CN105944692B - A kind of preparation method of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen - Google Patents
A kind of preparation method of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen Download PDFInfo
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- B01J20/26—Synthetic macromolecular compounds
- B01J20/268—Polymers created by use of a template, e.g. molecularly imprinted polymers
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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Abstract
Invention describes a kind of preparation methods of the molecular blotting polymer microsphere to nucleocapsid structure of the ADP- ribosylation protein with molecular recognition function.Pass through reversible addition-fracture chain tra nsfer first（RAFT）The polystyrene microsphere kernel that surface carries chain tra nsfer group is prepared in precipitation polymerization reaction；Then template, function monomer, crosslinking agent and initiator are added to progress second step polymerisation in the reaction dissolvent containing polystyrene microsphere kernel, so that microsphere surface forms the molecular engram outer shell of layer；After finally removing template molecule using eluant, eluent, the imprinted cavity of template molecule is left in the molecular engram thin layer of microsphere surface, obtains the molecular engram material that can be used for specific recognition and enrichment to ADP- ribosylation protein.
Description
Technical field
It is that one kind is prepared based on molecular imprinting technology to ADP- cores the invention belongs to function of molecular engram field of material technology
Glycosylating albumen has the polymer 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）A kind of posttranslational modification process of protein, i.e., it is logical
It crosses adenosine diphosphate ribosyltransferase and adenosine diphosphate ribose group is transferred to the target protein after translation from NAD+.This
Kind protein post-translational modification is related to many important cell processes, including DNA is repaired, transcription, is translated, cellular signal transduction
And Apoptosis, research finds that this process is also related to the generation of many 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, ADP- ribosylation protein, which is identified and is enriched with just, becomes extremely important.
Currently, the method developed identification or be enriched with ADP- ribosylation protein includes：Using NAD+ and its spread out
Biology [J. Zhang, S. H. Snyder, Biochemistry《Biochemistry》 1993, 32, 2228-2233；J.
Zhang, Methods Enzymol.《Method zymetology》1997,280,255-265], recombination macromolecular structure domain [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 is in progress》.
2009,106,4243-4248], anti-ADP- ribosyls antibody [T. Lischke, V. Schumacher, J.
Wesolowski, R. Hurwitz, F. Haag, F. Koch‐Nolte, H. W. Mittrücker, Eur. J.
Immunol.《European Journal of Immunology》2013,43,1828-1838], and have for ADP- ribosylation protein
The substance of high-affinity（Such as phosphate affinitive material and borate affinitive material）[H. Okayama, K. Ueda, O.
Hayaishi, Proc. Natl. Acad. Sci. U. S. A.《American Academy of Sciences is in progress》1978, 75, 1111-
1115].However, due to the dynamic of this protein post-translational modification, isomerism and unstability, it is single without any type
Method can be adapted for all types of ADP- ribosylation processes, therefore just need to study effectively identification or enrichment ADP- cores
The new method and new material of glycosylated protein.
Molecular imprinting technology（Molecular Imprintitng Technique, MIT）[G. Wulff, A.
Sarhan, Angew. Chern. Int. Ed.《German applied chemistry》1972, 11, 341-345；G. Vlatakis, L.
I. Andersson, R. MUller, K. Mosbach, Nature 《It is natural》1993,361,645-647] also it is molecule
Mould plate technique belongs to supramolecular chemistry research category, is a kind of side of high molecular polymer of the synthesis with bionical identification function
Method prepares the process for the polymer for having specific selectivity to the molecule that is, using a certain specific target molecule as template, this
High molecular polymer is referred to as molecularly imprinted polymer（Molecularly Imprinted Polymer, MIP）.Due to its tool
It is considered as potential biological identification molecule to have preferable stability and lower cost, molecularly imprinted polymer（Such as antibody
And receptor）Alternative materials, and have been observed that it can be applied to separation, sensing, catalysis and many aspects [ginger such as drug design
It is loyal and righteous, Wu Hong《Molecular imprinting technology》, Beijing:Chemical Industry Press, 2003].Molecule is utilized although having been reported
Engram technology prepare can specific recognition target protein molecularly imprinted polymer [W. Zhang, W. Liu, P. Li, H.
Xiao, H. Wang, B. Tang, Angew. Chem. Int. Ed. 《German 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. 《German application
It learns》 2012, 51, 8326-8329; Z. Bie, Y. Chen, J. Ye, S. Wang and Z. Liu, Angew.
