Nanoparticle protein MntC-rePO@LS, preparation method and application thereof

Abstract

This invention provides a nanoparticle protein, MntC-rePO@LS, its preparation method, and its applications. This protein is formed by the covalent binding of MntC@LS and recombinant V antigen-outer membrane protein OprI (rePO) via heteropeptide bonds, creating a dual-nanoparticle vaccine molecule. The recombinant protein SpyCatcher-MntC@LS of this invention can be induced to express in soluble form in *E. coli* and can bind efficiently to the recombinant protein rePO-SpyTag under mild conditions. The nanoparticle protein of this invention can induce a high level of immune response in animals, exerting an immunoprotective effect. Experiments show that its rate of antibody response induction and protective efficiency are significantly superior to those of monomeric MntC, monomeric rePO, and their physical mixtures, and it can be used for the prevention and treatment of *Pseudomonas aeruginosa* and / or *Staphylococcus aureus* infections.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nanoparticle protein MntC-rePO@LS for the prevention or treatment of Pseudomonas aeruginosa infection and Staphylococcus aureus infection, its preparation method, and its application. Background Technology

[0002] Globally, approximately 7-7.7 million people die from bacterial infections each year, of which 4.95 million are related to drug-resistant pathogens, and about 1.27 million are directly attributable to antibiotic-resistant infections. This grim situation highlights that drug-resistant bacterial infections have become a major threat to global public health. Compared to single-microorganism infections, multi-microorganism co-infections often lead to more severe tissue damage, more complex pathological processes, and, due to their synergistic or antagonistic microbial interactions, make pathogens more difficult to eradicate completely. Among various co-infection systems, Staphylococcus aureus (S. aureus) is a common culprit. Staphylococcus aureus SA) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Co-infections involving both pulmonary fibrosis (PF) and cystic fibrosis (PC) are particularly prominent, especially in patients with chronic wound infections and pulmonary infections. These two factors not only enhance each other's biofilm formation and promote antibiotic resistance, but also exacerbate the infection process through metabolic interactions and signal interference, significantly increasing treatment difficulty and leading to a worsening of patient prognosis.

[0003] Staphylococcus aureus is a Gram-positive opportunistic pathogen. Its pathogenicity relies primarily on a variety of virulence factors, including toxins and enzymes, and its strong biofilm-forming ability. It can cause infections ranging from localized skin infections to severe systemic infections such as sepsis and endocarditis. SA exhibits multidrug resistance, especially methicillin-resistant Staphylococcus aureus (MRSA), which has become a significant pathogen in hospital and community infections. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Its pathogenic mechanism revolves around the use of a complex array of virulence factors, including the type III secretion system, exotoxins, proteases, and biofilms, and relies on the quorum sensing system to regulate the infection process. It often causes acute or chronic infections in immunocompromised hosts. PA is extremely difficult to treat clinically due to both inherent drug resistance and resistance to a wide range of available antimicrobial agents.

[0004] Currently, the common clinical treatment strategy for SA and PA infections involves the combined use of anti-SA and anti-PA antibiotics, such as vancomycin or linezolid combined with anti-pseudomonas β-lactam drugs. However, in practice, such combination therapies have not significantly improved patients' pulmonary function indicators or effectively reduced the risk of acute exacerbations. Instead, they are often accompanied by increased treatment failure rates, prolonged disease course, and further development of drug resistance. This phenomenon is particularly common in patients with hospital-acquired pneumonia, chronic wounds, and cystic fibrosis. Therefore, relying solely on combination antibiotic therapy is often insufficient to address the synergistic pathogenicity and drug resistance mechanisms of SA and PA infections, suggesting a need for further research and development of novel treatment strategies targeting multiple microbial infections to improve the clinical outcomes of such refractory infections.

[0005] Vaccination is an effective strategy for preventing bacterial infections. In SA vaccine research, manganese transporter C (MntC), a key component of the MntABC high-affinity manganese uptake system, is highly conserved and plays a central role in bacterial pathogenesis, thus being considered a highly promising vaccine antigen. For PA, its type III secretion system key protein V antigen (PcrV) is responsible for mediating the transport of toxins to host cells, while the outer membrane lipoprotein OprI can not only activate the TLR2 / 4 immune pathway but also synergistically participate in bacterial nutrient transport and membrane stability maintenance with other outer membrane proteins; both are recognized immune targets. However, the above antigens still suffer from limited immunogenicity, making it difficult to induce a sufficient and durable protective immune response.

[0006] Nanoparticle vaccines, as novel antigen delivery platforms, can mimic the spatial conformation of natural pathogens, enhancing antigen stability and dendritic cell presentation efficiency while inducing potent and durable immune responses, thus attracting significant attention. Lumazine synthase (LS) can self-assemble into an icosahedral 6-hexamethylenetetramer structure, enabling high-density and precise arrangement of various heterologous antigens on its surface, making it an ideal platform for developing multivalent vaccines. Studies have successfully induced broad-spectrum neutralizing antibodies in mouse models by constructing nanoparticles simultaneously displaying the spike proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV, providing effective protection against SARS-CoV-2 infection, including variant strains. Based on LS fusion nanoparticles, the SpyCatcher / SnoopCatcher system was used to co-display the chikungunya virus E2 protein and the Zika virus EDIII antigen, simultaneously stimulating high levels of IgG and neutralizing antibodies against both pathogens in mice.

[0007] Based on the above analysis, we designed a dual nanovaccine, MntC-rePO@LS, using LS nanoparticles as a carrier platform. Through genetic engineering, SpyCatcher and MntC were linked to the N-terminus and C-terminus of LS nanoparticles, respectively. The rePO antigen was then directionally displayed on the surface of the particles using covalent linkage mediated by the SpyTag / SpyCatcher system. This dual nanovaccine aims to target both SA and PA infections simultaneously, providing a novel immune intervention strategy for addressing both single and co-infections. Summary of the Invention

[0008] In view of the above limitations, this invention addresses the serious harm caused by Pseudomonas aeruginosa and Staphylococcus aureus infections by using the SpyTag / SpyCatCher protein conjugation system to jointly display MntC and the fusion protein rePO on the surface of self-assembled nanoparticles LS, and provides a self-assembled nanoparticle protein MntC-rePO@LS for Pseudomonas aeruginosa and Staphylococcus aureus.

