CN105950576A - Method for extracting multiple proteins from bovine blood - Google Patents
Method for extracting multiple proteins from bovine blood Download PDFInfo
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- CN105950576A CN105950576A CN201610364078.0A CN201610364078A CN105950576A CN 105950576 A CN105950576 A CN 105950576A CN 201610364078 A CN201610364078 A CN 201610364078A CN 105950576 A CN105950576 A CN 105950576A
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- 241000283690 Bos taurus Species 0.000 title claims abstract description 77
- 210000004369 blood Anatomy 0.000 title claims abstract description 55
- 239000008280 blood Substances 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 44
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
- 210000002381 plasma Anatomy 0.000 claims abstract description 33
- 108090000190 Thrombin Proteins 0.000 claims abstract description 27
- 229960004072 thrombin Drugs 0.000 claims abstract description 27
- 108010049003 Fibrinogen Proteins 0.000 claims abstract description 26
- 102000008946 Fibrinogen Human genes 0.000 claims abstract description 26
- 229940012952 fibrinogen Drugs 0.000 claims abstract description 26
- 239000000843 powder Substances 0.000 claims abstract description 23
- 102000019197 Superoxide Dismutase Human genes 0.000 claims abstract description 21
- 108010012715 Superoxide dismutase Proteins 0.000 claims abstract description 21
- 102000006395 Globulins Human genes 0.000 claims abstract description 18
- 108010044091 Globulins Proteins 0.000 claims abstract description 18
- 102000004506 Blood Proteins Human genes 0.000 claims abstract description 16
- 108010017384 Blood Proteins Proteins 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 10
- -1 IgM Proteins 0.000 claims abstract description 9
- 238000000926 separation method Methods 0.000 claims abstract description 9
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 7
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 7
- 108010094028 Prothrombin Proteins 0.000 claims abstract description 6
- 102100027378 Prothrombin Human genes 0.000 claims abstract description 6
- 229940039716 prothrombin Drugs 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims description 71
- 238000001914 filtration Methods 0.000 claims description 69
- 239000000706 filtrate Substances 0.000 claims description 60
- 210000003677 hemocyte Anatomy 0.000 claims description 39
- 229940000351 hemocyte Drugs 0.000 claims description 39
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 28
- 238000003825 pressing Methods 0.000 claims description 26
- 239000006166 lysate Substances 0.000 claims description 22
- 108010067306 Fibronectins Proteins 0.000 claims description 17
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000000284 extract Substances 0.000 claims description 11
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 10
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 8
- 230000007850 degeneration Effects 0.000 claims description 7
- 238000000751 protein extraction Methods 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 238000001994 activation Methods 0.000 claims description 6
- 238000005571 anion exchange chromatography Methods 0.000 claims description 6
- 239000004606 Fillers/Extenders Substances 0.000 claims description 5
- 238000005277 cation exchange chromatography Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 5
- IRXRGVFLQOSHOH-UHFFFAOYSA-L dipotassium;oxalate Chemical compound [K+].[K+].[O-]C(=O)C([O-])=O IRXRGVFLQOSHOH-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000002002 slurry Substances 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical group [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 5
- 229940038773 trisodium citrate Drugs 0.000 claims description 5
- 239000011592 zinc chloride Substances 0.000 claims description 5
- 235000005074 zinc chloride Nutrition 0.000 claims description 5
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 4
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 claims description 4
- 229910001626 barium chloride Inorganic materials 0.000 claims description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 4
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- 102000016359 Fibronectins Human genes 0.000 claims 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 2
- 238000004458 analytical method Methods 0.000 claims 1
- 229910052788 barium Inorganic materials 0.000 claims 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 claims 1
- 238000005341 cation exchange Methods 0.000 claims 1
- 238000005660 chlorination reaction Methods 0.000 claims 1
- 238000004090 dissolution Methods 0.000 claims 1
- 239000002893 slag Substances 0.000 claims 1
- 210000000601 blood cell Anatomy 0.000 abstract description 5
- 238000004925 denaturation Methods 0.000 abstract description 3
- 230000036425 denaturation Effects 0.000 abstract description 3
- 101001027553 Bos taurus Fibronectin Proteins 0.000 abstract 2
- 238000011085 pressure filtration Methods 0.000 abstract 2
- 102000001554 Hemoglobins Human genes 0.000 abstract 1
- 108010054147 Hemoglobins Proteins 0.000 abstract 1
- 102100037362 Fibronectin Human genes 0.000 description 14
- 239000000243 solution Substances 0.000 description 9
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- 239000008351 acetate buffer Substances 0.000 description 4
