CN115838729A - Aptamer AS2-3 of human thrombin protein, and screening method and application thereof - Google Patents

Abstract

The invention discloses a nucleic acid aptamer AS2-3 of human thrombin protein and a screening method and application thereof, wherein the sequence of the nucleic acid aptamer AS2-3 is shown AS SEQ ID NO. 1; the screening method comprises the following steps: on the basis of the human thrombin aptamer obtained by previous screening, a specific sequence of the aptamer is reserved for library construction, and screening is carried out again by using the transcription process of a cell-free transcription and translation system after primary screening is carried out by adopting a Capture-SELEX method, so that a nucleic acid aptamer sequence AS2-3 which can be specifically combined with the human thrombin and can respond to 0.5 mu M of human thrombin in the cell-free transcription process in a linear and plasmid mode to inhibit and regulate the transcription of downstream genes is successfully obtained; by combining the two screening methods, the aptamer sequence screening method is hopeful to be applied to aptamer sequence screening of other target ligands, so that the aptamer sequence screening method has the opportunity of replacing a transcription regulation factor as a gene element to be applied to biological circuits for regulation.

Description

Aptamer AS2-3 of human thrombin protein, and screening method and application thereof

Technical Field

The invention specifically relates to a nucleic acid aptamer AS2-3 of human thrombin protein, a screening method and application thereof, and belongs to the technical field of molecular biology.

Background

Thrombin is Na ⁺ Activated allosteric serine proteases, containing a heparin binding site and a fibrin binding site, function as central proteases in the coagulation cascade, and after vascular injury, thrombin is rapidly generated from inactivated prothrombin by cleavage of a series of enzymes, and activated thrombin breaks down fibrinogen into fibrin and forms a clot at the vascular injury to prevent bleeding. Thrombin and inactivated prothrombin play a key role in physiological and pathological coagulation and are involved in a variety of diseases, such as alzheimer's disease and cancer.

An aptamer is a short, single-stranded oligonucleotide (ssDNA or RNA), usually consisting of 20 to 80 nucleotides, with high affinity and specificity for a target ligand. Aptamers are usually screened by the SELEX (systematic evolution of ligands by exponential enrichment) method. Since the aptamer was studied in 1990, 20% of the aptamer papers published to date involved the study of thrombin and its aptamer. Since thrombin aptamers are screened at an early stage and can show strong anticoagulant activity on the procoagulant function of thrombin at nanomolar concentrations, the combination of thrombin and its aptamers is the most common model system for proving aptamer affinity analysis-based conceptual validation.

Most human thrombin assays involve intact alpha-thrombin (295 amino acids), which can be proteolytically generated into beta-thrombin and gamma-thrombin, and the currently well-established aptamers capable of specifically binding thrombin are: 15mer DNA oligonucleotides (5 'GGTTGGTGTGGTTGG-3') interacting with a fibrinogen recognition site which is one of the two anion binding sites of thrombin with dissociation constant K _d 100nM and a 29mer DNA oligonucleotide (5 'AGTCCGTGGTAGGGCAGGTGGGTGACT-3') with higher affinity to the heparin-binding outer surface of thrombin _d ～0.5nM。

With the rapid development of synthetic biology, numerous genetic circuits have been designed and validated, wherein molecular tools for signal sensing and regulation of gene expression are essential for designing and constructing synthetic genetic circuits in cellular and cell-free transcription-translation systems. The existing transcription regulation factors are mature and stable in application, but the number and the types of the transcription regulation factors are limited, so that the construction of a synthetic gene loop in which the transcription regulation factors participate is limited. Therefore, there are researches to use aptamers as gene elements, to regulate downstream genes in a loop in a manner similar to that of a transcription regulatory factor, and to screen aptamer sequences with affinity to any target ligand in a SELEX manner, so that the combination of aptamers and ligands as a regulatory element has a great development space in future researches.

Experiments show that the two thrombin aptamers are not suitable for being directly used in a biological loop as a target ligand in a gene element response system to influence the transcription process, so that mature thrombin is necessarily researched as a model to screen and obtain the aptamer which can be specifically combined with the thrombin in vitro, and the aptamer can be used as a gene element to be applied to the biological loop for gene regulation, so that the limitation caused by the lack of natural regulatory factors is solved.

