CN103820458A - Group of aptamers for specific recognition of beta-bungatotoxin and use thereof - Google Patents
Group of aptamers for specific recognition of beta-bungatotoxin and use thereof Download PDFInfo
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Abstract
The invention provides a group of aptamers for specific recognition of beta-bungatotoxin and use thereof. The aptamers provided by the invention are any single-chain deoxyribonucleic acid (DNA) sequence shown in SEQ ID NO.1-4 in a sequence table. The aptamers and the beta-bungatotoxin have high affinity and specificity, and have broad application prospects in detection of the beta-bungatotoxin and diagnosis of bungarus multicintus bite.
Description
Technical field
The present invention relates to biotoxin technical field, specifically refer to that the SELEX technology (the Fas lignand system evolution technology of index concentration) of utilizing in Protocols in Molecular Biology prepares one group of aptamer of being combined with β-bungatotoxin high specific, for this aptamer provides scientific basis and theoretical basis in the detection of β-bungatotoxin and the diagnosis of Induced By Coral Snake Bite.
Background technology
β-bungatotoxin derives from coral snake (Bungarus multicinctus) snake venom, is the neural poison of a kind of presynaptic polypeptide, shows Ca
2+dependency Phospholipase A2 (Phospholipase A
2, PLA
2) activity, be the topmost toxic component of bungarotoxin, LD50(i.p. mouse) be 0.019 μ g/g.Molecular weight (Mr) is 20500, formed by two chains, i.e. and A chain and B chain, and couple together by a pair of interchain disulfide bond.The A chain (Mr=13500) that molecular weight is larger contains 120 amino acid, has 13 halfcystines, and its primary structure is extremely similar to the PLA identifying from snake venom and mammalian pancreas
2, there is more weak activity.The B chain (Mr=7000) of molecular weight is made up of 60 amino acid, there are some sequence homologies with protein kinase inhibitor, as β-bungatotoxin to particular target the identification subunit to cytolemma, in some the composition interphase interactions in neuromuscular junction presynaptic membrane, have critical function.When between subunit when disulfide bonds, or PLA
2active centre during by covalent modification, all can cause neurotoxicity and the PLA of β-bungatotoxin
2active forfeiture.
Snakebite is the torrid zone, the unhealthful major issue in one of Rural areas, subtropics, is often only in Asia, and the number lethal by snakebite just reaches 50,000 people.Induced By Coral Snake Bite accounts for 8.12% of all kinds of venomous snake bites in China, occupy the 5th, and death toll ranks first.China does not also have one snakebite reagent for clinical diagnosis and method fast and effectively at present, snakebite diagnosis main dependence medical history and clinical manifestation.Owing to being had similar symptom by different types of venomous snake bite, in the situation that medical history is not quite clear, clinician is difficult to judge down from patient's clinical manifestation the end is by which kind of venomous snake bite.
In the laboratory qualification technology of snake venom, immunodetection is topmost technique means wherein.Existing immune diagnostic technique is to utilize polyclonal antibody to detect toxin mostly, between resisting, has cross reaction because different snake venom, and therefore specificity is poor.The monoclonal antibody of preparing single snake venom composition can improve the specificity of detection greatly, but monoclonal antibody itself also exists some defects.A little less than monoclonal antibody is wanted with respect to how anti-avidity, good not to the adsorptivity of enzyme plate, simultaneously to carry out in enzyme or biotin labeled operation stability also poor.
