Nucleic acid aptamer for targeted combination with KIM-1 and application of nucleic acid aptamer

Abstract

The invention discloses a nucleic acid aptamer for targeted combination with KIM-1 and application of the nucleic acid aptamer. The nucleic acid aptamer for targeted combination with the KIM-1 is at least one of a nucleic acid aptamer K3 with a nucleic acid sequence as shown in SEQ ID NO.1 and a nucleic acid aptamer K34 with a nucleic acid sequence as shown in SEQ ID NO.2. Compared with a KIM-1 monoclonal antibody, the nucleic acid aptamer provided by the invention has the advantages of high specificity, high affinity, weak immunogenicity, small side effect, large-batch artificial synthesis, short period, low cost, strict and controllable quality and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nucleic acid aptamer that targets and binds to KIM-1 and an application thereof. Background Art

[0002] However, it is difficult for clinical medical workers to diagnose, intervene and treat early kidney disease in actual medical work, mainly because the clinical symptoms of patients with early kidney damage are not obvious, resulting in frequent misdiagnosis and missed diagnosis, which can easily lead to delayed treatment (Xiao Z, Huang Q, Yang Y, et al. Emerging early diagnostic methods for acute kidney injury. Theranostics. 2022; 12(6), 2963-2986.).

[0003] Clinical judgment of kidney damage uses several indicators such as urine routine, osmotic pressure, blood creatinine, urea nitrogen, and endogenous creatinine clearance. However, in most cases, when these indicators increase, many kidney damages are already very serious or irreversible damage has occurred (Oh DJ. A long journey for acute kidney injury biomarkers. RenFail. 2020 Nov; 42 (1), 154-165.). The best way to treat kidney damage is prevention. Early kidney damage is reversible. Timely detection, early intervention and treatment are beneficial to preventing the occurrence of the disease and controlling the progression of the disease. Therefore, a large number of scientists continue to study diagnostic methods or biomarkers that can be used for early kidney damage.

[0004] KIM-1 (also known as HAVCR1 or TIM-1) is a sugar-modified transmembrane protein. Under normal circumstances, it is not expressed or expressed at a low level in the kidney. When renal injury occurs, KIM-1 is highly expressed in proximal renal tubular cells (Karmakova capital TeCAC, Sergeeva NS, Kanukoev capital Ka C, Alekseev BYKaprin capital A C. Kidney Injury Molecule 1 (KIM-1): a Multifunctional Glycoprotein and Biological Marker (Review). Sovrem Tekhnologii Med. 2021; 13(3), 64-78.). In renal tissue biopsies of patients with acute kidney injury, KIM-1 expression in the proximal tubules was significantly increased (Yin W, Kumar T, Lai Z, et al. Kidney injury molecule-1, a sensitive and specific marker for identifying acute proximal tubular injury, can be used to predict renal functional recovery in native renal biopsies. Int Urol Nephrol. 2019 Dec; 51(12), 2255-2265.). Koyner JL et al. studied the expression of KIM-1 in various human kidney diseases and analyzed whether the concentration of KIM-1 in urine was correlated with the expression of KIM-1 in the kidney, kidney tissue damage, renal function and proteinuria. The results showed that except for kidney diseases with minimal changes, KIM-1 expression was increased in all kidney diseases, positively correlated with serum creatinine, negatively correlated with creatinine clearance, and had no correlation with urine protein (Koyner JL, Vaidya VS, Bennett MR, et al. Urinary biomarkers in the clinical prognosis and early detection of acute kidney injury. Clin J Am Soc Nephrol. 2010 Dec; 5(12), 2154-65.).KIM-1 is extremely stable in urine, and the level of urinary KIM-1 is consistent with the degree of pathological damage. These characteristics indicate that urinary KIM-1 has a high value for the early diagnosis of kidney injury (Vaidya VS, Ford GM, Waikar SS, et al. A rapid urine test for early detection of kidney injury. Kidney Int. 2009 Jul; 76(1), 108-14.).

