CN103820458B - The aptamer of one group of specific recognition β-bungarotoxin and purposes - Google Patents

Abstract

The invention provides aptamer and the purposes of a group-specific identification β bungarotoxin.The aptamer that the present invention provides, it it is the arbitrary single-stranded DNA sequence shown in SEQ ID NO.1～4 in sequence table, described aptamer and β bungarotoxin have higher affinity and specificity, are with a wide range of applications in the detection of β bungarotoxin and the diagnosis of Induced By Coral Snake Bite.

Description

The aptamer of one group of specific recognition β-bungarotoxin and purposes

Technical field

The present invention relates to biotoxin technical field, specifically refer to utilize the SELEX technology in Protocols in Molecular Biology (to refer to The Fas lignand system evolution technology of number enrichment) prepare one group of aptamer being combined with β-bungarotoxin high specific, for this Aptamer provides scientific basis and theoretical basis in the detection of β-bungarotoxin and the diagnosis of Induced By Coral Snake Bite.

Background technology

β-bungarotoxin derives from Agkistrodon (Bungarus multicinctus) snake venom, is that a kind of presynaptic is many Peptide Nervous toxicity, shows Ca²⁺Dependency phospholipase A2 (Phospholipase A₂, PLA₂) activity, it is the most main of bungarotoxin The toxic component wanted, LD50(i.p. mice) it is 0.019 μ g/g.Molecular weight (Mr) is 20500, is made up of two chains, i.e. A chain With B chain, and coupled together by a pair interchain disulfide bond.The A chain (Mr=13500) that molecular weight is bigger contains 120 aminoacid, tool Having 13 cysteine, its primary structure is sufficiently analogous to from snake venom and mammalian pancreas the PLA identified₂, have and compare Weak activity.The B chain (Mr=7000) of molecular weight is made up of 60 aminoacid, has some sequences with protein kinase inhibitor Homology, as the β-bungarotoxin identification subunit to specific targeting cell membrane, in neuromuscular junction presynaptic membrane Critical function is had in some composition interphase interactions.When intersubunit disulfide bonds ruptures, or PLA₂Active center is repaiied by covalency During decorations, all may result in neurotoxicity and the PLA of β-bungarotoxin₂The forfeiture of activity.

Venom is the torrid zone, the unhealthful major issue in one, rural area, subtropical zone, is often only in Asia, Serpentis stings Hinder lethal number and just reach 50,000 people.Induced By Coral Snake Bite accounts for the 8.12% of all kinds of venom in China, occupies the 5th, and dead people Number ranks first.China does not also have one snakebite reagent for clinical diagnosis and method fast and effectively, snakebite diagnosis to rely primarily at present Medical history and clinical manifestation.Owing to be there is similar symptom by different types of venom, in the case of medical history is not quite clear, clinical The clinical manifestation that doctor is difficult to from patient judges down the end is by which kind of venom.

In the laboratory qualification technology of snake venom, immune detection is the most topmost technological means.Existing immunity is examined Disconnected technology is to utilize polyclonal antibody to detect toxin mostly, owing to there is cross reaction between different snake venom multi-resistance, the most special Property is poor.The monoclonal antibody preparing single snake venom composition can be greatly improved the specificity of detection, but monoclonal antibody itself there is also Some defects.Monoclonal antibody is weak relative to the affinity of multi-resistance, the best to the adsorptivity of ELISA Plate, is carrying out enzyme or biology simultaneously In the operation of element labelling, stability is the most poor.