Aptamer be can with the oligonucleotide sequence of target molecule specific binding (RNA or ssDNA).Nineteen ninety, Gold and Ellington Liang Ge research group have almost set up a kind of technology of preparing specific oligonucleotide molecule simultaneously, and this technology is defined as to SELEX (systematic evolution of ligands by exponential enrichment, index concentration formula Fas lignand system is evolved), can prepare there is certain three-dimensional structure, can and target molecule oligonucleotide molecules-aptamers (aptamer) that high specific, high-affinity are combined by the adaptability complementation between three-dimensional conformation (be not base pairing).The appearance of SELEX technology comes from people's going deep into nucleic acid and protein-interacting research.Single-chain nucleic acid can form some stable three-D space structures by the pairing between some complementary base in chain, electrostatic interaction, hydrogen bond action etc., as hair fastener (hairpin), false knot (pseudoknot), bulge loop (stem loop), G-tetrad (G-quartet) etc.By these structures, single-chain nucleic acid can form the stable compound with high specific bonding force with protein or other molecules.Single-chain nucleic acid can also pass through the bonding force of the various interactions such as Van der Waals force, hydrogen bond action, electrostatic interaction and form fit and target molecule generation high specific.The same with antibody, aptamer and target have high-affinity and a specificity in conjunction with same, and therefore, aptamer is also called as " chemical antibody ".
Because aptamer is one section of little single stranded DNA or RNA, there are a lot of natural advantages with respect to antibody.For example, aptamer is easy to synthetic and can applies that conventional chemical process is modified, long-term stability (if the reversible denature and renature of long term maintenance), nontoxicity or immunogenicity.From the angle of engineering science, different from biological antibody production process in body, the difference between the chemosynthesis of external aptamer batch is very little.All of these factors taken together has all promoted aptamer to be widely used in the research in the field such as medical science and life science and the Clinics and Practices of disease.
Summary of the invention
The object of this invention is to provide a kind of can with the aptamer of β-bungatotoxin specific binding.
Another object of the present invention is to provide the application of above-mentioned aptamer in the detection of β-bungatotoxin and Induced By Coral Snake Bite.
The aptamers of described specific binding β-bungatotoxin and fragment thereof, be selected from one or more in the sequence shown in SEQ ID NO.1～4, structurally all meet the constitutional features shown in following general formula, 5 '-AGC AGC ACA GAG GTC AGA TG – Nx-CCT ATG CGT GCT ACC GTG AA-3 ' (general formula I).In general formula, N represents any in base A, G, C, T, and Nx represents that random fragment length is 40,39 or 51 bases.Above-mentioned sequence all exists with single stranded form.
Sequence shown in SEQ ID NO.1～4, can modify or transform, and obtains the derivative of described aptamer.Described modification or reforming mode are selected from one or more in following mode:
1), by described aptamer deletion or increase part Nucleotide, what obtain has the derivative of the aptamer of identical function with described aptamer.
2) described aptamer is vulcanized entirely, end-block or PEG modify, what obtain has the derivative of the aptamer of identical function with described aptamer.
3) described aptamer is connected or other functional group of mark or molecule, such as sulfydryl, amino, radio isotope, fluorescein, vitamin H, toxin or enzyme etc., what obtain has the derivative of the aptamer of identical function with described aptamer.
The screening method of described β-bungatotoxin aptamer is the screening that index concentration Fas lignand system evolution technology (being SELEX screening) is applied to β-bungatotoxin aptamer, and the concrete steps of described SELEX screening comprise:
1) synthesize the random single-stranded DNA banks for screening β-bungatotoxin aptamer, and the synthetic corresponding primer of design;
2) β-bungatotoxin is coated in to 96 hole enzyme plates, and hatches with described random single-stranded DNA banks, the DNA of separation and the combination of β-bungatotoxin;
3) DNA of separation is carried out to pcr amplification, amplified production is cut with lambda exonuclease enzyme, obtains time one-level single stranded DNA storehouse, and for next round SELEX screening, until obtain corresponding aptamer.
In step 1), the two ends of described random single-stranded DNA banks are the fixed sequence program of 20 bases, and centre is the stochastic sequence of 40 bases, that is: 5 '-AGC AGC ACA GAG GTC AGA TG-N40-CCT ATG CGT GCT ACC GTG AA-3 '.
In step 1), described primer is:
Primer 1:5 '-fluorescein-AGC AGC ACA GAG GTC AGA TG-3 ';
Primer 2: 5 '-phosphate group-TTC ACG GTA GCA CGC ATA GG-3 ';
Primer 3:5 '-AGC AGC ACA GAG GTC AGA TG-3 ';
Primer 4:5 '-TTC ACG GTA GCA CGC ATA GG-3 '.