Chem. Int. Ed. 《German applied chemistry》2015,54,10211-5], but for can recognize that ADP- ribosylation
There is not been reported for the molecularly imprinted polymer of protein.
Invention content
In order to overcome the shortcomings of existing method, the present invention provides it is a kind of can specific recognition ADP- ribosylation protein
The preparation method of hud typed molecular blotting polymer microsphere, it is to prepare to imitate antibody molecule recognition performance using molecular imprinting technology
Functional material.
For obtain it is above-mentioned can specific recognition ADP- ribosylation protein hud typed molecular blotting polymer microsphere, this hair
Bright technical solution is as follows：
Pass through reversible addition-fracture chain tra nsfer first（RAFT）Precipitation polymerization reaction prepares surface and carries chain tra nsfer base
The polystyrene microsphere kernel of group；Then template, function monomer, crosslinking agent and initiator are added to containing polystyrene microsphere
Second step polymerisation is carried out in the reaction dissolvent of kernel, so that microsphere surface forms the molecular engram shell of layer
Layer；After finally template is removed using eluant, eluent, it will leave the print of template molecule in the molecular engram thin layer of microsphere surface
Mark hole.
Preferably, reversible addition-fracture chain tra nsfer as described above（RAFT）Precipitation polymerization reaction can be it is light-initiated,
Or thermal initiation.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Polymerized monomer is added in method in the preparation of polystyrene microsphere kernel, the molar ratio of crosslinking agent is 1:3 ~ 5, function monomer, crosslinking
The percentage by volume 1 ~ 5% of agent in the reaction system；The amount of initiator is the 0.5 ~ 2% of polymerized monomer and crosslinking agent total amount, is caused
The molar ratio 1 of agent and chain-transferring agent:3 ~ 9, the total amount of chain-transferring agent and initiator be polymerized monomer and crosslinking agent total amount 3 ~
10%。
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, the preparation condition of polystyrene microsphere kernel：Before polymerisation, reaction system leads to 5 ~ 60min of argon gas or nitrogen, removes
Then oxygen reacts 3 ~ 12 hours, 60 ~ 70 °C of reaction temperature under argon gas or nitrogen atmosphere.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, the preparation of molecular engram shell are that template molecule, function monomer, crosslinking agent and initiator are added to containing polystyrene
In the reaction system of microballoon kernel, by second step polymerisation outside the molecular engram that polystyrene microsphere forms layer
Shell.Wherein function monomer, crosslinking agent molar ratio be 1:3 ~ 5, the volume basis of function monomer, crosslinking agent in the reaction system
Number 1 ~ 5%, the amount of initiator are the 0.3 ~ 2% of function monomer and crosslinking agent total amount, the molar ratio 1 of template molecule and function monomer:1
~ 4,50 ~ 200mg of dosage of polystyrene microsphere kernel, template used is adenosine（A）Or adenosine monophosphate（AMP）, or
Bis phosphoric acid adenosine（ADP）Or atriphos（ATP）In one kind.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, function monomer used are：Acrylamide, methacrylic acid, methyl methacrylate, 4-vinylpyridine, 2- ethylene pyrroles
One kind in pyridine.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, crosslinking agent used are：Ethylene glycol dimethacrylate (EGDMA), tetramethylene dimethacrylate (TDMA), three
In methacrylic acid trihydroxy methyl propyl ester (TRIM), N, N'- methylene-bisacrylamides or N, N'- vinyl bisacrylamide
It is a kind of.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, initiator used are：Azo such as AIBN or peroxide radical initiator.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, chain-transferring agent used are：Double thioesters such as BDC or three thioesters.
Preferably, a kind of preparation of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described above
Method, solvent used are：Acetonitrile, dimethyl sulfoxide, methanol or first alcohol and water mixture in one kind.
Compared with prior art, the present invention having the following advantages：
1, this method uses a part for adenosine-ADP- ribose groups, rather than entire modified albumen is as trace mould
Plate has 3 advantages using adenosine as imprinted templates：First, as the terminal portion structure of ADP- ribosyls, adenosine is
The common trait structure of all ADP- ribosylation protein, and select adenosine rather than entire ADP- ribosylation protein
The complexity of identification can be reduced as identification target；Second, compared with protein macromolecule, adenosine is as small point a kind of
Son, it is easier to carry out molecular engram；Third, adenosine or a kind of easy acquisition and cheap template molecule.
