Preparation method of specific inflammatory factor nucleic acid aptamer

Abstract

The invention relates to a preparation method of a specific inflammatory factor nucleic acid aptamer. The preparation method comprises the following steps: providing a high-purity active protein sample of inflammatory factors such as TNF-alpha, IL-16, C5a, MMP-8 or IL-3; designing an RNA library containing a random sequence, and covering a diversified secondary structure; the method comprises the following steps: eliminating a non-specific binding sequence by using a control protein (such as GM-CSF instead of IL-3) or a low-concentration target protein sample which is similar to a target protein in structure but irrelevant to a function through ultracentrifugation, magnetic bead sorting or ion-exchange chromatography technologies; incubating the library subjected to negative screening and high-purity target protein, and optimizing the binding specificity and affinity of the aptamer through multiple rounds of iterative screening by combining with a magnetic bead separation technology; high-throughput sequencing is adopted to analyze an enriched sequence, surface plasmon resonance (SPR) is utilized to determine affinity, interference of a conservative structural domain is eliminated through negative screening, functional sequences are accurately enriched in combination with positive screening, and the targeting of the aptamer to inflammatory factors is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of RNA screening, and in particular to a method for preparing a specific inflammatory factor nucleic acid aptamer. Background Art

[0002] SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is an in vitro evolution method for screening nucleic acid aptamers. This method constructs a library containing a large number of random nucleic acid sequences and interacts with the target protein to screen out nucleic acid sequences that can specifically bind to the target protein. Generally speaking, SELEX mainly includes several key steps: first, the random nucleic acid library is incubated with the target protein so that a part of the sequence binds to the target protein, while the unbound or weakly bound sequences are washed away. Then, the nucleic acid aptamers with strong binding force are eluted under specific conditions, and this part of the sequence is enriched by PCR amplification to enter the next round of screening. After multiple rounds of screening, aptamers with high affinity and high specificity are finally obtained.

[0003] However, since a large number of non-specific binding sequences may be generated during the SELEX process, researchers usually use negative selection to remove nucleic acid sequences that may non-specifically bind to components other than the target protein. In the negative selection process, the random nucleic acid library is first incubated with non-target components to remove sequences that may bind to these non-target substances, followed by positive screening to enrich nucleic acid sequences that truly have a high affinity for the target protein.

[0004] Although SELEX technology is widely used in the screening of nucleic acid aptamers, there are still many technical defects in the actual operation, especially in the screening of TNF-α aptamers, mainly including the following problems:

[0005] The source and purity of the target protein. Currently, SELEX screening often relies on exogenous proteins extracted from commercial channels or laboratories, such as TNF-α extracted from cell culture supernatants or recombinant expression systems. However, these proteins are often accompanied by a certain amount of impurities, such as non-target proteins, protein aggregates or degradation products, which may affect the screening effect of aptamers. For example, during the SELEX process, if TNF-α complexes or degradation fragments exist, the screened aptamers may tend to recognize these non-target components rather than free TNF-α.

[0006] The selection of negative screening samples plays a key role in the specificity of SELEX aptamer screening. Traditional negative screening methods usually use buffers or solutions containing similar protein components (such as serum or protein extracts) as negative screening samples to remove non-specific binding sequences. However, for the screening of TNF-α aptamers, the defect of conventional negative screening samples is that they cannot effectively distinguish between free TNF-α and TNF-α-antibody complexes. Many TNF-α-related physiological functions are affected by TNF-α-antibody complexes. For example, in a disease environment, TNF-α may bind to its natural receptor or antibody to form a stable complex. Since the SELEX process usually uses commercially purchased or extracted TNF-α, these samples may contain a certain proportion of TNF-α complexes, and conventional negative screening methods cannot remove the influence of these complexes, resulting in the final screened aptamers having a high affinity for TNF-α complexes, but a low affinity for free TNF-α, thereby reducing its practical application value.

[0007] Non-specific binding problem: During the SELEX process, the aptamer may not only bind to the target protein, but also have non-specific interactions with other proteins, lipids or small molecules in the solution. For example, complex biological fluids such as CSF (cerebrospinal fluid) or plasma contain a large number of proteins, such as albumin, globulin and cytokines, which may have weak non-specific binding with nucleic acid aptamers, resulting in the enrichment of sequences with high affinity for non-target proteins during the SELEX screening process, affecting the specificity of the final aptamer. Summary of the invention

[0008] The purpose of the present invention is to provide a method for preparing a specific inflammatory factor nucleic acid aptamer to solve the above technical problems.

