CN105505939A - High-affinity aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4) and application thereof - Google Patents
High-affinity aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4) and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and provides an aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4). The general formula is 5'-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3', wherein N represents any one of bases A, T, C and G, and N40 represents a random sequence with the length of 40bp. The high-affinity aptamer capable of being specifically combined with GTX1 and GTX4 is obtained by screening on the basis of graphene oxide SELEX technology. The single-chain DNA (deoxyribonucleic acid) adapter can be used for separation and enrichment of trace amounts of GTX1 and GRX4 in the sample, analysis detection of GTX1 and GRX4, preparation of neutral or antagonistic GTX1 and GRX4 drugs, and removal of toxins in a water body.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a high-affinity aptamers that is that obtain based on graphene oxide SELEX technology screening and paint ditch Algae toxins 1 and 4 specific binding, for the separation and concentration of trace paint ditch Algae toxins 1 and 4 in sample, the rapid detection of paint ditch Algae toxins 1 and 4, the preparation of neutralization or antagonism paint ditch Algae toxins 1 and 4 medicine, and in water body, the removal of toxin is laid a good foundation.
Background technology
Paralytic shellfish poisoning is one of three big world property marine organisms public hazards.Paint ditch Algae toxins 1 and 4 (Gonyautoxin1andGonyautoxin4, GTX1andGTX4) and analogue paint ditch Algae toxins 2 and 3 (GTX2andGTX3), saxitoxin (Saxitoxin, STX) and N-STX (neoSTX) be the representational neurotoxin of most in paralytic shellfish poisoning (PSP).GTX1 and GTX4 is by food chain in water at fish and shellfish body accumulation, and the mankind will cause paralytic shellfish poisoning after eating the fishery products of these contaminations by mistake.Poisoning symptom mainly comprises limb muscle paralysis, katzeniammer, salivation fever, fash etc.Poisoning serious time, then there will be expiratory dyspnea, throat is nervous; Increase the weight of along with muscular paralysis is constantly expanded, finally cause death.The toxicity of paralytic shellfish poisoning (PSP) is very strong, taking in 1mg just can causing death, high degree of specificity is had to animal nervous system and cardiovascular systems, between 600-5000Mu, (Mu is virulence unit to the scope making people poisoning, 1Mu is the virulence of instigating the small white mouse of 18-22g dead in 15min), lethal dose is 3000-30000Mu.There is the distribution of the paralytic shellfish poisoning (PSP) comprising GTX1 and GTX4 all over the world, especially Europe and some countries of North America.Also people's poisoning of edible poisonous clam class, snail, shellfish initiation is often there is in China's southeastern coast one with area.Because dye has the shellfish of GTX1 and GTX4 seriously to endanger the health of the mankind and the development of global shellfish industry, the detection therefore for marine biotoxins GTX1 and GTX4 seems particularly important.For a long time, the method always detected for GTX1 and GTX4 is the mouse biological method of generally acknowledging in the world.But the sensitivity of this method is low, and cost is high, and need to use a large amount of animals etc.Also the foundation such as high performance liquid chromatography (HPLC) of some other analytical procedure is had, mass spectrum (MS), fluorescence (fluorescence) and various technology couplings etc., particularly LC-MS technology is defined as primary detection method by European Union.But, these methods need expensive plant and instrument, technical professional, detection length consuming time and toxin standard substance be difficult to the problems such as acquisition.Therefore, still in the urgent need to researching and developing the on-the-spot rapid detection of a kind of sensitive, stable and cheap detection system for marine biotoxins GTX1 and GTX4.For a kind of field fast detection method, the part of most critical is molecular recognition probe, as conventional antibody and acceptor etc.
Aptamer is a kind of single stranded DNA or DNA with function, it is the oligonucleotide sequence obtained from random nucleic acid library by in-vitro screening technology SELEX (index concentration Fas lignand system is evolved, SystematicEvolutionofLigandsbyExponentialEnrichment).These aptamers sequences such as, by intermolecular reactive force, hydrogen bond, Van der Waals force, aromatic nucleus accumulation power etc. is folded into the three-dimensional space of stable uniqueness, and with target molecule high-affinity, strong specific combination.For antibody, there is many advantages in aptamers, comprises its stability high, immunogenicity is low, and tissue permeability is strong, and consistence is good, do not need immune animal etc., therefore aptamers is detecting as a kind of novel analysis recognition component, all there is prospect Diagnosis and Treat aspect very much.Aptamers is applied to marine biotoxins detection field by existing scholar in recent years, is combined, have developed many quick, novel detection methods and receive much concern with biosensor platform.
