Pseudomonas aeruginosa vaccine based on outer membrane matrix and application of pseudomonas aeruginosa vaccine

Abstract

The invention provides a recombinant pseudomonas aeruginosa cell adhesin protein CdA-F1 as well as a preparation method and application of the recombinant pseudomonas aeruginosa cell adhesin protein CdA-F1. According to the present invention, AlphaFold 2 and other biological information analysis tools are adopted to predict the three-dimensional structure, the antigenicity, the epitope distribution and other information of CCdA, detect the physicochemical properties of the recombinant protein and the like, and construct and screen to obtain the gene recombinant protein CCdA-F1; the recombinant protein CdA-F1 disclosed by the invention is subjected to induced expression in escherichia coli in a soluble form.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and immunology, and in particular to a preparation method and application of a recombinant Pseudomonas aeruginosa cell adhesin CdrA protein. Background Art

[0002] Pseudomonas aeruginosa (PA) is a major pathogen causing infections following wartime trauma and burns, and is also a leading cause of hospital-acquired infections (HAIs). The Lancet reported in 2022 that in 2019 alone, 33 bacterial species (excluding Mycobacterium tuberculosis) infected approximately 470 million people worldwide and resulted in approximately 7.7 million related deaths. Of these, P. aeruginosa caused 560,000 deaths, ranking among the top four. According to the National Bacterial Antimicrobial Resistance Surveillance Report, Gram-negative bacteria accounted for 71.5% of the 4.929 million bacterial strains analyzed in 2022, with P. aeruginosa accounting for 11.9% of these Gram-negative bacteria, ranking among the top three. PA also accounts for a significant proportion of respiratory infections. Among the pathogens causing burn wound infections, P. aeruginosa accounts for 15.0%, ranking second. The prevention and control of PA infections is a major public health issue worldwide.

[0003] Chronic persistent infection is one of the main characteristics of Pseudomonas aeruginosa infection, which is mainly manifested in chronic respiratory tract infections, chronic wound infections and chronic urinary tract infections in the elderly. The infection is stubborn and persistent and very difficult to treat. In the pathogen spectrum of hospital-acquired pneumonia in my country, PA accounts for 23.8-28.3% of patients with hospital-acquired pneumonia aged 65 and above, ranking first. It accounts for 27.6-34.6% of patients with ventilator-associated pneumonia (65 years old and above), ranking first. Globally, chronic trauma is common, and the global prevalence of mixed chronic trauma is estimated to be 2.21 cases per 1,000 people. According to data from 2022, the number of elderly people aged 60 and above in China reached 267 million, accounting for 18.9% of the total population, while the number of elderly people aged 65 and above exceeded 200 million, accounting for 14.2% of the total population. It is estimated that by around 2035, the number of people aged 60 and above will exceed 400 million, accounting for over 30% of the total population. By then, China will have entered a period of advanced aging. The elderly are at high risk for bacterial infections and are particularly susceptible to Pseudomonas aeruginosa infections. Persistent chronic infections, in particular, impose a significant burden on society in terms of medical care and nursing care. Therefore, PA infections, especially chronic ones, are a global public health issue that urgently needs to be addressed.

[0004] While the number of PA infections remains high, the problem of antibiotic resistance is becoming increasingly serious, especially with the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, making the prevention and control of PA infections extremely difficult. In February 2017, the World Health Organization (WHO) released the "List of Priority Pathogens of Antibiotic Resistance," which classified Pseudomonas aeruginosa (PA) as Level 1 (the most critical level). In 2019, PA was one of the six main pathogens responsible for 3.57 million resistance-related deaths. In May 2024, the World Health Organization (WHO) released its updated list of "highly virulent and highly lethal superbugs" that pose the greatest threat to human health, the "2024 List of Priority Bacterial Pathogens." The list includes 15 drug-resistant bacteria, among which PA has been listed as a "high priority" pathogen. According to the "2022 National Bacterial Resistance Detection Report", approximately 130,000 respiratory specimens were tested, and the total detection rate of Pseudomonas aeruginosa ranked third; the average resistance rate of Pseudomonas aeruginosa to carbapenems in my country is 17.7%.

[0005] Biofilms are organized bacterial communities encased in extracellular macromolecules attached to the surfaces of living or inanimate objects. They are composed of extracellular polymeric substances (EPS) secreted by bacteria, including polysaccharides, proteins, nucleic acids, and lipids. Bacteria within biofilms can firmly adhere to lesions or surfaces and evade the host's immune response, protecting them from external environmental stressors and hindering host phagocytosis and clearance, thereby achieving colonization and persistent infection. Furthermore, bacteria within biofilms are 1,000 times more resistant to antibiotics than bacteria in a planktonic state. According to the National Institutes of Health, biofilms are associated with more than 80% of microbial infections and more than 60% of nosocomial infections. The Centers for Disease Control (CDC) estimates that biofilms are the pathogens of 60% of chronic infections. Pseudomonas aeruginosa is the second leading cause of these infections. The "Chinese Expert Consensus on the Diagnosis and Treatment of Pseudomonas aeruginosa Infections" (2022 edition) states that the importance of biofilms is now greater than previously recognized.

[0006] Intervention of biofilm formation can block the pathogenicity of PA and mitigate the infectious diseases caused by PA. The biofilm formation process is divided into initial bacterial adhesion, exopolysaccharide secretion, microcolony formation, biofilm maturation, and spread. Studies on the formation, development, and life cycle of Pseudomonas aeruginosa biofilms have revealed that during initial aggregation and adhesion, the bacteria reversibly adhere to the surface, but once the bacteria begin to secrete exopolysaccharides, they enter an irreversible adhesion state. Therefore, intervention during the early stages of biofilm formation is particularly important. CdrA protein was the first biofilm matrix protein identified in Pseudomonas aeruginosa. As a cell adhesion protein, CdrA is transported to the extracellular space by the outer membrane transporter CdrB. In liquid culture, it has been shown to promote cell aggregation and adhesion. Defects in biofilm formation have been observed in strains lacking the CdrA gene. Furthermore, studies have shown that CdrA protein plays an important role as a biofilm structural matrix protein in cell aggregation, adhesion, and biofilm stability.

[0007] Pseudomonas aeruginosa vaccines can prevent bacterial infections and reduce the use of antibiotics. They are the most cost-effective means of preventing and controlling PA infections. However, no Pseudomonas aeruginosa vaccine has yet to be successfully marketed. Previous PA vaccines mostly used Pseudomonas aeruginosa outer membrane proteins, lipopolysaccharides, toxins, or flagellar components as antigens. In preclinical studies or clinical trials, they all had varying degrees of problems such as large differences in volunteer protection, poor stability, and few patient benefits. Structure-based vaccine design has been shown to have many advantages. It can accurately locate key antigenic epitopes of pathogens, making vaccine design more precise. Vaccine antigens can be precisely designed based on structural information, enhancing the immunogenicity and drugability of the vaccine and stimulating the immune system to produce an efficient response.

[0008] Previous PA vaccine efforts have overlooked the crucial role of biofilm formation in PA infection, which may be one of the reasons for their failure. Therefore, research integrating the biofilm formation process is urgently needed to address the high failure rate of vaccine development against P. aeruginosa infection. Summary of the Invention

[0009] In response to the above needs, this study focused on CdrA, a key protein in the PA biofilm formation process. Through bioinformatics analysis tools such as AlphaFold 2, the three-dimensional structure, antigenicity, epitope distribution and other information of CdrA were predicted. Based on this, a genetically engineered recombinant CdrA-F1 vaccine was designed and constructed, and its immunogenicity, protective properties and protective mechanisms were studied in animal models, laying the foundation for the successful development of a new PA vaccine.

[0010] The present invention first provides a recombinant protein based on the outer membrane matrix protein of Pseudomonas aeruginosa, which comprises a truncated peptide of the CdrA protein, wherein 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 of the present invention, its amino acid sequence 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 invention also provides a gene encoding the above-mentioned recombinant peptide, whose nucleotide sequence 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 invention 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 the pGEX series vectors, pET series vectors or pQE series vectors, preferably pGEX-6P-1.

[0015] The present invention also provides a recombinant engineered bacterium, which comprises the above-mentioned expression vector and a host bacterium; preferably, the host bacterium is selected from any one of Escherichia coli XL1-blue strain, BL21 series strain and HMS174 series strain, preferably Escherichia coli BL21 strain.

[0016] The present invention also provides the use of the recombinant protein, gene, expression vector, or recombinant engineered bacteria in the 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 invention provides a composition for preventing or treating Pseudomonas aeruginosa infection, comprising the above-mentioned recombinant protein based on the outer membrane matrix protein of Pseudomonas aeruginosa, and pharmaceutically acceptable excipients.

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

[0019] In one embodiment according to the present invention, it contains a pharmaceutically acceptable adjuvant, preferably, 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 Calmette-Guerin adjuvant.

[0020] The beneficial effects of the above technical solution of the present invention are as follows:

[0021] 1) This study focused on CdrA, a key protein in the PA biofilm formation process. Using bioinformatics analysis tools such as AlphaFold 2, we predicted the three-dimensional structure, antigenicity, epitope distribution, and other information of CdrA. Based on this, we designed and constructed a genetically engineered recombinant CdrA-F1 vaccine.

[0022] 2) The recombinant protein CdrA-F1 of the present invention is induced to be expressed in a soluble form in Escherichia coli, and 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 a purity greater than 90%.

[0023] 3) The CdrA-F1 vaccine molecule of the present invention is administered by intramuscular injection, which can induce the production of specific IgGs antibodies in mouse serum, not only significantly inhibiting the formation of PA biofilms, but also exerting a highly effective immune protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of CdrA structure and antigenicity prediction results;

[0025] Figure 2 Expression identification and preparation of SDS-PAGE images of CdrA truncations;

[0026] Figure 3 anti-CdrA-F1 total IgG and its subtype titers;

[0027] Figure 4 Survival curve of mice immunized with CdrA-F1;

[0028] Figure 5 Body weight change curve of CdrA-F1 immunized mice;

[0029] Figure 6 Wound healing in CdrA-F1 immunized mice;

[0030] Figure 7In vivo imaging of mice to detect bacterial colonization results;

[0031] Figure 8 Results of bacterial colonization tests in various organs of mice;

[0032] Figure 9 Survival curve of mice immunized with anti-CdrA-F1 antibody;

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

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

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

[0036] Figure 13 Anti-CdrA-F1 antibodies inhibited initial bacterial adhesion;

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

[0038] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, the following will be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0039] Throughout the specification and claims, the terms "including" and "comprising" are open-ended and should be interpreted as meaning "including, but not limited to." The specification subsequently describes preferred embodiments of the present application. However, such descriptions are intended for general purposes only and are not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the appended claims.

