CN103937781A

CN103937781A - Real-time monitoring systematic evolution technology of ligands by exponential enrichment

Info

Publication number: CN103937781A
Application number: CN201310082033.0A
Authority: CN
Inventors: 王方雨; 张改平; 胡骁飞; 赵东; 职爱民; 史西保; 邓瑞广
Original assignee: Henan Academy of Agricultural Sciences
Current assignee: Henan Academy of Agricultural Sciences
Priority date: 2013-03-15
Filing date: 2013-03-15
Publication date: 2014-07-23

Abstract

The invention discloses a real-time monitoring systematic evolution technology of ligands by exponential enrichment (SELEX). The technology comprises: fixing to-be screened targets to a solid carrier polystyrene microscale reaction plate, combining with a single-chain nucleotide library of which the downstream terminal is labeled with biotin, performing incubation reaction flushing, adding streptomycin avidin-fluorescent substance to identify the current-cycle screening effect, using a specific eluate to elute combined ssDNA, and performing amplification for next-cycle screening until an nucleic acid aptamer capable of specifically boning targets is finally screened out. The technology is capable of effectively overcoming the disadvantage that screening results cannot be detected in real time by employing conventional SELEX methods, no expensive equipment such as capillary electrophoresis equipment and the like is not needed, and the technology is widely applicable to nucleic acid aptamer rapid screening aiming at various targets.

Description

A kind of Fas lignand system evolution technology of solid phase index concentration that can Real-Time Monitoring

Technical field

The present invention relates to a kind of Fas lignand system evolution technology of improved index concentration, mainly improve the immobilization method of Real-Time Monitoring and the Screening target of its screening process, be mainly used in the rapid screening of high specific aptamer.

Background technology

The Fas lignand system evolution technology (SELEX) of index concentration refers to that oligonucleotide relies on self high-affinity and high specific to respective target material, in an oligonucleotide library that contains extremely many stochastic sequences, by selective enrichment repeatedly, obtain the process of needed ad hoc structure and function aptamer.This technology is applicable to the life small-molecule substance of any special requirement of enrichment.Mono-clonal, ribozyme, the round pcr of the development of SELEX technology based on the seventies in last century and the eighties.This technology has been widely used in the various fields such as gene regulating and signal conduction, protein function, nucleic acid, oncology, virusology and food safety detection at present.

But at present SELEX method still exists screening process longer, because the content of specific nucleotide in library is lower, there is blind sieve, the finishing screen drawbacks such as power is lower of hanking in screening in early stage; And at present screening still will by the expensive plant and instrument such as kapillary electric current, limited the widespread use of this technology.

Summary of the invention

The technical problem to be solved in the present invention: overcome the defect of prior art, provide a kind of fast, can Real-Time Monitoring, do not rely on the SELEX method of expensive device.

Technical scheme of the present invention:

Building length is the ssDNA pool of 60-100 nt, and two ends are fixed sequence program, and a middle 20-60 Nucleotide is stochastic sequence, and storage capacity is at least 10 ¹⁴.Design ssDNA pool: 5'-CCCCTGCAGGTGATTTTGCTCAAGT-(20-60 nt)-AGTATCGCT AATCAGGCGGAT-3'.Upstream primer design: 5'-CCCCTGCA GGTGATTTTGCTCAAGT-3', downstream 5' end mark vitamin H: 5'-vitamin H-ATCCGCCTGATTAGCGATACT-3 '.Taking ssDNA library as template, carry out pcr amplification and obtain the dsDNA library of 3' end with vitamin H, separate ssDNA through vitamin H-Streptomycin sulphate magnetic bead.

