Nucleic acid aptamers for specifically recognizing diffuse large B-cell lymphoma cells and application of nucleic acid aptamers

Abstract

The invention discloses a group of nucleic acid aptamers for specifically recognizing diffuse large B-cell lymphoma cells and application of the nucleic acid aptamers. The invention provides a nucleic acid aptamer capable of specifically recognizing diffuse large B-cell lymphoma cells. The nucleic acid aptamer is a single-stranded DNA molecule as shown in SEQ ID No.1. The aptamer provided by the invention can specifically detect WSU-DLCL2 cells, and is of great significance to early diagnosis and treatment of diffuse large B lymphoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a group of nucleic acid aptamers specifically recognizing diffuse large B cell lymphoma cells and application thereof. BACKGROUND

[0002] Diffuse large B cell lymphoma (DLBCL) is a mature B cell-derived aggressive tumor, which is the most common type of non-Hodgkin's lymphoma, accounting for about 25-50% of all non-Hodgkin's lymphoma. Moreover, DLBCL is a group of highly heterogeneous aggressive lymphoma, and currently 40% of patients cannot be cured. In addition, the probability of involving the central nervous system (CNS) is about 5%, and the overall survival (OS) of DLBCL patients with CNS involvement is less than 6 months, with extremely poor prognosis. Patients with incurable DLBCL are often associated with poor prognostic factors. Although researchers have explored many treatment options for DLBCL, the heterogeneity of DLBCL limits the clinical benefits of these options in DLBCL patients. Therefore, if early diagnosis and treatment of DLBCL patients can be achieved, it not only can better guide the selection of clinical treatment options, but also can improve the prognosis outcome. Nucleic acid aptamers not only have the function of inhibiting cell growth or promoting cell apoptosis as therapeutic drugs, but also can be used as carriers for molecular biological imaging, disease diagnosis and targeted drug therapy.

[0003] Nucleic acid aptamer is a piece of oligonucleotide sequence (RNA or ssDNA) with a length of 15-100 nt, which is a new type of molecular probe. It has a unique and stable three-dimensional structure, which enables it to form a specific three-dimensional structure by self-adaptive folding through various interaction forces such as nucleotide base complementary pairing, hydrogen bonding, π-π stacking, and electrostatic force, and bind to specific targets (such as ions, proteins, viruses, and even entire cells and tissues). Aptamers are also known as "chemical antibodies", which have the advantages of easy synthesis, low toxicity, easy modification, nanoscale, low immunogenicity, and wide range from metal ions to cell targets compared with antibodies. Through the Cell-SELEX technology of in vitro index enrichment of ligand system evolution with live cells as targets, nucleic acid aptamers specifically recognizing target cells can be effectively screened from a large number of random libraries.

[0004] Therefore, in order to achieve early and precise diagnosis and treatment of DLBCL patients, screening of nucleic acid aptamers specifically recognizing diffuse large B cell lymphoma cells and discovery of diffuse large B cell lymphoma markers are of great significance for early diagnosis and prognosis of tumors. SUMMARY

[0005] The purpose of the present application is to provide a nucleic acid aptamer specifically recognizing diffuse large B cell lymphoma cells and application thereof.

[0006] In a first aspect, the present application claims a nucleic acid aptamer.

[0007] The nucleic acid aptamer provided by the present application can be specifically any one of the following:

[0008] (A1) a single-stranded DNA molecule as shown in any one of SEQ ID No. 1 to SEQ ID No. 15;

[0009] As long as it is the same as the nucleic acid aptamer sequence (any one of SEQ ID No. 1 to SEQ ID No. 15) of the present application, regardless of the secondary structure, it should be considered to fall within the protection scope of the present application.

[0010] (A2) deleting or adding one or several nucleotides to the nucleic acid aptamer as shown in (A1) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer;

[0011] Further, with individual base modification of the nucleic acid aptamer sequence (any one of SEQ ID No. 1 to SEQ ID No. 15) of the present application, other sequences with a similarity of more than 80%, the same or extremely similar application as the nucleic acid aptamer of the present application, and no effect on the overall function, should also be considered to fall within the protection scope of the present application.

[0012] (A3) nucleotide substitution or modification of the nucleic acid aptamer as shown in (A1) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer;

[0013] Further, the modification can be phosphorylation, methylation, amination, sulfydrylation or isotopic modification, etc.

[0014] (A4) modifying the backbone of the nucleic acid aptamer as shown in (A1) to a phosphorothioate backbone to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer;

[0015] (A5) an RNA molecule encoded by the nucleic acid aptamer as shown in (A1) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer;

[0016] (A6) a peptide nucleic acid encoded by the nucleic acid aptamer as shown in (A1) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer;

[0017] (A7) connecting a chemical group and / or a fluorescein and / or an anti-tumor drug and / or a radioactive element and / or a biological enzyme and / or biotin and / or a nanomaterial to one end or the middle (one position or multiple positions) of the nucleic acid aptamer as shown in any one of (A1) to (A6) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer.

[0018] In a second aspect, the present invention claims protection for a nucleic acid aptamer-functionalized magnetic nanoparticle.

[0019] The nucleic acid aptamer-functionalized magnetic nanoparticles claimed in the present invention are loaded with the nucleic acid aptamer described in the first aspect above.

[0020] In the aptamer-functionalized magnetic nanoparticles, the aptamer can be coupled to the magnetic nanoparticles via biotin and streptavidin.

[0021] Furthermore, the aptamer-functionalized magnetic nanoparticles can be prepared according to a method comprising the following steps: incubating the biotin-modified aptamer described in the first aspect above with streptavidin-modified magnetic nanoparticles to obtain the aptamer-functionalized magnetic nanoparticles.

[0022] In a specific embodiment of the present invention, biotin may be modified at the 5' end of the nucleic acid aptamer.

[0023] In a third aspect, the present invention claims protection for the use of the nucleic acid aptamer described in the first aspect above or the nucleic acid aptamer-functionalized magnetic nanoparticles described in the second aspect above in any of the following:

[0024] (B1) preparing a product for recognizing and binding to diffuse large B-cell lymphoma cells, or recognizing and binding to diffuse large B-cell lymphoma cells;

[0025] (B2) preparing a product for capturing, enriching, or purifying diffuse large B-cell lymphoma cells, or capturing, enriching, or purifying diffuse large B-cell lymphoma cells;

[0026] (B3) preparing a product for detecting diffuse large B-cell lymphoma cells, or detecting diffuse large B-cell lymphoma cells;

[0027] (B4) preparing products for diagnosis or auxiliary diagnosis of diffuse large B-cell lymphoma;

[0028] (B5) Preparing products for the treatment or adjuvant treatment of diffuse large B-cell lymphoma.

