CN101402671A - Method simultaneously separating fibrinogen and immunoglobulin from livestock and poultry blood - Google Patents
Method simultaneously separating fibrinogen and immunoglobulin from livestock and poultry blood Download PDFInfo
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- 239000008280 blood Substances 0.000 title claims abstract description 52
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- 108010049003 Fibrinogen Proteins 0.000 title claims abstract description 27
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- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 238000010612 desalination reaction Methods 0.000 claims abstract description 13
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 12
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 11
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- 239000000243 solution Substances 0.000 claims description 22
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 14
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Abstract
The invention discloses a method for separating fibrinogen and immunoglobulin from blood of livestock simultaneously. The specific steps are as follows: 1) preparation of blood plasma: fresh blood is provided, centrifuged, and red blood cells are removed to obtain the blood plasma; 2) sedimentation of inorganic salt: an inorganic salt is added into the blood plasma to reach a certain concentration, centrifugal separation is carried out after generating sediment which is fibrinogen, and supernatant is taken for subsequent separation; 3) column chromatography: the supernatant is separated by using a chromatography column filled with a mixed-mode adsorbent, and eluting peak is collected; and 4) desalination and drying; after collected liquid is desalinated, lyophilization is carried out to obtain immunoglobulin with purity of about 70 percent. The method is characterized in that a new separation technology is designed so that the fibrinogen and the immunoglobulin can be separated from the blood of livestock simultaneously. The key of the method lies in that the immunoglobulin can be extracted directly from the supernatant of the fibrinogen separated from the sediment. The method has advantages of simple operation and high separation purification factor.
Description
Technical field
The present invention relates to a kind of method of from livestock and poultry blood, isolating Fibrinogen and immunoglobulin (Ig) simultaneously, belong to the separating and purifying technology field of biologically active substance.
Background technology
In recent years, the whole world is the aquaculture develop rapidly of characteristics with mass-producing, intensive management, has increased substantially people's living standard, but a large amount of dischargings of animal waste (ight soil, butcher tankage, blood etc.) also bring immense pressure to ecotope.These wastes, especially blood contain rich in protein, various trace elements, VITAMIN and mineral substance, have very high nutritive value, but discharging has but caused the serious environmental pollution arbitrarily, and may cause the generation of some diseases.Fully effectively utilize the livestock and poultry blood resource, can not only alleviate environmental pollution, and can turn waste into wealth, prepare high value added product.
China, the amount of the annual livestock and poultry blood that produces is very big, is example with the livestock and poultry blood pig blood of China's maximum, and China delivers about 400,000,000 of live pig every year for sale, and year pig blood output can reach 1,000,000 tons.Remove at present part pig blood and be processed to blood meal, as fodder additives and be used for outside the biochemical pharmacy, major part is not utilized and discharging naturally, has caused the resource serious waste and has increased the pressure of environmental treatment.Fibrinogen in the pig blood is the main skeleton that constitutes blood clotting, can prepare fibrin sealant, is biological hemostatic material of new generation, is used for the acute hemostasis of surgery.Immunoglobulin content has biological activity and immunocompetences such as antibiotic, antiviral up to 2% in the pig blood, and the potential medicinal use is arranged.Same fiber-enriched proteinogen of other livestock and poultry blood and immunoglobulin (Ig) are developed suitable separating technology, extract the protein product of multiple high added value from livestock and poultry blood, realize voluminous thing coproduction, have a good application prospect.
The blood constitutent complexity, very stable in vivo, a series of biochemical variations but can be taken place after exsomatizing very soon, as blood coagulation, haemolysis, oxidation etc.The separation and purification process of blood protein is very complicated, needs a series of effective biochemical separation means.Salt precipitation is the effective ways of protein separation, also is the former main method of separating fibrin from blood, and the salt concn of supernatant liquor is still not higher behind the oversalting, has had a strong impact on follow-up separation and purification, and directly discharging can cause environmental pollution again.Therefore, the separation method of seeking a kind of effective salt tolerant is crucial.The mixed mode adsorption chromatography is the novel chromatographic technique that development in recent years is got up, the function aglucon contains hydrophobic grouping and ion-exchange group simultaneously, can by static inhale mutually strengthen absorption or Coulomb repulsion assist desorb, aglucon density is usually than higher, loading capacity is big, have typical salt tolerant characterization of adsorption, be particularly suitable for large-scale separation and purification.The present invention combines the mixed mode adsorption chromatography with saltouing to separate, directly handle the supernatant liquor of salt precipitation with the mixed mode sorbent material, the high-adsorption-capacity, the antibody that make full use of mixed mode absorption are selected the characteristic of row and salt tolerant absorption, set up an economical and efficient method of extracting Fibrinogen and immunoglobulin (Ig) simultaneously from livestock blood.
