CN103833843A - Method for extracting and purifying prealbumin PA from plasma of normal people - Google Patents
Method for extracting and purifying prealbumin PA from plasma of normal people Download PDFInfo
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- CN103833843A CN103833843A CN201210489310.5A CN201210489310A CN103833843A CN 103833843 A CN103833843 A CN 103833843A CN 201210489310 A CN201210489310 A CN 201210489310A CN 103833843 A CN103833843 A CN 103833843A
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Abstract
The invention provides a method for extracting and purifying prealbumin PA from plasma of normal people. The method comprises the following steps of 1) adding NaCl into the human plasma to be adequately dissolved; 2) processing by utilizing a phenolic solution, and extracting a component containing the prealbumin (PA); 3) dialyzing and replacing a buffer solution by utilizing the component containing the prealbumin (PA); 4) rapidly and moderately purifying PA by utilizing ion exchange chromatography of an AKTA system; 5) subsequently purifying the PA by utilizing a Sephacryl series molecular sieve chromatography of the AKTA system; 6) finely purifying the PA by utilizing a Superdex series molecular sieve chromatography of the AKTA system. By adopting the method, the PA can be well separated, the prepared PA is high in purity and activity, the purity can reach 98 percent, after being multiply diluted for 64 times, the PA still can have clear precipitation reaction with antiserum; moreover, the method is safe and convenient to operate, short in extracting period and applicable to the industrialized production.
Description
Technical field
The present invention relates to biotechnology, be specifically related to one and from human normal plasma, extract fast the method for purifying prealbumin (PA).
Background technology
Prealbumin (Prealbumin, PA) be the synthetic a kind of serum protein of liver cell, formed the about 55KD of molecular weight, optical extinction coefficient (E280nm) 13.6 by 4 identical subunits, 1.9 days transformation period, when electrophoresis, migration is before albumin, therefore be called prealbumin, main Physiological Function is the transport that participates in Tiroidina and Vogan-Neu in serum, and there is thymine activity, can be by promoting the lymphocytic maturation of hedge to increase immunity of organisms.In serum, the measurement result of prealbumin is the important indicator of reaction liver function and body nutritional status clinically, is common in dietetic patient.Because the prealbumin transformation period is very short, can reflect that liver synthesizes and catabolic slight change, the amplitude that its serum-concentration reduces and the degree of liver parenchyma lesion are closely related, are therefore also common in clinically liver dysfunction, liver cirrhosis, injure infected patient outward.Clinically, using the variation that detects prealbumin content as weighing hepatic disorder and underfed a kind of responsive index reliably.Therefore the extraction purifying of prealbumin has good application prospect in diagnosis.The separation purification method of having reported for prealbumin, is to need a large amount of blood plasma mostly, complex steps, and complicated operation, is difficult to use in routine operation and the clinical study of production use.EP0711786A2, US5310878A disclose a kind of method of purifying Prealbumin from Human Plasma, and this method is used ion-exchange, immunodiffusion(ID), affinity chromatography, molecular exclusion chromatography and the displacement of multistep damping fluid, the 11 step extracting method that amount to such as concentrated under specific environment.But, change method steps loaded down with trivial details and complicated, thereby target protein yield reduces, and it is consuming time longer to extract flow process, is difficult to meet the needs of large-scale industrial production.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and research and design is easy, the method for rapid extraction purifying prealbumin (PA).
The invention provides a kind of method of extracting purifying prealbumin (PA) from human normal plasma.The method comprises the following steps:
1) in human plasma, add NaCl, fully dissolve;
2) use phenol solution processing, extracting and enriching is containing the component of prealbumin (PA);
3) replace damping fluid containing the component dialysis of prealbumin (PA);
4) GE AKTA
tMthe quick moderate purifying of protein chromatographic instrument purifier100 system ion exchange chromatography PA fast;
5) AKTA system Sephacryl Series Molecules sieve chromatography subsequent purification PA;
6) AKTA system Superdex Series Molecules sieve chromatography polishing purification PA, obtains highly purified PA.
The human plasma of described step 1) is the human normal plasma that healthy population is contributed, and the concentration that adds NaCl is 5%～30%(w/v), preferably 10 ~ 25%, the suitableeest 20%;
Described step 2) in the phenol solution that uses including but not limited to cresols, naphthols, chlorophenol, phenol or phenylic acid.
