CN108822205A - A kind of extracting method of sheep blood serum albumin - Google Patents
A kind of extracting method of sheep blood serum albumin Download PDFInfo
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- CN108822205A CN108822205A CN201810610560.7A CN201810610560A CN108822205A CN 108822205 A CN108822205 A CN 108822205A CN 201810610560 A CN201810610560 A CN 201810610560A CN 108822205 A CN108822205 A CN 108822205A
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- sheep blood
- blood serum
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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
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- Proteomics, Peptides & Aminoacids (AREA)
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention is a kind of extracting method of sheep blood serum albumin, is included the following steps：(1) fresh sheep blood is taken, former 12% anti-coagulants of serum volume is added, after inspection and quarantine is qualified, 2500rpm is centrifuged 15min at 4 DEG C, takes supernatant to freeze in -20 DEG C, takes the serum of freezing, after sufficiently melting at room temperature, 10000rpm is centrifuged 12min, and taking supernatant is sheep blood serum.A kind of extracting method of sheep blood serum albumin of the present invention, process is short, it is easy to operate, high production efficiency, and in the present invention organic solvent-free use, avoid albumen and aggregation or denaturation occur, while the insecurity factor in the pollution and production process to environment can be reduced, product purity is improved, it can be widely using fields such as medical treatment, biochemistry.
Description
Technical field
The present invention relates to seralbumins to extract field more particularly to a kind of extracting method of sheep blood serum albumin.
Background technique
Protein is the material base of life, and seralbumin is the most abundant protein of content in blood plasma, it can with permitted
Mostly endogenous and outer rim compound combines, and plays the role of storage and conversion.In animal cell non-serum culture, add white
Albumen can play the role of physiology and mechanical protection and carrier function.Sheep blood serum albumin (BSA), also known as fifth component are sheep blood
One of clear globulin includes 583 amino acid residues, and molecular weight 66.43kDa, isoelectric point 4.7 is biology, doctor
Widely applied biochemical reagents in the fields such as, pharmacy.
At this stage, the extraction of sheep blood serum albumin mainly has following three kinds of methods.Salting out method ammonium sulfate precipitation,
It is obtained through octanoic acid processing purification, the method is most traditional production method, but its technique is cumbersome, and production efficiency is not high；It is organic molten
The agent precipitation method are low-k property and protein using ethyl alcohol in certain temperature, pH, ionic strength and concentration conditions
Under different solubility in different concentration ethanol, to realize the separation of plasma proteins, this method can produce a variety of blood
Albumen is starched, but the purity of albumen is not high；Heat shock method is to obtain blood plasma after taking the anticoagulant centrifugation of sodium citrate of sheep blood, and heating adds
Enter Sodium Caprylate, adjust after pH value plus ethyl alcohol, using obtaining sheep blood serum albumin, meeting in the method seralbumin after multistep treatment
Residual fraction chemical reagent causes the purity of sheep blood serum albumin lower.
For this purpose, designing a kind of extracting method of sheep blood serum albumin, problem above is solved.
Summary of the invention
The present invention is to overcome the above deficiency, provides a kind of extracting method of sheep blood serum albumin, includes the following steps：
(1) fresh sheep blood is taken, former 12% anti-coagulants of serum volume is added, after inspection and quarantine is qualified, at 4 DEG C
2500rpm is centrifuged 15min, takes supernatant to freeze in -20 DEG C, takes the serum of freezing, after sufficiently melting at room temperature, 10000rpm centrifugation
12min, taking supernatant is sheep blood serum；
(2) it takes the sheep blood serum in reaction kettle, normal saline dilution is added, stabilizer is added, adds 1mol/L's
Solution ph is adjusted to 5.1~5.2 by hydrochloric acid, and heating water bath makes the temperature of solution in reaction kettle be maintained at 68~75 DEG C, and reaction is held
Continuous 60~80min after filtering, collects filtrate；
(3) filtrate is taken, is dialysed with the Tris-HCl buffer solution of 0.02mol/L in 4 DEG C, dialysis bag retention molecular weight
It is 20000；
(4) the group liquid separation after dialysis is taken, is further purified using anion-exchange chromatography, uses 0.2mol/L concentration chlorine
Change sodium and carries out gradient elution；
(5) elution fraction is collected, and carries out desalination and concentration, the eluent after concentration is filtered using filter to be removed
Bacterium, pore size filter 0.1um；
(6) solution after aseptic filtration is freeze-dried to get sheep blood serum albumin freeze-dried powder is arrived.
Further, the sodium citrate solution that anti-coagulants described in the step (1) is 4%.
Further, the volume ratio of physiological saline and sheep blood serum is 1 in the step (2):2.
Further, the stabilizer in the step (2) is sodium palmitate.
Further, stabilizer is added by the amount that 0.75g is added in every 100ml sheep blood serum in the step (2).
Further, the filler of anion-exchange chromatography is DEAE-cellulose in the step (4).
The beneficial effects of the invention are as follows：
A kind of extracting method of sheep blood serum albumin of the present invention, process is short, easy to operate, high production efficiency, and
The use of organic solvent-free in the present invention avoids albumen and aggregation or denaturation occurs, while can reduce the pollution and life to environment
Insecurity factor during production, improves product purity, can be widely using fields such as medical treatment, biochemistry.
Specific embodiment
It is described in detail below with reference to the embodiment of the present invention.
