CN114574497B - A nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients and its application - Google Patents

Abstract

The invention discloses a nucleic acid aptamer for identifying an extracellular vesicle of a patient with drug resistance to ovarian cancer and application thereof, belonging to the technical field of biology. The aptamer obtained by cell screening has the advantages of strong affinity and specificity, low immunogenicity, capability of in vitro chemical synthesis, low cost, small molecular weight, capability of modifying and replacing different parts of the aptamer, stable sequence and easiness in storage. The aptamer can be used for preparing anti-tumor drugs, ovarian cancer drug-resistant cell strains or ovarian cancer drug-resistant patient serum exosome detection kits or ovarian cancer drug-resistant patient serum exosome detection probes.

Description

A nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients and its application

technical field

The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients and its application.

Background technique

Ovarian cancer is one of the three most common malignant tumors in women, and it is also the most deadly malignant gynecological tumor in the world. At present, the main clinical treatment methods for ovarian cancer are surgery and chemotherapy, among which cisplatin and paclitaxel are commonly used chemotherapy drugs for the clinical treatment of ovarian cancer. Ovarian cancer lacks typical symptoms in the early stage, and the anatomical position of the ovary is deep, so it is mostly advanced when diagnosed. In addition, most patients will have varying degrees of drug resistance to chemotherapy, resulting in a very low long-term survival rate for ovarian cancer patients.

Aptamers are single-stranded DNA or RNA molecules capable of tightly binding to a specific target molecule. These oligonucleotide strands were obtained by phylogenetic evolution of ligands with exponential enrichment in vitro (SELEX). Nucleic acid aptamers, also known as "chemical antibodies", have many advantages when combined with proteins, including low immunogenicity, low synthesis cost, small batch variation, and easy chemical modification. Cell-SELEX refers to a SELEX technology that targets living cells to screen aptamers. Cell-SELEX technology offers the opportunity to identify aptamers against known biomarkers and new biomarkers on the cell membrane surface. In Cell-SELEX, the target molecule has its own native conformation, which facilitates the development of aptamers for therapeutic and diagnostic applications. It also reduces the complexity of dealing with target molecule conformations and increases the likelihood of finding effective aptamers.

At present, the research on nucleic acid aptamers is gradually increasing. Chinese patent CN111944819A discloses a nucleic acid aptamer for rapid screening of ovarian cancer and its application in the preparation of preparations for detecting ovarian cancer. Compared with traditional antibodies, the disclosed nucleic acid aptamer is more It has the characteristics of a wide range of recognized targets, strong specificity, strong affinity, low toxicity and low immunogenicity, but it cannot be used to detect drug resistance in ovarian cancer patients. Some studies have been conducted on nucleic acid aptamers for cancer drug resistance. For example, Chinese patent CN109337909A discloses a nucleic acid aptamer for detecting drug resistance of liver cancer cell lines and its application. However, there are few studies on nucleic acid aptamers for identifying drug resistance in ovarian cancer.

SUMMARY OF THE INVENTION

The invention provides a nucleic acid aptamer with good affinity, low immunogenicity, small molecular weight, in vitro chemical synthesis, stable sequence, convenient storage, convenient labeling and easy modification, which can be used for distinguishing ovarian cancer drug-resistant patients from chemotherapy Extracellular vesicles extracted from sensitive patient serum.

In order to achieve the above purpose of the invention, the following technical solutions are adopted.

A nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients, comprising at least one of the following sequences:

(1) The sequence shown in SEQIDNO.1;

(2) The complementary sequence of the sequence shown in SEQ ID NO.1;

(3) RNA sequences transcribed from the sequences in (1) or (2) above.

The present invention also provides a method for screening the above-mentioned nucleic acid aptamers, which is screening using Cell-SELEX.

Preferably, screening using Cell-SELEX comprises the steps of:

(1) Design of nucleic acid library and primers;

(2) Positive sieve;

(3) Amplification of the library;

(4) Preparation of ssDNA library;

(5) Reverse screening;

(6) Positive sieve;

(7) Nucleic acid aptamer circulating screening;

(8) Detection.

More preferably, the nucleic acid library designed in step (1) is:

SEQ ID NO. 2: 5'-AAGGAGCAGCGTGGAGGATA(N) _{45TTAGGGTGTGTCGTCGTGGT} -3'.

