Immunogenic composition for preventing pseudomonas aeruginosa infection and application thereof

Abstract

The invention relates to a novel immunogenic composition for preventing and treating pseudomonas aeruginosa infection, a fusion protein, a recombinant vaccine, a molecular architecture design and application. The invention provides a novel fusion molecular architecture, which comprises a fusion protein A coded by three genes of EF-Tu, DnaK and EF-G, or a fusion protein B coded by two genes of PcrV and PilA, or a combination of the fusion protein A and the fusion protein B, and can be used for research and development of nucleic acid vaccines or subunit vaccines. The invention finds that the novel fusion molecule has good immunogenicity, can provide an effective immune protection effect, remarkably inhibits colonization of pseudomonas aeruginosa, and has a 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 ( Pseudomonas aeruginosa 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 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 as the best potential measure to control P. aeruginosa infection and drug resistance spread. The current international and domestic P. aeruginosa research or commercial vaccines mainly include inactivated whole bacteria vaccine, toxoid vaccine, subunit vaccine (such as OprF / OprI fusion protein vaccine), polysaccharide conjugate vaccine, etc. The above vaccines can effectively reduce the acute mortality rate and lung bacterial load in animal models, but it is difficult to completely block the colonization of P. aeruginosa, infection and horizontal transfer of drug resistance genes. In recent years, domestic scholars have also been actively developing other new P. aeruginosa vaccines, such as patent CN118703546A discloses a recombinant lactococcus lactis expressing P. aeruginosa outer membrane protein I, a plasmid and a vaccine and its application, patent CN118048284A discloses a P. aeruginosa fur gene deletion mutant and its construction method and application, and patent CN116731209A discloses a recombinant P. aeruginosa nanoparticle protein rePO-FN and its application.

[0005] However, the existing vaccine strategy still faces significant challenges: the inactivated whole bacteria vaccine has high safety risk and weak immunogenicity; the polysaccharide vaccine has weak ability to activate T cell-dependent immune response and poor effect in infants; and the live attenuated vaccine has the risk of virulence recovery and pathogenicity in immunodeficient hosts. With the continuous progress of reverse vaccinology, structural biology and genetic engineering technology, the development of safe, broad-spectrum and efficient new genetic engineering vaccines for P. aeruginosa has become the main direction of vaccine development. There is still an urgent need in the art for a fusion protein, an immunological composition and a vaccine with good immunogenicity and capable of providing effective immune protection effect and significantly inhibiting P. aeruginosa colonization. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a new immunogenic composition, fusion protein, recombinant vaccine for preventing and treating P. aeruginosa infection, as well as a molecular architecture design and application. The present application provides a new fusion molecule architecture, which comprises a fusion protein A encoded by three genes of EF-Tu, DnaK and EF-G, or a fusion protein B encoded by two genes of PcrV and PilA, or a combination of fusion proteins A and B, which can be used for the development of nucleic acid vaccines or subunit vaccines. The present application finds that the new fusion molecule has good immunogenicity and can provide effective immune protection effect and significantly inhibit P. aeruginosa colonization, which has a wide application prospect. The present application also finds that the new molecular architecture can effectively improve the expression level of the fusion protein, thereby further improving the immune protection effect. The present application also provides corresponding recombinant nucleic acid, gene expression cassette, vector, host cell, pharmaceutical composition, vaccine, use, etc.

[0007] One aspect of the present application provides a fusion protein, characterized in that it is selected from any one of (1)-(3) below: (1) a fusion protein A comprising an Elongation factor Tu (EF-Tu) antigen or an antigenic fragment thereof, a Chaperone protein DnaK (DnaK) antigen or an antigenic fragment thereof, and an Elongation factor G (EF-G) antigen or an antigenic fragment thereof; (2) a fusion protein B comprising a PcrV (type III secretion protein PcrV) antigen and a PilA (type IV fimbrial precursor PilA) antigen, wherein the PcrV antigen has an amino acid sequence as set forth in SEQ ID NO: 4, and the PilA antigen has an amino acid sequence as set forth in SEQ ID NO: 5; (3) a combination of the fusion protein A and the fusion protein B.

[0008] Further, the EF-Tu, DnaK, EF-G antigen or an antigenic fragment thereof and the PcrV, PilA antigen are from a Gram-negative bacterium; preferably, the EF-Tu, DnaK, EF-G antigen or an antigenic fragment thereof and the PcrV, PilA antigen are from the order Pseudomonadales, the family Pseudomonadaceae; most preferably, the EF-Tu, DnaK, EF-G antigen or an antigenic fragment thereof and the PcrV, PilA antigen are from P. aeruginosa or Pseudomonas aeruginosa (P. aeruginosa). Pseudomonas aeruginosa ).

[0009] Further, the fusion protein A comprises the EF-Tu antigen or an antigenic fragment thereof, the DnaK antigen or an antigenic fragment thereof, and the EF-G antigen or an antigenic fragment thereof in sequence from N-terminus to C-terminus; and the fusion protein B comprises the PcrV antigen and the PilA antigen in sequence from N-terminus to C-terminus.