The Cell-Free Transcription-Translation System (TX-TL) is an in vitro expression System that utilizes crude Cell extracts in place of intact cells to perform the Transcription and Translation processes. Since the natural transcription and translation mechanisms are retained in cell extracts, transcription and translation can be achieved by the addition of exogenous energy (including amino acids, nucleotides, etc. and secondary energy substrates) in vitro. Cell-free transcription translation systems play an important role in the rapid design and testing of the intermediate layers of the prototype loop before the prototype is transferred to a more complex in vivo environment.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the aptamer AS2-3 capable of responding to the human thrombin protein in the biological loop of the cell-free transcription and translation system, and the screening method and the application thereof.

The technical scheme of the invention is as follows:

one of the purposes of the invention is to provide a nucleic acid aptamer AS2-3 of human thrombin protein, wherein the sequence of the nucleic acid aptamer AS2-3 is shown AS SEQ ID NO. 1.

Further, the 5 'end or the 3' end of the aptamer AS2-3 is chemically modified by FITC, amino, biotin or digoxin.

The invention also provides a nucleic acid aptamer AS2-3 which is a mutant or truncated sequence or an extended sequence of the sequence shown in SEQ ID NO. 1.

Further, the mutant or truncated sequence or extended sequence of the aptamer AS2-3 is chemically modified at the 5 'end or the 3' end by FITC, amino, biotin or digoxin.

The second purpose of the invention is to provide a method for screening the aptamer AS2-3 of human thrombin protein, which combines the in vitro SELEX screening mode based on the aptamer with the cell-free transcription process screening.

Further, the screening method comprises the following steps:

(1) Preparation of screening library: a random ssDNA library was prepared as shown by the following sequence:

5’-CGCAACCAGGTCTCTAAGC-N40-TAGGGCAGGTT-N10-AATGTGAGACCGGCCTGTG-3’；

(2) And (3) carrying out forward screening: complementarily pairing the ssDNA library with single-stranded DNA Capture Oligo with a biotin label at the 3' end to form partial double-stranded DNA;

(3) Incubating part of double-stranded DNA obtained in the step (2) with streptavidin magnetic beads overnight, so that the ssDNA library is immobilized on the streptavidin magnetic beads by taking DNA Capture Oligo as a medium, and washing off ssDNA sequences which cannot be immobilized;

(4) Incubating the magnetic bead-CO-ssDNA Library complex obtained in the step (3) with a target ligand human thrombin protein, and competitively binding with a ssDNA aptamer sequence with specific affinity;

(5) Magnetically separating the incubation mixture obtained in the step (4), collecting and eluting to obtain an ssDNA aptamer sequence specifically bound with human thrombin protein, namely an ssDNA enrichment library, and continuously reducing the concentration of a target ligand and increasing the screening pressure in the subsequent screening process;

(6) And (3) PCR amplification: and (3) carrying out PCR amplification on the ssDNA enrichment library obtained in the step (5), wherein the primers for PCR amplification are as follows:

primer P1:5 'CGCAACCAGGTCTCTAAGC-3';

and (3) primer P2:5 'CACACAGGGCCGGTCTCACTATT-3';

(7) ssDNA library acquisition and purification: carrying out enzyme digestion on the enriched dsDNA library with the phosphorylation markers obtained by amplification in the step (6) for 30min by utilizing Lambda Exonuclease in water bath at 37 ℃ to obtain an ssDNA library, and carrying out ethanol precipitation to obtain a secondary ssDNA library for next round of screening;

(8) And (3) circulating screening: taking the secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of (2) to (7) to complete the forward screening;

(9) Negative screening: and (3) taking the ssDNA Library subjected to the last positive screening as an initial Library subjected to negative screening, performing the steps (2) and (3), incubating the magnetic bead-CO-ssDNA Library complex in a cell-free transcription and translation system for 60min before adding the target ligand for incubation, magnetically separating and washing off non-specific sequences bound with the cell-free transcription and translation system, and repeating the screening steps (4) to (7).

