A nucleic acid aptamer that specifically binds to cd117 and uses thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, in particular to a nucleic acid aptamer specifically binding to CD117 and uses thereof. BACKGROUND

[0002] CD117 (c-KIT) is a type III receptor tyrosine kinase, which forms a dimer when binding to stem cell growth factor, thereby activating the downstream KIT signaling pathway and regulating cell survival, proliferation, differentiation and migration. Initially, CD117 was widely studied as a proto-oncogene in the field of oncology. Among them, tyrosine kinase inhibitors represented by imatinib mesylate tablets (Gleevec) can effectively inhibit the kinase activity of CD117. On the other hand, as an important leukocyte differentiation antigen molecule, CD117 is specifically expressed on the surface of hematopoietic stem cells and acute myeloid leukemia tumor cells, and is an important marker for hematopoietic stem cell and AML immunophenotyping. Therefore, the monoclonal antibody, antibody drug conjugate and CAR-T cell therapy targeting CD117 can selectively eliminate hematopoietic stem cells and tumor cells in the bone marrow of patients, creating a safer and more effective implantation space for hematopoietic stem cell transplantation. However, inhibitors against CD117 have drug resistance, and monoclonal antibodies have high production cost, long time-consuming, poor stability, and difficulty in achieving precise chemical modification, which to some extent hinders the clinical application of CD117. Therefore, the development of new recognition ligands targeting CD117 is of great significance for the study of clinically relevant diseases.

[0003] Nucleic acid aptamer is a kind of ssDNA or RNA composed of 20-100 bases, which can specifically recognize and bind to target molecules. Because the binding of nucleic acid aptamer to the target is similar to the immunological affinity binding between antigen and antibody, it is called "chemist's antibody" figuratively. But unlike antibody production, nucleic acid aptamer is obtained by exponential enrichment ligand system evolution technology in vitro, which not only has comparable affinity and specificity to antibody, but also has the following unique advantages: (1) relatively extensive binding range of target, including small molecules, proteins, microorganisms, viruses and cells, etc.; (2) successfully identified nucleic acid aptamer can be synthesized by chemical synthesis, with high production efficiency, relatively low cost, and small quality difference between batches; (3) relatively small molecular weight and low immunogenicity; (4) precise chemical modification and design can be achieved, with strong thermal stability and easy long-term storage. Therefore, developing a nucleic acid aptamer specifically binding to CD117 as a new recognition ligand and widely applying it to many fields such as molecular imaging, biosensing, cell separation, etc. shows great application prospect. SUMMARY

[0004] The present application aims to provide a nucleic acid aptamer specifically binding to CD117 and use thereof.

[0005] The technical scheme adopted by the present application to achieve the above-mentioned purpose is:

[0006] A screening method of a nucleic acid aptamer specifically binding to CD117, comprising,

[0007] S1, constructing a ssDNA library and primers;

[0008] S2, cell screening: the ssDNA library is first subjected to negative screening selection with K562 cells, and then subjected to positive screening selection with HEL cells, after high-temperature denaturation and PCR amplification, a cell screening enriched ssDNA library is obtained and used for the next round of cell screening;

[0009] S3, cell lysate screening: the cell screening enriched single-stranded ssDNA library is mixed with HEL cell membrane proteins, and immune magnetic beads are added for incubation, after high-temperature denaturation and PCR amplification, a cell lysate screening enriched ssDNA library is obtained and used for the next round of cell lysate screening;

[0010] S4, using flow analysis and high-throughput sequencing to obtain a nucleic acid aptamer specifically binding to CD117;

[0011] The immune magnetic beads comprise anti-CD117 antibody modified magnetic beads, or modified anti-CD117 antibody modified magnetic beads; the modified anti-CD117 antibody modified magnetic beads are surface-modified with amino groups and carboxyl groups.

[0012] The present application combines cell-SELEX technology and cell lysate-SELEX technology, takes HEL cells as positive screening cells and K562 cells as negative screening cells, and obtains a nucleic acid aptamer AptCD117 capable of specifically recognizing and high-affinity binding to CD117 antigen. The present application can improve the adsorption efficiency of modified anti-CD117 antibody modified magnetic beads to CD117 protein, separate and purify CD117 protein and ssDNA combined with CD117 protein, thereby improving the DNA recovery efficiency and obtaining a ssDNA library with better enrichment effect, by synergistically modifying a large number of active groups of the modified anti-CD117 antibody modified magnetic beads with 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline.

[0013] Preferably, the ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', the nucleotide sequence of which is shown as SEQ ID No. 1, and N45 is a sequence of 45 arbitrary nucleotide bases connected.

[0014] Preferably, the primers comprise an upstream primer and a downstream primer, the upstream primer modifies 5-FAM, and the downstream primer modifies Biotin.

[0015] Preferably, the upstream primer is 5'-ATCCAGAGTGACGCAGCA-3', and the nucleotide sequence thereof is shown as SEQ ID No. 2; and the downstream primer is 5'-ACTAAGCCACCGTGTCCA-3', and the nucleotide sequence thereof is shown as SEQ ID No. 3.

[0016] Preferably, the cell screening is repeated for at least 6 rounds, and the cell lysate screening is repeated for at least 6 rounds.

[0017] Preferably, 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-isopropenylphenyl aniline, and then coupled with the biotinylated CD117 antibody to obtain the modified anti-CD117 antibody modified magnetic beads.

[0018] Preferably, in the preparation of the anti-CD117 antibody modified magnetic beads, specifically,

[0019] The oleic acid modified ferroferric oxide nanoreagent is added with 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution, incubated at room temperature for 30-60 min, added with streptavidin solution and mixed, coupled at room temperature for 2-4 h, added with balanced salt solution for resuspension, reacted at room temperature for 1-2 h, magnetically separated to remove the supernatant, washed with phosphate buffered saline solution for 2-5 times, discarded the supernatant after standing for 30-60 s, added with biotinylated CD117 antibody, incubated at room temperature for 30-60 min, and the anti-CD117 antibody modified magnetic beads are obtained.