But about the molecular recognition probe with GTX1 and GTX4 specific binding, with the high-affinity aptamers of GTX1 and GTX4 specific binding, and the affinity costant qualification of this aptamers, optimization and application there is no relevant report.
Summary of the invention
The object of the present invention is to provide one with the high-affinity aptamers of paint ditch Algae toxins 1 and 4 specific binding.The second object of the present invention is to provide to be optimized this aptamers by the method for brachymemma and obtains a specific binding GTX1 and the higher aptamers (GO18-T-d) of GTX4 avidity.Another object of the present invention is to provide above-mentioned aptamers in the separation and concentration reagent preparing trace paint ditch Algae toxins 1 and 4 in sample, in preparation paint ditch Algae toxins 1 and 4 detection reagent or test kit, in preparation prevention or treatment paint ditch Algae toxins 1 and 4 poisoning medicine, and removes in water body in preparation and paint the medium many-sided application of ditch Algae toxins 1 and 4 preparation.
Main technical schemes of the present invention is: based on graphene oxide SELEX technology screening obtain one with the high-affinity aptamers of paint ditch Algae toxins 1 and 4 specific binding.This single stranded DNA aptamers can be used for the separation and concentration of trace GTX1 and GTX4 in sample, the analyzing and testing of GTX1 and GTX4, neutralization or the preparation of antagonism paint ditch Algae toxins 1 and 4 medicine, and the removal of toxin in water body.
A first aspect of the present invention, there is provided one with the high-affinity aptamers of paint ditch Algae toxins 1 and 4 (GTX1 and GTX4) specific binding, there is general formula as follows:
5 '-AGCAGCACAGAGGTCAGATG-N
40-CCTATGCGTGCTACCGTGAA-3 '; Wherein, N represents base A, in T, C, G any one, N
40represent the stochastic sequence that length is 40bp.
Preferably, described single stranded DNA aptamers is selected from arbitrary in following sequence:
GO9: as shown in SEQIDNO:2;
GO12: as shown in SEQIDNO:1;
GO17: as shown in SEQIDNO:4;
GO18: as shown in SEQIDNO:5;
GO34: as shown in SEQIDNO:6;
GO37: as shown in SEQIDNO:3.
A second aspect of the present invention is to provide based on above-mentioned single stranded DNA aptamers, and be optimized by the method for brachymemma and obtain a specific binding GTX1 and the higher aptamers of GTX4 avidity, called after GO18-T-d, as shown in SEQIDNO:14.
Described GO18-T-d aptamers, can carry out the chemically modifieds such as vitamin H, fluorescence molecule, isotropic substance, electrochemistry, enzyme or sulfydryl at its 5 ' end or 3 ' end.
A third aspect of the present invention, there is provided the application of above-mentioned aptamers in the separation and concentration reagent preparing GTX1 and GTX4 in sample (GTX1 and GTX4).
Present invention also offers the application of above-mentioned aptamers in preparation paint ditch Algae toxins 1 and 4 detection reagent or test kit.Described reagent or test kit can be used for GTX1 and GTX4 in rapid detection water body or fishery products.
Present invention also offers the application of above-mentioned aptamers in preparation prevention or the poisoning medicine for the treatment of paint ditch Algae toxins 1 and 4.Described medicine is also for the medicine of neutralization paint ditch Algae toxins 1 and 4 or for painting ditch Algae toxins 1 and 4 antagonist etc.Described medicine can be alleviated, cure the symptoms such as poisoning nausea,vomiting,diarrhea, neuromuscular paralysis, the breathing caused of paint ditch Algae toxins 1 and 4 is unable, blood pressure drops, decreased heart rate, irregular pulse.
Present invention also offers above-mentioned aptamers and remove in water body or fishery products the application of painting in ditch Algae toxins 1 and 4 preparation in preparation.Described preparation can remove the paint ditch Algae toxins 1 and 4 in water body or fishery products completely, or reduce the content painting ditch Algae toxins 1 and 4 in water body or fishery products make its reach United Nations's health organization and Food and Argriculture OrganizationFAO's regulation and recommend below the mark.
