CN105944692A - Preparation method of molecularly imprinted polymer microspheres capable of recognizing ADP-ribosylation protein - Google Patents
Preparation method of molecularly imprinted polymer microspheres capable of recognizing ADP-ribosylation protein Download PDFInfo
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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Abstract
The invention provides a preparation method of molecularly imprinted polymer microspheres which adopt core-shell structures and have a molecular recognition function on ADP-ribosylation protein. Firstly, polystyrene microsphere cores with chain transfer groups on the surface are prepared through an RAFT (reversible addition-fragmentation chain transfer) precipitation polymerization reaction; then, a template, functional monomers, a crosslinking agent and an initiator are added to a reaction solvent containing the polystyrene microsphere cores for a second polymerization reaction, and thinner molecularly imprinted shell layers are formed on the surfaces of the microspheres; finally, after template molecules are removed by means of an eluent, printed holes of the template molecules are left in the thinner molecular imprinting layers on the surfaces of the microspheres, and the molecular imprinting material which can be used for specific recognition and enrichment of ADP-ribosylation protein is obtained.
Description
Technical field
The invention belongs to function of molecular engram field of material technology, be that one is prepared ADP-core based on molecular imprinting
Glycosylation albumen has the polymeric material of special identification function, can be used for the specific recognition of ADP-ribosylation albumen and divides
From.
Background technology
Adenosine diphosphate ribose base (ADP-ribosylation) is the post translational modification process of a kind of protein, the most logical
Cross adenosine diphosphate ribosyltransferase and adenosine diphosphate ribose group is transferred to the target protein after translating from NAD+.This
Kind of protein post-translational modification relates to the most important cell processes, repairs including DNA, transcribes, translates, cellular signal transduction
And apoptosis, research finds that this process is also relevant to the generation of a lot of diseases, such as cancer, diabetes, nervus retrogression
Disease and heart failure.Therefore, ADP-ribosylation has important clinical treatment meaning.In order to understand ADP-ribosylation
Mechanism, is identified for ADP-ribosylation protein and is enriched with and just become extremely important.
At present, have been developed over identifying or be enriched with ADP-ribosylation method of protein to include: use NAD+ and spread out
Biological [J. Zhang, S. H. Snyder, Biochemistry " biochemistry " 1993,32,2228-2233；J.
Zhang, Methods Enzymol. " method zymetology ". 1997,280,255-265], recombinate macromolecular structure territory [N.
Dani, A. Stilla, A. Marchegiani, A. Tamburro, S. Till, A. G. Ladurner, D.
Corda, M. Di Girolamo, Proc. Natl. Acad. Sci. U. S. A. " American Academy of Sciences's progress ".
2009,106,4243-4248], anti-ADP-ribosyl antibody [T. Lischke, V. Schumacher, J.
Wesolowski, R. Hurwitz, F. Haag, F. Koch‐Nolte, H. W. Mittrücker, Eur. J.
Immunol. " Europe Journal of Immunology " 2013,43,1828-1838], and ADP-ribosylation protein is had
Material (such as phosphate affinitive material and borate affinitive material) [H. Okayama, the K. Ueda, O. of high-affinity
Hayaishi, Proc. Natl. Acad. Sci. U. S. A. " American Academy of Sciences's progress " 1978,75,1111-
1115].But, due to dynamic, isomerism and the unstability of this protein post-translational modification, do not have any single
Method go for all types of ADP-ribosylation process, be therefore accomplished by studying and effectively identify or enrichment ADP-core
The new method of glycosylated protein and new material.
Molecular imprinting (Molecular Imprintitng Technique, MIT) [G. Wulff, A.
Sarhan, Angew. Chern. Int. Ed. " Germany's applied chemistry " 1972,11,341-345；G. Vlatakis, L.