[0009] The present invention provides a method for preparing a specific inflammatory factor nucleic acid aptamer, comprising the following steps:

[0010] For the desired inflammatory factors, the target protein is expressed and purified in the E. coli expression system through the gene recombination technology Gibson Assembly homologous recombination;

[0011] constructing a library containing a large number of random nucleic acid sequences;

[0012] Use control protein to incubate with random RNA library to remove non-specific binding sequences;

[0013] The negatively screened RNA library is incubated with a high-purity, high-activity target protein, and the specificity and affinity of the aptamer are gradually optimized through multiple rounds of iterative screening;

[0014] Furthermore, the target inflammatory factor is selected from one or more of TNF-α, IL-16, C5a, MMP-8 and IL-3.

[0015] Furthermore, in the negative screening step, a control protein with a similar structure to the target protein but irrelevant function is used.

[0016] Furthermore, high-throughput sequencing (NGS) was used to analyze the sequence information of enriched aptamers and screen out high-frequency sequences.

[0017] Furthermore, the positive screening step includes incubating the negatively screened RNA library with a high-purity target protein sample, separating the specifically bound aptamers using magnetic beads, and optimizing the specificity and affinity of the aptamers through multiple rounds of iterative screening.

[0018] Furthermore, the affinity of the aptamer was determined using surface plasmon resonance technology.

[0019] Furthermore, the screened high-affinity aptamers are chemically modified to improve their stability, biological activity or in vivo circulation time.

[0020] Furthermore, the chemical modification is selected from one or more of 2'-fluorine modification, cholesterol modification, and LNA modification.

[0021] The beneficial effects achieved by the present invention using the above structure are as follows:

[0022] Through the negative screening step, control proteins with similar structures but irrelevant functions or low-content target protein samples are introduced to effectively remove non-specific binding sequences, avoid cross-reactions caused by conservative domains in traditional screening, and enhance the aptamer's ability to accurately identify target inflammatory factors. This method is applicable to a variety of inflammatory factors such as TNF-α, IL-16, C5a, MMP-8, IL-3, etc. By modularly adjusting control proteins and screening conditions, it can be quickly expanded to other targets, reducing the cost and time of aptamer development. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the predicted secondary structure diagram of the Apt TNF-α aptamer RNA of the present invention.

[0024] Figure 2 This is the predicted secondary structure of the Apt IL-16 aptamer RNA of the present invention.

[0025] Figure 3 This is the predicted secondary structure of the Apt C5a aptamer RNA of the present invention.

[0026] Figure 4 This is the predicted secondary structure of the Apt MMP-8 aptamer RNA of the present invention.

[0027] Figure 5 This is the predicted secondary structure of the Apt IL-3 aptamer RNA of the present invention. DETAILED DESCRIPTION

[0028] In order to make the contents of the present invention more clearly understood, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0029] The specific implementation of the present invention is as follows:

[0030] Example 1

[0031] A method for preparing a TNF-α aptamer, which adds a negative screening step to remove sequences that bind to TNF-α complexes or other non-specific bindings during the SELEX screening process.

[0032] The principle of negative screening is to use non-target proteins or proteins complexed with TNF-α to screen out non-specific binding sequences in the random nucleic acid library in advance, thereby improving the specificity of the final aptamer.

[0033] The following steps are involved:

[0034] First, humanized TNF-α target protein was prepared by Gibson Assembly homologous recombination technology.

[0035] The TNF-α gene fragment was amplified from the DNA library using specific upstream and downstream primers and cloned into the pET-28a(+) plasmid vector.

[0036] The recombinant plasmid was transformed into BL21(DE3) Escherichia coli competent cells, cultured by shaking and induced by IPTG for protein expression.

[0037] During the culture process, CO 2 The incubator maintains temperature and shaking speed control to ensure efficient protein expression.

[0038] After the cells were collected, they were lysed by ultrasonication and centrifuged at 12,000 g for 20 min at 4°C in a tabletop refrigerated centrifuge to remove cell debris and collect the soluble protein fraction.