The present invention in view of GTX1 and GTX4 stability high, the constructional feature of good water solubility, devises based on graphene oxide SELEX technology, and screening obtains the single stranded DNA aptamers with GTX1 and GTX4 high-affinity, strong specific binding.These aptamers as the molecular recognition probe with GTX1 and GTX4 specific binding, have that avidity is high, low, the easy preparation of high specificity, good stability, immunogenicity, the advantage such as modification and mark.Can be used for the separation and concentration of trace GTX1 and GTX4 in sample, the analyzing and testing of GTX1 and GTX4, the preparation of neutralization paint ditch Algae toxins 1 and 4 medicine, the preparation of antagonism paint ditch Algae toxins 1 and 4 medicine, and the removal of toxin in water body.
Therefore, aptamers of the present invention has huge potentiality towards practical application.
Accompanying drawing explanation
Fig. 1 is the schematic diagram screened based on the aptamers of graphene oxide.
Fig. 2 is that each takes turns the combination rate of aptamers and target molecule GTX1 and GTX4 in screening.
Fig. 3 is the secondary structure figure that the truncated sequence of GO18 obtains through mfold software prediction.
Fig. 4 is aptamers GO18-T-d avidity and specificity identification figure.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.
The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The structure of embodiment 1. ssDNA pool and primer thereof
1. build the ssDNA pool that length is 80 Nucleotide
5 '-AGCAGCACAGAGGTCAGATG-N
40-CCTATGCGTGCTACCGTGAA-3 '; Wherein, N represents base A, in T, C, G any one, N
40represent the stochastic sequence that length is 40bp.
2. the structure of primer
Upstream primer: 5 '-AGCAGCACAGAGGTCAGATG-3 ', SEQIDNO:15
Downstream primer 1:5 '-TTCACGGTAGCACGCATAGG-3 ', SEQIDNO:16
Downstream primer 2:5 '-poly (dA20)-Spacer18-TTCACGGTAGCACGCATAGG-3 '
The screening of embodiment 2.GTX1 and GTX4 nucleic acid adaptation
In order to obtain GTX1 and GTX4 high-affinity, the aptamer that high specific combines, the characteristic of non-specific adsorption ssDNA can carry out 8 and has taken turns screening altogether based on graphene oxide, as shown in Figure 1; Introduce reverse screening wherein taking turns from the 6th, namely ssDNA library is first hatched with reverse target (GTX2, GTX3, STX and neoSTX), but adds appropriate graphene oxide and hatch altogether; After centrifugal segregation supernatant, add free target toxin GTX1 and GTX4 and hatch again; Finally, the aptamers with GTX1 and GTX4 specific binding is reclaimed.Each takes turns screening, and the rate of recovery that aptamers is combined with target toxin GTX1 and GTX4 as shown in Figure 2.
Get the ssDNA initial libraries of 1nmol, add 200 μ L binding buffer liquid (20mMTris-HCl, 100mMNaCl, 2mMMgCl2 and 5mMKCl, pH7.5), 95 DEG C of water-bath 10min, the sudden cold 5min of ice bath, then room temperature 25 DEG C places 5min.But being joined in free GTX1 and GTX4 solution, 2h is hatched in room temperature 25 DEG C of rotations.Add 200 μ L again, the graphene oxide solution (U level, mean thickness 0.4nm, layer length 3-5 μm) of 2mg/mL makes the final concentration of graphene oxide solution be 0.4mg/mL, and room temperature 25 DEG C rotates again hatches 2h.Due to graphene oxide can be free in nonspecific adsorbent solution by pi-pi accumulation reactive force and hydrophobic forces ssDNA, and the potential aptamers sequence be combined with GTX1 and GTX4 is deposited in the solution with the form of GTX1 and GTX4 and ssDNA mixture, therefore by repeatedly centrifugal (4 DEG C, 15,000rpm, centrifugal 3 times of 15min), reclaim supernatant and just can obtain the aptamers sequence with GTX1 and GTX4 specific binding.The supernatant of recovery is carried out the quantitative Analysis rate of recovery, and alcohol settling carries out concentrated and purifying.
Add in the library in alcohol settling library as template after 10 μ L sterilized waters, the PCR reaction system of 50 μ L is as follows:
Amplification condition: 94 DEG C, denaturation 1min; 94 DEG C, sex change 30s; 60 DEG C, annealing 30s; 72 DEG C, extend 30s; 72 DEG C, extend 2min; 25 circulations.