2, this adenosine molecule imprinted polymer microballoon with nucleocapsid can carry out specific knowledge not only for adenosine
Not, and for the protein of ADP- ribosylation good selection identity is also shown, it can be from cell extract
It is identified in sample and is enriched with ADP- ribosylation protein.Description of the drawings
Fig. 1 is for the synthetic route of hud typed adenosine molecule imprinted polymer microballoon and its to ADP- ribosylation protein
The schematic diagram of identification.
The polystyrene microsphere core that Fig. 2 is obtained by experiment（a）, adenosine molecule trace microballoon (b) and non-trace microballoon (c)
Scanning electron microscope（SEM）Figure.
The transmission electricity for the hud typed adenosine molecule trace microballoon (MIP) and non-trace microballoon (NIP) that Fig. 3 is obtained by experiment
Mirror（TEM）Figure.
Fig. 4 is adsorption dynamics adsorption kinetics figure of the MIP/NIP microballoons to template molecule adenosine.
Fig. 5 is saturation absorption property figure of the MIP/NIP microballoons to template molecule adenosine.
Fig. 6 is isothermal adsorption figure of the MIP/NIP microballoons to template molecule adenosine.
Fig. 7 is selective absorption figure of the MIP/NIP microballoons to template molecule adenosine and its analogue, wherein
A, G, C and U indicate adenosine and the like guanosine, cytidine and uridine respectively.
Fig. 8 is that purified protein samples are adsorbed front and back SDS-PAGE by MIP/NIP microballoons, and that wherein gapA is indicated is ADP-
The glyceraldehyde-3-phosphate dehydrogenase of ribosylation（GAPDH）, what ck was indicated is control group（ADP- ribosylation is not carried out
GAPDH）, what BSA was indicated is bovine serum albumin.1st swimming lane is albumen Marker, and the gapA of purifying is shown in 2-4 swimming lanes
Sample and its respectively by MIP and NIP microballoons adsorb after protein band, 5-7 swimming lanes be shown purifying ck samples and
Its protein band after being adsorbed respectively by MIP and NIP microballoons, 8-10 swimming lanes are shown the BSA samples of purifying and its divide
Protein band after not adsorbed by MIP and NIP microballoons.
Fig. 9 is that mixed protein sample is adsorbed front and back SDS-PAGE by MIP microballoons, and what wherein gapA was indicated is ADP- ribose
What the GAPDH of base, ck were indicated is control group（The GAPDH of ADP- ribosylation is not carried out）, what BSA was indicated is cow's serum egg
In vain.1st swimming lane is albumen Marker, and 2-3 swimming lanes are shown the mixing sample of BSA and gapA and its respectively by MIP microballoons
Protein band after absorption, 4-5 swimming lanes are shown the mixing sample of BSA and ck and its adsorb it by MIP microballoons respectively
Protein band afterwards.
Figure 10 is cellular extract samples by the front and back SDS-PAGE of MIP/NIP microballoons absorption.Wherein gapA indicate be
What the GAPDH of ADP- ribosylation, BSA were indicated is bovine serum albumin.Wherein, the 1st swimming lane is albumen Marker, 2-4 swimming lanes
Protein band after cellular extract samples are shown and its are adsorbed respectively by MIP and NIP microballoons, what No. 5 swimming lanes were shown
It is the protein band of the BSA samples of purifying, the protein band of the gapA samples of purifying is shown in No. 6 swimming lanes.
Specific implementation method
Following embodiment is not used in and limits the scope of the invention for illustrating the present invention, if not otherwise specified, this hair
Bright method therefor is routine techniques, and agents useful for same is buying reagent.
Embodiment 1：The preparation of polystyrene microsphere
（1）Desired amount of polymerized monomer styrene, crosslinking agent EGDMA, chain-transferring agent BDC and initiator will be reacted
After AIBN is dissolved with appropriate acetonitrile solvent, it is placed in 30mL vials and is uniformly mixed, be then transferred to polymerization liquid
It is supplemented to 50mL in 100mL three neck round bottom flask and by solvent acetonitrile, upper end installs threeway glass additional after magnetic agitation 10min at room temperature
Glass pipe persistently vacuumizes and is passed through nitrogen, successively reciprocal 40min, to remove oxygen.
