Pseudomonas aeruginosa hyperproducing membrane vesicles, methods of preparation and uses

Abstract

The present application belongs to the field of biological science and technology, and particularly relates to Pseudomonas aeruginosa over-production membrane vesicles, a preparation method and application. prtN and prtR The present application first proposes that a Pseudomonas aeruginosa double-deletion strain is subjected to X-ray irradiation, so that the yield of outer-inner membrane vesicles can be significantly improved, and over-production membrane vesicles are obtained. The over-production membrane vesicles have great potential and medical value for preparation as vaccines.

Description

[0001] Priority application This application claims priority to PCT international application filed July 16, 2025 [PCT / CN2025 / 108793], “Supermembrane vesicle production, preparation method and application of Pseudomonas aeruginosa”, and PCT international application filed July 16, 2025 [PCT / CN2025 / 108808], “A novel mechanism of outer-inner membrane vesicle biogenesis in Pseudomonas aeruginosa "Under X-ray irradiation and its potential as a vaccine against acute pneumonia", two priority invention patent applications are incorporated by reference. Technical Field

[0002] This invention belongs to the field of bioscience and technology, specifically relating to the hyperproduced membrane vesicles of Pseudomonas aeruginosa, their preparation methods, and applications. Background Technology

[0003] Bacterial membrane vesicles (MVs) are nanoscale, spherical lipid bilayer structures released by bacteria during growth or stress. Most known bacteria can produce membrane vesicles with diameters ranging from 20 to 400 nanometers. MVs play a crucial role in various biological processes and have significant potential for biomedical applications. Studies have shown that membrane vesicles produced by Gram-negative bacteria are generally referred to as outer membrane vesicles (OMVs). However, some Gram-negative bacteria produce outer-inner membrane vesicles (OIMVs) containing cytoplasmic proteins and DNA. The formation mechanism of OIMVs remains poorly understood. One hypothesis suggests that ruptured cell membranes can trap cytoplasmic material released into the extracellular space and self-assemble to form OIMVs and explosive outer membrane vesicles; this view is supported by evidence of explosive cell lysis. However, reports on the artificial preparation of OIMVs are scarce.

[0004] Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that can cause serious infections such as pneumonia. Due to the increasing prominence of multidrug resistance, the development of effective vaccines against Pseudomonas aeruginosa is of great significance. Previous studies have shown that X-ray irradiation can promote the secretion of membrane vesicles by bacteria. For example, publication number CN112410240A, titled "Pseudomonas aeruginosa membrane vesicles and their preparation methods and applications," discloses the use of ionizing X-rays to irradiate Pseudomonas aeruginosa, isolate and purify its secreted vesicles (MVs), and the obtained MVs can be used as vaccines, vaccine adjuvants, and drug carriers. However, research on the production of outer-inner membrane vesicles by wild-type Pseudomonas aeruginosa under X-ray irradiation conditions has not yet been reported.

[0005] In summary, it is necessary to propose a new technology for the superproduction of membrane vesicles by Pseudomonas aeruginosa to supplement the shortcomings of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Pseudomonas aeruginosa hyperproduced membrane vesicles and its application, thereby partially solving or alleviating the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0007] A strain that produces hypermembrane vesicles, wherein the hypermembrane vesicle-producing strain is a *Pseudomonas aeruginosa* strain with double gene deletions; the *Pseudomonas aeruginosa* strain with double gene deletions is... prtN and prtR A *Pseudomonas aeruginosa* strain with double gene deletion; the supermembrane-producing vesicles are *Pseudomonas aeruginosa* outer-inner membrane vesicles. A method for producing superproductive membrane vesicles, the method comprising irradiating a *Pseudomonas aeruginosa* strain with double gene deletions; wherein the *Pseudomonas aeruginosa* strain with double gene deletions is... prtN and prtR A *Pseudomonas aeruginosa* strain with double gene deletion; the supermembrane-producing vesicles are *Pseudomonas aeruginosa* outer-inner membrane vesicles.

[0008] Furthermore, the superproductive membrane vesicles are outer-inner membrane vesicles with a double membrane structure; the outer-inner membrane vesicles are formed by the outward bubbling of the inner membrane of the Pseudomonas aeruginosa double-deleted gene strain induced by radiation irradiation, resulting in the extrusion and shedding of the outer membrane.

[0009] Furthermore, the radiation irradiation is X-ray irradiation.

[0010] Preferably, the X-ray irradiation dose rate is 7-8 Gy / min, and the total irradiation dose is 500-1000 Gy.

[0011] More preferably, the dose rate of the X-ray irradiation is 7-8 Gy / min, and the total irradiation dose is 800-1000 Gy.

[0012] Furthermore, the method includes the following steps: S01: Knockout of Pseudomonas aeruginosa prtN and prtR Genetic preparation of a Pseudomonas aeruginosa double-deleted strain; S02: Superproductive membrane vesicles were obtained by treating a Pseudomonas aeruginosa strain with double gene deletions using X-ray irradiation.

[0013] Application of the superproductive membrane vesicles prepared by the above method in vaccine preparation.

[0014] Furthermore, the vaccine is used to prevent or treat infections caused by Pseudomonas aeruginosa.

[0015] Furthermore, the infection includes lung infections caused by Pseudomonas aeruginosa.

[0016] Application of a *Pseudomonas aeruginosa* double-deleted gene strain in vaccine preparation, wherein the *Pseudomonas aeruginosa* double-deleted gene strain is... prtN and prtR A double-deleted Pseudomonas aeruginosa strain (the double-deleted Pseudomonas aeruginosa strain can produce Pseudomonas aeruginosa outer-inner membrane vesicles); the vaccine is used to prevent or treat infections caused by Pseudomonas aeruginosa.

[0017] Furthermore, the infection includes lung infections caused by Pseudomonas aeruginosa.

[0018] Beneficial technical effects: (1) This invention is the first to propose a method for treating Pseudomonas aeruginosa. prtN and prtR X-ray irradiation of double-deleted gene strains can significantly increase the yield of membrane vesicles, resulting in hyperproduced membrane vesicles. These hyperproduced membrane vesicles have great potential and medical value for vaccine preparation.

