Nucleic acid structure-based method for selectively separating and analyzing newborn extracellular vesicles after vaccination

Abstract

The invention discloses a method for selectively separating and analyzing newborn extracellular vesicles after vaccination based on a nucleic acid structure. The method comprises the following steps: step 1, acquiring a plasma sample; step 2, preparing a nucleic acid structure including a DNA triangular prism or tetrahedral structure or cubic structure or triangular splicing type reticular plane structure or regular icosahedron structure, wherein the nucleic acid structure is formed by assembling a framework nucleic acid chain and an aptamer recognition chain; the nucleic acid aptamer is used for capturing newborn extracellular vesicles after vaccination; 3, modifying the nucleic acid structure prepared in the step 2 on a chip; and 4, contacting a plasma sample with the chip interface modified in the step 3, so that the new extracellular vesicles are captured and separated by the nucleic acid structure after vaccination. The captured newborn extracellular vesicles can be used for further biological analysis.

Description

Technical Field

[0001] The present invention relates to a method for selectively separating and analyzing newborn extracellular vesicles after vaccination based on nucleic acid structure. Background Art

[0002] Extracellular vesicles (EVs) are a type of nano-sized membrane vesicles secreted by cells, which have rich membrane proteins and nucleic acid contents. Studies have found that extracellular vesicles are not only important in life activities such as intercellular communication, cell growth and differentiation, and tumor immune response, but also participate in the immune process of mRNA vaccines. The coronavirus (COVID-19) caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging on a large scale. In order to induce antiviral immunity in the body and reduce the severity of new coronavirus infection, the U.S. Food and Drug Administration (FDA) has approved the emergency use license of the mRNA vaccine developed by Pfizer-BioNTech and Moderna for SARS-CoV-2 virus infection. When this type of mRNA vaccine is administered, the body will produce humoral and cellular immune responses, reducing the risk of infection with the virus.

[0003] Traditional evaluation of vaccination effectiveness is generally performed by measuring the amount of antibodies targeting specific viral antigens for analysis and comparison. However, the study found that healthy adults who received two doses of SARS-CoV-2 mRNA vaccine (Pfizer-BioNTech) produced extracellular vesicles carrying SARS-CoV-2 spike protein on the 14th day after the first mRNA vaccine, while the corresponding antibodies were not produced at this time; only on the 14th day after the second booster dose of vaccination could the corresponding antibodies be detected, and the number of extracellular vesicles expressing spike protein increased dramatically. This indicates that extracellular vesicles are produced earlier than antibodies, revealing that extracellular vesicles expressing SARS-CoV-2 spike protein may be one of the necessary conditions for effective immunity after mRNA vaccination. In addition, the researchers immunized C57BL / 6 mice with extracellular vesicles expressing spike protein isolated from the plasma of vaccine recipients, while the control group immunized mice with total extracellular vesicles in plasma. Compared with the control group, extracellular vesicles expressing spike protein stimulated higher levels of cytokine expression in mice, and more antibodies targeting spike protein were detected in mice. This reveals the important role of extracellular vesicles expressing spike proteins in antiviral immunity.

[0004] After the body is immunized, the abundance of extracellular vesicles expressing spike proteins in the plasma is low, and there is currently a lack of corresponding efficient separation and analysis methods to monitor the dynamic changes of corresponding extracellular vesicles in the body after mRNA vaccination. Existing technologies make it difficult to achieve efficient separation and analysis of low-abundance new extracellular vesicles in plasma that express specific antigenic proteins after the body is vaccinated with mRNA vaccines. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for selectively isolating and analyzing newborn extracellular vesicles after vaccination based on nucleic acid structure.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure, comprising the following steps:

[0008] Step 1, obtaining a plasma sample;

[0009] Step 2, preparing a nucleic acid structure including a DNA triangular prism or tetrahedral structure or a cubic structure or a triangular splicing mesh planar structure or a regular icosahedral structure, wherein the nucleic acid structure is assembled from a framework nucleic acid chain and a nucleic acid aptamer recognition chain; the nucleic acid aptamer is used to capture newly generated extracellular vesicles after vaccination;

[0010] Step 3, modifying the nucleic acid structure prepared in step 2 on the chip;

[0011] Step 4: The plasma sample is brought into contact with the chip interface modified in step 3, so that the newly generated extracellular vesicles after vaccination can be captured and separated by the nucleic acid structure.

