CN106749626B - Method for extracting albumin and globulin from bovine serum and method for utilizing bovine serum - Google Patents
Method for extracting albumin and globulin from bovine serum and method for utilizing bovine serum Download PDFInfo
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- 239000012888 bovine serum Substances 0.000 title claims abstract description 79
- 238000000034 method Methods 0.000 title claims abstract description 67
- 102000006395 Globulins Human genes 0.000 title claims abstract description 51
- 108010044091 Globulins Proteins 0.000 title claims abstract description 51
- 102000009027 Albumins Human genes 0.000 title claims abstract description 49
- 108010088751 Albumins Proteins 0.000 title claims abstract description 49
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 19
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 19
- 102000005686 Serum Globulins Human genes 0.000 claims abstract description 16
- 108010045362 Serum Globulins Proteins 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims description 92
- 239000000243 solution Substances 0.000 claims description 64
- 238000003756 stirring Methods 0.000 claims description 49
- 239000006228 supernatant Substances 0.000 claims description 43
- 239000000706 filtrate Substances 0.000 claims description 32
- 241000283690 Bos taurus Species 0.000 claims description 22
- 210000004369 blood Anatomy 0.000 claims description 22
- 239000008280 blood Substances 0.000 claims description 22
- 238000011033 desalting Methods 0.000 claims description 12
- 229960005480 sodium caprylate Drugs 0.000 claims description 11
- BYKRNSHANADUFY-UHFFFAOYSA-M sodium octanoate Chemical group [Na+].CCCCCCCC([O-])=O BYKRNSHANADUFY-UHFFFAOYSA-M 0.000 claims description 11
- 238000004925 denaturation Methods 0.000 claims description 10
- 230000036425 denaturation Effects 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 8
- 238000000967 suction filtration Methods 0.000 claims description 7
- 229920005654 Sephadex Polymers 0.000 claims description 5
- 239000012507 Sephadex™ Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 239000003223 protective agent Substances 0.000 claims description 5
- 239000012047 saturated solution Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 abstract description 7
- 239000002158 endotoxin Substances 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 102000004506 Blood Proteins Human genes 0.000 abstract description 4
- 108010017384 Blood Proteins Proteins 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 4
- 239000012266 salt solution Substances 0.000 abstract description 4
- 238000005185 salting out Methods 0.000 abstract description 4
- 238000000751 protein extraction Methods 0.000 abstract description 2
- 229920006395 saturated elastomer Polymers 0.000 description 18
- 239000000203 mixture Substances 0.000 description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 238000000502 dialysis Methods 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
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- 108010067306 Fibronectins Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
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- 102000018358 immunoglobulin Human genes 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- 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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Abstract
The invention discloses a method for extracting albumin and globulin from bovine serum and a method for utilizing bovine serum, and relates to the technical field of protein extraction. The method for extracting albumin and globulin from bovine serum provided by the invention utilizes the principle that plasma protein is precipitated in a salt solution with a certain saturation degree and adopts (NH)4)2SO4Carrying out fractional precipitation and further purifying to obtain bovine serum albumin which has high purity and low endotoxin content and meets the requirements of Chinese biological product regulation; by saturation (NH)4)2SO4The method of salting out repeatedly in stages extracts the bovine serum globulin, removes the high molecular weight plasma impure protein to the maximum extent, and the extracted bovine serum globulin has high purity. The method for extracting albumin and globulin from the bovine serum provided by the invention has the advantages of simple process, no need of special equipment and suitability for expanded production, and the utilization rate of the bovine serum is improved by the method for utilizing the bovine serum provided by the invention.
Description
Technical Field
The invention relates to the technical field of protein extraction, in particular to a method for extracting albumin and globulin from bovine serum and a method for utilizing bovine blood.
Background
The bovine blood processing technology mainly refers to a process for extracting various proteins from bovine blood so as to provide additional value of the bovine blood and improve the utilization rate of the bovine blood. At present, many methods for separating and purifying bovine serum albumin are available at home and abroad, and the methods are commonly known as an ion exchange method, a gel filtration method, an affinity chromatography method, a zone electrophoresis method, an ultracentrifugation method and the like. The yield of the methods is ideal, but the method has harsh requirements and needs special equipment, so the method is difficult to popularize and has no production significance.
