CN106065029A - A kind of extraction method of bovine serum albumin and bovine serum albumin(BSA) - Google Patents
A kind of extraction method of bovine serum albumin and bovine serum albumin(BSA) Download PDFInfo
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- CN106065029A CN106065029A CN201610715134.0A CN201610715134A CN106065029A CN 106065029 A CN106065029 A CN 106065029A CN 201610715134 A CN201610715134 A CN 201610715134A CN 106065029 A CN106065029 A CN 106065029A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/765—Serum albumin, e.g. HSA
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Abstract
The invention discloses a kind of extraction method of bovine serum albumin, it includes, concentrates with the first time obtaining protein concentrated solution that protein dissolution liquid is concentrated by ultrafiltration by the first milipore filter and changes liquid step；Carry out chromatographic purifying to protein concentrated solution with weak anion exchange medium to collect and obtain the first time purification step of thick albumin solution；Liquid step is changed in the second time concentration carrying out being concentrated by ultrafiltration thick albumin concentrate with the second milipore filter to thick albumin solution；Carry out chromatographic purifying to thick albumin concentrate with strong cation exchange medium to collect and obtain the second time purification step of albumin solution.The extracting method of the present invention is concentrated by two times of ultrafiltration, the step that twice chromatographic purifies, and farthest removes the impurity in blood plasma, improves the purity of bovine serum albumin(BSA).In addition present invention also offers a kind of bovine serum albumin(BSA) being obtained by said method.
Description
Technical field
The present invention relates to bovine serum albumin(BSA) extractive technique field, extract in particular to a kind of bovine serum albumin(BSA)
Method and bovine serum albumin(BSA).
Background technology
At present, bovine serum albumin(BSA) (BSA), also known as BSA, is a kind of globulin in cow's serum, comprises 583
Amino acid residue, molecular weight is 66.43kDa, and isoelectric point is 4.7, is extensively should in biology subjects and doctor, each field of pharmacy
Biochemical reagents, it is possible to be used as medicines and health protection food, flavouring.
In cow's serum, albuminous preparation method is a lot, mainly uses salting out method, organic solvent precipitation method and heat shock method to carry
Take.After salting out method ammonium sulfate precipitation, process refined through octanoic acid and obtain；Organic solvent precipitation method is used for human blood system the earliest
The production of product, utilizes the low-k character of ethanol and protein under uniform temperature, pH, ionic strength and concentration conditions
Different solubility separated plasma protein in different concentration ethanol, can produce multiple plasma protein；Heat shock method is to take
Ox blood sodium citrate anti-freezing obtains blood plasma after centrifuging, heating, adds Sodium Caprylate, adds ethanol after adjusting pH value, then through filtering, taking off
Salt, concentration, lyophilized after albumin in cow's serum.
In a word, the bovine serum albumin(BSA) yield of the existing method gained for preparing bovine serum albumin(BSA) or purity all than
Relatively low, and containing the impurity such as aliphatic acid of higher concentration, protease, nuclease and interior in the bovine serum albumin(BSA) being extracted
Toxin.This kind of impurity limits the application further of bovine serum albumin(BSA).
Content of the invention
It is an object of the invention to provide a kind of extraction method of bovine serum albumin, the method can extract from ox blood
High-purity, the bovine serum albumin(BSA) of high yield, and extract the bovine serum albumin(BSA) obtaining without fat acid, albumen by this method
Enzyme, nuclease and the endotoxin containing only low concentration.
Another object of the present invention is to provide a kind of bovine serum albumin(BSA), this bovine serum albumin(BSA) is by using said extracted
Method is extracted and is obtained, and the purity of this bovine serum albumin(BSA) is high, without fat acid, protease, nuclease and containing only low concentration
Endotoxin.
The present invention solves it and technical problem is that and realize by the following technical solutions.
