CN105039273A - Method for purifying and preparing superoxide dismutase in rabbit blood - Google Patents
Method for purifying and preparing superoxide dismutase in rabbit blood Download PDFInfo
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- CN105039273A CN105039273A CN201510407291.0A CN201510407291A CN105039273A CN 105039273 A CN105039273 A CN 105039273A CN 201510407291 A CN201510407291 A CN 201510407291A CN 105039273 A CN105039273 A CN 105039273A
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- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
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Abstract
The invention belongs to the technical field of biological proteins, and particularly relates to a method for purifying and preparing superoxide dismutase in rabbit blood. The method mainly includes technological steps of adding anticoagulants into the rabbit blood and centrifugally collecting precipitates; washing the precipitates by the aid of normal saline, centrifuging the precipitates and removing hemoglobin; stirring and dissolving the blood by the aid of deionized water with equal volumes; adding 95% of precooled ethyl alcohol with the equal volumes and precooled chloroform with 0.2-times volume into the dissolved blood; stirring and centrifuging the blood and acquiring supernatants; heating the supernatants until the temperatures of the supernatants reach 60 DEG C, preserving heat for 15 minutes, quickly cooling the supernatants until the temperatures of the supernatants reach the room temperature, centrifuging the supernatants and acquiring second supernatants; cooling the second supernatants in normal saline, adding precooled acetone into the second supernatants, stirring the second supernatants, acquiring second precipitates and dissolving the second precipitates by the aid of deionized water; carrying out desalination and chromatography on the second precipitates; washing the second precipitates twice by the aid of HAC-NaAC liquor, then dissolving the second precipitates again by the aid of PBS liquor and filtering the second precipitates by the aid of 0.45 micrometer filter paper; carrying out ion exchange chromatography on the second precipitates; carrying out ultra-filtration on the second precipitates; centrifuging SOD eluent under the condition of temperature of 4 DEG C, acquiring third supernatants which are SOD stock liquid. The method has the advantages that the method is easy to implement, and the superoxide dismutase extracted by the aid of the method is high in purity.
Description
Technical field
The invention belongs to bioprotein technical field, be specifically related to the method for preparing purified of superoxide-dismutase in rabbit blood.
Background technology
Rabbit blood contains very high nutritive value, can be processed into multiple product, edible, medicinal, or as the animal feedstuff of livestock and poultry.Rabbit blood can extract multiple bio-pharmaceutical and biochemical reagents, as medical serum, serum antigen, zymoplasm, leucine, peptone, superoxide-dismutase etc., rabbit blood medical value is high, but rabbit blood extractive technique is only limitted to laboratory applications at present, cannot purification be reached, and industrialization can not extract and produce, the application of rabbit blood is had some limitations.
Superoxide-dismutase Orgotein (SuperoxideDismutase, SOD), another name orgotein, abbreviation: SOD.SOD is a kind of active substance coming from life entity, can eliminate the objectionable impurities that organism produces in metabolic processes.SOD is constantly supplemented to human body there is antidotal special-effect.Superoxide-dismutase (SuperoxideDismutase, EC1.15.1.1, SOD) within 1938, is separated first from ox red blood corpuscle to obtain superoxide-dismutase, it is wide at the distributed pole of organic sphere, almost from animal to plant, even from people to unicellular organism, there is its existence.SOD is regarded as the rubbish street cleaner in the enzyme of the magical magic power of most in Life Science, human body.SOD is the natural enemy of oxyradical, is the number one killer of oxyradical in body, is life and health basis.SOD is a kind of active protease containing metallic element.SOD is one of material with pharmic function of ministry of Health of China approval.
The technical process mainly blood anticoagulant-use brine-low temperature haemolysis-chloroform alcohol extracting oxyphorase-potassiumphosphate extraction-thermally denature-acetone precipitation-ion exchange chromatography-molecular sieve column chromatography of superoxide-dismutase is extracted at present from animal blood.This processing disadvantages is that flow process is complicated, with high costs, and chloroform, acetone and other organic solvent have stronger toxicity, have severe contamination to environment, also cause immeasurable infringement to operator's health.