[0009] This invention provides a nanoparticle protein MntC-rePO@LS, comprising covalently linked recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag; wherein, SpyCatcher-MntC@LS is composed of SpyCatcher, -(Linker)n-, dioxetine synthase (LS), -(Linker)n-, and MntC linked sequentially, wherein MntC is Staphylococcus aureus manganese ion transporter C, and rePO is a fusion protein of Pseudomonas aeruginosa PcrV and OprI, wherein each occurrence of Linker is independently selected from any one of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and each occurrence of n is independently selected from 1, 2, 3, or 4, preferably 3.

[0010] In one embodiment of the present invention, the amino acid sequence of SpyCatcher is SEQ ID NO:1, the amino acid sequence of LS is SEQ ID NO:2, and the amino acid sequence of rePO is SEQ ID NO:3.

[0011] In one embodiment of the present invention, the amino acid sequence of the MntC is SEQ ID NO:4; and / or, the amino acid sequence of the rePO-SpyTag is SEQ ID NO:8.

[0012] In one embodiment of the present invention, the amino acid sequence of the SpyCatcher-MntC@LS is SEQ ID NO:5.

[0013] Another aspect of the present invention provides a nucleotide that is the encoding gene of the above-mentioned nanoparticle protein MntC-rePO@LS@LS.

[0014] Another aspect of the present invention provides an expression vector comprising the above-mentioned nucleotides, and / or a backbone vector; preferably, the backbone vector is selected from one of the pGEX series vectors, the pET series vectors, or the pQE series vectors, preferably a pET series vector, and the expression vector containing the coding gene of SpyCatcher-MntC@LS is preferably pET-21a(+).

[0015] The present invention also provides a recombinant strain comprising the expression vector and host bacteria as described in claim 6: preferably, the host bacteria 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 further provides a method for preparing the above-mentioned recombinant protein MntC-rePO@LS, which includes the following steps: 1) The coding gene for MntC is fused with the coding gene for SpyCatcher to construct a first recombinant gene; the coding gene for rePO is fused with the coding gene for SpyTag to construct a second recombinant gene; preferably, the amino acid sequence encoded by the first recombinant gene is the peptide of SEQ ID NO:5; the amino acid sequence encoded by the second recombinant gene is SEQ ID NO:9; 2) The first recombinant gene is ligated into the vector plasmid pET-21a(+) to obtain the first expression vector; the second recombinant gene is ligated into the vector plasmid pET-21a(+) to obtain the second expression vector; 3) The first expression vector is transformed into the first host bacterium to obtain the first recombinant bacterium; the second expression vector is transformed into the first host bacterium to obtain the second recombinant bacterium; 4) After inducing expression in the first host bacterium, the recombinant protein SpyCatcher-MntC@LS was purified; after inducing expression in the second host bacterium, the recombinant protein rePO-SpyTag was purified. 5) Recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag were co-dissolved in PBS buffer at pH 7.5 at mass ratios of 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, and 1:2.5, and bound at 4°C for 12 h; preferably, the recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag were respectively subjected to Ni 2+ After affinity chromatography purification, the binding operation is then performed; 6) The MntC-rePO@LS recombinant protein is obtained after purification; preferably, the recombinant protein SpyCatcher-MntC@LS and the recombinant protein rePO-SpyTag are combined and then purified by size exclusion chromatography.

[0017] The present invention also provides the application of the above-mentioned nanoparticle protein MntC-rePO@LS, expression vector, or recombinant strain in the preparation of subunit vaccines against Pseudomonas aeruginosa and / or Staphylococcus aureus.

[0018] A vaccine for the simultaneous prevention or treatment of bacterial infections, comprising the aforementioned nanoparticle protein MntC-rePO@LS; wherein the bacteria are Pseudomonas aeruginosa and / or Staphylococcus aureus.

[0019] The beneficial effects of the above-described technical solution of the present invention are as follows: 1) This invention involves analyzing the charge, spatial structure, and energy kinetic parameters of key Staphylococcus aureus proteins MntC, PcrV (a key protein in the type III secretion system of Pseudomonas aeruginosa), outer membrane protein OprI, and dioxetine synthase LS, followed by the fusion expression of SpyCatcher with MntC and LS. The recombinant protein SpyCatcher-MntC@LS of this invention utilizes the SpyTag / SpyCatcher conjugation system, a highly efficient site-specific protein conjugation method. While this method still relies on gene manipulation and protein purification, it eliminates the need for de novo production, allowing for the production of modular protein components and assembly according to requirements. This method also minimizes disruption to the protein's spatial conformation and biological activity.

[0020] 2) The recombinant protein SpyCatcher-MntC@LS of this invention is expressed in soluble form in E. coli. The histidine tag at the N-terminus of the expression vector is retained, and after Ni... 2+ Recombinant protein SpyCatcher-MntC@LS, which is stable in buffer systems and has uniform particle size, was obtained by purification techniques such as packing affinity chromatography and size exclusion chromatography.

[0021] 3) The recombinant protein SpyCatcher-MntC@LS of the present invention can covalently bind to the recombinant protein rePO-SpyTag through isopeptide bonds to form MntC-rePO@LS dual nanoparticle protein. The binding conditions are simple and the coupling efficiency is high. Under transmission electron microscopy, it appears as dual nanoparticles with uniform particle size.

[0022] 4) The MntC-rePO@LS dual nanoparticle vaccine of the present invention is administered via intramuscular injection. It can induce the production of rePO-specific IgGs antibodies in mouse serum and exert a highly effective immune protection against Pseudomonas aeruginosa infection; it can also induce the production of MntC-specific IgGs antibodies in mouse serum and exert a highly effective immune protection against Staphylococcus aureus. The induced antibody response speed and protective efficiency are significantly better than those of monomeric rePO protein and MntC protein. Attached Figure Description

[0023] Figure 1 SDS-PAGE electrophoresis images of MntC@LS, rePO-ST, and MntC-rePO@LS.

[0024] Figure 2 These are molecular sieve chromatography images of rePO-ST, MntC@LS, and MntC-rePO@LS.

[0025] Figure 3 The images show the dynamic light scattering particle size distribution of rePO-ST, MntC@LS, and MntC-rePO@LS.

[0026] Figure 4 Transmission electron microscopy images of MntC@LS and MntC-rePO@LS.

[0027] Figures 5-6 The image shows the results of detecting the levels of MntC and rePO-specific IgG antibodies induced after immunization with MntC-rePO@LS.