Disclosure of Invention
The invention aims to provide a method for extracting albumin and globulin from bovine serum, which does not need special equipment and has high yield, high purity and high recovery rate of the extracted bovine serum albumin and the extracted bovine serum globulin.
Another object of the present invention is to provide a method for using bovine blood, which comprises separating bovine serum from the bovine blood and then extracting bovine serum albumin and bovine serum globulin by the above-mentioned method for extracting albumin and globulin from bovine serum.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
A method of extracting albumin and globulin from bovine serum comprising:
centrifuging bovine serum to obtain first supernatant, and adding 4-6mM PBS (pH 7.0-7.4) in an amount of 0.8-1.2 times the volume of the first supernatant; adding (NH) with stirring4)2SO4To 48% -52% saturation; refrigerating, standing, and filtering to obtain a first filtrate and a first precipitate;
albumin is extracted from the first filtrate.
Globulin is extracted from the first precipitate.
A method of utilizing bovine blood comprising:
bovine serum was isolated from bovine blood.
Bovine serum albumin and bovine serum globulin were extracted from bovine serum according to the above-described method for extracting albumin and globulin from bovine serum.
The method for extracting albumin and globulin from bovine serum and the method for utilizing bovine serum provided by the embodiment of the invention have the beneficial effects that: the method for extracting albumin and globulin from bovine serum provided by the invention utilizes the principle that plasma protein is precipitated in a salt solution with a certain saturation degree and adopts (NH)4)2SO4Fractional precipitation and further purification to obtain bovine serum albumin with high purity,the endotoxin content is low, which meets the requirements of Chinese biological product regulation; by saturation (NH)4)2SO4The method of salting out repeatedly in stages extracts the bovine serum globulin, removes the high molecular weight plasma impure protein to the maximum extent, and the extracted bovine serum globulin has high purity. The method for extracting albumin and globulin from the bovine serum provided by the invention has the advantages of simple process, no need of special equipment and suitability for expanded production, and the utilization rate of the bovine serum is improved by the method for utilizing the bovine serum provided by the invention.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a process flow chart of the process of extracting albumin and globulin from bovine serum in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Hereinafter, a method for extracting albumin and globulin from bovine serum and a method for using bovine serum according to an embodiment of the present invention will be described in detail.
A method of extracting albumin and globulin from bovine serum comprising:
the first step is as follows: and (5) separating bovine serum.
Taking bovine blood, and separating out bovine serum.
Preferably, the obtained fresh bovine serum is placed at 35-38 ℃ for heat preservation and standing for 2-4h, then refrigerated at 2-8 ℃ for 12-16h, the yellow liquid precipitated from the upper layer is absorbed to obtain the bovine serum, and the bovine serum is refrigerated at 2-8 ℃ for standby after being aseptically subpackaged.
Preferably, the bovine blood is fresh bovine blood.
The second step is that: and (4) a pretreatment step.
(1) Taking the above bovine serum, centrifuging to obtain a first supernatant, and adding 0.8-1.2 times volume of 4-6mM PBS with pH of 7.0-7.4 into the first supernatant.
Specifically, the centrifugation is carried out for 20-40min under the condition of 3000-4000 r/min. Standing at 2-8 deg.C for 12-16 h.
(2) Adding (NH) with stirring4)2SO4To 48% -52% saturation; refrigerating and standing, and then performing suction filtration to obtain a first filtrate and a first precipitate. The first filtrate and the first precipitate are both reserved at 2-8 ℃ for standby.
48% -52% saturation (NH)4)2SO4The globulin is precipitated, while the albumin is optionally dissolved in the solution, which is separated by suction filtration.
The third step: and (3) extracting albumin.
And taking the first filtrate, and extracting to obtain albumin.
(1) Adding a protective agent into the first filtrate, and heating to a denaturation temperature to denature the hybrid protein; and (4) performing suction filtration to obtain a second filtrate.
Preferably, the protective agent is sodium caprylate, and the final concentration of sodium caprylate in the first filtrate is 0.15% to 0.19% (m: v). The addition of a protective agent protects the albumin from denaturation during denaturation and remains in solution.
Preferably, 0.8% to 1.2% (v: v) of absolute ethanol is added to the first filtrate. The absolute ethyl alcohol at the concentration plays a role in assisting the denaturation.
Preferably, the denaturation temperature is 55-65 deg.C, and the hybrid protein is sufficiently denatured by heating at the denaturation temperature for 20-40 min. If the temperature is too low, the purpose of removing the albumin cannot be achieved, and if the temperature is too high, the albumin can be denatured.
