A vaccine for preventing salmonella infection and use thereof

Abstract

This invention relates to a fusion protein for preventing Salmonella infection, an immunogenic composition, a recombinant vaccine, and its molecular architecture design and applications. The invention selected three fusion proteins—PstS-LpfB, PstS-YidR, and LpfB-SinH—that can be successfully expressed in eukaryotic cells to design animal immunization experiments and verify their protective effects. This invention found that fusion protein molecules such as PstS-LpfB can attenuate tissue lesions caused by Salmonella infection, possess good immunogenicity, and play an effective preventive and immunoprotective role, highly preventing Salmonella infection and showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to a humoral immune vaccine for the prevention of Salmonella infection and its application. Background Technology

[0002] salmonella( Salmonella Salmonella is a Gram-negative bacillus widely distributed in nature and also present in the intestines of healthy humans and animals. Infections can occur when the body's immunity is weakened or when prolonged use of antibiotics leads to dysbiosis. Types of infection mainly include food poisoning, enteritis, typhoid fever, paratyphoid fever, and sepsis. In recent years, Salmonella has become an important foodborne pathogen and a significant potential cause of community-acquired and nosocomial infections.

[0003] Salmonella is classified based on the antigenic components on its cell surface, primarily including the O antigen (lipopolysaccharide antigen) and the H antigen (flagellate antigen). Currently, over 2600 serotypes of Salmonella have been identified. Among them, *Salmonella enteritidis* (…) Salmonella enteritidis ) and Salmonella typhimurium ( Salmonella typhimurium These are the two most common serotypes associated with human diseases. These two serotypes account for over 40% of human salmonellosis cases globally, and over 30% in China.

[0004] The World Health Organization (WHO) lists Salmonella as a significant global public health threat. According to the WHO, Salmonella causes more than 93.8 million cases of gastroenteritis and more than 155,000 deaths worldwide each year. The WHO also emphasizes the issue of Salmonella drug resistance, noting that its increasing resistance has become a serious public health problem. Therefore, developing an effective Salmonella vaccine is crucial for controlling its spread and infection.

[0005] Due to the lack of commercially available vaccines, current clinical treatment for Salmonella infections still primarily relies on antibiotics. However, antibiotic treatment has become a major driving force behind the emergence and spread of highly resistant and virulent strains. To overcome this predicament, researchers are actively developing new vaccines. For example, existing patent CN104736171B (publication date 2018-11-06) discloses an inactivated Salmonella vaccine, which prepares trivalent or quadrivalent vaccines by inactivating C1 or C2-3 serotypes of enteric Salmonella, providing some cross-immune protection. Patent CN119241728A (publication date 2024-11-14) discloses a method for preparing a fusion protein of Salmonella typhimurium flagellin and Seneca virus antigen, but does not provide relevant data on vaccine efficacy testing. There are too many pathogenic Salmonella subtypes, and prevention and control using traditional inactivated and attenuated vaccines have limited protective efficacy and are difficult to iterate upon. While some studies have proposed candidate targets for subunit vaccine development, most have not yet validated the efficacy of these targets and the subunit vaccines through animal experiments. Therefore, Salmonella vaccine development remains in a relatively early stage and cannot meet the current huge market demand.

[0006] Phosphate-binding protein PstS is part of the phosphate-specific transport (Pst) system, located in the bacterial periplasmic space, and has a high affinity for phosphate. Previous studies have demonstrated that PstS can serve as a biosensor for phosphate contamination. For example, patent CN114965403A (publication date 2022-08-30) discloses a method for constructing a phosphate biosensor using the Pseudomonas aeruginosa PstS protein. Furthermore, in some pathogenic bacteria, PstS expression is associated with the production of virulence factors and participates in biofilm formation and host cell adhesion; however, no studies have reported that PstS can be used as a protective antigen for Salmonella in vaccine development.

[0007] Molecular chaperone LpfB is a chaperone protein whose main function is to assist in the synthesis of long polar fimbriae (Lpf). Lpf is a fimbriae structure on the surface of Salmonella and plays an important role in Salmonella adhesion and colonization. Studies have shown that LpfB protein can assist in the assembly and transport of Lpf, enabling Salmonella to better adhere to host cells, especially on M cells (microfold cells) in the Peyer's patch region of the small intestine, thereby promoting Salmonella colonization and infection in the host. Although some studies have attempted to use LpfB alone as a vaccine antigen and found that LpfB alone has no immunoprotective effect, no studies have demonstrated whether the fusion of this protein with other antigens can help improve the immunoprotective efficacy of vaccines.

