Nucleic acid aptamer of ll37, and preparation method and application thereof

Abstract

The application discloses a nucleic acid aptamer of LL37 antibacterial peptide and a preparation method and application thereof. The nucleic acid aptamer screened by the application has good affinity with antibacterial peptide LL37, can be used for detecting LL37, and provides a new candidate tool for diagnosing psoriasis, systemic lupus erythematosus and other autoimmune diseases based on an LL37 target.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a nucleic acid aptamer of the LL37 antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] Nucleic acid aptamers are ssDNA or RNA obtained through exponential enrichment ligand system evolution technology. They can bind with high affinity and high specificity to targets such as small molecules, peptides, proteins, viruses, and cells. This technology can obtain highly specific nucleic acid aptamers targeting low immunogenicity targets, and the artificial synthesis is low-cost and stable. Currently, nucleic acid aptamers are used in the detection of small molecule peptides such as insulin and salivary histamine, and have high sensitivity.

[0003] LL37 is the only known antimicrobial peptide in the human cathelicidin family and plays a crucial role in the innate immune system. It not only possesses broad-spectrum antimicrobial activity but also participates in various physiological processes such as immune regulation, wound healing, and angiogenesis. Existing research indicates that LL37 has a pro-inflammatory effect in autoimmune diseases such as lesional psoriasis. LL37 binds to nucleic acids and stimulates plasma cells / myeloid dendritic cells to secrete type I interferon (IFN-I) and pro-inflammatory factors, making it a potential biomarker for autoimmune diseases such as psoriasis.

[0004] Currently, there are few reports on nucleic acid aptamers that specifically bind to the antimicrobial peptide LL37. George W. (Bill) Jackson et al. disclosed a DNA aptamer for LL37, Apt-222, in their conference paper "Topical DNA aptamers to the antimicrobial peptide LL37 for the potential treatment of psoriasis" (2013, Naples, Aptamers in Medicine). This aptamer has an affinity of KD ≈ 60 nM and can completely block the formation of the LL-37 / DNA complex and completely inhibit the production of IFN-α by pDC. This aptamer has a stronger affinity than the natural binding of LL-37 to genomic DNA. Therefore, the authors believe that it can be used as a lead agent for topical treatment of psoriasis.

[0005] US Patent 9044495B2 discloses a DNA aptamer that specifically binds to human LL-37, competitively blocking the binding of LL-37 to its own DNA / RNA, inhibiting plasmacytoid dendritic cell (pDC) activation and IFN-α release, with a representative sequence (e.g., SEQ 7) having a Kd ≈ 117.9 nM. Its related patent, US10927381B2, protects modified LL-37 aptamers (DNA / RNA), chemically modified variants (2'-fluoro, 2'-methoxy, locked nucleic acid LNA, etc.), and bispecific / multivalent aptamers. The modified aptamers exhibit enhanced affinity (Kd reaching the nM level), improved serum stability, and increased skin penetration. Both patents target the pro-inflammatory domain of LL-37, blocking its binding to its own nucleic acid and inhibiting the TLR / IFN pathway, providing a strategy for treating psoriasis. Summary of the Invention

[0006] This invention provides a nucleic acid aptamer for the LL37 antimicrobial peptide, its preparation method, and its application. The sequence of the nucleic acid aptamer is: CCACGCGGCATGTCTCTGCAATCATCGAAGCGTCCACTCG. The nucleic acid aptamer described in this invention does not require modification and possesses good affinity (Kd = 35.34±10.02 nM).

[0007] The specific technical solution of the present invention is as follows:

[0008] 1. The nucleic acid aptamer of the LL37 antimicrobial peptide, wherein the DNA sequence of the nucleic acid aptamer is: CCACGCGGCATGTCTCTGCAATCATCGAAGCGTCCACTCG.