[0019] (2) The results of this invention reveal that blistering of the inner membrane of Pseudomonas aeruginosa PAO1 under X-ray irradiation is a new mechanism for the occurrence of OIMVs. Using transmission electron microscopy, this invention demonstrates that X-ray irradiation induces blistering of the inner membrane of Pseudomonas aeruginosa PAO1, leading to the extrusion and shedding of the outer membrane to form OIMVs. It was also found that this process is precisely regulated by both PrtR and PrtN.

[0020] (3) In a mouse model of acute pneumonia, immunization with OIMVs induced a strong protective immune response, significantly improving the clearance rate of bacteria in the lungs, reducing lung injury, and increasing survival. This immunization not only provides strong protection against PAO1 strains, but also effectively reduces the bacterial load in the lungs caused by heterologous Pseudomonas aeruginosa serotype infection and improves survival, demonstrating the great potential of OIMVs as a broad-spectrum vaccine candidate for Pseudomonas aeruginosa infection. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0022] Figure 1 This is an analysis of the results of X-ray irradiation inducing the production of outer-inner membrane vesicles by Pseudomonas aeruginosa PAO1 in one embodiment of the present invention; Figure 2 In one embodiment of the present invention, X-ray irradiation induces budding of the inner membrane, promoting the production of outer-inner membrane vesicles by Pseudomonas aeruginosa PAO1. Figure 3 This is an example of the effect of X-ray irradiation on the survival rate of Pseudomonas aeruginosa PAO1 in one embodiment of the present invention. Figure 4 This is an analysis of the generation of outer-inner membrane vesicles of Pseudomonas aeruginosa PAO1 under X-ray irradiation, which is jointly regulated by PrtR and PrtN in one embodiment of the present invention. Figure 5 This is one embodiment of the invention showing the effects of PrtR and PrtN on the growth and survival of Pseudomonas aeruginosa PAO1; Figure 6 This is an example of gene expression in Pseudomonas aeruginosa PAO1 after X-ray irradiation in one embodiment of the present invention (X-ray- represents the unirradiated control group, and X-ray+ represents the irradiated experimental group). Figure 7 This is a SEM image of a double-deleted strain of Pseudomonas aeruginosa after X-ray irradiation in one embodiment of the present invention; Figure 8 This is the result of the total vesicle yield of a double-deleted strain of Pseudomonas aeruginosa after X-ray irradiation in one embodiment of the present invention; Figure 9 This is the zeta potential result of vesicles of a double-deleted strain of Pseudomonas aeruginosa after X-ray irradiation in one embodiment of the present invention; Figure 10 This is the average particle size result of vesicles of a *Pseudomonas aeruginosa* double-deletion strain after X-ray irradiation in one embodiment of the present invention; Figure 11This is the result of one embodiment of the present invention showing that the outer-inner membrane vesicles have a protective effect against acute Pseudomonas aeruginosa pneumonia in mice. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0024] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0025] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0026] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0027] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within that range. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range. Detailed description of some accompanying drawings: Figure 1(a) Transmission electron microscopy (TEM) image of the internal structure of PAO1 cells without X-ray irradiation. Scale bar: 500 nm. (b)-(f) TEM images of the internal structure of PAO1 cells after X-ray irradiation, where red arrows indicate vesicle formation within irradiated PAO1 cells, and white boxes indicate extracellular vesicles released from the extracellular space. Scale bar: 200 nm. (g) Purification of extracellular vesicles by size exclusion chromatography using a qEV column, followed by analysis of the obtained components by silver-stained SDS-polyacrylamide gel electrophoresis. (h) Scanning electron microscopy image of purified extracellular vesicles. Scale bar: 500 nm. (i) TEM image of purified extracellular vesicles. Scale bar: 200 nm. (j) Size distribution of purified extracellular vesicles as determined by nanoparticle tracking analysis. (k) The production of outer-inner membrane vesicles of PAO1 exposed to X-rays at doses of 100, 200, 500, 800, and 1000 Gy was quantitatively determined using nanoparticle tracking analysis. (l) The production of outer-inner membrane vesicles of PAO1 exposed to X-rays at a dose of 1000 Gy was quantitatively determined every 24 hours using nanoparticle tracking analysis. Significant differences between datasets are indicated by an asterisk (ns indicates no significance). P<0.05, P<0.01 **P<0.001; two-tailed unpaired t-test or one-way ANOVA were used. (m) Outer-inner membrane vesicles were identified by agarose gel electrophoresis after digestion with DNase I and RNase I. (n) Total protein was analyzed by SDS-polyacrylamide gel electrophoresis of outer membrane vesicles purified from PAO1 supernatant cultured under normal conditions and outer-inner membrane vesicles purified from PAO1 supernatant after X-ray irradiation.

[0028] Figure 2 (A) Scanning electron microscope image of the surface morphology of PAO1 cells without X-ray irradiation. Scale bar: 500 nm. (B)-(D) Scanning electron microscope images of the surface morphology of PAO1 cells after X-ray irradiation. White arrows indicate vesicular protrusions (B), releasing outer-inner membrane vesicles (C), and pore structures formed on the cell surface after irradiation (D), respectively.

