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

Abstract

The invention relates to a fusion protein, an immunogenic composition and a recombinant degenerate vaccine for preventing and treating infection of various pathogenic streptococci, as well as a molecular architecture design, application and the like. According to the invention, three immune antigens, namely an elongation factor Tu (Tuf), a molecular chaperone DnaK and an elongation factor G (fusA), are screened, and it is proved that fusion protein molecules of the three antigens can significantly inhibit tissue lesions caused by infection of different serotypes and different types of streptococci, have good immunogenicity, play roles in immune protection and effective prevention and treatment, and have good application prospects. The bacillus subtilis has the characteristics of broad-spectrum and high-efficiency prevention of streptococcus bacterial infection, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunotherapeutic drugs, and specifically relates to fusion proteins for the prevention and treatment of various pathogenic streptococcal infections and their applications. Background Technology

[0002] Streptococcus ( Streptococcus Streptococci are a common group of Gram-positive bacteria, widely distributed in nature and in the oral cavity, respiratory tract, and gastrointestinal tract of humans and animals. Some of these streptococci are pathogenic, causing various diseases in humans and animals. Common pathogenic streptococci include Streptococcus pyogenes, Streptococcus pneumoniae, and Streptococcus agalactiae.

[0003] Streptococcal infections can lead to a variety of serious illnesses. For example, pyogenic streptococci can cause acute infections such as tonsillitis, scarlet fever, erysipelas, and cellulitis. If left untreated, they can also cause serious complications such as rheumatic fever and acute glomerulonephritis, damaging vital organs such as the heart, joints, and kidneys. Streptococcus pneumoniae is a major pathogen causing pneumonia, otitis media, and meningitis, posing a serious threat to the health of children, the elderly, and those with weakened immune systems. In developing countries, Streptococcus pneumoniae infection is one of the leading causes of death in children. Streptococcus agalactiae primarily causes neonatal infections such as sepsis, meningitis, and pneumonia, with high mortality and disability rates.

[0004] In the past, antibiotics were the primary treatment for streptococcal infections. However, with the widespread use of antibiotics, the problem of streptococcal resistance has become increasingly serious. Many pathogenic streptococci have developed resistance to a variety of commonly used antibiotics, making treatment more difficult. For example, the resistance rate of Streptococcus pneumoniae to antibiotics such as penicillin and erythromycin is constantly rising, which not only increases treatment costs and patient suffering but may also lead to treatment failure and even endanger patients' lives. In addition, the aging population and the increase in immunocompromised individuals have also led to a rising trend in the incidence and mortality of streptococcal infections, making it one of the important threats to public health security.

[0005] Safe and effective vaccines are considered the best measure for preventing and controlling streptococcal infection. Currently, vaccines against streptococcus mainly include pneumococcal polysaccharide vaccines and conjugate vaccines. Pneumococcal polysaccharide vaccines were among the earliest developed vaccines; they can stimulate the body to produce antibodies, playing a role in preventing pneumococcal infection. However, polysaccharide vaccines have some limitations; they only induce humoral immunity, and their effectiveness is poor in children under 2 years old because their immune systems are not yet fully developed and their immune response to polysaccharide antigens is weaker.

[0006] To overcome the shortcomings of polysaccharide vaccines, scientists have developed pneumococcal conjugate vaccines. Conjugate vaccines combine pneumococcal polysaccharides with carrier proteins, inducing T-cell-dependent immune responses and improving immunization efficacy in children. For example, patent document (CN115120712A, publication date 2022-09-30) discloses a pneumococcal glycoprotein biological conjugate vaccine, its preparation method, and its application. This involves transferring pneumococcal K5 serotype CPS onto a carrier protein using the PglL system, resulting in a glycoprotein with pneumococcal vaccine function. However, conjugate vaccines also have some limitations. For instance, they only cover a limited number of serotypes and cannot provide truly broad-spectrum protection. Currently, commercially available pneumococcal conjugate vaccines mainly target common serotypes, offering limited protection against other serotypes. Furthermore, the high production cost of conjugate vaccines restricts their widespread use in developing countries. The promotion of serotype-related vaccines may also lead to a gradual increase in the proportion of pathogens from non-vaccine serotypes, raising the issue of serotype substitution.

