CN104941602A - Protein antigen determinant molecularly imprinted material as well as preparation and application thereof - Google Patents
Protein antigen determinant molecularly imprinted material as well as preparation and application thereof Download PDFInfo
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Abstract
The invention relates to an antigen determinant molecularly imprinted material capable of carrying out selective recognition on a target protein and a preparation process of the antigen determinant molecularly imprinted material. The molecularly imprinted material is prepared by the following steps: by utilizing a section of specific peptide fragment corresponding to the target protein as a template molecule, fixing the specific peptide fragment on a matrix material by employing a histidine tag as a connecting arm; and polymerizing on the matrix material through a functional monomer. The imprinted material is applied to selective recognition of a standard protein.
Description
Technical field
The invention belongs to Polymer materialspreparation technology and the application in protein identification thereof, a kind of novel proteantigen determines base molecular engram material and Synthesis and applications thereof specifically.
Background technology
Molecular imprinting is the technology adopting artificial synthesis preparation target molecule to be carried out to specific recognition material.Relevant report is the earliest the relevant report utilizing non-covalent bond to synthesize theophylline molecularly imprinted polymer that Klaus Mosbach etc. delivers for 1993 on " Nature ", and after this molecular engram material enters rapidly the visual field of global numerous scientist as a kind of Prof. Du Yucang high selectivity material.At present, the trace work for organic molecule and metal ion has achieved huge progress, and experimental technique and evaluation criterion are gradually ripe, and started field (Chen LX, Chemical Society Reviews, 40 such as being applied to clinical analysis, catalyze and synthesize, 2011,2922-2942).In these areas, molecular engram material is compared with natural molecule recognition system, not only has efficient selective, and has very high tolerance to various physics and chemistry influence factor, but also can reuse, compensate for natural identity system many deficiencies in use.Meanwhile, when the research of Small molecular trace is ripe gradually time, the research of molecular engram starts to turn to large biological molecule (especially protein) trace.Dream when this foundes at first to it, " Prof. Du Yucang antibody ", steps again and goes a step further.The protein-imprinted polymer preparation method of current bibliographical information mainly comprises (the Yang KG et.al. such as 3D molecular imprinting method (investment), 2D molecular imprinting method (surface imprinted method), metal-chelating immunoblot method, Langmuir monofilm blotting, surperficial micro-contact immunoblot method, Analytical and Bioanalytical Chemistry, 403,2012,2173-83.).Be usually template molecule with whole protein in these immunoblot method, but due to whole protein template molecule be difficult to obtain, expensive, because which limit the use (Sellergren of protein imprinted material, B.Nature Chemisty, 2,2010,7).
Epitope trace is for template molecule carries out trace with target protein the preceding paragraph specific polypeptide, due to specificity extremely strong between template peptide section and target protein, the imprinted sites formed all has specific recognition capability (Shea to template peptide section and target protein, KJ.Angwandte Chemie, 45,2006,2392-2396).This solves albumen in protein molecule engram and blame acquisition, expensive problem; Meanwhile, polypeptide template conformational stability, adds the specificity of imprinted sites identification.Owing to utilizing free peptide section extremely low for template utilization rate during template, therefore need to carry out immobilized to template; And the template process for fixation reported before this has the deficiency that template difficulty is removed, which has limited the application of epitope immunoblot method.
Therefore, we with end modified histidine-tagged epitope peptide section for template, by the chelation between histidine-tagged and metal ion, template is fixed, after being polymerized, utilize the competitive chelation of EDTA-Na to carry out wash-out to template, prepare the epitope molecular engram material that can carry out Selective recognition to target protein.
Summary of the invention
Having adopted histidine-tagged target protein epitope peptide section end modified is template molecule, utilizes metal-chelating effect to secure metal ion Ni by immobilized for template molecule in surface
2+host surface, form molecular engram layer by the polymerization of function monomer, remove template and form epitope imprinted material; And this molecular engram material is used in the identification of standard target albumen.For achieving the above object, the technical solution used in the present invention is:
(1) introduce iminodiacetic acid (IDA) group at host surface by chemical modification, utilize IDA group and Ni
2+chelation, by Ni
2+immobilized at host surface.
(2) utilize solid-phase synthetic peptide legal system for end modified histidine-tagged template molecule.
(3) at ambient temperature, in above-mentioned host material, 1:1000-100:1000 (template: host material) adds template peptide section solution in mass ratio, incubated at room temperature (2 minutes-5 hours), make template immobilized in host surface, wherein the mass concentration of host material is 0.2mg/ml-500mg/ml.