[0039] Use Ni 2+ The His-Tag was purified by affinity chromatography to remove non-specific binding proteins and ensure high purity of TNF-α.

[0040] Finally, the purity was verified by SDS-PAGE and Western Blot and stored at -80°C for subsequent SELEX screening.

[0041] First, negative screening was performed by adding the RNA random library at a concentration of 10-100 nM to the system treated with non-TNF-α target protein. 2 Incubate at 37°C in an incubator for 30-60 minutes.

[0042] Subsequently, the non-specifically bound sequences were removed by centrifugation at 10,000 g and 4° C. for 10 minutes using an ultracentrifuge, and the unbound nucleic acid sequences were collected in the supernatant.

[0043] Next, magnetic bead purification was performed using a magnetic stand and magnetic beads.

[0044] First, the magnetic beads were pre-washed and suspended in PBS buffer. Then, the nucleic acids in the supernatant were mixed with the magnetic beads and incubated at room temperature for 15 minutes. The magnetic beads were adsorbed using a magnetic stand to remove unbound nucleic acids. The beads were washed multiple times and the purified nucleic acid library was finally collected.

[0045] The nucleic acid library finally obtained was used for the next stage of positive screening to enrich aptamers that specifically bind to TNF-α.

[0046] The nucleic acid library after negative screening was added to the TNF-α protein solution expressed by Escherichia coli and purified by chromatography column, and incubated at 37°C for 1 hour to enrich high-affinity aptamers.

[0047] Then, TNF-α affinity magnetic beads are used to specifically bind the nucleic acid-TNF-α complex, and the conjugate is collected by magnetic separation to ensure that the screened aptamers have a high affinity for free TNF-α.

[0048] First, the magnetic beads were washed with a low-salt buffer (0.1 M NaCl) to remove non-specific binding sequences.

[0049] The specific nucleic acid aptamers bound to the magnetic beads were then eluted with a high salt buffer (gradually increasing to 1 M NaCl).

[0050] After PCR amplification of the eluted sequences, a new enriched library is obtained and enters the next round of SELEX screening, with a total of 8-12 rounds of screening.

[0051] The enriched sequences obtained after screening were cloned and sequenced and analyzed using high-throughput sequencing technology (NGS) to obtain candidate aptamer sequences.

[0052] Subsequently, the affinity of the aptamer to TNF-α was detected using the surface plasmon resonance (SPR) method, and high-affinity aptamers with Kd values ​​below 10 nM were determined.

[0053] In order to enhance the stability of the aptamer, 2'-fluorine (2'-F) modification was used to significantly improve the biological stability of the RNA aptamer.

[0054] In addition, the 5' or 3' end of the aptamer is fluorescently labeled (such as FAM or Cy5) or biotin-labeled for subsequent use in the development of biosensors or diagnostic kits.

[0055] The aptamer of this embodiment can specifically bind to free TNF-α, avoiding the interference of TNF-α-antibody complex during the screening process, ensuring that the aptamer has higher specificity and affinity for free TNF-α, and is suitable for TNF-α detection in complex body fluid environments such as CSF and serum, and has broad clinical diagnosis and treatment application prospects.

[0056] Example 2

[0057] A method for preparing an IL-16 aptamer, which adds a negative screening step to remove sequences that bind to IL-16 complexes or other non-specific bindings during the SELEX screening process.

[0058] The following steps are involved:

[0059] First, humanized IL-16 target protein was prepared by Gibson Assembly homologous recombination technology.

[0060] The IL-16 open reading frame (ORF) was amplified from the plasmid containing the IL-16 gene, homologous recombination arms were introduced using specific primers, and cloned into the pET-28a(+) plasmid vector.

[0061] The recombinant plasmid was transformed into BL21(DE3) Escherichia coli competent cells, cultured by shaking and induced by IPTG for protein expression.

[0062] During the culture process, CO 2 The incubator maintains temperature and shaking speed control to ensure efficient protein expression.

[0063] After the cells were collected, they were lysed by ultrasonication and centrifuged at 12,000 g for 20 min at 4°C in a tabletop refrigerated centrifuge to remove cell debris and collect the soluble protein fraction.