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

[0021] 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 ferroferric oxide nanoreagent and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 1:10-100.

[0022] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent and the N-hydroxysuccinimide solution is 1:10-100.

[0023] 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 nano reagent and the streptavidin solution is 1:10-30.

[0024] More preferably, the volume ratio of the balanced salt solution and the streptavidin solution is 1:1-2.

[0025] More preferably, the volume ratio of the biotinylated CD117 antibody and the streptavidin solution is 1:20-60.

[0026] Preferably, the preparation of the modified anti-CD117 antibody modified magnetic beads, in particular,

[0027] The oleic acid modified ferroferric oxide nano reagent is taken, 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline are added, deionized water is ultrasonically dispersed for 10-20 min, and is left to stand under nitrogen for 2-3 h. A potassium persulfate solution is added and left to stand for 1-2 h. Reaction is carried out at 60-80°C for 2-4 h. A 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and an N-hydroxysuccinimide solution are added, and incubation is carried out at room temperature for 30-60 min. A streptavidin solution is added and mixed, and coupling is carried out at room temperature for 2-4 h. A balanced salt solution is added to resuspend, and reaction is carried out at room temperature for 1-2 h. Magnetic separation is used to remove the supernatant, and washing is carried out 2-5 times with a phosphate buffered saline solution. The supernatant is discarded after standing for 30-60 s. A biotinylated CD117 antibody is added, and incubation is carried out at room temperature for 30-60 min. The modified anti-CD117 antibody modified magnetic beads are obtained.

[0028] More preferably, the oleic acid modified ferroferric oxide nano reagent comprises oleic acid modified ferroferric oxide nanoparticles and n-hexane, and the amount ratio of the oleic acid modified ferroferric oxide nanoparticles and the n-hexane is 1 g:10-100 mL.

[0029] More preferably, the amount ratio of the oleic acid modified ferroferric oxide nano reagent and the 3-allyl-4-methoxybenzoic acid is 1 mL:10-30 mg.

[0030] More preferably, the amount ratio of the oleic acid modified ferroferric oxide nano reagent and the 2-isopropenylphenyl aniline is 1 mL:10-30 mg.

[0031] More preferably, the amount ratio of the 3-allyl-4-methoxybenzoic acid and the deionized water is 1 mg:20-60 mL.

[0032] More preferably, the concentration of the potassium persulfate solution is 0.02-0.1 g / mL, and the amount ratio of the oleic acid modified ferroferric oxide nano reagent and the potassium persulfate solution is 1:0.1-0.2.

[0033] 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 ferroferric oxide nanoreagent and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 1:10-100.

[0034] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent and the N-hydroxysuccinimide solution is 1:10-100.

[0035] 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 and the streptavidin solution is 1:10-30.

[0036] More preferably, the volume ratio of the balanced salt solution and the streptavidin solution is 1:1-2.

[0037] More preferably, the volume ratio of the biotinylated CD117 antibody and the streptavidin solution is 1:20-60.

[0038] Preferably, in the preparation of the modified anti-CD117 antibody modified magnetic beads, glutaric anhydride is further used for modification, specifically,

[0039] The oleic acid modified ferroferric oxide nanoreagent is added with 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline, deionized water is added for ultrasonic dispersion for 10-20 min, and is left standing under nitrogen for 2-3 h, potassium persulfate solution is added and left standing for 1-2 h, and is reacted at 60-80°C for 2-4 h, oxalyl chloride is slowly added and stirred for 10-20 min, butanediol is added, and is reacted at 80-100°C for 6-9 h, glutaric anhydride and 4-dimethylaminopyridine are added, and is reacted at 70-90°C for 12-18 h, 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and N-hydroxysuccinimide solution are added, and is incubated at room temperature for 30-60 min, the streptavidin solution is added and mixed, and is coupled at room temperature for 2-4 h, the balanced salt solution is added to resuspend, and is reacted at room temperature for 1-2 h, magnetic separation is performed to remove the supernatant, and is washed with phosphate buffered saline solution for 2-5 times, the supernatant is discarded after standing for 30-60 s, the biotinylated CD117 antibody is added, and is incubated at room temperature for 30-60 min to obtain the modified anti-CD117 antibody modified magnetic beads. The use of glutaric anhydride can further modify the modified anti-CD117 antibody modified magnetic beads with carboxylation, thereby improving the protein adsorption efficiency and DNA recovery efficiency of the modified anti-CD117 antibody modified magnetic beads.

[0040] More preferably, the oleic acid modified ferroferric oxide nano-reagent comprises oleic acid modified ferroferric oxide nanoparticles and n-hexane, and the ratio of the amount of the oleic acid modified ferroferric oxide nanoparticles to the n-hexane is 1 g: 10-100 mL.

[0041] More preferably, the ratio of the amount of the oleic acid modified ferroferric oxide nano-reagent to the 3-allyl-4-methoxybenzoic acid is 1 mL: 10-30 mg.

[0042] More preferably, the ratio of the amount of the oleic acid modified ferroferric oxide nano-reagent to the 2-isopropenylphenyl aniline is 1 mL: 10-30 mg.

[0043] More preferably, the ratio of the amount of the 3-allyl-4-methoxybenzoic acid to the deionized water is 1 mg: 20-60 mL.

[0044] More preferably, the concentration of the potassium persulfate solution is 0.02-0.1 g / mL, and the ratio of the amount of the oleic acid modified ferroferric oxide nano-reagent to the potassium persulfate solution is 1: 0.1-0.2.

[0045] More preferably, the volume ratio of the oleic acid modified ferroferric oxide nano-reagent to the oxalyl chloride is 1: 1-2.

[0046] More preferably, the ratio of the amount of the oxalyl chloride to the butanediol is 1 mL: 0.02-0.1 g.

[0047] More preferably, the mass ratio of the butanediol to the glutaric anhydride is 1: 1-2.

[0048] More preferably, the mass ratio of the butanediol to the 4-dimethylaminopyridine is 1: 1-2.

[0049] 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 ferroferric oxide nano-reagent to the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 1: 10-100.

