CN106749626A - A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum - Google Patents
A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum Download PDFInfo
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Abstract
The invention discloses a kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum, it is related to Protein Extraction technical field.The method that albumin and globulin are extracted from cow's serum that the present invention is provided, the principle precipitated in the salting liquid of certain saturation degree using plasma protein, using (NH4)2SO4Fractional precipitation, and be further purified and obtain bovine serum albumin(BSA), the bovine serum albumin(BSA) purity is high, and endotoxin content is low, meets《Products in China code》Requirement；Using saturation (NH4)2SO4Salting-out method repeatedly is classified, bovine serum globulin is extracted, the blood plasma foreign protein of HMW is dispelled to greatest extent, the bovine serum globulin purity of extraction is high.The method process is simple that albumin and globulin are extracted from cow's serum that the present invention is provided, is not required to special installation, is adapted to expanding production, and the method for the utilization ox blood that the present invention is provided improves the utilization rate of ox blood.
Description
Technical field
The present invention relates to Protein Extraction technical field, extracted from cow's serum in particular to one kind albumin and
The method of globulin and the method using ox blood.
Background technology
Ox blood process technology refers mainly to be extracted from ox blood the technique of various albumen, to provide the surcharge of ox blood, carries
Its utilization rate high.At present, the isolation and purification method on bovine serum albumin is many both at home and abroad, common are ion-exchange, coagulates
Glue filtering method, affinity chromatography, zone electrophoresis method, supercentrifugation etc..The yield of these methods is all more satisfactory, but requires bar
Part is harsh, it is necessary to special installation, so be difficult to popularize, without production meaning.
The content of the invention
An object of the present invention is to provide a kind of method that albumin and globulin are extracted from cow's serum, using this
Method extracts albumin from ox blood and globulin does not need special installation, bovine serum albumin(BSA) and cow's serum ball that extraction is obtained
Protein yield is high, purity is high, the rate of recovery is high.
The second object of the present invention is to provide a kind of method of utilization ox blood, and the method isolates cow's serum from ox blood
Afterwards, bovine serum albumin(BSA) and cow's serum ball egg are extracted using the above-mentioned method that albumin and globulin are extracted from cow's serum
In vain.
The present invention is solved its technical problem and is realized using following technical scheme.
A kind of method that albumin and globulin are extracted from cow's serum, it includes：
Cow's serum is taken, the first supernatant is centrifuged to obtain, to 0.8-1.2 times of the first supernatant volume of addition in the first supernatant
4-6mM pH for 7.0-7.4 PBS；Stirring is lower to add (NH4)2SO4To 48%-52% saturation degrees；Taken out after refrigeration, standing
Filter to obtain the first filtrate and the first precipitation；
Albumin is extracted from the first filtrate.
Globulin is extracted from the first precipitation.
A kind of method of utilization ox blood, it includes：
Cow's serum is isolated from ox blood.
Bovine serum albumin is extracted from cow's serum according to the above-mentioned method that albumin and globulin are extracted from cow's serum
White and bovine serum globulin.
A kind of method and the method using ox blood that albumin and globulin are extracted from cow's serum of the embodiment of the present invention
Beneficial effect be：The method that albumin and globulin are extracted from cow's serum that the present invention is provided, using plasma protein one
The principle precipitated in the salting liquid of saturation degree is determined, using (NH4)2SO4Fractional precipitation, and be further purified and obtain bovine serum albumin
In vain, the bovine serum albumin(BSA) purity is high, and endotoxin content is low, meets《Products in China code》Requirement；Using saturation
(NH4)2SO4Salting-out method repeatedly is classified, bovine serum globulin is extracted, the blood plasma foreign protein of HMW is dispelled to greatest extent,
The bovine serum globulin purity of extraction is high.What the present invention was provided extracts the method technique letter of albumin and globulin from cow's serum
It is single, special installation is not required to, it is adapted to expanding production, the method for the utilization ox blood that the present invention is provided improves the utilization rate of ox blood.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be attached to what is used needed for embodiment
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, thus be not construed as it is right
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 be the embodiment of the present invention 1 in from cow's serum extract albumin and globulin process chart.