[0040] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was monitored by thin-layer chromatography.

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

[0042] 1. Test methods

[0043] Figure 1 At the same time, DNASTAR software was used to predict the antigenicity of CdrA. The results are shown in Figure 1 Based on the above analysis results, the present invention designed four CdrA truncations, which were named as: CdrA-F1 (Met41-Asp437, SEQ ID NO: 3), CdrA-F2 (Ala445-Thr824, SEQ ID NO: 4), CdrA-F3 (Thr824-Thr1148, SEQ ID NO: 5), and CdrA-F4 (Leu1960-Phe2154, SEQ ID NO: 6).

[0044] 2. Test results

[0045] The biofilm matrix protein CdrA (PA4625) of Pseudomonas aeruginosa PAO1 consists of 2154 amino acids with a molecular weight of approximately 219 kDa. It is difficult to express it recombinantly in Escherichia coli. In order to obtain a CdrA truncate that can be efficiently prepared, this study used AlphaFold 2 to predict the three-dimensional structure of CdrA. The results are as follows: Figure 1 As shown. CdrA is mainly composed of two domains, the TPS (two-partner secretion) domain rich in β sheets and the FhaB (Filamentous hemagglutinin B) domain from the N-terminus. At the same time, the antigenicity of CdrA was predicted using DNA STAR, and the results are shown as follows Figure 1 Based on the above analysis results, the present invention provides four CdrA truncations: CdrA-F1 (Met41-Asp437) nucleotide sequence is SEQ ID NO: 7, CdrA-F2 (Ala445-Thr824) nucleotide sequence is SEQ ID NO: 8, CdrA-F3 (Thr824-Thr1148) nucleotide sequence is SEQ ID NO: 9, and CdrA-F4 (Leu1960-Phe2154) nucleotide sequence is SEQ ID NO: 10.

[0046] Example 2 Expression, Identification and Preparation of Truncated Biofilm Matrix Protein CdrA

[0047] 1. Test methods

[0048] (1) Using Primer Premier 5.0 software, as shown in Table 1, the required primers were designed and four recombinant plasmids were constructed using PCR technology. The plasmids were sent to Jinkairui Biotechnology Co., Ltd. for sequencing, and the strains with correct sequencing were preserved.

[0049] (2) Take 20 μL of the correctly sequenced preserved bacteria and inoculate it into a shake flask containing 20 mL of LB medium. Add 10 μL of ampicillin (2000x) and culture at 37°C, 180 rpm overnight.

[0050] (3) Take 20 mL of overnight cultured bacteria and inoculate it into a shake flask containing 2000 mL of LB medium. Add 1000 μL of ampicillin (2000x) and culture at 37°C, 180 rpm for 3.5 hours.

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

[0052] (5) Remove the overnight culture and divide it into 50 mL centrifuge tubes. Centrifuge at 8500 rpm for 20 minutes.

[0053] (6) Resuspend the bacterial pellet in PBS buffer (1X) and disrupt the cells with ultrasound. The parameter settings are: power 38%, on for 8 seconds, off for 9 seconds, for a total of 15 minutes.

[0054] (7) Centrifuge the disrupted bacterial solution at low temperature with the following parameters: 12,000 rpm, 10°C, and centrifuge for 20 minutes.

[0055] (8) The supernatant was aliquoted, the precipitate was reselected with PBS buffer, and 50 μL of each sample was taken for subsequent detection.

[0056] (9) Pipette 10 mL of GST filler and equilibrate with PBS buffer (1X) three times.

[0057] (10) Mix the supernatant of the lysis solution with the GST filler, place it on a horizontal suspension instrument, and allow it to combine at room temperature for 3-4 hours.

[0058] (11) Purify the protein by affinity chromatography. The specific steps are as follows:

[0059] 1) Collect the binding supernatant and sample 50 μL for subsequent detection.

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

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

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

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

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

[0065] 7) Add PBS buffer with half the volume of the access solution, mix well and let it stand, aliquot the supernatant, and sample 50 μL for subsequent detection.

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

[0067] (12) SDS-PAGE electrophoresis was used to verify the protein and its purity.

[0068] Table 1. Primer design for recombinant CdrA truncations

[0069]

[0070] 2. Test results

[0071] The DNA sequences of CdrA-F1, CdrA-F2, CdrA-F3, and CdrA-F4 were amplified and ligated into the pGEX-6P-1 vector. The expression was tested in E. coli by IPTG induction. It was found that the CdrA-F1 fragment was expressed in a soluble form, while CdrA-F2, CdrA-F3, and CdrA-F4 were expressed in the form of inclusion bodies ( Figure 2 A). The supernatant of E. coli expressing CdrA-F1 was purified by affinity chromatography and ion exchange chromatography, and then analyzed by SDS-PAGE. The relative molecular mass was approximately 40 kDa, consistent with the theoretical value, and the purity was approximately 95%, meeting the standards for subsequent animal experiments ( Figure 2 B,C).

[0072] Example 3 Immunogenicity Detection of CdrA-F1 Protein

[0073] 1. Test methods

[0074] (1) Use the target protein to immunize mice (Balb / c mice, female, 6-8 weeks old) at a dose of 20 mg / mouse. The vaccination method is intramuscular injection. The immunization schedule is three injections on days 0, 7, and 14.

[0075] (2) One day before each immunization, blood was collected from the tail vein of the mice and the antibody titer was detected by enzyme-linked immunosorbent assay (ELISA) (indirect method).

[0076] 1) Coating antigen: Dilute the antigen to the optimal concentration with coating buffer and add 100 μL (6 μg) to each well of the ELISA plate. Incubate at 4°C overnight or in a 37°C water bath for 2-3 hours.

[0077] 2) Washing: Remove the coating solution and wash the plate using a plate washer: Wash the concave wells three times with washing buffer, each time for 5 minutes.

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

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

[0080] 5) Washing: Remove the coating solution and wash the plate using a plate washer: Wash the concave wells three times with washing buffer, each time for 5 minutes.

[0081] 6) Add enzyme-labeled antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody (after titration) to each reaction well. Incubate at 37°C for 0.5-1 hour and wash the plate.

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

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

[0084] 9) Reading: Measure the OD value of each well on a microplate reader at 450 nm, adjusting the blank control well to zero.

[0085] 2. Test results

[0086] To evaluate the immunogenicity of CdrA-F1, recombinant CdrA-F1 protein was mixed with AS03 adjuvant and mice were immunized on days 0, 7, and 14. Anti-CdrA-F1 antibody titers were measured 1 day before immunization and 7 days after each immunization. Figure 3 As shown in the figure, the specific antibody titer in the serum of mice immunized with CdrA-F1 increased significantly on day 7 after immunization and continued to increase on days 14 and 21, with geometric mean titers of 1:120,000 and 1:8,000,000, respectively. These results indicate that CdrA-F1 has good immunogenicity.

[0087] In order to evaluate the type of immune response induced by CdrA-F1 of AS03 preparation, anti-IgG1, IgG2a and IgG2b were used as secondary antibodies in ELISA to detect the titer of each subtype of antibody. Figure 2-4As shown in Figure A, CdrA-F1 immunization induced high titers of IgG1, IgG2a, and IgG2b subtype antibodies, significantly different from the PBS group. IgG1 antibody titers were significantly higher than those of IgG2a and IgG2b (P < 0.0001), with IgG1 titers approximately five times higher than those of IgG2a. These results indicate that the CdrA-F1-induced immune response is dominated by a Th2-type, or humoral, immune response.

[0088] Example 4 Detection of the protective effect of CdrA-F1 protein

[0089] 1. Test methods

[0090] (1) Agar bead-PAO1 production

[0091] 1) Streak the preserved bacteria onto a plate, pick a single colony, and activate the bacterial solution twice to expand the culture.

[0092] 2) Centrifuge and detect absorbance at a wavelength of 600 nm.

[0093] 3) Take 5 OD of bacteria [(5 / OD value) mL] and centrifuge.

[0094] 4) Discard the supernatant and resuspend in 100 μL of saline.

[0095] 5) Add 900 μL of LBA (LB + agar) and mix well (pay attention to the temperature).

[0096] 6) Add the mixture to a conical flask containing 120 mL of mineral oil (the conical flask is placed in a water bath at 45°C, and a vertical stirrer is placed in the conical flask at a speed of 240 rpm for 30 minutes).

[0097] 7) The formed beads are buried in ice and placed on a shaker at 100 rpm / min for 30 min (or placed in a vertical stirrer at 80 rpm for 30 min).

[0098] 8) Divide the mixture into two 50 mL centrifuge tubes and centrifuge at 3900 rpm for 15 min.

[0099] 9) Pour away the upper layer of mineral oil directly.

[0100] 10) Add 10 mL of physiological saline to each centrifuge tube to resuspend the beads, transfer them to new centrifuge tubes, and centrifuge at 3900 rpm for 15 min.

[0101] 11) Carefully aspirate and discard the supernatant with a pipette, then add 10 mL of saline to resuspend and wash the beads, transfer them to a new centrifuge tube, and centrifuge at 3900 rpm for 15 minutes.

[0102] 12) Carefully discard the supernatant with a pipette and add 10 mL of saline to resuspend the beads.

[0103] 13) Pipette 5 mL of the resuspension through a 100 μm filter. Rinse the filter several times with saline. Remove from a new 50 mL centrifuge tube and backwash with 3 mL of saline to recover the beads. Transfer the remaining 5 mL of the resuspension to the same 50 mL centrifuge tube, recovering the beads as described above, until the total volume of the beads recovery solution is 5-7 mL.