A kind of extraction method of bovine serum albumin, comprising:
Blood separation procedure: take fresh bovine blood, is separated into haemocyte and blood plasma；
Albumen precipitation dissolving step: add precipitating reagent to precipitate in blood plasma, obtain albumen precipitation, add water to dissolve egg
White precipitation, obtains protein dissolution liquid；
Concentrate for the first time and change liquid step: with the first milipore filter, protein dissolution liquid is concentrated by ultrafiltration, the first phosphoric acid
Salt buffer changes liquid, obtains protein concentrated solution；
Purification step for the first time: with weak anion exchange medium, chromatographic purifying is carried out to protein concentrated solution, use sodium chloride-containing
The second phosphate buffer carry out gradient elution, collect and obtain thick albumin solution；
Second time concentrates and changes liquid step: thick albumin solution is concentrated by ultrafiltration with the second milipore filter, uses citrate
Buffer solution changes liquid, obtains thick albumin concentrate；
Purification step for the second time: with strong cation exchange medium, chromatographic purifying is carried out to thick albumin concentrate, collect
To albumin solution.
A kind of bovine serum albumin(BSA), it is extracted by extraction method of bovine serum albumin described above and obtains.
Extraction method of bovine serum albumin and bovine serum albumin(BSA) that the present invention provides provide the benefit that: the ox of the present invention
The protein dissolution liquid that seralbumin extracting method prepares after dissolving from the blood plasma that fresh bovine blood separates through albumen precipitation,
Obtain protein concentrated solution with the first milipore filter to what protein dissolution liquid was concentrated by ultrafiltration；With weak anion exchange medium to albumen
Concentrate carries out chromatographic purifying collection and obtains thick albumin solution；With the second milipore filter, thick albumin solution is concentrated by ultrafiltration
To thick albumin concentrate；With strong cation exchange medium, chromatographic purifying collection is carried out to thick albumin concentrate and obtain albumin
Solution.Utilize repeatedly concentrate change liquid (concentrate for the first time and change liquid step, second time concentrates and changes liquid step) and repeatedly chromatographic purifying walk
Suddenly (purification step for the first time, for the second time purification step), bovine serum albumin(BSA), obtained cow's serum are purified with maximum
Albuminous purity be more than or equal to 99%, and make extracted bovine serum albumin(BSA) without fat acid, protease, nuclease with
And the endotoxin less than 0.2EU/mg.
Detailed description of the invention
Purpose, technical scheme and advantage for making the embodiment of the present invention are clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or instrument unreceipted production firm person, being can be by the commercially available conventional product bought and obtain
Product.
Below the extraction method of bovine serum albumin of the embodiment of the present invention is specifically described.
A kind of extraction method of bovine serum albumin, it comprises the steps.
S1 blood separates
Take fresh bovine blood, be isolated haematoblast or blood plasma.It is preferred that in order to prevent bovine blood from solidifying, can be
Separate and go to addition anti-coagulants such as sodium citrate or hirudin etc. in fresh bovine blood.In preferred embodiment of the present invention
In, using sodium citrate as anti-coagulants, and control sodium citrate adds final concentration of 3.6～4% in blood.
S2 albumen precipitation dissolves
Add precipitating reagent to precipitate in blood plasma, obtain albumen precipitation, then, add water to dissolve this albumen precipitation,
To protein dissolution liquid.
Wherein, the Main Function of precipitating reagent is the protein precipitation making in blood plasma, and it can be ammonium sulfate.Need explanation
It is, in order to ensure extracted bovine serum albumin(BSA) does not produce interference when being applied in diagnostic kit, in the present invention relatively
In good embodiment, precipitating reagent is preferably ammonium sulfate, control ammonium sulfate join the ammonium sulfate in blood plasma final concentration of 40%～
80%.It is preferred that control ammonium sulfate joins the ammonium sulfate in blood plasma final concentration of 30%～50%.Ammonium sulfate used is
The saturated ammonium sulfate solution (1L saturated ammonium sulfate solution liquid containing ammonium sulfate 685g) of 100%.Certainly, in other examples, may be used
According to the application of the bovine serum albumin(BSA) being extracted, selectively use precipitating reagent, also just say in other examples, heavy
Shallow lake agent is can be with the precipitating reagent of Sodium Caprylate or other classifications.
