Nucleic acid aptamer and application thereof

Abstract

The aptamer combined with the CD8 alpha polypeptide fragment or the CD8 alpha protein with high specificity and high affinity is obtained through three rounds of screening by a microbead display engineering screening method, and compared with a traditional SELEX (systematic evolution of ligands by exponential enrichment) screening method, the screening efficiency is remarkably improved. The screened CD8 alpha aptamer can be combined with a CD8 alpha polypeptide fragment or a CD8 alpha protein with high specificity and high affinity, so that the CD8 alpha aptamer can be used for detecting CD8 alpha and is expected to be applied to various other CD8 alpha related applications.

Description

Technical Field

[0001] The invention belongs to the field of nucleic acid technology, and in particular relates to a nucleic acid aptamer and a use thereof. Background Art

[0002] CD8α is a membrane-bound protein mainly expressed on the surface of cytotoxic T cells. It usually combines with CD8β chain to form a heterodimer, and together they serve as auxiliary receptors for major histocompatibility complex class I molecules (MHC I). The main function of CD8α is to enhance T cell recognition of antigens, and by binding to MHC I molecules, it transmits co-stimulatory signals and promotes T cell activation and proliferation. Under natural conditions, CD8 usually forms dimers, which can have two structures. The first is a heterodimer composed of one α chain and one β chain (CD8-αβ), and the second is a homodimer composed of two α chains (CD8-αα).

[0003] An aptamer is a short single-stranded nucleic acid molecule (ssDNA or RNA) that can bind to a target molecule with high specificity and affinity through a specific conformation of a three-dimensional structure. These target molecules can be small molecules, proteins, or even cells. Aptamers are usually screened out through a technology called Systematic Evolution of Ligands by EXponential enrichment (SELEX). In this process, a large library of nucleic acid molecules is gradually screened and enriched to find sequences that can specifically bind to a specific target. Aptamers have broad potential in biomedical research and clinical applications due to their high specificity and adjustability, such as as diagnostic tools, therapeutic agents, or drug delivery systems. They provide new possibilities for disease treatment and biological detection.

[0004] T cell populations contain not only CD8+T cells, but also CD4+T cells and other subsets, which complicates the accurate identification and quantification of CD8+T cells in mixed cell samples. The expression of CD8 may vary depending on the activation state, differentiation stage or microenvironment of T cells, which can affect the results of detection methods such as flow cytometry or immunohistochemistry. Different detection techniques (such as flow cytometry, immunofluorescence staining, ELISA, etc.) vary in sensitivity and specificity, which may lead to inconsistent test results. The quality and selection of antibodies used to detect CD8 can also affect the results. Antibodies from different sources and types may have different binding properties and background signals. Based on the high specificity and high affinity of aptamers to targets, in theory, aptamers that specifically bind to CD8 obtained after screening can be used for the detection of CD8. However, there are currently no reports of aptamers that specifically bind to CD8. Summary of the invention

[0005] The present invention uses an engineering screening method displayed by microbeads to obtain an aptamer that binds to a CD8α polypeptide fragment or a CD8α protein with high specificity and high affinity through three rounds of screening.

[0006] In one aspect, the present invention provides a nucleic acid aptamer, comprising a nucleotide sequence selected from SEQ ID NOs: 1 to 12 or a nucleotide sequence selected from SEQ ID NOs: 1 to 12 having one nucleotide inserted, substituted or deleted compared to SEQ ID NOs: 1 to 12.

[0007] Preferably, the nucleic acid aptamer has a nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 1 to 12 or a nucleotide sequence selected from the group consisting of a nucleotide insertion, substitution or deletion compared to SEQ ID NOs: 1 to 12.

[0008] More preferably, the nucleic acid aptamer has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-12.

[0009] In one or more embodiments, the nucleic acid aptamer comprises a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12 or comprises a nucleotide sequence having an insertion, substitution or deletion of one nucleotide compared to SEQ ID NO: 9 or SEQ ID NO: 12.

[0010] Preferably, the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12, or has a nucleotide sequence with one nucleotide inserted, substituted or deleted compared with SEQ ID NO: 9 or SEQ ID NO: 12.

[0011] More preferably, the nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12.

[0012] In one or more embodiments, the nucleic acid aptamer is coupled with a polypeptide tag, a biotin tag, an isotope tag or a fluorescent tag.