[0104] 14) Microscopic examination: The appropriate diameter of the beads observed under a microscope is 100-200 μm.

[0105] (2) Mouse back skin trauma with chronic infection model

[0106] 1) Adjust the bacterial solution concentration. Use physiological saline to adjust the concentration of the agar-bead-coated PAO1 bacterial solution (prepared in 2.2.4.5) to 5.0×104 CFUs / mL, 1.0×105 CFUs / mL, 2.5×105 CFUs / mL, 5.0×105 CFUs / mL, and 1.0×106 CFUs / mL, respectively. Place on ice and bring to the animal room for later use.

[0107] 2) Anesthetize the animal with 6-7 μL / g of 1% (w / v) sodium pentobarbital (i.e., 120-140 μL for a 20 g mouse); observe the toe pinch reflex after 5-7 minutes. If positive, add 10%-20% more sodium.

[0108] 3) Remove the hair on the back and apply depilatory cream for further hair removal. Use saline gauze to wipe off the depilatory cream. Disinfect the local skin with 75% alcohol. Make a 10x10 mm square skin incision in the middle of the back. Apply PAO1 solution wrapped with agar beads on the incision surface. Then cover the surface with Smith & Nephew medical breathable waterproof dressing

[48] to isolate it from external contamination. Inject carprofen (5 mg / kg) as a long-acting analgesic subcutaneously.

[0109] 4) The mouse is rewarmed and returned to its cage after awakening.

[0110] (3) Survival rate

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

[0112] 2. Test results

[0113] To investigate the protective effect of CdrA-F1 protein, this study used mice immunized with CdrA-F1 protein (immunization schedule: 0, 7, and 14 days, dose: 20 μg / mouse). A 10x10 mm square of skin was cut from the middle of the back and inoculated with a lethal dose (1.0×106 CFUs) of agar beads coated with PAO1 bacteria. The survival of the immunized and control groups was observed for 10 consecutive days. Figure 4 As shown, deaths began to occur in the control group on day 2 after inoculation and continued on days 3 and 4, resulting in a 100% mortality rate during the observation period. No deaths were observed in the CdrA-F1-immunized group during the observation period. Statistical analysis showed that the survival time of mice in the CdrA-F1-immunized group was significantly longer than that in the control group (P < 0.0001). These results demonstrate that CdrA-F1 has a strong immune protective effect in the mouse skin wound infection model.

[0114] Example 5 Immunization with CdrA-F1 significantly alleviates infection symptoms in a mouse skin wound infection model

[0115] 1. Test methods

[0116] The preparation of agar beads-PAO1 and the operation steps of the mouse back skin wound with chronic infection model were the same as those in Example 4. The weight changes of mice with skin wound and chronic infection (immune 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, this study applied a sublethal dose of PAO1 (1.0×10 5 CFUs) agar beads, and the weight changes were monitored daily. Figure 5 As shown, the weight of mice in the immunized group began to recover on the third day after challenge, and by the end of the observation period on the seventh day, their weight had returned to the pre-challenge level. In contrast, the weight of the control group decreased rapidly after challenge, but the rate of decline slowed after the third day, and no recovery trend was observed throughout the observation period.

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

[0120] 1. Test methods

[0121] To further evaluate the protective effect of CdrA-F1 protein immunization against bacterial infection in wounds, the back wounds of mice were treated with a sublethal dose of PAO1 (1.0×10 5 After infection with CFUs agar beads, the wound area was measured daily and the healing status was recorded, and the percentage of the unhealed area to the initial wound area was calculated.

[0122] 2. Test results

[0123] The results are as follows Figure 6 As shown in A, on the first day after the infection, bacterial infection changes such as congestion and exudation appeared in the back wounds of both groups of mice. On the 5th day, the wounds on the backs of the mice in the immunized group were significantly reduced, and at the end of the 10-day observation period, only a very small area of ​​wound surface remained unhealed. On the 5th day, the wounds on the backs of the mice in the control group not only did not heal, but the infection worsened. The surface of the wound was dark red to grayish white, and there was a lot of secretion. On the 10th day, the infection changes were more obvious, the wound was ulcerated, and the surface was covered with pus. The wound area also tended to expand. Summarizing the percentage data of the unhealed area to the initial area of ​​the wound, it was found that the proportion of the immunized group mice decreased significantly over time, while the proportion of the control group mice did not change significantly ( Figure 6 B) The above results indicate that CdrA-F1 protein immunization can exert a local protective effect on the wound.

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

[0125] 1. Test methods

[0126] The skin incision surface of mice in the immunized group and the negative control group was smeared with agar-bead-encapsulated NanoLuc luciferase-labeled PAO1 bacterial solution. On the 1st, 5th, and 10th days after modeling, the small animal living imaging system was used to take pictures and quantify the fluorescence to evaluate the bacterial colonization and clearance.

[0127] 2. Test results

[0128] To further verify the protective effect of CdrA-F1 protein immunization on the local wound, this study used Nanoluc luciferase-labeled PAO1-agar beads to apply to the wound surface in a mouse skin wound model. The bacterial fluorescence intensity was detected by in vivo imaging on the 1st, 5th, and 10th days to monitor the local bacterial colonization in the wound. Figure 7 As shown, on day 1, fluorescent bacteria were detected in both the control and immunization groups, indicating successful model establishment. On day 5, the fluorescence intensity in the control group significantly increased, indicating that bacteria were multiplying and increasing in number within the wound. However, almost no fluorescence was detected in the immunization group, suggesting that the bacterial load was low and the infection was under control. On day 10, the fluorescence intensity in the control group continued to increase, indicating a further increase in the local bacterial population and aggravated infection, consistent with the trend observed for local symptomatic infection. These results demonstrate that CdrA-F1 protein immunization exerts a strong protective effect locally within the wound.

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

[0130] 1. Test methods

[0131] On the 1st, 5th and 10th day after modeling, 3 mice were taken from each of the immune group and negative control group, and blood was collected by enucleation. After soaking in 75% alcohol, the mice were dissected and the spleen, lung and liver tissues were removed. The organs were placed in a sterile plate and weighed. The organs were then placed in a high-pressure sterilized glass homogenizer, 1 mL of sterile PBS was added and the organs were fully ground. After the grinding was completed, 100 μl of the homogenate was mixed with PBS at a ratio of 1:10 and 1:10. 2 , 1:10 3 , 1:10 4 , 1:10 5 , 1:10 6 Dilute in the same proportion, mix well, take 10 μl of homogenate for each dilution, drop it on the top of an antibody-free solid LB square dish, and then tilt it to allow it to naturally remain (be careful to prevent the bacterial solution from touching the upper and lower edges of the square dish to avoid affecting subsequent counting), place it in a 37°C incubator, culture overnight, and select the appropriate dilution ratio for colony counting.

[0132] 2. Test results

[0133] To further investigate the immune protection mechanism of CdrA-F1 protein, this study measured the number of bacterial colonization in organs and observed tissue pathological changes on days 1, 5, and 10 after infection in a mouse skin wound model. Figure 8 As shown, on day 1, bacterial colonization was observed in the liver, spleen, and lungs of both groups of mice, but the bacterial counts in each organ were significantly higher in the control group than in the immunized group. On days 5 and 10, bacterial counts in each organ of the control group continued to increase. In contrast, on day 5, bacterial counts in each organ of the immunized group decreased. On day 10, with the exception of a small amount of bacterial colonization in the lungs, no bacteria were detected in the spleen or 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 methods

[0136] Given the characteristics of CdrA-F1 protein inducing Th2 immune responses and anti-infection immunity against Pseudomonas aeruginosa, it is speculated that specific antibodies against CdrA-F1 are the key substances for its immune protection. To verify this hypothesis, this study purified anti-CdrA-F1 antibodies from mouse immune serum and used the above-mentioned mouse skin wound model to inoculate mice with a lethal dose (1.0×10 6 The mice were then inoculated with agar beads containing PAO1 (CFUs) and then given anti-CdrA-F1 antibodies, and the survival of the mice was observed.

[0137] 2. Test results

[0138] The results are as follows Figure 9 As shown, mice in the PBS group died on day 2 after vaccination, with no mice surviving by day 4. Three mice in the blank serum group died on day 2, and all seven died by day 3, resulting in a 100% mortality rate for both groups during the observation period. In the anti-CdrA-F1 antibody-immunized group, one mouse died on day 2, while none of the remaining nine mice died at the end of the observation period, day 10. Statistical analysis showed that mice vaccinated with anti-CdrA-F1 antibodies had significantly longer survival times than the control group. These results demonstrate that anti-CdrA-F1 antibodies have a strong immunoprotective effect in the mouse skin wound infection model, while nonspecific antibody serum from mice was not found to have any protective effect against skin wound infection.

[0139] Example 10 Anti-CdrA-F1 Antibodies Inhibit Pseudomonas aeruginosa Biofilm Formation in Vitro

[0140] 1. Test methods

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

[0142] (2) Aspirate the culture medium and add 200 μL of sterile PBS buffer to each well to wash the plate three times;

[0143] (3) Add 100 μL of methanol to each well for 15 min, then aspirate the methanol in the culture wells and allow them to 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 well, rinse off the excess dye with running water;

[0146] (6) Place the culture plate upside down on filter paper to remove any remaining water and dry in a 37°C oven or 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 well using a microplate reader at 590 nm;

[0149] (9) Each test was repeated three times for each strain, and the experimental value was taken as the average of three times (D value);

[0150] (10) The culture medium without inoculation 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 > 2xDc); (2) Weak biofilm-forming strains (DDc <UNK> Dc < D ≤ 2x 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 under in vitro culture conditions containing different concentrations of anti-CdrA-F1 antibody was detected by crystal violet staining method. 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 the formation of obvious biofilms (intuitive figures can be added). In contrast, after adding anti-CdrA-F1 antibody serum diluted in different multiples, the OD 590 value decreased significantly compared with the control group, and as the antibody concentration increased, the OD 590 value showed a downward trend, and the difference was statistically significant (P < 0.0001). To evaluate whether anti-CdrA-F1 antibody directly affects bacterial growth, the time-growth curve of PAO1 was measured in this study, and the area under the curve (AUC) was calculated. The results are as 590 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 <UNK>

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

[0155] 1. Test method

[0156] (1) Scanning electron microscopy method

[0157] In a sterile 6-well cell culture plate, place a sterile round coverslip with a diameter of 10 mm in each well; add 1.0 mL of bacterial solution and serum, and incubate statically at 37 °C for 36 h. Then, sampling, washing, fixing, dehydration, drying, sample adhesion, and conductive treatment are carried out. The specific steps are as follows:

[0158] 1) Sampling: Carefully remove the round coverslip from the culture well, avoiding pulling, cutting, and squeezing the sample during the process.