[0050] More preferably, the concentration of the N-hydroxysuccinimide solution is 1-10 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nano-reagent to the N-hydroxysuccinimide solution is 1: 10-100.

[0051] 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 nano-reagent to the streptavidin solution is 1: 10-30.

[0052] More preferably, the volume ratio of the balanced salt solution to the streptavidin solution is 1: 1-2.

[0053] More preferably, the volume ratio of the biotinylated CD117 antibody and the streptavidin solution is 1:20-60.

[0054] Preferably, a screening method of a nucleic acid aptamer specifically binding to CD117, in particular,

[0055] S1, constructing a single-stranded DNA (ssDNA) library and primers: the ssDNA library is synthesized as 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', the nucleotide sequence is shown as SEQ ID No. 1, N45 is a sequence of 45 arbitrary nucleotide bases connected; the primers include an upstream primer and a downstream primer, the upstream primer is modified by 5-carboxyfluorescein (5-FAM), the nucleotide sequence is shown as SEQ ID No. 2; the downstream primer is modified by biotin (Biotin), the nucleotide sequence is shown as SEQ ID No. 3. Sterile ultrapure water is added to the ssDNA library to fully dissolve, denatured at 94-96℃ for 5-10 min, and then cooled rapidly to add binding buffer (Binding Buffer), the temperature of the Binding Buffer is 4℃, vortexed and mixed, and ready for use.

[0056] S2, cell screening: the positive screening cell is human erythroleukemia cell (HEL), and the negative screening cell is human chronic myeloid leukemia cell (K562), the ssDNA library is added to the K562 cells, the number of K562 cells is 4×10 6 -6×10 6 , gently shaken and mixed, and incubated in the dark for 30-60 min, centrifuged at 300-400g at 22-28℃ for 5-10 min, the supernatant was collected and added to the HEL cells, the number of HEL cells was 9×10 6 -11×10 6The cells were incubated in the dark for 40-50 minutes, centrifuged at 300-400 g for 5-10 minutes at 22-28 ° C, the supernatant was discarded, and the cells were gently resuspended in Binding Buffer and washed 1-2 times. The cells were centrifuged at 300-400 g for 5-10 minutes at 22-28 ° C, and sterilized ultrapure water was added to resuspend the cells. The cells were denatured at 94-96 ° C for 10-15 minutes, cooled for 2-5 minutes, and centrifuged at 10000 g for 5-10 minutes at 22-28 ° C. The supernatant was collected and used for PCR amplification. The PCR reaction procedure was 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, and the cell screening enriched ssDNA library obtained in each round was stored and used for the next round of cell screening.

[0057] S3. Cell lysate screening: Use the cell membrane protein and cytoplasm extraction kit to extract HEL cell membrane protein, add the cell screening enrichment ssDNA library prepared in the last round of step S2, gently shake and mix, incubate in the dark for 30-40 minutes, then add the immunomagnetic beads, gently shake and mix, incubate in an ice bath for 30-40 minutes, and add Binding Gently resuspend and wash with buffer 1-2 times, discard the supernatant by magnetic separation, resuspend the magnetic beads in sterile ultrapure water, denature at 94-96℃ for 10-15 min, cool for 2-5 min, collect the supernatant by magnetic separation, and use the supernatant for PCR amplification. The PCR reaction program is as follows: pre-denaturation at 94-96℃ for 5-10 min, denaturation at 94-96℃ for 30-40 s, annealing at 55-60℃ for 30-40 s, extension at 70-75℃ for 30-40 s, amplification for 15-20 cycles, extension at 70-75℃ for 5-10 min, store the sample at 2-10℃, then denature with sodium hydroxide solution for 5-10 min to obtain cell lysate for screening and enriching the ssDNA library. Repeat this step for at least 6 rounds, save the cell lysate obtained in each round for screening and enriching the ssDNA library, and use it for the next round of cell lysate screening.

[0058] S4, obtaining nucleic acid aptamer specifically binding to CD117: performing high-throughput sequencing on the ssDNA library with the strongest binding ability to HEL cells and almost no binding to K562 cells to obtain nucleic acid aptamer specifically binding to CD117. The enriched ssDNA library includes the ssDNA library screened by the cells prepared in each round of step S3, and the ssDNA library screened by the cell lysate prepared in each round of step S4.

[0059] More preferably, the Binding Buffer is prepared, in particular,

[0060] Weigh bovine serum albumin (BSA), magnesium chloride hexahydrate, and glucose, add Dulbecco's phosphate buffered saline (DPBS) and mix to obtain the Binding Buffer.

[0061] More preferably, the mass ratio of BSA to magnesium chloride hexahydrate is 1:1-2.

[0062] More preferably, the mass ratio of BSA to glucose is 1:2-6.

[0063] More preferably, the amount ratio of BSA to DPBS is 1g:1-2L.

[0064] More preferably, the amount ratio of ssDNA library to sterile ultrapure water in step S1 is 1 nmol:5-10 μL.

[0065] More preferably, the amount ratio of ssDNA library to Binding Buffer in step S1 is 1 nmol:10-30 μL.

[0066] More preferably, the volume ratio of ssDNA library to Binding Buffer in step S2 is 1:20-60.

[0067] More preferably, the volume ratio of ssDNA library to sterile ultrapure water in step S2 is 1:20-60.

[0068] More preferably, the mass concentration of sodium hydroxide solution in step S2 is 150-250 mmol / L, and the volume ratio of sterile ultrapure water to sodium hydroxide solution is 1:1-2.

[0069] More preferably, the volume ratio of HEL cell membrane protein to ssDNA library in step S3 is 1:1-2.

[0070] More preferably, the immunomagnetic beads in step S3 are anti-CD117 antibody modified magnetic beads or modified anti-CD117 antibody modified magnetic beads.

[0071] More preferably, the volume ratio of the ssDNA library and the immunomagnetic beads in step S3 is 1:0.1-0.2.

[0072] More preferably, the volume ratio of the immunomagnetic beads and the Binding Buffer in step S3 is 1:20-60.