[0010] Further, the EF-Tu antigen has an amino acid sequence as set forth in SEQ ID NO: 1, the DnaK antigen has an amino acid sequence as set forth in SEQ ID NO: 2, and the EF-G antigen has an amino acid sequence as set forth in SEQ ID NO: 3.

[0011] Further, the antigens can be optionally connected by a linker sequence or a spacer sequence; preferably, the linker sequence has an amino acid sequence as set forth in SEQ ID NO: 10, and the spacer sequence has an amino acid sequence as set forth in SEQ ID NO: 11.

[0012] Further, the amino acid sequence of the fusion protein A is shown as SEQ ID NO: 6, and the amino acid sequence of the fusion protein B is shown as SEQ ID NO: 7.

[0013] Further, the N-terminal of the fusion protein B further comprises a signal peptide and / or an element for facilitating secretion; preferably, the signal peptide is derived from Azurocidin (AZU1), and the element for facilitating secretion is derived from the Fc domain of Immunoglobulin Heavy Constant Gamma (IGHG); more preferably, the amino acid sequence of the signal peptide is shown as SEQ ID NO: 8, and the amino acid sequence of the element for facilitating secretion is shown as SEQ ID NO: 9.

[0014] Another aspect of the present application provides a recombinant nucleic acid molecule, characterized by encoding the fusion protein of the present application; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

[0015] Another aspect of the present application provides a recombinant gene expression cassette, characterized by comprising the recombinant nucleic acid molecule of the present application; preferably, the recombinant gene expression cassette further comprises one or more of a promoter, a terminator, and a regulatory sequence.

[0016] Another aspect of the present application provides a recombinant vector, characterized by comprising the recombinant nucleic acid molecule of the present application, or the recombinant gene expression cassette of the present application.

[0017] Further, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0018] Further, the prokaryotic vector comprises, but is not limited to, an E. coli vector.

[0019] Further, the E. coli vector comprises, but is not limited to, a pET vector, a pGEX vector, a pMAL vector, a pBAD vector, a pUC vector, a pBR vector.

[0020] Further, the eukaryotic vector comprises, but is not limited to, a yeast expression vector, an insect expression vector, a mammalian cell expression vector.

[0021] Further, the yeast expression vector comprises, but is not limited to, a pPICZ vector, a pGAPZ vector, a pYES vector, a pGAP vector, a pAO815 vector, a pPIC9 vector.

[0022] Another aspect of the present application provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule of the present application, or the recombinant gene expression cassette of the present application, or the recombinant vector of the present application.

[0023] Further, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0024] Further, the eukaryotic cell comprises a mammalian cell, an insect cell, a yeast cell.

[0025] Further, the yeast cell comprises but is not limited to Saccharomyces cerevisiae, Pichia pastoris, Hansenula.

[0026] Further, the prokaryotic cell comprises but is not limited to Escherichia coli cell, Bacillus subtilis cell, Pseudomonas cell.

[0027] Further, the Escherichia coli cell comprises but is not limited to BL21 (DE3), DH5a, TOP10, Rosetta.

[0028] Another aspect of the present application provides an immunogenic composition or a pharmaceutical composition, characterized in that it comprises one or more fusion proteins of the present application, and / or one or more recombinant nucleic acid molecules of the present application, and / or one or more recombinant gene expression cassettes of the present application, and / or one or more recombinant vectors of the present application, and / or one or more recombinant host cells of the present application; preferably, the immunogenic composition or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0029] Further, the immunogenic composition or the pharmaceutical composition comprises 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 the pharmaceutical composition is 1:1.

[0030] Another aspect of the present application provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins of the present application, and / or one or more recombinant nucleic acid molecules of the present application, and / or one or more recombinant gene expression cassettes of the present application, and / or one or more recombinant vectors of the present application, and / or one or more recombinant host cells of the present application, and / or one or more immunogenic compositions or pharmaceutical compositions of the present application; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; preferably, the recombinant vaccine comprises the fusion protein A and the fusion protein B; more preferably, the mass ratio of the fusion protein A and the fusion protein B in the recombinant vaccine is 1:1.

[0031] Another aspect of the present application provides the use of one or more fusion proteins of the present application, and / or one or more recombinant nucleic acid molecules of the present application, and / or one or more recombinant gene expression cassettes of the present application, and / or one or more recombinant vectors of the present application, and / or one or more recombinant host cells of the present application, and / or one or more immunogenic compositions or pharmaceutical compositions of the present application, and / or one or more recombinant vaccines of the present application in the manufacture of a medicament for prophylaxis, treatment and / or vaccination.