Compare with existing antibody technique, the invention has the advantages that: the aptamer that screening obtains, with respect to traditional antibody, has easier screening conditions, nontoxicity, molecular weight is little, is easy to chemically modified and mark, synthetic cost compared with antibody prepare low, the cycle is short.The binding affinity of aptamer provided by the invention and β-bungatotoxin is high, Kd value reaches nM level, and only specific recognition β-bungatotoxin and the bungarotoxin that contains β-bungatotoxin, to other homology toxin or snake venom, as α-bungatotoxin, Bungarus toxin, agkistrodon acutus venom, viper venom, cobra venom etc. do not have recognition function.Above-mentioned advantage makes described aptamer become the powerful that β-bungatotoxin detects and Induced By Coral Snake Bite is diagnosed.
Accompanying drawing explanation
Fig. 1. the 1st takes turns the pilot PCR of screening.1:20bp DNA ladder; The PCR product of 2-9:12-26 circulation.
The secondary structure of aptamers β B-1, β B-19, β B-32 and the β B-20 of Fig. 2 .Mfold software prediction.
Fig. 3 .SELEX screens each takes turns the combination rate of single stranded DNA storehouse and β-bungatotoxin.
Fig. 4. Media by Fluorescence Anisotropy is measured the dissociation constant Kd value of aptamers β B-1, β B-20 and the combination of β-bungatotoxin.
Fig. 5. nitrocotton membrane filter method is measured the combination rate of aptamers β B-1 and bungarotoxin, Bungarus toxin, ahylysantinfarctase, cobra venom and viper venom.1: bungarotoxin; 2: Bungarus toxin; 3: ahylysantinfarctase; 4: viper venom; 5: cobra venom.
Fig. 6. pass through ELISA method with aptamers β B-1 and identify bungarotoxin and β-bungatotoxin.1. β-bungatotoxin; 2.BSA; 3. casein; 4. α-bungatotoxin; 5. bungarotoxin; 6. cobra venom; 7. Bungarus toxin; 8. ahylysantinfarctase; 9. viper venom; 10. contrast.
Embodiment
Following examples are used for setting forth explanation the present invention, but should not be construed as limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The screening of the aptamer of embodiment 1 specific binding β-bungatotoxin
1. build random single-stranded DNA banks and primer: design two ends are the fixed sequence program of 20 bases, centre is the random single-stranded DNA banks of 40 base stochastic sequences, that is: 5 '-AGC AGC ACA GAG GTC AGA TG-N40-CCT ATG CGT GCT ACC GTG AA-3 '.Wherein N represents any in base A, G, C, T, and N40 represents that random fragment length is 40 bases.Primer 1:5 '-fluorescein-AGC AGC ACA GAG GTC AGA TG-3 ', primer 2: 5 '-TTC ACG GTA GCA CGC ATA GG – phosphate group-3 '.Library and primer are synthetic by Shanghai Sheng Gong Bioisystech Co., Ltd.By binding buffer liquid (20mM HEPES, pH7.4 for random library; 150mM NaCl; 5mM KCl; 2mM MgCl
2; 2mM CaCl
2), primer ddH
2it is for subsequent use that O is mixed with ℃ storage of 100 μ M stock solution-20.
