CN107937402A - A kind of specific recognition glutathione aptamer GSH1 and its derivative - Google Patents

A kind of specific recognition glutathione aptamer GSH1 and its derivative Download PDF

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CN107937402A
CN107937402A CN201711180508.4A CN201711180508A CN107937402A CN 107937402 A CN107937402 A CN 107937402A CN 201711180508 A CN201711180508 A CN 201711180508A CN 107937402 A CN107937402 A CN 107937402A
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aptamer
glutathione
gsh1
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王颖皓
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Abstract

The present invention discloses specific recognition glutathione aptamer GSH1 and its derivative.The nucleotides sequence of glutathione aptamer is classified as：Ggtggaggca agagatcggc cgaggttttc, are named as GSH1.Glutathione aptamer preparation method includes：Random Sequence Library is built, screens target substance nucleic acid complexes, separates purpose oligonucleotide aptamers, PCR amplification purpose oligonucleotide aptamers, cycle sieve selects high degree of specificity aptamer, the modification of glutathione aptamer.The aptamer prepared is non-toxic, and molecular weight is small, is readily synthesized and marks, a specific recognition glutathione, other special-shaped cysteines or its analog nonrecognition are combined, and can become the molecular beacon of detection glutathione content.

Description

A kind of specific recognition glutathione aptamer GSH1 and its derivative
Technical field
The invention belongs to amino acid detection techniques field, is related to a kind of nucleic acid, affine more particularly to high specific and height Glutathione aptamer of power and preparation method thereof.
Background technology
Glutathione (glutathione, r-glutamyl cysteingl+glycine, GSH) is that one kind contains γ-acyl The tripeptides of amine key and sulfydryl, is made of glutamic acid, cysteine and glycine.It is present in each cell of almost body.Paddy The sweet peptide of Guang can assist in keeping the function of normal immune system, and with antioxidation and integrate detoxication, cysteine On sulfydryl be its active group（Therefore often it is abbreviated as G-SH）, easily and some drugs（Such as paracetamol）, toxin（As free radical, The heavy metal such as iodoacetic acid, mustard gas, lead, mercury, arsenic）Deng combination, and with integration detoxication.Glutathione detoxifies with wide spectrum Effect, cannot be only used for medicine, more can be as the base-material of functional food, in the work(such as anti-aging, strengthen immunity, antitumor Can property food extensive use.
The detection method of currently used glutathione has three major types：Chromatography, Immunological Method, Enzymatic cycling.Chromatography High sensitivity, specificity are good, but sample treatment, separation condition, chromatographic column prepare all multi-Varis, make it be difficult to standardize；And Hplc device is expensive, technical conditions require height, needs special maintenance personnel, makes its Difficulty.Immunological Method needs also The free GSH forms of original, antibody fluorescence analytic approach and turbidimetry cannot directly detect sequestered glutathione, it is total can only to detect blood plasma Glutathione, is educated in 37 DEG C of half an hour ovum with reducing agent and carries out reduction treatment to blood sample.Immunological Method needs more than one hour ability Go out as a result, complex for operation step, since it is desired that carrying out reduction treatment can be influenced by some uncertain factors.Enzymatic cycling process It is cumbersome, and test limit is low, it is larger to produce error, it is expensive, therefore cannot promote.
Aptamer is a kind of new identification molecule that developed recently gets up, and is subject to the extensive pass of scientist in recent years Note, largely is screened out for the aptamer of important physiological activity molecule；The various analysis sides based on aptamer Method and technology have been reported；Aptamer medicine " Macugen " has also been listed in 2005 by FDA official approvals.SELEX skills The oligonucleotide sequence that art is screened is referred to as aptamer, and domestic its, which is translated as aptamer, nucleic acid aptamer, nucleic acid, to be known Body or aptamer etc..SELEX technologies refer to the random oligonucleotide of applied chemistry method synthesis large capacity (by consolidating for both ends Sequencing arranges and the random sequence of centre composition) library, by applying selection pressure, (with reference to target, elutriation and target are highly The process of specific bond fragment), and Amplification Technologies are combined, the circulation selection through excessively taking turns is enriched with, acquisition and target substance The oligonucleotide molecules that high special combines, can be that RNA can also be DNA, length is generally 25 ~ 60 nucleotide.