Specific embodiment
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, are the conventional product that can be obtained by commercially available purchase
Product.
A kind of method that albumin and globulin are extracted from cow's serum following to the embodiment of the present invention and utilize ox blood
Method be specifically described.
A kind of method that albumin and globulin are extracted from cow's serum, it includes：
The first step：Cow's serum separating step.
Ox blood is taken, cow's serum is isolated.
Preferably, it is incubated at the fresh bovine serum of acquirement being placed in into 35-38 DEG C after standing 2-4h, 12- is refrigerated at 2-8 DEG C
16h, draws the yellow liquid that upper strata separates out, and obtains cow's serum, is refrigerated at aseptic subpackaged latter 2-8 DEG C standby.
Preferably, above-mentioned ox blood is fresh ox blood.
Second step：Pre-treatment step.
(1) above-mentioned cow's serum is taken, the first supernatant is centrifuged to obtain, to the 0.8-1.2 times of 4- of volume of addition in the first supernatant
The pH of 6mM is the PBS of 7.0-7.4.
Specifically, above-mentioned centrifugation be 3000-4000r/min under the conditions of 20-40min is centrifuged.It is 2-8 DEG C that above-mentioned refrigeration stands
Under, stand 12-16h.
(2) lower addition (NH is stirred4)2SO4To 48%-52% saturation degrees；Suction filtration obtains the first filtrate and the after being stood through refrigeration
One precipitation.Retain standby at first filtrate is equal 2-8 DEG C with the above-mentioned first precipitation.
(the NH of 48%-52% saturation degrees4)2SO4Globulin is precipitated, and albumin times is so dissolved in solution, by taking out
The mode of filter separates the two.
3rd step：Extract albumin step.
The first filtrate is taken, extraction obtains albumin.
(1) above-mentioned first filtrate is taken, denaturation temperature is heated to after adding protective agent, be denatured foreign protein；Suction filtration obtains second
Filtrate.
Preferably, above-mentioned protective agent is Sodium Caprylate, and final concentration of in the first filtrate of Sodium Caprylate is 0.15%-
0.19% (m:v).Protective agent is added, can protect albumin from denaturation in degenerative process, stayed in the solution.
Preferably, toward addition 0.8%-1.2% (v in the first filtrate:V) absolute ethyl alcohol.Absolute ethyl alcohol under the concentration
Play a part of auxiliary denaturation.
Preferably, denaturation temperature is 55-65 DEG C, and heats 20-40min under denaturation temperature, foreign protein is fully become
Property.The too low purpose not reached except foreign protein of temperature, temperature is too high, then can be denatured albumin.
(2) toward addition (NH in above-mentioned second filtrate4)2SO4To 60%-64% saturation degrees, and pH to 4.4-4.8 is adjusted, it is quiet
Put and the second precipitation is centrifuged to obtain.
Specifically, above-mentioned time of repose be 2-4h, above-mentioned centrifugation be 3000-4000r/min under the conditions of 20-40min is centrifuged.
(3) above-mentioned second precipitation is dissolved, centrifugation obtains the second supernatant after dialysis, and above-mentioned second supernatant of dialysis concentration is obtained
To concentrate.
Specifically, second is dissolved with the PBS that the pH of 4-6mM is 7.0-7.4 to precipitate.
(4) above-mentioned concentrate is carried out into desalting processing, obtains albumin solution, obtained after albumin solution is freeze-dried
Albumin.
Preferably, above-mentioned concentrate carries out desalting processing by Sephadex G-50 posts.
4th step：Extract globulin step.
Above-mentioned first precipitation is taken, extraction obtains globulin.