[0008] In summary, developing a vaccine that provides broad-spectrum protection against Salmonella, is safe and effective, and is easy to produce, has significant scientific and practical value. Such a vaccine could not only prevent various human infections but also potentially be used for animal disease prevention, aiming to reduce the incidence of Salmonella infections and bringing significant benefits to public health and the economy. Therefore, this invention addresses the aforementioned issues by proposing a novel fusion protein design scheme to develop a highly effective, broad-spectrum Salmonella infection prevention vaccine, potentially capable of simultaneously combating infections from multiple different Salmonella serotypes. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a novel fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention of Salmonella infection. This invention adopts a reverse vaccinology approach, screening for highly conserved target-binding immunotopes of Salmonella to identify candidate antigens for fusion protein design. After in vitro expression verification, three fusion proteins—PstS-LpfB, PstS-YidR, and LpfB-SinH—that can be successfully expressed in eukaryotic cells were selected for animal immunization experiments to verify their protective efficacy. This invention reveals that fusion protein molecules such as PstS-LpfB can attenuate tissue lesions caused by Salmonella infection, exhibiting good immunogenicity and providing effective prevention and immune protection, thus highly preventing Salmonella infection. This invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and applications.

[0010] One aspect of the present invention provides a fusion protein, characterized in that it is a fusion protein selected from any one of the following (1)-(3):

[0011] (1) Fusion protein A; the fusion protein A contains a phosphate-binding protein PstS antigen or a fragment thereof; preferably, the fusion protein further contains a molecular chaperone LpfB antigen or a fragment thereof.

[0012] (2) Fusion protein B: The fusion protein B comprises the PstS antigen or a fragment thereof and the YidR antigen or a fragment thereof;

[0013] (3) Fusion protein C: The fusion protein C contains the LpfB antigen or a fragment thereof and the SinH antigen or a fragment thereof.

[0014] Furthermore, the PstS, LpfB, YidR, SinH antigens or fragments thereof are derived from bacteria.

[0015] Furthermore, the bacteria are Gram-negative.

[0016] Furthermore, the Gram-negative bacteria are Enterobacteriaceae.

[0017] Furthermore, the Enterobacteriaceae bacteria mentioned are Salmonella.

[0018] Furthermore, the fusion protein A comprises, from N-terminus to C-terminus, the PstS antigen or a fragment thereof, and the LpfB antigen or a fragment thereof; the fusion protein B comprises, from N-terminus to C-terminus, the PstS antigen or a fragment thereof, and the YidR antigen or a fragment thereof; and the fusion protein C comprises, from N-terminus to C-terminus, the LpfB antigen or a fragment thereof, and the SinH antigen or a fragment thereof.

[0019] Further, the amino acid sequence of the PstS antigen or its fragment is shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen or its fragment is shown in SEQ ID NO: 2, the amino acid sequence of the YidR antigen or its fragment is shown in SEQ ID NO: 9, and the amino acid sequence of the SinH antigen or its fragment is shown in SEQ ID NO: 10.

[0020] Furthermore, the N-terminus of the fusion protein further includes a signal peptide and / or the Fc domain of the human immunoglobulin heavy chain constant region γ1 protein (IGHG1); or, the C-terminus of the fusion protein further includes the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit.

[0021] Furthermore, the signal peptide is derived from human azurocidin protein, the amino acid sequence of which is shown in SEQ ID NO: 3; the amino acid sequence of the Fc domain of IGHG1 is shown in SEQ ID NO: 4; and the amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0022] Furthermore, different components may optionally be connected via GGS linker sequences, spacer sequences, and / or GS linker sequences.

[0023] Further, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 8.

[0024] Furthermore, the fusion protein comprises any one of the following five fusion proteins:

[0025] (1) Fusion protein A: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen protein or fragment thereof, spacer sequence, LpfB antigen or fragment thereof, GS linker, STABILON;

[0026] (2) Fusion protein B: From the N-terminus to the C-terminus, it contains the following components in sequence: signal peptide, Fc domain, GGS linker, PstS antigen protein or fragment thereof, spacer sequence, YidR antigen protein or fragment thereof, GS linker, and STABILON.

[0027] (3) Fusion protein C: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, LpfB antigen protein or fragment thereof, spacer sequence, SinH antigen protein or fragment thereof, GS linker, STABILON;

[0028] (4) Fusion protein D: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, PstS antigen protein or fragment thereof, spacer sequence, SthB antigen protein or fragment thereof, GS linker, STABILON;

[0029] (5) Fusion protein E: From N-terminus to C-terminus, it contains: signal peptide, Fc domain, GGS linker, SinH antigen protein or fragment thereof, spacer sequence, SthB antigen protein or fragment thereof, GS linker, STABILON;

[0030] Further, the amino acid sequences of the PstS antigen or its fragments are shown in SEQ ID NO: 1, the amino acid sequences of the LpfB antigen or its fragments are shown in SEQ ID NO: 2, the amino acid sequences of the YidR antigen or its fragments are shown in SEQ ID NO: 9, the amino acid sequences of the SinH antigen or its fragments are shown in SEQ ID NO: 10, the amino acid sequences of the SthB antigen or its fragments are shown in SEQ ID NO: 11, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain is shown in SEQ ID NO: 4; the amino acid sequence of the STABILON is shown in SEQ ID NO: 5, the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO: 8.

