CN112410240B

CN112410240B - Pseudomonas aeruginosa membrane vesicle and preparation method and application thereof

Info

Publication number: CN112410240B
Application number: CN201910777606.9A
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: 2022-10-18
Anticipated expiration: 2039-08-22
Also published as: CN112410240A

Abstract

The invention discloses a pseudomonas aeruginosa membrane vesicle and a preparation method and application thereof, belongs to the field of microbiology, and provides a membrane vesicle separated from inactivated pseudomonas aeruginosa. 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 radiation X-ray to irradiate pseudomonas aeruginosa, separates and purifies MVs secreted by the pseudomonas aeruginosa, and the prepared MVs can be used as vaccines, vaccine adjuvants and drug carriers.

Description

Pseudomonas aeruginosa membrane vesicle and preparation method and application thereof

Technical Field

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

Background

Membrane Vesicles (MVs) are vesicular structural products 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 bacteria can be promoted to generate the membrane vesicles 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 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 of the vesicle.

In summary, the present invention provides a method for isolating Pseudomonas aeruginosa MVs, and a method for preparing Pseudomonas aeruginosa 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 and better immunogenicity, and have wide subsequent further development and application prospects.

Disclosure of Invention

In view of the above, the present invention provides a pseudomonas aeruginosa membrane vesicle.

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

A pseudomonas aeruginosa vaccine comprising membrane vesicles isolated from inactivated pseudomonas aeruginosa.

Inactivated pseudomonas aeruginosa containing the separated membrane vesicles, and a composition formed by the separated membrane vesicles and the inactivated pseudomonas aeruginosa.

Further, the inactivated pseudomonas aeruginosa is combined with the vaccine and/or vaccine adjuvant and/or membrane vesicle of the drug carrier.

The invention also aims to provide a method for separating the pseudomonas aeruginosa membrane vesicles.

A method for separating membrane vesicles from pseudomonas aeruginosa, comprising the steps of:

1) Culturing bacteria to logarithmic growth phase and then fermenting;

2) Collecting bacterial liquid, centrifuging the bacterial liquid and collecting supernatant, and filtering and sterilizing the supernatant by using a 0.3-0.5 mu M filter;

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 speed of the step 2) is 100-10000g; the centrifugation time is 10-60min.

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

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

Preferably, the high-speed centrifugation speed is 10000-20000g; the centrifugation time is 30-60min.

Further, the ultra-high speed centrifugation speed of the step 4) is 5000-150000g; the centrifugation time is 60-600min.

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

Further, the pseudomonas aeruginosa is inactivated pseudomonas aeruginosa.

The pseudomonas aeruginosa membrane vesicle is obtained by adopting the separation method.

The invention also aims to provide a preparation method.

A process for preparing the pseudomonas aeruginosa vaccine described above, the process comprising the steps of:

1) Culturing the bacteria to logarithmic growth phase;

2) Fermenting to further enrich the thalli;

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 irradiated bacteria liquid, centrifuging by a centrifuge and collecting the supernatant, filtering the supernatant by a 0.3-0.5 mu M filter for sterilization;

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

6) Centrifuging the supernatant with the flagella removed by an ultra-high speed centrifuge, and precipitating to obtain the 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 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.

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

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

Further, the centrifugal rate in the step 4) is 100-10000g; the centrifugation time is 10-60min.

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 5000-25000g; the centrifugation time is 10-100min.

Further, the ultra-high speed centrifugation speed in the step 6) is 5000-150000g; the centrifugation time is 60-600min.

The pseudomonas aeruginosa membrane vesicle and the vaccine prepared by the method are adopted.

Furthermore, compared with bacterial membrane vesicles which are not irradiated by ionizing rays, the content of nucleic acid and the content of protein in the pseudomonas aeruginosa membrane vesicles are increased by 10-20 times.

The method for improving the content of the pseudomonas aeruginosa membrane vesicle contents comprises nucleic acid and protein, adopts irradiation equipment to treat pseudomonas aeruginosa bacteria liquid, and adopts X-ray radiation with the irradiation dose of 500-1000Gy.

A method for reducing the content of endotoxin in pseudomonas aeruginosa membrane vesicles adopts irradiation equipment to treat pseudomonas aeruginosa bacterial liquid, the rays of the irradiation equipment are X-ray, and the irradiation dose is 500-1000Gy.

The invention also aims to provide an application of the pseudomonas aeruginosa membrane vesicle and inactivated pseudomonas aeruginosa.

The pseudomonas aeruginosa membrane vesicle prepared by the invention is applied to the preparation of a vaccine for resisting bacterial infection, and 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 pseudomonas aeruginosa membrane vesicle can also be used as a carrier of a vaccine.

The pseudomonas aeruginosa membrane vesicle prepared by the invention is applied as immunogen.