(1) above-mentioned first precipitation is taken, dissolving first is precipitated, and adds (NH4) 2SO4 to 30%-35% saturation degrees, and adjust
PH to 6.5-7.0, the 3rd precipitation is obtained after agitated, standing centrifugation.
(the NH of 30%-35% saturation degrees4)2SO4In solution, globulin is separated out with foreign protein.
(2) dissolving the 3rd is precipitated, and adds (NH4)2SO4To 30%-35% saturation degrees, the 4th is obtained after agitated, standing centrifugation
Precipitation.
(3) dissolving the 4th is precipitated, and adds (NH4)2SO4To 18%-22% saturation degrees, the 3rd is obtained after agitated, standing centrifugation
Supernatant.
(the NH of 18%-22% saturation degrees4)2SO4In solution, foreign protein is separated out from solution, and is dissolved in globulin molten
In liquid.Globulin solution is obtained by centrifugation.
(4) toward addition (NH in the 3rd supernatant4)2SO4To 30%-35% saturation degrees, it is agitated, stand after centrifugation the
Five precipitations.
(the NH of 30%-35% saturation degrees4)2SO4In solution, globulin is separated out from solution, and centrifugation obtains containing globulin
Precipitation.
(5) precipitation of dissolving the 5th obtains thick globulin solution, and thick globulin solution is obtained after desalting processing and freeze-drying
To globulin.
Preferably, in this step (the 4th step), mentioned standing is standing 2-4h.Mentioned centrifugation is
20-40min is centrifuged under the conditions of 3000-4000r/min.The pH that medium used by dissolution precipitation is 4-6mM is 7.0-7.4's
PBS。
Preferably, what the present invention was provided extracts albumin and (NH used in the method for globulin from cow's serum4)2SO4
It is its saturated solution.(NH4)2SO4Saturated solution in, (NH4)2SO4Equal distributed mutually, when being added in protein solution, its energy
Rapid reaction, separates out albumen.Also to be avoided that and cause the excessive inconvenience of the liquor capacity for adding using unsaturated solution.
It should be noted that the 3rd step in this method：Extract albumin step and the 4th step：Extract globulin step it
Between do not have absolute sequencing point.That is the 3rd step：Extract albumin step and the 4th step：Extracting globulin step can hand over
Change sequencing.
Feature of the invention and performance are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of method that albumin and globulin are extracted from cow's serum, and the technological process of the method is such as
Shown in Fig. 1.Specifically, it includes：
The first step, carries out cow's serum separating step.
Take the fresh bovine bloods of 1L to be positioned in container, be incubated at 37 DEG C after standing 3h, 12h is refrigerated at 4 DEG C, in absorption
The yellow liquid for chromatographing out, obtains cow's serum, it is aseptic subpackaged after refrigerate at 4 DEG C it is standby.
Second step, carries out pre-treatment step.
Above-mentioned cow's serum is taken, 30min is centrifuged under the conditions of 3500r/min, discard precipitation, obtain the first supernatant, toward first
The 5mM PBS of isometric pH7.0 are added in supernatant, is stirred.Saturation (NH is slowly added under stirring4)2SO4Solution is extremely
(NH4)2SO4Saturation degree be 50%.Refrigerated at 4 DEG C after stirring and stand 12h, then suction filtration obtains the first filtrate and first
Precipitation, the first filtrate retains standby with the first precipitation.
3rd step, carries out extracting albumin step.
(1) above-mentioned first filtrate is taken, stirring is lower to add Sodium Caprylate to the final concentration of 0.17% (m of Sodium Caprylate:V), octanoic acid
After sodium is completely dissolved, the lower addition absolute ethyl alcohol of stirring to the concentration of absolute ethyl alcohol is 1% (v:V), heating water bath is maintained to 60 DEG C
30min, and pH to 6.3 is adjusted, continue to stir 45min.