[0028] Figures 7-8 Figure 1 shows the results of detecting MntC and rePO-specific IgG antibody subtypes induced after immunization with MntC-rePO@LS. Figures 9-13 To evaluate the immunoprotective effect of MntC-rePO@LS in a mouse model of pneumonia caused by Pseudomonas aeruginosa. Figure 9 Results of mouse body weight changes within 168 h after XN-1 infection; Figure 10 The results of infection status scoring in mice within 168 h after XN-1 infection; Figure 11 Results of XN-1 bacterial colonization detection in lung tissue; Figures 12-13 The results of H&E staining and scoring of lung tissue 24 hours after XN-1 infection.

[0029] Figures 14-18 To evaluate the immunoprotective effect of MntC-rePO@LS in a mouse model of Staphylococcus aureus-infected pneumonia. Figure 14 The results of body weight changes in mice within 168 hours after infection with USA300; Figure 15 The infection status scores of mice within 168 hours after infection with USA300; Figure 16 Results of the detection of USA300 bacterial colonization in lung tissue; Figures 17-18 The results of H&E staining and scoring of lung tissue 24 hours after infection with USA300.

[0030] Figures 19-24 To evaluate the immunoprotective effect of MntC-rePO@LS in a mouse pneumonia model co-infected with Pseudomonas aeruginosa and Staphylococcus aureus. Figure 19 The results show the changes in body weight of mice within 168 h after co-infection with XN-1 and USA300. Figure 20 The infection status scores of mice within 168 h after co-infection with XN-1 and USA300; Figure 21 Results of XN-1 bacterial colonization detection in lung tissue; Figure 22 Results of the detection of USA300 bacterial colonization in lung tissue; Figure 23 The results of H&E staining of lung tissue 24 hours after infection; Figure 24 The results of H&E staining of lung tissue 24 hours after infection. Detailed Implementation

[0031] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0032] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0033] Example 1 pET-21a(+)_ SpyCatcher-MntC@Lumazine Synthase Construction of recombinant plasmids 1. Test Methods (1) The amino acid sequences of MntC, LS and SpyCatcher were retrieved from the NCBI official website database (the amino acid sequence of SpyCatcher is SEQ ID NO:1, the amino acid sequence of LS is SEQ ID NO:2, the amino acid sequence of rePO is SEQ ID NO:3, and the amino acid sequence of MntC is SEQ ID NO:4). The amino acid sequence of SpyCatcher-MntC@LS was designed and obtained as SEQ ID NO:5. It was then synthesized by Nanjing Genscript Biotech Co., Ltd. to obtain SpyCatcher-MntC@LS recombinant DNA, which was cloned into the pET-21a(+) vector.

[0034] (2) Transformation: Take out pET-21a(+)_ stored at -20℃ SpyCatcher-MntC@Lumazine Synthase The plasmid powder was centrifuged at 5000 rpm for 2 min at 4 °C, dissolved in 40 μL of sterile ddH2O, gently pipetted to mix, and briefly centrifuged to collect the liquid. 1 μL of the plasmid solution was added to 50 μL of BL21(DE3) competent cells (Solarbio), gently mixed, and incubated on ice for 30 min. Then, the cells were heat-shocked in a 42 °C metal bath for 90 s and quickly transferred to ice for 3 min to cool. 1 mL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C with shaking at 220 rpm for 60 min. After incubation, the cells were centrifuged at 5000 rpm for 2 min, 800 μL of supernatant was discarded, and 200 μL of medium was retained to resuspend the cells. 50 μL of the bacterial suspension was spread onto LB agar plates containing ampicillin sodium (1:4000), incubated upright for 30 min, and then inverted at 37 °C for overnight incubation for 12–16 h.

[0035] (3) Cultivating engineered bacteria: In a clean bench, pick a single colony from the plate with a sterile 10 μL pipette tip, inoculate it into a shake flask containing 10 mL LB medium, add 5 μL of ampicillin sodium solution, and incubate overnight (12~16 h) at 37℃ and 220 rpm with shaking.

[0036] (4) Plasmid extraction: Plasmids were extracted from the culture medium using the TIANGEN DP103 plasmid mini-prep kit.

[0037] 2. Experimental Results The extracted plasmid was sent to Wuhan Jinkairui Co., Ltd., and the sequencing results were consistent with the target sequence.

[0038] Example 2: Preparation and biochemical characterization of recombinant protein SpyCatcher-MntC@LS and binding protein MntC-rePO@LS 1. Test Methods 1.1 Preparation of recombinant protein SpyCatcher-MntC@LS 1.1.1 Expression identification of recombinant protein SpyCatcher-rePO@LS (1) Activation culture: In a clean bench, add 10 mL of antibiotic-free LB liquid medium and 5 μL of ampicillin sodium solution (final concentration 100 μg / mL) to the inoculation bottle, pick a single colony from the transformation plate and inoculate it into the bottle, and culture overnight at 37℃ and 220 rpm with shaking (about 12-15 h). (2) Secondary activation culture: The bacterial culture from step (1) was inoculated into 20 mL of antibiotic-free LB liquid medium at a ratio of 1:100 (v / v), and ampicillin sodium solution was added to a final concentration of 100 μg / mL. The culture was then incubated at 37℃ and 220 rpm for about 4 h until OD was reached. 600 It reaches 0.6~0.8.

[0039] (3) Induction of expression: The culture temperature was adjusted to 16℃, and after cooling for 2 h, IPTG was added to a final concentration of 200 μM. Expression was induced at 16℃ and 160 rpm for 12-15 h.

[0040] (4) Ultrasonic disruption: After induction, 1 mL of bacterial culture was collected from each treatment group for later use. The remaining bacterial culture was transferred to a 10 mL centrifuge tube and centrifuged at 8000 rpm for 10 min. The supernatant was discarded, and the precipitate was resuspended in 2 mL of PBS buffer. The resuspended solution was placed in an ice-water mixture and disrupted using an ultrasonic disruptor with the following parameters: 5 s sonication, 6 s interval, 6 min total duration, and 15% power. After disruption, the sample was centrifuged at 12000 rpm for 10 min to separate the supernatant and precipitate. The precipitate was resuspended in 900 μL of PBS buffer and kept together with the supernatant for subsequent protein expression detection.

[0041] (5) SDS-PAGE gel electrophoresis to identify protein expression: Prepare protein gels in advance. Take 40 µL of each sample and mix with 10 µL of 5× protein loading buffer. Gently pipette to mix and heat in a 100℃ metal bath for 5 min. Load the samples sequentially, adding 10 µL of sample to each well and 5 µL of protein molecular weight standard (Protein Marker, 26616). Set the initial electrophoresis voltage to 80 V. After the samples enter the separating gel, adjust the voltage to 180 V and continue electrophoresis until the end.