[0013] Preferably, the polypeptide tag is a His tag.

[0014] In another aspect, the present invention provides a method for detecting the level of CD8α in a sample, the method comprising using the nucleic acid aptamer as described in any embodiment herein.

[0015] In another aspect, the present invention provides a kit comprising the nucleic acid aptamer as described in any embodiment herein.

[0016] In another aspect, the present invention provides use of the nucleic acid aptamer or kit as described in any embodiment herein in detecting CD8α protein.

[0017] In another aspect, the present invention provides use of the nucleic acid aptamer as described in any embodiment herein in the preparation of a drug or a kit for diagnosing or treating a disease or disorder associated with CD8α protein.

[0018] The present invention uses an engineering screening method displayed by microbeads to obtain aptamers that bind to CD8α polypeptide fragments or CD8α proteins with high specificity and high affinity through three rounds of screening, and the screening efficiency is significantly improved compared to the traditional SELEX screening method. The CD8α aptamers obtained by screening can bind to CD8α polypeptide fragments or CD8α proteins with high specificity and high affinity, so they can be used for the detection of CD8α and are expected to be applied to various other CD8α-related uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the distribution diagram of flow sorting results of CD8α protein (His-OG488)-aptamer-magnetic beads and CD8α polypeptide fragment (FITC)-aptamer-magnetic beads.

[0020] Figure 2 This is the flow cytometry result of aptamer-magnetic beads binding to CD8α polypeptide fragment (FITC).

[0021] Figure 3 This is a flow cytometry result diagram of aptamer-magnetic beads binding to CD8α protein (His-OG488).

[0022] Figure 4 This is the flow cytometry result of aptamer (Cy3) binding to CD8α protein (His)-magnetic beads.

[0023] Figure 5 Aptamer (Cy3) binds to CD8 + Fluorescence intensity-concentration diagram of positive cells. Figure 5 A is the aptamer C6 binding to CD8 + Fluorescence intensity-concentration graph of positive cells; Figure 5 B is the aptamer C7 binding to CD8 + Fluorescence intensity-concentration graph of positive cells; Figure 5 C is the aptamer C9 binding to CD8 + Fluorescence intensity-concentration graph of positive cells; Figure 5 D is aptamer C10 binding to CD8 + Fluorescence intensity-concentration graph of positive cells; Figure 5 E is aptamer C11 binding to CD8 + Fluorescence intensity-concentration graph of positive cells; Figure 5 F is aptamer C12 binding to CD8 + Fluorescence intensity-concentration graph of positive cells.

[0024] Figure 6 It is the map of the positive plasmid. DETAILED DESCRIPTION

[0025] Unless otherwise specified, the magnetic beads used in the present invention are solids with a tiny particle size. The magnetic beads are quantitatively measured or quantified according to their volume (total volume) when used; the magnetic beads can be separated by centrifugation or magnetic attraction (i.e., magnetic separation, using magnetic force to adsorb the magnetic beads in a container while discarding other components).

[0026] The “CD8α protein (His)” in the present invention represents a CD8α protein coupled with a His tag, the “CD8α polypeptide fragment” represents a hinge polypeptide fragment of the CD8α protein, the “CD8α polypeptide fragment (FITC)” represents a hinge polypeptide fragment of the CD8α protein coupled with a green fluorescent tag, and the “CD8α protein (His)”, “CD8α polypeptide fragment” and “CD8α polypeptide fragment (FITC)” are all commercially available.

[0027] Example 1: Preparation of CD8α protein (His)-magnetic beads

[0028] S1: Use Dynabeads TM His-Tag Isolation and Pulldown magnetic beads (purchased from ThermoFisher) were firstly used with binding buffer solution (containing 2.5 mM MgCl 2 , 1 mM CaCl 2 , 0.1% (w / v) BSA, 0.1% (w / v) Tween-20, 0.1% (w / v) salmon sperm DNA in PBS) and then resuspend the beads in binding buffer;

[0029] S2: Add an appropriate amount of CD8α protein (His) to the magnetic bead resuspension and incubate at room temperature for 1 h;

[0030] S3: Perform magnetic separation on the magnetic beads, discard the incubation liquid, and wash the magnetic beads three times with a binding buffer solution. Perform magnetic separation on the magnetic beads each time, discard the liquid, retain the magnetic beads, and obtain CD8α protein (His)-magnetic beads after washing.