(2) suction filtration, discards precipitation, obtains the second filtrate.Stirring is lower toward addition saturation (NH in the second filtrate4)2SO4Solution
To (NH4)2SO4Saturation degree is 62%, and adds 6mol/L HCl regulation pH to 4.6 under agitation, stands 2h.
(3) 20min is centrifuged under the conditions of 3500r/min, abandoning supernatant obtains the second precipitation.Added toward the second precipitation
The precipitations of 5mM PBS to second of pH7.0 are completely dissolved, and then will dissolve the solution after the second precipitation and be transferred to bag filter (molecule section
Allowance is 5KDa) dialyse 12h in the 5mM PBS of pH7.0, and change a 5mM PBS per 4h.
(4) solution after dialysing is centrifuged 30min under the conditions of 3500r/min, discards precipitation, obtains the second supernatant.Will
Second supernatant loads bag filter (molecule interception be 5KDa), and carries out during bag filter is imbedded into Macrogol 6000 (solid)
Dialysis concentration 12h, obtains concentrate.
(5) by Sephadex G-50 post desalinations on concentrate, eluted with the 5mM PBS of pH7.0, collected eluent,
Freeze-drying, obtains bovine serum albumin(BSA).
4th step, carries out globulin extraction step.
(1) above-mentioned first precipitation is taken, adds the precipitations of 5mM PBS to first of pH7.0 to be completely dissolved.Stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 33%, and adds 1mol/L NaOH regulation pH to 6.5 under agitation, is stood
20min is centrifuged after 2h under the conditions of 3500r/min, abandoning supernatant obtains the 3rd precipitation.
(2) precipitations of 5mM PBS to the 3rd toward addition pH7.0 in the 3rd precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 33%, and 20min, supernatant discarded is centrifuged under the conditions of 3500r/min after standing 2h
Liquid, obtains the 4th precipitation.
(3) precipitations of 5mM PBS to the 4th toward addition pH7.0 in the 4th precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 20%, and 30min is centrifuged under the conditions of 3500r/min after standing 2h, discards precipitation,
Obtain the 3rd supernatant.
(4) stir lower toward addition saturation (NH in the 3rd supernatant4)2SO4Solution is to (NH4)2SO4Saturation degree is 33%, quiet
Put and 20min is centrifuged under the conditions of 3500r/min after 2h, abandoning supernatant obtains the 5th precipitation.
(5) precipitations of 5mM PBS to the 5th toward addition pH7.0 in the 5th precipitation are completely dissolved, the precipitation gained of dissolving the 5th
Solution on Sephedex G-50 post desalinations, eluted with the 5mMPBS of pH7.0, collect eluent, freeze-drying obtains
Bovine serum globulin.
It should be noted that the 3rd step in the present embodiment：Extract albumin step and the 4th step：Extract globulin step
Between do not have absolute sequencing point.In other embodiments, the 3rd step：Extract albumin step and the 4th step：Extract
Globulin step can exchange sequencing.
Embodiment 2
The present embodiment provides a kind of method that albumin and globulin are extracted from cow's serum.It includes：
The first step, carries out cow's serum separating step.
Take the fresh bovine bloods of 1L to be positioned in container, be incubated at 35 DEG C after standing 2h, 14h is refrigerated at 2 DEG C, in absorption
The yellow liquid for chromatographing out, obtains cow's serum, it is aseptic subpackaged after refrigerate at 2 DEG C it is standby.
Second step, carries out pre-treatment step.
Above-mentioned cow's serum is taken, 20min is centrifuged under the conditions of 3000r/min, discard precipitation, obtain the first supernatant, toward first
0.8 times of 4mM PBS of the pH7.2 of volume is added in supernatant, is stirred.Saturation (NH is slowly added under stirring4)2SO4Solution
To (NH4)2SO4Saturation degree be 48%.Refrigerated at 2 DEG C after stirring and stand 14h, then suction filtration obtains the first filtrate and the
One precipitation, the first filtrate retains standby with the first precipitation.