[0073] More preferably, the volume ratio of the immunomagnetic beads and the sterilized ultrapure water in step S3 is 1:20-60.

[0074] More preferably, the mass concentration of the sodium hydroxide solution in step S3 is 150-250 mmol / L, and the volume ratio of the sterilized ultrapure water and the sodium hydroxide solution is 1:1-2.

[0075] The application further discloses a nucleic acid aptamer specifically combined with CD117 screened by the method.

[0076] Preferably, the nucleic acid aptamer specifically combined with CD117 is 5'-TCCAGTGACGCAGCATCGAGCGGGGGACCCTATTAGCTGAATGAGATGCAATTACAAGCGTGGACACTGGC-3', and the nucleotide sequence is shown as SEQ ID No. 4.

[0077] The application further discloses a use of the nucleic acid aptamer specifically combined with CD117 in preparation of an AML tumor cell detection reagent.

[0078] The application further discloses a use of the nucleic acid aptamer specifically combined with CD117 in preparation of a tyrosine kinase inhibitor for blocking the interaction between SCF and CD117.

[0079] The application has the following beneficial effects: in the screening of the nucleic acid aptamer specifically combined with CD117, the modified anti-CD117 antibody modified magnetic beads are used for screening of the cell lysate, the adsorption efficiency of the modified anti-CD117 antibody modified magnetic beads to the CD117 protein is improved, the CD117 protein and the ssDNA combined with the CD117 protein are separated and purified, the DNA recovery efficiency is improved, and the ssDNA library with better enrichment effect is obtained. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 The figure is a graph of the binding level of the selected pool of the ssDNA library in Example 1.

[0081] Figure 2 The figure is a graph of the binding level of the selected pool of the ssDNA library in Example 1.

[0082] Figure 3 Flow cytometry analysis of the binding level of AptCD117 to K562 cells.

[0083] Figure 4 Expression of CD117 on HEL cells after siRNA interference.

[0084] Figure 5 Binding of AptCD117 to HEL cells after siRNA interference.

[0085] Figure 6 Analysis of the binding affinity of AptCD117 to CD117.

[0086] Figure 7 Flow cytometry analysis of the binding of AptCD117 to CD117 + Tumor cell situation.

[0087] Figure 8 Difference in binding sites of AptCD117 and SCF.

[0088] Figure 9 Difference in binding sites of anti-CD117 antibody and SCF. DETAILED DESCRIPTION

[0089] The present application will be further described in conjunction with the specific embodiments, and the examples given are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0090] The experimental methods in the following examples are all routine methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0091] Example 1:

[0092] Preparation of Binding Buffer, including,

[0093] Weigh BSA, magnesium chloride hexahydrate, 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 amount ratio of BSA to DPBS is 1g:1L.

[0094] Preparation of anti-CD117 antibody modified magnetic beads, including,

[0095] 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, let stand for 30 seconds, discard the supernatant, 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 ferroferric 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 ferroferric 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.

[0096] Screening of nucleic acid aptamers that specifically bind to CD117, including:

[0097] S1, constructing ssDNA library and primer: the ssDNA library is 5'-ATCCAGAGTGACGCAGCA-N45-TGGACACGGTGGCTTAGT-3', the nucleotide sequence is shown as SEQ ID No. 1, N45 is a sequence of 45 arbitrary nucleotide bases connected; the primer includes an upstream primer and a downstream primer, the upstream primer is modified by 5-FAM, the nucleotide sequence is shown as SEQ ID No. 2; the downstream primer is modified by Biotin, the nucleotide sequence is shown as SEQ ID No. 3. The ssDNA library and primer are synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. Sterile ultrapure water is added to the ssDNA library to fully dissolve, denatured at 95℃ for 5 min, then Binding Buffer is added after rapid cooling, the temperature of Binding Buffer is 4℃, vortexed and mixed, ready for use. The dosage ratio of ssDNA library and sterile ultrapure water is 1 nmol: 5 μL; the dosage ratio of ssDNA library and Binding Buffer is 1 nmol: 20 μL.

[0098] S2, cell screening: the positive screening cell is human erythroleukemia (HEL) cell, the negative screening cell is human chronic myelogenous leukemia (K562) cell, the ssDNA library is added to K562 cell, the number of K562 cell is 5×10 6 , gently mixed, incubated in dark for 30 min, centrifuged at 300 g at 25℃ for 5 min, the supernatant was collected and added to HEL cell, the number of HEL cell is 10×10 6 , incubated in dark for 45 min, centrifuged at 300 g at 25℃ for 5 min, the supernatant was discarded, Binding Buffer was added to gently resuspend the cell and washed once, centrifuged at 300 g at 25℃ for 5 min, the cell was resuspended with sterile ultrapure water, denatured at 95℃ for 10 min, cooled for 2 min, centrifuged at 10000 g at 25℃ for 5 min, the supernatant was collected, PCR amplification was carried out using the supernatant, the PCR reaction program is: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 15 cycles of amplification, 72℃ extension for 5 min, 4℃ storage of sample, then sodium hydroxide solution was added to denature for 5 min, to obtain the cell screening enriched ssDNA library; the step is repeated for 6 rounds, the cell screening enriched ssDNA library obtained in each round is stored and used for the next round of cell screening. The volume ratio of ssDNA library and Binding Buffer is 1:40; the volume ratio of ssDNA library and sterile ultrapure water is 1:40; the mass concentration of sodium hydroxide solution is 200 mmol / L, the volume ratio of sterile ultrapure water and sodium hydroxide solution is 1:1.