[0031] Example 2: Preparation of aptamer library and acquisition of random library

[0032] The universal nucleic acid sequence of the aptamer was submitted to Shanghai Bioengineering Co., Ltd. to synthesize the aptamer library. The universal nucleic acid sequence of the aptamer was: 5'-TGACAGTCGACCTGCTCT-N30-AAGCACGGCAGGGACTAT-3'; wherein N30 was a random sequence of 30 bases.

[0033] Other DNA sequence libraries with a molar mass of 5 times that of the aptamer of the present invention are used as random libraries, which do not bind to CD8α protein or CD8α polypeptide fragments. Random libraries can be ordered from Shanghai Bioengineering Co., Ltd.

[0034] Example 3: Library-CD8α protein (His)-magnetic beads preparation

[0035] S1: Denature the aptamer library at 95°C for 5 min, then place it in an ice bath for 5 min;

[0036] S2: adding the denatured and ice-bathed aptamer library to the CD8α protein (His)-magnetic beads prepared in Example 1, and adding the random library as a competitive library, and incubating in a metal bath at 37° C. and 1500 rpm for 1 h;

[0037] S3: Perform magnetic separation on the magnetic beads, discard the incubation liquid, and wash the magnetic beads three times with binding buffer solution. Perform magnetic separation on the magnetic beads each time, discard the liquid, retain the magnetic beads, and obtain the library-CD8α protein (His)-magnetic beads after washing.

[0038] Example 4: Secondary library preparation

[0039] DEPC water was added to the library-CD8α protein (His)-magnetic beads, placed in a 95°C environment for denaturation treatment for 5 minutes, and the magnetic beads were magnetically separated, the liquid was discarded, and the magnetic beads were recovered to obtain a secondary library-CD8α protein (His)-magnetic beads; wherein the library bound to the magnetic beads was used as the secondary library.

[0040] Aptamers that bind to CD8α protein with high affinity are more likely to be retained in the washing steps of Example 3 and in this Example than aptamers that bind to CD8α protein with weak affinity or non-specifically.

[0041] Example 5: Library Amplification

[0042] The concentration of the secondary library obtained by Example 4 was small and below the detection limit of the instrument, so its concentration was not measured, and the secondary library (secondary library-CD8α protein (His)-magnetic beads) was quantified only by volume. Since the aptamer with high affinity binding to CD8α protein will have more copies in the amplification pool and will undergo more rounds of enrichment than the weak affinity or non-specific aptamer, this example optimizes the PCR cycle conditions to achieve the best amplification effect of the binding aptamer.

[0043] S1: Initial library amplification:

[0044] The secondary library was subjected to PCR preliminary amplification for 10 rounds to obtain preliminary amplification products.

[0045] The PCR system is shown in Table 1 below; FP represents the forward primer, RP represents the reverse primer, and RP-P represents the phosphorylated reverse primer, which can be synthesized by Shanghai Bioengineering Co., Ltd. The sequence of the forward primer is: TGACAGTCGACCTGCTCT, and the reverse primer can be complementary to the AAGCACGGCAGGGACTAT-3' sequence of the aptamer. The concentrations in Table 1 are the concentrations or purities of each component, not the final concentrations of the components in the preparation system.

[0046] Table 1: Library initial amplification PCR system

[0047]

[0048] S2: Library amplification optimization:

[0049] The concentration of the preliminary amplification product was still small and below the detection limit of the instrument, so its concentration was not measured, and the preliminary amplification product (including magnetic beads) was quantified only by volume.

[0050] The preliminary amplification products were subjected to PCR amplification optimization, i.e., amplification for 2, 4, 6, 8 and 10 rounds respectively. The number of optimized amplification rounds for aptamers of different sequences in the library was different. After the amplification was completed, the amplification products of different amplification rounds were measured by 4% agarose gel electrophoresis, and the most appropriate PCR optimization amplification rounds for different sequences were determined by the standard of the highest band brightness or the highest DNA concentration after recovery, and the optimized amplification products were obtained.

[0051] The PCR system is shown in Table 2 below; wherein FP represents the forward primer, RP represents the reverse primer, and RP-P represents the phosphorylated reverse primer, which can be synthesized by Shanghai Shenggong Company. The sequence of the forward primer is: TGACAGTCGACCTGCTCT, and the reverse primer can be complementary to the AAGCACGGCAGGGACTAT-3' sequence of the aptamer. As mentioned above, the concentration of the secondary library was not measured, so it was directly added to the primary amplification PCR system. The concentrations in Table 2 are the concentrations or purities of each component, not the final concentrations of the components in the preparation system.

