Corynebacterium veitae extracellular vesicle as well as preparation method and application thereof

Abstract

The invention discloses a corynebacterium striatum extracellular vesicle as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the corynebacterium striatum extracellular vesicle provided by the invention comprises the following steps: (1) transferring a single colony of corynebacterium striatum to a culture medium, and culturing for 14 hours at the constant temperature of 37 DEG C at the speed of 220rpm to obtain a bacterial solution; (2) pouring the bacterial liquid into a culture medium, uniformly shaking, culturing for 48 hours at the constant temperature of 37 DEG C under 5% CO2, centrifugally filtering and sterilizing, and enriching and concentrating the filtrate to obtain a concentrated solution; and (3) carrying out ultra-high-speed centrifugation on the concentrated solution, and reserving precipitates, so as to obtain the corynebacterium striatum extracellular vesicles. According to the preparation method of the corynebacterium striatum extracellular vesicles, the extraction efficiency can be optimized, and meanwhile high yield, high purity and low pollution of the corynebacterium striatum extracellular vesicles are guaranteed. And on the basis, gene engineering modification can be carried out subsequently, so that the vaccine has vaccine development potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to extracellular vesicles of Corynebacterium striatum and a preparation method and application thereof. Background Art

[0002] Bacterial extracellular vesicles (BEVs) are nanoscale membrane particles produced by Gram-negative or Gram-positive bacteria, consisting of a lipid bilayer encapsulating proteins, nucleic acids and other biomolecules. They not only carry out communication between bacteria, but also play a cross-border regulatory role between bacteria and host cells.

[0003] Extracellular vesicles have unique characteristics such as the ability to deliver substances in a targeted manner and high biocompatibility. They are currently receiving great attention in the field of nanomedicine as advanced drug delivery carriers. These characteristics give natural extracellular vesicles a significant performance advantage over synthetic nanoparticles (such as liposomes used for the delivery of biological drugs). Some natural bacterial-derived cellular vesicles have been reported to have a variety of uses, such as the construction of targeted delivery carriers for anti-tumor drugs, vaccine development, and the treatment of infectious diseases. Bacterial extracellular vesicles obtained through various process modifications have broader application prospects. Further broadening our understanding of them will help the development of new vaccines or drugs.

[0004] Corynebacterium streaks is a common prokaryotic cell-type microorganism that colonizes human skin and mucous membranes. There are currently no reports on the function of its extracellular vesicles. Based on current literature reports, Corynebacterium streaks has strong adhesion or biofilm formation ability, and it is speculated that it can secrete or express certain adhesion factors to participate in its biological activities. These factors can also be secreted in the form of extracellular vesicles and then participate in subsequent biological activities mediated by Corynebacterium streaks, but this is not yet clear. At the same time, the secretion efficiency and influencing factors of Corynebacterium streaks extracellular vesicles are unknown. Summary of the invention

[0005] The purpose of the present invention is to provide extracellular vesicles of Corynebacterium striatum and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The Corynebacterium striatum prepared by the present invention has high membrane purity and less protein pollution, and has the advantages of wide practicability.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical solution 1: A method for preparing extracellular vesicles of Corynebacterium striatum, comprising the following steps:

[0008] (1) After transferring a single colony of Corynebacterium striatum to a culture medium, the culture was placed in a constant temperature culture at 220 rpm and 37° C. for 14 h to obtain a bacterial solution;

[0009] (2) Pour the bacterial solution into the culture medium, shake well, and place at 37°C and 5% CO 2 After culturing at a constant temperature for 48 hours, the filtrate was centrifuged and sterilized, and the filtrate was enriched and concentrated to obtain a concentrate;

[0010] (3) The concentrated solution is subjected to ultrahigh-speed centrifugation to collect the precipitate to obtain the extracellular vesicles of Corynebacterium striatum.

[0011] Furthermore, in step (1), the preparation method of the single colony is: the strain of Corynebacterium striatum is heated to 5% CO 2 , obtained by constant temperature culture at 37℃ for 48h.

[0012] Furthermore, in steps (1) and (2), the culture medium is tryptic soy broth.