[0029] In a specific embodiment of the present invention, the diffuse large B-cell lymphoma cells are WSU-DLCL2 cells. Accordingly, in (B1), the identification and binding of diffuse large B-cell lymphoma cells is the identification and binding of WSU-DLCL2 cells in a mixed cell. In (B2), the capture, enrichment or purification of diffuse large B-cell lymphoma cells is the capture, enrichment or purification of WSU-DLCL2 cells in a mixed cell. In (B3), the detection of diffuse large B-cell lymphoma cells is the detection of WSU-DLCL2 cells in a mixed cell. Wherein, the mixed cell specifically comprises (or is) WSU-DLCL2 cells and all or part of the following cells: CCRF-CEM (human acute lymphoblastic leukemia cells), Jurkat (human T lymphocytic leukemia cells), THP-1 (human acute monocytic leukemia cells).

[0030] Wherein, the product may be a reagent or a kit.

[0031] In a fourth aspect, the present invention claims the use of the nucleic acid aptamer described in the first aspect above in the preparation of a diagnostic probe or imaging agent for imaging diffuse large B-cell lymphoma.

[0032] In a fifth aspect, the present invention claims protection for the use of the nucleic acid aptamer described in the first aspect above in the preparation of an indicator reagent for guiding the resection of diffuse large B-cell lymphoma lesions and / or metastases during fluorescence laparoscopic surgery.

[0033] Wherein, the excision indicator reagent can be an ICG-type molecule-modified nucleic acid aptamer or a derivative thereof.

[0034] In a sixth aspect, the present invention claims protection for a product.

[0035] The product claimed in the present invention has as its active ingredient (or comprises) the nucleic acid aptamer described in the first aspect above;

[0036] The product has any of the following functions:

[0037] (C1) recognizes and binds to diffuse large B-cell lymphoma cells;

[0038] (C2) capturing, enriching, or purifying diffuse large B-cell lymphoma cells;

[0039] (C3) detection of diffuse large B-cell lymphoma cells;

[0040] (C4) Diagnosis or auxiliary diagnosis of diffuse large B-cell lymphoma;

[0041] (C5) Treatment or adjuvant treatment of diffuse large B-cell lymphoma;

[0042] (C6) Imaging of diffuse large B-cell lymphoma cells.

[0043] In a specific embodiment of the present invention, the diffuse large B-cell lymphoma cells are WSU-DLCL2 cells. Accordingly, in (C1), the identification and binding of diffuse large B-cell lymphoma cells is the identification and binding of WSU-DLCL2 cells in a mixed cell. In (C2), the capture, enrichment or purification of diffuse large B-cell lymphoma cells is the capture, enrichment or purification of WSU-DLCL2 cells in a mixed cell. In (C3), the detection of diffuse large B-cell lymphoma cells is the detection of WSU-DLCL2 cells in a mixed cell. Wherein, the mixed cell specifically comprises (or is) WSU-DLCL2 cells and all or part of the following cells: CCRF-CEM (human acute lymphoblastic leukemia cells), Jurkat (human T lymphocytic leukemia cells), THP-1 (human acute monocytic leukemia cells).

[0044] Wherein, the product may be a reagent or a kit.

[0045] In a seventh aspect, the present invention claims a diagnostic probe or imaging agent for imaging diffuse large B-cell lymphoma.

[0046] The diagnostic probe or imaging agent for imaging diffuse large B-cell lymphoma claimed in the present invention has as its active ingredient (or comprises) the nucleic acid aptamer described in the first aspect above.

[0047] In an eighth aspect, the present invention claims protection for a resection indicator reagent for guiding the resection of diffuse large B-cell lymphoma lesions and / or metastases during fluorescence laparoscopic surgery.

[0048] The present invention claims protection for the resection indicator reagent for guiding the resection of diffuse large B-cell lymphoma lesions and / or metastases in fluorescence laparoscopic surgery, the active ingredient of which is (or includes) the nucleic acid aptamer described in the first aspect above.

[0049] In a ninth aspect, the present invention claims a set of nucleic acid aptamers.

[0050] The set of nucleic acid aptamers claimed in the present invention may specifically be any of the following:

[0051] (D1) consists of all or part of the 16 single-stranded DNA molecules shown in SEQ ID No. 1 to SEQ ID No. 15; the part contains at least the single-stranded DNA molecule shown in SEQ ID No. 1;

[0052] (D2) deleting or adding one or more nucleotides to each nucleic acid aptamer shown in (D1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer;

[0053] (D3) performing nucleotide substitution or modification on each nucleic acid aptamer shown in (D1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer;

[0054] (D4) transforming the backbone of each nucleic acid aptamer shown in (D1) into a phosphothioate backbone to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer;

[0055] (D5) obtaining an aptamer derivative having the same function as the aptamer from an RNA molecule encoded by each aptamer shown in (D1);

[0056] (D6) obtaining a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer from the peptide nucleic acid encoded by each nucleic acid aptamer shown in (D1);

[0057] (D7) Connecting a chemical group and / or fluorescein and / or anti-tumor drug and / or radioactive element and / or biological enzyme and / or biotin and / or nanomaterial to one end or the middle of each nucleic acid aptamer shown in any one of (D1)-(D6) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer.

[0058] In the tenth aspect, the present invention further claims protection for the use of the set of nucleic acid aptamers described in the ninth aspect above in the preparation of a product for molecular typing of cells; the cells are selected from WSU-DLCL2 cells, THP-1 cells or CCRF-CEM cells.

[0059] The present invention uses the Cell-SELEX screening method, with diffuse large B-cell lymphoma cells WSU-DLCL2 as target cells, CCRF-CEM (human acute lymphoblastic leukemia cells), Jurkat (human T lymphocytic leukemia cells), and THP-1 (human acute monocytic leukemia cells) as control cells, to perform cell-to-cell difference screening. Based on the flow cytometry monitoring results, the 1st and 9th rounds of library PCR amplification are selected and then high-throughput sequencing is performed to select a nucleic acid aptamer (SEQ ID No. 1) that specifically recognizes diffuse large B-cell lymphoma cells.