[0029] Figure 4 (a) Quantitative analysis of the relative transcription levels of lexA, prtR, and alpR genes in PAO1 strain under X-ray irradiation using qRT-PCR. (b) Quantitative determination of wild-type PAO1 (WT) and its mutant strains under X-ray irradiation using nanoparticle tracking analysis. lexA S125A , prtR S162A and alpR S153A(c) Regulation of PrtR on the production of outer-inner membrane vesicles in Pseudomonas aeruginosa PAO1 under X-ray irradiation. (d) Quantitative determination of WT by nanoparticle tracking analysis under standard TSB medium conditions. lexA S125A , prtR S162A and alpR S153A (e) Quantitative analysis of outer membrane vesicle production in mutant strains under X-ray irradiation using qRT-PCR. prtR S162A mutant strains prtN (f) Under X-ray irradiation, enhanced PrtN expression and PrtR loss jointly promoted the formation of PAO1 outer-inner membrane vesicles. (g) WT and Δ under X-ray irradiation were determined by nanoparticle tracking analysis. prtN Δ prtR Mean size of outer-inner membrane vesicles produced by mutant strains. (h) WT and Δ under X-ray irradiation were measured using nanoparticle tracking analysis. prtN Δ prtR Zeta potentials of outer-inner membrane vesicles produced by mutant strains. Significant differences between data groups are indicated by an asterisk (ns indicates no significance). P<0.05, P<0.01 **P<0.001; use two-tailed unpaired t-test, one-way ANOVA or two-way ANOVA. (i) Δ without X-ray irradiation prtN Δ prtR Scanning electron microscopy characterization of the cell surface morphology of the mutant strain. Scale bar: 1 μm. (j) and (k) Δ after X-ray irradiation prtN Δ prtR Scanning electron microscopy characterization of the surface morphology of mutant cells. Representative microscopic images are shown, with white arrows indicating spherical cells and vesicular protrusions. Scale bar: 500 nm. (l) Δ without X-ray irradiation. prtN Δ prtR Transmission electron microscopy observation of the internal structure of mutant cells. Scale bar: 500 nm. (m)-(o) Δ after X-ray irradiation prtN Δ prtR Transmission electron microscopy observation of the internal structure of mutant cells. Representative microscopic images are shown, with red arrows indicating the formation of outer-inner membrane vesicles.

[0030] Figure 5 (A) Wild-type PAO1 (WT) and lexA S125A , prtR S162A , alpR S153AThe growth curve of the mutant strain was obtained by measuring the OD600 value every 2 hours. (B) WT, lexA S125A , prtR S162A , alpR S153A Survival rate of mutant strains exposed to 500 Gy X-rays was calculated as the ratio of viable bacteria to total colony-forming units. (C) Both PrtR deletion and PrtN expression inhibited the growth of Pseudomonas aeruginosa PAO1. (D) Inhibition of PrtN expression increased the survival rate of PAO1 strains exposed to 500 Gy X-rays. Significant differences between data groups are indicated by an asterisk (ns: not significant, ...). P<0.05， *P<0.01; one-way ANOVA was used.

[0031] Figure 11 (a) Schematic diagram of the experimental design for evaluating the in vivo anti-pneumonia effect of endothelial vesicles. C57BL / 6 mice were subcutaneously immunized with endothelial vesicles in the left inguinal region. (b) Mice immunized with different concentrations of endothelial vesicles (0.1, 1, 2, 5, 10 μg) were subjected to a lethal dose of PAO1 (1.0 × 10⁻⁶ mcg). 8 (c) Survival rate after CFU attack and (d) Weight change (initial body weight percentage). 7 Bacterial load in lung tissue of mice immunized with different concentrations of extracellular-intramembrane vesicles (0.1, 1, 2, 5, 10 μg) after CFU challenge. (e) Bacterial load in lung tissue of mice immunized with different doses of extracellular-intramembrane vesicles (2 μg) at a lethal dose of PAO1 (1.0 × 10⁻⁶ mcg). 8 (f) Survival rate after CFU attack. (f) Survival rate after sublethal dose of PAO1 (2.0 × 10⁻⁶) 7 Following CFU challenge, bacterial load in lung tissue of mice immunized with different doses of extracellular-intramembrane vesicles (2 μg) was measured. (g) Mice immunized with extracellular-intramembrane vesicles (2 μg), extracellular vesicles (2 μg), double-membrane vesicles (2 μg), or formalin-inactivated bacteria (2 μg) were compared with PAO1 (1.0 × 10⁻⁶). 8 Survival rate after CFU attack. (h) Survival rate after PAO1 (2.0×10) attack. 7 Bacterial load in lung tissue of mice immunized with external-inner membrane vesicles (2 μg), external membrane vesicles (2 μg), double-membrane vesicles (2 μg), or formalin-inactivated bacteria (2 μg) after CFU challenge. (i) Representative hematoxylin-eosin stained pathological images of lung tissue sections from each group. Scale bar: 100 μm. (j) Bacterial load in the lung tissue of mice immunized with external-inner membrane vesicles (2 μg) at a lethal dose of heterologous serotype (O10) strain PA14 (1.0 × 10⁻⁶). 8Survival rate after CFU attack. (k) Survival rate after sublethal dose of PA14 (2.0 × 10⁻⁶) 7 Bacterial load in lung tissue of mice immunized with extracellular vesicles (2 μg) after CFU challenge. (l) Bacterial load in lung tissue of mice immunized with extracellular vesicles (2 μg) at a lethal dose of heterologous serotype (O11) strain W19 (1.0 × 10⁻⁶). 10 Survival rate after CFU (Cellular Fuel Injection) challenge. (m) Survival rate of strain W19 (2.0 × 10⁻⁶) after sublethal dose heterologous serotype (O11) challenge. 9 Bacterial load in mouse lung tissue immunized with extracellular-intracellular vesicles (2 μg) after CFU challenge. Survival curves were compared using the log-rank test (Mantel-Cox test, conservative estimate). Significant differences between datasets are indicated by an asterisk (ns indicates not significant, *). P<0.05， ** P<0.01， *** P< 0.001 P < 0.0001; a two-tailed unpaired t-test was used.

[0032] Example 1 Strains, plasmids, and culture conditions: The strains and plasmids used in this study were commercially purchased or conventional strains or plasmids preserved in the laboratory. Unless otherwise specified, *Pseudomonas aeruginosa*, *Escherichia coli*, *Klebsiella pneumoniae* ATCC 700603, and *Salmonella typhimurium* ATCC 13311 were cultured overnight at 37°C in TSB liquid medium or aerobic TSA plates (BD Biosciences). The overnight cultures were inoculated into TSB medium with an initial OD630 value of 0.05 and cultured at 37°C with continuous shaking (220 rpm) until the early exponential growth stage (OD630 = 1.0 ± 0.1). The antibiotics used and their final concentrations are as follows: Gentamicin (10 μg / mL for *Escherichia coli*). -1 Pseudomonas aeruginosa 30 μg·mL -1 ).