[0159] 2) Cleaning: Wash the sample slowly and carefully three times with PBS buffer (1X) to remove impurities on the observation surface and fully expose the observation surface without damaging it.

[0160] 3) Fixation: Fix with 2.5% glutaraldehyde at 4°C for 4 hours, aspirate the fixative, add PBS buffer (1X), rinse for 1 hour, and change the buffer 3-4 times.

[0161] 4) Dehydration: Aspirate the buffer and dehydrate using 30%, 50%, 70%, 80%, 90%, 95% and anhydrous ethanol in a stepwise gradient, with a residence time of 15 minutes per step. Dehydrate again using 30%, 50%, 70%, 80%, 90%, 95% and anhydrous tert-butyl alcohol in a stepwise gradient, with a residence time of 15 minutes per step.

[0162] 5) Drying: Use a critical point dryer to prevent surface morphology distortion caused by water evaporation. Make sure that the drying is thorough.

[0163] 6) Gluing the sample: Use a toothpick to apply a small amount of conductive glue to the sample table. The glue surface should be slightly smaller than the sample ground. Use tweezers to gently clamp the side of the sample to ensure that the observation surface is firmly attached to the glue. After pasting, wait for the conductive glue to dry thoroughly before vacuum coating and SEM microscopy.

[0164] 7) Conductive Treatment: Biological samples are composed of low-atomic-number elements such as carbon, hydrogen, oxygen, and nitrogen. Their secondary electron emission rates are very low, resulting in weak signals and difficulty in achieving the necessary image contrast. Therefore, biological samples require surface conductive treatment before SEM observation.

[0165] (2) Laser confocal method

[0166] 1.0 mL of BFP-labeled PAO1 bacteria and serum containing anti-CdrA-F1 antibodies at varying dilutions were added to a sterile glass-bottom confocal culture dish and incubated at 37°C for 36 hours. The upper layer of planktonic bacteria was then slowly aspirated and the dish was carefully washed three times with 1X PBS buffer to remove impurities while ensuring the observation surface was not damaged. Biofilm formation was then observed using a confocal laser scanning microscope.

[0167] 2. Test results

[0168] The results are as follows Figure 11As shown, after one day of culture, a large number of bacteria adhered to the slide surface in the PBS and blank mouse serum groups, with a large amount of mucus-like biofilm exopolysaccharide between the bacteria, which encapsulated the bacteria and firmly adhered them to the surface. In contrast, in the serum group containing a 1:20 dilution of anti-CdrA-F1 antibodies, although the bacterial numbers were high and growth was good, less mucus-like material was present between the bacteria, in stark contrast to the PBS and blank serum groups. After four days of culture, the bacterial numbers in the PBS and blank serum groups increased significantly compared to the first day, and the amount of mucus-like material continued to increase. Biofilms further developed, with bacteria overlapping and clinging to each other, forming compact aggregates. In contrast, although the bacterial numbers in the antibody serum group increased further, no obvious biofilm formation was observed, with the bacteria mechanically overlapping and lacking a firm morphology. After seven days of culture, the mucus-like exopolysaccharide between the bacteria in the PBS and blank serum groups increased further, resulting in a more compact and tough texture. Meanwhile, bacteria in the experimental group continued to multiply, with further increases in number, as evidenced by an increase in the number of bacterial layers, but no signs of spreading and spreading, and no obvious biofilm formation was observed. This suggests that in the absence of obvious biofilm formation, bacterial spread along the surface is limited. The group containing anti-CdrA-F1 antibody serum at a 1:10 dilution showed a similar trend to the 1:20 dilution, with bacteria growing and accumulating at the initial adhesion sites, but no biofilm formation and inability to spread and extend across the slide surface. At a 1:5 dilution, however, no biofilm formation was observed. Unlike the other two higher dilution groups, the number of bacteria observed microscopically after one day of incubation was low. After four and seven days of incubation, the bacteria showed normal growth and proliferation, but with clear outlines and loose connections between the bacteria, and no biofilm formation. This suggests that at this dilution, anti-CdrA-F1 antibodies inhibited bacterial adhesion, biofilm formation, and spread during the initial adhesion phase of P. aeruginosa.

[0169] Figure 12 As shown, further examination by laser confocal microscopy revealed that biofilm thickness in the PBS and blank serum groups increased continuously during the observation period, reaching 30±3μm on day 1, 70±5μm on day 4, and approximately 120μm on day 7. The biofilm thickness in the group containing anti-CdrA-F1 antibody serum was significantly thinner than that in the control group, reaching 18±3μm on day 1, 35±4μm on day 4, and approximately 38±2μm on day 7. At the same time point, no statistically significant differences were observed between the different antibody concentrations, but the anti-CdrA-F1 antibody group showed significant differences compared with both the PBS and blank serum groups (P < 0.0001).

[0170] Combined scanning electron microscopy and laser confocal microscopy results showed that anti-CdrA-F1 antibodies could inhibit the formation of Pseudomonas aeruginosa PAO1 biofilm in vitro, and higher concentrations of antibodies could inhibit the initial adhesion of bacteria on the surface of objects.

[0171] Example 12 Anti-CdrA-F1 Antibody Inhibits Initial Adhesion of Pseudomonas aeruginosa

[0172] 1. Test methods

[0173] Pseudomonas aeruginosa was cultured overnight with shaking. 200 μL of a suspension containing anti-CdrA-F1 antibody serum at varying dilutions was added to a 96-well microtiter plate. Three replicate wells were set up for each concentration and incubated at 37°C for 2 h. After carefully removing floating bacteria, 200 μL of 1× PBS buffer was added to each well. The suspension was thoroughly mixed using a micropipette and diluted to 1×10, 1×100, 1×1000, and 1×10000 times, respectively. 10 μL of each suspension was titrated in a square Petri dish containing solid LB and incubated at 37°C for 12 h. Samples with colony counts between 30 and 300 CFUs were counted to test the inhibitory effect of the antibody serum on initial bacterial adhesion.

[0174] 2. Test 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 the initial adherent bacteria under different culture conditions. Figure 13 As shown, after 3 hours of culture, the initial number of adhered bacteria in the PBS group and the blank serum group was 1×10 6 In contrast, the serum group containing anti-CdrA-F1 antibodies significantly reduced the number of bacteria adhering to the culture dish surface (P < 0.0001), and the 1:10 dilution group further reduced the number of bacteria compared to the 1:20 dilution group (P < 0.0001). These results suggest that anti-CdrA-F1 antibodies can inhibit the initial adhesion of P. aeruginosa PAO1.

[0176] Example 13: Anti-CdrA-F1 Antibodies Inhibit Transcription of Genes Related to Exopolysaccharide Formation in Pseudomonas aeruginosa PAO1 Biofilms

[0177] 1. Test methods

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

[0179] 1) Sample lysis: Collect bacteria by centrifugation and add Buffer RL. For every <5 × 106 cells, add 500 μl Buffer RL and vortex until no obvious cell clumps are observed.

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

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

[0182] 4) Transfer the entire mixture from step ③ to FastPure RNA Columns III (FastPure RNA Columns III have been placed in the collection tube), centrifuge at 12,000 rpm (13,400 x g) for 30 seconds, and discard the filtrate.

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

[0184] 6) Add 700 μl of Buffer RW2 (containing anhydrous ethanol) to FastPure RNA Columns III, centrifuge at 12,000 rpm (13,400 x g) for 30 seconds, and discard the filtrate.

[0185] 7) Add 500 μl of Buffer RW2 (with anhydrous ethanol) to FastPure RNA Columns III and centrifuge at 12,000 rpm (13,400 x g) for 2 min. Carefully remove the column from the collection tube to avoid contact with the filtrate, which may cause contamination.

[0186] 8) If there is liquid residue in the adsorption column or it comes into contact with the filtrate, discard the filtrate, return the FastPure RNA Columns III to the collection tube, and centrifuge at 12,000 rpm (13,400 x g) for 1 min to prevent ethanol contamination.

[0187] 9) Carefully transfer the adsorption column to a new RNase-free Collection Tube (1.5 mL). Add 50-200 μl of RNase-free ddH2O to the center of the adsorption column. Let stand at room temperature for 1 minute. Centrifuge at 12,000 rpm (13,400 x g) for 1 minute to elute the RNA.

[0188] 10) Measure RNA concentration. The extracted total RNA can be used directly in downstream experiments or stored at -80°C.

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

[0190] 1) Genomic DNA removal.

[0191] 2) Prepare the reverse transcription reaction system by directly adding 5×HiScript III qRTSuperMix to the reaction tube in step 1.

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

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

[0194]

[0195]

[0196] 1) Prepare the following mixture in a qPCR tube

[0197] Table 3 Reaction system

[0198]

[0199] 2) Perform qPCR reaction according to the following conditions:

[0200] Table 4 Program settings

[0201]

[0202] 3) Finally, collect the fluorescence signal.

[0203] 2. Test results

[0204] The main component of biofilm is exopolysaccharide. The matrix protein CdrA strengthens the biofilm structure by interacting with exopolysaccharides Pel and Psl. At the same time, the quorum sensing system of Pseudomonas aeruginosa is involved in regulating the formation and development of biofilm. Therefore, it is speculated that anti-CdrA-F1 antibodies can inhibit the expression of key genes for biofilm formation. To verify this hypothesis, this study used RT-PCR to detect changes in the transcription levels of genes related to biofilm exopolysaccharide formation (algD, algU, pelA, pslA) and quorum sensing system-related genes lasR, lasI, rhlR, and rhlI regulated by PAO1.

