Aptamer specifically combined with CD117 and application thereof

Abstract

The invention discloses an aptamer specifically bound with CD117 and application thereof, belongs to the technical field of biochemistry, and particularly relates to a screening method of the aptamer specifically bound with CD117, which comprises the following steps: constructing an ssDNA library and primers, screening cells, screening cell lysis buffer, and obtaining the aptamer specifically bound with CD117 by using flow analysis and high-throughput sequencing. The CD117 protein and DNA combined on the CD117 protein are separated and purified by using immunomagnetic beads in the cell lysis solution screening; the immunomagnetic beads comprise anti-CD117 antibody modified magnetic beads or modified anti-CD117 antibody modified magnetic beads; the surface of the modified anti-CD117 antibody modified magnetic bead is modified with an amino group and a carboxyl group. According to the screening method disclosed by the invention, the aptamer specifically combined with CD117 is obtained and can be used for specifically targeting and detecting leukemia cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a nucleic acid aptamer specifically binding to CD117 and a use thereof. Background Art

[0002] CD117 (c-KIT) is a type III receptor tyrosine kinase that forms a dimer when bound to stem cell growth factors, thereby activating the downstream KIT signaling pathway and regulating cell survival, proliferation, differentiation and migration. Initially, CD117 was widely studied in the field of oncology as a proto-oncogene. Among them, tyrosine kinase inhibitors represented by imatinib mesylate tablets (Gleevec) can effectively inhibit the kinase activity of CD117. On the other hand, CD117, as an important leukocyte differentiation antigen molecule, is specifically expressed on the surface of hematopoietic stem cells and acute myeloid leukemia tumor cells, and is an important marker for the immunophenotyping of hematopoietic stem cells and AML. Therefore, monoclonal antibodies, antibody-drug conjugates, and CAR-T cell therapies developed with CD117 as the target can selectively eliminate hematopoietic stem cells and tumor cells in the patient's bone marrow, creating a safer and more effective implantation space for hematopoietic stem cell transplantation. However, the inhibitors targeting CD117 have drug resistance, and the monoclonal antibodies have the disadvantages of high production cost, long production time, poor stability, and difficulty in achieving precise chemical modification, which to some extent hinder the clinical application of CD117. Therefore, the development of new recognition ligands targeting CD117 is of great significance for the research of clinically related diseases.

[0003] Aptamers are a type of ssDNA or RNA composed of 20-100 bases that can specifically recognize and bind to target molecules. Since the binding of aptamers to targets is similar to the immunoaffinity binding between antigens and antibodies, they are vividly called "chemists' antibodies". However, unlike antibody production, aptamers are obtained by rapid in vitro screening through exponential enrichment ligand system evolution technology. They not only have affinity and specificity comparable to antibodies, but also have the following unique advantages: (1) The binding target range is relatively wide, including small molecules, proteins, microorganisms, viruses, and cells; (2) Successfully identified aptamers can be synthesized chemically, with high production efficiency, relatively low cost, and small quality differences between batches; (3) The molecular weight is relatively small and the immunogenicity is low; (4) Precise chemical modification and design can be achieved, with strong thermal stability and easy long-term storage. Therefore, developing a nucleic acid aptamer that specifically binds to CD117 as a new type of recognition ligand and widely applying it to many fields such as molecular imaging, biosensing, and cell separation shows great application prospects. Summary of the invention

[0004] The object of the present invention is to provide a nucleic acid aptamer that specifically binds to CD117 and its use.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for screening a nucleic acid aptamer that specifically binds to CD117, comprising: S1, construction of ssDNA library and primers; S2. Cell screening: The ssDNA library is first subjected to negative screening with K562 cells, and then subjected to positive screening with HEL cells. After high temperature denaturation and PCR amplification, the cell screening enriched ssDNA library is obtained and used for the next round of cell screening; S3, cell lysate screening: the cell screening enriched single-stranded ssDNA library is mixed with HEL cell membrane protein, and incubated with immunomagnetic beads. After high temperature denaturation and PCR amplification, the cell lysate screening enriched ssDNA library is obtained and used for the next round of cell lysate screening; S4. Using flow cytometry and high-throughput sequencing, we obtained nucleic acid aptamers that specifically bind to CD117; The immunomagnetic beads include anti-CD117 antibody modified magnetic beads, or modified anti-CD117 antibody modified magnetic beads; the surface of the modified anti-CD117 antibody modified magnetic beads is modified with amino groups and carboxyl groups.

[0006] The present invention combines cell screening (cell-SELEX) technology and cell lysate screening technology (cell lysate-SELEX), using HEL cells as positive screening cells and K562 cells as negative screening cells, and the nucleic acid aptamer AptCD117 obtained by screening can specifically recognize and bind to CD117 antigen with high affinity. The present invention can improve the adsorption efficiency of CD117 protein by modified anti-CD117 antibody modified magnetic beads by cooperating 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline, and separate and purify CD117 protein and ssDNA bound to CD117 protein, thereby improving DNA recovery efficiency and obtaining a ssDNA library with better enrichment effect.

[0007] Preferably, the ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', and its nucleotide sequence is shown in SEQ ID No. 1, and N45 is a sequence formed by connecting 45 arbitrary nucleotide bases.

[0008] Preferably, the primers include an upstream primer and a downstream primer, the upstream primer is modified with 5-FAM, and the downstream primer is modified with Biotin.

[0009] Preferably, the upstream primer is 5'-ATCCAGAGTGACGCAGCA-3', and its nucleotide sequence is shown in SEQ ID No.2; the downstream primer is 5'-ACTAAGCCACCGTGTCCA-3', and its nucleotide sequence is shown in SEQ ID No.3.

[0010] Preferably, cell screening is repeated for at least 6 rounds, and cell lysate screening is repeated for at least 6 rounds.

[0011] Preferably, in the preparation of modified anti-CD117 antibody modified magnetic beads, the oleic acid modified ferrosoferric oxide nanoreagent is first reacted with 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, and then coupled with biotinylated CD117 antibody to obtain modified anti-CD117 antibody modified magnetic beads.

[0012] Preferably, the preparation of anti-CD117 antibody modified magnetic beads comprises: Take oleic acid-modified ferrosoferric oxide nanoreagent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30-60 minutes, add streptavidin solution and mix well, couple at room temperature for 2-4 hours, add balanced salt solution to resuspend, react at room temperature for 1-2 hours, remove the supernatant by magnetic separation, wash 2-5 times with phosphate buffered saline solution, discard the supernatant after standing for 30-60 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30-60 minutes, and obtain anti-CD117 antibody modified magnetic beads.

[0013] More preferably, the oleic acid-modified ferroferric oxide nanoreagent comprises oleic acid-modified ferroferric oxide nanoparticles and n-hexane, and the usage ratio of the oleic acid-modified ferroferric oxide nanoparticles to n-hexane is 1 g: 10-100 mL.

[0014] More preferably, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1:10-100.