[0009] Furthermore, the nucleic acid aptamer is a mutated or modified nucleic acid aptamer, wherein the mutation or modification is selected from any one or more of the following:

[0010] (1) The nucleotide sequence is modified by deleting, inserting or replacing one or more nucleotides, and the modified nucleotide sequence has ≥25% sequence homology with the sequence, preferably ≥80% sequence homology, and retains the activity of specific binding to the target;

[0011] (2) At least one nucleotide in the nucleotide sequence is chemically modified, wherein the chemical modification is selected from at least one of the following: phosphorylation, methylation, amination, thiolation, isotope labeling, 2'-fluorine modification, 2'-methoxy modification, locked nucleic acid (LNA) modification, and peptide nucleic acid (PNA) modification;

[0012] (3) The nucleotide sequence is coupled with at least one of the following:

[0013] Biotin, digoxigenin, radioactive substances, fluorescent substances, colloidal gold, folic acid;

[0014] Solid support: magnetic beads;

[0015] Carrier / delivery modification materials: liposomes, nanomaterials;

[0016] Functionally modified molecules: peptides, proteins, enzymes, polyethylene glycol;

[0017] The above are collectively referred to as functional modifiers;

[0018] (4) The corresponding RNA sequence transcribed from the DNA sequence.

[0019] 2. The present invention also provides a pharmaceutical composition comprising the above-mentioned nucleic acid aptamer and pharmaceutically acceptable excipients.

[0020] Furthermore, the dosage form of the pharmaceutical composition is selected from any one of topical preparations, injections, and oral preparations; the topical preparation is selected from at least one of creams, gels, lotions, and liniments; the injection is selected from at least one of intravenous injections, subcutaneous injections, and intramuscular injections; and the oral preparation is selected from at least one of tablets, capsules, granules, and oral liquids.

[0021] 3. The present invention also provides the use of the above-mentioned nucleic acid aptamer in the preparation of drugs for treating psoriasis, dermatitis or systemic lupus erythematosus-related diseases, and in the preparation of detection reagents for detecting psoriasis, dermatitis or systemic lupus erythematosus-related diseases.

[0022] 4. The present invention also provides the application of the above-mentioned nucleic acid aptamer in the preparation of reagents for detecting LL37 antimicrobial peptide.

[0023] In the above applications, nucleic acid aptamers can be used directly, as delivery vectors, or as detection probes.

[0024] 5. The present invention also provides a detection kit comprising the above-mentioned nucleic acid aptamer and auxiliary reagents for detection; the auxiliary reagents for detection are selected from at least one of buffer solution, washing solution, and chromogenic agent.

[0025] 6. The present invention also provides a method for preparing nucleic acid aptamers, comprising the following steps:

[0026] (1) Biotin-modified LL37 antimicrobial peptide was coupled with streptavidin magnetic beads to obtain LL37-magnetic bead conjugate;

[0027] (2) After denaturing the random single-stranded DNA library, it is incubated with the LL37-magnetic bead conjugate to separate unbound single-stranded DNA; the nucleotide sequence of the random single-stranded DNA library is 5'-GTGCTT-N40-AGAGCA-3', where N represents any one of the bases A, T, G, and C; the sequence length of the random single-stranded DNA library is 46 nt, of which the middle 40 nt is a random sequence; the library capacity is 10. 13 ~10 15 ;

[0028] (3) Dissociate and collect the single-stranded DNA bound to the LL37 antimicrobial peptide, amplify by ePCR, purify and prepare the single-stranded DNA to obtain a secondary library; the upstream primer nucleotide sequence of the ePCR amplification is 5'-GTGCTT-3', and the downstream primer nucleotide sequence is 5'-AGAGCA-3'.

[0029] (4) Repeat the screening steps (2) to (3) for 3 to 8 rounds, and then perform high-throughput sequencing and bioinformatics analysis on the finally obtained single-stranded DNA to obtain candidate nucleic acid aptamers for LL37.

[0030] Furthermore, in step (2), the denaturation conditions for the random single-stranded DNA library are: heating in a 95°C water bath for 10 min, followed immediately cooling in an ice bath for 10 min; the total volume of the first round of incubation system is 500 μL, which contains 10 μg of random single-stranded DNA library, the incubation temperature is 25°C, and the incubation time is 1 h.