In addition, in step s 2, water used is ultra-pure water.It is preferred that water used with the mass ratio of albumen precipitation is
4.8～5.3:1.
S3 concentrates for the first time and changes liquid
With the first milipore filter, protein dissolution liquid is concentrated by ultrafiltration, changes liquid with the first phosphate buffer, obtain albumen
Concentrate.
Wherein, the molecular cut off of the first milipore filter is 4.8～5KD, ensure that protein especially bovine serum albumin
(its molecular weight is 66.43kDa) macromolecular substances can be trapped within the first milipore filter effectively in vain, and small-molecule substance
Removal can be actively filtered, improve the purity of bovine serum albumin(BSA).In addition, transmembrane pressure control when protein dissolution liquid concentrates
At about 0.1psi.
In addition, for the phosphate concn for example, 18～21mM of the first phosphate buffer changing liquid, pH is 6.8～
7.2.It should be noted that the first phosphate buffer can be to be configured by disodium hydrogen phosphate and sodium dihydrogen phosphate to form, it is also possible to
It is to be configured by dipotassium hydrogen phosphate and potassium dihydrogen phosphate to form.The ox in protein concentrated solution after changing liquid with the first phosphate buffer
Seralbumin is electronegative.Changing the operating procedure of liquid is to join the first phosphate buffer just concentrated with constant flow pump
In protein dissolution liquid in journey, and the volume of protein dissolution liquid is made to tie up by the flow velocity of constant flow pump control the first phosphate buffer
It is fixed to keep steady.
Further, it is preferred that in step s3 the cycles of concentration of protein dissolution liquid be 9～11, say, that protein dissolution liquid
The volume ratio of volume and protein concentrated solution be 9～11:1.
S4 purifies for the first time
With weak anion exchange medium, chromatographic purifying is carried out to protein concentrated solution, by the second phosphate-buffered of sodium chloride-containing
Liquid carries out gradient elution, collects and obtains thick albumin solution.
Wherein, weak anion exchange medium uses diethylamino ethyl (DEAE) Ion Exchange Medium to protein concentrated solution
Carrying out chromatographic purifying, it can adsorb electronegative molecule on chromatographic column, is particularly suited for isolated and purified large biological molecule
Material such as bovine serum albumin(BSA).
In addition, the phosphate concn for example, 18～21mM of the second phosphate buffer for gradient elution, pH is 6.8
～7.2.It is 0.8～1.2mol that every liter of second phosphate buffer contains sodium chloride.It should be noted that the second phosphate-buffered
Liquid can be to be configured by disodium hydrogen phosphate and sodium dihydrogen phosphate to form, it is also possible to is to be configured by dipotassium hydrogen phosphate and potassium dihydrogen phosphate
Form.
S5 second time concentrates changes liquid
With the second milipore filter, thick albumin solution is concentrated by ultrafiltration, changes liquid with citrate buffer, obtain thick white
Protein concentrated solution.
Wherein, wherein, the molecular cut off of the second milipore filter is 4.8～5KD, ensure that protein especially cow's serum
Albumin (its molecular weight is 66.43kDa) macromolecular substances can be trapped within the second milipore filter effectively, through twice
The purity of the bovine serum albumin(BSA) after ultrafiltration is further enhanced.
In addition, in this step, being 18-21mM for changing the citrate buffer citric acid salt concentration of liquid, pH is 4.8
～5.2.This citrate buffer is mainly configured by citric acid and trisodium citrate and forms.Change liquid with this citrate buffer
After thick albumin concentrate in bovine serum albumin(BSA) electronegative.
Further, the cycles of concentration of thick albumin lysate is 9～11 in step s 5, say, that thick albumin solution
The volume ratio of volume and thick albumin concentrate be 9～11:1.
S6 second time purifies
With strong cation exchange medium, chromatographic purifying is carried out to thick albumin concentrate, collect and obtain albumin solution.
Wherein, strong cation exchange medium is sulfopropyl Ion Exchange Medium, and it can adsorb positively charged molecule, enter
And the materials such as some the positively charged foreign proteins in thick albumin concentrate can be adsorbed.