2.SELEX screening: β-carbonate buffer solution for bungatotoxin (CBS, pH9.6) is mixed with to 10 μ g/mL concentration, gets coated 96 orifice plates of 100 μ L, 4 ℃ are spent the night.Outwell supernatant, add 100 μ L binding buffer liquid (containing 1mg/mL BSA, replacing BSA with 1mg/mL casein while sealing from the 5th takes turns) sealing 2h.Outwell supernatant, with 200 μ L binding buffer liquid (containing 0.05%tween20) cleaning 6 times.SsDNA library (being dissolved in binding buffer liquid, containing 0.05%tween20) is placed in PCR instrument, and 90 ℃ of 10min, are placed in rapidly 10min on ice after taking-up, be then placed in room temperature 10min.Get 100 μ L and join in 96 orifice plates, incubated at room 1h.The concentration in ssDNA library reduces to the 10th 0.03 μ M taking turns gradually from the 1st 10 μ M that take turns.Outwell supernatant, with 200 μ L binding buffer liquid (containing 0.05%tween20) cleaning 6 times, then use ddH
2o washes 3 times, finally adds 100 μ L ddH
2o, is placed in 95 ℃, and 10min takes out supernatant elutriant, carries out PCR reaction as template.
Each is taken turns in the process of screening, and the elutriant that first takes a morsel carries out pilot PCR and determines the optimum cycle number increasing.PCR reaction system is: 50 μ L Premix Taq solution(are precious biological, Dalian), 4 μ L elutriants, 1 μ L10 μ M primer 1,1 μ L10 μ M primer 2,44 μ L ddH
2o.PCR reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of sex change 0.5min, 56 ℃ of annealing 0.5min, 72 ℃ are extended 0.5min, 26 circulations; Last 72 ℃ are extended 10min.From the 12nd circulation, from PCR pipe, take out 10 μ L PCR products every 2 circulations.After PCR completes, the product of different cycle numbers is carried out to 3% agarose gel electrophoresis, analytical electrophoresis band, optimum cycle number is the largest loop number that does not produce short chain or long-chain by product.Determine after optimum cycle number, under optimum cycle number, carry out PCR reaction, reaction system and condition are the same, carry out altogether 8 tube reactions, and cumulative volume is 800 μ L.PCR product carries out enzyme with lambda exonuclease to be cut, and the antisense strand of labeled phosphorus acid groups is fallen in hydrolysis, obtains the positive-sense strand of mark fluorescent element.After ethanol deposition and purification, the ssDNA of acquisition is for the SELEX screening of next round.
3. Cloning and sequencing: take turns screening through 10, with the primer PCR amplification elutriant of mark fluorescent element and phosphate group not, PCR product is connected to cloning vector pMD19-T, clones and serves Hai Sheng work Bioisystech Co., Ltd and check order, and obtains 4 aptamers sequences.Result, as shown in SEQ ID NO.1～4, is distinguished called after β B-1, β B-19, β B-32 and β B-20.With Mfold software (
http:// mfold.rit.albany.edu) secondary structure of aptamers is predicted, the secondary structure of 4 aptamers is as shown in Figure 1.Loop-stem structure in every aptamer and hairpin structure may be the combining sites of aptamers and β-bungatotoxin.
(0.22 μ m), with after 0.5MKOH solution soaking 30min, uses ddH to nitrocellulose membrane for HAWP02500nitrocellulose filters, Millipore
2o fully cleans, and then with being placed in binding buffer liquid, shake 40min, is placed in new binding buffer liquid, 4 ℃ of preservations.Fluorescein-labeled each ssDNA of wheel storehouse is mixed with 1 μ M concentration, gets 10 μ L and joins in 1mL binding buffer liquid, is placed in 90 ℃ of 10min, is then placed in 10min on ice, and room temperature is placed 10min.Add 1mg/mL β-bungatotoxin 4 μ L, incubated at room 1h, contrast does not add β-bungatotoxin, and then filtering membrane, with 1mL binding buffer liquid flushing filter membrane.Merging filtrate, by the fluorescence intensity of Perkin Elmer LS55 fluorescent spectrophotometer assay filtrate.Location parameter is: applied sample amount=2mL, excitation wavelength=492nm, emission wavelength=518nm, integration time=10sec, inlet and outlet slit width=10nm.The fluorescence intensity of contrast deducts the fluorescence intensity of sample for being attached to the fluorescence intensity of the ssDNA on β-bungatotoxin.In conjunction with fluorescence intensity and the ratio of the fluorescence intensity contrasting be each and take turns the combination rate of aptamer storehouse and β-bungatotoxin.Result as shown in Figure 2, can see, along with the increase of screening wheel number, the combination rate of aptamers and β-bungatotoxin increases gradually.While wheel to the 10th, combination rate no longer increases, and shows that aptamers is highly enriched, and screening finishes.
