Bacterial outer membrane vesicle and application thereof in preparation of pseudomonas aeruginosa vaccine

Abstract

The invention discloses a bacterial outer membrane vesicle. The bacterial outer membrane vesicle is obtained through induced expression and purification of a pseudomonas aeruginosa recombinant expression vector. The pseudomonas aeruginosa recombinant expression vector is obtained by knocking out an htrB1 gene on a wild type pseudomonas aeruginosa PAO1 strain; or the pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene and the tolB gene on the wild type pseudomonas aeruginosa PAO1 strain. The invention also discloses an application of the bacterial outer membrane vesicle in preparation of a pseudomonas aeruginosa vaccine. The pseudomonas aeruginosa vaccine can cause humoral immune response and cell-mediated immune response. According to the invention, a genome of pseudomonas aeruginosa is edited by utilizing a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology, and an OMV attenuated pseudomonas aeruginosa strain and an OMV attenuated and productive pseudomonas aeruginosa strain are constructed. The OMV of the constructed strain is taken as an antigen, and broad-spectrum protective pseudomonas aeruginosa vaccines aiming at different serotypes can be developed.

Description

Technical Field

[0001] The present application relates to the technical field of vaccine preparation, and specifically to a bacterial outer membrane vesicle and its application in the preparation of Pseudomonas aeruginosa vaccine. Background Art

[0002] Pseudomonas aeruginosa, also known as Pseudomonas aeruginosa, is a common conditional pathogen and a Gram-negative bacterium. The size of Pseudomonas aeruginosa bacteria is (1.5-5.0)um × width (0.5-1)um, slender and of varying lengths, sometimes in the shape of a club or thread, arranged in pairs or short chains. The bacteria are commonly found in hospital-acquired infections and community-acquired infections, including infections of the lower respiratory tract, surgical wounds, burn wounds, urinary tract, cornea, etc. It may also cause severe infections in any organ, causing pneumonia, enteritis, meningitis and sepsis.

[0003] As one of the common conditional pathogens of hospital infection, infection with Pseudomonas aeruginosa seriously threatens human public health. Antibiotics are the main treatment for Pseudomonas aeruginosa infection in clinical practice. However, due to its natural and complex drug resistance and acquired drug resistance, clinical antibiotic treatment has become very difficult. Therefore, there is still a lack of effective treatment and control strategies, and new treatment methods need to be found. In recent years, component vaccines, such as lipopolysaccharide (LPS), exopolysaccharide (EPS), flagella, etc., recombinant subunit vaccines: such as outer membrane protein (OMP), whole bacterial inactivated vaccine, attenuated live vaccine, etc. Pseudomonas aeruginosa vaccines have been studied. There are multiple vaccine products entering the clinical research stage, but so far, no approved Pseudomonas aeruginosa vaccine has been marketed.

[0004] Outer membrane vesicles (OMVs) are bacterial nanoparticles that are spontaneously released by Gram-negative bacteria during their growth in vitro and in vivo. They are spherical double-layer membrane nanostructures with a diameter of approximately 20 to 250 nm. OMVs contain almost all biological components of bacteria, such as peptidoglycan, phospholipids, LPS, proteins, etc. On the one hand, OMVs are a mechanism for bacteria to deliver DNA, RNA, proteins, and toxins, as well as promote biofilm formation and reshape the outer membrane during growth. On the other hand, because OMVs have an optimal size for uptake by immune cells, can be engulfed by immune cells through membrane fusion, have strong immune delivery capabilities, and have almost all the components of bacteria, they can be used as an important direction for the development of Pseudomonas aeruginosa vaccines. At present, no OMV-based Pseudomonas aeruginosa vaccines are on the market. Summary of the invention

[0005] In order to solve the above technical problems, the first object of the present application is to provide a bacterial outer membrane vesicle, which is obtained by inducing expression and purification of a Pseudomonas aeruginosa recombinant expression vector; the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene in the wild-type Pseudomonas aeruginosa PAO1 strain; or, the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene and the tolB gene in the wild-type Pseudomonas aeruginosa PAO1 strain.

[0006] Furthermore, the wild-type Pseudomonas aeruginosa PAO1 strain is an ATCC15692 strain.

[0007] Furthermore, the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene in the wild-type Pseudomonas aeruginosa PAO1 strain, and the method for constructing the Pseudomonas aeruginosa recombinant expression vector comprises the following steps:

[0008] S1: electroporate the pCasPA plasmid into the wild-type Pseudomonas aeruginosa PAO1 strain;

[0009] S2: The electroporated strain was cultured on a tetracycline resistance plate;

[0010] S3: The above bacterial solution was inoculated into tetracycline-resistant LB medium, and cultured until OD600 was 0.6. Arabinose was added and cultured for 3 hours. The concentration of arabinose after addition was 2 mg / ml.