[0032] Further, the medicament is used for preventing and / or treating infection or disease caused by Pseudomonas aeruginosa; preferably, the infection or disease comprises pneumonia, skin and soft tissue infection, urinary tract infection, eye infection, ear infection, bacteremia, sepsis, wound infection, chronic obstructive pulmonary disease (COPD), chronic pulmonary infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, stubborn mastitis, fibrinous emphysema, endocarditis, bloodstream infection, central nervous system infection, bone and joint infection.

[0033] Another aspect of the present application provides a method for preventing and / or treating infection or disease caused by Pseudomonas aeruginosa, characterized in that, comprising administering to a subject one or more fusion proteins of the present application, and / or one or more recombinant nucleic acid molecules of the present application, and / or one or more recombinant gene expression cassettes of the present application, and / or one or more recombinant vectors of the present application, and / or one or more recombinant host cells of the present application, and / or one or more immunogenic compositions or pharmaceutical compositions of the present application, and / or one or more recombinant vaccines of the present application; preferably, the infection or disease comprises pneumonia, skin and soft tissue infection, urinary tract infection, eye infection, ear infection, bacteremia, sepsis, wound infection, chronic obstructive pulmonary disease (COPD), chronic pulmonary infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, stubborn mastitis, fibrinous emphysema, endocarditis, bloodstream infection, central nervous system infection, bone and joint infection.

[0034] The fusion protein molecule architecture of the present application and the nucleic acid architecture encoding the same, the pharmaceutical composition, the recombinant vaccine, etc. have the following beneficial technical effects: 1. Antigen screening: a plurality of Pseudomonas aeruginosa antigens are selected, and through bioinformatics technologies such as protein three-dimensional structure prediction and immunological epitope screening, the antigens are selected and fusion expression design, including N / C terminal truncation, full-length selection, fusion protein stability prediction, etc., and finally the selected EF-Tu antigen, DnaK antigen, EF-G antigen, PcrV antigen and PilA antigen of the application are obtained.

[0035] 2. The glycosylation site of the PcrV antigen in the application is mutated to T265A, in order to avoid glycosylation of prokaryotic proteins when expressed in eukaryotic cells, which affects the correct presentation of epitopes. The mutated PcrV antigen has high expression abundance and more accurate epitope presentation.

[0036] 3. Example 4 shows that vaccine A (EF-Tu antigen-DnaK antigen-EF-G antigen fusion protein) can be expressed significantly in cells, proving that the antigen fusion expression molecular architecture provided by the application can enable the smooth translation and correct folding of the bacterial-derived multi-antigen combination in eukaryotic cells, and the structure is stable and has a long half-life; vaccine B (PcrV antigen-PilA antigen fusion protein) can be expressed significantly in the supernatant and cell lysate, proving that the molecular architecture provided by the application, which increases the Fc domain at the N terminus of the antigen, can enable the smooth translation and correct folding of the bacterial-derived fusion protein in eukaryotic cells, and promote the efficient secretion of the fusion protein to the extracellular. Therefore, the vaccine designed based on the application can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immunological epitopes.

[0037] 4. Example 5 shows that the Pseudomonas aeruginosa bacterial content in the lung tissue of mice in the vaccine A immunization group, vaccine B immunization group and A and B mixed immunization group (vaccine A + vaccine B) is significantly reduced, among which the bacterial load in the lung tissue of mice in the mixed immunization group is the lowest, and the bacterial load in the lung tissue of mice in the vaccine A immunization group is the second. In addition, the comparison results of the tissue sections after challenge show that whether vaccine A and vaccine B are mixed or immunized alone, they can effectively prevent Pseudomonas aeruginosa infection and reduce the degree of tissue lesions, achieving the expected results. Among them, the mixed immunization has the best preventive effect, and vaccine A has a suboptimal preventive effect.

[0038] 5. The antigen and fusion molecular architecture provided by the application successfully induce an effective immune response in the model animal mouse, and show good preventive effect on Pseudomonas aeruginosa infection. This breakthrough fills the gap in the current Pseudomonas aeruginosa vaccine research and development field, provides a new solution for the production and research and development of animal immunological drugs, and has very high commercial value and broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1This is a schematic diagram of the molecular structure of the fusion protein expressed by vaccine A and vaccine B of the present invention.

[0040] Figure 2 This is a schematic diagram of a template plasmid containing the gene expression cassette of the present invention.

[0041] Figures 3A-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.

[0042] Figures 4A-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.

[0043] 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.

[0044] Figures 6A-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.

[0045] 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

[0046] Terms and Definitions The term "Pseudomonas aeruginosa" refers to Pseudomonas aeruginosaPseudomonas aeruginosa, abbreviated as PA, is 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.

[0047] The term "PcrV" in Pseudomonas aeruginosa ( Pseudomonas aeruginosa The term "PcrV" refers to type III secretion protein, 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.

[0048] 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.