[0013] Furthermore, in step (3), the extracellular vesicles of Corynebacterium striatum include free extracellular vesicles of Corynebacterium striatum and membrane-bound extracellular vesicles of Corynebacterium striatum.

[0014] Furthermore, when the extracellular vesicles of Corynebacterium striatum are free extracellular vesicles, in step (2), the bacterial solution is poured into the culture medium and shaken evenly, and placed at 220 rpm, 37° C., 5% CO 2 The cells were cultured at a constant temperature with shaking for 48 h.

[0015] Furthermore, when the extracellular vesicles of Corynebacterium striatum are membrane-bound extracellular vesicles, in step (2), the bacterial solution is poured into a culture medium and shaken, and placed at 37° C. and 5% CO 2 The culture was incubated at a constant temperature for 48 h.

[0016] Furthermore, in step (3), the number of ultra-high-speed centrifugation is 2 times; the conditions of the first ultra-high-speed centrifugation are: centrifugal force 100,000×g, centrifugal time 4 hours, centrifugal temperature 4°C; the conditions of the second ultra-high-speed centrifugation are: centrifugal force 100,000×g, centrifugal time 1 hour, centrifugal temperature 4°C.

[0017] Technical Solution 2: The free extracellular vesicles of Corynebacterium striatum prepared by the preparation method have an average particle size of 109.5 nm.

[0018] Technical Solution Three: The membrane-bound extracellular vesicles of Corynebacterium striatum obtained by the preparation method have an average particle size of 121.0 nm.

[0019] Technical Solution 4: Application of the free extracellular vesicles of Corynebacterium striatum or the membrane-bound extracellular vesicles of Corynebacterium striatum in vaccine development and / or specific drug delivery.

[0020] The present invention discloses the following technical effects:

[0021] The preparation method provided by the invention can successfully separate free and membrane-state BEVs of Corynebacterium taeniae, which present spherical vesicles with clear double-layer membrane structure under transmission electron microscopy; the test results show that the average particle density and particle size (7.2E+9Particles / mL, 109.5nm) of free BEVs of Corynebacterium taeniae are both smaller than those of membrane-state BEVs of Corynebacterium taeniae (6.3E+10Particles / mL, 121.0nm); the results of BCA protein quantitative detection method show that the average protein concentration (0.478μg / μL) of membrane-state BEVs of Corynebacterium taeniae is significantly higher than the average protein concentration (0.213μg / μL) of free BEVs of Corynebacterium taeniae. The membrane-state extracellular vesicles of Corynebacterium taeniae prepared by the present invention have high purity and less protein contamination, and have the advantages of wide practicality.

[0022] The separation and extraction process of the extracellular vesicles of Corynebacterium striatum provided by the present invention can optimize the extraction efficiency while ensuring its high yield, high purity and low pollution. Subsequently, on this basis, genetic engineering can be used to improve its immunogenicity, reduce its toxicity, stimulate the body's inherent immunity and adaptive immune response, and make it have the potential for vaccine development. At the same time, BEVs, as nano-scale particles, have good diffusion ability and bioavailability and can carry exogenous substances. Through surface modification and design and addition of functional ligands to enhance its targeting transformation, it can be used as a drug delivery carrier. BEVs that have been genetically engineered and have specific targeting capabilities can provide new technical means and platforms for the preparation of new vaccines for various infectious diseases, specific drug delivery, and anti-tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 A conical flask with a vent hole for preparing a free bacterial solution of Corynebacterium striatum;

[0025] Figure 2 T225 breathable cover oblique mouth cell bottle for preparing membrane-state bacterial liquid of Corynebacterium striatum;

[0026] Figure 3 The figure is a schematic diagram of the process of separating and extracting the extracellular vesicles of Corynebacterium striatum;

[0027] Figure 4This is a transmission electron microscopy image of the free extracellular vesicles of Corynebacterium striatum obtained in Example 1 at a scale of 100 nm;

[0028] Figure 5 This is a transmission electron microscopy image of the free extracellular vesicles of Corynebacterium striatum obtained in Example 1 at a scale of 200 nm;