[0052] Table 2: Optimized PCR system for library amplification

[0053]

[0054] Example 6: Regeneration of aptamers

[0055] In order to proceed to the next screening cycle, the ssDNA aptamer was first regenerated from the optimized amplification product, that is, λ-nuclease was added to the optimized amplification product to digest the dsDNA therein into ssDNA; then the regenerated ssDNA was purified using a DNA purification kit (purchased from Novozymes, DC301) to obtain the regenerated product of the aptamer.

[0056] Example 7: Forward primer-magnetic bead preparation

[0057] Take 100 μL of carboxyl magnetic beads (purchased from MCE, product number HY-K0225), wash them three times with N,N-dimethylformamide (DMF), separate the magnetic beads each time, and discard the liquid; then add 100 μL of 2M propargylamine (purchased from Adamas, product number 01049519), 100 μL of 2.8M 1,3-diisopropylcarbodiimide (DIC) and 200 μL of 1M 1-hydroxy-7-azabenzotriazole (HOAt), respectively, place in a metal bath at 50°C, incubate at 1500 rpm for 1 hour, discard the liquid, retain the magnetic beads, and obtain propargyl-magnetic beads.

[0058] The propargyl-magnetic beads were washed with DMF for 3 times, and the magnetic beads were separated by magnetic separation each time, and the liquid was discarded; the magnetic beads were resuspended with DMF to obtain a propargyl-magnetic bead resuspension. The propargyl-magnetic beads in the resuspension were magnetically separated, the liquid was discarded, and the magnetic beads were retained; according to the order of Table 3 below, each component was added to 100 μL of propargyl-magnetic beads in sequence for reaction; after the reaction, the magnetic beads were magnetically separated to discard the reaction liquid, and the magnetic beads were washed with ultrapure water for 2 times, and the magnetic separation magnetic beads were washed each time to discard the liquid, and the forward primer-magnetic beads were obtained after washing.

[0059] Among them, 5'-N3-PEG18-FP is a modified forward primer, which can be synthesized by Beijing Haijing High-tech Co., Ltd. At the 5' end of the primer, PEG18 (ethylene glycol octadecyl derivative group) and N3 (azido group) are connected in sequence, and the sequence of the FP part is TGACAGTCGACCTGCTCT; the CuAAC solution is 12μL 12mM Tris(3-hydroxypropyltriazolylmethyl)amine(THPTA), 10μL 10mM CuSO 4 and 6 μL of 100 mM sodium ascorbate copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) product. The concentrations in Table 3 are the concentrations or purity of each component, not the final concentration of the component in the preparation system.

[0060] Table 3: Forward primer-magnetic bead preparation system

[0061] Components and order Concentration or purity Volume / μL 5'-N3-PEG18-FP 100μM 20 CuAAC solution / 28 DMSO 100％ 5 Tween 20 1％ 10 Triethylamineacetic acid (TEAA) 2.2M 10 <![CDATA[ddH 2 The]]> / 27 total / 100

[0062] Example 8: Emulsion PCR

[0063] In this embodiment, the regeneration product of the aptamer is amplified onto magnetic beads with forward primers by emulsion PCR, and these PCR amplification reactions are carried out in tiny droplets. Since each droplet usually contains only one DNA template (aptamer), cross contamination between different templates during the amplification process can be avoided.

[0064] S1: Preparation of oil phase system: DMF-A-6cs (purchased from Shin-Etsu Co., Ltd.) and KF-6038 (purchased from Shin-Etsu Co., Ltd.) were uniformly mixed in a ratio of 93:7 (volume ratio) to obtain an oil phase system.

[0065] S2: PCR amplification: Add the components of the emulsion PCR system to the oil phase system to perform PCR amplification. The components are shown in Table 4 below. Among them, the forward primer-magnetic beads are prepared by Example 7; RP is a reverse primer, synthesized by Shanghai Shenggong Company; the regeneration product of the aptamer is prepared by Example 6. The sequence of the forward primer is: TGACAGTCGACCTGCTCT, and the reverse primer can be complementary to the AAGCACGGCAGGGACTAT-3' sequence of the aptamer.

