A vaccine based on an outer membrane matrix of pseudomonas aeruginosa and its use

Abstract

The application provides a recombinant Pseudomonas aeruginosa cell adhesion protein CdrA-F1, and a preparation method and application thereof. The application predicts the three-dimensional structure, antigenicity, epitope distribution and other information of CdrA and the detection of the physicochemical properties of the recombinant protein by using a biological information analysis tool such as AlphaFold 2, and constructs and screens the gene recombinant protein CdrA-F1. The recombinant protein CdrA-F1 of the application is expressed in a soluble form in Escherichia coli.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine and immunology, and particularly relates to a preparation method and application of recombinant Pseudomonas aeruginosa cell adhesion factor CdrA protein. BACKGROUND

[0002] Pseudomonas aeruginosa (PA) is the main pathogenic bacterium of war trauma and burn infection, and is also one of the main pathogens of hospital acquired infection (HAI). According to the report of the Lancet in 2022, about 4.7 million people were infected by 33 kinds of bacteria (except Mycobacterium tuberculosis) worldwide in 2019, and about 7.7 million related deaths occurred, among which 560,000 people died of Pseudomonas aeruginosa, ranking the top four. According to the data of the National Bacterial Drug Resistance Monitoring Report, among the 4,929,000 bacteria included in the analysis in 2022, gram-negative bacteria accounted for 71.5%, and Pseudomonas aeruginosa accounted for 11.9% of gram-negative bacteria, ranking the top three. At the same time, PA also accounts for a large proportion in respiratory tract infections, and Pseudomonas aeruginosa accounts for 15.0% in burn wound infection pathogens, ranking the second. The prevention and control of its infection is an important public health problem worldwide.

[0003] Chronic persistent infection is one of the main characteristics of Pseudomonas aeruginosa infection, mainly manifested as chronic respiratory tract infection, chronic wound infection and chronic urinary system infection in the elderly, and the infection is stubborn and persistent, which is very difficult to treat. In the pathogen spectrum of hospital acquired pneumonia, PA accounts for 23.8-28.3% in patients aged 65 and above with hospital acquired pneumonia, ranking the first. In patients with ventilator-associated pneumonia (65 years and above), PA accounts for 27.6-34.6%, ranking the first. Globally, chronic wounds are common, and the global prevalence of mixed chronic wounds is estimated to be 2.21 cases per 1,000 people. According to the data in 2022, the number of elderly population aged 60 and above in China reached 267 million, accounting for 18.9% of the total population, and the number of elderly population aged 65 and above exceeded 200 million, accounting for 14.2% of the total population. It is predicted that by around 2035, the number of elderly population aged 60 and above will break through 400 million, accounting for more than 30%, and China will enter the stage of severe aging. The elderly are a high-risk group of bacterial infection and a good population of Pseudomonas aeruginosa infection, especially persistent chronic infection, which will bring serious medical and nursing burden to the society. Therefore, PA infection, especially chronic infection, is an urgent global public health problem.

[0004] While the number of PA infection cases remains high, the problem of antibiotic resistance is becoming increasingly serious, especially the emergence of multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains, making it very difficult to prevent and control PA infection. In February 2017, the World Health Organization (WHO) released the 'List of Priority Pathogens for Antibiotic Resistance' and listed Pseudomonas aeruginosa (PA) as the first level (the most critical level). In 2019, PA was one of the six major pathogens that caused 3.57 million drug-resistant related deaths. In May 2024, the World Health Organization (WHO) released its updated list of 'highly virulent and lethal superbugs' that pose the greatest threat to human health, the '2024 Bacterial Priority Pathogen Directory', which includes 15 drug-resistant bacteria, and PA has been listed as a 'high priority' priority pathogen. According to the '2022 National Bacterial Drug Resistance Detection Report', about 130,000 respiratory tract specimens were detected, and the total detection rate of Pseudomonas aeruginosa ranked third; the average drug resistance rate of Pseudomonas aeruginosa to carbapenems in China was 17.7%.

[0005] Biofilm is a structured bacterial population attached to the surface of living or non-living objects and wrapped by bacterial extracellular macromolecules. They are composed of extracellular polymeric substances (EPS) secreted by bacteria, including polysaccharides, proteins, nucleic acids, and lipid substances. Bacteria in biofilms can firmly adhere to lesions or object surfaces and can evade host immune responses, protect them from external environmental pressures, and hinder host phagocytic clearance, thereby achieving colonization and persistent infection. The antibiotic resistance of bacteria in biofilms is 1000 times higher than that of bacteria in planktonic state. According to data from the National Institutes of Health, biofilms are associated with more than 80% of microbial infections and more than 60% of hospital infections. The Centers for Disease Control and Prevention (CDC) estimates that biofilms are the cause of 60% of chronic infections. Pseudomonas aeruginosa is the second largest cause of these infections. The 'China Pseudomonas aeruginosa Infection Diagnosis and Treatment Expert Consensus' (2022 Edition) points out that the importance of biofilms has been found to be more important than previously recognized.

[0006] The pathogenic process of PA can be blocked by interfering with biofilm formation, slowing down the infectious diseases caused by PA. The formation of biofilm includes the initial adhesion of bacteria, the secretion of exopolysaccharide, the formation of microcolonies, the maturation and spread of biofilm, etc. Through the study of the development and life cycle of P. aeruginosa biofilm formation, it is found that when the bacteria initially aggregate and adhere, the bacteria and the object surface are reversibly adhered, and when the bacteria begin to secrete exopolysaccharide, it enters an irreversible adhesion state. Therefore, it is particularly important to intervene in the early stage of biofilm formation. CdrA protein is the first biofilm matrix protein identified in P. aeruginosa. As a cell adhesion factor, CdrA is transported to the extracellular by outer membrane transporter CdrB, and it is found to promote cell aggregation and adhesion in liquid culture. In CdrA gene deletion strains, biofilm formation defects can be observed. Studies have shown that CdrA protein has been proven to play an important role in cell aggregation and adhesion and biofilm structure stability as a biofilm structural matrix protein.