[0015] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the N-hydroxysuccinimide solution is 1:10-100.

[0016] More preferably, the concentration of the streptavidin solution is 0.02-0.1 mg / mL, and the volume ratio of the oleic acid-modified ferroferric oxide nanoreagent to the streptavidin solution is 1:10-30.

[0017] More preferably, the volume ratio of the balanced salt solution to the streptavidin solution is 1:1-2.

[0018] More preferably, the volume ratio of the biotinylated CD117 antibody to the streptavidin solution is 1:20-60.

[0019] Preferably, the preparation of modified anti-CD117 antibody modified magnetic beads is specifically as follows: Take oleic acid modified ferroferric oxide nanoreagent, add 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, add deionized water for ultrasonic dispersion for 10-20 minutes, let stand for 2-3 hours under nitrogen conditions, add potassium persulfate solution and let stand for 1-2 hours, react at 60-80°C for 2-4 hours, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30-60 minutes, add streptavidin solution to mix, couple at room temperature for 2-4 hours, add balanced salt solution to resuspend, react at room temperature for 1-2 hours, remove the supernatant by magnetic separation, wash with phosphate buffered saline solution for 2-5 times, discard the supernatant after standing for 30-60 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30-60 minutes, and obtain modified anti-CD117 antibody modified magnetic beads.

[0020] More preferably, the oleic acid-modified ferroferric oxide nanoreagent comprises oleic acid-modified ferroferric oxide nanoparticles and n-hexane, and the usage ratio of the oleic acid-modified ferroferric oxide nanoparticles to n-hexane is 1 g: 10-100 mL.

[0021] More preferably, the dosage ratio of the oleic acid-modified ferroferric oxide nanoreagent to 3-allyl-4-methoxybenzoic acid is 1 mL: 10-30 mg.

[0022] More preferably, the usage ratio of the oleic acid-modified ferroferric oxide nanoreagent and 2-isopropenylphenylaniline is 1 mL: 10-30 mg.

[0023] More preferably, the usage ratio of 3-allyl-4-methoxybenzoic acid to deionized water is 1 mg: 20-60 mL.

[0024] More preferably, the concentration of the potassium persulfate solution is 0.02-0.1 g / mL, and the dosage ratio of the oleic acid-modified ferroferric oxide nanoreagent to the potassium persulfate solution is 1:0.1-0.2.

[0025] More preferably, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1:10-100.

[0026] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the N-hydroxysuccinimide solution is 1:10-100.

[0027] More preferably, the concentration of the streptavidin solution is 0.02-0.1 mg / mL, and the volume ratio of the oleic acid-modified ferroferric oxide nanoreagent to the streptavidin solution is 1:10-30.

[0028] More preferably, the volume ratio of the balanced salt solution to the streptavidin solution is 1:1-2.

[0029] More preferably, the volume ratio of the biotinylated CD117 antibody to the streptavidin solution is 1:20-60.

[0030] Preferably, in the preparation of the modified anti-CD117 antibody modified magnetic beads, glutaric anhydride is further used for modification, specifically, Take oleic acid modified ferroferric oxide nanoreagent, add 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, add deionized water and ultrasonically disperse for 10-20 minutes, let stand for 2-3 hours under nitrogen, add potassium persulfate solution and let stand for 1-2 hours, react at 60-80°C for 2-4 hours, slowly add oxalyl chloride and stir for 10-20 minutes, add butanediol, react at 80-100°C for 6-9 hours, add glutaric anhydride and 4-dimethylaminopyridine, react at 70-90°C for 12-18 hours, and then Add 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30-60 minutes, add streptavidin solution and mix, couple at room temperature for 2-4 hours, add balanced salt solution to resuspend, react at room temperature for 1-2 hours, remove supernatant by magnetic separation, wash 2-5 times with phosphate buffered saline solution, discard supernatant after standing for 30-60 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30-60 minutes, and obtain modified anti-CD117 antibody modified magnetic beads. The use of glutaric anhydride can further carboxyl modify the modified anti-CD117 antibody modified magnetic beads, thereby improving the protein adsorption efficiency and DNA recovery efficiency of the modified anti-CD117 antibody modified magnetic beads.

[0031] More preferably, the oleic acid-modified ferroferric oxide nanoreagent comprises oleic acid-modified ferroferric oxide nanoparticles and n-hexane, and the usage ratio of the oleic acid-modified ferroferric oxide nanoparticles to n-hexane is 1 g: 10-100 mL.

[0032] More preferably, the dosage ratio of the oleic acid-modified ferroferric oxide nanoreagent to 3-allyl-4-methoxybenzoic acid is 1 mL: 10-30 mg.

[0033] More preferably, the usage ratio of the oleic acid-modified ferroferric oxide nanoreagent and 2-isopropenylphenylaniline is 1 mL: 10-30 mg.

[0034] More preferably, the usage ratio of 3-allyl-4-methoxybenzoic acid to deionized water is 1 mg: 20-60 mL.

[0035] More preferably, the concentration of the potassium persulfate solution is 0.02-0.1 g / mL, and the dosage ratio of the oleic acid-modified ferroferric oxide nanoreagent to the potassium persulfate solution is 1:0.1-0.2.

[0036] More preferably, the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to oxalyl chloride is 1:1-2.

[0037] More preferably, the usage ratio of oxalyl chloride to butanediol is 1 mL: 0.02-0.1 g.

[0038] More preferably, the mass ratio of butanediol to glutaric anhydride is 1:1-2.

[0039] More preferably, the mass ratio of butanediol to 4-dimethylaminopyridine is 1:1-2.

[0040] More preferably, the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1:10-100.

[0041] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid-modified ferrosoferric oxide nanoreagent to the N-hydroxysuccinimide solution is 1:10-100.

[0042] More preferably, the concentration of the streptavidin solution is 0.02-0.1 mg / mL, and the volume ratio of the oleic acid-modified ferroferric oxide nanoreagent to the streptavidin solution is 1:10-30.

[0043] More preferably, the volume ratio of the balanced salt solution to the streptavidin solution is 1:1-2.

[0044] More preferably, the volume ratio of the biotinylated CD117 antibody to the streptavidin solution is 1:20-60.

[0045] Preferably, a method for screening a nucleic acid aptamer that specifically binds to CD117 comprises: S1. Construction of single-stranded DNA (ssDNA) library and primers: The synthetic ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', and its nucleotide sequence is shown in SEQ ID No.1, and N45 is a sequence formed by connecting 45 arbitrary nucleotide bases; the primers include upstream primers and downstream primers, the upstream primer is modified with 5-carboxyfluorescein (5-FAM), and its nucleotide sequence is shown in SEQ ID No.2; the downstream primer is modified with biotin, and its nucleotide sequence is shown in SEQ ID No.3. Sterile ultrapure water is added to the ssDNA library to fully dissolve, denature at 94-96℃ for 5-10min, quickly cool, add binding buffer, the temperature of Binding Buffer is 4℃, vortex and shake to mix, and set aside.

