CN103480346B - A kind of novel Human serum protein material preparation method containing macrocyclic compound and application - Google Patents
A kind of novel Human serum protein material preparation method containing macrocyclic compound and application Download PDFInfo
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- CN103480346B CN103480346B CN201310464363.6A CN201310464363A CN103480346B CN 103480346 B CN103480346 B CN 103480346B CN 201310464363 A CN201310464363 A CN 201310464363A CN 103480346 B CN103480346 B CN 103480346B
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Abstract
The invention provides a kind of preparation method and application of the novel Human serum protein material containing macrocyclic compound.The invention provides a kind of manufacture method of Human serum protein material, this manufacture method comprises: macrocyclic compound and polymerizable material are combined the operation forming blended material in blended mode, polymerizable material comprises ethylene-vinyl alcohol copolymer (EVAL) and polyether sulfone (PES) etc.; Utilize the chemical reaction in aglucon (reactive dye) and blended material between macrocyclic compound by immobilized for aglucon to blended material, thus obtain the operation of Human serum protein material; The present invention provides a kind of application of Human serum protein material simultaneously: this novel Human serum protein material is for adsorbing the application of the large biological molecules such as bovine serum albumin (BSA).
Description
Technical field
The present invention relates to preparation method and the application of Human serum protein material, concrete be exactly by macrocyclic compound with polymerizable material (PES, EVAL etc.) blended preparation is blended be separated material after, aglucon is utilized to realize the immobilized of aglucon with the blended chemical reaction be separated in material between macrocyclic compound, finally prepare Human serum protein material, and this Human serum protein material is used for the adsorption applications of the large biological molecules such as bovine serum albumin (BSA).Raw material mostly is commercially produced product, operation is succinct and productive rate is higher, and the expansion simultaneously for the deep understanding of macrocyclic compound specific function and structure, Human serum protein technology is significant.
Background technology
In recent ten years, along with life science with bionicly to develop rapidly, the requirement be separated for biological macromolecule purifyings such as protein, enzyme, nucleotides is strict all the more.Traditional separation method, as precipitation, crystallization, centrifugal etc. is difficult to meet clinical requirement under many circumstances.Human serum protein material technology have selective strong, treating capacity large, be easy to the remarkable advantages such as amplification, be widely used in the separation of protein and other, developed into an important branch of Human serum protein.
Reactive dye are used for Human serum protein as aglucon and not only have good binding ability with the large biological molecule such as BSA, and solving the limitation such as native ligand is expensive, poor universality, triasine dyes is separated as ciba blue (CibacronBlueF3GA) has been widely used in protein purification with reactive blue 5 (ProcionBlueMX-R) etc.
Up to the present, host material selected by dyestuff Human serum protein material is mostly the polymer containing a large amount of active group (as hydroxyl) such as cellulose, shitosan, and reason is to be given by the covalent effect between active group with dye ligand to be separated the affine performance of material (adsorbed proteins etc.).And macrocyclic compound is as cyclodextrin (Cyclodextrin, be called for short CD) similar to cellulosic molecular structural formula, on the one hand, cyclodextrin cyclodextrin contains abundant active group (hydroxyl), the specific group needed when coupling aglucon can be provided to react, on the other hand, the character of its special cavity structure and external hydrophilic, inner hydrophobic, is adsorbed with certain " induction " and clathration to protein and other.
Summary of the invention
The object of this invention is to provide a kind of containing macrocyclic compound and with the preparation method of the novel Human serum protein material taking dyestuff as aglucon and application thereof, this invention is significant for the expansion of the deep understanding of cyclodextrin specific function and structure, Human serum protein material technology.Simultaneously the method is quick, easy, cheap, efficient, can the large biological molecule such as efficient adsorption bovine serum albumin, is applicable to large-scale production.
The present invention relates to dyestuff ProcionBlueMX-R is aglucon containing the preparation method of the novel Human serum protein material of macrocyclic compound, comprises the following steps:
One. blended separation material preparation:
Macrocyclic compound and polymerizable material EVAL or PES are mixed in dimethyl sulfoxide solvent, under heating and the condition that stirs, utilizes phase inversion to prepare blended separation material.
Two. prepared by Human serum protein material:
(1) the above-mentioned blended separation material prepared is put in appropriate volume, certain density dyestuff ProcionBlueMX-R solution react;
(2) add appropriate volume, certain density NaCI solution-treated, adsorption reaction is easily carried out, dye molecule is immobilized in blended separation material the most at last;
(3) regulate gas bath temperature, add appropriate volume, certain density Na
2cO
3fixation reaction is carried out in solution;
(4) reactant liquor is cooled, after fully washing with deionized water, methyl alcohol, NaCl solution, urea liquid successively, finally again use deionized water rinsing, and be stored in Tris-HCI buffer solution, obtaining with dyestuff ProcionBlueMX-R is aglucon containing the novel Human serum protein material of macrocyclic compound.