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- 230000008014 freezing Effects 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 3
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 206010020633 Hyperglobulinaemia Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical class [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003811 acetone extraction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003918 blood extract Substances 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 102000005525 fibrillarin Human genes 0.000 description 1
- 108020002231 fibrillarin Proteins 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000021419 vinegar Nutrition 0.000 description 1
- 239000000052 vinegar Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
- C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
- C12N9/004—Cytochrome-b5 reductase (1.6.2.2)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4717—Plasma globulins, lactoglobulin
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- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
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- 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
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- 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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
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- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- C12Y304/21005—Thrombin (3.4.21.5)
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Abstract
The invention discloses a method for extracting multiple proteins from bovine blood. The method comprises a blood separating step of taking bovine blood and adding an anticoagulant, and separating into blood cells and plasma; a blood cell extracting step of subjecting the blood cells to crushing, denaturation, pressure filtration and purification treatment to obtain at least one of superoxide dismutase, protoheme, and blood globulin powder; and a plasma extracting step of subjecting the plasma to pressure filtration and purification to obtain at least one of fibrinogen, prothrombin, thrombin, bovine IgM, bovine fibronectin , bovine IgG, and plasma protein powder. According to the method, by fully utilizing a biological separation technology, the superoxide dismutase, hemoglobin, fibrinogen, prothrombin, thrombin, IgM, bovine fibronectin and bovine IgG are extracted one by one in high purity according to a principle of separating similar components first and then finely separating, so that the potential value of blood is fully found and the utilization rate of bovine blood is improved.
Description
Technical field
The present invention relates to Sanguis Bovis seu Bubali processing technique field, carry from Sanguis Bovis seu Bubali in particular to one
The method taking multiple protein.
Background technology
At present, Sanguis Bovis seu Bubali process technology refers mainly to extract the technique of various albumen from Sanguis Bovis seu Bubali, to carry
For the surcharge of Sanguis Bovis seu Bubali, improve its utilization rate.But existing extraction process efficiency is low, extraction
Few and extract component the content also low-purity of protein varieties the highest, and it is a large amount of to there is filtrate filtering residue
The phenomenon abandoned, strongly limit the deep processing to Sanguis Bovis seu Bubali and utilization rate.
Summary of the invention
It is an object of the invention to provide a kind of method extracting multiple protein from Sanguis Bovis seu Bubali, this side
Method can extract multiple proteins from bovine blood sample one by one, and the purity extracted is high, fills
Divide the potential value having excavated bovine blood, improve the utilization rate of bovine blood.
The present invention solves it and technical problem is that and realize by the following technical solutions.
A kind of method extracting multiple protein from Sanguis Bovis seu Bubali, comprising:
Blood separation procedure: take fresh bovine blood, adds anticoagulant, separates, and collects hemocyte
And blood plasma；
Hemocyte extraction step: hemocyte is difference after broken, degeneration, filter pressing, purification process
Obtain superoxide dismutase, haemachrome and blood globulin powder at least one；
Blood plasma extraction step: blood plasma is through filter pressing, respectively obtain Fibrinogen, blood coagulation after purification
Proenzyme, thrombin, IgM, cattle fibronectin, cattle IgG and spray-dried plasma protein are at least
One of them.
A kind of method extracting multiple protein from Sanguis Bovis seu Bubali that the present invention provides provides the benefit that:
Relative to traditional extraction process, the Sanguis Bovis seu Bubali extracting method of the present invention include blood separation procedure,
Hemocyte extraction step and blood plasma extraction step, make full use of bioseparation technology, by first separating
Close component, then carry out the principle of fine separation, can be expeditiously the super oxygen in bovine blood
Compound dismutase, haemachrome, Fibrinogen, thrombinogen, thrombin, IgM, cattle fibre
Dimension connects albumen, extracts to cattle IgG high-purity seriatim, and processing obtains hyperglobulinemia
The effective ingredient such as powder and spray-dried plasma protein, fully excavates the potential value of blood, improves Sanguis Bovis seu Bubali
The utilization rate of liquid.