The fluorescently-labeled aptamers β B-1 of synthetic, β B-19, β B-32 and β B-20, get respectively 4 μ L aptamers (1 μ M) and join in 2mL binding buffer liquid, measures fluorescence anisotropy value.Then carry out titration with the β-bungatotoxin of different concns, measure the fluorescence anisotropy value under each concentration.Location parameter is with embodiment 2.By the concentration of β-bungatotoxin, as X-coordinate, the increment of fluorescence anisotropy value is as ordinate zou, does non-linear regression with SigmaPlot10.0 software, analyzes dissociation constant (Kd) value of aptamers.
The Kd value of aptamers β B-1 and β B-20, all lower than 100nM, is distinguished 65.9nM and 83.8nM, and measurement result as shown in Figure 3.The Kd value of β B-19 is 540nM, and the Kd value of β B-32 is 1200nM.Aptamers β B-1 and β B-20 and β-bungatotoxin have higher avidity, show its using value in β-bungatotoxin detects.
Bungarotoxin, Bungarus toxin, ahylysantinfarctase, viper venom and cobra venom are mixed with 10mg/mL concentration with binding buffer liquid respectively, and the centrifugal 5min of 12000rpm, gets supernatant.Get the fluorescein-labeled aptamers β B-1 of 1 μ M or β B-206 μ L, join in 1mL binding buffer liquid, be placed in 90 ℃ of 10min, be then placed in 10min on ice, room temperature is placed 10min.Then add respectively various snake venom 2 μ L, incubated at room 1h, contrast does not add snake venom.Filtering membrane, and with 1mL binding buffer liquid rinse filter membrane.Merging filtrate, the fluorescence intensity of mensuration filtrate.Location parameter is with embodiment 2.The fluorescence intensity that the fluorescence intensity of contrast deducts sample is for being attached to the fluorescence intensity of the aptamers on various snake venom, in conjunction with fluorescence intensity and the ratio of the fluorescence intensity contrasting be the combination rate of aptamer and various snake venom.Measurement result as shown in Figure 4.The combination rate of aptamers β B-1 or β B-20 and bungarotoxin is all greater than 80%, and and the combination rate of Bungarus toxin, ahylysantinfarctase, cobra venom and viper venom all lower than 10%, show only specific identification bungarotoxin of aptamers β B-1 and β B-20.The aptamers of that therefore, utilize SELEX technology screening and β-bungatotoxin high-affinity, high specific combination has application prospect very widely in the diagnosis of snakebite.
The aptamers β B-1 of synthetic 5 ' end mark vitamin H.CBS damping fluid for β-bungatotoxin, BSA, casein, α-bungatotoxin, bungarotoxin, cobra venom, Bungarus toxin, ahylysantinfarctase and viper venom (pH9.6) is mixed with 10 μ g/mL separately, get respectively the coated elisa plate of 100 μ L, 4 ℃ are spent the night.Abandon supernatant, elisa plate cleans 3 times with 200 μ L cleaning buffer solutions (binding buffer liquid+0.05%tween20), adds 100 μ L1mg/mL BSA sealings, room temperature, 2h.Abandon supernatant, elisa plate cleans 3 times with 200 μ L cleaning buffer solutions, adds the biotin labeled β B-1(of 100 μ L concentration 10pM, is dissolved in binding buffer liquid), room temperature is in conjunction with 40min.Abandon supernatant, elisa plate cleans 3 times with 200 μ L cleaning buffer solutions, adds the peroxidase labelling avidin (with the dilution of binding buffer liquid) of 100 μ L1:1000 dilutions, room temperature, 40min.Abandon supernatant, elisa plate cleans 3 times with 200 μ L cleaning buffer solutions, adds the freshly prepared substrate OPD of 100 μ L solution, and room temperature lucifuge reaction 15min, finally adds 50 μ L2M H
2sO
4termination reaction, the colour-change of observing every hole.Coated β-the bungatotoxin of contrast, but do not add aptamers.Result as shown in Figure 6.β-bungatotoxin and bungarotoxin hole color are brown, and wherein β-bungatotoxin hole color is darker, and other albumen and snake venom hole color and contrast are basic identical, and obvious colour-change does not occur.Do not need instrument interpretation, only the color in each hole, just can significantly from associated protein, identify β-bungatotoxin, from various snake venom, identify bungarotoxin.
