DNA (deoxyribonucleic acid) aptamer enzyme for detecting high-expression glycosylated PD-L1 cell line

Abstract

The invention discloses a DNA (deoxyribonucleic acid) aptamer enzyme for detecting a high-expression glycosylated PD-L1 cell line, and relates to the field of biological and medical detection. The method comprises the following steps: connecting a DNA pool with a shearing substrate under the assistance of a template, reacting with A549Degly, separating to generate sheared and unsheared molecules by using polyacrylamide gel, retaining the unsheared molecules, taking a lung cancer cell line A549 as a background cell, reacting with a connecting product, and separating the sheared molecules by using the polyacrylamide gel, thereby obtaining the lung cancer cell line A549. And the RFD probe for detecting the glycosylated PD-L1 is screened out. The probe is the first RFD probe for detecting the glycosylated protein; the RFD probe has the potential to be expanded and applied to detection of other glycosylation receptor proteins; the RFD probe is used for clinical pathology PD-L1 detection of carcinoma in situ, and the CTC capture magnetic nano device can be established by using nucleic acid aptamers of different tumors and is used for PD-L1 detection of various metastatic cancers such as liver cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma and bladder cancer.

Description

Technical Field

[0001] The present invention relates to the field of biological and medical detection technology, and in particular to a DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1. Background Art

[0002] In recent years, anti-PD-1 / PD-L1 cancer immunotherapy has been widely recognized and has become one of the most important treatment methods. The current method to determine whether a patient is suitable for anti-PD-1 / PD-L1 treatment is to detect the expression of PD-L1 in cancer tissues. This method has been approved by the FDA and applied in clinical practice. However, according to the results of clinical trials, the PD-L1 expression level of some patients is not positively correlated with the efficacy of anti-PD-1 / PD-L1 treatment. According to the current PD-L1 detection method, there are 10-20% of PD-L1-negative tumor patients who still show a positive response to anti-PD-1 / PD-L1 treatment in several independent clinical trials. This fact is obviously contrary to the theoretical prediction that only PD-L1-positive cancer cells will have a positive response to this immunotherapy. In other words, according to the current detection method, whether the expression of PD-L1 is positive or negative, patients may benefit from PD-L1 inhibitor treatment, and its predictive value is controversial. Therefore, how to screen patients who are suitable for anti-PD-1 / PD-L1 immunotherapy and achieve relief or even cure of their condition is a crucial issue.

[0003] PD-L1 is a highly glycosylated protein, and the N-glycosylation on the cell surface accounts for 52% of the apparent molecular weight of PD-L1. In 2019, Professor Hong Mingqi's team found that in the detection of lung cancer and breast cancer tumor tissues, the outside of the PD-L1 protein was covered with a specific glycosylation structure, so that its polypeptide antigen could not be recognized and bound by the PD-L1 antibody, resulting in inaccurate PD-L1 immunohistochemistry results in some patients' tumor tissues, which in turn caused false negative misjudgments and ultimately presented a phenomenon that contradicted the treatment results. This result shows that if glycosylated PD-L1 can be detected, these tumor patients who express PD-L1 protein but are not recognized by antibodies can be selected clinically and receive anti-PD-1 / PD-L1 treatment, avoiding these specific patients from missing the precious opportunity for this immunotherapy due to false negative results in the test.

[0004] To this end, we provide a DNA nucleic acid aptamer for detecting cell lines with high expression of glycosylated PD-L1 to solve the above problems. Summary of the invention

[0005] In view of the problems existing in the above-mentioned prior art, the present invention provides a DNA nucleic acid aptamer for detecting a cell line with high expression of glycosylated PD-L1, which can bypass the sugar molecules, thereby more accurately identifying PD-L1 and avoiding false negatives. At the same time, since the aptamer itself can catalyze the cleavage of the substrate after recognizing PD-L1 and generate reporter fluorescence, the "mix-read" one-step method for detecting the level of glycosylated PD-L1 is realized, which is simpler to operate and has lower cost than the existing inventions.

