CN106977596A - A kind of method for extracting bovine serum albumin - Google Patents
A kind of method for extracting bovine serum albumin Download PDFInfo
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- CN106977596A CN106977596A CN201710365717.XA CN201710365717A CN106977596A CN 106977596 A CN106977596 A CN 106977596A CN 201710365717 A CN201710365717 A CN 201710365717A CN 106977596 A CN106977596 A CN 106977596A
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- serum albumin
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- 238000000034 method Methods 0.000 title claims abstract description 38
- 108091003079 Bovine Serum Albumin Proteins 0.000 title claims abstract description 36
- 229940098773 bovine serum albumin Drugs 0.000 title claims abstract description 36
- 239000000463 material Substances 0.000 claims abstract description 78
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 51
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 51
- 239000007787 solid Substances 0.000 claims abstract description 29
- 210000004369 blood Anatomy 0.000 claims abstract description 24
- 239000008280 blood Substances 0.000 claims abstract description 24
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 43
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 38
- 239000006228 supernatant Substances 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 33
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 28
- 210000002381 plasma Anatomy 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 150000001875 compounds Chemical class 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 17
- 239000002184 metal Substances 0.000 claims description 16
- 229910052751 metal Inorganic materials 0.000 claims description 16
- 239000003146 anticoagulant agent Substances 0.000 claims description 15
- 229940127219 anticoagulant drug Drugs 0.000 claims description 15
- 230000008021 deposition Effects 0.000 claims description 15
- 239000007800 oxidant agent Substances 0.000 claims description 15
- 230000001590 oxidative effect Effects 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 14
- 239000012024 dehydrating agents Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 14
- 230000006920 protein precipitation Effects 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 14
- 239000011260 aqueous acid Substances 0.000 claims description 12
- 238000001556 precipitation Methods 0.000 claims description 12
- 241000283690 Bos taurus Species 0.000 claims description 11
- 239000012266 salt solution Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims description 7
- 102000002932 Thiolase Human genes 0.000 claims description 7
- 108060008225 Thiolase Proteins 0.000 claims description 7
- 230000003750 conditioning effect Effects 0.000 claims description 7
- 235000011187 glycerol Nutrition 0.000 claims description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 7
- 229910052742 iron Inorganic materials 0.000 claims description 7
- -1 iron ion Chemical class 0.000 claims description 7
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 claims description 7
- 230000003647 oxidation Effects 0.000 claims description 7
- 238000007254 oxidation reaction Methods 0.000 claims description 7
- 239000012460 protein solution Substances 0.000 claims description 7
- 230000035484 reaction time Effects 0.000 claims description 7
- 210000003462 vein Anatomy 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000002270 dispersing agent Substances 0.000 claims description 4
- 229920002401 polyacrylamide Polymers 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 3
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims 2
- 239000007788 liquid Substances 0.000 claims 2
- 240000004307 Citrus medica Species 0.000 claims 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Natural products CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims 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 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 235000009508 confectionery Nutrition 0.000 claims 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052938 sodium sulfate Inorganic materials 0.000 claims 1
- 235000011152 sodium sulphate Nutrition 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 4
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000002253 acid Substances 0.000 description 8
- 239000001509 sodium citrate Substances 0.000 description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 8
- 125000001931 aliphatic group Chemical group 0.000 description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 6
- 229930195725 Mannitol Natural products 0.000 description 6
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 239000000594 mannitol Substances 0.000 description 6
- 235000010355 mannitol Nutrition 0.000 description 6
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical class ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 5
- 239000012535 impurity Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 244000144977 poultry Species 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- WXHLLJAMBQLULT-UHFFFAOYSA-N 2-[[6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-yl]amino]-n-(2-methyl-6-sulfanylphenyl)-1,3-thiazole-5-carboxamide;hydrate Chemical compound O.C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1S WXHLLJAMBQLULT-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000004035 construction material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229960005480 sodium caprylate Drugs 0.000 description 1
- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical compound [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 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)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to protein extracting process field, more particularly to a kind of method for extracting bovine serum albumin；Comprise the following steps：The processing of blood separation solid protein material, the processing of solution material, batch mixing；Using the extracting method of the present invention, the effective recovery rate for improving bovine serum albumin, the extraction rate reached of bovine serum albumin is to 95.6%, purity is up to more than 99.6%, the significant quality for improving bovine serum albumin, demand of the existing market to bovine serum albumin effectively is met, effectively the medical value of lifting bovine serum albumin.