[0042] (6) Preservation: Based on the SDS-PAGE gel results, preserve the bacterial strains that meet the purification conditions. Prepare the bacterial solution after two activations. In a clean bench, draw 500 μL of 50% glycerol into the preservation tube, then draw 500 μL of bacterial solution into the preservation tube. Mix well, ignite the tube opening with the outer flame of an alcohol lamp, tighten the tube, and store at -80℃.

[0043] 1.1.2 Purification of recombinant protein SpyCatcher-MntC@LS (1) First activation culture: Take the MntC-rePO@LS culture medium from -80℃, and after it is completely dissolved, take 10 μL and inoculate it into 10 mL LB medium. Add 5 μL of ampicillin sodium solution and incubate overnight (12~16 h) at 37℃ and 220 rpm with shaking. (2) Secondary activation culture: The bacterial culture from step (1) was inoculated into 2L of antibiotic-free LB liquid medium at a ratio of 1:100 (v / v), and ampicillin sodium solution was added to a final concentration of 100 μg / mL. The culture was carried out at 37℃ and 220 rpm for about 4 h with shaking until OD was reached. 600 It reaches 0.6~0.8.

[0044] (3) Induction of expression: The culture temperature was adjusted to 16℃, and after cooling for 2 h, 400 μL of 1M IPTG inducer (final concentration of 200 μM) was added. Expression was induced at 16℃ and 160 rpm for 12-15 h.

[0045] (4) Collection of bacterial cells: After induction, transfer the bacterial solution to a special centrifuge bottle, centrifuge at 10℃ and 6000 rpm for 20 min, discard the supernatant, collect the wet bacterial cells in a 50 mL centrifuge tube, and store at -20℃ or use immediately for subsequent disruption.

[0046] (5) Ultrasonic disruption: Add 40 mL of PBS buffer to the wet bacterial cells, vortex until homogeneous, place in a beaker, and sonicate under ice bath conditions. The parameters are set as follows: sonication for 8 s, interval for 9 s, total duration for 15 min, and power for 38%. After disruption, centrifuge at 4℃ and 12000 rpm for 17 min to separate the supernatant and precipitate.

[0047] (6) Purification: Take 5 mL of Ni 2+ Affinity chromatography column was packed with the packing material and repeatedly equilibrated with PBS buffer. The supernatant from sonication lysis was then loaded onto the column, mixed thoroughly, and placed on a rotary mixer for binding at 4°C for 30 min. After binding, the flow-through was collected. The packing material was washed repeatedly with PBS buffer until the eluent was clear. A gradient elution was performed sequentially using 15 mL each of PBS solutions containing 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, and 500 mM imidazole, controlling the flow rate to allow the liquid to flow down naturally. Finally, the packing material was equilibrated three times with PBS buffer and washed twice with 25% ethanol solution. The packing material was then immersed in 25% ethanol and stored at 4°C.

[0048] (7) SDS-PAGE gel electrophoresis to identify protein purification status: Prepare protein gels in advance. Take 40 µL of each sample and mix with 10 µL of 5× protein loading buffer. Gently pipette to mix and heat in a 100℃ metal bath for 5 min. Load the samples sequentially, adding 10 µL of sample to each well and 5 µL of protein molecular weight standard (Protein Marker, 26616). Set the initial electrophoresis voltage to 80 V. After the sample enters the separating gel, adjust the voltage to 180 V and continue electrophoresis until the end.

[0049] (8) Replacement buffer: Based on the SDS-PAGE results, the imidazole-containing protein solution was replaced with PBS buffer (pH 8.0) using gel filtration chromatography. An ÄKTA avant purification system was used, with a G25 desalting column installed. The column inlet pressure was set to 0.5 MPa, the wall pressure to 0.3 MPa, and the flow rate to 10 mL / min. The flow path was flushed with PBS as the mobile phase to equilibrate the system and column until the conductivity (Cond) and UV280 absorption signal stabilized at baseline. The protein solution was then loaded, and elution was performed at the same flow rate, with real-time monitoring of the UV280 signal. The target protein elution peak was collected when the absorbance rose above 10 mAU, and collection was stopped when the signal dropped below 10 mAU, ultimately yielding the MntC-rePO@LS protein solution.

[0050] 1.2 Preparation and biochemical characterization of the binding protein MntC-rePO@LS (1) Preparation of the binding protein MntC-rePO@LS: Using 50 μL of MntC-rePO@LS protein as the reaction system, the corresponding volumes of rePO-ST protein solution were added at molar ratios of 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, and 1:2.5 (MntC-rePO@LS:rePO-ST). Each reaction system was brought to the same final volume with PBS buffer and incubated overnight at 4°C. After the reaction, samples were taken for SDS-PAGE analysis, and the target bands were analyzed using ImageJ to determine the optimal ratio for complex formation.

[0051] (2) Molecular sieve chromatography: Start the ÄKTA avant protein purification system, equilibrate the system pathway with PBS buffer, set the flow rate to 10 mL / min, and after the conductivity (Cond) and UV280 absorption signal stabilize to baseline, install a Superose™ 6Increase 10 / 300 GL gel filtration chromatography column. Set the column inlet pressure to 4 MPa, the tube wall pressure to 2 MPa, and the column flow rate to 0.5 mL / min. Continue to equilibrate the chromatography column with PBS buffer until the system conductivity stabilizes at approximately 16 mS / cm, and zero the UV280 absorbance signal. Inject 500 μL of MntC-rePO@LS sample through the sample loop, monitor the UV280 absorption signal in real time, and record the elution peak appearance time.

[0052] (3) Dynamic light scattering particle size detection: Dynamic light scattering of samples was measured using a Zetasizer Nano particle size analyzer. After the instrument was preheated for 20 min, the detection type was set to Size, the sample material to Protein, the dispersant to Water, the temperature to 25℃, and the detection angle to 90°. The other parameters were kept at their default settings. Each sample was measured three times.

[0053] (4) Transmission electron microscopy: The protein sample was diluted with PBS to 0.01 mg / mL, and the carbon-supported copper mesh (200 mesh) was treated with a PELCO easiGlow™ glow discharge instrument at 0.26 mBar and 15 mA for 40 seconds to complete the hydrophilication treatment. 3.5 μL of protein solution was added to the copper mesh, and after adsorption for 1 min, the residual droplets were removed. The mesh was washed with 10 μL of PBS and then dried. 50 μL of uranium acetate staining solution was applied in three drops onto plastic wrap, and the copper mesh was then dipped into the staining solution on the front side in turn. The first two drops were quickly removed, and the third drop was allowed to stand for 30 s before being dried. The mesh was then dried at room temperature and stored for later use. The morphology and dispersion of the protein were observed using a JEM-1400Plus transmission electron microscope, and images were acquired and analyzed.