[0006] In order to achieve the above object, the present invention adopts a DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1, and the DNA aptamer is screened by the following steps:

[0007] The DNA pool was ligated with the cleavage substrate under template assistance and then with A549 Degly Reaction, using dPAGE to separate sheared and unsheared molecules, retaining unsheared molecules, using lung cancer cell line A549 as background cells, reacting with the ligation product and separating sheared molecules by dPAGE; amplifying these sheared molecules by PCR1, extending the complementary chains of these sheared molecules by PCR2, separating sheared molecules and their complementary chains by dPAGE, and connecting the sheared molecules with the shearing substrate under the assistance of a template after ethanol purification to enter the next round of screening process. The screening process can be performed until the fluorescence signal generated by the sheared molecules is no longer significantly enhanced, and the screened DNA library is subjected to deep sequencing after specificity verification;

[0008] The sequencing results were analyzed and compared, and sequences with more repetitions and higher homology were selected as candidate probes;

[0009] First, the reactivity of the candidate probes was tested: the probes targeting hyperglycosylated PD-L1 were incubated with A549 at room temperature, and the real-time fluorescence intensity changes of each candidate probe were detected by an ELISA instrument to test its reactivity. The probe concentration corresponding to half of the maximum fluorescence signal change under the same cell concentration, i.e., P1 / 2, was calculated; the sequences with smaller P1 / 2 were selected as candidate probes with better activity for subsequent experiments;

[0010] The candidate probes were tested with cell lines expressing hyperglycosylated PD-L1 and their deglycosylated counterparts to detect their specificity; the hyperglycosylated cell lines included A549 and breast cancer cell line BT-549, and the deglycosylated PD-L1 cell line was A549. Degly 、BT-549 Degly , the cell line without PD-L1 expression was MCF-7, which served as a negative control, and the splicing rate of the sequences obtained by screening was detected by dPAGE as a specificity verification of the candidate probe;

[0011] The RFD molecule PDL01 targeting glycosylated PD-L1 has a strong cleavage effect on A549. PDL01 was selected as a candidate probe to detect its ability to recognize A549 and BT549 cells expressing glycosylated PD-L1.

[0012] As a further optimization of the above invention, it is suitable for PD-L1 detection in exosomes.

[0013] As a further optimization of the above invention, the nucleic acid aptamer of the DNA nucleic acid aptamer is a single-stranded DNA or RNA obtained through an in vitro screening process.

[0014] As a further optimization of the above invention, the DNA aptamer is used for the "mix-and-read" one-step method to detect the level of glycosylated PD-L1, and the specific operation steps include:

[0015] S1: Phosphorylation reaction of substrate:

[0016] Mix 10 pmol of substrate, 100 nmol of ATP, 10 μL of 10×T4PNK buffer, and 20 U of T4PNK, and add ddH20 to 100 μL; react at 37°C for 30 minutes, and heat to 90°C for 5 minutes;

[0017] S2: Phosphorylated substrate and Lib ligation reaction:

[0018] Add 12 pmol of Lib and 12 pmol of Temp to the substrate obtained in S1, react at 90°C for 50 seconds, and cool at 25°C for 15 minutes; add 20 μL of 10×T4 ligase buffer, 5 U of T4 ligase and add ddH20 to 200 μL, and heat at 25°C for 90 minutes;

[0019] S3: Ethanol precipitation of the ligation product:

[0020] Add 1 / 10 volume of 0.3M NaOAc to the ligation system, mix well, add 3 times volume of ethanol, invert to mix well, let stand at -20℃ for 20min; centrifuge at 15,000rpm, 4℃ for 15min; remove the supernatant, add 500μL 75% ethanol, invert several times, let stand at room temperature for 10min, centrifuge at 15,000rpm, 4℃ for 10min; remove the supernatant, and vacuum dry at room temperature for 10min;

[0021] S4: Purify the ligation product using 10% dPAGE gel:

[0022] Pour 40 mL of 10% dPAGE gel storage solution, 40 μL of tetramethylethylenediamine and 400 μL of 10% ammonium persulfate into a gel plate, solidify at room temperature for 30 min, pre-electrophoresis for 20 min, add 15 μL of 1× gel loading buffer to the ligation product obtained in S3, mix well and heat at 90°C for 2 min, add the sample to the 10% dPAGE gel after pre-electrophoresis, the voltage used in the electrophoresis process is 500 volts, and react for 90 min;

[0023] After electrophoresis, remove the gel plate, cover the 10% dPAGE gel with clean plastic wrap, and cut the product with a disposable blade according to the position of the positive control shear band under a fluorescent imager;

