Fusion protein for preventing and treating various pathogenic enterobacter infections and application thereof

Abstract

The invention relates to a fusion protein, an immunogenic composition and a recombinant degenerate vaccine for preventing and treating various pathogenic enterobacter infections, as well as a molecular architecture design, application and the like. According to the invention, three cellular immune antigens Tuf, DnaK and fusA are screened, and the constructed fusion protein molecules of the three antigens can significantly inhibit tissue lesions caused by various enterobacter infections, have good immunogenicity, play roles in effective prevention and immune protection, and broad-spectrum and efficient prevention of various pathogenic enterobacter infections, and have broad industrial 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 fusion proteins for the prevention and treatment of various pathogenic Enterobacter infections and their applications. Background Technology

[0002] Enterobacteriaceae ( Enterobacteriaceae Gram-negative bacilli are widely distributed in the external environment and in the intestines of animals. Some of these bacteria exhibit pathogenicity, such as Escherichia coli, Klebsiella pneumoniae, Salmonella, and Shigella, which can infect both humans and animals, causing a variety of serious diseases.

[0003] Unlike viral infections, bacterial infections are difficult to eradicate once bacteria colonize the body. Their reproduction process produces large amounts of toxins, which can lead to death in severe cases. In the past, antibiotics were the only way to control bacterial infections in clinical treatment. However, in recent years, with the increase in antibiotic resistance and the aging population, pathogenic Enterobacteriaceae infections have been on the rise globally, becoming a significant threat to public health.

[0004] Safe and effective vaccines are considered the best measure for preventing and controlling bacterial infections. However, current vaccine development against Enterobacteriaceae still revolves around inactivated vaccines or capsular polysaccharide vaccines, unable to escape the limitations of single bacterial species or single serotypes. Although some polysaccharide vaccines enhance their protective spectrum by combining multiple Enterobacterial polysaccharides or binding virulence factor proteins to achieve broad-spectrum protection, multivalent polysaccharide vaccines or conjugate vaccines not only have limitations in manufacturing processes, but their immunoprotective range is mostly limited to one bacterium, such as Escherichia coli (Escherichia coli) multivalent polysaccharide vaccines and Klebsiella pneumoniae multivalent polysaccharide vaccines. These vaccines have not yet been successfully launched on the market. In addition, there are some glycoprotein conjugate vaccines. For example, the patent "A Neonatal Escherichia coli Meningitidis Glycoprotein Conjugate Vaccine and Its Application" (Publication No.: CN117771349A) discloses a neonatal Escherichia coli glycoprotein conjugate vaccine and its application, in which the immunoconjugate is formed by coupling neonatal Escherichia coli surface polysaccharide with a carrier protein; the patent "Glycoconjugate Vaccine Containing Basic Units of a Molecular Construct Expressing Multiple Built-in Epitopes for Preparing a Broad-Spectrum Vaccine Against Infections Caused by Enteropathogenic Bacteria" (Publication No.: CN106659799B) discloses a novel glycoconjugate antigen expressing multiple built-in epitopes and a multivalent glycoconjugate vaccine intended for protecting mammals, the main components of which are Clostridium difficile toxoid proteins and multiple bacterial polysaccharides conjugate. These vaccines can effectively reduce the clinical symptoms caused by some Enterobacter infections, but they still have some limitations. First, they can only cover a limited number of serotypes and cannot provide truly broad-spectrum protection. Second, the production cost of conjugate vaccines is high, limiting their widespread use in developing countries. Furthermore, the widespread adoption of serotype-related vaccines will lead to a gradual increase in the proportion of pathogens with non-vaccine serotypes, subsequently triggering the issue of serotype substitution. Therefore, developing a novel Enterobacter vaccine that can provide broad-spectrum protection, has low production costs, and high safety is imperative.

[0005] With the development of genomics, proteomics, and immunology, reverse vaccinology has become an important method for vaccine antigen screening. Through reverse vaccinology screening, non-glycoproteins and non-virulence factors with good conservation and immunogenicity can be obtained from the vast bacterial protein pool, making them highly promising for novel vaccine development. Novel vaccines can use conserved proteins from Enterobacteriaceae as antigens and can achieve multi-antigen combinations, avoiding serotype limitations while providing as many immunotopes as possible, thus offering broad-spectrum protection.

[0006] Compared with traditional polysaccharide and conjugate vaccines, vaccines designed based on conserved antigen proteins have several advantages: (1) they are not limited by serotype and are expected to provide broad-spectrum protection; (2) the production process is relatively simple and the cost is low; and (3) they can be combined with antigens of other pathogens to develop multivalent vaccines.

[0007] In conclusion, the development of a broad-spectrum vaccine against pathogenic Enterobacteriaceae is urgently needed, as it will be key to solving various problems associated with current traditional vaccines. New vaccines not only promise to provide broader protection but may also reduce production costs, making them easier to distribute globally. These studies are crucial not only for controlling Enterobacteriaceae infection but also for providing new ideas and methods for the development of other broad-spectrum bacterial vaccines. Therefore, although various Enterobacteriaceae vaccines have been disclosed in existing technologies, there is still a pressing need in the field for a fusion protein, immune composition, or vaccine that attenuates the tissue damage caused by Enterobacteriaceae infection, possesses good immunogenicity, provides effective prevention and immune protection, and offers highly efficient and broad-spectrum prevention against Enterobacteriaceae infection. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a fusion protein, immunogenic composition, recombinant degenerate vaccine, molecular architecture design, and applications for the prevention and treatment of various pathogenic Enterobacter infections. This invention screened three cellular immune antigens—Tuf, DnaK, and fusA—and constructed a fusion protein molecule containing these three antigens. This fusion protein molecule significantly inhibits tissue lesions caused by various Enterobacter infections, exhibits good immunogenicity, and provides effective prevention and immune protection. It offers broad-spectrum and highly effective prevention of various pathogenic Enterobacter infections and has broad prospects for industrial application.

[0009] One aspect of the present invention provides a fusion protein, characterized in that it comprises the following antigen: (a) Elongation factor Tu (Tuf) antigen or a selected fragment of its antigen; (b) Chaperone protein DnaK (DnaK) antigen or a selected fragment thereof; and (c) Elongation factor G (fusA) antigen or its antigenic selection fragment.

[0010] Further, the antigen or a selected fragment thereof is derived from Enterobacteriaceae bacteria; preferably, the Enterobacteriaceae bacteria are pathogenic Enterobacteriaceae; more preferably, the pathogenic Enterobacteriaceae are derived from Salmonella (…). Salmonella enterica ) and / or Klebsiella pneumoniae ( Klebsiella pneumoniae Most preferably, the Tuf antigen or a selected fragment thereof is derived from Salmonella ( ); Salmonella enterica The DnaK antigen or its antigen-selected fragment is derived from Salmonella ( Salmonella enterica The fusA antigen or a selected fragment thereof is derived from Klebsiella pneumoniae (…). Klebsiella pneumoniae ).

