℃ of incubator 1 h of test tube 45° angle, then put standing 3～4 h in 4 ℃ of refrigerators, after blood coagulation blood clot retraction, with capillary burette, draw serum, then with centrifugal 10 min of 1000 * g, get in rearmounted-20 ℃ of refrigerators of supernatant serum packing and add NaN
3save backup;
(2) preparation of serum high-density LP: get serum 1.0 ml and add 1.0 ml precipitation agent I to mix, standing 10 min, centrifugal 15 min of 1000 * g, in sucking-off, honest and upright and thrifty 1.8 ml add 0.42 ml precipitation agent II to mix, standing 5 min, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is dissolved in 0.1 ml 12%NaCl and 1.0 ml 0.02 mol/L Tris-HCL liquid, PH=8.6, add standing 5 min of 0.26 ml precipitation agent II, 1000 * g is centrifugal, and 10 min abandon supernatant liquor, precipitation is carried out to redissolve, precipitation and again dissolution process are once, finally obtain the solution of crude product that contains HDL,
(3) degreasing of HDL: treat that by prepared degreasing HDL solution is in the situation that constantly stirring, with liquid-transfering gun, be dropwise incorporated in 60 mL acetone-ethanol of-20 ℃ of precoolings, volume ratio is in 1:1 mixed solution, and degreasing 2.5 h abandon supernatant after centrifugal 15 min of 1000 * g, under similarity condition again after degreasing 1 time, centrifugal 15 min of 1000 * g, abandon supernatant, finally add 50 mL ether defatting 2.5 h, the centrifugal supernatant of abandoning, gained white precipitate is just degreasing HDL goods;
(4) dissolving of degreasing HDL: the HDL after degreasing is dissolved in 6 mol/L urea, 10mmol/L Tris-HCl 10 mmol/L dithiothreitol (DTT) 100 mL mixing solutionss, and pH 8.0, are put in 4 ℃ of refrigerator overnight;
(5) dialysis of degreasing HDL solution and concentrated: above-mentioned HDL solution is dialysed with 6 mol/L urea-10 mmol/L Tris-HCl-1 mmol/L EDTA mixing solutionss, after removing organic solvent wherein, with the polyethylene glycol 6000 of 500 g/L, be concentrated into 2-3mL, to improve the concentration of HDL solution;
(6) extraction of chicken ApoA1: column volume 1.5 * 40.0 cm, after DEAE Sepharose CL-6B dress post, with 0.03 mol/L Tris-6 mol/L urea, regulating pH is 8.0, and mixing solutions balance, gets after approximately 10 mL sample upper props, first with the 0.03 mol/L Tris-6 mol/L urea mixed solution that is equivalent to 2 times of bed volumes, wash post, then with 0～0.5 mol/L NaCl, carry out gradient elution, wash fast 1 mL/min, every pipe is collected 6 mL; Gradient liquid composition: A liquid: 0.03 mol/L Tris-HC-6 mol/L urea, pH is 8.0, mixed solution 500 mL; B liquid: 0.03 mol/L Tris-HC-6 mol/L urea-0.5 mol/L NaCl, pH is 8.0, mixed solution 500 mL;
280 nm Ultraviolet Detectors detect, and merge near several pipes of each summit, after concentrating with sucrose, at-20 ℃ of freezer storages.
2. a kind of separation and purification of chicken ApoA1 and authentication method according to claim 1, is characterized in that its Purity method method is: adopt separation gel T=12.5%, and C=2.6%, pH 8.8; Concentrated glue: T=5%, C=2.6%, pH 6.8; In 1 L electrode buffer, contain 3 g Tris, 14.4 g glycine, 1 g SDS, pH 8.3;
While starting electrophoresis, concentrated glue is used 120 V voltage stabilizings, uses 240 V voltage stabilizings after entering separation gel;
Use 1 g/L coomassie brilliant blue R_250 3 h that dye after electrophoresis completes, with 500 mL/L methyl alcohol and the decolouring of 100 mL/L glacial acetic acids, urea-SDS-PAGE electrophoresis result ApoA1 only presents single band, illustrate that the ApoA1 of preparing is that electrophoresis is pure subsequently.
3. a kind of separation and purification of chicken ApoA1 and authentication method according to claim 1, is characterized in that described precipitation agent I is by phospho-wolframic acid 0.44 g, MgCl
26H
2o 1.1 g and 1mol/L NaOH 1.3 ml add distilled water to 100 ml, regulate PH=6.2.
4. a kind of separation and purification of chicken ApoA1 and authentication method according to claim 1, is characterized in that described precipitation agent II is by phospho-wolframic acid 4.4 g, MgCl
26H
2o 11.0 g and 1 mol/L NaOH 13.0 ml add distilled water to 100 ml, regulate PH=6.2.
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| CN201310708396.0A CN103641910A (en) | 2013-06-27 | 2013-12-20 | Separation and purification method and identification method of chicken apolipoprotein AI |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320086A (en) * | 2019-07-09 | 2019-10-11 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
| CN112665950A (en) * | 2021-01-28 | 2021-04-16 | 安徽佳创生物科技有限公司 | Method for detecting high-density lipoprotein in serum |
-
2013
- 2013-12-20 CN CN201310708396.0A patent/CN103641910A/en active Pending
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| 李浩棠等: "鸡血清载脂蛋白AI的分离纯化及其抗血清的制备", 《中国兽医科技》 * |
| 胡国良等: "蛋鸡血清载脂蛋白AII的分离纯化及其抗血清的制备", 《中国动物检疫》 * |
| 胡国良等: "蛋鸡血清载脂蛋白AII的分离纯化及初步鉴定", 《江西农业大学学报》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110320086A (en) * | 2019-07-09 | 2019-10-11 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
| CN110320086B (en) * | 2019-07-09 | 2020-04-14 | 上海宝藤生物医药科技股份有限公司 | Lipoprotein electrophoresis staining solution and preparation method and application thereof |
| CN112665950A (en) * | 2021-01-28 | 2021-04-16 | 安徽佳创生物科技有限公司 | Method for detecting high-density lipoprotein in serum |
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Application publication date: 20140319 |