CN105646699B - A method of aPoA-I being extracted from human plasma using fumed silica - Google Patents
A method of aPoA-I being extracted from human plasma using fumed silica Download PDFInfo
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- CN105646699B CN105646699B CN201610024664.0A CN201610024664A CN105646699B CN 105646699 B CN105646699 B CN 105646699B CN 201610024664 A CN201610024664 A CN 201610024664A CN 105646699 B CN105646699 B CN 105646699B
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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
- C07K14/775—Apolipopeptides
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Abstract
The invention discloses a kind of method for extracting aPoA-I from human plasma using fumed silica, specific steps are as follows: (1) fumed silica is added in blood plasma and is uniformly mixed, mixture room temperature stands 5-100 minutes；(2) obtained mixture centrifugal treating collection solid sediment is spare；(3) obtained solid sediment is washed with the sodium chloride solution that molar concentration is 0.5mol/L, it is spare to collect solid sediment for centrifugal treating after washing；(4) obtained solid sediment is washed with apoA1 eluent, the liquid that centrifugal treating obtains after washing is aPoA-I solution.The present invention obtains the apoA1 ingredient in human plasma by simple and quick separation process, and separation process can be completed using only centrifuge, therefore is a kind of quick and selective solid-liquid separating method.Absorption compared to diatomite to apoA1, added fumed silica composition is single, preferable to the selectivity of purpose product absorption, and product is more controllable.
Description
Technical field
The invention belongs to Protein Extraction technical fields, and in particular to a kind of to be mentioned from human plasma using fumed silica
The method for taking aPoA-I.
Background technique
Apolipoprotein A1 (apoA1) is in content in blood plasma between 1.06-1.21g/L.ApoA1 and body's cholesterol generation
Thank closely related, the apoA1 content in human plasma is higher, and the risk for suffering from cardiovascular and cerebrovascular disease is lower.
It is at present cohn method, also known as cold ethanol precipitation from the main method that human plasma prepares protein in pharmaceuticals industry
Method, aPoA-I mainly is difficult to recycle by kieselguhr adsorption in traditional cohn method.It is existing to be prepared from human plasma
The method of Apolipoprotein A1 mainly has high speed density-gradient centrifugation method and hplc chromatography method.Cohn method can not be recycled effectively
Using Apolipoprotein A1, high speed density-gradient centrifugation method relies on Large expensive equipment, and liquid chromatography complex procedures handle the time
It is longer.
Since apoA1 possesses higher hydrophobicity relative to other protein in blood plasma, higher chaotropic is shown,
In the case where being added with the solid absorbent of bigger serface in blood plasma, it is more likely to be adsorbed precipitating.Diatomite is blood
It is used to clarify the common solid absorbent of blood plasma in Products Industry, diatomite main component is silica, but diatomite is made
For a kind of minerals, containing more impurity, specific surface area is relatively small.Containing preceding white in diatomite filter cake after clarifying blood plasma
The more impurity component such as albumen.Fumed silica possesses great specific surface area, and adsorption capacity is stronger, is used as drug or epidemic disease
Seedling slow-released carrier, used also as thickener or thixotropic agent.But there has been no pertinent literature reports to be used for protein at present
Purification process.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of extracted from human plasma using fumed silica to carry rouge egg
The method of white A-1, this method are a kind of principle of bigger serface adsorbent based on fumed silica, under given conditions plus
Enter into blood plasma adsorption precipitation apoA1 therein, the apoA1 of higher degree is obtained by the washing desorption to sediment composite
Albumen.
The present invention adopts the following technical scheme that solve above-mentioned technical problem, a kind of to utilize fumed silica from human plasma
The middle method for extracting aPoA-I, it is characterised in that specific steps are as follows:
(1) fumed silica is added in blood plasma and is uniformly mixed, wherein 1mL blood plasma corresponds to the matter of fumed silica
Amount is 2 ~ 35mg, and mixture room temperature stands 5-100 minutes；
(2) mixture that step (1) obtains the centrifugal treating 10 under conditions of 2000 ~ 14000g is placed in a centrifuge to divide
It is spare to collect solid sediment for clock；
(3) solid sediment that step (2) obtains is washed with the sodium chloride solution that molar concentration is 0.5mol/L,
Washing be placed in centrifuge with step (2) centrifugal treating 10 minutes under the same conditions, it is spare to collect solid sediment；
(4) solid sediment that step (3) obtains is washed with apoA1 eluent, wherein apoA1 eluent is
The mixed solution of Tris-HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 10%-40% and pH=7.4, washing are placed on centrifugation
Centrifugal treating 10 minutes, the liquid being centrifuged are aPoA-I solution under the same conditions with step (2) in machine.
It further limits, it is 5 ~ 20mg that 1mL blood plasma, which corresponds to the quality of fumed silica, in step (1).
It further limits, it is 40 ~ 80 minutes that the time is stored at room temperature in step (1).
It further limits, the centrifugal treating condition in step (2) is 12000g.