(4) in the above-mentioned immobilized host material of template, add function monomer with certain mass ratio subsequently, after pre-assembled certain hour (2 minutes-5 hours), add initator, be polymerized under stirring state; Wherein radical type monomer initator azo two isobutyl (AIBN) as corresponding in methacrylic acid (MAA), N, N'-methylene-bisacrylamide (MBA), hydroxyethyl methacrylate (HEMA) etc., ammonium persulfate, dibenzoyl peroxide etc.; The corresponding acidity of the monomers such as dopamine, aniline, silane or alkaline matter are as initator.
(5) after being polymerized, repeatedly use the EDTA-Na aqueous cleaning polymeric material of 20mM-1M, make template molecule elution wherein out, until can not detect template molecule in eluent, and then obtain epitope molecular engram material (MIP).
(6) not immobilized template molecule on host material, prepares non-trace microballoon (NIP) by above-mentioned identical step.
(7) this molecular engram material is used in the Selective recognition of target protein.
Tool of the present invention has the following advantages:
(1) the present invention has utilized histidine-tagged epitope peptide section end modified is template, by metal-chelating effect, template peptide section is fixed on finishing Ni
2+host surface (Zhang LH, et.al., Chemical Communication, 47,2011,3969-3971).This method is simple, and efficiently, ensure that the immobilized efficiency of all peptide sections is consistent, without discrimination time immobilized while of different peptide section is a kind of general template solid support method simultaneously.
(2) the present invention adopts artificial synthesis peptide's section to be template, instead of protein molecule, solves albumen in protein molecule engram and blames acquisition, expensive problem; Meanwhile, polypeptide template conformational stability, adds the specificity of imprinted sites identification.
(3) the present invention can adopt multiple different function monomer to carry out polymerization and form imprinted sites.The utilization of several functions monomer can high degree multiple abundant interaction is provided, ensure the imprinted sites that the formed specific recognition to target peptide section and target protein.
(4) the present invention adopts EDTA-Na solution to carry out wash-out to template, and the method does not relate to the use of strong acid, highly basic, Strong oxdiative reducing condition, and mild condition can not destroy imprinted sites, can further improve the specificity of the identification of imprinted sites.
(5) host material preparation process of the present invention, the immobilized process of template, polymerization process and template removal processes all have versatility, high degree ensure that the method determines versatility in the preparation of base imprinted material and universality at different target proteantigen.
Accompanying drawing explanation
Fig. 1. the transmission electron microscope picture of human serum albumins epitope imprinted material (MIP) and non-imprinted material (NIP);
Fig. 2 (a). human serum albumins epitope imprinted material (MIP) and non-imprinted material (NIP) are to the adsorption capacity figure of human serum albumins epitope;
Fig. 2 (b). human serum albumins epitope imprinted material (MIP) and non-imprinted material (NIP) are to the adsorption capacity figure of human serum albumins;
Fig. 3. human serum albumins epitope imprinted material (MIP) and non-imprinted material (NIP) are to the Selective recognition of human serum albumins (HSA), bovine serum albumin(BSA) (BSA), cytochrome c (Cyc), and ordinate is imprinting factor;
Fig. 4 .MIP2, NIP2 are to the adsorption capacity figure of human serum albumins epitope;
Fig. 5 .MIP3, NIP3 are to the adsorption capacity figure of human serum albumins epitope;
Fig. 6 .MIP4, NIP4 are to the adsorption capacity figure of human serum albumins;
Fig. 7 .MIP5, NIP5 are to the adsorption capacity figure of human serum albumins.
Embodiment 1
(1) preparation of human serum albumins epitope trace micro-sphere material
Solvent-thermal method preparation is adopted to be coated with the magnetic nano-particle of silicon layer as host material.Get host material 50mg, add 1ml0.5mg/ml human serum albumins template peptide section (AASQAALGLHHHHHH) (the biochemical Shanghai Co., Ltd of gill) solution, hatch at 25 DEG C and make template be fixed on stromal surface (mass ratio of template and host material is 10:1000).After rinsing loose template peptide section, resuspended matrix is in 25ml water, add 15mg dopamine (monomer and substrate material quality are than being 0.3:1), pre-assembled 30min under 30 DEG C of mechanical agitation, add 0.5ml1M tris-HCl buffer solution (pH8.0), auto polymerization 5h under mechanical agitation, the unpolymerized dopamine monomer of the auxiliary lower cleaning of magnet.Subsequently, repeatedly clean magnetic-particle with 200mM EDTA-Na, make Ni
2+elute, and then make chelating template peptide section thereon be able to wash-out, until high performance liquid chromatography (the high-new chromaster of Hitachi, 10*4.6mm, C18 post) examine the template molecule do not measured in eluent, vacuum drying, obtains albumin antigen and determines base molecular engram material (MIP).