[0011] S4: Collect the above bacterial solution, and electroporate the sgRNA and homology arms of the htrB1 gene into the above strain;

[0012] S5: The electroporated strain was cultured on a tetracycline and carbenicillin resistance plate overnight. The next day, a single clone was picked and transferred to 5 mL of tetracycline and carbenicillin resistant LB medium and cultured overnight. The single clone with successful htrB1 gene knockout was identified by bacterial liquid PCR and genome sequencing to obtain the htrB1 gene knockout PAO1 strain.

[0013] Furthermore, the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene and the tolB gene in the wild-type Pseudomonas aeruginosa PAO1 strain; the method for constructing the Pseudomonas aeruginosa recombinant expression vector comprises the following steps:

[0014] S1: electroporate the pCasPA plasmid into the wild-type Pseudomonas aeruginosa PAO1 strain;

[0015] S2: The electroporated strain was cultured on a tetracycline resistance plate;

[0016] S3: The above bacterial solution was inoculated into tetracycline-resistant LB medium, and cultured until OD600 was 0.6. Arabinose was added and cultured for 3 hours. The concentration of arabinose after addition was 2 mg / ml.

[0017] S4: Collect the above bacterial solution, and electroporate the sgRNA and homology arms of the htrB1 gene into the above strain;

[0018] S5: The electroporated strains were cultured on tetracycline and carbenicillin resistance plates overnight. On the second day, single clones were picked and transferred to 5 mL of tetracycline and carbenicillin resistance LB medium and cultured overnight. Single clones with successful htrB1 gene knockout were identified by bacterial liquid PCR and genome sequencing to obtain the htrB1 gene knockout PAO1 strain.

[0019] S6: Knock out the tolB gene in the htrB1 gene knockout PAO1 strain according to steps S1 to S5 to obtain a PAO1 strain with both htrB1 and tolB genes knockout.

[0020] The second object of the present application is to provide an application of bacterial outer membrane vesicles in the preparation of Pseudomonas aeruginosa vaccine, which can induce humoral immune response and cell-mediated immune response.

[0021] Furthermore, the Pseudomonas aeruginosa vaccine uses an adjuvant that activates Th1 or Th2 cell immunity to adsorb the bacterial outer membrane vesicles.

[0022] Furthermore, the adjuvant includes one or more of CPG, alum and aluminum hydroxide.

[0023] Lipopolysaccharide (LPS) is a major virulence factor on the outer membrane of Gram-negative bacteria. Lipid A is the biologically active center of LPS, and the htrB1 gene is an important gene for lipid A acylation. Therefore, the knockout of htrB1 can effectively reduce the toxicity of the bacterial outer membrane. The Tol-Pal system is a group of protein complexes produced by most Gram-negative bacteria, which are involved in maintaining the stability of the bacterial outer membrane. Defects in the components of the Tol-Pal system will cause the outer membrane to separate from the peptidoglycan layer, leading to membrane bending and blistering. It has been reported in the literature that the loss of any gene in the Tol-Pal system will induce a higher OMV yield. The tolB gene is an important component of the Tol-Pal system, and compared with other components of the Tol-Pal system, the knockout of the tolB gene induces the most OMVs. Therefore, constructing PAO1 with double knockouts of htrB1 and tolB can obtain attenuated and high-yield OMVs.

[0024] Compared with the existing technology, outer membrane vesicles (OMVs) are bacterial nanoparticles spontaneously released by Gram-negative bacteria during their growth, which have: (1) an optimal size for uptake by immune cells; (2) a series of surface-exposed antigens in natural conformations; and (3) activation components of Toll-like receptors (TLRs) in the innate immune system. Therefore, by constructing an attenuated Pseudomonas aeruginosa strain that produces a lot of OMVs, we can obtain attenuated and high-yield OMVs, and use the multi-pathway immune activation characteristics of OMVs to prepare an effective vaccine against multidrug-resistant Pseudomonas aeruginosa.

[0025] The present invention uses CRISPR-Cas9 technology to edit the genome of Pseudomonas aeruginosa, constructs an OMV attenuated Pseudomonas aeruginosa strain and an OMV attenuated prolific Pseudomonas aeruginosa strain. By using the attenuated prolific OMV of the strain constructed by the present invention as an antigen, a broad-spectrum protective Pseudomonas aeruginosa vaccine against different serotypes can be developed. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Electrophoresis results of ΔhtrB1 PAO1 strain.