[0031] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding a fusion protein according to any one of the present invention.

[0032] 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.

[0033] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0034] 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.

[0035] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0036] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0037] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0038] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0039] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0040] 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.

[0041] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0042] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0043] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0044] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0045] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0046] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention.

[0047] Furthermore, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0048] Furthermore, the immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Salmonella.

[0049] Furthermore, the other medications used to prevent diseases caused by Salmonella include antibiotics.

[0050] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention.

[0051] Furthermore, the recombinant vaccine is a nucleic acid vaccine or a subunit vaccine.

[0052] Furthermore, the recombinant vaccine is a nucleic acid vaccine.

[0053] Another aspect of the present invention provides the use of one or more fusion proteins of any one 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 a medicament for the prevention of diseases caused by Salmonella.

[0054] Another aspect of the present invention provides a method for preventing diseases caused by Salmonella, characterized in that it includes administering to a subject one or more fusion proteins of any one 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.

[0055] Furthermore, the diseases caused by the Salmonella include, but are not limited to, gastroenteritis, typhoid fever, bacteremia, localized purulent infections, reactive arthritis, enteric fever, intravascular infections, and focal infections.

[0056] Furthermore, the focal infections include osteomyelitis, arthritis, meningitis, endocarditis, liver abscess, cholecystitis, and pyelonephritis.

[0057] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0058] 1. This invention discovers that novel fusion molecules possess excellent immunogenicity and can provide immune protection, making them suitable for the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of this invention can provide effective immune protection against Salmonella infection.

[0059] 2. This invention can serve as a universal antigenic framework for nucleic acid vaccines or subunit vaccines, exhibiting strong immunogenicity. Compared to traditional vaccines, it is not limited by serotype and can induce broad-spectrum immune protection.

[0060] 3. Through systematic screening and immunoepitaxy prediction analysis, this invention identified five candidate antigens: PstS, LpfB, YidR, SthB, and SinH. Fusion proteins were then designed and experimentally validated. Furthermore, to improve the expression of the candidate antigens and enhance their immunogenicity, N-terminal truncation or amino acid mutations at specific sites were performed on the known PstS, LpfB, YidR, SthB, and SinH antigens.

[0061] 4. This invention utilizes reverse vaccinology technology to screen candidate targets with broad-spectrum protective effects, and selects PstS antigen and LpfB antigen for vaccine design. Through comparative experiments, it is verified that the fusion protein composed of the two antigens can induce mice to produce immune protection against Salmonella.

[0062] 5. Example 5 of the present invention shows that the fusion molecule based on the present invention can effectively promote the high abundance expression and secretion of bacterial proteins in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes, thereby further improving the immune protection effect and providing a new technology for the design of immunotherapeutic drugs for Salmonella multivalent vaccines.

[0063] 6. The mouse gavage infection model experiment in Example 6 of this invention showed that, in lung tissue, the bacterial load in the mouse group vaccinated with the vaccine of this invention was significantly lower than that in the PBS control group. Statistical analysis showed that this reduction was statistically significant (P<0.001). This result demonstrates that the vaccine based on this invention can effectively reduce the colonization of Salmonella in the intestine, spleen, and liver, providing effective multi-tissue immune protection.

[0064] 7. The fusion protein vaccine provided by this invention exhibits excellent immunogenicity and protective efficacy, showing great potential in preventing Salmonella infection. This novel vaccine design strategy provides a reference for the development of vaccines against other bacterial pathogens. Attached Figure Description

[0065] Figure 1 This is a schematic diagram of the molecular structure of the vaccines used in the comparative experiments in the examples, including schematic diagrams of the molecular structures of vaccine A, vaccine B, vaccine C, vaccine D, and vaccine E.

[0066] Figure 2 This is a schematic diagram of a circular plasmid containing the molecular structure of the fusion protein of the present invention.

[0067] Figures 3A-3E This invention provides quality control peak diagrams and purity test results of the nucleic acid vaccines used in comparative experiments; wherein, Figure 3A The image shows the quality control peaks and purity test results for vaccine A. Figure 3B The results show the quality control peaks and purity test results for vaccine B. Figure 3C The results show the quality control peaks and purity test results for vaccine C. Figure 3D This is a quality control peak diagram and purity test results for vaccine D. Figure 3E This is a peak diagram of the quality control of vaccine E and the results of purity testing.