[0033] X-ray irradiation procedure: The simplified X-ray irradiation procedure is as follows: Bacterial cells were collected by centrifugation (5000 × g, 30 minutes, 4°C) and resuspended in 0.9% sodium chloride solution to the desired concentration. The bacterial suspension was divided into two portions: one portion was irradiated using an RS2000 biological X-ray irradiator (Rad Source Technologies) at a dose rate of 7.086 Gy / min at 160 kV and 25 mA; the other portion served as a non-irradiated control sample.

[0034] Results analysis: Under X-ray irradiation, Pseudomonas aeruginosa PAO1 produces outer-inner membrane vesicles.

[0035] Ultrathin sections of Pseudomonas aeruginosa PAO1 cells irradiated with X-rays were analyzed using transmission electron microscopy. The results showed that the inner membrane of irradiated PAO1 cells underwent budding, and outer membrane constriction formed extracellular-inner membrane vesicles with a double-membrane structure. This process was significantly different from the conventional secretion pattern of outer membrane vesicles. Figure 1 af). Scanning electron microscopy also revealed vesicle-like protrusions on the surface of PAO1 cells. Figure 2 ).

[0036] To separate and purify the outer-inner membrane vesicles, the supernatant of the PAO1 suspension after irradiation was first subjected to ultracentrifugation, followed by size exclusion chromatography using a qEV column. The second fraction with the highest vesicle concentration was collected for subsequent analysis. Figure 1 g). Morphological and size distribution analysis showed that the purified outer-inner membrane vesicles were spherical with an average diameter of approximately 150 nm. Figure 1 It is noteworthy that, with increasing X-ray irradiation dose, the production of outer-inner membrane vesicles showed a significant dose-dependent increasing trend (hj). Figure 1 Correspondingly, the survival rate of PAO1 cells decreased with increasing irradiation dose, and at an irradiation dose of approximately 1000 Gy, they completely lost their ability to form colonies. Figure 3 ).

[0037] Agarose gel electrophoresis confirmed that the purified outer-inner membrane vesicles contained both RNA and DNA. Figure 1 Furthermore, SDS-PAGE analysis showed that the protein composition of the outer-inner membrane vesicles differed significantly from that of the outer membrane vesicles produced by PAO1 under conventional culture conditions. Figure 1 n).

[0038] Based on this embodiment, we further explore the mechanism of gene regulation of outer-inner membrane vesicle formation.

[0039] Previous studies have shown that when DNA is damaged, the RecA protein binds to single-stranded DNA to form nucleoprotein filaments, thereby activating the potential serine protease activity of three structure-related regulatory factors (LexA, PrtR, and AlpR) in *Pseudomonas aeruginosa*. qPCR analysis in this study showed that under X-ray irradiation, the transcriptional levels of these regulatory factors in *Pseudomonas aeruginosa* PAO1 were significantly increased, indicating their activation. Figure 4 a). To investigate their role in the biosynthesis of outer-inner membrane vesicles, this study constructed site-directed mutant strains by replacing the catalytic serine codon with the alanine codon. lexA S125A , prtR S162A and alpRS153A Quantitative analysis of the outer-inner membrane vesicle production in these mutant strains revealed that, compared to wild-type PAO1, prtR S162A The strain exhibited a significant decrease in the production of outer-inner membrane vesicles, while lexA S125A and alpR S153A No significant differences were observed in the mutant strains. Figure 4 b). In prtR S162A PrtR reintroduction in mutant strains restored outer-inner membrane vesicle production to wild-type levels, while overexpression of PrtR in wild-type PAO1 did not further increase production. Figure 4 c), indicating that PrtR precisely regulates the biosynthesis of PAO1's outer and inner membrane vesicles under X-ray irradiation.

[0040] Under liquid culture conditions, the growth rate and outer membrane vesicle production of these mutants were comparable to those of wild-type PAO1. Figure 5 A, Figure 4 d). But prtR S162A The survival rate of the mutant strain was significantly increased after X-ray irradiation. Figure 5 B). qPCR analysis further showed that X-ray irradiation... prtR S162A mutant strains prtN The significantly reduced transcriptional level suggests that this mutation may affect the autocleavage activity of PrtR, leading to the inability to relieve the inhibition of PrtN. Figure 4 e). Furthermore, whether in wild-type PAO1 or... prtR S162A In mutant strains, overexpression of PrtN significantly increased the production of outer-inner membrane vesicles under X-ray irradiation. Figure 4 f).

[0041] Further in Δ prtN Introduced in the context of genetic background prtR Gene mutation, constructing Δ prtN Δ prtR S162A Mutant strain. Under X-ray irradiation, Δ prtN Δ prtR S162A With Δ prtN There was no significant difference in the production of outer and inner membrane vesicles among mutant strains. Figure 4 f). But in Δ prtN Knockout in the background prtR Genes can make Δ prtN Δ prtR The mutant strain showed a significant increase in outer-inner membrane vesicle production under X-ray irradiation, while the production of these vesicles was significantly increased by plasmid pME6032- prtRAfter re-introducing PrtR expression, its yield recovered to the level of Δ. prtN The mutant strain is at a comparable level ( Figure 4 f). Nanoparticle tracking analysis shows that Δ prtN Δ prtR The mutant strain produced outer-inner membrane vesicles that were not significantly different from wild-type PAO1 in terms of mean size and zeta potential. Figure 4 g, h).

[0042] Scanning electron microscopy revealed that the irradiated Δ prtN Δ prtR Some cells exhibit spherical shapes, and vesicle structures are visible on the cell surface. Figure 4 Transmission electron microscopy further revealed damage to some cell wall structures, accompanied by cell rupture and signs of outer-inner membrane vesicle release. Figure 4 (lo). The above results indicate that under X-ray irradiation, PrtR and PrtN jointly regulate the formation of outer-inner membrane vesicles in Pseudomonas aeruginosa PAO1.

[0043] Example 2 1. Expression levels of PrtR and PrtN genes before and after quantitative irradiation using qRCR 1.1 Bacterial culture and collection (1) Take out the Pseudomonas aeruginosa PAO1 strain from the -80℃ ultra-low temperature freezer and streak it onto a TSA plate. Incubate at 37℃ overnight (16~18 h).

[0044] (2) Select a single clone and inoculate it into 3 mL TSB, and incubate overnight (16-18 h) at 37℃ and 220 rpm.