More preferably, the primers designed in step (1) are:

Upstream primer: SEQ ID NO. 3: 5'-fluorescein isothiocyanate-AAGGAGCAGCGTGGAGGATA-3';

Downstream primer: SEQ ID NO. 4: 5'-Biotin-ACCACGACGACACACCCTAA-3'.

More preferably, the specific steps of step (2) include:

The nucleic acid library is incubated with the target cell line, and after the incubation is completed, the nucleic acid library of the first round of screening is obtained by heat denaturation and separation.

More preferably, the specific steps of step (3) include:

The nucleic acid library obtained by screening in step (2) is subjected to PCR amplification using the primers designed in step (1).

More preferably, the specific steps of step (4) include:

Use streptavidin dextran microspheres to separate the biotin-labeled PCR amplification product in step (3); then use sodium hydroxide to denature and melt the double-stranded DNA, and collect the fluorescein isothiocyanate-labeled DNA single. Strand library; final desalting column desalts single-stranded DNA.

More preferably, the specific steps of step (5) include:

The DNA single-stranded library obtained in step (4) is incubated with the control cells, and then the supernatant after the incubation of the cells is collected, that is, the non-specifically bound nucleic acid sequences are excluded.

More preferably, the specific steps of step (6) include:

The supernatant prepared in step (5) is incubated with the target cells, and the nucleic acid sequences bound to the target cells are eluted and retained to obtain the nucleic acid library for the second screening.

More preferably, the specific steps of step (7) include:

The amplification product obtained in step (2) is replaced with the nucleic acid library obtained in step (6), and the screening process of steps (4)-(6) is repeated until a nucleic acid library with strong binding ability to the target cell line is obtained by screening.

More preferably, a total of 10-20 rounds of screening are performed in step (7).

More preferably, step (7) carries out a total of 15 rounds of screening.

More preferably, the specific steps of step (8) include:

The enriched nucleic acid library obtained from multiple rounds of screening was used to detect the binding selectivity of the target cell line and the control cell line by flow cytometry.

More preferably, the above-mentioned target cell line is ovarian cancer paclitaxel-resistant cell line A2780/PTX.

More preferably, the above-mentioned control cell line is a paclitaxel-sensitive cell line A2780.

The invention utilizes Cell-SELEX technology, takes the ovarian cancer taxol-resistant cell line A2780/PTX as the target cell, and uses the taxol-sensitive cell line A2780 as the control cell to perform cell differential screening, thereby obtaining a nucleic acid aptamer that specifically recognizes the target cell. The obtained nucleic acid aptamer has strong affinity and specificity, small molecular weight, easy modification and storage.

The present invention also provides a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients, characterized in that, the nucleic acid aptamer comprises a DNA sequence with more than 60% homology with the sequence in (1).

The present invention also provides a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients, characterized in that the nucleic acid aptamer comprises a sequence obtained by modifying the above-mentioned nucleic acid aptamer, and the modification includes at least one of the following modification methods A sort of:

(1) phosphorylation;

(2) methylation;

(3) Amination;

(4) Sulfhydrylation;

(5) Isotope;

(6) Replace oxygen with sulfur;

(7) Replace oxygen with selenium.

The present invention also provides a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients, and the nucleic acid aptamer includes a sequence obtained by modifying the above-mentioned nucleic acid aptamer.

Preferably, the transformation comprises at least one of the following transformation methods:

(1) Connect a fluorescent label to the nucleic acid aptamer;

(2) Connect radioactive substances to nucleic acid aptamers;

(3) Linking therapeutic substances to nucleic acid aptamers;

(4) Connect biotin to the nucleic acid aptamer;

(5) Connect digoxigenin to the nucleic acid aptamer;

(6) Connect the nano-luminescent material to the nucleic acid aptamer;

(7) Linking small peptides to nucleic acid aptamers;

(8) Connect siRNA to the nucleic acid aptamer.

The present invention also provides a derivative of a nucleic acid aptamer for recognizing extracellular vesicles of ovarian cancer drug-resistant patients, the derivative includes at least one of the following:

(1) The phosphorothioate backbone sequence derived from the above-mentioned nucleic acid aptamer backbone;

(2) A peptide nucleic acid sequence transformed from the above-mentioned nucleic acid aptamer.