[0024] S5: Grind the cut gel pieces until they are viscous, add an appropriate amount of DNA elution buffer according to the size of the gel pieces, heat at 65°C for 5 min, shake for 5 min, and repeat 3 cycles; precipitate the product according to step S3;

[0025] S6: Incubate the dried ligation product with A549 cell lysate at room temperature for 2 hours; Separate the DNA molecules that have not been sheared using 10% polyacrylamide gel according to steps S3 to S5, and then incubate the selected DNA molecules with A549 degly Incubate for 2 hours, repeat steps S3 to S5 to separate the sheared DNA molecules;

[0026] S7: PCR1 amplification:

[0027] Take 5 μL of the screening product, add 0.5 μL of FP1, 0.5 μL of RP1, 5 μL of 10× PCR buffer, 1 μL of dNTPs, 0.5 μL of Taq enzyme, add ddH2O to 50 μL, heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, cool at 4°C for 5 minutes, and use 2% agarose gel for identification;

[0028] S8: PCR2 amplification:

[0029] Take 3 μL of PCR1 product, add 5 μL of FP1, 5 μL of RP2, 50 μL of 10× PCR buffer, 10 μL of dNTPs, 5 μL of Taq enzyme, add ddH2O to 500 μL, heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, cool at 4°C for 5 minutes, and use 2% agarose gel for identification;

[0030] The PCR2 products were separated using 10% dPAGE gel according to steps S3 to S3, and entered the next round of in vitro screening after being connected to the phosphorylated substrate.

[0031] The DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1 of the present invention has the following beneficial effects:

[0032] The DNA aptamer enzyme for detecting a cell line with high expression of glycosylated PD-L1 of the present invention will screen out an RFD probe for detecting glycosylated PD-L1, which will be the first RFD probe for detecting glycosylated proteins; and the RFD probe will have the potential to be expanded to the detection of other glycosylated receptor proteins;

[0033] In addition to using the RFD probe for clinical pathological PD-L1 detection of carcinoma in situ, the present invention can also use nucleic acid aptamers of different tumors to establish a CTC capture magnetic nanodevice for PD-L1 detection of various metastatic cancers such as liver cancer, melanoma, renal cell carcinoma, head and neck squamous cell carcinoma, bladder cancer, etc.

[0034] In addition to being used to screen tumor patients suitable for anti-PD-1 / PD-L1 immunotherapy, it can also be used to predict the efficacy of immunotherapy in tumor patients by comparing the PD-L1 expression level detected by the RFD probe with the prognosis of patients receiving immunotherapy.

[0035] With reference to the following description and drawings, a specific embodiment of the present invention is disclosed in detail, indicating the manner in which the principles of the present invention can be adopted. It should be understood that the scope of the embodiments of the present invention is not limited thereby, and within the spirit and scope of the appended claims, the embodiments of the present invention include many changes, modifications and equivalents. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a flow chart of the DNA aptamer enzyme for detecting a cell line with high expression of glycosylated PD-L1 according to the present invention;

[0037] Figure 2 This is a schematic diagram of the RFD probe molecule library obtained after screening of the present invention;

[0038] Figure 3 This is a schematic diagram of the 10% dPAGE results of the RFD molecule of PDL01 of the present invention against glycosylated PD-L1 on different cells;

[0039] Figure 4 Schematic diagram of the recognition ability of the candidate probe of the present invention to A549 expressing glycosylated PD-L1. DETAILED DESCRIPTION

[0040] Please refer to the instruction manual Figure 1-4 The present invention provides a technical invention: a DNA nucleic acid aptamer for detecting a cell line with high expression of glycosylated PD-L1.

[0041] Nucleic acid aptamers are single-stranded DNA or RNA obtained through an in vitro screening process that can bind to specific target molecules. Compared with traditional protein antibodies, aptamers have the following advantages:

[0042] (1) Aptamers have high affinity, and the binding capacity can be increased to the nanomolar (nM) range through structural design and optimization.

[0043] (2) Compared with antibodies, aptamers are nucleic acids, which are more chemically stable and have lower storage and transportation temperature requirements.

[0044] (3) Nucleic acids can be synthesized artificially, without the need for biological systems or immune responses. They are cheaper than protein antibodies, suitable for mass production, and will not vary between production batches.

