CN104156739B - Electronic tag affinity ligand library and purposes - Google Patents
Electronic tag affinity ligand library and purposes Download PDFInfo
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- CN104156739B CN104156739B CN201310178037.9A CN201310178037A CN104156739B CN 104156739 B CN104156739 B CN 104156739B CN 201310178037 A CN201310178037 A CN 201310178037A CN 104156739 B CN104156739 B CN 104156739B
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- electronic tag
- affinity ligand
- library
- ligand
- tag affinity
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- IJJCUITVBLOBCS-UHFFFAOYSA-N NC1=CC=CC(N(C2=CC=CC=C2)S)=C1N Chemical compound NC1=CC=CC(N(C2=CC=CC=C2)S)=C1N IJJCUITVBLOBCS-UHFFFAOYSA-N 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- BHHGXPLMPWCGHP-UHFFFAOYSA-N Phenethylamine Chemical compound NCCC1=CC=CC=C1 BHHGXPLMPWCGHP-UHFFFAOYSA-N 0.000 description 1
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- 239000012507 Sephadex™ Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- DZGWFCGJZKJUFP-UHFFFAOYSA-N Tyramine Natural products NCCC1=CC=C(O)C=C1 DZGWFCGJZKJUFP-UHFFFAOYSA-N 0.000 description 1
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- 230000004308 accommodation Effects 0.000 description 1
- BGLGAKMTYHWWKW-UHFFFAOYSA-N acridine yellow Chemical compound [H+].[Cl-].CC1=C(N)C=C2N=C(C=C(C(C)=C3)N)C3=CC2=C1 BGLGAKMTYHWWKW-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000007825 activation reagent Substances 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 229960001441 aminoacridine Drugs 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 229960000212 aminophenazone Drugs 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- PRQROPMIIGLWRP-BZSNNMDCSA-N chemotactic peptide Chemical compound CSCC[C@H](NC=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PRQROPMIIGLWRP-BZSNNMDCSA-N 0.000 description 1
- HBGGBCVEFUPUNY-UHFFFAOYSA-N cyclododecanamine Chemical compound NC1CCCCCCCCCCC1 HBGGBCVEFUPUNY-UHFFFAOYSA-N 0.000 description 1
- SSJXIUAHEKJCMH-UHFFFAOYSA-N cyclohexane-1,2-diamine Chemical compound NC1CCCCC1N SSJXIUAHEKJCMH-UHFFFAOYSA-N 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910000071 diazene Inorganic materials 0.000 description 1
- 229940043279 diisopropylamine Drugs 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical class C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 description 1
- ZZTCPWRAHWXWCH-UHFFFAOYSA-N diphenylmethanediamine Chemical compound C=1C=CC=CC=1C(N)(N)C1=CC=CC=C1 ZZTCPWRAHWXWCH-UHFFFAOYSA-N 0.000 description 1
- JRBPAEWTRLWTQC-UHFFFAOYSA-N dodecylamine Chemical compound CCCCCCCCCCCCN JRBPAEWTRLWTQC-UHFFFAOYSA-N 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 229940031098 ethanolamine Drugs 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ZWJINEZUASEZBH-UHFFFAOYSA-N fenamic acid Chemical compound OC(=O)C1=CC=CC=C1NC1=CC=CC=C1 ZWJINEZUASEZBH-UHFFFAOYSA-N 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007306 functionalization reaction Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229960004452 methionine Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N n-propyl alcohol Natural products CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- LRTFPLFDLJYEKT-UHFFFAOYSA-N para-isopropylaniline Chemical compound CC(C)C1=CC=C(N)C=C1 LRTFPLFDLJYEKT-UHFFFAOYSA-N 0.000 description 1
- DPBLXKKOBLCELK-UHFFFAOYSA-N pentan-1-amine Chemical compound CCCCCN DPBLXKKOBLCELK-UHFFFAOYSA-N 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- CBPYOHALYYGNOE-UHFFFAOYSA-M potassium;3,5-dinitrobenzoate Chemical compound [K+].[O-]C(=O)C1=CC([N+]([O-])=O)=CC([N+]([O-])=O)=C1 CBPYOHALYYGNOE-UHFFFAOYSA-M 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- HDOUGSFASVGDCS-UHFFFAOYSA-N pyridin-3-ylmethanamine Chemical compound NCC1=CC=CN=C1 HDOUGSFASVGDCS-UHFFFAOYSA-N 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- VHNQIURBCCNWDN-UHFFFAOYSA-N pyridine-2,6-diamine Chemical compound NC1=CC=CC(N)=N1 VHNQIURBCCNWDN-UHFFFAOYSA-N 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
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- 239000013049 sediment Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 229960002898 threonine Drugs 0.000 description 1
- PZJJKWKADRNWSW-UHFFFAOYSA-N trimethoxysilicon Chemical compound CO[Si](OC)OC PZJJKWKADRNWSW-UHFFFAOYSA-N 0.000 description 1
- 229960003732 tyramine Drugs 0.000 description 1
- 229960004441 tyrosine Drugs 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of electronic tag affinity ligand library, construction method and purposes；Provide synthetic technology on Tag Packaging material of electronic tag technology, ligand based on ligand structure information, target biomolecule label, unmarked screening technique, the affinity ligand for quick, special high flux screening target biomolecule；Electronic tag ligand library of the invention can be used for researching and developing the Non-covalent binding durative action preparation auxiliary material of the affinitive material of protein purification, disease target spot inhibitor and drug for the affinity ligand of target biomolecule screening.