[0205] The results are as follows Figure 14 As shown, on day 1, the transcript levels of the biofilm-forming genes algD, algU, pelA, and pslA decreased significantly compared to the control group, falling to 0.4-fold, 0.3-fold, 0.4-fold, and 0.3-fold, respectively. However, the transcript levels of the quorum sensing-related genes lasR, lasI, rhlR, and rhlI did not change significantly (P>0.05). The same trend was observed on days 4 and 7. Taken together, these results suggest that the anti-CdrA-F1 antibody inhibits biofilm formation by suppressing the expression of algD, algU, pelA, and pslA in P. aeruginosa PAO1.

[0206] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0207] Sequence Listing

[0208] <SEQ ID NO:1

[0209] MVRPDSQRGKRTSSKAGREGRFVLAASSTLMFAIASINAAMAAPTGAQFNPNEIKISQQGKTTLIDQS

[0210] TQRAI INWKGFDVSADEAVRFNQPGVTSSTLNRVTAGQESVIAGRISAPGQVIIYNSNGVVFSGSAKV

[0211] DVGSLITTTANISDEHFRQGKLIFDQPGNPDARIVNDGSISVAEKGLAAFVAPSVANNGVINARLGTV

[0212] AMAAGNAATIDLYGDGLVSIAVTDPVTRKPQDAQALVSNGGAIQADGGSVLITAEQASRVVDNAVNLS

[0213] GVILARGTEVREGSVALVSKSGDIQIAGKIDVSGPKNGGDVLVSGQQVALASTASIDARGTAQGGSVR

[0214] IGGDFQGRGELPRAKNATLAKGASIDVSATGKGNGGLAVVWSDGNTRMDGRILARGGAQGGNGGLVET

[0215] SGKVNLSIADSAYVSVAAPYGNGGTWLLDPTTLRIVASGGTSGSVGGANGASGDATVNASVVTGALAG

[0216] GKVTLSASDRLSVEAPLITSNLGGASRGLELIATGPAGAVDISAPILFRNGSLAIRAGNNIFLSGGT

[0217] PQTSGIVDLGSGTLWMQTSTAGKISQQAGTALIAANLAGRAGSIDLASWDNYAGNLALQTFNGTLKYR

[0218] QSNATGVTTSGTVFDPFINQSMTGTAQNIVSSVGTRILEANSVGTTGNYTLTADGNSEFDRLVFTALP

[0219] YRRVSGSASFPTNDSSDYLVTNLRYQVNGSNVTATPNGGAPSGFTVAAGNGSVTTWTGNWGTSWGVKG

[0220] FGGVIGVTDELQYDVGTGLTEELIFGLGGKTSRVDTRLDFMREGAFNSFAERAQVEMFKTTTTAGDI

[0221] LSRQQTATLTANDATRVYGDVNPTLTATMSGINAIDAYVNSQFNDLYQATASTTATQASNVGQYAITG

[0222] NANGSEYFSQRYQLVRQDGRLTVTPAQLIVSADAKTKVYGDADPTLTYQVSGLKNSDTAAGVLSGNLG

[0223] RVAGENVGNYGILQGGLGLNTANYTLSYVGNDLRITPAQLNVIADAKTKVYGDLDPALTYQVSGLKRG

[0224] DTAGAVLNGGSLSRVAGENVGVYGINQGGLGLVSSNYTLNYQGNNLTITKALLNVIADAKTKVYGDAD

[0225] PALTYQVSGLKNGDTAGAVLNGGSLSRVAGENVGVYGINQGGLGLLSANYDLSYQGNNLTITKALLNV

[0226] IADAKTKVYGDADPSLTYQVSGLKNGDTAGSILTGGLNRAAGENVGVYGINQGDLALNSGNYDLSYQG

[0227] NNLTITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDTAGAVLNGGGLVRVSGENVGNYAIQQGGL

[0228] GLVGSNYDLAYQGNNLTITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDSAGS ILTGGNLRAAGE

[0229] NVGVYGINQGDLALNSGNYDLSYQGNNLTITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDTAGA

[0230] VLNGGGLVRVSGENVGNYAIQQGGLGLVSGNYDLAYQGNNLTITKALLNVIADAKTKVYGDADPSLTY

[0231] QVSGLKNGDTAGAVLNGGSLSRVAGENVGVYGINQGDLALNSGNYDLSYQGNNLTITKALLNVIADAK

[0232] TKVYGDADPSLTYQVSGLKNGDTAGAVLNGGGLVRVSGENVGNYAIQQGGLGLVSGNYDLAYQGNNLT

[0233] ITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDSAGSILTGGLNRAAGENVGVYGINQGDLALNSG

[0234] NYDLSYQGNNLTITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDTAGAVLNGGGLVRVSGENVGN

[0235] YAIQQGGLGLVSGNYDLAYQGNNLTITKALLNVIADAKTKVYGDADPSLTYQVSGLKNGDTAGAVLNG

[0236] GSLSRVAGENVGVYGINQGGLGLVSGNYDLAYQGNNLTITKALLNVIADGKTKVYGDADPSLTYQVSG

[0237] LKNGDSAGS ILTGGLNRDAGENVGVYGINQGGLVLTSGNYDLAYQGNDLTITKALLNVFADAKSKQVG

[0238] TADPALTYQVSGLKNGDSAGQVLAGGLGRVGGEAVGQYDILQGGLALTSGNYQLNYQGNLLSILPLPV

[0239] TPGDLGQLAALSDLRELQKGRDPDTPGDAVYRTTTLENPFLENPFLRAYALGMDVSDPNLLPATAAGP

[0240] AEDASAKRVGQFTDRPLRAEAESGAGCSNQSYLADYWSCFNKPLNF

[0241] <SEQ ID NO:2

[0242] GCAGCGCGCTGCCGCGATCCAGGCGGCCATGGCTGAACACCCCCGGCAGGCTCACCACCTGCAGCTCG

[0243] CCATCGGCCAGCGGCAGGCTGTAGCGCTGGGCCAGGGCGTGCAGGTCGGGCTCGGCCGGCGCCTGTTC

[0244] GATACGCACCTGCCAGAGCTGGCAATGCCGCGCGCTATCCAGTTTGCGGGGCTTGCCGTAGGCGGCCA

[0245] ACTGCTTGCTCGCGCGCTCGATCCCGCCGCGCTTTTCGCCGACCAGGAAGAGTTCGCCGCCGGGCAGG

[0246] CGGGCGGCCAGGCTGGCCAGCAGGTAGTCGGTCAGTTCGCGGGACTTGGGCAGGAACAGAACGGCCGA

[0247] AGTCCAGGCGCCAGTCGGCGCGCGCGTGTCGAAACGGCTACGGCCGGGGAAGCGGGCGTCCAACTGCG

[0248] CCTGCTCGCCGGCGTGCCAGCTCCAGCCCTGGGCCTGCGGTAGCTCGGCGAGCAGGTCGTCGGCGGGC

[0249] AGGCCGGCCAGCAGCAGCGGGGTCGCGAAGAGGTGGCGCTGGCGCAGCAGCACTTCACTGCGAGGGTC

[0250] CATGCGGGCTCCGGAAAAAATGGGAAATCTTAACAGGCTTGGGCCTGGCATACCCGCATGGATTGTCG

[0251] GCGAGGATCGCCGTTCATCCGTGCGCGGTGCCCGCAGCGCCGTATCAGCCGGCGTTGACTACCCGCAG

[0252] GGCGCGCCCGGCCTTCCATGCCTCTGCGTTTTCCGCCAGTTGGCCGACGATGCGCTGCCGCGCCTCGC

[0253] GACTGCCCCAGGCGTTATGGGGGGTGACGATCAGGCGCGGGATGTCGGGGGCCAGCAGCGGATTGCCG

[0254] TTTCTCGGCGGCTCGACGCTGAGCACGTCGGTGGCGGCGCCGCCCAGGTGGCCGCCGCGCAGGGCGTC

[0255] GGCCAGCGCCTGTTCGTCGACCAGGCCTCCACGGGCGGTATTGACCAGGAAGGCGCCAGGCTTCATCA

[0256] GCGCCAGTTCGGCGGAGCCGAGCATCCCGCGGGTGTCCTCGGTCAGCGGGCAGTGCAGGGTCAGGGCG