[0045] (4) Most importantly, the molecular weight and volume of aptamers are much smaller than those of protein antibodies. For highly glycosylated proteins such as PD-L1, they can bypass the steric hindrance caused by a large amount of glycogen and thus detect the target glycan epitope.

[0046] At present, some nucleic acid aptamers have been screened for detecting protein glycosylation. For example, aptamers are used to identify glycosylated prostate cancer-specific antigen (PSA) and to quantitatively detect the glycosylation level of PSA in prostate cancer at different stages. Through forward and reverse screening, aptamers with high affinity and excellent selectivity for N-glycosylated peptide fragments of vascular endothelial growth factor (VEGF) are obtained. The dissociation constant reaches the μM level. Compared with the non-glycosylated form, it can preferentially bind to glycosylated peptides with a specificity of up to 50 times. In addition, aptamers are used to detect N-acetylneuraminic acid modified with sugar molecules and changes in fibrin glycosylation sites. These studies show that nucleic acid aptamers are suitable for detecting glycosylated proteins.

[0047] RNA-cleaving fluorogenic DNAzyme (RFD) is a molecule composed of an aptamer domain and a catalytically active unit (i.e., DNAzyme). The aptamer domain acts as a molecular switch that can regulate the catalytic activity of the DNAzyme. This regulation is triggered by the binding of the aptamer to the target molecule, resulting in significant structural changes in the aptamer and the DNAzyme. The generation of the fluorescent signal is achieved through a chimeric RNA substrate, which contains an RNA unit, the nucleotides on both sides of which are modified by a fluorescent group and a quenching group, respectively. The RNA unit is designed as a cleavage site, and the cleavage of the unit by the DNAzyme will lead to the separation of the fluorescent group and the quenching group, resulting in an increase in the fluorescent signal. Therefore, this RFD molecule has the ability to generate fluorescent signals and can be used to develop "hybrid read" type analysis to detect targets of interest.

[0048] A549 lung cancer cell line expressing highly glycosylated PD-L1 was selected, and 100 U / μL PNGaseF was used to remove the glycosyl units of PD-L1 in A549 cells to obtain A549 Degly , immunoblotting experiments were performed to detect whether the sugar units were completely removed; A549 Degly As counter-screening objects, molecules that can react with non-glycosylated PD-L1 are removed through the counter-screening process; lung cancer cell line A549 expressing highly glycosylated PD-L1 is used as a positive screening object to obtain molecules that can react with glycosylated PD-L1.

[0049] The SELEX process (a technique for selecting specific nucleic acid aptamers with high affinity to target substances from a random single-stranded nucleic acid sequence library) is as shown in the technical route: the DNA pool is connected to the shearing substrate under the assistance of the template, and then connected to A549 Degly Reaction, use dPAGE to separate the sheared and unsheared molecules, retain the unsheared molecules, use A549 as background cells, and use dPAGE to separate the sheared molecules by reacting with the ligation product. Amplify these sheared molecules by PCR1, and extend the complementary chains of these sheared molecules by PCR2. Use dPAGE to separate the sheared molecules and their complementary chains. After ethanol purification, connect the sheared molecules to the shearing substrate with the assistance of the template and enter the next round of screening. The screening process can be carried out until the fluorescence signal generated by the sheared molecules is no longer significantly enhanced, and specificity verification can be carried out. After specificity verification, the screened DNA library is deeply sequenced.

[0050] The sequencing results were analyzed and compared, and sequences with more repetitions and higher homology were selected as candidate probes. First, the reactivity of the candidate probes was tested: the probes for hyperglycosylated PD-L1 were incubated with A549 at room temperature, and the real-time fluorescence intensity changes of each candidate probe were detected by an enzyme-labeled instrument to test its reactivity. Under the same cell concentration, the probe concentration corresponding to half of the maximum change in the fluorescence signal, i.e., P1 / 2, was calculated. Sequences with smaller P1 / 2 were selected as candidate probes with better activity for subsequent experiments.