Claims (6)
1. the aptamer of specific binding β-bungatotoxin, it is selected from one or more in the sequence shown in SEQ ID NO.1～4, called after β B-1, β B-19, β B-32 and β B-20 respectively, its sequence is as follows:
βB-1：
agcagcacag aggtcagatg gttttcccct tgtcgctttt ggttcgttct gcctctatct 60
cctatgcgtg ctaccgtgaa 80
βB-19：
agcagcacag aggtcagatg tttggtgtgg atcctgaaca tttatattct ttcgtttttt 60
cctatgcgtg ctaccgtgaa 80
βB-32：
agcagcacag aggtcagatg gcaatgcacc tttgtctctt atagtttatt ttttgccttc 60
ctatgcgtgc taccgtgaa 79
βB-20：
agcagcacag aggtcagatg attagtcatg tttgtttgtc tggctttttg ggtttgtgca 60
gtattatgaa ccctatgcgt gctaccgtga a 91
2. the purposes of aptamers in β-bungatotoxin detects described in claim 1.
3. the purposes of aptamers in snakebite diagnosis described in claim 1.
4. the aptamer of specific binding β-bungatotoxin, comprises aptamers described in claim 1 is deleted or increased part Nucleotide, and has the aptamers of identical function with aptamers described in claim 1.
5. the aptamer of specific binding β-bungatotoxin, comprise the aptamers to aptamers connection or other functional group of mark or molecule described in claim 1, and thering is the aptamers of identical function with aptamers described in claim 1, described other functional group or molecule are selected from: sulfydryl, amino, radio isotope, fluorescein, vitamin H, toxin or enzyme enzyme labelling.
6. aptamers described in claim 1, it can be strengthened its transformation period, improve its stability, prevent that the compound of endonuclease or excision enzyme cutting from modifying.
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Cited By (4)
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| CN105821044A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Aptamer of L-serine and application of aptamer |
| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| CN114047341A (en) * | 2021-11-18 | 2022-02-15 | 云南大学 | Biological probe and biological immunosensor for detecting bungarus venosus and method for detecting bungarus venosus based on proportional signal |
| CN117487813A (en) * | 2023-12-19 | 2024-02-02 | 江南大学 | Single-stranded DNA aptamer sequence for specifically recognizing azithromycin and application thereof |
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2014
- 2014-03-26 CN CN201410116477.6A patent/CN103820458B/en not_active Expired - Fee Related
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105821044A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Aptamer of L-serine and application of aptamer |
| CN105821046A (en) * | 2016-05-24 | 2016-08-03 | 上海敬元投资有限公司 | Specific L-serine aptamer and application thereof |
| CN114047341A (en) * | 2021-11-18 | 2022-02-15 | 云南大学 | Biological probe and biological immunosensor for detecting bungarus venosus and method for detecting bungarus venosus based on proportional signal |
| CN114047341B (en) * | 2021-11-18 | 2022-10-18 | 云南大学 | Biological probe and biological immunosensor for detecting bungarus venosus and method for detecting bungarus venosus based on proportional signal |
| CN117487813A (en) * | 2023-12-19 | 2024-02-02 | 江南大学 | Single-stranded DNA aptamer sequence for specifically recognizing azithromycin and application thereof |
| CN117487813B (en) * | 2023-12-19 | 2024-06-07 | 江南大学 | Single-stranded DNA aptamer sequence for specifically recognizing azithromycin and application thereof |
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