[0012] Furthermore, the nucleic acid structure is a DNA triangular prism, each of which includes three nucleic acid aptamers and three framework nucleic acid chains, and the sequences of the three framework nucleic acid chains and the three nucleic acid aptamers are as follows:

[0013] The first framework nucleic acid chain A includes five sequences A1, A2, A3, A4 and A5 in sequence;

[0014] The second framework nucleic acid chain B includes five sequences, B1, B2, B3, B4 and B5 in sequence;

[0015] The first framework nucleic acid chain C includes five sequences, C1, C2, C3, C4 and C5;

[0016] A1 and A5 are complementary to B3, respectively; B1 and B5 are complementary to C3, respectively; C1 and C5 are complementary to A3, respectively;

[0017] The first of the three nucleic acid aptamers binds to A2 or A4; the second binds to B2 or B4, and the third binds to C2 or C4.

[0018] Furthermore, there are sequences between the five sequences A1, A2, A3, A4 and A5 that are not complementary to other framework nucleic acid chains or nucleic acid aptamers. There are sequences between the five sequences B1, B2, B3, B4 and B5 that are not complementary to other framework nucleic acid chains or nucleic acid aptamers, and there are sequences between the five sequences C1, C2, C3, C4 and C5 that are not complementary to other framework nucleic acid chains or nucleic acid aptamers.

[0019] Furthermore, the chip is a fishbone chip.

[0020] Furthermore, the method also includes step five, releasing the extracellular vesicles captured by the chip.

[0021] Furthermore, the release is carried out by hydrolyzing the nucleic acid structure with nuclease.

[0022] Furthermore, the method also includes step six, performing biological analysis on the released extracellular vesicles.

[0023] Furthermore, the biological analysis includes the evaluation of the receptor-ligand mediated recognition function and immunogenicity of the nascent extracellular vesicles.

[0024] The present invention also provides the application of the method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure in vaccine preparation.

[0025] The advantages of the present invention are as follows:

[0026] After the body is immunized, the abundance of extracellular vesicles expressing spike proteins in the plasma is low, and there is currently a lack of corresponding efficient separation and analysis methods to monitor the dynamic changes of corresponding extracellular vesicles in the body after mRNA vaccination. The present invention proposes a selective separation and analysis method for newly born extracellular vesicles after vaccination based on nucleic acid structure. This method utilizes the synergistic recognition of controllable valence states of heterogeneous nucleic acid aptamers, and based on the valence-conformation double matching strategy, in a trace plasma system, it can achieve efficient separation and analysis of newly born extracellular vesicles expressing corresponding proteins after vaccination (for example, SARS-CoV-2 mRNA vaccination in the embodiment expresses SARS-CoV-2 spike protein Spike). In addition, the vesicles can be further enriched and their receptor-ligand mediated recognition function and antigen immune activation function can be retained through mild nuclease hydrolysis, providing a scientific basis for subsequent vaccine immune mechanism research and immune intervention and analytical detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0028] Figure 1 It is a schematic diagram of the principle of the present invention.

[0029] Figure 2 It is a schematic diagram of the principle of the DNA triangular prism of the present invention.

[0030] Figure 3 Characterization of extracellular vesicles captured by microfluidic affinity chips for modified nucleic acid structures. (a) Schematic diagram of HEK293T-Spike cells transfected with SARS-CoV-2 Spike plasmid to produce extracellular vesicles expressing Spike; signal of extracellular vesicles captured on the chip based on (b) CD63 primary antibody characterization and (c) Spike primary antibody characterization; (d) Working curve of the proportion of extracellular vesicles captured on the chip based on CD63 primary antibody characterization (detection limit of 5.79 ng / μL). The concentration of extracellular vesicles was quantitatively determined based on BCA.

[0031] Figure 4 Schematic diagram of the recovery of 293T-Spike-EVs after nucleic acid hydrolysis by DNase I in the chip. After the release of 293T-Spike-EVs, the fluorescence signal of the chip changes.

[0032] Figure 5 (a) Dynamic light scattering and (b) transmission electron microscopy characterization of the properties of 293T-Spike-EV before and after capture.