[0049] The term "EF-Tu" refers to Elongation Factor Tu, which is an important GTPase in prokaryotes that plays a central role in the translation process of protein synthesis. In addition to its role in protein synthesis, EF-Tu can also have "moonlighting" functions as a surface protein and antigen of bacteria involved in host-pathogen interactions. The EF-Tu antigens of the present application include full-length EF-Tu antigens and truncated fragments thereof, such as N-terminal truncations, C-terminal truncations, partial truncations of the full-length, etc., all of which are capable of stimulating an immune response against the EF-Tu antigens. Preferably, the amino acid sequence of the EF-Tu antigen is set forth in SEQ ID NO: 1.

[0050] The term "DnaK" refers to DnaK chaperone, which is one of the major chaperones in bacteria, belonging to the DnaK chaperone system (also including DnaJ and GrpE). DnaK functions under the driving force of ATP, and its core function is to protect newly synthesized or denatured polypeptides under stress conditions (such as heat stress) from misfolding and aggregation. The DnaK antigens of the present application include full-length DnaK antigens and truncated fragments thereof, such as N-terminal truncations, C-terminal truncations, partial truncations of the full-length, etc., all of which are capable of stimulating an immune response against the DnaK antigens. Preferably, the amino acid sequence of the DnaK antigen is set forth in SEQ ID NO: 2.

[0051] The term "EF-G" refers to Elongation Factor G, which is a GTPase in prokaryotes that has two functions in protein synthesis, Translocation and Ribosome Recycling, and can drive the structural changes of ribosomes through GTP hydrolysis to ensure the efficiency and accuracy of protein synthesis and play a key role in the end stage of translation. The EF-G antigens of the present application include full-length EF-G antigens and truncated fragments thereof, such as N-terminal truncations, C-terminal truncations, partial truncations of the full-length, etc., all of which are capable of stimulating an immune response against the EF-G antigens. Preferably, the amino acid sequence of the EF-G antigen is set forth in SEQ ID NO: 3.

[0052] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. Immune responses can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and / or assays that measure the modulation of activity or expression of one or more cytokines.

[0053] The term "administration" or "inoculation" refers to the preferential intramuscular or subcutaneous route of delivery of the nucleic acid vaccine or vaccine composition of the present application, although other routes of administration can be used, e.g., oral, intranasal (e.g., aerosol or other non-needle administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Intramuscular administration in the neck of the animal is preferred. Accelerated regimens (boosting regimens) can be employed to adjust the administration regimen to provide optimal immunity.

[0054] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0055] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0056] The term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. The recombinant expression vector can include, for example, a transcriptional unit comprising (1) a collection of genetic elements having a regulatory role in gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and (3) appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner, can use any vector, including plasmids, viruses, bacteriophages, and transposons. Possible vectors for use in the present 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, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0057] The term "mRNA" refers to messenger RNA, a type of single-stranded RNA that carries genetic information from DNA to the ribosome for protein synthesis.

[0058] The term "5'-UTR" refers to a "5' untranslated region" or "5' UTR" and is a portion of a gene transcribed into a primary RNA transcript (pre-mRNA) and located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced from the transcription of DNA. Many primary transcripts must undergo RNA processing to form an RNA that is physiologically active. The process of processing to form mature mRNA includes modification of the ends, excision of introns, capping, and / or splicing out of individual rRNA molecules from the precursor RNA. Thus, the 5' UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In genomic sequences, the 5' UTR is usually defined as the region between the transcription start site and the start codon. The 5' untranslated region (5' UTR) of a vertebrate mRNA can be tens to hundreds of bases in length.

[0059] The term "3'-UTR" refers to a "3'-untranslated region" or "3' UTR" and relates to a region located at the 3' end of a gene, downstream of the stop codon of the protein coding region, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region usually extends from the stop codon of the translated product to a poly(A) sequence that is usually attached after the process of transcription. The 3'-untranslated region of a mammalian mRNA usually has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence can be the poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-untranslated region can contain one or more inverted repeats that can fold to produce stem-loop structures that act as a barrier to exonucleases or interact with proteins known to increase RNA stability (e.g., RNA binding proteins).

[0060] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of the cell. Host cells include "transformants" and "transformed cells," which include both the primary transformed cells and progeny derived therefrom. Host cells are any type of cellular system that can be used to produce a recombinant vaccine based on the present application, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0061] The term "individual," "patient" or "subject" includes a mammal. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0062] The term "transformation, transfection, transduction" has the meaning commonly understood by those of ordinary skill in the art, i.e., the process of introducing foreign DNA, RNA into a host.

[0063] The term "pharmaceutical combination" or "pharmaceutical composition" refers to a pharmaceutical formulation containing an auxiliary material widely used in the field of pharmaceutical production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and to provide a method for obtaining effective absorption in the body of a subject. The pharmaceutically acceptable carrier can be an inert filler or a functional ingredient that provides certain functions (e.g., stabilizes the overall pH of the composition or prevents degradation of the active ingredients in the composition) to the pharmaceutical combination. Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifying agents, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, gelling agents, absorption retardants, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorings, and sweeteners, etc.