The invention discloses the use of the above-mentioned nucleic acid aptamer in binding the serum exosomes of ovarian cancer drug-resistant patients.

The present invention discloses the use of the nucleic acid aptamer or the derivative of the nucleic acid aptamer in the preparation of antitumor drugs.

The invention discloses that the nucleic acid aptamer or the derivative of the nucleic acid aptamer is used in the preparation of ovarian cancer drug resistance cell lines and/or ovarian cancer drug resistance patient serum exosome detection kits and/or ovarian cancer drug resistance patient serum exosomes Use in body detection probes.

The beneficial effects of the present invention are: the nucleic acid aptamer obtained by the cell screening of the present invention has strong affinity and specificity, low immunogenicity, can be chemically synthesized in vitro, low cost, small molecular weight, and can be used for nucleic acid aptamers. Modifications and substitutions are made in different parts, the sequence is stable, and it is easy to preserve. The aptamer of the invention has good recognition ability for the extracellular vesicles of the ovarian cancer drug-resistant patients, and can efficiently bind the serum extracellular vesicles of the ovarian cancer drug-resistant patients. Using the nucleic acid aptamer of the invention can more conveniently and efficiently carry out the characterization of human ovarian cancer drug-resistant cells and the detection of ovarian cancer drug-resistant patients, and provide an effective molecular tool for future drug resistance target search and overcoming ovarian cancer drug resistance.

Description of drawings

Fig. 1 is the flow cytometric detection result of nucleic acid aptamer library in the process of cell screening;

Figure 2 shows the binding of candidate nucleic acid aptamers to target cells and non-target cells;

Fig. 3 is the laser confocal result of nucleic acid aptamer ZH-A2 binding to target cells and non-target cells;

Figure 4 shows the Kd values of nucleic acid aptamer ZH-A2 on drug-resistant cells A2780/PTX and A2780/DDP;

Figure 5 shows the particle size determination results of extracellular vesicles extracted from the culture supernatants of drug-resistant cells A2780/PTX, A2780/DDP and non-drug-resistant cells A2780 and IOSE-80;

Figure 6 shows the electron microscope observation results of extracellular vesicles extracted from the culture supernatants of drug-resistant cells A2780/PTX, A2780/DDP and non-drug-resistant cells A2780 and IOSE-80;

Figure 7 shows the western blot characterization results of extracellular vesicles extracted from the culture supernatants of drug-resistant cells A2780/PTX, A2780/DDP and non-drug-resistant cells A2780 and IOSE-80;

Figure 8 shows the binding of nucleic acid aptamer ZH-A2 to extracellular vesicles of drug-resistant cells A2780/PTX, A2780/DDP and non-drug-resistant cells A2780 and IOSE-80;

Figure 9 shows the fluorescence statistics of the binding of nucleic acid aptamer ZH-A2 to extracellular vesicles of drug-resistant cells A2780/PTX, A2780/DDP and non-drug-resistant cells A2780 and IOSE-80;

Figure 10 shows the characterization of the difference in the binding of nucleic acid aptamer ZH-A2 to extracellular vesicles in the serum of drug-resistant patients and the serum of chemotherapy-sensitive patients; among which, A is the extracellular vesicles of the nucleic acid aptamer ZH-A2 and the serum of drug-resistant patients and the serum of chemotherapy-sensitive patients The flow chart of the experimental operation of the vesicle binding differential characterization; B is the binding differential characterization result of the nucleic acid aptamer ZH-A2 with the serum of the drug-resistant patients and the serum of the chemotherapy-sensitive patients.

Detailed ways

Exemplary embodiments will be described in detail herein, and the implementations described in the following exemplary embodiments are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of approaches consistent with some aspects of the present disclosure.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, or according to the conditions suggested by the manufacturer. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The cells used in the following examples and test examples were obtained from the Wuhan Chinese Academy of Sciences Cell Bank.