By identical method, but do not add template molecule, prepare non-imprinted material (NIP).
As Fig. 1, MIP, NIP size evenly, good dispersion, be 200-300nm, this provides very large specific area for particle, and then has higher adsorption capacity.Meanwhile, the about thick 4nm of MIP and NIP outer surface trace shell, it reduces the resistance to mass tranfer of imprinted sites, enables target protein and peptide section enter recognition site at short notice, reaches adsorption equilibrium.
(2) albumin antigen determines that the recognition performance of base imprinted material to epitope peptide section and target protein thereof is investigated
Albumin antigen determines that sill is identified in 25 DEG C to human serum albumins epitope peptide section, carries out in aqueous phase.In experiment, the molecular engram material of the above-mentioned preparation of 0.4mg or non-imprinted material are respectively at 0.3ml, the albumin antigen of 0.05mg/ml determines to hatch 3h in base, go to hatch supernatant, high performance liquid chromatography (the high-new chromaster of Hitachi, 10*4.6mm, C18 post) peak area quantification method detects the concentration of wherein epitope, and then calculates adsorption capacity.As Fig. 2 (a), to albumin antigen, MIP determines that the recognition factor of base can reach 4.3, can reach 3.11mg/g to the adsorption capacity of template molecule, the adsorption capacity of contrast MIP and NIP to epitope calculates the template utilization rate of imprinted material, template utilization rate nearly 100%.Illustrate that the imprinted material surface by this legal system is standby has complete recognition site, good identification can be carried out to epitope peptide section.
Albumin antigen determines that sill is identified in 25 DEG C to human serum albumins, carries out in aqueous phase.In experiment, 0.4mg molecular engram material or non-imprinted material respectively with 0.3ml, the albumin antigen of 0.3mg/ml determines that base hatches 3h, get and hatch supernatant, high performance liquid chromatography (the high-new chromaster of Hitachi, 10*4.6mm, C8 post) peak area quantification method detection wherein albuminous concentration.Recognition factor as Fig. 2 (b), MIP human serum albumins is 2.2, can reach 51.7mg/g to albuminous adsorption capacity, and this illustrates that the imprinted sites that epitope is formed also can have obvious recognition effect to target protein.Meanwhile, contrast imprinted material and the adsorption capacity of non-imprinted material calculate and identify that the template utilization rate in protein of interest is 15%, and by the template utilization rate of the material identification albumen of the method gained of non-covalent fixed form less than 2%; This explanation, surface imprinted site is defined at host surface by surface orientation fixed form, by controlling shell thickness, these imprinted sites fully being exposed, making site to greatest extent for the identification of target protein, having fully demonstrated the advantage of covalency directional at-tachment template.
(3) albumin antigen determines selective to target protein of base imprinted material
The molecular engram material of the above-mentioned preparation of 0.4mg or non-imprinted material respectively with 0.3ml, the human serum albumins (HSA) of 0.5mg/ml, bovine serum albumin(BSA) (BSA), cytochrome c (Cyc) hatch 3h, get and hatch supernatant, high performance liquid chromatography (the high-new chromaster of Hitachi, 10*4.6mm, C8 post) peak area quantification method detects the concentration of wherein each albumen.As shown in Figure 3, the migration index of material to HSA, BSA, Cyc is respectively 1.57,1.45,1.11, the strongest to the recognition capability of target protein HSA.It is worth mentioning that, relative to BSA, material can better identify HSA, and this is be difficult to realize for traditional intact proteins, has fully demonstrated the identification advantage of epitope molecular engram material compared with protein molecule engram material.
(4) embodiment 2
More immobilized than carrying out template for 40:1000 by template and substrate material quality, other can obtain MIP2 and NIP2 with embodiment 1, and it to the adsorption capacity of human serum albumins epitope as shown in Figure 4.
(5) embodiment 3
More immobilized than carrying out template for 2.5:1000 by template and substrate material quality, other can obtain MIP3 and NIP3 with embodiment 1, and it to the adsorption capacity of human serum albumins epitope as shown in Figure 5.
(6) embodiment 4
Be polymerized than for 0.5:1 adds function monomer with substrate material quality by function monomer, other can obtain MIP4 and NIP4 with embodiment 1, and it to the adsorption capacity of human serum albumins as shown in Figure 6.
(7) embodiment 5
Be polymerized than for 0.2:1 adds function monomer with substrate material quality by function monomer, other can obtain MIP5 and NIP5 with embodiment 1, and it to the adsorption capacity of human serum albumins as shown in Figure 7.