[0060] Compared with the prior art, the advantages of the present invention are:

[0061] 1. Provides a nucleic acid aptamer that specifically targets diffuse large B-cell lymphoma with high affinity.

[0062] 2. The screened aptamers exhibit low immunogenicity, are easily modified and labeled, exhibit stable and reproducible properties, and offer excellent stability compared to antibodies, with low synthesis costs. Further characterization of aptamer properties and target identification will facilitate the exploration and discovery of tumor markers. Aptamers can also serve as highly effective and penetrating molecular probes for early diagnosis of tumors and can be combined with drugs for targeted tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Cell screening monitoring. A shows the binding of aptamer-enriched libraries to target cells WSU-DLCL2 at different rounds; B shows the length distribution of aptamer libraries in the ninth round of enrichment. In A, BLK represents a blank cell group incubated without aptamers; R2, R4, R6, R8, and R9 represent single-stranded libraries screened in rounds 2, 4, 6, 8, and 9, respectively. In B, the horizontal axis represents the sequence numbers of the top 100 sequences.

[0064] Figure 2 Figure 2 shows the binding of different aptamers to different cells. A: WSU-DLCL2 cells; B: Jurkat cells; C: THP-1 cells; D: CCRF-CEM cells (denoted by CEM in the figure). E: Confocal images of SWU-2a binding to different cells. In Figures A and D, blank cells were incubated without aptamers; the remaining cells represent the 12 sequences selected for screening. In Figure E, CEM represents CCRF-CEM cells.

[0065] Figure 3 This is a heatmap of the binding between different cells and different aptamers. From white to blue, darker colors indicate stronger binding between the cell and the aptamer. CEM in the figure represents CCRF-CEM cells.

[0066] Figure 4 is the equilibrium dissociation constant (K) between different aptamers and different cells at 4°C and 37°C d A is the Kd of the aptamer and different cells at 4°C, and B is the Kd of the aptamer and different cells at 37°C. d In the figure, CEM represents CCRF-CEM cells.

[0067] Figure 5 Flow cytometric characterization of truncated aptamers and cells. CEM in the figure represents CCRF-CEM cells.

[0068] Figure 6Figure 2 shows the capture of target cells by functionalized magnetic beads. Figure A shows the capture of WSU-DLCL2 cells by magnetic beads coupled with the nucleic acid aptamer SWU-2a and the control sequence RAM, respectively. Figure 2 shows the cell capture by magnetic beads functionalized with the nucleic acid aptamer SWU-2a and the control sequence RAM, respectively. Figure 2 shows the cell capture efficiency of magnetic beads functionalized with the nucleic acid aptamer SWU-2a and the control sequence RAM, respectively. Figure 2 shows the cell capture efficiency of magnetic beads functionalized with the nucleic acid aptamer SWU-2a and the control sequence RAM, respectively. Figure 2 shows the specific capture of target cells by magnetic beads coupled with the nucleic acid aptamer SWU-2a (capture efficiency exceeding 80%). Figure 2 shows the capture of different cell types by magnetic beads functionalized with the nucleic acid aptamer SWU-2a, respectively. Figure 2 shows the capture efficiency of magnetic beads functionalized with the nucleic acid aptamer SWU-2a, respectively. Figure 2 shows the capture of WSU-DLCL2 cells by magnetic beads coupled with the nucleic acid aptamer SWU-2a in a cell mixture. Figure 2 shows the capture of WSU-DLCL2 cells by magnetic beads coupled with the nucleic acid aptamer SWU-2a in a cell mixture. Figure 2 shows the capture of WSU-DLCL2 cells by magnetic beads functionalized ...

[0069] Figure 7 The standard curves for detecting WSU-DLCL2 cells using functionalized magnetic beads are shown in Figure 1. A is the standard curve for detecting single cells (WSU-DLCL2) using SWU-2a functionalized magnetic beads, and B is the standard curve for detecting WSU-DLCL2 in a mixed sample using SWU-2a functionalized magnetic beads. DETAILED DESCRIPTION

[0070] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0071] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0072] Example 1. Screening and synthesis of nucleic acid aptamers

[0073] In this example, the Cell-SELEX technology was used to screen randomly synthesized ssDNA sequences, using the human diffuse large B-cell lymphoma cell line WSU-DLCL2 as the positive screening cell line, and CCRF-CEM (human acute lymphoblastic leukemia cells), Jurkat (human T lymphocytic leukemia cells), and THP-1 (human acute monocytic leukemia cells) as the counter-screening cells. The aim was to screen for nucleic acid aptamers that can specifically recognize the WSU-DLCL2 cell line and have strong affinity binding.

[0074] 1. Cell Culture

[0075] All cells used in the experiments were obtained from the American Type Culture Collection (ATCC). Specific culture conditions are shown in Table 1. The culture medium used was supplemented with 10% FBS and 100 U / mL of penicillin and streptomycin. All cells were cultured in a 37°C incubator with a CO2 concentration of 5%. A commercial serum-free freezing medium was used for cryopreservation.

[0076] Table 1. Cell culture conditions

[0077] Cell lines Chinese name Culture conditions WSU-DLCL2 Human diffuse large B-cell lymphoma cells RPMI1640 + 10% FBS CCRF-CEM Human acute lymphoblastic leukemia cells RPMI1640 + 10% FBS Jurkat Human T lymphoblastic leukemia cells RPMI1640 + 10% FBS THP-1 Human acute monocytic leukemia cells RPMI1640 + 10% FBS

[0078] 2. Solution Preparation

[0079] ① Washing buffer solution: DPBS buffer (pH=7.4) + 5mM MgCl2 + 4.5g / L glucose.

[0080] ② Binding buffer solution: washing buffer solution + 1 mg / mL bovine serum albumin (BSA) + 0.5 mg / mL herring sperm DNA (hsDNA).

[0081] ③5× Mix: Dilute primers PZJAB and PZJS (for specific sequences, see step 3) to 50 μM in ddH2O. This mixture is as follows: 500 μL of 10× buffer (Takara, 9152A) + 150 μL of dNTPs (Takara, 639132) + 120 μL of primer PZJAB + 120 μL of primer PZJS + 25 μL of Taq enzyme (Takara, RR001) + 85 μL of ddH2O to make a 5× Mix.