Description
Technical field
The present invention relates to protein extracting process field, more particularly to a kind of method for extracting bovine serum albumin.
Background technology
Livestock and poultry blood is one of important byproduct of livestock and poultry, includes abundant nutriment and various bioactivators,
Haemocyanin is protein most abundant in blood plasma, is the carrier of aliphatic acid in blood, and each protein molecular can carry seven
Individual fatty acid molecule, these fatty acid molecules are combined in the gap of albumen, and their afterbodys rich in carbon are imbedded in the inside safety
The hydrone of surrounding is avoided on ground, and aliphatic acid is the composition for building lipid, and lipid constitutes cell peripheral and intracellular all
Biomembrane, aliphatic acid is the carrier of the important sources of energy, aliphatic acid or liposoluble vitamin again, when somagenic need energy
Or when needing construction material, fat cell is just discharged into aliphatic acid in blood, and aliphatic acid is obtained by haemocyanin, and transported
The position of needs is sent to, haemocyanin can equally carry many other water insoluble molecules, just because of haemocyanin is
So generally it is present in blood and is easily purified, so it turns into one of protein that scientist studies earliest, ox
Haemocyanin is widely used in terms of animal cell culture, diagnostic reagent, food emulsification, can carried as a kind of function factor
High T lymphocytes in human body quantity and antioxidation activity, with great medical science Development volue.
The preparation method of albumin is many in cow's serum, is mainly carried using salting out method, organic solvent precipitation method and heat shock method
Take, after salting out method ammonium sulfate precipitation, refine and obtain through octanoic acid processing；Organic solvent precipitation method is used for human blood system earliest
The production of product, using the low-k property and protein of ethanol under certain temperature, pH, ionic strength and concentration conditions
Different solubility separated plasma protein in different concentration ethanol, can produce a variety of plasma proteins；Heat shock method is to take
Ox blood after sodium citrate anti-freezing centrifugation with blood plasma is obtained, and heating adds Sodium Caprylate, adjusts after pH value plus ethanol, then through filtering, taking off
Salt, concentration, it is lyophilized after albumin in cow's serum, it is still, existing to be used to prepare the ox obtained by the method for bovine serum albumin(BSA)
Seralbumin yield or purity are all than relatively low, and such as fat of the impurity containing higher concentration in the bovine serum albumin(BSA) extracted
Fat acid, protease, nuclease and endotoxin, this kind of impurity limit further applying for bovine serum albumin(BSA).
The content of the invention
The present invention is in order to solve the above technical problems, there is provided a kind of method for extracting bovine serum albumin.
Realized particular by following technical scheme：
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20~30% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12~18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.5~5 to add hydrochloric acid regulation supernatant pH value, is added
The dehydrating agent of supernatant quality 4~8%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
(3) solution material is handled：By solution material, dispersant, oxidant by 1.2~1.8: 0.1~0.2: 0.35~0.45
Mass ratio is mixed, and obtains compound, is carried out oxidation processes, is added 0.12~0.15% metal agent of mixture quality, is centrifuged,
Supernatant is taken, the protein precipitation agent of supernatant quality 10~15% is added, 12~18h is stood, centrifuging and taking precipitation is dried
Material 2；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Described protein deposition agent, be 12~18% by content sulfosalisylic aqueous acid and content be 10~20%
Trichloroacetic acid solution be mixed to prepare by 0.8~1.2: 1.2~1.8 mass ratio.
Described anti-coagulants, be by sodium citrate and concentration for 0.012~0.18% salt solution by 1: 0.3~0.6 matter
Amount is than mixing.
Described dehydrating agent, is to mix mannitol and glycerine by 0.2~0.3: 0.12~0.18 mass ratio.
Described dispersant is one kind in lauryl sodium sulfate, polyacrylamide and methyl anyl alcohol.
Described oxidant, it by concentration is 22~28ml/L ozone to be, thiolase is by 1.2~1.6: 0.25~0.35
Mass ratio is mixed.
Described oxidation processes, are that material is heated into 34~36 DEG C, the reaction time is 2.5~3.5h.