[0033] Figure 6 (a) Anti-CD63 antibody recognition was used to analyze the binding of released 293T-Spike-EVs to 293T-ACE2 cells by flow cytometry; (b) Based on the interaction between ACE2 and Spike, the mechanism by which released 293T-Spike-EVs can bind to 293T-ACE2 cells was proposed.

[0034] Figure 7 (a) Flow cytometry analysis of the expression of the immunostimulatory marker CD40; (b) Proposed mechanism by which the released 293T-Spike-EVs can induce immunostimulation of mouse macrophages.

[0035] Figure 8 Spike-EV secretion during mouse mRNA vaccination. (a) Schematic diagram of mouse immunization and blood sample collection; (b) Analysis of Anti-Spike IgG secretion in mouse plasma after vaccination; (c) Analysis of Spike-EV secretion in mouse plasma after vaccination. (n=6, mRNA vaccine group, n=4, control group). DETAILED DESCRIPTION

[0036] See also Figure 1, which is a schematic diagram of the principle of the present invention. First, the DNA triangular prism nucleic acid structure functionalized with nucleic acid aptamers is modified in the fishbone chip channel, and the affinity recognition ability of nucleic acid aptamers is used to enrich Spike-EVs in plasma; then, the content change curve of Spike-EVs after the initial immunization of mRNA vaccine is obtained through the fluorescence signal generated by the combination of CD63 antibody, β-galactosidase antibody and FDG; finally, the Spike-EVs are released and recovered by nuclease hydrolysis of the nucleic acid structure, and whether Spike-EVs interact with host cells through the recognition of Spike and ACE2 and whether Spike-EVs have the immunogenicity to activate macrophages are further explored.

[0037] See also Figure 2 , which is a schematic diagram of the principle of forming a DNA triangular prism according to the present invention.

[0038] The following three nucleic acid aptamers (X-S1, Y-S2 and Y-S3) and three framework nucleic acid chains (S1, S2 and S3) are used in the following examples of the present invention;

[0039] Table 1

[0040]

[0041] Note: / iBiodT / indicates that the middle T base is modified with biotin

[0042] Among them, the sequences of S1, S2, and S3 are represented by five brackets representing the regions A1 to A5, B1 to B5, and C1 to C5, respectively;

[0043] That is, S1 includes five sequences, A1, A2, A3, A4 and A5;

[0044] S2 includes five sequences: B1, B2, B3, B4 and B5;

[0045] S3 includes five sequences: C1, C2, C3, C4 and C5;

[0046] A1 and A5 are complementary to B3, respectively; B1 and B5 are complementary to C3, respectively; C1 and C5 are complementary to A3, respectively;

[0047] A portion of the sequence in the first X-S1 of the three nucleic acid aptamers (i.e., in the brackets) binds to A2; a portion of the sequence in the second (Y-S2) (i.e., in the brackets) binds to B2, and a portion of the sequence in the third (Y-S3) (i.e., in the brackets) binds to C2.

[0048] After these six sequences are mixed, they form Figure 2 The triangular prism structure shown.

[0049] In other embodiments, according to the above principles, other sequences can be used to implement Figure 2 The triangular prism structure shown.

[0050] Example 1

[0051] 1. Extraction of extracellular vesicles from model cells

[0052] HEK293T cells expressing SARS-CoV-2 spike protein (Spike) and HEK293T cells were selected as positive and negative model cell lines, respectively. The culture medium was DMEM high glucose medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured in a cell culture incubator (37°C, 5% CO2). Before extracellular vesicle extraction, the cell culture medium was replaced with DMEM high glucose medium (containing 10% serum from which fetal bovine extracellular vesicles were removed and 1% penicillin-streptomycin), and the cells were cultured for at least 60 hours. Subsequently, the cell culture supernatant was collected, first centrifuged at 3000g for 20 minutes to remove the bottom sediment cell debris and dead cells; secondly, the supernatant was recovered and centrifuged at 16500g for 45 minutes in an ultracentrifuge to discard the larger microvesicles in the bottom sediment; then the recovered supernatant was centrifuged at 100000g for 2 hours in an ultracentrifuge, the supernatant was removed, and the extracellular vesicles in the bottom sediment were retained. Finally, the extracellular vesicles were washed with PBS buffer and recovered after centrifugation at 100,000 g for 2 hours. The above steps were all performed at 4°C unless otherwise specified.