[0011] Further, the amino acid sequence of the Tuf antigen has at least 97% sequence identity with SEQ ID NO: 1, preferably 98%, 99%, or 100%; the amino acid sequence of the DnaK antigen has at least 95% sequence identity with SEQ ID NO: 2, preferably 96%, 97%, 98%, 99%, or 100%; and the amino acid sequence of the fusA antigen has at least 92% sequence identity with SEQ ID NO: 3, preferably 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0012] Further, the Tuf antigen selected fragment contains a conserved domain of the elongation factor Tu antigen, preferably, the amino acid sequence of the Tuf antigen selected fragment is shown in SEQ ID NO: 4; the DnaK antigen selected fragment contains a conserved domain of the chaperone factor DnaK, the amino acid sequence of the DnaK antigen selected fragment is shown in SEQ ID NO: 5; the fusA antigen selected fragment contains a conserved domain of the elongation factor G, the amino acid sequence of the fusA antigen selected fragment is shown in SEQ ID NO: 6.

[0013] Further, the homology of the Tuf antigen selected fragment, the DnaK antigen selected fragment, and the fusA antigen selected fragment in Enterobacteriaceae is not less than 97%; preferably, the Tuf antigen selected fragment has at least 97% sequence identity with SEQ ID NO: 4, and more preferably, the sequence identity is 98%, 99%, or 100%; the DnaK antigen selected fragment has at least 97% sequence identity with SEQ ID NO: 5, and more preferably, the sequence identity is 98%, 99%, or 100%; the fusA antigen selected fragment has at least 85% sequence identity with SEQ ID NO: 6, and more preferably, the sequence identity is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0014] Further, the antigens (a), (b), and (c) can be arranged and combined in any order; preferably, the fusion protein contains, from the N-terminus to the C-terminus, the Tuf antigen or its antigen-selected fragment, the DnaK antigen or its antigen-selected fragment, and the fusA antigen or its antigen-selected fragment; optionally, different antigens or fragments can be linked by a linker; preferably, the amino acid sequence of the linker is shown in SEQ ID NO: 7.

[0015] Another aspect of the present invention provides a recombinant nucleic acid molecule characterized in that it encodes the fusion protein described in any one of the present invention.

[0016] 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.

[0017] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

[0018] 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.

[0019] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0020] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0021] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0022] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0023] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0024] 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.

[0025] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0026] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0027] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0028] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0029] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0030] 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; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0031] 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; preferably, the recombinant vaccine is a nucleic acid vaccine; more preferably, the nucleic acid vaccine is a DNA vaccine or an RNA vaccine.

[0032] Another aspect of the present invention provides the use of 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, and / or one or more recombinant vaccines according to the present invention in the preparation of medicaments for the prevention and / or treatment of diseases caused by Enterobacteriaceae bacteria; preferably, the Enterobacteriaceae are pathogenic Enterobacteriaceae.

[0033] Another aspect of the present invention provides a method for preventing and / or treating diseases caused by Enterobacteriaceae bacteria, characterized in that it includes administering to a subject 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, and / or one or more recombinant vaccines according to the present invention.

[0034] Furthermore, the Enterobacteriaceae family of bacteria includes, but is not limited to, Escherichia coli, Klebsiella pneumoniae, Salmonella, Shigella, and Proteus mirabilis.

[0035] Furthermore, the diseases include urinary tract infections, breast infections, abdominal infections, dysentery, enteritis, respiratory infections, pneumonia, wound infections, sepsis, and meningitis.

[0036] The fusion protein molecular structure and the nucleic acid structure encoding it, pharmaceutical compositions, recombinant vaccines, etc. of the present invention have the following beneficial technical effects: 1. In order to develop a vaccine that can be used for broad-spectrum prevention of pathogenic Enterobacter infections, the reverse vaccinology method was first used to screen antigens. After multiple experiments, the preferred Tuf antigen, DnaK antigen, and fusA antigen were selected. It was shown that the three antigens have multiple cellular immune epitopes and can be used for the development of cellular immune vaccines.

[0037] 2. Based on the predicted immunoepitope information, and combined with protein structure analysis and hydrophobicity analysis, Tuf, DnaK, and fusA antigen fragments containing stable domains and conserved sequences were selected for constructing fusion proteins. Further, the most conserved sequences from Enterobacterial pathogens were selected as template sequences for final vaccine design. Combining full-length sequence alignment and sequence alignment of the selected fragments, the Tuf antigen sequence of Salmonella enteritidis, the DnaK antigen sequence of Salmonella enteritidis, and the fusA antigen sequence of Klebsiella pneumoniae were ultimately selected as template sequences for vaccine design.

[0038] 3. This invention constructed three nucleic acid vaccines expressing DnaK-Tuf-fusA, Tuf-DnaK-fusA, and Tuf-fusA-DnaK with different fusion sequences. The purity of the recombinant nucleic acids was greater than 80%, meeting the quality requirements for cell transfection experiments and vaccine production. All three combinations exhibited high abundance of expression in eukaryotic cells, with the Tuf-DnaK-fusA combination showing the highest expression level. The "Tuf-DnaK-fusA" vaccine, with the highest expression level, was selected to verify whether the antigen possesses broad-spectrum immunoprotective effects, and further used for subsequent animal experiments.

[0039] 4. Example 7 shows the preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Escherichia coli infection by gavage. The results show that the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by pathogenic Escherichia coli infection and play a good preventive role.

[0040] 5. Example 8 shows the preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Salmonella enteritis infection. The results show that the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by Salmonella infection and play a good preventive role.

[0041] 6. Example 9 shows the preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Klebsiella pneumoniae infection. The results show that the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by Klebsiella pneumoniae infection and play a good preventive role.

[0042] 7. Example 10 shows the preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Shigella infection. The results show that the recombinant nucleic acid vaccine based on the present invention can provide effective immune protection in a mouse model of Shigella infection. The vaccinated mice can generate protective immunity and prevent Shigella infection.

[0043] 8. Example 11 shows the therapeutic effect of the recombinant nucleic acid vaccine of the present invention in a rat model of Escherichia coli urinary tract infection. The results show that the white blood cell level of the rats in the vaccine immunization group remained the same as that in the negative control group throughout the entire treatment period, proving that the vaccine based on the present invention can significantly inhibit urinary tract inflammation caused by Escherichia coli infection, and that the vaccine based on the present invention can treat urinary tract infection caused by Escherichia coli, achieving the effect of clearing bacterial colonization.

[0044] 9. The antigen combination, fusion protein, and immunogenic composition provided by this invention can induce effective and broad-spectrum protective immunity in mouse model animals, and have good preventive effects against representative pathogenic bacteria in the Enterobacteriaceae family: Escherichia coli, Salmonella, Klebsiella pneumoniae, and Shigella dysenteriae.

[0045] 10. In the field of therapeutic applications, the antigen combination, fusion protein, and immunogenic composition of this invention also show excellent performance. Experiments using a rat urinary tract infection model have confirmed that this technical solution can effectively eliminate colonizing bacteria in the host, demonstrating ideal therapeutic effects. It provides a novel approach and theoretical basis for the innovative development of therapeutic bacterial vaccines, possessing extremely high academic value and application potential.