[0007] P. aeruginosa vaccine can prevent bacterial infection and reduce antibiotic use, which is the most economical and effective means of preventing and controlling PA infection. However, there is no successful P. aeruginosa vaccine on the market. Previous PA vaccines mostly used P. aeruginosa outer membrane proteins, lipopolysaccharides, toxins or flagella components as antigens. In preclinical studies or clinical trials, there were problems such as large differences in volunteer protection, poor stability, and few patient benefits. Structure-based vaccine design has been proven to have many advantages, as it can precisely target key antigen epitopes of pathogens, making vaccine design more precise. Based on structural information, vaccine antigens can be precisely designed to enhance the immunogenicity and drugability of vaccines and stimulate the immune system to produce efficient responses.

[0008] Previous PA vaccines ignored the important role of biofilm formation in PA infection, which may be one of the reasons for their failure. Therefore, there is an urgent need to study the formation process of biofilm to solve the high failure rate of PA vaccine development. SUMMARY

[0009] To address the above needs, this study focuses on the key protein CdrA in the PA biofilm formation process, predicts the three-dimensional structure, antigenicity, epitope distribution, etc. of CdrA using bioinformatics analysis tools such as AlphaFold 2, and designs and constructs a genetically engineered recombinant CdrA-F1 vaccine based on this information. The immunogenicity, protection and protection mechanism of the vaccine were studied in animal models, laying the foundation for the successful development of a new PA vaccine.

[0010] The present application first provides a recombinant protein based on Pseudomonas aeruginosa outer membrane matrix protein, which comprises a truncated peptide of CdrA protein, the amino acid sequence of the CdrA protein is SEQ ID NO: 1 or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 1, and the truncated peptide is selected from any one of 41Met-437Asp, 445Ala-824Thr, 824Thr-1148Thr and 1960Leu-2154Phe.

[0011] In one embodiment according to the present application, the amino acid sequence thereof is selected from any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or has at least 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; preferably, the amino acid sequence of the recombinant peptide is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0012] The present application also provides a gene encoding the above-mentioned recombinant peptide, the nucleotide sequence of which is selected from any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

[0013] The present application further provides an expression vector comprising an expression plasmid and the above-mentioned gene.

[0014] Preferably, the expression plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors, preferably pGEX-6P-1.

[0015] The present application also provides a recombinant engineering bacterium comprising the above-mentioned expression vector and a host bacterium; preferably, the host bacterium is selected from any one of E. coli XL1-blue strain, BL21 series strain and HMS174 series strain, preferably E. coli BL21 strain.

[0016] The present application also provides use of the above-mentioned recombinant protein, gene, expression vector or recombinant engineering bacterium in preparation of a preparation for detecting, preventing or treating Pseudomonas aeruginosa infection; preferably, the preparation is a subunit vaccine for preventing or treating Pseudomonas aeruginosa infection.

[0017] Another aspect of the present application provides a composition for preventing or treating Pseudomonas aeruginosa infection, which comprises the above-mentioned recombinant protein based on Pseudomonas aeruginosa outer membrane matrix protein and a pharmaceutically acceptable adjuvant.

[0018] The present application also provides a vaccine for preventing or treating P. aeruginosa infection, which contains the recombinant protein based on the outer membrane matrix protein of P. aeruginosa or the composition.

[0019] In one embodiment according to the present application, it comprises a pharmaceutically acceptable adjuvant, preferably the adjuvant is selected from any one of the adjuvants aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant and Mycobacterium BCG adjuvant.

[0020] The beneficial effects of the above technical solutions of the present application are as follows:

[0021] 1) The present study focuses on the key protein CdrA in the process of PA biofilm formation. The three-dimensional structure, antigenicity, epitope distribution and other information of CdrA are predicted by bioinformatics analysis tools such as AlphaFold 2, and based on this, the genetic engineering recombinant CdrA-F1 vaccine is designed and constructed.

[0022] 2) The recombinant protein CdrA-F1 of the present application is induced to express in E. coli in a soluble form, and is purified by GST filler affinity chromatography, size exclusion chromatography and other purification techniques to obtain a recombinant protein with stable properties in a buffer system, uniform particle size and purity greater than 90%.

[0023] 3) The CdrA-F1 vaccine molecule of the present application adopts a muscle injection immunization method, which can induce the production of specific IgG antibodies in mouse serum, not only can significantly inhibit the formation of PA biofilm, but also can exert high-efficiency immune protection effect. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 CdrA structure schematic diagram and antigenicity prediction result graph;

[0025] Figure 2 Expression identification and preparation SDS-PAGE graph of CdrA truncation;

[0026] Figure 3 Anti-CdrA-F1 total IgG and subtype titer;

[0027] Figure 4 CdrA-F1 immune mouse survival curve graph;

[0028] Figure 5 CdrA-F1 immune mouse weight change curve;

[0029] Figure 6 CdrA-F1 immune mouse wound healing;

[0030] Figure 7 The results of bacterial colonization in mice were detected by in vivo imaging;

[0031] Figure 8 The results of bacterial colonization in each organ of mice were detected;

[0032] Figure 9 The survival curve of anti-CdrA-F1 antibody immunized mice;

[0033] Figure 10 The results of in vitro inhibition of bacterial biofilm formation by anti-CdrA-F1 antibody;

[0034] Figure 11 The effect of anti-CdrA-F1 antibody on PA biofilm formation was observed under scanning electron microscope;

[0035] Figure 12 The effect of anti-CdrA-F1 antibody on PA biofilm formation was observed under laser confocal microscope;

[0036] Figure 13 Anti-CdrA-F1 antibody inhibits initial bacterial adhesion;

[0037] Figure 14 The results of detection of biofilm regulation related genes affected by anti-CdrA-F1 antibody. DETAILED DESCRIPTION

[0038] To make the technical problems, technical solutions and advantages of the present application clearer, specific embodiments will be described in detail below with reference to the drawings. These embodiments are provided to enable a more thorough understanding of the present application and to enable the scope of the present application to be fully conveyed to those skilled in the art.

[0039] As mentioned throughout the specification and claims, "comprising" or "including" is an open term, and should be interpreted to "including but not limited to". The subsequent description is provided as a preferred embodiment of the present application, and is intended to illustrate the general principles of the specification, and is not intended to limit the scope of the present application. The scope of protection of the present application is defined by the appended claims.

[0040] Unless otherwise specified, the reagents used in the present embodiment are of analytical purity, and the progress of all chemical reactions is detected by thin layer chromatography.

