CN104945495A - Production process for separating and purifying fiber linking proteins from pig blood - Google Patents
Production process for separating and purifying fiber linking proteins from pig blood Download PDFInfo
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- CN104945495A CN104945495A CN201410120015.1A CN201410120015A CN104945495A CN 104945495 A CN104945495 A CN 104945495A CN 201410120015 A CN201410120015 A CN 201410120015A CN 104945495 A CN104945495 A CN 104945495A
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- damping fluid
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Abstract
The invention relates to a production process for separating and purifying fiber linking proteins from pig blood. The production process comprises the following steps: (1) separating blood plasma and blood cells from whole blood; (2) salting out: charging an acidic Na2HPO4-NaH2PO4 buffer solution into an upper layer of plasma, uniformly mixing, charging ammonium sulfate while stirring the solution so that the saturation degree reaches 20 percent, adjusting pH to be alkaline, placing the mixed solution into a refrigerator, refrigerating the mixed solution for 1.5 to 2.5 hours, sufficiently precipitating, finally centrifuging the solution, and obtaining a main precipitation component, i.e., fiber linking proteins; (3) dialyzing and desalting: dissolving precipitates in the step (2) into an acidic PBS buffer solution, putting the mixed solution into a dialysis bag, carrying out the dialysis and concentration, thereby obtaining a crude product of the fiber linking proteins; and (4) purifying: carrying out the affinity chromatography for an upper sample of the fiber linking protein crude product obtained in the step (3) by virtue of a gelatin sepharose-4B column, and eluting by virtue of a specific volume to obtain the purified fiber linking proteins. The production process is simple, low in requirement on needed equipment and suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of extraction process of biotechnological formulation, particularly a kind of utilize centrifugal, saltout, dialyse and the production technique of method separation and purification fibronectin from pig blood that affinity chromatography combines.
Background technology
Fibronectin is the polymer glycoprotein being extensively present in various zooblast surface, extracellular fluid, reticular tissue and most of basilar membrane, it has various biological activity, such as participate in cell adhesion, differentiation, migration, propagation etc., occur at maintenance vascular integrity and permeability, wound healing, tumour and play an important role in transfer, blood coagulation and reproductive development.Distribute in vivo according to it, be divided into blood plasma type and cellular type 2 kinds.Blood plasma type is mainly present in blood plasma and body fluid, and also known as soluble fibronectin, the diagnosis and prognosis of its content to various diseases is significant.At present, Chinese scholars is to the fundamental research of fibronectin and disease detection, and even clinical treatment has all carried out a large amount of reports.
Containing biologically active substances such as abundant fibronectins in animal blood, adopt gelatin affinity chromatography can extract the carrying out of fibronectin in chicken blood, ox blood.At present, China's live pig amount of delivering for sale day by day increases, but a large amount of pig blood resource utilizations of butchering rear generation are lower, main as the low value product such as fodder additives or edible blood bean curd, this not only causes the waste of protein resource, and causes serious environmental pollution.
Summary of the invention
Object of the present invention provide a kind of utilize centrifugal, saltout, dialyse and the producing and manufacturing technique of method separation and purification fibronectin from pig blood that affinity chromatography combines.
As above conceive, technical scheme of the present invention is: a kind of production technique of separation and purification fibronectin from pig blood, is characterized in that: comprise the steps:
1. from whole blood, blood plasma and hemocyte is isolated;
2. saltout: in upper plasma, add acid Na
2hPO
4-NaH
2pO
4damping fluid also fully mixes, and then adds ammonium sulfate while stirring, makes its saturation ratio reach 20%, and regulates pH to alkalescence, then puts into refrigerator cold-storage 1.5-2.5h and makes it abundant precipitation, is finally fibronectin through centrifugal gained precipitation main component again;
3. to dialyse desalination: 2. will walk precipitation and be dissolved in acid PBS damping fluid, load in dialysis tubing and dialyse, every 8-10h changes a dialyzate, until use 10%BaCl
2detect without SO
4 2-till, collect liquid in dialysis tubing and carry out concentration operation, obtain fibronectin crude product;
4. purifying: first use Tris-HCl damping fluid as balance liquid, alkaline TB damping fluid as elutriant, 3. will walk gained fibronectin crude product to be splined on gelatin Sepharose-4B post and to carry out affinity chromatography, not absorbed portion wash-out is incited somebody to action with TB damping fluid, then albumen is connected with 6mol/L urea eluting fibers, collect elutriant, merge elutriant, carry out dialysis removing urea with TB damping fluid, concentrate and obtain fibronectin sterling after dialysis.