Accompanying drawing explanation
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be in embodiment
The required accompanying drawing used is briefly described, it will be appreciated that the following drawings illustrate only this
Some bright embodiment, is therefore not construed as the restriction to scope, common for this area
From the point of view of technical staff, on the premise of not paying creative work, it is also possible to according to these accompanying drawings
Obtain other relevant accompanying drawings.
Fig. 1 is the flow chart of the method extracting multiple protein from Sanguis Bovis seu Bubali of the embodiment of the present invention.
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below by right
Technical scheme in the embodiment of the present invention is clearly and completely described.In embodiment unreceipted
Actual conditions person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument
Device unreceipted production firm person, being can be by the commercially available conventional products bought and obtain.
Below the method extracting multiple protein from Sanguis Bovis seu Bubali of the embodiment of the present invention is carried out specifically
Explanation.
Refer to Fig. 1, the side extracting multiple protein from Sanguis Bovis seu Bubali that the embodiment of the present invention provides
Method, comprises the following steps.
S1: blood separation procedure
Take fresh bovine blood, add anticoagulant, separated by blood cell seperator, and it is thin to collect blood
Born of the same parents and blood plasma.Need explanation, when first processing hemocyte, can be by blood plasma in-20 DEG C of freezing guarantors
Deposit, if first processing blood plasma, then can be by hemocyte in-20 DEG C of freezen protective.First process hemocyte
The most first process blood plasma to select according to practical situation.
Wherein, it is preferred that anticoagulant is trisodium citrate.Further, press in blood
Final concentration of 3.6～4% amount add trisodium citrate, prevent bovine blood from solidifying.
S2: hemocyte extraction step
Hemocyte can respectively obtain superoxides discrimination after broken, degeneration, filter pressing, purification process
Change enzyme, haemachrome and blood globulin powder at least one.In other words, hemocyte extracts
Step includes that superoxide dismutase extraction step, haemachrome extraction step and blood globulin powder carry
Take step at least one.Hereinafter superoxide dismutase extraction step, haemachrome are extracted
Step and blood globulin powder extraction step are specifically described.
(1) superoxide dismutase extraction step
With ultrasonic disruption hemocyte (KQ-100 type Ultrasound Instrument, power 100W, frequency 60K,
Ultrasonic 10 minutes), discharge born of the same parents' inclusion, obtain hemocyte and crush liquid.Break toward hemocyte
Broken liquid adds copper chloride and zinc chloride, after thermal denaturation processes, obtains hemocyte degeneration liquid.
Filter pressing (model SHXB-Z, pressure 0.2Mpa, double-filtration, first order aperture be 6 μm,
Aperture, the second level is 1.2 μm, first crosses the first order after the second level) hemocyte degeneration liquid, collect
First filtrate and the first filtering residue.Purify the first filtrate with anion-exchange chromatography, collect super oxidation
Thing dismutase, i.e. obtains highly purified superoxide dismutase.
Wherein, it is preferred that by final concentration of 0.48 in crushing liquid at hemocyte～0.52% (matter
Amount than) amount add copper chloride and adding by the amount of final concentration of 0.28～0.32% (mass ratio)
Enter zinc chloride.
Wherein, during it is preferred that thermal denaturation process hemocyte crushes liquid, under the conditions of 72～77 DEG C
Heat treatment hemocyte crushes liquid 30～35min.
(2) obtained in haemachrome extraction step water or buffer solution above-mentioned steps
One filtering residue, regulates pH to 1.8～2.2, adds sodium carboxymethyl cellulose, obtains the first filtering residue molten
Solve liquid.Wherein, sodium carboxymethyl cellulose can adsorb the purification of haemachrome, beneficially haemachrome.