[0041] Example 1 Design of CdrA truncation of biofilm matrix protein

[0042] 1. Test method

[0043] Figure 1 Figure 1 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 2 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 1 Figure 1 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 2 shows the results of the prediction of CdrA antigenicity using DNA STAR.

[0044] 2. Test results

[0045] Biofilm matrix protein CdrA (PA4625) of Pseudomonas aeruginosa PAO1 is composed of 2154 amino acids, with a molecular weight of about 219 kDa, and is difficult to express in E. coli. In order to obtain CdrA truncates that can be efficiently prepared, the three-dimensional structure of CdrA was predicted by AlphaFold 2, and the results are shown in Figure 1. Figure 1 Figure 1 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 2 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 1 Figure 1 shows the results of the prediction of CdrA antigenicity using DNA STAR. Figure 2 shows the results of the prediction of CdrA antigenicity using DNA STAR.

[0046] Example 2 Expression identification and preparation of biofilm matrix protein CdrA truncates

[0047] 1. Test method

[0048] (1) The required primers were designed by means of Primer Premier 5.0 software, as shown in Table 1, and four recombinant plasmids were constructed by means of PCR technology. The plasmids were sent to Jin Kai Rui Biological Technology Co., Ltd. for sequencing, and the strains with correct sequencing were preserved.

[0049] (2) Take 20 μL of the aforementioned sequencing-corrected preservation bacteria, inoculate in a shaking flask containing 20 mL of LB medium, add ampicillin (2000x) 10 μL, and incubate at 37°C, 180 rpm overnight.

[0050] (3) Take 20 mL of the overnight cultured bacteria, inoculate in a shaking flask containing 2000 mL of LB medium, add ampicillin (2000x) 1000 μL, and incubate at 37°C, 180 rpm for 3.5 hours.

[0051] (4) Cool to 16°C, add IPTG (1M) 400 μL, and incubate overnight.

[0052] (5) Take out the overnight cultured bacteria, dispense in 50 mL centrifuge tubes, and centrifuge at 8500 rpm for 20 minutes.

[0053] (6) Resuspend the bacterial precipitate with PBS buffer (1X), and sonicate the cells with the following parameters: power 38%, on for 8 seconds and off for 9 seconds, for a total of 15 minutes.

[0054] (7) Centrifuge the bacteria solution after breaking the bacteria at low temperature with the following parameters: 12000 rpm, temperature 10°C, and centrifugation for 20 minutes.

[0055] (8) Dispense the supernatant, resuspend the precipitate with PBS buffer, and take 50 μL of each sample for subsequent detection.

[0056] (9) Take 10 mL of GST filler, and equilibrate with PBS buffer (1X) for 3 times.

[0057] (10) Mix the supernatant after breaking the bacteria solution with the GST filler, and place it on a horizontal suspension instrument for 3-4 hours at room temperature.

[0058] (11) Affinity chromatography purification of protein, the specific steps are as follows:

[0059] 1) Take the combined supernatant, and take 50 μL of each sample for subsequent detection.

[0060] 2) Rinse the filler with PBS buffer (1X) once, add PBS buffer to the same height as the filler, mix thoroughly, and take 50 μL of each sample for subsequent detection.

[0061] 3) Equilibrate the filler with high-salt PBS buffer (containing 1M sodium chloride) for 3 times to remove non-specific binding.

[0062] 4) Equilibrate the filler with PBS buffer (1X) for 3 times to remove the high-salt PBS buffer, add PBS buffer to the same height as the filler, mix thoroughly, and take 50 μL of each sample for subsequent detection.

[0063] 5) Add PBS buffer (1X) and PPase in the ratio of 1:1:0.2 of buffer:filler:PPase, place on a horizontal suspension instrument, and incubate overnight at 4°C.

[0064] 6) Aliquot the supernatant after enzyme digestion, and take 50 μL for subsequent detection.

[0065] 7) Add half the volume of PBS buffer to the lysis solution, mix, and then stand. Aliquot the supernatant, and take 50 μL for subsequent detection.

[0066] 8) Treat the filler with 0.1M sodium hydroxide solution, and then store the filler in 20% ethanol solution.

[0067] (12) Verify the protein and its purity by SDS-PAGE electrophoresis.

[0068] Table 1. Primer design table for recombinant CdrA truncates

[0069]

[0070] 2. Test results

[0071] Amplify the DNA sequences of CdrA-F1, CdrA-F2, CdrA-F3, and CdrA-F4, and connect them to the pGEX-6P-1 vector. Test the expression in E. coli induced by IPTG, and find that CdrA-F1 is expressed in a soluble form, and CdrA-F2, CdrA-F3, and CdrA-F4 are expressed in an inclusion body form. Figure 2 A). After the supernatant of the broken E. coli expressing CdrA-F1 is purified by affinity chromatography and ion exchange chromatography, it is detected by SDS-PAGE, and the relative molecular mass is about 40 kDa, which is consistent with the theoretical value. The purity is about 95%, which meets the standard for subsequent animal experiments. Figure 2 B,C).

[0072] Example 3. Immunogenicity detection of CdrA-F1 protein

[0073] 1. Test method

[0074] (1) Immunize mice (Balb / c mice, female, 6-8 weeks old) with the target protein at a dose of 20 mg per mouse, and inoculate by intramuscular injection. The immunization program is three injections at 0, 7, and 14 days.

[0075] (2) One day before each immunization, take the tail vein blood of the mice, and detect the antibody titer by enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA) (indirect method).

[0076] 1) Coating antigen: Dilute antigen to the optimal concentration with coating buffer, add 100 μL (6 μg) to each well of the enzyme-labeled plate, and incubate at 4°C overnight or at 37°C for 2-3 h.

[0077] 2) Washing plate: Remove the coating solution, and wash the plate with the washing buffer for 3 times, 5 min each time.

[0078] 3) Adding blocking solution: Add 200 μL of the blocking solution to each well, and incubate at 37°C for 2 h.

[0079] 4) Adding serum: Add 100 μL of the diluted serum to each well, and incubate at 37°C for 1-2 h.

[0080] 5) Washing plate: Remove the coating solution, and wash the plate with the washing buffer for 3 times, 5 min each time.