Further, the step of screening the cell-free transcription process of step (10) is as follows:

101. after the positive and negative screening is completed, screening in the cell-free transcription process is carried out: amplifying the ssDNA library subjected to positive and negative screening into a dsDNA library by PCR, and connecting a vector with a promoter, an output signal and a terminator with the dsDNA library by using a Golden Gate Assembly method;

102. performing transformation, coating, bacterium selection, colony PCR and gel electrophoresis verification on the ligation product inserted with the dsDNA library in the step 101, selecting a correct inserted strip, recovering a corresponding Colony PCR linear DNA product from the PCR product, and numbering;

103. and (3) carrying out a transcription process on all recovered linear DNA containing a promoter, an aptamer sequence, an output signal and a terminator in a cell-free transcription and translation system containing or not containing the target ligand human thrombin protein, and observing the change of the output signal, thereby selecting the aptamer sequence which can respond to the target ligand and influence the transcription of downstream genes.

The invention also aims to apply the aptamer AS2-3 of the human thrombin protein to the identification, analysis and detection of the human thrombin protein.

The fourth purpose of the invention is to apply the aptamer AS2-3 of human thrombin protein to the anticoagulant activity in the coagulation enzymatic coagulation function.

Compared with the prior art, the invention has the beneficial effects that:

1. the aptamer AS2-3 provided by the invention is non-toxic, small in molecular weight and easy to synthesize and mark;

2. the synthesis cost of the aptamer AS2-3 provided by the invention is lower than that of antibody preparation, and the aptamer AS2-3 has the advantages of short period and good reproducibility.

3. The aptamer AS2-3 provided by the invention can be 'plug and play' AS a gene element, has small influence on transcription after being inserted into the downstream of a promoter, and can specifically recognize a target ligand thrombin protein in a complex cell-free transcription and translation system so AS to play a role in regulation. A

4. The invention combines the SELEX screening mode and the cell-free transcription process screening, the obtained human thrombin protein aptamer AS2-3 not only ensures the specific combination with a target ligand, but also is suitable for the regulation and control of the transcription level of a gene loop in a cell-free transcription and translation system, provides a new method and thought for screening other target aptamers and ligand combinations for the regulation and control of the gene loop, and has wide application prospect and important scientific, social and economic values in the fields of using the aptamer AS a gene element and the like.

Drawings

FIG. 1 is a bioinformatics mimetic diagram of the spatial structure of the aptamer AS2-3 of the present invention;

FIG. 2 is a schematic diagram of the screening process of the aptamer AS2-3 of the present invention;

FIG. 3 shows the output results of fluorescence reports of different concentrations of human thrombin protein in a linear DNA response cell-free system obtained when the aptamer AS2-3 of the present invention is inserted AS a genetic element into the downstream of pJ23151 promoter and 3WJdB is used AS a reporter gene: wherein, the graph (A) is the change of the fluorescence peak value before and after adding human thrombin with different concentrations, the graph (B) is the real-time fluorescence change, and the graph (C) is the fluorescence rate change;

FIG. 4 shows the output results of the fluorescence reports of the obtained plasmid DNA responding to human thrombin proteins with different concentrations in a cell-free system when the aptamer AS2-3 of the invention is inserted AS a genetic element into the downstream of pJ23151 promoter and 3WJdB is used AS a reporter gene: wherein, the figure (A) is the result of the plasmid DNA of the control group without the aptamer inserted, and the figure (B) is the result of the experimental group;

FIG. 5 is a full-length and truncated circular dichroism CD map of the aptamer AS2-3 of the invention: wherein, the graph (A) is the CD map of 40mer AS2-3 with N40 part reserved after 21nt truncation from the 3 'end, the graph (B) is the CD map of 51mer AS2-3 with N40 and Docking Sequence part reserved after 10nt truncation from the 3' end, the graph (C) is the CD map of full-length 61mer AS2-3, and the graph (D) is the CD map of the control group 29mer TBA;

FIG. 6 is a schematic diagram of the analysis of the affinity isothermal titration calorimeter for binding of aptamer AS2-3 to human thrombin protein in the present invention.

Detailed Description

The invention will be further described with reference to the accompanying drawings and preferred embodiments, which are given for illustration only and are not intended to limit the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified;

in the quantitative tests in the following examples, three replicates were set up and the results averaged.