(2) Adding (NH) to the second filtrate4)2SO4To 60 percent-64% saturation, adjusting the pH to 4.4-4.8, standing and centrifuging to obtain a second precipitate.
Specifically, the standing time is 2-4h, and the centrifugation is carried out for 20-40min under the condition of 3000-4000 r/min.
(3) Dissolving the second precipitate, dialyzing, centrifuging to obtain a second supernatant, and dialyzing and concentrating the second supernatant to obtain a concentrated solution.
Specifically, the second precipitate was dissolved with 4-6mM PBS at pH 7.0-7.4.
(4) Desalting the concentrated solution to obtain albumin solution, and freeze-drying the albumin solution to obtain albumin.
Preferably, the concentrated solution is desalted by a Sephadex G-50 column.
The fourth step: and (3) extracting globulin.
And (4) extracting the first precipitate to obtain globulin.
(1) Dissolving the first precipitate, adding (NH4)2SO4 to 30% -35% saturation, adjusting pH to 6.5-7.0, stirring, standing, and centrifuging to obtain a third precipitate.
30% -35% saturation of (NH)4)2SO4In the solution, both globulin and hetero-protein are separated out.
(2) Dissolving the third precipitate, adding (NH)4)2SO4And (4) stirring, standing and centrifuging to obtain a fourth precipitate after the saturation degree is 30-35%.
(3) Dissolving the fourth precipitate, adding (NH)4)2SO4And (4) stirring, standing and centrifuging to obtain a third supernatant after the saturation degree of 18-22%.
18% -22% saturation (NH)4)2SO4In the solution, the hetero-proteins are precipitated from the solution and dissolved in the solution together with the globulin. The globulin solution was obtained by centrifugation.
(4) (NH) is added to the third supernatant4)2SO4And (4) stirring, standing and centrifuging to obtain a fifth precipitate after the saturation degree of 30-35%.
30% -35% saturation of (NH)4)2SO4In the solution, globulin is precipitated from the solution, and a precipitate containing globulin is obtained by centrifugation.
(5) Dissolving the fifth precipitate to obtain a crude globulin solution, desalting the crude globulin solution, and freeze-drying to obtain the globulin.
Preferably, in this step (fourth step), all standing mentioned is from 2 to 4 hours. The centrifugation is carried out for 20-40min under the condition of 3000-4000 r/min. The medium used for dissolving the precipitate was 4-6mM PBS with pH 7.0-7.4.
Preferably, the (NH) used in the method for extracting albumin and globulin from bovine serum provided by the invention4)2SO4All are saturated solutions thereof. (NH)4)2SO4In a saturated solution of (NH)4)2SO4Homogeneous phase distribution, when added into protein solution, it can react quickly to separate out protein. And the inconvenience of overlarge volume of the added solution caused by using an unsaturated solution can be avoided.
It should be noted that, the third step in the method is: the step of extracting albumin and the fourth step: there is no absolute sequencing between the steps of extracting the globulin. Namely, the third step: the step of extracting albumin and the fourth step: the steps for extracting the globulin can be exchanged in sequence.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a method for extracting albumin and globulin from bovine serum, the process flow of which is shown in fig. 1. Specifically, it comprises:
in the first step, a bovine serum separation step is performed.
Placing 1L fresh bovine blood into a container, standing at 37 deg.C for 3 hr, refrigerating at 4 deg.C for 12 hr, sucking yellow liquid separated from upper layer to obtain bovine serum, aseptically packaging, and refrigerating at 4 deg.C for use.
And a second step of performing a pretreatment step.
Centrifuging the above bovine serum at 3500r/min for 30min, discarding precipitate,to the first supernatant was added an equal volume of 5mM PBS (pH7.0) and stirred well. Slowly add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation of (a) is 50%. Stirring, refrigerating at 4 deg.C, standing for 12 hr, filtering to obtain first filtrate and first precipitate, and keeping the first filtrate and the first precipitate.
And thirdly, carrying out a step of extracting albumin.
(1) Taking the first filtrate, adding sodium caprylate under stirring until the final concentration of sodium caprylate is 0.17% (m: v), adding anhydrous ethanol under stirring until the concentration of anhydrous ethanol is 1% (v: v), heating in water bath to 60 deg.C, maintaining for 30min, adjusting pH to 6.3, and continuing stirring for 45 min.