It further limits, apoA1 eluent is that Tris-HCl molar concentration 50mmol/L, ethyl alcohol volume are dense in step (4)
The mixed solution of degree 20% and pH=7.4.
The present invention obtains the apoA1 ingredient in human plasma by simple and quick separation process, separation process Jin Shiyong from
Scheming can be completed, therefore be a kind of quick and selective solid-liquid separating method.Compared to common column chromatography or sulphur
Sour ammonium sedimentation, this method equipment relies on less, easy to operate.Absorption compared to diatomite to apoA1, added gas phase
Silica composition is single, preferable to the selectivity of purpose product absorption, and product is more controllable.
Detailed description of the invention
Fig. 1 is that fumed silica adding proportion detects the adsorption effect SDS-PAGE of apoA1 in the embodiment of the present invention 1
Figure；
Fig. 2 is that fumed silica adsorption time detects the SDS-PAGE of apoA1 adsorption effect in the embodiment of the present invention 2
Figure；
Fig. 3 is the influence that ethyl alcohol volumetric concentration elutes effect to apoA1 from fumed silica in the embodiment of the present invention 4
Figure.
Specific embodiment
Above content of the invention is described in further details by the following examples, but this should not be interpreted as to this
The range for inventing above-mentioned theme is only limitted to embodiment below, and all technologies realized based on above content of the present invention belong to this hair
Bright range.
Embodiment 1
(1) it takes 1mL blood plasma to be respectively put into the centrifuge tube of 8 1.5mL, is then separately added into different centrifuge tubes
2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg and 35mg fumed silica, and be uniformly mixed, mixture room temperature
80 minutes；
(2) mixture that step (1) obtains is placed in a centrifuge centrifugal treating 10 minutes under conditions of 12000g, received
It is spare to collect solid sediment；
(3) solid sediment that step (2) obtains is washed with the sodium chloride solution that molar concentration is 0.5mol/L,
Washing is placed in centrifuge centrifugal treating 10 minutes under conditions of 12000g, and it is spare to collect solid sediment；
(4) solid sediment that step (3) obtains is washed with apoA1 eluent, wherein apoA1 eluent is
The mixed solution of Tris-HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 20% and pH=7.4, washing are placed in centrifuge
Centrifugal treating 10 minutes under conditions of 12000g, the liquid being centrifuged are aPoA-I solution.
The purity (Fig. 1) for the apoA1 that SDS-PAGE detection is eluted from fumed silica.Wherein track sequence is
1, protein molecular weight standard；2, human plasma；3, fumed silica 2mg apoA1 eluent；4, fumed silica 5mg
ApoA1 eluent；5, fumed silica 10mg apoA1 eluent；6, fumed silica 15mg apoA1 eluent；7,
Fumed silica 20mg apoA1 eluent；8, fumed silica 25mg apoA1 eluent；9, fumed silica
30mg；10, fumed silica 35mg apoA1 eluent.
Embodiment 2
(1) it takes 1mL blood plasma to be respectively put into the centrifuge tube of 7 1.5mL, is then separately added into different centrifuge tubes
10mg fumed silica, and be uniformly mixed, mixture room temperature is placed 5,10,20,30,40,50,60,80,100 minutes respectively；
(2) mixture that step (1) obtains is placed in a centrifuge centrifugal treating 10 minutes under conditions of 12000g, received
It is spare to collect solid sediment；
(3) solid sediment that step (2) obtains is washed with the sodium chloride solution that molar concentration is 0.5mol/L,
Washing is placed in centrifuge centrifugal treating 10 minutes under conditions of 12000g, and it is spare to collect solid sediment；
(4) solid sediment that step (3) obtains is washed with apoA1 eluent, wherein apoA1 eluent is
The mixed solution of Tris-HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 20% and pH=7.4, washing are placed in centrifuge
Centrifugal treating 10 minutes under conditions of 12000g, the liquid being centrifuged are aPoA-I solution.
The purity (Fig. 2) for the apoA1 that SDS-PAGE detection is eluted from fumed silica.Wherein track 1, albumen
Matter molecular weight standard；2, fumed silica adsorbs 100 minutes apoA1 and elutes result；3, fumed silica adsorbs 80 minutes
ApoA1 elutes result；4, fumed silica adsorbs 60 minutes apoA1 and elutes result；5, fumed silica adsorbs 50 minutes
ApoA1 elutes result；6, fumed silica adsorbs 40 minutes apoA1 and elutes result；7, fumed silica adsorbs 30 minutes
ApoA1 elutes result；8, fumed silica adsorbs 20 minutes apoA1 and elutes result；9, fumed silica adsorbs 10 minutes
ApoA1 elutes result；10, fumed silica adsorbs 5 minutes apoA1 and elutes result.