Summary of the invention
In order to overcome the technological deficiency that prior art exists, the invention provides the method for preparing purified of superoxide-dismutase in a kind of rabbit blood.
The technical scheme that the present invention solves the employing of its technical problem is: the method for preparing purified of superoxide-dismutase in rabbit blood, mainly comprises following processing step:
1, fresh for 50L rabbit blood is added Sodium Citrate or the Trisodium Citrate of 4.0% in the ratio of 16:1, stir evenly;
2, continuous flow centrifuge is centrifugal, and centrifugal force is about 400g; Centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell, collecting precipitation;
3, the precipitation 4 times of normal saline flushings will collected, Continuous Flow 4000 leaves the heart, in triplicate, removes oxyphorase; Then deionized water equimultiple volume stirs haemolysis;
4, in hemolysate, the pre-cooled ethanol of equimultiple volume 95% is added, the precooling chloroform of 0.2 times of volume; Stir after 20 minutes and leave standstill 1 hour, then 4000 turns of continuous flow centrifugations, get supernatant, and ultraviolet spectrophotometer measures absorbancy;
5, supernatant is heated to 60 DEG C, is incubated 15 minutes, is then cooled to room temperature rapidly, 4000 turns of continuous flow centrifugations, collect supernatant, measure absorbancy; Then cool in physiological saline, add pre-cold acetone, stir, collecting precipitation, then uses deionized water dissolving;
6, after above-mentioned precipitate with deionized water is dissolved, G25 desalting column chromatography is adopted; The main component of desalination chromatography gained cryoprecipitate is superoxide-dismutase;
7, centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, the centrifugal force of 15,000g, centrifugal about 30min, precipitation, supernatant separate collection;
8, precipitation 5mMHAC-NaAC solution is cleaned after 2 times heavy molten in the PBS solution with 10mMpH being 7.0, by the filter paper filtering of heavy molten superoxide-dismutase by 0.45um;
9, after filtering, solution is through DE32 ion exchange chromatography; And then through ultrafiltration;
10, by the SOD elutriant after ultrafiltration under the condition of 4 DEG C, the centrifugal force of 15000g, centrifugal about 30min, collect supernatant, be SOD stoste.
In rabbit blood provided by the invention, the method for preparing purified of superoxide-dismutase, simple to operate, and the superoxide-dismutase purity of extraction is high.
Embodiment
Technical scheme of the present invention is described in detail below by embodiment.
The method for preparing purified of superoxide-dismutase in rabbit blood, mainly comprises following processing step:
1, fresh for 50L rabbit blood is added the Sodium Citrate (Trisodium Citrate) of 4.0% in the ratio of 16:1, stir evenly.
2, continuous flow centrifuge is centrifugal, and centrifugal force is about 400g.Centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell.Collecting precipitation.
3, the precipitation 4 times of normal saline flushings will collected, Continuous Flow 4000 leaves the heart, in triplicate, removes oxyphorase.Then deionized water equimultiple volume stirs haemolysis.
4, in hemolysate, the pre-cooled ethanol of equimultiple volume 95% is added, the precooling chloroform of 0.2 times of volume.Stir after 20 minutes and leave standstill 1 hour, then 4000 turns of continuous flow centrifugations, get supernatant, and ultraviolet spectrophotometer measures absorbancy.
5, supernatant is heated to 60 DEG C, is incubated 15 minutes, is then cooled to room temperature rapidly, 4000 turns of continuous flow centrifugations, collect supernatant, measure absorbancy.Then cool in physiological saline, add pre-cold acetone, stir, collecting precipitation, then uses deionized water dissolving.
6, filtrate desalination: G25 desalination chromatography
Chromatography column 450*600, filler is DE32, and loading height is 50cm, about needs the G25 filler of 80L.Buffer：2.5mMPBS，pH5.2。
(1): extracted by water influent pipeline 1 by Buffer, balance chromatography column, until pH is 5.2, and UV-detector signal is flat.
(2): by the filtrate of 20L by loading pipeline loading, after completion of the sample, be switched to water influent pipeline 1.