[0064] The term "treatment" refers to contacting (e.g., administering) a subject with a recombinant vaccine, composition, etc. based on the present application after the subject has contracted a disease, so as to alleviate the symptoms of the disease compared to not contacting, and does not mean that it is necessary to completely inhibit the symptoms of the disease. Contracting a disease means that the body has symptoms of the disease.

[0065] The term "prevention" refers to contacting (e.g., administering) a subject with a recombinant vaccine, composition, etc. based on the present application before the subject has contracted a disease, so as to alleviate the symptoms after contracting the disease compared to not contacting, and does not mean that it is necessary to completely inhibit the disease.

[0066] Unless otherwise defined or indicated by context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0067] The present application discloses a fusion molecule architecture for preventing Pseudomonas aeruginosa infection, a preparation method and application of the recombinant vaccine based on the architecture. Those skilled in the art can refer to the content herein to appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art and are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0068] The raw materials and reagents used in the fusion protein, the encoding nucleic acid and elements thereof, the preparation method and the application provided by the present application can be obtained from the market. According to the basic knowledge of molecular cloning, expression construction, vaccine preparation and immunization in the prior art, those skilled in the art can implement the embodiment methods of the present application.

[0069] The present application will be further illustrated below in conjunction with examples. As a preferred option, a nucleic acid vaccine architecture is selected for preparing a recombinant vaccine.

[0070] Example 1 Construction of recombinant nucleic acid vaccine of the present application Antigen screening: a plurality of Pseudomonas aeruginosa antigens are selected, and biological information technology such as protein three-dimensional structure prediction and immunological epitope screening is used to design the antigen for truncation and fusion expression, including N / C terminal truncation, full-length truncation, fusion protein stability prediction, etc., and finally the selected EF-Tu antigen, DnaK antigen, EF-G antigen, PcrV antigen and PilA antigen of the present application are obtained. In addition, the glycosylation site of the PcrV antigen is mutated to T265A in order to avoid glycosylation of prokaryotic proteins when expressed in eukaryotic cells, which affects the correct presentation of epitopes. The mutated PcrV antigen has high expression abundance and more accurate epitope presentation.

[0071] In order to prepare a recombinant nucleic acid vaccine containing the antigen of the present application, a non-limiting architecture schematic diagram of the nucleic acid vaccine of the present application is shown in Figure 1 , and an exemplary Figure 1 molecular structure schematic diagram of the fusion protein expressed by the vaccine A and the vaccine B of the present application is shown. In order to prepare a recombinant nucleic acid vaccine capable of producing a molecular structure as shown in Figure 1 , a gene expression cassette is first constructed for expressing the antigen sequence described in the present application. The schematic diagram of the template plasmid containing the gene expression cassette of the present application is shown in Figure 2 , which contains 5'UTR, CDS region, 3'UTR and PolyA from 5' end to 3' end, wherein the CDS region contains the fusion molecular architecture described in the present application. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (synthesized by Jinsu Company). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription to obtain a vector plasmid for preparing a recombinant nucleic acid vaccine.

[0072] According to the above method, the vectors used in the subsequent examples are prepared: (1) Preparation of a vector based on the recombinant nucleic acid vaccine A of the present application Step a: synthesis of "EF-Tu antigen-DnaK antigen-EF-G antigen" gene fragment, connected by linker sequence with amino acid 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 as shown in SEQ ID NO: 6. Among them, the amino acid sequence of the EF-Tu antigen is as shown in SEQ ID NO: 1, the amino acid of the DnaK antigen is as shown in SEQ ID NO: 2, and the amino acid of the EF-G antigen is as shown in SEQ ID NO: 3.

[0073] Step b: construction of nucleic acid vaccine architecture vector.

[0074] The nucleic acid vaccine architecture vector comprises 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0075] Step c: preparation of recombinant plasmid.

[0076] Insert the gene synthesized in step a into the vector architecture of step b to obtain a recombinant nucleic acid vaccine A preparation vector based on the application.

[0077] (2) Recombinant nucleic acid vaccine B preparation vector based on the application Step a: synthesis of "signal peptide-IGHG Fc domain-PcrV antigen-PilA antigen" gene fragment, wherein the signal peptide is the signal peptide of Azurocidin (AZU1), the amino acid sequence is as shown in SEQ ID NO: 8, the amino acid of the Fc domain of IGHG is as shown in SEQ ID NO: 9, the amino acid of the PcrV antigen is as shown in SEQ ID NO: 4, the amino acid of the PilA antigen is as shown in SEQ ID NO: 5, the Fc domain of IGHG is connected with the PcrV antigen through the spacer sequence with amino acid sequence as shown in SEQ ID NO: 11, the PcrV antigen and the PilA antigen are connected through the linker sequence with amino acid sequence as shown in SEQ ID NO: 10, and the amino acid sequence of the fusion protein encoded by the PcrV antigen-PilA antigen fusion gene is as shown in SEQ ID NO: 7. Among them, the PcrV antigen comprises a glycosylation site mutation T265A.