(8) embodiment 6
With methacrylic acid (MAA), methylene-bisacrylamide (MBA) for monomer, the electrostatic interaction of MAA, MBA and amino silicon ball surface is utilized double bond to be modified in silicon matrix particle surface, with N-(4-vinyl)-benzyl imido grpup diacetic acid (VBIDA) for linking arm is at matrix granule finishing metal ion Ni
2+, be fixed having modified histidine-tagged template peptide section by method described in embodiment 1, respectively with MAA, MBA for function monomer, azodiisobutyronitrile (AIBN) is initator, by heat cause be polymerized; Utilize 200mM EDTA-Na to carry out template removal afterwards, obtain MIP6 and NIP6 respectively.
Claims (8)
1. proteantigen determines a base molecular engram material, it is characterized in that:
Utilize and histidine-taggedly for linking arm, template is fixed on host material, be polymerized by function monomer, then eluted template molecule, form imprinted sites, prepare proteantigen and determine base imprinted material;
Described template is the epitope peptide section of target protein;
Described histidine-tagged be one section of continuous print histidine section.
2. proteantigen as claimed in claim 1 determines base molecular engram material, it is characterized in that:
Described template molecule is the epitope peptide section of target protein, is a continuous print 2-40 amino acid sequence on target protein sequence, or naturally occurring epitope peptide section sequence on protein;
Described histidine-tagged be 2-15 continuous print histidine, the method for being synthesized by chemiluminescent polypeptide modifies c-terminus in template peptide section or aminoterminal;
Described target protein is all proteins that nature exists.
3. proteantigen as claimed in claim 1 determines base molecular engram material, it is characterized in that:
Described host material is that surface is by metal-chelating mode supported metal ion Ni
2+silicon matrix material or matrix material; Function monomer refers to, polymerizable forms the monomer of polymer.
4. a preparation method for epitope molecular engram material described in claim 1,2 or 3, is characterized in that: end modified histidine-tagged template is fixed on finishing Ni by metal-chelating effect
2+host surface, form polymeric layer by the polymerisation of function monomer at host surface; Utilize the competitive wash-out Ni of disodium ethylene diamine tetraacetate (EDTA-Na) solution subsequently
2+, thus make the template molecule be fixed thereon be able to wash-out, form imprinted sites, obtain molecular engram material.
5., according to preparation method according to claim 4, it is characterized in that:
Template fixing method: by end modified histidine-tagged template by template: the mass ratio 1:1000-100:1000 of host material adds in the suspension of host material, incubated at room temperature.
6., according to preparation method according to claim 4, it is characterized in that:
Function monomer is polymerized: in the host material suspension having added by function monomer template immobilized, after pre-assembled, add initator, be polymerized under stirring condition.
7., according to preparation method according to claim 6, it is characterized in that:
Described EDTA-Na solution refers to that concentration is the EDTA-Na aqueous solution of 20mM-1M.
8. the epitope imprinted material described in a claim 1,2 or 3 is used for the Selective recognition of target protein epitope peptide section or the target protein corresponding to it.
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| CN109721759A (en) * | 2017-10-30 | 2019-05-07 | 中国科学院大连化学物理研究所 | A kind of acetylglucosamine imprinted material and its identification for acetylglucosamine and acetylglucosamine modification peptide fragment |
| CN109897144A (en) * | 2019-02-28 | 2019-06-18 | 南开大学 | The protein molecule engram polymer and its preparation method and application of polypeptide crosslinking |
| JPWO2022064960A1 (en) * | 2020-09-23 | 2022-03-31 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109721759A (en) * | 2017-10-30 | 2019-05-07 | 中国科学院大连化学物理研究所 | A kind of acetylglucosamine imprinted material and its identification for acetylglucosamine and acetylglucosamine modification peptide fragment |
| CN109721759B (en) * | 2017-10-30 | 2021-08-03 | 中国科学院大连化学物理研究所 | Acetyl glucosamine imprinted material and application thereof in recognition of acetyl glucosamine and acetyl glucosamine modified peptide fragment |
| CN109897144A (en) * | 2019-02-28 | 2019-06-18 | 南开大学 | The protein molecule engram polymer and its preparation method and application of polypeptide crosslinking |
| JPWO2022064960A1 (en) * | 2020-09-23 | 2022-03-31 | ||
| WO2022064960A1 (en) * | 2020-09-23 | 2022-03-31 | 国立大学法人神戸大学 | Base material for manufacturing sensor for analyzing detection object, sensor for analyzing detection object, and method for analyzing detection object |
| JP7216460B2 (en) | 2020-09-23 | 2023-02-01 | 国立大学法人神戸大学 | Base material for producing sensor for analysis of detection target, sensor for analysis of detection target, and analysis method for detection target |
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Application publication date: 20150930 |