The target that the library of separation and solid phase is incorporated into polystyrene micro-reaction plate reacts under proper temperature condition, after reaction certain hour, washes away unconjugated ssDNA with dcq buffer liquid; Add appropriate Streptomycin sulphate-fluorescein, in microplate reader, detect its fluorescence intensity, preliminary judgement screening efficiency; In PCR system, the 5' end of downstream primer is marked with vitamin H.Add elution buffer in 80 DEG C of effect 10min, through phenol-chloroform extracting, ethanolic soln, ssDNA is dissolved in 20 μ l TE damping fluids, ssDNA is become to the dsDNA of one end band vitamin H with the primer of mark vitamin H through pcr amplification.Separate ssDNA through vitamin H-Streptomycin sulphate magnetic bead, for the ssDNA library of next round screening.

positive beneficial effect of the present invention:

1. SELEX method of the present invention has following advantages:

(1) can Real-Time Monitoring: utilize the high sensitivity of Streptomycin sulphate coupling fluorescent substance, can judge preliminary screening effect early stage in screening, to accelerate to greatest extent SELEX screening process;

(2) equipment is simple: solid phase SELEX method only needs to detect the microplate reader of fluorescence, can greatly reduce the dependence to main equipments such as capillary electrophoresis, promotes the popularization and application of SELEX method.

2. detection method of the present invention can be widely used in the aptamer screening of target, specifically comprises: enzyme, somatomedin, antibody, the generegulation factor, cell adhesion factor, phytohemagglutinin, complete virion, pathogenic bacteria etc.; Also include the screening of the small-molecule substances such as organic dye, metal ion, medicine, amino acid.

Brief description of the drawings

Fig. 1: solid phase SELEX method schematic diagram that can Real-Time Monitoring of the present invention.

Fig. 2: the present invention is for the process schematic diagram of heavy metal screening aptamer.

Embodiment

Detection method of the present invention can be widely used in the detection of specific gene in various invasive organisms or pathogenic agent, specifically comprises: enzyme, somatomedin, antibody, the generegulation factor, cell adhesion factor, phytohemagglutinin, complete virion, pathogenic bacteria etc.; Also include the detection of the small-molecule substances such as organic dye, metal ion, medicine, amino acid.

example 1:

Aptamer screening process with heavy metal lead is illustrated.

(1) coupling of lead ion and carrier proteins and DTPA

This experiment mainly obtains corresponding conjugate by metal ion by difunctional metal-chelate mixture DTPA and carrier protein couplet by the cruel method of isothiocyanic acid and glutaraldehyde method.

The cruel method of different sulphur cyanogen: take respectively BSA 10mg and be dissolved in 0.5ml HEPES damping fluid (10mM Hepes, pH 9.0), 9.0 mg p-NH ₂-Bn-DTPA is dissolved in the water of 200 μ L, claims 8mgPb (NO ₃) ₂be dissolved in 200 μ L 2%HNO ₂in.

200 μ l p-SCN-Bn-DTPA solution are under agitation added drop-wise in 0.5 mL protein solution, add subsequently 100 μ L tri-second butylamine (1.5 M), and room temperature reaction 24h, removed unreacted small molecules with processing Centricon-30 ultrafiltration.First use HEPES damping fluid (10 mM, pH9.0) to wash three times, then use HBS (10mM, pH7.4) to wash twice.In the BSA-Bn-DTPA of purifying, add 200 μ LPb (No ₃) ₂solution room temperature reaction 4h, removes unreacted metal ion with above-mentioned same method ultrafiltration.Gained end product is Pb-CN-Bn-DTPA.

Take 30 mg bovine serum albumins (BSA) and be dissolved in 6 mLHBS (10 mM Hepes, 137mM NaCl, 3mM KCI, pH7.4), take 18mg metal-chelate mixture Pb-NH2-Bn-DTPA and be dissolved in l.8 in mL ultrapure water.6mL BSA is dropwise added drop-wise to 1.8mL metal-chelate mixture, then adds the 20 mM glutaraldehyde of 1.8 mL, stir gently 24h under room temperature, purifying is removed wherein unreacted small-molecule substance (metal-chelate mixture).Reactant BSA-DTPA is divided into two parts, and portion adds 14.6 μ L Pb (NO3) 2 (424.25mM), room temperature reaction 4h, purified acquisition Pb-NH2-Bn-DTPA-BSA.

The same principle and step are prepared NH2-Bn-DTPA-BSA.