[0029] Figure 6 This is a transmission electron microscopy image of the free extracellular vesicles of Corynebacterium striatum obtained in Example 1 at a scale of 500 nm;

[0030] Figure 7 This is the particle size-particle density distribution diagram of the free extracellular vesicles of Corynebacterium striatum obtained in Example 1;

[0031] Figure 8 This is a transmission electron microscopy image of the membrane-bound extracellular vesicles of Corynebacterium striatum obtained in Example 2 at a scale of 100 nm;

[0032] Fig. 9 This is a transmission electron microscopy image of the membrane-bound extracellular vesicles of Corynebacterium striatum obtained in Example 2 at a scale of 200 nm;

[0033] Fig.10 This is a transmission electron microscopy image of the membrane-bound extracellular vesicles of Corynebacterium striatum obtained in Example 2 at a scale of 500 nm;

[0034] Fig.11 This is the particle size-particle density distribution diagram of the membrane-bound extracellular vesicles of Corynebacterium striatum obtained in Example 2;

[0035] Fig.12 This is the standard curve of BCA detection. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0042] The Corynebacterium striatum was the standard strain ATCC 6940 purchased from the American Type Culture Collection, and the tryptic soy broth (TSB) was from OXOID, a subsidiary of Thermo Fisher Scientific.

[0043] Example 1

[0044] 1. Isolation and concentration of free BEVs of Corynebacterium striatum

[0045] 1.1 Method for preparing Corynebacterium striata bacterial solution

[0046] Corynebacterium striata was inoculated on Columbia blood agar medium for recovery and 5% CO 2 , incubate at 37℃ for 48h, pick a single colony, transfer it to 10mL tryptic soy broth (TSB), place it in a constant temperature shaking incubator, 220rpm, 37℃, and culture for 14h; after 14h, pour 4mL of bacterial solution into a breathable conical flask containing 400mL tryptic soy broth, shake well, and place it at 37℃, 5% CO 2 The culture was cultured in a constant temperature shaking incubator at 220 rpm for 48 h to obtain free bacterial liquid.

[0047] 1.2 Method for centrifugal filtration of bacterial solution to sterilize and enrich and concentrate the filtrate

[0048] The free bacterial liquid prepared in 1.1 was centrifuged (centrifugal force 7000 × g, centrifugal time 5 min), and the precipitate was discarded after centrifugation to retain the supernatant; the supernatant was filtered for the first time, and the filter membrane particle size was 0.22 μm; the filtrate was enriched and concentrated using an ultrafiltration cup (including a magnetic stirrer); the enrichment and concentration multiple was 10 times; the molecular weight cutoff under the enrichment and concentration conditions was 100 kDa; the speed of the magnetic stirrer was 320-350 rpm; a certain pressure was applied to the ultrafiltration cup using a sterile nitrogen bottle device; the pressure indication was: the drip rate of the waste liquid in the ultrafiltration cup (liquid with a molecular weight less than 100 kDa) was 1 drop / 5-7 seconds.

[0049] The concentrated solution obtained in the above steps was poured into an ultracentrifuge tube, and the volume was adjusted to 29.9 mL with sterile PBS buffer. The ultracentrifuge tube was balanced and the first ultracentrifugation was performed (centrifugation conditions: centrifugal force 100,000 × g, centrifugation time 4 h, centrifugation temperature 4 ° C). After the centrifugation was completed, the supernatant was discarded and the precipitate was retained, and the precipitate was resuspended with sterile PBS buffer, and ultracentrifugation was performed again (centrifugation conditions 100,000 × g, centrifugation time 1 h, centrifugation temperature 4 ° C). After the centrifugation was completed, the supernatant was discarded and the precipitate was retained, and the precipitate was resuspended with 1 mL of sterile PBS buffer, and the agglomerated precipitate attached to the wall of the centrifuge tube was crushed with a disposable inoculation loop; the resuspended solution was filtered for a second time, and the filter membrane particle size was 0.22 μm, and the precipitate was retained to obtain BEVs of free-state Corynebacterium striatum.