I. Andersson, R. MUller, K. Mosbach, Nature " naturally " 1993,361,645-647] also it is molecule
Mould plate technique, belongs to supramolecular chemistry research category, is a kind of synthesis side with the high molecular polymer of bionical identification function
Method, i.e. with a certain specific target molecule as template, prepares the process of the polymer having specific selectivity to this molecule, this
High molecular polymer is referred to as molecularly imprinted polymer (Molecularly Imprinted Polymer, MIP).Owing to it has
Having preferable stability and relatively low cost, molecularly imprinted polymer is considered as potential biological identification molecule (such as antibody
And receptor) substitution material, and have been observed that it can be applicable to separate, senses, is catalyzed and the many aspects [Rhizoma Zingiberis Recens such as drug design
Loyal and righteous, Wu Hong. " molecular imprinting ", and Beijing: Chemical Industry Press, 2003].Although, have been reported and utilize molecule
Engram technology preparation can molecularly imprinted polymer [W. Zhang, W. Liu, the P. Li, H. of specific recognition target protein
Xiao, H. Wang, B. Tang, Angew. Chem. Int. Ed. " Germany's applied chemistry " 2014,53,12489-
12493; X.L. Zhao, D.Y. Li, X.W. He, W.Y. Li, Y.K. Zhang, J. Mater. Chem. B
" materials chemistry magazine " 2014,2,7575-7582, S. Shinde, A. Bunschoten, J. A. W.
Kruijtzer, R. M. J. Liskamp, B. Sellergren, Angew. Chem. Int. Ed. " Germany's application
Learn " 2012,51,8326-8329; Z. Bie, Y. Chen, J. Ye, S. Wang and Z. Liu, Angew.
Chem. Int. Ed. " Germany's applied chemistry " 2015,54,10211-5], but for recognizable ADP-ribosylation
There is not been reported for the molecularly imprinted polymer of protein.
Summary of the invention
In order to overcome the most methodical deficiency, the invention provides a kind of can specific recognition ADP-ribosylation protein
The preparation method of hud typed molecular blotting polymer microsphere, it is to utilize the imitative antibody molecule recognition performance of molecular imprinting preparation
Functional material.
For obtain above-mentioned can the hud typed molecular blotting polymer microsphere of specific recognition ADP-ribosylation protein, this
Bright technical scheme is as follows:
First pass through the reaction of RAFT (RAFT) precipitation polymerization and prepare surface with chain tra nsfer group
Polystyrene microsphere kernel；Then template, function monomer, cross-linking agent and initiator are joined containing polystyrene microsphere kernel
Reaction dissolvent in carry out second step polyreaction so that microsphere surface formed layer molecular engram outer shell；
After finally using eluant template to be removed, it will the trace leaving template molecule in the molecular engram thin layer of microsphere surface is empty
Cave.
Preferably, RAFT as above (RAFT) precipitation polymerization reaction can be light-initiated,
Or thermal initiation.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, in the preparation of polystyrene microsphere kernel, addition polymerization monomer, the mol ratio of cross-linking agent are 1:3 ~ 5, function monomer, crosslinking
Agent percentage by volume 1 ~ 5% in reaction system；The amount of initiator is polymerization monomer and the 0.5 ~ 2% of cross-linking agent total amount, causes
Mol ratio 1:3 ~ 9 of agent and chain-transferring agent, chain-transferring agent and the total amount of initiator be polymerized monomer and cross-linking agent total amount 3 ~
10%。
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, the preparation condition of polystyrene microsphere kernel: before polyreaction, reaction system is led to argon or nitrogen 5 ~ 60min, is removed
Oxygen, then reacts 3 ~ 12 hours, 60 ~ 70 ° of C of reaction temperature under argon or nitrogen atmosphere.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, the preparation of molecular engram shell is to join template molecule, function monomer, cross-linking agent and initiator containing polystyrene
In the reaction system of microsphere kernel, by second step polyreaction outside polystyrene microsphere forms the molecular engram of layer
Shell.Wherein function monomer, the mol ratio of cross-linking agent are 1:3 ~ 5, function monomer, the cross-linking agent volume basis in reaction system
Several 1 ~ 5%, the amount of initiator is function monomer and the 0.3 ~ 2% of cross-linking agent total amount, template molecule and mol ratio 1:1 of function monomer
~ 4, the consumption 50 ~ 200mg of polystyrene microsphere kernel, template used for adenosine (A), or AMP (AMP), or
One in bis phosphoric acid adenosine (ADP), or adenosine triphosphate (ATP).