The concentration range of the phenol solution using is at 0.5 ~ 10%(w/v), preferably 2 ~ 7%, most preferably 5.5%;
Described step 2) in extraction PA method steps be pending blood plasma dropwise to be added under the condition of the lasting stirring of magnetic stirring apparatus to phenol solution, the speed that splashes into of phenol solution is 1 ~ 10 ml/min;
Described step 2) the middle method that extracts PA, blood plasma and phenol solution mixture leave standstill 0.5 ~ 2 hour at 4 DEG C, by the centrifugal taking-up precipitation of supercentrifuge, stay supernatant liquor, and centrifugal force is 3000 ~ 12000g;
The dialyzate using in described step 3 is phosphoric acid buffer, concentration range 10 ~ 100mM, preferably 50mM; PH scope 7-10, preferably pH7.4;
Step 3) be 8000 ~ 20000 daltonian dialysis tubings containing the component dialysis process of prealbumin PA for packing component into molecular weight cut-off, immerse in above-mentioned phosphoric acid buffer, every 3 ~ 6 hours exchange buffering liquid once, change number of times 5 ~ 8 times at 4 DEG C;
The AKTA purification system using in described step 4 is anion-exchange chromatography, and the selection of chromatography column is including but not limited to HiTrap Q FF, HiPrep Q FF, HiPrep DEAE FF or HiTrap Capto Q FF, preferably HiTrap Capto Q FF;
In described step 4, the operational condition of anion-exchange chromatography is: A liquid: 50mM phosphoric acid buffer, pH7.4; B liquid: 50mM phosphoric acid buffer, 1M sodium chloride solution, pH 7.4; Flow velocity is 1 ~ 3 ml/min; First use A liquid to cross 3 column volumes, must penetrate peak one; Rear 0 ~ 20%(v/v) B liquid crosses 3 column volumes and obtains elution peak two; Finally use 20% ~ 100%B liquid to cross 3 column volumes, obtain elution peak three;
The AKTA purification system using in described step 5 is sieve chromatography, and the selection of chromatography column is including but not limited to Sephacryl S-100, S-200, S-300; Sephadex G-100, G-200, G-300; SuperdexG-100, G-200 or G-300; Preferably Sephacryl S-100;
In described step 5, the chromatography condition of Sephacryl S-100 sieve chromatography is: chromatographic solution: 50mM phosphoric acid buffer, pH 7.4; Flow velocity is 1 ~ 3 ml/min; Elution volume is 1 ~ 2 column volume;
The AKTA purification system using in described step 6 is sieve chromatography, the selection of chromatography column including but not limited to Sephadex G-75, G-100; Superdex G-75, G-100; Preferably Superdex G-75.
In described step 6, the chromatography condition of Sephacryl S-100 sieve chromatography is: chromatographic solution: 50mM phosphoric acid buffer, pH 7.4; Flow velocity is 1 ~ 3 ml/min; Elution volume is 1 ~ 2 column volume;
Method of the present invention can be good at separating PA, and the PA of preparation has very high purity and activity, and purity is more than 98%, still can be there is clear precipitin reaction with antiserum(antisera) by after 64 times of doubling dilutions, and easy-to-operate, extracting cycle is short, is suitable for suitability for industrialized production.
Brief description of the drawings
Fig. 1 HiTrap Capto Q FF strong cation exchange chromatography purifying PA: middle main peak is for containing PA component;
Fig. 2 Sephacryl S200 sieve chromatography purifying PA collection of illustrative plates: the second peak is for containing PA component
Fig. 3 Superdex G75 sieve chromatography purifying PA collection of illustrative plates: middle main peak is for containing PA component;
PA component agarose double diffusion result after Fig. 4 purifying: interstitial hole: anti-PA antibody; Hole around: from top counterclockwise: 1mg/ml PA is followed successively by: former times, 1:2,1:4,1:8,1:16,1:32 doubly dilute;
Fig. 5 BCA method is surveyed total protein content fitting a straight line collection of illustrative plates:
X-axis: standard protein concentration (ug/ml); Y-axis: standard protein A570 absorbance;
Fitting formula: y=0.0011x+0.1757; Linearly dependent coefficient R
2=0.9928
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Following raw material is commercially available to be obtained.
