CN112402601B

CN112402601B - Staphylococcus aureus membrane vesicle and preparation method and application thereof

Info

Publication number: CN112402601B
Application number: CN201910777473.5A
Authority: CN
Inventors: 王震玲; 魏于全
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-08-22
Filing date: 2019-08-22
Publication date: 2022-09-13
Anticipated expiration: 2039-08-22
Also published as: CN112402601A

Abstract

The invention discloses a staphylococcus aureus membrane vesicle and a preparation method and application thereof, belongs to the field of microbiology, and provides a membrane vesicle separated from inactivated staphylococcus aureus. The invention also provides methods of isolation and preparation, and use as vaccines. And the use of the bacterial vaccine. The invention firstly adopts ionizing rays X rays to irradiate the staphylococcus aureus, separates and purifies MVs secreted by the staphylococcus aureus, and the prepared MVs can be used as vaccines, vaccine adjuvants and drug carriers.

Description

Staphylococcus aureus membrane vesicle and preparation method and application thereof

Technical Field

The invention belongs to the field of microbiology, and particularly relates to preparation, separation and purification of a staphylococcus aureus membrane vesicle and application of the membrane vesicle.

Background

Membrane Vesicles (MVs) are vesicular structures secreted by the outer membrane of bacterial cells, including gram-positive and gram-negative bacteria. Mostly spherical, with a diameter of about 20-250 nm (Structures of gram-negative cell walls and the same derived membrane vesicles/gram-negative cell wall structure and membrane vesicles secreted thereby).

Bacterial membrane vesicles contain a variety of bioactive macromolecules such as nucleic acids, lipopolysaccharides, outer membrane proteins, and the like, as well as metal ions, enzymes, signal molecules, and the like (the Biological function and Biological origin of secreted bacterial outer membrane vesicles). It plays an important role in various vital activities of bacteria, such as secretion of virulence factors, stress response, nutrient uptake, and as a carrier for information exchange between bacteria, bacteria and host cells.

The secretion of the membrane vesicles occurs at any growth stage of bacteria, is different from cell lysis and apoptosis and is an independent secretion path, and researches show that the membrane vesicles can be promoted to be produced by the bacteria under the conditions of pressure stimulation, hypoxia, antibiotic compression and the like. However, the yield of naturally-produced membrane vesicles is low, a large amount of bacteria needs to be cultured to obtain a certain amount of membrane vesicles, and an additional purification process is subsequently required to obtain membrane vesicles with a certain quality.

The modern process technology has the following problems in the production, preparation and purification of bacterial membrane vesicles: 1) although the secretion of membrane vesicles can be promoted by means of antibiotics, detergents, oxidants and the like, the method is accompanied with the problem of toxic residues, and uncertainty is brought to the application of the method. 2) The intervention factors mentioned above also have a relatively low efficiency in stimulating the bacteria to produce membrane vesicles, and the preparation process does not allow for standardized production. 3) The method can change the antigenicity, conformation and the like of the outer membrane of the thallus, further influence the vesicle and limit the subsequent application.

In summary, the present invention provides a method for isolating staphylococcus aureus MVs, and a method for preparing staphylococcus aureus MVs. The invention adopts the international leading process technology without adding chemical stimulating substances, thereby having no adverse effect, simple process flow, high vesicle yield, high efficiency and good amplification effect, and can be used for the mass preparation of vesicles. Compared with normal vesicles, the prepared vesicles have lower endotoxin content, better immunogenicity and wide subsequent further development and application prospects.

Disclosure of Invention

In view of the above, the present invention provides a staphylococcus aureus membrane vesicle.

In order to achieve the purpose, the technical scheme of the invention is as follows:

staphylococcus aureus secretes biological particles that are membrane vesicles isolated from inactivated Staphylococcus aureus.

Inactivated Staphylococcus aureus containing the above biological particles.

Further, the inactivated staphylococcus aureus is combined with the biological particles of the active vaccine and/or vaccine adjuvant and/or drug carrier

The invention also aims to provide a method for separating staphylococcus aureus membrane vesicles.