[0066] Table 4: Emulsion PCR system

[0067]

[0068] S3: Collect the amplified product of S2 into a 1.5mL EP tube, centrifuge at 1000rpm for 1min, and discard the upper oily liquid; add 1mL BrB, vortex to mix, centrifuge at 1000rpm for 1min, combine the upper and lower liquid layers, and discard the clarified oily liquid in the middle layer; add BrB again and centrifuge at 1000rpm for 1min, discard the upper and middle liquid layers, and only retain the magnetic beads in the lower layer; add BrB again and centrifuge at 1000rpm for 1min, discard the upper liquid, take the lower layer of magnetic beads, add 200ul TE, mix by pipetting, and transfer to a new tube to obtain the emulsion PCR product, and store it in a dark environment at 4℃.

[0069] Example 9: Aptamer-magnetic bead preparation

[0070] The emulsion PCR product obtained in Example 8 was placed in a metal bath at 95°C and heated for 5 min, and then the supernatant was aspirated and discarded while hot, and the emulsion PCR product was washed three times with TE buffer, and the magnetic separation beads were washed each time, the supernatant was discarded, and the magnetic beads were retained; 2 mM MgCl was added to the washed magnetic beads 2 The solution was made to cover all the magnetic beads and provide Mg for the aptamer. 2+ Ions help it maintain its three-dimensional structure; PCR products in MgCl 2 After the solution was ice-bathed for 5 minutes, the beads were magnetically separated to discard the liquid and obtain aptamer-magnetic beads.

[0071] Example 10: Preparation of CD8α protein (His-OG488)-aptamer-magnetic beads and CD8α polypeptide fragment (FITC)-aptamer-magnetic beads

[0072] S1: CD8α protein (His) and CD8α peptide fragment (FITC) were mixed with protein buffer (containing 2.5 mM MgCl 2 , 0.1 mg / mL tRNA (purchased from Solarbio), 0.5 mΜ competitive library, 1 mg / mL BSA, 0.1% Tween-20) were prepared into 200 μL protein solution; 200 μL protein solution was added to about 5 μL aptamer-magnetic beads obtained in Example 9, and the mixture was incubated at 37°C and 1500 rpm for 40 min, and the magnetic beads were magnetically separated, the incubation liquid was discarded, and the aptamer-magnetic beads were retained;

[0073] S2: Add 200 μL Wash buffer to the incubated aptamer-magnetic beads, wash at 37°C and 1500 rpm for 2 min, magnetically separate the aptamer-magnetic beads, discard the liquid, and retain the aptamer-magnetic beads; repeat the washing process once; after washing, CD8α protein (His)-aptamer-magnetic beads and CD8α polypeptide fragment (FITC)-aptamer-magnetic beads are obtained;

[0074] S3: Take 1 μL of 10nM HIS Lite OG488-Tris NTA-Ni complex dye (referred to as OG488 herein, i.e., a dye combined with a His tag, purchased from AAT Bioquest, catalog number 12615), dissolve it in 200 μL of Wash buffer to obtain 200 μL of staining solution; add 200 μL of staining solution to CD8α protein (His)-aptamer-magnetic beads, stain for 30 min at 37°C and 1500 rpm, magnetically separate CD8α protein (His)-aptamer-magnetic beads, discard the liquid, and obtain CD8α protein (His-OG488)-aptamer-magnetic beads; CD8α polypeptide fragment (FITC)-aptamer-magnetic beads are not incubated with the staining solution;

[0075] S4: Add 200 μL of Sorting buffer (containing MgCl) to CD8α protein (His-OG488)-aptamer-magnetic beads and CD8α polypeptide fragment (FITC)-aptamer-magnetic beads respectively. 2 2.5 mM DPBS solution), placed at 37°C, 1500 rpm environment for washing for 2 min, magnetic beads were separated by magnetism, the liquid was discarded, and the magnetic beads were retained; the washing process was repeated once.

[0076] Example 11: Flow sorting

[0077] The washed CD8α protein (His-OG488)-aptamer-magnetic beads and CD8α polypeptide fragment (FITC)-aptamer-magnetic beads CD8α polypeptide fragment (FITC)-aptamer obtained in Example 10 were resuspended in 200 μL sorting buffer to finally obtain about 200 μL of liquid, which was diluted to 2 mL with sorting buffer as samples for flow sorting. According to different targets and concentrations, 4 groups of samples were prepared as shown in Table 5 below.