[0053] 2. Obtaining plasma samples from mice after vaccination:

[0054] Female Balb / c mice (6 weeks old) were randomly divided into a vaccination immunization experimental group and a control group. The experimental group was vaccinated with two doses of periodic mRNA vaccination, and 50 μL DPBS buffer (containing 10 μg mRNA-LNP) was injected intramuscularly into the right hind limb of the mouse, while the control group was vaccinated with an equal volume of PBS buffer for the same period. Plasma samples were collected on the 3rd, 7th and 14th days after the first vaccination, and the second vaccination was performed on the 15th day after the first vaccination, and then plasma samples were collected on the 21st and 28th days after the first vaccination, for a total of 5 times.

[0055] Before collecting mouse blood samples, 80-100 μL (per dose) of 3% sodium pentobarbital was injected intraperitoneally to anesthetize the mice. After anesthesia, the mice were blooded from the heart and stored in EDTA K2 anticoagulant tubes (BDmicrotainer, 365974). After centrifugation at 500 g for 5 minutes, the supernatant was taken in a 1.5 mL centrifuge tube, and then centrifuged at 1100 g for 20 minutes to obtain the supernatant, i.e., plasma. The above centrifugation steps were all performed at 4°C, and the plasma samples were stored in a minus 80°C refrigerator before use.

[0056] 3. DNA triangular prism nanostructure assembly

[0057] The DNA in Table 1 was obtained by Shanghai Shenggong Company using chemical synthesis and purified by high performance liquid chromatography. The absorbance of each DNA chain at 260nm was measured by ultraviolet spectrophotometer, and the molar extinction coefficient of the corresponding DNA chain was obtained from the IDT DNA website to calculate the actual concentration of each DNA chain. The equimolar ratio of each DNA assembly chain S1, S2, S3 (S1, S2, S3 are biotin-modified intermediate T bases) and the equimolar ratio of X-S1, Y-S2, Y-S3 were mixed in TM buffer (20mMTris-acetic acid, 2mM EDTA, 12.5mM magnesium acetate), and the final concentration of the DNA triangular prism was 1μM. Then the PCR annealing program was performed: the first step was to react at 95℃ for 5 minutes, the second step was to react at 80℃ for 30 minutes, the third step was to react at 79℃ for 30 minutes, and the fourth step was to react at 77℃ for 30 minutes, and then the temperature was slowly lowered to 4℃ for 1.5 hours.

[0058] 4. Microfluidic Chip Preparation

[0059] (1) Molding process of fishbone chip: The chip is manufactured using standard photolithography and polydimethylsiloxane (PDMS) casting technology. The manufacturing process of PDMS chip is as follows: 50g PDMS monomer and 5g initiator are uniformly mixed in a ratio of 10:1 (the amount can be increased according to the weight ratio according to the required amount), and the bubbles generated after mixing and stirring are removed by a vacuum pump to facilitate the subsequent glue pouring. The prepared glue is poured on the fishbone chip mold and gently blown with an ear bulb to remove the bubbles generated by the glue pouring in the mold to prevent affecting the subsequent capture and observation of the chip. Then, the mold filled with glue is placed in an oven with the oven temperature set at 75°C. After 15 minutes of shaping, it is taken out and cooled for a while. The PDMS block with the mold pattern is cut from the mold with a scalpel, and holes are punched at the inlet and outlet of the PDMS block channel with a punch pen to allow the inlet and outlet to flow. After punching, it is placed on a clean PDMS film and waits for subsequent use.

[0060] (2) Bonding process of fishbone chip: First, a clean glass sheet and a PDMS film are bonded using a plasma cleaning machine to make the PDMS film and the glass sheet fit well together. Then, the previously prepared PDMS block and the glass sheet with the PDMS film are put into a plasma cleaning machine for activation. After taking them out, the two are put together to complete the bonding and make a fishbone chip.