[0082] 3. Cell Screening Steps

[0083] When screening nucleic acid aptamers, the nucleic acid library and primers used are designed as follows

[0084] Random nucleic acid library (LibZJ1):

[0085] 5'-ACCGACCGTGCTGGACTCta-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-acTATGAGCGAGCCTGGCGt-3'.

[0086] Upstream primer (PZJS): 5′-fluorescein isothiocyanate-ACCGACCGTGCTGGACTCta-3′;

[0087] Downstream primer (PZJAB): 5'-biotin-acTATGAGCGAGCCTGGCGt-3'.

[0088] Wherein, N represents A, T, C, G randomly any base.

[0089] 1. First round of screening

[0090] (1) Library preparation: 2 OD random nucleic acid library (LibZJ1) was dissolved in 200 μL DPBS, shaken and mixed, denatured at 95 °C for 5 min, placed on ice for 5 min, and renatured at room temperature for 15 min.

[0091] (2) Cell preparation: WSU-DLCL2 cells with good growth potential were taken, counted, centrifuged at 1200 rpm for 3 min, the supernatant was removed, washed once with 3 mL of washing buffer solution, and resuspended with 800 μL of binding buffer solution.

[0092] (3) Cell incubation: The denatured and renatured 2 OD random nucleic acid library (LibZJ1) was added to the cells, incubated on ice for one hour, and shaken and mixed every 3-5 min. After incubation, the supernatant was removed, washed 4 times with 1 mL of washing buffer solution, and 200 μL of ddH2O was added to resuspend the cells, which were used as templates for PCR amplification.

[0093] (4) PCR amplification:

[0094] PCR1 system: 200 μL template 100 μL 5×Mix + 100 μL ddH2O, cycle number 9. PCR program: 95 °C for 3 min, 9 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 72 °C for 5 min, and 12 °C for termination.

[0095] Optimized cycle number PCR2: 2 μL PCR1 product + 10 μL 5×Mix + 38 μL ddH2O, divided into 5 tubes, 10 μL per tube. Set the cycle number to 8, 10, 12, 14, and 16. PCR program: 95 °C for 3 min, 16 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, 72 °C for 5 min, and 12 °C for termination. At the end of 8, 10, 12, 14, and 16 cycles, the corresponding samples were taken out and subjected to denaturing polyacrylamide gel electrophoresis, and the gel bands were observed to determine the optimal cycle number 12.

[0096] PCR3: Perform PCR3 at the optimal cycle number (PCR3 system: 20 μL PCR1 product + 100 μL 5× Mix + 380 μL ddH2O). PCR program: 95°C for 3 min, 12 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 72°C for 5 min, and terminate at 12°C.

[0097] (5) ssDNA preparation:

[0098] a) Add 60 μL of the streptavidin agarose bead suspension to an empty microcolumn tube and filter under pressure to dry the liquid. The beads will remain on the top of the filter cartridge.

[0099] b) Wash the column twice with 100 μL DPBS.

[0100] c) The product obtained from PCR3 is passed through a microcolumn.

[0101] d) Wash twice with DPBS, 100 μL each time.

[0102] e) The microcolumn was rinsed twice with 50 μL of 0.2 M NaOH solution, and the eluate was collected.

[0103] f) Desalting using NAP-5 desalting columns (GE Healthcare, UK). Pre-wash the desalting columns with deionized water.

[0104] g) Add 100 μL of ssDNA-NaOH solution to the column and wait for the liquid to flow out.

[0105] h) Add 400 μL of ultrapure water and wait for the liquid to flow out.

[0106] i) Add 600 μL of ultrapure water to elute the ssDNA and collect the flow-through in a 1.5 mL EP tube. The volume is approximately 600 μL.

[0107] k) The ssDNA content was calibrated using a UV spectrophotometer.

[0108] l) Vacuum dry and store at -20℃.

[0109] m) Label the prepared single-stranded DNA for use in the next round of screening.

[0110] 2. Screening from the 2nd to 9th rounds

[0111] (1) The single-chain prepared in the previous round was dissolved in DPBS to a concentration of 2 μM and renatured for use in the next round of screening.

[0112] (2) To further improve the affinity and specificity of aptamer, gradually increase the screening pressure in the screening process: including reducing the number of positive screening cells, reducing the incubation time of positive screening cells, increasing the number of negative screening cells, increasing the incubation time of negative screening cells, and increasing the number of positive screening cell washing. After 9 rounds of screening, after characterization by flow cytometry, the PCR products of the 1st and 9th were used for high-throughput sequencing.

[0113] Four, flow cytometry monitoring and high-throughput sequencing

[0114] 1. Flow cytometry monitoring

[0115] Library preparation: The prepared ssDNA library of different rounds was heated at 95°C for 5 min, cooled on ice for 5 min, and renatured at room temperature for 15 min for standby.

[0116] Cell suspension preparation: Prepare WSU-DLCL2 cells, centrifuge to remove culture medium, add 3 mL washing buffer and centrifuge twice, and resuspend with binding buffer for standby.

[0117] Flow sample preparation: Take 20 pmol of ssDNA of different rounds, add to different cell suspensions, incubate on ice for 30 min, wash twice with washing buffer, and finally resuspend with washing buffer for machine.

[0118] Real-time monitoring of library enrichment during screening by flow cytometry, as shown in Figure 1 A, with the increase of screening pressure, the binding of enriched library to target cell WAU-DLCL2 gradually becomes stronger.

[0119] 2. High-throughput sequencing

[0120] Based on the flow cytometry monitoring results, we selected the ssDNA library obtained by the 1st and 9th screening for PCR amplification, and selected the PCR bands without non-specific bands, clear target bands, and bright lanes for amplification. After amplification, sequencing was performed. Through high-throughput sequencing, the sequence distribution of random sequences is shown in Figure 1 B.