(2) Suction filtration is carried out, and the precipitate is discarded to obtain a second filtrate. Adding saturated (NH) into the second filtrate under stirring4)2SO4Solution to (NH)4)2SO4The saturation is 62%, and 6mol/L HCl is added to adjust the pH to 4.6 under stirring, and the mixture is kept stand for 2 hours.
(3) Centrifuging at 3500r/min for 20min, and discarding the supernatant to obtain a second precipitate. To the second precipitation, 5mM PBS (pH7.0) was added until the second precipitation was completely dissolved, and then the solution after dissolving the second precipitation was transferred to a dialysis bag (molecular cut-off: 5kDa) and dialyzed for 12 hours in 5mM PBS (pH7.0), and 5mM PBS was changed every 4 hours.
(4) Centrifuging the dialyzed solution at 3500r/min for 30min, and discarding the precipitate to obtain a second supernatant. And filling the second supernatant into a dialysis bag (with a molecular interception of 5KDa), embedding the dialysis bag into polyethylene glycol 6000 (solid), and dialyzing and concentrating for 12h to obtain a concentrated solution.
(5) Desalting the concentrated solution with Sephadex G-50 column, eluting with 5mM PBS (pH7.0), collecting eluate, and freeze drying to obtain bovine serum albumin.
And fourthly, performing a globulin extraction step.
(1) The first precipitate was taken and 5mM PBS pH7.0 was added until the first precipitate was completely dissolved. Add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 33 percent, 1mol/L NaOH is added under stirring to adjust the pH value to 6.5, the mixture is kept stand for 2 hours and then centrifuged for 20 minutes under the condition of 3500r/min, and the supernatant is discarded to obtain a third precipitate.
(2) Add 5mM PBS pH7.0 to the third precipitate until the third precipitate is completely dissolved, and add saturated (NH) under stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 33 percent, the mixture is kept stand for 2h and then centrifuged for 20min under the condition of 3500r/min, and the supernatant is discarded to obtain a fourth precipitate.
(3) Add 5mM PBS pH7.0 to the fourth precipitate until the fourth precipitate is completely dissolved, and add saturated (NH) under stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 20%, the mixture is kept stand for 2h and then centrifuged for 30min under the condition of 3500r/min, and the precipitate is discarded to obtain a third supernatant.
(4) Adding saturated (NH) to the third supernatant with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 33 percent, the mixture is kept stand for 2h and then centrifuged for 20min under the condition of 3500r/min, and the supernatant is discarded to obtain a fifth precipitate.
(5) And adding 5mM PBS (pH7.0) into the fifth precipitate until the fifth precipitate is completely dissolved, desalting the solution obtained by dissolving the fifth precipitate on a Sephedex G-50 column, eluting with 5mM PBS (pH7.0), collecting the eluent, and freeze-drying to obtain bovine serum albumin.
It should be noted that, in this embodiment, the third step: the step of extracting albumin and the fourth step: there is no absolute sequencing between the steps of extracting the globulin. In other embodiments, the third step: the step of extracting albumin and the fourth step: the steps for extracting the globulin can be exchanged in sequence.
Example 2
This example provides a method for extracting albumin and globulin from bovine serum. It includes:
in the first step, a bovine serum separation step is performed.
Placing 1L fresh bovine blood into a container, standing at 35 deg.C for 2 hr, refrigerating at 2 deg.C for 14 hr, sucking yellow liquid separated from upper layer to obtain bovine serum, aseptically packaging, and refrigerating at 2 deg.C for use.
And a second step of performing a pretreatment step.
Centrifuging the above bovine serum at 3000r/min for 20min, discarding precipitate to obtain first supernatant, adding 0.8 volume of 4mM PBS (pH7.2) into the first supernatant, and stirring. Slowly add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation of (a) is 48%. Stirring, refrigerating at 2 deg.C, standing for 14 hr, filtering to obtain first filtrate and first precipitate, and keeping the first filtrate and the first precipitate.
And thirdly, carrying out a step of extracting albumin.
(1) Taking the first filtrate, adding sodium caprylate under stirring until the final concentration of sodium caprylate is 0.15% (m: v), adding anhydrous ethanol under stirring until the concentration of anhydrous ethanol is 0.8% (v: v), heating in water bath to 55 deg.C, maintaining for 40min, adjusting pH to 6.0, and continuing stirring for 30 min.