[0081] 6) Adding enzyme-labeled antibody: Add 0.1 mL of the freshly diluted enzyme-labeled antibody (dilution after titration) to each well, and incubate at 37°C for 0.5-1 h, and then wash the plate.

[0082] 7) Color development: Add 100 μL of TMB substrate solution to each well, and incubate at 37°C for 10-30 min.

[0083] 8) Stopping reaction: Add 50 μL of 2 M sulfuric acid to each well.

[0084] 9) Reading: On the enzyme-labeled instrument, measure the OD value of each well at 450 nm after setting the blank control well to zero.

[0085] 2. Test results

[0086] In order to evaluate the immunogenicity of CdrA-F1, the recombinant CdrA-F1 protein was mixed with AS03 adjuvant, and mice were immunized at days 0, 7, and 14. The anti-CdrA-F1 antibody titers were detected 1 day before immunization and 7 days after each immunization. As shown in Figure 3 the results, the serum specific antibody titers of the CdrA-F1 immunized mice significantly increased at day 7 after immunization, and continuously increased at days 14 and 21, with the geometric mean titers of 1:120000 and 1:8000000, respectively. These results show that CdrA-F1 has good immunogenicity.

[0087] In order to evaluate the type of immune response induced by CdrA-F1 in the AS03 preparation, the antibody titers of each subtype were detected using anti-IgG1, IgG2a, and IgG2b as the secondary antibodies for ELISA. The results are shown in Figure 3As shown, CdrA-F1 immunization was able to induce high titer of IgGl, IgG2a and IgG2b subtype antibodies, with significant difference from the PBS group. And the IgGl antibody titer was significantly higher than IgG2a and IgG2b P <0.0001), and the IgGl antibody titer was about 5 times of IgG2a. The above results showed that the Th2 type immune response, i.e. the humoral immune response, dominated in the immune response induced by CdrA-F1.

[0088] Example 4 CdrA-F1 protein protection effect detection

[0089] 1. Test method

[0090] (1) Agar beads-PAOl preparation

[0091] 1) The stock bacteria were plated, and single colony was picked and cultured by 2 times of activation.

[0092] 2) Centrifugation, and the absorbance was detected at wavelength of 600 nm.

[0093] 3) 5 OD amount of bacteria (5 / OD value) mL was taken and centrifuged.

[0094] 4) The supernatant was discarded, and 100 μL of normal saline was added for resuspension.

[0095] 5) 900 μL of LBA (LB + agar) was further added, and mixed evenly (note the temperature).

[0096] 6) The conical flask containing 120 mL of mineral oil was added by suspension (the conical flask was placed in a water bath with temperature of 45°C, and the vertical stirrer was placed in the conical flask, with rotation speed of 240 rpm and time of 30 min).

[0097] 7) The shaped beads were buried in ice, and placed on the shaker with rotation speed of 100 rpm / min and time of 30 min (or placed in the vertical stirrer with rotation speed of 80 rpm and time of 30 min).

[0098] 8) Allocated to 2 50 mL centrifuge tubes, and centrifuged with parameters of 3900 rpm / min and time of 15 min.

[0099] 9) The upper mineral oil was directly poured off.

[0100] 10) 10 mL of normal saline was added to each centrifuge tube for resuspension of the beads, and transferred to a new centrifuge tube, and centrifuged with 3900 rpm and time of 15 min.

[0101] 11) Carefully pipette the supernatant and resuspend the beads in 10 mL of normal saline and transfer to a new centrifuge tube, centrifuge at 3900 rpm for 15 min.

[0102] 12) Carefully pipette the supernatant and resuspend the beads in 10 mL of normal saline.

[0103] 13) Pipette 5 mL of the resuspension through a 100 μm filter screen, rinse the screen several times with normal saline, and recover the beads in a new 50 mL centrifuge tube using 3 mL of normal saline backwash. Repeat the process with the remaining 5 mL of resuspension and recover the beads into the same 50 mL centrifuge tube, to a total volume of 5-7 mL.

[0104] 14) Microscopy. Observe the beads under a microscope to ensure that the diameter is 100-200 μm.

[0105] (2) Mouse model of skin wound with chronic infection

[0106] 1) Adjust the bacterial concentration. Adjust the concentration of the agar bead-encapsulated PAOl bacterial solution (prepared in 2.2.4.5) to 5.0 x 104 CFUs / mL, 1.0 x 105 CFUs / mL, 2.5 x 105 CFUs / mL, 5.0 x 105 CFUs / mL, and 1.0 x 106 CFUs / mL using normal saline, and place on ice in the animal room for use.

[0107] 2) Anesthetize the animals using 1% (w / v) sodium pentobarbital at 6-7 μL / g (i.e., 120-140 μL for a 20 g mouse); observe the toe-pinch reflex after 5-7 min, and add 10-20% if the reflex is positive.

[0108] 3) Shave the back of the mouse, apply depilatory cream to further remove the hair, and wipe the depilatory cream off with a normal saline gauze. Disinfect the local skin with 75% alcohol, and cut a 10 x 10 mm square skin incision in the middle of the back. Apply the agar bead-encapsulated PAOl bacterial solution to the surface of the incision, and then cover the surface with a 3M breathable waterproof dressing to isolate the wound from external contamination. Inject carprofen long-acting analgesic (5 mg / kg) subcutaneously.

[0109] 4) After the mouse has recovered, return it to the cage.

[0110] (3) Survival rate

[0111] Observe the survival of the mice with skin wounds with chronic infection (immune group and negative control group) daily for 10 consecutive days.

[0112] 2. Test results

[0113] To explore the protective effect of CdrA-F1 protein, the mice immunized with CdrA-F1 protein (immunization procedure: 0, 7, 14 days, dosage: 20 μg per mouse) were used, the dorsal median 10x10mm square skin was excised, and the agar beads wrapped with PAO1 bacterial solution at a lethal dose (1.0x106 CFUs) was inoculated, and the survival of the immunized group and the control group was observed for 10 days. The results are shown in Figure 4 The control group began to die on the 2nd day after inoculation, and continued to die on the 3rd and 4th days, and the mortality rate of the mice was 100% during the observation period. No death was found in the CdrA-F1 immunized group during the observation period, and the statistical analysis results showed that the survival time of the CdrA-F1 immunized mice was significantly higher than that of the control group (P<0.0001). The above results showed that CdrA-F1 had good immune protection effect in the mouse skin wound infection model. P

[0114] Example 5: CdrA-F1 immunization significantly reduces the symptoms of infection in the mouse skin wound infection model

[0115] 1. Test method

[0116] The agar beads-PAO1 preparation and the operation steps of the mouse dorsal skin wound infection model were the same as in Example 4, and the body weight changes of the mice with chronic skin wound infection (immunized group and negative control group) were observed daily for 10 consecutive days.