[0068] Figures 4A-4DThe results of in vitro expression WB (Western blot) detection in HEK293 cells transfected with vaccines A, B, C, D, and E of this invention; among them, Figure 4A The in vitro expression results of HEK293 cells transfected with vaccine A were obtained by Western blot analysis. Figure 4B The in vitro expression Western blot results of HEK293 cells transfected with vaccine B correspond to the results of Western blot analysis. Figure 4C The in vitro expression Western blot results of HEK293 cells transfected with vaccines C and D correspond to the results of Western blot analysis. Figure 4D The in vitro expression of the corresponding vaccine E transfected HEK293 cells was detected by Western blot analysis.

[0069] Figure 5 This is a schematic diagram of the immunization, challenge, and sampling process in mouse infection experiments.

[0070] Figures 6A-6B The results show the bacterial load in the feces of mice in each experimental group after immunization and challenge; among them Figure 6A These are images of bacterial load in fecal samples from mice in each experimental group after immunization and challenge. Figure 6B This is a comparative chart showing the bacterial load analysis in the feces of mice in different experimental groups after immunization and viral challenge.

[0071] Figures 7A-7B The results show the bacterial load in the spleen tissue of mice in each experimental group after immunization and challenge; among them Figure 7A These are plate smear images showing the bacterial load in the spleen tissue of mice in each experimental group after immunization and challenge. Figure 7B This is a comparative analysis of bacterial load in the spleen tissue of mice in different experimental groups after immune challenge.

[0072] Figures 8A-8B The results show the bacterial load in the liver tissues of mice in each experimental group after immunization and challenge; among them Figure 8A These are plate smear images showing the bacterial load in the liver tissue of mice in each experimental group after immunization and challenge. Figure 8B This is a comparative analysis of bacterial load in the liver tissues of mice in different experimental groups after immune challenge. Detailed Implementation

[0073] Terms and Definitions

[0074] The term "Salmonella" refers to Salmonella, a common foodborne pathogen belonging to the Enterobacteriaceae family. It is a Gram-negative bacillus with peritrichous flagella, enabling motility, and most species have pili. Salmonella is widely found in the intestines of humans, poultry, and livestock, primarily transmitted through contaminated food such as raw poultry, eggs, beef, and certain fruits and vegetables.

[0075] The term "Salmonella infection" refers to various diseases caused by Salmonella, including but not limited to gastroenteritis, typhoid fever, bacteremia, localized purulent infections, reactive arthritis, etc.

[0076] The term "PstS" refers to the phosphate-specific transport system substrate-binding protein (PstS), an important component of the Salmonella phosphate transport system. Preferably, the amino acid sequence of PstS is shown in SEQ ID NO: 1.

[0077] The term "LpfB" refers to molecular chaperone LpfB (LpfB for short), a chaperone protein whose main function is to assist in the synthesis of long polar fimbriae (Lpf). Lpf is a fimbriae structure on the surface of Salmonella and plays an important role in the adhesion and colonization of Salmonella. Preferably, the amino acid sequence of LpfB is shown in SEQ ID NO: 2.

[0078] The term "YidR" refers to a conserved protein in Salmonella containing the DUF3748 domain. Its function is not fully elucidated, but it may be involved in bacterial stress responses and biofilm formation. Preferably, the amino acid sequence of YidR is shown in SEQ ID NO: 9.

[0079] The term "SinH" refers to a protein in Salmonella that may be involved in regulating bacterial virulence and adaptive responses. This protein plays an important role in bacterial-host interactions and may promote bacterial survival and pathogenicity by regulating biofilm formation or influencing specific stress response pathways. Preferably, the amino acid sequence of SinH is shown in SEQ ID NO: 10.

[0080] The term "SthB" refers to a protein in Salmonella that may be involved in bacterial homeostasis and environmental adaptation. Its specific function is not fully understood, but it may play a role in stress response, cellular metabolic regulation, or pathogen adaptation. Preferably, the amino acid sequence of SthB is shown in SEQ ID NO: 11.

[0081] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immune responses 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.

[0082] The terms "administration" or "vaccination" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous route, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injection), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes.

[0083] 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.

[0084] 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 used to transform into a suitable host cell.

[0085] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide that is used to express, for example, a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate 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) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences.

[0086] The term "mRNA" refers to messenger RNA, also known as "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.

[0087] 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) located upstream of the coding sequence.

[0088] 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.

[0089] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells.

[0090] 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).