（2）Closed container equipped with reaction solution is placed on magnetic stirring apparatus, appropriate rotating speed is adjusted and persistently stirs, 70 DEG C
Lower oil bath heating is drawn off after reaction to required time, and polymerization occurs for polymerized monomer to prepare polystyrene microsphere（CP）,
The nitrogen atmosphere of closed container is remained in reaction process using the balloon apparatus of three-way pipe.
（3）The CP prepared is filtered by vacuum using 0.45 μm of organic phase filter membrane, to remove acetonitrile solvent, then by filter membrane
On white solid be transferred in the conical flask equipped with 50mL methanol, magnetic agitation elute 30 ~ 60min, be repeated in elution four
It is secondary, to remove the monomer residue of microsphere surface, finally collect the white powder dried on filter membrane.
Embodiment 2：The preparation of molecular engram and non-molecular engram shell
（1）Desired amount of template molecule adenosine, function monomer acrylamide, crosslinking agent EGDMA and initiation will be reacted
After agent AIBN is dissolved with appropriate DMSO solvents, weigh 0.15g CP microballoons and be placed on together in 30mL vials mix it is equal
It is even, then polymerization liquid is transferred in 100mL three neck round bottom flask and solvent DMSO is supplemented to 50mL, at room temperature magnetic force
Upper end installs threeway glass tube additional after stirring 10min, nitrogen is persistently vacuumized and be passed through, successively reciprocal 40min, to remove oxygen.
（2）Closed container equipped with reaction solution is placed on magnetic stirring apparatus, appropriate rotating speed is adjusted and persistently stirs, 70 DEG C
Lower oil bath heating is drawn off after reaction to required time, and polymerization occurs for reaction solution to prepare adenosine molecule imprinted polymer
（MIP）, the nitrogen atmosphere of closed container is remained in reaction process using the balloon apparatus of three-way pipe.
（3）The MIP prepared is filtered by vacuum using 0.45 μm of organic phase filter membrane, to remove dimethylsulfoxide solvent, then
White solid on filter membrane is transferred to equipped with 50mL methanol/acetic acid（9/1, v/v）In the 100mL conical flasks of mixed liquor, magnetic force
Stirring elution 30min, is repeated in elution five times, and to remove template molecule, last again with methanol elution is primary, then collects
The white solid dried on filter membrane.
Non-molecularly imprinted polymer is prepared using same method（NIP）And wherein it is added without adenosine template molecule.
Embodiment 3：Template molecule heavily adsorb is tested
（1）2mg adenosine solid samples are weighed, are dissolved in 2mL deionization pure water, ultrasonic mixing is configured to 1mg/mL's
Then adenosine aqueous solution therefrom draws 100 μ L with liquid-transfering gun, be added in 19.9mL deionization pure water, ultrasonic mixing is configured to 5
The adenosine aqueous solution of μ g/mL.
（2）2mL centrifuge tubes and marking serial numbers are taken, the microsphere solid that 20mg is prepared then is weighed respectively and places wherein.
（3）It is added the adenosine aqueous solution of 1mL a concentration of 5 μ g/mL into each centrifuge tube, after ultrasonic mixing, microballoon concentration
For 20mg/mL, 25 DEG C of incubation 16h on constant-temperature table are placed it in.
（4）Supernatant is taken after centrifugation, after 0.45 μm of water phase membrane filtration, with its 260nm of US-Vis spectrophotometric determinations
The absorbance at place.
Embodiment 4：The adsorption kinetic data
（1）2mg adenosine solid samples are weighed, are dissolved in 2mL deionization pure water, ultrasonic mixing is configured to 1mg/mL's
Then adenosine aqueous solution therefrom draws 100 μ L with liquid-transfering gun, be added in 19.9mL deionization pure water, ultrasonic mixing is configured to 5
The adenosine aqueous solution of μ g/mL.
（2）16 2mL centrifuge tubes and marking serial numbers are taken, then weigh the MIP microballoons and NIP microballoons that 20mg is prepared respectively
Solid is placed wherein, each 8 pipe of two kinds of microballoons.
（3）It is added the adenosine aqueous solution of 1mL a concentration of 5 μ g/mL into each centrifuge tube, after ultrasonic mixing, microballoon concentration
For 20mg/mL, places it on constant-temperature table and incubated at 25 DEG C.