[0046] S2. Cell screening: Positive screening cells are human erythroleukemia cells (HEL), and negative screening cells are human chronic myeloid leukemia cells (K562). The ssDNA library is added to K562 cells. The number of K562 cells is 4×10 6 -6×10 6 The cells were gently shaken and mixed, incubated in the dark for 30-60 min, centrifuged at 300-400 g for 5-10 min at 22-28 °C, and the supernatant was collected and added to HEL cells. The number of HEL cells was 9 × 10 6 -11×10 6 , incubate in dark for 40-50 min, centrifuge at 300-400g for 5-10 min at 22-28°C, discard the supernatant, add Binding Buffer to gently resuspend the cells, wash 1-2 times, centrifuge at 300-400g for 5-10 min at 22-28°C, resuspend the cells in sterile ultrapure water, denature at 94-96°C for 10-15 min, cool for 2-5 min, centrifuge at 10000g for 5-10 min at 22-28°C, collect the supernatant, and use the supernatant for PCR amplification. The PCR reaction procedure is as follows: pre-denaturation at 94-96°C for 5-10 min, denaturation at 94-96°C for 30-40s, annealing at 55-60°C for 30-40s, extension at 70-75°C for 30-40s, amplification for 15-20 cycles, extension at 70-75°C for 5-10min, storage of the sample at 2-10°C, and then denaturation with sodium hydroxide solution for 5-10min to obtain a cell screening enriched ssDNA library; this step was repeated for at least 6 rounds, the cell screening enriched ssDNA library obtained in each round was stored, and used for the next round of cell screening.

[0047] S3, cell lysate screening: extract HEL cell membrane protein using cell membrane protein and cytoplasm extraction kit, add the cell screening enriched ssDNA library prepared in the last round of step S2, gently shake and mix, incubate in dark for 30-40 minutes, then add immunomagnetic beads, gently shake and mix, incubate in ice bath for 30-40 minutes, add Binding The beads were gently resuspended and washed 1-2 times with buffer, and the supernatant was discarded by magnetic separation. Sterile ultrapure water was added to resuspend the magnetic beads, and the beads were denatured at 94-96°C for 10-15 min, cooled for 2-5 min, and the supernatant was collected by magnetic separation. The supernatant was used for PCR amplification. The PCR reaction program was as follows: pre-denaturation at 94-96°C for 5-10 min, denaturation at 94-96°C for 30-40 s, annealing at 55-60°C for 30-40 s, extension at 70-75°C for 30-40 s, amplification for 15-20 cycles, extension at 70-75°C for 5-10 min, and the sample was stored at 2-10°C. Then, sodium hydroxide solution was added for denaturation for 5-10 min to obtain a cell lysate for screening and enrichment of the ssDNA library. This step was repeated for at least 6 rounds, and the cell lysate obtained in each round was stored to screen and enrich the ssDNA library, and used for the next round of cell lysate screening.

[0048] S4. Obtaining nucleic acid aptamers that specifically bind to CD117: The enriched ssDNA library is subjected to flow cytometry analysis, and the ssDNA library with the strongest binding ability to HEL cells and almost no binding to K562 cells is selected for high-throughput sequencing to obtain nucleic acid aptamers that specifically bind to CD117. The enriched ssDNA library includes screening the enriched ssDNA library with cells prepared in each round in step S3, and screening the enriched ssDNA library with cell lysate prepared in each round in step S4.

[0049] More preferably, the preparation of Binding Buffer is specifically as follows: Weigh bovine serum albumin (BSA), magnesium chloride hexahydrate, and glucose, add them into Dulbecco's phosphate buffered saline (DPBS), and mix well to obtain Binding Buffer.

[0050] More preferably, the mass ratio of BSA to magnesium chloride hexahydrate is 1:1-2.

[0051] More preferably, the mass ratio of BSA to glucose is 1:2-6.

[0052] More preferably, the usage ratio of BSA to DPBS is 1 g:1-2 L.

[0053] More preferably, in step S1, the usage ratio of ssDNA library to sterile ultrapure water is 1 nmol:5-10 μL.

[0054] More preferably, in step S1, the usage ratio of ssDNA library to Binding Buffer is 1 nmol:10-30 μL.

[0055] More preferably, in step S2, the volume ratio of the ssDNA library to the Binding Buffer is 1:20-60.

[0056] More preferably, in step S2, the volume ratio of the ssDNA library to sterile ultrapure water is 1:20-60.

[0057] More preferably, the mass concentration of the sodium hydroxide solution in step S2 is 150-250 mmol / L, and the volume ratio of the sterile ultrapure water to the sodium hydroxide solution is 1:1-2.

[0058] More preferably, in step S3, the volume ratio of the HEL cell membrane protein to the ssDNA library is 1:1-2.

[0059] More preferably, the immunomagnetic beads in step S3 are anti-CD117 antibody-modified magnetic beads, or modified anti-CD117 antibody-modified magnetic beads.

[0060] More preferably, in step S3, the volume ratio of the ssDNA library to the immunomagnetic beads is 1:0.1-0.2.

[0061] More preferably, in step S3, the volume ratio of the immunomagnetic beads to the Binding Buffer is 1:20-60.

[0062] More preferably, in step S3, the volume ratio of the immunomagnetic beads to sterile ultrapure water is 1:20-60.

[0063] More preferably, the mass concentration of the sodium hydroxide solution in step S3 is 150-250 mmol / L, and the volume ratio of the sterile ultrapure water to the sodium hydroxide solution is 1:1-2.

[0064] The invention also discloses a nucleic acid aptamer that specifically binds to CD117 and is screened by the method.

[0065] Preferably, the nucleic acid aptamer that specifically binds to CD117 is 5′-TCCAGTGACGCAGCATCGAGCGGGGGACCCTATTAGCTGAATGAGATGCAATTACAAGCGTGGACACTGGC-3′, and its nucleotide sequence is shown in SEQ ID No.4.

[0066] The invention also discloses the use of a nucleic acid aptamer specifically binding to CD117 in preparing an AML tumor cell detection reagent.

[0067] The invention also discloses the use of a nucleic acid aptamer specifically binding to CD117 in preparing a tyrosine kinase inhibitor for blocking the interaction between SCF and CD117.

[0068] The present invention adopts 3-allyl-4-methoxybenzoic acid, 2-isopropenylphenylaniline and glutaric anhydride to prepare modified anti-CD117 antibody-modified magnetic beads, and uses the modified anti-CD117 antibody-modified magnetic beads for cell lysate screening to screen and obtain nucleic acid aptamers that specifically bind to CD117, thereby having the following beneficial effects: in the screening of nucleic acid aptamers that specifically bind to CD117, the present invention improves the adsorption efficiency of the modified anti-CD117 antibody-modified magnetic beads on the CD117 protein, separates and purifies the CD117 protein and the ssDNA bound to the CD117 protein, thereby improving the DNA recovery efficiency and obtaining an ssDNA library with better enrichment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the binding level of the selection pool for enriching the ssDNA library in Example 1.