[0054] 2. Experimental Results The recombinant plasmid pET-21a(+)-SpyCatcher-MntC@LS was transformed into *E. coli* to prepare the pET-21a(+)-SpyCatcher-MntC@LS / BL21(DE3) engineered strain. The strain was induced with IPTG at 16℃, followed by ultrasonic lysis, and the supernatant and precipitate were separated by centrifugation. A 68 kDa band was observed in the lysed supernatant, indicating that the protein was expressed in a soluble form in *E. coli*. To obtain the purified SpyCatcher-MntC@LS protein, the pET-21a(+)-SpyCatcher-MntC@LS / BL21(DE3) engineered strain was induced with IPTG, and the supernatant after ultrasonic lysis and centrifugation was mixed with Ni...2+ After binding with the packing material and washing away contaminating proteins, SpyCatcher-MntC@LS protein was eluted with PBS buffer containing 500 mM imidazole. The obtained SpyCatcher-MntC@LS protein was then desalted using a HiPrep™ 26 / 10 Desalting column, and the results are as follows. Figure 1 (Lane 2).

[0055] Mix the protein at molar ratios of 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, and 1:2.5 (SpyCatcher-MntC@LS:rePO-ST). A 1:1 ratio is considered the optimal binding ratio. Figure 1 As shown in lane 6, a complex of approximately 120 kDa is formed. Figure 2 As shown, the peak shapes of rePO-ST, MntC@LS, and MntC-rePO@LS are all symmetrical, with peak volumes of 14.73 mL, 11.22 mL, and 9.56 mL, respectively. Compared to MntC@LS nanoparticles, the peak volume of MntC-rePO@LS protein shifts forward by approximately 1.66 mL, indicating a significant increase in its molecular weight and suggesting that MntC-rePO@LS protein forms uniform nanoparticles. Figure 3 As shown, the fluid diameters of rePO-ST, MntC@LS, and MntC-rePO@LS are 13.55 nm, 56.49 nm, and 65.69 nm, respectively. Compared to MntC@LS, the nanoparticle size of MntC-rePO@LS increased by approximately 9.20 nm, suggesting that the MntC-rePO@LS protein forms uniform nanoparticles. Figure 4 As shown, transmission electron microscopy images further revealed that both MntC@LS and MntC-rePO@LS exhibit a uniform spherical structure.

[0056] Example 3: Evaluation of the immune response to MntC-rePO@LS 1. Test Methods 1.1 Humoral immune response detection (1) Vaccine preparation and mouse immunization: 6-8 week old SPF-grade female BALB / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 6 groups of 5 mice each. The groups were: ① PBS group; ② LS group (containing 8.57 μg); ③ rePO group (containing 9.43 μg); ④ MntC group (containing 10 μg); ⑤ MntC, rePO, LS mix group (containing 8.57 μg LS, 10 μg MntC and 9.43 μg rePO); ⑥ MntC-rePO@LS group (containing 10.86 μg rePO-ST and 18.57 μg MntC@LS). The mice were immunized by intramuscular injection on days 0, 7 and 14. The injection volume for each mouse was 200 μL. All preparations were freshly prepared and used immediately, and the immunization dose was consistent across different batches.

[0057] (2) Blood collection from mouse tail vein: Blood was collected from mice via tail vein on the 7th day after each immunization. The mice were incubated at 37°C for 2 hours, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected to obtain serum. The serum was then aliquoted and stored at -80°C for later use.

[0058] (3) Monitoring the levels of MntC-specific and rePO-specific IgG antibodies in mouse serum using indirect ELISA. 1) Coating antigen: Dissolve rePO and MntC antigen at 6 μg / mL in coating solution, add 100 μL to each well of a 96-well microplate, wrap with aluminum foil and coat overnight at 4°C.

[0059] 2) Washing: Place the overnight coated microplate on a plate washer and wash with PBST washing buffer. The program is set as follows: add 300 μL of buffer to each well, aspirate for 2.5 seconds, shake for 5 seconds, wash 3 times, and gently pat dry on clean absorbent paper.

[0060] 3) Blocking: Add 250 μL of blocking solution to each well and incubate in a 37℃ constant temperature incubator for 2 h.

[0061] 4) Washing the plate: Same as above.

[0062] 5) Primary antibody incubation: Add 100 μL of PBST solution to each well in rows B-H of the ELISA plate, and add 200 μL of diluted serum sample to row A. Transfer 100 μL of serum solution from row A to row B, mix thoroughly by pipetting 15-20 times, and then serially dilute to row H. Finally, discard the 100 μL solution. Incubate at 37°C for 1 h.

[0063] 6) Washing the plate: Same as above.

[0064] 7) Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse IgG secondary antibody with PBST at a ratio of 1:7500, mix thoroughly, add 100 μL to each well, and incubate at 37℃ for 45 min.

[0065] 8) Washing the plate: Same as above.

[0066] 9) Color development: Under light-protected conditions, add 100 μL of TMB substrate color development solution to each well and react in a constant temperature incubator at 37℃ for 10 min.

[0067] 10) Termination: Under light-protected conditions, add 50 μL of termination solution to each well.

[0068] 11) Plate reading: The absorbance of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0069] 12) Results analysis: Cut-off values ​​were calculated based on the results of the negative control group, and the serum antibody titer levels of mice in each group were analyzed.

[0070] 1.2 Detection of IgG subtypes IgG1, IgG2a, and IgG2b (1) Mouse immunization: Same as above (2) Blood collection from mouse tail vein: Serum from mice after the second immunization was collected. (3) The MntC-specific and rePO-specific IgG antibody subtypes (IgG1, IgG2a and IgG2b) induced in serum were detected by the same indirect ELISA method as described above, except that the secondary antibody was replaced with IgG1, IgG2a and IgG2b, and the antibody dilution factor remained unchanged.

[0071] 2. Experimental Results To evaluate the immunogenicity of MntC-rePO@LS, the levels of specific total IgG and its subtypes against MntC and rePO in the serum of immunized mice were detected by indirect ELISA. Figures 5-6 As shown, at three testing points (days 7, 14, and 21), the total titers of anti-MntC and anti-rePO IgG in the MntC-rePO@LS group were significantly higher than those in the monomer group and the physical mixture group. P <0.05%. The total titer of anti-MntC IgG reached its peak on day 14, significantly earlier than that of the monomer group and the physical mixture group. P <0.05). For example... Figures 7-8 As shown, the levels of anti-MntC and anti-rePO IgG2b induced by the MntC-rePO@LS group were significantly higher than those of IgG1 and IgG2a. PThe value was <0.05, indicating that the vaccine primarily elicited a Th1-type immune response. The levels of anti-rePO IgG2b in both the monomeric and physical mixture groups were significantly lower than those of IgG1. In conclusion, MntC-rePO@LS induced a stronger specific antibody response, primarily IgG2b, than either the monomeric or physical mixture groups.