The experimental methods in the following examples are all conventional methods unless otherwise specified;

example 1

An aptamer AS2-3 of human thrombin protein, wherein the sequence of the aptamer AS2-3 is shown AS SEQ ID NO. 1; carrying out any chemical modification of FITC, amino, biotin or digoxin at the 5 'end or the 3' end of the aptamer AS2-3;

SEQ ID NO:1：

TACAGGCAGTAAGGCCGTGTCATCAGGATGGGCACTTTCCTAGGGCAGGTTAGCAACAGGA

the linear DNA for transcription corresponding to the aptamer AS2-3 with the most obvious change of the output signal before the addition of the human thrombin protein is obtained by the screening method, the Shanghai biological technology limited company is entrusted with sequencing to obtain the aptamer sequence shown AS SEQ ID NO. 1, and the aptamer sequence is analyzed by an RNAFld network platform at the temperature of 25 ℃ and the content of 100mM Na ⁺ ，1mM Mg ⁺ The space structure diagram of the sequence of the aptamer AS2-3 under the conditions is shown in figure 1.

Magnetic shoe, a nucleic acid aptamer AS2-3 of human thrombin protein, is a mutant or truncated sequence or an extended sequence of a sequence shown in SEQ ID NO. 1; and carrying out any chemical modification of FITC, amino, biotin or digoxin on the 5 'end or the 3' end of the mutant or the truncated sequence or the extension sequence of the aptamer AS 2-3.

Example 2

As shown in FIG. 2, the method for screening the aptamer AS2-3 of human thrombin protein in example 1 comprises the following steps:

(1) Preparation of screening library: designing a middle fixed region to be 11 nucleotides, wherein the design reserves the conserved sequence part of the G-tetramer of the existing thrombin aptamer, random regions at two ends of the fixed part are 40 and 10 nucleotides respectively, and a random ssDNA library of 19 nucleotides of a 5 'fixed primer part and a 3' fixed primer part (5 '-CGCAACCAGGTTCTAAGC-N40-TAGGCAGGTT-N10-AATGTGTGAGACCGGCCTGTG-3', wherein N40 is 40 random bases, N10 is 10 random bases, and N40 and N10 in the synthesized library have more than ten million different base sequence combinations), and committing to the company Limited of bioengineering, and synthesizing the antibody with the biotin label;

(2) And (3) carrying out forward screening: determining the concentration of the synthesized initial ssDNA library and single-stranded DNA Capture Oligo (CO) with a biotin tag at the 3' end, annealing the ssDNA library with CO at an equimolar ratio in a Washing Buffer (Washing Buffer:5mM Tris-HCl,1M NaCl,0.5mM EDTA) system using 200. Mu.L of streptavidin magnetic beads (1 mg of magnetic beads can bind >400pmol of biotin-tagged Capture Oligo DNA) for each round of screening to approximately 320pmol CO; and (3) annealing procedure: denaturation at 95 deg.C for 10min at-0.1 deg.C/sec and x 350 times, standing at 60 deg.C for 5min and 4 deg.C;

(3) Adsorbing 200 mu L of magnetic beads on a magnetic frame to remove supernatant, repeatedly Washing the magnetic beads for three times by using 200 mu L of Washing Buffer, and carrying out overnight rotary incubation on the annealed ssDNA Library-CO compound and the magnetic beads on a rotator at 35rpm/min to ensure that the ssDNA Library-CO compound and the magnetic beads are combined through biotin labeling;

(4) Adsorbing the mixture incubated overnight in the step (3) on a magnetic frame to collect a supernatant, repeatedly Washing the magnetic bead mixture for 4 times by using 200 mu L Washing Buffer, and collecting and numbering the supernatant each time;

(5) mu.L Binding Buffer (0 mM Tris-HCl,100mM NaCl,1mM MgCl) containing 1. Mu.M human thrombin target ligand ₂ ) Adding the mixture into a cleaned ssDNAlibrary-CO-magnetic bead compound, uniformly mixing, placing the mixture on a rotator for 35r/min, incubating at 4 ℃ for 60min, competitively Binding a target ligand with an aptamer sequence with affinity, separating the target ligand from the CO-magnetic bead, placing the mixture on a magnetic frame for adsorption, collecting supernatant, eluting the supernatant by using the ligand with the same concentration contained in the equivalent-volume Binding Buffer, reserving the supernatant, repeating the elution for 2 times, and numbering and eluting 1/2/3 respectively (continuously reducing the concentration of the target ligand in the system to 0.25 mu M in the forward screening process, and increasing the screening pressure);