[0121] Example 2, nucleic acid aptamer specifically recognizing WSU-DLCL2 cells and its application in cell molecular typing

[0122] According to the enrichment of high-throughput sequencing, according to the sequence enrichment degree and homology analysis, we obtained 12 potential nucleic acid aptamer sequences by truncation and optimization. The 5' end of the aptamer was labeled with fluorescein isothiocyanate (FITC), and we determined their binding to different cells by flow cytometry. As shown in Figure 2As shown, the nucleic acid aptamer SWU-2a strongly binds to WSU-DLCL2 cells and essentially does not bind to THP-1, CCRF-CEM, or Jurkat cells. To more intuitively demonstrate the specificity of SWU-2a, we labeled the aptamer's 5' end with AF647 fluorescence. The aptamer was incubated with WSU-DLCL2, THP-1, CCRF-CEM, and Jurkat cells on ice for 30 minutes. The cells were washed twice with wash buffer, resuspended in 250 μL of wash buffer, and evenly plated on a confocal microscopy dish. The cells were observed and photographed under a laser confocal microscope. This further demonstrates that the screened nucleic acid aptamer SWU-2a specifically recognizes non-diffuse large B-cell lymphoma cells and that its target may be a potential tumor biomarker. The nucleotide sequence of the nucleic acid aptamer SWU-2a is shown in Table 2.

[0123] Table 2. Aptamer nucleic acid sequences

[0124]

[0125]

[0126] Note: Lowercase letters in the aptamer sequence represent the mutated base sites. A mutation caused by the substitution of one base pair in a DNA molecule with another different base pair is also called a point mutation.

[0127] 2. Aptamers can be used for molecular typing

[0128] A total of 12 nucleic acid aptamers were obtained from cell screening (Table 2). There were differences in the binding of these nucleic acid aptamers to cells. We measured the flow cytometry binding displacement of different cells and different nucleic acid aptamers, and drew a heat map based on the positive rate of flow cytometry displacement, as shown in Figure 2. Figure 3 Based on the different binding conditions, we can further understand the expression of different proteins in cells, which can be used for molecular typing of blood tumors in the future.

[0129] 3. Aptamer Affinity Determination

[0130] The affinity of the screened FITC-labeled aptamers SWU-2a, SWU-5a, SWU-7a, SWU-14a, and SWU-17a to different cells was characterized. 5WSU-DLCL2, CCRF-CEM, THP-1, and Jurkat cells were incubated at 4°C and 37°C for 30 min, respectively. The cells were centrifuged, washed, and resuspended before analysis using a flow cytometer. Flow cytometry data were processed using FlowJo software and plotted using GraphPad Prism 10 software. The horizontal axis represents ssDNA concentration (nM) and the vertical axis represents mean fluorescence intensity after subtracting cellular autofluorescence. The formula Y = B was used. max The apparent equilibrium dissociation constant of the aptamer was calculated using X / (Kd+X) (Bmax is the maximum specific binding, and Kd is the equilibrium dissociation constant).

[0131] like Figure 4 As shown in Figure 3, the apparent equilibrium dissociation constant (Kd) of the nucleic acid aptamer SWU-2a (SEQ ID No. 1) was 11.04 ± 3.44 nM at 4°C and 40.77 ± 6.01 nM at 37°C. The affinities of other nucleic acid aptamers are supplemented in Table 3. The above results indicate that the nucleic acid aptamer SWU-2a has a strong affinity for WSU-DLCL2 cells.

[0132] Table 3. Affinity of different nucleic acid aptamers to different cells

[0133] Cells Sequence Kd (4°C) Kd (37°C) WSU-DLCL2 SWU-2a 11.04 ± 3.44 nM 40.77 ± 6.01 nM WSU-DLCL2 SWU-17a 0.56 ± 0.04 nM 5.07 ± 1.15 nM THP-1 SWU-5a 0.25 ± 0.08 nM 9.44 ± 1.34 nM THP-1 SWU-7a 0.144 ± 0.029 nM 1.38 ± 0.41 nM CEM SWU-5a 0.335 ± 0.117 nM 9.48 ± 3.76 nM CEM SWU-7a 0.209 ± 0.038 nM 10.17 ± 2.16 nM CEM SWU-14a 0.812 ± 0.168 nM 1707.47 ± 1384.37 nM

[0134] 4. Aptamer Truncation Optimization

[0135] Flow cytometric characterization of aptamer after truncation optimization The presence of redundant base sequences on the aptamer will form a large steric hindrance, which will affect the formation of the recognition region structure, thereby having a negative impact on the recognition of the target molecule and weakening the binding ability of the aptamer to the target molecule. At the same time, a smaller number of bases facilitates synthesis, design and modification during the application process, which is conducive to in-depth research on its function. In addition, shorter aptamers will greatly reduce the synthesis cost of aptamers. Therefore, we optimized the truncation of aptamers with stronger affinity. The sequence information after truncation optimization is shown in Table 4, and their binding to cells was determined by flow cytometry. Figure 5 As shown, SWU-2a has undergone three truncations ( Figure 5 The truncated sequences (SWU-2b, SWU-2c, and SWU-2d) no longer bound to WSU-DLCL2 cells, so we used the full-length SWU-2a sequence in subsequent experiments. SWU-5a, SWU-7a, and SWU-14a still showed good binding to cells after truncation, so we can use these truncated sequences in subsequent applications.

[0136] Table 4. Sequence information of different truncated aptamers

[0137]

[0138]

[0139] Example 3: SWU-2a specifically captures WSU-DLCL2 cells

[0140] 1. Capture of Diffuse Large B-cell Lymphoma WSU-DLCL2 Cells by Magnetic Beads Conjugated with the Nucleic Acid Aptamer SWU-2a

[0141] 1. Preparation of magnetic nanoparticles coupled with SWU-2a nucleic acid aptamer

[0142] (1) The 5' end of the nucleic acid aptamer SWU-2a was modified with a biotin group, dissolved and diluted with DPBS and calibrated to a concentration of 2 μM according to the UV spectrophotometer, then heated at 95°C for denaturation for 5 minutes, placed on ice for 5 minutes, and placed at room temperature for 15 minutes for use.

[0143] (2) Take 1 μL of 10 mg / mL streptavidin-modified magnetic nanoparticles (200 nm) (Premier, Cat. No.: PMG015) and 10 pmol (based on aptamer concentration) of biotin-labeled aptamer SWU-2a solution, incubate at room temperature for 30 min, and perform magnetic separation to remove unbound aptamers. Wash three times with 1 mL of washing buffer solution (see Example 1 for the formula, the same below), and perform magnetic separation to obtain magnetic nanoparticles coupled with SWU-2a aptamer (named SWU-2a-MBs).

[0144] 2. Preparation of magnetic nanoparticles coupled with RAM (control sequence) nucleic acid aptamers

[0145] (1) The nucleotide sequence of the control sequence RAM is as follows:

[0146] 5'-TTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'.