Description
Technical field
The present invention relates to the affinity ligands of biomedicine field, and in particular to a kind of electronic tag affinity ligand library, building
Method and the purposes in biological medicine.
Background technique
The combination of drug molecule and large biological molecule (such as protein) be with good biological availability, metabolic stability and
The premise of hypotoxicity.Searching out can be with the ligand molecular of bio-target molecule high affinity and selective binding (commonly referred to as
Affinity ligand, lead compound, lead compound) it is the important starting of new drug development, and to prepare bio-target molecule affine
The basis of purifies and separates material.In order to rapidly research and develop target molecule affinity ligand, many sides have been developed over nearly more than 20 years
Method, including the MOLECULE DESIGN based on structure and with bio-target molecule screening compounds library, such as U.S. Patent application
US20090240033A1 " Affinity Matrix Library and Its Use " is disclosed in agarose, silica gel, fiber
Element, glass, Toyopearl, Hydroxyethymethacrylate, Polyacrylamide, Hyper D,
The affinity ligand library synthesized on Stryrenedivinylbenzene Perfluorocarbons medium.Screening affine match
Body molecule is to need every kind of medium filling chromatographic column respectively, and with sample, pillar carries out affinity chromatography operation, assessment purifying effect one by one
Fruit；The sample size for screening affinity ligand needs is more, and operating process is cumbersome, the time is long, manpower and material resources demand is big.Therefore, it is necessary to build
Stand that easy to operate, the time is short, speed is fast, high-throughput high for the affinity ligand library of bio-target molecule and screening system.
Summary of the invention
The electronic tag that the present invention is stored and read using electronic chip as affinity ligand structures information, in wrapping and encapsulating
Affinity ligand library is synthesized on material, carries out sieve library using target biomolecule (marking and unmarked), and selecting can be with target molecule knot
The affinity ligand of conjunction reads affinity ligand molecular structure information in chip, forms a kind of quick, high-throughput affinity ligand screening system
System.Electronic tag is encapsulated by encapsulation, such as glass tube, is carried out functionalization derivative, activation to encapsulating material surface group, is directly used
Covalent coupling ligand molecule is chemically reacted, and/or parent is synthesized by the molecule of the skeleton coupled stepwise different kinds of molecules of more active sites
With ligand library.It is quickly screened in screening stage using natural or gene engineering expression albumen label and/or unmarked screening
To the affinity ligand that can combine target biomolecule.Screening process is easy to operate, quick.
Therefore, it is an object of the present invention to construct an electronic tag affinity ligand library, the storage of affinity ligand information
In the electronic tag where ligand；
It is another object of the present invention to establish the method that electronic tag affinity ligand is quickly screened with biomolecule, target
Biomolecule (marks and/or unmarked) sample and affinity ligand library to be incubated for, and reads the target biology on each affinity ligand label
Molecular signal chooses the highest affinity ligand label of signal, with reader direct reading ligand structure information, to realize fast
The special affinity ligand screening of speed, high-throughput target biomolecule.
Another object of the present invention is to screen the affinity ligand of native protein or gene engineering expression albumen, is developed affine
Purifies and separates material and purifying process.
In the present invention, the affinity ligand library is small point of the glass-encapsulated pipe surface chemical coupling in built-in electronic tag
Sub- ligand compound, ligand compound contain multiple functions group, constitute a variety of special space conformations, can specifically bind it is a variety of
Albumen, comprising: using the electronic tag glass tube with reading and writing function as matrix of packages, and function is carried out to glass pipe surface
Change processing, and coupling and the small molecule of fabricated in situ various structures match based compound, constitute affinity ligand library, can be used for high pass
It measures, the protein screening of scale.
High-throughput, the quick screening and the preparation of affinitive material of heretofore described target protein affinity ligand, separation with
The foundation of purification process, incubation, washing, positive label including label and/or unmarked target protein and affinity ligand library are chosen
Choosing, the reading of ligand structure information and the zeolite regeneration of label；
Heretofore described screening technique label or unmarked native protein or gene engineering expression protein screening,
Application in isolation and purification is incubated for the electronic tag glass tube in a small amount of target protein sterling and affinity ligand library, and use is glimmering
Optical signal or other non-marked signals select adsorption target protein positive signal electron label glass tube, read the structure of affinity ligand
Information；
The affinity chromatography method of heretofore described target protein is that the affinity ligand information obtained according to screening is chromatographing
Corresponding affinity ligand is coupled and/or synthesized on medium, quick screening and isolation and purification for albumen.