(6) Using 100. Mu.L SSC Buffer (20 mM Tris-HCl,2mM MgCl) ₂ ，5mM KCl，1mM CaCl ₂ 100mM NaCl), incubating for 10min at 95 ℃, and dissociating the base complementary pairing between the CO and the ssDNAlibrary which is not combined with the target ligand at high temperature, repeating for 3 times, and collecting the supernatant with the number HOT1/2/3 each time;

(7) RT-qPCR absolute quantitative calculation elution rate monitoring and screening process, diluting ssDNA Library used in the screening to 2 ng/mu L and using the diluted ssDNA Library as an amplification template for establishing a standard curve, and performing gradient dilution 10 ^-1 ～10 ^-7 Simultaneously, eluting 1/2/3 and HOT1/2/3 of the serial numbers and finally diluting the magnetic beads to proper concentration for RT-qPCR amplification; calculating R according to the standard curve ² Values and amplification efficiencies, calculating the copy numbers of elution 1/2/3, HOT1/2/3 and final beads, and then substituting into the formula: calculating the elution rate by { (elution 1+ elution 2+ elution 3) copy number/(HOT 1+ HOT2+ HOT3+ final bead) copy number }. Times.100%; 20 μ L amplification system: 10 μ L of 2 XSSYBR qPCR Supermix, 2 μ L each of primers P1 and P2, 1 μ L of DNA template, made up to 20 μ L with DEPC water; and (3) amplification procedure: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 20s, annealing at 58 ℃ for 30s, reading data, extension at 72 ℃ for 30s, and running for 39 cycles; melting curve program: reading data at 95 ℃ for 10s,65 ℃ for → 95 ℃ for 0.5 ℃/0.5 s;

wherein, the RT-qPCR amplification primer is as follows:

primer P1:5 'CGCAACCAGGTCTCTAAGC-3';

and (3) primer P2:5 'CACACAGGGCCGGTCTCACTATT-3';

(8) Mixing 1/2/3 of elution obtained by competitive binding of targets in each round of screening process uniformly into a 1.5mL centrifuge tube, adding a 3M sodium acetate solution (pH = 5.2) with the volume of 1/10 of the mixed liquid volume, adding absolute ethyl alcohol with the volume of 2.5 times of the mixed liquid volume, mixing uniformly, placing the mixture in a refrigerator at minus 20 ℃ for 60min, then performing refrigerated centrifugation at 12000rpm and 4 ℃ for 30min to precipitate DNA, carefully taking out the supernatant, sucking out the supernatant by using a pipette gun completely, adding 19 mu L DEPC water into the centrifuge tube after the ethanol is volatilized completely, oscillating to completely dissolve the precipitated DNA into water, and amplifying by using high fidelity ExTaq enzyme;

wherein, the amplification primers are as follows: SE-XP3-F:5' CGCAACCAGGTCTTAAGC-3 ' and SE-XP4-5' P:5 'CACACAGGGCCGGTCTCACTATT-3';

the 50 μ L amplification system was: 19 μ L DEPC water + ssDNA template, 1 μ L dNTP Mix, 25 μ L2 XBuffer Mix, 2 μ L each of primers SE-XP3-F and SE-XP4-5' P, 1 μ L High-Fidelity DNA polymerase; the amplification procedure was: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 10s, running for 8-10 cycles, and final extension at 72 ℃ for 5min (note that due to the sequence diversity of ssDNA libraries, the number of amplification cycles should be maintained at 8-10 cycles in order to reduce non-specific amplification);

(9) Precipitating the amplified product again according to the method to obtain a dsDNA library with a 5' phosphorylation marker, dissolving the dsDNA library with a proper amount of water, measuring the concentration, carrying out enzyme digestion on the dsDNA for 30min by using Lambda Exonuclease in water bath at 37 ℃, and precipitating the ssDNA library with ethanol again to obtain a ssDNA library which is used for the next round of screening;

wherein, the 50 mu L enzyme digestion system comprises: 5 μ g dsDNA,5 μ L10 XExonuclease Reaction Buffer,1 μ L Lambda Exonuclease, made up to 50 μ L with DEPC water;