(3): when UV-detector detects that albumen flows out, switch to collecting 1, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat, repeat loading complete.
(4): 1MNaOH is extracted by water influent pipeline 2, CIP(incumbent firms is carried out to chromatography column), until pH is 13 or more and UV-detector signal is flat, and keep 30min.
(5): ddH2O is extracted by water influent pipeline 3, chromatography column is rinsed, until its pH value is 7 or following and UV-detector signal is flat.
(6): 20% ethanol is extracted by water influent pipeline 4, chromatography column is rinsed, about 1.5 column volumes.Cryoprecipitate: spent the night by the environment that the collection liquid of G25 desalting column is placed in 4 DEG C, it can separate out a large amount of throw outs, and this throw out composition is mainly superoxide-dismutase SOD.
7, centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, the centrifugal force of 15,000g, centrifugal about 30min, precipitation supernatant separate collection.
8, precipitation 5mMHAC-NaAC solution is cleaned after 2 times molten in the PB solution weight with 10mMpH being 7.0, by the filter paper filtering of heavy molten superoxide-dismutase by 0.45um.
9, DE32 ion exchange chromatography: chromatography column 100*300, filler is DE32, and loading height is 10cm, about needs 0.6L filler.BufferA：10mMPB，pH7.0；BufferB：BufferA+250mMNaCl。
(1): extracted by water influent pipeline 1 by BufferA, balance chromatography column, until pH is 7.0, and UV-detector signal is flat.
(2): by all superoxide-dismutases by loading pipeline loading, after completion of the sample, be switched to water influent pipeline 1.
(3): continue to rinse chromatography column until UV-detector signal is flat with BufferA.
(4): extracted by water influent pipeline 2 by BufferB, when UV-detector detects that albumen flows out, switch to collecting 1, until UV-detector signal value is 1/10 of its peak value, switch to waste liquid, until UV-detector signal is flat.
(5): 1MNaOH is extracted by water influent pipeline 3, CIP(incumbent firms is carried out to chromatography column), until pH is 13 or more and UV-detector signal is flat.
(6): BufferB is extracted by water influent pipeline 2, chromatography column is rinsed, until its pH value is 7 or following and UV-detector signal is flat.
(7): 20% ethanol is extracted by water influent pipeline 4, chromatography column is rinsed, about 1.5 column volumes.
10, ultrafiltration: the molecular weight cut-off of ultra-fine filter is 10KD, and membrane area is 200cm2.
(1): installed by operational manual by ultrafiltration system, and run 5min under the pressure of 1Bar, check the whether leakage of film bag and holder contacts place.
(2): rinse until pH is about 7 with water after film bag 0.1MNaOH being rinsed 30min, then rinse with 10mMPB+250mMNaCl solution.
(3): fluid inlet is put into SOD elutriant, backflow end 1 is closed with leaching to hold, and opens backflow end pipeline 2 pipeline, after total system runs 5min, open and leach end, and regulate and leach end valve door, make to leach end flow velocity and be about 1/10 of backflow end and transmembrane pressure is 1Bar.
(4): before SOD effluent volume is about 1/10 time, close ultrafiltration system, and the SOD elutriant in water influent pipeline and return line collected as far as possible.
(5): rinse until pH is about 7 with water after film bag 0.1MNaOH being rinsed 30min, film bag is taken out, is placed in conserving liquid.
11, by the SOD elutriant after ultrafiltration under the condition of 4 DEG C, the centrifugal force of 15000g, centrifugal about 30min, collect supernatant, be SOD stoste.