[0007] With the development of genomics, proteomics, and immunology, reverse vaccinology has become an important means of screening vaccine antigens. Through reverse vaccinology, non-glycoproteins and non-virulence factors with good conservation and immunogenicity can be screened from a vast pool of bacterial proteins for the development of novel vaccines. Vaccines designed based on conserved antigen proteins have several advantages: (1) They are not limited by serotype: they are expected to provide broader protection and reduce the risk of serotype substitution. (2) The production process is relatively simple: the cost is low, which is conducive to the global promotion and use of vaccines. (3) Multivalent vaccines can be developed: they can be combined with antigens from other pathogens to develop multivalent vaccines that can simultaneously prevent multiple diseases.

[0008] In conclusion, the development of a broad-spectrum vaccine against pathogenic streptococci is urgently needed. Novel vaccines not only promise to provide broader protection and reduce the morbidity and mortality of streptococcal infections, but may also lower production costs, making them easier to distribute globally. These studies are crucial not only for controlling streptococcal infections but also provide new ideas and methods for the development of other broad-spectrum bacterial vaccines. Although several streptococcal vaccines have been disclosed in existing technologies, there is still a pressing need in the field for a fusion protein, immune composition, and vaccine that can attenuate tissue lesions caused by pathogenic streptococcal infections, possesses good immunogenicity, provides effective prevention and immune protection, and provides highly efficient and broad-spectrum prevention of pathogenic streptococcal infections. Summary of the Invention

[0009] 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 streptococcal infections. This invention screened three immunogenic antigens: extension factor Tu (Tuf), molecular chaperone DnaK, and extension factor G (fusA), and demonstrated that the fusion protein molecule of these three antigens can significantly inhibit tissue lesions caused by different serotypes and species of streptococci, exhibiting good immunogenicity and providing immune protection and effective prevention and treatment. It possesses broad-spectrum and highly effective characteristics for preventing streptococcal infections and has broad application prospects.

[0010] One aspect of the present invention provides a fusion protein for the prevention and treatment of pathogenic bacterial infections, characterized in that it comprises the following antigens: (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 truncated fragment.

[0011] Further, the antigen or a selected fragment thereof is derived from Streptococcus bacteria; preferably, the Streptococcus bacteria are pathogenic Streptococcus bacteria; more preferably, the pathogenic Streptococcus bacteria are derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae ) and / or agalactiae (Streptococcus agalactiae) Streptococcus agalactiae Most preferably, the Tuf antigen or a selected fragment thereof is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The DnaK antigen or its antigen-selected fragment is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The fusA antigen or a selected fragment thereof is derived from Streptococcus agalactiae (Streptococcus agalactiae). Streptococcus agalactiae ).

[0012] Further, the amino acid sequence of antigen Tuf has at least 94% identity with the sequence of SEQ ID NO: 1, preferably 95%, 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of antigen DnaK has at least 93% identity with the sequence of SEQ ID NO: 2, preferably 94%, 95%, 96%, 97%, 98%, 99%, or 100%; and the amino acid sequence of antigen fusA has at least 95% identity with the sequence of SEQ ID NO: 3, preferably 96%, 97%, 98%, 99%, or 100%.

[0013] 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.

[0014] Further, the homology of the Tuf antigen selected fragment, the DnaK antigen selected fragment, and the fusA antigen selected fragment in Streptococcus is not less than 93%; preferably, the Tuf antigen selected fragment has at least 96% sequence identity with SEQ ID NO: 4, and more preferably, the sequence identity is 97%, 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 93% sequence identity with SEQ ID NO: 6, and more preferably, the sequence identity is 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0015] Further, the antigens (a), (b), and (c) can be arranged and combined in any order; preferably, the fusion protein comprises, 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.

[0016] 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.

[0017] 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.