[0051] The candidate probes were tested with cell lines expressing hyperglycosylated PD-L1 and deglycosylated PD-L1 to test their specificity. The hyperglycosylated cell lines included lung cancer cell line A549 and breast cancer cell line BT-549, while the deglycosylated PD-L1 cell line was A549. Degly 、BT-549 Degly The cell line without PD-L1 expression was MCF-7, which was used as a negative control. The splicing rate of the obtained sequences was detected by dPAGE to verify the specificity of the candidate probe.

[0052] Table 1. DNA sequences used in the screening process

[0053]

[0054] Among them, S9 is a triethylene glycol linker; F, R and Q represent fluorescein-labeled dT, adenine ribonucleotide, and 4-(4'-dimethylaminoazophenyl)benzoic acid (DABCYL)-labeled dT, respectively; N40 is a random sequence composed of A, T, C, and G, with each base accounting for 25%.

[0055] Figure 2 A is the result of the reaction between the molecular library obtained after the 12th, 15th, 20th, 25th, 28th, and 31st rounds of screening and A549 cells; Figure 2 B is Figure 2 A bar graph is shown. The shear rate (Clv%) is calculated according to the following formula:

[0056] Clv%=(Int [clv] / 6) / (Int [clv] / 6+Int [unclv] )×100%

[0057] Int [clv] Indicates the grayscale of the sheared DNA, Int [unclv] Gray represents DNA that has not been sheared.

[0058] Table 2. Sequencing results

[0059]

[0060] Figure 3 Schematic diagram of the recognition ability of candidate probes for A549 expressing glycosylated PD-L1.

[0061] Figure 4 10% dPAGE results of PDL01 targeting glycosylated PD-L1 RFD molecules on different cells.

[0062] The RFD molecule PDL01 targeting glycosylated PD-L1 has a strong cleavage on A549. PDL01 was selected as a candidate probe to detect its recognition ability for A549 and BT549 cells expressing glycosylated PD-L1. It can be seen that PDL01 has strong cleavage on A549, BT549 and deglycosylated A549 Degly and BT-549 Degly This does not affect the subsequent application of the probe, because regardless of whether glycosylation exists, the RFD probe only needs to be able to identify the expression of PD-L1.

[0063] Currently, there are tests for the level of glycosylated PD-L1 in tumor exosomes, but this invention only detects PD-L1 in exosomes and cannot detect PD-L1 in in situ tumor tissues. In addition, there are also in situ PD-L1 detection methods for tumor tissues, such as FRET strategy and RCA strategy, but this method requires the additional introduction of lectins and combined with real-time quantitative PCR amplification, and the operation steps are relatively cumbersome. The present invention can realize the "mix-read" one-step method to detect the level of glycosylated PD-L1, which is simpler to operate and cheaper than the current existing inventions.

[0064] Among them, SELEX operation steps:

[0065] (1) Substrate phosphorylation reaction: 10 pmol of substrate, 100 nmol of ATP (adenosine triphosphate), 10 μL of 10×T4PNK buffer, and 20 U of T4PNK were mixed, and 20 to 100 μL of ddH (double distilled deionized water) was added; the reaction was carried out at 37°C for 30 minutes, and then heated to 90°C for 5 minutes.

[0066] (2) Ligation reaction between phosphorylated substrate (P-substrate) and Lib (screening molecule library):

[0067] Add 12 pmol of Lib and 12 pmol of Temp to the P-substraste obtained in the previous step, react at 90°C for 50 seconds, and cool at 25°C for 15 minutes; add 20 μL of 10×T4ligase buffer, 5 U of T4ligase, and add ddH20 to 200 μL, and heat at 25°C for 90 minutes.

[0068] (3) Ethanol precipitation of the ligation product:

[0069] Add 1 / 10 volume of 0.3M NaOAc (sodium acetate) to the connection system, mix well, add 3 volumes of ethanol, invert to mix well, let stand at -20°C for 20 min; centrifuge at 15,000rpm, 4°C for 15 min; remove the supernatant, add 500μL 75% ethanol, invert several times, let stand at room temperature for 10 min, centrifuge at 15,000rpm, 4°C for 10 min; remove the supernatant, and dry under vacuum at room temperature for 10 min.