[0070] Figure 2 This is a flow cytometry analysis of the binding level of AptCD117 to HEL cells.

[0071] Figure 3 This is a flow cytometry analysis of the binding level of AptCD117 to K562 cells.

[0072] Figure 4 This is the expression of CD117 in HEL cells after siRNA interference.

[0073] Figure 5 This is the binding of AptCD117 to HEL cells after siRNA interference.

[0074] Figure 6 This is the binding affinity analysis between AptCD117 and CD117.

[0075] Figure 7 Flow cytometry analysis of AptCD117 binding to CD117 + Tumor cell status.

[0076] Figure 8 The difference in the binding sites between AptCD117 and SCF.

[0077] Fig. 9 The difference in binding sites between anti-CD117 antibody and SCF. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0079] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0080] Embodiment 1: Preparation of Binding Buffer, including: Weigh BSA, magnesium chloride hexahydrate, and glucose, add DPBS and mix well to obtain Binding Buffer. The mass ratio of BSA to magnesium chloride hexahydrate is 1:1, the mass ratio of BSA to glucose is 1:4, and the dosage ratio of BSA to DPBS is 1g:1L.

[0081] The preparation of anti-CD117 antibody modified magnetic beads comprises: Take oleic acid-modified ferrosoferric oxide nanoreagent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30 minutes, add streptavidin solution and mix well, couple at room temperature for 2 hours, add balanced salt solution to resuspend, react at room temperature for 1 hour, remove the supernatant by magnetic separation, wash 3 times with phosphate buffered saline solution, discard the supernatant after standing for 30 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30 minutes, and obtain anti-CD117 antibody modified magnetic beads. The oleic acid-modified ferroferric oxide nanoreagent includes oleic acid-modified ferroferric oxide nanoparticles and n-hexane. The oleic acid-modified ferroferric oxide nanoparticles are purchased from Nanjing Jicang Nanotechnology Co., Ltd. The dosage ratio of oleic acid-modified ferroferric oxide nanoparticles and n-hexane is 1 g:100 mL; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1 mg / mL, the volume ratio of oleic acid-modified ferroferric oxide nanoreagent and 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 1:10; the concentration of N-hydroxysuccinimide solution is 1 mg / mL The volume ratio of oleic acid-modified ferric oxide nanoreagent and N-hydroxysuccinimide solution was 1:10; the concentration of streptavidin solution was 0.05 mg / mL, and the volume ratio of oleic acid-modified ferric oxide nanoreagent and streptavidin solution was 1:20; balanced salt solution was purchased from Shanghai ELISA Biotechnology Co., Ltd., and the volume ratio of balanced salt solution and streptavidin solution was 1:1; biotinylated CD117 antibody was purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd., and the volume ratio of biotinylated CD117 antibody and streptavidin solution was 1:40.

[0082] Screening of nucleic acid aptamers that specifically bind to CD117, including, S1. Construction of ssDNA library and primers: The synthetic ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', and its nucleotide sequence is shown in SEQ ID No.1, and N45 is a sequence formed by connecting 45 arbitrary nucleotide bases; the primers include upstream primers and downstream primers, the upstream primer is modified with 5-FAM, and its nucleotide sequence is shown in SEQ ID No.2; the downstream primer is modified with Biotin, and its nucleotide sequence is shown in SEQ ID No.3. The ssDNA library and primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd. Add sterile ultrapure water to the ssDNA library to fully dissolve, denature at 95°C for 5 minutes, quickly cool and add Binding Buffer, the temperature of Binding Buffer is 4°C, vortex and shake to mix, and set aside. The usage ratio of ssDNA library and sterile ultrapure water is 1nmol:5μL; the usage ratio of ssDNA library and BindingBuffer is 1nmol:20μL.

[0083] S2. Cell screening: Positive screening cells are human erythroleukemia (HEL) cells, and negative screening cells are human chronic myeloid leukemia (K562) cells. The ssDNA library was added to K562 cells. The number of K562 cells was 5×10 6 , gently shake and mix, incubate in the dark for 30 min, centrifuge at 300 g for 5 min at 25 °C, collect the supernatant and add it to HEL cells. The number of HEL cells was 10 × 10 6, incubate in dark for 45 min, centrifuge at 300 g for 5 min at 25 ° C, discard the supernatant, add Binding Buffer to gently resuspend the cells and wash once, centrifuge at 300 g for 5 min at 25 ° C, resuspend the cells in sterile ultrapure water, denature at 95 ° C for 10 min, cool for 2 min, centrifuge at 10000 g for 5 min at 25 ° C, collect the supernatant, and use the supernatant for PCR amplification. The PCR reaction program is: pre-denaturation at 95 ° C for 5 min, denaturation at 95 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 30 s, amplification for 15 cycles, extension at 72 ° C for 5 min, store the sample at 4 ° C, and then add sodium hydroxide solution for denaturation for 5 min to obtain a cell screening enriched ssDNA library; repeat this step for 6 rounds, save the cell screening enriched ssDNA library obtained in each round, and use it for the next round of cell screening. The volume ratio of ssDNA library to Binding Buffer is 1:40; the volume ratio of ssDNA library to sterile ultrapure water is 1:40; the mass concentration of sodium hydroxide solution is 200 mmol / L, and the volume ratio of sterile ultrapure water to sodium hydroxide solution is 1:1.

[0084] S3, cell lysate screening: extract HEL cell membrane proteins using a cell membrane protein and cytoplasm extraction kit, add the cell screening enriched ssDNA library prepared in the sixth round of step S3, gently shake and mix, incubate in the dark for 30 minutes, then add anti-CD117 antibody modified magnetic beads, gently shake and mix, incubate in an ice bath for 30 minutes, and add Binding The beads were gently resuspended and washed once with buffer, and the supernatant was discarded by magnetic separation. Sterile ultrapure water was added to resuspend the magnetic beads, and the beads were denatured at 95°C for 10 min, cooled for 2 min, and the supernatant was collected by magnetic separation. The supernatant was used for PCR amplification. The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, amplification for 15 cycles, extension at 72°C for 5 min, and the sample was stored at 4°C. Then, sodium hydroxide solution was added for denaturation for 5 min to obtain the cell lysate for screening the enriched ssDNA library. This step was repeated for 6 rounds, and the cell lysate obtained in each round was saved for screening the enriched ssDNA library, and used for the next round of cell lysate screening. The cell membrane protein and cytoplasm extraction kits were purchased from Shanghai Biotime Biotechnology Co., Ltd.; the volume ratio of HEL cell membrane protein to cell screening and enrichment ssDNA library was 1:1; the volume ratio of cell screening and enrichment ssDNA library to anti-CD117 antibody modified magnetic beads was 1:0.1; the volume ratio of anti-CD117 antibody modified magnetic beads to Binding Buffer was 1:40; the volume ratio of anti-CD117 antibody modified magnetic beads to sterile ultrapure water was 1:40; the mass concentration of sodium hydroxide solution was 200 mmol / L, and the volume ratio of sterile ultrapure water to sodium hydroxide solution was 1:1.