Filter pressing the first filtering residue lysate again, collects the second filtrate and the second filtering residue.In second filtering residue
Containing haemachrome, with water or buffer solution the second filtering residue.Collect the residue after washing and be height
The haemachrome of purity, and collect the cleaning mixture after washing the second filtering residue.
(3) blood globulin powder extraction step
It is spray-dried above-mentioned cleaning mixture and collects the mixed liquor of the second filtrate obtained, i.e. obtaining blood
Globulin powder.
It should be noted that above-mentioned steps is optionally carried out, such as, super oxidation is only carried out
Thing dismutase extraction step, obtains superoxide dismutase；Or only carry out superoxide dismutase
Enzyme extraction step and haemachrome extraction step, obtain superoxide dismutase and haemachrome；Certainly,
Superoxide dismutase extraction step, haemachrome extraction step and hyperglobulinemia can also be carried out
Powder extraction step, obtains superoxide dismutase, haemachrome and blood globulin powder.
S3: blood plasma extraction step
Blood plasma through filter pressing, after purification respectively obtain Fibrinogen, thrombinogen, thrombin,
Cattle IgM, cattle fibronectin, cattle IgG and spray-dried plasma protein at least one.Change
Sentence talk about, blood plasma extraction step include Fibrinogen extraction step, thrombinogen extraction step,
Thrombin extraction step, cattle IgM and cattle fibronectin extraction step, cattle IgG extract
Step and spray-dried plasma protein extraction step at least one.Hereinafter Fibrinogen is extracted
Step, thrombinogen extraction step, thrombin extraction step, cattle IgM and cattle fiber connect
Protein extraction step, cattle IgG extraction step and spray-dried plasma protein extraction step at least within it
One is specifically described.
(1) Fibrinogen extraction step
It is 0.85～the sodium chloride solution diluting plasma of 0.9% (mass ratio) by concentration, obtains blood
Slurry diluent.If it is understood that blood plasma is frozen process, should first melt the blood of freezing
Slurry, then it is diluted operation.
Filter pressing plasma extender, collects the 3rd filtrate and the 3rd filtering residue.Containing fibre in 3rd filtering residue
Fibrillarin is former, with water or buffer solution the 3rd filtering residue, collects the residue after washing and is high-purity
The Fibrinogen of degree.
Wherein, it is preferred that be by volume 4～6:1 amount toward blood plasma adds sodium chloride solution.
(2) thrombinogen extraction step
Take part the 3rd filtrate, and add potassium oxalate and barium chloride, stir 18～20min, make
It fully dissolves.Wherein, it is preferred that by the 3rd filtrate final concentration of 0.08～0.12%
The amount of (mass ratio) adds potassium oxalate and adds barium chloride by the amount of 0.18～0.22%.
This part the 3rd filtrate of filter pressing again, collects the 4th filtrate and the 4th filtering residue, with pH6.8～7.2
0.018～0.022M phosphate buffer dissolve the 4th filtering residue, obtain the 4th filtering residue lysate.
(DEAE Beads 6FF, balance pH6.8～7.2 are purified with anion-exchange chromatography
0.018～0.022M phosphate buffer, eluting is with 0.1-1mol/L NaCl's and pH6.8～7.2
0.018～0.022M phosphate buffer, regeneration 2mol/LNaCl) the 4th filtering residue lysate,
Collection obtains highly purified thrombinogen.
Mass ratio
(3) thrombin extraction step
Adding calcium chloride in thrombinogen and carry out activation process, after being activated, thrombinogen is molten
Liquid, then chromatograph (SP Beads 6FF, balance pH5.2～the 0.018 of 6.0～0.022M phosphoric acid
Salt buffer, the eluting 0.018～0.022M phosphorus of 0.1-1mol/L NaCl and pH5.2～6.0
Phthalate buffer, regeneration 2mol/LNaCl) prothrombin solution after this activation, collect
To highly purified thrombin.
(4) cattle IgM and cattle fibronectin extraction step
Final concentration of 18～22% (mass ratio) ammonium sulfate are added, stirring in the 3rd filtrate
18～20min so that it is fully dissolve.This 3rd filtrate separately taken of filter pressing again, collects the 5th filter
Liquid and the 5th filtering residue.The is dissolved with the 0.008～0.012M acetate buffer of pH5.0～5.5
Five filtering residues, obtain the 5th filtering residue lysate.
