CN110759992A - Method for extracting and purifying albumin in rabbit blood - Google Patents
Method for extracting and purifying albumin in rabbit blood Download PDFInfo
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- CN110759992A CN110759992A CN201910891515.8A CN201910891515A CN110759992A CN 110759992 A CN110759992 A CN 110759992A CN 201910891515 A CN201910891515 A CN 201910891515A CN 110759992 A CN110759992 A CN 110759992A
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- 210000004369 blood Anatomy 0.000 title claims abstract description 58
- 239000008280 blood Substances 0.000 title claims abstract description 58
- 102000009027 Albumins Human genes 0.000 title claims abstract description 45
- 108010088751 Albumins Proteins 0.000 title claims abstract description 45
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 30
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 30
- 238000011033 desalting Methods 0.000 claims abstract description 28
- 239000000706 filtrate Substances 0.000 claims abstract description 27
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 24
- 239000003480 eluent Substances 0.000 claims abstract description 20
- 230000006920 protein precipitation Effects 0.000 claims abstract description 17
- 238000003756 stirring Methods 0.000 claims abstract description 13
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 238000011097 chromatography purification Methods 0.000 claims abstract description 8
- 238000012799 strong cation exchange Methods 0.000 claims abstract description 8
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 7
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 7
- 238000010790 dilution Methods 0.000 claims abstract description 7
- 239000012895 dilution Substances 0.000 claims abstract description 7
- 239000012535 impurity Substances 0.000 claims abstract description 7
- 239000012528 membrane Substances 0.000 claims abstract description 7
- 239000011259 mixed solution Substances 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- 239000012716 precipitator Substances 0.000 claims abstract description 7
- 230000001112 coagulating effect Effects 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 3
- 238000010828 elution Methods 0.000 claims description 25
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 17
- 210000002966 serum Anatomy 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000001035 drying Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 7
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- 239000008213 purified water Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical group N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 238000001291 vacuum drying Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 2
- 238000010612 desalination reaction Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a first step, rabbit blood pretreatment, fresh blood is taken out from a rabbit body, anticoagulant is added into the blood to prevent the blood from coagulating, impurities are filtered, the blood is placed in a centrifuge for centrifugal treatment, after physiological saline is added for dilution, precipitator is added while stirring is carried out until complete precipitation, protein precipitation liquid is obtained, and the protein precipitation liquid is passed through a deep filter to prepare filtrate; desalting the filtrate, and performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium; desalting again; pouring the mixture into a centrifuge for centrifugation; step four, chromatography, namely performing chromatography operation on the supernatant in the step three; and fifthly, performing ultrafiltration, namely performing ultrafiltration on the mixed solution. The purity of the albumin extracted by the method is high, the purity of the extracted albumin can reach 99%, and compared with the traditional method, the extraction efficiency can be improved by at least 25%.
Description
Technical Field
The invention relates to the technical field of protein extraction, in particular to a method for extracting and purifying albumin in rabbit blood.
Background
Albumin, also known as albumin, is the most important protein in human plasma and maintains the nutrition and osmotic pressure of the body. The concentration reaches 38-48g/L, and accounts for about 50% of the total plasma protein. The rabbit blood has high nutritive value, can be processed into various products for eating and medicine, or can be used as animal feed of livestock and poultry, the albumin in the rabbit blood is less extracted, and the prior art has insufficient extraction purity of the albumin in the rabbit blood, so that the method for extracting and purifying the albumin in the rabbit blood is provided.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for extracting and purifying albumin in rabbit blood.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting and purifying albumin in rabbit blood is designed, and comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent the blood from coagulating, filtering impurities, then placing the blood in a centrifuge for centrifugal treatment at the temperature of 4-10 ℃, the rotating speed of 3000 plus 4500r/min, and the centrifugation time of 10-20min, collecting serum on the upper layer, placing the serum at room temperature for standing and storing for 30-60min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation is achieved to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, and heating the filtrate in the step one in a water area at the temperature of 55-70 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating the rabbit blood albumin, standing the fine elution liquid in the step two for 15-24h at 4-10 ℃, pouring the solution into a centrifuge, centrifuging the solution at the rotating speed of 5000-
Step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
Preferably, in step one, the precipitating agent is ammonium sulfate, and the final concentration of ammonium sulfate added into serum is 35-70%.
Preferably, in step two, after desalting by G25 desalting chromatography, desalting and concentrating the fine eluate, filtering the concentrated fine eluate with a filter to remove bacteria, wherein the filter pore diameter is 0.05-1.2 μm, desalting and sterilizing.
Preferably, in the fifth step, the drying method is one of a vacuum drying method, a low-temperature freeze drying method or a low-temperature freeze vacuum drying method.
The method for extracting and purifying albumin in rabbit blood provided by the invention has the beneficial effects that: the method for extracting and purifying albumin from rabbit blood comprises the steps of firstly carrying out centrifugal separation on fresh blood to obtain protein precipitation liquid, then sequentially carrying out ultrafiltration membrane filtration and strong cation exchange medium chromatography purification on the protein precipitation liquid in the second step, and carrying out desalination, centrifugal layer chromatography, ultrafiltration and drying on the protein precipitation liquid by a G25 desalination chromatography method to obtain pure albumin.
Detailed Description
The invention is further illustrated by the following examples.