[0147] Among them, N represents any random base among A, T, C, and G.

[0148] The 5' end of the control nucleic acid sequence RAM was modified with a biotin group, dissolved and diluted with DPBS, and calibrated to a concentration of 2 μM according to the UV spectrophotometer. It was then heated at 95°C for denaturation for 5 minutes, placed on ice for 5 minutes, and placed at room temperature for 15 minutes for use.

[0149] (2) Take 1 μL of 10 mg / mL streptavidin-modified magnetic nanoparticles (200 nm) and 10 pmol of biotin-labeled RAM random sequences and incubate them at room temperature for 30 min. Then perform magnetic separation to remove unbound sequences. Wash the mixture three times with 1 mL of washing buffer and perform magnetic separation to obtain magnetic nanoparticles coupled with RAM nucleic acid aptamers (named RAM-MBs).

[0150] 3. Aptamer-functionalized magnetic nanoparticles capture diffuse large B-cell lymphoma WSU-DLCL2 cells

[0151] (1) Cell preparation

[0152] The WSU-DLCL2 cells were centrifuged to remove the culture medium, washed twice with 3 mL of washing buffer solution, and resuspended in washing buffer solution (the formula is shown in Example 1, the same below) for later use.

[0153] (2) Cell staining

[0154] Add 1 μL of 100× Hoechst 33342 staining solution to the WSU-DLCL2 cell suspension, stain for 10 min at room temperature in the dark, centrifuge, wash twice with washing buffer solution, and resuspend in the combined buffer solution.

[0155] (3) Functionalized magnetic beads capture diffuse large B-lymphoma cells

[0156] Take 10 μL of SWU-2a-MBs and RAM-MBs, and mix with 1×10 5 WSU-DLCL2 cells were incubated at 4° C. for 30 min, then washed three times with a washing buffer solution containing 0.01% (volume percentage) Tween-80, and magnetically separated for later use.

[0157] (3) Microscope observation

[0158] The magnetic beads-cell complex was resuspended in 10 μL of washing buffer solution, shaken to make it evenly dispersed, and observed and photographed under a confocal microscope.

[0159] 4. Data Analysis

[0160] like Figure 6 As shown in Figure A, SWU-2a-functionalized streptavidin magnetic beads (SWU-2a-MBs) effectively capture diffuse large B-cell lymphoma WSU-DLCL2 cells, while control sequence-functionalized magnetic beads (RAM-MBs) fail to capture the target cells. This demonstrates that the SWU-2a aptamer can be used to capture and enrich diffuse large B-cell lymphoma cells and further demonstrates that the optimal aptamer SWU-2a screened by this invention has great potential for application in tumor recognition and detection, or in diagnostic preparations for diffuse large B-cell lymphoma.

[0161] 2. Magnetic beads coupled with nucleic acid aptamer SWU-2a specifically recognize and capture diffuse large B-cell lymphoma cells

[0162] 1. Cell Preparation

[0163] Take cultured WSU-DLCL2 cells, centrifuge to remove the culture medium, wash twice with wash buffer, and resuspend in 99 μL of wash buffer for later use. Take exponentially growing Jurkat cells, CCRF-CEM cells, THP-1 cells, and Jurkat cells, centrifuge, wash twice with wash buffer, resuspend in 99 μL of wash buffer for later use. Count and set aside.

[0164] 2. Cell staining

[0165] Add 1 μL of 100× Hoechst 33342 dye solution to the cell suspension of WSU-DLCL2 cells, Jurkat cells, THP-1 cells, and CCRF-CEM cells prepared in step 1, stain for 10 min at room temperature in the dark, centrifuge, wash twice with wash buffer, and resuspend in combined buffer.

[0166] 3. Aptamer-functionalized magnetic nanoparticles specifically recognize and capture WSU-DLCL2 cells

[0167] 10 μL of magnetic beads coupled to the SWU-2a aptamer was added to a cell suspension containing 1000 WSU-DLCL2 cells, CCRF-CEM cells, THP-1 cells, or Jurkat cells, respectively, and incubated at 4°C with shaking for 30 minutes. The incubation products were placed on a magnetic separation rack for magnetic separation to remove unbound cells, washed twice with wash buffer, and resuspended in 10 μL of wash buffer for confocal microscopy.

[0168] 3. Data Analysis

[0169] The results are as follows Figure 6 As shown in Figure B, SWU-2a-functionalized magnetic beads can specifically recognize and capture human diffuse large B-cell lymphoma cells, WSU-DLCL2, with a capture rate exceeding 80%. However, they cannot capture other non-targeted cells, with capture rates below 10%. Therefore, by coupling the nucleic acid aptamer SWU-2a, specific capture of human diffuse large B-cell lymphoma cells can be achieved, providing a possibility for accurate tumor diagnosis.

[0170] 3. Magnetic beads coupled with SWU-2a nucleic acid aptamers capture diffuse large B-cell lymphoma cells in a mixed cell population

[0171] 1. Cell Preparation

[0172] (1) Preparation of WSU-DLCL2 cell suspension: Take the cultured WSU-DLCL2 cells, centrifuge to remove the culture medium, wash twice with DPBS buffer, and resuspend in 99 μL DPBS for later use.

[0173] (2) Preparation of Jurkat cell suspension: Jurkat cells in the exponential growth phase were centrifuged and washed twice with washing buffer solution, and then resuspended with the washing buffer solution for later use.

[0174] 2. Cell staining

[0175] Add 1 μL of 100× Hoechst 33342 dye solution to the WSU-DLCL2 cell suspension, stain at 37°C in the dark for 10 min, centrifuge and wash twice with washing buffer solution, resuspend in the buffer solution and count, taking 1000 and 1×10 6 cells.

[0176] Dil cell membrane dye (Biyuntian, C1036) was added to the Jurkat cell suspension and stained at 37°C in the dark for 15 minutes. After centrifugation, the cells were washed twice with washing buffer solution and resuspended in the washing buffer solution for counting.

[0177] 3. Preparation of cell mixture

[0178] Different numbers of WSU-DLCL2 cells stained with Hoechst 33342 were added to 1×10 6 To Jurkat cells stained with Dil cell membrane dye, 10 μL of magnetic beads coupled to the SWU-2a aptamer was added and incubated at 4°C with shaking for 30 minutes. The incubation product was placed on a magnetic separation rack for magnetic separation to further remove unbound cells, washed twice with wash buffer, resuspended in 10 μL of binding buffer, and observed under a microscope.