[0072] Example 4: Evaluation of the immunoprotective effect of immunization with MntC-rePO@LS in a mouse model of acute pneumonia caused by Pseudomonas aeruginosa infection 1. Test Methods (1) Immunization of mice: 6-8 week old SPF-grade female BALB / c mice were randomly divided into 5 groups of 10 mice each. The groups were: ① PBS group; ② LS group (containing 8.57 μg); ③ rePO group (containing 9.43 μg); ④ MntC, rePO, LS mix group (containing 8.57 μg LS, 10 μg MntC and 9.43 μg rePO); ⑤ MntC-rePO@LS group (containing 10.86 μg rePO-ST and 18.57 μg MntC@LS). The mice were immunized by intramuscular injection on days 0, 7 and 14. The injection volume for each mouse was 200 μL. All preparations were freshly prepared and used immediately. The immunization dose was consistent across different batches.

[0073] (2) Resuscitation, culture and conditioning of clinical strain PA XN-1 1) Streaking: After the PA XN-1 strain stored at -80℃ was naturally thawed, it was inoculated into LB agar plates containing ampicillin sodium (1:2000) using the three-zone streak method. The plates were then inverted and incubated overnight in a 37℃ incubator. After the incubation was completed, the plates were temporarily stored at 4℃ for later use.

[0074] 2) Activation: In a clean bench, pick a single colony from the plate and inoculate it into 10 mL of antibiotic-free LB liquid medium. Incubate overnight at 37°C and 220 rpm.

[0075] 3) Secondary activation: In a clean bench, take 200 μL of live bacterial culture and inoculate it into 20 mL of antibiotic-free LB liquid medium. Incubate at 37℃ and 220 rpm until the bacterial culture reaches the logarithmic growth phase, which takes about 3.5 h.

[0076] 4) Bacterial conditioning: In a clean bench, take the optimally grown bacterial culture and aliquot it into 10mL centrifuge tubes. Centrifuge at 8500 rpm for 5 min, discard the supernatant, and wash the precipitate three times with 5 mL of sterile physiological saline. Measure the OD using a UV spectrophotometer. 600 Adjust the bacterial concentration to the range of 1.19~1.23, then dilute with physiological saline to 15 times the volume, dispense into 1.5mL centrifuge tubes and store on an ice-water bath.

[0077] (3) Sublethal dose clinical strain PA XN-1 was used to challenge mice with acute pneumonia. 1) Tracheal challenge: Mice were anesthetized by intraperitoneal injection of 150 μL of anesthetic, and 20 μL of prepared bacterial culture was injected through tracheal intubation (the inoculation volume per mouse was 1.33 × 10⁻⁶). 6 CFU / each).

[0078] 2) Post-infection monitoring: After challenge, the weight changes of mice in each group were recorded every 12 hours and the infection status was scored. The monitoring continued for 168 hours. The infection status scoring criteria included 5 indicators: hair condition, body posture, respiratory status, motor activity and eye secretions. Each indicator was scored from 0 to 2 points according to the severity, with a total score of 10 points.

[0079] (3) Assessment of bacterial colonization in lung tissue 1) 24 hours after the virus challenge, 5 mice from each group were euthanized by blood collection from their eyes, and their bodies were disinfected with 75% alcohol. Their lung tissue was then dissected and removed in a clean bench.

[0080] 2) Place the lung tissue in a sterile glass homogenizer containing 1 mL PBS and homogenize it. Transfer the resulting homogenate into a 1.5 mL sterile EP tube and store it on ice for later use.

[0081] 3) Add 180 μL of PBS to each well of a 96-well plate. Add 20 μL of lung tissue homogenate to the first well, mix thoroughly, and then add 20 μL to the second well. Perform serial dilutions sequentially, with a dilution factor of 1×10⁻⁶. -1 1×10 -2 1×10 -3 1×10 -4 1×10 -5 and 1×10 -6 Take 10 μL of each grade of bacterial culture and drop it onto LB agar plates containing ampicillin sodium (1:2000). Invert the plates and incubate overnight at 37°C. Count the number of colonies on the plates and convert them to bacterial load per gram of lung tissue (CFU / g).

[0082] (4) Assessment of lung tissue pathological damage: 24 h after challenge, lung tissue from one mouse in each group was taken and fixed in 4% paraformaldehyde for 24 h. After dehydration, paraffin embedding, sectioning and hematoxylin-eosin (H&E) staining, histopathological changes were observed under an optical microscope.

[0083] 2. Experimental Results To evaluate the protective effect of MntC-rePO@LS immunization against Pseudomonas aeruginosa lung infection, 6-8 week old SPF-grade female BALB / c mice were inoculated via endotracheal intubation on day 7 after the last immunization with a sublethal dose of the clinical PA strain XN-1 (diluted 15-fold) to establish a mouse pneumonia infection model. Lung bacterial load, body weight changes, and infection status scores were monitored. Figures 9-13 As shown, the MntC-rePO@LS group recovered better than the LS group and the physical mixture group. Mouse infection status scores showed that the MntC-rePO@LS group had the mildest infection symptoms and the fastest recovery, with scores significantly lower than other groups. Lung bacterial colonization results further indicated that the number of bacteria per milligram of lung tissue in the MntC-rePO@LS group was significantly lower than that in the PBS group, LS group, single-component group, and physical mixture group. P <0.05). Pathological results of lung tissue sections showed that the lung tissue structure of mice in the MntC-rePO@LS group was basically normal, while the control group showed obvious pathological changes such as inflammatory cell infiltration and alveolar structure destruction.

[0084] Example 5: Evaluation of the immunoprotective effect of immunization with MntC-rePO@LS in a mouse model of acute pneumonia caused by Staphylococcus aureus infection. 1. Test Methods (1) Immunization of mice: 6-8 week old SPF-grade female BALB / c mice were randomly divided into 5 groups of 10 mice each. The groups were: ① PBS group; ② LS group (containing 8.57 μg); ③ MntC group (containing 10 μg); ④ MntC, rePO, LS mix group (containing 8.57 μg LS, 10 μg MntC and 9.43 μg rePO); ⑤ MntC-rePO@LS group (containing 10.86 μg rePO-ST and 18.57 μg MntC@LS). The mice were immunized by intramuscular injection on days 0, 7 and 14. The injection volume for each mouse was 200 μL. All preparations were freshly prepared and used immediately. The immunization dose was consistent across different batches.