Owing to the bovine serum albumin(BSA) in the thick albumin concentrate in step S5 is in its isoelectric point, neutral,
In this step, bovine serum albumin(BSA) does not combine with the chromatographic column absorption with strong cation exchange medium, therefore, can wash
De-, can directly collect the albumin solution that efflux obtains highly purified low impurity content.
Therefore, the present invention is concentrated and twice chromatographic purification step by two times of ultrafiltration, can farthest remove blood plasma
In impurity such as aliphatic acid, protease, nuclease and endotoxin, obtain the bovine serum albumin(BSA) of higher degree, it can be wide
Apply the field such as medical treatment, biochemistry generally.
Below in conjunction with embodiment, inventive feature and performance are described in further detail.
Embodiment
The extraction method of bovine serum albumin that the present embodiment provides, comprises the following steps:
1. blood separates
Take fresh bovine blood 6000 milliliters, add the trisodium citrate of 667 milliliter 38%, make trisodium citrate at ox blood
Whole weight concentration in liquid is 3.8%, then passes through blood cell seperator (GFX-105 blood separator, the grand pharmaceutical machine of Liaoyang Xiang
Co., Ltd), carry out to bovine blood separating (rotating speed 16300rpm/min), isolated haemocyte and blood plasma.
2. albumen precipitation dissolves
2.1 take the blood plasma 4000 milliliters in step 1, add the saturated ammonium sulfate of 6000 milliliters, make ammonium sulfate in blood plasma
Final concentration of 60%, and stir 1h, make the protein precipitation in blood plasma, remove supernatant, retain albumen precipitation.
2.2 add ultra-pure water 6000 milliliters in the ratio that the mass ratio with albumen precipitation is 5:1, dissolve above-mentioned albumen and sink
Form sediment, obtain protein dissolution liquid 7000 milliliters.
3. concentrate for the first time and change liquid
It is concentrated by ultrafiltration to step 2.2 obtains protein dissolution liquid by ultrafilter, milipore filter bag used on ultrafilter
The molecular cut off of milipore filter be 5KD, parameter (Mini Pellicon ultrafiltration system, the merk of regulation ultrafilter
Millipore company), flow velocity be 400ml/min, transmembrane pressure be 0.1psi, concentrate 10 times, meanwhile, with the phosphorus of 20mM, pH7.0
Acid sodium buffer solution carries out changing liquid.The protein dissolution i.e. just joining the first phosphate buffer in concentration process with constant flow pump
In liquid, and make the volume of protein dissolution liquid remain stable by the flow velocity of constant flow pump control the first phosphate buffer, obtain egg
White concentrate 700 milliliters.
4. purify for the first time
With chromatographic purifying instrument (APPS MV 100D, profit fringe (Suzhou) Science and Technology Ltd.), above-mentioned protein concentrated solution is carried out
Chromatographic purifying, using diethylamino ethyl-cellulose (diethylamino ethyl Ion Exchange Medium) as chromatographic column filler.On
Before post, with the sodium phosphate buffer balance of 20mM, pH7.0, flow velocity 10ml/min, equilibrium volume 5CV；After balance terminates, by egg
White concentrate Column chromatography, coutroi velocity 8ml/min；After end of the sample, balancing 5CV with equilibrium liquid, flow velocity is 10ml/min；With
The phosphate buffer of 20mM, pH7.0 of sodium chloride containing 1mol/L carries out gradient elution and (draws gradient to wash from 0%B to 100%B
De-, 10CV altogether), collect and obtain thick albumin solution 400ml milliliter.After wash-out, with the sodium chloride solution of 2mol/L to chromatographic column
Carry out regeneration process.
5. second time carries out changing liquid
It is concentrated by ultrafiltration to step 4 obtains thick albumin solution by ultrafilter, milipore filter bag used on ultrafilter
The molecular cut off of milipore filter be 5KD, the parameter of regulation ultrafiltration system, flow velocity be 400ml/min, transmembrane pressure be 0.1psi,
Carry out changing liquid with the sodium citrate buffer solution of 20mM, pH5.0.Obtain crude protein liquid 600 milliliters.