[0046] 11. From the perspective of industrial development, the results of this invention can be directly applied to the production and research and development of immunotherapeutic drugs, successfully filling the current technological gap in the field of Enterobacterial broad-spectrum vaccine research and development. With its outstanding technical advantages and significant application effects, this invention not only possesses extremely high commercial development value but also demonstrates extremely broad application prospects in the biomedical field, and is expected to bring revolutionary breakthroughs to the prevention and control of bacterial infectious diseases. Attached Figure Description

[0047] Figure 1A-1C This invention involves a sequence homology comparison of the Enterobacterial pathogenic bacterial antigens Tuf, DnaK, and fusA, wherein... Figure 1A Homology comparison of Tuf sequences of pathogenic Enterobacteriaceae bacteria antigens. Figure 1BHomology comparison of DnaK sequences of pathogenic Enterobacteriaceae bacteria antigens. Figure 1C This is a sequence homology comparison of fusA antigens from pathogenic Enterobacteriaceae.

[0048] Figure 2A-2C This invention presents the three-dimensional conformations of the full-length Tuf, DnaK, and fusA antigens and their selected fragments, wherein... Figure 2A This is the three-dimensional conformation of the full-length Tuf antigen and the selected fragment screened in this invention. Figure 2B This is a three-dimensional conformation of the full-length DnaK antigen and the selected fragment screened in this invention. Figure 2C This is a three-dimensional conformation of the full-length fusA antigen and the selected fragment screened in this invention.

[0049] Figures 3A-3C This invention relates to a homology comparison of selected antigen fragments Tuf, DnaK, and fusA from pathogenic Enterobacteriaceae bacteria, wherein... Figure 3A Homology comparison of Tuf, a selected fragment of pathogenic Enterobacteriaceae antigen. Figure 3B Homology comparison of the selected fragment DnaK of pathogenic Enterobacteriaceae antigens. Figure 3C Homology comparison of fusA, a selected fragment of pathogenic Enterobacteriaceae antigen.

[0050] Figure 4 This is a three-dimensional conformation of the fusion protein molecule containing Tuf, DnaK, and fusA antigens selected by the present invention.

[0051] Figure 5 This is a non-limiting molecular structure diagram of the fusion protein expressed by the vaccine containing Tuf, DnaK, and fusA selected antigens of the present invention.

[0052] Figure 6 This is a schematic diagram of a template plasmid containing the present invention.

[0053] Figures 7A-7C This is a peak diagram of the quality control of the vaccine of the present invention, wherein... Figure 7A The quality control peak diagram for a nucleic acid vaccine expressing DnaK-Tuf-fusA. Figure 7B The quality control peak diagram for the nucleic acid vaccine expressing Tuf-DnaK-fusA is shown. Figure 7C Quality control peak diagram for nucleic acid vaccines expressing Tuf-fusA-DnaK.

[0054] Figure 8 This is the expression effect of the vaccine of the present invention after in vitro transfection into HEK293T cells.

[0055] Figure 9This document outlines the immunization and sampling procedures for the vaccine of this invention in mouse models of Escherichia coli (Escherichia coli) infection via gavage, mouse Salmonella enteritis infection, mouse Klebsiella pneumoniae infection, and mouse Shigella infection.

[0056] Figures 10A-10C To demonstrate the protective effect of the vaccine of this invention in a mouse Escherichia coli (Escherichia coli) gavage infection model after immunization, wherein... Figure 10A To compare the bacterial load (number of colonies on agar plates) in the ileum tissue of mice after immunization and challenge. Figure 10B To compare the bacterial load (number of colonies on agar plates) in colon tissue of mice after immunization and challenge. Figure 10C The results of microscopic observation of the histopathological changes in the colon, spleen, and kidney tissues of mice after immunization with the virus.

[0057] Figures 11A-11D To demonstrate the protective effect of the vaccine of this invention in a mouse model of Salmonella enteritis infection after immunization, wherein... Figure 11A To compare the bacterial load (number of bacterial colonies on agar plates) in the feces of mice after immunization and challenge. Figure 11B To compare the bacterial load (number of colonies on agar plates) in liver tissue of mice after immunization and challenge. Figure 11C To compare the bacterial load (number of colonies on agar plates) in spleen tissue of mice after immunization and challenge. Figure 11D The results of microscopic observation of the pathological changes in the jejunum, liver, and spleen tissues of mice after immunization with the virus.

[0058] Figures 12A-12B To demonstrate the protective effect of the vaccine of this invention in a mouse model of Klebsiella pneumoniae infection after immunization, wherein... Figure 12A To compare the bacterial load (number of colonies on agar plates) in lung tissue of mice after immunization and challenge. Figure 12B The results of microscopic observation of lung tissue pathological changes in mice after immunization with the virus.

[0059] Figures 13A-13C To demonstrate the protective effect of the vaccine of this invention in a mouse model of Shigella infection after immunization, wherein... Figure 13A To compare the bacterial load (number of colonies on agar plates) in the ileum tissue of mice after immunization and challenge. Figure 13B To compare the bacterial load (number of colonies on agar plates) in colon tissue of mice after immunization and challenge. Figure 13C The results show the pathological changes in the colon tissue of mice after immune challenge.

[0060] Figure 14 This is the immunization schedule for the vaccine of the present invention in a rat Escherichia coli (Escherichia coli) urinary tract infection model.

[0061] Figures 15A-15B To illustrate the therapeutic effect of the vaccine of this invention in a rat model of Escherichia coli (Escherichia coli) urinary tract infection, wherein... Figure 15AThe results are from the detection of white blood cell count in rat urine. Figure 15B Results of bacterial load in rat urine. Detailed Implementation

[0062] Terms and Definitions The term "Enterobacteriaceae" refers to Enterobacteriaceae Gram-negative bacilli commonly reside in the intestines of humans and animals, and are also found in water, soil, and decaying matter.

[0063] The term "pathogenic enterobacteria" refers to some types of bacteria in the Enterobacteriaceae family that can cause diseases in humans or animals, such as Salmonella (e.g., Salmonella typhi), Shigella, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Shigella, etc.

[0064] The term "Escherichia coli" refers to Escherichia coli Also known as Escherichia coli, abbreviated as E. coli, it belongs to the Enterobacteriaceae family and is widely found in nature, including the intestines of humans and animals. It can cause infection in humans and animals and is an important zoonotic pathogen.

[0065] The term "Salmonella" refers to Salmonella Salmonella is a Gram-negative bacterium that can cause infection in humans and animals. It belongs to the Enterobacteriaceae family and is an important zoonotic pathogen. The infection it induces can be called Salmonellosis.

[0066] The term "Klebsiella pneumoniae" refers to Klebsiella pneumoniae Klebsiella pneumoniae, also known as Kp or KP, belongs to the Enterobacteriaceae family and can cause infection in humans and animals. It is an important zoonotic pathogen.

