An immunogenic composition for preventing pseudomonas aeruginosa infection and use thereof

Abstract

The present application relates to a kind of new immunogenic compositions for preventing and treating pseudomonas aeruginosa infection, fusion protein, recombinant vaccine, and molecular architecture design and application.The present application provides new fusion molecular architecture, including EF-Tu, DnaK, EF-G three gene coding fusion protein A, or PcrV, PilA two gene coding fusion protein B, or the combination of fusion protein A and B, can be used in the development of nucleic acid vaccine or subunit vaccine.The present application finds that new fusion molecule has good immunogenicity, and can provide effective immunoprotective effect, significantly inhibits pseudomonas aeruginosa colonization, has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to an immunogenic composition of Pseudomonas aeruginosa that can induce immune protection and its applications. Background Technology

[0002] Pseudomonas aeruginosa (PA), also known as Pseudomonas aeruginosa, is a zoonotic pathogen that is both environmentally persistent and multidrug resistant. It not only causes serious nosocomial infections (collectively known as Pseudomonas aeruginosa-associated diseases, PAADs), but also triggers highly lethal outbreaks in livestock. In human medicine, PAADs mainly manifest as chronic pulmonary infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), and catheter-related bloodstream infection (CRBSI), with CPI having a mortality rate exceeding 30% in cystic fibrosis patients. In livestock, PA can cause acute septicemia in calves and lambs (mortality rate 30%-60%), refractory mastitis in dairy cows (milk yield reduction of over 40%), and fibrinous airsacculitis in broilers (increasing culling rate by 15%-25%), causing global economic losses to the livestock industry exceeding US$2 billion annually.

[0003] Pseudomonas aeruginosa damages the host's immune system, leading to immunosuppression and inflammatory imbalance, which in turn triggers other opportunistic infections. Pseudomonas aeruginosa often forms mixed biofilm infections with Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans. In livestock farms, the immunosuppressive effect exacerbates secondary infections such as porcine reproductive and respiratory syndrome (PRRS) and avian Escherichia coli, raising the mortality rate of mixed infections to 70%. Livestock farming has become a breeding ground for Pseudomonas aeruginosa resistance evolution; the multidrug resistance rate of farmed isolates in my country exceeds 65%, and the resistance rate to commonly used veterinary antibiotics such as enrofloxacin and ceftiofur is >50%. More seriously, plasmid-borne resistance genes can spread to humans through the food chain via fecal contamination of the environment. Monitoring data from 2023 showed that the Pseudomonas aeruginosa contamination rate in raw milk in my country reached 8.7%, and the contamination rate in poultry meat reached 12.3%, posing a significant public health threat. In recent years, the detection rate of multidrug-resistant (MDR) Pseudomonas aeruginosa strains in Chinese hospitals has continued to rise, and it has become one of the important drug-resistant pathogens that endanger public health in key departments such as ICU, burn ward, and respiratory department.

[0004] Safe and effective vaccines are considered the best potential measure to control Pseudomonas aeruginosa infection and the spread of drug resistance. Currently, existing international and domestic research or commercialized vaccines against Pseudomonas aeruginosa mainly include inactivated whole-cell vaccines, toxoid vaccines, subunit vaccines (such as OprF / OprI fusion protein vaccines), and polysaccharide conjugate vaccines. These vaccines can effectively reduce acute mortality and pulmonary bacterial load in animal models, but none can completely block the colonization, infection, and horizontal transfer of drug resistance genes in Pseudomonas aeruginosa. In recent years, domestic scholars have also been actively developing other novel Pseudomonas aeruginosa vaccines, such as the recombinant lactococcus expressing Pseudomonas aeruginosa outer membrane protein I, plasmid, and vaccine disclosed in patent CN118703546A, and their applications; patent CN118048284A discloses a Pseudomonas aeruginosa fur gene deletion mutant strain and its construction method and applications; and patent CN116731209A discloses recombinant Pseudomonas aeruginosa nanoparticle protein rePO-FN and its applications.