This bionical affinity ligand of small molecule is more more stable than bio-molecules property, and specificity and repeatability are more preferable, especially
Be for an advantage outstanding: can disposably synthesize the ligand library of a large amount of small molecule compound, for protein screening, point
From with purifying.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis of the bionical affinitive material antibody purification of embodiment three
Fig. 2 is the SDS-PAGE electrophoresis of the bionical affinitive material purifying protein A of embodiment three
1,100 × 100 electronics of table (label) glass tube smaller ligand number and attribute column (table)
The present invention is achieved by the following technical solutions, specifically comprises the following steps:
Step 1: the building in electronic tag affinity ligand library: including by a certain number of electronic tag glass tubes and activation
Reagent is placed under low temperature environment and stirs 1-12 hours, reaction solution pH5-10 is adjusted with acid or alkaline reagent in reaction process, with having
Solvent or water clean electronic tag glass tube 2-20 times, reject unbonded activating reagent, finally soak electronic tag glass tube
The aqueous solution of the ethyl alcohol or acetone in 10%-30% is steeped, cryo-conservation is stand-by.
The behaviour that the electronic tag of the read-write two-dimensional numerals of reaction kinetic is write and read by the rule of experimental design
Make, makes each electronic tag glass tube that there is corresponding independent number；According to the size in quasi- building ligand library, by certain amount
Electronic tag glass tube be divided in the container with (body) reaction solution of certain concentration.It is fixed in vertical rotating baking oven, adjusts
Saving temperature is 20-55 DEG C, revolving speed 5-100rpm, is reacted 5-48 hours, and the Ph value of reaction solution is adjusted with acid or alkaline solution
To pH5-9.To use ethyl alcohol, acetone after reaction, DMF (N ' dinethylformamide) or water equal solvent wash electronic tag glass
Glass pipe 5-20 times removes unbonded free small molecule aglucon.
It has been write that, read and be coupled the electronic tag glass tube of first small molecule aglucon, it only need to can reading and writing to its second
The number of code area carries out read and write operation.
By a certain number of electronics marks for being coupled first small molecule aglucon and having completed second small molecule aglucon number
It is divided in the container for including certain density specific ligand solution after label glass grouping, is fixed in vertical rotating baking oven, adjusts
Oven temperature is saved to 45-98 (85-95) DEG C, revolving speed 10-200rpm reacts 24-72 hours, 4-8 hours every in reaction process
The pH value of reaction solution is measured, and is adjusted to pH5-10 with acid or alkaline solution；To be cooled to room temperature after reaction.With ethyl alcohol,
Acetone, DMF (N ' dinethylformamide) or other organic solvents or water washing electronic tag glass tube 5-20 times, removing are not tied
The free small molecule aglucon closed.Finally by electronic tag glass tube be soaked in the ethyl alcohol of 5-20%, N ' N-dimethylformamide or
In the aqueous solution of acetone, cryo-conservation is stand-by.
Step 2: in the present invention, high-throughput, the quick screening of the protein, the foundation of isolation and purification method；It
Range including target protein is that label, the native protein of non-marked or gene engineering expression albumen and coupling have different ligands electronics
Label glass tube is incubated for 0.1-6h at 4-70 DEG C, is rinsed with the buffer that the ion concentration of pH3-12 is 0.005-2M unbonded
Albumen, choose strongest 10 labels of protein signal, read ligand structure information.It measures on label
Specific embodiment
Below with reference to embodiment, the present invention is further elaborated, and following implementation only describes this hair by way of example
It is bright.It is obvious that those of ordinary skill in the art can carry out various flexible and repair in the scope of the present invention and essence to the present invention
Change.It is to be understood that this invention is intended to cover the accommodation and modification that include in the dependent claims.
Embodiment one:
The building in high-throughput bionical affinity ligand library
On electronic label chip can reading and writing code area by the bar code writable area of 10 digit and the item of 4 digits
Code writable area composition.The bar code writable area of first 10 digit in this ligand library records, and writes the operation with reading with frequency read/write.
Coding and information write-in rule: the 0th bit digital in first 10 digit reading and writing code area indicates building batch the 1st time of ligand library
The write-in 1 of the building information in smaller ligand library.Amino-compound is successively numbered according to from No. 001 starting to No. 100, as
When first functional group is coupled to electronic tag, last 6-9 in the first bar code writable area are written in compound code；As
When being coupled to electronic tag for second functional group, 1-3, the 0th of the second bar code writable area are written in compound code
Write-in 1.