[0085] S4. Obtaining nucleic acid aptamers that specifically bind to CD117: The enriched ssDNA library is subjected to flow cytometry analysis, and the enriched ssDNA library with the strongest binding ability to HEL cells and almost no binding to K562 cells is selected for high-throughput sequencing to obtain a nucleic acid aptamer (AptCD117) that specifically binds to CD117. The enriched ssDNA library includes screening the enriched ssDNA library with cells prepared in each round in step S3, and screening the enriched ssDNA library with cell lysate prepared in each round in step S4.

[0086] Embodiment 2: The difference between this embodiment and embodiment 1 lies in the preparation of modified anti-CD117 antibody modified magnetic beads and the screening of nucleic acid aptamers that specifically bind to CD117.

[0087] The preparation of modified anti-CD117 antibody modified magnetic beads comprises: Take oleic acid modified ferroferric oxide nanoreagent, add 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, add deionized water for ultrasonic dispersion for 10 minutes, let it stand for 2 hours under nitrogen conditions, add potassium persulfate solution and let it stand for 1 hour, react at 70°C for 2 hours, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30 minutes, add streptavidin solution to mix, couple at room temperature for 2 hours, add balanced salt solution to resuspend, react at room temperature for 1 hour, remove the supernatant by magnetic separation, wash 3 times with phosphate buffered saline solution, discard the supernatant after standing for 30 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30 minutes, and obtain modified anti-CD117 antibody modified magnetic beads. The oleic acid-modified ferroferric oxide nanoreagent includes oleic acid-modified ferroferric oxide nanoparticles and n-hexane. The oleic acid-modified ferroferric oxide nanoparticles are purchased from Nanjing Jicang Nanotechnology Co., Ltd. The dosage ratio of oleic acid-modified ferroferric oxide nanoparticles and n-hexane is 1 g:100 mL; the dosage ratio of oleic acid-modified ferroferric oxide nanoreagent and 3-allyl-4-methoxybenzoic acid is 1 mL:20 mg; the dosage ratio of oleic acid-modified ferroferric oxide nanoreagent and 2-isopropenylphenylaniline is 1 mL:20 mg; the dosage ratio of 3-allyl-4-methoxybenzoic acid and deionized water is 1 mg:40 mL; the concentration of potassium persulfate solution is 0.05 g / mL, and the dosage ratio of oleic acid-modified ferroferric oxide nanoreagent and potassium persulfate solution is 1:0.1; 1-ethyl-(3-dimethylaminopropyl)carbodiimide The concentration of the solution was 1 mg / mL, and the volume ratio of the oleic acid-modified ferric oxide nanoreagent to the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution was 1:10; the concentration of the N-hydroxysuccinimide solution was 1 mg / mL, and the volume ratio of the oleic acid-modified ferric oxide nanoreagent to the N-hydroxysuccinimide solution was 1:10; the concentration of the streptavidin solution was 0.05 mg / mL, and the volume ratio of the oleic acid-modified ferric oxide nanoreagent to the streptavidin solution was 1:20; the balanced salt solution was purchased from Shanghai ELISA Biotechnology Co., Ltd., and the volume ratio of the balanced salt solution to the streptavidin solution was 1:1; the biotinylated CD117 antibody was purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd., and the volume ratio of the biotinylated CD117 antibody to the streptavidin solution was 1:40.

[0088] The screening of nucleic acid aptamers that specifically bind to CD117 was carried out under the same conditions as in Example 1, except that the anti-CD117 antibody-modified magnetic beads were replaced with the modified anti-CD117 antibody-modified magnetic beads prepared in this example.

[0089] Embodiment 3: The difference between this embodiment and embodiment 2 is only the preparation of modified anti-CD117 antibody modified magnetic beads.

[0090] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 2 except that the dosage ratio of oleic acid modified ferroferric oxide nanoreagent and 3-allyl-4-methoxybenzoic acid was changed to 1 mL:10 mg.

[0091] Embodiment 4: The difference between this embodiment and embodiment 2 is only the preparation of modified anti-CD117 antibody modified magnetic beads.

[0092] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 2 except that the ratio of oleic acid modified ferroferric oxide nanoreagent and 2-isopropenylphenylaniline was changed to 1 mL:10 mg.

[0093] Embodiment 5: The difference between this embodiment and embodiment 1 lies in the preparation of modified anti-CD117 antibody modified magnetic beads and the screening of nucleic acid aptamers that specifically bind to CD117.

[0094] The preparation of modified anti-CD117 antibody modified magnetic beads comprises: Take oleic acid modified ferroferric oxide nanoreagent, add 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, add deionized water and ultrasonically disperse for 10 minutes, let it stand for 2 hours under nitrogen, add potassium persulfate solution and let it stand for 1 hour, react at 70°C for 2 hours, slowly add oxalyl chloride and stir for 10 minutes, add butanediol, react at 90°C for 6 hours, add glutaric anhydride and 4-dimethylaminopyridine, react at 80°C for 12 hours, and then add 1-ethyl -(3-dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution, incubate at room temperature for 30 minutes, add streptavidin solution to mix, couple at room temperature for 2 hours, add balanced salt solution to resuspend, react at room temperature for 1 hour, remove supernatant by magnetic separation, wash 3 times with phosphate buffered saline solution, discard supernatant after standing for 30 seconds, add biotinylated CD117 antibody, incubate at room temperature for 30 minutes, and obtain modified anti-CD117 antibody modified magnetic beads. Oleic acid modified ferrous oxide nanoreagent includes oleic acid modified ferrous oxide nanoparticles and n-hexane. Oleic acid modified ferrous oxide nanoparticles are purchased from Nanjing Jicang Nanotechnology Co., Ltd. The dosage ratio of oleic acid modified ferrous oxide nanoparticles and n-hexane is 1g:100mL; the dosage ratio of oleic acid modified ferrous oxide nanoreagent and 3-allyl-4-methoxybenzoic acid is 1mL:20mg; the dosage ratio of oleic acid modified ferrous oxide nanoreagent and 2-isopropylphenylaniline ... The dosage ratio is 1mL:20mg; the dosage ratio of 3-allyl-4-methoxybenzoic acid and deionized water is 1mg:40mL; the concentration of potassium persulfate solution is 0.05g / mL, the dosage ratio of oleic acid-modified ferrous oxide nanoreagent and potassium persulfate solution is 1:0.1; the volume ratio of oleic acid-modified ferrous oxide nanoreagent and oxalyl chloride is 1:1; the dosage ratio of oxalyl chloride and butanediol is 1mL:0.05g; the mass ratio of butanediol and glutaric anhydride is 1:1 ; The mass ratio of butanediol and 4-dimethylaminopyridine was 1:1; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution was 1 mg / mL, and the volume ratio of oleic acid-modified ferric oxide nanoreagent and 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution was 1:10; the concentration of N-hydroxysuccinimide solution was 1 mg / mL, and the volume ratio of oleic acid-modified ferric oxide nanoreagent and N-hydroxysuccinimide solution was 1:10; the concentration of streptavidin solution was 0.05 mg / mL, and the volume ratio of oleic acid-modified ferric oxide nanoreagent and streptavidin solution was 1:20; the balanced salt solution was purchased from Shanghai ELISA Biotechnology Co., Ltd., and the volume ratio of the balanced salt solution and the streptavidin solution was 1:1; the biotinylated CD117 antibody was purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd., and the volume ratio of the biotinylated CD117 antibody and the streptavidin solution was 1:40.