[0061] (3) Modification process of fishbone chip: In order to achieve the subsequent capture of extracellular vesicles on the chip, the prepared fishbone chip needs to be modified. After the fishbone chip is made, it is modified immediately. First, 3-mercaptopropylmethyldimethoxysilane (MPTS, 4% in anhydrous ethanol) is injected into the chip from the inlet for 1 hour to form a sulfurized microfluidic surface on the chip channel, and then the chip is rinsed two or three times with anhydrous ethanol to remove the remaining MPTS, and the chip is placed in an oven at 100°C for 1 hour. After drying, the chip is taken out of the oven, and after cooling for a while, N-γ-maleimidobutylhydroxysuccinimide (GMBS, 0.5 mg / mL in anhydrous ethanol) is continuously injected into the chip for 30 minutes to form an NH2 ester-modified microfluidic surface. After that, the chip is rinsed twice with anhydrous ethanol and PBS, and streptavidin (50 ng / μL) is injected into the chip. After modification, it is stored at 4°C. The chip was rinsed with PBS before use to remove excess streptavidin, and then 1 μM biotin-modified DNA triangular prism nanostructures were added to the chip twice, each incubated for 1 hour.

[0062] 5. Characterization of Samples

[0063] Isolation and analysis of extracellular vesicles:

[0064] 1) Microfluidic affinity separation and characterization of extracellular vesicles expressing Spike protein:

[0065] 2.5 μg of HEK 293T-Spike extracellular vesicles were added to a final volume of 25 μL of BB binding buffer (PBS solution containing 0.55 mM MgCl2, 0.5% BSA). After capture by the chip, the channel was washed with PBS (0.5% BSA PBS) to remove unbound extracellular vesicles. First, the extracellular vesicles captured on the chip were detected with CD63 antibody or Spike antibody (25 μg / mL), and then the β-galactosidase-modified goat anti-mouse secondary antibody was diluted in binding buffer (PBS containing 0.55 mM MgCl2 and 0.5% BSA) and passed into the chip and incubated for 30 minutes. After incubation, the excess β-galactosidase-modified goat anti-mouse IgG in the channel was washed with PBS. Then, 20 μL FDG (100 μM, PBS containing 0.55 mM MgCl2 and 0.5% BSA) was added and reacted in the dark for 0.5 h, and the fluorescence signal was detected by amplification of the enzyme signal. The fluorescence image was taken by an inverted fluorescence microscope, and the captured image was processed by Image J software to calculate the fluorescence value. Different concentrations of extracellular vesicles will show different fluorescence intensities, so the concentration of extracellular vesicles is used as the horizontal axis and the fluorescence intensity is used as the vertical axis to draw the detection curve. After linear fitting, the detection limit of the chip can be determined.

[0066] 2) Characterization of downstream recognition and immune effects after release of extracellular vesicles expressing Spike protein:

[0067] 20U / mL DNase I (NEB, USA) was injected into the chip through a syringe pump and reacted at 37°C for 15 minutes. After being rinsed with PBS buffer at a high flow rate, the released extracellular vesicles were collected into a centrifuge tube through the connection of a polytetrafluoroethylene tube at the outlet. Fluorescence microscopy was used to compare the changes in the fluorescence intensity of the microfluidic chip before and after release, and dynamic light scattering and transmission electron microscopy were used to characterize the size and morphology of the released extracellular vesicles.

[0068] To investigate whether the physicochemical properties of extracellular vesicles change after release, studies have shown that SARS-CoV-2Spike binds to the human cell receptor protein ACE2, which prompts the SARS-CoV-2 virus to bind to and infect human cells. The released HEK293T-Spike extracellular vesicles were incubated with HEK 293T cells (293T-ACE2) transfected with ACE2 protein to investigate whether the extracellular vesicles released from the chip still have the ability to bind to receptor cells. 2×10 5293T-ACE2 cells were incubated with HEK 293T-Spike extracellular vesicles (293T-Spike-EV) released from the microfluidic chip in a final volume of 100 μL for 2 hours. After centrifugation at 1500 rpm for 3 minutes, the cells were washed twice with PBS. APC-CD63 antibody (8 μL) was added to the cell suspension in PBS (0.5% BSA) in a final volume of 100 μL. After washing twice with PBS (0.5% BSA), the cells were resuspended in 200 μL PBS for flow cytometry analysis.

[0069] RAW 264.7 cells were seeded in 96-well culture wells (2×10 4 / well), and immune stimulation was performed after incubation for 24 hours to explore the immune effect of the released 293T-Spike-EV. Fresh culture medium containing the released and recovered 293T-Spike-EV was added to the wells and incubated in the cell culture incubator for another 20 hours. The negative control was fresh culture medium with PBS or DNaseⅠ added. After stimulation, the cells were separated and washed once with 100μL PBS. Then they were incubated with APC anti-mouse CD40 antibody (1:100) in 100μL PBS containing 2% FBS for 1 hour. After washing twice with PBS containing 2% FBS, the cells were resuspended in 100μL PBS for flow cytometry analysis (FACSVerse, BD). All the above experiments were performed at room temperature unless otherwise specified, and centrifuged at 1500rpm for 3min.