By 20000 electronic tag glass tubes (1.5mm × 7mm), the 1st write-in 1 represents the 1st batch and synthesizes biradical group
Ligand.Be fitted into the tapered plastic bottle of 2L, be added 500ml dry toluene and 7.5mmol3- TSL 8330 and
The tetraethyl orthosilicate reagent of 92.5mmol, 50 DEG C are stirred to react for 24 hours, the cooling dereaction liquid that inclines, with countless ethanol washings 10 times,
It is saved backup with 20% ethyl alcohol.
Compound concentration is the trichloride and triazine (C of 0.5mg/ml3N3Cl3)-acetone soln 1000ml is placed in the pre-cooling of -20 refrigerators
After 1 hour, 10000 electronic tag glass tubes to be activated are fitted into the plastic bottle of the solution containing cold acetone, 4 DEG C of blenders are placed in
It is upper to react after five minutes, by the trichloride and triazine (C of prepared 0.5mg/ml3N3Cl3)-acetone soln is added in plastic bottle, makes
Trichloride and triazine (C3N3Cl3) final concentration of 0.1mg/ml, stirring measures solution ph after twenty minutes, and constantly with saturation Na2CO3
Solution adjusts trichloride and triazine (C3N3Cl3)-acetone soln pH value is pH7-8, and continuation is stirred to react 4.5 hours on blender
Afterwards, it is washed with distilled water electronic tag glass tube 10 times, obtains in 4 DEG C of refrigerators of electronic tag (glass tube) of activation and save for use.
Amino-compound is successively numbered according to from No. 001 starting to No. 100；100 electronic tags are taken, 1001 write-ins
2-4 of the first bar code of electronic tag writable area are then charged into the high-temperature resistance plastice bottle of 100ml, are added prepared No. 1 small
Molecule amino-compound solution, sealing, which is fixed on, to be stirred to react in case, is stirred to react at 50 DEG C 24 hours, tests reaction process
In, first 8 hours or so, the pH value of every 2 hours measurement primary first-order equation liquid, with saturation Na2CO3Solution (reaction solution that water is solvent)
Maintain pH7-10.To successively be washed with acetone, DMF (the amino-containing small molecule aglucon for being dissolved in organic solvent) or water after reaction
Wash electronic tag glass tube 5-15 times.Every bottle of electronic tag glass tube individually washs in washing process, avoids mixing；4 DEG C of preservations
For use；Successively remaining amino-compound is numbered, each batch electronic tag is written, synthesis corresponding construction write-in electronic tag is matched
Body, respective compound in synthesis obtain the electronic tag word bank of all compounds couplings.
One is respectively taken from the electronic tag character library for being coupled first compound, totally 100 electronic tags, 001
5-7 of the first bar code of electronic tag writable area are written, are then charged into the high-temperature resistance plastice bottle of 100ml, are added prepared 1
Number small molecule amino-compound solution, sealing, which is fixed on, to be stirred to react in case, is stirred to react at 95 DEG C 24 hours, and experiment was reacted
Cheng Zhong, first 8 hours or so, the pH value of every 2 hours measurement primary first-order equation liquid, with saturation Na2CO3(water is the reaction of solvent to solution
Liquid) maintain pH7-10.To successively wash electronic tag glass tube 5-15 times with N ' dinethylformamide and water after reaction.
Every bottle of electronic tag glass tube individually washs in washing process, avoids mixing；4 DEG C save for use；Continue from being coupled first
One is respectively taken in the electronic tag character library of a compound, totally 100 electronic tags, successively remaining amino-compound is numbered and is written
Each batch electronic tag synthesizes respective compound, electronic tag ligand library 1.
Embodiment two:
The building in high-throughput bionical affinity ligand library
By 300 electronic tag glass tubes (1.5mm × 7mm), the 1st write-in 2 represents the single group of the 2nd batch synthesis and matches
Body.It is fitted into the tapered plastic bottle of 500mL, 200ml dry toluene and 7.5mmol2,3- epoxypropyl trimethoxy silicon is added
The tetraethyl orthosilicate reagent of alkane and 92.5mmol, 50 DEG C are stirred to react for 24 hours, the cooling dereaction liquid that inclines, with countless ethanol washings
It 10 times, is saved backup with 20% ethyl alcohol.
3 electronic tags are taken every time, and 2-4 write-ins 001 of the first bar code writable area are then charged into the high temperature resistant of 50ml
Prepared No. 1 small molecule amino-compound solution is added in plastic bottle, and sealing, which is fixed on, to be stirred to react in case, stirs at 60 DEG C
Reaction 24 hours is tested in reaction process, first 8 hours or so, the pH value of every 2 hours measurement primary first-order equation liquid, with saturation Na2CO3
Solution (reaction solution that water is solvent) maintains pH7-10.To successively wash electronic tag glass tube with DMF and water after reaction
5-15 times.Every bottle of electronic tag glass tube individually washs in washing process, avoids mixing；4 DEG C save for use；Successively by remaining ammonia
Based compound number write-in each batch electronic tag, synthesizes the compound of corresponding construction, obtains the electronics mark of all compound couplings
Ligand library 2.