[0031] Furthermore, in step (3), the oil phase surfactant mixture used for ePCR amplification needs to be pre-cooled at 4°C in advance; the ePCR aqueous phase system consists of the following components: 2×Taq enzyme buffer, forward primer, reverse primer, template DNA (single-stranded DNA collected in step 3) and sterile nuclease-free water; the ePCR amplification program is: 94°C pre-denaturation for 3 min; followed by 22 cycles, each cycle including 94°C denaturation for 30 s, 55°C annealing for 1 min, and 72°C extension for 1 min; after the cycle, 72°C final extension for 5 min, and storage at 4°C for later use;

[0032] The purification process of the ePCR product is as follows: 2-butanol is added and vortexed, followed by 5 times the volume of aqueous buffer and shaking. The mixture is then centrifuged at 12,000 rpm for 2 min. The lower aqueous phase is collected and 1.2 times the volume of anhydrous ethanol is added. The mixture is then transferred to a DNA purification column and centrifuged at 11,000 g for 1 min. After rinsing and elution, the purified DNA is stored at -20℃.

[0033] The beneficial effects of this invention are as follows:

[0034] The nucleic acid aptamer that specifically binds to LL37 provided by this invention has a dissociation constant Kd of 35.34±10.02 nM, indicating high affinity. Furthermore, experiments show that it exhibits good sensitivity and specificity. In its secondary structure, the double-stranded stem region ensures structural stability and resistance to degradation, the flexible loop region enables precise target epitope matching, and the negatively charged groove mediates electrostatic recognition. These three elements synergistically endow it with high affinity and high specificity (35.34±10.02 nM), providing a reliable molecular basis for the treatment and detection of LL37-related diseases. It has the potential for targeted therapy of LL37-mediated autoimmune / inflammatory diseases such as psoriasis, dermatitis, and lupus erythematosus.

[0035] In addition, a fluorescent microsphere detection system based on the LL37 nucleic acid aptamer was used for the detection of the antimicrobial peptide LL37, with a detection limit of 0.1 ng / mL, providing a new method for the diagnosis and monitoring of autoimmune diseases.

[0036] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0038] Figure 1 This is a flowchart illustrating the screening of LL37 nucleic acid aptamers based on magnetic bead SELEX technology;

[0039] Figure 2 The binding-dissociation curves of nucleic acid aptamers Apt-01 and LL37, as determined by biolayer interferometry;

[0040] Figure 3 The figure shows the results of the binding specificity analysis between the candidate aptamer and the LL37 protein.

[0041] Figure 4 Sensitivity analysis of LL37 was performed to construct an LL37 detection system using aptamers.

[0042] Figure 5 To construct an LL37 detection system using aptamers and determine the specificity of LL37;

[0043] Figure 6The diagram shows the predicted secondary structure of aptamer Apt-01, predicted by Mfold, with ΔG = -1.74 kcal / mol. The diagram is labeled with stem region 1 (3-6 paired with 30-33), stem region 2 (10-12 paired with 23-25), the internal closed loop region (unpaired bases, approximately 17 nt), and the open loop region (terminus, approximately 9 nt). Detailed Implementation

[0044] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should also be noted that technical means not described in detail in this invention can be implemented using conventional technical means.

[0045] Example 1: Screening of candidate nucleic acid aptamers for LL37 using magnetic bead SELEX technology

[0046] SELEX buffer formulation: 90mM NaCl, 10mM KCl, 2mM CaCl2, 5mM MgCl2, 30mM Tris, 0.02% Tween 20, pH=7.4. The random single-stranded DNA (ssDNA) library sequence used is approximately 40 nt in length. The two ends are fixed primer sequences (forward primer: 5'-GTGCTT-3', reverse primer: 5'-AGAGCA-3'), and the middle 40 nt is a random sequence. The random ssDNA library sequence is: 5'-GTGCTT-N40-AGAGCA-3', where N represents any one of the bases A, T, G, or C. The random ssDNA library capacity is 10... 13 ~10 15 In addition, the upstream primer for ePCR amplification of the above random ssDNA library sequence is the forward primer with a nucleotide sequence of 5'-GTGCTT-3'; the downstream primer has a nucleotide sequence of 5'-AGAGCA-3'.