Described separation material is matrix in order to polymer EVA L or PES, adopts phase inversion to be prepared from;
Described macrocyclic compound is cyclodextrin, comprises α, β, gamma-cyclodextrin;
The reaction temperature that in described step one prepared by blended separation material is 60 DEG C, is 10 ~ 20% with content, and the reaction time is 4 ~ 6h.
The concentration of described dyestuff ProcionBlueMX-R is 10mg/ml, and consumption is 100ml;
Reaction in described step (1) is the gas bath oscillating reactions 20 ~ 50min of 40 ~ 60 DEG C;
NaCI solution-treated in described step (2) uses the NaCI solution of 20ml, 20wt% at 40 ~ 60 DEG C of gas bath oscillation treatment 50 ~ 80min;
Na in described step (3)
2cO
31wt%Na is used in fixation reaction
2cO
3at 60 ~ 90 DEG C of gas bath oscillating reactions 20 ~ 40min;
Cleaning solution in described step (4) to be content be 99.5 ~ 100% methyl alcohol, the NaCI solution of 2mol/L, the urea liquid of 6mol/L.
Urea washes, washing degree be wash after cleaning solution be colourless;
With the absorption that can be applicable to the large biological molecules such as BSA containing the novel Human serum protein material of macrocyclic compound that dyestuff ProcionBlueMX-R is aglucon.
Invention effect:
(1) the present invention is significant for the expansion of the deep understanding of macrocyclic compound specific function and structure, Human serum protein technology;
(2) method of the present invention is simple to operate, and consuming time less, adsorption effect is good;
(3) raw material used in the present invention mostly are industrialization product, cheap and easy to get, reduce production cost.Containing abundant active group (as hydroxyl) in selected polymer substrate EVAL, be conducive to the carrying out of the immobilized reaction of dyestuff, there is good mechanical strength and resistance tocrocking simultaneously;
(4) verify the impact of various influence factor for affine absorption property, establish the corresponding relation of various factors and absorption.
Accompanying drawing explanation
Fig. 1 is the influence curve of change to absorption property of bovine serum albumin initial concentration;
Fig. 2 is the influence curve that the change of reaction pH is adsorbed bovine serum albumin;
Detailed description of the invention:
Below in conjunction with specific embodiment, set forth the present invention further.Should understand, these embodiments are only not used in for illustration of the present invention and limit the scope of the invention, in addition should understand, after the content of having read the present invention's instruction, those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1:
(1) blended separation material preparation:
Matrix material EVAL and solvent DMAc is mixed by a certain percentage, add appropriate beta-schardinger dextrin-, wherein the same content of EVAL is 20%, the solid content of beta-schardinger dextrin-is that at 2%, 40 DEG C, mechanical agitation 4h extremely dissolves completely, vacuum defoamation, adopt phase inversion preparative separation material, put into coagulating bath (deionized water) and leave standstill 3 days, period changes water 5 times, puts into drier and stores for subsequent use.
(2) Human serum protein material preparation:
The blended separation material of getting preparation in 3 (1) puts into the dye solution of 100ml, 1g/L; Mixed liquor, at 60 DEG C of lower seal vibration 30min, then adds the NaCl solution of 20%wt, reaction 60min.Water-bath is heated to 80 DEG C gradually, adds the Na of 1%wt
2cO
3solution makes the pH value of mixed liquor remain in the scope of 10-11, cools after continuing reaction 30min, spends deionized water to colourless, then adds methyl alcohol, the NaCl solution of 2mol/L and the urea liquid of 6mol/L successively, finally again use deionized water rinsing.And use Tris-HCl buffer preserving.
Embodiment 2:
With the application containing the novel Human serum protein material of macrocyclic compound that dyestuff ProcionBlueMX-R is aglucon, concrete steps are as follows:
(1) determination of bovine serum albumin concentration initial concentration:
Take 5 groups of each 0.1g Human serum protein materials, the buffer solution added and concentration be respectively 0,0.2,0.4,0.6, in the bovine serum albumin of 1.2mg/ml, vibrate 4h at 25 DEG C.Adopt Coomassie Brilliant Blue to measure the concentration of bovine serum albumin, and then calculate adsorption capacity.Fig. 1 is the influence curve of change to absorption property of bovine serum albumin initial concentration, and result shows that adsorption capacity increases along with the increase of bovine serum albumin initial concentration, considers, and the initial concentration selecting bovine serum albumin is 1.0mg/ml.
(2) determination of pH value in reaction:
Take 5 groups of each 0.06g Human serum protein materials, the bovine serum albumen solution adding 10ml phosphate buffered saline carries out adsorption experiment, and the pH value of solution is respectively 3.8,4.4,5.0,5.8 and 6.5, and vibrate 4h at 25 DEG C.Adopt Coomassie Brilliant Blue to measure the concentration of bovine serum albumin, and then calculate adsorption capacity.Fig. 2 is the influence curve that the change of reaction pH is adsorbed bovine serum albumin, and when the pH value of reaction system is 5.0, adsorbance is the highest.