Embodiment three:
Application of the electronic tag affinity ligand library in protein purification
By the total 11.8mg of human serum immunoglobulin IgG (5ml, 2.36mg/ml) PBS (0.01M PB+0.15M
NaCl, pH7.4) to be diluted to concentration be 1.2mg/ml to buffer, it stirs 5~10 minutes, takes on the magnetic stirring apparatus in 4 DEG C of refrigerators
FITC (fluorescein isothiocynate) 0.12mg is marked with antibody to be marked to react: the FITC fluorescein weighed is slowly added to
It in antibody after dilution, is all added in 10 minutes, stirs 15h, 12000g is centrifuged 10min, removes a small amount of sediment, will mark
The antibody-solutions of note are fitted into bag filter, with 0.01M PBS (0.01M PB+0.15M NaCl, pH7.4), are dialysed at 4 DEG C
Night is finally removed by filtration free fluorescein with SepHadex G-25 desalting column, collects the fluorescence antibody of label.
It is 3mg/ml that human plasma protein fraction 0.01M PBS (0.15M NaCl, pH7.4), which is diluted to concentration, and corresponding dense
The albumen of the FITC label of degree is according to marked IgG: human plasma=1: the mixture that 9 ratio is prepared；
200 electronic tag glass tubes are taken out from electronics standard configuration body library 1, are placed in 100ml plastic bottle, are added 30ml's
The human plasma protein fraction for having prepared the IgG containing FITC label, under the conditions of 4 DEG C, slight concussion is incubated for 30min, then takes out IgG sample
Product add 30ml 0.01M PBS (0.01M PB+0.15M NaCl, pH7.4) equilibration buffer slightly to shake at room temperature
10min takes out equilibration buffer, repeats this operation 10 times.Finally electronic tag glass tube is placed in fluorescence ELISA Plate, through protecting
After fresh film package, be placed in FLA-5100 multifunction scanner carry out fluorescence imaging scanning (exciting light 473nm absorbs light 532nm,
Resolution ratio is 100um), fluorescent value scanning and data processing and the analysis of electronic tag glass tube.
The high electronic tag glass tube of fluorescent value is picked out, selecting the salt ionic concentration of pH10 and pH3 respectively is 1-
(fliud flushing) concussion washing 20min is delayed in the washing of 1.5M, repeats this operation twice, the electronic tag glass after collecting washing twice respectively
Glass pipe carries out second-order fluorescence scanning, rejects the high electronic tag glass tube of fluorescent value, retains the lower electronic tag glass of fluorescent value
Pipe, and the aglucon information of electronic tag glass tube is read, it is (1000000079,1039), (1000000079,1074),
(1000000027,1049), corresponding construction are respectively undecylamine and 4- (2 aminoethyl)-benzsulfamide, Serine and 4- (2 ammonia
Ethyl)-benzsulfamide, double group ligands of 2,4,6- Triaminopyrimidines and ethylenediamine composition.Distinguish in subsequent synthesis verifying
Labeled as bionical affinity ligand 7, bionical affinity ligand 11 and bionical affinity ligand 14.
Sepharose 6B trichloride and triazine activates (operation reference " affinity chromatography introduction " (Lip river (C.R.Lowe) work；Liu
Yu Xiuyi Science Press, May nineteen eighty-three the 1st edition), 3 parts of each 50ml are measured, three nitrogen piperazine molal quantitys is estimated, weighs 5 times respectively
Amino-compound (R1) 4- (2 aminoethyl)-benzsulfamide of molar excess, 4- (2 aminoethyl)-benzsulfamide, ethylenediamine, dissolution
It in the distilled water, DMF of 100ml amount, is separately added into three nitrogen piperazine activated medias, mixing is stirred to react for 24 hours at 50 DEG C, is reacted
NaHCO is saturated in journey3Solution maintains pH in 7-8, takes out after the reaction was completed, successively with 10 times of volume DMF (N ' N dimethyl formyl
Amine) and water washing medium.
Weigh amino-compound (R2) undecylamine of 5 times of molar excess respectively again, Serine, 2,4,6- triamidos are phonetic
Pyridine is dissolved in the distilled water of 100ml amount, in DMF, is separately added into, has been coupled 4- (2 aminoethyl)-benzsulfamide, 4- (2 ammonia second
Base)-benzsulfamide, three nitrogen piperazine activated medias of ethylenediamine, mixing, 95 DEG C are stirred to react 24 hours, reaction process saturation
NaHCO3Maintain pH between 7.0-8.0, take out after the reaction was completed successively with 10 times of volume DMF (N ' N-dimethylformamide) and
Water washing medium obtains bionical affinity media 7, bionical affinity media 11 and bionical affinity media 14.It is 20% with volume fraction
Ethyl alcohol saves stand-by.