[0085] (2) Resuscitation, culture and conditioning of clinical strain SA USA300 1) Streaking: After the SA USA 300 strain stored at -80℃ is naturally thawed, it is inoculated onto MHA plates using the three-zone streak method, inverted in a 37℃ constant temperature incubator for overnight culture, and then temporarily stored at 4℃ for later use after culture.

[0086] 2) Activation: In a clean bench, pick a single colony from the plate and inoculate it into 10 mL of MHB medium. Incubate overnight (15-19 h) at 37°C and 220 rpm.

[0087] 3) Secondary activation: In a clean bench, inoculate 200 μL of live bacterial culture into 20 mL of MHB medium and incubate at 37°C and 220 rpm until the bacterial culture reaches OD500. 600 Within the range of 0.6 to 0.8, it takes approximately 3 hours and 40 minutes.

[0088] 4) Bacterial conditioning: In a clean bench, take the optimal growth status of the two live bacteria suspension, aliquot it into 10 mL centrifuge tubes, centrifuge at 8500 rpm for 5 min, discard the supernatant, resuspend the precipitate in 5 mL of sterile physiological saline, combine the precipitates, and add physiological saline to a final volume of 10 mL. Repeat the washing process three times. Measure the OD using a UV spectrophotometer. 600 Adjust the bacterial concentration to 1.0±0.1, then concentrate to 5 times the volume, dispense, and store in an ice bath.

[0089] (3) Sublethal dose clinical strain SA USA300 was used to challenge mice with acute pneumonia. 1) Tracheal challenge: Mice were anesthetized by intraperitoneal injection of 150 μL of anesthetic, and 40 μL of prepared bacterial culture was injected through tracheal intubation (the inoculation volume per mouse was 2.00 × 10⁻⁶). 8 CFU / each).

[0090] 2) Post-infection surveillance: Same as above.

[0091] (4) Assessment of bacterial colonization in lung tissue: Except for the use of MHA plates for dripping, the specific methods are the same as those described above.

[0092] (5) Assessment of pathological damage to lung tissue: Same as above.

[0093] 2. Experimental Results To evaluate the protective effect of MntC-rePO@LS immunization against Staphylococcus aureus lung infection, this study established a Staphylococcus aureus pneumonia model in mice by inoculating them with a sublethal dose (5-fold concentrated) of the clinical SA strain USA300 via endotracheal intubation on day 7 after the last immunization. Lung bacterial load, body weight changes, and infection status scores were monitored. Figures 14-18 As shown, the bacterial load per milligram of lung tissue in the MntC-rePO@LS group was significantly lower than that in the control groups. P <0.05%. 72 h post-infection, mice in this group recovered body weight more quickly ( P The infection status score was <0.05, and the score was also significantly lower than that of other groups. Histological analysis further showed that the lung tissue pathological damage in the MntC-rePO@LS group was significantly reduced.

[0094] Example 6: Evaluation of the immunoprotective effect of immunization with MntC-rePO@LS in a mouse model of acute pneumonia co-infected with Pseudomonas aeruginosa and Staphylococcus aureus. 1. Test Methods (1) Immunization of mice: 6-8 week old SPF-grade female BALB / c mice were randomly divided into 6 groups of 10 mice each. The groups were: ① PBS group; ② LS group (containing 8.57 μg); ③ rePO group (containing 9.43 μg); ④ MntC group (containing 10 μg); ⑤ MntC, rePO, LS mix group (containing 8.57 μg LS, 10 μg MntC and 9.43 μg rePO); ⑥ MntC-rePO@LS group (containing 10.86 μg rePO-ST and 18.57 μg MntC@LS). The mice were immunized by intramuscular injection on days 0, 7 and 14. The injection volume for each mouse was 200 μL. All preparations were freshly prepared and used immediately. The immunization dose was consistent across different batches.

[0095] (2) Resuscitation, culture and conditioning of clinical strain PA XN-1: Same as above. (3) Resuscitation, culture and conditioning of clinical strain SA USA300: Same as above. (4) Sublethal doses of clinical strains PA XN-1 and SA USA300 were used to challenge mice with acute pneumonia. 1) Tracheal challenge: Same as above. Administer 10 μL of PA XN-1 per mouse and 20 μL of SA USA300 per mouse (the XN-1 inoculation dose per mouse is 4.76 × 10⁻⁶). 5 CFU / animal, USA300 inoculation dose is 1.72 × 10⁻⁶ 7 CFU / each).

[0096] 2) Post-infection surveillance: Same as above.

[0097] (5) Assessment of bacterial colonization in lung tissue: LB agar plates and MHA plates were used for the drop plate operation, and the specific method was the same as above.

[0098] (6) Assessment of lung tissue pathological damage: Same as above.

[0099] 2. Experimental Results To evaluate the protective effect of the dual nanoparticle vaccine MntC-rePO@LS against co-infection with Pseudomonas aeruginosa and Staphylococcus aureus, 6-8 week old SPF-grade female BALB / c mice were selected and inoculated via endotracheal intubation with a sublethal dose of PA clinical strain XN-1 (diluted 21-fold) and SA clinical strain USA300 (concentrated 0.86-fold) to establish a co-infection pneumonia model. Figures 19-24 As shown, the pulmonary PA and SA loads in the MntC-rePO@LS group were significantly lower than those in the control groups. P(<0.05). After 48 h of co-infection, the weight recovery of this group was better than that of the LS group and the physical mixture group ( P (<0.05), and the infection status score was also significantly lower. Further histopathological analysis of the lung tissue showed that MntC-rePO@LS could effectively reduce the lung tissue damage caused by co-infection.

[0100] The above results indicated that after immunization with the dual nanoparticle vaccine MntC-rePO@LS, it could exert a more potent protective effect in both the single and co-infection models of Pseudomonas aeruginosa and Staphylococcus aureus in mice.