Aptamer is oligonucleotide sequence (RNA or ssDNA) that can be specific binding with target molecule.Nineteen ninety, Two research groups of Gold and Ellington establish a kind of technology preparing specific oligonucleotide molecule the most simultaneously, and This technology is defined as SELEX (systematic evolution of ligands by exponential Enrichment, index concentration formula Fas lignand system evolve), can prepare have certain three dimensional structure, can with target molecule by solid Adaptability between conformation complementary (not base pairing) and oligonucleotide molecules-adaptation that high specific, high-affinity combine Body (aptamer).The appearance of SELEX technology comes from people and gos deep into nucleic acid with protein-interacting research.Single-chain nucleic acid energy Some stable three-D space structures are formed by 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 Structure, single-chain nucleic acid can form the stable compound with high specific adhesion with protein or other molecules.Strand core Acid can also be passed through the various interactions such as Van der Waals force, hydrogen bond action, electrostatic interaction and form fit and produce height with target molecule Specific adhesion.As antibody, aptamer and target have high-affinity and a specificity as combining, therefore, Aptamer also referred to as " chemistry antibody ".

Owing to aptamer is one section of little single stranded DNA or RNA, relative to antibody, there is the most natural advantage. Such as, aptamer is readily synthesized and can apply the chemical method of routine to carry out modifying, long-term stability is (if long term maintenance Reversible degeneration and renaturation), avirulence or immunogenicity.From the perspective of engineering, with vivo biodistribution antibody generation process Difference, the difference between the chemosynthesis batch of external aptamer is the least.It is wide that all of these factors taken together all promotes aptamer It is applied in the researchs in field such as medical science and life sciences and the Clinics and Practices of disease generally.

Summary of the invention

It is an object of the invention to provide a kind of aptamer that can be specific binding with β-bungarotoxin.

Further object is that and provide above-mentioned aptamer at β-bungarotoxin and Induced By Coral Snake Bite Application in detection.

The aptamers of described specific binding β-bungarotoxin and fragment thereof, selected from the sequence shown in SEQ ID NO.1～4 One or more in row, the most all meets the architectural feature shown in following formula, 5 '-AGC AGC ACA GAG GTC AGA TG Nx-CCT ATG CGT GCT ACC GTG AA-3 ' (formula I).During in formula, N represents base A, G, C, T Any one, Nx represents a length of 40,39 or 51 bases of random fragment.Above-mentioned sequence all exists with single stranded form.

Sequence shown in SEQ ID NO.1～4, can be modified or transform, and obtains the derivative of described aptamer Thing.Described modification or one or more in the following manner of reforming mode:

1) described aptamer being deleted part or increases partial nucleotide, obtain has with described aptamer The derivant of the aptamer of identical function.

2) entirely vulcanized by described aptamer, end-block or PEG modify, that obtain with described aptamer There is the derivant of the aptamer of identical function.

3) described aptamer is connected or other functional group of labelling or molecule, such as sulfydryl, amino, radioactivity with Position element, fluorescein, biotin, toxin or enzyme etc., obtain has the aptamer of identical function with described aptamer Derivant.

The screening technique of described β-bungarotoxin aptamer is by index concentration Fas lignand system evolution technology (i.e. SELEX screens) it is applied to the screening of β-bungarotoxin aptamer, the concrete steps of described SELEX screening include:

1) synthesis is for screening the random single-stranded DNA banks of β-bungarotoxin aptamer, and it is corresponding to design synthesis Primer；

2) β-bungarotoxin is coated in 96 hole ELISA Plate, and hatches with described random single-stranded DNA banks, separate and β- The DNA that bungarotoxin combines；

3) DNA of separation is carried out PCR amplification, amplified production lambda exonuclease enzyme action, obtain time one-level single stranded DNA Storehouse, and screen for next round SELEX, until obtaining 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 40 alkali The random sequence of base, it may be assumed that 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, it is an advantage of the current invention that: the aptamer that screening is obtained is relative to tradition Antibody, has easier screening conditions, avirulence, and molecular weight is little, it is easy to chemical modification and labelling, synthesizes cost relatively antibody system Standby low, the cycle is short.Aptamer and the binding affinity of β-bungarotoxin that the present invention provides are high, and Kd value reaches nM level, And only specific recognition β-bungarotoxin and the bungarotoxin containing β-bungarotoxin, to other cognate toxin or Serpentis Poison, does not identify function such as α-bungarotoxin, Bungarus toxin, agkistrodon acutus venom, viper venom, cobra venom etc..Above-mentioned advantage makes Described aptamer becomes β-bungarotoxin detection and the powerful of Induced By Coral Snake Bite diagnosis.