A method of separating membrane vesicles from staphylococcus aureus comprising the steps of:

1) culturing bacteria to logarithmic phase and fermenting;

2) collecting bacterial liquid, centrifuging the bacterial liquid, removing supernatant, and filtering the supernatant with 0.3-0.5 μ M filter for sterilization;

3) centrifuging the filtered supernatant by using a high-speed centrifuge, collecting the supernatant, and removing flagella;

4) centrifuging the supernatant with the flagella removed by an ultra-high speed centrifuge, and precipitating to obtain the membrane vesicle.

Further, the supernatant of step 2) was sterilized by filtration through a 0.45 μ M filter.

Further, the centrifugation rate of the step 2) is 10000 g; the centrifugation time is 10-60 min.

Preferably, the centrifugation rate is 400-8000 g; the centrifugation time is 10-30 min.

Further, the high-speed centrifugation rate of the step 3) is 5000-25000 g; the centrifugation time is 10-100 min.

Preferably, the high-speed centrifugation rate is 10000-; the centrifugation time is 30-60 min.

Further, the ultra-high speed centrifugation rate of the step 4) is 5000-; the centrifugation time is 60-600 min.

Preferably, the ultra-high speed centrifugation rate is 15000-150000 g; the centrifugation time is 60-180 min.

Further, the staphylococcus aureus is inactivated staphylococcus aureus.

The staphylococcus aureus membrane vesicle is obtained by the separation method.

The invention also aims to provide a preparation method.

A method for preparing the above biological particles, the method comprising the steps of:

1) culturing the bacteria to logarithmic growth phase;

2) fermenting to further enrich the thallus;

3) collecting thallus, re-suspending the thallus with proper amount of phosphate buffer solution or sterile physiological saline and treating with ionizing radiation below the deactivating threshold;

4) collecting the bacteria liquid after irradiation treatment, centrifuging, removing the supernatant, and filtering and sterilizing the supernatant by using a 03-0.5 mu M filter;

5) centrifuging the filtered supernatant by using a high-speed centrifuge, collecting the supernatant, and removing flagella;

6) centrifuging the supernatant with flagella removed at ultra high speed, and precipitating to obtain membrane vesicle.

Further, the OD600 value of the bacteria in the logarithmic growth phase in the step 1) is 0.3-0.8.

Further, it is characterized in that the ratio of the amount of the phosphate buffer or the sterile physiological saline added in the step 3) to the total amount of the bacterial cells is such that the bacterial cell amount per 1ml of the solution is 20 to 80 as the OD600 value.

Further, the ray used for the irradiation treatment in the step 3) is an X-ray; the irradiation dose is 100-2000 Gy. The irradiation dose specifically includes: 200Gy at temperature of 100, 300Gy at temperature of 200, 400Gy at temperature of 300, 500Gy at temperature of 500, 600Gy at temperature of 600, 800Gy at temperature of 700, 900Gy at temperature of 800, 1000Gy at temperature of 900, 1100Gy at temperature of 1000, 1200Gy at temperature of 1100, 1300Gy at temperature of 1200, 1400Gy at temperature of 1300, 1400, 1500Gy at temperature of 1400, 1600Gy at temperature of 1600, 1700Gy at temperature of 1700, 1800Gy at temperature of 1800, 1900Gy at temperature of 1900, 2000Gy at temperature of 1900.

Preferably, the irradiation dose is 500-1000 Gy. The irradiation dose specifically includes: 600Gy at 500, 700Gy at 600, 800Gy at 700, 900Gy at 800, and 1000Gy at 900.

Further, the centrifugation rate in the step 4) is 10000 g; the centrifugation time is 10-60 min.

Further, the supernatant in step 4) was sterilized by filtration using a 0.45. mu.M filter.

Further, the high-speed centrifugation rate in the step 5) is 5000-25000 g; the centrifugation time is 10-100 min.