3rd step, carries out extracting albumin step.
(1) above-mentioned first filtrate is taken, stirring is lower to add Sodium Caprylate to the final concentration of 0.15% (m of Sodium Caprylate:V), octanoic acid
After sodium is completely dissolved, the lower addition absolute ethyl alcohol of stirring to the concentration of absolute ethyl alcohol is 0.8% (v:V), heating water bath is tieed up to 55 DEG C
Hold 40min and adjust pH to 6.0, continue to stir 30min.
(2) suction filtration, discards precipitation, obtains the second filtrate.Stirring is lower toward addition saturation (NH in the second filtrate4)2SO4Solution
To (NH4)2SO4Saturation degree is 60%, and adds 6mol/L HCl regulation pH to 4.4 under agitation, stands 3h.
(3) 30min is centrifuged under the conditions of 3000r/min, abandoning supernatant obtains the second precipitation.Added toward the second precipitation
The precipitations of 4mM PBS to second of pH7.2 are completely dissolved, and then will dissolve the solution after the second precipitation and be transferred to bag filter (molecule section
Allowance is 5KDa) dialysed in the 4mM PBS of pH7.2, and change a 4mM PBS per 4h.
(4) solution after dialysing is centrifuged 20min under the conditions of 3300r/min, discards precipitation, obtains the second supernatant.Will
Second supernatant loads bag filter (molecule interception be 5KDa), and carries out during bag filter is imbedded into Macrogol 6000 (solid)
Dialysis concentration 12h, obtains concentrate.
(5) by Sephadex G-50 post desalinations on concentrate, eluted with the 4mM PBS of pH7.2, collected eluent,
Freeze-drying, obtains bovine serum albumin(BSA).
4th step, carries out globulin extraction step.
(1) above-mentioned first precipitation is taken, adds the precipitations of 4mM PBS to first of pH7.2 to be completely dissolved.Stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 30%, and adds 1mol/L NaOH regulation pH to 6.7 under agitation, is stood
30min is centrifuged after 3h under the conditions of 3000r/min, abandoning supernatant obtains the 3rd precipitation.
(2) precipitations of 4mM PBS to the 3rd toward addition pH7.2 in the 3rd precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 30%, and 30min, supernatant discarded is centrifuged under the conditions of 3000r/min after standing 3h
Liquid, obtains the 4th precipitation.
(3) precipitations of 4mM PBS to the 4th toward addition pH7.2 in the 4th precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 18%, and 20min is centrifuged under the conditions of 3000r/min after standing 3h, discards precipitation,
Obtain the 3rd supernatant.
(4) stir lower toward addition saturation (NH in the 3rd supernatant4)2SO4Solution is to (NH4)2SO4Saturation degree is 30%, quiet
Put and 30min is centrifuged under the conditions of 3000r/min after 3h, abandoning supernatant obtains the 5th precipitation.
(5) precipitations of 4mM PBS to the 5th toward addition pH7.2 in the 5th precipitation are completely dissolved, the precipitation gained of dissolving the 5th
Solution on Sephedex G-50 post desalinations, eluted with the 4mM PBS of pH7.2, collect eluent, freeze-drying obtains
Bovine serum globulin.
Embodiment 3
The present embodiment provides a kind of method that albumin and globulin are extracted from cow's serum.It includes：
The first step, carries out cow's serum separating step.
Take the fresh bovine bloods of 1L to be positioned in container, be incubated at 38 DEG C after standing 4h, 16h is refrigerated at 8 DEG C, in absorption
The yellow liquid for chromatographing out, obtains cow's serum, it is aseptic subpackaged after refrigerate at 8 DEG C it is standby.
Second step, carries out pre-treatment step.