[0045] (3) Take the overnight bacterial culture and measure the OD600 value, calculate the overnight bacterial culture concentration, and inoculate it into 100 mL of TSB medium so that the initial OD600 = 0.05. Incubate at 37℃ and 220 rpm until the logarithmic phase (OD600 is 0.4~0.8).

[0046] (4) Centrifuge the above bacterial solution at 3,000 g for 10 min, collect the bacterial cells, wash them three times with 0.9% sodium chloride injection and resuspend them, adjust the OD600 to 10, and take 100 μL to calculate the number of viable bacteria.

[0047] 1.2 X-ray irradiation treatment The bacterial suspension was divided into two groups. 1 mL of each group was added to a 1.5 mL sterile centrifuge tube. One group was placed in an irradiator at 160 kV, 25 mA, a dose rate of 7.068 Gy / min, and a total irradiation dose of 1000 Gy. The other group served as a control and was not irradiated.

[0048] 1.3 RNA Extraction RNA extraction was performed according to the instructions for the bacterial total RNA extraction kit.

[0049] (1) Take 100 μL of bacterial suspensions that have been irradiated (experimental group) and those that have not been irradiated (control group), respectively, and centrifuge at 12,000 rpm for 2 min at 4℃ to collect the bacterial cells. Carefully remove all supernatant.

[0050] (2) Add 150 μL of TE buffer containing lysozyme (400 μg / mL) to completely resuspend the bacterial cells and incubate at room temperature for about 5 min.

[0051] (3) Add 350 μL of lysis buffer RL (containing 1% β-mercaptoethanol) and vortex to mix.

[0052] (4) Add 250 μL of anhydrous ethanol, mix thoroughly, transfer to the adsorption column CR3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column CR3 back into the collection tube.

[0053] (5) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0054] (6) Preparation of DNase I working solution: Take 10 μL of DNase I stock solution and put it into an RNase-Free centrifuge tube. Add 70 μL of RDD buffer and mix gently.

[0055] (7) Add 80 μL of DNase I working solution to the center of the adsorption column CR3 and incubate at room temperature for 15 min.

[0056] (8) Add 350 μL of protein removal solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the collection tube.

[0057] (9) Add 500 μL of washing solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, put the adsorption column CR3 back into the collection tube, and repeat twice.

[0058] (10) Centrifuge at 12,000 rpm for 2 min and discard the waste liquid. Place the adsorption column CR3 at room temperature for 15 min to thoroughly dry any residual rinsing liquid in the adsorption material.

[0059] (11) Transfer the adsorption column CR3 into a new RNase-Free centrifuge tube, add 40 μL of RNase-Free ddH2O to the middle of the adsorption membrane, place at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min to obtain the RNA solution.

[0060] (12) Concentration determination: The concentration of RNA sample was determined by Nanodrop 2000, and the ratio of 260 / 280 was about 2.0.

[0061] 1.4 cDNA Synthesis cDNA synthesis references PrimeScript TM The RT kit was used.

[0062] (1) Removal of genomic DNA: Prepare the reaction mixture on ice according to the components in Table 1, then dispense it into each reaction tube, and finally add the RNA sample to obtain the first reaction solution; the reaction program is 42℃, 2 min.

[0063] Table 1. Genomic DNA Removal Reaction System (2) Reverse transcription reaction: The second reaction solution was prepared on ice according to the system shown in Table 2. The reaction program was 37°C for 15 min, 85°C for 5 sec, and the reverse transcription product was stored at -20°C.

[0064] Table 2 Reverse transcription reaction system 1.5 qPCR reaction (1) Design qPCR primers. The primer sequences are shown in Table 3.

[0065] Table 3 qRCR primer sequence listing (2) The reagent used in the experiment, 2×SYBR Green qPCR Master Mix, was prepared in a biosafety cabinet according to Table 4.

[0066] Table 4 qPCR reaction system (3) Add the mixture to the eight-tube set, cover it, mix it in the dark, centrifuge it to the bottom of the tube, put the eight-tube set into the real-time PCR instrument, and carry out the reaction according to the procedure in Table 5.

[0067] Table 5 qPCR reaction procedure (4) After the experiment, the data was analyzed using QuantStudio™ Design & Analysis software. See Figure 6 See Table 6.

[0068] The results showed that Pseudomonas aeruginosa PAO1, after X-ray irradiation, prtN and prtR The expression levels were significantly improved.

[0069] Table 6 prtN and prtR level of expression Example 3 Construction of a double-deleted strain of Pseudomonas aeruginosa PrtR and PrtN This embodiment describes the construction of the PAO1 double deletion strain using a two-step allele exchange method. The deletion strain PAO1Δ... prtN Taking the construction of [the product] as an example, first construct the recombinant suicide plasmid (pEX18GM:: prtN ud ) and donor bacteria (S17-1pEX18GM:: prtN ud The first step, allele exchange, involves conjugation transfer and antibiotic selection to integrate homologous sequences from the donor bacteria into the recipient bacteria genome. The second step, allele exchange, involves sucrose reverse selection to remove sucrose-sensitive genes. sacB Final realization prtN Gene deletion. Then, in the deletion strain PAO1Δ prtN Based on the construction of PAO1Δ prtN Δ prtR Double-deleted strains 。

[0070] First, construct a strain lacking PrtN, following these steps: 1.1 Donor bacteria S17-1 pEX18GM:: prtNud Construction (1) Primer design Based on the homologous arm design principle, sequences of approximately 800 bp were selected as homologous arms from both the upstream (before the start codon) and downstream (after the stop codon) of the target gene (Pseudomonas aeruginosa PAO1 prtN sequence). Deletion primers were designed, and double enzyme digestion for linearization was selected. The suicide plasmid pEX18GM sequence used in this experiment was linearized along the 5'–3' direction, and the Bam restriction enzyme site was selected for linearization. H I / X For baI, select the restriction enzyme sites to be preserved and choose the most suitable primer sequence according to primer design principles.

[0071] Table 7 Primers used for all gene deletions. (2) PAO1 genome extraction, according to the method in Example 1.

[0072] (3) Amplification of homologous arm fragments: Prepare the following reaction solution in a PCR tube.