[0018] Furthermore, the recombinant gene expression cassette also includes one or more of a promoter, a terminator, and a regulatory sequence.

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

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

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

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

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

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

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

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

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

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

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

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

[0031] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins 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.

[0032] 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.

[0033] 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 Streptococcus bacteria; preferably, the Streptococcus is a pathogenic Streptococcus.

[0034] Another aspect of the present invention provides a method for preventing and / or treating diseases caused by Streptococcus bacteria, characterized in that it includes administering to a subject one or more fusion proteins according to any one of the present inventions, and / or one or more recombinant nucleic acid molecules according to the present inventions, and / or one or more recombinant gene expression cassettes according to the present inventions, and / or one or more recombinant vectors according to the present inventions, and / or one or more recombinant host cells according to the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present inventions, and / or one or more recombinant vaccines according to the present inventions; preferably, the Streptococcus is a pathogenic Streptococcus.

[0035] Furthermore, the bacteria of the genus Streptococcus include, but are not limited to, Streptococcus pneumoniae, group A Streptococcus, group B Streptococcus, and Streptococcus suis; preferably, the group A Streptococcus includes Streptococcus pyogenes, the group B Streptococcus includes Streptococcus agalactiae, and the Streptococcus pneumoniae includes Streptococcus pneumoniae type 2 and / or Streptococcus pneumoniae type 5.

[0036] Furthermore, the diseases include purulent inflammation, urinary tract infection, skin infection, respiratory tract infection, abdominal infection, vaginal infection, wound infection, pneumonia, meningitis, otitis media, and sepsis.

[0037] The fusion protein molecular structure, the nucleic acid structure encoding it, the pharmaceutical composition, and the recombinant vaccine of the present invention have the following beneficial technical effects: 1. In order to develop a vaccine that can be used for broad-spectrum prevention of pathogenic streptococcal infection, 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.

[0038] 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. Furthermore, the most conserved sequence from streptococcal pathogens was selected as the template sequence for final vaccine design. Combining full-length sequence alignment and sequence alignment of the selected fragments, the selected Tuf, DnaK, and fusA antigen sequences were ultimately selected as template sequences for vaccine design.

[0039] 3. Nucleic acid vaccines containing three antigens have high expression levels of fusion proteins, which can be used to verify whether the antigens have broad-spectrum immunoprotective effects and further validate them in subsequent animal experiments.

[0040] 4. Example 7 shows the preventive effect of the recombinant nucleic acid vaccine based on the present invention in a mouse model of nasal infection with Streptococcus pneumoniae type 2. The results show that the recombinant nucleic acid vaccine based on the present invention can provide effective immune protection in a mouse model of nasal infection with Streptococcus pneumoniae type 2. The vaccinated mice can generate protective immunity and prevent Streptococcus pneumoniae type 2 infection.

[0041] 5. Example 8 illustrates the preventive effect of the recombinant nucleic acid vaccine based on the present invention in a mouse model of nasal infection with Streptococcus pneumoniae serotype 5. The results show that the recombinant nucleic acid vaccine based on the present invention can provide effective immune protection in a mouse model of nasal infection with Streptococcus pneumoniae serotype 5. Vaccinated mice can develop protective immunity and prevent infection with Streptococcus pneumoniae serotype 5. Furthermore, combined with the experimental results of Example 7, the recombinant nucleic acid vaccine based on the present invention can provide cross-immune protection and prevent infection with different serotypes of Streptococcus pneumoniae.

[0042] 6. 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 in the genus Streptococcus: Streptococcus pneumoniae type 2, Streptococcus pneumoniae type 5, and Streptococcus suis. Although the vaccine sequence is derived from Streptococcus pneumoniae type 19F and Streptococcus agalactiae, it still exhibits good immunoprotective effects against Streptococcus suis, which has a homology range of 93%-99%. Furthermore, based on the results of challenge experiments with two different serotypes of Streptococcus pneumoniae, it is demonstrated that the antigen combination provided by this invention is not limited by serotype differences in bacteria, possessing the potential to develop a broad-spectrum vaccine and promising to provide an efficient and broad-spectrum immune protection strategy for clinical use.