[0064] Use Ni 2+ His-Tag purification was performed using an affinity chromatography column to remove non-specific binding proteins and ensure high purity of IL-16.

[0065] Finally, the purity was verified by SDS-PAGE and Western Blot and stored at -80°C for subsequent SELEX screening.

[0066] First, negative screening was performed by adding RNA random library to BSA or blank buffer at a concentration of 10-100 nM and incubating in CO2 Incubate at 37°C in an incubator for 30-60 minutes.

[0067] After incubation with the control protein, the unbound nucleic acid was directly collected in the filtrate by ultrafiltration centrifugation (10 kDa molecular weight cutoff).

[0068] First, the magnetic beads were pre-washed and suspended in PBS buffer. Then, the nucleic acids in the supernatant were mixed with the magnetic beads and incubated at room temperature for 15 minutes. The magnetic beads were adsorbed using a magnetic stand to remove unbound nucleic acids. The beads were washed multiple times and the purified nucleic acid library was finally collected.

[0069] The nucleic acid library finally obtained was used for the next stage of positive screening to enrich aptamers that specifically bind to IL-16.

[0070] The nucleic acid library after negative screening was added to the IL-16 protein solution expressed in Escherichia coli and purified by chromatography column, and incubated at 37°C for 1 hour to enrich high-affinity aptamers.

[0071] Then, IL-16 affinity magnetic beads were used to specifically bind the nucleic acid-IL-16 complex, and the conjugate was collected by magnetic separation to ensure that the screened aptamers had a high affinity for free IL-16.

[0072] First, the magnetic beads were washed with a low-salt buffer (0.1 M NaCl) to remove non-specific binding sequences.

[0073] The specific nucleic acid aptamers bound to the magnetic beads were then eluted with a high salt buffer (gradually increasing to 1 M NaCl).

[0074] After PCR amplification of the eluted sequences, a new enriched library is obtained and enters the next round of SELEX screening, with a total of 8-12 rounds of screening.

[0075] The enriched sequences obtained after screening were cloned and analyzed using high-throughput sequencing technology (NGS) to obtain candidate aptamer sequences.

[0076] Subsequently, surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC) were used to detect the affinity of the aptamer to IL-16, and high-affinity aptamers with Kd values ​​below 10 nM were determined.

[0077] In order to enhance the stability of the aptamer, 2'-fluorine (2'-F) modification was used to significantly improve the biological stability of the RNA aptamer.

[0078] In addition, the 5' or 3' end of the aptamer is fluorescently labeled (such as FAM or Cy5) or biotin-labeled for subsequent use in the development of biosensors or diagnostic kits.

[0079] The aptamer of this embodiment can specifically bind to free IL-16, avoiding the interference of IL-16-antibody complex during the screening process, ensuring that the aptamer has higher specificity and affinity for free IL-16, and is suitable for IL-16 detection in complex body fluid environments such as serum, and has broad clinical diagnosis and treatment application prospects.

[0080] Example 3

[0081] A method for preparing a C5a aptamer comprises the following steps:

[0082] The C5a open reading frame (ORF) was amplified from the plasmid containing the C5a gene and homologous recombination arms were introduced.

[0083] The gene was cloned into pET-28a(+) vector (C-terminal His tag) and transformed into BL21(DE3) Escherichia coli.

[0084] Expression was induced using IPTG and the ThermoFisherForma TM 3110CO 2 Incubate in an incubator (37°C, 200 rpm) for 4-6 hours.

[0085] The cells were disrupted by ultrasonication and centrifuged at 4°C (12,000 g × 20 min) to collect the supernatant.

[0086] Ni 2+ The protein was purified by affinity chromatography, and its purity was verified by SDS-PAGE and Western Blot.

[0087] Store in PBS (pH 7.4) and keep at -80°C until use.

[0088] A control protein C5a mutant Δ71-73, which is structurally similar to C5a but functionally irrelevant, was used.

[0089] The RNA random library (10-100 nM) was incubated with the control protein (1 μM) in binding buffer (PBS, 0.1% BSA) at 37°C for 30 minutes.

[0090] Unbound nucleic acids in the filtrate were collected by ultrafiltration centrifugation (10 kDa molecular weight cutoff, 4°C x 3000 g x 15 min).