The pseudomonas aeruginosa membrane vesicle prepared by the invention is used as an antigen presenting cell function promoter.

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

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

The pseudomonas aeruginosa membrane vesicle prepared by the invention is applied as a DC cell antigen presentation capability promoter.

A method for improving the proliferation of CD + T cells is characterized in that DCs which are prepared by irradiation and stimulate pseudomonas aeruginosa membrane vesicles and phagocytose OVA antigens and CD4+ T lymphocytes marked by CFSE are co-cultured in vitro.

The pseudomonas aeruginosa membrane vesicle prepared by the invention is applied to preparation of veterinary drugs.

The invention relates to application of inactivated pseudomonas aeruginosa as a bacterial vaccine.

Advantageous effects

The invention adopts ionizing radiation X-ray to irradiate the pseudomonas aeruginosa for the first time to separate and purify MVs secreted by the pseudomonas aeruginosa, and the process technology is internationally advanced. Antibiotics and other chemical irritants are not added, so that the adverse effects of irritant residues and irritants on membrane 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. Compared with normal vesicles, the prepared vesicles have the advantages that the yield is improved by tens of times, and the prepared vesicles have better immunogenicity. The bacterial membrane vesicles obtained after the optimization have wide development and application prospects subsequently.

Drawings

FIG. 1 is a transmission electron microscope image of Pseudomonas aeruginosa membrane vesicles (A: control membrane vesicles; B: irradiated membrane vesicles; scale bar: 200 nm).

FIG. 2 is a diagram for measuring the content of the pseudomonsa aeruginosa membrane vesicle.

FIG. 3 is a diagram showing the particle size distribution of the Pseudomonas aeruginosa membrane vesicle.

FIG. 4 shows that the membrane vesicles after irradiation treatment promote the marked up-regulation of the marrow-derived dendritic cell surface molecules CD80, CD86 and MHCII.

FIG. 5 bar graph of phagocytic capacity of antigen-stimulated DC cells following irradiation treatment of membrane vesicles.

Figure 6 percentage proliferation of CD4+ T cells after interaction with DCs after different treatment regimes.

FIG. 7 flow charts of proliferation of CD4+ T cells after interaction with DCs following different treatment regimes.

FIG. 8 irradiation treated membrane vesicles enhance the 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. 9 is a system for producing P.aeruginosa membrane vesicles.

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 pseudomonas aeruginosa 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 kind of isotope generator orSeveral kinds of the raw materials; the centrifugal device comprises one or more of a centrifuge, a high speed centrifuge, and an ultra high speed centrifuge, as shown in fig. 9.

Example 2

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

1) Culturing the bacteria to logarithmic growth phase, wherein the OD600 value of the bacteria in the logarithmic growth phase is 0.3-0.8; preferably, the OD600 value is selected to be 0.5-0.8;

2) Fermenting to further enrich the thalli;

3) Collecting thalli, resuspending the thalli with a proper amount of phosphate buffer solution and irradiating the thalli with ionizing rays lower than an inactivation threshold value; the ratio of the amount of the added phosphate buffer solution to the total amount of the thallus is that the bacterial amount contained in each 1ml of solution is 20-80 of OD600 value; preferably, the OD600 value is selected to be 40-60;

4) Irradiating with X-ray below the inactivation threshold at an irradiation dose of 100-2000Gy, which comprises: 100-200 Gy,200-300 Gy,300-400 Gy,400-500 Gy,500-600 Gy,600-700 Gy,700-800 Gy,800-900 Gy,900-1000 Gy,1000-1100 Gy,1100-1200 Gy,1200-1300 Gy,1300-1400 Gy,1400-1500 Gy,1500-1600 Gy,1600-1700 Gy,1700-1800 Gy,1800-1900 Gy,1900-2000 Gy;

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

6) Centrifuging the supernatant without flagella at a high speed, and precipitating membrane vesicles; the ultra-high speed centrifugation rate is 5000-150000g; centrifuging for 60-600min;

7) The membrane vesicles were collected.

Example 3

The invention provides a method for separating and purifying pseudomonas aeruginosa membrane vesicles, which comprises the following steps:

1) Culturing bacteria to logarithmic phase and fermenting;

2) Collecting bacterial liquid, centrifuging the bacterial liquid and collecting supernatant, and filtering and sterilizing the supernatant by using a 0.45 mu M filter; 400-8000g; centrifuging for 10-30min;

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

4) Centrifuging the supernatant without flagella by an ultra-high speed centrifuge, and precipitating membrane vesicles; the ultra-high speed centrifugation rate is 15000-150000g; centrifuging for 60-180min;

5) The membrane vesicles were collected.