[0179] 4. Data Analysis

[0180] The results are as follows Figure 6 As shown in C, at 1×10 6 Different numbers of target cells (WSU-DLCL2 cells) were added to non-target cells (Jurkat cells). Through confocal microscopy, the capture rate of target cells was above 80%, which further proved that this method can specifically capture and enrich diffuse large B-cell lymphoma cells WSU-DLCL2 from different cells. This also shows that the SWU-2a aptamer-functionalized magnetic beads can specifically identify and capture target cells in complex samples, indicating that it has good prospects in achieving accurate diagnosis of diffuse large B-cell lymphoma.

[0181] Example 4: Detection of Diffuse Large B-Cell Lymphoma Using SWU-2a

[0182] 1. SWU-2a for single cell WSU-DLCL2 detection

[0183] 1. Preparation of magnetic nanoparticles coupled with SWU-2a nucleic acid aptamer

[0184] (1) The 5' end of the nucleic acid aptamer SWU-2a (SEQ ID No. 1) was modified with a biotin group, dissolved and diluted with DPBS and calibrated to a concentration of 2 μM according to the UV spectrophotometer, then heated at 95°C for 5 min, placed on ice for 5 min, and placed at room temperature for 15 min for use.

[0185] (2) Take 15 μL of 10 mg / mL streptavidin-modified magnetic nanoparticles (200 nm) and 150 pmol biotin-labeled nucleic acid aptamer SWU-2a solution, incubate at room temperature for 30 min, and magnetically separate to remove unbound nucleic acid aptamer. Wash three times with 1 mL of washing buffer solution (see Example 1 for the formula, the same below), and magnetically separate to obtain magnetic nanoparticles coupled with SWU-2a nucleic acid aptamer (SWU-2a-MBs).

[0186] 2. Cell Preparation

[0187] The WSU-DLCL2 cells were centrifuged to remove the culture medium, washed twice with 3 mL of washing buffer solution, resuspended in a buffer solution (the formula is shown in Example 1, the same below), and counted, taking 0, 10, 20, 50, 100, 200, and 500 cells, respectively.

[0188] 3. Functionalized magnetic beads capture diffuse large B-lymphoma cells

[0189] 10 μL of SWU-2a-MBs were incubated with 0, 10, 20, 50, 100, 200, and 500 target cells (WSU-DLCL2 cells) at 4°C for 30 min, then washed twice with a washing buffer solution containing 0.01% (volume percentage) Tween-80, magnetically separated, and resuspended in a binding buffer solution for later use.

[0190] 4. Cell-magnetic bead coupled nucleic acid aptamer

[0191] 20 pmol of biotin-labeled SWU-2a nucleic acid aptamer (SEQ ID No. 1) was added to each cell-magnetic bead sample and incubated at 4°C for 30 min. The cells were then washed twice with a washing buffer solution containing 0.01% (volume percentage) Tween-80, magnetically separated, and resuspended in a binding buffer solution for later use.

[0192] 5. Enzymatic substrate colorimetric assay for detection of diffuse large B-cell lymphoma WSU-DLCL2

[0193] (1) Magnetic bead cell complex linked to alkaline phosphatase

[0194] The complexes with different cell numbers were incubated with 200 μL 1.5 μg / mL streptavidin-modified alkaline phosphatase at 4° C. for 30 min, washed twice with washing buffer containing 0.01% (volume percentage) Tween-80, and resuspended in binding buffer for later use.

[0195] (2) Colorimetry

[0196] Add 100 μL of 1 mg / mL pNpp (disodium p-nitrophenyl phosphate) and incubate at room temperature for 45 min. Detect the absorbance directly at a wavelength of 405 nm using a microplate reader and record the absorbance.

[0197] 6. Data Analysis

[0198] The results are as follows Figure 7 As shown in Figure A, as the number of target cells (WSU-DLCL2 cells) increases, the measured sample absorbance value also increases, and a good linear relationship is shown, demonstrating the feasibility of the method of using the nucleic acid aptamer SWU-2a (SEQ ID No. 1) to detect a single small number of target cells (WSU-DLCL2 cells), and having good development and application value in the early diagnosis of diffuse large B-cell lymphoma.

[0199] 2. SWU-2a is used for the detection of WSU-DLCL2 in mixed cells

[0200] 1. Preparation of magnetic nanoparticles coupled with SWU-2a nucleic acid aptamer

[0201] (1) The 5' end of the nucleic acid aptamer SWU-2a (SEQ ID No. 1) was modified with a biotin group, dissolved and diluted with DPBS and calibrated to a concentration of 2 μM according to the UV spectrophotometer, then heated at 95°C for 5 min, placed on ice for 5 min, and placed at room temperature for 15 min for use.

[0202] (2) Take 15 μL of 10 mg / mL streptavidin-modified magnetic nanoparticles (200 nm) and 150 pmol of biotin-labeled nucleic acid aptamer SWU-2a solution, incubate at room temperature for 30 min, and magnetically separate to remove unbound nucleic acid aptamers. Wash three times with 1 mL of washing buffer solution and magnetically separate to obtain magnetic nanoparticles coupled with SWU-2a nucleic acid aptamers (SWU-2a-MBs).

[0203] 2. Cell Preparation

[0204] WSU-DLCL2 cells were centrifuged to remove the culture medium, washed twice with 3 mL of wash buffer, resuspended in the buffer solution, and counted. 0, 10, 20, 50, 100, 200, and 500 cells were taken at different concentrations. Jurkat cells were centrifuged to remove the culture medium, washed twice with 3 mL of wash buffer, and counted.

[0205] 3. Functionalized magnetic beads capture diffuse large B-lymphoma cells

[0206] 10 μL of SWU-2a-MBs and 1×10 6 Jurkat cells were incubated together at 4° C. for 30 min; then washed twice with a washing buffer solution containing 0.01% (volume percentage) Tween-80, magnetically separated, and resuspended in a binding buffer solution for later use.

[0207] 4. Cell-magnetic bead coupled nucleic acid aptamer

[0208] 20 pmol of biotin-labeled SWU-2a nucleic acid aptamer was added to each cell-magnetic bead sample and incubated at 4°C for 30 min. The cells were then washed twice with a washing buffer solution containing 0.01% (volume percentage) Tween-80, magnetically separated, and resuspended in a binding buffer solution for later use.