[0117] 2. Test results

[0118] To further clarify the protective effect of CdrA-F1 protein, the mice immunized with CdrA-F1 protein were smeared with agar beads containing sub-lethal dose of PAO1 (1.0x10 5 CFUs) on the back wound, and the body weight changes were monitored daily. The results are shown in Figure 5 The body weight of the immunized mice began to recover on the 3rd day after challenge, and the body weight had recovered to the pre-challenge level by the end of the 7th day of observation. The body weight of the control group decreased rapidly after challenge, and the decrease slowed down after the 3rd day, and there was no recovery trend during the entire observation period.

[0119] Example 6: CdrA-F1 immunization significantly reduces the wound infection in the mouse skin wound infection model

[0120] 1. Test method

[0121] To further evaluate the protective effect of CdrA-F1 protein immunization on bacterial infection in the wound, the mice were smeared with sub-lethal dose of PAO1 (1.0x10 5 ​Wound area was measured daily and healing was recorded after infection with CFUs) agar beads, and the percentage of unhealed area to the initial wound area was calculated.

[0122] 2. Test results

[0123] Results as shown in Figure 6 A, on the first day after challenge, the back wounds of mice in both groups showed changes of bacterial infection such as hyperemia and exudation. On the 5th day, the back wounds of mice in the immunized group were significantly reduced, and at the end of the 10-day observation period, only a very small area of the wound remained unhealed. On the 5th day, the back wounds of mice in the control group not only did not heal, but the infection worsened, with the wound surface appearing dark red to grayish white and secreting more. On the 10th day, the infection changes were more obvious, with the wound ulcerating, covered with pus, and the wound area also showing a tendency to expand. The data on the percentage of unhealed area to the initial wound area showed that the percentage of mice in the immunized group decreased significantly over time, while the percentage of mice in the control group did not change significantly. Figure 6 B). The above results show that CdrA-F1 protein immunization can exert a protective effect in the local wound.

[0124] Example 7 CdrA-F1 immunization reduces bacterial colonization in a mouse skin wound infection model

[0125] 1. Test method

[0126] The skin incision surface of mice in the immunized and negative control groups was coated with agar beads wrapped with NanoLuc luciferase-labeled PAO1 bacterial solution. On the 1st, 5th, and 10th days after modeling, a small animal live imaging system was used to take photos and quantify fluorescence to evaluate bacterial colonization and clearance.

[0127] 2. Test results

[0128] To further verify the protective effect of CdrA-F1 protein immunization in the local wound, in this study, Nanoluc luciferase-labeled PAO1-agar beads were used to coat the wound in a mouse skin wound model, and on the 1st, 5th, and 10th days, the bacterial fluorescence intensity was detected by live imaging to monitor the bacterial colonization in the local wound. Results as shown in Figure 7 On the 1st day, both the control and immunized groups detected fluorescent bacteria, indicating successful modeling. On the 5th day, the fluorescence intensity of the control group increased significantly, indicating that the bacteria were proliferating in the wound and their number was increasing, while the immunized group had almost no fluorescence, indicating that the bacterial amount was small and the infection had been controlled. On the 10th day, the fluorescence intensity of the control group continued to increase, indicating that the number of local bacteria further increased and the infection further worsened, which was consistent with the observation trend of local symptoms. The above results show that CdrA-F1 protein immunization has a good protective effect in the local wound.

[0129] Example 8 CdrA-F1 immunization significantly reduces organ bacterial colonization and inflammation in a mouse skin wound infection model

[0130] 1. Test method

[0131] On the 1st, 5th, 10th day after modeling, 3 mice were taken from each of the immunization group and the negative control group, blood was taken by enucleation, and after soaking in 75% alcohol, the mice were dissected, the complete spleen, lung, and liver tissues were taken out, placed in a sterile dish, and weighed. Then the organs were placed in a high-pressure sterile glass homogenizer, 1 mL of sterile PBS was added, and it was ground thoroughly. After grinding, 100 μl of homogenate was diluted with PBS at a ratio of 1:10, 1:10 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 , 1:10. After mixing, 10 μl of homogenate was taken from each dilution and dropped on the top of the solid LB square dish. Then it was tilted and left naturally (note that the bacterial solution should not touch the upper and lower edges of the square dish to avoid affecting subsequent counting). It was placed in a 37°C incubator and incubated overnight. The appropriate dilution ratio was selected for colony counting.

[0132] 2. Test results

[0133] To further study the immune protection mechanism of CdrA-F1 protein, the number of bacterial colonization in organs was measured on the 1st, 5th, 10th day after infection in the mouse skin wound model, and histopathological changes were observed. The results are shown in Figure 8 On the 1st day, bacterial colonization was observed in the liver, spleen, and lung of both groups of mice, but the number of bacteria in the control group was significantly higher than that in the immunization group. On the 5th and 10th day, the number of bacteria in the control group continued to increase. While in the immunization group, the number of bacteria in the organs decreased on the 5th day, and on the 10th day, only a small amount of bacteria was detected in the lung, while no bacteria was detected in the spleen and liver.

[0134] Example 9 Anti-CdrA-F1 antibody significantly improves the survival rate of mice in a mouse skin wound infection model

[0135] 1. Test method

[0136] In view of the fact that CdrA-F1 protein induces Th2-type immune response and the characteristics of P. aeruginosa anti-infection immunity, it is speculated that specific antibodies against CdrA-F1 are the key substances for its immune protection. To verify this hypothesis, anti-CdrA-F1 antibodies in mouse immune serum were purified and prepared, and the above-mentioned mouse skin wound model was used for inoculation with a lethal dose (1.0 x 10 6Agar beads of PAOl CFUs) and then administered with anti-CdrA-F1 antibody, and the survival of the mice was observed.