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 0.8~1.2: 0.1~0.2:
0.36~0.48.
Described protein precipitation agent, be by mass mixings such as methanol and ethanol, with hydrochloric acid conditioning solution pH value be 4.5~
5, mix thoroughly.
In summary, the beneficial effects of the present invention are：It is effective to improve cow's serum egg using the extracting method of the present invention
White recovery rate, the extraction rate reached of bovine serum albumin is to 95.6%, and purity significantly improves cow's serum egg up to more than 99.6%
White quality, effectively meets demand of the existing market to bovine serum albumin, effectively the medical value of lifting bovine serum albumin.
Wherein, the separation of blood plasma and haemocyte is first carried out to blood, reduces the difficulty extracted, first pass through sulfosalicylic acid and
Protein deposition agent is made by suitable concn proportioning in trichloroacetic acid, passes through the combination between the positive and negative charge of institute's band so that blood
Protein in slurry largely settles down, then the anti-coagulants being made up of sodium citrate and salt solution, forms soluble chelating thing,
Increase solution osmotic pressure, it is to avoid fibrinogen formation fibrin, it is to avoid blood plasma solidification, meanwhile, also destroy blood
Fibrinogen in slurry, the purity for raising bovine serum albumin provides basis；When handling solution material, wherein also containing portion
Point protein and substantial amounts of impurity, therefore, by solution material carry out oxidation processes, by regulate and control oxidation be by temperature, with
And using suitable oxidant so that the aliphatic acid in blood plasma obtains oxidation Decomposition to greatest extent, improves bovine serum albumin
Purity, then by situation of the specific metal to the specific destruction of enzyme, it is effective to remove in blood plasma using the metal of proper ratio
The impurity such as original protease, nuclease, further increase the purity of bovine serum albumin.
Embodiment
The embodiment to the present invention is described in further detail below, but the invention is not limited in these realities
Mode is applied, any improvement or replacement on the present embodiment essence spirit still falls within the claims in the present invention claimed
Scope.
Embodiment 1
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 12% by content and three chloroethenes that content is 10%
Aqueous acid is mixed to prepare by 0.8: 1.2 mass ratio；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.5 to add hydrochloric acid regulation supernatant pH value, adds supernatant
The dehydrating agent of quality 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.3 mass ratio for 0.012% salt solution；
Described dehydrating agent, is to mix mannitol and glycerine by 0.2: 0.12 mass ratio；
(3) solution material is handled：Solution material, lauryl sodium sulfate, oxidant are mixed by 1.2: 0.1: 0.35 mass ratio
Close, obtain compound, by mixed material heating to 34 DEG C, the reaction time is 2.5h, add 0.12% metal of mixture quality
Agent, centrifugation, takes supernatant, adds the protein precipitation agent of supernatant quality 10%, stands 12h, and centrifuging and taking precipitation is dried
Material 2；
Described oxidant, is to mix concentration by 1.2: 0.25 mass ratio for 22ml/L ozone, thiolase；
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 0.8: 0.1: 0.36；
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.5 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 2
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 30% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 18% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1.2: 1.8 mass ratio；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 8%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.6 mass ratio for 0.18% salt solution；
Described dehydrating agent, is to mix mannitol and glycerine by 0.3: 0.18 mass ratio；
(3) solution material is handled：Solution material, polyacrylamide, oxidant are mixed by 1.8: 0.2: 0.45 mass ratio, obtained
Compound, by mixed material heating to 36 DEG C, the reaction time is 3.5h, adds 0.15% metal agent of mixture quality, from
The heart, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 18h, and centrifuging and taking is precipitated, and 2 must be expected by drying；
Described oxidant, is to mix concentration by 1.6: 0.35 mass ratio for 28ml/L ozone, thiolase；
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.2: 0.48；