[0050] 1.3 Characteristics of free extracellular vesicles of Corynebacterium striatum

[0051] 1.3.1 Take 20 μL of free BEVs of Corynebacterium fasciatum prepared in step 1.2 and drop them into the copper grid of electron microscope in the form of water droplets and let them stand for 2 minutes. Fix with phosphotungstic acid solution for 10 minutes and leave to dry at room temperature.

[0052] 1.3.2 The morphological characteristics of BEVs were observed using transmission electron microscopy (TEM). The electron microscopy images of free BEVs of Corynebacterium fasciatum were as follows: Figure 4 , 5 and 6.

[0053] 1.3.3 Analyze the particle characteristics using nanoparticle tracking analysis (NTA) technology: dilute the free BEVs of Corynebacterium fasciatum with 100-fold, 1000-fold, and 10000-fold concentrations using sterile PBS buffer. Wash the analyzer's sample pool with 10 mL of sterile buffer and draw 1 mL of the diluted sample for loading. 6 —10 7The obtained free extracellular vesicles of Corynebacterium fasciatum had a particle size range of 20-450nm; the particle concentration was 7.2E+9Particles / mL; the extracellular vesicles with a particle size range of 22-325nm accounted for 88.6%; the average particle size was 109.5nm. The particle size-particle density distribution diagram is shown in Figure 7 shown.

[0054] 1.3.4 BCA protein quantitative detection method to determine the protein concentration: Pipette 25 μL of free BEVs of Corynebacterium fasciatum and BSA standard into the microwells of a 96-well plate, and perform three parallel reactions. Add 200 μL of BCA working solution to each well, mix thoroughly, and incubate at 37°C for 30 min. Cool to room temperature and use a microplate reader to read the OD 620nm Measure the absorbance of each sample and BSA standard under 4 °C. Draw a standard curve and calculate the protein concentration in the sample. The BCA detection standard curve is as follows: Fig.12 The free extracellular vesicle protein concentration of Corynebacterium striatum is shown in Table 1.

[0055] Table 1

[0056]

[0057] Example 2

[0058] 1. Isolation and concentration of membrane-bound BEVs of Corynebacterium striatum

[0059] 1.1 Method for preparing Corynebacterium striata bacterial solution

[0060] Corynebacterium striata was inoculated on Columbia blood agar medium for recovery and 5% CO 2 , incubate at 37℃ for 48h, pick a single colony, transfer to 10mL tryptic soy broth (TSB), place in a constant temperature shaking incubator, 220rpm, 37℃, incubate for 14h; after 14h, pour 4mL of bacterial solution into a T225 breathable cap oblique mouth cell bottle containing 400mL tryptic soy broth, place at 37℃, 5% CO 2 The cells were placed in a constant temperature incubator and cultured for 48 hours to obtain membrane bacterial liquid.

[0061] 1.2 Method for centrifugal filtration of bacterial solution to sterilize and enrich and concentrate the filtrate

[0062] The membrane bacterial liquid prepared in 1.1 was centrifuged (centrifugal force 7000 × g, centrifugal time 5 min), and the precipitate was discarded after centrifugation to retain the supernatant; the supernatant was filtered for the first time, and the filter membrane particle size was 0.22 μm; the filtrate was enriched and concentrated using an ultrafiltration cup (including a magnetic stirrer); the enrichment and concentration multiple was 10 times; the molecular weight cutoff under the enrichment and concentration conditions was 100 kDa; the speed of the magnetic stirrer was 320-350 rpm; a certain pressure was applied to the ultrafiltration cup using a sterile nitrogen bottle device; the pressure indication was: the drip rate of the waste liquid in the ultrafiltration cup (liquid with a molecular weight less than 100 kDa) was 1 drop / 5-7 seconds.