[0027] Figure 2 :ELECTROPHORESIS RESULTS OF ΔhtrB1&ΔtolB PAO1 Strain.

[0028] Figure 3 : Bacterial growth curve.

[0029] Figure 4 : Comparison of characteristics of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV: (A) Schematic diagram of experimental design; (B) SDS-PAGE analysis results of OMVs in each group; (C) Morphology of OMVs under transmission electron microscopy; (D) NTA analysis of OMVs; (E) Concentration of OMVs in each group; (F) Particle size of OMVs in each group; (G) Concentration of OMVs in each group.

[0030] Figure 5 : OMV in vitro cytotoxicity test.

[0031] Figure 6 : Serum IgG antibody levels.

[0032] Figure 7: WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV induce protection against Pseudomonas aeruginosa PAO1 in a bloodstream infection model: (A) Schematic diagram of vaccination, bacterial challenge (intravenous injection) and pathological study of Balb / c mice; (B) Survival curves of WT OMV-immunized mice and control mice in the bacteremia model; (C) Survival curves of ΔhtrB1 OMV-immunized mice and control mice in the bacteremia model; (D) Survival curves of ΔhtrB1&ΔtolB OMV-immunized mice and control mice in the bacteremia model; (E) Body weight change curves of WT, ΔhtrB1 and ΔhtrB1&ΔtolB OMVs-immunized mice and control mice in the bacteremia model; (F) Bacterial load in lung tissue of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection; (G) Bacterial load in lung tissue of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection Bacterial load in kidney tissue of mice immunized with OMV, ΔhtrB1&ΔtolB OMV, and control mice; (H) Representative images of H&E staining of lung tissue of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV, and control mice on the fourth day after infection; (I) Lung injury score calculated as percentage of area.

[0033] Figure 8: WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV induce protection against clinical strains of Pseudomonas aeruginosa in blood infection models: (A) Schematic diagram of vaccination, bacterial challenge (intravenous injection) and pathological study of Balb / c mice; (B) Survival curves of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV and control mice in the PA0062815 bacteremia model; (C) Body weight change curves of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV and control mice in the PA0062815 bacteremia model; (D) Survival curves of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV and control mice in the PA4029629 bacteremia model; (E) Body weight change curves of OMV-immunized mice and control mice in the PA4029629 bacteremia model; (F) Bacterial load in lung tissues of WT OMV-, ΔhtrB1 OMV-, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection (PA0062815 bacteremia model); (G) Bacterial load in lung tissues of WT OMV-, ΔhtrB1 OMV-, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection (PA4029629 bacteremia model); (H) Representative images of H&E staining of lung tissues of WT OMV-, ΔhtrB1 OMV-, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection (PA0062815 bacteremia model); (J) Representative images of H&E staining of lung tissues of WT OMV-, ΔhtrB1 OMV-, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection (PA0062815 bacteremia model); Representative images of H&E staining of lung tissues of OMV-, ΔhtrB1&ΔtolBOMV-immunized mice and control mice (PA4029629 bacteremia model); (I) Lung injury score calculated as area percentage (PA0062815 bacteremia model); (K) Lung injury score calculated as area percentage (PA4029629 bacteremia model).

[0034] Fig. 9: WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV induce protection against Pseudomonas aeruginosa PAO1, PA0062815 and PA4029629 in pneumonia model: (A) Schematic diagram of vaccination, bacterial challenge (intravenous injection) and pathological study of Balb / c mice; (B) Survival curves of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice in PAO1 pneumonia model; (C) Survival curves of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice in PA0062815 pneumonia model; (D) Survival curves of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice in PA4029629 pneumonia model; (E) Survival curves of WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV-immunized mice and control mice on the fourth day after infection. (F) Bacterial load in lung tissues of mice immunized with OMV, ΔhtrB1 & ΔtolB OMV and control mice on the fourth day after infection (PA0062815); (G) Bacterial load in lung tissues of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1 & ΔtolB OMV and control mice on the fourth day after infection (PA4029629); (H) Representative images of H&E staining of lung tissues of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1 & ΔtolB OMV and control mice on the fourth day after infection (PAO1); (J) Representative images of H&E staining of lung tissues of mice immunized with WT OMV, ΔhtrB1 OMV, ΔhtrB1 & ΔtolB OMV and control mice on the fourth day after infection. Representative images of H&E staining of lung tissues of OMV-immunized mice and control mice (PA0062815); (L) Representative images of H&E staining of lung tissues of WT OMV-immunized mice, ΔhtrB1 OMV-immunized mice, ΔhtrB1&ΔtolBOMV-immunized mice and control mice on the fourth day after infection (PA4029629); (I) Lung injury score calculated as area percentage (PAO1); (K) Lung injury score calculated as area percentage (PA0062815); (M) Lung injury score calculated as area percentage (PA4029629). DETAILED DESCRIPTION

[0035] The present application is further described below with reference to embodiments, but is not limited thereto.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] The experimental materials and equipment involved in the present invention are as follows:

[0038] 1. Bacterial strains and plasmids

[0039] (1) Wild-type Pseudomonas aeruginosa PAO1 strain (ATCC 15692 strain).

