CN103725685B - AFB 1aptamer AFB 1-14 and application - Google Patents

Abstract

AFB ₁aptamer AFB ₁-14 and application, relate to a kind of nucleic acid.Described AFB ₁aptamer AFB ₁-14 is by the sterile S ELEX method screening preparation based on Agarose microbead separation-flow cytometry.Described AFB ₁aptamer AFB ₁-14 are rich in G base, and have loop-stem structure, avidity is high, and good stability is nontoxic, are easy to synthesis and mark, can be used as AFB in sample ₁the potential detection reagent of one.

Description

AFB 1aptamer AFB 1-14 and application

Technical field

The present invention relates to a kind of nucleic acid, particularly relate to AFB ₁aptamer AFB ₁-14 and application.

Background technology

In the biotic pollution problem of oil and foodstuffs, the pollution of mycotoxins is one of topmost factor, and aflatoxin (Aflatoxins, AF) is the natural pollutant that the toxicity that finds so far and carinogenicity are the strongest.Aflatoxin is by the similar secondary metabolite of one group of chemical structure of the generation such as Aspergillus flavus and Aspergillus parasiticus bacterium, has the basic structure of two furan nucleuss and coumarin (tonka bean camphor), has found kind more than 20 at present, mainly comprise B ₁, B ₂, G ₁, G ₂, M ₁and M ₂deng.Wherein, AFB ₁toxicity the strongest, amount is maximum, and stability is also the highest, its toxicity is 10 times of potassium cyanide, 68 times of arsenic, mainly act on the organs such as liver, primary hepatocarcinoma, cancer of the stomach and lung cancer etc. can be brought out, and to be delimited by World Health Organization Agency for Research on Cancer in 1993 be a class carcinogens.AFB ₁extensively be present in soil and animals and plants, especially plant food, as peanut, corn, rice, wheat, milk and various nuts etc.AFB ₁physico-chemical property more stable, fat-soluble, heat-resisting, be difficult to detoxification, harm greatly produced to the health of humans and animals.AFB ₁content is the required Inspection Index in international food health and Agricultural Products Trade, and many countries have all formulated the national standard of its allowance in food.Therefore, to AFB ₁carry out effective, quick, highly sensitive detection to have great importance.（1.Wogan,G.N.Chemicalnatureandbiologicaleffectsoftheaflatoxins.BacteriolRev1966,30,460-470;2.Shankaran,R.;Raj,H.G.;Venkitasubramanian,T.A.Biochemicalchangesinliverduetoaflatoxin.BrJExpPathol1970,51,487-491）。

At present, conventional AFB ₁detection method overview gets up to mainly contain chemical analysis and immunoassay.Chemical analysis mainly contains thin-layer chromatography chromatography (TLC), high performance liquid chromatography (HPLC) and microtrabeculae method etc., these common detection methods have possessed higher detection sensitivity and accuracy, but have that numerous and diverse sample pre-treatments, separation detection are consuming time, the expensive heaviness of plant and instrument and be difficult to realize on-the-spot real-time analysis, need the shortcomings such as professional and technical personnel.Immunoassay is mainly based on the high-affinity of aspergillus flavus resisting toxin monoclone antibody and a series of immunological detection methods of high specific development, enzyme-linked immunosorbent assay (ELISA) is wherein highly sensitive, security good, interference is few, fast easy and simple to handle, be the AFB of the most general practicality at present ₁detection method.But AFB ₁be a kind of hypertoxic small molecules, there is larger difficulty in the preparation of its antibody, the reagent life-span, shorter being difficult to ensure was deposited, and differences between batches are also larger etc., and these bottlenecks limit the range of application of immunological detection method and fast-developing (3.JinHwanDo; Dong-KugChoi.Aflatoxins:Detection, Toxicity, andBiosynthesis.BiotechnologyandBioprocessEngineering200 7,12,585-593; 4.LiuZ.; GaoJ..Advancesinresearchondetectionmethodsforaflatoxins. JournalofAnhuiAgriculturalUniversity2004,31,223-226).Therefore, towards AFB ₁novel detection reagent and detection method urgently develop.