[0067] The term "Tuf" refers to elongation factor Tu, abbreviated as Tuf or EFTU, which is one of the conserved proteins expressed by bacteria. Studies have shown that it can exist both intracellularly and extracellularly in bacteria. The Tuf antigen of this invention comprises the full-length Tuf antigen and a selected fragment, both of which can stimulate an immune response against the Tuf antigen. The Tuf antigen is preferably derived from Salmonella enterica. Preferably, the amino acid sequence of the full-length Tuf antigen is shown in SEQ ID NO: 1, and the amino acid sequence of the selected fragment of the Tuf antigen is shown in SEQ ID NO: 4.

[0068] The term "DnaK" refers to the chaperone protein DnaK, one of the conserved proteins expressed by bacteria. The DnaK antigen of this invention comprises both the full-length DnaK antigen and a selected fragment, both of which can stimulate an immune response against the DnaK antigen. The DnaK antigen is preferably derived from Salmonella enterica. Preferably, the amino acid sequence of the full-length DnaK antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the selected fragment of the DnaK antigen is shown in SEQ ID NO: 5.

[0069] The term "fusA" refers to elongation factor G (EFG), abbreviated as fusA or EFG, which is one of the conserved proteins expressed by bacteria. The fusA antigen of this invention comprises both the full-length fusA antigen and a selected fragment, both of which can stimulate an immune response against the fusA antigen. The fusA antigen is preferably derived from Klebsiella pneumoniae. Preferably, the amino acid sequence of the full-length fusA antigen is shown in SEQ ID NO: 3, and the amino acid sequence of the selected fragment of the fusA antigen is shown in SEQ ID NO: 6.

[0070] The term "immune response" refers to a humoral response, a cellular response, or both in an organism. Immunity should be measurable 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.

[0071] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

[0072] 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.

[0073] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," a "recombinant protein," or a "fusion protein."

[0074] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

[0075] The term "mRNA" refers to messenger RNA, which is a type of single-stranded ribonucleic acid transcribed from one strand of DNA as a template. It carries genetic information and can guide protein synthesis.

[0076] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In the genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0077] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0078] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0079] 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).

[0080] The terms “transformation,” “transfection,” and “transduction” have the meanings generally understood by those skilled in the art: the process of introducing exogenous DNA or RNA into a host.

[0081] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0082] 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.

[0083] 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.

[0084] This invention discloses a novel method for preparing a fusion protein and degenerate vaccine for preventing and treating infections caused by various pathogenic Enterobacteriaceae, as well as 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 methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0085] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0086] 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.

[0087] Example 1: Antigen Screening and Homology Analysis of the Present Invention In order to develop a vaccine that can be used for broad-spectrum prevention of pathogenic Enterobacter infections, the reverse vaccinology approach is first used for antigen screening.

[0088] Omics data of all pathogenic Enterobacteriaceae were downloaded from NCBI. Homology analysis identified several highly homologous proteins, including Tuf, DnaK, and fusA, as candidate antigens. Transcriptome analysis was performed on all candidate antigens to calculate their transcriptional levels. Functional annotation analysis was then used to eliminate virulence factors, resulting in the selection of Tuf, DnaK, and fusA antigens. IEDB epitope database alignment and immunoepitaph prediction analysis revealed that these three antigens possess multiple cellular immune epitopes, making them suitable for cellular immune vaccine development.

[0089] Klebsiella pneumoniae, Escherichia coli, Shigella, and Salmonella, belonging to the Enterobacteriaceae family, are some of the most prevalent pathogenic bacteria and are representative examples. They are respectively as follows: Figure 1A , Figure 1B , Figure 1C As shown, the Tuf antigen, DnaK antigen, and fusA antigen exhibit extremely high homology in the aforementioned bacteria, with similarities all exceeding 92%. The inventors have discovered that if the sequence similarity of antigen proteins reaches over 90%, they will show high similarity in cellular immune epitopes, with some key immune epitope regions potentially being identical. Therefore, the Tuf antigen, DnaK antigen, and fusA antigen possess the potential for developing broad-spectrum cellular immune vaccines.

[0090] Example 2: Selection of conserved antigen fragments and construction of fusion proteins according to the present invention From the perspective of developing broad-spectrum vaccines, numerous studies have confirmed that multivalent vaccines offer superior protection compared to monovalent vaccines. However, through multiple experiments, the inventors discovered that the full-length sequences of the Tuf antigen, DnaK antigen, and fusA antigen obtained in Example 1 have relatively large molecular weights. Directly constructing them into fusion proteins would result in excessively large protein molecular weights and overly complex protein structures, making them unsuitable for intracellular translation and expression. Therefore, this invention, referring to the predicted immunoepitope information obtained in Example 1 and combining protein structure analysis and hydrophobicity analysis, has selected antigen fragments containing stable domains and conserved sequences through multiple screening and optimization processes (the three-dimensional conformations of the full-length Tuf, DnaK, and fusA antigens and the selected fragments selected in this invention are shown below). Figure 2A , Figure 2B , Figure 2C (As shown), used to construct fusion proteins.

[0091] like Figure 3A , Figure 3B , Figure 3C(The homology comparisons of the selected antigen fragments Tuf, DnaK, and fusA from the Enterobacterial pathogenic bacteria antigens of this invention are shown.) The antigen fragments selected in this invention still exhibit high homology across different Enterobacteriaceae: the Tuf antigen shows greater than 97% similarity across different pathogenic Enterobacteriaceae; the DnaK antigen shows greater than 97% similarity across different pathogenic Enterobacteriaceae; and the fusA antigen shows greater than 85% similarity across different pathogenic Enterobacteriaceae. Still from the perspective of developing a broad-spectrum vaccine, this invention selects the most conserved sequence among these pathogenic bacteria as the template sequence for the final vaccine design. Combined with full-length sequence alignment (… Figure 1A-1C ), and sequence alignment of selected fragments ( Figures 3A-3C Finally, the following sequences were selected as template sequences for vaccine design: the Tuf antigen sequence of Salmonella enteritidis, the DnaK antigen sequence of Salmonella enteritidis, and the fusA antigen sequence of Klebsiella pneumoniae. Figures 3A-3C (As indicated by the arrow).

[0092] When the above-mentioned selected antigens are fused for expression, three different combination sequences exist without distinguishing between the N-terminus and C-terminus: DnaK-Tuf-fusA, Tuf-DnaK-fusA, and Tuf-fusA-DnaK. Through comparison, the inventors found that even with different combination sequences, the resulting fusion proteins containing the selected Tuf, DnaK, and fusA antigens all possess stable conformations (e.g., ...). Figure 4 As shown in the figure, theoretically, all of them can be stably expressed in eukaryotic cells. The presentation of cellular immune antigen epitopes requires intracellular degradation to generate polypeptide fragments containing the epitopes before they can enter the MHC-mediated cellular immune process. Therefore, from an immunological perspective, as long as the fusion protein molecule can be stably expressed, even if the combination sequence is different, the antigen sequences used to compose the fusion protein are the same, so there will not be significant differences in antigen epitope presentation. Figure 5 This is a non-limiting molecular structure diagram of the fusion protein expressed by the vaccine containing Tuf, DnaK, and fusA selected antigens of the present invention.