Described protein precipitation agent, is, by mass mixings such as methanol and ethanol, to be 5 with hydrochloric acid conditioning solution pH value, mix
It is even；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 3
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 25% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 15% by content and three chloroethenes that content is 15%
Aqueous acid is mixed to prepare by 1: 1.5 mass ratio；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
15% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 4.8 to add hydrochloric acid regulation supernatant pH value, adds supernatant
The dehydrating agent of quality 6%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.5 mass ratio for 0.015% salt solution；
Described dehydrating agent, is to mix mannitol and glycerine by 0.25: 0.15 mass ratio；
(3) solution material is handled：Solution material, methyl anyl alcohol, oxidant are mixed by 1.5: 0.15: 0.4 mass ratio, obtain mixed
Material is closed, by mixed material heating to 35 DEG C, the reaction time is 3h, adds 0.13% metal agent of mixture quality, centrifugation takes
Supernatant, adds the protein precipitation agent of supernatant quality 13%, stands 15h, and centrifuging and taking is precipitated, and 2 must be expected by drying；
Described oxidant, is to mix concentration by 1.4: 0.3 mass ratio for 5ml/L ozone, thiolase；
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1: 0.15: 0.42；
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.8 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 4
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 18% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1: 1.2 mass ratio；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
12% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.3 mass ratio for 0.18% salt solution；
Described dehydrating agent, is to mix mannitol and glycerine by 0.2: 0.15 mass ratio；
(3) solution material is handled：Solution material, polyacrylamide, oxidant are mixed by 1.2: 0.1: 0.45 mass ratio, obtained
Compound, by mixed material heating to 34 DEG C, the reaction time is 3.5h, adds 0.12% metal agent of mixture quality, from
The heart, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 12h, and centrifuging and taking is precipitated, and 2 must be expected by drying；
Described oxidant, is to mix concentration by 1.6: 0.35 mass ratio for 22ml/L ozone, thiolase；
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.1: 0.4；
Described protein precipitation agent, is, by mass mixings such as methanol and ethanol, to be 5 with hydrochloric acid conditioning solution pH value, mix
It is even；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Embodiment 5
A kind of method for extracting bovine serum albumin, comprises the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma are separated into through blood separator, is added in blood plasma
Enter 20% protein deposition agent of plasma purity, separate, obtain solid protein material and solution material；
Described protein deposition agent, is the sulfosalisylic aqueous acid for being 12% by content and three chloroethenes that content is 20%
Aqueous acid is mixed to prepare by 1.2: 1.2 mass ratio；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, solid protein material quality is added
18% anti-coagulants, stirring shakes up, and centrifuges, and takes supernatant, and it is 5 to add hydrochloric acid regulation supernatant pH value, adds supernatant matter
The dehydrating agent of amount 4%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
Described anti-coagulants, is to mix sodium citrate and concentration by 1: 0.6 mass ratio for 0.18% salt solution；
Described dehydrating agent, is to mix mannitol and glycerine by 0.3: 0.12 mass ratio；
(3) solution material is handled：Solution material, lauryl sodium sulfate, oxidant are mixed by 1.5: 0.2: 0.35 mass ratio
Close, obtain compound, by mixed material heating to 34 DEG C, the reaction time is 3.5h, add 0.12% metal of mixture quality
Agent, centrifugation, takes supernatant, adds the protein precipitation agent of supernatant quality 15%, stands 12h, and centrifuging and taking precipitation is dried
Material 2；
Described oxidant, is to mix concentration by 1.2: 0.35 mass ratio for 22ml/L ozone, thiolase；
The mass ratio of described metal agent, wherein zinc ion, lead ion and iron ion is 1.2: 0.1: 0.48；
Described protein precipitation agent, it, by mass mixings such as methanol and ethanol, is 4.5 with hydrochloric acid conditioning solution pH value to be,
Mix thoroughly；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then 2~3 are washed with ether
It is secondary, drying.