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, function monomer used is: acrylamide, methacrylic acid, methyl methacrylate, 4-vinylpyridine, 2-ethylene pyrrole
One in pyridine.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, cross-linking agent used is: Ethylene glycol dimethacrylate (EGDMA), tetramethylene dimethacrylate (TDMA), three
In methacrylic acid trihydroxy methyl propyl ester (TRIM), N, N'-methylene-bisacrylamide or N, N'-vinyl bisacrylamide
A kind of.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, initiator used is: azo such as AIBN or peroxide radical initiator.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, chain-transferring agent used is: double thioesters such as BDC or three thioesters.
Preferably, the preparation of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as above
Method, solvent used is: the one in the mixture of acetonitrile, dimethyl sulfoxide, methanol or first alcohol and water.
The present invention compared with prior art, has the advantage that
1, this method use the part of adenosine A DP-ribose groups rather than whole modified albumen as imprinted templates,
Using adenosine to have 3 advantages as imprinted templates: first, as the terminal portion structure of ADP-ribosyl, adenosine is all
The common trait structure of ADP-ribosylation protein, and select adenosine rather than whole ADP-ribosylation protein conduct
Identify that target can reduce the complexity of identification；Second, compared with protein macromolecule, adenosine is as a kind of little molecule, more
Easily carry out molecular engram；3rd, adenosine or a kind of easy acquisition and cheap template molecule.
2, this adenosine molecule imprinted polymer microsphere with nucleocapsid structure can carry out specificity knowledge not only for adenosine
Not, and the protein for ADP-ribosylation also shows good selection identity, and it can be from cell extract
Sample identifies and is enriched with ADP-ribosylation protein.Accompanying drawing explanation
Fig. 1 is synthetic route and the identification to ADP-ribosylation protein thereof of hud typed adenosine molecule imprinted polymer microsphere
Schematic diagram.
Polystyrene microsphere core (a), adenosine molecule trace microsphere (b) and non-trace microsphere (c) that Fig. 2 is obtained by experiment
Scanning electron microscope (SEM) figure.
Hud typed adenosine molecule trace microsphere (MIP) and the transmission of non-trace microsphere (NIP) that Fig. 3 is obtained by experiment are electric
Mirror (TEM) figure.
Fig. 4 is the MIP/NIP microsphere adsorption dynamics adsorption kinetics figure to template molecule adenosine.
Fig. 5 is the MIP/NIP microsphere saturated absorption property figure to template molecule adenosine.
Fig. 6 is the MIP/NIP microsphere isothermal adsorption figure to template molecule adenosine.
Fig. 7 is the MIP/NIP microsphere selective absorption figure to template molecule adenosine and analog thereof, wherein
A, G, C and U represent adenosine and the like guanosine, cytidine and uridnine respectively.
Fig. 8 is the SDS-PAGE before and after purified protein samples is adsorbed by MIP/NIP microsphere, and that wherein gapA represents is ADP-
The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of ribosylation, what ck represented is that control group (does not carries out ADP-ribosylation
GAPDH), what BSA represented is bovine serum albumin.1st swimming lane is albumen Marker, and 2-4 swimming lane is shown that the gapA of purification
Sample and respectively by MIP and NIP microsphere adsorb after protein band, 5-7 swimming lane be shown that purification ck sample and
It is respectively by the protein band after the absorption of MIP and NIP microsphere, and 8-10 swimming lane is shown that the BSA sample of purification and divides
Protein band after not adsorbed by MIP and NIP microsphere.
Fig. 9 is the SDS-PAGE before and after mixed protein sample is adsorbed by MIP microsphere, and what wherein gapA represented is ADP-ribose
What the GAPDH of base, ck represented is control group (not carrying out the GAPDH of ADP-ribosylation), and what BSA represented is Ox blood serum egg
In vain.1st swimming lane is albumen Marker, and 2-3 swimming lane is shown that the biased sample of BSA Yu gapA and respectively by MIP microsphere
Protein band after absorption, 4-5 swimming lane is shown that the biased sample of BSA Yu ck and is adsorbed it by MIP microsphere respectively
After protein band.