[0257] TCGACCCGTGGCAGGAGTTCGCCAAGCGGCAGGCGGTCCGCCCGGGCGGGCCGTCCTGGCAACTGACC

[0258] GAGCAGCACGCGCATGCCGAACGCCTCTGCCAGCCGCGCCACCGCCCCGCCCAGTTCGCCATGGCCGA

[0259] GCAGGCCGAGGGTCTTGCCTTCCAGTTCGACGATGGGGAAGTCCAGCAGGCAGAACTGGCTGGATTGC

[0260] TGCCAGCGACCGCTGCGTACCGCCTGCTGGTAGTCCGGCAGGCGCGTAGCCAGGGCCAGTAGCAGCGC

[0261] GAGGGTGTGCTGGGCTACCGAGGGCGTGCCGTAGCCATGGCAGTTGGCGACGACGATACCGCGCTCGC

[0262] GAGCGGCAGCGAGATCGATGTTGTTGGTGCCGGTAGCGCTGATCAGGATCAGTTCCAGCTCTGGGCAG

[0263] GCGGCGATGCATTGCGCGTCGATCGGCACCTTGTTGCTGATCGCGACCCGCGCATCGTGCAGCCGCGC

[0264] GGCGACTTCCTGCGGTTGGCTGGCGGCATGCAGTTGCAGCTCGTCGAAGGCGGCGCGCAGGGGGCGCA

[0265] TGTCCAGGTCGCCGAGGTCGAGGGAGGCATGGTCGAGGAAAACGGCGCGCAGGGCATGGCTCATGGAA

[0266] GGTTCCTTGGCGGCAGCGGACGGGGGCTGCCTAGCCTAGCAAACCCGCGTGCCAGGGAGGTCTTCCGG

[0267] TTTTCTTGACAGTGATTATCTCGAAAGCATTGTCGGTTTTTTGACGGTATTACATATTTAACCTCCGT

[0268] AGTGTCATTTTTGAGTCACTCCGACGAACGGTCGCCCTTTCAAGTGGACAATTTTTGATCTGAAAGGC

[0269] ATTTTTTTGGCACACAAGGCGCAATGTGAATTGCTTCTCAGATTTTCGTCATTTAACTGACGAAATGC

[0270] AGTGCTGGAAAACTGACGTGCGCCACGCAGATAGGGAGATTTTCATGGTCCGTCCGGATAGCCAGCGC

[0271] GGCAAGCGCACCTCTTCGAAAGCTGGTAGGGAAGGGCGTTTCGTCCTTGCCGCCAGCAGCACCCTCAT

[0272] GTTCGCCATCGCCAGCATCAACGCGGCCATGGCGGCACCTACCGGTGCCCAGTTCAACCCCAACGAGA

[0273] TCAAGATCAGCCAGCAGGGCAAGACCACGCTGATCGACCAGAGCACCCAGCGCGCGATCATCAACTGG

[0274] AAGGGCTTCGACGTCTCCGCCGACGAGGCGGTGCGCTTCAACCAGCCCGGTGTGACCTCGTCGACCCT

[0275] GAACCGGGTCACCGCAGGCCAGGAAAGCGTGATCGCCGGGCGCATCTCCGCGCCCGGCCAGGTGATCA

[0276] TCTACAACTCCAACGGCGTGGTCTTCAGCGGCTCGGCCAAGGTCGACGTGGGCTCCCTGATCACCACC

[0277] ACGGCGAACATCAGCGACGAACACTTCCGCCAGGGCAAGCTGATCTTCGACCAGCCCGGAAACCCCGA

[0278] CGCGCGGATCGTCAACGACGGCAGCATCAGCGTCGCGGAGAAAGGCCTGGCGGCCTTCGTCGCGCCGT

[0279] CCGTGGCCAACAACGGCGTGATCAACGCCCGCCTCGGCACCGTGGCGATGGCCGCCGGCAATGCCGCG

[0280] ACCATCGACCTCTACGGCGATGGCCTGGTGTCGATCGCCGTGACCGACCCGGTTACCCGCAAGCCGCA

[0281] GGATGCCCAGGCGCTGGTCAGCAACGGTGGCGCGATCCAGGCCGACGGCGGTAGCGTGCTGATCACCG

[0282] CCGAGCAGGCTTCCCGGGTGGTCGACAACGCGGTCAACCTGTCCGGCGTGATCCTTGCCCGCGGCACC

[0283] GAGGTGCGCGAGGGCAGCGTGGCGCTGGTAAGCAAGTCCGGGGACATCCAGATCGCCGGCAAGATCGA

[0284] CGTCAGCGGTCCGAAGAACGGCGGCGACGTGCTGGTCAGCGGGCAGCAGGTGGCCCTGGCGAGCACCG

[0285] CCAGCATCGACGCCCGCGGCACCGCACAGGGCGGCAGCGTGCGCATCGGCGGCGACTTCCAGGGCCGC

[0286] GGCGAGCTGCCACGGGCGAAGAACGCTACGCTGGCCAAGGGCGCCAGCATCGACGTCTCGGCTACCGG

[0287] CAAGGGCAATGGCGGCCTGGCGGTGGTCTGGTCCGACGGCAATACCCGCATGGACGGGCGGATCCTCG

[0288] CGCGCGGCGGCGCCCAGGGCGGCAACGGCGGCCTGGTGGAGACCTCCGGCAAGGTCAACCTGAGCATC

[0289] GCCGACAGCGCCTACGTCAGCGTCGCCGCACCCTACGGCAACGGTGGCACCTGGCTGCTCGACCCGAC

[0290] CACCCTGCGCATCGTCGCCTCCGGCGGCACCTCCGGCTCGGTGGGGGGCGCCAACGGCGCCAGCGGCG

[0291] ACGCGACGGTGAATGCCTCGGTGGTTACCGGTGCGCTGGCAGGCGGCAAGGTGACCCTCAGCGCCAGC

[0292] GACCGGCTGTCGGTGGAAGCGCCGCTGATCACCAGCAACCTGGGCGGCGCCAGTCGCGGCCTGGAGCT

[0293] GATCGCCACCGGACCTGCCGGTGCGGTGGATATCTCTGCGCCGATCCTGTTCCGCAACGGCAGCCTGG

[0294] CAATCCGCGCCGGCGGCAACATCAATTTCCTCAGCGGCGGCACGCCGCAGACCAGCGGCATCGTCGAC

[0295] CTGGGCAGCGGCACCCTGTGGATGCAGACCAGCACCGCCGGCAAGATCAGCCAGCAGGCCGGTACCGC

[0296] GCTGATCGCCGCCAACCTCGCCGGCCGTGCTGGTAGCATCGACCTGGCTTCCTGGGACAACTACGCCG

[0297] GCAACCTGGCGCTGCAGACCTTCAACGGTACGCTCAAGTACCGGCAGAGCAACGCCACCGGCGTGACC

[0298] ACGAGCGGCACGGTGTTCGATCCGTTCATCAACCAGAGCATGACCGGCACCGCGCAGAACATCGTCTC

[0299] CAGCGTCGGTACGCGGATTCTCGAGGCCAACAGCGTTGGCACCACCGGCAACTACACGCTGACTGCCG

[0300] ACGGCAACAGCGAGTTCGACCGCCTGGTGTTCACCGCGCTGCCCTATCGGCGGGTCAGCGGTTCCGCC

[0301] AGTTTCCCTACCAACGACTCGTCGGACTACCTGGTGACCAACCTGCGCTACCAGGTCAACGGCAGCAA

[0302] CGTCACCGCCACCCCGAACGGCGGTGCGCCCAGCGGGTTCACCGTCGCCGCCGGCAACGGCAGCGTCA

[0303] CCACCTGGACCGGAAACTGGGGCACCAGTTGGGGCGTCAAGGGCTTCGGCGGCGTGATCGGCGTGACC

[0304] GACGAGCTGCAGTACGACGTCGGCACCGGCCTGACCGAGGAACTGATCTTCGGCCTCGGCGGCAAGAC

[0305] CAGCCGCGTCGATACCCGCCTGGACCTGTTCATGCGCGAGGGAGCGTTCAACAGCTTCGCCGAGCGCG

[0306] CCCAGGTGGAGATGTTCAAGACCACCACCACTGCCGGCGACATCCTTTCCCGCCAGCAGACCGCCACC

[0307] CTGACCGCCAATGACGCTACCCGCGTCTACGGTGATGTGAATCCGACTCTGACGGCCACGATGTCCGG

[0308] CATCAACGCCATCGACGCCTACGTCAACAGTCAGTTCAACGACCTCTACCAGGCGACCGCCAGCACCA

[0309] CGGCGACCCAGGCGTCGAATGTCGGGCAGTACGCGATCACCGGTAACGCCAATGGCAGCGAGTATTTC

[0310] AGCCAGCGTTACCAACTGGTGCGCCAGGACGGCAGGCTGACCGTCACGCCGGCGCAATTGATCGTCAG

[0311] CGCCGACGCCAAGACCAAGGTCTATGGCGACGCCGATCCGACCCTCACCTACCAGGTCAGTGGCCTGA

[0312] AGAACAGCGATACCGCCGCTGGCGTGCTGAGCGGCAACCTCGGCCGGGTGGCCGGGGAGAACGTCGGC

[0313] AACTACGGGATACTCCAGGGCGGGCTGGGCCTCAACACGGCCAATTACACCCTCAGCTACGTCGGCAA

[0314] CGACCTGCGCATCACCCCGGCACAACTGAACGTGATCGCCGATGCCAAGACCAAGGTCTACGGCGACC