(2) Suction filtration is carried out, and the precipitate is discarded to obtain a second filtrate. Adding saturated (NH) into the second filtrate under stirring4)2SO4Solution to (NH)4)2SO4The saturation is 60%, and 6mol/L HCl is added to adjust the pH to 4.4 under stirring, and the mixture is kept stand for 3 hours.
(3) Centrifuging at 3000r/min for 30min, and discarding supernatant to obtain second precipitate. To the second precipitate, 4mM PBS (pH7.2) was added until the second precipitate was completely dissolved, and then the solution after dissolving the second precipitate was transferred to a dialysis bag (molecular cut-off: 5kDa) and dialyzed against 4mM PBS (pH7.2), and 4mM PBS was changed every 4 hours.
(4) Centrifuging the dialyzed solution at 3300r/min for 20min, and discarding the precipitate to obtain a second supernatant. And filling the second supernatant into a dialysis bag (with a molecular interception of 5KDa), embedding the dialysis bag into polyethylene glycol 6000 (solid), and dialyzing and concentrating for 12h to obtain a concentrated solution.
(5) Desalting the concentrated solution with Sephadex G-50 column, eluting with 4mM PBS (pH7.2), collecting eluate, and freeze drying to obtain bovine serum albumin.
And fourthly, performing a globulin extraction step.
(1) The first precipitate was taken and 4mM PBS pH7.2 was added until the first precipitate was completely dissolved. Add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 30 percent, 1mol/L NaOH is added under stirring to adjust the pH value to 6.7, the mixture is stood for 3 hours and then centrifuged for 30 minutes under the condition of 3000r/min, and the supernatant is discarded to obtain a third precipitate.
(2) Adding 4mM PBS (pH7.2) to the third precipitate until the third precipitate is completely dissolved, and adding saturated (NH) solution under stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 30%, the mixture is stood for 3 hours and then centrifuged for 30min under the condition of 3000r/min, and the supernatant is discarded to obtain a fourth precipitate.
(3) Add 4mM PBS pH7.2 to the fourth precipitate until the fourth precipitate is completely dissolved, add saturated (NH) solution with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 18%, the mixture is stood for 3 hours and then centrifuged for 20min at the speed of 3000r/min, and the precipitate is discarded to obtain a third supernatant.
(4) Adding saturated (NH) to the third supernatant with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 30%, the mixture is stood for 3 hours and then centrifuged for 30min under the condition of 3000r/min, and the supernatant is discarded to obtain a fifth precipitate.
(5) Adding 4mM PBS (pH7.2) into the fifth precipitate until the fifth precipitate is completely dissolved, desalting the solution obtained by dissolving the fifth precipitate on a Sephedex G-50 column, eluting with 4mM PBS (pH7.2), collecting the eluate, and freeze-drying to obtain bovine serum globulin.
Example 3
This example provides a method for extracting albumin and globulin from bovine serum. It includes:
in the first step, a bovine serum separation step is performed.
Placing 1L fresh bovine blood into a container, standing at 38 deg.C for 4 hr, refrigerating at 8 deg.C for 16 hr, sucking yellow liquid separated from upper layer to obtain bovine serum, aseptically packaging, and refrigerating at 8 deg.C for use.
And a second step of performing a pretreatment step.
Taking the bovine serum, centrifuging for 40min at 4000r/min, discarding the precipitate to obtain a first supernatant, adding equal volume of 6mM PBS (pH7.4) into the first supernatant, and stirring uniformly. Slowly add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation of (a) is 52%. Stirring, refrigerating at 8 deg.C, standing for 16h, filtering to obtain first filtrate and first precipitate, and keeping the first filtrate and the first precipitate.
And thirdly, carrying out a step of extracting albumin.
(1) Taking the first filtrate, adding sodium caprylate under stirring until the final concentration of sodium caprylate is 0.19% (m: v), completely dissolving sodium caprylate, adding anhydrous ethanol under stirring until the concentration of anhydrous ethanol is 1.2% (v: v), heating in water bath to 65 deg.C, maintaining for 20min, adjusting pH to 6.5, and continuing stirring for 40 min.