Accompanying drawing explanation

Fig. 1. the 1st takes turns the pilot PCR of screening.1:20bp DNA ladder；The PCR primer of 2-9:12-26 circulation.

The secondary structure of aptamers β B-1, β B-19, β B-32 and β B-20 of Fig. 2 .Mfold software prediction.

Fig. 3 .SELEX screens each combination rate taking turns single stranded DNA storehouse and β-bungarotoxin.

Fig. 4. Media by Fluorescence Anisotropy measures dissociation constant Kd that aptamers β B-1, β B-20 and β-bungarotoxin combines Value.

Fig. 5. nitrocellulose membrane filter method measures aptamers β B-1 and bungarotoxin, Bungarus toxin, Agkistrodon acutus venom, glasses Snake venom and the combination rate of viper venom.1: bungarotoxin；2: Bungarus toxin；3: Agkistrodon acutus venom；4: viper venom；5: cobra venom.

Fig. 6. identify bungarotoxin and β-bungarotoxin with aptamers β B-1 by ELISA method.1. β-bungarotoxin Element；2.BSA；3. casein；4. α-bungarotoxin；5. bungarotoxin；6. cobra venom；7. Bungarus toxin；8. Agkistrodon acutus Poison；9. viper venom；10. comparison.

Detailed description of the invention

Following example are used for illustrating the explanation present invention, but should not be construed as limiting the scope of the invention.If not Specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.

The screening of the aptamer of the specific binding β-bungarotoxin of embodiment 1

1. build random single-stranded DNA banks and primer: design two ends are the fixed sequence program of 20 bases, and centre is 40 alkali The random single-stranded DNA banks of base random sequence, it may be assumed that 5 '-AGC AGC ACA GAG GTC AGA TG-N40-CCT ATG CGT GCT ACC GTG AA-3′.Any one during wherein N represents base A, G, C, T, N40 represents a length of 40 alkali of random fragment Base.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 synthesized by Shanghai Sheng Gong Bioisystech Co., Ltd.Random library is tied Close buffer (20mM HEPES, pH7.4;150mM NaCl;5mM KCl;2mM MgCl₂;2mM CaCl₂), primer ddH₂O It is configured to 100 μMs of stock solutions-20 DEG C storage standby.

2.SELEX screens: β-bungarotoxin carbonate buffer solution (CBS, pH9.6) is configured to 10 μ g/mL concentration, Taking 100 μ L and be coated 96 orifice plates, 4 DEG C overnight.Outwell supernatant, add 100 μ L combine buffer (containing 1mg/mL BSA, from the 5th take turns BSA is replaced with 1mg/mL casein during closing) close 2h.Outwell supernatant, combine buffer (containing 0.05%tween20) with 200 μ L Clean 6 times.SsDNA library (be dissolved in and combine buffer, containing 0.05%tween20) is placed in PCR instrument, and 90 DEG C of 10min, after taking-up It is immediately placed in 10min on ice, is subsequently placed in room temperature 10min.Take 100 μ L and join in 96 orifice plates, incubated at room 1h.SsDNA literary composition The concentration in storehouse is gradually reduced to the 10th 0.03 μM taken turns from the 1st 10 μMs taken turns.Outwell supernatant, combine buffer with 200 μ L and (contain 0.05%tween20) clean 6 times, then use ddH₂O washes 3 times, is eventually adding 100 μ L ddH₂O, is placed in 95 DEG C, 10min, takes out Supernatant eluent, carries out PCR reaction as template.