[0093] Example 3: Construction of the recombinant nucleic acid vaccine of the present invention To compare whether there are differences in expression levels among the three fusion proteins with different combinations of sequences, three corresponding recombinant nucleic acid vaccines were prepared. To prepare recombinant nucleic acid vaccines capable of producing the three fusion proteins, a gene expression cassette was first constructed for expression. 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. Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy, and the DNA sequence was directly obtained through gene synthesis (commissioned by GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector suitable for in vitro RNA transcription. A schematic diagram of the template plasmid containing this invention is shown below. Figure 6 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine was obtained.

[0094] According to the above method, a carrier for use in subsequent embodiments is prepared: (1) Nucleic acid vaccine preparation vector expressing DnaK-Tuf-fusA Step a: Synthesize a fusion gene fragment of “DnaK antigen selected fragment-Tuf antigen selected fragment-fusA antigen selected fragment”, wherein the amino acid sequence of the DnaK antigen selected fragment is shown in SEQ ID NO: 5, the amino acid sequence of the Tuf antigen selected fragment is shown in SEQ ID NO: 4, the amino acid sequence of the fusA antigen selected fragment is shown in SEQ ID NO: 6, and the antigen selected fragments are linked by a linker sequence with an amino acid sequence shown in SEQ ID NO: 7.

[0095] Step b: Construct a nucleic acid vaccine architecture carrier.

[0096] 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.

[0097] Step c: Prepare recombinant plasmids.

[0098] The gene synthesized in step a is inserted into the vector architecture in step b to obtain a nucleic acid vaccine preparation vector expressing DnaK-Tuf-fusA.

[0099] (2) Nucleic acid vaccine preparation vector expressing Tuf-DnaK-fusA Step a: Synthesize the fusion gene fragment “Tuf antigen selected fragment-DnaK antigen selected fragment-fusA antigen selected fragment”, wherein the amino acid sequence of the Tuf antigen selected fragment is shown in SEQ ID NO: 4, the amino acid sequence of the DnaK antigen selected fragment is shown in SEQ ID NO: 5, the amino acid sequence of the fusA antigen selected fragment is shown in SEQ ID NO: 6, and the antigen selected fragments are linked by a linker sequence with an amino acid sequence shown in SEQ ID NO: 7.

[0100] Step b: Construct a nucleic acid vaccine architecture carrier.

[0101] 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.

[0102] Step c: Prepare recombinant plasmids.

[0103] The gene synthesized in step a is inserted into the vector architecture in step b to obtain a nucleic acid vaccine preparation vector expressing Tuf-DnaK-fusA.

[0104] (3) Nucleic acid vaccine preparation vector expressing Tuf-fusA-DnaK Step a: Synthesize the fusion gene fragment “Tuf antigen selected fragment-fusA antigen selected fragment-DnaK antigen selected fragment”, wherein the amino acid sequence of the Tuf antigen selected fragment is shown in SEQ ID NO: 4, the amino acid sequence of the fusA antigen selected fragment is shown in SEQ ID NO: 6, the amino acid sequence of the DnaK antigen selected fragment is shown in SEQ ID NO: 5, and the antigen selected fragments are linked by a linker sequence with an amino acid sequence shown in SEQ ID NO: 7.

[0105] Step b: Construct a nucleic acid vaccine architecture carrier.

[0106] 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.

[0107] Step c: Prepare recombinant plasmids.

[0108] The gene synthesized in step a is inserted into the vector architecture in step b to obtain a nucleic acid vaccine preparation vector expressing Tuf-fusA-DnaK.

[0109] Table 1. Protein amino acid sequences involved in this invention.

[0110] Example 4: Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccines Step a: Linearize the vector plasmid used in Example 3 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0111] 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.

[0112] (2) Preparation of uncapped mRNA vaccines Step a: Linearize the vector plasmid used in Example 3 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0113] Step b: Perform an in vitro uncapped transcription reaction on the linearized plasmid and degrade the template DNA.

[0114] (3) DNA vaccine preparation Step a: Amplify the vector plasmid used in Example 3 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0115] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0116] Example 5: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention Nucleic acid vaccines expressing DnaK-Tuf-fusA, Tuf-DnaK-fusA, and Tuf-fusA-DnaK were prepared using the capped mRNA vaccine preparation method described in Example 4. The purity of the produced recombinant nucleic acids was tested, and the purity of all recombinant nucleic acids used in the experiments was greater than 80%. The quality control peak diagram of the recombinant nucleic acids based on this invention is shown below. Figures 7A-7C As shown. Specifically, it is described as follows: (1) A nucleic acid vaccine expressing DnaK-Tuf-fusA with a purity of 81.1%, the quality control peak diagram and purity test results are as follows. Figure 7A (2) The nucleic acid vaccine expressing Tuf-DnaK-fusA had a purity of 86.3%. The quality control peak diagram and purity test results are as follows: Figure 7B (3) The nucleic acid vaccine expressing Tuf-fusA-DnaK had a purity of 89.9%. The quality control peak diagram and purity test results are as follows: Figure 7C The purity levels described above all meet the quality requirements for cell transfection experiments and vaccine production.

[0117] Example 6: In vitro expression effect of the recombinant nucleic acid of the present invention The vaccine from Example 5 was 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 three fusion proteins had the same molecular weight, all of which was 68.5 kDa.

[0118] Figure 8 The results of in vitro expression WB (Western blot) detection of the vaccine transfected HEK293 cells were presented. The three fusion proteins expressed only differed in the antigen sequence, and theoretically the detected signal positions were the same, with the only difference being the expression level.

[0119] The results showed that the three fusion proteins of this invention had the same molecular weight and all met expectations. Among them, the Tuf-DnaK-fusA combination showed the highest expression level. This result confirms the correctness of the inference in Example 2, that is, all three combinations can be expressed at high abundance in eukaryotic cells, with only relative differences in expression levels.

[0120] To verify whether the antigen has a broad-spectrum immunoprotective effect, we selected the "Tuf-DnaK-fusA" vaccine with the highest expression level, as shown in the non-restrictive molecular structure diagram below. Figure 5 The fusion protein architecture shown is used for subsequent animal testing and validation.

[0121] Example 7: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse Escherichia coli (Escherichia coli) gavage infection model. To verify whether the nucleic acid vaccine based on the present invention has an immune protective effect against Escherichia coli (Escherichia coli) infection, this embodiment will conduct an immunization and challenge comparison experiment on vaccine-immunized mice (immunization group) and unimmunized mice (PBS group), while setting up unimmunized mice as negative controls.

[0122] Eleven Balb / c strain mice, aged 6-8 weeks and weighing 18-25 g, were used in the experiment. All mice were housed in individual cages with constant temperature and humidity, and were acclimatized to the environment for 3-7 days prior to the experiment. 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, 10 mice were randomly divided into two groups of five, with one mouse serving as a negative control. Each mouse was ear-tagged. Details are shown in Table 2. Dosages in this table and below refer to the amount of active ingredient.