[0091] The terms "transformation," "transfection," and "transduction" have the meanings commonly understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0092] The term "drug 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, as well as to provide a method for its effective absorption in a subject.

[0093] 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.

[0094] 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.

[0095] This invention discloses a novel method for preparing a Salmonella vaccine and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. 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 method and application 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.

[0096] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all use 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.

[0097] 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.

[0098] Example 1 Antigen Screening

[0099] Because Salmonella vaccines are limited by serotypes, this invention employs a reverse vaccinology strategy to screen for potential cross-protective antigens against Salmonella. The primary goal of the screening is to identify antigens that are highly expressed in the transcriptome and are highly conserved across different Salmonella subtypes.

[0100] The specific screening steps are as follows:

[0101] (1) Collect transcriptome data of different serotypes of Salmonella.

[0102] (2) Bioinformatics analysis of transcriptome data to identify genes with high abundance expression.

[0103] (3) The conservation of these highly expressed genes across different serotypes was analyzed using comparative genomics methods.

[0104] Through the above systematic screening and combined with immune epitope prediction analysis, this invention finally identified five antigens—PstS, LpfB, YidR, SthB, and SinH—for subsequent fusion protein design and experimental verification.

[0105] In addition, in order to improve the expression of candidate antigens and enhance their immunogenicity, N-terminal truncation or amino acid mutations at individual sites were performed on known PstS, LpfB, YidR, SthB, and SinH antigens.

[0106] Example 2: Design of Fusion Protein Vaccine and Construction of Recombinant Nucleic Acid Vaccine

[0107] Based on the screening results in Example 1, a total of 5 fusion protein vaccines were obtained through fusion protein design: PstS-LpfB, PstS-YidR, LpfB-SinH, PstS-SthB, and SinH-SthB.

[0108] To prepare the above five recombinant nucleic acid vaccines, an exemplary schematic diagram of the vaccine molecular structure involved in the embodiments is shown below. Figure 1 As shown, from the N-terminus to the C-terminus, the following elements are sequentially included: signal peptide, Fc domain, target antigen region, and STABILON. These elements can be directly linked or linked through different linkers, such as GGS linker, spacer sequence, or GS linker. Figure 1 The five vaccines illustrated (A, B, C, D, and E) differ in their target antigen regions. Vaccine A targets PstS-LpfB (linked by a spacer sequence), vaccine B targets PstS-YidR (linked by a spacer sequence), vaccine C targets LpfB-SinH (linked by a spacer sequence), vaccine D targets PstS-SthB (linked by a spacer sequence), and vaccine E targets SinH-SthB (linked by a spacer sequence). To prepare vaccines capable of producing... Figure 1The recombinant nucleic acid vaccine for the protein shown first constructs a gene expression cassette to express the antigen sequence of the fusion protein molecular structure described in this invention. The expression cassette, from the 5' end to the 3' end, sequentially includes: a 5' UTR, a CDS region, a 3' UTR, and PolyA, wherein the CDS region contains the fusion protein molecular structure described in this invention. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector suitable for in vitro RNA transcription, such as... Figure 2 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine was obtained.

[0109] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0110] (1) Recombinant nucleic acid vaccine A preparation vector containing PstS-LpfB

[0111] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-LpfB antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGS linker sequence shown in SEQ ID NO: 7. The PstS antigen and LpfB antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The LpfB antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the LpfB antigen is shown in SEQ ID NO: 2. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0112] Step b: Construct a nucleic acid vaccine architecture carrier.

[0113] 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.

[0114] Step c: Prepare recombinant plasmids.

[0115] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector A containing PstS-LpfB. Figure 2 (As shown).

[0116] (2) Preparation vector of recombinant nucleic acid vaccine B containing PstS-YidR

[0117] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-YidR antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The PstS antigen and YidR antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The YidR antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the YidR antigen is shown in SEQ ID NO: 9. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0118] Step b: Construct a nucleic acid vaccine architecture carrier.

[0119] 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.

[0120] Step c: Prepare recombinant plasmids.

[0121] 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 containing PstS-YidR. Figure 2 (As shown).

[0122] (3) Preparation vector of recombinant nucleic acid vaccine containing LpfB-SinH C

[0123] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-LpfB antigen-SinH antigen-STABILON”. The human IGHG Fc domain and LpfB antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The LpfB antigen and SinH antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The SinH antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the LpfB antigen is shown in SEQ ID NO: 2. The amino acid sequence of the SinH antigen is shown in SEQ ID NO: 10. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0124] Step b: Construct a nucleic acid vaccine architecture carrier.

[0125] 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.

[0126] Step c: Prepare recombinant plasmids.