[0047] The operation steps are as follows:

[0048] (1) Coupling of biotin-labeled peptides with streptavidin magnetic beads:

[0049] Take an appropriate amount of streptavidin magnetic beads (SA magnetic beads), add biotin-modified antimicrobial peptide LL37, mix well and incubate at room temperature for 30 minutes. Separate the magnetic bead-LL37 conjugate (LL37-MB) using a magnetic rack and wash three times with SELEX buffer.

[0050] (2) Target and ssDNA library incubation:

[0051] First, denature the ssDNA library at 95°C for 10 min, then immediately incubate on ice for 10 min. Next, mix it with the eluted DNA and incubate. In the first round of screening, the incubation system consisted of 500 μL, containing 10 μg of the ssDNA library and an appropriate amount of LL37-MB. Incubate gently at 25°C for 1 h to allow the ssDNA library to fully bind to the target.

[0052] (3) Separation of ssDNA that has not bound to the target:

[0053] After incubation, the DNA-LL37-MB conjugate was collected using a magnetic rack, and the magnetic beads were washed with SELEX buffer to remove unbound or weakly bound ssDNA.

[0054] (4) Dissociation of ssDNA bound to the target:

[0055] Add 100 μL of 2% SDS solution to the DNA-LL37-MB conjugate, heat and incubate at 94 °C for 10 min, separate the magnetic beads with a magnetic rack, and collect the supernatant, which is the ssDNA that can bind to the target, and use it as the first round of aptamer library screening.

[0056] (5) ePCR amplification:

[0057] ePCR amplification was performed using the first-round screened aptamer library as a template. An oil-surfactant mixture was prepared, vortexed, and stored on ice. The PCR aqueous phase was prepared as follows: 2×Taq, forward primer (100 μM), reverse primer (100 μM), template DNA, and sterile nuclease-free water. 300 μL of the pre-chilled oil-surfactant mixture and 50 μL of the pre-chilled PCR aqueous phase were mixed and vortexed at maximum speed for 5 min.

[0058] The PCR program was 94℃ for 3 min, 94℃ for 30 s, 55℃ for 1 min, 72℃ for 1 min, for 22 cycles, with a final cycle of 72℃ for 5 min.

[0059] (6) Purification of ePCR products:

[0060] After amplification, add 1.0 mL of 2-butanol and vortex to mix. Add 5 volumes of buffer to the aqueous phase, gently vortex for 2 min, and centrifuge at 12000 rpm for 2 min to separate the phases. Remove the upper organic phase, transfer the lower aqueous phase to a new EP tube, add 1.2 volumes of anhydrous ethanol, mix well, transfer to a DNA purification column, incubate at room temperature for 2 min, and centrifuge at 11000g for 1 min. Rinse with wash buffer, then elute with elution buffer. Store the purified DNA at -20℃ for subsequent analysis.

[0061] (7) Preparation of single-stranded DNA:

[0062] The PCR product obtained by purification and recovery in step (6) is dsDNA, which is denatured and used as a secondary library for the next round of screening.

[0063] (8) Repeated filtering:

[0064] Mix 1-3 μg dsDNA with 50-100 μL 10×SELEX buffer, and add nuclease-free water to a final volume of 500-1000 μL. Denature at 95°C for 3 min, then immediately incubate on ice. Add 50 μg blank SA-magnetic beads and incubate at 37°C with gentle shaking for 30 min. Collect the supernatant and add it to a fresh LL37-MB container. Incubate at 37°C with gentle shaking for 60 min. Wash twice with 500 μL-1 mL SELEX buffer; add an additional washing step for each additional selection round. Add 2% SDS solution, incubate the magnetic beads at 70-94°C, collect the eluent, precipitate the DNA, and determine its concentration. After each round of selection, perform ePCR amplification and purification to prepare secondary libraries for the next round. After a total of 10 rounds of selection, the final LL37 candidate nucleic acid aptamer library is obtained.

[0065] The flowchart of the LL37 nucleic acid aptamer screening based on magnetic bead SELEX technology in this embodiment is as follows: Figure 1 As shown in the figure, electrophoretic analysis of the products from each round of screening showed that the target size DNA fragment could be successfully amplified in each round, and the amount of product recovered showed an enrichment trend with the increase of screening rounds.