（4）According to label, each microballoon incubates absorption 0.5,1,2,4,8,12,16,20h successively.
（5）Supernatant is taken after centrifugation, after 0.45 μm of water phase membrane filtration, with its 260nm of US-Vis spectrophotometric determinations
The absorbance at place, is as a result shown in Fig. 4.
Embodiment 5：Adsorption capacity is tested
（1）2mg adenosine solid samples are weighed, are dissolved in 2mL deionization pure water, ultrasonic mixing is configured to 1mg/mL's
Then adenosine aqueous solution therefrom draws 100 μ L with liquid-transfering gun, be added in 19.9mL deionization pure water, ultrasonic mixing is configured to 5
The adenosine aqueous solution of μ g/mL.
（2）Take 8 2mL centrifuge tubes and marking serial numbers, then weigh 8 respectively, 12,16, the microsphere solids that prepare of 20mg
It places wherein, MIP microballoons and each 4 pipe of NIP microballoons.
（3）It is added the adenosine aqueous solution of 1mL a concentration of 5 μ g/mL into each centrifuge tube, after ultrasonic mixing, each microballoon
Concentration is followed successively by 8,12,16,20mg/mL, then place it on constant-temperature table and incubate 16h at 25 DEG C.
（4）Supernatant is taken after centrifugation, after 0.45 μm of water phase membrane filtration, with its 260nm of US-Vis spectrophotometric determinations
The absorbance at place, is as a result shown in Fig. 5.
Embodiment 6：Adsorption isotherm experiment
（1）2mg adenosine solid samples are weighed, are dissolved in 2mL deionization pure water, ultrasonic mixing is configured to 1mg/mL's
Then adenosine aqueous solution therefrom draws 200 μ L with liquid-transfering gun, be added in 19.8mL deionization pure water, ultrasonic mixing is configured to
The adenosine aqueous solution of 10 μ g/mL.
（2）8 4mL centrifuge tubes and marking serial numbers are taken, it is respectively 1 μ g/mL, 2 μ g/mL, 3 μ g/mL, 4 μ g/ to be configured to concentration
The adenosine aqueous solution of mL, 5 μ g/mL, 6 μ g/mL, 7 μ g/mL, 8 μ g/mL.
（3）Then MIP microballoons that 20mg is prepared are weighed respectively and NIP microsphere solids are placed wherein, two kinds of microballoons each 8
Pipe.
（4）The adenosine aqueous solution of a concentration of 1-8 μ g/mL of 1mL, ultrasonic mixing is added into each centrifuge tube successively according to label
Afterwards, a concentration of 20mg/mL of microballoon is placed it on constant-temperature table and is incubated 16h at 25 DEG C.
（5）Supernatant is taken after centrifugation, after 0.45 μm of water phase membrane filtration, with its 260nm of US-Vis spectrophotometric determinations
The absorbance at place, is as a result shown in Fig. 6.
Embodiment 7：Selective absorption is tested
（1）Each 2mg of solid sample for weighing adenosine, guanosine, cytidine, uridine respectively, is dissolved in 2mL deionization pure water,
Ultrasonic mixing is configured to adenosine, guanosine, cytidine, the uridine aqueous solution of 1mg/mL, then therefrom draws 100 μ respectively with liquid-transfering gun
L is added in 19.9mL deionization pure water, it is configured to adenosine, guanosine, cytidine, the uridine water of 5 μ g/mL by ultrasonic mixing respectively
Solution.
（2）8 2mL centrifuge tubes and marking serial numbers are taken, then weigh the MIP microballoons and NIP microballoons that 20mg is prepared respectively
Solid is placed wherein, each 4 pipe of two kinds of microballoons.
（3）Adenosine, guanosine, cytidine, the uridine of a concentration of 5 μ g/mL of 1mL are separately added into every group of centrifuge tube according to label
Aqueous solution, after ultrasonic mixing, then a concentration of 20mg/mL of microballoon is placed it on constant-temperature table and is incubated 16h at 25 DEG C.
（4）It takes supernatant after centrifugation, after 0.45 μm of water phase membrane filtration, adenosine is measured respectively with US-Vis spectrophotometers
260nm, guanosine 252nm, cytidine 270nm, the absorbance at uridine 261nm, are as a result shown in Fig. 7.