[0099] S3, cell lysate screening: HEL cell membrane proteins were extracted using a cell membrane protein and cytoplasm extraction kit, and the cell screening ssDNA library prepared in step S3 was added and mixed gently, and incubated in the dark for 30 min. Anti-CD117 antibody modified magnetic beads were added and mixed gently, and incubated in an ice bath for 30 min. Binding Buffer was added to resuspend and wash the magnetic beads once, and then the supernatant was removed by magnetic separation. The magnetic beads were resuspended in sterilized ultrapure water, 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, and the PCR reaction program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 30 s, 15 cycles of amplification, 72°C extension for 5 min, and 4°C storage of the sample. Then, sodium hydroxide solution was added to denature for 5 min, and the cell lysate screening ssDNA library was obtained. This step was repeated for 6 rounds, and the cell lysate screening ssDNA library obtained in each round was saved and used for the next round of cell lysate screening. The cell membrane protein and cytoplasm extraction kit was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. The volume ratio of HEL cell membrane proteins to cell screening ssDNA library was 1:1. The volume ratio of cell screening 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 sterilized ultrapure water was 1:40. The mass concentration of sodium hydroxide solution was 200 mmol / L, and the volume ratio of sterilized ultrapure water to sodium hydroxide solution was 1:1.

[0100] S4, obtaining a nucleic acid aptamer specifically binding to CD117: The enriched ssDNA library was 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 was selected for high-throughput sequencing to obtain a nucleic acid aptamer (AptCD117) specifically binding to CD117. The enriched ssDNA library included the cell screening ssDNA library prepared in each round of step S3 and the cell lysate screening ssDNA library prepared in each round of step S4.

[0101] Example 2:

[0102] Compared with Example 1, the difference of this example is the preparation of modified anti-CD117 antibody modified magnetic beads and the screening of nucleic acid aptamer specifically binding to CD117.

[0103] The preparation of modified anti-CD117 antibody modified magnetic beads includes,

[0104] The oleic acid modified ferroferric oxide nanoreagent is added with 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline, and deionized water is ultrasonically dispersed for 10 min, and then placed under nitrogen for 2 h. A potassium persulfate solution is added and placed for 1 h, and then reacted at 70°C for 2 h. A 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution and an N-hydroxysuccinimide solution are added, and incubated at room temperature for 30 min. A streptavidin solution is added and mixed, and then coupled at room temperature for 2 h. An equilibrium salt solution is added to resuspend, and then reacted at room temperature for 1 h. The supernatant is removed by magnetic separation, and then washed with a phosphate buffered saline solution for 3 times. The supernatant is discarded after standing for 30 s. A biotinylated CD117 antibody is added and incubated at room temperature for 30 min to obtain modified anti-CD117 antibody modified magnetic beads. The oleic acid modified ferroferric oxide nanoreagent comprises oleic acid modified ferroferric oxide nanoparticles and n-hexane, and the oleic acid modified ferroferric oxide nanoparticles are purchased from Nanjing Jicang Nanotechnology Co., Ltd. The amount ratio of the oleic acid modified ferroferric oxide nanoparticles to n-hexane is 1 g:100 mL. The amount ratio of the oleic acid modified ferroferric oxide nanoreagent to 3-allyl-4-methoxybenzoic acid is 1 mL:20 mg. The amount ratio of the oleic acid modified ferroferric oxide nanoreagent to 2-isopropenylphenyl aniline is 1 mL:20 mg. The amount ratio of 3-allyl-4-methoxybenzoic acid to deionized water is 1 mg:40 mL. The concentration of the potassium persulfate solution is 0.05 g / mL, and the amount ratio of the oleic acid modified ferroferric oxide nanoreagent to the potassium persulfate solution is 1:0.1. The concentration of the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 1 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to the 1-ethyl-(3-dimethylaminopropyl) carbodiimide solution is 1:10. The concentration of the N-hydroxysuccinimide solution is 1 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to the N-hydroxysuccinimide solution is 1:10. The concentration of the streptavidin solution is 0.05 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to the streptavidin solution is 1:20. The equilibrium salt solution is purchased from Shanghai Ziliano Biotechnology Co., Ltd. The volume ratio of the equilibrium salt solution to the streptavidin solution is 1:1. The biotinylated CD117 antibody is purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd. The volume ratio of the biotinylated CD117 antibody to the streptavidin solution is 1:40.

[0105] The screening of the nucleic acid aptamer specifically binding to CD117 is the same as in Example 1, except that the anti-CD117 antibody modified magnetic beads are replaced by the modified anti-CD117 antibody modified magnetic beads prepared in this example.

[0106] Example 3:

[0107] The difference between this embodiment and embodiment 2 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0108] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as that in embodiment 2, except that the ratio of the amount of the oleic acid modified ferroferric oxide nanoreagent and 3-allyl-4-methoxybenzoic acid is changed to 1 mL:10 mg.

[0109] Embodiment 4:

[0110] The difference between this embodiment and embodiment 2 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0111] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as that in embodiment 2, except that the ratio of the amount of the oleic acid modified ferroferric oxide nanoreagent and 2-isopropenylphenyl aniline is changed to 1 mL:10 mg.

[0112] Embodiment 5:

[0113] The difference between this embodiment and embodiment 1 is in the preparation of the modified anti-CD117 antibody modified magnetic beads and the screening of the nucleic acid aptamer specifically binding to CD117.

[0114] The preparation of the modified anti-CD117 antibody modified magnetic beads comprises,