Summary of the invention
The purpose of this invention is to provide a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin simultaneously.
Comprise the steps:
1) preparation blood plasma
The sodium citrate solution of configuration 0.1M is as antithrombotics, fresh livestock and poultry blood and antithrombotics is even according to 9: 1 mixed, and usefulness whizzer centrifugal 10～20 minutes with 2500～3000rpm rotating speed is removed red corpuscle, obtain blood plasma, place-20～-40 ℃ of refrigerators standby;
2) inorganic salt precipitator method separating fibrin is former
In the fresh plasma that step 1) obtains, add saturated inorganic salt solution, make its saturation ratio reach 16～22%, left standstill under the room temperature 30 minutes, treat that precipitation fully, with whizzer centrifugal 10～20 minutes with 4000～5000rpm rotating speed, collecting precipitation is a Fibrinogen, obtains supernatant liquor, is the immunoglobulin (Ig) crude product solution;
3) mixed mode adsorption chromatography separating immune globulin
PH to 7～8 of the immunoglobulin (Ig) crude product solution that regulating step 2) obtains, make its pH identical with level pad, behind 0.2 μ m membrane filtration, last sample is in the chromatography column that is filled with the mixed mode sorbent material, the level pad flushing, the elution buffer wash-out is collected elution peak, obtains immunoglobulin solution;
4) desalination and drying
With the immunoglobulin solution desalination that step 3) obtains, lyophilize obtains immunoglobulin (Ig).
Described livestock and poultry blood is fresh pig blood, ox blood, chicken blood, duck blood or goose blood.Inorganic salt are ammonium sulfate or sodium sulfate.The mixed mode sorbent material is MEP Hypercel or is the chromatography media of function aglucon with 4-mercapto ethylpyridine.Level pad is a sodium citrate buffer solution, and the pH value is 7.0～8.0.Elutriant is a sodium citrate buffer solution, and pH is 3.5～4.5.
The present invention is directed to the comprehensive utilization of livestock and poultry blood resource, will saltout to separate combines with the mixed mode adsorption chromatography, is used for the separation and purification of Fibrinogen and immunoglobulin (Ig).Directly handle the supernatant liquor of salt precipitation separating fibrin after former with the mixed mode sorbent material, the highly selective that the high-adsorption-capacity that makes full use of mixed mode absorption improves the treatment capacity of process, antibody improves the characteristic simplification separating step of isolating efficient, salt tolerant absorption, obtains the extraction Fibrinogen of an economical and efficient and the method for immunoglobulin (Ig).The invention has the advantages that: 1) extract Fibrinogen and immunoglobulin (Ig) simultaneously; 2) technology is simple, is easy to amplify lower cost; 3) the process treatment capacity is big, the separation efficiency height; 4) purity of gained immunoglobulin (Ig) is higher.
Description of drawings
Accompanying drawing is the electrophoresis spectrogram that MEP Hypercel mixed mode adsorption chromatography of the present invention separates the porcine blood plasma immunoglobulin (Ig).
Embodiment
The invention provides a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin simultaneously.With the centrifugal removal red corpuscle of fresh anticoagulation, obtain blood plasma after, obtain its Fibrinogen with saturated inorganic salt precipitation, supernatant liquor is separated by mixed mode adsorption chromatography post, obtain immunoglobulin (Ig).Separate the immunoglobulin purity about 70% that obtains by the present invention.
Separating fibrin is former simultaneously from livestock and poultry blood comprises the steps: with method immunoglobulin (Ig)
1) preparation blood plasma
The sodium citrate solution of configuration 0.1M is as antithrombotics, new livestock and poultry blood liquid and antithrombotics is even according to 9: 1 mixed, and usefulness whizzer centrifugal 10～20 minutes with 2500～3000rpm rotating speed is removed red corpuscle, obtain blood plasma, place-20～-40 ℃ of refrigerators standby.
2) inorganic salt precipitator method separating fibrin is former
In the fresh plasma that step 1) obtains, add saturated inorganic salt solution, make its saturation ratio reach 16～22%, left standstill under the room temperature 30 minutes, treat that precipitation fully, with whizzer centrifugal 10～20 minutes with 4000～5000rpm rotating speed, collecting precipitation is a Fibrinogen, obtains supernatant liquor, is the immunoglobulin (Ig) crude product solution.
3) mixed mode adsorption chromatography separating immune globulin
PH to 7～8 of the immunoglobulin (Ig) crude product solution that regulating step 2) obtains, make its pH identical with level pad, behind 0.2 μ m membrane filtration, last sample is in the chromatography column that is filled with the mixed mode sorbent material, the level pad flushing, the elution buffer wash-out is collected elution peak, obtains immunoglobulin solution; Level pad and elutriant are sodium citrate buffer solution, and the pH value is respectively 7.0～8.0 and 3.5～4.5.