Example 4

Preparation, separation and purification of membrane vesicles by ionizing ray irradiation of pseudomonas aeruginosa PAO1

1) Recovering pseudomonas aeruginosa PAO1 from minus 80 ℃ and drawing the pseudomonas aeruginosa PAO1 to an LB plate, and culturing the pseudomonas aeruginosa PAO1 in an incubator at 37 ℃ for 16 to 18 hours;

2) Picking a monoclonal colony from an LB plate, inoculating the colony in 20mL of LB liquid culture medium, and culturing at 37 ℃ and 250 rpm for 16-18 h at constant temperature;

3) Inoculating overnight bacterial liquid into 1L LB culture medium until the initial concentration is 0.05 OD600/mL, culturing at 37 deg.C and 250 rpm until logarithmic phase, and measuring OD600 value;

4) Transferring the bacterial liquid obtained in the step 3) to a centrifugal barrel, centrifuging for 20min at 5,000 g, collecting thalli, re-suspending with physiological saline, and adjusting the concentration of the thalli to about 50 OD;

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

6) Centrifuging the irradiated bacterium liquid for 20min at 8,000 Xg twice, and collecting 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 a high-speed centrifuge, and removing flagella in the supernatant;

8) Centrifuging the supernatant obtained in the step 7) by using an ultra-high speed centrifuge, and precipitating membrane vesicles;

9) Discarding the supernatant, resuspending the precipitate with MV buffer, and storing at-80 deg.C;

10 The extracted normal and experimental membrane vesicles of the present invention were subjected to transmission electron microscopy. And simultaneously measuring the content of the extracted normal and experimental membrane vesicles, including DNA content, RNA content and protein content. And finally, measuring the particle sizes of the extracted normal membrane vesicles and the experimental membrane vesicles.

The experimental results are as follows:

according to electron microscope results, ionizing rays can stimulate pseudomonas aeruginosa PAO1 to secrete membrane vesicles, and the number of experimental group membrane vesicles is large; the vesicle shape and size are not obviously different from those of a normal control group. See fig. 1.

Content measurement results: compared with a normal control group, the nucleic acid content and the protein content of the membrane vesicle prepared by the experimental group are improved by 10 to 20 times. See fig. 2. The specific measurement data are shown in table 1.

TABLE 1 determination of content of Membrane vesicle

Irradiation dose Gy	DNA ng/μL	RNA ng/μL	Protein μ g/mL	Endotoxin (EU/ml)
					-	24.8	19.7	262.7	1.28×10 ⁵

980

469.0

364.0

4551.0

1.07×10 ⁶

The data of the particle size distribution diagram show that the sizes of the membrane vesicles prepared by the normal control group and the experimental group have no obvious difference, and the average size is about 150 nm. See fig. 3. The specific measurement data are shown in Table 2.

TABLE 2 particle size

Irradiation dose Gy	Average particle diameter (nm)	Peak(nm)
			-	152.2	192.6
980	146.5	222.9

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. Is a known professional antigen presenting cell with the strongest function and the only ability to activate 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 = CD 86) on the surface of DCs can bind to CD28 or CD152 molecules on the surface of T cells, either enhancing or attenuating MHC-TCR signaling between DCs and T cells. The primary manifestations of the mature DCs include altered expression of the costimulatory molecules CD80 and CD86, reduced ability to phagocytose and process presented antigens, and interaction with T lymphocytes.

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

Taking a C57 female mouse with 6-8 weeks, carrying out aseptic separation on femurs of the mouse, removing muscles on the femurs, cutting two ends of the femurs off, washing bone tube cavities by PBS until the femurs are white, filtering PBS suspension, separating at 1200rpm for 5min, removing supernatant, and adding 5ml of erythrocyte lysate for resuspending cells. After standing for 15min, centrifugation at 1200rpm for 5min, supernatant was removed, and cells were resuspended by adding 50ml of 1640 complete medium (20 ng/ml GM-CSF, 10% FBS, 50mM 2-mercaptoethanol). 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 1100 rpm for 5min, removing supernatant, adding 1ml culture medium to resuspend cells, counting viable cells, and adjusting cell concentration to 1 × 10 ⁶ Perml, inoculate 2ml into a new 6-well plate. Adding each stimulant and mixing evenly, respectively: whole bacteria, whole bacteria + vesicles and vesiclesTo a final concentration of 15. Mu.g/mL (protein basis), 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 enable the cells to fall off, collecting cell suspension to a flow tube, centrifuging at 1500 rpm for 3 min, removing supernatant, adding 1ml of PBS, continuing to centrifuge at 1500 rpm for 3 min, 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 establishing an isotype control group as a negative control group and adding a cocurrent control 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 the 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. In conclusion, the vesicle can obviously promote the differentiation and maturation of the DCs. See fig. 4.

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 at 1 × 10 cells per well ⁶ Adding stimulators into each cell, adding equal volume of P into GC groupBS, control and Treatment groups were incubated at 37 ℃ for 24h after addition of the same concentration of membrane vesicles (at the protein level).