[0115] The oleic acid modified ferroferric oxide nanoreagent is added with 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline, and deionized water is ultrasonically dispersed for 10 min, and then left to stand under nitrogen for 2 h, and then a potassium persulfate solution is added and left to stand for 1 h, and then reacted at 70°C for 2 h, and then oxalyl chloride is slowly added and stirred for 10 min, and then butanediol is added, and then reacted at 90°C for 6 h, and then glutaric anhydride and 4-dimethylaminopyridine are added, and then reacted at 80°C for 12 h, and then 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution and N-hydroxysuccinimide solution are added, and then incubated at room temperature for 30 min, and then streptavidin solution is added and mixed, and then coupled at room temperature for 2 h, and then an equilibrium salt solution is added to resuspend, and then reacted at room temperature for 1 h, and then the supernatant is removed by magnetic separation, and then washed with phosphate buffered saline solution for 3 times, and then the supernatant is discarded after standing for 30 s, and then a biotinylated CD117 antibody is added, and then incubated at room temperature for 30 min, and then a modified anti-CD117 antibody modified magnetic bead is obtained. The oleic acid modified ferroferric oxide nanoreagent comprises oleic acid modified ferroferric oxide nanoparticles and n-hexane, the oleic acid modified ferroferric oxide nanoparticles are purchased from Nanjing Jicang Nanotechnology Co., Ltd., and the amount ratio of the oleic acid modified ferroferric oxide nanoparticles to n-hexane is 1 g:100 mL; the amount ratio of the oleic acid modified ferroferric oxide nanoreagent to 3-allyl-4-methoxybenzoic acid is 1 mL:20 mg; the amount ratio of the oleic acid modified ferroferric oxide nanoreagent to 2-isopropenylphenyl aniline is 1 mL:20 mg; the amount ratio of 3-allyl-4-methoxybenzoic acid to deionized water is 1 mg:40 mL; the concentration of the potassium persulfate solution is 0.05 g / mL, and the amount ratio of the oleic acid modified ferroferric oxide nanoreagent to the potassium persulfate solution is 1:0.1; the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to oxalyl chloride is 1:1; the amount ratio of oxalyl chloride to butanediol is 1 mL:0.05 g; the mass ratio of butanediol to glutaric anhydride is 1:1; the mass ratio of butanediol to 4-dimethylaminopyridine is 1:1; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution is 1 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide solution is 1:10; the concentration of N-hydroxysuccinimide solution is 1 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to N-hydroxysuccinimide solution is 1:10; the concentration of streptavidin solution is 0.05 mg / mL, and the volume ratio of the oleic acid modified ferroferric oxide nanoreagent to streptavidin solution is 1:20; the equilibrium salt solution is purchased from Shanghai Ziliano Biotechnology Co., Ltd., and the volume ratio of the equilibrium salt solution to streptavidin solution is 1:1; the biotinylated CD117 antibody is purchased from BIOLEGEND (Beijing) Biotechnology Co., Ltd., and the volume ratio of the biotinylated CD117 antibody to streptavidin solution is 1:40.

[0116] Example 6:

[0117] The difference between this example and Example 5 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0118] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as Example 5 except that the mass ratio of butanediol and glutaric anhydride is changed to 1:0.5.

[0119] Comparative Example 1:

[0120] The difference between this comparative example and Example 2 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0121] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as Example 2 except that 3-allyl-4-methoxybenzoic acid is not added.

[0122] Comparative Example 2:

[0123] The difference between this comparative example and Example 2 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0124] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as Example 2 except that 2-isopropenylphenyl aniline is not added.

[0125] Comparative Example 3:

[0126] The difference between this comparative example and Example 5 is only in the preparation of the modified anti-CD117 antibody modified magnetic beads.

[0127] The preparation of the modified anti-CD117 antibody modified magnetic beads is the same as Example 5 except that glutaric anhydride is not added.

[0128] Experimental Example:

[0129] 1. Flow analysis of the enrichment process of the nucleic acid aptamer library

[0130] The enriched ssDNA library is subjected to flow analysis. 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 referred to as the 1st-6th round of ssDNA library; the cell lysate screening enriched ssDNA library prepared in each round of step S4 is sequentially referred to as the 7th-12th round of ssDNA library.

[0131] Figure 1The selection pool binding level of the enriched ssDNA library of Example 1 is shown. As the ssDNA library is screened and enriched, the selection pool binding level of the enriched ssDNA library gradually increases, and the selection pool binding level of the 12th round of ssDNA library is the highest, which indicates that the 12th round of ssDNA library can be selected for high-throughput sequencing. By analyzing the homology and abundance difference of the enriched sequences in the screened library, a candidate aptamer can be selected.

[0132] 2. Flow analysis of the binding ability of AptCD117 to HEL cells and K562 cells

[0133] 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 blank group, the control group and the experimental group were set up, and each of the blank group, the control group and the experimental group included 150 μL of HEL cells with a cell number of 5×10 5 The blank group, the control group and the experimental group were set up, and each of the blank group, the control group and the experimental group included 150 μL of HEL cells with a cell number of 5×10

[0134] According to the above method, the HEL cells were replaced by K562 cells, and the binding ability of AptCD117 to K562 cells was detected by flow cytometry.

[0135] Figure 2 The flow analysis of the binding level of AptCD117 to HEL cells is shown. There is no difference in the binding level between the blank group and the control group, and the binding level of the experimental group is higher than that of the blank group and the control group. Figure 3 The flow analysis of the binding level of AptCD117 to K562 cells is shown. There is no difference in the binding level between the blank group and the blank group, and the control group. This indicates that the AptCD117 screened in Example 1 of the present application has strong binding ability to HEL cells, and does not bind to K562 cells.

[0136] 3. siRNA gene interference experiment to identify the binding target of AptCD117

[0137] HEL cells were seeded in 24-well plates at a cell number of 6 × 10 5 / well, then add 1640 culture medium (without serum and double antibody) to a final volume of 1mL, and then place the 24-well plate in a 37℃ incubator for use. Set up a blank control group (NC), siRNA1 group (siRNA1), siRNA2 group (siRNA2), and siRNA3 group (siRNA3), each group includes 1mL HEL cells, 150μL 1640 culture medium (without serum and double antibody), 40μL transfection buffer (Transfection buffer), siRNA with a concentration of 20μmol / L and 1.8μL transfection reagent (GenMute TM Reagent, wherein the siRNA in the NC group is 5'-CCCAACACAACUUCCUUAUTT-3', whose nucleotide sequence is shown in SEQ ID No. 5; the nucleotide sequence of siRNA1 is 5'-GCCCACAAUAGAUUGGUAUTT-3', whose nucleotide sequence is shown in SEQ ID No. 6; the nucleotide sequence of siRNA2 is 5'-CUGGCAUGAUGUGCAUUAUTT-3', whose nucleotide sequence is shown in SEQ ID No. 7; and the nucleotide sequence of siRNA3 is 5'-GACCAACAUUCAAGCAAAUTT-3', whose nucleotide sequence is shown in SEQ ID No. 8. Cells were centrifuged at 800 g for 75 minutes at 37°C and incubated at 37°C for 5 hours. After the incubation period, the culture medium was discarded and 2 mL of fresh 1640 medium (containing 10% FBS and 1% double-antibody) was added. The cells were incubated in a 37°C incubator for another 48 hours. After the incubation period, the cells in each group were collected and resuspended in Binding Buffer. Afterwards, the cells in 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 placed in an ice box and incubated for 45 minutes. During this period, the cells were gently shaken and mixed once every 15 minutes. After incubation, 400 μL of Washing Buffer was added to wash once, and the cells were centrifuged at 300 g for 5 minutes at 25°C. The supernatant was discarded, and 400 μL of Washing Buffer was added to resuspend the cells and transferred to a flow cytometer. The expression of CD117 protein on the HEL cell membrane surface was analyzed by flow cytometry, and the binding ability of AptCD117 to HEL cells after siRNA interference was analyzed.