[0040] (2) pCasPA plasmid: from Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

[0041] (3) pACRISPR plasmid: from Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

[0042] (4) DH5α competent cells: purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0043] 2. Main materials, as shown in Table 1

[0044] Table 1 Main materials

[0045]

[0046]

[0047] 3. Main equipment, as shown in Table 2:

[0048] Table 2 Main equipment

[0049]

[0050] Embodiment 1:

[0051] This embodiment provides a recombinant expression vector of Pseudomonas aeruginosa. Specifically, an attenuated Pseudomonas aeruginosa PAO1 strain (ΔhtrB1 PAO1 strain) and an attenuated prolific Pseudomonas aeruginosa PAO1 strain (ΔhtrB1&ΔtolBPAO1 strain) are constructed, comprising the following steps:

[0052] Step S0: Before formally constructing the Pseudomonas aeruginosa recombinant expression vector, it is necessary to design primers, sgRNA and homology arms.

[0053] S0-1: Primer and sgRNA design

[0054] The whole genome sequence of the wild-type Pseudomonas aeruginosa PAO1 strain was obtained through the ATCC database. The coding regions of the htrB1 and tolB genes were obtained by bioinformatics analysis. Primers and sgRNAs for each target gene were designed based on the sequences of the htrB1 and tolB gene coding regions, respectively (where the sgRNA design website is http: / / www.rgenome.net / cas-designer / ), as shown in Table 3, and the primers were synthesized by BGI.

[0055] Table 3 Primer list

[0056]

[0057] S0-2: sgRNA vector construction

[0058] The pACRISPR plasmid was digested with BsaI-HF. The digestion system was referred to the instructions. The digestion products were purified and recovered for later use. Each pair of single-stranded sgRNA was annealed into a double strand, and a phosphate group was added to the 5' end of the primer. The reaction system was referred to the product instructions. The product was diluted 1:200 and connected to pACRISPR according to the T4 ligase instructions. After transformation and culture, the universal primer M13-R was used for sequencing.

[0059] S0-3: Homology arm construction

[0060] First, the left and right homology arm sequence fragments of the target gene were amplified using H1F / H1R and H2F / H2R primers, respectively, to obtain purified PCR products. Subsequently, Overlap PCR was performed using H1F / H2R primers to seamlessly clone the upstream homology arm and downstream homology arm fragments of the target gene.

[0061] Then, the recombinant expression vector of Pseudomonas aeruginosa is constructed, which includes the following steps:

[0062] S1: The pCasPA plasmid was electroporated into the wild-type Pseudomonas aeruginosa PAO1 strain (WTPAO1 strain) at an electroporation parameter of 2.5Kv.

[0063] S2: Culture the electroporated strain on a tetracycline-resistant plate in a 37°C incubator overnight. The next day, pick a single clone and transfer it to 5 mL of tetracycline-resistant LB medium, and culture it in a shaker at 37°C, 200 rpm overnight.

[0064] S3: Take the above bacterial solution and inoculate it into 15 mL of tetracycline-resistant LB medium at a ratio of 1:100, place it in a shaker at 37°C and 200 rpm and culture it until OD600 is 0.6. Add arabinose (L-arabinose) with a final concentration of 2 mg / ml and continue culturing for 3 hours.

[0065] S4: Collect the above bacterial solution, and electroporate the sgRNA and homology arms of the htrB1 gene into the above strain at an electroporation parameter of 2.5Kv.

[0066] S5: The electroporated bacteria were cultured on tetracycline and carbenicillin resistant plates in a 37°C incubator overnight. The next day, a single clone was picked and placed in 5 mL of tetracycline and carbenicillin resistant LB medium, and cultured overnight in a shaker at 37°C, 200 rpm. The single clone with successful htrB1 gene knockout was confirmed by bacterial liquid PCR and genome sequencing, and the attenuated PAO1 strain was obtained, named ΔhtrB1 PAO1 strain.