From the foregoing, it will be observed that aptamer is combined presented hypersensitivity and high specific with target substance, it is set to be examined in disease Have a good application prospect in disconnected, although clinical practice report ripe at present is less, application aptamer detection amino acid Research be but on the increase, the new detecting technique based on aptamer also continuously emerges.But it is currently based on being directed to for aptamer The efficient specific recognition research of glutathione also lacks very much, and is directed to aptamer and its screening preparation side of glutathione Method is there is not yet report.
The content of the invention
The technical problem to be solved in the present invention is existing glutathione detection technique is overcome the shortcomings of, fill up and yet there are no paddy The aptamer and its screening preparation method blank of the sweet peptide of Guang, there is provided a kind of glutathione aptamer and preparation method thereof, this The there is provided aptamer name GSH1 of invention.
The solution of the present invention is realized in this way：A kind of specific recognition glutathione aptamer GSH1, it is described Its sequence of the nucleotide sequence of aptamer is：ggtggaggca agagatcggc cgaggttttc.
The derivative of specific recognition glutathione aptamer GSH1 described above, the derivative include following four In any one：
（1）Base A, T, C or G on aptamer optional position described in claim 1 is replaced as rare bases to methylate After purine, dihydrouracil or hypoxanthine, obtained nucleic acid aptamer derivative；
（2）The phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 derives；
（3）The peptide nucleic acid that aptamer described in claim 1 is transformed into；
（4）The lock nucleic acid that aptamer described in claim 1 is transformed into.
More than one described specific recognition glutathione aptamer GSH1 or derivatives thereof are in identification, detection gluathione Application in terms of peptide, or the kit of preparation detection glutathione.
Abundant in order to disclose the present invention, the preparation method step of glutathione aptamer of the present invention is as follows：
The preparation method of glutathione aptamer comprises the following steps：
1）Synthesizing single-stranded DNA random sequence oligonucleotides storehouse：The both ends in the single stranded DNA random oligonucleotide storehouse are fixed sequence Row, as PCR amplification primer binding zone, the middle random sequence for 60 bases, storage capacity 1015More than.The PCR primer For：
Primer 1：5’-ATACCAGCTTATTCAATT-3’
Primer 2：5’-Biotin-AGATTGCACTTACTATCT-3’
The PCR primer 2 is with 5 ' end biotin labelings.
2）Prepare the solid-phase matrix of connection glutathione：Using micro- magnetic bead as matrix, glutathione is led to by chemical method Its carboxyl is crossed to be covalently attached on micro- magnetic bead.
3）First screening purpose oligonucleotide sequence：DNA random oligonucleotides storehouse is mixed with glutathione, screening removes Go in DNA random oligonucleotides storehouse not combining the oligonucleotide sequence with non-specific binding glutathione, recycle specific bond paddy The nucleotide sequence of the sweet peptide of Guang.
4）Prepare secondary single strand dna oligonucleotide storehouse：By gained in step 3 and the few nucleosides of glutathione specific bond Acid sequence carries out PCR amplification, and pcr amplification product is separated as matrix using the micro- magnetic bead of Streptavidin, unwind by alkaline denaturation, Filtering, purifying, obtains secondary DNA oligonucleotide libraries, is screened for next round.
5）Screen and identify glutathione aptamer：Secondary single strand dna oligonucleotide storehouse obtained by step 4 is carried out Next round is screened, and target oligonucleotide sequence is obtained after 15 wheel screenings.Clone and the target oligonucleotide sequence is sequenced, pass through Enzyme-linked aptamer adsorption measurement identifies its specificity and affinity for being combined with glutathione.
6）The glutathione aptamer can be used as detection reagent to be used to detect glutathione.
Beneficial effects of the present invention are as follows：（1）The Nucleic acid aptamer molecules amount screened is small, non-toxic, is conducive to molecule The design of probe, is readily synthesized and marks；（2）Aptamer only special identification glutathione, do not combine non-glutathione and Other amino acid moleculars, the ability with reference to glutathione are 20 ~ 80 times of the ability with reference to non-gluathione Peptide C y, can become mirror The reagent of other glutathione, improves specificity and the sensitivity of detection method, simplification detection method, reduces cost.