[0123] Table 2. Grouping and Immunization Procedure for Mouse Immunization Experiment in Example 7

[0124] Each group of mice was immunized twice according to the immunization protocol in Table 2. After immunization, the mice were fed normally. On day 32, the mice were administered 100 μL (50 mg / mL) of a penicillin-streptomycin antibiotic mixture by gavage once daily for 3 days. On day 35, the mice were administered 500 μL of E. coli bacterial solution (1×10⁻⁶ mg / mL) via intragastric gavage. 7 CFU was administered, and the mice were then fed normally, with daily observation and monitoring of their clinical manifestations. On day 40, the mice were sacrificed, and samples were collected to detect bacterial load in the ileum and colon. Histopathological sections of the colon, spleen, and kidney tissues were prepared to evaluate the immunoprotective effect of the vaccine. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0125] The ileum and colon of each mouse were harvested, and equal weights were cut off, ground, diluted 1000 times, and spread and cultured in solid culture dishes to detect the bacterial load in the tissues. The results are as follows: Figure 10A and Figure 10B As shown. Figure 10A The study compared the bacterial load (number of colonies on a plate) in the ileum tissue of mice after immunization and challenge. The results showed that, compared with the PBS group, the pathogenic Escherichia coli load per mg of tissue in the immunized group was significantly reduced, by about 4.8 times. Figure 10B A comparison of bacterial load (colony count) in colon tissue of mice after immunization showed that, compared with the PBS group, the pathogenic Escherichia coli load per mg of tissue in the immunized group was significantly reduced, approximately 5-fold. This result demonstrates that the recombinant nucleic acid vaccine based on this invention can provide effective immune protection in a mouse Escherichia coli infection model, and that vaccinated mice can develop protective immunity, preventing pathogenic Escherichia coli infection.

[0126] A portion of colon, spleen, and kidney tissue was harvested from each mouse, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin dye. Pathological changes in the colon, spleen, and kidney tissues were observed under a microscope. Results are as follows: Figure 10C As shown, in the PBS group, the intestinal villi of the colon tissue of mice were broken and dissolved, and a large number of inflammatory cells infiltrated and accumulated in the mucosal layer; in the immunized group, a small number of intestinal villi were broken and dissolved, and a small number of inflammatory cells infiltrated; in the negative control group, the intestinal villi structure was intact, and no obvious lesions or inflammatory infiltration were observed. No significant differences were observed in the spleen and kidney tissues of the three groups of mice.

[0127] This result proves that: (1) the PBS group showed obvious infection symptoms, high bacterial load and tissue lesions, and the challenge model was established; (2) the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by pathogenic Escherichia coli infection and play a good preventive role.

[0128] Example 8: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Salmonella enteritis. To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect against Salmonella enteritidis infection, this embodiment will conduct an immunization and challenge comparison experiment on vaccine-immunized mice (immunization group) and unimmunized mice (PBS group), while setting up unimmunized mice as a negative control. The mice selected for the experiment and the immunization procedure are the same as in Example 7.

[0129] Each group of mice was immunized twice according to the immunization protocol in Table 2. After immunization, the mice were fed normally. On day 34, mice were administered 100 μL (200 mg / mL) of streptomycin solution via gavage to enhance the bacterial challenge effect. On day 35, mice were administered 500 μL of Salmonella Typhimurium bacterial suspension (1×10⁻⁶ mg / mL) via abdominal gavage. 7 CFU was administered, and the mice were then fed normally, with daily observation and monitoring of their clinical manifestations. On day 40, the mice were sacrificed, and samples were collected to test bacterial load in feces, liver tissue, and spleen tissue. Pathological sections of jejunum, liver, and spleen tissue were prepared to evaluate the immunoprotective effect of the vaccine. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0130] Feces, liver tissue, and spleen tissue were collected from each mouse, weighing the same amount. Each sample was ground separately, diluted 1000 times, and spread onto solid culture dishes for incubation. The bacterial load in the tissues was then measured. Results are as follows: Figure 11A , Figure 11B , Figure 11C As shown. Figure 11A The study compared the fecal bacterial load (number of colonies on plates) of mice after immunization and challenge. The results showed that, compared with the PBS group, the Salmonella enteritidis load per mg of feces in the immunized group was significantly reduced, by about 5.2 times. Figure 11B The study compared the bacterial load (number of colonies on plates) in the liver tissue of mice after immunization and challenge. The results showed that, compared with the PBS group, the Salmonella enteritidis load per mg of tissue in the liver of the immunized group mice was significantly reduced, by about 32 times. Figure 11CThe comparison of bacterial load (colony count) in spleen tissue of mice after immunization showed that, compared with the PBS group, the bacterial load of Salmonella enteritidis per mg of tissue in the immunized group was significantly reduced, approximately 7.3 times lower. This result demonstrates that the recombinant nucleic acid vaccine based on this invention can provide effective immune protection in a mouse Salmonella infection model, and that vaccinated mice can develop protective immunity to prevent Salmonella infection.

[0131] Partial jejunum, liver, and spleen tissues were harvested from each mouse, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin dye. Pathological changes in the jejunum, liver, and spleen tissues were observed under a microscope. Results are as follows: Figure 11D As shown, the jejunal tissue comparison revealed that the PBS group mice exhibited abnormal morphology and structure of the intestinal villi in the mucosal layer, tissue damage, and inflammatory cell infiltration near the central lacteal vessels; the immunized group mice showed only a small amount of inflammatory cell infiltration near the central lacteal vessels; and the negative control group mice had intact intestinal villi structure without obvious lesions or inflammatory infiltration. Liver tissue comparison showed that the PBS group mice had loosely stained, pale hepatocytes with small vacuoles, a small number of neutrophils and eosinophils, and inflammatory cell infiltration with hemorrhage in the portal area; the immunized group mice had only a small number of neutrophils and eosinophils in their liver tissue, with a small amount of inflammatory cell infiltration in the portal area; and the negative control group mice had normal liver tissue without obvious tissue lesions. Spleen tissue comparison showed that the PBS group mice had abundant erythrocyte infiltration and reduced splenic bodies; the immunized group mice had a small amount of erythrocyte infiltration in their spleen tissue; and the negative control group mice had normal spleen tissue without obvious tissue lesions.

[0132] This result proves that: (1) the PBS group showed obvious infection symptoms, high bacterial load and tissue lesions, and the challenge model was established; (2) the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by Salmonella infection and play a good preventive role.

[0133] Example 9: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Klebsiella pneumoniae infection. To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect against Klebsiella pneumoniae infection, this embodiment will conduct an immunization and challenge comparison experiment on vaccine-immunized mice (immunized group) and unimmunized mice (PBS group), while setting up unimmunized mice as a negative control. The mice selected for the experiment and the immunization procedure are the same as in Example 7.