[0138] Figure 4 To analyze 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 the NC group, which indicated that siRNA1, siRNA2 and siRNA3 groups could effectively knock down the expression of CD117 protein in HEL cells.

[0139] Figure 5 To analyze the binding of AptCD117 to HEL cells after siRNA interference, the binding level of AptCD117 in siRNA1, siRNA2 and siRNA3 groups was lower than that in the NC group, which indicated that the binding ability between AptCD117 and HEL cells was significantly weakened after the siRNA knockdown of CD117 protein in HEL cells, and was positively correlated with the down-regulation level of CD117. Therefore, AptCD117 specifically targets CD117 protein.

[0140] 4. Analysis of the binding affinity of nucleic acid aptamer AptCD117 to CD117

[0141] The cultured HEL cells were washed once with 5 mL DPBS, centrifuged at a speed of 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 and number of HEL cell samples in each group were 200 μL and 5 x 10 5 The 5-FAM modified AptCD117 with a concentration gradient of 0, 25, 50, 75, 100, 150, 250, 400 and 600 nmol / L was added to the 9 groups of HEL cell samples, respectively, each concentration gradient was repeated for 3 groups, and the cells were mixed and incubated on ice for 45 min, during which the cells were gently mixed once every 15 min. After incubation, the cells were washed once with 400 μL Washing Buffer, centrifuged at a speed of 300 g for 5 min at 25°C, the supernatant was discarded, and 400 μL 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 = dissociation rate of antibody and antigen / binding rate of antibody and antigen.

[0142] Figure 6 To analyze the binding affinity of AptCD117 to CD117, AptCD117 had strong binding affinity to CD117 protein, and the Kd value was 2.83 ± 1.44 nM.

[0143] 5. Application of nucleic acid aptamer AptCD117 in detecting tumor cells in bone marrow samples of AML patients.

[0144] AML bone marrow samples were collected at Zhejiang Cancer Hospital (Ethics Number: IRB-2023-1159), 200 μL blood samples were taken, 1 mL 1x ACK lysis buffer was added and mixed thoroughly, and then lysed at room temperature for 10 min. After centrifugation at 200 g for 5 min at 25°C, the supernatant was discarded. Then 500 μL DPBS and 10 μL fetal bovine serum were added for washing once. Finally, 1 mL Binding Buffer was added to resuspend the cells, 5 μL of anti-CD45 antibody modified with biotin and 2.5 μL of anti-CD117 antibody modified with phycoerythrin were added, and the mixture was shaken and mixed, incubated in an ice bath for 30 min, and gently shaken and mixed once every 10 min during the incubation. Then 400 μL Washing Buffer was added for washing once, and centrifuged at 300 g for 5 min at 25°C. The supernatant was discarded, and 400 μL Binding Buffer was added to resuspend the cells. The control group was added with 50 pmol of 5-FAM modified initial ssDNA library, and the experimental group was added with 50 pmol of 5-FAM modified AptCD117. The mixture was shaken and mixed, incubated in an ice bath for 30 min, and gently shaken and mixed once every 15 min during the incubation. Then 400 μL Washing Buffer was added to the two groups of samples for washing once, and centrifuged at 300 g for 5 min at 25°C. The supernatant was discarded, and 600 μL Washing Buffer was added to resuspend the cells. Then 400 μL of cell sample was taken into a flow tube, and the recognition and binding ability of AptCD117 to AML tumor cells were detected by flow cytometry.

[0145] Figure 7 For flow cytometry analysis of AptCD117 binding to CD117+ tumor cells, the experimental group was added with 50 pmol of 5-FAM modified initial ssDNA library, and the control group was added with 50 pmol of 5-FAM modified AptCD117. The mixture was shaken and mixed, incubated in an ice bath for 30 min, and gently shaken and mixed once every 15 min during the incubation. Then 400 μL Washing Buffer was added to the two groups of samples for washing once, and centrifuged at 300 g for 5 min at 25°C. The supernatant was discarded, and 600 μL Washing Buffer was added to resuspend the cells. Then 400 μL of cell sample was taken into a flow tube, and the recognition and binding ability of AptCD117 to AML tumor cells were detected by flow cytometry. + The percentage of aptamer in tumor cells was higher than that in the control group, which indicated that AptCD117 could specifically recognize and bind CD117 + tumor cells.

[0146] 6. Flow cytometry analysis of the binding site difference between AptCD117 and SCF.

[0147] 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 cell samples were set in two groups, one group was added with 50 pmol of 5-FAM fluorescent modified AptCD117 and 2 μg SCF; the other group was added with 2.5 μL of fluorescent modified anti-CD117 antibody and 2 μg SCF, and incubated in an ice bath for 30 min, during which the cells were gently shaken and mixed once every 10 min, washed once with 400 μL Washing Buffer, centrifuged at 300 g for 5 min at 25 °C, the supernatant was discarded, 400 μL Washing Buffer was added to resuspend the cells and transferred to a flow tube, and the binding site differences of AptCD117, anti-CD117 antibody and SCF were detected by flow cytometry.