[0067] S6: Knock out the tolB gene in the ΔhtrB1 PAO1 strain according to steps S1 to S5, thereby obtaining an attenuated PAO1 strain with high OMV production, named ΔhtrB1&ΔtolB PAO1 strain. It can be understood that the steps and parameters for knocking out the tolB gene are exactly the same as steps S1 to S5, except that the sgRNA and homology arms are replaced with the sgRNA and homology arms corresponding to the tolB gene.

[0068] Embodiment 2:

[0069] This embodiment discloses a method for extracting bacterial outer membrane vesicles, specifically, a method for extracting bacterial outer membrane vesicles of WT PAO1 strain, ΔhtrB1 PAO1 strain, ΔhtrB1 & ΔtolB PAO1 strain, comprising the following steps:

[0070] A1: WT PAO1 strain, ΔhtrB1 PAO1 strain, ΔhtrB1&ΔtolB PAO1 strain were spread on the surface of non-resistant plates respectively and placed in a 37°C incubator to grow overnight.

[0071] A2: Pick single clones separately into LB medium without resistance, and culture them in a shaker at 37°C and 200 rpm for 6 hours.

[0072] A3: Inoculate the bacterial suspension into 1L of LB medium without resistance at a ratio of 1:100, and culture it in a shaker at 37°C and 200 rpm overnight.

[0073] A4: Centrifuge the bacterial solution at 8000rpm, 4℃, 30min. Pass the collected bacterial solution supernatant through a filter with a 0.45μm filter membrane, collect the filtered supernatant, and use a high-speed centrifuge to centrifuge the supernatant at 18000rpm, 4℃, 2 hours. After centrifugation, collect the precipitate, which is the bacterial outer membrane vesicle. The bacterial outer membrane vesicles extracted from the three strains were named WTOMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV, respectively.

[0074] Verification example:

[0075] The present invention detects the concentration of the target protein by using a BCA kit.

[0076] In order to verify the various properties of WT PAO1 strain, ΔhtrB1 PAO1 strain, ΔhtrB1&ΔtolB PAO1 strain and WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV, animal experiments were conducted, bacteremia model and pneumonia model were constructed, and bacterial load experiment and cytotoxicity experiment were conducted. The specific process is as follows:

[0077] (1) Animal experiments

[0078] Each Balb / c mouse was immunized with 50 μg OMV plus aluminum adjuvant by subcutaneous injection in the neck. The total volume injected per animal was 100 μL, of which aluminum adjuvant (1 mg / ml) accounted for 10%. Immunization was performed once every 14 days, for a total of three times. The negative control was PBS buffer plus aluminum adjuvant, and the total injection volume per animal was 100 μL.

[0079] (2) Bacteremia Model

[0080] The mice that had been immunized with protein three times were injected with PAO1 strain via tail vein, with a dose of 5×10 6 CFU, injection volume was 100 μL. The mice were monitored daily for two weeks.

[0081] (3) Pneumonia model

[0082] After the mice were anesthetized by intraperitoneal injection of avertin (ready-to-use tribromoethanol), 5×10 6 BALB / c mice were challenged intranasally with CFU PAO1 via the left and right trachea with an injection volume of 100 μL, 50 μL each in the left and right trachea. The health of the infected mice was monitored daily for two weeks.

[0083] (4) Bacterial load

[0084] Each mouse was injected with a half-lethal dose of 2.5 × 10 6 To determine the number of PAO1 CFU, spleen, kidney and lung tissues were collected from mice 4 days after the challenge. The tissues were ground evenly using a tissue grinder. Tissue samples were graded and the number of PAO1 CFU was counted on LB plates.

[0085] (5) Cytotoxicity assay

[0086] Mouse macrophages RAW264.7, human acute promyelocytic leukemia cells HL-60, microglial cells BV-2, human embryonic kidney cells 293T, and macrophages RAW264.7 were respectively cultured at 1×104 Each well was added to a 96-well plate, and OMVs of wild-type, attenuated, and attenuated prolific PAO1 were added to the above 4 cell culture solutions at 0, 5, and 20 μg / ml, respectively. The total volume of each well was 100 μL, and three replicates were set for each protein sample. The bacteria and cell mixture was incubated in a 37°C incubator for 16 hours. After the incubation, the culture supernatant was removed, 100 μL of cell activity detection reagent (CellTiter-Go Luminescent Cell Viability Assay, G7572) was added, and the cell plate was placed on a microplate oscillator and incubated at room temperature for 10 minutes, and the fluorescence of each well was detected under a fluorescence microplate reader.