(2) optimization of the synthetic and amplification condition of the structure of ssDNA pool, primer

Building length is the ssDNA pool of 60-100 nt, and two ends are fixed sequence program, and a middle 20-60 Nucleotide is stochastic sequence, and storage capacity is at least 1014.Design ssDNA pool: 5'-CCCCTGCAGGTGATTTTGCTCAAGT-(20-60 nt)-AGTATCGCTAATCAGGCGGAT-3'.Upstream primer design: 5'-CCCCTGCA GGTGATTTTGCTCAAGT-3', downstream 5' end mark vitamin H I: 5'-biotin-ATCC GCCTGATTAGCGATACT-3 '.SsDNA pool and primer are synthetic by Synbiotics AB.By synthetic primer, optimize library pcr amplification condition.

In PCR system, the 5' end of downstream primer is marked with vitamin H.Taking ssDNA library as template, carry out pcr amplification and obtain the dsDNA library of 3' end with vitamin H.DsDNA library product with separation and purification after, be combined with Streptomycin sulphate-avidin magnetic bead, now dsDNA by vitamin H the Streptomycin sulphate on magnetic bead be combined, then with the NaOH of 0.15 mol/L, dsDNA sex change is unwind, be combined and stay on magnetic bead with Streptomycin sulphate with a chain of vitamin H, and do not dissociate out with a chain of vitamin H, precipitate with ethanol, be dissolved in TE damping fluid, measure absorbancy, as the ssDNA library of next round screening.

(3) SELEX screening method

Metal ion-sequestrant-carrier proteins is coated on 96 hole elisa plates, ssDNA pool and a certain amount of tRNA first in binding buffer liquid SHCMK liquid with 37 DEG C, blank hole through 5%BSA sealing in conjunction with 40min, anti-sieve is removed the ssDNA of being combined with BSA-sequestrant, then transfers to metal chelator carrier proteins and is coated with 37 DEG C, hole in conjunction with 40min.Wash 6 times with dcq buffer liquid (SHCMK liquid+0.05% Tween 20), wash away unconjugated ssDNA; Add the Streptomycin sulphate-fluorescence of appropriate amount, under microplate reader, carry out the Screening and Identification that this is taken turns; Then add elution buffer in 80 DEG C of effect 10min, through phenol-chloroform extracting, ethanolic soln, ssDNA is dissolved in 20 μ l TE damping fluids, ssDNA is become to the dsDNA of one end band vitamin H with the primer of mark vitamin H through pcr amplification.Separate ssDNA through vitamin H-Streptomycin sulphate magnetic bead, for the ssDNA library of next round screening, until finishing screen is selected the aptamer of energy specific combination lead ion.

Claims

One kind can Real-Time Monitoring solid phase SELEX method, it is characterized in that: by Screening target being fixed in solid phase carrier polystyrene micro-reaction plate, the strand Nucleotide storehouse of downstream end mark being crossed to vitamin H combines with it, after rinsing through incubation reaction, add the effect of Streptomycin sulphate avidin-fluorescent substance qualification epicycle screening, with specific elutriant by the ssDNA wash-out of combination and amplification, for next round screening, until finishing screen is selected the aptamer of energy specific combination target.
2. screening method according to claim 1, is characterized in that: biotin labeled strand Nucleotide storehouse.
3. screening method according to claim 1, it is characterized in that: described fluorescein is fluorescein isothiocyanate, Tetrachlorofluorescein, chlordene fluorescein, carboxyl-4 ', 5 '-bis-chloro-2 ', 7 '-dimethyl fluoresceins, CY3, CY5, tetraethylrhodamine, TRITC.
4. according to the screening method described in claim 1-3 any one, it is characterized in that: the number that the size of fluorescence intensity combines with target with biotin labeled strand Nucleotide with Streptomycin sulphate avidin-fluorescent substance becomes positive correlation.
5. Screening target comprises enzyme, somatomedin, antibody, the generegulation factor, cell adhesion factor, phytohemagglutinin, complete virion, pathogenic bacteria etc.; Also include the detection of the small-molecule substances such as organic dye, metal ion, medicine, amino acid.
6. the detection of screening method small molecular material according to claim 5, its feature comprises the coupling of small molecules and respective carrier, and wherein carrier proteins mainly comprises the poly-lysine of bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, human serum albumin and synthetic.
7. the application of screening method claimed in claim 5 in the various targets of screening.