[0066] Example 2 High-throughput sequencing and analysis

[0067] The ssDNA library obtained in 10 rounds of screening in Example 1 was amplified by PCR using upstream and downstream primers. The amplified products (ssDNA concentration greater than 50 ng / μL) were subjected to high-throughput sequencing, and a total of 4,717,945 valid sequences were obtained. The sequence with the most repetitions was selected and named Apt-01, with the following nucleotide sequence: CCACGCGGCATGTCTCTGCAATCATCGAAGCGTCCACTCG.

[0068] Example 3 Affinity Analysis of Nucleic Acid Aptamers

[0069] The aptamer Apt-01 obtained in Example 2 was dissolved in SELEX buffer and then incubated at 95°C for 10 min in a metal bath followed by an ice bath for 10 min. The equilibrium dissociation constant of the candidate aptamer binding to LL37 protein was determined using a high-throughput biomolecular interaction analyzer. The nucleic acid aptamer was diluted to 500 nM and the LL37 protein was diluted to 20 μg / mL with PBST buffer, and then added sequentially to 96-well plates. The following program was set: Equilibration 1 (90 s) → Solidification (180 s) → Equilibration 2 (90 s) → Binding (120 s) → Dissociation (180 s). Equilibration steps 1 and 2, as well as the dissociation step, were all performed in PBST buffer. Through the above operation, the nucleic acid aptamer was bound to the streptavidin sensor in the solidification step, then the LL37 protein was captured in the binding step, and finally the weakly bound portion was washed away in the dissociation step.

[0070] The Kd assay results of the candidate nucleic acid aptamers are shown in Table 1 below. Figure 2 As shown, it can be seen that Apt-01 has a smaller equilibrium dissociation constant and a stronger affinity for LL37.

[0071] Table 1. Kd assay results for nucleic acid aptamers

[0072] Apt-01 35.34 0.112

[0073] Example 4: Determination of candidate aptamer specificity with LL37 using biolayer interferometry

[0074] This embodiment utilizes biolayer interferometry to analyze the affinity between the candidate aptamer Apt-01 and LL37. The specific steps are as follows: Apt-01 was diluted with PBST to a concentration of 200 nM, while LL37, BSA, and Protegrin-1 were diluted to a concentration of 20 μg / mL, and then added sequentially to a 96-well plate. Biolayer interferometry was used to detect the binding ability of Apt-01 to the above three proteins, and the results are as follows: Figure 3 As shown, Apt-01 shows no obvious binding signal with BSA and Protegrin-1, but has a strong binding signal with LL37, indicating that the above nucleic acid aptamers can specifically bind to LL37.

[0075] Example 5: Construction of an LL37 detection system based on the Apt-01 nucleic acid aptamer

[0076] In this embodiment, the magnetic microspheres are streptavidin-coated magnetic beads at a concentration of 10 mg / mL. The aptamer is Apt-01, with biotin-modified 5' end and Cy3 fluorescently labeled 3' end. The complementary sequence is a cDNA sequence complementary to the aptamer Apt-01, and its 5' end is labeled with the quencher group BHQ-2, with the sequence: 5'-BHQ-2-CGAG TGGA CGCT-3'.

[0077] (1) Sensitivity detection: Take 50 μL of SA-magnetic beads into a 1.5 mL centrifuge tube, wash 3 times with 200 μL binding buffer, and discard the supernatant after magnetic separation. Add 100 μL of 200 nM labeled nucleic acid aptamer Apt-01 (Biotin-Apt-01-Cy3), incubate at room temperature with shaking for 30 minutes, discard the supernatant after magnetic separation, and wash 3 times with 200 μL binding buffer to remove unbound aptamers. Add a series of concentration gradient LL37 solutions respectively, and replace LL37 with PBS for the blank control. Mix well and incubate at room temperature for 1 h to allow the aptamers to bind to the target protein. Then add 100 μL of 200 nM aptamer cDNA-BHQ to each tube and mix well. Incubate at room temperature in the dark with shaking for 30 minutes to allow the unbound aptamers to hybridize with cDNA-BHQ. After magnetic separation for 1 min, the supernatant was discarded, and the cells were washed three times with 200 μL binding buffer to remove unbound cDNA-BHQ. The magnetic beads were resuspended in 200 μL binding buffer and transferred to a 96-well black opaque plate. The fluorescence intensity of each well was measured using a microplate reader (excitation wavelength 550 nm, emission wavelength 570 nm). The fluorescence recovery value ΔF = F - F0 (where F0 is the fluorescence value without LL37 protein) was calculated. A standard curve was plotted with ΔF as the ordinate and LL37 protein concentration (μg / mL) as the abscissa. The results are shown in Figure 4. ΔF showed a good linear relationship with LL37 protein concentration in the range of 0.02–20 μg / mL, with a linear regression equation of y = 612.74x + 23.968 (R² = 0.9941) and a limit of detection (LOD) of 0.102 ng / mL.