4) desalination and drying
With the immunoglobulin solution desalination that step 3) obtains, lyophilize obtains immunoglobulin (Ig).
The invention will be further described by the following examples:
Embodiment 1
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 20 minutes with the 2500rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 1.9ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 16%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 4000rpm centrifugal 10 minutes, obtain moist precipitate 0.22g, be the Fibrinogen crude product, supernatant liquor 10.8ml, standby.Supernatant liquor is got 1ml as sample introduction sample with 0.2 μ m membrane filtration.Fill about 5ml MEP Hypercel mixed mode medium in the chromatography column (internal diameter 1cm), level pad is the sodium citrate buffer solution (pH7.0) of 40mM, elutriant is the sodium citrate buffer solution (pH4.0) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 68.2%.
Embodiment 2
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 10 minutes with the 3000rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 2.0ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 17%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 4000rpm centrifugal 15 minutes, obtain moist precipitate 0.27g, be the Fibrinogen crude product, supernatant liquor 11ml, standby.Supernatant liquor is got 2ml as sample introduction sample with 0.2 μ m membrane filtration.Fill about 5ml MEP Hypercel mixed mode medium in the chromatography column (internal diameter 1cm), level pad is the sodium citrate buffer solution (pH7.0) of 40mM, elutriant is the sodium citrate buffer solution (pH4.0) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 71.4%.
Embodiment 3
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 15 minutes with the 3000rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 2.2ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 18%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 5000rpm centrifugal 10 minutes, obtain moist precipitate 0.30g, be the Fibrinogen crude product, supernatant liquor 10.8ml, standby.Supernatant liquor is got 2ml as sample introduction sample with 0.2 μ m membrane filtration.Filling about 5ml in the chromatography column (internal diameter 1cm) is the chromatography media of function aglucon with 4-mercapto ethylpyridine, level pad is the sodium citrate buffer solution (pH8.0) of 40mM, elutriant is the sodium citrate buffer solution (pH4.0) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 77.4%.
Embodiment 4
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 20 minutes with the 3000rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 2.4ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 19%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 4000rpm centrifugal 20 minutes, obtain moist precipitate 0.48g, be the Fibrinogen crude product, supernatant liquor 11.4ml, standby.Supernatant liquor is got 5ml as sample introduction sample with 0.2 μ m membrane filtration.Fill about 5ml MEP Hypercel mixed mode medium in the chromatography column (internal diameter 1cm), level pad is the sodium citrate buffer solution (pH8.0) of 40mM, elutriant is the sodium citrate buffer solution (pH4.0) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 69.2%.
Embodiment 5
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 10 minutes with the 3000rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 2.5ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 20%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 4000rpm centrifugal 20 minutes, obtain moist precipitate 0.45g, be the Fibrinogen crude product, supernatant liquor 11.5ml, standby.Supernatant liquor is got 2ml as sample introduction sample with 0.2 μ m membrane filtration.Fill about 5ml MEP Hypercel mixed mode medium in the chromatography column (internal diameter 1cm), level pad is the sodium citrate buffer solution (pH7.0) of 40mM, elutriant is the sodium citrate buffer solution (pH3.5) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 68.4%.
Embodiment 6
Get fresh pig blood, according to the sodium citrate solution anti-freezing of 9: 1 ratios adding 0.1M, usefulness whizzer centrifugal 10 minutes with the 3000rpm rotating speed is removed red corpuscle, obtains blood plasma.Get 10ml blood plasma, add 2.8ml saturated ammonium sulphate solution, the ammonium sulfate saturation ratio reaches 22%, leaves standstill under the room temperature 30 minutes, treat that fibrinogen deposition is complete, with 4000rpm centrifugal 20 minutes, obtain moist precipitate 0.65g, be the Fibrinogen crude product, supernatant liquor 10.9ml, standby.Supernatant liquor is got 2ml as sample introduction sample with 0.2 μ m membrane filtration.Fill about 5ml MEP Hypercel mixed mode medium in the chromatography column (internal diameter 1cm), level pad is the sodium citrate buffer solution (pH7.0) of 40mM, elutriant is the sodium citrate buffer solution (pH4.5) of 20mM, collect elution fraction, desalination, lyophilize obtains immunoglobulin (Ig), and the purity of electrophoretic analysis immunoglobulin (Ig) is 70.5%.