Embodiment 1: phenol precipitator method enrichment PA
Get in 100ml human plasma and dissolve 20g NaCl, fully dissolve and make NaCl solubleness reach 20%(w/v); Configuration 120ml 5.5%(w/v) phenol solution, in stink cupboard, slowly dropwise splash into, fully mix, leave standstill 30min; 10000g(8500 rev/min) after centrifugal 15min, discard precipitation, retain supernatant liquor; Supernatant liquor, after the water filter of 0.8 μ m filters, packed dialysis tubing into, and a dialyzate was changed in 4 DEG C of dialysis (50mMPBS pH7.4) 48 hours every 6 ~ 12 hours;
Embodiment 2: reinforcing yin essence ion exchange chromatography moderate purifying PA
HiTrap Capto Q FF(U.S. GE medical company product) reinforcing yin essence ion exchange chromatography: A liquid (50mM phosphoric acid buffer (pH7.4); B liquid (50mM phosphoric acid buffer, 1M sodium-chlor, pH7.4), pumps into after chromatography column until 100ml component to be purified, first at AKTA
tMin protein chromatographic instrument purifier100 system, rinse chromatography column with 50ml A liquid fast, wash the foreign protein of non-specific adsorption off, then setting program, it is 0 ~ 20%(v/v of 5 column volumes that the A of wash-out 50ml, B mixed solution form cumulative volume) linear elution of B liquid (100% ~ 80%A liquid), except foreigh protein removing, after use 50ml (5 column volumes) 20% ~ 100% B liquid (80% ~ 0%A liquid) linear elution, in elution peak, containing PA, collect the about 45ml of PA elutriant;
Embodiment 3: sieve chromatography precision purifying PA
First purify by Sephacryl S200 molecular sieve (U.S. GE medical company product) chromatography column moderate, purification condition is: the about 12ml of sample applied sample amount to be purified, and elution flow rate is 1 ml/min, Fig. 2 is shown in by wash-out 400ml(wash-out collection of illustrative plates), collect absorption peak two 30ml, abandon peak one;
Superdex G75 (U.S. GE medical company product) sieve chromatography polishing purification, purification condition is: the about 5ml of sample applied sample amount to be purified, elution flow rate is 1 ml/min, Fig. 3 is shown in by wash-out 250ml(wash-out collection of illustrative plates) collect maximum absorption peak component 20ml, obtain highly purified prealbumin.
Embodiment 4: the qualification of purifying PA purity
Will be with Superdex G75(U.S. GE medical company product) BCA method protein concentration quantification kit (the ThermoFisher company for protein ingredient content of sieve chromatography polishing purification
bCA ProteinAssay Kit) detect; BCA method is one of protein concentration monitoring method of commonly using in the world; reference: Smith; P.K.etal.Anal.Biochem.; 150,76-85 (1985). after matched curve, to record component total protein concentration after purifying be 1.01mg/ml to (see figure 5) substitution formula (y=0.0011x+0.1757);
Simultaneously will be with Superdex G75(U.S. GE medical company product) the human apolipoprotein A/B immue quantitative detection reagent box produced with commercially available Shanghai Foxing Changzheng medical science Co., Ltd of the protein ingredient of sieve chromatography polishing purification; detect the upper detection of analyser (AU400 of Hitachi) at full-automatic biochemical; detect on analyser and detect at full-automatic biochemical, detect that PA content is 0.997mg/ml;
PA purity is (0.997/1.01) * 100%=98.7%
Table 1BCA method is surveyed the A570OD value of total protein content standard protein and testing protein
Claims (10)
1. a method of extracting purifying prealbumin PA from human normal plasma, is characterized in that, the method comprises the following steps:
1) in human plasma, add NaCl, fully dissolve;
2) use phenol solution processing, extracting and enriching is containing the component of prealbumin PA;
3) replace damping fluid containing the component dialysis of prealbumin PA;
4) GE AKTA
tMthe quick moderate purifying of protein chromatographic instrument purifier100 system ion exchange chromatography PA fast;
5) AKTA system Sephacryl Series Molecules sieve chromatography subsequent purification PA;
6) AKTA system Superdex Series Molecules sieve chromatography polishing purification PA, obtains highly purified PA.
2. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, is characterized in that, it is 5% ~ 30% w/v that described step 1) adds the concentration of NaCl, preferably 10 ~ 25%, most preferably 20%.
3. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, is characterized in that, described step 2) in the phenol solution that uses be selected from cresols, naphthols, chlorophenol, phenol or phenylic acid; The concentration range of phenol solution is 0.5% ~ 10% w/v, preferably 2% ~ 7%, most preferably 5.5%.
4. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, it is characterized in that, described step 2) in extraction PA method steps be pending blood plasma dropwise to be added under the condition of the lasting stirring of magnetic stirring apparatus to phenol solution, the speed that splashes into of phenol solution is 1 ~ 10 ml/min; Blood plasma and phenol solution mixture leave standstill 0.5 ~ 2 hour at 4 DEG C, by the centrifugal taking-up precipitation of supercentrifuge, stay supernatant liquor, and centrifugal force is 3000 ~ 12000g.
5. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, is characterized in that, the dialyzate using in described step 3) is phosphoric acid buffer, concentration range 10 ~ 100mM, preferably 50mM; PH scope 7-10, preferably pH7.4.
6. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, it is characterized in that, described step 3) is 8000 ~ 20000 daltonian dialysis tubings containing the component dialysis process of prealbumin PA for packing described component into molecular weight cut-off, immerse in above-mentioned phosphoric acid buffer, at 4 DEG C, every 3 ~ 6 hours exchange buffering liquid once, changing number of times is 5 ~ 8 times.
7. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, it is characterized in that, the AKTA purification system using in described step 4) is anion-exchange chromatography, chromatography column is selected from HiTrap Q FF, HiPrep Q FF, HiPrep DEAE FF or HiTrap Capto Q FF, preferably HiTrap Capto Q FF.
8. the method for extracting purifying prealbumin PA from human normal plasma according to claim 7, is characterized in that, in described step 4), the operational condition of anion-exchange chromatography is: A liquid: 50mM phosphoric acid buffer, pH 7.4; B liquid: 50mM phosphoric acid buffer, 1M sodium chloride solution, pH 7.4; Flow velocity is 1 ~ 3 ml/min; First use A liquid to cross 3 column volumes, must penetrate peak one; Rear 0 ~ 20%B liquid is crossed 3 column volumes and is obtained elution peak two; Finally use 20% ~ 100%B liquid to cross 3 column volumes, obtain elution peak three.
9. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, is characterized in that, the AKTA purification system using in described step 5) is sieve chromatography, and chromatography column is selected from Sephacryl S-100, S-200, S-300; Sephadex G-100, G-200, G-300; Superdex G-100, G-200 or G-300; Preferably Sephacryl S-100; The chromatography condition of sieve chromatography is: chromatographic solution: 50mM phosphoric acid buffer, pH 7.4; Flow velocity is 1 ~ 3 ml/min; Elution volume is 1 ~ 2 column volume.
10. the method for extracting purifying prealbumin PA from human normal plasma according to claim 1, it is characterized in that, the AKTA purification system that described step 6) is used is molecular sieve layer analysis system, and chromatography column is selected from Sephadex G-75, G-100, Superdex G-75 or G-100; Preferably Superdex G-75; In described step 6, the condition of Sephacryl S-100 sieve chromatography is: chromatographic solution: 50mM phosphoric acid buffer, pH7.4; Flow velocity is 1 ~ 3 ml/min; Elution volume is 1 ~ 2 column volume.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200099A (en) * | 2014-06-10 | 2015-12-30 | 中国人民解放军第二军医大学 | Microbial sourced antimicrobial protein separated from cyanea capillata and preparation method and application thereof |
| CN107312082A (en) * | 2017-07-06 | 2017-11-03 | 四川新健康成生物股份有限公司 | A kind of method that natural prealbumin is obtained from serum |
| CN114058636A (en) * | 2021-11-16 | 2022-02-18 | 大连润生康泰医学检验实验室有限公司 | Method for cloning, expressing and purifying transthyretin gene |
-
2012
- 2012-11-27 CN CN201210489310.5A patent/CN103833843A/en active Pending
Non-Patent Citations (3)
| Title |
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| GORAN FEX 等: "Purification of Prealbumin from Human and Canine Serum Using a Two-step Affinity Chromatographic Procedure", 《EUR. J. BIOCHEM.》 * |
| MARK M BASHOR 等: "Purification of prealbumin from human serum", 《PREPARATIVE BIOCHEMISTRY》 * |
| 赵霞 等: "人血浆前白蛋白的分离和纯化", 《上海第二医科大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200099A (en) * | 2014-06-10 | 2015-12-30 | 中国人民解放军第二军医大学 | Microbial sourced antimicrobial protein separated from cyanea capillata and preparation method and application thereof |
| CN105200099B (en) * | 2014-06-10 | 2018-10-19 | 中国人民解放军第二军医大学 | A kind of microbe-derived antibacterial protein and the preparation method and application thereof being isolated from Cyanea capillata |
| CN107312082A (en) * | 2017-07-06 | 2017-11-03 | 四川新健康成生物股份有限公司 | A kind of method that natural prealbumin is obtained from serum |
| CN114058636A (en) * | 2021-11-16 | 2022-02-18 | 大连润生康泰医学检验实验室有限公司 | Method for cloning, expressing and purifying transthyretin gene |
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Application publication date: 20140604 |