[0005] However, existing vaccine strategies still face significant challenges: inactivated whole-cell vaccines have high safety risks and weak immunogenicity; polysaccharide vaccines have weak activation capacity for T-cell-dependent immune responses and are ineffective in infants and young children; and live attenuated vaccines carry the risk of virulence reversion and pathogenicity in immunodeficient hosts. With the continuous advancements in reverse vaccinology, structural biology, and genetic engineering technologies, the development of safe, broad-spectrum, and highly effective novel genetically engineered vaccines against Pseudomonas aeruginosa has become a major direction in vaccine development. There is still an urgent need in this field for a fusion protein, immune composition, and vaccine with good immunogenicity that can provide effective immune protection and significantly inhibit Pseudomonas aeruginosa colonization. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a novel immunogenic composition, fusion protein, recombinant vaccine, molecular architecture design, and applications for the prevention and treatment of *Pseudomonas aeruginosa* infection. This invention provides a novel fusion molecular architecture comprising fusion protein A encoded by three genes (EF-Tu, DnaK, and EF-G), or fusion protein B encoded by two genes (PcrV and PilA), or a combination of fusion proteins A and B, which can be used in the development of nucleic acid vaccines or subunit vaccines. This invention reveals that the novel fusion molecule exhibits good immunogenicity and provides effective immune protection, significantly inhibiting *Pseudomonas aeruginosa* colonization, and has broad application prospects. This invention also finds that the novel molecular architecture can effectively increase the expression level of the fusion protein, thereby further enhancing the immune protection effect. This invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and uses.

[0007] One aspect of the present invention provides a fusion protein, characterized in that it is selected from any one of the following (1)-(3):

[0008] (1) Fusion protein A, wherein the fusion protein A comprises an elongation factor Tu (EF-Tu) antigen or an antigen-selected fragment thereof, a chaperone protein DnaK (DnaK) antigen or an antigen-selected fragment thereof, and an elongation factor G (EF-G) antigen or an antigen-selected fragment thereof.

[0009] (2) Fusion protein B, wherein the fusion protein B comprises PcrV (type III secretion protein PcrV) antigen and PilA (type IV fimbrial precursor PilA) antigen, wherein the amino acid sequence of the PcrV antigen is shown in SEQ ID NO: 4 and the amino acid sequence of the PilA antigen is shown in SEQ ID NO: 5.

[0010] (3) The combination of the fusion protein A and the fusion protein B.

[0011] Further, the EF-Tu, DnaK, EF-G antigens or selected fragments thereof, as well as the PcrV and PilA antigens, are derived from Gram-negative bacteria; preferably, the EF-Tu, DnaK, EF-G antigens or selected fragments thereof, as well as the PcrV and PilA antigens, are derived from the order Pseudomonas and family Pseudomonas; most preferably, the EF-Tu, DnaK, EF-G antigens or selected fragments thereof, as well as the PcrV and PilA antigens, are derived from Pseudomonas aeruginosa or Pseudomonas aeruginosa.

[0012] Furthermore, the fusion protein A comprises, from N-terminus to C-terminus, the EF-Tu antigen or its antigen-selected fragment, the DnaK antigen or its antigen-selected fragment, and the EF-G antigen or its antigen-selected fragment; the fusion protein B comprises, from N-terminus to C-terminus, the PcrV antigen and the PilA antigen.

[0013] Furthermore, the amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 1, the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 3.

[0014] Furthermore, the antigens may optionally be linked by a linker sequence or a spacer sequence; preferably, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 10, and the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 11.

[0015] Furthermore, the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 6, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO: 7.

[0016] Furthermore, the N-terminus of the fusion protein B further comprises a signal peptide and / or any secretion-promoting element; preferably, the signal peptide is derived from azurocidin (AZU1), and the secretion-promoting element is derived from the Fc domain of the immunoglobulin heavy constant gamma (IGHG) protein; more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 8, and the amino acid sequence of the secretion-promoting element is as shown in SEQ ID NO: 9.

[0017] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the fusion protein of the present invention; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

[0018] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention; preferably, the recombinant gene expression cassette further comprises one or more of a promoter, a terminator, and a regulatory sequence.

[0019] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0020] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0021] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0022] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0023] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0024] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0025] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0026] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0027] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0028] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0029] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0030] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0031] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0032] Further, the immunogenic composition or pharmaceutical composition contains the fusion protein A and the fusion protein B; preferably, the mass ratio of the fusion protein A and the fusion protein B in the immunogenic composition or pharmaceutical composition is 1:1.

[0033] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins described in the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; preferably, the recombinant vaccine comprises fusion protein A and fusion protein B; more preferably, the mass ratio of fusion protein A to fusion protein B in the recombinant vaccine is 1:1.