Then (SP Beads 6FF, balance pH5.0～5.5 are purified with cation-exchange chromatography
0.018～0.022M acetate buffer, eluting 0.1-1mol/L NaCl and pH5.0～5.5
0.018～0.022M acetate buffer, regeneration 2mol/LNaCl) the 5th filtering residue dissolve
Liquid, collects and obtains cattle IgM and cattle fibronectin.
Wherein, it is preferred that add acid ammonium by the amount of final concentration of 20% in the 3rd filtrate.
(5) cattle IgG extraction step
Final concentration of 33～the ammonium sulfate of 37% (mass ratio) are added, stirring in the 3rd filtrate
18～22min so that it is fully dissolve.This 3rd filtrate the most separately taken of filter pressing, collects the 6th filter
Liquid and the 6th filtering residue.Phosphate solution with the 0.008～0.012M of pH6.8～7.2 dissolves the
Six filtering residues, obtain the 6th filtering residue lysate.
(SP Beads 6FF, balance pH6.8～7.2 are purified with cation-exchange chromatography
0.018～0.022M phosphate buffer, eluting 0.1-1mol/L NaCl and pH6.8～7.2
0.018～0.022M phosphate buffer, regeneration 2mol/LNaCl) the 6th filtering residue dissolve
Liquid, collects and obtains cattle IgG.
Wherein, it is preferred that add acid ammonium by the amount of final concentration of 35% in the 3rd filtrate.
(6) spray-dried plasma protein extraction step
It is spray-dried the 3rd filtrate, the 4th filtrate, the 5th filtrate, the 6th filtrate and the 7th filtrate
Mixed liquor, i.e. obtain spray-dried plasma protein.
Being also required to explanation, above-mentioned steps is optionally carried out, and such as, only carries out fiber
Proteinogen extraction step, obtains Fibrinogen；Or only carry out Fibrinogen extraction step
With thrombinogen extraction step, obtain Fibrinogen and thrombinogen；Or only carry out fiber
Proteinogen extraction step, thrombinogen extraction step and thrombin extraction step, obtain fiber egg
Bai Yuan, thrombinogen and thrombin；Or only carry out Fibrinogen extraction step, thrombin
Former extraction step, thrombin extraction step and cattle IgM and cattle fibronectin are extracted
Step, obtains Fibrinogen, thrombinogen extraction step, thrombin, cattle IgM and cattle
Fibronectin；Or above-mentioned steps is all carried out, carry out Fibrinogen the most successively and carry
Take step, thrombinogen extraction step, thrombin extraction step, cattle IgM and cattle fiber even
Connect protein extraction step and cattle IgG extraction step, obtain Fibrinogen, thrombinogen
Extraction step, thrombin, cattle IgM, cattle fibronectin and cattle IgG.
The method extracting multiple protein from Sanguis Bovis seu Bubali of the present invention, makes full use of bioseparation technology,
Especially filter pressing isolation technics and ion-exchange chromatography, by first separating close component, then enter
The principle of row fine separation, first isolates superoxide dismutase, blood red from hemocyte one by one
The albumen such as element, and obtain blood globulin powder；Then isolate one by one from blood plasma Fibrinogen,
The albumen such as thrombinogen, thrombin, cattle IgM, fibronectin, cattle IgG, and obtain
Spray-dried plasma protein.Relative to traditional extraction process, the extracting method of the present invention can be from portion
Blood sample goes out to extract multiple proteins, has saved the cost of raw material and time, has fully excavated
The potential value of blood, improves the utilization rate of bovine blood.
Below in conjunction with embodiment, inventive feature and performance are described in further detail.
Embodiment
This example with from bovine blood extract superoxide dismutase, haemachrome, Fibrinogen,
Thrombinogen, thrombin, IgM, cattle fibronectin, cattle IgG, blood globulin powder with
And as a example by spray-dried plasma protein, describe present invention offer in detail extracts multiple protein from Sanguis Bovis seu Bubali
The operating process of method.
First, blood separation procedure is carried out.