[0043] 7. 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 technological gap in the current field of broad-spectrum streptococcal 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, potentially bringing revolutionary breakthroughs to the prevention and control of bacterial infectious diseases. Attached Figure Description

[0044] Figures 1A-1C This invention relates to a sequence homology comparison of the pathogenic streptococcal antigens Tuf, DnaK, and fusA. Figure 1A Homology comparison of Tuf sequences of pathogenic streptococcal antigens. Figure 1B This is a homology comparison of the DnaK sequence of pathogenic streptococcal antigens. Figure 1C This is a sequence homology comparison of the fusA antigen of pathogenic streptococci.

[0045] Figures 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.

[0046] Figures 3A-3C This invention relates to a homology comparison of selected fragments of pathogenic streptococcal antigens, wherein... Figure 3A Homology comparison of Tuf, a selected fragment of pathogenic streptococcal antigen. Figure 3B Homology comparison of the selected fragment DnaK of pathogenic streptococcal antigen. Figure 3C Homology comparison of the selected fragment fusA of pathogenic streptococcal antigen.

[0047] Figure 4 This is a three-dimensional conformation of the fusion protein molecule containing Tuf, DnaK, and fusA antigens selected by the present invention.

[0048] 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.

[0049] Figure 6 This is a schematic diagram of a template plasmid containing the present invention.

[0050] Figure 7 This is a quality control peak diagram of the nucleic acid vaccine expressing Tuf-DnaK-fusA according to the present invention.

[0051] Figure 8 This is the expression effect of the vaccine of the present invention after in vitro transfection into HEK293T cells.

[0052] Figure 9 This describes the immunization and sampling process of the vaccine of the present invention in a mouse infection model.

[0053] Figures 10A-10B To demonstrate the protective effect of the vaccine of this invention in a mouse model of nasal infection with Streptococcus pneumoniae type 2 after immunization, wherein... Figure 10A To compare the bacterial load (number of colonies on agar plates) in lung tissue of mice after immunization and challenge. Figure 10B The results show the pathological changes in lung tissue observed under a microscope after immune challenge in mice.

[0054] Figures 11A-11B To demonstrate the protective effect of the vaccine of this invention in a mouse model of nasal infection with Streptococcus pneumoniae type 5 after immunization, wherein... Figure 11A To compare the bacterial load (number of colonies on agar plates) in lung tissue of mice after immunization and challenge. Figure 11B The results show the pathological changes in lung tissue observed under a microscope after immune challenge in mice.

[0055] Figures 12A-12B To demonstrate the protective effect of the vaccine of this invention in a mouse model of intraperitoneal infection with Streptococcus suis 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 show the pathological changes in lung tissue observed under a microscope after immune challenge in mice. Detailed Implementation

[0056] Terms and Definitions The term "Streptococcus" refers to StreptococcusGram-positive bacteria are arranged in chains and are widely distributed in nature and in specific parts of humans and animals. Some of them are pathogenic and can cause a variety of diseases in humans and animals, ranging from respiratory infections to serious systemic diseases.

[0057] The term "pathogenic streptococci" refers to a group of bacteria in the genus *Streptococcus* that possess pathogenicity. These bacteria are widely distributed in nature and in humans and animals, and can cause various infectious diseases in humans and animals, such as tonsillitis, pneumonia, rheumatic fever, and neonatal sepsis. Common pathogenic streptococci include *Streptococcus pyogenes*, *Streptococcus pneumoniae*, and *Streptococcus agalactiae*.

[0058] The term "Streptococcus pneumoniae" refers to Streptococcus pneumoniae It is a Gram-positive coccus, often arranged in pairs in a spearhead shape. It is widely found in nature and the human upper respiratory tract and is an important pathogen that causes a variety of diseases such as pneumonia, otitis media, meningitis, and sepsis.

[0059] The term "Streptococcus pneumoniae type 2" refers to a serotype of Streptococcus pneumoniae that can cause pneumonia, meningitis, otitis media, and other diseases, and is more common in children, the elderly, and people with weakened immune systems.

