CN109395616A - Bionical albumen transmission film of the equal hole polycarbonate of a kind of nucleoporin grafting and preparation method thereof - Google Patents
Bionical albumen transmission film of the equal hole polycarbonate of a kind of nucleoporin grafting and preparation method thereof Download PDFInfo
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- CN109395616A CN109395616A CN201811501825.6A CN201811501825A CN109395616A CN 109395616 A CN109395616 A CN 109395616A CN 201811501825 A CN201811501825 A CN 201811501825A CN 109395616 A CN109395616 A CN 109395616A
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- polycarbonate
- nucleoporin
- film
- grafted
- diisocyanate
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
- B01D71/06—Organic material
- B01D71/50—Polycarbonates
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0002—Organic membrane manufacture
- B01D67/0006—Organic membrane manufacture by chemical reactions
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- Chemical Kinetics & Catalysis (AREA)
- Engineering & Computer Science (AREA)
- Manufacturing & Machinery (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of nucleoporins to be grafted bionical albumen transmission film of equal hole polycarbonate and preparation method thereof.Method includes the following steps: the irradiation of (1) using plasma or persulfide oxidation make polycarbonate homopore membrane surface hydroxylation；(2) polycarbonate membrane of surface hydroxylation and di-isocyanate reaction make surface terminal hydroxy group be changed into isocyanates (- NCO) group；(3) film handled through diisocyanate keeps film surface aminated again with diamine reactant；(4) film of surface amine groups is reacted with bifunctional protein crosslinking agent 4- (p- maleimide phenyl) butyric acid succinimide ester makes film surface have high activity maleimide；(5) sulfydryl (SH) of the end nucleoporin (NSP1) is bonded with the maleimide of film surface is grafted to polycarbonate homopore membrane surface for nucleoporin, nucleoporin is made and is grafted equal hole polycarbonate Biomimetic membranes, which can be used for selectivity and transmit protein.
Description
Technical field
The present invention relates to bio-separation field, specifically a kind of nucleoporin is grafted the equal bionical albumen of hole polycarbonate
Transmit film and preparation method thereof.
Background technique
Pharmaceutical grade protein is to be diagnosed to disease in protein level, prevented and treated, the pharmaceutical grade protein packet of broad sense
Include the product that all chemical natures are protein or polypeptide, as hormone, growth factor/cell factor, protease, acceptor molecule,
Monoclonal antibody and antibody relevant molecule, part albumen or polypeptide vaccine etc..Compared with previous small-molecule drug, pharmaceutical grade protein
Have the characteristics that high activity, high specificity, hypotoxicity, biological function are clear, be conducive to clinical application.Due to its success rate is high,
Securely and reliably, it has also become important component in medical product.However, naturally occurring protein, or pass through fermentation, culture
Or synthesis protein, be always made of Multiple components in the initial stage, must through separation and after purification could be in medicine, food etc.
It is applied in field.Therefore, the high efficiency of protein and Selective Separation research have important theory significance and practical application valence
Value, becomes modern medicines analysis, life science, bioengineering and the hot spot to chemical engineering research.
Means currently used for Separation of Proteins are mainly chromatography, including gel chromatography, ion-exchange chromatography, efficient liquid phase
Chromatography etc..However equipment needed for these technologies is expensive and treating capacity is small, it is difficult to realize industrialized production and application.In recent years, close
Albumen, nucleic acid are widely used in its good separating property with chromatography, the large biological molecules such as polypeptide isolate and purify, the party
Method is based on the effect that is mutually distinguishable between aglucon and target biological molecules, such as antibody and antigen, sugar and agglutinin, enzyme-to-substrate,
Reach separation purpose, be one of most effective means in current macromolecular separation mechanism, however its operation vulnerable to mass transfer and limit,
Treating capacity is few, 1 is soaked in acid esters film, and in hexamethylene-diisocyanate solution, reacting 10min at 60 DEG C turns surface terminal hydroxy group
Become isocyanates-NCO group；(3) acetonitrile solution for the p-phenylenediamine that configuration quality score is 5%, by two isocyanide of surface
The film of acid esters processing is soaked in p-phenylenediamine solution, and 10min is reacted at 60 DEG C keeps film surface aminated；It (4) will be difunctional
Protein-crosslinking agent 4- (p- maleimide phenyl) butyric acid succinimide ester (SMPB) is dissolved in the phosphoric acid buffer that pH value is 7.4
In salting liquid, the concentration of SMPB is 2mmol/L, and then surface is placed in SMPB solution with the polycarbonate membrane of active amine
React 15min at room temperature to get the polycarbonate membrane of maleimide amino-functionalization；(5) by nucleoporin (NSP1) stock solution
It is dissolved in the PBS buffer solution that pH is 7.4, the concentration of NSP1 is 0.2mg/mL, then by the poly- of maleimide amino-functionalization
Carbonic ester film, which is soaked in NSP1 solution, reacts 20min to get the equal hole polycarbonate Biomimetic membranes of nucleoporin grafting.