A kind of extracting method of sheep blood serum albumin, includes the following steps：
(1) fresh sheep blood is taken, former 12% anti-coagulants of serum volume is added, after inspection and quarantine is qualified, at 4 DEG C
2500rpm is centrifuged 15min, takes supernatant to freeze in -20 DEG C, takes the serum of freezing, after sufficiently melting at room temperature, 10000rpm centrifugation
12min, taking supernatant is sheep blood serum；
(2) it takes the sheep blood serum in reaction kettle, normal saline dilution is added, stabilizer is added, adds 1mol/L's
Solution ph is adjusted to 5.1~5.2 by hydrochloric acid, and heating water bath makes the temperature of solution in reaction kettle be maintained at 68~75 DEG C, and reaction is held
Continuous 60~80min after filtering, collects filtrate；
(3) filtrate is taken, is dialysed with the Tris-HCl buffer solution of 0.02mol/L in 4 DEG C, dialysis bag retention molecular weight
It is 20000；
(4) the group liquid separation after dialysis is taken, is further purified using anion-exchange chromatography, uses 0.2mol/L concentration chlorine
Change sodium and carries out gradient elution；
(5) elution fraction is collected, and carries out desalination and concentration, the eluent after concentration is filtered using filter to be removed
Bacterium, pore size filter 0.1um；
(6) solution after aseptic filtration is freeze-dried to get sheep blood serum albumin freeze-dried powder is arrived.
The sodium citrate solution that anti-coagulants described in the step (1) is 4%.
The volume ratio of physiological saline and sheep blood serum is 1 in the step (2):2.
Stabilizer in the step (2) is sodium palmitate.
Stabilizer is added by the amount that 0.75g is added in every 100ml sheep blood serum in the step (2).
The filler of anion-exchange chromatography is DEAE-cellulose in the step (4).
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.The above is only a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications should also regard
For protection scope of the present invention.
Claims (6)
1. a kind of extracting method of sheep blood serum albumin, which is characterized in that
Include the following steps：
(1) take fresh sheep blood, former 12% anti-coagulants of serum volume be added, after inspection and quarantine is qualified, at 4 DEG C 2500rpm from
Heart 15min takes supernatant to freeze in -20 DEG C, takes the serum of freezing, and after sufficiently melting at room temperature, 10000rpm is centrifuged 12min, takes
Supernatant is sheep blood serum；
(2) it takes the sheep blood serum in reaction kettle, normal saline dilution is added, stabilizer is added, adds the hydrochloric acid of 1mol/L
Solution ph is adjusted to 5.1~5.2, heating water bath makes the temperature of solution in reaction kettle be maintained at 68~75 DEG C, and reaction continues 60
~80min after filtering, collects filtrate；
(3) filtrate is taken, is dialysed with the Tris-HCl buffer solution of 0.02mol/L in 4 DEG C, dialysis bag retention molecular weight is
20000；
(4) the group liquid separation after dialysis is taken, is further purified using anion-exchange chromatography, uses 0.2mol/L concentration sodium chloride
Carry out gradient elution；
(5) elution fraction is collected, and carries out desalination and concentration, the eluent after concentration is filtered degerming, mistake using filter
Filter opening diameter is 0.1um；
(6) solution after aseptic filtration is freeze-dried to get sheep blood serum albumin freeze-dried powder is arrived.
2. a kind of extracting method of sheep blood serum albumin according to claim 1, which is characterized in that in the step (1)
The sodium citrate solution that the anti-coagulants is 4%.
3. a kind of extracting method of sheep blood serum albumin according to claim 2, which is characterized in that in the step (2)
The volume ratio of physiological saline and sheep blood serum is 1:2.
4. a kind of extracting method of sheep blood serum albumin according to claim 3, which is characterized in that in the step (2)
Stabilizer be sodium palmitate.
5. a kind of extracting method of sheep blood serum albumin according to claim 4, which is characterized in that in the step (2)
Stabilizer is added by the amount that 0.75g is added in every 100ml sheep blood serum.
6. a kind of extracting method of sheep blood serum albumin according to claim 5, which is characterized in that in the step (4)
The filler of anion-exchange chromatography is DEAE-cellulose.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810610560.7A CN108822205A (en) | 2018-06-14 | 2018-06-14 | A kind of extracting method of sheep blood serum albumin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810610560.7A CN108822205A (en) | 2018-06-14 | 2018-06-14 | A kind of extracting method of sheep blood serum albumin |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734796A (en) * | 2019-02-01 | 2019-05-10 | 广州蕊特生物科技有限公司 | A kind of technique separating albumin from haemolysis serum |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104136040A (en) * | 2012-01-20 | 2014-11-05 | 费尔南多·托姆·克罗伊茨 | Autologous cancer cell vaccine |
| CN107365376A (en) * | 2017-07-31 | 2017-11-21 | 天津市正江现代生物技术有限公司 | A kind of extracting method of deer haemocyanin |
| CN107383185A (en) * | 2017-07-31 | 2017-11-24 | 天津市正江现代生物技术有限公司 | A kind of extracting method of high-purity bovine serum albumin |
-
2018
- 2018-06-14 CN CN201810610560.7A patent/CN108822205A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104136040A (en) * | 2012-01-20 | 2014-11-05 | 费尔南多·托姆·克罗伊茨 | Autologous cancer cell vaccine |
| CN107365376A (en) * | 2017-07-31 | 2017-11-21 | 天津市正江现代生物技术有限公司 | A kind of extracting method of deer haemocyanin |
| CN107383185A (en) * | 2017-07-31 | 2017-11-24 | 天津市正江现代生物技术有限公司 | A kind of extracting method of high-purity bovine serum albumin |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734796A (en) * | 2019-02-01 | 2019-05-10 | 广州蕊特生物科技有限公司 | A kind of technique separating albumin from haemolysis serum |
| CN109734796B (en) * | 2019-02-01 | 2022-04-15 | 广州蕊特生物科技有限公司 | Process for separating albumin from haemolytic serum |
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