Claims (1)
1. the method for preparing purified of superoxide-dismutase in rabbit blood, mainly comprises following processing step:
(1), by fresh for 50L rabbit blood in the ratio of 16:1 add Sodium Citrate or the Trisodium Citrate of 4.0%, stir evenly;
(2), continuous flow centrifuge is centrifugal, and centrifugal force is about 400g; Centrifugal supernatant is blood plasma, and throw out is the cells such as blood cell, collecting precipitation;
(3), will collect precipitation 4 times of normal saline flushings, Continuous Flow 4000 leaves the heart, in triplicate, removal oxyphorase; Then deionized water equimultiple volume stirs haemolysis;
(4), in hemolysate, the pre-cooled ethanol of equimultiple volume 95%, the precooling chloroform of 0.2 times of volume is added; Stir after 20 minutes and leave standstill 1 hour, then 4000 turns of continuous flow centrifugations, get supernatant, and ultraviolet spectrophotometer measures absorbancy;
(5), by supernatant be heated to 60 DEG C, be incubated 15 minutes, be then cooled to room temperature rapidly, 4000 turns of continuous flow centrifugations, collect supernatant, measure absorbancy; Then cool in physiological saline, add pre-cold acetone, stir, collecting precipitation, then uses deionized water dissolving;
(6), after above-mentioned precipitate with deionized water dissolves, G25 desalting column chromatography is adopted; The main component of desalination chromatography gained cryoprecipitate is superoxide-dismutase;
(7), centrifugal: by the solution of cryoprecipitate under the condition of 4 DEG C, the centrifugal force of 15,000g, centrifugal about 30min, precipitation, supernatant separate collection;
(8), by precipitation 5mMHAC-NaAC solution clean after 2 times heavy molten in the PBS solution with 10mMpH being 7.0, by the filter paper filtering of heavy molten superoxide-dismutase by 0.45um;
(9) after, filtering, solution is through DE32 ion exchange chromatography; And then through ultrafiltration;
(10) by the SOD elutriant after ultrafiltration under the condition of 4 DEG C, the centrifugal force of 15000g, centrifugal about 30min, collect supernatant, be SOD stoste.
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| CN201510407291.0A CN105039273A (en) | 2015-07-13 | 2015-07-13 | Method for purifying and preparing superoxide dismutase in rabbit blood |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011091A (en) * | 2016-06-22 | 2016-10-12 | 绍兴文理学院 | Method for extracting and purifying SOD freeze-dried powder from meat of hyriopsis cumingii |
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
| CN102337252A (en) * | 2010-07-19 | 2012-02-01 | 天津宝迪农业科技股份有限公司 | Method for extracting superoxide dismutase (SOD) from pig blood |
| CN102888386A (en) * | 2012-11-02 | 2013-01-23 | 中国水产科学研究院南海水产研究所 | Method for purifying superoxide dismutase from tilapia mossambica blood |
| CN103436500A (en) * | 2013-08-22 | 2013-12-11 | 慈溪市迈康生物科技有限公司 | Method for extracting superoxide dismutase from animal blood |
| CN104450656A (en) * | 2014-09-09 | 2015-03-25 | 青岛康大食品有限公司 | Method for preparing and purifying thrombin in rabbit blood |
-
2015
- 2015-07-13 CN CN201510407291.0A patent/CN105039273A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034369A (en) * | 1987-01-08 | 1989-08-02 | 苏州医学院 | The method of purification of superoxide-dismutase |
| CN102337252A (en) * | 2010-07-19 | 2012-02-01 | 天津宝迪农业科技股份有限公司 | Method for extracting superoxide dismutase (SOD) from pig blood |
| CN102888386A (en) * | 2012-11-02 | 2013-01-23 | 中国水产科学研究院南海水产研究所 | Method for purifying superoxide dismutase from tilapia mossambica blood |
| CN103436500A (en) * | 2013-08-22 | 2013-12-11 | 慈溪市迈康生物科技有限公司 | Method for extracting superoxide dismutase from animal blood |
| CN104450656A (en) * | 2014-09-09 | 2015-03-25 | 青岛康大食品有限公司 | Method for preparing and purifying thrombin in rabbit blood |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011091A (en) * | 2016-06-22 | 2016-10-12 | 绍兴文理学院 | Method for extracting and purifying SOD freeze-dried powder from meat of hyriopsis cumingii |
| CN108486074A (en) * | 2018-04-03 | 2018-09-04 | 西北工业大学 | A method of utilizing crystallisation separating-purifying superoxide dismutase |
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