[0034] Another aspect of the present invention provides the use of one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention in the preparation of vaccines or medicaments for prevention, treatment and / or inoculation.

[0035] Furthermore, the drug is used to prevent and / or treat infections or diseases caused by Pseudomonas aeruginosa; preferably, the infections or diseases include pneumonia, skin and soft tissue infections, urinary tract infections, eye infections, ear infections, bacteremia, sepsis, wound infections, chronic obstructive pulmonary disease (COPD), chronic lung infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, refractory mastitis, fibrinous airsacculitis, endocarditis, bloodstream infection, central nervous system infection, and bone and joint infection.

[0036] Another aspect of the present invention provides a method for preventing and / or treating infections or diseases caused by *Pseudomonas aeruginosa*, characterized in that it includes administering to a subject one or more fusion proteins described in the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention, and / or one or more recombinant vaccines described in the present invention; preferably, the infection or disease includes pneumonia, skin and soft tissue infections, urinary tract infections, eye infections, ear infections, bacteremia, sepsis, wound infections, chronic obstructive pulmonary disease (COPD), chronic lung infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, refractory mastitis, fibrinous airsacculitis, endocarditis, bloodstream infection, central nervous system infection, and bone and joint infection.

[0037] The fusion protein molecular structure, the nucleic acid structure encoding it, the pharmaceutical composition, and the recombinant vaccine of the present invention have the following beneficial technical effects:

[0038] 1. Antigen screening: Multiple Pseudomonas aeruginosa antigens were selected, and bioinformatics techniques such as protein three-dimensional structure prediction and immunoepitaxy were used to select antigens and design fusion expression, including N / C-terminal truncation, full-length truncation, and fusion protein stability prediction, to finally obtain the EF-Tu antigen, DnaK antigen, EF-G antigen, PcrV antigen and PilA antigen selected in this invention.

[0039] 2. This invention mutates the glycosylation site of the PcrV antigen to T265A to prevent glycosylation of the prokaryotic protein during expression in eukaryotic cells, which would affect the correct presentation of epitopes. The mutated PcrV antigen exhibits high expression abundance and more accurate epitope presentation.

[0040] 3. Example 4 shows that vaccine A (EF-Tu antigen-DnaK antigen-EF-G antigen fusion protein) can be significantly expressed in cells, proving that the antigen fusion expression molecular architecture provided by the present invention enables the smooth translation and correct folding of bacterial-derived multi-antigen combinations in eukaryotic cells, and that the structure is stable and has a long half-life. Vaccine B (PcrV antigen-PilA antigen fusion protein) can be significantly expressed in supernatant and cell lysate, proving that the molecular architecture provided by the present invention, which adds an Fc domain to the N-terminus of the antigen, enables the smooth translation and correct folding of bacterial-derived fusion proteins in eukaryotic cells, and promotes the efficient secretion of fusion proteins to the extracellular space. Therefore, the vaccines designed based on the present invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial for the presentation of immunoepitaxes.

[0041] 4. Example 5 shows that the bacterial load of *Pseudomonas aeruginosa* was significantly reduced in the lung tissue of mice immunized with vaccine A, vaccine B, and a combination of vaccines A and B (vaccine A + vaccine B). The combined immunization group had the lowest bacterial load in its lung tissue, followed by the vaccine A immunization group. Furthermore, comparison of tissue sections after challenge showed that both combined and individual immunization with vaccines A and B effectively prevented *Pseudomonas aeruginosa* infection, reduced the severity of tissue lesions, and achieved the expected results. Combined immunization showed the best preventive effect, while vaccine A showed the second best preventive effect.

[0042] 5. The antigen and fusion molecular structure provided by this invention successfully induced an effective immune response in mouse model animals, demonstrating a good preventive effect against Pseudomonas aeruginosa infection. This breakthrough fills the gap in the current field of Pseudomonas aeruginosa vaccine development, provides a novel solution for the production and development of animal immunotherapies, and has extremely high commercial value and broad application prospects. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine A and vaccine B of the present invention.

[0044] Figure 2 This is a schematic diagram of a template plasmid containing the gene expression cassette of the present invention.

[0045] Figure 3A-Figure 3B The table shows the quality control peak diagrams and purity test results for nucleic acid vaccines A and B containing the antigen sequences of this invention; wherein... Figure 3A The image shows the quality control peaks and purity test results for vaccine A. Figure 3B This is a peak diagram of the quality control for vaccine B and the results of its purity test.