Figure 10 is the SDS-PAGE before and after cellular extract samples is adsorbed by MIP/NIP microsphere.That wherein gapA represents is ADP-
What the GAPDH of ribosylation, BSA represented is bovine serum albumin.Wherein, the 1st swimming lane is albumen Marker, and 2-4 swimming lane shows
Be cellular extract samples and respectively by MIP and NIP microsphere adsorb after protein band, No. 5 swimming lanes are shown that pure
The protein band of BSA sample changed, No. 6 swimming lanes are shown that the protein band of the gapA sample of purification.
Specific implementation method
Following example are used for illustrating the present invention, but are not used in restriction the scope of the present invention, if not otherwise specified, and institute of the present invention
Being routine techniques by method, agents useful for same is buying reagent.
Embodiment 1: the preparation of polystyrene microsphere
(1) desired amount of polymerization monomer styrene, cross-linking agent EGDMA, chain-transferring agent BDC and initiator A IBN will be reacted
After dissolving with appropriate acetonitrile solvent, it is placed on mix homogeneously in 30mL vial, then polymerization liquid is transferred to 100mL
Being supplemented to 50mL in three neck round bottom flask and by solvent acetonitrile, under room temperature, after magnetic agitation 10min, upper end installs threeway glass tubing additional,
Continue evacuation and be passed through nitrogen, successively reciprocal 40min, to remove oxygen.
(2) hermetic container that will be equipped with reactant liquor is placed on magnetic stirring apparatus, regulates suitable rotating speed continuously stirred, 70 DEG C
Lower oil bath is heated, and reaction is drawn off to required time, and polymerization monomer occurs polymerization thus prepares polystyrene microsphere (CP),
The balloon apparatus utilizing tee T in course of reaction remains the nitrogen atmosphere of hermetic container.
(3) use the CP that 0.45 μm organic facies filter membrane vacuum filtration prepares, thus remove acetonitrile solvent, then by filter membrane
On white solid be transferred in the conical flask equipped with 50mL methanol, magnetic agitation eluting 30 ~ 60min, be repeated in eluting four
Secondary, to remove the monomer residue of microsphere surface, finally collect the white powder being dried on filter membrane.
Embodiment 2: molecular engram and the preparation of non-molecular engram shell
(1) desired amount of template molecule adenosine, function monomer acrylamide, cross-linking agent EGDMA and initiator will be reacted
After AIBN dissolves with appropriate DMSO solvent, weigh 0.15g CP microsphere and be placed on mix homogeneously in 30mL vial together,
Then being transferred to polymerization liquid be supplemented to 50mL in 100mL three neck round bottom flask and by solvent DMSO, under room temperature, magnetic force stirs
After mixing 10min, upper end installs threeway glass tubing additional, continues evacuation and is passed through nitrogen, successively reciprocal 40min, to remove oxygen.
(2) hermetic container that will be equipped with reactant liquor is placed on magnetic stirring apparatus, regulates suitable rotating speed continuously stirred, 70 DEG C
Lower oil bath is heated, and reaction is drawn off to required time, and reactant liquor occurs polymerization thus prepares adenosine molecule imprinted polymer
(MIP), course of reaction utilize the balloon apparatus of tee T remain the nitrogen atmosphere of hermetic container.
(3) use the MIP that 0.45 μm organic facies filter membrane vacuum filtration prepares, thus remove dimethylsulfoxide solvent, then
White solid on filter membrane is transferred in the 100mL conical flask equipped with 50mL methanol/acetic acid (9/1, v/v) mixed liquor, magnetic force
Stirring eluting 30min, is repeated in eluting five times, thus removes template molecule, and last again with methanol eluting once, is then collected
The white solid being dried on filter membrane.
Same method is used to prepare non-molecularly imprinted polymer (NIP) and be wherein added without adenosine template molecule.