[0005] Given the clinical demand for KIM-1 detection, various commercial companies have developed monoclonal antibody-based test kits for detecting KIM-1 protein in serum and urine specimens. However, after purchasing nearly every KIM-1 test kit available on the market, we found that most were used solely for research and suffered from low specificity and sensitivity. We also attempted to generate monoclonal antibodies, but found their effectiveness to be limited. This is primarily due to the fact that KIM-1 is a membrane protein and is highly glycosylated, resulting in a weak immune prototype. Increasing the amount of the adjuvant or changing its type has had limited success. Summary of the Invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a nucleic acid aptamer that targets and binds to KIM-1.

[0007] Another object of the present invention is to provide an application of the aforementioned nucleic acid aptamer that targets and binds to KIM-1.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A nucleic acid aptamer that targets and binds to KIM-1, which is at least one of the nucleic acid aptamer K3 having a nucleic acid sequence as shown in SEQ ID NO.1 and the nucleic acid aptamer K34 having a nucleic acid sequence as shown in SEQ ID NO.2;

[0010] K3: 5'-CACGTCCGTCCCCTCACCGCTGCGTCCCCCTCATC-3';

[0011] K34: 5'-CCCCTCCCGCCGATCCCTCGCTCTTTAGCCTGACCT-3'.

[0012] The nucleic acid aptamer targeting and binding to KIM-1 is a labeled nucleic acid aptamer.

[0013] The labeled substances include colloidal gold, biotin and fluorescent molecules.

[0014] The fluorescent molecule is preferably at least one of FAM and Cy3.

[0015] Application of the above-mentioned nucleic acid aptamer that targets and binds to KIM-1 in the preparation of a reagent for detecting KIM-1.

[0016] The present invention has the following advantages and effects compared to the prior art:

[0017] The DNA aptamers provided by this invention, which recognize and bind to the KIM-1 protein, exhibit high specificity and affinity compared to our previously developed monoclonal antibodies. They also exhibit weak immunogenicity and minimal side effects. Aptamers can be synthesized in large quantities, with rapid production cycles, low costs, and strict quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the identification results of target cells.

[0019] Figure 2 This is a graph showing the efficiency identification results of each round of aptamer screening.

[0020] Figure 3 This is the result of flow cytometry screening of nucleic acid aptamers that can bind to target cells.

[0021] Figure 4 This figure shows the results of flow cytometry detection of the binding of different concentrations of FAM-labeled nucleic acid aptamers to target cells.

[0022] Figure 5 This is a graph showing the detection results of the Kd value of the nucleic acid aptamer.

[0023] Figure 6 It is a fluorescence photograph of target cells and control cells labeled with Cy3-labeled nucleic acid aptamers. DETAILED DESCRIPTION

[0024] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0025] Example 1

[0026] The screening is performed using CELL-SELEX technology, and the steps include: first, incubating the nucleic acid aptamer library with reverse screening cells that do not express or low-express KIM-1, retaining unbound nucleic acid aptamers; second, incubating the nucleic acid aptamers retained in the first step with target cells that highly express KIM-1, removing unbound oligonucleotides, and collecting nucleotides bound to the target cells; third, PCR amplifying the nucleic acid aptamers that can bind to the target cells obtained in the second step, preparing single strands by asymmetric PCR, and obtaining a library that can be used for further screening; the above is a screening cycle. Through the above cyclic steps, several rounds of screening are performed to enrich high-affinity nucleic acid aptamers, and the nucleic acids obtained are sequenced and ranked according to the abundance of enrichment, and then the top 70 are selected for verification and identification, and finally nucleic acid aptamers that target KIM-1 are obtained. The specific screening steps used in the present invention are as follows:

[0027] 1. Library Information:

[0028] All nucleic acid sequences involved in the present invention (such as libraries, primers, etc.) were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in Table 1.

[0029] Table 1

[0030]

[0031] 2. Construction of target cells:

[0032] Human renal tubular epithelial HK-2 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) were seeded in F12 medium supplemented with 10% v / v fetal bovine serum and cultured for three passages in vitro. After stabilization, cells were cultured for 24 hours in F12 medium supplemented with 10, 20, 30, 40, and 50 μg / mL vancomycin. A portion of the cells was analyzed for KIM-1 protein expression by Western blotting, and KIM-1 mRNA expression by qPCR.