[0137] 2. Test results

[0138] The results are shown in Table 1. Figure 9 As shown in Table 1, the PBS group had 100% mortality on the 2nd day after inoculation, and no mice survived on the 4th day. The blank serum group had 3 deaths on the 2nd day, and the remaining 7 mice all died on the 3rd day. The mortality rate of the above two groups of mice was 100% within the observation period. The anti-CdrA-F1 antibody immunized group had 1 death on the 2nd day, and the remaining 9 mice were not found to have died at the end of the 10-day observation period. Statistical analysis showed that the survival time of the mice inoculated with anti-CdrA-F1 antibody was significantly higher than that of the control group. The above results show that the anti-CdrA-F1 antibody has good immunoprotective effect on the skin wound infection model of mice, and no non-specific antibody serum of mice was found to have immunoprotective effect on the skin wound infection.

[0139] Example 10 Anti-CdrA-F1 antibody inhibits Pseudomonas aeruginosa biofilm formation in vitro

[0140] 1. Test method

[0141] (1) Add 100 μL of culture solution to each well of a 96-well polystyrene microplate, inoculate 10 μL of overnight culture solution, and incubate at 37°C for 36 h;

[0142] (2) Aspirate the culture solution, and wash the plate wells with 200 μL of sterile PBS buffer three times per well;

[0143] (3) Add 100 μL of methanol to each well and incubate for 15 min, then aspirate the methanol from the culture wells and air dry naturally;

[0144] (4) Add 100 μL of 1% crystal violet solution to each well, and stain at room temperature for 5 min;

[0145] (5) After aspirating the crystal violet staining solution from the culture wells, rinse off the excess dye with running water;

[0146] (6) Invert the plate on filter paper to remove residual water, and dry in a 37°C oven or air dry at room temperature;

[0147] (7) After complete drying, add 100 μL of 33% glacial acetic acid solution to each well, and incubate in a 37°C incubator for 30 min to dissolve the crystal violet;

[0148] (8) Measure the OD value of the solution in the culture wells at 590 nm using a microplate reader;

[0149] (9) For each strain in each experiment, three replicates were made for each well, and the experimental values were the average of three measurements (D value).

[0150] (10) The culture medium without inoculated bacteria was used as the negative control, and twice the negative value was used as the cut-off value (Dc).

[0151] Result determination: Based on the D value, the strains can be divided into three categories: (1) Strong biofilm-forming strains (D > 2 × Dc); (2) Weak biofilm-forming strains (Dc < D ≤ 2 × Dc); (3) Non-biofilm-forming strains (D ≤ Dc).

[0152] 2. Test results

[0153] The promotion of biofilm development and maturation by CdrA protein is a key factor in the pathogenesis of Pseudomonas aeruginosa infection. Therefore, it is speculated that anti-CdrA-F1 antibody may exert a protective effect by inhibiting bacterial biofilm formation. To verify this hypothesis, in this study, the biofilm formation of PAO1 was detected by crystal violet staining method under in vitro culture conditions containing different concentrations of anti-CdrA-F1 antibody. The results are as Figure 10 shown in Figure A. The OD values after crystal violet decolorization in the PBS control group and the blank mouse serum control group were both around 0.8, indicating that obvious biofilms were formed (an intuitive figure can be added). In contrast, after adding serums of anti-CdrA-F1 antibody diluted in different ratios, the OD 590 values were significantly decreased compared with the control group, and with the increase of antibody concentration, the OD 590 values showed a downward trend, and the difference was statistically significant ( 590 < 0.0001). To evaluate whether anti-CdrA-F1 antibody directly affects bacterial growth, in this study, the time-growth curve of PAO1 was measured, and the area under the curve (AUC) was calculated. The results are as P shown in Figure B. In the culture medium containing different concentrations of anti-CdrA-F1 antibody, there was no significant statistical difference in the AUC of the growth curve of PAO1 compared with the PBS group and the blank serum group, indicating that anti-CdrA-F1 antibody does not affect bacterial growth. The above results show that in vitro, anti-CdrA-F1 antibody does not affect the growth and proliferation of Pseudomonas aeruginosa PAO1, but inhibits biofilm formation, and this inhibitory effect is concentration-dependent. Figure 10

[0154] Example 11 Observation of the effect of anti-CdrA-F1 antibody on PA biofilm formation by scanning electron microscopy and confocal microscopy

[0155] <000036,2>1. Test method

[0156] (1) Scanning electron microscopy method [[ID= ,34]]

[0157] In sterile 6-well cell culture plates, each well is placed in a sterile round cover glass with a diameter of 10 mm; 1.0 mL of bacterial solution and serum is added, and incubated at 37°C for 36 h. Then sampling, washing, fixing, dehydrating, drying, sticking, and conducting treatment are carried out, and the specific steps are as follows:

[0158] 1) Sampling: the round cover glass in the culture hole is carefully taken out to avoid pulling, squeezing and damaging the sample.

[0159] 2) Washing: the sample is slowly and carefully washed with PBS buffer (1X) for 3 times to remove the impurities on the observation surface, and the observation surface is fully exposed under the condition of not damaging the observation surface.

[0160] 3) Fixing: the sample is fixed at 4°C for 4 h using 2.5% glutaraldehyde, the fixing solution is then absorbed, PBS buffer (1X) is added, and the sample is rinsed for 1 h with 3-4 times of liquid change.

[0161] 4) Dehydrating: the buffer solution is absorbed, and 30%, 50%, 70%, 80%, 90%, 95% and anhydrous ethanol are sequentially used for gradient dehydration, and each step stays for 15 min. Then 30%, 50%, 70%, 80%, 90%, 95% and anhydrous tert-butanol are sequentially used for gradient dehydration, and each step stays for 15 min.

[0162] 5) Drying: critical point drying instrument is used to prevent the distortion of the surface morphology caused by water evaporation, and it is necessary to ensure that the drying is complete.

[0163] 6) Sticking: a small amount of conductive glue is applied to the sample stage with a toothpick, the glue surface should be slightly smaller than the sample surface, the sample is gently clamped with tweezers, the observation surface is ensured to be upwardly and firmly attached to the glue, and after the conductive glue is dry, vacuum coating and SEM inspection are carried out.