[0046] Figure 4A-Figure 4B The results of Western blot analysis of the expression of vaccines A and B of the present invention after in vitro transfection into HEK293T cells are shown; wherein Figure 4A The results of WB detection for in vitro expression of vaccine A are as follows. Figure 4B The results of WB detection for in vitro expression of vaccine B.

[0047] Figure 5 This document outlines the immunization and sampling procedures for vaccines A, B, and combinations thereof in a mouse model of Pseudomonas aeruginosa infection.

[0048] Figure 6A-Figure 6B This invention relates to vaccines A, B, and combinations thereof, and the changes in bacterial load after challenge with *Pseudomonas aeruginosa* in mice. Figure 6A Plate colony counts to show changes in bacterial load after challenge.Figure 6B This is a quantitative result of colony counts showing changes in bacterial load after challenge.

[0049] Figure 7 This is a comparison of tissue sections after challenge with Pseudomonas aeruginosa in mice using vaccines A, B, and combinations thereof of the present invention. Detailed Implementation

[0050] Terms and Definitions

[0051] The term "Pseudomonas aeruginosa" refers to Pseudomonas aeruginosa, abbreviated as PA, a common Gram-negative bacillus, also known as Pseudomonas aeruginosa. It is widely distributed in nature, such as in soil and water, and is also commonly found in the humid environments of hospitals. It can cause a variety of infections, including pneumonia, urinary tract infections, wound infections, and sepsis, and is a significant nosocomial pathogen, especially among hospitalized patients. Infectious diseases caused by Pseudomonas aeruginosa include, but are not limited to, pneumonia, urinary tract infections, wound infections, sepsis, chronic obstructive pulmonary disease (COPD), chronic pulmonary infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, refractory mastitis, fibrinous airsacculitis, endocarditis, skin and soft tissue infections, ear infections, eye infections, bloodstream infections, central nervous system infections, and bone and joint infections.

[0052] The term "PcrV" in *Pseudomonas aeruginosa* refers to type III secretion protein PcrV, a key component of the bacterial type III secretion system (T3SS). The PcrV antigen of this invention comprises the full-length PcrV antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the PcrV antigen. Preferably, the amino acid sequence of the PcrV antigen is shown in SEQ ID NO: 4.

[0053] The term "PilA" refers to Pilin A, a major subunit protein constituting type IV Pili (T4P). PilA facilitates bacterial adhesion to host cells or object surfaces and is a key initiation step in biofilm formation. The PilA antigen of this invention comprises the full-length PilA antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the PilA antigen. Preferably, the amino acid sequence of the PilA antigen is shown in SEQ ID NO: 5.

[0054] The term "EF-Tu" refers to Elongation Factor Tu, an important GTPase in prokaryotes that plays a central role in the translation process of protein synthesis. Besides its role in protein synthesis, EF-Tu may also have "moonlighting" functions, participating in host-pathogen interactions as a bacterial surface protein and antigen. The EF-Tu antigen of this invention comprises the full-length EF-Tu antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the EF-Tu antigen. Preferably, the amino acid sequence of the EF-Tu antigen is as shown in SEQ ID NO: 1.

[0055] The term "DnaK" refers to the DnaK chaperone protein, one of the main molecular chaperones in bacteria, belonging to the DnaK chaperone system (which also includes DnaJ and GrpE). DnaK functions under ATP-driven conditions, its core function being to protect newly synthesized polypeptides or those denatured under stress conditions (such as heat stress) from misfolding and aggregation. The DnaK antigen of this invention comprises the full-length DnaK antigen and its selected fragments, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the DnaK antigen. Preferably, the amino acid sequence of the DnaK antigen is as shown in SEQ ID NO: 2.

[0056] The term "EF-G" refers to Elongation Factor G, a GTPase in prokaryotes that functions in protein synthesis through both translocation and ribosome recycling. It drives structural changes in ribosomes via GTP hydrolysis, ensuring the efficiency and accuracy of protein synthesis and playing a crucial role in the final stage of translation. The EF-G antigen of this invention comprises the full-length EF-G antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the EF-G antigen. Preferably, the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 3.

[0057] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immunity can be measured by assays, including but not limited to assays measuring the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays measuring T cell activation or proliferation, and / or assays measuring the regulation of the activity or expression of one or more cytokines.