[0095] Embodiment 6: The difference between this embodiment and embodiment 5 is only the preparation of modified anti-CD117 antibody modified magnetic beads.

[0096] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 5 except that the mass ratio of butanediol to glutaric anhydride was changed to 1:0.5.

[0097] Comparative Example 1: The difference between this comparative example and Example 2 is only the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0098] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 2 except that 3-allyl-4-methoxybenzoic acid was not added.

[0099] Comparative Example 2: The difference between this comparative example and Example 2 is only the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0100] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 2 except that 2-isopropenylphenylaniline was not added.

[0101] Comparative Example 3: The difference between this comparative example and Example 5 is only the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0102] The preparation of modified anti-CD117 antibody modified magnetic beads was carried out under the same conditions as in Example 5 except that glutaric anhydride was not added.

[0103] Experimental example: 1. Enrichment process of nucleic acid aptamer screening library by flow cytometry The enriched ssDNA library is subjected to flow cytometry analysis, and the enriched ssDNA library includes the cell screening enriched ssDNA library prepared in each round of step S3, and the cell lysate screening enriched ssDNA library prepared in each round of step S4. The cell screening enriched ssDNA library prepared in each round of step S3 is sequentially recorded as the 1st to 6th round ssDNA library; the cell lysate screening enriched ssDNA library prepared in each round of step S4 is sequentially recorded as the 7th to 12th round ssDNA library.

[0104] Figure 1This is the binding level of the selection pool of the enriched ssDNA library in Example 1. As the screening and enrichment of the ssDNA library proceed, the binding level of the selection pool of the enriched ssDNA library gradually increases, wherein the binding level of the selection pool of the 12th round of ssDNA library is the highest, which indicates that the present invention can select the 12th round of ssDNA library for high-throughput sequencing, and select candidate nucleic acid aptamers by analyzing the homology and abundance differences of the enriched sequences in the screening library.

[0105] 2. Flow cytometry analysis of the binding ability of AptCD117 to HEL cells and K562 cells The cultured HEL cells were washed once with 5 mL DPBS, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, and 1 mL Binding Buffer was added to resuspend the cells. A blank group, a control group, and an experimental group were set up. The blank group, the control group, and the experimental group all included 150 μL of 5 × 10 cells. 5 HEL cells; 50 pmol of the initial ssDNA library modified with 5-FAM was also added to the control group, and 50 pmol of AptCD117 modified with 5-FAM was also added to the experimental group. AptCD117 was screened in Example 1, and the cells were gently shaken and mixed, and incubated for 45 minutes. During the incubation period, the cells were shaken and mixed once every 15 minutes. After the incubation, 400 μL of washing buffer (Washing Buffer) was added to wash once, and centrifuged at 300g for 5 minutes at 25°C, and then 400 μL of Washing Buffer was added to resuspend the cells, and the binding ability of AptCD117 to HEL cells was detected by flow cytometry. Washing Buffer includes magnesium chloride hexahydrate, glucose and DPBS, the mass ratio of magnesium chloride hexahydrate to glucose is 1:4.5, and the dosage ratio of magnesium chloride hexahydrate to DPBS is 1g:980mL.

[0106] According to the above method, HEL cells were replaced with K562 cells, and the binding ability of AptCD117 to K562 cells was detected by flow cytometry.

[0107] Figure 2 The flow cytometry analysis of the binding level of AptCD117 to HEL cells showed that there was no difference in the binding level between the blank group and the control group, and the binding level of the experimental group was higher than that of the blank group and the control group. Figure 3 The figure is a flow cytometry analysis of the binding level of AptCD117 to K562 cells. There is no difference in the binding level between the blank group and the control group. This shows that the AptCD117 screened in Example 1 of the present invention has a strong binding ability with HEL cells and does not bind to K562 cells.

[0108] 3. siRNA gene interference experiment to identify the binding target of AptCD117 HEL cells were seeded in 24-well plates at a cell number of 6 × 10 5 / well, and then add 1640 culture medium (without serum and double antibody) to a final volume of 1 mL, and then place the 24-well plate in a 37°C incubator for use. Set up a blank control group (NC), siRNA1 group (siRNA1), siRNA2 group (siRNA2), and siRNA3 group (siRNA3), each group including 1 mL HEL cells, 150 μL 1640 culture medium (without serum and double antibody), 40 μL transfection buffer (Transfection buffer), siRNA with a concentration of 20 μmol / L, and 1.8 μL transfection reagent (GenMute TM Reagent), wherein the siRNA of the NC group is 5'-CCCAACACAACUUCCUUAUTT-3', and its nucleotide sequence is shown in SEQ ID No.5; the nucleotide sequence of siRNA1 is 5'-GCCCACAAUAGAUUGGUAUTT-3', and its nucleotide sequence is shown in SEQ ID No.6; the nucleotide sequence of siRNA2 is 5'-CUGGCAUGAUGUGCAUUAUTT-3', and its nucleotide sequence is shown in SEQ ID No.7; the nucleotide sequence of siRNA3 is 5'-GACCAACAUUCAAGCAAAUTT-3', and its nucleotide sequence is shown in SEQ ID No.8. Centrifuge at 800g for 75min at 37℃, and then culture at 37℃ for 5h. After the culture, the culture medium is discarded, 2mL of fresh 1640 medium (containing 10% FBS and 1% double antibody) is added, and the cells are placed in a 37℃ incubator for further culture for 48h. After the culture is completed, the cells of each group are collected and resuspended in Binding Buffer. Afterwards, the cells of the NC, siRNA1, siRNA2, and siRNA3 groups were divided into two groups, 2.5 μL of phycoerythrin-modified anti-CD117 antibody was added to one group, and 50 pmol of 5-FAM-modified AptCD117 was added to the other group. They were incubated in an ice box for 45 min, during which the cells were gently shaken and mixed every 15 min. After the incubation, 400 μL of Washing Buffer was added for washing, and the cells were centrifuged at 300 g for 5 min at 25°C. The supernatant was discarded, and 400 μL of Washing Buffer was added to resuspend the cells and transferred to a flow tube. The expression of CD117 protein on the surface of the HEL cell membrane was analyzed by flow cytometry, and the binding ability of AptCD117 to the HEL cells after siRNA interference was analyzed.