[0063] The concentrated solution obtained in the above steps was poured into an ultracentrifuge tube, and the volume was adjusted to 29.9 mL with sterile PBS buffer. The ultracentrifuge tube was balanced and the first ultracentrifugation was performed (centrifugation conditions: centrifugal force 100,000 × g, centrifugation time 4 h, centrifugation temperature 4 ° C). After the centrifugation was completed, the supernatant was discarded and the precipitate was retained, and the precipitate was resuspended with sterile PBS buffer, and ultracentrifugation was performed again (centrifugation conditions 100,000 × g, centrifugation time 1 h, centrifugation temperature 4 ° C). After the centrifugation was completed, the supernatant was discarded and the precipitate was retained, and the precipitate was resuspended with 1 mL of sterile PBS buffer, and the agglomerated precipitate attached to the wall of the centrifuge tube was crushed with a disposable inoculation loop; the resuspended solution was filtered for a second time, and the filter membrane particle size was 0.22 μm), and the precipitate was retained to obtain the membrane-state BEVs of Corynebacterium striatum.

[0064] 1.3 Characteristics of membrane-bound extracellular vesicles of Corynebacterium striatum

[0065] 1.3.1 Take 20 μL of the membrane-state BEVs of Corynebacterium fasciatum prepared in step 1.2 and drop them into the copper mesh grid of the electron microscope in the form of water drops and let them stand for 2 minutes. Fix with phosphotungstic acid solution for 10 minutes and leave to dry at room temperature.

[0066] 1.3.2 The morphological characteristics of BEVs were observed using transmission electron microscopy (TEM). Figure 8 , 9 and 10.

[0067] 1.3.3 Analyze the particle characteristics using nanoparticle tracking analysis (NTA) technology: dilute the membrane-state BEVs of Corynebacterium striatum with sterile PBS buffer at 100 times, 1000 times, and 10000 times. Wash the analyzer's injection pool with 10 mL of sterile buffer and draw 1 mL of the diluted sample for loading. 6 -10 7The dilutions within the range were used for data statistics and analysis. The particle size range of the obtained membrane-bound extracellular vesicles of Corynebacterium striatum was 20-460nm; the particle concentration was 6.3E+10Particles / mL; the extracellular vesicles with a particle size of 22-302nm accounted for 98.4%; the average particle size was 121.0nm. The particle size-particle density distribution diagram is shown in Fig.11 shown.

[0068] 1.3.4 BCA protein quantitative detection method to determine the protein concentration: Pipette 25 μL of membrane-bound BEVs of Corynebacterium fasciatum and BSA standard into the microwells of a 96-well plate, and perform three parallel reactions. Add 200 μL of BCA working solution to each well, mix thoroughly, and incubate at 37°C for 30 min. Cool to room temperature and use a microplate reader to read the OD 620nm Measure the absorbance of each sample and BSA standard under 4 °C. Draw a standard curve and calculate the protein concentration in the sample. Fig.12 The concentration of membrane-bound extracellular vesicle proteins of Corynebacterium striatum is shown in Table 2.

[0069] Table 2

[0070]

[0071] Experimental Results

[0072] The schematic diagram of the process for separating and extracting the extracellular vesicles of Corynebacterium striatum in Examples 1 and 2 is shown in Figure 3 . The present invention, according to the above-mentioned method for preparing the bacterial liquid of Corynebacterium streaks, comprises centrifuging and filtering the obtained bacterial liquid for sterilization, enriching and concentrating the filtrate, ultracentrifuging the retained concentrated liquid, and retaining the precipitate to obtain free and membrane-bound extracellular vesicles of Corynebacterium streaks. The present invention also analyzes and compares the differences in morphological characteristics, particle size analysis, particle density, protein content and other characteristics of the free state of Corynebacterium streaks in Example 1 and the membrane-bound extracellular vesicles of Corynebacterium streaks in Example 2 by using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) technology and BCA protein quantitative detection method.

[0073] The ultrahigh-speed centrifugation method provided by the present invention can successfully separate the free and membrane-state BEVs of Corynebacterium streaks, which present spherical vesicles with clear double-layer membrane structure under transmission electron microscopy; the test results show that the average particle density and particle size (7.2E+9Particles / mL, 109.5nm) of the free BEVs of Corynebacterium streaks are smaller than those of the membrane-state BEVs of Corynebacterium streaks (6.3E+10Particles / mL, 121.0nm); the results of the BCA protein quantitative detection method show that the average protein concentration of the membrane-state BEVs of Corynebacterium streaks (0.478μg / μL) is significantly higher than the average protein concentration of its free BEVs (0.213μg / μL).