Embodiment 3: template molecule weight adsorption experiment
(1) weigh 2mg adenosine solid sample, be dissolved in 2mL deionization pure water, ultrasonic mixing, be configured to the adenosine of 1mg/mL
Aqueous solution, then, therefrom draws 100 μ L with liquid-transfering gun, adds in 19.9mL deionization pure water, and ultrasonic mixing is configured to 5 μ g/
The adenosine aqueous solution of mL.
(2) take 2mL centrifuge tube marking serial numbers, weigh the microsphere solid that 20mg prepares the most respectively and place wherein.
(3) in each centrifuge tube, the adenosine aqueous solution that 1mL concentration is 5 μ g/mL, after ultrasonic mixing, microsphere concentration are added
For 20mg/mL, place it in 25 DEG C of incubation 16h on constant-temperature table.
(4) supernatant is taken, after 0.45 μm aqueous phase membrane filtration, with its 260nm of US-Vis spectrophotometric determination after being centrifuged
The absorbance at place.
Embodiment 4: the adsorption kinetic data
(1) weigh 2mg adenosine solid sample, be dissolved in 2mL deionization pure water, ultrasonic mixing, be configured to the adenosine of 1mg/mL
Aqueous solution, then, therefrom draws 100 μ L with liquid-transfering gun, adds in 19.9mL deionization pure water, and ultrasonic mixing is configured to 5 μ g/
The adenosine aqueous solution of mL.
(2) take 16 2mL centrifuge tubes marking serial numbers, weigh MIP microsphere and NIP microsphere that 20mg prepares the most respectively
Solid is placed wherein, two kinds of each 8 pipes of microsphere.
(3) in each centrifuge tube, the adenosine aqueous solution that 1mL concentration is 5 μ g/mL, after ultrasonic mixing, microsphere concentration are added
For 20mg/mL, place it in and on constant-temperature table, at 25 DEG C, carry out incubation.
(4) according to label every kind microsphere successively incubation absorption 0.5,1,2,4,8,12,16,20h.
(5) supernatant is taken, after 0.45 μm aqueous phase membrane filtration, with its 260nm of US-Vis spectrophotometric determination after being centrifuged
The absorbance at place, result is shown in Fig. 4.
Embodiment 5: adsorption capacity is tested
(1) weigh 2mg adenosine solid sample, be dissolved in 2mL deionization pure water, ultrasonic mixing, be configured to the adenosine of 1mg/mL
Aqueous solution, then, therefrom draws 100 μ L with liquid-transfering gun, adds in 19.9mL deionization pure water, and ultrasonic mixing is configured to 5 μ g/
The adenosine aqueous solution of mL.
(2) take 8 2mL centrifuge tubes marking serial numbers, weigh 8 the most respectively, 12,16, the microsphere solid for preparing of 20mg
Place wherein, MIP microsphere and each 4 pipes of NIP microsphere.
(3) in each centrifuge tube, the adenosine aqueous solution that 1mL concentration is 5 μ g/mL, after ultrasonic mixing, every kind of microsphere are added
Concentration is followed successively by 8,12,16,20mg/mL, then place it on constant-temperature table incubation 16h at 25 DEG C.
(4) supernatant is taken, after 0.45 μm aqueous phase membrane filtration, with its 260nm of US-Vis spectrophotometric determination after being centrifuged
The absorbance at place, result is shown in Fig. 5.
Embodiment 6: adsorption isotherm experiment
(1) weigh 2mg adenosine solid sample, be dissolved in 2mL deionization pure water, ultrasonic mixing, be configured to the adenosine of 1mg/mL
Aqueous solution, then, therefrom draws 200 μ L with liquid-transfering gun, adds in 19.8mL deionization pure water, and ultrasonic mixing is configured to 10 μ g/
The adenosine aqueous solution of mL.
(2) take 8 4mL centrifuge tubes marking serial numbers, be configured to concentration and be respectively 1 μ g/mL, 2 μ g/mL, 3 μ g/mL, 4 μ g/
ML, 5 μ g/mL, 6 μ g/mL, 7 μ g/mL, the adenosine aqueous solution of 8 μ g/mL.
(3) weigh MIP microsphere that 20mg prepares the most respectively and NIP microsphere solid is placed wherein, two kinds of microspheres each 8
Pipe.