[0109] Figure 4 Figure 3 is the expression of CD117 in HEL cells after siRNA interference. The expression of CD117 in siRNA1, siRNA2, and siRNA3 groups was lower than that in NC group, which indicates that siRNA1, siRNA2, and siRNA3 groups can effectively knock down the expression of CD117 protein on HEL cells.

[0110] Figure 5 Figure 2 is the binding of AptCD117 to HEL cells after siRNA interference. The binding levels of AptCD117 in the siRNA1, siRNA2, and siRNA3 groups were all lower than those in the NC group, indicating that after the CD117 protein on HEL cells was knocked down by siRNA, the binding ability between AptCD117 and HEL cells was significantly weakened, and was positively correlated with the downregulation level of CD117. Therefore, AptCD117 specifically targets the CD117 protein.

[0111] 4. Analysis of the binding affinity between nucleic acid aptamer AptCD117 and CD117 The cultured HEL cells were washed once with 5 mL DPBS, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, and 1 mL Binding Buffer was added to resuspend the cells. The HEL cell samples were divided into 9 groups, and the volume of each group of HEL cell samples was 200 μL and the number of cells was 5×10 5 5-FAM-modified AptCD117 was added to 9 groups of HEL cell samples in a concentration gradient of 0, 25, 50, 75, 100, 150, 250, 400, and 600 nmol / L, and each concentration gradient was repeated for 3 groups. The cells were mixed by oscillation and incubated in an ice bath for 45 min. During this period, the cells were gently mixed by oscillation every 15 min. After the incubation, 400 μL of Washing Buffer was added for washing once, and the cells were centrifuged at 300 g for 5 min at 25°C. The supernatant was discarded, and 400 μL of Washing Buffer was added to resuspend the cells and transferred to a flow tube. The fluorescence intensity of the cells was detected by flow cytometry and the Kd value was calculated. Kd value = antibody and antigen dissociation rate / antibody and antigen binding rate.

[0112] Figure 6 The binding affinity analysis of AptCD117 and CD117 showed that AptCD117 had a strong binding affinity with CD117 protein, and the Kd value was 2.83±1.44 nM.

[0113] 5. Application of nucleic acid aptamer AptCD117 in the detection of tumor cells in bone marrow samples of AML patients.

[0114] AML bone marrow samples were collected in Zhejiang Cancer Hospital (Ethics Number: IRB-2023-1159), 200 μL of blood sample was drawn, 1 mL of 1×ACK lysis buffer was added and mixed thoroughly, lysed at room temperature for 10 min, centrifuged at 200 g for 5 min at 25 °C, the supernatant was discarded, and 500 μL of DPBS and 10 μL of fetal bovine serum were added to wash once, and finally 1 mL of BindingBuffer was added to resuspend the cells, 5 μL of allelic protein-modified anti-CD45 antibody and 2.5 μL of phycoerythrin-modified anti-CD117 antibody were added, vortexed and mixed, and incubated in an ice bath for 30 min. During this period, the cells were gently vortexed and mixed once every 10 min, and 400 μL of WashingBuffer was added to wash once, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, and 400 μL of The cells were resuspended in BindingBuffer, and a control group and an experimental group were set up. 50 pmol of the initial ssDNA library modified with 5-FAM was added to the control group, and 50 pmol of AptCD117 modified with 5-FAM was added to the experimental group. The cells were shaken and mixed, and incubated in an ice bath for 30 min. During this period, the cells were gently shaken and mixed once every 15 min. 400 μL of Washing Buffer was added to each of the two groups of samples for washing once. The samples were centrifuged at 300 g for 5 min at 25°C, the supernatant was discarded, and 600 μL of Washing Buffer was added to resuspend the cells. 400 μL of cell sample was aspirated into a flow tube, and the recognition and binding ability of AptCD117 to AML tumor cells was detected by flow cytometry.

[0115] Figure 7 To analyze the binding of AptCD117 to CD117+ tumor cells by flow cytometry, CD117 + The percentage of nucleic acid aptamers in tumor cells was higher than that in the control group, indicating that AptCD117 can specifically recognize and bind to CD117 + Tumor cells.

[0116] 6. Flow cytometry analysis of the differences in the binding sites between AptCD117 and SCF.

[0117] The cultured HEL cells were washed once with 5 mL DPBS, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, and the cells were resuspended in 1 mL Binding Buffer. Two groups of cell samples were set up, one group was added with 50 pmol of 5-FAM fluorescently modified AptCD117 and 2 μg SCF; the other group was incubated in an ice bath for 30 min, during which the cells were gently shaken and mixed once every 10 min, and washed once with 400 μL Washing Buffer, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, and the cells were resuspended in 400 μL Washing Buffer and transferred to a flow tube, and the differences in the binding sites of AptCD117, anti-CD117 antibody and SCF were detected by flow cytometry.

[0118] Figure 8 The difference in the binding sites between AptCD117 and SCF, Fig. 9 The difference in the binding sites between anti-CD117 antibody and SCF. Figure 8 and Fig. 9 It can be seen that SCF has a competitive effect on the binding of AptCD117 to CD117 protein, that is, the two have a common binding site, while the binding sites of SCF and anti-CD117 antibody are inconsistent. Therefore, AptCD117 can be used as a tyrosine kinase inhibitor to block the binding of SCF to CD117.

[0119] 7. DNA recovery efficiency of anti-CD117 antibody modified magnetic beads The ssDNA library prepared in the sixth round of the screening step S3 of the nucleic acid aptamer specifically binding to CD117 in Example 1 was collected, and its DNA content was determined by ultraviolet spectrophotometry, which was recorded as the initial DNA content (ng / μL); the HEL cell membrane protein was extracted using a cell membrane protein and cytoplasm extraction kit, and the ssDNA library prepared in the sixth round of the screening step S3 of the nucleic acid aptamer specifically binding to CD117 in Example 1 was added, gently shaken and mixed, incubated in the dark for 30 min, anti-CD117 antibody-modified magnetic beads were added, gently shaken and mixed, incubated in an ice bath for 30 min, added Binding Buffer, gently resuspended and washed once, magnetic separation was discarded, and sterile ultrapure water was added to resuspend the magnetic beads, denatured at 95° C. for 10 min, cooled for 2 min, and magnetic separation was performed to collect the supernatant. The DNA content in the supernatant was determined by ultraviolet spectrophotometry (ng / μL); the DNA recovery efficiency was calculated, and the DNA recovery efficiency (%) = DNA content in the supernatant / initial DNA content × 100%. Using the anti-CD117 antibody modified magnetic beads prepared in Example 1-6 and Comparative Example 1-3, the DNA recovery efficiency of Example 1-6 and Comparative Example 1-3 was obtained accordingly, and the measurement results are shown in Table 1.