6. second time purifies
With chromatographic purifying instrument to above-mentioned crude protein concentrate chromatographic purifying, with sulfopropyl-cellulose, (sulfopropyl ion exchanges
Medium) as chromatographic column filler.Before upper prop, by the sodium citrate buffer solution balance of 20mM, pH5.0, flow velocity 6ml/min, balance
Volume 10CV；After balance terminates, by protein concentrated solution Column chromatography, flow velocity 4ml/min, directly collect efflux, obtain high-purity
The bovine serum albumin solution 1000ml milliliter of the low impurity content of degree.Freeze-dried postscript obtains bovine serum albumin white powder.
(detection of aliphatic acid utilizes determination of fatty acid instrument to detect, and protease utilizes protease elisa kit to examine after testing
Surveying, nuclease utilizes elisa kit to detect, and endotoxin uses gel method detection), the ox blood using the present embodiment to provide is pure
The purity of the bovine serum albumin(BSA) obtained by protein extracting method is more than or equal to 99%, and not fatty acids, protease and nucleic acid
Enzyme, endotoxin content is less than 0.2EU/mg.It can apply the necks such as medical research, Biochemical Research and genetic engineering widely
Territory.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (10)
1. an extraction method of bovine serum albumin, it is characterised in that comprising:
Blood separation procedure: take fresh bovine blood, is separated into haemocyte and blood plasma；
Albumen precipitation dissolving step: add precipitating reagent to precipitate in described blood plasma, obtain albumen precipitation, add water to dissolve institute
State albumen precipitation, obtain protein dissolution liquid；
Concentrate for the first time and change liquid step: with the first milipore filter, described protein dissolution liquid is concentrated by ultrafiltration, uses the first phosphate
Buffer solution changes liquid, obtains protein concentrated solution；
Purification step for the first time: with weak anion exchange medium, chromatographic purifying is carried out to described protein concentrated solution, use sodium chloride-containing
The second phosphate buffer carry out gradient elution, collect and obtain thick albumin solution；
Second time concentrates and changes liquid step: described thick albumin solution is concentrated by ultrafiltration with the second milipore filter, uses citrate
Buffer solution changes liquid, obtains thick albumin concentrate；
Purification step for the second time: with strong cation exchange medium, chromatographic purifying is carried out to described thick albumin concentrate, collect
To albumin solution.
2. extraction method of bovine serum albumin according to claim 1, it is characterised in that described precipitating reagent is ammonium sulfate,
Described ammonium sulfate joins in described blood plasma final concentration of 40%～80%.
3. extraction method of bovine serum albumin according to claim 1, it is characterised in that dissolve step at described albumen precipitation
In Zhou, described water is 4.8～5.3:1 with the mass ratio of described albumen precipitation.
4. extraction method of bovine serum albumin according to claim 3, it is characterised in that described protein concentrated solution with described
The volume ratio of protein dissolution liquid is 1:8～11.
5. extraction method of bovine serum albumin according to claim 1, it is characterised in that described first phosphate buffer
Phosphate concn be 18～21mM, pH is 6.8～7.2, and the phosphate concn of the second phosphate buffer is 18～21mM, pH
Being 6.8～7.2, the citric acid salt concentration of described citrate buffer is 18～21mM, and pH is 4.8～5.2.
6. extraction method of bovine serum albumin according to claim 1, it is characterised in that every liter of described second phosphate delays
Rushing sodium chloride described in liquid is 0.8～1.2mol.
7. extraction method of bovine serum albumin according to claim 1, it is characterised in that retaining of described first milipore filter
Molecular weight is 4.8～5KD, and the molecular cut off of described second milipore filter is 4.8～5KD.
8. extraction method of bovine serum albumin according to claim 1, it is characterised in that described weak anion exchange medium
It is diethylamino ethyl Ion Exchange Medium；Described strong cation exchange medium is sulfopropyl Ion Exchange Medium.
9. extraction method of bovine serum albumin according to claim 1, it is characterised in that in described blood separation procedure
In, in described fresh bovine blood, first adding anti-coagulants, then be separated into described haemocyte and described blood plasma, described anti-coagulants is lemon
Lemon acid sodium, described sodium citrate adds final concentration of 3.8% in described blood.