Claims (6)
1. the method for the former and immunoglobulin (Ig) of separating fibrin simultaneously from livestock and poultry blood is characterized in that comprising the steps:
1) preparation blood plasma
The sodium citrate solution of configuration 0.1M is as antithrombotics, fresh livestock and poultry blood and antithrombotics is even according to 9: 1 mixed, and usefulness whizzer centrifugal 10～20 minutes with 2500～3000rpm rotating speed is removed red corpuscle, obtain blood plasma, place-20～-40 ℃ of refrigerators standby;
2) inorganic salt precipitator method separating fibrin is former
Saturated inorganic salt solution is joined in the fresh plasma that step 1) obtains, make the saturation ratio of inorganic salt reach 16～22%, left standstill under the room temperature 30 minutes, treat that precipitation fully, with whizzer centrifugal 10～20 minutes with 4000～5000rpm rotating speed, collecting precipitation is a Fibrinogen, obtains supernatant liquor, is the immunoglobulin (Ig) crude product solution;
3) mixed mode adsorption chromatography separating immune globulin
PH to 7～8 of the immunoglobulin (Ig) crude product solution that regulating step 2) obtains, make the immunoglobulin (Ig) crude product solution identical with the pH of level pad, behind 0.2 μ m membrane filtration, last sample is in the chromatography column that is filled with the mixed mode sorbent material, the level pad flushing, the elution buffer wash-out is collected elution peak, obtains immunoglobulin solution;
4) desalination and drying
With the immunoglobulin solution desalination that step 3) obtains, lyophilize obtains immunoglobulin (Ig).
2. according to claim 1 a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin, it is characterized in that described livestock and poultry blood is fresh pig blood, ox blood, chicken blood, duck blood or goose blood.
3. according to claim 1 a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin, it is characterized in that described inorganic salt are ammonium sulfate or sodium sulfate.
4. according to claim 1 a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin, it is characterized in that described mixed mode sorbent material is MEP Hypercel or is the chromatography media of function aglucon with 4-mercapto ethylpyridine.
5. according to claim 1 a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin, it is characterized in that described level pad is a sodium citrate buffer solution, the pH value is 7.0～8.0.
6. according to claim 1 a kind of from livestock and poultry blood the method for the former and immunoglobulin (Ig) of separating fibrin, it is characterized in that described elution buffer is a sodium citrate buffer solution, the pH value is 3.5～4.5.
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| CN1563091A (en) * | 2004-03-31 | 2005-01-12 | 浙江金大地生物工程股份有限公司 | Method for extracting high active immunoglobulin in blood |
| CN1294147C (en) * | 2005-06-22 | 2007-01-10 | 王德亮 | Combined process ofr producing animal blood fiber protein |
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| CN101948535A (en) * | 2010-09-27 | 2011-01-19 | 浙江大学 | Method for separating immunoglobulin IgY from chicken blood |
| CN101948535B (en) * | 2010-09-27 | 2012-11-07 | 浙江大学 | Method for separating immunoglobulin IgY from chicken blood |
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| CN102268090B (en) * | 2011-07-13 | 2014-12-10 | 北京洪源澳达生物技术发展有限公司 | Siamese crocodile blood extract and extraction method and application thereof |
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| CN103412119A (en) * | 2013-07-31 | 2013-11-27 | 中国农业科学院兰州兽医研究所 | Preparation method and application of colloidal gold test strip for antibody detection |
| CN104231074A (en) * | 2014-09-09 | 2014-12-24 | 青岛康大食品有限公司 | Purification and preparation method of albumin in cony blood |
| CN107106928A (en) * | 2014-11-12 | 2017-08-29 | 彭特科尔有限公司 | Citric acid solution is used for the purposes that affinity chromatography purifies CRP by phosphocholine and its derivative |
| CN107106928B (en) * | 2014-11-12 | 2018-12-18 | 彭特科尔有限公司 | Purposes of the citric acid solution by phosphocholine and its derivative for affinity chromatography purifying CRP |
| CN108779166A (en) * | 2015-10-21 | 2018-11-09 | 科博锐恩生物制品有限责任公司 | From the method for plasma purification protein |
| EP3365365A4 (en) * | 2015-10-21 | 2019-06-19 | Cambryn Biologics, LLC | Processes for purifying proteins from plasma |
| CN105950576A (en) * | 2016-05-26 | 2016-09-21 | 成都远睿生物技术有限公司 | Method for extracting multiple proteins from bovine blood |
| CN112250757A (en) * | 2020-11-13 | 2021-01-22 | 广东深蓝生物科技有限公司 | Method for extracting and purifying protein in porcine plasma |
| CN112480246A (en) * | 2020-12-22 | 2021-03-12 | 中国科学院过程工程研究所 | Separation and purification method of dog immunoglobulin and application thereof |
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