[0073] Table 8 PCR reaction system After mixing and briefly centrifuging, proceed with the reaction according to the reaction procedure in Table 9.

[0074] Table 9 PCR reaction procedure Electrophoresis detection: 5 μL of PCR product was subjected to 1% agarose gel electrophoresis. The product size was all around the 800 bp band position, which met the expected size requirements.

[0075] (4) Purification of homologous arm fragments The homologous arm fragments obtained from amplification were purified and recovered using a DNA purification and recovery kit, and their concentrations were measured using Nanodrop2000. They were then stored at -20°C for later use.

[0076] (5) Linearization of suicide plasmids by enzyme digestion The suicide plasmid pEX18GM (concentration 200ng / μL, 1μg required) was linearized and digested with enzymes according to the following reaction system. The reaction conditions were 37℃ for 2-4 h. After the reaction was completed, the linearized enzyme digestion product was purified and recovered using a DNA purification and recovery kit, and the concentration was measured by Nanodrop 2000. It was then stored at -20℃ for later use.

[0077] Table 10 Enzyme digestion reaction system (50 μL) (6) Construction of recombinant plasmids Recombinant plasmids were constructed using the seamless cloning kit inNova Recombinase Enzyme III One-Step Cloning Mix (REIII Mix). The reaction solution was prepared according to the following ligation system, and the reaction program was to ligate at 37°C for 30 min, and then place on ice.

[0078] Table 11 REIII Mix reaction system (10 μL) Note: The molar ratio of plasmid to fragment is generally selected in the range of 1:1 to 1:5. In this study, 1:3 was selected.

[0079] (7) Transformation Remove competent DH5α cells from -80℃ and place them on ice to thaw. Add 10 µL of reaction solution to 100 μL of competent cells and mix well. Place on ice for 30 min. Heat shock in a 42℃ water bath for 90 sec, then place on ice for 2 min. Add 800 μL of LB medium and incubate at 37℃ on a shaker for 60 min. After centrifugation at 10000 rpm for 1 min, remove 600 μL of supernatant. Spread the cells evenly on LB plates containing gentamicin (10 μg / mL) and incubate inverted at 37℃ overnight.

[0080] (8) Screening and identification of recombinant positive clones After 18-24 hours of culture, gentamicin resistance (Gm) was observed. r Clear single-clonal colonies grew on LB plates; 6 single colonies were randomly selected and resuspended in 100 μL of sterile H2O for PCR identification. The primers used for identification were Up F / Down R (the specific primer sequences are shown in Table 7). The reaction solution was prepared according to the reaction system in Table 12, and the PCR reaction procedure is shown in Table 9.

[0081] Table 12 PCR reaction system (10 μL) Electrophoresis detection: After the reaction was completed, 5 μL of PCR product was taken and subjected to 1% agarose gel electrophoresis. The product size was around 1000bp~2000bp, which met the expected size requirements.

[0082] (9) Extraction of recombinant plasmids Positive clones were inoculated into 3 mL LB medium (containing 10 μg / mL Gm) and cultured overnight at 37°C and 220 rpm. Recombinant plasmids were extracted according to the instructions of the plasmid miniprep kit, and the concentration was determined by Nanodrop 2000. The plasmids were stored at -20°C for later use.

[0083] (10) Preparation of S17-1 competent cells Resuscitate Escherichia coli S17-1 at -80℃ onto LB agar plates and incubate overnight at 37℃. Pick a single colony and inoculate it into 3 mL of LB medium, incubate overnight at 37℃ and 220 rpm. Transfer 1 mL of the overnight bacterial culture to 100 mL of fresh LB medium (1:100) and incubate at 37℃ and 220 rpm for 2–2.5 h. Centrifuge at 5000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and resuspend them in 10 mL of pre-chilled 0.1 M CaCl2. Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the bacterial cells in 2 mL of pre-chilled 0.1 M CaCl2, aliquot into 1.5 mL sterile centrifuge tubes (100 μL / tube), and store at -80℃ for later use.

[0084] (11) Transformation Remove competent cells S17-1 from -80℃ and place them on ice to thaw; add 1 μg of recombinant plasmid to 100 μL of competent cells and mix well. The transformation process is shown in step (7); after single-clone colonies grow, randomly select two single clones for PCR identification and sequencing. The sequencing was completed by Qingke Company; expand the positive clones with correct sequencing and resuspend them in TSB medium containing 20% ​​glycerol, and store them at -80℃ for later use.

[0085] 1.2 Bonding Transfer (1) The recipient bacteria PAO1 and the donor bacteria S17-1 pEX18GM:: were removed from -80℃. prtNud The samples were revived separately on TSA and LB plates (containing 10 μg / mL Gm) and incubated overnight at 37°C in an inverted incubator.

[0086] (2) Pick single colonies and inoculate them into 3 mL TSB and 3 mL LB (containing 10 μg / mL Gm) medium, respectively, and incubate overnight at 37℃ and 220 rpm.

[0087] (3) PAO1 overnight bacterial culture was supplemented with 3 mL of fresh TSB medium and then placed in a 42℃ metal bath for heat blockage for 4 h.

[0088] (4) After heat inactivation, aspirate 500 μL of the donor bacteria S17-1 pEX18GM:: prtN ud Mix the bacterial culture thoroughly, draw up the suspension with a 10 mL disposable syringe, filter it through a 0.22 μm filter, squeeze out as much filtrate as possible, carefully remove the filter membrane with tweezers, gently place it on a pre-prepared fresh TSA plate (crystal side up), seal with sealing glue, and incubate upright in a 30℃ incubator for 8-10 h.

[0089] In the deletion strain PAO1Δ prtN Based on the construction of PAO1Δ prtN Δ prtR For double-deleted strains, the experimental procedure is the same as above.

[0090] Example 4 Screening and identification of Pseudomonas aeruginosa double-deletion strains PrtR and PrtN (1) Take 1 mL of 0.9% sodium chloride injection and add it to a 50 mL centrifuge tube. Carefully peel off the filter membrane covered with bacteria with tweezers and suspend it in 0.9% sodium chloride injection. Vortex and wash off the bacteria on the filter membrane. At this time, the bacterial suspension can be seen to be resuspended in the liquid. Take 200 μL of bacterial suspension and spread it on a VBMM (containing Gm 30 μg / mL) plate with a disposable spreader. Incubate overnight at 37℃ in an inverted incubator.