Experimental example
The present invention is counted to the recovery rate and purity of bovine serum albumin respectively, with tradition to bovine serum albumin
Extracting method is as control, as a result as shown in table 1：
Table 1
| Project | The inventive method | Conventional method |
| Average recoveries % | 95.6 | 93.2 |
| Purity % | 99.6 | 99.3 |
Claims (9)
1. a kind of method for extracting bovine serum albumin, it is characterised in that comprise the following steps：
(1) fresh bovine blood is collected at ox vein, haemocyte and blood plasma is separated into through blood separator, blood is added in blood plasma
20~30% protein deposition agent of quality is starched, separation obtains solid protein material and solution material；
(2) solid protein material is handled：Solid protein material is added water stirring, protein solution is obtained, the 12 of solid protein material quality are added
~18% anti-coagulants, stirring shakes up, centrifugation, takes supernatant, and it is 4.5~5 to add hydrochloric acid regulation supernatant pH value, adds supernatant
The dehydrating agent of liquid quality 4~8%, is mixed thoroughly, is stood, centrifuging and taking precipitation, and 1 must be expected by drying；
(3) solution material is handled：By solution material, dispersant, oxidant press 1.2~1.8: 0.1~0.2: 0.35~0.45 quality
Than mixing, compound is obtained, oxidation processes is carried out, adds 0.12~0.15% metal agent of mixture quality, centrifuges, takes
Clear liquid, adds the protein precipitation agent of supernatant quality 10~15%, stands 12~18h, and centrifuging and taking is precipitated, and 2 must be expected by drying；
(4) material 1 is mixed with material 2, obtains compound, compound is first washed with ethanol 2~3 times, then washed 2~3 times with ether, dried
It is dry.
2. as claimed in claim 1 extract bovine serum albumin method, it is characterised in that described protein deposition agent, be by
The trichloroacetic acid solution that the sulfosalisylic aqueous acid and content that content is 12~18% are 10~20% is by 0.8~1.2:
1.2~1.8 mass ratio is mixed to prepare.
3. the method for bovine serum albumin is extracted as claimed in claim 1, it is characterised in that described anti-coagulants, is by citron
Sour sodium and concentration are mixed for 0.012~0.18% salt solution by 1: 0.3~0.6 mass ratio.
4. the method for bovine serum albumin is extracted as claimed in claim 1, it is characterised in that described dehydrating agent, is by sweet dew
Alcohol and glycerine are mixed by 0.2~0.3: 0.12~0.18 mass ratio.
5. the method for bovine serum albumin is extracted as claimed in claim 1, it is characterised in that described dispersant is dodecyl
One kind in sodium sulphate, polyacrylamide and methyl anyl alcohol.
6. the method for bovine serum albumin is extracted as claimed in claim 1, it is characterised in that described oxidant, is by concentration
Mixed for 22~28ml/L ozone, thiolase by 1.2~1.6: 0.25~0.35 mass ratio.
7. the method for bovine serum albumin is extracted as claimed in claim 1, it is characterised in that described oxidation processes, are by thing
Material is heated to 34~36 DEG C, and the reaction time is 2.5~3.5h.
8. as claimed in claim 1 extract bovine serum albumin method, it is characterised in that described metal agent, wherein zinc from
The mass ratio of son, lead ion and iron ion is 0.8~1.2: 0.1~0.2: 0.36~0.48.
9. extracting the method for bovine serum albumin as claimed in claim 1, it is characterised in that described protein precipitation agent, it is
By mass mixings such as methanol and ethanol, it is 4.5~5 with hydrochloric acid conditioning solution pH value, mixes thoroughly.
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| CN114209059A (en) * | 2021-12-20 | 2022-03-22 | 黑龙江乌苏里江制药有限公司 | Acanthopanax senticosus polysaccharide calcium and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | Continuous separation method for three proteins in bovine plasma |
| WO2010128142A1 (en) * | 2009-05-07 | 2010-11-11 | Novozymes Biopharma Dk A/S | Method for purifying albumin |
| CN106065029A (en) * | 2016-08-24 | 2016-11-02 | 成都远睿生物技术有限公司 | A kind of extraction method of bovine serum albumin and bovine serum albumin(BSA) |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010128142A1 (en) * | 2009-05-07 | 2010-11-11 | Novozymes Biopharma Dk A/S | Method for purifying albumin |
| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | Continuous separation method for three proteins in bovine plasma |
| CN106065029A (en) * | 2016-08-24 | 2016-11-02 | 成都远睿生物技术有限公司 | A kind of extraction method of bovine serum albumin and bovine serum albumin(BSA) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114209059A (en) * | 2021-12-20 | 2022-03-22 | 黑龙江乌苏里江制药有限公司 | Acanthopanax senticosus polysaccharide calcium and preparation method and application thereof |
| CN114209059B (en) * | 2021-12-20 | 2024-01-19 | 黑龙江乌苏里江制药有限公司 | Acanthopanax polysaccharide calcium and preparation method and application thereof |
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