[0101] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

[0102] Sequence Listing

[0103] <SEQ ID NO:1 SpyCatcher (Molecular type: AA)

[0104] AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

[0105] <SEQ ID NO:2 LS (Molecular type: AA)

[0106] MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR​​​​​GSEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGGGGSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK

[0109] <SEQ ID NO:4 MntC (Molecular type: AA)

[0110] SSDKSNGKLKVVTTNSILYDMAKNVGGDNVDIHSIVPVGQDPHEYEVKPKDIKKLTDADVILYNGLNLETGNGWFEKALEQAGKSLKDKKVIAVSKDVKPIYLNGEEGNKDKQDPHAWLSLDNGIKYVKTIQQTFIDNDKKHKADYEKQGNKYIAQLEKLNNDSKDKFNDIPKEQRAMITSEGAFKYFSKQYGITPGYIWEINTEKQGTPEQMRQAIEFVKKHKLKHLLVETSVDKKAMESLSEETKKDIFGEVYTDSIGKEGTKGDSYYKMMKSNIETVHGSMK

[0111] <SEQ ID NO:5 SpyCatcher-MntC@LS (Molecular type: AA)

[0112] AMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGGSGGGGSMQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLRGGGGSGGGGSGGGGSSSDKSNGKLKVVTTNSILYDMAKNVGGDNVDIHSIVPVGQDPHEYEVKPKDIKKLTDADVILYNGLNLETGNGWFEKALEQAGKSLKDKKVIAVSKDVKPIYLNGEEGNKDKQDPHAWLSLDNGIKYVKTIQQTFIDNDKKHKADYEKQGNKYIAQLEKLNNDSKDKFNDIPKEQRAMITSEGAFKYFSKQYGITPGYIWEINTEKQGTPEQMRQAIEFVKKHKLKHLLVETS

[0113] [[ID=③]]<SEQ ID NO:6 LINKER1 (Molecular type: AA)

[0114] GGGGS

[0115] [[ID=⑨]]<SEQ ID NO:7 LINKER2 (Molecular type: AA)

[0116] GGSGG

[0117] [[ID=⑮]]<SEQ ID NO:8 LINKER3 (Molecular type: AA)

[0118] YAPVDV

[0119] [[ID=㉑]]<SEQ ID NO:9 rePO-SpyTag (Molecular type: DNA)

[0120] Note: The Chinese characters in the translation of item 3, 9, 15 and 21 are used to match the format of the original text with Chinese characters in tags. In actual English patent texts, they should be in English. For example, item 3 should be "<SEQ ID NO:6 LINKER1 (Molecular type: AA)".

Claims

1. A nanoparticle protein MntC-rePO@LS, comprising covalently linked recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag; wherein, The SpyCatcher-MntC@LS consists of SpyCatcher and -(Linker). n - Dioxetine synthase (LS), - (Linker) n - MntC is sequentially linked together, wherein MntC is Staphylococcus aureus manganese ion transporter C, and rePO is a fusion protein of Pseudomonas aeruginosa PcrV and OprI. Each Linker is independently selected from any one of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 each time it appears, and n is independently selected from 1, 2, 3 or 4 each time it appears, preferably 3.

2. The nanoparticle protein MntC-rePO@LS as described in claim 1, wherein, The amino acid sequence of SpyCatcher is SEQ ID NO:1, the amino acid sequence of LS is SEQ ID NO:2, and the amino acid sequence of rePO is SEQ ID NO:

3.

3. The nanoparticle protein MntC-rePO@LS as described in claim 1 or 2, wherein, The amino acid sequence of the MntC is SEQ ID NO:4; and / or, the amino acid sequence of the rePO-SpyTag is SEQ ID NO:

9.

4. The nanoparticle protein MntC-rePO@LS as described in any one of claims 1-3, wherein, The amino acid sequence of SpyCatcher-MntC@LS is SEQ ID NO:

5.

5. A nucleotide that is the gene encoding the nanoparticle protein MntC-rePO@LS@LS as described in any one of claims 1-4.

6. An expression carrier, characterized in that, The vector contains the nucleotides as described in claim 5, and / or a backbone vector; preferably, the backbone vector is selected from one of the pGEX series vectors, the pET series vectors, or the pQE series vectors, preferably the pET series vector, and the expression vector containing the coding gene of SpyCatcher-MntC@LS is preferably pET-21a(+).

7. A recombinant bacterial strain, characterized in that, It includes the expression vector and host bacteria as described in claim 6: preferably, the host bacteria is selected from any one of Escherichia coli XL1-blue strain, BL21 series strain and HMS174 series strain, preferably Escherichia coli BL21 strain.

8. A method for preparing the recombinant protein MntC-rePO@LS as described in any one of claims 1-4, characterized in that, Includes the following steps: 1) The coding gene for MntC is fused with the coding gene for SpyCatcher to construct a first recombinant gene; the coding gene for rePO is fused with the coding gene for SpyTag to construct a second recombinant gene; preferably, the amino acid sequence encoded by the first recombinant gene is the peptide of SEQ ID NO:5; the amino acid sequence encoded by the second recombinant gene is SEQ ID NO:9; 2) The first recombinant gene is ligated into the vector plasmid pET-21a(+) to obtain the first expression vector; the second recombinant gene is ligated into the vector plasmid pET-21a(+) to obtain the second expression vector; 3) The first expression vector is transformed into the first host bacterium to obtain the first recombinant bacterium; the second expression vector is transformed into the first host bacterium to obtain the second recombinant bacterium; 4) After inducing expression in the first host bacterium, the recombinant protein SpyCatcher-MntC@LS was purified; after inducing expression in the second host bacterium, the recombinant protein rePO-SpyTag was purified. 5) Recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag were co-dissolved in PBS buffer at pH 7.5 at mass ratios of 1:1, 1:1.25, 1:1.5, 1:1.75, 1:2, and 1:2.5, and bound at 4°C for 12 h; preferably, the recombinant proteins SpyCatcher-MntC@LS and recombinant protein rePO-SpyTag were respectively subjected to Ni 2+ After affinity chromatography purification, the binding operation is then performed; 6) The MntC-rePO@LS recombinant protein is obtained after purification; preferably, the recombinant protein SpyCatcher-MntC@LS and the recombinant protein rePO-SpyTag are combined and then purified by size exclusion chromatography.

9. The use of the nanoparticle protein MntC-rePO@LS according to any one of claims 1-4, the expression vector according to claim 5, or the recombinant strain according to claim 6 in the preparation of subunit vaccines against Pseudomonas aeruginosa and Staphylococcus aureus.

10. A drug for the prevention or treatment of bacterial infections, characterized in that, Contains the nanoparticle protein MntC-rePO@LS as described in any one of claims 1-4; the bacteria are infected with Pseudomonas aeruginosa and / or Staphylococcus aureus.

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