Aptamer (aptamer) is a class new type functional nucleic acid molecular probe, obtain based on index concentration Fas lignand system evolution technology (SELEX) in-vitro screening from random oligonucleotide library, be described as " artificial antibody ", can be combined with target molecule high-affinity and high specific, there is screening cycle short, the convenient economy of chemosynthesis and high without lot size variance, stability, be easy to the plurality of advantages such as pinpoint modification, be potential antibody surrogate thing, demonstrate huge application prospect at biochemical analysis detection field.At present, a kind of blanket technology (5.Liu, J. is newly become gradually based on the identification of aptamer and detection method; Cao, Z.; Lu, Y.Functionalnucleicacidsensors.ChemRev2009,109,1948-1998; 6.Clark, S.L.; Remcho, V.T.Aptamersasanalyticalreagents.Electrophoresis2002,23,1335-1340).The biosensor that aptamer technology is applied to mycotoxins detection analysis is in active development development.Such as, ochratoxin A (OchratoxinA, OTA) and fumonisin B ₁(FumonisinB ₁, FB ₁) etc. the aptamer of multiple mycotoxins screened; and developed multiple detection system (7.ScreeningandInitialBindingAssessmentofFumonisinB1Aptam ersInt.J.Mol.Sci.2010; 11; 4864-4881.8.DeterminationofOchratoxinAwithaDNAAptamer.J. Agric.FoodChem.2008; 56; 10456 – 10461.9.Yang, C.; Wang, Y.; Marty, J.; Yang, X.Aptamer-BasedColorimetricBiosensingofOchratoxinAusingU nmodifiedGoldNanoparticlesIndicator.Biosens.Bioelectron. 2010.10.Kuang, H.; Chen, W.; Xu, D.; Xu, L.; Zhu, Y.; Liu, L.; Chu, H.; Peng, C.; Xu, C.; Zhu, S.FabricatedAptamer-BasedElectrochemical " signal-Off " SensorofOchratoxinA.Biosens.Bioelectron.2010,26,710 – 716).Therefore, AFB is screened ₁specific nucleic acid fit and develop aptamer technology for AFB ₁new detecting method there is important Research Significance and market using value.

Summary of the invention

The first object of the present invention is to provide AFB ₁aptamer AFB ₁-14, the application of this aptamer obtains based on the sterile S ELEX technology screening of Agarose microbead separation-flow cytometry, can high-affinity identification AFB ₁.

The second object of the present invention is to provide AFB ₁aptamer AFB ₁-14 are preparing AFB in sample ₁application in detection reagent.

Described AFB ₁aptamer AFB ₁-14, its sequence is as follows:

ATACCAGCTTATTCAATTTTGGTGGCATTGGGGGGGGGGGTTTGGTGGCCGAGTTGATGTTTAAGATAGTAAGTGCAATCT

Described AFB ₁aptamer AFB ₁-14, utilize its secondary structure of the online software simulation of OligoAnalyzer3.1, have a kind of possible loop-stem structure, its loop-stem structure is as follows:

Described AFB ₁aptamer AFB ₁-14, with target molecule AFB ₁in conjunction with after the mixture that formed there is fluorescence polarization response, and AFB ₁fluorescence itself can by quencher, therefore based on fluorescence polarization technology or fluorescence spectrum method, and described AFB ₁aptamer AFB ₁-14 can prepare AFB in sample ₁apply in detection reagent.

The invention has the advantages that: 1) aptamer is by obtaining based on the sterile S ELEX technology screening of Agarose microbead separation-flow cytometry, in-vitro screening and detect fast easy; 2) aptamer itself is one section of oligonucleotide, can chemosynthesis in a large number, and without lot size variance, good stability, is easy to preserve; 3) aptamer energy high-affinity identification AFB ₁, can match in excellence or beauty with antibody; 4) aptamer is easy to pointed decoration mark, and application form is versatile and flexible, and AFB ₁for organic molecule, itself has photoluminescent property, and both character combine and can develop a series of biosensor, is detecting having a extensive future of analysis field.

Accompanying drawing explanation

Fig. 1 is AFB ₁the fluorescent microscope image of-beads.Figure a is the light field imaging of control group Control-beads, and figure b is sample sets AFB ₁the light field imaging of-beads, figure c is the fluorescent dark field imaging of control group Control-beads, and figure d is sample sets AFB ₁the fluorescent dark field imaging of-beads.