[0074] Through the above method of the present invention, it can be seen that the particle size, particle density and protein content of the membrane-state extracellular vesicles of Corynebacterium striatum are significantly higher than those of the free extracellular vesicles of Corynebacterium striatum. This result is consistent with the results of electron microscopy technology. The obtained membrane-state extracellular vesicles of Corynebacterium striatum have high purity and less protein contamination, and have the advantages of wide practicality.

[0075] The separation and extraction process of the extracellular vesicles of Corynebacterium striatum provided by the present invention can optimize the extraction efficiency while ensuring its high yield, high purity and low pollution. On this basis, it can be genetically engineered to improve its immunogenicity, reduce its toxicity, stimulate the body's inherent immunity and adaptive immune response, and make it have the potential for vaccine development. At the same time, BEVs, as nano-scale particles, have good diffusion ability and bioavailability and can carry exogenous substances. Subsequently, through surface modification and design and addition of functional ligands to enhance its targeting transformation, it can be used as a drug delivery carrier. In summary, BEVs with genetic engineering and specific targeting capabilities can provide new technical means and platforms for the preparation of new vaccines for various infectious diseases, specific drug delivery, and anti-tumor treatment.

[0076] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing extracellular vesicles of Corynebacterium striatum, characterized in that: The following steps are involved: (1) After transferring a single colony of Corynebacterium striatum to a culture medium, the culture was placed in a constant temperature culture at 220 rpm and 37° C. for 14 h to obtain a bacterial solution; (2) pouring the bacterial solution into the culture medium and shaking it evenly, culturing it at 37° C. and 5% CO 2 for 48 hours, centrifuging and filtering to sterilize it, and enriching and concentrating the filtrate to obtain a concentrate; (3) The concentrated solution is subjected to ultrahigh-speed centrifugation to collect the precipitate to obtain the extracellular vesicles of Corynebacterium striatum.

2. The preparation method according to claim 1, characterized in that: In step (1), the single colony is prepared by culturing the strain of Corynebacterium striatum at a constant temperature of 5% CO2 and 37°C for 48 hours.

3. The preparation method according to claim 1, characterized in that: In steps (1) and (2), the culture medium is tryptic soy broth.

4. The preparation method according to claim 1, characterized in that: In step (3), the extracellular vesicles of Corynebacterium striatum include free extracellular vesicles of Corynebacterium striatum and membrane-bound extracellular vesicles of Corynebacterium striatum.

5. The preparation method according to claim 4, characterized in that: When the extracellular vesicles of Corynebacterium striatum are free extracellular vesicles, in step (2), the bacterial solution is poured into the culture medium and shaken, and then cultured at 220 rpm, 37° C., and 5% CO 2 for 48 hours under constant temperature shaking.

6. The preparation method according to claim 4, characterized in that: When the extracellular vesicles of Corynebacterium striatum are membrane-bound extracellular vesicles, in step (2), the bacterial solution is poured into a culture medium and shaken evenly, and then placed in a constant temperature static culture at 37° C. and 5% CO 2 for 48 hours.

7. The preparation method according to claim 1, characterized in that: In step (3), the ultra-high-speed centrifugation is performed twice; the conditions for the first ultra-high-speed centrifugation are: centrifugal force 100,000×g, centrifugal time 4 hours, and centrifugal temperature 4°C; the conditions for the second ultra-high-speed centrifugation are: centrifugal force 100,000×g, centrifugal time 1 hour, and centrifugal temperature 4°C.

8. The free extracellular vesicles of Corynebacterium striatum obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The average particle size of the free extracellular vesicles of Corynebacterium striatum is 109.5 nm.

9. The membrane-bound extracellular vesicles of Corynebacterium striatum obtained by the preparation method according to any one of claims 1 to 7, characterized in that: The average particle size of the membrane-bound extracellular vesicles of Corynebacterium striatum is 121.0 nm.

10. Use of the free extracellular vesicles of Corynebacterium striatum according to claim 8 or the membrane-bound extracellular vesicles of Corynebacterium striatum according to claim 9 in vaccine development and / or specific drug delivery.

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