[0087] Verification process and results:

[0088] 1. Target gene knockout verification

[0089] like Figure 1 and Figure 2 As shown, the successfully electroporated monoclonal bacterial solution was used as a template, and the polymerase chain reaction (PCR) reaction solution was prepared according to the proportion in the DNA polymerase manual. The amplification program was run on a PCR instrument, and the gene knockout situation was determined by the electrophoresis results. The genome of the monoclonal gene that was successfully knocked out by PCR was further extracted and sent to a sequencing company for sequencing verification. Finally, the htrB1 gene knockout and htrB1 and tolB gene double knockout Pseudomonas aeruginosa strains were successfully obtained.

[0090] 2. Analysis of growth characteristics of wild-type and gene-deficient strains

[0091] The above-mentioned WT PAO1 strain, ΔhtrB1 PAO1 strain, and ΔhtrB1&ΔtolB PAO1 strain were placed in a 37°C incubator and incubated for 20 hours, and the growth curve was measured. Figure 3 As shown, the ΔhtrB1 PAO1 strain and the ΔhtrB1&ΔtolB PAO1 strain had similar growth curves to the WT PAO1 strain, indicating that the gene deletion had no significant effect on bacterial growth.

[0092] 3. Characterization of bacterial outer membrane vesicles

[0093] The extracted WT OMV, ΔhtrB1 OMV, ΔhtrB1&ΔtolB OMV were analyzed by SDS-PAGE, scanning electron microscopy (TEM) and NTA to analyze the characteristics of OMV extracted from each strain ( Figure 4 A). Figure 4 As shown in B, the protein composition of OMV of each strain was displayed after protein gel silver staining. Figure 4C shows the morphology of OMVs of each group of strains under transmission electron microscopy (TEM). The OMVs extracted from each group are round, and the outer membrane vesicles have clear outlines. The concentration, proportion and particle size of the OMVs of each strain were further analyzed by nanoparticle tracking analysis (NTA). Figure 4 As shown in D, the average diameter is between 130-180 nm. ΔhtrB1&ΔtolB OMVs show good stability. Figure 4 E, 4F and 4G respectively counted the concentration, diameter and proportion of OMVs in each group. Compared with the OMVs secreted by the WT PAO1 strain and the ΔhtrB1 PAO1 strain, the concentration of OMVs secreted by the ΔhtrB1&ΔtolB PAO1 strain was significantly increased, the diameter was significantly reduced, and the overall proportion of OMVs was also significantly increased.

[0094] IV. OMV in vitro cytotoxicity test

[0095] Mouse macrophages RAW264.7, human acute promyelocytic leukemia cells HL-60, microglial cells BV-2, human embryonic kidney cells 293T, and macrophages RAW264.7 were respectively cultured at 1×10 4 / well was added to a 96-well plate, WT OMV, ΔhtrB1OMV, ΔhtrB1&ΔtolB OMV were added to the above five cell culture solutions at 0, 5, and 20μg / ml, respectively. The total volume of each well was 100μL, and three replicate wells were set for each protein sample. The bacteria and cell mixture was placed in a 37°C incubator and incubated for 16 hours. After the incubation, the culture supernatant was removed, 100μL of cell activity detection reagent (CellTiter-Go LuminescentCell Viability Assay, G7572) was added, the cell plate was placed on a microplate oscillator and incubated at room temperature for 10 minutes, and the fluorescence of each well was detected under a fluorescence microplate reader. The results are as follows Figure 5 As shown, after 48 h of incubation, the cytotoxicity of ΔhtrB1 OMV and ΔhtrB1&ΔtolB OMV was significantly reduced in all five cell types compared with WT OMV (p<0.001).

[0096] 5. Immunogenicity test of OMV in mice

[0097] Blood was collected from mice on days 7, 21, and 35 after immunization, and serum was separated. The immunogenicity of OMVs in each group was evaluated by ELISA. Figure 6As shown, WT OMV, ΔhtrB1 OMV and ΔhtrB1&ΔtolB OMV can all induce higher antibody titers in mice, and the antibody titers increase with the number of immunizations.