Example 1

Cell-SELEX screening process

(1) Design of nucleic acid library and primers:

Random nucleic acid library:

5'-AAGGAGCAGCGTGGAGGATA(N)45TTAGGGTGTGTCGTCGTGGT-3';

Upstream primer: 5'-fluorescein isothiocyanate-AAGGAGCAGCGTGGAGGATA-3';

Downstream primer: 5'-biotin-ACCACGACGACACACCCTAA-3';

(2) Positive sieve:

①Library processing:

The above random nucleic acid library was placed in a centrifuge at 10,000 rpm, centrifuged for 10 min, fully dissolved in DPBS, denatured at 95°C for 5 min, and quickly placed in ice for 10 min.

②Incubate:

The treated random library was incubated with the cultured and pretreated ovarian cancer paclitaxel-resistant cell line A2780/PTX at 37°C for 60 min.

③Separation:

After the incubation, remove the solution in the incubation dish, wash the cells in the incubation dish with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride); scrape the cells in the incubation dish with 600 μL of pre-cooled sterile water , placed in a 1.5ml centrifuge tube, heated at 95°C for denaturation for 10min, centrifuged at 5500rpm per second, and the supernatant was taken, which was the nucleic acid library for the first round of screening of A2780/PTX cells.

(3) Amplification of the library:

Take the nucleic acid library screened in step (2) for pre-amplification,

Upstream primer: 5'-fluorescein isothiocyanate-AAGGAGCAGCGTGGAGGATA-3';

Downstream primer: 5'-biotin-ACCACGACGACACACCCTAA-3';

The amplification system was: 65 μL of sterile water, 4 μL of 2.5 mM MgCl ₂ , 10 μL of 10×Buffer, 6 μL of 0.2 mM dNTPs, 2 μL of 0.4 μM upstream primer, 0.4 μM, 2 μL of downstream primer, 0.5 μL of 1U Taq enzyme, and 10 μL of template;

Amplification conditions were: 95°C, 3 min; 95°C, 30s; 58°C, 30s, 72°C, 30s, after 8 cycles, 72°C, 5min;

Amplification is performed using the amplification product as a template,

The primers and amplification system used were the same as the pre-amplification, and the amplification conditions were: 95°C, 3 min; 95°C, 30s; 58°C, 30s, 72°C, 30s, after 20 cycles, 72°C, 5min.

(4) Preparation of ssDNA library:

Use a denaturing column of streptavidin dextran microspheres to separate the biotin-labeled PCR amplification products in step (3); then use 0.2M sodium hydroxide to denature and melt the double-stranded DNA, and collect the isothiocyanate fluorescence The single-stranded DNA library labeled with phospholipids; the final desalting column desalts the single-stranded DNA.

(5) Reverse sieve:

The DNA single-stranded library obtained in step (4) was incubated with the control cell A2780, and then the supernatant after the cell incubation was collected, that is, the non-specifically bound nucleic acid sequences were excluded.

(6) Positive sieve:

The supernatant prepared in step (5) is incubated with the target cells, and the nucleic acid sequences bound to the target cells are eluted and retained, which is the nucleic acid library of the second screening.

(7) Nucleic acid aptamer circulating screening:

Replace the amplification product obtained in step (2) with the nucleic acid library obtained in step (6), and repeat the screening process of steps (4)-(6), until screening obtains a product with strong binding ability to the target cell A2780/PTX cell line. nucleic acid library.

(8) Detection:

The enriched nucleic acid library obtained from multiple rounds of screening is used to detect the binding selectivity of A2780/PTX and A2780 cell lines by flow cytometry. To avoid redundancy, only the detection results of the 12th, 14th, and 15th rounds of screening are displayed, such as As shown in Figure 1;

It can be seen from Figure 1 that the nucleic acid library obtained in each round of screening is biased compared with the unscreened control library (Library). The shift value gradually increased, indicating that the affinity with the A2780/PTX cell line gradually increased in each round of screening.

Example 2

Determination of optimal nucleic acid aptamers

According to the nucleic acid library screened in Example 1, 6 candidate nucleic acid sequences were selected through computer analysis and comparison, and were named ZH-A2, ZH-A4, ZH-A6, ZH-A9, ZH-A10, ZH- A11 and determine the optimal nucleic acid aptamer.