Above-mentioned steps is Na 2.
2hPO
4-NaH
2pO
4the pH=6.3 of damping fluid, and Na
2hPO
4-NaH
2pO
4the add-on of damping fluid and upper plasma equal-volume.
Above-mentioned steps is middle Na 2.
2hPO
4-NaH
2pO
4the concentration of damping fluid is 0.0175mol/L.
Above-mentioned steps 2. in centrifugal speed be 3000r/min, the time is 15min.
Above-mentioned steps 3. in the pH=6.3 of PBS damping fluid.
Above-mentioned steps 4. in the concentration of TB damping fluid be 0.05mol/L, pH=7.5; Containing 5mmol/L benzene carbon amidine in Tris-HCl damping fluid.
The blood plasma liquid obtained after blood separation is obtained fibronectin crude product by the present invention after salt precipitation and dialysis desalting process, again after gelatin post affinity chromatography, the fibronectin sterling after purifying is obtained via specific volume wash-out, therefore present invention process is simple, required equipment requires lower, is applicable to suitability for industrialized production.Not only can meet the needs of the biologically active substance large-scale productions such as fibronectin, and animal blood comprehensive resource utilization rate can be improved and improve added value of product.
Embodiment
Below in conjunction with embodiment, the present invention will be further described:
From pig blood, a production technique for separation and purification fibronectin, comprises the steps:
1, being separated of blood plasma and hemocyte: get the whole blood that 500mL is added with 6 ‰ sodium citrate anticoagulants, through the centrifugal 20min of 3000r/min, separated plasma and hemocyte.
2, saltout: in upper plasma, add isopyknic 0.0175mol/LNa
2hPO
4-NaH
2pO
4damping fluid (pH6.3) also fully mixes, and then adds 280g ammonium sulfate while stirring, makes its saturation ratio reach 20%, regulates pH to 7.0, put into 4 DEG C of refrigerator 2h and make it abundant precipitation with 5%NaOH.Again through the centrifugal 15min of 3000r/min, gained precipitation main component is fibronectin.
3, dialysis desalination: be dissolved in PBS damping fluid (pH6.3) by 2 steps precipitations, load in dialysis tubing and dialyse, every 8-10h changes a dialyzate, until use 10%BaCl
2detect without SO
4 2-till.Collect liquid in dialysis tubing and carry out concentration operation, obtain fibronectin crude product.
4, purifying: first with containing the Tris-HCl damping fluid of 5mmol/L benzene carbon amidine as balance liquid, 0.05mol/L(pH7.5) TB damping fluid as elutriant, 3 step gained fibronectin crude products are splined on gelatin Sepharose-4B post and carry out affinity chromatography.Will not absorbed portion wash-out with TB damping fluid, then connects albumen with 6mol/L urea eluting fibers, collect elutriant, merge elutriant, carry out dialysis remove urea with TB damping fluid, dialysing concentrates and obtains fibronectin sterling afterwards.