Each take turns screening during, first take a small amount of eluent and carry out pilot PCR and determine the optimum cycle number of amplification. PCR reaction system is: 50 μ L Premix Taq solution(treasured are biological, Dalian), 4 μ L eluents, 1 μ L10 μM primer 1,1 μ L10 μM of primer 2,44 μ L ddH₂O.PCR reaction condition is: 94 DEG C of degeneration 5min；94 DEG C of degeneration 0.5min, 56 DEG C of annealing 0.5min, 72 DEG C extend 0.5min, 26 circulations；Last 72 DEG C extend 10min.From the 12nd circulation, every 2 circulation from PCR pipe is taken out 10 μ L PCR primer.After PCR completes, the product of different periods is carried out 3% agarose gel electrophoresis, analyzes Electrophoretic band, optimum cycle number is the largest loop number not producing short chain or long-chain by-product.After determining optimum cycle number, Carrying out PCR reaction, reaction system and condition ibid under good period, carry out 8 tube reactions altogether, cumulative volume is 800 μ L.PCR primer Carrying out enzyme action with lambda exonuclease, the antisense strand of labeled phosphorus acid groups is fallen in hydrolysis, obtains the positive-sense strand of mark fluorescent element.Through second Alcohol precipitate after purification, it is thus achieved that ssDNA for next round SELEX screening.

3. clone and order-checking: take turns screening through 10, with the primer PCR amplification eluting of not mark fluorescent element and phosphate group Liquid, PCR primer is connected to cloning vehicle pMD19-T, and clone serves Hai Sheng work Bioisystech Co., Ltd and checks order, it is thus achieved that 4 Bar aptamers sequence.Result, as shown in SEQ ID NO.1～4, is respectively designated as 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, 4 aptamers Secondary structure is as shown in Figure 1.Loop-stem structure and hairpin structure in every aptamer are probably aptamers and β-bungarotoxin Binding site.

Embodiment 2 measures each adhesion taking turns aptamer storehouse and β-bungarotoxin with digestion fiber embrane method

Nitrocellulose membrane (HAWP02500nitrocellulose filters, Millipore, 0.22 μm) uses 0.5MKOH After solution soaking 30min, use ddH₂O fully cleans, and then combines in buffer with being placed in, and shakes 40min, is placed in new combination In buffer, 4 DEG C of preservations.The fluorescein-labeled each ssDNA of wheel storehouse is configured to 1 μM of concentration, takes 10 μ L and joins 1mL combination buffering In liquid, being placed in 90 DEG C of 10min, be subsequently placed in 10min on ice, room temperature places 10min.Add 1mg/mL β-bungarotoxin 4 μ L, Incubated at room 1h, comparison is not added with β-bungarotoxin, then filter membrane, combines wash buffer filter membrane with 1mL.Merging filtrate, Fluorescence intensity with 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 comparison deduct the fluorescence intensity of sample then for be attached to β- The fluorescence intensity of the ssDNA on bungarotoxin.In conjunction with fluorescence intensity be each wheel with the ratio of the fluorescence intensity compareed Aptamer storehouse and the combination rate of β-bungarotoxin.Result is as shown in Figure 2, it can be seen that along with the increase of screening wheel number, The combination rate of aptamers and β-bungarotoxin is gradually increased.To the 10th take turns time combination rate be not further added by, show that aptamers is Highly enriched, screening terminates.

Embodiment 3 measures the dissociation constant Kd value of aptamer with Media by Fluorescence Anisotropy

Synthetic fluorescently-labeled aptamers β B-1, β B-19, β B-32 and β B-20, takes 4 μ L aptamers (1 μM) respectively Join 2mL and combine in buffer, measure fluorescence anisotropy value.Then carry out dripping with the β-bungarotoxin of variable concentrations Fixed, measure the fluorescence anisotropy value under each concentration.Location parameter is with embodiment 2.Do by the concentration of β-bungarotoxin For abscissa, the increment of fluorescence anisotropy value, as vertical coordinate, does nonlinear regression with SigmaPlot10.0 software, analyzes Dissociation constant (Kd) value of aptamers.

The Kd value of aptamers β B-1 and β B-20 is below 100nM, respectively 65.9nM and 83.8nM, measurement result such as Fig. 3 institute Show.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 Yu β-bungarotoxin tool There is higher affinity, show its using value in β-bungarotoxin detection.