[0070] (4) Purify the ligation product using 10% dPAGE (polyacrylamide gel):

[0071] Pour 40mL of 10% dPAGE gel storage solution, 40μL of TEMED (tetramethylethylenediamine) and 400μL of 10% ammonium persulfate into the gel plate, solidify at room temperature for 30min, and pre-electrophoresis for 20min. Add 15μL of 1× gel loading buffer to the ligation product obtained in the previous step, mix well and heat at 90℃ for 2min, add the sample to the 10% dPAGE gel after pre-electrophoresis, 500V (the voltage used in the electrophoresis process is 500 volts), and react for 90min.

[0072] After the electrophoresis, remove the gel plate, cover the 10% dPAGE gel with a clean plastic wrap, and cut the product with a disposable blade according to the position of the positive control shear band under the fluorescent imager.

[0073] (5) Elute the ligated product from the gel:

[0074] Grind the cut gel pieces until they become viscous, add an appropriate amount of DNA elution buffer according to the size of the gel pieces, heat at 65°C for 5 min, shake for 5 min, and repeat 3 cycles. Precipitate the product according to step (3).

[0075] (6) Incubate the dried ligation product with A549 cell lysate at room temperature for 2 hours; separate the DNA molecules that have not been sheared using 10% polyacrylamide gel according to steps S3 to S5; and separate the selected DNA molecules with A549 degly Incubate for 2 hours and repeat steps S3-S5 to separate the sheared DNA molecules.

[0076] (7) PCR1 amplification: Take 5 μL of the screening product (total volume 10 μL), add 0.5 μL of FP1 (concentration 100 μM), 0.5 μL of RP1 (concentration 100 μM), 5 μL of 10× PCR buffer, 1 μL of dNTPs (concentration 10 mM), 0.5 μL of Taq enzyme, and add ddH2O to 50 μL. Heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, and cool at 4°C for 5 minutes. Use 2% agarose gel for identification.

[0077] (8) PCR2 amplification: Take 3 μL of PCR1 product, add 5 μL of FP1 (concentration 100 μM), 5 μL of RP2 (concentration 100 μM), 50 μL of 10× PCR buffer, 10 μL of dNTPs (concentration 10 mM), 5 μL of Taq enzyme, and add ddH2O to 500 μL. Heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, and cool at 4°C for 5 minutes. Use 2% agarose gel for identification.

[0078] The PCR2 product was separated using 10% dPAGE gel according to steps (3) to (5), and then connected to the phosphorylated substrate and entered the next round of in vitro screening.

Claims

1. A DNA aptamer for detecting a cell line that highly expresses glycosylated PD-L1, characterized in that: The DNA aptamer is screened using the following steps: The DNA pool was ligated with the cleavage substrate under template assistance and then with A549 Degly reaction, using polyacrylamide gel to separate the sheared and unsheared molecules, retaining the unsheared molecules, using lung cancer cell line A549 as background cells, reacting with the connection product to separate the sheared molecules using polyacrylamide gel; amplifying these sheared molecules through PCR1, extending the complementary chains of these sheared molecules through PCR2, separating the sheared molecules and their complementary chains using polyacrylamide gel, and connecting the sheared molecules with the shearing substrate under the assistance of a template after ethanol purification to enter the next round of screening process. The screening process can be performed until the fluorescence signal generated by the sheared molecules is no longer significantly enhanced, and the screened DNA library can be deeply sequenced after specificity verification; The sequencing results were analyzed and compared, and sequences with more repetitions and higher homology were selected as candidate probes; First, the reactivity of the candidate probes was tested: the probes targeting hyperglycosylated PD-L1 were incubated with A549 at room temperature, and the real-time fluorescence intensity changes of each candidate probe were detected by an ELISA instrument to test its reactivity. The probe concentration corresponding to half of the maximum fluorescence signal change under the same cell concentration, i.e., P1 / 2, was calculated; the sequences with smaller P1 / 2 were selected as candidate probes with better activity for subsequent experiments; The candidate probes were reacted with cell lines expressing hyperglycosylated PD-L1 and deglycosylated cell lines to detect their specificity; the hyperglycosylated cell lines included A549 and breast cancer cell line BT-549, and the deglycosylated PD-L1 cell line was A549. Degly 、BT-549 Degly , the cell line without PD-L1 expression was MCF-7, which served as a negative control, and the shear rate of the sequences obtained by screening was detected by polyacrylamide gel as a specificity verification of the candidate probe; The RFD molecule PDL01 targeting glycosylated PD-L1 has a strong cleavage effect on A549. PDL01 was selected as a candidate probe to detect its ability to recognize A549 and BT549 cells expressing glycosylated PD-L1.