[0078] Step b: construction of nucleic acid vaccine architecture vector.

[0079] The nucleic acid vaccine architecture vector comprises 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccine.

[0080] Step c: preparation of recombinant plasmid.

[0081] The gene synthesized in step a is inserted into the vector framework of step b to obtain a recombinant nucleic acid vaccine B preparation vector based on the present application.

[0082] Table 1 Amino acid sequences of the framework elements involved in the present application Amino acid sequences and sequence numbers 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 ERNKPHVNVGTIGHVDHGKTTLTAALTKVCSDTWGGSARAFDQIDNAPEEKARGITINTSHVEYDSAVRHYAHVDCPGHADYVKNMITGAAQMDGAILVCSAADGPMPQTREHILLSRQVGVPYIVVFLNKADMVDDAELLELVEMEVRDLLNTYDFPGDDTPIIIGSALMALEGKDDNGIGVSAVQKLVETLDSYIPEPV (SEQ ID NO: 6) PcrV antigen - PilA antigen fusion protein MTRLTVLALLAGLLASSRAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGSGGSGGSGMEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDASSRYNSAVEALNRFIQKYDSVLRDILSAIGGSGGGGSGGPQYQNYVARSEGASALATINPLKTTVEESLSRGIAGSKIKIGTTASTATETYVGVEPDANKLGVIAVAIEDSGAGDITFTFQTGTSSPKNATKVITLNRTADGVWACKSTQDPMFTPKGCDN (SEQ ID NO: 7) signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 8) IGHG protein Fc domain EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) linker sequence GGSGGGGSGG (SEQ ID NO: 10) spacer sequence GGSGGSGGSG (SEQ ID NO: 11) Example 2 Preparation of recombinant nucleic acid vaccines of the present application (1) Preparation of capped mRNA vaccine Step a: linearize the vector plasmid used for producing the capped mRNA vaccine in Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0083] Step b: perform an in vitro co-transcription capping reaction on the linearized plasmid to add a 7-methylated guanosine cap structure to the 5' end of the transcribed mRNA, and degrade the template DNA.

[0084] (2) Preparation of uncapped mRNA vaccine Step a: linearize the vector plasmid used for producing the uncapped mRNA vaccine in Example 1 by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0085] Step b: perform an in vitro uncapped transcription reaction on the linearized plasmid, and degrade the template DNA.

[0086] (3) Preparation of DNA vaccine Step a: amplify the vector plasmid used for producing the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0087] Step b: extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0088] Example 3 In vitro transcription quality control of recombinant nucleic acids of the present application and vaccine preparation Vaccine A (recombinant nucleic acid vaccine A based on the present application) and vaccine B (recombinant nucleic acid vaccine B based on the present application) were prepared using the method for preparing the capped mRNA vaccine in Example 2. The purity of the produced recombinant nucleic acids was detected, and the purity of the recombinant nucleic acids used in the experiment was all greater than 85%. The quality control peak chart and purity detection results of the recombinant nucleic acid vaccines A and B based on the present application are shown in Figure 3A , Figure 3B . The specific description is as follows: (1) the purity of the recombinant nucleic acid vaccine A based on the present application is 89%; (2) the purity of the recombinant nucleic acid vaccine B based on the present application is 85.1%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0089] Example 4 In vitro expression effect of recombinant nucleic acids of the present application The vaccines A and B in Example 3 were transfected into HEK293T cells by a cell transfection reagent, and proteins were collected after in vitro culture for 48 hours, and Western blot detection was performed.

[0090] Figure 4A 、 Figure 4B The in vitro expression WB (Western blot) detection results of the vaccines A and B transfected into HEK293 cells are shown respectively, wherein the antigen expressed by the vaccine A is a cellular immune antigen, and theoretically, significant expression can be detected in the cell lysate; the antigen expressed by the vaccine B is a humoral immune antigen, and theoretically, significant expression can be detected in the supernatant and the cell lysate. The protein molecular weights of the vaccines A and B are shown in Table 2.

[0091] Table 2 Protein molecular weights of vaccines A and B Name Protein molecular weight (kDa) Vaccine A 71.6 Vaccine B 77.1 The results show that the vaccine A can be significantly expressed in the cells, proving that the antigen fusion expression molecular architecture provided by the application can enable the bacterial-derived multi-antigen combination to be successfully translated and correctly folded in eukaryotic cells, and the structure is stable and has a long half-life; the vaccine B can be significantly expressed in the supernatant and the cell lysate, proving that the molecular architecture of adding an Fc domain at the N terminus of the antigen provided by the application can enable the bacterial-derived fusion protein to be successfully translated and correctly folded in eukaryotic cells, and promote the fusion protein to be efficiently secreted to the extracellular. Therefore, the vaccine designed based on the application can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes.

[0092] Example 5 Preventive effect of the recombinant nucleic acid vaccine based on the application in a P. aeruginosa infection model of mice In order to verify whether the recombinant nucleic acid vaccine based on the application can produce effective preventive protection effect in a model animal after immunization, the vaccines A and B were used for immunization and challenge experiments in a mouse model in this embodiment.