[0078] (2) Specificity detection: After treating the magnetic beads as described above, the following were added: LL37 group: 100 μL 20 ng / mL LL37 protein; BSA group: 100 μL 20 ng / mL BSA; Protegrin-1 group: 100 μL 20 ng / mL Protegrin-1. Blank control group: 100 μL binding buffer. The mixture was incubated at room temperature with shaking for 1 h to allow the aptamers to bind to the target proteins. Afterwards, cDNA-BHQ was added according to the sensitivity assay method, and the fluorescence intensity was measured using a microplate reader. The results are as follows: Figure 5 As shown, the fluorescence intensity reached 9982, close to the unquenched background, indicating that the aptamer specifically binds to LL37, undergoes a conformational change, and cannot hybridize with cDNA-BHQ, thus preserving fluorescence. In the BSA and Protegrin-1 groups, the fluorescence intensity showed no significant difference from the blank control group (both below 900), indicating that the aptamer did not bind to these proteins, and that cDNA-BHQ hybridization with the aptamer led to fluorescence quenching.

[0079] This embodiment uses a magnetic microsphere-fluorescence detection system to confirm that the nucleic acid aptamer Apt-01 of the present invention can recognize LL37 protein with high specificity and has no obvious cross-reactivity with BSA and Protegrin-1.

[0080] Example 6: Prediction of the secondary structure of nucleic acid aptamer Apt-01 and analysis of its binding site with LL37

[0081] In this embodiment, Mfold software was used to predict the secondary structure of the Apt-01 aptamer, and the results are as follows: Figure 6 As shown, Apt-01 forms a typical stem-loop structure under predicted conditions, with the following specific characteristics: The stem region is formed by reverse complementary pairing of the 5' and 3' end sequences. Specifically, the 5' bases ACGC (positions 3-6) and TGCG (positions 30-33) at the 3' end form a stable double-stranded stem region via hydrogen bonds. This stem region contains a total of four base pairs (including three GC pairs and one AT pair), located near the 3' and 5' ends of the sequence, providing a stable framework for the overall structure. Unpaired bases, including positions 7-9, 13-22, and 26-29, form an internal closed loop region with high conformational flexibility. Positions 13-22 are rich in T and C, forming a negatively charged trench that can bind positively charged amino acid residues on the LL37 surface, serving as a potential binding site for LL37.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The nucleic acid aptamer of the LL37 antimicrobial peptide, characterized in that, The DNA sequence of the nucleic acid aptamer is CCACGCGGCATGTCTCTGCAATCATCGAAGCGTCCACTCG.

2. The nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid aptamer is a mutated or modified nucleic acid aptamer, and the mutation or modification is selected from any one or more of the following: (1) Based on the nucleotide sequence of claim 1, one or more nucleotides are deleted, inserted or replaced, and the modified nucleotide sequence has ≥25% sequence homology with the sequence of claim 1, preferably ≥80% sequence homology, and retains the activity of specific binding to the target; (2) At least one nucleotide in the nucleotide sequence of claim 1 is chemically modified, wherein the chemical modification is selected from at least one of phosphorylation, methylation, amination, thiolation, isotope labeling, 2'-fluorine modification, 2'-methoxy modification, locked nucleic acid modification, and peptide nucleic acid modification; (3) The nucleotide sequence of claim 1 is coupled with a functional modifier, wherein the functional modifier is selected from at least one of: biotin, digoxigenin, radioactive substances, fluorescent substances, nanomaterials, magnetic beads, colloidal gold, liposomes, polyethylene glycol, peptides, proteins, enzymes or folic acid; (4) The corresponding RNA sequence transcribed from the DNA sequence of claim 1.