[0033] The results are as follows Figure 1 As shown, the expression level of KIM-1 is positively correlated with the concentration of vancomycin. Since vancomycin causes certain damage to cells and affects cell growth, HK-2 cells treated with 30 μg / mL vancomycin were selected for cloning and growth to obtain target cells.

[0034] 3. Related Experimental Systems

[0035] The composition of the QPCR mixed solution is as follows: 866 μL of ddH2O, 100 μL of 10× enzyme buffer, 20 μL of dNTP mixture (10 mM), 5 μL of Lib1S1 (100 μM), 5 μL of Lib2A2 (100 μM), 4 μL of Taq Plus enzyme (50 U), and 40 μL of EvaGreen.

[0036] The composition of the PCR mixed solution used to prepare single-stranded DNA (ssDNA) is as follows: 866 μL of ddH2O, 100 μL of 10× enzyme buffer, 20 μL of dNTP mix (10 mM), 5 μL of Lib1S1-FAM (100 μM), 5 μL of Lib2A2-ployA (100 μM), and 4 μL of Taq Plus enzyme (50 U).

[0037] 4. Specific screening process

[0038] 1. Recovery of HK-2 cells and target cells prepared in step 2 (i.e., HK-2 cells incubated with 30 μg / mL vancomycin for 24 h):

[0039] (1) Prepare F12 complete medium: inactivate fetal bovine serum (PAN, Argentina) in a 65°C water bath for 30 minutes. Add the inactivated fetal bovine serum to a final concentration of 10% v / v based on the volume of F12 medium in a clean bench. Store the prepared medium in a 4°C refrigerator until use.

[0040] (2) Quickly remove the frozen HK-2 and target cells from the -80°C freezer, quickly place them in a 37°C water bath to thaw the cells, and carefully shake the cryotubes to accelerate cell lysis; in a clean bench, transfer the thawed cells to a 15 mL centrifuge tube pre-filled with 2 mL of F12 complete medium and carefully pipette to mix;

[0041] (3) Centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant in a clean bench, resuspend the cells in 1 mL of F12 complete medium, transfer to a 60 mm culture dish, and add medium to 4 mL.

[0042] (4) Label the culture medium with the cell name, date, operator, and cell generation number;

[0043] (5) Observe the cells under an inverted microscope. When the cell density is appropriate, transfer the culture dish containing the cells to a 37°C, 5% CO2 constant temperature incubator for culture.

[0044] 2. Cell passaging:

[0045] (1) The target cells and counter-screen cells (untreated HK-2 cells) were removed from the constant temperature incubator and examined under an inverted microscope. The cells were normal.

[0046] (2) Transfer the cells to a sterile 15 mL centrifuge tube in a clean bench and centrifuge at 1000 rpm for 5 min at room temperature.

[0047] (3) Transfer the centrifuge tube to a clean bench, discard the cell supernatant, resuspend the cells with 1 mL of fresh complete culture medium, transfer the cells to a culture dish, and add fresh culture medium to 4 mL.

[0048] (4) Label the culture dish with the cell name, date, operator, and cell generation number.

[0049] (5) Observe the cells under an inverted microscope. When the cell density is appropriate, transfer the culture dish containing the cells to a 37°C, 5% CO2 constant temperature incubator for further culture. Observe the cells under a microscope every day and passage the cells regularly. The experiment begins with P3 target cells (treated with 30 μg / mL vancomycin for 24 hours) and counter-screening cells (PBS control). Each subsequent generation will be used for the next round of screening. The target cells and counter-screening cells were digested with trypsin and counted on a cell counter. The unified count was 3×10 5 / mL, the screening experiment was started.

[0050] 3. Screening process: First round of screening

[0051] (1) Take a tube of synthetic lib2-76nt library powder, centrifuge at 14000g for 10 min, add 137μL DPBS, vortex to dissolve the powder, centrifuge at 14000g for 10 min, and dispense into PCR tubes.

[0052] (2) Place the cells in a PCR instrument for denaturation and renaturation. The procedure is: 95°C for 10 min, followed by an ice-water bath for 5 min, and then equilibrate to room temperature.

[0053] (3) Add the denatured library to the reverse screening cells, mix thoroughly by slowly pipetting, and incubate on an ice shaker for 60 min.