Embodiment 3
(1) it takes 1mL blood plasma to be respectively put into the centrifuge tube of 5 1.5mL, is then separately added into different centrifuge tubes
10mg fumed silica, and be uniformly mixed, mixture room temperature 60 minutes；
(2) by the mixture that step (1) obtains be placed in a centrifuge respectively 14000g, 12000,10000,8000,
It is spare to collect solid sediment for centrifugal treating 10 minutes under conditions of 6000；
(3) solid sediment that step (2) obtains is washed with the sodium chloride solution that molar concentration is 0.5mol/L,
Washing be placed in centrifuge with step (2) centrifugal treating 10 minutes under the same conditions, it is spare to collect solid sediment；
(4) solid sediment that step (3) obtains is washed with apoA1 eluent, wherein apoA1 eluent is
The mixed solution of Tris-HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 20% and pH=7.4, washing are placed in centrifuge
It is step (2) centrifugal treating 10 minutes under the same conditions, the liquid being centrifuged is aPoA-I solution.
Using spectrophotometer, the turbidity of blood plasma, is examined after measurement is adsorbed by fumed silica under 600nm wavelength with this
Survey the deposition degree (see Table 1) of solid phase silica in supernatant.
Embodiment 4
(1) it is taking 1mL blood plasma to be respectively put into 5 1.5mL centrifuge tubes, is then being separately added into different centrifuge tubes
10mg fumed silica, and be uniformly mixed, mixture room temperature 60 minutes；
(2) above-mentioned five parts of mixtures are placed in a centrifuge centrifugal treating 10 minutes under conditions of 12000g, make solid-liquid
It is separated；
(3) to the solid sediment being centrifugally separating to obtain with the molar concentration of 1mL be 0.5mol/L sodium chloride solution into
Row washing, washing are placed in centrifuge centrifugal treating 10 minutes under conditions of 12000g, collect solid sediment；
(4) each solid precipitating that step (3) obtains is washed using solution listed in Table respectively, and addition volume is 1mL.
Washing is placed in centrifuge centrifugal treating 10 minutes under conditions of 12000g, apoA1 be present in the resulting supernatant of centrifugation it
In, apoA1 in supernatant is detected by SDS-PAGE electrophoresis, track sequence is corresponding with serial number in Fig. 3.
Embodiment above describes basic principles and main features of the invention and advantage, the technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, under the range for not departing from the principle of the invention, various changes and improvements may be made to the invention, these changes and improvements are each fallen within
In the scope of protection of the invention.
Claims (5)
1. a kind of method for extracting aPoA-I from human plasma using fumed silica, it is characterised in that specific steps
Are as follows:
(1) it is added and fumed silica and is uniformly mixed in blood plasma, wherein 1mL blood plasma corresponds to the quality of fumed silica and is
2 ~ 35mg, mixture room temperature stand 5-100 minutes；
(2) mixture that step (1) obtains is placed in a centrifuge centrifugal treating 10 minutes under conditions of 2000 ~ 14000g,
It is spare to collect solid sediment；
(3) solid sediment that step (2) obtains is washed with the sodium chloride solution that molar concentration is 0.5mol/L, is washed
Be placed in centrifuge with step (2) centrifugal treating 10 minutes under the same conditions, it is spare to collect solid sediment；
(4) solid sediment that step (3) obtains is washed with apoA1 eluent, wherein apoA1 eluent is Tris-
The mixed solution of HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 10%-40% and pH=7.4, washing are placed in centrifuge
Centrifugal treating 10 minutes, the liquid being centrifuged are aPoA-I solution under the same conditions with step (2).
2. the method according to claim 1 for extracting aPoA-I from human plasma using fumed silica, special
Sign is: it is 5 ~ 20mg that 1mL blood plasma, which corresponds to the quality of fumed silica, in step (1).
3. the method according to claim 1 for extracting aPoA-I from human plasma using fumed silica, special
Sign is: it is 40 ~ 80 minutes that the time is stored at room temperature in step (1).
4. the method according to claim 1 for extracting aPoA-I from human plasma using fumed silica, special
Sign is: the condition of centrifugal treating is 12000g in step (2).
5. the method according to claim 1 for extracting aPoA-I from human plasma using fumed silica, special
Sign is: apoA1 eluent is Tris-HCl molar concentration 50mmol/L, ethyl alcohol volumetric concentration 20% and pH=7.4 in step (4)
Mixed solution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396479A (en) * | 2007-08-17 | 2013-11-20 | Csl百灵有限公司 | Method for purification of alpha-1-antitrypsin and apolipoprotein A-I |
| CN104513306A (en) * | 2014-12-15 | 2015-04-15 | 山西瑞亚力科技有限公司 | Apolipoprotein A1 purification method and ApoAI protein injection antigen |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396479A (en) * | 2007-08-17 | 2013-11-20 | Csl百灵有限公司 | Method for purification of alpha-1-antitrypsin and apolipoprotein A-I |
| CN104513306A (en) * | 2014-12-15 | 2015-04-15 | 山西瑞亚力科技有限公司 | Apolipoprotein A1 purification method and ApoAI protein injection antigen |
Non-Patent Citations (1)
| Title |
|---|
| 高分散的气相法二氧化硅在医学领域的应用;吴春蕾;《有机硅氟资讯》;20021231(第12期);第20-21页 |
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