(4) in each centrifuge tube, the adenosine aqueous solution that 1mL concentration is 1-8 μ g/mL, ultrasonic mixing are added successively according to label
After, microsphere concentration is 20mg/mL, places it on constant-temperature table incubation 16h at 25 DEG C.
(5) supernatant is taken, after 0.45 μm aqueous phase membrane filtration, with its 260nm of US-Vis spectrophotometric determination after being centrifuged
The absorbance at place, result is shown in Fig. 6.
Embodiment 7: selective absorption is tested
(1) weigh each 2mg of solid sample of adenosine, guanosine, cytidine, uridnine respectively, be dissolved in 2mL deionization pure water, ultrasonic
Mixing, is configured to the adenosine of 1mg/mL, guanosine, cytidine, uridnine aqueous solution, then draws 100 μ L with liquid-transfering gun the most respectively, add
Enter in 19.9mL deionization pure water, ultrasonic mixing, it is configured to respectively the adenosine of 5 μ g/mL, guanosine, cytidine, uridnine water-soluble
Liquid.
(2) take 8 2mL centrifuge tubes marking serial numbers, weigh MIP microsphere and NIP microsphere that 20mg prepares the most respectively
Solid is placed wherein, two kinds of each 4 pipes of microsphere.
(3) according to label, to often organizing, to be separately added into 1mL concentration in centrifuge tube be the adenosine of 5 μ g/mL, guanosine, cytidine, uridnine
Aqueous solution, after ultrasonic mixing, microsphere concentration is 20mg/mL, then places it on constant-temperature table incubation 16h at 25 DEG C.
(4) take supernatant after being centrifuged, after 0.45 μm aqueous phase membrane filtration, measure adenosine respectively with US-Vis spectrophotometer
Absorbance at 260nm, guanosine 252nm, cytidine 270nm, uridnine 261nm, result is shown in Fig. 7.
Embodiment 8: the adsorptivity experiment of protein of interest
(1) weighing 0.6055g Tris solid to be dissolved in 80mL deionization pure water, then dropping HCl regulates pH to 7.5, finally
Graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solution of pH7.5.
Each with tri-kinds of albumen of gapA, ck, BSA that 50mM Tris-HCl buffer solution compound concentration respectively is 0.3mg/mL
2mL。
(2) take 6 2mL centrifuge tubes marking serial numbers, weigh the microsphere solid that 12mg prepares the most respectively and place wherein,
Each three pipes of MIP and NIP microsphere.
(3) in the centrifuge tube equipped with MIP and NIP microsphere, it is separately added into three kinds of albumen that 600 μ L concentration are 0.3mg/mL
After solution, fully mixing, microsphere concentration is 20mg/mL, then places it in 25 DEG C of incubation 16h on constant-temperature table.
(4) taking supernatant after centrifugal, carry out SDS-PAGE electrophoresis to judge the concentration of albumen after albumen sample preparation, result is shown in figure
8。
Embodiment 9: the adsorptivity experiment of target protein in egg mix white liquor
(1) weighing 0.6055g Tris solid to be dissolved in 80mL deionization pure water, then dropping HCl regulates pH to 7.5, finally
Graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solution of pH7.5.
(2) with 50mM Tris-HCl buffer solution prepare respectively two kinds of egg mix white liquor (0.5mg/mL BSA+
0.2mg/mL gapA) and (0.5mg/mL BSA+0.2mg/mL ck) each 2mL.
(3) take two 2mL centrifuge tubes marking serial numbers, weigh the MIP microsphere solid placement that 10mg prepares the most respectively
Wherein.
(4) in two centrifuge tubes equipped with MIP microsphere solid, it is separately added into two kinds of egg mix white liquor (0.5mg/mL BSA
+ 0.2mg/mL gapA) and (0.5mg/mL BSA+0.2mg/mL ck) each 500 μ L, after fully mixing, microsphere concentration is
20mg/mL, then places it in 25 DEG C of incubation 16h on constant-temperature table.