Fig. 2 is AFB ₁the fluorescence spectrum scintigram of-beads.Curve a is sample sets AFB ₁-beads.Curve b is control group Control-beads.

Fig. 3 be in flow cytometry monitoring screening process DNA initial libraries for target molecule AFB ₁the fluorescence shift figure of-beads enrichment.Curve a is blank AFB ₁-beads, curve b are DNA initial libraries, and curve c the 4th takes turns enrichment storehouse, and curve d the 6th takes turns enrichment storehouse, and curve e the 8th takes turns enrichment storehouse, and curve f is 11th round enrichment storehouse.

Fig. 4 be in flow cytometry monitoring screening process DNA initial libraries for the fluorescence shift figure of anti-sieve element COOH-beads enrichment.Curve a is blank COOH-beads, and curve b is DNA initial libraries, and curve c the 4th takes turns enrichment storehouse, and curve d the 6th takes turns enrichment storehouse, and curve e the 8th takes turns enrichment storehouse, and curve f is 11th round enrichment storehouse.

Fig. 5 is Flow Cytometry Assay aptamer AFB ₁-14 couples of target molecule AFB ₁the binding curve figure of-beads.

Fig. 6 is AFB ₁with aptamer AFB ₁-14 fluorescence polarization response diagrams combined.A is the free AFB of blank group ₁, b is control group stochastic sequence Random, c is sample sets aptamer AFB ₁-14.

Fig. 7 is AFB ₁with aptamer AFB ₁-13 fluorescence spectrum scintigrams combined.Curve a is blank group AFB ₁, curve b is control group stochastic sequence Random, and curve c is sample sets aptamer AFB ₁-14.

Embodiment

Embodiment 1 target AFB ₁the syntheses and properties of-beads

Using Agarose microbead as the solid phase carrier of target molecule, be conducive to being separated and do not combine or weak binding and non-specific binding sequence, and be applicable to Flow cytometry.By AFB ₁the synthetic route being coupled to Agarose microbead is specific as follows:

Get 7.6mgAFB ₁be dissolved in 4mL pyridine, add 35mg carboxymethyl hydroxylamine hydrochloride, 80 DEG C of backflows are spent the night, and through vacuum concentration and silica gel column chromatography (chloroform/methanol=10:1), isolate AFB ₁-oxime is also dissolved in 3mL anhydrous methylene chloride, adds 5.4mgNHS, 9.6mg dicyclohexylcarbodiimide and 5mg Dimethylamino pyridine, and magnet rotor stirs and spends the night, and after filtration and evaporation concentration, obtains AFB1-oxime Acibenzolar (11.Chu, F.S.; Hsia, M.T.; Sun, P.S.:Preparationandcharacterizationofaflatox-nB1-1-(O-carboxymethyl) oxime.Journal-AssociationofOfficialAnalyticalChemists197 7,60,791-4.).Get 0.107gNHS-beads to be dispersed in 900 μ L dipotassium hydrogen phosphate damping fluid (50mM, pH9.0), add 100 μ L quadrols, magnet rotor stirs and spends the night.Centrifugally remove supernatant, and clean microballon with PB damping fluid (0.1M, pH8.0) and obtain NH ₂-beads.By AFB ₁-oxime Acibenzolar is dissolved in 0.8mLDMF, NH ₂-beads is scattered in 0.8mL dipotassium hydrogen phosphate damping fluid (50mM, pH9.0), both mixing, and magnet rotor stirs and spends the night, and then obtains AFB with 50%DMF and PB buffer solution for cleaning microballon ₁-beads, 4 DEG C keep in Dark Place.By fluorescence spectrum scanning (Ex:365nm; Em:380-600nm) and fluorescent microscope imaging AFB1-beads is characterized (see Fig. 1 and 2).