[0134] Each group of mice was immunized twice according to the immunization protocol in Table 2. After immunization, the mice were fed normally. On day 35, mice were given 500 μL of Klebsiella pneumoniae bacterial solution (1×10⁻⁶) via intranasal instillation.8 CFU was administered, and the mice were then fed normally, with daily observation and monitoring of their clinical manifestations. On day 38, the mice were sacrificed, samples were collected, lung tissue bacterial load was measured, lung tissue pathological sections were prepared, and the immunoprotective effect of the vaccine was evaluated. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0135] Lung tissue was taken from each mouse, weighed in the same amount, ground separately, diluted 1000 times, spread and cultured in solid culture dishes, and the bacterial load in the tissue was detected. Figure 12A The comparison of bacterial load (number of colonies on agar plates) in lung tissue of mice after immunization showed that, compared with the PBS group, the Klebsiella pneumoniae load per mg of lung tissue in the immunized group was significantly reduced, approximately 30-fold. This result demonstrates that the recombinant nucleic acid vaccine based on this invention can provide effective immune protection in a mouse model of Klebsiella pneumoniae infection, and that vaccinated mice can develop protective immunity, preventing Salmonella infection.

[0136] Partial lung tissue was harvested from each mouse, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin dye. Pathological changes in the lung tissue were observed under a microscope. Results are as follows: Figure 12B As shown, in the PBS group, the alveolar septa of the lung tissue of mice were thickened, the alveolar tissue degenerated and almost disappeared, and there was a large amount of inflammatory cell infiltration. The bronchial smooth muscle was significantly thickened. In the immunized group, the lung tissue of mice showed only mild alveolar fibrosis, with a small amount of inflammatory hemorrhage and mild bronchial smooth muscle thickening. The lung tissue of mice in the negative control group was normal, with no obvious lesions or inflammatory infiltration.

[0137] This result proves that: (1) the PBS group showed obvious infection symptoms, high bacterial load and tissue lesions, and the challenge model was established; (2) the immune protection induced by the recombinant nucleic acid vaccine of the present invention in the mouse model can weaken the tissue lesions caused by Klebsiella pneumoniae infection and play a good preventive role.

[0138] Example 10: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Shigella infection. To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect against Shigella infection, this embodiment will conduct an immunization and challenge comparison experiment on vaccine-immunized mice (immunization group) and unimmunized mice (PBS group). The mice used in the experiment and the immunization procedure are the same as in Example 7.

[0139] Each group of mice was immunized twice according to the immunization protocol in Table 2. After immunization, the mice were fed normally. On day 35, the mice were administered 500 μL of Shigella bacteria solution (1×10⁻⁶) by gavage. 8CFU was administered, and the mice were then fed normally, with daily observation and monitoring of their clinical manifestations. On day 40, the mice were sacrificed, samples were collected, lung tissue bacterial load was measured, lung tissue pathological sections were prepared, and the immunoprotective effect of the vaccine was evaluated. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0140] Ileal and colon tissues were taken from each mouse, weighed in the same amount, ground separately, diluted 1000 times, spread and cultured in solid culture dishes, and the bacterial load in the tissues was detected. Figure 13A The results showed that, compared with the PBS group mice, the bacterial load of Shigella bacteria per mg of lung tissue in the immunized group mice was significantly reduced, by about 5 times. Figure 13B The study compared the bacterial load (number of colonies on a plate) in the colon tissue of mice after immunization and challenge. The results showed that, compared with the PBS group, the bacterial load of Shigella bacteria per mg of lung tissue in the immunized group was significantly reduced, by about 4.5 times.

[0141] A portion of colonic tissue was harvested from each mouse, fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin dye. Pathological changes in the colonic tissue were observed under a microscope. Results are as follows: Figure 13C As shown in the comparison of colon tissue sections, the PBS group mice showed a large number of inflammatory cells infiltrating the mucosal layer of the colon tissue, with damage to the upper mucosa and poor continuity; the immunized group mice showed a significant reduction in inflammatory cell infiltration at the same location in the colon, and the upper mucosal layer had better continuity.

[0142] This result proves that: (1) the PBS group showed obvious infection symptoms, high bacterial load and tissue lesions, and the challenge model was established; (2) the recombinant nucleic acid vaccine based on the present invention can provide effective immune protection in the mouse Shigella infection model, and the vaccinated mice can generate protective immunity and prevent Shigella infection.

[0143] Example 11: Therapeutic effect of the recombinant nucleic acid vaccine of the present invention in a rat model of Escherichia coli (Escherichia coli) urinary tract infection. To verify whether the nucleic acid vaccine based on the present invention has an immunotherapeutic effect against Escherichia coli (Escherichia coli) urinary tract infection, this embodiment will conduct a comparative experiment in a rat urinary tract infection model.

[0144] Female Sprague-Dawley rats, 6–8 weeks old and weighing 180–220g, were used in the experiment. After 3 days of acclimatization feeding, they were randomly divided into 3 groups. The specific grouping information is shown in Table 3.

[0145] Table 3. Grouping of rats in Example 11 Immunological Experiment

[0146] Rats in the vaccine treatment group and the blank control group were subjected to urinary tract infection on day 0 of the experiment, and urinary tract re-infection on day 7 (immunization schedule as follows). Figure 14 (As shown), the bacterial strain was a clinically isolated urinary tract pathogenic Escherichia coli (CFT073). The mold-making procedure included: (1) Rats were deprived of water overnight.

[0147] (2) Intraperitoneal anesthesia of rats.

[0148] (3) Gently press on the abdomen to empty the bladder.

[0149] (4) Disinfect the external urethral opening and surrounding area with 75% medical alcohol.

[0150] (5) Insert the indwelling needle tubing into the bladder and inject 100 μl of bacterial suspension. The bacterial count for modeling is 10. 9 CFU.

[0151] (6) After the inoculation is completed, remove the indwelling needle, put the rat back in the cage in a supine position, keep it warm, and resume drinking water on the second day after the model is established.

[0152] Vaccine immunization was administered on days 0, 5, 10, and 15 of the experiment, according to the specific schedule as follows: Figure 14 As shown in Table 3. Urine samples were collected from rats on days 5, 10, and 15 before vaccine treatment to detect urinary bacterial load and white blood cell count, and to evaluate the effectiveness of the vaccine treatment.

[0153] The results of rat urine white blood cell count detection are as follows Figure 15A As shown, on day 5 of the experiment, the white blood cell level in the PBS group was significantly higher than that in the negative control group, proving that the model can evaluate the vaccine efficacy using white blood cell count. The results showed that throughout the treatment period, the white blood cell level in the vaccine-immunized group remained the same as that in the negative control group, demonstrating that the vaccine based on this invention can significantly inhibit urinary tract inflammation caused by Escherichia coli infection.

[0154] Results of bacterial load in rat urine as follows Figure 15B As shown, on day 5 of the experiment, the urinary bacterial load in the PBS group was higher than that in the negative control group, proving that the vaccine efficacy can be evaluated using urinary bacterial load in this model. The results showed that throughout the treatment period, the urinary bacterial load in the vaccine-immunized group remained the same as that in the negative control group, with a bacterial detection rate of approximately 0 on days 10 and 15. This demonstrates that the vaccine based on this invention can treat urinary tract infections caused by Escherichia coli and achieve the effect of clearing bacterial colonization.