[0070] 3) Analysis of extracellular vesicles expressing Spike protein in mouse plasma after mRNA vaccination:

[0071] 10 μL of mouse plasma sample was diluted into 25 μL of BB binding buffer (PBS solution containing 0.55 mM MgCl2, 0.5% BSA) and passed into the chip modified with DNA triangular prism nanostructure. After incubation for 30 minutes, it was also washed with BB binding buffer, and CD63 antibody, β-galactosidase-modified goat anti-rat secondary antibody and FDG substrate were passed in turn. After the reaction, a fluorescence microscope was used to detect the fluorescence signal of the extracellular vesicles captured on the chip, and then Image J software was used to measure the fluorescence intensity of the captured fluorescence images.

[0072] Figure 3 The results showed that extracellular vesicles derived from HEK293T cells expressing the SARS-CoV-2 spike protein could be successfully and specifically captured by a microfluidic affinity chip modified with a nucleic acid structure, with a detection limit as low as 5.79 ng / μL.

[0073] Figure 4-7The results showed that the extracellular vesicles derived from HEK293T cells expressing the SARS-CoV-2 spike protein (Spike) could be gently released after the nucleic acid structure was hydrolyzed by DNA hydrolase, and the size and morphology of the vesicles did not change significantly. The released Spike-EVs can still bind to ACE2-HEK293T cells based on the interaction between Spike and ACE2, and can also stimulate antigen presenting cells (mouse macrophages).

[0074] Figure 8 The results showed that the method of the present invention can be used to monitor the secretion of Spike-EVs during mRNA inoculation in mice.

[0075] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure, comprising the following steps: Step 1, obtaining a plasma sample; Step 2, preparing a nucleic acid structure including a DNA triangular prism or tetrahedral structure or a cubic structure or a triangular splicing mesh planar structure or a regular icosahedral structure, wherein the nucleic acid structure is assembled from a framework nucleic acid chain and a nucleic acid aptamer recognition chain; the nucleic acid aptamer is used to capture newly generated extracellular vesicles after vaccination; Step 3, modifying the nucleic acid structure prepared in step 2 on the chip; Step 4: The plasma sample is brought into contact with the chip interface modified in step 3, so that the newly generated extracellular vesicles after vaccination can be captured and separated by the nucleic acid structure.

2. A method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 1, characterized in that: The nucleic acid structure is a DNA triangular prism, each of which includes three nucleic acid aptamers and three framework nucleic acid chains. The sequences of the three framework nucleic acid chains and the three nucleic acid aptamers are as follows: The first framework nucleic acid chain A includes five sequences A1, A2, A3, A4 and A5 in sequence; The second framework nucleic acid chain B includes five sequences, B1, B2, B3, B4 and B5 in sequence; The first framework nucleic acid chain C includes five sequences, C1, C2, C3, C4 and C5; A1 and A5 are complementary to B3, respectively; B1 and B5 are complementary to C3, respectively; C1 and C5 are complementary to A3, respectively; The first of the three nucleic acid aptamers binds to A2 or A4; the second binds to B2 or B4, and the third binds to C2 or C4.

3. A method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 2, characterized in that: The binding between the three nucleic acid aptamers and the three framework nucleic acid chains is the binding of partial regions of the three nucleic acid aptamers.

4. The method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 1, characterized in that: The chip is a fishbone chip.

5. The method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 1, characterized in that: It also includes step five, releasing the extracellular vesicles captured by the chip.

6. A method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 5, characterized in that: The release is carried out by hydrolyzing the nucleic acid structure with nuclease.

7. The method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 5, characterized in that: The method also includes step six, performing biological analysis on the released extracellular vesicles.

8. The method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to claim 7, characterized in that: The biological analysis includes receptor-ligand mediated recognition function and immunogenicity assessment of nascent extracellular vesicles.

9. Use of a method for selectively isolating and analyzing newly formed extracellular vesicles after vaccination based on nucleic acid structure according to any one of claims 1 to 8 in vaccine preparation.

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