[0127] The gene synthesized in step a is inserted into the vector architecture of step b to obtain the recombinant nucleic acid vaccine preparation vector C containing LpfB-SinH. Figure 2 (As shown).

[0128] (4) Preparation vector of recombinant nucleic acid vaccine containing PstS-SthB

[0129] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-PstS antigen-SthB antigen-STABILON”. The human IGHG Fc domain and PstS antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The PstS antigen and SthB antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The SthB antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1. The amino acid sequence of the SthB antigen is shown in SEQ ID NO: 11. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0130] Step b: Construct a nucleic acid vaccine architecture carrier.

[0131] 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.

[0132] Step c: Prepare recombinant plasmids.

[0133] The gene synthesized in step a is inserted into the vector architecture of step b to obtain the recombinant nucleic acid vaccine preparation vector D containing PstS-SthB. Figure 2 (As shown).

[0134] (5) Preparation vector of recombinant nucleic acid vaccine containing SinH-SthB

[0135] Step a: Synthesize the fusion gene fragment “signal peptide-human IGHG Fc domain-SinH antigen-SthB antigen-STABILON”. The human IGHG Fc domain and SinH antigen are linked by the GGSlinker sequence shown in SEQ ID NO: 7. The SinH antigen and SthB antigen are linked by the spacer sequence shown in SEQ ID NO: 6. The SthB antigen and STABILON are linked by the GS linker sequence shown in SEQ ID NO: 8. The amino acid sequence of the signal peptide is shown in SEQ ID NO: 3. The amino acid sequence of the human IGHG Fc domain is shown in SEQ ID NO: 4. The amino acid sequence of the SinH antigen is shown in SEQ ID NO: 10. The amino acid sequence of the SthB antigen is shown in SEQ ID NO: 11. The amino acid sequence of STABILON is shown in SEQ ID NO: 5.

[0136] Step b: Construct a nucleic acid vaccine architecture carrier.

[0137] 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.

[0138] Step c: Prepare recombinant plasmids.

[0139] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine E preparation vector containing SinH-SthB. Figure 2 (As shown).

[0140] Table 1. Protein amino acid sequences involved in this invention.

[0141]

[0142] Example 3: Preparation of the recombinant nucleic acid vaccine of the present invention

[0143] (1) Preparation of capped mRNA vaccines

[0144] Step a: Linearize the vector plasmid used in Example 2 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0145] 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.

[0146] (2) Preparation of uncapped mRNA vaccines

[0147] Step a: Linearize the vector plasmid used in Example 2 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0148] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0149] (3) DNA vaccine preparation

[0150] Step a: Amplify the vector plasmid used in Example 2 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0151] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0152] Example 4: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0153] Vaccine A (recombinant nucleic acid vaccine A containing PstS-LpfB), vaccine B (recombinant nucleic acid vaccine B containing PstS-YidR), vaccine C (recombinant nucleic acid vaccine C containing LpfB-SinH), vaccine D (recombinant nucleic acid vaccine D containing PstS-SthB), and vaccine E (recombinant nucleic acid vaccine E containing SinH-SthB) were prepared using the capped mRNA vaccine preparation method described in Example 3. 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 or equal to 85%. The recombinant nucleic acid quality control peak diagram based on this invention is shown below. Figures 3A-3E As shown. Specifically, it is described as follows: (1) Recombinant nucleic acid vaccine A containing PstS-LpfB, with a purity of 90.1%, and the quality control peak diagram and purity test results are as follows. Figure 3A (2) Recombinant nucleic acid vaccine B containing PstS-YidR, with a purity of 85.1%, has the following quality control peak diagram and purity test results: Figure 3B (3) Recombinant nucleic acid vaccine C containing LpfB-SinH, with a purity of 89.3%, has the following quality control peak diagram and purity test results: Figure 3C (4) Recombinant nucleic acid vaccine D containing PstS-SthB, with a purity of 85.4%, has the following quality control peak diagram and purity test results: Figure 3D (5) Recombinant nucleic acid vaccine E containing SinH-SthB, with a purity of 90.7%, and the quality control peak diagram and purity test results are as follows: Figure 3E The purity levels described above all meet the quality requirements for cell transfection experiments and vaccine production.

[0154] Example 5: In vitro expression effect of the recombinant nucleic acid of the present invention

[0155] The vaccines from Example 4 were transfected into HEK293T cells using cell transfection reagents. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot. The molecular weights of the proteins of vaccines A, B, C, D, and E were calculated, as shown in Table 2.