2. The DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1 according to claim 1, characterized in that: Suitable for PD-L1 detection in exosomes.

3. The DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1 according to claim 1, characterized in that: The nucleic acid aptamer of the DNA nucleic acid aptamer is a single-stranded DNA or RNA obtained through an in vitro screening process.

4. The DNA aptamer for detecting a cell line with high expression of glycosylated PD-L1 according to claim 1, characterized in that: The DNA aptamer is used for the one-step "mix-and-read" method to detect the level of glycosylated PD-L1, and the specific operation steps include: S1: Phosphorylation reaction of substrate: Mix 10 pmol of substrate, 100 nmol of ATP, 10 μL of 10×T4PNK buffer, and 20 U of T4PNK, and add double-distilled deionized water to 100 μL; react at 37°C for 30 minutes, and heat to 90°C for 5 minutes; S2: Phosphorylated substrate and Lib ligation reaction: Add 12 pmol of Lib and 12 pmol of Temp to the substrate obtained in S1, react at 90°C for 50 seconds, and cool at 25°C for 15 minutes; add 20 μL of 10×T4 ligase buffer, 5 U of T4 ligase and double distilled deionized water to 200 μL, and heat at 25°C for 90 minutes; S3: Ethanol precipitation of the ligation product: Add 1 / 10 volume of 0.3M sodium acetate to the ligation system, mix well, add 3 times volume of ethanol, invert to mix well, let stand at -20℃ for 20min; centrifuge at 15,000rpm, 4℃ for 15min; remove the supernatant, add 500μL 75% ethanol, invert several times, let stand at room temperature for 10min, centrifuge at 15,000rpm, 4℃ for 10min; remove the supernatant, and vacuum dry at room temperature for 10min; S4: Purify the ligation product using 10% polyacrylamide gel: Pour 40 mL of 10% polyacrylamide gel storage solution, 40 μL of tetramethylethylenediamine and 400 μL of 10% ammonium persulfate into a gel plate, solidify at room temperature for 30 min, pre-electrophoresis for 20 min, add 15 μL of 1× gel loading buffer to the ligation product obtained in S3, mix well and heat at 90°C for 2 min, add the sample to the 10% polyacrylamide gel after pre-electrophoresis, the voltage used in the electrophoresis process is 500 volts, and react for 90 min; After the electrophoresis, remove the gel plate, cover the 10% d polyacrylamide gel with a clean plastic wrap, and cut the product with a disposable blade according to the position of the positive control shear band under the fluorescent imager; S5: Grind the cut gel pieces until they are viscous, add an appropriate amount of DNA elution buffer according to the size of the gel pieces, heat at 65°C for 5 min, shake for 5 min, and repeat 3 cycles; precipitate the product according to step S3; S6: Incubate the dried ligation product with A549 cell lysate at room temperature for 2 hours; Separate the DNA molecules that have not been sheared using 10% polyacrylamide gel according to steps S3 to S5, and then incubate the selected DNA molecules with A549 degly Incubate for 2 hours, repeat steps S3 to S5 to separate the sheared DNA molecules; S7: PCR1 amplification: Take 5 μL of the screening product, add 0.5 μL of FP1, 0.5 μL of RP1, 5 μL of 10× PCR buffer, 1 μL of dNTPs, 0.5 μL of Taq enzyme, add double distilled deionized water to 50 μL, heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, cool at 4°C for 5 minutes, and use 2% agarose gel for identification; S8: PCR2 amplification: Take 3 μL of PCR1 product, add 5 μL of FP1, 5 μL of RP2, 50 μL of 10× PCR buffer, 10 μL of dNTPs, 5 μL of Taq enzyme, add ddH2O to 500 μL, heat shock reaction at 94°C for 1 minute; heat at 94°C for 30 seconds, react at 56°C for 45 seconds, react at 72°C for 45 seconds, 12-17 cycles; react at 72°C for 1 minute, cool at 4°C for 5 minutes, and use 2% agarose gel for identification; The PCR2 products were separated using 10% polyacrylamide gel according to steps S3 to S3 and entered the next round of in vitro screening after being ligated to the phosphorylated substrate.

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