[0058] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0059] The term “expression” includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0060] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," a "recombinant protein," or a "fusion protein."

[0061] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

[0062] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0063] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In the genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0064] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0065] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0066] The terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0067] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0068] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0069] The term "treatment" refers to exposing a subject to (e.g., administering medication) a recombinant vaccine, composition, etc., based on the present invention after contracting a disease, thereby reducing the symptoms of the disease compared to when not exposed, and does not imply the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body has developed symptoms of a disease.

[0070] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0071] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0072] This invention discloses a fusion molecular architecture for the prevention and treatment of Pseudomonas aeruginosa infection, a method for preparing a recombinant vaccine based on this architecture, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0073] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0074] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0075] Example 1: Construction of the recombinant nucleic acid vaccine of the present invention

[0076] Antigen screening: Multiple *Pseudomonas aeruginosa* antigens were selected, and bioinformatics techniques such as protein three-dimensional structure prediction and immunoepitope screening were used to truncate and design fusion expression, including N / C-terminal truncation, full-length selection, and fusion protein stability prediction. The final results yielded the EF-Tu, DnaK, EF-G, PcrV, and PilA antigens selected in this invention. Furthermore, this invention mutated the glycosylation site of the PcrV antigen to T265A to prevent glycosylation of the prokaryotic protein during expression in eukaryotic cells, which could affect the correct presentation of epitopes. The mutated PcrV antigen exhibits high expression abundance and more accurate epitope presentation.

[0077] To prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, a non-limiting schematic diagram of the nucleic acid vaccine of the present invention is shown below. Figure 1 As shown, an exemplary Figure 1 This is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine A and vaccine B of the present invention. In order to prepare vaccines capable of producing... Figure 1 The recombinant nucleic acid vaccine with the molecular structure shown first involves constructing a gene expression cassette to express the antigen sequence described in this invention. A schematic diagram of a template plasmid containing the gene expression cassette of this invention is shown below. Figure 2 As shown, from the 5' end to the 3' end, it sequentially includes: 5'UTR, CDS region, 3'UTR, and PolyA, wherein the CDS region contains the fusion molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy, and the DNA sequence was directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing recombinant nucleic acid vaccines.

[0078] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0079] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0080] Step a: Synthesize the “EF-Tu antigen-DnaK antigen-EF-G antigen” gene fragments, which are linked together by a linker sequence as shown in SEQ ID NO: 10. The amino acid sequence of the fusion protein encoded by the EF-Tu antigen-DnaK antigen-EF-G antigen fusion gene is shown in SEQ ID NO: 6. Specifically, the amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 1, the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 3.

[0081] Step b: Construct a nucleic acid vaccine architecture carrier.

[0082] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0083] Step c: Prepare recombinant plasmids.

[0084] The gene synthesized in step a is inserted into the vector structure in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention.

[0085] (2) Preparation vector of recombinant nucleic acid vaccine B based on the present invention

[0086] Step a: Synthesize the gene fragment “signal peptide-IGHG Fc domain-PcrV antigen-PilA antigen”, wherein the signal peptide is the signal peptide of azurocidin (AZU1), and its amino acid sequence is shown in SEQ ID NO: 8; the amino acid sequence of the Fc domain of IGHG is shown in SEQ ID NO: 9; the amino acid sequence of the PcrV antigen is shown in SEQ ID NO: 4; and the amino acid sequence of the PilA antigen is shown in SEQ ID NO: 5. The Fc domain of IGHG and the PcrV antigen are linked by a spacer sequence as shown in SEQ ID NO: 11, and the PcrV antigen and the PilA antigen are linked by a linker sequence as shown in SEQ ID NO: 10. The amino acid sequence of the fusion protein encoded by the PcrV antigen-PilA antigen fusion gene is shown in SEQ ID NO: 7. The PcrV antigen contains the glycosylation site mutation T265A.

[0087] Step b: Construct a nucleic acid vaccine architecture carrier.

[0088] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0089] Step c: Prepare recombinant plasmids.

[0090] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector B based on the present invention.

[0091] Table 1. Amino acid sequences of the architectural elements involved in this invention.