[0155] In summary, the antigen combination, fusion protein, and immunogenic composition provided by this invention can induce effective and broad-spectrum protective immunity in mouse model animals, demonstrating good preventive effects against representative pathogenic bacteria of the Enterobacteriaceae family: Escherichia coli, Salmonella, and Klebsiella pneumoniae. In-depth sequence homology comparison analysis revealed that although the homology of the Tuf, DnaK, and fusA proteins of the three pathogenic Enterobacteriaceae was at least 85%, this invention, by scientifically extracting and fusing the Tuf and DnaK fragments from Salmonella and the fusA antigen fragment from Klebsiella pneumoniae, successfully induced a broad-spectrum immune protective effect against Escherichia coli in mice. Based on the above, this invention has verified its preventive effect against multiple representative pathogenic Enterobacteriaceae families. Therefore, under the condition of sequence homology not less than 85%, the fusion protein composed of three key antigens screened by this invention has the potential to be developed into a vaccine for preventing other Enterobacterial infections, and is expected to provide an efficient and broad-spectrum immune protection strategy for clinical use.

[0156] In the field of therapeutic applications, the antigen combination, fusion protein, and immunogenic composition of this invention also show excellent performance. Experiments using a rat urinary tract infection model have confirmed that this technical solution can effectively eliminate colonizing bacteria in the host, demonstrating ideal therapeutic effects. It provides a novel approach and theoretical basis for the innovative development of therapeutic bacterial vaccines, possessing extremely high academic value and application potential.

[0157] From an industrial development perspective, the results of this invention can be directly applied to the production and research and development of immunotherapeutic drugs, successfully filling the current technological gap in the field of Enterobacterial broad-spectrum vaccine development. With its outstanding technical advantages and significant application effects, this invention not only possesses extremely high commercial development value but also demonstrates a very broad application prospect in the biomedical field, potentially bringing revolutionary breakthroughs to the prevention and control of bacterial infectious diseases.

[0158] 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, It contains the following antigens: Elongation factor Tu (Tuf) antigen or its antigenic truncated fragment; Chaperone protein DnaK (DnaK) antigen or a selected fragment thereof; and Elongation factor G (fusA) antigen or its antigenic truncated fragment.

2. The fusion protein according to claim 1, characterized in that, The antigen or a selected fragment thereof is derived from Enterobacteriaceae bacteria; preferably, the Enterobacteriaceae bacteria are pathogenic Enterobacteriaceae; more preferably, the pathogenic Enterobacteriaceae are derived from Salmonella. (Salmonella enterica) ) and / or Klebsiella pneumoniae ( Klebsiella pneumoniae Most preferably, the Tuf antigen or a selected fragment thereof is derived from Salmonella ( ); Salmonella enterica The DnaK antigen or its antigen-selected fragment is derived from Salmonella ( Salmonella enterica The fusA antigen or a selected fragment thereof is derived from Klebsiella pneumoniae (…). Klebsiella pneumoniae ).

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of the Tuf antigen has at least 97% identity with the sequence of SEQ ID NO: 1, preferably 98%, 99%, or 100%; the amino acid sequence of the DnaK antigen has at least 95% identity with the sequence of SEQ ID NO: 2, preferably 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of the fusA antigen has at least 92% identity with the sequence of SEQ ID NO: 3, preferably 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

4. The fusion protein according to any one of claims 1-3, characterized in that, The Tuf antigen selected fragment contains a conserved domain of the elongation factor Tu antigen, and preferably, the amino acid sequence of the Tuf antigen selected fragment is shown in SEQ ID NO: 4; the DnaK antigen selected fragment contains a conserved domain of the chaperone factor DnaK, and the amino acid sequence of the DnaK antigen selected fragment is shown in SEQ ID NO: 5; the fusA antigen selected fragment contains a conserved domain of the elongation factor G, and the amino acid sequence of the fusA antigen selected fragment is shown in SEQ ID NO:

6.

5. The fusion protein according to any one of claims 1-4, characterized in that, The selected fragments of the Tuf antigen, the DnaK antigen, and the fusA antigen show at least 97% homology in Enterobacteriaceae; preferably, the selected fragment of the Tuf antigen has at least 97% sequence identity with SEQ ID NO: 4, and more preferably, the sequence identity is 98%, 99%, or 100%; the selected fragment of the DnaK antigen has at least 97% sequence identity with SEQ ID NO: 5, and more preferably, the sequence identity is 98%, 99%, or 100%; the selected fragment of the fusA antigen has at least 85% sequence identity with SEQ ID NO: 6, and more preferably, the sequence identity is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

6. The fusion protein according to any one of claims 1-5, characterized in that, The antigens (a), (b), and (c) can be arranged in any order; preferably, the fusion protein comprises, from the N-terminus to the C-terminus, the Tuf antigen or a selected fragment thereof, the DnaK antigen or a selected fragment thereof, and the fusA antigen or a selected fragment thereof; optionally, different antigens or fragments can be linked by a linker; preferably, the amino acid sequence of the linker is shown in SEQ ID NO:

7.

7. A recombinant nucleic acid molecule, characterized in that, Encodes the fusion protein according to any one of claims 1-6.

8. A recombinant gene expression cassette, characterized in that, It includes the recombinant nucleic acid molecule of claim 7.

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

10. A recombinant host cell, characterized in that, It comprises the recombinant nucleic acid molecule of claim 7, or the recombinant gene expression cassette of claim 8, or the recombinant vector of claim 9.

11. An immunogenic composition or pharmaceutical composition, characterized in that, The composition comprises one or more of the fusion proteins of any one of claims 1-6, and / or one or more of the recombinant nucleic acid molecules of claim 7, and / or one or more of the recombinant gene expression cassettes of claim 8, and / or one or more of the recombinant vectors of claim 9, and / or one or more of the recombinant host cells of claim 10; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

12. A recombinant vaccine, characterized in that, The recombinant vaccine comprises one or more fusion proteins according to any one of claims 1-6, and / or one or more recombinant nucleic acid molecules according to claim 7, and / or one or more recombinant gene expression cassettes according to claim 8, and / or one or more recombinant vectors according to claim 9, and / or one or more recombinant host cells according to claim 10, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 11; preferably, the recombinant vaccine is a nucleic acid vaccine; more preferably, the nucleic acid vaccine is a DNA vaccine or an RNA vaccine.

13. Use of one or more fusion proteins according to any one of claims 1-6, and / or one or more recombinant nucleic acid molecules according to claim 7, and / or one or more recombinant gene expression cassettes according to claim 8, and / or one or more recombinant vectors according to claim 9, and / or one or more recombinant host cells according to claim 10, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 11, and / or one or more recombinant vaccines according to claim 12 in the preparation of medicaments for the prevention and / or treatment of diseases caused by Enterobacteriaceae bacteria; preferably, the Enterobacteriaceae are pathogenic Enterobacteriaceae.

14. The use according to claim 13, characterized in that, The Enterobacteriaceae family includes, but is not limited to, Escherichia coli, Klebsiella pneumoniae, Salmonella, Shigella, and Proteus mirabilis.

15. The use according to claim 13 or 14, characterized in that, The diseases mentioned include urinary tract infections, breast infections, abdominal infections, dysentery, enteritis, respiratory infections, pneumonia, wound infections, sepsis, and meningitis.

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