(10) And (3) carrying out negative screening: taking an ssDNA Library obtained by enrichment after 8 rounds of positive screening as an initial Library of negative screening, wherein the difference of the screening process is that the ssDNA Library-CO-magnetic bead complex is incubated for 60min at normal temperature in a Cell-free system before adding a target ligand for incubation, removing a non-specific sequence combined with the Cell-free system, replacing the system with a Binding Buffer, and washing magnetic beads;

(11) Screening in the gene loop transcription process of the cell-free transcription and translation system; firstly amplifying ssDNA library after 12 rounds of primary screening into dsDNA with correct enzyme cutting sites and recognition sites, then inserting the library sequence into a vector fragment containing a framework, a promoter pJ23151, a promoter 3WJdB and a terminator by a Golden Gate Assembly method to construct a complete circular ligation product, and dividing the ligation product into 10 groups of transformed clone bacteria E.coli DH5 alpha competent cells.

(12) 1000 single colonies are picked on 10 plates with successful transformation and are subjected to Colony PCR, agarose gel electrophoresis shows that the connection is successful and 832 linear DNAs for transcription with a complete promoter pJ23151, a dsDNA aptamer sequence, an output signal 3WJdB and a terminator can be amplified, the linear DNAs for transcription are recovered, and the single colonies corresponding to the single colonies are stored.

(13) Preparing a cell-free transcription and translation system, screening 832 transcripts inserted with aptamer sequences in the cell-free system in the presence of target ligands in the transcription process by using linear DNA, and observing the difference of fluorescence change of 3WJdB of output signals before and after adding human thrombin protein.

Example 3

The aptamer AS2-3 is used for regulating and controlling the gene element, and meanwhile, the robustness of the aptamer AS2-3 used for regulating and controlling the gene element is verified;

RNA aptamer 3WJdB is taken AS report output, and after successful transcription, the RNA aptamer 3WJdB is specifically combined with small molecule DFHBI to emit fluorescence, so that the regulation effect of representing the nucleic acid aptamer AS2-3 AS a gene element is realized; selecting pJ23151-AS2-3-3WJdB-T500 AS the linear DNA for transcription obtained by purifying a product after colony PCR in the embodiment 1, adding the linear DNA for transcription into a TX-TL reaction system, reacting for 4 hours at 29 ℃ with the final concentration of 50nM and the concentrations of human thrombin protein of 0, 0.5 and 1 μ M respectively, and monitoring the fluorescence characterization transcription efficiency under the conditions of exciting light 472nM and emitting light 507nM in real time by using a multifunctional microplate reader;

as shown in figure 3, the linear DNA for transcription corresponding to the aptamer AS2-3 can respond to the addition of a target ligand human thrombin in the transcription process, the fluorescence peak value is obviously reduced, the real-time fluorescence and fluorescence rate of an experimental group added with human thrombin protein are always lower than those of a control group not added, and the inhibition effect is more obvious when the ligand concentration is increased. Compared with a control group, the nucleic acid aptamer AS2-3 serving AS a gene element is inserted into a gene loop, downstream transcription is not influenced, the sequence structure of the nucleic acid aptamer AS2-3 is relatively loose, and transcription can be obviously inhibited only in the presence of a thrombin ligand, which is probably because the structure of the thrombin ligand becomes more compact due to the combination of thrombin and the loose aptamer sequence, so that RNA polymerase is difficult to perform downstream, and inhibition and regulation of a transcription process are expressed;

selecting a preservation strain corresponding to the nucleic acid aptamer AS2-3 in the example 1, shaking the bacteria and culturing overnight to extract plasmids to obtain stable plasmid DNA for transcription: V-pJ23151-AS2-3-3WJdB-T500 and control plasmid DNA: V-pJ23151-3WJdB-T500, adding plasmid DNA for transcription into a TX-TL reaction system, reacting for 6h at 29 ℃ with the final concentration of 30nM and the concentrations of human thrombin protein of 0, 0.5 and 1 μ M respectively, and monitoring the fluorescence characterization transcription efficiency under excitation light 472nM and emission light 507nM in real time by using a multifunctional microplate reader;

as shown in FIG. 4, the results show that the transcription efficiency of the aptamer AS2-3 in the system of the experimental group shows different reductions in the presence of human thrombin, while the fluorescence of the control group without aptamer sequence inserted downstream of the promoter has no major influence, and further show that the human thrombin can specifically recognize the aptamer sequence in the plasmid DNA for transcription to regulate the transcription efficiency of downstream genes, and the inhibition effect is obvious along with the increase of the ligand concentration in the system.