Brief description of the drawings
Fig. 1 is the binding ability of aptamer of the present invention and glutathione, and abscissa is aptamer dna concentration in Fig. 1, Ordinate is dissociation constant (Kd) relative value, and GSH curves represent the binding curve of aptamer GSH1 and glutathione, Cy in figure Curve represents aptamer GSH1 and non-glutathione（Cysteine Cy）Binding curve.
Embodiment
Implement the preparation of the aptamer GSH1 of 1 glutathione specific bond
Build random sequence oligonucleotides storehouse：Artificial synthesized single-stranded DNA sequence, structure storage capacity are 1 × 105Single stranded DNA with Machine sequence oligonucleotides storehouse, the DNA sequence dna that single stranded DNA random oligonucleotide storehouse includes are：
5’-GGATCCACCAGCGTCATCAGCA-N25~40-AGATAGTAAGTGCAATCTGGC-3’
The DNA sequence dna in the single stranded DNA random oligonucleotide storehouse includes middle random sequence N25~60With both ends fixed sequence program, institute State middle random sequence N25~40For the random sequence of 25 ~ 40 bases, the both ends fixed sequence program is：5’- GGATCCACCAGCGTCATCAGCA, 3 '-CGGTCTAACGTGAATGATAGA, the both ends fixed sequence program are PCR amplification primer Land
1）By 0.5ml (1x109Particulate) Invitrogen companies with activation amino micro- magnetic bead and 1 mol/L paddy Guangs Sweet peptide is in coupling buffer（20mM potassium phosphate buffer solutions, 0.15M NaCl, 1mM DTT, pH 5.5）Middle mixing, adds 200ul coupling agent solutions [57% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC)], By above-mentioned reaction be placed under the conditions of 25oC it is light it is mixed 24 it is small when.Lotus root is connected to the magnetic bead magnetic bead separating device and cleaning solution of glutathione （PBS, 1mM DTT, crowd H7.3）After cleaning, it is resuspended in 0.5ml PBS.
2）5nmol single-stranded DNA banks are dissolved in combination buffer（100 mM NaCl, 20 mM Tris-HCl pH 7.6, 2 mM MgCl2, 5mM KCl, 1 mM CaCl2,0.02% Tween 20, 1 mM DTT）, heated： 90 oC heat 10min, are placed in 10min on ice, and then temperature places 5min.
3）By the single-stranded DNA banks handled well and be combined with cysteine micro- magnetic bead be incubated after, collection do not combine magnetic bead Liquid.
4）By step 3）The liquid of collection and 25ul steps 1）Obtained glutathione-magnetic bead is together in combination buffer 37oC incubates 30min.
5）The magnetic bead after incubating is washed with combination buffer, then adds 200ul elution buffers（20 mM Tris-HCl pH7.6, 200 mM NaCl, 10 mM EDTA）, after 92oC is incubated 5min, elution of the recycling with single strand oligonucleotide acid sequence Buffer solution, carries out PCR reactions.
6）PCR response procedures are：5 min of 94oC pre-degenerations;94oC 30s, 47oC 1min, 72oC 1min, amplification 20 circulations；Final extension is 72oC 10min.
7）PCR product is 5 ' double-stranded DNAs of the end with biotin labeling, product is mixed with Streptavidin MagneSphere, 25o After C is incubated 30min, double-stranded DNA denaturation is set to be single stranded DNA with 0.15 mol/L NaOH, it is next up to arriving by desalination column purification Take turns the single-stranded DNA banks of screening.
8）Use 200pmol steps 7）Obtained new single-stranded DNA banks, repeat step 2）To step 8）Screening journey Sequence, carries out 15 wheel screenings altogether.Finally clone and the 15th wheel single-stranded DNA banks are sequenced, obtain GSH1 nucleotide sequences：gggaggcatg atggtgtagc cgttacgagc actggtaaca ttggaaccc。
Embodiment 2 detects the binding ability of aptamer GSH1 and glutathione with flow cytometer showed method
1）Glutathione aptamer of the end of synthesis 5 ' with fluorophor FAM marks.
2）Use 0 nml/L, 5 nml/L, 10 nml/L, 20 nml/L, 50 nml/L, 100 nml/L, 200 Micro- magnetic bead of the aptamer connection glutathione (GSH) of the FAM marks of nml/L concentration gradients is fitted to measure glutathione nucleic acid The dissociation constant of body（kd）.With the diluted above-mentioned various concentration nucleic acid aptamers of 200 μ L combination buffers, add 150 nmol/L and connect Micro- magnetic bead of glutathione is connect, 37oC incubates 30min.After washing magnetic bead with combination buffer, 250 μ L combinations are resuspended in In buffer solution.The oligonucleotide fragment of random sequence and micro- magnetic bead of connection cysteine (Cy) is set to be used as control.
3）Fluoremetry is carried out to microballon using the flow cytometer of BD companies, is then mapped with Sigma plot softwares, Calculate screened aptamer and the dissociation constant of glutathione interaction（kd）Relative value, the results are shown in Figure 1.
Sequence table
<110>Wang Yinghao
<120>A kind of specific recognition glutathione aptamer GSH1 and its derivative
<130> 2017
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence ()
<400> 1
ggtggaggca agagatcggc cgaggttttc 30