Specific steps:

(1) Streaming inspection

Adherent state A2780/PTX cells were digested from culture dishes with 0.2% EDTA, cells were collected into centrifuge tubes, and washed twice with DPBS; were added to A2780/PTX cells soaked in binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg/L tRNA, 1g/L BSA); then placed in a shaker at 37°C and incubated for 45min; the incubation was completed After that, it was centrifuged and washed twice with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride), and finally the fluorescence was detected by flow cytometry;

The same method was used to detect the binding of candidate sequences to cell lines A2780, HO-8910, OVCAR3, Caov3 and IOSE-80.

The results of flow cytometry are shown in Figure 2. The results show that in the affinity measurement results with the A2780/PTX cell line, ZH-A2 has the highest offset, indicating that among all candidate sequences, ZH-A2 The affinity with the A2780/PTX cell line is the best, and from the results of the affinity determination with several other cell lines, it can be seen that ZH-A2 has almost no deviation, indicating that the nucleic acid aptamer ZH-A2 only recognizes drug resistance The cells do not recognize common cell lines and normal ovarian epithelial cells, so the nucleic acid aptamer ZH-A2 is determined to be the optimal nucleic acid aptamer.

(2) Laser confocal inspection

The adherent A2780/PTX cells were digested from the culture dish with 0.2% EDTA, the cells were collected into centrifuge tubes, and washed twice with DPBS; the final concentration of 250nM, FITC-labeled ZH-A2 and random library were added to A2780/PTX cells soaked in binding buffer (D-PBS, containing 0.45% glucose, 5mM magnesium chloride, 100mg/L tRNA, 1g/L BSA); then placed in a shaker at 37°C and incubated for 45min; the incubation was completed After that, the cells were centrifuged and washed twice with washing buffer (PBS, containing 0.45% glucose, 5mM magnesium chloride), and fluorescence detection was performed by laser confocal; ZH-A2 and cell lines A2780, HO-8910, OVCAR3, Caov3, Combination of IOSE-80.

The results of laser confocal detection are shown in Figure 3. The results show that ZH-A2 has affinity with A2780/PTX cells, and the fluorescence distribution surrounds the cell membrane; while there is no fluorescence inside and outside other cells, indicating that ZH-A2 is closely related to other cells. The cells tested were all incompatible, which was consistent with the results of flow cytometry.

Test Example 1

Determination of the affinity of nucleic acid aptamer ZH-A2 to drug-resistant ovarian cancer cell lines

The equilibrium dissociation constant Kd is used to characterize the binding affinity of nucleic acid aptamers to target cells. The specific operation steps are as follows:

(1) The ovarian cancer drug-resistant cells (A2780/PTX, A2780/DDP, SKOV3/DDP) were inoculated in a cell culture dish in advance. When the cell confluence reached about 90%, the culture medium was aspirated and the cells were washed twice with DPBS. , and digested the cells from the dish with 0.2% EDTA;

(2) Count the cells and divide them into 8 tubes, each with 300,000 cells;

(3) Nucleic acid aptamer ZH-A2 pretreatment: DNA was denatured in a 95°C metal bath for 5min, and placed on ice for 10min;

(4) The pretreated nucleic acid aptamer ZH-A2 is divided into 7 tubes, each tube has a final concentration of 0, 5, 10, 20, 50, 100, 200, and 500 nM in a 200 μL system;

(5) The nucleic acid aptamer ZH-A2 was added to the cell tube correspondingly, mixed well, incubated in the dark for 30 minutes, washed twice by WB, and detected by upflow;

(6) After the flow detection is completed, use FlowJO software to calculate the fluorescence intensity of each tube, and then use Graphpad to fit the binding affinity curve to calculate the Kd value.

The calculation results are shown in Figure 4. The results show that the Kd values of ZH-A2 on ovarian cancer drug-resistant cells (A2780/PTX, A2780/DDP, SKOV3/DDP) are all in the nanomolar level, indicating that ZH-A2 has a certain effect on these cells. Excellent affinity.