[0091] (2) Randomly select single clones grown on VBMM plates and isolate them on salt-free NSLB (containing 15% sucrose) plates. Incubate overnight at 37°C with the incubator inverted.

[0092] (3) Prepare three types of plates: TSA, PIA, and LB (containing 30 μg / mL Gm); use a pipette tip to pick up the single colonies grown on the NSLB plate and resuspend them in 50 μL of PBS. Take 5 μL of each and drop it onto the three types of plates. Select TSA and PIA for growth, and LB (containing 30 μg / mL Gm) for growth. r Clones that do not grow long on the plate were identified by PCR and sequenced.

[0093] (4) Expand the culture of the positive clones and resuspend them in TSB medium containing 20% ​​glycerol and store them at -80℃ for later use.

[0094] 1. Observation of bacterial cell surface morphology using scanning electron microscopy 1) Coverslip coating: Use a circular coverslip with a diameter of 8 mm. First, clean and sonicate with anhydrous ethanol and air dry, then immerse in 2.5% CS solution for about 5 seconds and dry at room temperature.

[0095] (2) Sample preparation: Bacterial culture and collection are described in Example 1. The X-ray irradiation procedure is described in Example 1 (placed in an irradiator, 160 kV, 25 mA, dose rate 7.068 Gy / min, total irradiation dose 1000 Gy). Take 100 μL of bacterial suspension and add 900 μL of physiological saline (i.e., dilute 10 times).

[0096] (3) Fixation: Prepare a 24-well plate, place a coverslip coated with CS in the well, use a pipette to draw 10 μL of the prepared bacterial solution and drop it onto the center of the coverslip, let it stand for 30 min to allow the bacteria to fully adhere to the coverslip, add 1 mL of 2.5% glutaraldehyde to completely soak the coverslip, and let it stand at 4℃ overnight for fixation.

[0097] (4) Gradient dehydration: Discard glutaraldehyde and wash three times with PBS, 5 min each time; then use different concentrations of ethanol for gradient dehydration: 30% (1 time), 50% (1 time), 70% (1 time), 90% (1 time), 100% (3 times), 5 min / time; finally, soak the sample in a small amount of 100% anhydrous ethanol.

[0098] (5) Drying: The critical point drying method was used to dry the sample. The dryer was first pre-cooled for about 30 minutes. The coverslip with the sample was carefully placed into the dryer with tweezers. Liquid CO2 was injected, followed by CO2 displacement, heating and vaporization, and finally discharge. The dryer was then turned on and the sample was taken out.

[0099] (6) Coating: Before metal coating, the sample is attached with Leit C Plast™ carbon conductive adhesive of appropriate size and placed on the metal sample stage of the ion sputtering instrument. Then, the sample is sprayed with gold for 120 seconds using vacuum sputtering to make the sample conductive.

[0100] (7) Image acquisition: After sample preparation, the cell surface morphology of bacteria before and after irradiation was observed using SEM. See Figure 7 .

[0101] The results showed that after X-ray irradiation, the double-deleted strain of Pseudomonas aeruginosa changed from rod-shaped to spherical, and a large number of vesicle structures formed on the surface of the bacterial cells.

[0102] 2. Nanoparticle size tracking analyzer detects the particle size distribution and concentration of MVs. The particle size and concentration of OIMVs were measured using NTA and analyzed using Zetaview software. Purified OIMVs samples were thawed on ice and diluted in ultrapure water (1:200–1:8000). One mL of the diluted sample was injected using Zetaview software to quantify the particle size distribution and concentration of OIMVs. Specific analytical parameters were: maximum particle size: 2000, minimum particle size: 0. All measurements were performed at room temperature (24.5℃ ± 0.1). Results are shown below. Figure 8 , Figure 9 and Figure 10 .in, Figure 8 The total vesicle yield of *Pseudomonas aeruginosa* strains after X-ray irradiation was measured. The results showed that the total vesicle yield of double-deletion strains was significantly increased after X-ray irradiation treatment. Figure 9 The results showed little difference in the zeta potential of vesicles of *Pseudomonas aeruginosa* strains after X-ray irradiation. Figure 10The average particle size of vesicles after X-ray irradiation of *Pseudomonas aeruginosa* strains was shown in Table 13; the zeta potential of vesicles after X-ray irradiation was shown in Table 14; and the average particle size of vesicles after X-ray irradiation was shown in Table 15.

[0103] Table 13 Total vesicle yield of *Pseudomonas aeruginosa* strains after X-ray irradiation Table 14. Zeta potentials (mV) of vesicles in *Pseudomonas aeruginosa* strains after X-ray irradiation. Table 15. Average particle size (nm) of vesicles from *Pseudomonas aeruginosa* strains after X-ray irradiation. Example 5 Verify the immune effects of OIMVs Mouse immunization and challenge experiments: This study used 6-7 week old female C57BL / 6J mice for immunization experiments. All animal experimental procedures followed the approved protocols of the Animal Experiment Ethics Committee of West China Hospital, Sichuan University. Mice were housed in a standard laboratory environment at 23°C, with a 12-hour light-dark cycle, and free access to food and water. Animal stress was minimized as much as possible during the experiment.

[0104] To assess the immunogenicity of endothelial vesicles, mice were subcutaneously injected with different concentrations (0.1, 1, 2, 5, 10 µg / 100 µL) of endothelial vesicles into the left inguinal region every two weeks. Fourteen days after the last immunization, anesthetized mice were challenged intratracheally with approximately 2 × 10⁶ vesicles. 7 CFU / mL (PAO1), 2×10 7 CFU / mL (PA14), 2×10 9 50 µL of bacterial suspension at CFU / mL (W19) was administered. Mice (n=6) were sacrificed 24 hours after sublethal dose challenge, and lung tissue was collected for bacterial load determination. In the lethal dose challenge experiment, PAO1, PA14, and W19 bacterial suspensions (concentrations of 1×10⁻⁶) were used respectively. 8 1×10 8 1×10 10 After challenge with CFU / mL (n=10), the survival rate and weight of mice were monitored for 10 days.