Further, the ultra-high speed centrifugation speed in the step 6) is 5000-; the centrifugation time is 60-600 min.

The staphylococcus aureus membrane vesicle is prepared by the method.

The method for improving the content of the staphylococcus aureus membrane vesicle comprises nucleic acid and protein, adopts irradiation equipment to treat staphylococcus aureus liquid, and adopts X-ray as the ray of the irradiation equipment, wherein the irradiation dose is 500-1000 Gy-.

A method for reducing the content of endotoxin in a staphylococcus aureus membrane vesicle comprises the step of treating staphylococcus aureus bacteria liquid by adopting irradiation equipment, wherein the ray of the irradiation equipment is an X-ray, and the irradiation dose is 500-1000 Gy.

The invention also aims to provide application of the staphylococcus aureus membrane vesicle and the inactivated bacteria.

The staphylococcus aureus membrane vesicle prepared by the invention is applied to preparation of a vaccine for resisting bacterial infection, and the staphylococcus aureus membrane vesicle can be used as an adjuvant of the vaccine.

Further, the vaccine adjuvant may non-specifically alter or enhance the body's specific immune response to the antigen.

Further, the bacterial infection disease includes pneumonia, urinary tract infection, meningitis, septicemia, or skin and soft tissue infection.

Furthermore, the staphylococcus aureus membrane vesicle can also be used as a carrier of a vaccine.

The staphylococcus aureus membrane vesicle prepared by the invention is applied as immunogen.

The staphylococcus aureus membrane vesicle prepared by the invention is applied as an antigen presenting cell function promoter.

Further, antigen presenting cells include dendritic cells, macrophages, and B cells.

The staphylococcus aureus membrane vesicles prepared by irradiation can stimulate co-stimulatory molecules CD80, CD86 and MHCII on the surface of a DC cell to be remarkably upregulated, and promote maturation and differentiation of the DC cell.

The staphylococcus aureus membrane vesicle prepared by the invention is applied as an accelerant of DC cell antigen presentation capacity.

A composition for promoting proliferation of CD4 and T cells, the composition comprising the staphylococcus aureus membrane vesicles and DC cells described above.

A method for improving the proliferation of CD + T cells comprises the step of co-culturing DCs which are prepared by irradiation and used for stimulating staphylococcus aureus membrane vesicles and phagocytosing OVA antigens and CD4+ T lymphocytes marked by CFSE in vitro.

The invention discloses application of a staphylococcus aureus membrane vesicle in preparation of veterinary drugs.

The inactivated staphylococcus aureus is applied as a bacterial vaccine.

Advantageous effects

The invention adopts ionizing ray X-ray to irradiate staphylococcus aureus for the first time to separate and purify the MVs secreted by the staphylococcus aureus, and the process technology is internationally advanced. Antibiotics and other chemical irritants are not added, so that the bad effects of irritant residues and irritants on the vesicles are avoided. Meanwhile, the process flow is simple and is suitable for industrial amplification and standardized production; the vesicle has high yield, high efficiency, good amplification effect and purification effect, and can be used for the mass preparation of vesicles. The prepared vesicle has lower endotoxin content compared with normal vesicle and better immunogenicity. The bacterial membrane vesicles obtained after the optimization have wide development and application prospects subsequently.

Drawings

FIG. 1 is a graph for measuring the content of Staphylococcus aureus membrane vesicles.

Fig. 2 the membrane vesicles after irradiation treatment promote significant upregulation of the myeloid-derived dendritic cell fine surface molecules CD80, CD86, and MHCII molecules.

FIG. 3 is a bar graph of the phagocytic capacity of antigen-stimulated DC cells following irradiation of treated membrane vesicles.

Figure 4 percentage proliferation of CD4+ T cells after interaction with DC after different treatment modalities.

FIG. 5 flow chart of proliferation of CD4+ T cells after interaction with DCs after different treatment regimes.

FIG. 6 irradiation-treated membrane vesicles enhance interaction of DC cells with T cells (GC: growth control, dendritic Cell growth control (unstimulated group); Cell + MVs (whole Cell + vesicle-treated group); MVs (vesicle-treated group)).