Nucleoporin modification polycarbonate membrane is fixed among diffusion cell as shown in Figure 1 and two Room of left and right are isolated,
Right ventricle is put into albumen to be separated (caryoplasm transport protein Kaps95 or bovine serum albumin BSA) sample solution, and (stoste, protein concentration are
0.5mg/mL), left room is put into buffer solution, and two building volumes are 5mL.According to concentration difference principle, protein will transmit through film from right ventricle
Room is spread to the left.In order to expand the concentration difference of two Room, left room is passed through into the biggish diluting tank of the external volume of pipeline
(1000mL), to increase concentration difference driving force, enabling protein, faster room is spread to the left.At the different experiments moment respectively from a left side
Right two Room sampling, and with microplate reader test protein concentration.After diffusion for 24 hours, the concentration of left room Kaps95 is increased to from 0mg/mL
0.22mg/mL, and BSA hardly spread to the left by room under the same terms.The result shows that the nucleoporin is grafted equal hole polycarbonate
Biomimetic membranes have selectivity to Kaps95 albumen, are expected to the Selective Separation applied to protein.
Embodiment 2
(1) use potassium persulfate oxidation polycarbonate homopore membrane, by potassium peroxydisulfate be made into mass concentration be 20% it is water-soluble
Polycarbonate homopore membrane is soaked in potassium persulfate solution and reacts 5h at 80 DEG C to get the poly- carbonic acid of surface hydroxylation by liquid
Ester film；(2) toluene 2 that configuration quality score is 5%, the acetonitrile solution of 6- diisocyanate, by the poly- carbonic acid of surface hydroxylation
Ester film is soaked in toluene 2, and in 6- diisocyanate solution, reacting 10min at 60 DEG C makes surface terminal hydroxy group be changed into isocyanic acid
Ester-NCO group；(3) acetonitrile solution for the m-phenylene diamine (MPD) that configuration quality score is 5%, surface is handled through diisocyanate
Film is soaked in m-phenylenediamine solution, and 10min is reacted at 60 DEG C keeps film surface aminated；(4) by bifunctional protein crosslinking agent
4- (p- maleimide phenyl) butyric acid succinimide ester (SMPB) is dissolved in the phosphate buffered saline solution that pH value is 7.4,
The concentration of SMPB is 2mmol/L, and then surface is placed in SMPB solution at room temperature with the polycarbonate membrane of active amine
15min is reacted to get the polycarbonate membrane of maleimide amino-functionalization；(5) nucleoporin (NSP1) stock solution is dissolved in pH
For in 7.4 PBS buffer solution, the concentration of NSP1 is 0.2mg/mL, then by the polycarbonate membrane of maleimide amino-functionalization
It is soaked in NSP1 solution and reacts 20min to get the equal hole polycarbonate Biomimetic membranes of nucleoporin grafting.
Nucleoporin modification polycarbonate membrane is fixed among diffusion cell as shown in Figure 1 and two Room of left and right are isolated,
Right ventricle is put into albumen to be separated (caryoplasm transport protein Kaps95 or bovine serum albumin BSA) sample solution, and (stoste, protein concentration are
0.5mg/mL), left room is put into buffer solution, and two building volumes are 5mL.According to concentration difference principle, protein will transmit through film from right ventricle
Room is spread to the left.In order to expand the concentration difference of two Room, left room is passed through into the biggish diluting tank of the external volume of pipeline
(1000mL), to increase concentration difference driving force, enabling protein, faster room is spread to the left.At the different experiments moment respectively from a left side
Right two Room sampling, and with microplate reader test protein concentration.After diffusion for 24 hours, the concentration of left room Kaps95 is increased to from 0mg/mL
0.18mg/mL, and BSA hardly spread to the left by room under the same terms.The result shows that the nucleoporin is grafted equal hole polycarbonate
Biomimetic membranes have selectivity to Kaps95 albumen, are expected to the Selective Separation applied to protein.