Take the fresh bovine blood of 10L, add trisodium citrate and make its final concentration in bovine blood
It it is 3.8% anticoagulant.Utilize hemocyte and the blood of 5.2L of blood cell seperator isolated 5.0kg
Slurry.In the present embodiment, first processing hemocyte, blood plasma is in-20 DEG C of freezen protective.
Secondly, from hemocyte, extract superoxide dismutase, haemachrome successively, and obtain blood
Globulin powder.Its concrete extraction step is as follows.
(1) superoxide dismutase is extracted
After brine hemocyte, then with sonicator, hemocyte is carried out ultrasonic
Ripple crushes so that it is discharges born of the same parents' inclusion, obtains hemocyte and crush liquid.
Filter pressing hemocyte crushes liquid, removes cell debris residue, after removing cell debris residue
Hemocyte crush liquid and proceed to reactor is carried out heat treatment, and add final concentration of 0.5%
Copper chloride and the zinc chloride of 0.3%, be warming up to 75 DEG C, is incubated 20min.
Hemocyte after filter pressing heat treatment crushes liquid and obtains the first filtrate and the first filtering residue.
First filtrate is accompanied with normal saline dilution 5 and is i.e. arrived by the volume ratio addition normal saline of 4:1
In first filtrate, and regulate its pH to 7.6, purify with anion-exchange chromatography after diluting
First filtrate, collects after eluting and obtains superoxide dismutase sterling 200mg.
(2) haemachrome is extracted
The first filtering residue obtained by water or buffer solution above-mentioned steps, regulates pH to 2.0,
Add sodium carboxymethyl cellulose absorption, obtain the first filtering residue lysate.
Filter pressing the first filtering residue lysate, collects and obtains the second filtrate and the second filtering residue.
Second filtering residue, with acid acetone extraction, reclaims acetone after backflow, and concentrate diluted alkaline is molten
Xie Hou, obtains haemachrome lysate, regulates haemachrome lysate pH to 4.0, then sucking filtration,
Obtain haemachrome filtering residue, wash this haemachrome filtering residue with water, obtain highly purified haemachrome 50g.And
Collect the cleaning mixture after washing the second filtering residue.
(3) blood globulin powder is extracted
It is spray-dried cleaning mixture and the mixed liquor of the second filtrate that above-mentioned steps obtains, i.e. obtains blood
Globulin powder.
Then, from blood plasma extract Fibrinogen, thrombinogen, thrombin, cattle IgM,
Cattle fibronectin and cattle IgG.Its concrete extraction step is as follows.
(4) Fibrinogen is extracted
Melt the blood plasma of freezing, take 5L blood plasma, with the sodium chloride solution dilution that concentration is 0.9%
It is that the amount of 5:1 adds sodium chloride solution in blood plasma the most by volume, obtains plasma extender.
Filter pressing plasma extender, collects and obtains the 3rd filtrate and the 3rd filtering residue, wash the 3rd filtering residue with water,
Collect the residue after washing and i.e. obtain highly purified Fibrinogen.
(5) thrombinogen is extracted
In the 3rd filtrate and add the potassium oxalate of final concentration of 0.01% and the barium chloride of 0.2%,
Stirring 20min so that it is fully dissolve, filter pressing, collects and obtains the 4th filtrate and the 4th filtering residue.
Dissolve the 4th filtering residue with the 0.02M phosphate buffer of pH7, obtain the 4th filtering residue lysate.
(DEAE Beads 6FF, the 0.02M of balance pH7.0 is purified with anion-exchange chromatography
Phosphate buffer, eluting 0.1-1mol/L NaCl and pH7.0 0.02M phosphate buffer,
Regeneration 2mol/L NaCl) the 4th filtering residue lysate, collect and obtain highly purified thrombin
Former.
(6) thrombin is extracted
In thrombinogen, add 0.05M calcium chloride carry out activation process, solidifying after being activated
Hemase original solution, then chromatograph prothrombin solution after this activation, collect and obtain highly purified blood coagulation
Enzyme 50mg.