[0315] TGGATCCGGCGCTGACCTACCAGGTCTCCGGTCTCAAGCGCGGCGATACCGCCGGTGCCGTGCTCAAC

[0316] GGCGGCAGCCTGAGCCGCGTGGCTGGCGAGAACGTCGGCGTGTACGGCATCAACCAGGGTGGACTGGG

[0317] CCTGGTCAGCAGCAATTACACCCTGAACTACCAGGGCAATAACCTGACCATCACCAAGGCACTGCTCA

[0318] ATGTGATCGCCGATGCCAAGACCAAGGTCTATGGCGATGCCGATCCGGCACTGACCTACCAGGTGTCC

[0319] GGGCTGAAGAACGGCGACACCGCCGGTGCCGTGCTCAACGGCGGCAGCCTGAGCCGCGTGGCTGGCGA

[0320] GAATGTCGGCGTGTACGGCATCAACCAGGGTGGACTGGGCCTGCTCAGCGCGAACTACGACCTGAGCT

[0321] ACCAGGGCAATAACCTGACCATCACCAAGGCGCTGCTGAATGTGATCGCCGACGCCAAGACCAAGGTC

[0322] TACGGCGATGCCGATCCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAACGGTGATACCGCCGGTTC

[0323] GATCCTGACCGGTGGGTTGAACCGCGCCGCAGGCGAGAACGTGGGTGTGTATGGCATCAACCAGGGCG

[0324] ACCTGGCATTGAACAGCGGTAACTACGACCTGAGCTACCAGGGCAATAACCTGACCATCACCAAGGCA

[0325] CTGCTGAATGTGATCGCCGACGCCAAGACCAAGGTCTACGGCGATGCCGATCCGAGCCTGACCTACCA

[0326] GGTCAGCGGCCTGAAGAATGGCGATACCGCTGGCGCGGTGCTCAATGGCGGCGGCCTGGTTCGTGTCT

[0327] CCGGCGAAAACGTAGGCAACTATGCGATTCAGCAGGGTGGCCTGGGTCTGGTCAGCGGTAACTATGAC

[0328] CTGGCTTACCAGGGCAACAACCTGACCATCACCAAGGCACTGCTGAATGTGATCGCTGATGCCAAGAC

[0329] CAAGGTCTACGGCGATGCCGATCCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAACGGCGACAGCG

[0330] CCGGTTCGATCCTGACCGGCGGGCTGAACCGCGCCGCAGGCGAGAACGTGGGTGTGTATGGCATCAAC

[0331] CAGGGCGACCTGGCATTGAACAGCGGTAACTACGACCTGAGCTACCAGGGCAATAACCTGACCATCAC

[0332] CAAGGCACTGCTGAATGTGATCGCCGATGCCAAGACCAAGGTCTATGGCGATGCCGACCCGAGCCTGA

[0333] CCTACCAGGTCAGCGGCCTGAAGAATGGCGATACCGCTGGCGCGGTGCTCAATGGCGGCGGCCTGGTT

[0334] CGTGTCTCCGGCGAAAACGTAGGCAACTATGCGATTCAGCAGGGTGGCCTGGGTCTGGTCAGCGGTAA

[0335] CTATGACCTGGCTTACCAGGGCAACAACCTGACCATCACCAAGGCACTGCTGAATGTGATCGCCGACG

[0336] CCAAGACCAAGGTCTACGGCGATGCCGATCCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAACGGT

[0337] GATACCGCTGGCGCGGTGCTCAACGGTGGCAGCCTGAGCCGCGTGGCTGGCGAGAATGTCGGCGTGTA

[0338] CGGCATCAACCAGGGCGACCTGGCATTGAACAGCGGTAACTACGACCTGAGCTACCAGGGCAATAACC

[0339] TGACCATCACCAAGGCGCTGCTGAATGTGATCGCTGACGCCAAGACCAAGGTCTACGGCGATGCCGAT

[0340] CCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAATGGCGATACCGCTGGCGCGGTGCTCAATGGCGG

[0341] CGGCCTGGTTCGTGTCTCCGGCGAAAACGTAGGCAACTATGCGATTCAGCAGGGTGGCCTGGGTCTGG

[0342] TCAGCGGTAACTATGACCTGGCTTACCAGGGCAACAACCTGACCATCACCAAGGCACTGCTGAATGTG

[0343] ATCGCTGATCCAAGACCAAGGTCTACGGCGATGCCGATCCGAGCCTGACCTACCAGGTCAGCGGCCT

[0344] GAAGAACGCGCGACAGCGCCGGTTCGATCCTGACGGCGGGCTGAACCGCGCCGCAGGCGAGAACGTGG

[0345] GTGTGTATGGCATCAACCAGGGCGACCTGGCATTGAACAGCGTAACTACGACCTGAGCTACCAGGGC

[0346] AATAACCTGACCATCACCAAGGCACTGCTGAATGTGATCGCCGATGCCAAGACCAAGGTCTATGGCGA

[0347] TGCCGACCCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAATGGCGATACCGCTGGCGCGGTGCTCA

[0348] ATGGCGGCGGCCTGGTTCGTGTCTCCGGGCGAAAACGTAGGCAACTATGCGATTCAGCAGGGTGGCCTG

[0349] GGTCTGGTCAGCGGTAACTATGACCTGGCTTACCAGGCAACAACCTGACCATCACCAAGGCACTGCT

[0350] GAATGTGATCGCCGACGCCAAGACCAAGGTCTACGGCGATGCCGATCCGAGCCTGACCTACCAGGTCA

[0351] GCGGCCTGAAGAACGGTGATACCGCTGGCGCGGTGCTCAACGGTGGCAGCCTGAGCCGCGTGGCTGGC

[0352] GAGAATGTCGGCGTGTACGGCATCAACCAGGGTGGACTGGGCCTGGTCAGCGGTAACTATGACCTGGC

[0353] TTACCAGGGCAACAACCTGACCATCACCAAGGCGCTGCTCAATGTGATCGCCGACGGCAAGACCAAGG

[0354] TCTATGGCGACGCTGACCCGAGCCTGACCTACCAGGTCAGCGGCCTGAAGAACGGTGACAGCGCCGGC

[0355] TCGATCCTGACCGGCGGGCTGAACCGCGACGCAGGCGAGAACGTGGGTGTGTATGGCATCAACCAGGG

[0356] CGGCCTGGTGCTGACCAGTGGCAACTATGACCTGGCCTACCAGGGTAATGACCTGACCATCACCAAGG

[0357] CGCTGCTCAACGTATTCGCCGATGCCAAGAGCAAGCAGGTCGGCACCGCCGACCCGGCGCTGACCTAT

[0358] CAGGTTTCCGGCCTGAAGAATGGCGATAGCGCCGGTCAGGTGCTCGCCGGCGGGCTCGGTCGGGTGGG

[0359] CGGCGAAGCCGTAGGCCAGTACGACATCCTCCAGGGTGGGCTTGCCCTCACCAGCGGCAACTACCAGC

[0360] TCAACTACCAGGGCAACCTGCTGAGCATCCTGCCGCTGCCGGTCACGCCGGGCGACCTGGGGCAACTG

[0361] GCAGCGCTCTCCGACTTGCGCGAACTGCAGAAGGGGCGCGATCCGGATACCCCCGGGGATGCGGTCTA

[0362] CCGCACCACCACGCTGGAAAACCCGTTCCTCGAAAACCCGTTCCTGCGCGCCTACGCTCTGGGCATGG

[0363] ACGTCAGCGATCCTAACCTGCTGCCGGCTACCGCCGCGGGTCCGGCGGAGGACGCCAGCGCCAAGCGG

[0364] GTGGGGCAGTTCACCGATCGTCCGCTCAGGGCAGAGGCCGAGAGTGGGGCGGGTTGCTCGAACCAGAG

[0365] CTACCTGGCCGACTACTGGAGCTGCTTCAACAAGCCTCTGAAT

[0366] <SEQ ID NO:3

[0367] MAAPTGAQFNPNEIKISQQGKTTLIDQSTQRAI INWKGFDVSADEAVRFNQPGVTSSTLNRVTAGQES

[0368] VIAGRISAPGQVI IYNSNGVVFSGSAKVDVGSLITTTANISDEHFRQGKLIFDQPGNPDARIVNDGSI

[0369] SVAEKGLAAFVAPSVANNGVINARLGTVAMAAGNAATIDLYGDGLVSIAVTDPVTRKPQDAQALVSNG

[0370] GAIQADGGSVLITAEQASRVVDNAVNLSGVILARGTEVREGSVALVSKSGDIQIAGKIDVSGPKNGGD

[0371] VLVSGQQVALASTAS IDARGTAQGGSVRIGGDFQGRGELPRAKNATLAKGASIDVSATGKGNGGLAVVWSDGNTRMDGRILARGGAQGGNGGLVETSGKVNLSIADSAYVSVAAPYGNGGTWLLD