[0092] EF-Tu antigen ERNKPHVNVGTIGHVDHGKTTLTAALTKVCSDTWGGSARAFDQIDNAPEEKARGITINTSHVEYDSAVRHYAHVDCPGHADYVKNMITGAAQMDGAILVCSAADGPMPQTREHILLSRQVGVPYIVVFLNKADMVDDAELLELVEMEVRDLLNTYDFPGDDTPIIIGSALMALEGKDDNGIGVSAVQKLVETLDSYIPEPV (SEQ ID NO: 1) DnaK antigen VTPLTLGIETLGGVMTGLIEKNTTIPTKKSQVFSTADDNQGAVTIHVLQGERKQAAQNKSLGKFDLADIPPAPRGVPQIEVTFDIDANGILHVSAKDKATGKQQSIVIKASS (SEQ ID NO: 2) EF-G antigen PQVAYRETITKDNVEIEGKFVRQSGGRGQFGHCWIRFSAADVDEKGNITEGLVFENEVVGGVVPKEYIPAIQKGIEEQMKNGVVAGYPLIGLKATVFDGSYHDVDSNEMAFKIAASMATKQLAQKGGGKVLEPIMKVEVVTPEDYMGDVMGDLNRRRGLIQGMEDTVSGKVIRAEVPLGEMFGYATDVRSMSQGRASYSMEFSKYAEAPSNIVEALVKKQG (SEQ ID NO: 3) PcrV antigen MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDASSRYNSAVEALNRFIQKYDSVLRDILSAI (SEQ ID NO: 4) PilA antigen PQYQNYVARSEGASALATINPLKTTVEESLSRGIAGSKIKIGTTASTATETYVGVEPDANKLGVIAVAIEDSGAGDITFTFQTGTSSPKNATKVITLNRTADGVWACKSTQDPMFTPKGCDN(SEQ ID NO: 5) EF-Tu antigen-DnaK antigen-EF-G antigen fusion protein (SEQ ID NO: 6) PcrV antigen-PilA antigen fusion protein MTRLTVLALLAGLLASSRAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSGGSGGSGMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDASSRYNSAVEALNRFIQKYDSVLRDILSAIGGSGGGGSGGPQYQNYVARSEGASALATINPLKTTVEESLSRGIAGSKIKIGTTASTATETYVGVEPDANKLGVIAVAIEDSGAGDITFTFQTGTSSPKNATKVITLNRTADGVWACKSTQDPMFTPKGCDN(SEQ ID NO: 7) Signal peptide MTRLTVLALLAGLLASSRA(SEQ ID NO: 8) Fc domain of IGHG protein EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 9) linker sequence GGSGGGGSGG (SEQ ID NO: 10) Interval sequence GGSGGSGGSG (SEQ ID NO: 11)

[0093] Example 2: Preparation of the recombinant nucleic acid vaccine of the present invention

[0094] (1) Preparation of capped mRNA vaccines

[0095] Step a: Linearize the vector plasmid used in Example 1 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0096] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0097] (2) Preparation of uncapped mRNA vaccines

[0098] Step a: Linearize the vector plasmid used in Example 1 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0099] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0100] (3) DNA vaccine preparation

[0101] Step a: Amplify the vector plasmid used in Example 1 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0102] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0103] Example 3: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0104] Vaccine A (recombinant nucleic acid vaccine A based on the present invention) and vaccine B (recombinant nucleic acid vaccine B based on the present invention) were prepared using the method for preparing capped mRNA vaccines in Example 2. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiments was greater than 85%. The quality control peak diagrams and purity test results of recombinant nucleic acid vaccines A and B based on the present invention are as follows: Figure 3A , Figure 3BAs shown. Specifically, it is described as follows: (1) Recombinant nucleic acid vaccine A based on the present invention has a purity of 89%; (2) Recombinant nucleic acid vaccine B based on the present invention has a purity of 85.1%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0105] Example 4: In vitro expression effect of the recombinant nucleic acid of the present invention

[0106] Using cell transfection reagents, vaccines A and B from Example 3 were transfected into HEK293T cells. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot.

[0107] Figure 4A , Figure 4B The in vitro expression (WB) results of vaccines A and B transfected into HEK293 cells are presented. The antigen expressed by vaccine A is a cellular immune antigen, which theoretically should be significantly expressed in cell lysates. The antigen expressed by vaccine B is a humoral immune antigen, which theoretically should be significantly expressed in both the supernatant and cell lysates. The protein molecular weights of vaccines A and B are shown in Table 2.