[0054] (4) After centrifugation at 1000 rpm for 5 min at room temperature, the supernatant was aspirated with a pipette tip and recorded as pool-;

[0055] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash1-;

[0056] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash2-;

[0057] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash3-;

[0058] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash4-;

[0059] Add 200 μL of ultrapure water to the cells and boil in a water bath for 10 min. Centrifuge at 10,000 rpm for 2 min at room temperature. The supernatant is recorded as Elution-.

[0060] (5) Add pool- to target cells, mix thoroughly by slowly pipetting, and incubate on an ice shaker for 60 min;

[0061] After centrifugation at 1000 rpm for 5 min at room temperature, the supernatant was aspirated with a pipette tip and recorded as pool+.

[0062] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash1+;

[0063] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash2+;

[0064] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash3+;

[0065] The cells were rinsed with 200 μL DPBS and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was designated as wash4+;

[0066] Add 200 μL of ultrapure water to the cells and boil in a water bath for 10 min. Centrifuge at 10,000 rpm at room temperature for 2 min. The supernatant is recorded as Elution+.

[0067] (6) Take 8-well PCR tubes from Roche and add 30 μL of QPCR mixed solution to each well. Then add 2 μL of Elution- and Elution+ respectively. Perform fluorescence quantitative PCR as follows: 95°C for 2 min; 95°C for 0.5 min, 60°C for 0.5 min, and 72°C for 0.5 min, for 25 cycles.

[0068] 4. Preparation of single chain:

[0069] (1) Remove 2 mL of the PCR mixture used to prepare single-stranded DNA from -20°C and add it to the remaining Elution+. Transfer the mixture to a 50 mL centrifuge tube and mix thoroughly. Add 8 mL of EM90 oil and vortex on a high-power vortex to prepare an emulsion.

[0070] (2) Aliquot the emulsion into PCR tubes, 90 μL per tube, and perform 25 cycles of PCR. The program is: 95°C for 2 min; 95°C for 1 min, 60°C for 1 min, 72°C for 1 min, for 25 cycles.

[0071] (3) Recover the PCR product.

[0072] (4) Concentrate the PCR product with n-butanol, transfer the PCR product to a 10 mL centrifuge tube, fill it up with n-butanol, mix thoroughly, and centrifuge at 10,000 g for 10 min. After centrifugation, separate the layers, remove the upper clear layer, and transfer the lower amplified product to a small EP tube (approximately 100 μL). Transfer 90 μL to a small centrifuge tube, add 100 μL of urea loading buffer, mix thoroughly, and heat at 95°C for 10 min in a PCR instrument.

[0073] (5) Separate single strands by denaturing PAGE electrophoresis, cut the gel, boil the gel, and concentrate ssDNA with n-butanol; then dialyze the ssDNA overnight using a 3.5KD dialysis bag and DPBS, and measure the concentration using a micro-UV microscope.

[0074] In rounds 2, 3, 4, 5, and 6, the screening conditions were changed in each round, as shown in Table 2, and the other steps were the same as above.

[0075] Table 2

[0076]

[0077] 5. Library retention rate:

[0078] The retention rates of the six-round libraries are shown in Table 3 below:

[0079] Table 3

[0080]

[0081] The analysis of the retention rate in each round of screening is as follows: Figure 2 As shown, from Figure 2 It can be seen that each round of screening can effectively obtain the corresponding nucleic acid aptamers. As the rounds increase, the nucleic acid aptamers bound to the target cells are gradually enriched, and the nucleic acid aptamers that are not bound or have low binding affinity gradually decrease.

[0082] 6. Flow cytometry FC-MS detection of library affinity:

[0083] 1. Sample preparation: Digest the P10 target cells with trypsin and divide them into 6 groups. Count them on a cell counter. Each group is counted as 3×10 5 / mL for later use, add control1 (PBS) and 50 μL of 400 nM enriched library control2 (Pool0), pool1 (library obtained in the first round of screening), pool4 (library obtained in the fourth round of screening), pool5 (library obtained in the fifth round of screening) and pool6 (library obtained in the sixth round of screening) into HK-2 cells, mix well, and incubate on ice for 40 min for use;

[0084] 2. Flow cytometer power-on cleaning process: Power on the instrument, open the software, initialize it, and clean the pipes with ultrapure water for 10 minutes;

[0085] 3. On the machine: Perform flow cytometry on the above samples in order; during each test, the pipeline is cleaned with PBS solution, and PBS solution is used for blank correction. Control 1 and control 2 are used as negative controls;

[0086] 4. After the test is completed, the flow cytometer enters the shutdown process and analyzes the experimental data.