Embodiment 8：The adsorptivity of protein of interest is tested
（1）It weighs 0.6055g Tris solids to be dissolved in 80mL deionization pure water, HCl is then added dropwise and adjusts pH to 7.5,
Last graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solutions of pH7.5.
It is each with 50mM Tris-HCl buffer solutions tri- kinds of albumen of gapA, ck, BSA that compound concentration is 0.3mg/mL respectively
2mL。
（2）6 2mL centrifuge tubes and marking serial numbers are taken, the microsphere solid that 12mg is prepared then is weighed respectively and places wherein,
Each three pipe of MIP and NIP microballoons.
（3）Three kinds of albumen of 600 a concentration of 0.3mg/mL of μ L are separately added into the centrifuge tube equipped with MIP and NIP microballoons
Solution, after mixing well, then a concentration of 20mg/mL of microballoon places it in 25 DEG C of incubation 16h on constant-temperature table.
（4）Supernatant is taken after centrifugation, and SDS-PAGE electrophoresis is carried out after albumen sample preparation to judge the concentration of albumen, as a result sees figure
8。
Embodiment 9：The adsorptivity experiment of target protein in egg mix white liquor
（1）It weighs 0.6055g Tris solids to be dissolved in 80mL deionization pure water, HCl is then added dropwise and adjusts pH to 7.5,
Last graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solutions of pH7.5.
（2）Two kinds of egg mix white liquors are prepared respectively with 50mM Tris-HCl buffer solutions（0.5mg/mL BSA +
0.2mg/mL gapA）And（0.5mg/mL BSA + 0.2mg/mL ck）Each 2mL.
（3）Two 2mL centrifuge tubes and marking serial numbers are taken, the MIP microsphere solids that 10mg is prepared then is weighed respectively and places
Wherein.
（4）It is separately added into two kinds of egg mix white liquors into two centrifuge tubes equipped with MIP microsphere solids（0.5mg/mL BSA
+ 0.2mg/mL gapA）And（0.5mg/mL BSA + 0.2mg/mL ck）Each 500 μ L, after mixing well, microballoon is a concentration of
Then 20mg/mL places it in 25 DEG C of incubation 16h on constant-temperature table.
（5）Supernatant is taken after centrifugation, and SDS-PAGE electrophoresis is carried out after albumen sample preparation to judge the concentration of albumen, as a result sees figure
9。
Embodiment 10：The adsorptivity experiment of target protein in clasmatosis liquid
（1）It weighs 0.6055g Tris solids to be dissolved in 80mL deionization pure water, HCl is then added dropwise and adjusts pH to 7.5,
Last graduated cylinder is settled to 100mL, is configured to the 50mM Tris-HCl buffer solutions of pH7.5.
（2）The clasmatosis liquid for expressing the GAPDH containing ADP- ribosylation is carried with 50mM Tris-HCl buffer solutions
It takes object to dilute 3 times, is configured to 2mL solution.
（3）Two 2mL centrifuge tubes and marking serial numbers are taken, then weigh MIP the and NIP microsphere solids that 10mg is prepared respectively
It places wherein.
（4）The dilution of above-mentioned prepared cell extract is separately added into the centrifuge tube equipped with MIP and NIP microballoons
Liquid, after mixing well, then a concentration of 20mg/mL of microballoon places it in 25 DEG C of incubation 16h on constant-temperature table.
（5）Supernatant is taken after centrifugation, and SDS-PAGE electrophoresis is carried out after albumen sample preparation to judge the concentration of albumen, as a result sees figure
10。
Claims (9)
1. a kind of preparation method of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen, it is characterised in that：It should
Method includes the following steps：
（1）The preparation of polystyrene microsphere：It is prepared by reversible addition-fracture chain tra nsfer precipitation polymerization reaction of polymerized monomer
Go out the polystyrene microsphere kernel that surface carries chain tra nsfer group, the polymerized monomer is styrene, acrylamide, ethylene pyrrole
One kind in pyridine；
（2）The preparation of molecular engram shell：Template molecule, function monomer, crosslinking agent and initiator are added to containing polyphenyl second
Polymerisation is carried out in the reaction dissolvent of alkene microballoon kernel 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 adenosine monophosphate（AMP）, Huo Zheshuan
Adenosine phosphate（ADP）Or atriphos（ATP）In one kind；
（3）Template molecule removes：Using eluant, eluent by step（2）Shell in template molecule remove, microsphere surface point
The imprinted cavity of template molecule is left in sub- trace thin layer.
2. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described in claim 1
Method, it is characterised in that：Step（1）The preparation method of polystyrene microsphere be：
（1）Polymerized monomer, crosslinking agent, chain-transferring agent and initiator dissolving after, be uniformly mixed reaction system is made, at room temperature
After magnetic agitation 10min, nitrogen or argon gas 5-60min are persistently vacuumized and are passed through；
（2）By step（1）Reaction system continue to be placed on magnetic stirring apparatus and persistently stir, 3 ~ 12h is reacted at 60-70 DEG C,
Prepare polystyrene microsphere；
（3）Step is filtered by vacuum using organic phase filter membrane（2）Polystyrene microsphere, remove solvent, then will be white on filter membrane
Color solid is transferred in the container equipped with methanol, and magnetic agitation elutes 30 ~ 60min, repeats elution four times, is finally collected on filter membrane
The white powder dried.
3. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described in claim 1
Method, it is characterised in that：Step（2）The preparation method of molecular engram shell be：
（1）After template molecule, function monomer, crosslinking agent, initiator dissolving, it is uniformly mixed with polystyrene microsphere and reactant is made
System, at room temperature after magnetic agitation 10min, persistently vacuumizes and is passed through nitrogen or argon gas 5-60min；
（2）By step（1）Reaction system continue to be placed on magnetic stirring apparatus and persistently stir, 3 ~ 12h is reacted at 60-70 DEG C,
Prepare adenosine molecule imprinted polymer.
4. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described in claim 1
Method, it is characterised in that：Step（3）Template molecule remove method be：The gland prepared is filtered by vacuum using organic phase filter membrane
Glycosides molecularly imprinted polymer removes solvent, and then the white solid on filter membrane is transferred to equipped with methanol and acetate mixture
In container, magnetic agitation elutes 30min, repeats elution 5 times, removes template molecule, last again with methanol elution is primary, then receives
The white solid dry on collection filter membrane, methanol in the methanol, acetate mixture, acetic acid volume ratio be 9:1.
5. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as claimed in claim 2
Method, it is characterised in that：Polymerized monomer, the crosslinking agent molar ratio of the preparation addition of polystyrene microsphere kernel are 1:3 ~ 5, polymerization is single
The percentage by volume 1 ~ 5% of body, crosslinking agent in the reaction system；The amount of initiator be polymerized monomer and crosslinking agent total amount 0.5 ~
2%, the molar ratio 1 of initiator and chain-transferring agent:3 ~ 9, the total amount of chain-transferring agent and initiator is polymerized monomer and crosslinking agent total amount
3 ~ 10%.
6. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as claimed in claim 3
Method, it is characterised in that：Function monomer, crosslinking agent molar ratio be 1:3 ~ 5, the body of function monomer, crosslinking agent in the reaction system
Product percentage 1 ~ 5%, the amount of initiator are the 0.3 ~ 2% of function monomer and crosslinking agent total amount, and template molecule and function monomer rub
That ratio 1:1 ~ 4,50 ~ 200mg of dosage of polystyrene microsphere kernel.
7. a kind of preparation side of the molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as described in claim 1
Method, it is characterised in that：The function monomer is acrylamide and its derivative, any one of acrylic acid and its derivative；
The crosslinking agent is ethylene glycol dimethacrylate, tetramethylene dimethacrylate, sour three hydroxyls of trimethacrylate
Methyl propyl ester, N, any one of N'- methylene-bisacrylamides or N, N'- vinyl bisacrylamide；
The initiator is azo or peroxide radical initiator；
The solvent is:Acetonitrile, dimethyl sulfoxide, methanol or any one of methanol and aqueous mixtures.
8. a kind of molecule of recognizable ADP- ribosylation albumen prepared by the preparation method as described in claim any one of 1-7
Imprinted polymer microballoon, it is characterised in that：Polyalcohol microspherulite diameter is distributed 100nm-5 μm.
9. a kind of molecular blotting polymer microsphere of recognizable ADP- ribosylation albumen as claimed in claim 8, feature
It is：Application of the polymer microballoon in terms of separation, enrichment ADP- ribosylation albumen.
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| 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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| 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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| "Synthesis and evaluation of multiply templated molecularly imprinted polyaniline";K.Sreenivasan;《J.Mater.Sci》;20071231;第42卷;7575-7578 * |
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