Embodiment 2 AFB ₁the fit screening of specific nucleic acid

(1) Design and synthesis of random oligonucleotide library

Design and synthesize the random oligonucleotide library that two ends FX is 18 Nucleotide, middle random areas is 45 Nucleotide as follows: 5'-ATACCAGCTTATTCAATT-N45-AGATAGTAAGTGCAATCT-3', storage capacity is 1015.The primer sequence is respectively forward primer (FP): 5'-ATACCAGCTTATTCAATT-3'; reverse primer (RP): 5'-AGATTGCACTTACTATCT-3'; fluorescent dye primer (FFP): 5'-FAM-ATACCAGCTTATTCAATT-3'; biotin labeling primer (BRP): 5'-Bio-AGATTGCACTTACTATCT-3'(12.Shangguan, D.; Li, Y.; Tang, Z.; Cao, Z.C.; Chen, H.W.; Mallikaratchy, P.; Sefah, K.; Yang, C.J.; Tan, W.Aptamersevolvedfromlivecellsaseffectivemolecularprobes forcancerstudy.ProcNatlAcadSciUSA2006,103,11838-11843).

(2) screening of aptamer

AFB ₁-beads is as target molecule, and the COOH-beads that hydrolyzing N HS-beads is formed is as anti-sieve element.

Get 10nmol initial libraries and be dissolved in 200 μ L binding buffer liquid (20mMTris-HCl, pH8.0,100mMNaCl, 5mMKCl, 2mMMgCl ₂, 1mMCaCl ₂) in, 95 DEG C of 5min, ice bath 5min, room temperature 30min.Secondary one-level library all uses 200pmol.Anti-sieve operates from third round, by the library of denaturing treatment and 2 μ LCOOH-beads incubated at room 5min.Its collecting by filtration liquid and 2 μ LAFB ₁-beads incubated at room 60min.Use binding buffer liquid cleaning microballon, will the AFB of DNA be combined ₁-beads directly carries out PCR amplification and (uses FP and BRP, 94 DEG C of 3min; 94 DEG C of 15s; 50 DEG C of 15s; 72 DEG C of 15s; 72 DEG C of 3min), and obtain single-chain nucleic acid library with the absorption of streptavidin microballon and the NaOH wash-out through 0.1M, screen for next round.Screening process increases proof strength by wheel, carries out 11 altogether and takes turns.By the enrichment condition (see Fig. 3 and 4) in Flow cytometry library.Finally enrichment is restrained storehouse and carry out cloning and sequencing (13.Hu, J.; Wu, J.; Li, C.; Zhu, L.; Zhang, W.Y.; Kong, G.; Lu, Z.; Yang, C.J.AG-quadruplexaptamerinhibitsthephosphataseactivityof oncogenicproteinShp2invitro.Chembiochem.2011,12,424-430).

The avidity of embodiment 3 aptamer characterizes

By Nucleic acid aptamer molecules at 5' end mark Fluoresceincarboxylic acid (FAM), in 200 μ L binding buffer liquid, prepare 0-7000nM gradient solution, thermally denature process, with 0.4 μ LAFB ₁-beads incubated at room 30min.After the cleaning of binding buffer liquid, be suspended in binding buffer liquid, by cells were tested by flow cytometry bead surface fluorescence intensity.With fluorescence intensity to the mapping of aptamer concentration, use formula Y=BmaxX/ (K _d+ X) carry out binding curve matching measure aptamer in conjunction with dissociation constant K _d.(see Fig. 5).

Embodiment 4 fluorescence polarization and fluorescence spectrum study aptamer and AFB ₁between interaction

With binding buffer liquid (20mMTris-HCl, pH8.0,100mMNaCl, 5mMKCl, 2mMMgCl ₂, 1mMCaCl ₂) preparation 2 μMs of unmarked aptamer solution, carry out thermally denature process, 95 DEG C of 5min, ice bath 5min, room temperature places 30min.Then AFB is added wherein ₁make its final concentration be 0.2 μM, and at room temperature hatch 30min.Blank group is AFB independent under the same terms ₁solution, control group is stochastic sequence Random.Measure the fluorescence anisotropy value (Ex:365nm of each group of solution system; Em:442nm); Fluorescence spectrum scanning (Ex:365nm is carried out to each group of solution system; Em:380-650nm) (see Fig. 6 and Fig. 7).