(7) cattle IgM and cattle fibronectin are extracted
In the 3rd filtrate and add final concentration of 20% ammonium sulfate, stir 20min so that it is
Fully dissolve, then filter pressing, collect and obtain the 5th filtrate and the 5th filtering residue.With the 0.01M of pH5.0
Acetate buffer dissolves the 5th filtering residue, obtains the 5th filtering residue lysate.
(SP Beads 6FF, the 0.01M vinegar of balance pH5.0 is purified with cation-exchange chromatography
Phthalate buffer, the eluting 0.01M acetate salt buffer of 0.1-1mol/L NaCl and pH5.0
Liquid, regeneration 2mol/LNaCl) the 5th filtering residue lysate, collect and obtain cattle IgM10g and cattle
Fibronectin 1 g.
(8) cattle IgG is extracted
In the 3rd filtrate, add the ammonium sulfate of final concentration of 35%, stir 20min so that it is fill
Divide and dissolve.
Filter pressing the 3rd filtrate, collects and obtains the 6th filtrate and the 6th filtering residue.With the 0.01M of pH7
Phosphate salt solution the 6th filtering residue, obtain the 6th filtering residue lysate.
(SP Beads 6FF, the 0.01M phosphorus of balance pH7.0 is purified with cation-exchange chromatography
Phthalate buffer, the eluting 0.018～0.022M phosphoric acid of 0.1-1mol/L NaCl and pH7.0
Salt buffer, regeneration 2mol/LNaCl) the 6th filtering residue lysate, collect and obtain cattle IgG40g.
(9) spray-dried plasma protein is extracted
It is spray-dried the 3rd filtrate, the 4th filtrate, the 5th filtrate and the mixed liquor of the 6th filtrate,
I.e. obtain spray-dried plasma protein.
It should be noted that in other examples, above-mentioned steps can all be carried out,
Can carry out with selectivity.Extract according to the actual requirements.
To sum up, the method extracting multiple protein from Sanguis Bovis seu Bubali of the present embodiment, make full use of biological point
From technology, by first separating close component, then carry out the principle of fine separation, expeditiously cattle
Superoxide dismutase in blood, haemachrome, Fibrinogen, thrombinogen, thrombin,
Extract to IgM, cattle fibronectin, cattle IgG high-purity seriatim, and process
To the effective ingredient such as blood globulin powder and spray-dried plasma protein, the filtrate of each of which step or filtering residue
Can all be obtained by, do not have waste liquid or waste residue abandons situation, and it fully excavates blood
Potential value, improves the utilization rate of bovine blood.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention,
For a person skilled in the art, the present invention can have various modifications and variations.All at this
Within the spirit of invention and principle, any modification, equivalent substitution and improvement etc. made, all should
Within being included in protection scope of the present invention.
Claims (10)
1. the method extracting multiple protein from Sanguis Bovis seu Bubali, it is characterised in that comprising:
Blood separation procedure: take fresh bovine blood, adds anticoagulant, is separated into hemocyte and blood
Slurry；
Hemocyte extraction step: described hemocyte is after broken, degeneration, filter pressing, purification process
Respectively obtain superoxide dismutase, haemachrome and blood globulin powder at least one；
Blood plasma extraction step: described blood plasma through filter pressing, after purification respectively obtain Fibrinogen,
Thrombinogen, thrombin, cattle IgM, cattle fibronectin, cattle IgG and plasma protein
Powder at least one.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 1, it is special
Levying and be, described hemocyte extraction step includes superoxide dismutase extraction step, haemachrome
Extraction step and blood globulin powder extraction step at least one, wherein
Described superoxide dismutase extraction step is with hemocyte described in ultrasonic disruption, obtains
Hemocyte crushes liquid, crushes toward described hemocyte and adds copper chloride and zinc chloride in liquid, becomes through heat
Property process after obtain hemocyte degeneration liquid, hemocyte degeneration liquid described in filter pressing, collect the first filtrate
With the first filtering residue, purify described first filtrate with anion-exchange chromatography, obtain described super oxidation
Thing dismutase；
Described haemachrome extraction step is with the first filtering residue described in water dissolution, regulates pH extremely
1.8～2.2, add sodium carboxymethyl cellulose, obtain the first filtering residue lysate, described in filter pressing first
Filtering residue lysate, collects the second filtrate and the second filtering residue, washs described second filtering residue, obtains institute
State haemachrome；