Example 4

Performing circular dichroism chromatogram characterization on the aptamer AS2-3 and the target ligand human thrombin protein and affinity characterization on the aptamer AS2-3 and the target ligand human thrombin protein respectively;

the circular dichroism chromatogram characterization of the aptamer AS2-3 and the target ligand human thrombin protein is specifically carried out AS follows:

(1) Respectively preparing a mixture containing 0.5 mu M human thrombin,5 mu M aptamer AS2-3 ssDNA and the same concentration by using 1 x Binding Buffer, wherein the total volume is 200 mu L, respectively incubating groups containing ssDNA at 95 ℃ for 10min before adding no ligand, immediately placing the groups on ice to denature the ssDNA and reduce the secondary structure of the groups, and adding a target ligand for incubation for 1h and then scanning; setting parameters of a circular dichroism spectrum instrument: the nitrogen flow rate is 4-5L/min, the scanning wavelength is 220-320 nm, the scanning speed is 50nm/min, the bandwidth is 1nm, and the accumulated 3 times of scanning data are averaged;

(2) Truncating the nucleic acid aptamer AS2-3, and reserving 40 mers of an N40 part, 51 mers of N40 and DS sequence parts and a full-length aptamer sequence 61mer respectively; as shown in FIG. 5, the CD profile shows the maximum peak at about 260-270nm and the minimum peak at about 245nm for aptamer AS2-3, while the CD profile of the existing 29mer TBA shows a typical G-tetramer structure with two maximum peaks at 260/295nm and a minimum peak at 245nm, which shows a change in signal after the addition of 0.5. Mu.M human thrombin protein at 220-240nm (FIG. 5D), indicating that the existing 29mer TBA can bind to the ligand;

(3) When the aptamer AS2-3 obtained by screening is reserved with the N40 part, signals are not obviously different after the ligand is added (figure 5A), and after the aptamer AS2-3 reserved with a full-length 61mer is added with 0.5 mu M human thrombin protein, the signals are obviously changed at 220-240nm and 245nm peak values (figure 5C), which indicates that the 61mer AS2-3 aptamer sequence can be combined with the ligand, and simultaneously indicates that the main combination site is positioned near the 3' end of the sequence;

thus, the circular dichroism data again show that the candidate aptamer screened by the research can not only be combined with a specific ligand, but also play a role in regulation in the transcription process

In addition, the affinity characterization of the aptamer AS2-3 and the target ligand human thrombin protein is specifically performed AS follows: an ITC experiment of human thrombin-bound aptamer was performed using a Nano ITC isothermal titration calorimeter of Waters corporation, 1500. Mu.L of 1. Mu.M aptamer candidate was heated in a 1. Mu.M Binding Buffer at 95 ℃ for 10min at 25 ℃ in each experiment, immediately placed on ice for cooling, and 100. Mu.L of a 1. Mu.M Binding Buffer containing 10. Mu.M human thrombin was loaded into a syringe; carrying out injection titration with the volume of 2 mu L each time, carrying out 25 times of continuous injection at the interval of 300s and the rotating speed of 300rpm; data collected in ITC experiments were analyzed using the Launch NanoAnalyze software and data fitting using the independent model as shown in figure 6 gave its Kd =3.356 μ M.

The fourth purpose of the invention is to apply the aptamer AS2-3 of human thrombin protein to the anticoagulant activity in the coagulation enzymatic coagulation function.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (9)

1. An aptamer AS2-3 of human thrombin protein, wherein the sequence of the aptamer AS2-3 is shown AS SEQ ID NO. 1.

2. The aptamer AS2-3 of human thrombin protein according to claim 1, wherein the aptamer AS2-3 is chemically modified at the 5 'end or the 3' end by any one of FITC, amino, biotin and digoxin.

3. Aptamer AS2-3 of human thrombin protein, which is a mutant or truncated or extended sequence of the sequence shown in SEQ ID NO. 1 of claim 1.