Example one
A method for extracting and purifying albumin in rabbit blood comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent the blood from coagulating, filtering impurities, centrifuging the blood in a centrifuge at the temperature of 4 ℃, the rotating speed of 3000 r/min for 11min, collecting serum on the upper layer, standing and storing the serum at room temperature for 31min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, and heating the filtrate in the step one in a water area at 55 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating rabbit blood albumin, standing the fine elution liquid in the step two for 15 hours at the standing temperature of 4 ℃, pouring the fine elution liquid into a centrifuge, centrifuging the fine elution liquid for 32 minutes at the rotating speed of 5000r/min, and taking supernatant;
step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
Example two
A method for extracting and purifying albumin in rabbit blood comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent the blood from coagulating, filtering impurities, centrifuging the blood in a centrifuge at the temperature of 6 ℃, the rotation speed of 3500 r/min for 14min, collecting serum on the upper layer, standing and storing the serum at room temperature for 40min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, and heating the filtrate in the step one in a water area at 63 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating rabbit blood albumin, standing the fine elution liquid in the step two for 18 hours at the standing temperature of 4-10 ℃, pouring the fine elution liquid into a centrifuge, centrifuging the fine elution liquid for 30-50min at the rotating speed of 5300r/min, and taking supernate
Step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
EXAMPLE III
A method for extracting and purifying albumin in rabbit blood comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent blood coagulation, filtering impurities, centrifuging the blood in a centrifuge at the temperature of 6 ℃, the rotation speed of 4100 r/min for 15min, collecting serum on the upper layer, standing and storing the serum at room temperature for 43min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, namely heating the filtrate in the step one in a water area at the heating temperature of 62 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating rabbit blood albumin, standing the fine elution liquid in the step two for 20 hours at 7 ℃, pouring the fine elution liquid into a centrifuge, centrifuging for 40min at a rotating speed of 5500r/min, and taking supernatant
Step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
Example four
A method for extracting and purifying albumin in rabbit blood comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent the blood from coagulating, filtering impurities, centrifuging the blood in a centrifuge at the temperature of 9 ℃, the rotating speed of 4400 r/min for 20min, collecting serum on the upper layer, standing and storing the serum at room temperature for 60min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, and heating the filtrate in the step one in a water area at 65 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating the rabbit blood albumin, standing the fine elution liquid in the step two for 23 hours at the standing temperature of 10 ℃, pouring the fine elution liquid into a centrifuge, centrifuging the fine elution liquid for 50 minutes at the rotating speed of 5900r/min, and taking the supernatant
Step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts of the present invention are equivalent to or changed within the technical scope of the present invention.
Claims (4)
1. A method for extracting and purifying albumin in rabbit blood is characterized by comprising the following steps: the method comprises the following steps:
step one, pretreatment of rabbit blood, namely taking fresh blood from a rabbit body, adding an anticoagulant into the blood to prevent the blood from coagulating, filtering impurities, then placing the blood in a centrifuge for centrifugal treatment at the temperature of 4-10 ℃, the rotating speed of 3000 plus 4500r/min, and the centrifugation time of 10-20min, collecting serum on the upper layer, placing the serum at room temperature for standing and storing for 30-60min, adding physiological saline for dilution, adding a precipitator while stirring until complete precipitation is achieved to obtain a protein precipitation solution, and passing the protein precipitation solution through a deep filter to obtain a filtrate;
step two, desalting the filtrate, and heating the filtrate in the step one in a water area at the temperature of 55-70 ℃; performing ultrafiltration concentration on the filtrate by using an ultrafiltration membrane, and using citrate as a buffer solution to obtain a crude eluent; carrying out chromatographic purification on the crude eluent by using a strong cation exchange medium to prepare a fine eluent; the fine elution liquid contains protein with higher purity, and then the fine elution liquid is desalted by a G25 desalting chromatography method until the desalting is complete;
step three, separating the rabbit blood albumin, standing the fine elution liquid in the step two for 15-24h at 4-10 ℃, pouring the solution into a centrifuge, centrifuging the solution at the rotating speed of 5000-
Step four, chromatography, namely performing chromatography operation on the supernatant in the step three, and collecting protein separated out by layers;
and fifthly, ultrafiltration, namely diluting the protein collected in the fourth step with purified water, stirring the mixture at room temperature, performing ultrafiltration on the mixed solution to obtain an albumin precipitate, and drying the albumin precipitate to obtain the albumin.
2. The method of claim 1, wherein the precipitating agent is ammonium sulfate, and the final concentration of ammonium sulfate added to the serum is 35-70%.
3. The method for extracting and purifying albumin in rabbit blood as claimed in claim 1, wherein in step two, after desalting by desalting chromatography with G25, desalting and concentrating the concentrated fine eluate, filtering and sterilizing the concentrated fine eluate with a filter having a pore size of 0.05-1.2 μm, and desalting and sterilizing again.
4. The method of claim 1, wherein in the step five, the drying method is one of a vacuum drying method, a low-temperature freeze drying method or a low-temperature freeze vacuum drying method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910891515.8A CN110759992A (en) | 2019-09-20 | 2019-09-20 | Method for extracting and purifying albumin in rabbit blood |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910891515.8A CN110759992A (en) | 2019-09-20 | 2019-09-20 | Method for extracting and purifying albumin in rabbit blood |
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| CN110759992A true CN110759992A (en) | 2020-02-07 |
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| CN201910891515.8A Withdrawn CN110759992A (en) | 2019-09-20 | 2019-09-20 | Method for extracting and purifying albumin in rabbit blood |
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- 2019-09-20 CN CN201910891515.8A patent/CN110759992A/en not_active Withdrawn
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