Above-mentioned cow's serum is taken, 40min is centrifuged under the conditions of 4000r/min, discard precipitation, obtain the first supernatant, toward first
The 6mM PBS of isometric pH7.4 are added in supernatant, is stirred.Saturation (NH is slowly added under stirring4)2SO4Solution is extremely
(NH4)2SO4Saturation degree be 52%.Refrigerated at 8 DEG C after stirring and stand 16h, then suction filtration obtains the first filtrate and first
Precipitation, the first filtrate retains standby with the first precipitation.
3rd step, carries out extracting albumin step.
(1) above-mentioned first filtrate is taken, stirring is lower to add Sodium Caprylate to the final concentration of 0.19% (m of Sodium Caprylate:V), octanoic acid
After sodium is completely dissolved, the lower addition absolute ethyl alcohol of stirring to the concentration of absolute ethyl alcohol is 1.2% (v:V), heating water bath is tieed up to 65 DEG C
20min is held, and adjusts pH to 6.5, continue to stir 40min.
(2) suction filtration, discards precipitation, obtains the second filtrate.Stirring is lower toward addition saturation (NH in the second filtrate4)2SO4Solution
To (NH4)2SO4Saturation degree is 64%, and adds 6mol/L HCl regulation pH to 4.8 under agitation, stands 4h.
(3) 40min is centrifuged under the conditions of 4000r/min, abandoning supernatant obtains the second precipitation.Added toward the second precipitation
The precipitations of 6mM PBS to second of pH7.4 are completely dissolved, and then will dissolve the solution after the second precipitation and be transferred to bag filter (molecule section
Allowance is 5KDa) dialysed in the 6mM PBS of pH7.4, and change a 6mM PBS per 4h.
(4) solution after dialysing is centrifuged 20min under the conditions of 4000r/min, discards precipitation, obtains the second supernatant.Will
Second supernatant loads bag filter (molecule interception is 5KDa), and bag filter is imbedded into the middle dialysis of Macrogol 6000 (solid)
Concentration 12h, obtains concentrate.
(5) by Sephadex G-50 post desalinations on concentrate, eluted with the 6mM PBS of pH7.4, collected eluent,
Freeze-drying, obtains bovine serum albumin(BSA).
4th step, carries out globulin extraction step.
(1) above-mentioned first precipitation is taken, adds the precipitations of 6mM PBS to first of pH7.4 to be completely dissolved.Stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 35%, and adds 1mol/L NaOH regulation pH to 7.0 under agitation, is stood
40min is centrifuged after 4h under the conditions of 4000r/min, abandoning supernatant obtains the 3rd precipitation.
(2) precipitations of 6mM PBS to the 3rd toward addition pH7.4 in the 3rd precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 35%, and 40min, supernatant discarded is centrifuged under the conditions of 4000r/min after standing 4h
Liquid, obtains the 4th precipitation.
(3) precipitations of 6mM PBS to the 4th toward addition pH7.4 in the 4th precipitation are completely dissolved, and stirring is lower to add saturation
(NH4)2SO4Solution is to (NH4)2SO4Saturation degree is 22%, and 40min is centrifuged under the conditions of 4000r/min after standing 4h, discards precipitation,
Obtain the 3rd supernatant.
(4) stir lower toward addition saturation (NH in the 3rd supernatant4)2SO4Solution is to (NH4)2SO4Saturation degree is 35%, quiet
Put and 40min is centrifuged under the conditions of 4000r/min after 4h, abandoning supernatant obtains the 5th precipitation.
(5) precipitations of 6mM PBS to the 5th toward addition pH7.4 in the 5th precipitation are completely dissolved, the precipitation gained of dissolving the 5th
Solution on Sephedex G-50 post desalinations, eluted with the 6mM PBS of pH7.4, collect eluent, freeze-drying obtains
Bovine serum globulin.
Embodiment 4
The present embodiment provides the method extraction ox blood that albumin and globulin are extracted from cow's serum in Application Example 2
The purity of pure albumen and bovine serum globulin, the rate of recovery and recovery rate are detected.