Test Example 2

Binding assay of nucleic acid aptamer ZH-A2 to extracellular vesicles

(1) Extraction and characterization of extracellular vesicles

Take A2780/PTX, A2780/DDP, A2780, and IOSE-80 cells and culture them in normal serum-containing medium for a certain period of time. When the cell confluence is about 60%-70%, remove the original serum-containing culture medium. The base was replaced with fresh serum-free medium, and the culture was continued for 24-48 h. The supernatant was collected when the cell confluence reached about 80%-95%. Outer vesicles were extracted from the supernatant by ultracentrifugation. Electron microscopy, western blot, and nano-flow were used to characterize extracellular vesicles; the characterization results are shown in Figures 5, 6, and 7. Figure 5 is the result of measuring the particle size of extracellular vesicles using nano-flow, and Figure 6 is The results of electron microscope observation of extracellular vesicles, Figure 7 shows the results of western blot characterization. The results of western blot characterization showed that extracellular vesicles were successfully extracted.

(2) Binding assay

After the extracellular vesicles were extracted, the binding to the nucleic acid aptamer ZH-A2 was detected by nanoflow. The results are shown in Figure 8 and Figure 9. It can be seen from the offset in Figure 8 and the fluorescence statistics in Figure 9 The nucleic acid aptamer ZH-A2 has good affinity with A2780/PTX extracellular vesicles and A2780/DDP extracellular vesicles, poor affinity for A2780 extracellular vesicles, and almost no affinity for IOSE80 extracellular vesicles, so the nucleic acid The aptamer ZH-A2 has good specificity and can effectively distinguish extracellular vesicles of drug-resistant cells and non-drug-resistant cells.

Test Example 3

Characterization of the binding of extracellular vesicles to ZH-A2 in patient serum

In order to further verify the differentiation of nucleic acid aptamers in patient samples, the serum of ovarian cancer chemotherapy-resistant patients and chemotherapy-sensitive patients were collected, and the extracellular vesicles in the patient's serum were extracted and examined by nanoflow cytometry. The flow is shown in Figure 10A.

The results are shown in Figure 10B, indicating that the nucleic acid aptamer ZH-A2 has stronger binding ability to extracellular vesicles of drug-resistant patients, and has good specificity.

Routine operations in the operation steps of the present invention are well known to those skilled in the art and will not be repeated here.

The above embodiments describe the technical solutions of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Anything done within the scope of the principles of the present invention Any modifications, additions or substitutions in similar manners, etc., shall be included within the protection scope of the present invention.

sequence listing

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Claims (6)

1. An aptamer for identifying an extracellular vesicle of an ovarian cancer drug-resistant patient, which has a sequence shown as SEQ ID No. 1.

2. An aptamer for recognizing an extracellular vesicle in a patient resistant to ovarian cancer, wherein the aptamer is a modified sequence of the aptamer according to claim 1.

3. The nucleic acid aptamer according to claim 2, wherein the modification comprises at least one of the following modification methods:

(1) phosphorylation;

(2) methylation;

(3) amination;

(4) sulfhydrylation;

(5) isotopic ization;

(6) replacing oxygen with sulfur;

(7) selenium is used to replace oxygen.

4. An aptamer for recognizing extracellular vesicles of a patient with ovarian cancer resistance, wherein the aptamer is a sequence modified from the aptamer according to claim 1; the modification comprises at least one of the following modification methods:

(1) connecting a fluorescent marker to the aptamer;

(2) attaching a radioactive substance to the aptamer;

(3) attaching a therapeutic substance to the nucleic acid aptamer;

(4) attaching biotin to the aptamer;

(5) connecting digoxin to the aptamer;

(6) connecting a nano luminescent material on the aptamer;

(7) linking a small peptide to the aptamer;

(8) linking siRNA to the aptamer.

5. A derivative of an aptamer that recognizes an extracellular vesicle in an ovarian cancer-resistant patient, wherein the derivative is at least one of:

(1) a phosphorothioate backbone sequence derived from the aptamer backbone of claim 1;

(2) a peptide nucleic acid sequence modified from the nucleic acid aptamer of claim 1.

6. Use of the nucleic acid aptamer as claimed in claim 1 or a derivative of the nucleic acid aptamer as claimed in claim 5, comprising at least one of:

(1) the application in the preparation of an ovarian cancer drug-resistant cell strain detection kit;

(2) the application in preparing ovarian cancer drug-resistant cell strain detection probes;

(3) the application in preparing a kit for detecting the serum exosomes of the drug-resistant patients of ovarian cancer;

(4) the application of the probe in preparing the serum exosome detection probe for the drug-resistant patients of ovarian cancer.

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