[0120] Table 1 DNA recovery efficiency (%)

[0121] As can be seen from Table 1, the DNA recovery efficiency of Example 2-4 is higher than that of Example 1 and Comparative Example 1-2. This is because in the cell lysate screening, Example 2-4 uses 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline to prepare modified anti-CD117 antibody-modified magnetic beads, while 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline are not used in Example 1, only 3-allyl-4-methoxybenzoic acid is used in Comparative Example 1, and only 2-isopropenylphenylaniline is used in Comparative Example 2. This shows that the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline to prepare modified anti-CD117 antibody-modified magnetic beads can improve their DNA recovery efficiency. The DNA recovery efficiency of Examples 5-6 is higher than that of Examples 2-3 and Comparative Example 3 because, in the cell lysate screening, Examples 5-6, on the basis of the coordinated use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline, further use glutaric anhydride to prepare modified anti-CD117 antibody modified magnetic beads, while Comparative Example 3 only uses glutaric anhydride to prepare modified anti-CD117 antibody modified magnetic beads. This shows that glutaric anhydride can further improve the recovery efficiency of modified anti-CD117 antibody modified magnetic beads for DNA.

[0122] 8. Protein adsorption performance of anti-CD117 antibody modified magnetic beads HEL cell membrane protein was extracted using a cell membrane protein and cytoplasm extraction kit, and the HEL cell membrane protein content was determined using the biuret method, recorded as the initial protein content (mg / mL), anti-CD117 antibody-modified magnetic beads were added, gently shaken and mixed, incubated in an ice bath for 30 min, added Binding Buffer and gently resuspended and washed once, magnetic separation was discarded, and sterile ultrapure water was added to resuspend the magnetic beads, and the supernatant was collected by magnetic separation. The protein content in the supernatant was determined using the biuret method (mg / mL); the protein adsorption efficiency was calculated, and the protein adsorption efficiency (%) = protein content in the supernatant / initial protein content × 100%. The anti-CD117 antibody-modified magnetic beads prepared in Example 1-6 and Comparative Example 1-3 were used to obtain the protein adsorption efficiencies of Example 1-6 and Comparative Example 1-3, and the measurement results are shown in Table 2.

[0123] Table 2 Protein adsorption efficiency (%)

[0124] As can be seen from Table 2, the protein adsorption efficiency of Example 2-4 is higher than that of Example 1 and Comparative Example 1-2. This is because in the cell lysate screening, Example 2-4 uses 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline to prepare modified anti-CD117 antibody modified magnetic beads, while 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline are not used in Example 1, only 3-allyl-4-methoxybenzoic acid is used in Comparative Example 1, and only 2-isopropenylphenylaniline is used in Comparative Example 2. This shows that the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline to prepare modified anti-CD117 antibody modified magnetic beads can improve their protein adsorption efficiency. The protein adsorption efficiency of Examples 5-6 is higher than that of Examples 2-3 and Comparative Example 3 because, in the cell lysate screening, Examples 5-6, in addition to the coordinated use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenylaniline, further use glutaric anhydride to prepare modified anti-CD117 antibody modified magnetic beads, while Comparative Example 3 only uses glutaric anhydride to prepare modified anti-CD117 antibody modified magnetic beads. This shows that glutaric anhydride can further improve the adsorption efficiency of modified anti-CD117 antibody modified magnetic beads for proteins.

[0125] The conventional operations in the operating steps of the present invention are well known to those skilled in the art and will not be described in detail here.

[0126] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for screening a nucleic acid aptamer that specifically binds to CD117, comprising: S1, construction of ssDNA library and primers; S2. Cell screening: The ssDNA library is first subjected to negative screening with K562 cells, and then subjected to positive screening with HEL cells. After high temperature denaturation and PCR amplification, the cell screening enriched ssDNA library is obtained and used for the next round of cell screening; S3, cell lysate screening: the cell screening enriched single-stranded ssDNA library is mixed with HEL cell membrane protein, and incubated with immunomagnetic beads. After high temperature denaturation and PCR amplification, the cell lysate screening enriched ssDNA library is obtained and used for the next round of cell lysate screening; S4. Using flow cytometry and high-throughput sequencing, we obtained nucleic acid aptamers that specifically bind to CD117; The immunomagnetic beads include anti-CD117 antibody modified magnetic beads, or modified anti-CD117 antibody modified magnetic beads; the surfaces of the modified anti-CD117 antibody modified magnetic beads are modified with amino groups and carboxyl groups.

2. The method for screening a nucleic acid aptamer that specifically binds to CD117 according to claim 1, characterized in that: The ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', and its nucleotide sequence is shown in SEQ ID No. 1, and the N45 is a sequence formed by connecting 45 arbitrary nucleotide bases.

3. The method for screening a nucleic acid aptamer that specifically binds to CD117 according to claim 1, characterized in that: The primers include an upstream primer and a downstream primer, the upstream primer is modified with 5-FAM, and the downstream primer is modified with Biotin.

4. The method for screening a nucleic acid aptamer that specifically binds to CD117 according to claim 3, characterized in that: The upstream primer is 5'-ATCCAGAGTGACGCAGCA-3', and its nucleotide sequence is shown in SEQ ID No.2; the downstream primer is 5'-ACTAAGCCACCGTGTCCA-3', and its nucleotide sequence is shown in SEQ ID No.

3.

5. The method for screening a nucleic acid aptamer that specifically binds to CD117 according to claim 1, characterized in that: The cell screening was repeated for at least 6 rounds, and the cell lysate screening was repeated for at least 6 rounds.

6. The method for screening a nucleic acid aptamer that specifically binds to CD117 according to claim 1, characterized in that: In the preparation of the modified anti-CD117 antibody modified magnetic beads, the oleic acid modified ferroferric oxide nanoreagent is first reacted with 3-allyl-4-methoxybenzoic acid and 2-isopropylphenylaniline, and then coupled with biotinylated CD117 antibody to obtain the modified anti-CD117 antibody modified magnetic beads.

7. The nucleic acid aptamer that specifically binds to CD117 obtained by screening according to any one of the methods of claims 1 to 6.

8. The nucleic acid aptamer that specifically binds to CD117 according to claim 7, characterized in that: The nucleic acid aptamer that specifically binds to CD117 is 5'-TCCAGTGACGCAGCATCGAGCGGGGGACCCTATTAGCTGAATGAGATGCAATTACAAGCGTGGACACTGGC-3', and its nucleotide sequence is shown in SEQ ID No.

4.

9. Use of the nucleic acid aptamer that specifically binds to CD117 according to claim 7 in the preparation of an AML tumor cell detection reagent.

10. Use of the nucleic acid aptamer that specifically binds to CD117 according to claim 7 in the preparation of a tyrosine kinase inhibitor that blocks the interaction between SCF and CD117.

Images