The principle that this method is precipitated using plasma protein in the salting liquid of certain saturation degree, using (NH4)2SO4Classification is heavy
Form sediment, and be further purified and obtain bovine serum albumin(BSA).Ox blood is extracted respectively from 5 fresh ox bloods of different batches with this method
Pure albumen is detected that testing result is as shown in table 1.
The testing result of the bovine serum albumin(BSA) that table 1 is extracted from 5 fresh ox bloods of batch
| Batch | Purity (%) | The rate of recovery (%) |
| 1 | 98.6 | 81.0 |
| 2 | 98.1 | 81.3 |
| 3 | 98.9 | 80.6 |
| 4 | 98.8 | 80.9 |
| 5 | 98.6 | 80.2 |
As shown in Table 1, the purity of the bovine serum albumin(BSA) for being extracted from 5 ox bloods of batch is all higher than 98%, and returns
Yield is all higher than 80%.
Endotoxin content in the bovine serum albumin(BSA) of extraction is determined with limulus reagent test, is as a result shown, in bovine serum albumin(BSA)
Endotoxic content is less than 0.25EU/mL, and quality meets《Products in China code》Requirement.
This method chooses saturated ammonium sulfate classification salting-out method repeatedly, carries out separation to the immunoglobulin in cow's serum and carries
Take, the blood plasma foreign protein of HMW is dispelled to greatest extent (such as：Fibrinogen, fibronectin splicing variants, IgM etc.), obtain
Bovine serum globulin, the yield for making the extraction bovine serum globulin of this method is 9.03g/L, and the rate of recovery is 90%.After testing, carry
The purity of the bovine serum globulin for taking is more than 95%.
To sum up, the method that albumin and globulin are extracted from cow's serum that the present invention is provided, using plasma protein
The principle precipitated in the salting liquid of saturation degree is determined, using (NH4)2SO4Fractional precipitation, and be further purified and obtain bovine serum albumin
In vain, the bovine serum albumin(BSA) purity is high, and endotoxin content is low, meets《Products in China code》Requirement；Using saturation
(NH4)2SO4Salting-out method repeatedly is classified, bovine serum globulin is extracted, the blood plasma foreign protein of HMW is dispelled to greatest extent,
The bovine serum globulin purity of extraction is high.What the present invention was provided extracts the method technique letter of albumin and globulin from cow's serum
It is single, special installation is not required to, it is adapted to expanding production, the method for the utilization ox blood that the present invention is provided improves the utilization rate of ox blood.
Embodiments described above is a part of embodiment of the invention, rather than whole embodiments.Reality of the invention
The detailed description for applying example is not intended to limit the scope of claimed invention, but is merely representative of selected implementation of the invention
Example.Based on the embodiment in the present invention, what those of ordinary skill in the art were obtained under the premise of creative work is not made
Every other embodiment, belongs to the scope of protection of the invention.
Claims (10)
1. a kind of method that albumin and globulin are extracted from cow's serum, it is characterised in that it includes：
Cow's serum is taken, the first supernatant is centrifuged to obtain, to 0.8-1.2 times of first supernatant of addition in first supernatant
The pH of the 4-6mM of volume is the PBS of 7.0-7.4；Stirring is lower to add (NH4)2SO4To 48%-52% saturation degrees；Through refrigeration, stand
Suction filtration obtains the first filtrate and the first precipitation afterwards；
Albumin is extracted from first filtrate；
Globulin is extracted from the described first precipitation.
2. the method that albumin and globulin are extracted from cow's serum according to claim 1, it is characterised in that from described
The albumin is extracted in first filtrate to be included：
Protective agent is added to first filtrate and be heated to denaturation temperature, be denatured foreign protein；Suction filtration obtains the second filtrate；
(NH is added toward second filtrate4)2SO4To 60%-64% saturation degrees, and pH to 4.4-4.8 is adjusted, stand centrifugation
Obtain the second precipitation；
Second precipitation is dissolved, centrifugation obtains the second supernatant after dialysis, and dialysis concentration second supernatant is concentrated
Liquid；
The concentrate obtains albumin solution through desalting processing, and the albumin solution is freeze-dried to obtain the white egg
In vain.