[0098] VI. Determination of the protective efficacy of OMVs against the mouse PAO1 bacteremia model

[0099] Seven days after the third immunization with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV, mice were injected with wild-type Pseudomonas aeruginosa PAO1 strain (ATCC15692) via the tail vein at a dose of 5×10 6 CFU / mouse. The mice were observed for 14 consecutive days, and the survival and weight changes of the mice were recorded every day ( Figure 7 A). After infection with Pseudomonas aeruginosa blood, all mice in the control group died within three days after infection. The protection rates of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV after infection with a lethal dose of Pseudomonas aeruginosa blood reached 100% ( Figure 7 B- Figure 7 D). The body weight of mice in the three OMV-immunized groups showed a downward trend within two days after challenge, and began to gradually recover on the third day after challenge ( Figure 7 E). In order to detect the bacterial load in the lung and kidney tissues of each group of mice in the bacteremia model, the lung and kidney tissues of each group of mice were collected on the fourth day after the blood infection with a sublethal dose of Pseudomonas aeruginosa, and the bacterial load in each tissue was determined by the plate colony forming units (CFU) method. Compared with the control group, the bacterial load in the lung and kidney tissues of mice immunized with WT OMV, ΔhtrB1 OMV and ΔhtrB1&ΔtolB OMV was significantly reduced. Among them, the bacterial load in the lung and kidney tissues of mice immunized with ΔhtrB1OMV and ΔhtrB1&ΔtolB OMV was less than that in the tissues of mice immunized with WT OMV ( Figure 7 F- Figure 7 G). In addition, unlike the pathological changes in lung tissue of the control group, there were no substantive differences in the pathological changes in lung tissue of each OMV vaccination group ( Figure 7 H- Figure 7 I).

[0100] VII. Determination of the protective efficacy of OMV against the mouse Pseudomonas aeruginosa clinical strains PA0062815 and PA4029629 infection model

[0101] Seven days after the third immunization with different OMVs, mice were injected with Pseudomonas aeruginosa clinical strain PA0062815 through the tail vein at a dose of 5×10 6CFU / unit; PA4029629, dose 1×10 6 CFU / mouse. The mice were observed for 14 consecutive days, and the survival and weight changes of the mice were recorded every day ( Figure 8 A). After blood infection with a lethal dose of clinical strain PA0062815, all mice in the control group died within two days after infection, while the survival rates of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV all reached 100% ( Figure 8 B). The body weight of mice in the three OMV-immunized groups showed a downward trend within two days after challenge, and began to gradually recover on the third day after challenge ( Figure 8 C). After blood infection with a lethal dose of clinical strain PA4029629, all mice in the control group died within two days after infection, while the survival rates of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV were all 66.7% ( Figure 8 D). The body weight of mice in the three OMV-immunized groups showed a downward trend within six days after challenge, and began to gradually recover on the seventh day after challenge ( Figure 8 E) Compared with the control group, the bacterial load in the lung tissue of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV was significantly reduced ( Figure 8 F- Figure 8 G). In addition, unlike the pathological changes in lung tissue of the control group, there were no substantive differences in the pathological changes in lung tissue of each OMV vaccination group after blood infection with the two clinical strains ( Figure 8 H- Figure 8 K).

[0102] 8. Determination of the protective efficacy of OMV against Pseudomonas aeruginosa PAO1, PA0062815 and PA4029629 strains pneumonia model in mice

[0103] Seven days after the third immunization with different OMVs, mice were injected with PAO1 via the lung bronchi at a dose of 5×10 6 CFU / unit; PA0062815, dose 1×10 6 CFU / unit; PA4029629, dose 8×10 5 CFU / mouse. The mice were observed for 14 consecutive days, and the survival and weight changes of the mice were recorded every day ( Fig. 9 A). After infection with a lethal dose of PAO1 in the lungs and bronchi, the survival rate of mice in the control group was 16.7% within 14 days, the survival rate of mice immunized with WT OMV and ΔhtrB1 OMV was 83.3%, and the survival rate of mice immunized with ΔhtrB1&ΔtolB OMV was 100% ( Fig. 9B). After infection of the lungs and bronchi with a lethal dose of PA0062815, all mice in the control group died 10 days after infection. The survival rates of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV were 33.3%, 66.7%, and 83.3%, respectively ( Fig. 9 C). After infection of the lungs and bronchi with a lethal dose of the clinical strain PA4029629, all mice in the control group died within eight days after infection, while the survival rates of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV were all 100% ( Fig. 9 D). Compared with the control group, the bacterial load in the lung tissues of mice immunized with WT OMV, ΔhtrB1 OMV, and ΔhtrB1&ΔtolB OMV was significantly reduced ( Fig. 9 E- Fig. 9 G). In addition, unlike the pathological changes in lung tissue of the control group, there were no substantive differences in the pathological changes in lung tissue of each OMV vaccination group after bronchial infection with the two clinical strains ( Fig. 9 H- Fig. 9 M).

[0104] Embodiment 3:

[0105] This embodiment provides an application of bacterial outer membrane vesicles in the preparation of Pseudomonas aeruginosa vaccine.