FIG. 7 is a diagram of a Staphylococcus aureus production system.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1

The invention provides a system for separating and preparing staphylococcus aureus membrane vesicles, which is sequentially provided with a fermentation tank, irradiation equipment, ultraviolet spectrophotometry equipment and centrifugal equipment; the radiation generator of the irradiation equipment is an X-ray generator, a gamma-ray generator, or Co ⁶⁰ One or more isotope generators; the centrifugal device comprises one or more of a centrifuge, a high speed centrifuge, and an ultra high speed centrifuge, see fig. 7.

Example 2

The invention provides a method for preparing staphylococcus aureus membrane vesicles, which comprises the following steps:

1) culturing the bacteria to a logarithmic growth phase, wherein the OD600 value of the bacteria in the logarithmic growth phase is 0.3-0.8;

2) fermenting to further enrich the thallus;

3) collecting thallus, and resuspending thallus with appropriate amount of phosphate buffer solution or sterile physiological saline, preferably sterile physiological saline, wherein the ratio of the amount of physiological saline added to the total amount of thallus is that the thallus amount per 1ml solution is OD600 value of 20-80;

4) the X-ray irradiation treatment is carried out by using X-ray lower than an inactivation threshold value, the irradiation dose is 100-2000Gy, and the method specifically comprises the following steps: 200Gy at temperature of 100, 300Gy at temperature of 200, 400Gy at temperature of 300, 500Gy at temperature of 500, 600Gy at temperature of 600, 700Gy at temperature of 700, 900Gy at temperature of 800, 1000Gy at temperature of 900, 1000Gy at temperature of 1000, 1100Gy at temperature of 1100, 1200, 1300Gy at temperature of 1300, 1400Gy at temperature of 1400, 1600Gy at temperature of 1500, 1600Gy at temperature of 1600, 1700Gy at temperature of 1700, 1800Gy at temperature of 1800, 1900Gy at temperature of 1900, 1900Gy at temperature of 2000;

5) centrifuging the irradiated bacterial liquid at a centrifugation rate of 100-10,000 g; the centrifugation time is 10-60 min. Centrifuging the supernatant again by a high-speed centrifuge, collecting the supernatant, and removing flagella; the high-speed centrifugation speed is 5000-25000 g; centrifuging for 10-100 min;

6) centrifuging the supernatant without flagella at a high speed, and precipitating membrane vesicles; the ultra-high speed centrifugation speed is 5000-; the centrifugation time is 60-600 min.

7) The membrane vesicles were collected.

Example 3

The invention provides a method for separating staphylococcus aureus membrane vesicles, which comprises the following steps:

1) culturing bacteria to logarithmic phase and fermenting;

2) collecting bacterial liquid, centrifuging the bacterial liquid, removing supernatant, and filtering the supernatant with 03-0.5 μ M filter for sterilization; 400-8000 g;

centrifuging for 10-30 min;

3) centrifuging the filtered supernatant by using a high-speed centrifuge, collecting the supernatant, and removing flagella; the high-speed centrifugation rate is 10000-20000 gg; centrifuging for 30-60 min;

4) centrifuging the supernatant without flagella by an ultra-high speed centrifuge, and precipitating membrane vesicles; the ultra-high speed centrifugation speed is 15000-; centrifuging for 60-180 min;

5) the membrane vesicles were collected.