The preparation of strand secondary library: add in the library of pcr amplification sample-loading buffer fully mix after refolding strategy process, i.e. 95 DEG C of thermally denature 10min, the sudden cold 5min of ice bath, room temperature places 5min; The sample handled well is joined in 12% urea-denatured polyacrylamide gel loading hole; Connect electrophoresis apparatus, under 200V constant voltage, carry out electrophoresis; When tetrabromophenol sulfonphthalein migrates to the lower end 1/3 of gel, cut off the electricity supply; 20mlddH is added in clean plate
2o and 5 μ LSafeViewDNA dyestuffs, fully place gel wherein after mixing, horizontal shaker shake gently, dyeing 15min; Polyacrylamide gel is placed on imaging on gel imaging system, cuts the simply connected library that glue reclaims lower end; The ddH of 1.5mL is added in the test tube that glue is housed
2o, boiling water boiling glue 30min, centrifugal 5min, reclaim supernatant; Supernatant precipitation concentration is the secondary library of next round screening.
Next round screening is repeated according to screening method above, take turns to the 8th, stop screening, Cloning and sequencing is carried out in the library of enrichment, obtain sequence as table 1, the affinity constant Kd value be combined with target toxin GTX1 and GTX4 by the sequence in microbial film interference technique analytical table 1.
Table 1: sequence table and affinity constant Kd value thereof
The brachymemma optimization of embodiment 3.GO18 aptamers
The sequence of all brachymemmas is as table 2, and after being clipped by the two ends primer of aptamers GO18, measure binding affinity by microbial film interference technique, result shows that GO18-T can not only be combined with target toxin GTX1 and GTX4, and has good avidity.Based on the secondary structure of GO18-T as Fig. 3, form a, b, c tri-footpath rings.The avidity that aptamers GO18-T-a is combined with target toxin significantly improves, and GO18-T-b and GO18-T-c display does not combine.May be that after clipping because of footpath ring b and c, the footpath ring a on aptamers GO18-T-a is fully exposed, and be combined finer and close with target molecule GTX1 and GTX4, the avidity that result in both increases significantly.Therefore, the aptamers GO18-T-d only containing 25 Nucleotide is obtained after further brachymemma.
Table 2: aptamers GO18 truncated sequence and affinity constant Kd value thereof
Embodiment 4. microbial film interference technique measures intermolecular interaction
It is a kind of molecular interaction analysis technology exempting to mark that microbial film is interfered.Its principle is that instrument transmitting white collects reflected light to sensor surface, the reflection spectrum of different frequency is subject to the impact of the optical film thickness of biosensor, the reflection spectrum of some frequencies is subject to the impact of the optical film thickness of biosensor, the reflected light of some frequencies defines constructive interference (blueness), and other receive destructive interference (redness).These interference lights detected by spectrograph, and form a width interference spectrum, and show with the phase-shifted intensity (nm) of interference spectrum.Therefore, be attached to the molecule of sensor surface once there be quantitative increase and decrease, spectrograph just can detect the displacement of interference spectrum in real time, and this displacement directly reflects the biomembranous thickness of sensor surface.
1) biotin labeling aptamers GO18-T-d binding buffer liquid is dissolved and is diluted to 5 μMs.All aptamers before use all will through 95 DEG C of water-bath 10min, the refolding strategy treating processes of the sudden cold 5min of ice bath, are again folded to form best space structure to urge them.
2) by aptamers, certain density target molecule, the each 200 μ L of damping fluid join after in each reacting hole respectively, Streptavidin chip is immersed in each reacting hole successively according to the program of setting, through overbalance (2min), aptamers coupling (5min), rinsing (3min), in conjunction with (5min) and (5min) five steps that dissociates.