Bionical affinity media 7 is taken respectively, and bionical affinity media 11 and each 2ml of bionical affinity media 14 load 10ml modeling respectively
Expect in chromatographic column, and marked in gravity column outer wall, balances each bionical affinity chromatography with 10ml PB (10mM, pH7.4) buffer
Column.By 10 times of dilution human plasma protein fraction samples of 0.01M PBS buffer solution, 2ml is taken to be loaded to each affinity column, with 20ml0.01M's
PB solution equilibria finally elutes adhesion protein with the 50mMGly-HCl (pH3) of 10ml and 0.1N NaOH solution, uses 12%SDS-
The detection of PAG (E) gel electrophoresis is with the bionical affinity separation polymer of analysis to the adsorption effect of IgG antibody in human plasma.Bionical affine material
The purity of specific isolation IgG purification antibody from human serum sample of material 7,11 and 14 is respectively 95%, 91% and 86%.
Example IV:
Albumin A 20mg 20ml PBS (0.01M PB+0.15M NaCl, pH7.4) buffer solution is taken, adds FITC (different
Thiocyanic acid fluorescein) 0.15mg dissolution after mix, 4 DEG C of stirring 15h；Taking-up is fitted into bag filter, with 0.01M PBS (0.01M
PB+0.15M NaCl, pH7.4), in 4 DEG C of dialysed overnights, collect fluorescent marker protein A.
500 electronic tag glass tubes are taken out from electronics standard configuration body library 1, are placed in 200ml plastic bottle, and it is glimmering that 50ml is added
Signal albumin A (0.1mg/ml), under the conditions of 4 DEG C, slight concussion is incubated for 30min, and it is effective to then take out electronic tag glass
0.01M PBS (0.01M PB+0.15M NaCl, pH7.4) buffer washs 10 times.Finally electronic tag glass tube is placed in glimmering
In light ELISA Plate, after preservative film wraps up, it is placed in FLA-5100 multifunction scanner and carries out fluorescence imaging scanning (exciting light
473nm absorbs light 532nm, resolution ratio 100um), fluorescent value scanning and data processing and the analysis of electronic tag glass tube.
The high electronic tag glass tube of fluorescent value is picked out, selecting the salt ionic concentration of pH10 and pH3 respectively is 1-
Washing 20min is swung in the washing bradyseism of 1.5M, repeats this operation twice, collects the electronic tag glass collected after washing twice respectively
Pipe carries out second-order fluorescence scanning, rejects the high electronic tag glass tube of fluorescent value, retains the lower electronic tag glass tube of fluorescent value,
And the aglucon information of electronic tag glass tube is read, it is (1000000002,0000), (1000000031,0000),
(1000000065,0000), corresponding construction are respectively n-octyl amine and histamine list group ligand.It is marked respectively in subsequent synthesis verifying
It is denoted as bionical affinity ligand CL3 and bionical affinity ligand CL31.
Sepharose 6B trichloride and triazine activates (operation reference " affinity chromatography introduction " (Lip river (C.R.Lowe) work；Liu
Yu Xiuyi Science Press, May nineteen eighty-three the 1st edition), 3 parts of each 50ml are measured, three nitrogen piperazine molal quantitys is estimated, weighs 5 times respectively
Amino-compound (R1) n-octyl amine of molar excess, histamine and carbaniloyl hydrazine, are dissolved in 100mlDMF, are separately added into three
In nitrogen piperazine activated media, mixing is stirred to react for 24 hours at 50 DEG C, and NaHCO is saturated in reaction process3Solution maintains pH in 7-8, reaction
It takes out after the completion successively with 10 times of volume DMF (N ' N-dimethylformamide) and water washing medium.Bionical affinity media CL3 and
Bionical affinity media CL31 is saved stand-by with the ethyl alcohol that volume fraction is 20%.
Bionical affinity media CL3 and bionical each 2ml of affinity media CL31 is taken to be loaded in 10ml plastics chromatographic column respectively respectively,
And marked in gravity column outer wall, each bionical affinity column is balanced with 10ml PB (10mM, pH7.4) buffer.Take 2ml large intestine
The albumin A sample (according to Chinese patent application: 201110087262.2 preparations) of bacillus fermentation recombinant expression is loaded to each affine
Column is finally inhaled with the 50mMGly-HCl (pH3) of 10ml and the elution of 0.1N NaOH solution with the PB solution equilibria of 20ml 0.01M
Attached albumen detects the effect with the bionical affinity separation polymer purifying protein A of analysis with 12%SDS-PAG (E) gel electrophoresis.Bionical parent
Purity with medium CL3 and bionical affinity media CL31 purifying protein A is respectively 96%, 97%.