(5) taking supernatant after centrifugal, carry out SDS-PAGE electrophoresis to judge the concentration of albumen after albumen sample preparation, result is shown in figure
9。
Embodiment 10: the adsorptivity experiment of target protein in cell breakage liquid
(1) weighing 0.6055g Tris solid to be dissolved in 80mL deionization pure water, then dropping HCl regulates pH to 7.5, finally
Graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solution of pH7.5.
(2) with 50mM Tris-HCl buffer solution, the cell breakage liquid expressing the GAPDH containing ADP-ribosylation is carried
Take thing and dilute 3 times, be configured to 2mL solution.
(3) take two 2mL centrifuge tubes marking serial numbers, weigh MIP and the NIP microsphere solid that 10mg prepares the most respectively
Place wherein.
(4) in the centrifuge tube equipped with MIP and NIP microsphere, it is separately added into the dilution of the above-mentioned cell extract prepared
After liquid, fully mixing, microsphere concentration is 20mg/mL, then places it in 25 DEG C of incubation 16h on constant-temperature table.
(5) taking supernatant after centrifugal, carry out SDS-PAGE electrophoresis to judge the concentration of albumen after albumen sample preparation, result is shown in figure
10。
Claims (9)
1. the preparation method of the molecular blotting polymer microsphere of a recognizable ADP-ribosylation albumen, it is characterised in that: should
Method comprises the steps:
(1) preparation of polystyrene microsphere: by the RAFT precipitation polymerization reaction preparation of polymerization monomer
Going out the surface polystyrene microsphere kernel with chain tra nsfer group, described polymerization monomer is styrene, acrylamide, ethylene pyrrole
One in pyridine；
(2) preparation of molecular engram shell: template molecule, function monomer, cross-linking agent and initiator are joined containing polyphenyl second
The reaction dissolvent of alkene microsphere kernel carries out polyreaction so that microsphere surface forms one layer of molecular engram outer shell, described
Template molecule is the ribosyl moieties of ADP-ribosylation protein, is adenosine (A), or AMP (AMP), or double
One in adenosine phosphate (ADP), or adenosine triphosphate (ATP)；
(3) template molecule removes: use eluant to be removed by the template molecule in the shell of step (2), dividing at microsphere surface
Sub-trace thin layer stays the imprinted cavity of template molecule.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 1
Method, it is characterised in that: the preparation method of the polystyrene microsphere of step (1) is:
(1) after polymerization monomer, cross-linking agent, chain-transferring agent and initiator dissolve, mix homogeneously make reaction system, at room temperature
After magnetic agitation 10min, continue evacuation and be passed through nitrogen or argon 5-60min；
(2) reaction system of step (1) is continued to be placed on magnetic stirring apparatus continuously stirred, at 60-70 DEG C, reacts 3～12h,
Prepare polystyrene microsphere；
(3) use the polystyrene microsphere of organic facies filter membrane vacuum filtration step (2), remove solvent, then white by filter membrane
Color solid is transferred to equipped with in the container of methanol, magnetic agitation eluting 30～60min, repeats eluting four times, finally collects filter membrane
On be dried white powder.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 1
Method, it is characterised in that: the preparation method of the molecular engram shell of step (2) is:
(1), after template molecule, function monomer, cross-linking agent, initiator dissolve, mix homogeneously with polystyrene microsphere and make reactant
System, at room temperature after magnetic agitation 10min, continues evacuation and is passed through nitrogen or argon 5-60min；
(2) reaction system of step (1) is continued to be placed on magnetic stirring apparatus continuously stirred, at 60-70 DEG C, reacts 3～12h,
Prepare adenosine molecule imprinted polymer.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 1
Method, it is characterised in that: the method that the template molecule of step (3) removes is: use the gland that organic facies filter membrane vacuum filtration prepares
Glycosides molecularly imprinted polymer, removes solvent, is then transferred to by the white solid on filter membrane equipped with methanol and acetate mixture
In container, magnetic agitation eluting 30min, repeat eluting 5 times, remove template molecule, last again with methanol eluting once, is then received
The white solid being dried on collection filter membrane, in described methanol, acetate mixture, methanol, the volume ratio of acetic acid are 9:1.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 2
Method, it is characterised in that: function monomer, cross-linking agent mol ratio that the preparation of polystyrene microsphere kernel adds are 1:3～5, function
Monomer, the cross-linking agent percentage by volume 1～5% in reaction system；The amount of initiator is polymerization monomer and cross-linking agent total amount
0.5～2%, mol ratio 1:3～9 of initiator and chain-transferring agent, the total amount of chain-transferring agent and initiator is for being polymerized monomer and friendship
The 3～10% of connection agent total amount.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 3
Method, it is characterised in that: function monomer, the mol ratio of cross-linking agent are 1:3～5, function monomer, the cross-linking agent body in reaction system
Long-pending percent 1～5%, the amount of initiator is function monomer and the 0.3 of cross-linking agent total amount～2%, template molecule and function monomer
Mol ratio 1:1～4, the consumption 50～200mg of polystyrene microsphere kernel.