[0156] Table 2. Protein molecular weight of vaccines A, B, C, and D

[0157] vaccine Protein molecular weight (kDa) A 89.3 B 111.8 C 84.6 D 97 E 92.3

[0158] Figures 4A-4D The results of in vitro expression WB (Western blot) detection of vaccines A, B, C, D, and E transfected into HEK293 cells are presented. Figure 4A The in vitro expression results of HEK293 cells transfected with vaccine A were obtained by Western blot analysis. Figure 4B The in vitro expression Western blot results of HEK293 cells transfected with vaccine B correspond to the results of Western blot analysis. Figure 4C The in vitro expression Western blot results of HEK293 cells transfected with vaccines C and D correspond to the results of Western blot analysis. Figure 4D The in vitro expression of the corresponding vaccine E transfected HEK293 cells (WB detection results) showed that the expressed antigens were all humoral immune antigens, which theoretically could be significantly expressed in the supernatant.

[0159] As shown in Figure 4, the target protein signal was detected in the supernatant of vaccines A, B, and C, and the molecular weight was consistent with expectations. This demonstrates that the fusion proteins based on this invention can not only be successfully translated and correctly folded in eukaryotic cells, but also have a stable structure and can be secreted extracellularly. No target protein signal was detected in the supernatant of vaccines D and E, indicating that the PstS-SthB and SinH-SthB fusion protein designs could not be normally secreted and expressed.

[0160] Therefore, vaccines D and E, which could not be expressed or secreted normally, were excluded. Vaccines A, B, and C were selected for subsequent animal experiments to verify their immune protection effects.

[0161] Example 6: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse gavage infection model.

[0162] To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect, this embodiment conducted immunization and challenge comparison experiments on mice immunized with vaccine A (vaccine A immunization group), mice immunized with vaccine B (vaccine B immunization group), mice immunized with vaccine C (vaccine C immunization group), and unimmunized mice (PBS group). One mouse was not immunized or challenged and served as a negative control group.

[0163] Twenty-one 6-8 week old BALB / c mice, weighing 18-25g, were selected for the experiment. All mice were housed in individual cages with constant temperature and humidity, and were acclimatized to the environment for 7 days prior. The temperature in the housing was 20-26℃, and the humidity was 40-70%. A day-night cycle was implemented, with light from 8:00 AM to 8:00 PM and darkness from 8:00 PM to 8:00 AM the following day. Sufficient feed was continuously provided, with unlimited access to sterile water via a continuous water bottle. After acclimatization, the mice were randomly assigned to groups, and each mouse was ear-tagged. Details are shown in Table 3. Dosages in this table and below refer to the amount of active ingredient.

[0164] Table 3. Grouping and Immunization Procedure for Mouse Immunization Experiment in Example 5

[0165]

[0166] Mice in each group were immunized twice according to the immunization protocol in Table 3. On Day 34, mice were administered 100 μL (200 mg / mL) of streptomycin solution by gavage to enhance the bacterial challenge effect. Challenge was performed on Day 35. Mice were fasted for 4 hours prior to bacterial challenge and were given 100 μL of Salmonella bacterial suspension (1×10⁻⁶). 7 CFU was administered via gavage. Mice were then fed normally after gavage, and their clinical manifestations were observed and recorded daily. On Day 40, mice were sacrificed and tissue samples were collected to detect bacterial load in different tissues and assess the immunoprotective effect of the vaccine. The immunization, challenge, and sampling schedule is as follows: Figure 5 As shown.

[0167] Equal amounts of feces, spleen, and liver tissue were taken from each mouse and ground on ice with sterile PBS buffer until no obvious precipitation occurred. Blood was diluted 1:1000 with sterile PBS buffer, and 100 μl of the diluted solution was evenly spread on a solid culture medium and incubated for 24 h. The culture dish was then removed to count and photograph bacterial clones to observe the protective effect of the vaccine on mice. The results are shown in Figures 6, 7, and 8.

[0168] Figure 6 shows the comparison of fecal bacterial load in different experimental groups after mouse immunization challenge. Figure 6A These are images of bacterial load in fecal samples from mice in each experimental group after immunization and challenge. Figure 6B This is a comparative analysis of fecal bacterial load in mice from different experimental groups after immunization and challenge. The results show that the bacterial load in the negative control group was 0, confirming the validity of the comparative experiment. Compared with the PBS group, the Salmonella load in the vaccine A immunization group was significantly reduced, approximately three-fold; the bacterial load in the vaccine B and vaccine C immunization groups did not show a decrease.

[0169] Figure 7 shows the comparison of bacterial load in spleen tissue of different experimental groups after mouse immunization challenge. Figure 7A These are plate smear images showing the bacterial load in the spleen tissue of mice in each experimental group after immunization and challenge. Figure 7BThis is a comparative analysis of bacterial load in the spleen tissue of mice in different experimental groups after immunization and challenge. The results show that the bacterial load in the negative control group was 0, confirming the validity of the comparative experiment. Compared with the PBS group, the Salmonella load in the vaccine A immunization group was significantly reduced, approximately 7-fold; the bacterial load in the vaccine B immunization group did not decrease; and the bacterial load in the vaccine C immunization group decreased.