Claims (6)
1. the production technique of separation and purification fibronectin from pig blood, is characterized in that: comprise the steps:
1. from whole blood, blood plasma and hemocyte is isolated;
2. saltout: in upper plasma, add acid Na
2hPO
4-NaH
2pO
4damping fluid also fully mixes, and then adds ammonium sulfate while stirring, makes its saturation ratio reach 20%, and regulates pH to alkalescence, then puts into refrigerator cold-storage 1.5-2.5h and makes it abundant precipitation, is finally fibronectin through centrifugal gained precipitation main component again;
3. to dialyse desalination: 2. will walk precipitation and be dissolved in acid PBS damping fluid, load in dialysis tubing and dialyse, every 8-10h changes a dialyzate, until use 10%BaCl
2detect without SO
4 2-till, collect liquid in dialysis tubing and carry out concentration operation, obtain fibronectin crude product;
4. purifying: first use Tris-HCl damping fluid as balance liquid, alkaline TB damping fluid as elutriant, 3. will walk gained fibronectin crude product to be splined on gelatin Sepharose-4B post and to carry out affinity chromatography, not absorbed portion wash-out is incited somebody to action with TB damping fluid, then albumen is connected with 6mol/L urea eluting fibers, collect elutriant, merge elutriant, carry out dialysis removing urea with TB damping fluid, concentrate and obtain fibronectin sterling after dialysis.
2. the production technique of separation and purification fibronectin from pig blood according to claim 1, is characterized in that: above-mentioned steps is Na 2.
2hPO
4-NaH
2pO
4the pH=6.3 of damping fluid, and Na
2hPO
4-NaH
2pO
4the add-on of damping fluid and upper plasma equal-volume.
3. the production technique of separation and purification fibronectin from pig blood according to claim 1, is characterized in that: above-mentioned steps is middle Na 2.
2hPO
4-NaH
2pO
4the concentration of damping fluid is 0.0175mol/L.
4. the production technique of separation and purification fibronectin from pig blood according to claim 1, is characterized in that: above-mentioned steps 2. in centrifugal speed be 3000r/min, the time is 15min.
5. the production technique of separation and purification fibronectin from pig blood according to claim 1, is characterized in that: above-mentioned steps 3. in the pH=6.3 of PBS damping fluid.
6. the production technique of separation and purification fibronectin from pig blood according to claim 1, is characterized in that: above-mentioned steps 4. in the concentration of TB damping fluid be 0.05mol/L, pH=7.5; Containing 5mmol/L benzene carbon amidine in Tris-HCl damping fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410120015.1A CN104945495A (en) | 2014-03-27 | 2014-03-27 | Production process for separating and purifying fiber linking proteins from pig blood |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410120015.1A CN104945495A (en) | 2014-03-27 | 2014-03-27 | Production process for separating and purifying fiber linking proteins from pig blood |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104945495A true CN104945495A (en) | 2015-09-30 |
Family
ID=54160610
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410120015.1A Pending CN104945495A (en) | 2014-03-27 | 2014-03-27 | Production process for separating and purifying fiber linking proteins from pig blood |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107412878A (en) * | 2017-08-07 | 2017-12-01 | 上海交通大学医学院附属第九人民医院 | Composite fibrous scaffold and preparation method thereof |
| TWI674903B (en) * | 2016-09-26 | 2019-10-21 | 國立陽明大學 | Process for a preparation of the modified porcine plasma fibronectin for enhance wound healing |
| CN114558118A (en) * | 2022-02-28 | 2022-05-31 | 河南省健达动保有限公司 | Prescription for repairing intestinal mucosa and promoting digestion and preparation process thereof |
-
2014
- 2014-03-27 CN CN201410120015.1A patent/CN104945495A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| TWI674903B (en) * | 2016-09-26 | 2019-10-21 | 國立陽明大學 | Process for a preparation of the modified porcine plasma fibronectin for enhance wound healing |
| CN107412878A (en) * | 2017-08-07 | 2017-12-01 | 上海交通大学医学院附属第九人民医院 | Composite fibrous scaffold and preparation method thereof |
| CN107412878B (en) * | 2017-08-07 | 2018-04-24 | 上海交通大学医学院附属第九人民医院 | Composite fibrous scaffold and preparation method thereof |
| CN114558118A (en) * | 2022-02-28 | 2022-05-31 | 河南省健达动保有限公司 | Prescription for repairing intestinal mucosa and promoting digestion and preparation process thereof |
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Application publication date: 20150930 |