[0093] Twenty-one 6-8-week-old male BALB / C mice were selected, which were similar in size and weight, and were adaptively fed for 7 days, and then the mice were grouped: 5 mice in the vaccine A immunization group, 5 mice in the vaccine B immunization group, 5 mice in the mixed immunization group (vaccines A+B), 5 mice in the PBS group (negative control), and 1 mouse in the blank control group. The specific grouping is shown in Table 3.

[0094] Table 3 Immunization process of experimental animals in Example 5

[0095] Note: The group D is replaced by PBS solution instead of vaccine, which is a negative control group; the group E does not perform immunization and challenge, which is a blank control group.

[0096] The mice in each group were immunized according to the immunization process in Table 3, twice on day 0 (Day 0) and day 21 (Day 21). On day 35 (Day 35), 50 μl of P. aeruginosa bacterial solution (concentration 2x10 7 CFU / ml) was dropped into the nose, and then the mice were normally fed, and the changes in body temperature, body weight and survival rate of the mice were observed and recorded. On day 14 (Day 49) after the challenge, the mice were sacrificed, the lungs were collected, the bacterial load was determined, and the histopathological sections were prepared for comprehensive evaluation of the protective effect of the vaccine on the mice. The immunization and sampling process is shown in Figure 5 .

[0097] The changes in the lung tissue bacterial load of the mice in different groups after the challenge are shown in Figure 6A , Figure 6B , Figure 6A is the plate colony count, and Figure 6B is the colony count result. According to the results, it can be seen that a large number of P. aeruginosa bacteria were detected in the PBS group, and no P. aeruginosa bacteria were detected in the blank control group, proving that the challenge model is established; compared with the PBS group, the P. aeruginosa bacterial content in the lungs of the mice in the vaccine A immunization group, the vaccine B immunization group and the A and B mixed immunization group (vaccine A + vaccine B) is significantly reduced, and the lung tissue bacterial load of the mice in the mixed immunization group is the lowest, and the lung tissue bacterial load of the mice in the vaccine A immunization group is the second.

[0098] In addition, after the autopsy, the lung tissue was sectioned and HE stained to further determine the degree of tissue lesions. The comparison results of the tissue sections of the P. aeruginosa infection experiments of the mice in different groups after the challenge are shown in Figure 7 . The alveolar septum of the mice in the PBS group is obviously thickened, the alveolar structure disappears, the bronchial epithelial smooth muscle is thickened, and a large number of inflammatory cells are infiltrated; the alveolar structure of the blank control group is complete, and no inflammatory cells are infiltrated, proving that the challenge model is established. In addition, compared with the PBS group, the alveolar structure of the mixed immunization group is better, and the thickness of the alveolar epithelial septum is obviously reduced; the alveolar septum of the mice in the vaccine A immunization group is thickened, and slight pulmonary fibrosis and inflammatory symptoms occur. Therefore, whether the vaccine A and the vaccine B are mixed or immunized alone, they can effectively prevent P. aeruginosa infection and reduce the degree of tissue lesions, achieving the expected results. Among them, the mixed immunization has the best preventive effect, and the vaccine A has a suboptimal preventive effect.

[0099] In summary, the antigen and fusion molecule architecture provided by the present application successfully induce an effective immune response in the model animal mice, and show good preventive effect on P. aeruginosa infection. This breakthrough fills the gap in the current P. aeruginosa vaccine research and development field, provides a new solution for the production and research and development of animal immunization drugs, and has very high commercial value and broad application prospect.

[0100] The above-described embodiments of the present disclosure are merely for clear illustration of the present disclosure and are not intended to limit the implementation of the present disclosure. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, it is not necessary or possible to exhaust all the implementations. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that, selected from any one of the following (1)-(3): (1) a fusion protein A comprising an Elongation factor Tu (EF-Tu) antigen or an antigenic fragment thereof, a Chaperone protein DnaK (DnaK) antigen or an antigenic fragment thereof, and an Elongation factor G (EF-G) antigen or an antigenic fragment thereof; (2) a fusion protein B comprising a PcrV (type III secretion protein PcrV) antigen and a PilA (type IV fimbrial precursor PilA) antigen, wherein the PcrV antigen has an amino acid sequence as set forth in SEQ ID NO: 4, and the PilA antigen has an amino acid sequence as set forth in SEQ ID NO: 5; (3) a combination of the fusion protein A and the fusion protein B.

2. The fusion protein according to claim 1, characterized in that, The EF-Tu, DnaK, EF-G antigens or antigenic fragments thereof and the PcrV, PilA antigens are from a Gram-negative bacterium; preferably, the EF-Tu, DnaK, EF-G antigens or antigenic fragments thereof and the PcrV, PilA antigens are from the order Pseudomonadales, the family Pseudomonadaceae; most preferably, the EF-Tu, DnaK, EF-G antigens or antigenic fragments thereof and the PcrV, PilA antigens are from Pseudomonas aeruginosa (P. aeruginosa) Pseudomonas aeruginosa ).