Claims (3)

  1. A kind of 1. specific recognition glutathione aptamer GSH1, it is characterised in that the nucleotide sequence of the aptamer Its sequence is：
    ggtggaggca agagatcggc cgaggttttc。
  2. 2. the derivative of any specific recognition glutathione aptamer GSH1, the derivative in a kind of claim 1 Thing includes any one in following four：
    （1）Base A, T, C or G on aptamer optional position described in claim 1 is replaced as rare bases to methylate After purine, dihydrouracil or hypoxanthine, obtained nucleic acid aptamer derivative；
    （2）The phosphorothioate backbone that the skeleton of the nucleotide sequence of aptamer described in claim 1 derives；
    （3）The peptide nucleic acid that aptamer described in claim 1 is transformed into；
    （4）The lock nucleic acid that aptamer described in claim 1 is transformed into.
  3. 3. specific recognition glutathione aptamer GSH1 described in a kind of claim 1 or 2 or derivatives thereof is in identification, inspection Survey the application in terms of glutathione, or the kit of preparation detection glutathione.
CN201711180508.4A 2017-11-23 2017-11-23 A kind of specific recognition glutathione aptamer GSH1 and its derivative Withdrawn CN107937402A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021177657A1 (en) * 2020-03-03 2021-09-10 주식회사 넥스모스 Dna aptamer specifically binding to glutathione and use thereof

Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101537189A (en) * 2009-04-28 2009-09-23 谭蔚泓 Aptamer and new use of derivative thereof
CN104561008A (en) * 2014-11-30 2015-04-29 陈燕婷 Homocysteine aptamer HCy1 and preparation method thereof
CN106480042A (en) * 2016-10-17 2017-03-08 柳州立洁科技有限公司 A kind of quick identification insulin aptamer INS2 and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101537189A (en) * 2009-04-28 2009-09-23 谭蔚泓 Aptamer and new use of derivative thereof
CN104561008A (en) * 2014-11-30 2015-04-29 陈燕婷 Homocysteine aptamer HCy1 and preparation method thereof
CN106480042A (en) * 2016-10-17 2017-03-08 柳州立洁科技有限公司 A kind of quick identification insulin aptamer INS2 and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JYOTI BALA等: "In vitro selected RNA aptamer recognizing glutathione induces ROS-mediated apoptosis in the human breast cancer cell line MCF 7", 《RNA BIOLOGY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021177657A1 (en) * 2020-03-03 2021-09-10 주식회사 넥스모스 Dna aptamer specifically binding to glutathione and use thereof
JP2023517008A (en) * 2020-03-03 2023-04-21 ネクスモス カンパニー リミテッド DNA aptamer that specifically binds to glutathione and use thereof

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Application publication date: 20180420