[0078] Table 5: Flow cytometry samples

[0079] Magnetic beads Protein / peptide concentration Magnetic beads volume CD8α peptide fragment (FITC)-aptamer-magnetic beads 200pm 70μL CD8α protein (His-OG488)-aptamer-magnetic beads 200pm 70μL CD8α peptide fragment (FITC)-aptamer-magnetic beads 100pm 40μL CD8α protein (His-OG488)-aptamer-magnetic beads 100pm 30μL

[0080] The samples were placed on the flow sorter, and the FITC fluorescence channel was set. The collection conditions were limited to collecting the top 0.1% of the nucleic acid magnetic beads with fluorescence intensity. The flow rate was 8000-10000 particles per second. The flow sorting diagrams of the four groups of samples are shown in Figure 2. Figure 1 The magnetic beads that have reached the collection conditions are submitted to a sequencing company for sequencing.

[0081] Example 12: Analysis of sequencing results

[0082] The sequencing results were analyzed by aptasuite software. All aptamer sequences were divided into twelve families according to sequence similarity, and each family had more than 100,000 sequences. The first sequence of each family was selected as a candidate sequence, and a total of 12 candidate sequences were obtained as shown in Table 6 for further verification.

[0083] Table 6: Candidate sequences of aptamers

[0084]

[0085]

[0086] Example 13: Aptamer-magnetic beads binding to CD8α polypeptide fragment (FITC)

[0087] For the aptamer-magnetic beads corresponding to C1 to C12 obtained in Example 9, as well as blank magnetic beads (blank) not bound to nucleic acids and random magnetic beads (random) bound to random sequence nucleic acids (not bound to CD8α polypeptide fragments), 1 μL of each was taken and added to 199 μL of sorting buffer containing 500 nM CD8α polypeptide fragment (FITC), and incubated for 30 minutes at 37°C and 1500 rpm in a metal bath. After incubation, the magnetic beads were magnetically separated to discard the incubation liquid, and washed twice with TE buffer. The magnetic beads were magnetically separated each time and the liquid was discarded. After washing, the aptamer-magnetic beads were retained and measured by flow cytometry. The measurement results are shown in Figure 2 As shown, compared with the control group, the 12 candidate aptamers had more shifts toward high fluorescence values, confirming that the candidate aptamer-magnetic beads have a significant ability to bind to the CD8α polypeptide fragment.

[0088] Example 14: Aptamer-magnetic beads binding CD8α protein (His-OG488)

[0089] For the aptamer-magnetic beads corresponding to C1 to C12 obtained in Example 9, as well as blank magnetic beads (blank) not bound to nucleic acids and random magnetic beads (random) bound to random sequence nucleic acids (not bound to CD8α protein), 1 μL of each was taken and added to 199 μL of sorting buffer containing 500 nM CD8α protein (His-OG488), and incubated for 30 minutes at 37°C and 1500 rpm in a metal bath. After incubation, the magnetic beads were magnetically separated to discard the incubation liquid, and washed twice with TE buffer. The magnetic beads were magnetically separated each time and the liquid was discarded. After washing, the aptamer-magnetic beads were retained and measured by flow cytometry. The measurement results are shown in Figure 3As shown, compared with the control group, the 12 candidate aptamers had more shifts in the positive fluorescence value direction, confirming that the candidate aptamer-magnetic beads have a significant ability to bind to CD8α protein.

[0090] Wherein, the CD8α protein (His-OG488) is prepared by co-incubating CD8α protein (His) and OG488.

[0091] Example 15: Aptamer (Cy3) binding to CD8α protein-magnetic beads

[0092] Aptamers (Cy3) C1-C12 modified with dye Cy3 were synthesized by Shanghai Bioengineering Corporation.

[0093] DynabeadsTM His-Tag Isolation and Pulldown magnetic beads were coupled to CD8 protein (His), and the coupled magnetic beads were magnetically separated, and the supernatant was discarded; 200 μL of sorting buffer containing 500 nM of each aptamer (Cy3) was added to the magnetic beads, and incubated in a metal bath at 37°C and 1500 rpm for 30 minutes. After incubation, the magnetic beads were magnetically separated, and the incubation liquid was discarded, and the beads were washed twice with TE buffer, and the magnetic beads were magnetically separated each time, and the liquid was discarded. After washing, the magnetic beads were retained and measured by flow cytometry. The measurement results are shown in Figure 4 As shown, compared with the control group, the 12 candidate aptamers had more shifts in the direction of positive fluorescence values, confirming that the candidate aptamers have significant ability to bind to CD8α protein.