[0164] 7) Conducting treatment: biological samples are composed of low atomic number elements such as carbon, hydrogen, oxygen and nitrogen, and the secondary electron emission rate is very low, so the signal is weak, and it is difficult to obtain the necessary image contrast. Before the biological sample is observed by scanning electron microscope, surface conductive treatment is required.

[0165] (2) Laser confocal method

[0166] In a sterile glass bottom confocal culture dish, 1.0 mL of BFP-labeled PAO1 bacterial solution and different dilution ratios of anti-CdrA-F1 antibody-containing serum are added, and incubated at 37°C for 36 h. Then the supernatant bacterial solution is slowly absorbed, and the sample is slowly and carefully washed with PBS buffer (1X) for 3 times to remove the impurities under the condition of not damaging the observation surface. Then the biological film formation is observed by using a confocal laser scanning microscope.

[0167] 2. Test results

[0168] Results as shown in Figure 11 Figure 6 shows that after 1 day of culture, the PBS group and the blank mouse serum group had a large number of bacteria adhering to the surface of the glass slide, and there was a large amount of mucus-like biofilm extracellular polysaccharide between the bacteria, which wrapped the bacteria and firmly adhered to the surface of the object. The serum group containing 1:20 dilution of anti-CdrA-F1 antibody showed that although the number of bacteria was large and the growth state was good, there was less mucus-like substance between the bacteria, which was in sharp contrast to the PBS group and the blank serum group. After 4 days of culture, the number of bacteria in the PBS group and the blank serum group increased significantly compared to 1 day of culture, and the mucus-like substance also continued to increase, and the biofilm further developed, with the bacteria overlapping and adhering to each other to form a tight aggregate. Although the number of bacteria in the experimental group containing the antibody serum further increased, no obvious biofilm formation was observed, and the bacteria mechanically overlapped each other, with an unstable morphology. After 7 days of culture, the mucus-like extracellular polysaccharide between the bacteria in the PBS group and the blank serum group further increased compared to before, and the texture was more compact and tough. At the same time, the number of bacteria in the experimental group continued to increase, and the number of layers of bacteria increased, but there was no sign of spreading, and no obvious biofilm formation was observed. This also suggests that the ability of bacteria to spread along the surface is limited without obvious biofilm formation. The change trend of the serum group containing 1:10 dilution of anti-CdrA-F1 antibody was similar to the 1:20 dilution group, showing that the bacteria grew and reproduced at the initial adhesion site, accumulated continuously, but did not form a biofilm, and could not spread and extend on the surface of the glass slide. However, under the 1:5 dilution ratio, it was observed that the bacteria still did not form a biofilm, and unlike the other two high dilution groups, the number of bacteria observed under the microscope was less after 1 day of culture, and after 4 days and 7 days of culture, the bacteria showed normal growth and reproduction, but the outline between the bacteria was clear and the connection was loose, and no biofilm was formed, which suggests that under this dilution ratio, the anti-CdrA-F1 antibody inhibited the adhesion and biofilm formation and spreading ability of P. aeruginosa at the initial adhesion stage.

[0169] Figure 12 As shown in Figure 7, further detection by laser confocal microscopy found that the PBS group and the blank serum group were similar, and the thickness of the biofilm increased continuously during the observation period, reaching 30 ± 3 μm on the first day, 70 ± 5 μm on the fourth day, and about 120 μm on the seventh day. The biofilm thickness of the serum group containing anti-CdrA-F1 antibody was significantly thinner than that of the control group, with a thickness of 18 ± 3 μm on the first day, 35 ± 4 μm on the fourth day, and about 38 ± 2 μm on the seventh day. There was no significant statistical difference between different concentrations of antibody groups at the same time point, but the experimental group of anti-CdrA-F1 antibody had a significant statistical difference compared to the PBS group and the blank serum group (P < 0.0001). P <0.0001).

[0170] Combined results from scanning electron microscopy and laser confocal microscopy indicate that the anti-CdrA-F1 antibody can inhibit the formation of Pseudomonas aeruginosa PAO1 biofilm in vitro, and that higher concentrations of the antibody can inhibit the initial adhesion of bacteria to object surfaces.

[0171] Example 12 Anti-CdrA-F1 antibody inhibits initial adhesion of Pseudomonas aeruginosa

[0172] 1. Test Methods

[0173] Pseudomonas aeruginosa was cultured overnight using a shaker. 200 μL of bacterial suspension containing different dilutions of anti-CdrA-F1 antibody serum was added to each well of a 96-well microplate, with three parallel wells for each concentration. The plates were incubated at 37°C for 2 h. After carefully removing airborne bacteria, 200 μL of 1× PBS buffer was added to each well and thoroughly mixed using a micropipette. The plates were then diluted sequentially to 1×10, 1×100, 1×1000, and 1×10000 times. 10 μL of each diluted buffer was titrated in a square petri dish containing solid LB broth and incubated at 37°C for 12 h. Samples with colony counts between 30 and 300 CFUs were used for colony counting to assess the inhibitory effect of the antibody-containing serum on initial bacterial adhesion.

[0174] 2. Experimental Results

[0175] The CdrA protein of *Pseudomonas aeruginosa* promotes biofilm formation by mediating initial bacterial adhesion. To further clarify whether anti-CdrA-F1 antibodies can inhibit initial bacterial adhesion, this study counted and statistically analyzed the initial adhesion bacteria under different culture conditions. The results are as follows: Figure 13 As shown, after 3 hours of incubation, the initial number of bacteria adhering to both the PBS group and the blank serum group was 1×10⁻⁶. 6 CFUs. In comparison, the serum group containing anti-CdrA-F1 antibody showed a significantly reduced amount of bacteria adhering to the surface of the culture dish. P The anti-CdrA-F1 antibody showed a decrease of <0.0001, and the 1:10 serial dilution group was further reduced compared to the 1:20 dilution group (P<0.0001). These results suggest that the anti-CdrA-F1 antibody can inhibit the initial adhesion of Pseudomonas aeruginosa PAO1.

[0176] Example 13: Anti-CdrA-F1 antibody inhibits the transcriptional level of genes related to the formation of extracellular polysaccharides in Pseudomonas aeruginosa PAO1 biofilm.

[0177] 1. Test Methods

[0178] (1) Extraction of total bacterial RNA (FastPure® Cell / Tissue Total RNA Isolation Kit, RC112, Vazyme)

[0179] 1) Sample lysis: centrifuge to collect bacteria directly, add Buffer RL, 500 μl Buffer RL per <5x106 cells, vortex until no obvious cell mass.