[0372] <SEQ ID NO:4

[0373] ASGGTSGSVGGANGASGDATVNASVVTGALAGGKVTLSASDRLSVEAPLITSNLGGASRGLELIATGP

[0374] AGAVDISAPILFRNGSLAIRAGGNINFLSGGTPQTSGIVDLGSGTLWMQTSTAGKISQQAGTALIAAN

[0375] LAGRAGSIDLASWDNYAGNLALQTFNGTLKYRQSNATGVTTSGTVFDPFINQSMTGTAQNIVSSVGTR

[0376] ILEANSVGTTGNYTLTADGNSEFDRLVFTALPYRRVSGSASFPTNDSSDYLVTNLRYQVNGSNVTATP

[0377] NGGAPSGFTVAAGNGSVTTWTGNWGTSWGVKGFGGVIGVTDELQYDVGTGLTEELIFGLGGKTSRVDT

[0378] RLDLFMREGAFNSFAERAQVEMFKTTTTAGDILSRQQTAT

[0379] <SEQ ID NO:5

[0380] TLTANDATRVYGDVNPTLTATMSGINAIDAYVNSQFNDLYQATASTTATQASNVGQYAITGNANGSEY

[0381] FSQRYQLVRQDGRLTVTPAQLIVSADAKTKVYGDADPTLTYQVSGLKNSDTAAGVLSGNLGRVAGENV

[0382] GNYGILQGGLGLNTANYTLSYVGNDLRITPAQLNVIADAKTKVYGDLDPALTYQVSGLKRGDTAGAVL

[0383] NGGSLSRVAGENVGVYGINQGGLGLVSSNYTLNYQGNNLTITKALLNVIADAKTKVYGDADPALTYQV

[0384] SGLKNGDTAGAVLNGGSLSRVAGENVGVYGINQGGLGLLSANYDLSYQGNNLT

[0385] <SEQ ID NO:6

[0386] LNVFADAKSKQVGTADPALTYQVSGLKNGDSAGQVLAGGLGRVGGEAVGQYDILQGGLALTSGNYQLN

[0387] YQGNLLSILPLPVTPGDLGQLAALSDLRELQKGRDPDTPGDAVYRTTTLENPFLENPFLRAYALGMDV

[0388] SDPNLLPATAAGPAEDASAKRVGQFTDRPLRAEAESGAGCSNQSYLADYWSCFNKPLNF

[0389] <SEQ ID NO:7

[0390] GTCGAGCAGCCAGGTGCCACCGTTGCCGTAGGGTGCGGCGACGCTGACGTAGGCGCTGTCGGCGATGC

[0391] TCAGGTTGACCTTGCCGGAGGTCTCCACCAGGCCGCCGTTGCCGCCCTGGGCGCCGCCGCGCGCGAGG

[0392] ATCCGCCCGTCCATGCGGGTATTGCCGTCGGACCAGACCACCGCCAGGCCGCCATTGCCCTTGCCGGT

[0393] AGCCGAGACGTCGATGCTGGCGCCCTTGGCCAGCGTAGCGTTCTTCGCCCGTGGCAGCTCGCCGCGGC

[0394] CCTGGAAGTCGCCGCCGATGCGCACGCTGCCGCCCTGTGCGGTGCCGCGGGCGTCGATGCTGGCGGTG

[0395] CTCGCCAGGGCCACCTGCTGCCCGCTGACCAGCACGTCGCCGCCGTTCTTCGGACCGCTGACGTCGAT

[0396] CTTGCCGGCGATCTGGATGTCCCCGGACTTGCTTACCAGCGCCACGCTGCCCTCGCGCACCTCGGTGC

[0397] CGCGGGCAAGGATCACGCCGGACAGGTTGACCGCGTTGTCGACCACCCGGGAAGCCTGCTCGGCGGTG

[0398] ATCAGCACGCTACCGCCGTCGGCCTGGATCGCGCCACCGTTGCTGACCAGCGCCTGGGCATCCTGCGG

[0399] CTTGCGGGTAACCGGGTCGGTCACGGCGATCGACACCAGGCCATCGCCGTAGAGGTCGATGGTCGCGG

[0400] CATTGCCGGCGGCCATCGCCACGGTGCCGAGGCGGGCGTTGATCACGCCGTTGTTGGCCACGGACGGC

[0401] GCGACGAAGGCCGCCAGGCCTTTCTCCGCGACGCTGATGCTGCCGTCGTTGACGATCCGCGCGTCGGG

[0402] GTTTCCGGGCTGGTCGAAGATCAGCTTGCCCTGGCGGAAGTGTTCGTCGCTGATGTTCGCCGTGGTGG

[0403] TGATCAGGGAGCCCACGTCGACCTTGGCCGAGCCGCTGAAGACCACGCCGTTGGAGTTGTAGATGATC

[0404] ACCTGGCCGGGCGCGGAGATGCGCCCGGCGATCACGCTTTCCTGGCCTGCGGTGACCCGGTTCAGGGT

[0405] CGACGAGGTCACACCGGGCTGGTTGAAGCGCACCGCCTCGTCGGCGGAGACGTCGAAGCCCTTCCAGT

[0406] TGATGATCGCGCGCTGGGTGCTCTGGTCGATCAGCGTGGTCTTGCCCTGCTGGCTGATCTTGATCTCG

[0407] TTGGGGTTGAACTGGGCACCGGTAGGTGCCGCCAT

[0408] <SEQ ID NO:8

[0409] GGTGGCGGTCTGCTGGCGGGAAAGGATGTCGCCGGCAGTGGTGGTGGTCTTGAACATCTCCACCTGGG

[0410] CGCGCTCGGCGAAGCTGTTGAACGCTCCCTCGCGCATGAACAGGTCCAGGCGGGTATCGACGCGGCTG

[0411] GTCTTGCCGCCGAGGCCGAAGATCAGTTCCTCGGTCAGGCCGGTGCCGACGTCGTACTGCAGCTCGTC

[0412] GGTCACGCCGATCACGCCGCCGAAGCCCTTGACGCCCCAACTGGTGCCCCAGTTTCCGGTCCAGGTGG

[0413] TGACGCTGCCGTTGCCGGCGGCGACGGTGAACCCGCTGGGCGCACCGCCGTTCGGGGTGGCGGTGACG

[0414] TTGCTGCCGTTGACCTGGTAGCGCAGGTTGGTCACCAGGTAGTCCGACGAGTCGTTGGTAGGGAAACT

[0415] GGCGGAACCGCTGACCCGCCGATAGGGCAGCGCGGTGAACACCAGGCGGTCGAACTCGCTGTTGCCGT

[0416] CGGCAGTCAGCGTGTAGTTGCCGGTGGTGCCAACGCTGTTGGCCTCGAGAATCCGCGTACCGACGCTG

[0417] GAGACGATGTTCTGCGCGGTGCCGGTCATGCTCTGGTTGATGAACGGATCGAACACCGTGCCGCTCGT

[0418] GGTCACGCCGGTGGCGTTGCTCTGCCGGTACTTGAGCGTACCGTTGAAGGTCTGCAGCGCCAGGTTGC

[0419] CGGCGTAGTTGTCCCAGGAAGCCAGGTCGATGCTACCAGCACGGCCGGCGAGGTTGGCGGCGATCAGC

[0420] GCGGTACCGGCCTGCTGGCTGATCTTGCCGGCGGTGCTGGTCTGCATCCACAGGGTGCCGCTGCCCAG

[0421] GTCGACGATGCCGCTGGTCTGCGGCGTGCCGCCGCTGAGGAAATTGATGTTGCCGCCGGCGCGGATTG

[0422] CCAGGCTGCCGTTGCGGAACAGGATCGGCGCAGAGATATCCACCGCACCGGCAGGTCCGGTGGCGATC

[0423] AGCTCCAGGCCGCGACTGGCGCCGCCCAGGTTGCTGGTGATCAGCGGCGCTTCCACCGACAGCCGGTC

[0424] GCTGGCGCTGAGGGTCACCTTGCCGCCTGCCAGCGCACCGGTAACCACCGAGGCATTCACCGTCGCGT

[0425] CGCCGCTGGCGCCGTTGGCGCCCCCCACCGAGCCGGAGGTGCCGCCGGAGGC

[0426] <SEQ ID NO:9

[0427] GGTCAGGTTATTGCCCTGGTAGCTCAGGTCGTAGTTCGCGCTGAGCAGGCCCAGTCCACCCTGGTTGA

[0428] TGCCGTACACGCCGACATTCTCGCCAGCCACGCGGCTCAGGCTGCCGCCGTTGAGCACGGCACCGGCG

[0429] GTGTCGCCGTTCTTCAGCCCGGACACCTGGTAGGTCAGTGCCGGATCGGCATCGCCATAGACCTTGGT

[0430] CTTGGCATCGGCGATCACATTGAGCAGTGCCTTGGTGATGGTCAGGTTATTGCCCTGGTAGTTCAGGG

[0431] TGTAATTGCTGCTGACCAGGCCCAGTCCACCCTGGTTGATGCCGTACACGCCGACGTTCTCGCCAGCC

[0432] ACGCGGCTCAGGCTGCCGCCGTTGAGCACGGCACCGGCGGTATCGCCGCGCTTGAGACCGGAGACCTG

[0433] GTAGGTCAGCGCCGGATCCAGGTCGCCGTAGACCTTGGTCTTGGCATCGGCGATCACGTTCAGTTGTG

[0434] CCGGGGTGATGCGCAGGTCGTTGCCGACGTAGCTGAGGGTGTAATTGGCCGTGTTGAGGCCCAGCCCG

[0435] CCCTGGAGTATCCCGTAGTTGCCGACGTTCTCCCCGGCCACCCGGCCGAGGTTGCCGCTCAGCACGCC

[0436] AGCGGCGGTATCGCTGTTCTTCAGGCCACTGACCTGGTAGGTGAGGGTCGGATCGGCGTCGCCATAGA

[0437] CCTTGGTCTTGGCGTCGGCGCTGACGATCAATTGCGCCGGCGTGACGGTCAGCCTGCCGTCCTGGCGC

[0438] ACCAGTTGGTAACGCTGGCTGAAATACTCGCTGCCATTGGCGTTACCGGTGATCGCGTACTGCCCGAC

[0439] ATTCGACGCCTGGGTCGCCGTGGTGCTGGCGGTCGCCTGGTAGAGGTCGTTGAACTGACTGTTGACGT

[0440] AGGCGTCGATGGCGTTGATGCCGGACATCGTGGCCGTCAGAGTCGGATTCACATCACCGTAGACGCGG

[0441] GTAGCGTCATTGGCGGTCAGGGT

[0442] <SEQ ID NO:10

[0443] GAAATTCAGAGGCTTGTTGAAGCAGCTCCAGTAGTCGGCCAGGTAGCTCTGGTTCGAGCAACCCGCCC

[0444] CACTCTCGGCCTCTGCCCTGAGCGGACGATCGGTGAACTGCCCCACCCGCTTGGCGCTGGCGTCCTCC

[0445] GCCGGACCCGCGGCGGTAGCCGGCAGCAGGTTAGGATCGCTGACGTCCATGCCCAGAGCGTAGGCGCG

[0446] CAGGAACGGGTTTTCGAGGAACGGGTTTTCCAGCGTGGTGGTGCGGTAGACCGCATCCCCGGGGGTAT

[0447] CCGGATCGCGCCCCTTCTGCAGTTCGCGCAAGTCGGAGAGCGCTGCCAGTTGCCCCAGGTCGCCCGGC

[0448] GTGACCGGCAGCGGCAGGATGCTCAGCAGGTTGCCCTGGTAGTTGAGCTGGTAGTTGCCGCTGGTGAG

[0449] GGCAAGCCCACCCTGGAGGATGTCGTACTGGCCTACGGCTTCGCCGCCCACCCGACCGAGCCCGCCGG

[0450] CGAGCACCTGACCGGCGCTATCGCCATTCTTCAGGCCGGAAACCTGATAGGTCAGCGCCGGGTCGGCG

[0451] GTGCCGACCTGCTTGCTCTTGGCATCGGCGAATACGTTGAG。

Claims

1. A recombinant protein based on the outer membrane matrix protein of Pseudomonas aeruginosa, comprising a truncated peptide of a CdrA protein, wherein 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.

2. The recombinant protein according to claim 1, wherein the amino acid sequence 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.

3. A gene encoding the recombinant peptide according to claim 1 or 2, wherein the nucleotide sequence thereof is selected from any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:

10.

4. An expression vector comprising an expression plasmid and the gene according to claim 3; Preferably, the expression plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors, preferably pGEX-6P-1.

5. A recombinant engineered bacterium, characterized in that: Comprising the expression vector as claimed in claim 4 and a host bacterium; preferably, the host bacterium is selected from any one of Escherichia coli XL1-blue strain, BL21 series strain and HMS174 series strain, preferably Escherichia coli BL21 strain.

6. Use of the recombinant protein according to claim 1 or 2, or the gene according to claim 3, or the expression vector according to claim 4, or the recombinant engineered bacteria according to claim 5 in the 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.

7. A composition for preventing or treating Pseudomonas aeruginosa infection, characterized in that: The invention comprises the recombinant protein based on the outer membrane matrix protein of Pseudomonas aeruginosa as claimed in claim 1 or 2, and pharmaceutically acceptable excipients.

8. A vaccine for preventing or treating Pseudomonas aeruginosa infection, characterized in that: Contains the recombinant protein based on the outer membrane matrix protein of Pseudomonas aeruginosa as described in claim 1 or 2, or the composition as described in claim 7.

9. The vaccine according to claim 8, characterized in that The invention comprises a pharmaceutically acceptable adjuvant, and preferably, 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 BCG adjuvant.

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