(2) Suction filtration is carried out, and the precipitate is discarded to obtain a second filtrate. Adding saturated (NH) into the second filtrate under stirring4)2SO4Solution to (NH)4)2SO4The saturation is 64%, and 6mol/L HCl is added to adjust the pH to 4.8 under stirring, and the mixture is kept stand for 4 hours.
(3) Centrifuging at 4000r/min for 40min, and discarding the supernatant to obtain a second precipitate. To the second precipitate, 6mM PBS at pH7.4 was added until the second precipitate was completely dissolved, and then the solution after dissolving the second precipitate was transferred to a dialysis bag (molecular cut-off of 5kDa) and dialyzed against 6mM PBS at pH7.4, and 6mM PBS was changed every 4 hours.
(4) Centrifuging the dialyzed solution at 4000r/min for 20min, and discarding the precipitate to obtain a second supernatant. And filling the second supernatant into a dialysis bag (with a molecular interception of 5KDa), embedding the dialysis bag into polyethylene glycol 6000 (solid), and dialyzing and concentrating for 12h to obtain a concentrated solution.
(5) Desalting the concentrated solution with Sephadex G-50 column, eluting with 6mM PBS (pH7.4), collecting eluate, and freeze drying to obtain bovine serum albumin.
And fourthly, performing a globulin extraction step.
(1) The first precipitate was taken and 6mM PBS pH7.4 was added until the first precipitate was completely dissolved. Add saturated (NH) with stirring4)2SO4Solution to (NH)4)2SO4The saturation is 35 percent, 1mol/L NaOH is added under stirring to adjust the pH value to 7.0, the mixture is kept stand for 4 hours and then centrifuged for 40 minutes under the condition of 4000r/min, and the supernatant is discarded to obtain a third precipitate.
(2) Add 6mM PBS pH7.4 to the third precipitate until the third precipitate is completely dissolved, add saturated (NH) solution with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 35 percent, the mixture is kept stand for 4 hours and then centrifuged for 40min at 4000r/min, and the supernatant is discarded to obtain a fourth precipitate.
(3) Add 6mM PBS pH7.4 to the fourth precipitate until the fourth precipitate is completely dissolved, add saturated (NH) under stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 22%, the mixture is stood for 4 hours and then centrifuged for 40min at 4000r/min, and the precipitate is discarded to obtain a third supernatant.
(4) Adding saturated (NH) to the third supernatant with stirring4)2SO4Solution to (NH)4)2SO4The saturation degree is 35%, the mixture is stood for 4 hours and then centrifuged for 40min at 4000r/min, and the supernatant is discarded to obtain a fifth precipitate.
(5) Adding 6mM PBS (pH7.4) into the fifth precipitate until the fifth precipitate is completely dissolved, desalting the solution obtained by dissolving the fifth precipitate on a Sephedex G-50 column, eluting with 6mM PBS (pH7.4), collecting the eluate, and freeze-drying to obtain bovine serum globulin.
Example 4
This example provides the purity, recovery rate and extraction rate of bovine serum albumin and bovine serum globulin extracted by the method of example 2.
Method for producing a composite materialBy using the principle that plasma protein is precipitated in a salt solution with certain saturation degree, adopts (NH)4)2SO4And (4) carrying out fractional precipitation, and further purifying to obtain bovine serum albumin. The method is used for extracting bovine serum albumin from 5 different batches of fresh bovine blood respectively for detection, and the detection results are shown in table 1.
TABLE 1 results of measurement of bovine serum albumin extracted from 5 batches of fresh bovine blood
| Batches of | Purity (%) | 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 can be seen from Table 1, the purities of bovine serum albumin extracted from 5 batches of bovine blood were all greater than 98%, and the recovery rates were all greater than 80%.
The content of endotoxin in the extracted bovine serum albumin is determined by a limulus reagent method, and the result shows that the content of endotoxin in the bovine serum albumin is lower than 0.25EU/mL, and the quality meets the requirement of Chinese biological product regulation.
The method selects a saturated ammonium sulfate grading repeated salting-out method to separate and extract immune globulin in the bovine serum, removes high molecular weight plasma impure proteins (such as fibrinogen, fibronectin, IgM and the like) to the maximum extent to obtain bovine serum globulin, and ensures that the yield of the extracted bovine serum globulin by the method is 9.03g/L and the recovery rate is 90 percent. The purity of the extracted bovine serum globulin is higher than 95 percent through detection.