3) each chip respectively with target molecule GTX1 and GTX4 (5 μMs of different concns, 10 μMs and 20 μMs) and non-specific target molecule GTX2/3 (10 μMs), STX (10 μMs) and neoSTX (10 μMs) interact, result as shown in Figure 4, the combination of aptamers GO18-T-d and GTX1, GTX4 high-affinity, and the affinity constant combined is 21.9nM.Aptamers is not combined with analogue GTX2, GTX3, STX and neoSTX of target toxin simultaneously.In addition, a random sequence is not also combined with target toxin GTX1 and GTX4, indicates aptamers GO18-T-d and GTX1, GTX4 is high-affinity, strong specific binding.The damping fluid adopted in microbial film interference technique is binding buffer liquid.Each test is provided with and interactionally with damping fluid contrasts chip, deducting from the response value of sample chip by Octet data analysis software CFRPart11Version6.x in conjunction with dissociation curve of contrast chip, adopt the binding pattern of 1:1 to carry out matching such as Fig. 4 to response data simultaneously, obtain aptamers and target molecule affinity costant Kd value, result is as table 1.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (9)
1., with the aptamers painting ditch Algae toxins 1 and 4 specific binding, there is general formula as follows: 5 '-AGCAGCACAGAGGTCAGATG-N
40-CCTATGCGTGCTACCGTGAA-3 '; Wherein, N represents base A, in T, C, G any one, N
40represent the stochastic sequence that length is 40bp.
2. the aptamers with painting ditch Algae toxins 1 and 4 specific binding according to claim 1, is characterized in that: described aptamers is selected from arbitrary in following sequence SEQIDNO:1-SEQIDNO:6.
3., with the aptamers painting ditch Algae toxins 1 and 4 specific binding, its nucleotide sequence is as shown in SEQIDNO:14.
4. the aptamers with painting ditch Algae toxins 1 and 4 specific binding according to claim 3, is characterized in that: described aptamers, carries out vitamin H, fluorescence molecule, isotropic substance, electrochemistry, enzyme or sulfydryl modification at its 5 ' end or 3 ' end.
5. according to claim 1 or 3 with the application of aptamers in the separation and concentration reagent preparing trace GTX1 and GTX4 in sample of paint ditch Algae toxins 1 and 4 specific binding.
6. according to claim 1 or 3, preparing the application in GTX1 and GTX4 detection reagent or test kit with the aptamers of paint ditch Algae toxins 1 and 4 specific binding.
7. according to claim 1 or 3, preparing with the aptamers of paint ditch Algae toxins 1 and 4 specific binding the application preventing or treat in the poisoning medicine of GTX1 and GTX4.
8. according to claim 1 or 3 with the aptamers of paint ditch Algae toxins 1 and 4 specific binding in preparation and or antagonism GTX1 and GTX4 medicine in application.
9. according to claim 1 or 3, preparing the application of removing in water body or fishery products in GTX1 and GTX4 preparation with the aptamers of paint ditch Algae toxins 1 and 4 specific binding.
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| CN105886512A (en) * | 2016-03-07 | 2016-08-24 | 江南大学 | A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity |
| CN105886512B (en) * | 2016-03-07 | 2020-06-09 | 江南大学 | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine |
| CN106282193A (en) * | 2016-08-08 | 2017-01-04 | 湖南大学 | HPP's aptamer and screening technique thereof and application |
| CN106282193B (en) * | 2016-08-08 | 2019-08-30 | 湖南大学 | Human pancreatic polypeptide aptamer and its screening technique and application |
| CN109337908A (en) * | 2018-10-15 | 2019-02-15 | 复旦大学附属眼耳鼻喉科医院 | The aptamer of one group-specific combination gliotoxin and its application |
| CN109337908B (en) * | 2018-10-15 | 2021-10-15 | 复旦大学附属眼耳鼻喉科医院 | Nucleic acid aptamer group specifically binding to gliotoxin and application thereof |
| CN112852824A (en) * | 2021-02-08 | 2021-05-28 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Aptamer specifically recognizing FPV |
| CN112852824B (en) * | 2021-02-08 | 2023-05-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Nucleic acid aptamer capable of specifically recognizing FPV |
| CN112986212A (en) * | 2021-03-10 | 2021-06-18 | 中国人民解放军海军军医大学 | Research method for interaction of G-quadruplex aptamer and small molecule |
| CN113720819A (en) * | 2021-08-30 | 2021-11-30 | 安徽师范大学 | Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor |
| CN113720819B (en) * | 2021-08-30 | 2023-10-17 | 安徽师范大学 | Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor |
| CN113686818A (en) * | 2021-09-01 | 2021-11-23 | 南开大学 | Method for determining affinity of graphene oxide to chlorella extracellular polymeric substances by utilizing biofilm interference technology |
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