Claims (10)
1. the manufacture method of a Human serum protein material, it is characterized in that, it comprises: blended separation material preparation section: macrocyclic compound and polymerizable material ethylene-vinyl alcohol copolymer (EVAL) or polyether sulfone (PES) mixed in dimethyl sulfoxide solvent, under heating and the condition that stirs, utilize phase inversion to prepare blended separation material; And Human serum protein material preparation section: utilize reactive dye to be separated in material by immobilized for aglucon to blended with the blended chemical reaction be separated in material between macrocyclic compound, the blended separation material of above-mentioned preparation is placed in appropriate certain density dye solution, react under pH=10.0 ~ 11.0, gas bath constant temperature oscillation condition, product, after the washing of deionized water, methyl alcohol, sodium chloride, urea reagent, finally prepares Human serum protein material.
2. the manufacture method of Human serum protein material according to claim 1, is characterized in that, comprise the following steps:
Step one: blended separation material preparation:
Macrocyclic compound and polymerizable material EVAL or PES are mixed in dimethyl sulfoxide solvent, under heating and the condition that stirs, utilizes phase inversion to prepare blended separation material;
Step 2: prepared by Human serum protein material:
A1, blended separation material step one prepared is put in appropriate volume, certain density dyestuff ProcionBlueMX-R solution and is reacted;
A2, adds appropriate volume, certain density NaCl solution process, adsorption reaction is easily carried out, and dye molecule is immobilized in blended separation material the most at last;
A3, regulates gas bath temperature, adds appropriate volume, certain density Na
2cO
3fixation reaction is carried out in solution;
A4, reactant liquor is cooled, after fully washing with deionized water, methyl alcohol, NaCl solution, urea liquid successively, finally again uses deionized water rinsing, and be stored in Tris-HCl buffer solution, obtaining with dyestuff ProcionBlueMX-R is aglucon containing macrocyclic compound Human serum protein material.
3. the manufacture method of Human serum protein material according to claim 2, is characterized in that, described macrocyclic compound is cyclodextrin.
4. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the concentration of described dyestuff ProcionBlueMX-R is 10mg/ml, and consumption is 100ml.
5. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the reaction in described steps A 1 is the gas bath oscillating reactions 20 ~ 50min of 40 ~ 60 DEG C.
6. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the NaCl solution process in described steps A 2 uses the NaCl solution of 20ml, 20wt% at 40 ~ 60 DEG C of gas bath oscillation treatment 50 ~ 80min.
7. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the Na in described steps A 3
2cO
31wt%Na is used in fixation reaction
2cO
3at 60 ~ 90 DEG C of gas bath oscillating reactions 20 ~ 40min.
8. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the cleaning solution in described steps A 4 to be content be 99.5 ~ 100% methyl alcohol, the NaCl solution of 2mol/L, the urea liquid of 6mol/L.
9. the manufacture method of Human serum protein material according to claim 2, is characterized in that, the urea washes in described steps A 4, washing degree be wash after cleaning solution be colourless.
10. the manufacture method of a blended separation material, it is characterized in that, it comprises: blended separation material preparation section: macrocyclic compound and polymerizable material ethylene-vinyl alcohol copolymer (EVAL) or polyether sulfone (PES) mixed in dimethyl sulfoxide solvent, under heating and the condition that stirs, utilize phase inversion to prepare blended separation material.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557538A (en) * | 2004-01-30 | 2004-12-29 | 浙江科锐生物科技有限公司 | Method for preparing macromolecule resin type bilirubin sorbent |
| CN101224410A (en) * | 2007-10-19 | 2008-07-23 | 东华大学 | Preparing method of cellulose affinity membrane with dyestuff as petunidin and applications thereof |
| CN101259405A (en) * | 2007-12-13 | 2008-09-10 | 东华大学 | Preparation and application of nylon affinity membrane with reactive blue 4 as ligand |
| CN101862610A (en) * | 2010-05-05 | 2010-10-20 | 大连理工大学 | Absorption film for eliminating bilirubin and preparation method thereof |
| WO2013075740A1 (en) * | 2011-11-23 | 2013-05-30 | Sanofi | Antibody purification method |
-
2013
- 2013-10-08 CN CN201310464363.6A patent/CN103480346B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557538A (en) * | 2004-01-30 | 2004-12-29 | 浙江科锐生物科技有限公司 | Method for preparing macromolecule resin type bilirubin sorbent |
| CN101224410A (en) * | 2007-10-19 | 2008-07-23 | 东华大学 | Preparing method of cellulose affinity membrane with dyestuff as petunidin and applications thereof |
| CN101259405A (en) * | 2007-12-13 | 2008-09-10 | 东华大学 | Preparation and application of nylon affinity membrane with reactive blue 4 as ligand |
| CN101862610A (en) * | 2010-05-05 | 2010-10-20 | 大连理工大学 | Absorption film for eliminating bilirubin and preparation method thereof |
| WO2013075740A1 (en) * | 2011-11-23 | 2013-05-30 | Sanofi | Antibody purification method |
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