[0108] Table 2. Protein molecular weight of vaccines A and B

[0109] Vaccine A 71.6 Vaccine B 77.1

[0110] The results showed that vaccine A was significantly expressed in cells, demonstrating that the antigen fusion expression molecular architecture provided by this invention enables the smooth translation and correct folding of bacterial-derived multi-antigen combinations in eukaryotic cells, and exhibits structural stability and a long half-life. Vaccine B was significantly expressed in supernatant and cell lysate, demonstrating that the molecular architecture provided by this invention, which adds an Fc domain to the N-terminus of the antigen, enables the smooth translation and correct folding of bacterial-derived fusion proteins in eukaryotic cells, and promotes the efficient secretion of fusion proteins to the extracellular space. Therefore, the vaccines designed based on this invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial for immunoepitaxy presentation.

[0111] Example 5: Preventive effect of the recombinant nucleic acid vaccine based on the present invention in a mouse model of Pseudomonas aeruginosa infection.

[0112] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, this embodiment uses vaccine A and vaccine B to conduct immunization and challenge experiments in mouse models.

[0113] Twenty-one male BALB / C mice aged 6-8 weeks with similar body size and weight were used in the experiment. After 7 days of acclimatization, the mice were divided into the following groups: vaccine A immunization group (n=5), vaccine B immunization group (n=5), mixed immunization group (vaccine A+B) (n=5), PBS group (n=5) (negative control), and blank control group (n=1). Details are shown in Table 3.

[0114] Table 3. Immunization procedure for experimental animals in Example 5

[0115] Note: Group D used PBS solution instead of vaccine and served as the negative control group; Group E did not undergo immunization or challenge and served as the blank control group.

[0116] The mice in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 3. On day 35, 50 μl of Pseudomonas aeruginosa bacterial suspension (concentration 2×10⁻⁶) was administered intranasally. 7 (CFU / ml) was then fed normally, and changes in body temperature, weight, and survival rate were observed and recorded. Mice were sacrificed on day 49 post-challenge, lungs were collected for bacterial load determination, and histopathological sections were prepared to comprehensively evaluate the protective effect of the vaccine on mice. The immunization and sampling procedures are as follows: Figure 5 As shown.

[0117] Changes in bacterial load in lung tissue of mice in different groups after viral challenge are as follows: Figure 6A , Figure 6B As shown, Figure 6A For plate colony counting, Figure 6B The results were used to quantify the colony count. The results showed that a large number of *Pseudomonas aeruginosa* were detected in the PBS group, while no *P. aeruginosa* was detected in the blank control group, confirming the success of the challenge model. Compared to the PBS group, the *P. aeruginosa* bacterial load was significantly reduced in the lung tissue of mice immunized with vaccine A, vaccine B, and a mixed immunization group (vaccine A + vaccine B). The mixed immunization group had the lowest bacterial load in its lung tissue, followed by the vaccine A immunization group.

[0118] In addition, after necropsy, lung tissue was sectioned and stained with hematoxylin and eosin (HE) to further determine the degree of tissue lesions. The results of tissue section comparisons after challenge in different groups of mice with Pseudomonas aeruginosa infection are as follows: Figure 7As shown, in the PBS group, the alveolar septa of mice were significantly thickened, alveolar morphology and structure disappeared, bronchial epithelial smooth muscle was thickened, and there was a large amount of inflammatory cell infiltration; the alveolar structure of the blank control group was intact, and there was no inflammatory cell infiltration, proving that the challenge model was established. In addition, compared with the PBS group, the alveolar structure of the mixed immunization group was better intact, and the thickness of the alveolar epithelial septa was significantly reduced; the alveolar septa of mice immunized with vaccine A were thickened, and mild pulmonary fibrosis and inflammatory symptoms appeared. Therefore, whether it is a mixed immunization with vaccine A and vaccine B or a single immunization, it can effectively prevent Pseudomonas aeruginosa infection, reduce the degree of tissue lesions, and achieve the expected results. Among them, mixed immunization has the best preventive effect, and vaccine A has the second best preventive effect.

[0119] In summary, the antigen and fusion molecular architecture provided by this invention successfully induced an effective immune response in mouse model animals, demonstrating a good preventive effect against Pseudomonas aeruginosa infection. This breakthrough fills a gap in the current field of Pseudomonas aeruginosa vaccine development, provides a novel solution for the production and development of animal immunotherapies, and has extremely high commercial value and broad application prospects.