The preparation side of the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 1
Method, it is characterised in that: described function monomer is any one in acrylamide and derivant thereof, acrylic acid and derivant thereof；
Described cross-linking agent is Ethylene glycol dimethacrylate, tetramethylene dimethacrylate, trimethacrylate acid three hydroxyls
Methyl propyl ester, N, N'-methylene-bisacrylamide or N, any one in N'-vinyl bisacrylamide；
Described initiator is azo or peroxide radical initiator；
Described chain-transferring agent is double thioesters or three thioesters；
Described solvent is: acetonitrile, dimethyl sulfoxide, methanol or methanol and any one in aqueous mixtures.
8. the molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen as prepared by claim 1-7, its
It is characterised by: polyalcohol microspherulite diameter distribution 100nm-5 μm.
The molecular blotting polymer microsphere of a kind of recognizable ADP-ribosylation albumen the most as claimed in claim 8, its feature
Be: this polymer microballoon separating, enrichment ADP-ribosylation albumen, ADP-ribosylation protein science, metabolism give financial aid to students, disease
Application in terms of sick diagnosis, Mycobacterium tuberculosis drug-resistant Journal of Sex Research.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108918483A (en) * | 2018-05-08 | 2018-11-30 | 青岛大学 | A kind of photocatalysis RAFT polymerization prepares the method and its application of molecular engram sensor |
| CN109821513A (en) * | 2019-03-09 | 2019-05-31 | 河南工业大学 | A kind of cell blots polymer microballoon and preparation method thereof of targeting filtering cancer cell |
| CN115368522A (en) * | 2022-09-01 | 2022-11-22 | 江苏大学 | Preparation method of post-modified surface imprinted polymer based on double covalent bonds and application of post-modified surface imprinted polymer in selective separation of adenosine monophosphate |
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|---|---|---|---|---|
| CN102924645A (en) * | 2012-11-21 | 2013-02-13 | 安徽农业大学 | Preparation method and application of molecularly imprinted polymer of penicillin antibiotics and intermediate of penicillin antibiotics |
| CN104289186A (en) * | 2014-09-10 | 2015-01-21 | 济南大学 | Preparation of palm tree bark surface S-adenosylmethionine molecular imprinted adsorption material |
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| CN102924645A (en) * | 2012-11-21 | 2013-02-13 | 安徽农业大学 | Preparation method and application of molecularly imprinted polymer of penicillin antibiotics and intermediate of penicillin antibiotics |
| CN104289186A (en) * | 2014-09-10 | 2015-01-21 | 济南大学 | Preparation of palm tree bark surface S-adenosylmethionine molecular imprinted adsorption material |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918483A (en) * | 2018-05-08 | 2018-11-30 | 青岛大学 | A kind of photocatalysis RAFT polymerization prepares the method and its application of molecular engram sensor |
| CN109821513A (en) * | 2019-03-09 | 2019-05-31 | 河南工业大学 | A kind of cell blots polymer microballoon and preparation method thereof of targeting filtering cancer cell |
| CN115368522A (en) * | 2022-09-01 | 2022-11-22 | 江苏大学 | Preparation method of post-modified surface imprinted polymer based on double covalent bonds and application of post-modified surface imprinted polymer in selective separation of adenosine monophosphate |
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