[0091] The negative selection library (10-100 nM) was incubated with C5a protein (100 nM) in binding buffer at 37°C for 1 hour.

[0092] Add anti-His tag magnetic beads (pre-washed and resuspended in PBS), incubate at room temperature for 15 minutes, separate on a magnetic stand, and discard the supernatant.

[0093] The elution condition was low salt washing (0.1 M NaCl, pH 7.4) × 3 times to remove non-specific binding.

[0094] The specific aptamers were collected by high salt elution (1 M NaCl, pH 7.4).

[0095] The eluted products were amplified by PCR and screened for 8-12 rounds, with the incubation time gradually shortened in each round (down to 30 min after the 8th round).

[0096] High-throughput sequencing (NGS) was used to screen high-frequency sequences, and RNAstructure was used to predict secondary structures.

[0097] SPR was used to detect the Kd value of candidate aptamers with C5a (target Kd < 10 nM).

[0098] ITC verified the binding thermodynamic parameters (ΔG, ΔH, ΔS).

[0099] High affinity sequences were modified with 2'-fluoro (2'-F).

[0100] The 5' end is labeled with FAM / Cy5 or the 3' end is biotinylated for subsequent sensing or diagnostic applications.

[0101] Example 4

[0102] A method for preparing a MMP-8 aptamer

[0103] The catalytic domain (catalytic domain + hinge region, excluding the propeptide domain) was amplified from the plasmid containing the MMP-8 gene.

[0104] The protein was cloned into the pFastBac vector (N-terminal GST tag) and expressed in Sf9 insect cells using the Bac-to-Bac system.

[0105] Culture conditions: 27°C constant temperature shaker (120 rpm), multiplicity of infection (MOI) = 2, supernatant was collected 72 hours after infection.

[0106] After purification by GST affinity chromatography, the GST tag was removed using PreScission protease.

[0107] The enzyme was further purified by Superdex200 gel filtration chromatography and its activity was verified (fluorescent substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH 2 ).

[0108] Store in 10 mM CaCl 2 The samples were stored in Tris buffer (pH 7.5) at -80°C.

[0109] TIMP-1, a protein with conserved structure but unrelated function in the MMP family, was used to verify that it had no cross-reaction with MMP-8.

[0110] The ssDNA / RNA random library (10-100 nM) and the control protein (1 μM) were mixed in a binding buffer (50 mM Tris, 150 mM NaCl, 10 mM CaCl 2 , pH 7.5) at 37°C for 1 h.

[0111] Negatively charged sequences bound to the control protein were removed by anion exchange chromatography (HiTrapQHP, low salt elution).

[0112] The negative selection library (10-100 nM) was incubated with active MMP-8 (100 nM) in binding buffer at 4°C for 2 hours (low temperature reduces enzyme activity interference).

[0113] Add anti-GST magnetic beads (block non-specific sites after pre-washing), incubate at room temperature for 30 minutes, separate on a magnetic stand, and discard the supernatant.

[0114] Excess unlabeled library sequences (10 μM) were used for competitive elution to collect specific aptamers.

[0115] 10-15 rounds of iterative screening were performed, with the incubation time gradually shortened in each round (reduced to 30 minutes after the 8th round), and MMP-8 inhibitors (such as Marimastat) were introduced after the 6th round for reverse screening.

[0116] High-throughput sequencing (NGS) was used to screen high-frequency sequences, and binding pockets were predicted using RNAcomposer.

[0117] SPR was used to detect the Kd value (target Kd < 50 nM) and to verify the inhibitory ability of the aptamer on MMP-8 enzyme activity (IC 50 <1 μM).

[0118] Molecular docking simulation (AutoDock) predicted the interaction between the aptamer and the catalytic domain of MMP-8.

[0119] Spiegelmer modification was performed on high affinity sequences.

[0120] The 5' end is labeled with Cy5 or TAMRA for subsequent cell imaging or tissue distribution studies.

[0121] Example 5

[0122] A method for preparing an IL-3 aptamer, which excludes sequences that non-specifically bind to the hematopoietic factor family through negative screening, enriches aptamers that specifically bind to active IL-3 through positive screening, and improves the stability and biological activity regulation ability of the aptamer by combining functional modification. The specific steps are as follows:

[0123] The full-length coding sequence (including the signal peptide to promote native folding) was amplified from a plasmid containing the IL-3 gene.