[0087] VII. High-throughput sequencing sample preparation and sequencing results:

[0088] 1. Add the single-stranded template (the library after each round of screening is ssDNA) to 400 μL of PCR mixture at a concentration of (0.5 μM * 10 μL). Aliquot the mixture into eight strips of PCR tubes, 100 μL per well, and perform 25 cycles of PCR amplification on a Bio-Rad instrument. The PCR program is as follows: 95°C initial denaturation for 1 minute; 95°C for 60 seconds, 60°C for 60 seconds, and 72°C for 60 seconds, for 25 cycles.

[0089] 3. After PCR, take 20 μL of each sample for storage and electrophoresis detection, and divide the remaining mixture into two 15 mL centrifuge tubes and concentrate with n-butanol to a volume of about 100 μL.

[0090] 4. Recover the double-stranded DNA using the μNIQ-10 oligonucleotide purification kit.

[0091] 8. Collation and Analysis of Sequencing Results

[0092] 1. Use analysis software to extract sequences from the raw sequencing results using the tag primers, resulting in a total of 1,048,576 sequences. Note: The extracted sequences are random, meaning the primer regions at both ends are removed. When synthesizing the Aptamer, the primer regions must be added before and after the extracted sequences.

[0093] 2. Analyze the resulting sequences, select the test sequences, and synthesize monoclonal clones. The library is a closed-loop library. When synthesizing monoclonal clones, the open loop regions at both ends of the library are removed. Sequencing results are obtained and ranked and sorted according to sequence abundance.

[0094] IX. Verification of specific binding to KIM-1 DNA aptamers:

[0095] The nucleic acid sequences were ranked according to their abundance, and then Qingke Biotechnology Co., Ltd. was commissioned to synthesize 5'-labeled fluorescein FAM nucleic acid aptamers for subsequent verification. The first 10 nucleic acid sequences were taken as an example, as shown in Table 4 below:

[0096] Table 4

[0097]

[0098] In order to reduce workload and unnecessary waste, ssDNA with high binding affinity was screened for further dissociation constant determination:

[0099] 1. Based on the screening sequencing results, the top 10 sequences were synthesized to synthesize FAM-labeled ssDNA nucleic acid aptamers.

[0100] 2. 100 pM FAM-labeled aptamer was mixed with 1×10 6 The target cells were gently mixed in 200 μL PBS solution, incubated at 37°C in the dark for 30 min, and 20 μL 3% BSA was added; three replicates were set up for each group.

[0101] 3. Centrifuge at 350g for 5 min at 4°C, discard the supernatant, and wash twice with 2-3 mL of PBS to remove unbound and weakly bound ssDNA.

[0102] 4. Centrifuge at 350 g for 5 min at 4°C, discard the supernatant, resuspend the cell pellet in 200 μL of pre-cooled PBS, filter the cells through a filter membrane, and collect them into a flow cytometry tube.

[0103] 5. Use flow cytometry to detect the fluorescence intensity of nucleic acid aptamer binding, and perform statistical analysis on the data obtained by FCM to rank the 10 ssDNA sequences.

[0104] The results are as follows Figure 3 As shown, it can be seen that the K1-K10 we screened can effectively bind to target cells and can be used for flow cytometry detection or screening of target cells. Based on this method, K11-K70 was further screened to obtain nucleic acids with strong binding affinity for further testing of dissociation constants.