[0120] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein, characterized in that, The fusion protein is fusion protein A, which consists of EF-Tu antigen, linker, DnaK antigen, linker, and EF-G antigen from N-terminus to C-terminus. The amino acid sequence of the EF-Tu antigen is shown in SEQ ID NO: 1, the amino acid sequence of the DnaK antigen is shown in SEQ ID NO: 2, the amino acid sequence of the EF-G antigen is shown in SEQ ID NO: 3, and the amino acid sequence of the linker is shown in SEQ ID NO:

10.

2. A recombinant nucleic acid molecule, characterized in that, Encodes the fusion protein of claim 1.

3. The recombinant nucleic acid molecule according to claim 2, characterized in that, The recombinant nucleic acid molecule is an mRNA or DNA molecule.

4. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 2 or 3.

5. The recombinant gene expression cassette according to claim 4, characterized in that, The recombinant gene expression cassette also includes one or more of a promoter, terminator, and regulatory sequence.

6. A recombinant vector, characterized in that, It comprises the recombinant nucleic acid molecule of claim 2 or 3, or the recombinant gene expression cassette of claim 4 or 5.

7. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 2 or 3, or the recombinant gene expression cassette of claim 4 or 5, or the recombinant vector of claim 6.

8. A pharmaceutical composition, characterized in that, It comprises the fusion protein of claim 1, or the recombinant nucleic acid molecule of claim 2 or 3, or the recombinant gene expression cassette of claim 4 or 5, or the recombinant vector of claim 6, or the recombinant host cell of claim 7.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 8 or 9, characterized in that, The pharmaceutical composition comprises fusion protein A and fusion protein B, wherein fusion protein B comprises, from N-terminus to C-terminus, a signal peptide, an Fc domain, a spacer sequence, a PcrV antigen, a linker, and a PilA antigen, wherein the amino acid sequence of the PcrV antigen is shown in SEQ ID NO: 4, the amino acid sequence of the PilA antigen is shown in SEQ ID NO: 5, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 10, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 11, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc domain is shown in SEQ ID NO:

9.

11. The pharmaceutical composition according to claim 10, characterized in that, The mass ratio of fusion protein A to fusion protein B in the pharmaceutical composition is 1:

1.

12. A recombinant vaccine, characterized in that, The pharmaceutical composition comprises the fusion protein of claim 1, or the recombinant nucleic acid molecule of claim 2 or 3, or the recombinant gene expression cassette of claim 4 or 5, or the recombinant vector of claim 6, or the recombinant host cell of claim 7, or any one of claims 8-11.

13. The recombinant vaccine according to claim 12, characterized in that, The recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

14. The recombinant vaccine according to claim 12 or 13, characterized in that, The recombinant vaccine comprises fusion protein A and fusion protein B, wherein fusion protein B, from N-terminus to C-terminus, consists of a signal peptide, an Fc domain, a spacer sequence, a PcrV antigen, a linker, and a PilA antigen. The amino acid sequence of the PcrV antigen is shown in SEQ ID NO: 4, the amino acid sequence of the PilA antigen is shown in SEQ ID NO: 5, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 10, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 11, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc domain is shown in SEQ ID NO:

9.

15. The recombinant vaccine according to claim 14, characterized in that, The mass ratio of fusion protein A to fusion protein B in the recombinant vaccine is 1:

1.

16. Use of the fusion protein of claim 1, or the recombinant nucleic acid molecule of claim 2 or 3, or the recombinant gene expression cassette of claim 4 or 5, or the recombinant vector of claim 6, or the recombinant host cell of claim 7, or the pharmaceutical composition of any one of claims 8-11, or the recombinant vaccine of any one of claims 12-15 in the preparation of a medicament for the prevention and / or treatment of infections or diseases caused by Pseudomonas aeruginosa.

17. The use according to claim 16, characterized in that, Infections or diseases caused by *Pseudomonas aeruginosa* include pneumonia, skin and soft tissue infections, urinary tract infections, eye infections, ear infections, bacteremia, sepsis, wound infections, chronic obstructive pulmonary disease (COPD), chronic lung infection (CPI), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), refractory mastitis, fibrinous airsacculitis, endocarditis, bloodstream infections, central nervous system infections, and bone and joint infections.

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