Example 4

Preparation of membrane vesicle by ionizing radiation staphylococcus aureus ATCC 25923

1) The staphylococcus aureus ATCC 25923 is recovered from the temperature of minus 80 ℃ and streaked to an LB flat plate, and the culture is carried out in an incubator at the temperature of 37 ℃ for 16-18 h;

2) picking a monoclonal colony from an LB plate, inoculating the colony in 100mL of LB liquid culture medium, and carrying out constant-temperature culture at 37 ℃ and 250rpm for 16-18 h;

3) inoculating overnight bacterial liquid into 2L LB culture medium until the initial concentration is 0.05 OD600/mL, culturing at 37 deg.C and 250rpm until logarithmic phase, and measuring OD600 value;

4) transferring the bacterial liquid 3) into a 2L centrifugal barrel, centrifuging for 20min at 5,000g, collecting thallus, re-suspending with normal saline, adjusting thallus concentration to about 50OD, and taking 100 μ L diluted 107 coated plates to calculate viable count;

5) placing the bacterial liquid in an irradiator with the irradiation dose of 1000 Gy;

6) centrifuging the irradiated bacterial liquid for 20min at 8,000 Xg twice, and collecting the supernatant; filtering the supernatant with 0.45 μ M filter, sterilizing, collecting the supernatant, spreading a small amount of the supernatant on LB plate, and culturing at 37 deg.C for 24-72 hr to confirm no viable bacteria;

7) centrifuging the supernatant obtained in the step 6) by an ultra-high speed centrifuge for 90min at 39,000 Xg, and precipitating MVs;

8) discarding the supernatant, resuspending the precipitate with 2mL MV buffer, and storing at-80 ℃;

9) and (3) measuring the content of the extracted normal and experimental membrane vesicles, including the DNA content, the RNA content and the protein content.

The experimental results are as follows:

the determination result shows that the nucleic acid content and the protein content of the membrane vesicle prepared by the experimental group are obviously improved for staphylococcus aureus ATCC27853, and the ionizing radiation irradiation of gram-positive bacteria can stimulate the generation of the membrane vesicle. See fig. 1.

Example 5

Immunomodulation of irradiated bacterial membrane vesicles-promotion of dendritic cell maturation

Dendritic Cells (DCs) are the main antigen presenting Cells of the body, and their main functions are phagocytosis, processing and processing of antigen molecules, and presentation to T Cells. The cell is a known professional antigen presenting cell with the strongest function and the only function of activating resting T cells in vivo, and is a central link for starting, regulating and maintaining immune response. The maturation of dendritic cells determines the body's development of an immune response or tolerance. Costimulatory molecules B7(B7-1 ═ CD80 and B7-2 ═ CD86) on DCs surfaces can bind to CD28 or CD152 molecules on T cell surfaces, either enhancing or attenuating MHC-TCR signaling between DCs and T cells. The expression of costimulatory molecules CD80 and CD86 is changed, the antigen phagocytosis capacity is weakened, the antigen presentation capacity is improved (the expression of MHCII molecules is increased), and the interaction with T lymphocytes is mainly shown on mature DCs.

1) Mouse bone marrow-derived dendritic cell (BMDC) cell induction culture

Taking a C57 female mouse with 6-8 weeks, aseptically separating the femur of the mouse, cleaning the muscle on the femur, cutting the two ends of the femur, washing the bone lumen with PBS until the femur is whitish, filtering the PBS suspension, separating at 1200rpm for 5min, removing the supernatant, and adding 5ml of erythrocyte lysate for resuspending the cells. After standing for 15min, centrifugation is carried out at 1200rpm for 5min, the supernatant is removed, and 50ml of 1640 complete medium (20 ng/ml GM-CSF, 10% FBS, 50mM 2-mercaptoethanol) is added to resuspend the cells. After mixing uniformly, the mixture is divided into 5 culture dishes to be cultured in an incubator, the culture solution is changed every 2 days, and cells are collected on the 7 th day.

2) BMDC stimulation

Repeatedly blowing BMDC cells for inducing for 7 days into a 6-hole plate to make adherent cells fall off, collecting cell suspension, centrifuging at 1100rpm for 5min, removing supernatant, adding 1ml culture medium to resuspend cells, counting viable cells, and adjusting cell concentration to 1 × 10 ⁶ Per ml, inoculate 2ml to new6 well plates. Adding each stimulant and mixing evenly, respectively: whole bacteria, whole bacteria + vesicles and vesicles were added to a final concentration of 15. mu.g/mL (protein standard), incubation was continued for 24 hours, and an equal volume of PBS was added to the growth control.