Claims (2)
1. a kind of nucleoporin is grafted bionical albumen transmission film of equal hole polycarbonate and preparation method thereof, it is characterised in that this method
The following steps are included: the irradiation of (1) using plasma or persulfide oxidation make polycarbonate homopore membrane surface hydroxylation；(2)
The polycarbonate membrane and di-isocyanate reaction of surface hydroxylation make surface terminal hydroxy group be changed into isocyanates-NCO group；(3)
The film handled through diisocyanate keeps film surface aminated again with alkalescent diamine reactant；(4) film of surface amine groups and double function
Energy protein-crosslinking agent 4- (p- maleimide phenyl) butyric acid succinimide ester reaction makes film surface have high activity Malaysia acyl
Imines；(5) sulfydryl (SH) of the end nucleoporin (NSP1) is bonded with the maleimide of film surface is grafted to nucleoporin
Polycarbonate homopore membrane surface is made nucleoporin and is grafted equal hole polycarbonate Biomimetic membranes；
The plasma source of the plasma radiation be hydrogen and helium mixture, the two volume ratio 0.5~2: 99, flow 10~
40L/min, power are 120~220W；
The persulfide is the mixture of one of ammonium persulfate, potassium peroxydisulfate, sodium peroxydisulfate or arbitrary proportion；
The average pore size scope of the polycarbonate homopore membrane is 10nm~2000nm；
The diisocyanate includes 1, hexamethylene-diisocyanate, toluene 2, in 6- diisocyanate, 1,5- naphthalene diisocyanate
One kind or arbitrary proportion mixture；
The alkalescent diamines is p-phenylenediamine or m-phenylene diamine (MPD).
2. a kind of nucleoporin according to claim 1 is grafted the bionical albumen transmission film of equal hole polycarbonate and its preparation side
Method, it is characterised in that the selectivity transport to caryoplasm transport protein Kaps95 may be implemented.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811501825.6A CN109395616B (en) | 2018-12-10 | 2018-12-10 | Preparation method of nucleoporin grafted homogeneous pore polycarbonate bionic protein transport membrane |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811501825.6A CN109395616B (en) | 2018-12-10 | 2018-12-10 | Preparation method of nucleoporin grafted homogeneous pore polycarbonate bionic protein transport membrane |
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| CN109395616A true CN109395616A (en) | 2019-03-01 |
| CN109395616B CN109395616B (en) | 2021-03-09 |
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| CN103240006A (en) * | 2013-05-10 | 2013-08-14 | 天津大学 | Bovine serum albumin-polycarbonate composite membrane and preparation method thereof |
| CN104136106A (en) * | 2011-12-29 | 2014-11-05 | 通用电气公司 | Porous membranes having a polymeric coating and methods for their preparation and use |
| CN105038151A (en) * | 2015-08-04 | 2015-11-11 | 上海弘睿化工产品有限公司 | Biodegradable film based on irradiation modification and preparing method thereof |
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2018
- 2018-12-10 CN CN201811501825.6A patent/CN109395616B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003072641A1 (en) * | 2002-02-28 | 2003-09-04 | Universität Stuttgart | Oligomers and polymers containing sulfinate groups, and methods for producing the same |
| JP2003327658A (en) * | 2002-05-07 | 2003-11-19 | Nitto Denko Corp | Sequence defined polyurethane urea polymer and its sheet |
| US20100196922A1 (en) * | 2004-07-27 | 2010-08-05 | Life Technologies Corporation | Fluorescent metal ion indicators with large stokes shifts |
| CN104136106A (en) * | 2011-12-29 | 2014-11-05 | 通用电气公司 | Porous membranes having a polymeric coating and methods for their preparation and use |
| US20130171619A1 (en) * | 2011-12-30 | 2013-07-04 | General Electric Company | Porous membranes having a hydrophilic coating and methods for their preparation and use |
| CN103172988A (en) * | 2013-03-25 | 2013-06-26 | 山东汇盈新材料科技有限公司 | Method for increasing capacity of biodegradable polyester blend film |
| CN103240006A (en) * | 2013-05-10 | 2013-08-14 | 天津大学 | Bovine serum albumin-polycarbonate composite membrane and preparation method thereof |
| KR20170014642A (en) * | 2015-07-30 | 2017-02-08 | 서울대학교산학협력단 | Renewable water-treatment membranes and method for manufacturing the same |
| CN105038151A (en) * | 2015-08-04 | 2015-11-11 | 上海弘睿化工产品有限公司 | Biodegradable film based on irradiation modification and preparing method thereof |
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| CN109395616B (en) | 2021-03-09 |
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