Result

Embodiment 1 is according at AFB ₁on add a carboxyl, with NH ₂-beads carries out coupling by the basic ideas forming stable amido linkage and successfully synthesizes AFB ₁-beads, and by fluorescent microscope imaging and fluorescent scanning spectrum, its pattern and coupling content are characterized.As shown in Figure 1, in details in a play not acted out on stage, but told through dialogues fluorescence imaging, relative to not through AFB ₁the Control-beads modified, AFB ₁-beads sends obvious fluorescence; As shown in Figure 2, under 365nm UV-irradiation, AFB ₁-beads sends fluorescence at 425nm place, higher than the fluorescent value of Control-beads about 6 times.It can thus be appreciated that, AFB ₁-beads coupling success.AFB on Agarose microbead ₁coupling content by measuring based on fluorescent scanning establishment of spectrum standard working curve, estimate for 4.5fmolAFB ₁/ beads.

Embodiment 2 successfully obtains based on the sterile S ELEX technology screening of Agarose microbead separation-flow cytometry can high-affinity identification AFB ₁aptamer.As shown in Figure 3, for just sieving target molecule AFB ₁-beads, initial storehouse does not have obvious fluorescence shift, substantially not with AFB ₁-beads combine, along with the increase of screening wheel number, the 4th, 6,8,11 fluorescence shift of taking turns enrichment storehouse increase gradually, and the fluorescence shift intensity in 11th round enrichment storehouse reaches 300 times of initial storehouse, illustrate in enrichment storehouse with AFB ₁sequence enrichment gradually in screening process that-beads combines; As shown in Figure 4, for anti-sieve element COOH-beads, enrichment storehouse is taken turns in initial storehouse, the 4th, 6,8,11 does not all have obvious fluorescence shift, is not combined with COOH-beads.Illustrate that from random oligonucleotide library, screen enrichment obtains and AFB ₁the nucleotide sequence that-beads combines, therefore sends to cloning and sequencing by 11th round enrichment storehouse.

Embodiment 3 passes through Flow Cytometry Assay aptamer to target molecule AFB ₁the binding ability of-beads.As shown in Figure 5, AFB ₁aptamer AFB ₁-14 in conjunction with dissociation constant K _dbe 2.04 ± 0.22 μMs, there is higher avidity.

Embodiment 4 have studied aptamer and target molecule AFB by fluorescence polarization assay and fluorescence spectrum scanning analysis ₁between binding interactions, thus demonstrating described aptamer can as AFB in sample ₁the potential detection reagent of one.As shown in Figure 6, there is not aptamer AFB ₁when-14, AFB freely ₁owing to itself being organic molecule, molecular weight is only 312.27g/mol, rotates very fast, records its fluorescence anisotropy value and be about 0.013; As aptamer AFB ₁-14 when existing, and records its fluorescence anisotropy value and be increased to and be about 0.051; And when contrasting stochastic sequence Random and existing, its fluorescence anisotropy value is about 0.016, is not significantly increased.Described aptamer and target molecule AFB are described thus ₁between specific combination interact.As shown in Figure 7, under buffer system condition used, AFB ₁under UV-light 365nm irradiates, at 442nm place emitting fluorescence; As aptamer AFB ₁-14 when existing, and the reduction of highly significant has appearred in its maximum emission wavelength place fluorescent value; And when contrasting stochastic sequence Random and existing, corresponding fluorescent value is only in a slight decrease.Illustrate thus and state aptamer and target molecule AFB ₁in conjunction with after, quenching of fluorescence.

Claims (2)

1. AFB ₁aptamer AFB ₁-14, it is characterized in that its sequence is as follows:

ATACCAGCTTATTCAATTTTGGTGGCATTGGGGGGGGGGGTTTGGTGGCCGAGTTGATGTTTAAGATAGTAAGTGCAATCT；

Described AFB ₁aptamer AFB ₁-14 have a kind of loop-stem structure, and its structural formula is as follows:

Described aptamer AFB ₁-14 application obtain based on the sterile S ELEX technology screening of Agarose microbead separation-flow cytometry.

2. AFB as claimed in claim 1 ₁aptamer AFB ₁-14 are preparing AFB in sample ₁application in detection reagent.