3) Flow type detection mature marker

And taking out the 6-hole plate after 24h, repeatedly blowing cells to make the cells fall off, collecting cell suspension to a flow tube, centrifuging at 1500rpm for 3min, removing supernatant, adding 1ml of PBS, continuing to centrifuge at 1500rpm for 3min, removing supernatant, and repeatedly cleaning for 3 times. Adding CD11c/CD80/CD86/MHCII antibody, incubating for 30min at room temperature in the dark, and simultaneously setting up an isotype control group as a negative control group and adding a peer control group of CD11c/CD80/CD 86/MHCII. After incubation was completed, PBS was added to wash 2 times, 200. mu.l PBS was added to resuspend the cells, and detection was performed by flow cytometry.

4) Result processing

Flow cytometry software analyzed the CD80/CD86/MHCII ratio in CD11c cells.

The experimental results are as follows:

compared with whole thallus, the X-ray treated experimental group (MVs) vesicles can obviously up-regulate DCs surface co-stimulatory molecules CD80, CD86, MHCII and the like after stimulation, and the surface molecules are markers of dendritic cell maturation. Taken together, vesicles were shown to significantly promote differentiation and maturation of DCs. See fig. 2.

Example 6

Phagocytic capacity of DC cells was examined by measuring FITC-labeled dextran fluorescence intensity

The DC cells have extremely strong antigen endocytosis and processing capacity. The test determines the amount of phagocytic glucan of the DC by detecting the fluorescence intensity of FITC labeled glucan so as to detect whether the phagocytic capacity of the DC is enhanced or not.

1) BMDC cell induction culture

2) Stimulation of

Cells were harvested on day 7, blown down and centrifuged to resuspend and count, and plated into 6-well plates 1 x 10 cells per well ⁶ The cells were cultured at 37 ℃ for 24 hours after adding the same volume of PBS, Control and Treatment to the GC group and the membrane vesicles (protein level) to the GC group, respectively.

3) Phagocytosis and detection

Dextran (5. mu.g/ml) is added, after the culture is continued for 1h, the cells are sucked out to a flow tube, after 3 times of PBS washing, CD11c antibody is added, and after the incubation is carried out for 30min in the dark at room temperature, the FITC fluorescence is detected in a flow mode after 3 times of PBS washing.

4) Result processing

Flow cytometry software analyzed the FITC ratio in CD11c cells.

Experimental data:

to examine the phagocytic function of DCs, we used FITC-dextran as a model antigen for DCs phagocytosis and measured the FITC mean fluorescence intensity value of CD11c + DCs. As a result of the experiment, dendritic cells (growth control group) of GC group had substantially no FITC-dextran uptake, but the FITC mean fluorescence intensity values were significantly reduced compared to those of GC group regardless of whether DCs were stimulated. This experimental result again demonstrates that vesicles can promote maturation of DCs, thereby reducing the uptake capacity of antigen. See fig. 3.

Example 7

Mature DCs after X-ray treatment with bacterial membrane vesicle stimulation interact with T cells:

A. mature DCs interact with CD4+ T cells

Effective cross-antigen presentation of extracellular proteins by DCs plays an important role in inducing specific cellular immune responses. Thus, cross-presentation of OVA antigen by DCs following vesicle stimulation was examined. At 72h after DCs-T cell co-culture, we examined the proliferation of OT-II CD4+ T lymphocytes by CFSE flow cytometry. The fluorescent dye CFSE (CFDA-SE), namely hydroxy fluorescein diacetate succinimide ester, is a cell staining reagent which can carry out fluorescent labeling on living cells. It can be coupled to a cell protein irreversibly by binding to an amino group in the cell after entering the cell. During cell division and proliferation, CFSE marker fluorescence can be equally distributed to two daughter cells, with half the intensity of the parent cell. Therefore, we can use flow cytometry to count the percentage of cells with weak CFSE fluorescence, and thus obtain the proportion of proliferating cells.