In summary, the method for extracting albumin and globulin from bovine serum provided by the invention utilizes the principle that plasma protein is precipitated in a salt solution with a certain saturation degree and adopts (NH)4)2SO4Carrying out fractional precipitation and further purifying to obtain bovine serum albumin which has high purity and low endotoxin content and meets the requirements of Chinese biological product regulation; by saturation (NH)4)2SO4The method of salting out repeatedly in stages extracts the bovine serum globulin, removes the high molecular weight plasma impure protein to the maximum extent, and the extracted bovine serum globulin has high purity. The method for extracting albumin and globulin from the bovine serum provided by the invention has the advantages of simple process, no need of special equipment and suitability for expanded production, and the utilization rate of the bovine serum is improved by the method for utilizing the bovine serum provided by the invention.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (8)
1. A method for extracting albumin and globulin from bovine serum, comprising:
taking bovine serum, centrifuging to obtain a first supernatant, and adding 0.8-1.2 times of the volume of the first supernatant into 4-6mM PBS with pH of 7.0-7.4; adding (NH) with stirring4)2SO4To 48% -52% saturation; refrigerating, standing, and filtering to obtain a first filtrate and a first precipitate;
extracting albumin from the first filtrate;
extracting globulin from said first precipitate;
extracting the globulin from the first precipitate comprises:
dissolving the first precipitate and adding (NH)4)2SO4Adjusting the pH value to 6.5-7.0 when the saturation degree is 30% -35%, stirring, standing and centrifuging to obtain a third precipitate;
dissolving the third precipitate and adding (NH)4)2SO4Stirring, standing and centrifuging to obtain a fourth precipitate after the saturation degree of 30-35 percent is reached;
dissolving the fourth precipitate and adding (NH)4)2SO4Stirring, standing and centrifuging to 18-22% of saturation to obtain a third supernatant;
(NH) is added to the third supernatant4)2SO4Stirring, standing and centrifuging to obtain a fifth precipitate after the saturation degree is 30-35%;
dissolving the fifth precipitate to obtain a crude globulin solution, and desalting and freeze-drying the crude globulin solution to obtain the globulin;
extracting the albumin from the first filtrate comprises:
adding a protective agent to the first filtrate and heating to a denaturation temperature to denature the hybrid protein; performing suction filtration to obtain a second filtrate;
(NH) is added to the second filtrate4)2SO4To 60-64% saturation, adjusting pH to 4.4-4.8, standing and centrifugingObtaining a second precipitate;
dissolving the second precipitate, dialyzing, centrifuging to obtain a second supernatant, and dialyzing and concentrating the second supernatant to obtain a concentrated solution;
desalting the concentrated solution to obtain an albumin solution, and freeze-drying the albumin solution to obtain the albumin.
2. The method of extracting albumin and globulin from bovine serum according to claim 1, wherein said denaturation temperature is 55-65 ℃.
3. The method of claim 2, wherein the denaturation temperature is maintained after heating to the denaturation temperature, and heating is continued for 20-40 min.
4. The method of claim 1, wherein the protective agent is sodium caprylate, and the final concentration of the sodium caprylate in the first filtrate m: v is 0.15% -0.19%.
5. The method for extracting albumin and globulin from bovine serum according to claim 1, wherein said desalting is: desalting the concentrated solution by a Sephadex G-50 column.
6. The method for extracting albumin and globulin from bovine serum according to any one of claims 1 to 5, wherein said (NH)4)2SO4Are all (NH)4)2SO4And (4) saturated solution.
7. A method of using bovine blood, comprising:
separating bovine serum from bovine blood;
the method of extracting albumin and globulin from bovine serum according to any one of claims 1 to 6 wherein bovine serum albumin and bovine serum globulin are extracted from said bovine serum.
8. The method of claim 7, wherein the step of separating the bovine serum from the bovine blood comprises: and (3) keeping the bovine blood at 35-38 ℃ for 2-4h, refrigerating at 2-8 ℃ for 12-16h, and absorbing yellow liquid precipitated from the upper layer to obtain the bovine serum.
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| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | A method for the continuous separation of three proteins in bovine plasma |
| 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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| CN1110279A (en) * | 1994-04-11 | 1995-10-18 | 余国华 | Method for extracting albumen from animal's blood by precipitating method |
| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | A method for the continuous separation of three proteins in bovine plasma |
| 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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