[0105] Experimental results: This embodiment established a mouse model of acute Pseudomonas aeruginosa pneumonia. C57BL / 6J mice (6-10 mice per group) were subcutaneously inoculated with different doses of outer-inner membrane vesicles on days 0, 14, and 28. Figure 11 a). Fourteen days after the last immunization, mice were challenged with a lethal dose of Pseudomonas aeruginosa PAO1. All mice in the PBS-treated control group died within 3 days, while mice in the outer-inner membrane vesicle immunization group showed a dose-dependent protective effect: the survival rate in the 0.1 µg dose group reached 70%, and the survival rate in the 1 µg and above dose groups reached 100%, demonstrating a strong protective immune response. Figure 11 b). Surviving outer-inner membrane vesicle-immunized mice gradually recovered to their baseline weight after infection. Figure 11 c).

[0106] To assess bacterial clearance capacity, mice were challenged with a sublethal dose of PAO1, and lung tissue was collected on day 1 post-challenge for bacterial load analysis. Results showed that, compared to the PBS control group, extracellular-endocellular vesicle immunization significantly reduced lung bacterial load, with mice immunized at doses greater than 2 µg having the lowest bacterial load. Figure 11 d). Further studies found that a single 2 µg dose of extra-endothelial vesicle immunization provided 90% protection against lethal dose PAO1 challenge, while two immunizations provided complete protection. Figure 11 e). Furthermore, compared to the PBS control group, mice immunized twice with a 2 µg dose showed significantly lower bacterial loads after sublethal PAO1 challenge ( Figure 11 f).

[0107] Notably, the protective effect of outer-inner membrane vesicles was superior to that of outer membrane vesicles. Mice immunized with 2 µg of outer-inner membrane vesicles achieved 100% survival and significantly reduced bacterial load after challenge with a lethal dose of PAO1, while mice immunized with the same dose of outer membrane vesicles only achieved 50% protection. Figure 11 g, h). Histopathological analysis further validated these results: compared with the severe lung pathological damage observed after challenge in the PBS control group and other immunization groups, the pulmonary edema, alveolar hemorrhage, and tissue structural damage were significantly reduced in the outer-inner membrane vesicle immunization group mice. Figure 11 i). The above results indicate that extracellular-endocellular vesicle vaccination can induce a strong protective immune response, enhance bacterial clearance, and reduce lung tissue damage caused by Pseudomonas aeruginosa infection.

[0108] Given the multiple and serious challenges posed by Pseudomonas aeruginosa infection, including its serotype diversity and multidrug resistance, this study further explored whether extracellular-endocellular vesicle vaccination has broad-spectrum protective potential. The results showed that, compared with the PBS control group, mice immunized via extracellular-endocellular vesicle vaccination exhibited significantly higher survival rates and significantly lower lung bacterial loads when challenged with highly virulent Pseudomonas aeruginosa PA14 (O10 serotype). Figure 11 (j, k). Furthermore, considering that serotype O11 is one of the most common serotypes in Pseudomonas aeruginosa pneumonia cases, the protective effect of outer-inner membrane vesicles against clinical isolate W19 (serotype O11) was further evaluated using this as a representative. Experiments showed that, compared with the PBS control group, mice immunized with outer-inner membrane vesicles exhibited significantly reduced lung bacterial load and significantly improved survival rate. Figure 11 These results demonstrate that extracellular-intramembrane vesicle immunization not only provides strong protection against PAO1 strains but also effectively reduces the pulmonary bacterial load and improves survival rate caused by heterologous Pseudomonas aeruginosa serotype infection, highlighting its great potential as a broad-spectrum vaccine candidate.

[0109] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A strain that produces hypermembrane vesicles, characterized in that, The strain that produces supercapillary membrane vesicles is a *Pseudomonas aeruginosa* strain with double gene deletions; the *Pseudomonas aeruginosa* strain with double gene deletions is... prtN and prtR A *Pseudomonas aeruginosa* strain with double gene deletion; the supermembrane-producing vesicles are *Pseudomonas aeruginosa* outer-inner membrane vesicles.

2. A method for producing superproductive membrane vesicles, characterized in that, The method involves irradiating a *Pseudomonas aeruginosa* strain with double gene deletions; the *Pseudomonas aeruginosa* strain with double gene deletions is... prtN and prtR A *Pseudomonas aeruginosa* strain with double gene deletion; the supermembrane-producing vesicles are *Pseudomonas aeruginosa* outer-inner membrane vesicles.

3. The method as described in claim 2, characterized in that, The superproductive membrane vesicles are outer-inner membrane vesicles with a double membrane structure; the outer-inner membrane vesicles are formed by the outward bubbling of the inner membrane of the Pseudomonas aeruginosa double-deleted gene strain induced by radiation irradiation, resulting in the extrusion and shedding of the outer membrane.

4. The method as described in claim 2, characterized in that, The radiation is X-ray radiation.

5. The method as described in claim 4, characterized in that, The dose rate of the X-ray irradiation is 7-8 Gy / min, and the total irradiation dose is 500-1000 Gy.

6. The method as described in claim 4, characterized in that, The method includes the following steps: S01: Knockout of Pseudomonas aeruginosa prtN and prtR Genetic preparation of a Pseudomonas aeruginosa double-deleted strain; S02: Superproductive membrane vesicles were obtained by treating a Pseudomonas aeruginosa strain with double gene deletions using X-ray irradiation.

7. The use of the superproductive membrane vesicles prepared by the method of any one of claims 2-6 in the preparation of vaccines.

8. The application as described in claim 7, characterized in that, The vaccine is used to prevent or treat infections caused by Pseudomonas aeruginosa.

9. The application as described in claim 8, characterized in that, The infections include lung infections caused by Pseudomonas aeruginosa.

10. The application of a *Pseudomonas aeruginosa* strain with double gene deletions in vaccine preparation, characterized in that, The *Pseudomonas aeruginosa* strain with double gene deletion is... prtN and prtR A double-deleted strain of *Pseudomonas aeruginosa*; the vaccine is used to prevent or treat infections caused by *Pseudomonas aeruginosa*.

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