Described blood globulin powder extraction step is the second residue collection described in spray-dried laundry
Cleaning mixture and the mixed liquor of described second filtrate, obtain described blood globulin powder.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special
Levy and be, be respectively 0.48～0.52% He by the final concentration crushed in liquid at described hemocyte
0.28～0.32% amount add described copper chloride and described zinc chloride.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special
Levying and be, described thermal deformation field crushes for hemocyte described in heat treatment under the conditions of 72～77 DEG C
Liquid 30～35min.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 2, it is special
Levying and be, described blood plasma extraction step includes that Fibrinogen extraction step, thrombinogen extract
Step, thrombin extraction step, cattle IgM and cattle fibronectin extraction step, cattle IgG
Extraction step and spray-dried plasma protein extraction step at least one, wherein
Described Fibrinogen extraction step be with concentration be 0.85～0.9% sodium chloride solution dilute
Release described blood plasma, obtain plasma extender, plasma extender described in filter pressing, collect the 3rd filtrate
With the 3rd filtering residue, wash described 3rd filtering residue, obtain described Fibrinogen；
Described thrombinogen extraction step is to add potassium oxalate and chlorination in described 3rd filtrate
Barium, stirs 18～20min, then filter pressing, collects the 4th filtrate and the 4th filtering residue, with pH6.8～7.2
0.018～0.022M phosphate buffer dissolve described 4th filtering residue, obtain the 4th filtering residue molten
Solve liquid, purify described 4th filtering residue lysate with anion-exchange chromatography, obtain described thrombin
Former；
Described thrombin extraction step is to add calcium chloride to described thrombinogen to carry out at activation
Reason, prothrombin solution after being activated, then chromatograph prothrombin solution after described activation,
To described thrombin；
Described cattle IgM and cattle fibronectin extraction step are to add in described 3rd filtrate
Enter final concentration of 18～22% ammonium sulfate, stir 18～20min, then the 3rd filtrate described in filter pressing,
Collect the 5th filtrate and the 5th filtering residue, delay with the 0.008～0.012M acetate of pH6.8～7.2
Rush liquid and dissolve described 5th filtering residue, obtain the 5th filtering residue lysate, then use cation exchange layer
5th filtering residue lysate described in analysis purification, obtains described cattle IgM and described cattle fibronectin
In vain；
Described cattle IgG extraction step is to add final concentration of 33～37% in described 3rd filtrate
Ammonium sulfate, stirs 18～22min, three filtrates described in filter pressing, collects the 6th filtrate and the 6th filter
Slag, the Acetate Solution with the 0.008～0.012M of pH6.8～7.2 dissolves described 6th filtering residue,
Obtain the 6th filtering residue lysate, purify described 6th filtering residue lysate with cation-exchange chromatography,
Obtain described cattle IgG；
Described spray-dried plasma protein extraction step be spray-dried collect obtain described 3rd filtrate,
Described 4th filtrate, described 5th filtrate and the mixed liquor of described 6th filtrate, obtain described blood
Slurry protein powder.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special
Levy and be, be that the amount of 4～6:1 adds described sodium chloride solution in described blood plasma by volume.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special
Levy and be, be respectively 0.08～0.12% He by the final concentration in described 3rd filtrate
0.18～0.22% amount be separately added into described potassium oxalate and described barium chloride.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special
Levy and be, add ammonium sulfate by the amount of final concentration of 20% in described 3rd filtrate.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 5, it is special
Levying and be, the amount of final concentration of 35% in described 3rd filtrate adds ammonium sulfate.
The method extracting multiple protein from Sanguis Bovis seu Bubali the most according to claim 1, it is special
Levying and be, described anticoagulant is trisodium citrate, final concentration of by described blood
3.6～4% amount add described trisodium citrate.
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| CN114558371B (en) * | 2022-03-23 | 2023-08-11 | 长睿生物技术(成都)有限公司 | Bacterial fermentation liquid separation device |
| WO2024119285A1 (en) * | 2022-12-05 | 2024-06-13 | Guangzhou Bioseal Biotech Co., Ltd. | Fibrinogen preparation |
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