[0124] The protein was cloned into pHLsec vector (C-terminal His tag) and transfected into HEK293F suspension cells for mammalian expression.

[0125] Culture conditions: 37°C constant temperature shaker (120 rpm), 5% CO 2 , the supernatant was collected 72 hours after transfection.

[0126] Ni 2+ After affinity chromatography purification, the product was further purified by Superdex 75 gel filtration chromatography and the purity was verified (>95%).

[0127] The TF-1 cell proliferation assay was used to verify the biological activity of IL-3 (EC 50 <10 ng / mL).

[0128] Store in PBS (pH 7.4) containing 0.1% BSA at -80°C.

[0129] Use the hematopoietic factor GM-CSF.

[0130] The RNA random library (10-100 nM) was incubated with the control protein (1 μM) in binding buffer (PBS, 0.5% Tween-20) at room temperature for 1 hour.

[0131] Sequences binding to positively charged proteins were removed by cation exchange chromatography (HiTrap SP HP, low salt elution).

[0132] The negative selection library (10-100 nM) was incubated with active IL-3 (100 nM) in binding buffer overnight at 4°C (conformation maintained at low temperature).

[0133] Add anti-His tag magnetic beads (block non-specific sites after pre-washing), incubate at room temperature for 1 hour, separate on a magnetic stand, and discard the supernatant.

[0134] Competitive elution of the extracellular segment of the IL-3 receptor α chain (CD123) was used to collect aptamers that overlapped with the receptor binding site.

[0135] 12-15 rounds of iterative screening were performed, with the incubation time gradually shortened in each round (reduced to 2 hours after the 8th round), and IL-3 neutralizing antibodies were introduced for reverse screening after the 5th round.

[0136] High-throughput sequencing (NGS) was used to screen high-frequency sequences, and RNAfold was used to predict binding regions and stem-loop structures.

[0137] SPR was used to detect the Kd value (target Kd < 5 nM) and to verify the inhibitory ability of the aptamer on TF-1 cell proliferation (IC 50 <100nM).

[0138] The binding blocking efficiency of the aptamer to the CD123 receptor was verified by BLI (biolayer interferometry).

[0139] Cholesterol modification (5' end) of high affinity sequences prolongs in vivo circulation time.

[0140] The introduction of LNA (locked nucleic acid) modification improves nuclease resistance.

[0141] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a specific inflammatory factor nucleic acid aptamer, characterized in that: The following steps are involved: Prepare high-purity protein samples of target inflammatory factors; preparing an RNA library comprising random nucleic acid sequences; Negative screening is performed using a control protein that is similar in structure to the target protein but has no functional relation to the target protein or a sample with a low content of the target protein to remove non-specifically bound nucleic acid sequences; The RNA library after negative screening is positively screened with high-purity target protein samples to enrich specific binding aptamers; The enriched aptamers are identified and high-affinity aptamers are screened out.

2. The preparation method according to claim 1, characterized in that: The target inflammatory factor is selected from one or more of TNF-α, IL-16, C5a, MMP-8 and IL-3.

3. The preparation method according to claim 1 or 2, characterized in that: The negative screening step includes incubating the RNA library with a control protein or a low-content target protein sample, and removing non-specifically bound nucleic acid sequences using ultracentrifugation, magnetic bead sorting, ultrafiltration centrifugation or anion / cation exchange chromatography technology.

4. The preparation method according to claim 1 or 2, characterized in that: The positive screening step includes incubating the RNA library after negative screening with a high-purity target protein sample, separating the specifically bound aptamers using magnetic beads, and optimizing the specificity and affinity of the aptamers through multiple rounds of iterative screening.

5. The preparation method according to claim 1 or 2, characterized in that: The aptamer identification step includes using high-throughput sequencing technology to analyze the sequence information of the enriched aptamers, and using surface plasmon resonance technology to determine the affinity of the aptamers.

6. The preparation method according to claim 1 or 2, characterized in that: The method also includes a step of chemically modifying the screened high-affinity aptamers to improve their stability, biological activity or in vivo circulation time.

7. The preparation method according to claim 6, characterized in that: The chemical modification is selected from one or more of 2'-fluorine modification, cholesterol modification, and LNA modification.

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