[0170] Figure 8 shows the comparison of bacterial load in the livers of different experimental groups after mouse immunization challenge. Figure 8A These are plate smear images showing the bacterial load in the liver tissue of mice in each experimental group after immunization and challenge. Figure 8B This is a comparative analysis of bacterial load in the liver tissues of mice in different experimental groups after immunization. The results show that the bacterial load in the negative control group was 0, confirming the validity of the comparative experiment. Compared with the PBS group, the Salmonella load in the vaccine A immunization group was significantly reduced, approximately 50-fold; the bacterial load in the vaccine B immunization group did not decrease; and the bacterial load in the vaccine C immunization group decreased.

[0171] In summary, among the three fusion proteins PstS-LpfB, PstS-YidR, and LpfB-SinH provided by this invention, PstS-LpfB is the optimal combination, capable of inducing effective protective immunity in mouse model animals and exhibiting good preventive efficacy against Salmonella infection. The PstS-LpfB fusion protein is a preferred vaccine target. Therefore, this invention provides an optimal antigen combination that can be applied to vaccine development, filling a gap in current Salmonella vaccine research and development, and possessing extremely high commercial value and broad application prospects.

[0172] 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, From N-terminus to C-terminus, the sequence is as follows: signal peptide, Fc domain, GGS linker, PstS (Phosphate-binding protein PstS) antigen, spacer sequence, LpfB (molecular chaperone LpfB) antigen, GS linker, and STABILON, a C-terminal polypeptide fragment of the human S5a / PSMD4 proteasome subunit; wherein, the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, the amino acid sequence of the LpfB antigen is shown in SEQ ID NO: 2, the amino acid sequence of STABILON is shown in SEQ ID NO: 5, and the amino acid sequence of the Fc domain is shown in SEQ ID NO:

4.

2. The fusion protein according to claim 1, characterized in that, The PstS and LpfB antigens are derived from Salmonella.

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO:

3.

4. The fusion protein according to claim 3, characterized in that, The amino acid sequence of the spacer sequence is shown in SEQ ID NO: 6, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 7, and the amino acid sequence of the GS linker sequence is shown in SEQ ID NO:

8.

5. A recombinant nucleic acid molecule, characterized in that, The nucleic acid comprising encoding the fusion protein of any one of claims 1-4.

6. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 5.

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

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

9. An immunogenic composition or pharmaceutical composition, characterized in that, It comprises one or more of the fusion proteins of any one of claims 1-4, and / or one or more of the recombinant nucleic acid molecules of claim 5, and / or one or more of the recombinant gene expression cassettes of claim 6, and / or one or more of the recombinant vectors of claim 7, and / or one or more of the recombinant host cells of claim 8.

10. The immunogenic composition or pharmaceutical composition according to claim 9, characterized in that, The immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

11. The immunogenic composition or pharmaceutical composition according to claim 9 or 10, characterized in that, The immunogenic composition or pharmaceutical composition may also contain other drugs for the prevention of diseases caused by Salmonella.

12. The immunogenic composition or pharmaceutical composition according to claim 11, characterized in that, Other medications used to prevent diseases caused by Salmonella include antibiotics.

13. A recombinant vaccine, characterized in that, The composition comprises one or more of the fusion proteins of any one of claims 1-4, and / or one or more of the recombinant nucleic acid molecules of claim 5, and / or one or more of the recombinant gene expression cassettes of claim 6, and / or one or more of the recombinant vectors of claim 7, and / or one or more of the recombinant host cells of claim 8, and / or one or more of the immunogenic compositions or pharmaceutical compositions of any one of claims 9-12.

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

15. Use of one or more fusion proteins according to any one of claims 1-4, or one or more recombinant nucleic acid molecules according to claim 5, or one or more recombinant gene expression cassettes according to claim 6, or one or more recombinant vectors according to claim 7, or one or more recombinant host cells according to claim 8, or one or more immunogenic compositions or pharmaceutical compositions according to any one of claims 9-12, or one or more recombinant vaccines according to any one of claims 13-14 in the preparation of a medicament for the prevention of diseases caused by Salmonella.

16. The use according to claim 15, characterized in that, The diseases caused by Salmonella include gastroenteritis, typhoid fever, bacteremia, localized purulent infections, enteric fever, intravascular infections, and focal infections.

17. The use according to claim 16, characterized in that, The focal infections include osteomyelitis, arthritis, meningitis, endocarditis, liver abscess, cholecystitis, and pyelonephritis.

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