3. A pharmaceutical composition, characterized in that, It comprises the nucleic acid aptamer as described in claim 1 or 2, and pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is selected from any one of topical preparations, injections, and oral preparations; the topical preparation is selected from at least one of creams, gels, lotions, and liniments; the injection is selected from at least one of intravenous injections, subcutaneous injections, and intramuscular injections; and the oral preparation is selected from at least one of tablets, capsules, granules, and oral liquids.

5. The use of the nucleic acid aptamer according to claim 1 or 2 in the preparation of a medicament for treating psoriasis, dermatitis or systemic lupus erythematosus-related diseases, and in the preparation of a diagnostic reagent for detecting psoriasis, dermatitis or systemic lupus erythematosus-related diseases.

6. The use of the nucleic acid aptamer according to claim 1 or 2 in the preparation of reagents for detecting LL37 antimicrobial peptide.

7. A test kit, characterized in that, It comprises the nucleic acid aptamer as described in claim 1 or 2, and an auxiliary reagent for detection; the auxiliary reagent for detection is selected from at least one of buffer solution, washing solution, and chromogenic agent.

8. The method for preparing the nucleic acid aptamer according to claim 1, characterized in that, Includes the following steps: (1) Biotin-modified LL37 antimicrobial peptide was coupled with streptavidin magnetic beads to obtain LL37-magnetic bead conjugate; (2) After denaturing the random single-stranded DNA library, it is incubated with the LL37-magnetic bead conjugate to separate unbound single-stranded DNA; the nucleotide sequence of the random single-stranded DNA library is 5'-GTGCTT-N40-AGAGCA-3', where N represents any one of the bases A, T, G, and C; the sequence length of the random single-stranded DNA library is 46 nt, of which the middle 40 nt is a random sequence; the library capacity is 10. 13 ~10 15 ; (3) Dissociate and collect the single-stranded DNA bound to the LL37 antimicrobial peptide, amplify by ePCR, purify and prepare the single-stranded DNA to obtain a secondary library; the upstream primer nucleotide sequence of the ePCR amplification is 5'-GTGCTT-3', and the downstream primer nucleotide sequence is 5'-AGAGCA-3'. (4) Repeat the screening steps (2) to (3) for 3 to 8 rounds, and then perform high-throughput sequencing and bioinformatics analysis on the finally obtained single-stranded DNA to obtain candidate nucleic acid aptamers for LL37.

9. The method according to claim 8, characterized in that, In step (2), the denaturation conditions for the random single-stranded DNA library are: heating in a 95°C water bath for 10 min, followed immediately cooling in an ice bath for 10 min; the total volume of the first incubation system is 500 μL, which contains 10 μg of random single-stranded DNA library, the incubation temperature is 25°C, and the incubation time is 1 h.

10. The method according to claim 8 or 9, characterized in that, In step (3), the oil phase surfactant mixture used for ePCR amplification needs to be pre-cooled at 4°C in advance; the ePCR aqueous phase system consists of the following components: 2×Taq enzyme buffer, forward primer, reverse primer, template DNA and sterile nuclease-free water; the ePCR amplification program is: 94°C pre-denaturation for 3 min; followed by 22 cycles, each cycle including 94°C denaturation for 30 s, 55°C annealing for 1 min, and 72°C extension for 1 min; after the cycle, 72°C final extension for 5 min, and storage at 4°C for later use; The purification process of the ePCR product is as follows: 2-butanol is added and vortexed, followed by 5 times the volume of aqueous buffer and shaking. The mixture is then centrifuged at 12,000 rpm for 2 min. The lower aqueous phase is collected and 1.2 times the volume of anhydrous ethanol is added. The mixture is then transferred to a DNA purification column and centrifuged at 11,000 g for 1 min. After rinsing and elution, the purified DNA is stored at -20℃.

Images