1. BMDC cell induction culture

Same as in the maturation experiment.

2. Antigen phagocytosis

DCs cultured for 7 days were cultured in OVA medium containing 10. mu.g/ml for 24 hours as GC (growth control), and in MVs group, vesicles were additionally added, followed by centrifugation to collect antigen-phagocytized DCs and resuspension in normal medium at 2X 10 ⁴ The density of cells/well was plated in 96-well plates, 100. mu.l per well, 3 replicates per group.

3. T cell extraction

On the next day, OT-II mice spleen OVA-specific CD4+ T lymphocytes were isolated and enriched using the Stem Cell Technologies negative magnetic bead screening kit.

4. Co-culture of DC and T cells

Selected CD4+ T cells were labeled with 1 μ M CFSE according to the kit instructions. After labeling, wash 3 times with PBS at 10 ⁵ The density of cells/well was added to a 96-well plate to give a final culture volume of 200. mu.l (CD4: DC: 5: 1).

5. On day 3 after co-culture, proliferation of the CD4+ T cell population was detected by CFSE depletion using flow cytometry.

DCs that vesicle-stimulated and phagocytized OVA antigen were co-cultured in vitro with CFSE-labeled OT-II mouse CD4+ T lymphocytes. Flow analysis of CFSE fluorescence intensity results indicated an increased proportion of proliferating CD4+ T cells. As shown, vesicles (14.05%) significantly increased proliferation of specific CD4+ T cells by DCs phagocytosing OVA antigens (6.80%). See fig. 4, 5 for details.

B. Vesicle-treated DCs promote T cell proliferation

Efficient cross-antigen presentation of extracellular proteins by DCs plays an important role in inducing specific cellular immune responses. Thus, cross-presentation of OVA antigen by DCs following vesicle stimulation was examined. At 72h after DCs-T cell co-culture, we examined T lymphocyte proliferation by CFSE flow cytometry. The fluorescent dye CFSE (CFDA-SE), namely hydroxyfluorescein diacetate succinimide ester, is a cell staining reagent capable of carrying out fluorescent labeling on living cells. It can be coupled to a cell protein irreversibly by binding to an amino group in the cell after entering the cell. During cell division and proliferation, CFSE marker fluorescence can be equally distributed to two daughter cells, with half the intensity of the parent cell. Therefore, we can use flow cytometry to count the percentage of cells with weak CFSE fluorescence, and thus obtain the proportion of proliferating cells.

1. BMDC cell induction culture

Same as in the maturation experiment.

2. Antigen phagocytosis

DCs cultured for 7 days were cultured in a medium for 24 hours as GC (growth control), and the MVs group was separately added with vesicles, followed by centrifugation to collect antigen-phagocytosed DCs and resuspension in a normal medium at 4X 10 ⁴ Cell/well density was plated in 96-well plates at 100. mu.l per well, 3 replicates per group.

3. T cell extraction

On the following day, T cells enriched in mice were isolated using a negative magnetic bead screening kit from mouse spleen Stem Cell Technologies one week after one immunization with MVs.

4. Co-culture of DCs with T cells

Selected T cells were labeled with 1 μ M CFSE according to kit instructions. After labeling, wash 3 times with PBS at 4X 10 ⁵ The density of cells/well was added to a 96-well plate to make the final culture volume 200. mu.l (CD3: DC ═ 10: 1).

5. On day 3 after co-culture, proliferation of CD3+, CD8+, CD4+ T cell populations was detected by CFSE depletion using flow cytometry.