[0105] 10. Determination of the dissociation constant of a single nucleic acid aptamer by flow cytometry (FCM)

[0106] 1. Based on the results of step 9, select a nucleic acid aptamer with strong binding ability to measure the dissociation constant as follows:

[0107] 2. Set different concentrations of FAM-labeled nucleic acid aptamers (9 ssDNA concentration gradients of 0, 0.25, 0.50, 1.0, 1.25, 1.50, 1.75, 2.0, and 2.25 μM) and add 1×10 6 HK-2 cells were incubated at 37° C. in the dark for 30 min, and 20 μL of 3% w / v BSA solution was added (three replicates were set for each group).

[0108] 3. Centrifuge at 350g for 5 minutes at 4°C, discard the supernatant, and wash twice with 2-3 mL of PBS to remove unbound and weakly bound ssDNA.

[0109] 4. Centrifuge at 350g for 5 minutes at 4°C, remove as much supernatant as possible, resuspend the cell pellet with 200 μL pre-cooled PBS, filter the cells with a filter membrane, and collect them into a flow cytometry tube.

[0110] 5. Use flow cytometry to detect the fluorescence intensity of aptamer binding, and perform statistical analysis on the data obtained by FCM to calculate the dissociation constant (Kd value) of the aptamer.

[0111] The results are as follows Figure 4 As can be seen, the binding rate of the aptamer to the cells at each concentration gradient. As the concentration of the aptamer increases, the binding rate of the aptamer to the cells also increases, reaching a plateau at a certain concentration and then no longer increasing. The dissociation constant Kd of the aptamer was calculated using Formula (1) and GraPHPad Prism 9.0.

[0112] Y=Bmax X / (Kd+X) (1);

[0113] Where Y represents the average absorbance value, Bmax represents the maximum absorbance value measured, Kd is the dissociation constant of the aptamer, and X is the concentration of a single aptamer.

[0114] In the detection of single aptamer binding ability, such as Figure 5 As shown: FAM-labeled aptamers K3 and K34 have good affinity with target cells (Kd value reaches nM level), indicating that the aptamers have high affinity with cells that highly express KIM-1.

[0115] 11. Fluorescence microscopy observation of the labeling effect of Cy3-labeled aptamers on KIM-1-overexpressing cells

[0116] 1. Based on the previous screening results, we commissioned Qingke Biotechnology Co., Ltd. to synthesize Cy3-labeled ssDNA aptamers Cy3-K3 and Cy3-K34.

[0117] 2. 3×10 5HK-2 cells were plated per well in a 12-well cell culture plate and divided into groups 1, 2, 3, and 4, with 3 wells in each group. Groups 1 and 2 were treated with culture medium containing 30 μg / mL vancomycin for 24 hours, while groups 3 and 4 were treated with PBS as a control.

[0118] 3. After 24 hours, the cells were washed three times with PBS. Groups 1 and 3 were added with 200 μL of PBS containing 100 pM Cy3-K3 and incubated in the dark for 30 min. Groups 2 and 4 were added with 200 μL of PBS containing 100 pM Cy3-K34 and incubated in the dark for 30 min.

[0119] 4. After incubation, wash with PBS three times and add 100 μL PBS to keep moist.

[0120] 5. Use a fluorescent microscope to observe and take pictures.

[0121] The results are as follows Figure 6 As shown, the Cy3-labeled nucleic acid aptamers we screened can be used to label target cells for fluorescence microscopy detection.

[0122] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer targeting KIM-1, characterized in that: The nucleic acid aptamer that targets and binds to KIM-1 is at least one of the nucleic acid aptamer K3 having a nucleic acid sequence as shown in SEQ ID NO.1 and the nucleic acid aptamer K34 having a nucleic acid sequence as shown in SEQ ID NO.

2.

2. The nucleic acid aptamer targeting and binding to KIM-1 according to claim 1, characterized in that: The nucleic acid aptamer that targets and binds to KIM-1 is a labeled nucleic acid aptamer.

3. The nucleic acid aptamer targeting and binding to KIM-1 according to claim 2, characterized in that: The labeled substance is at least one of colloidal gold, biotin and fluorescent molecules.

4. The nucleic acid aptamer targeting and binding to KIM-1 according to claim 3, characterized in that: The fluorescent molecule is at least one of FAM and Cy3.

5. Use of the nucleic acid aptamer targeting and binding to KIM-1 according to any one of claims 1 to 4 in the preparation of a reagent for detecting KIM-1.

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