Embodiment 4 differentiates different snake venom with nitrocellulose embrane method

Bungarotoxin, Bungarus toxin, Agkistrodon acutus venom, viper venom and cobra venom become with combining buffer respectively 10mg/mL concentration, 12000rpm is centrifuged 5min, takes supernatant.Take 1 μM of fluorescein-labeled aptamers β B-1 or β B-206 μ L, add Entering and combine in buffer to 1mL, be placed in 90 DEG C of 10min, be subsequently placed in 10min on ice, room temperature places 10min.Add the most respectively Entering various snake venom 2 μ L, incubated at room 1h, comparison is not added with snake venom.Filter membrane, and combine wash buffer filter membrane with 1mL.Merge filter Liquid, measures the fluorescence intensity of filtrate.Location parameter is with embodiment 2.The fluorescence intensity of comparison deducts the fluorescence intensity of sample The fluorescence intensity of the aptamers being attached on various snake venom, in conjunction with fluorescence intensity be core with the ratio of the fluorescence intensity compareed Acid aptamers and the combination rate of various snake venom.Measurement result is as shown in Figure 4.Aptamers β B-1 or β B-20 and the knot of bungarotoxin Conjunction rate is all higher than 80%, and and the combination rate of Bungarus toxin, Agkistrodon acutus venom, cobra venom and viper venom be below 10%, show Aptamers β B-1 and β B-20 the most specific identification bungarotoxin.Therefore, utilize SELEX technology screening and β-bungarotoxin The aptamers that element high-affinity, high specific combine has application prospect widely in the diagnosis of venom.

Embodiment 5 ELISA method identifies snake venom and associated protein

The aptamers β B-1 of synthesis 5 ' end labelling biotin.β-bungarotoxin, BSA, casein, α-bungarotoxin, Bungarotoxin, cobra venom, Bungarus toxin, Agkistrodon acutus venom and viper venom CBS buffer (pH9.6) are each formulated into 10 μ G/mL, takes 100 μ L respectively and is coated elisa plate, and 4 DEG C overnight.Abandon supernatant, elisa plate with 200 μ L cleaning buffer solutions (in conjunction with buffering Liquid+0.05%tween20) clean 3 times, add 100 μ L1mg/mL BSA and close, room temperature, 2h.Abandoning supernatant, elisa plate is with 200 μ L Cleaning buffer solution cleans 3 times, adds 100 μ L biotin labeled β B-1(concentration 10pM, is dissolved in and combines buffer), room temperature combines 40min.Abandoning supernatant, elisa plate cleans 3 times with 200 μ L cleaning buffer solutions, adds the peroxidase of 100 μ L1:1000 dilutions Labelling Avidin (with combining buffer dilution), room temperature, 40min.Abandoning supernatant, elisa plate cleans 3 with 200 μ L cleaning buffer solutions Secondary, add 100 μ L freshly prepared substrate OPD solution, room temperature lucifuge reaction 15min, be eventually adding 50 μ L2M H₂SO₄Terminate anti- Should, observe the color change in every hole.Comparison is coated β-bungarotoxin, but is added without aptamers.Result is as shown in Figure 6.β-silver Bungarotoxin and bungarotoxin hole color are sepia, and wherein β-bungarotoxin hole color is deeper, and other albumen and snake venom Hole color and comparison are essentially identical, do not occur obvious color to change.Need not instrument interpretation, only from the color in each hole just Significantly can identify β-bungarotoxin from associated protein, from various snake venom, identify bungarotoxin.

Claims (3)

1. The aptamer of the most specific binding β-bungarotoxin, its sequence is as follows:

   β B-1:

   agcagcacag aggtcagatg gttttcccct tgtcgctttt ggttcgttct gcctctatct 60

   cctatgcgtg ctaccgtgaa 80。
2. 2. aptamers described in claim 1, it is characterised in that described aptamers connects sulfydryl, amino, radiosiotope, glimmering Light element, biotin or toxic label.
3. 3. aptamers described in claim 1, its by can be strengthened its half-life, improve its stability, prevent Cobra venom endonuclease or The compound of person's excision enzyme cutting is modified.