The experimental results are as follows:

DCs that vesicle-stimulated and phagocytized OVA antigen were co-cultured in vitro with CFSE-labeled OT-II mouse CD4+ T lymphocytes. Flow analysis CFSE fluorescence intensity results indicated an increased proportion of proliferating CD4+ T cells. As shown in the figure, the fluorescence intensity of the bacteria and vesicle stimulation group was 63.5%, and that of the vesicle stimulation group was 71%. Indicating that DCs after vesicle processing can remarkably stimulate the proliferation of CD4+ T cells. See fig. 6.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. A method of making a staphylococcus aureus membrane vesicle, comprising the steps of:

1) culturing the bacteria to logarithmic growth phase;

2) fermenting to further enrich the thallus;

3) collecting thalli, resuspending the thalli by using a proper amount of phosphate buffer solution or sterile normal saline, and irradiating the thalli by using X-ray with the irradiation dose of 100-2000 Gy;

4) collecting the bacteria liquid after irradiation treatment, centrifuging, removing the supernatant, and filtering the supernatant with a 0.3-0.5 μ M filter for sterilization;

6) and centrifuging the supernatant without flagellum at a high speed, and precipitating to obtain the membrane vesicle vaccine.

2. The method as claimed in claim 1, wherein the OD600 value of the bacteria in the logarithmic growth phase in step 1) is 0.3-0.8.

3. The method according to claim 1, wherein the ratio of the amount of the phosphate buffer or the sterile physiological saline added in step 3) to the total amount of the bacterial cells is such that the OD600 value of the bacterial cell content per 1ml of the solution is 20 to 80.

4. The method according to claim 1, wherein the centrifugation rate in step 4) is 100-10000 g; the centrifugation time is 10-60 min.

5. The method of claim 1, wherein the supernatant of step 4) is sterile filtered through a 0.45 μ M filter.

6. The method as claimed in claim 1, wherein the high-speed centrifugation rate in step 5) is 5000-25000 g; the centrifugation time is 10-100 min.

7. The method as claimed in claim 1, wherein the ultra high speed centrifugation rate in step 6) is 5000-150000 g; the centrifugation time is 60-600 min.

8. Staphylococcus aureus membrane vesicle produced according to any one of claims 1-7, having an increased nucleic acid and protein content.

9. The method for improving the content of the staphylococcus aureus membrane vesicle comprises nucleic acid and protein and is characterized in that irradiation equipment is adopted to treat staphylococcus aureus liquid, the radiation of the irradiation equipment is X-ray, and the irradiation dose is 1000 Gy.

10. The method for reducing the content of endotoxin in a staphylococcus aureus membrane vesicle is characterized in that irradiation equipment is adopted to treat staphylococcus aureus liquid, the ray of the irradiation equipment is an X-ray, and the irradiation dose is 500-1000 Gy-.

11. The use of staphylococcus aureus membrane vesicles as in claim 8 in the preparation of a vaccine against bacterial infection, wherein the staphylococcus aureus membrane vesicles are used as an adjuvant for the vaccine.

12. The use according to claim 11, wherein the vaccine adjuvant can non-specifically alter or enhance the body's specific immune response to an antigen.

13. The use of claim 11, wherein the bacterial infection disease comprises pneumonia, urinary tract infection, meningitis, sepsis, or skin and soft tissue infection.

14. The use according to claim 11, wherein the s.aureus membrane vesicles are also used as carriers for vaccines.

15. Use of a staphylococcus aureus membrane vesicle according to claim 8 as an immunogen.

16. The use of the staphylococcus aureus membrane vesicle of claim 8 as an antigen-presenting cell function promoter, wherein the antigen-presenting cell is a dendritic cell.

17. The use of the staphylococcus aureus membrane vesicle in preparing a DC cell growth promoter according to claim 8, wherein the staphylococcus aureus membrane vesicle can stimulate the co-stimulatory molecules CD80, CD86 and MHCII on the surface of a DC cell to be significantly up-regulated, and promote the maturation and differentiation of the DC cell.

18. Use of the staphylococcus aureus membrane vesicles as defined in claim 8 as an agent for promoting antigen presentation in DC cells.

19. A method for increasing the proliferation of CD + T cells, wherein DCs that stimulate and phagocytose OVA antigens using the Staphylococcus aureus membrane vesicles of claim 8 are co-cultured in vitro with CFSE-labeled CD4+ T lymphocytes.