[0148] Figure 8 For the binding site difference of AptCD117 and SCF, Figure 9 For the binding site difference of anti-CD117 antibody and SCF. By Figure 8 and Figure 9 It can be seen that SCF has a competitive effect on the binding of AptCD117 to CD117 protein, that is, both have a common binding site, while the binding site of SCF and anti-CD117 antibody is not consistent. Therefore, AptCD117 can act as a tyrosine kinase inhibitor to block the binding of SCF to CD117.

[0149] 7. DNA recovery efficiency of anti-CD117 antibody modified magnetic beads

[0150] The ssDNA library prepared in the 6th round of screening step S3 of the nucleic acid aptamer specifically binding to CD117 of Example 1 was determined for DNA content using ultraviolet spectrophotometry, and the initial DNA content (ng / μL) was recorded. HEL cell membrane proteins were extracted using a cell membrane protein and cytoplasm extraction kit, and the ssDNA library prepared in the 6th round of screening step S3 of the nucleic acid aptamer specifically binding to CD117 of Example 1 was added. After gentle shaking and mixing, the mixture was incubated in the dark for 30 min. Anti-CD117 antibody modified magnetic beads were added, and the mixture was gently shaken and mixed. The mixture was incubated in an ice bath for 30 min. Binding Buffer was added to resuspend the magnetic beads, and the mixture was washed once. The supernatant was removed by magnetic separation. The magnetic beads were resuspended in sterilized ultrapure water, denatured at 95°C for 10 min, cooled for 2 min, and the supernatant was collected by magnetic separation. The DNA content in the supernatant (ng / μL) was determined using ultraviolet spectrophotometry. The DNA recovery efficiency was calculated, and the DNA recovery efficiency (%) = the DNA content in the supernatant / the initial DNA content × 100%. The anti-CD117 antibody modified magnetic beads prepared in Examples 1-6 and Comparative Examples 1-3 were used to obtain the DNA recovery efficiencies of Examples 1-6 and Comparative Examples 1-3, respectively. The determination results are shown in Table 1.

[0151] Table 1 DNA recovery efficiency (%)

[0152]

[0153] As shown in Table 1, the DNA recovery efficiencies of Examples 2-4 were higher than those of Example 1 and Comparative Examples 1-2. This was because, in the cell lysate screening, 3-allyl-4-methoxybenzoic acid and 2-isopropenylaniline were used to prepare modified anti-CD117 antibody modified magnetic beads in Examples 2-4, while 3-allyl-4-methoxybenzoic acid was not used in Example 1, 3-allyl-4-methoxybenzoic acid was used in Comparative Example 1, and 2-isopropenylaniline was used in Comparative Example 2. This indicates that the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylaniline to prepare modified anti-CD117 antibody modified magnetic beads can improve the DNA recovery efficiency. The DNA recovery efficiencies of Examples 5-6 were higher than those of Examples 2-3 and Comparative Example 3. This was because, in the cell lysate screening, glutaric anhydride was further used to prepare modified anti-CD117 antibody modified magnetic beads on the basis of the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylaniline in Examples 5-6, while glutaric anhydride was used to prepare modified anti-CD117 antibody modified magnetic beads in Comparative Example 3. This indicates that glutaric anhydride can further improve the DNA recovery efficiency of modified anti-CD117 antibody modified magnetic beads.

[0154] 8. Protein adsorption performance of anti-CD117 antibody modified magnetic beads

[0155] HEL cell membrane protein was extracted using cell membrane protein and cytoplasm extraction kit, HEL cell membrane protein content was determined using biuret method, recorded as initial protein content (mg / mL), anti-CD117 antibody modified magnetic beads were added, gently shaken and mixed, incubated in ice bath for 30 min, added Binding Buffer, gently resuspended and washed once, magnetic separation discarded supernatant, added sterilized ultrapure water to resuspend magnetic beads, magnetic separation collected supernatant, biuret method was used to determine protein content (mg / mL) in supernatant; protein adsorption efficiency was calculated, protein adsorption efficiency (%) = supernatant protein content / initial protein content x 100%. Anti-CD117 antibody modified magnetic beads prepared using examples 1-6 and comparative examples 1-3 were used, corresponding to obtain protein adsorption efficiency of examples 1-6 and comparative examples 1-3, the determination results are shown in Table 2.

[0156] Table 2 Protein adsorption efficiency (%)

[0157]

[0158] As can be seen from Table 2, the protein adsorption efficiency of examples 2-4 is higher than that of examples 1 and comparative examples 1-2, because in the cell lysate screening, 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline are used in combination to prepare modified anti-CD117 antibody modified magnetic beads in examples 2-4, while 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline are not used in example 1, only 3-allyl-4-methoxybenzoic acid is used in comparative example 1, and only 2-isopropenylphenyl aniline is used in comparative example 2. This shows that the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline in combination to prepare modified anti-CD117 antibody modified magnetic beads can improve the protein adsorption efficiency thereof. 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, glutaric anhydride is further used on the basis of the use of 3-allyl-4-methoxybenzoic acid and 2-isopropenylphenyl aniline in combination to prepare modified anti-CD117 antibody modified magnetic beads in examples 5-6, while only glutaric anhydride is used to prepare modified anti-CD117 antibody modified magnetic beads in comparative example 3. This shows that glutaric anhydride can further improve the protein adsorption efficiency of modified anti-CD117 antibody modified magnetic beads.

[0159] The routine operations in the operation steps of the present application are well known to those skilled in the art, and will not be described here.

[0160] The above examples have described the technical solutions of the present application in detail, and it should be understood that the above description is only a specific embodiment of the present application and is not intended to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application shall be included in the protection scope of the present application.

Claims

1. A nucleic acid aptamer that specifically binds to CD117, characterized in that: The nucleic acid aptamer that specifically binds to CD117 is 5'-TCCAGTGACGCAGCATCGAGCGGGGACCCTATTAGCTGAATGAGATGCAATTACAAGCGTGGACACTGGC-3'.

2. Use of the nucleic acid aptamer that specifically binds to CD117 according to claim 1 in the preparation of an AML tumor cell detection reagent.

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