4. The aptamer AS2-3 of human thrombin protein AS claimed in claim 3, wherein 5 'end or 3' end of the mutant or truncated or extended sequence of the aptamer AS2-3 is chemically modified by FITC, amino, biotin or digoxin.

5. The method for screening aptamer AS2-3 of human thrombin protein according to claim 1 or 3, wherein the aptamer-based in vitro SELEX screening method and the cell-free transcription process screening method are combined.

6. The method for screening the aptamer AS2-3 of human thrombin protein according to claim 5, comprising the following steps:

(1) Preparation of screening library: a random ssDNA library was prepared as shown by the following sequence:

5’-CGCAACCAGGTCTCTAAGC-N40-TAGGGCAGGTT-N10-AATGTGAGACCGGCCTGTG-3’

(2) And (3) carrying out forward screening: complementarily pairing the ssDNA library with single-stranded DNA Capture Oligo with a biotin label at the 3' end to form partial double-stranded DNA;

(3) Incubating part of double-stranded DNA obtained in the step (2) with streptavidin magnetic beads overnight, so that the ssDNA library is immobilized on the streptavidin magnetic beads by taking DNA Capture Oligo as a medium, and washing off ssDNA sequences which cannot be immobilized;

(4) Incubating the magnetic bead-CO-ssDNA Library complex obtained in the step (3) with a target ligand human thrombin protein, and competitively binding with a ssDNA aptamer sequence with specific affinity;

(5) Magnetically separating the incubation mixture obtained in the step (4), collecting and eluting to obtain an ssDNA aptamer sequence specifically bound with human thrombin protein, namely an ssDNA enrichment library, and continuously reducing the concentration of a target ligand and increasing the screening pressure in the subsequent screening process;

(6) And (3) PCR amplification: and (3) carrying out PCR amplification on the ssDNA enrichment library obtained in the step (5), wherein the primers for PCR amplification are as follows:

a primer P1:5 'CGCAACCAGGTCTCTAAGC-3';

and (3) primer P2:5 'CACACAGGGCCGGTCTCACTATT-3';

(7) ssDNA library acquisition and purification: carrying out enzyme digestion on the enriched dsDNA library with the phosphorylation markers obtained by amplification in the step (6) for 30min by utilizing Lambda Exonuclease in water bath at 37 ℃ to obtain an ssDNA library, and carrying out ethanol precipitation to obtain a secondary ssDNA library for next round of screening;

(8) And (3) circulating screening: taking the secondary ssDNA library obtained in the step (7) as a secondary library for the next round of screening, and repeating the screening processes of (2) to (7) to complete the forward screening;

(9) Negative screening: taking the ssDNA Library subjected to the last positive screening as an initial Library subjected to the negative screening, performing the steps (2) and (3), incubating the magnetic bead-CO-ssDNA Library complex in a cell-free transcription and translation system for 60min before adding a target ligand for incubation, magnetically separating and washing off non-specific sequences bound with the cell-free transcription and translation system, and repeating the screening steps (4) to (7);

(10) After the positive and negative screening is completed, the screening of the cell-free transcription process is performed.

7. The method for screening the aptamer AS2-3 of human thrombin protein according to claim 6, wherein the cell-free transcription process screening of step (10) comprises the following steps:

101. amplifying the ssDNA library subjected to positive and negative screening into a dsDNA library by PCR, and connecting a vector with a promoter, an output signal and a terminator with the dsDNA library by using a Golden Gate Assembly method;

102. performing transformation, coating, bacterium selection, colony PCR and gel electrophoresis verification on the ligation product inserted with the dsDNA library in the step 10-1, selecting a correct inserted strip, recovering a corresponding Colony PCR linear DNA product from the PCR product, and numbering;

103. and (3) carrying out a transcription process on all recovered linear DNA containing a promoter, an aptamer sequence, an output signal and a terminator in a cell-free transcription and translation system containing or not containing the target ligand human thrombin protein, and observing the change of the output signal, so as to select the aptamer sequence which can respond to the target ligand and influence the transcription of downstream genes.

8. Use of the aptamer AS2-3 of human thrombin protein AS claimed in claim 1 or 3 for identification, analysis and detection of human thrombin protein.

9. Use of the aptamer AS2-3 of human thrombin protein AS claimed in claim 1 or 3 for anticoagulant activity in the coagulation enzymatic coagulation function.

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