[0209] 5. Enzymatic substrate colorimetric assay for detection of diffuse large B-cell lymphoma WSU-DLCL2

[0210] (1) Magnetic bead cell complex linked to alkaline phosphatase

[0211] The complexes with different cell numbers were incubated with 200 μL 1.5 μg / mL streptavidin-modified alkaline phosphatase at 4° C. for 30 min, washed twice with washing buffer containing 0.01% (volume percentage) Tween-80, and resuspended in binding buffer for later use.

[0212] (2) Colorimetry

[0213] Add 100 μL of 1 mg / mL pNPP (disodium p-nitrophenyl phosphate), incubate at room temperature for 45 min, and directly detect the absorbance at a wavelength of 405 nm using a microplate reader and record the absorbance.

[0214] 6. Data Analysis

[0215] The results are as follows Figure 7As shown in Figure B, in a complex environment simulated by adding Jurkat lymphocytes, the measured sample absorbance value increases with the increase in the number of target cells (WSU-DLCL2 cells), and still shows a good linear relationship. This indicates that the aptamer-functionalized magnetic beads can specifically detect WSU-DLCL2 cells in complex samples, further demonstrating the feasibility of the aptamer SWU-2a (SEQ ID No. 1) in detecting actual tumor samples, and is expected to achieve early diagnosis of diffuse large B-cell lymphoma.

[0216] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A nucleic acid aptamer, which is any of the following: (A1) a single-stranded DNA molecule represented by any one of SEQ ID No. 1 to SEQ ID No. 15; (A2) deleting or adding one or more nucleotides to the nucleic acid aptamer shown in (A1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (A3) performing nucleotide substitution or modification on the nucleic acid aptamer shown in (A1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (A4) transforming the backbone of the nucleic acid aptamer shown in (A1) into a phosphothioate backbone to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (A5) obtaining an aptamer derivative having the same function as the aptamer from an RNA molecule encoded by the aptamer shown in (A1); (A6) obtaining a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer represented by (A1) from a peptide nucleic acid encoded by the nucleic acid aptamer; (A7) attaching a chemical group and / or fluorescein and / or anti-tumor drug and / or radioactive element and / or biological enzyme and / or biotin and / or nanomaterial to one end or the middle of any nucleic acid aptamer shown in (A1)-(A6) to obtain a derivative of the nucleic acid aptamer having the same function as the nucleic acid aptamer.

2. A nucleic acid aptamer-functionalized magnetic nanoparticle, characterized in that: The aptamer-functionalized magnetic nanoparticles are loaded with the aptamer according to claim 1.

3. The aptamer-functionalized magnetic nanoparticles according to claim 2, characterized in that: The nucleic acid aptamer according to claim 1 is coupled to the magnetic nanoparticles via biotin and streptavidin.

4. Use of the nucleic acid aptamer according to claim 1 or the nucleic acid aptamer-functionalized magnetic nanoparticles according to claim 2 or 3 in any of the following: (B1) preparing a product for recognizing and binding to diffuse large B-cell lymphoma cells, or recognizing and binding to diffuse large B-cell lymphoma cells; (B2) preparing a product for capturing, enriching, or purifying diffuse large B-cell lymphoma cells, or capturing, enriching, or purifying diffuse large B-cell lymphoma cells; (B3) preparing a product for detecting diffuse large B-cell lymphoma cells, or detecting diffuse large B-cell lymphoma cells; (B4) preparing products for diagnosis or auxiliary diagnosis of diffuse large B-cell lymphoma; (B5) Preparing products for the treatment or adjuvant treatment of diffuse large B-cell lymphoma.

5. Use of the nucleic acid aptamer according to claim 1 in preparing a diagnostic probe or imaging agent for imaging diffuse large B-cell lymphoma.

6. Use of the nucleic acid aptamer according to claim 1 in the preparation of an indicator reagent for guiding the resection of diffuse large B-cell lymphoma lesions and / or metastases during fluorescence laparoscopic surgery.

7. A product, the active ingredient of which is the nucleic acid aptamer according to claim 1; The product has any of the following functions: (C1) recognizes and binds to diffuse large B-cell lymphoma cells; (C2) capturing, enriching, or purifying diffuse large B-cell lymphoma cells; (C3) detection of diffuse large B-cell lymphoma cells; (C4) Diagnosis or auxiliary diagnosis of diffuse large B-cell lymphoma; (C5) Treatment or adjuvant treatment of diffuse large B-cell lymphoma; (C6) Imaging of diffuse large B-cell lymphoma cells.

8. A diagnostic probe or developer for imaging diffuse large B-cell lymphoma, wherein the active ingredient is the nucleic acid aptamer according to claim 1.

9. A reagent for guiding the resection of diffuse large B-cell lymphoma lesions and / or metastases during fluorescence laparoscopic surgery, wherein the active ingredient is the nucleic acid aptamer according to claim 1.

10. Complete set of nucleic acid aptamers or their uses: The set of nucleic acid aptamers is any one of the following: (D1) consists of all or part of the 16 single-stranded DNA molecules shown in SEQ ID No. 1 to SEQ ID No. 15; the part contains at least the single-stranded DNA molecule shown in SEQ ID No. 1; (D2) deleting or adding one or more nucleotides to each nucleic acid aptamer shown in (D1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (D3) performing nucleotide substitution or modification on each nucleic acid aptamer shown in (D1) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (D4) transforming the backbone of each nucleic acid aptamer shown in (D1) into a phosphothioate backbone to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; (D5) obtaining an aptamer derivative having the same function as the aptamer from an RNA molecule encoded by each aptamer shown in (D1); (D6) obtaining a derivative of a nucleic acid aptamer having the same function as the nucleic acid aptamer from the peptide nucleic acid encoded by each nucleic acid aptamer shown in (D1); (D7) attaching a chemical group and / or fluorescein and / or an anti-tumor drug and / or a radioactive element and / or an enzyme and / or biotin and / or a nanomaterial to one end or the middle of each nucleic acid aptamer shown in any one of (D1) to (D6) to obtain a nucleic acid aptamer derivative having the same function as the nucleic acid aptamer; The use is: use of the set of nucleic acid aptamers in preparing a product for molecular typing of cells; the cells are selected from WSU-DLCL2 cells, THP-1 cells or CCRF-CEM cells.

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