3. The fusion protein according to claim 1 or 2, characterized in that, The fusion protein A comprises the EF-Tu antigen or the antigenic fragment thereof, the DnaK antigen or the antigenic fragment thereof, and the EF-G antigen or the antigenic fragment thereof, in order from N-terminus to C-terminus; and the fusion protein B comprises the PcrV antigen and the PilA antigen, in order from N-terminus to C-terminus.

4. The fusion protein according to claims 1-3, characterized in that, The EF-Tu antigen has an amino acid sequence as set forth in SEQ ID NO: 1, the DnaK antigen has an amino acid sequence as set forth in SEQ ID NO: 2, and the EF-G antigen has an amino acid sequence as set forth in SEQ ID NO:

3.

5. The fusion protein according to any one of claims 1 to 4, characterized in that, The antigens can be optionally connected by a linker sequence or a spacer sequence; preferably, the linker sequence has an amino acid sequence as set forth in SEQ ID NO: 10, and the spacer sequence has an amino acid sequence as set forth in SEQ ID NO:

11.

6. The fusion protein according to any one of claims 1 to 5, characterized in that, The fusion protein A has an amino acid sequence as set forth in SEQ ID NO: 6, and the fusion protein B has an amino acid sequence as set forth in SEQ ID NO:

7.

7. The fusion protein according to any one of claims 1 to 6, characterized in that, The N-terminus of the fusion protein B further comprises a signal peptide and / or an element for facilitating secretion; preferably, the signal peptide is derived from Azurocidin (AZU1), and the element for facilitating secretion is derived from the Fc domain of Immunoglobulin Heavy Constant Gamma (IGHG); more preferably, the signal peptide has an amino acid sequence as set forth in SEQ ID NO: 8, and the element for facilitating secretion has an amino acid sequence as set forth in SEQ ID NO:

9.

8. A recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule encodes the fusion protein according to any one of claims 1-7; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

9. A recombinant gene expression cassette comprising, comprises the recombinant nucleic acid molecule of claim 8; preferably, the recombinant genetic expression cassette further comprises one or more of a promoter, a terminator, a regulatory sequence.

10. A recombinant vector, characterized in that, comprises the recombinant nucleic acid molecule of claim 8, or the recombinant genetic expression cassette of claim 9.

11. A recombinant host cell, characterized in that, comprises the recombinant nucleic acid molecule of claim 8, or the recombinant genetic expression cassette of claim 9, or the recombinant vector of claim 10.

12. An immunogenic or pharmaceutical composition comprising, comprises one or more of the fusion protein of any one of claims 1-7, and / or one or more of the recombinant nucleic acid molecule of claim 8, and / or one or more of the recombinant genetic expression cassette of claim 9, and / or one or more of the recombinant vector of claim 10, and / or one or more of the recombinant host cell of claim 11; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

13. The immunogenic or pharmaceutical composition according to claim 12, characterized in that, the immunogenic composition or pharmaceutical composition comprises 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.

14. A recombinant vaccine, characterized in that, comprises one or more of the fusion protein of any one of claims 1-7, and / or one or more of the recombinant nucleic acid molecule of claim 8, and / or one or more of the recombinant genetic expression cassette of claim 9, and / or one or more of the recombinant vector of claim 10, and / or one or more of the recombinant host cell of claim 11, and / or one or more of the immunogenic composition or pharmaceutical composition of claim 12 or 13; preferably, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine; preferably, the recombinant vaccine comprises the fusion protein A and the fusion protein B; more preferably, the mass ratio of the fusion protein A and the fusion protein B in the recombinant vaccine is 1:

1.

15. Use of one or more of the fusion protein of any one of claims 1-7, and / or one or more of the recombinant nucleic acid molecule of claim 8, and / or one or more of the recombinant genetic expression cassette of claim 9, and / or one or more of the recombinant vector of claim 10, and / or one or more of the recombinant host cell of claim 11, and / or one or more of the immunogenic composition or pharmaceutical composition of claim 12 or 13, and / or one or more of the recombinant vaccine of claim 14 in the preparation of a medicament for prophylaxis, treatment and / or vaccination.

16. Use according to claim 15, characterized in that, the medicament is for the prevention and / or treatment of infection or disease caused by Pseudomonas aeruginosa; preferably, the infection or disease comprises pneumonia, skin and soft tissue infection, urinary tract infection, ocular infection, otic infection, bacteremia, sepsis, wound infection, chronic obstructive pulmonary disease (COPD), chronic pulmonary infection (CPI), acute pneumonia (AP), burn wound sepsis (BWS), catheter-related bloodstream infection (CRBSI), acute sepsis, stubborn mastitis, fibrinous emphysematous cystitis, endocarditis, bloodstream infection, central nervous system infection, bone and joint infection.

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