[0180] 2) Transfer the lysed sample to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 xg) for 30 sec. Discard the FastPure gDNA-Filter Columns III and collect the filtrate.

[0181] 3) Add 0.5 times the volume of anhydrous ethanol to the filtrate, mix well;

[0182] 4) Transfer the mixture of step 3 to FastPure RNA Columns III (FastPure RNA Columns III has been placed in the collection tube), centrifuge at 12,000 rpm (13,400 xg) for 30 sec, discard the filtrate.

[0183] 5) Add 700 μl Buffer RW1 to the FastPure RNA Columns III, centrifuge at 12,000 rpm (13,400 xg) for 30 sec, discard the filtrate.

[0184] 6) Add 700 μl Buffer RW2 (with anhydrous ethanol added) to the FastPure RNA Columns III, centrifuge at 12,000 rpm (13,400 xg) for 30 sec, discard the filtrate.

[0185] 7) Add 500 μl Buffer RW2 (with anhydrous ethanol added) to the FastPure RNA Columns III, centrifuge at 12,000 rpm (13,400 xg) for 2 min, carefully remove the adsorption column from the collection tube to avoid contamination of the filtrate.

[0186] 8) If there is residual liquid in the adsorption column or it has been in contact with the filtrate, discard the filtrate, place the FastPure RNA Columns III back into the collection tube, centrifuge at 12,000 rpm (13,400 xg) for 1 min to prevent contamination by ethanol.

[0187] 9) Carefully transfer the column to a new RNase-free Collection Tubes 1.5 mL centrifuge tube, add 50-200 μl RNase-free ddH2O to the center of the column, let stand for 1 min at room temperature, centrifuge at 12,000 rpm (13,400 xg) for 1 min to elute the RNA.

[0188] 10) Determine the concentration of RNA, the extracted total RNA can be directly used for downstream experiments or stored at -80°C.

[0189] (2) Synthesize cDNA (HiScript® III RT SuperMix for qPCR (+gDNA wiper), Vazyme)

[0190] 1) Remove genomic DNA.

[0191] 2) Prepare the reverse transcription reaction system, directly add 5 × HiScript III qRT SuperMix to the reaction tube of step 1.

[0192] (3) Perform RT-qPCR reaction (ChamQ Universal SYBR qPCR MasterMix, Vazyme) according to Table 2, 3, 4 parameters.

[0193] Table 2 RT-qPCR reaction gene primer sequence

[0194]

[0195] 1) Prepare the following mixture in a qPCR tube

[0196] Table 3 reaction system

[0197]

[0198] 2) Perform qPCR reaction under the following conditions:

[0199] Table 4 program settings

[0200]

[0201] 3) Finally collect the fluorescence signal.

[0202] 2. Test results

[0203] The main component of biofilms is extracellular polysaccharides. The matrix protein CdrA strengthens the biofilm structure by interacting with the extracellular polysaccharides Pel and Psl. Meanwhile, the quorum sensing system of Pseudomonas aeruginosa is involved in regulating the formation and development of biofilms. Therefore, it is hypothesized that anti-CdrA-F1 antibodies can inhibit the expression of key genes in biofilm formation. To verify this hypothesis, this study used RT-PCR to detect changes in the transcriptional levels of PAO1-regulated genes related to biofilm extracellular polysaccharide formation (algD, algU, pelA, pslA) and genes related to quorum sensing system regulation (lasR, lasI, rhlR, rhlI).

[0204] The results are as follows Figure 14 As shown, on day 1, compared with the control group, the transcriptional levels of biofilm formation-related genes algD, algU, pelA, and pslA were significantly decreased, decreasing to 0.4-fold, 0.3-fold, 0.4-fold, and 0.3-fold of the control group, respectively. However, the transcriptional levels of quorum sensing regulation-related genes lasR, lasI, rhlR, and rhlI did not show significant changes. P >0.05). The same trend was observed on days 4 and 7. These results indicate that the anti-CdrA-F1 antibody inhibits biofilm formation by suppressing the expression of Pseudomonas aeruginosa PAO1 algD, algU, pelA, and pslA.

[0205] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant protein based on Pseudomonas aeruginosa outer membrane matrix protein, which is a truncated peptide CdrA-F1 of CdrA, and has an amino acid sequence of SEQ ID NO:

3.

2. A gene encoding the recombinant peptide of claim 1, which has a nucleotide sequence of SEQ ID NO:

7.

3. An expression vector comprising an expression plasmid and the gene of claim 2.

4. The expression vector of claim 3, wherein, The expression plasmid is selected from any one of pGEX series vectors, pET series vectors, or pQE series vectors.

5. The expression vector of claim 4, wherein, The expression plasmid is pGEX-6P-1.

6. A host cell, characterized in that, The expression vector of claim 3 is included.

7. The host cell of claim 6, wherein The host bacteria are selected from any one of E. coli XL1-blue strain, BL21 series strains, and HMS174 series strains.

8. The host cell of claim 7, wherein The host bacteria are E. coli BL21 strain.

9. Use of the recombinant protein of claim 1, or the gene of claim 2, or the expression vector of claim 3, in the preparation of a preparation for preventing Pseudomonas aeruginosa infection.

10. Use according to claim 9, wherein The preparation is a subunit vaccine for preventing Pseudomonas aeruginosa infection.

11. A composition for preventing Pseudomonas aeruginosa infection, characterized by, The recombinant protein based on Pseudomonas aeruginosa outer membrane matrix protein of claim 1, and a pharmaceutically acceptable adjuvant are included.

12. A vaccine for preventing Pseudomonas aeruginosa infection, characterized by, The recombinant protein based on Pseudomonas aeruginosa outer membrane matrix protein of claim 1, or the composition of claim 11 is included.

13. The vaccine as described in claim 12, characterized in that, A pharmaceutically acceptable adjuvant is included.

14. The vaccine as described in claim 13, characterized in that, The adjuvant is selected from any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, aluminum monostearate adjuvant, MF59, complete Freund's adjuvant, incomplete Freund's adjuvant, and Mycobacterium bovis bacillus Calmette-Guerin adjuvant.

Images