[0060] The term "Streptococcus pneumoniae type 5" refers to a serotype of Streptococcus pneumoniae that can cause infections in the lungs and other parts of the body, and is an important pathogen that causes diseases such as pneumonia.

[0061] The term "Streptococcus suis" refers to Streptococcus suis These are bacteria that primarily infect pigs, but some strains can also infect humans. Humans infected with these bacteria may develop serious illnesses such as meningitis, septicemia, and endocarditis, making them an important zoonotic pathogen.

[0062] 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 Streptococcus pneumoniae. 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.

[0063] 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 Streptococcus pneumoniae. 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.

[0064] 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 *Streptococcus agalactiae*. 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 fusA antigen fragment is shown in SEQ ID NO: 6.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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."

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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).

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] This invention discloses a novel method for preparing a fusion protein and degenerate vaccine for preventing and treating various pathogenic streptococcal infections, 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.

[0080] 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.

[0081] 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.

[0082] 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 streptococcal infection, the reverse vaccinology approach was first used for antigen screening.

[0083] Omics data of all pathogenic streptococci 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 vaccine development.

[0084] Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus agalactiae, and Streptococcus suis are some of the most prevalent zoonotic pathogens 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 93%. The inventors have discovered that if the sequence similarity of antigen proteins reaches over 90%, they will share 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.

[0085] 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, selected antigen fragments containing stable domains and conserved sequences (the three-dimensional conformations of the full-length Tuf, DnaK, and fusA antigens selected in this invention and the selected fragments are shown below). Figure 2A , Figure 2B , Figure 2C (As shown), used to construct fusion proteins.

[0086] like Figure 3A , Figure 3B , Figure 3C(The homology comparisons of the selected antigen fragments Tuf, DnaK, and fusA from the Streptococcus pathogenic bacteria of this invention are shown respectively.) The antigen fragments selected in this invention still exhibit high homology across different streptococci: the Tuf antigen shows greater than 96% similarity among the three streptococci; the DnaK antigen shows greater than 97% similarity; and the fusA antigen shows greater than 93% similarity. 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 (… Figures 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 Streptococcus pneumoniae, the DnaK antigen sequence of Streptococcus pneumoniae, and the fusA antigen sequence of Streptococcus agalactiae. Figures 3A-3C (As indicated by the arrow).

[0087] When the above-mentioned selected antigens are fused and expressed, the fusion protein formed by DnaK containing Tuf, DnaK, and fusA selected antigens has a stable conformation. Figure 4 (A schematic diagram of the non-restricted molecular structure is shown below) Figure 5 As shown.

[0088] Example 3: Construction of the recombinant nucleic acid vaccine of the present invention To verify whether the fusion protein designed in the above embodiments can be normally expressed in eukaryotic cells, a corresponding recombinant nucleic acid vaccine was prepared. First, a gene expression cassette was 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.

[0089] Based on the above method, a nucleic acid vaccine preparation vector for expressing Tuf-DnaK-fusA was prepared: 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.

[0090] Step b: Construct a nucleic acid vaccine architecture carrier.

[0091] 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.

[0092] Step c: Prepare recombinant plasmids.

[0093] 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.

[0094] Table 1. Protein amino acid sequences involved in this invention.

[0095] Example 4: Preparation of the recombinant nucleic acid vaccine of the present invention

[0096] (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.

[0097] 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.

[0098] (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.

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

[0100] (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.

[0101] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0102] Example 5: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention A nucleic acid vaccine expressing Tuf-DnaK-fusA was prepared using the method for preparing the capped mRNA vaccine described in Example 4. The purity of the produced recombinant nucleic acid was tested, such as... Figure 7 As shown, the purity of the Tuf-DnaK-fusA recombinant nucleic acid is 84%, which meets the quality requirements for cell transfection experiments and vaccine production.

[0103] 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 24 hours of in vitro culture, the protein was collected and analyzed by Western blot. The estimated molecular weight of the fusion protein was 90.7 kDa.