3) Phagocytosis and detection

Dextran (5. Mu.g/ml) is added, the culture is continued for 1 h, the cells are sucked out to a flow tube, the cells are washed for 3 times by PBS, then the CD11c antibody is added, the cells are incubated for 30min in a dark place at room temperature, and the FITC fluorescence is detected by flow after the cells are washed for 3 times by PBS.

4) Result processing

Flow cytometry software analyzed the proportion of FITC 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, the dendritic cells (growth control group) of the GC group had substantially no FITC-dextran uptake, but the FITC mean fluorescence intensity values were significantly reduced compared to the GC group regardless of the DCs after stimulation. The results of this experiment again demonstrate that vesicles can promote maturation of DCs, thereby reducing the uptake capacity of antigen. See fig. 5.

Example 7

Mature DCs after stimulation of X-ray treatment group bacterial membrane vesicles interact with T cells:

A. mature DC interacting with CD4+ T cells

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 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 proliferation, CFSE labeled fluorescence can be equally distributed to two daughter cells with half the intensity of fluorescence as 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 the MVs group was separately added with vesicles, followed by centrifugation to collect antigen-phagocytosed DCs and resuspension in normal medium at 2X 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 next day, OT-II mouse spleen OVA specific CD4+ T lymphocytes were isolated and enriched using a negative magnetic bead screening kit from Stem Cell Technologies, inc.

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 make the final culture volume 200 μ l (CD 4: DC = 5.

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 phagocytosed 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. 6 and 7 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 hydroxy fluorescein diacetate succinimide ester, is a cell staining reagent which can carry out fluorescent labeling on living cells. After entering cells, the protein can be irreversibly combined with amino groups in the cells and coupled to cell proteins. During cell division proliferation, CFSE labeled fluorescence can be equally distributed to two daughter cells with half the intensity of fluorescence as 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 DC and 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 μ l (CD 3: DC = 10.

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 of CFSE fluorescence intensity results indicated an increased proportion of proliferating CD4+ T cells. As shown in the figure, the fluorescence intensity of the cell and vesicle stimulation group was 63.5%, and the fluorescence intensity of the vesicle stimulation group was 71%. Indicating that DCs after vesicle treatment can remarkably stimulate the proliferation of CD4+ T cells. See fig. 8.

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 process for preparing pseudomonas aeruginosa membrane vesicles, comprising the steps of:

culturing the bacteria to logarithmic growth phase;

fermenting to further enrich the thallus;

collecting thalli, resuspending the thalli with a proper amount of phosphate buffer solution or sterile normal saline, and irradiating with X-ray lower than an inactivation threshold value, wherein the irradiation dose is 980-1000Gy;

centrifuging the collected bacteria liquid after irradiation treatment by using a centrifuge, collecting supernatant, and filtering and sterilizing the supernatant by using a 0.3-0.5 mu M filter;

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

centrifuging the supernatant fluid after flagellum removal by using an ultrahigh speed machine, and precipitating to obtain the membrane vesicle; compared with bacterial membrane vesicles which are not irradiated by X-ray, the content of nucleic acid and the content of protein in the membrane vesicles are improved by 10-20 times.

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 100-10000g; the centrifugation time is 10-60min.

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 according to claim 1, wherein the high speed centrifugation rate in step 5) is 5000-25000g; the centrifugation time is 10-100min.

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

8. Pseudomonas aeruginosa membrane vesicles obtainable by any one of the processes of claims 1 to 7.

9. The method for improving the content of the pseudomonas aeruginosa membrane vesicle contents comprises nucleic acid and protein and is characterized in that irradiation equipment is adopted to treat pseudomonas aeruginosa bacterial liquid, the rays of the irradiation equipment are X-ray rays, and the irradiation dose is 980-1000Gy.

10. The method for reducing the endotoxin content in the pseudomonas aeruginosa membrane vesicle is characterized in that irradiation equipment is adopted to treat pseudomonas aeruginosa bacterial liquid, the radiation of the irradiation equipment is X-ray, and the irradiation dose is 980-1000Gy.

11. The use of the pseudomonas aeruginosa membrane vesicles as claimed in claim 8 in the preparation of a vaccine against bacterial infection, wherein the pseudomonas aeruginosa membrane vesicles are useful as an adjuvant for a 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 pseudomonas aeruginosa membrane vesicles are also useful as carriers for vaccines.

15. Use of the pseudomonas aeruginosa membrane vesicle of claim 8 in the preparation of an immunogen.

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

17. The use of the Pseudomonas aeruginosa membrane vesicle of claim 8 in the preparation of an accelerant of DC cell antigen presentation ability.

18. A method for increasing the proliferation of CD + T cells, wherein DCs which are stimulated by the Pseudomonas aeruginosa membrane vesicles and phagocytose OVA antigens are co-cultured with CD4+ T lymphocytes in vitro.