[0106] In Example 1 of the present invention, an attenuated Pseudomonas aeruginosa PAO1 strain (ΔhtrB1 PAO1 strain) and an attenuated prolific Pseudomonas aeruginosa PAO1 strain (ΔhtrB1&ΔtolB PAO1 strain) were constructed, and in Example 2, WT OMV, ΔhtrB1 OMV and ΔhtrB1&ΔtolB OMV were induced. Animal experiments have verified that ΔhtrB1 OMV and ΔhtrB1&ΔtolBOMV can induce humoral immune responses and cell-mediated immune responses. Therefore, the bacterial outer membrane vesicles ΔhtrB1 OMV and ΔhtrB1&ΔtolB OMV induced by the present invention can be applied to the preparation of Pseudomonas aeruginosa vaccines, wherein the Pseudomonas aeruginosa vaccine can use adjuvants that activate Th1 or Th2 cell immunity to adsorb bacterial outer membrane vesicles. Specifically, the present invention uses aluminum hydroxide adjuvant to adsorb OMV. OMV can also be adsorbed using adjuvants such as CPG and alum.

[0107]

Claims

1. A bacterial outer membrane vesicle, characterized in that: The bacterial outer membrane vesicles are obtained by inducing expression and purifying a Pseudomonas aeruginosa recombinant expression vector; the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene on a wild-type Pseudomonas aeruginosa PAO1 strain; or the Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene and the tolB gene on a wild-type Pseudomonas aeruginosa PAO1 strain.

2. The bacterial outer membrane vesicle according to claim 1, characterized in that: The wild-type Pseudomonas aeruginosa PAO1 strain is an ATCC15692 strain.

3. The bacterial outer membrane vesicle according to claim 1, characterized in that: The Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene on the wild-type Pseudomonas aeruginosa PAO1 strain. The method for constructing the Pseudomonas aeruginosa recombinant expression vector comprises the following steps: S1: electroporate the pCasPA plasmid into the wild-type Pseudomonas aeruginosa PAO1 strain; S2: The electroporated strain was cultured on a tetracycline resistance plate; S3: The above bacterial solution was inoculated into tetracycline-resistant LB medium, and cultured until OD600 was 0.

6. Arabinose was added and cultured for 3 hours. The concentration of arabinose after addition was 2 mg / ml. S4: Collect the above bacterial solution, and electroporate the sgRNA and homology arms of the htrB1 gene into the above strain; S5: The electroporated strain was cultured on a tetracycline and carbenicillin resistance plate overnight. The next day, a single clone was picked and transferred to 5 mL of tetracycline and carbenicillin resistant LB medium and cultured overnight. The single clone with successful htrB1 gene knockout was identified by bacterial liquid PCR and genome sequencing to obtain the htrB1 gene knockout PAO1 strain.

4. The bacterial outer membrane vesicle according to claim 1, characterized in that: The Pseudomonas aeruginosa recombinant expression vector is obtained by knocking out the htrB1 gene and the tolB gene on the wild-type Pseudomonas aeruginosa PAO1 strain; the method for constructing the Pseudomonas aeruginosa recombinant expression vector comprises the following steps: S1: electroporate the pCasPA plasmid into the wild-type Pseudomonas aeruginosa PAO1 strain; S2: The electroporated strain was cultured on a tetracycline resistance plate; S3: The above bacterial solution was inoculated into tetracycline-resistant LB medium, and cultured until OD600 was 0.

6. Arabinose was added and cultured for 3 hours. The concentration of arabinose after addition was 2 mg / ml. S4: Collect the above bacterial solution, and electroporate the sgRNA and homology arms of the htrB1 gene into the above strain; S5: The electroporated strains were cultured on tetracycline and carbenicillin resistance plates overnight. On the second day, single clones were picked and transferred to 5 mL of tetracycline and carbenicillin resistance LB medium and cultured overnight. Single clones with successful htrB1 gene knockout were identified by bacterial liquid PCR and genome sequencing to obtain the htrB1 gene knockout PAO1 strain. S6: Knock out the tolB gene in the htrB1 gene knockout PAO1 strain according to steps S1 to S5 to obtain a PAO1 strain with both htrB1 and tolB genes knockout.

5. Use of bacterial outer membrane vesicles in the preparation of a Pseudomonas aeruginosa vaccine, wherein the Pseudomonas aeruginosa vaccine can induce humoral immune response and cell-mediated immune response.

6. The use according to claim 5, characterized in that: The Pseudomonas aeruginosa vaccine uses an adjuvant that activates Th1 or Th2 cell immunity to adsorb the bacterial outer membrane vesicles.

7. The use according to claim 6, characterized in that: The adjuvant includes one or more of CPG, alum and aluminum hydroxide.

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