Table 1
| Coding 1 | Coding 2 | Chinese |
| 1000000001 | 1001 | Para aminoacetophenone |
| 1000000002 | 1002 | N-octyl amine |
| 1000000003 | 1003 | One water hydrochloride of 9-aminoacridine |
| 1000000004 | 1004 | 1- amino anthraquinones |
| 1000000005 | 1005 | Two positive ethamine |
| 1000000006 | 1006 | 2- amino-Isosorbide-5-Nitrae-dicarboxylic acids (2 amino terephthalic acid (TPA)) |
| 1000000007 | 1007 | P-aminobenzoic acid |
| 1000000008 | 1008 | Hexylamine |
| 1000000009 | 1009 | N- (3- aminopropyl) imidazoles |
| 1000000010 | 1010 | 1- amino 1- hydrochloric acid |
| 1000000011 | 1011 | 2- aminobenzimidazole |
| 1000000012 | 1012 | Tyramine hydrochloride |
| 1000000013 | 1013 | Metaphenylene diamine hydrochloride |
| 1000000014 | 1014 | Adenine |
| 1000000015 | 1015 | Acridine yellow (nitrogen uh Huang) |
| 1000000016 | 1016 | Meta-aminophenol |
| 1000000017 | 1017 | Aminoguanidine hydrochloride glucose |
| 1000000018 | 1018 | Hydroxysuccinimide |
| 1000000019 | 1019 | L-cysteine |
| 1000000020 | 1020 | L-tyrosine |
| 1000000021 | 1021 | Trimethylamine |
| 1000000022 | 1022 | L-threonine |
| 1000000023 | 1023 | L- asparagine |
| 1000000024 | 1024 | L-methionine |
| 1000000025 | 1025 | L- aspartic acid |
| 1000000026 | 1026 | Diaminobenzoic acid |
| 1000000027 | 1027 | 2,4,6- Triaminopyrimidines |
| 1000000028 | 1028 | 2,6- diamino-anthraquinones |
| 1000000029 | 1029 | 1,5- diamino-anthraquinone |
| 1000000030 | 1030 | 6- Amino-n-hexanoic acid |
| 1000000031 | 1031 | Maxamine |
| 1000000032 | 1032 | Phenyl ethylamine |
| 1000000033 | 1033 | Diamino benzothiazole |
| 1000000034 | 1034 | 4,4 '-diaminodiphenyl ethers |
| 1000000035 | 1035 | 1,2- cyclohexanediamine |
| 1000000036 | 1036 | P-aminobenzene sulfonic acid |
| 1000000037 | 1037 | Ortho-nitraniline |
| 1000000038 | 1038 | 2- amino terephthalic acid (TPA) |
| 1000000039 | 1039 | Undecylamine |
| 1000000040 | 1040 | 4-ASA sodium |
| 1000000041 | 1041 | Cyclohexylamine |
| 1000000042 | 1042 | Tricyclic sunflower |
| 1000000043 | 1043 | Allylamine |
| 1000000044 | 1044 | 1- aspartic acid |
| 1000000045 | XX045 | P-phenylenediamine |
| 1000000046 | 1046 | O-phenylenediamine |
| 1000000047 | 1047 | Thiamine hydrochloride |
| 1000000048 | 1048 | 3- pyridyl-methanamine |
| 1000000049 | 1049 | Ethylenediamine |
| 1000000050 | 1050 | Valine |
| 1000000051 | 1051 | L-lysine |
| 1000000052 | 1052 | Diphenylamines |
| 1000000053 | 1053 | Pidolidone mono-sodium salt |
| 1000000054 | 1054 | N- phenylanthranilic acid |
| 1000000055 | 1055 | Cycloheptyl imines |
| 1000000056 | 1056 | 4- aminobenzophenone |
| 1000000057 | 1057 | 5- amino -2,2,4- trimethyl -1- cyclopenta methylamine |
| 1000000058 | 1058 | Diaminodiphenylmethane |
| 1000000059 | 1059 | Aminopyrine |
| 1000000060 | 1060 | N, N- diethyl Isosorbide-5-Nitrae phenylenediamine |
| 1000000061 | 1061 | 4- aminobenzaldehyde |
| 1000000062 | 1062 | 2- naphthylamines -1- sulfonic acid Tobias acid (tobias acid) |
| 1000000063 | 1063 | 1- amino anthraquinones |
| 1000000064 | 1064 | 4,4 diamino-diphenylamine sulfide |
| 1000000065 | 1065 | Carbaniloyl hydrazine |
| 1000000066 | 1066 | 1-amino-2-naphthol-4-sulfonic acid |
| 1000000067 | 1067 | 3- 5 amido benzotrifluoride |
| 1000000068 | 1068 | 2,6-diaminopyridine |
| 1000000069 | 1069 | N-butylamine |
| 1000000070 | 1070 | Triethylamine |
| 1000000071 | 1071 | Aniline |
| 1000000072 | 1072 | 3- amino, 1- propyl alcohol |
| 1000000073 | 1073 | Chaff amine |
| 1000000074 | 1074 | Serine |
| 1000000075 | 1075 | Ethanol amine |
| 1000000076 | 1076 | 4- isopropyl aniline |
| 1000000077 | 1077 | N ' N diisopropylethylamine |
| 1000000078 | 1078 | P-phenylenediamine |
| 1000000079 | 1079 | 4- (2 aminoethyl)-benzsulfamide |
| 1000000080 | 1080 | Benzylamine |
| 1000000081 | 1081 | Lauryl amine |
| 1000000082 | 1082 | Diethylamine hydrochloride |
| 1000000083 | 1083 | Dibenzylamine |
| 1000000084 | 1084 | Diisopropylamine |
| 1000000085 | 1085 | Di-n-butylamine |
| 1000000086 | 1086 | Di-iso-butylmanice |
| 1000000087 | 1087 | T-octanylamine |
| 1000000088 | 1088 | 3- aminoethyl pyrimidine |
| 1000000089 | 1089 | L-Histidine |
| 1000000090 | 1090 | Cyclo-dodecyl amine |