3. the method that albumin and globulin are extracted from cow's serum according to claim 2, it is characterised in that the change
Degree warm in nature is 55-65 DEG C.
4. the method that albumin and globulin are extracted from cow's serum according to claim 3, it is characterised in that be heated to
After the denaturation temperature, the denaturation temperature is maintained, continue to heat 20-40min.
5. the method that albumin and globulin are extracted from cow's serum according to claim 2, it is characterised in that the guarantor
Shield agent is Sodium Caprylate, final concentration of 0.15%-0.19% (m of the Sodium Caprylate in first filtrate:v).
6. the method that albumin and globulin are extracted from cow's serum according to claim 2, it is characterised in that described de-
Salt treatment is：By the concentrate by Sephadex G-50 post desalinations.
7. the method that albumin and globulin are extracted from cow's serum according to claim 1, it is characterised in that from described
Extracting the globulin in first precipitation includes：
First precipitation is dissolved, (NH is added4)2SO4To 30%-35% saturation degrees, and pH to 6.5-7.0 is adjusted, agitated,
The 3rd precipitation is obtained after standing centrifugation；
The 3rd precipitation is dissolved, (NH is added4)2SO4The 4th is obtained to 30%-35% saturation degrees, after agitated, standing centrifugation to sink
Form sediment；
The 4th precipitation is dissolved, (NH is added4)2SO4To 18%-22% saturation degrees, obtained on the 3rd after agitated, standing centrifugation
Clear liquid；
(NH is added toward the 3rd supernatant4)2SO4The 5th is obtained to 30%-35% saturation degrees, after agitated, standing centrifugation to sink
Form sediment；
Dissolve the 5th precipitation and obtain thick globulin solution, the thick globulin solution is obtained after desalting processing and freeze-drying
To the globulin.
8. the method that albumin and globulin are extracted from cow's serum according to any one of claim 1-7, its feature
It is, (the NH4)2SO4It is (NH4)2SO4Saturated solution.
9. a kind of method of utilization ox blood, it is characterised in that it includes：
Cow's serum is isolated from ox blood；
According to the method for extraction albumin and globulin from cow's serum any one of claim 1-8 from the ox blood
Bovine serum albumin(BSA) and bovine serum globulin are extracted in clear.
10. the method for utilization ox blood according to claim 9, it is characterised in that the ox is isolated from the ox blood
The step of serum, includes：By the ox blood at 35-38 DEG C, insulation stands 2-4h, 2-8 DEG C of refrigeration 12-16h, is chromatographed in absorption
The yellow liquid for going out, obtains the cow's serum.
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| RU2696480C1 (en) * | 2018-03-12 | 2019-08-02 | Александр Владимирович Захаров | Method of producing a gamma-globulin semi-product with high content of antiviral antibodies from slaughter blood of vaccinated cows |
| CN111138517A (en) * | 2020-01-10 | 2020-05-12 | 武汉科前生物股份有限公司 | Method for improving safety of recombinant protein vaccine and application thereof |
| CN111205362A (en) * | 2020-03-19 | 2020-05-29 | 贾芳苗 | Bovine serum albumin and preparation method thereof |
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| CN102952187A (en) * | 2012-11-30 | 2013-03-06 | 深圳市美凯特科技有限公司 | Preparation method of high-purity bovine serum albumin |
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| CN111138517A (en) * | 2020-01-10 | 2020-05-12 | 武汉科前生物股份有限公司 | Method for improving safety of recombinant protein vaccine and application thereof |
| CN111205362A (en) * | 2020-03-19 | 2020-05-29 | 贾芳苗 | Bovine serum albumin and preparation method thereof |
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