[0094] Example 16: Aptamer Binding to CD8 + Positive cells

[0095] 2×10 6 The 293T cells were transferred to a 100 mm culture dish and cultured for 24 hours. The cells were then transfected with PEI and a positive plasmid for 48 hours to obtain CD8 expressing a CAR protein containing the CD8α hinge region and overexpressing GFP. + Positive 293T cells. The coding gene sequence of the CAR protein is shown in SEQ ID NO: 13, the sequence of the positive plasmid is shown in SEQ ID NO: 14, and the map of the positive plasmid is shown in Figure 6 shown.

[0096] Take the six aptamers (Cy3) (C6, C7, C9, C10, C11 and C12) in Example 15 and add them to CD8 +Positive cells were placed in a metal bath at 37°C and 1500rpm and incubated for 30 minutes. After incubation, the incubation liquid was discarded, the cells were digested with enzyme-free cell digestion solution, and then the cells were washed twice with PBS, centrifuged at 4°C and 12000rpm for 3 minutes each time, and the supernatant was removed; the cells were washed twice with 200μL sorting buffer, centrifuged at 4°C and 12000rpm for 3 minutes each time, and the supernatant was removed; finally, the cells were resuspended with 200μL Sorting Buffer and measured by flow cytometer. The measurement results are as follows: Figure 5 As shown in the figure, at the same concentration, the fluorescence intensity of the C9 and C12 aptamers binding to the sample was significantly higher than that of other aptamers. It can be seen that C9 and C12 aptamers exhibit excellent CD8 + Positive cell affinity.

[0097] In summary, the present invention uses an engineering screening method based on microbead display to obtain aptamers that bind to CD8α polypeptide fragments or CD8α proteins with high specificity and high affinity through three rounds of screening, which is expected to be used for the detection of CD8α and various other related uses.

[0098] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A nucleic acid aptamer, characterized in that: The nucleic acid aptamer comprises a nucleotide sequence selected from SEQ ID NOs: 1 to 12 or a nucleotide sequence selected from SEQ ID NOs: 1 to 12 having one nucleotide inserted, replaced or deleted compared to SEQ ID NOs: 1 to 12.

2. The nucleic acid aptamer according to claim 1, characterized in that The nucleic acid aptamer has a nucleotide sequence selected from the group consisting of nucleotide sequences shown in SEQ ID NOs: 1 to 12 or a nucleotide sequence selected from the group consisting of a nucleotide inserted, replaced or deleted compared to SEQ ID NOs: 1 to 12.

3. The nucleic acid aptamer according to claim 2, characterized in that The nucleic acid aptamer has a nucleotide sequence selected from SEQ ID NOs: 1 to 12.

4. The nucleic acid aptamer according to claim 1, characterized in that The nucleic acid aptamer comprises a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12, or comprises a nucleotide sequence having one nucleotide inserted, replaced or deleted compared with SEQ ID NO: 9 or SEQ ID NO:

12.

5. The nucleic acid aptamer according to claim 4, characterized in that The nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 12, or has a nucleotide sequence with one nucleotide inserted, replaced or deleted compared with SEQ ID NO: 9 or SEQ ID NO:

12.

6. The nucleic acid aptamer according to claim 5, characterized in that The nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO: 9 or SEQ ID NO:

12.

7. The nucleic acid aptamer according to any one of claims 1 to 6, characterized in that The nucleic acid aptamer is coupled with a polypeptide tag, a biotin tag, an isotope tag or a fluorescent tag.

8. A method for detecting the level of CD8α in a sample, characterized in that: The method comprises using the nucleic acid aptamer according to any one of claims 1 to 6.

9. A kit comprising the nucleic acid aptamer according to any one of claims 1 to 6.

10. Use of the nucleic acid aptamer according to any one of claims 1 to 6 or the kit according to claim 9 in detecting CD8α protein. 11 . Use of the nucleic acid aptamer according to any one of claims 1 to 6 in preparing a drug or a kit for diagnosing or treating a disease or condition associated with CD8α protein.

Images