| 1000000091 | 1091 | Octadecylamine |
| 1000000092 | 1092 | L-arginine |
| 1000000093 | 1093 | 5- amino isophthalic acid |
| 1000000094 | 1094 | L-sodium |
| 100000095 | 1095 | 4- amino -1,8- benzene-naphthalene diimide |
| 100000096 | 1096 | 4- morpholine propylamine |
| 1000000097 | 1097 | Amylamine, |
| 1000000098 | 1098 | 4- amino -1- naphthalenol hydrochloride |
| 1000000099 | 1099 | Paraphenetidine |
| 1000000100 | 1100 | 1- amino -5- naphthols |
Claims (11)
1. a kind of electronic tag affinity ligand library, which is characterized in that the skeleton of the ligand in the ligand library is multi-active base group bone
Frame, and the multi-active base group skeleton is trichloride and triazine skeleton, and the structural information of each ligand is stored in electronics
It is convenient quickly to directly read in label, and for high-throughput, the quick screening of protein, disposable synthesis is a large amount of small
Molecular compound.
2. electronic tag affinity ligand according to claim 1 library, which is characterized in that the electronic tag has information reading
Function is taken, is written and wipes, working method contains to be carried out by radio frequency reading and writing code device.
3. electronic tag affinity ligand according to claim 1 library, which is characterized in that the electronic tag is by resistance to organic molten
The material package of agent.
4. electronic tag affinity ligand according to claim 3 library, which is characterized in that the material of the organic solvent-resistant
Including inorganic material, high molecular material.
5. electronic tag affinity ligand according to claim 4 library, which is characterized in that the inorganic material is glass, institute
Stating high molecular material is polypropylene.
6. electronic tag affinity ligand according to claim 1 library, which is characterized in that construction step includes but is not limited to electricity
The surface-functionalized processing of subtab glass tube, priming reaction, the reading and writing coding of electronic tag glass tube and small molecule aglucon
Coupling reaction.
7. according to electronic tag affinity ligand library described in claim l, which is characterized in that screen the affine of target molecule and match
Body, target molecule fluorescein when screening, enzyme, labelled with radioisotope.
8. according to electronic tag affinity ligand library described in claim l, which is characterized in that screen the affine of target molecule and match
Body, target molecule is the native protein of non-marked or the fusion protein of gene engineering expression when screening.
9. electronic tag affinity ligand according to claim 1 library, which is characterized in that the affinity ligand screened is for sending out
Exhibition target molecule isolates and purifies material.
10. electronic tag affinity ligand according to claim 1 library, which is characterized in that the affinity ligand screened is used for
The inhibitor of R&D target molecule.
11. electronic tag affinity ligand according to claim 1 library, which is characterized in that the affinity ligand screened is used for
The durative action preparation auxiliary material of R&D target molecule.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181720A (en) * | 1995-04-25 | 1998-05-13 | 伊萝莉公司 | Remotely programmable matrices with memories and uses thereof |
| CN101512016A (en) * | 2006-06-30 | 2009-08-19 | 埃姆比特生物科学公司 | Detectable nucleic acid tag |
| CN102719430A (en) * | 2012-06-13 | 2012-10-10 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detecting histidine-tag recombinant proteins and detection method thereof |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181720A (en) * | 1995-04-25 | 1998-05-13 | 伊萝莉公司 | Remotely programmable matrices with memories and uses thereof |
| CN101512016A (en) * | 2006-06-30 | 2009-08-19 | 埃姆比特生物科学公司 | Detectable nucleic acid tag |
| CN102719430A (en) * | 2012-06-13 | 2012-10-10 | 湖南大学 | Nucleic acid aptamer molecular beacon probe for detecting histidine-tag recombinant proteins and detection method thereof |
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