10. a bovine serum albumin(BSA), it is characterised in that it is by the bovine serum albumin as according to any one of claim 1～9
White extracting method prepares.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106749626A (en) * | 2017-01-24 | 2017-05-31 | 西藏康威科技发展有限公司 | A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum |
| CN106977596A (en) * | 2017-05-22 | 2017-07-25 | 南宁学院 | A kind of method for extracting bovine serum albumin |
| CN108864278A (en) * | 2018-07-27 | 2018-11-23 | 珠海宝锐生物科技有限公司 | A method of preparing molecular biosciences grade bovine serum albumin(BSA) |
| CN110204610A (en) * | 2019-06-24 | 2019-09-06 | 长春市中保牧生物科技有限公司 | A kind of preparation method of hemoglobin conjugate |
| CN110229229A (en) * | 2019-07-10 | 2019-09-13 | 甘肃养泰和生物科技有限公司 | A kind of production method and its application of the extraction purification cell culture grade bovine serum albumin(BSA) from ox blood slurry |
| CN111205362A (en) * | 2020-03-19 | 2020-05-29 | 贾芳苗 | Bovine serum albumin and preparation method thereof |
| CN113735962A (en) * | 2021-08-27 | 2021-12-03 | 常熟纳微生物科技有限公司 | Method for purifying recombinant human serum albumin from pig blood and application |
| CN113968905A (en) * | 2021-10-27 | 2022-01-25 | 哈尔滨国生生物科技股份有限公司 | Bovine serum albumin and application thereof |
| CN117143222A (en) * | 2023-09-11 | 2023-12-01 | 江苏帆博生物制品有限公司 | Bovine serum albumin production method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106749626B (en) * | 2017-01-24 | 2020-05-19 | 西藏森威医疗科技有限公司 | Method for extracting albumin and globulin from bovine serum and method for utilizing bovine serum |
| CN106749626A (en) * | 2017-01-24 | 2017-05-31 | 西藏康威科技发展有限公司 | A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum |
| CN106977596A (en) * | 2017-05-22 | 2017-07-25 | 南宁学院 | A kind of method for extracting bovine serum albumin |
| CN106977596B (en) * | 2017-05-22 | 2020-11-06 | 南宁学院 | Method for extracting bovine serum albumin |
| CN108864278B (en) * | 2018-07-27 | 2021-02-19 | 珠海宝锐生物科技有限公司 | Method for preparing molecular biological grade bovine serum albumin |
| CN108864278A (en) * | 2018-07-27 | 2018-11-23 | 珠海宝锐生物科技有限公司 | A method of preparing molecular biosciences grade bovine serum albumin(BSA) |
| CN110204610A (en) * | 2019-06-24 | 2019-09-06 | 长春市中保牧生物科技有限公司 | A kind of preparation method of hemoglobin conjugate |
| CN110229229A (en) * | 2019-07-10 | 2019-09-13 | 甘肃养泰和生物科技有限公司 | A kind of production method and its application of the extraction purification cell culture grade bovine serum albumin(BSA) from ox blood slurry |
| CN111205362A (en) * | 2020-03-19 | 2020-05-29 | 贾芳苗 | Bovine serum albumin and preparation method thereof |
| CN113735962A (en) * | 2021-08-27 | 2021-12-03 | 常熟纳微生物科技有限公司 | Method for purifying recombinant human serum albumin from pig blood and application |
| CN113735962B (en) * | 2021-08-27 | 2023-11-10 | 常熟纳微生物科技有限公司 | Method for purifying recombinant human serum albumin from pig blood and application thereof |
| CN113968905A (en) * | 2021-10-27 | 2022-01-25 | 哈尔滨国生生物科技股份有限公司 | Bovine serum albumin and application thereof |
| CN117143222A (en) * | 2023-09-11 | 2023-12-01 | 江苏帆博生物制品有限公司 | Bovine serum albumin production method |
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Application publication date: 20161102 |