[0104] Figure 8 The results of in vitro expression analysis (WB) on HEK293 cells transfected with the vaccine are presented, such as... Figure 8 As shown, the molecular weight and expression level of the fusion protein are in line with expectations, proving that the fusion protein designed based on this invention can form a stable conformation and be expressed at high abundance in eukaryotic cells.

[0105] To verify whether the fusion protein has a broad-spectrum immunoprotective effect, the present invention uses the Tuf-DnaK-fusA recombinant nucleic acid vaccine for subsequent animal experiments.

[0106] Example 7: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of nasal infection with Streptococcus pneumoniae type 2. To verify whether the nucleic acid vaccine based on the present invention has an immune protective effect against Streptococcus pneumoniae infection type 2, 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.

[0107] 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.

[0108] Table 2. Grouping and Immunization Procedure for Mouse Immunization Experiment in Example 7

[0109] 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 40 μL of Streptococcus pneumoniae (type 2) (5 × 10⁻⁶) intranasally. 6 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, and lung tissue bacterial load was measured. Lung tissue pathological sections were prepared to evaluate the immunoprotective effect of the vaccine. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0110] Lungs were harvested from each mouse, and equal weights were cut, ground, diluted 1000 times, and spread and cultured in solid culture dishes to detect tissue bacterial load. Results are as follows: Figure 10A and Figure 10B As shown. Figure 10A The study compared the bacterial load (number of colonies on agar plates) in the lung tissue of mice after immunization and challenge. The results showed that, compared with the PBS group, the bacterial load of Streptococcus type 2 pneumoniae per mg of tissue in the lungs of the immunized mice was significantly reduced, with an average decrease of about 22 times.

[0111] 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 10B As shown, the alveolar septa of mice in the PBS group were significantly thickened, the alveolar morphology and structure were basically destroyed, and there was inflammatory cell infiltration; the alveolar septa of mice in the vaccine immunization group were partially thickened, the degree of pulmonary fibrosis was lower, and there were mild inflammatory symptoms.

[0112] 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 model of Streptococcus pneumoniae nasal drop infection, and the vaccinated mice can generate protective immunity and prevent Streptococcus pneumoniae nasal infection.

[0113] Example 8: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of nasal infection with Streptococcus pneumoniae type 5. To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect against Streptococcus pneumoniae type 5 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 negative controls. The mice selected for the experiment and the immunization procedure are the same as in Example 7.

[0114] 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 40 μL of Streptococcus pneumoniae (type 5) (5 × 10⁻⁶) intranasally. 6 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, and lung tissue bacterial load was measured. Lung tissue pathological sections were prepared to evaluate the immunoprotective effect of the vaccine. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown.

[0115] Lungs were harvested from each mouse, and equal weights were cut, ground, diluted 1000 times, and spread and cultured in solid culture dishes to detect tissue bacterial load. Results are as follows: Figure 11A and Figure 11B As shown. Figure 11A The study compared the bacterial load (number of colonies on agar plates) in lung tissue of mice after immunization and challenge. The results showed that, compared with the PBS group, the bacterial load of Streptococcus pneumoniae type 5 in the lung tissue of the immunized group mice was significantly reduced per mg of tissue, with an average decrease of about 4.3 times.

[0116] 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 11B As shown, the alveolar septa of mice in the PBS group were significantly thickened, the alveolar morphology and structure were basically destroyed, and there was inflammatory cell infiltration; the alveolar septa of mice in the vaccine immunization group were partially thickened, the degree of pulmonary fibrosis was lower, and there were mild inflammatory symptoms.

[0117] 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 serotype 5 pneumococcal intranasal infection model, and the vaccinated mice can generate protective immunity to prevent serotype 5 pneumococcal infection; (3) Combined with the experimental results of Example 7, the recombinant nucleic acid vaccine based on the present invention can provide cross-immune protection to prevent infection of different serotypes of pneumococcus.

[0118] Example 9: Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse model of Streptococcus suis infection. To verify whether the nucleic acid vaccine based on the present invention has an immunoprotective effect against Streptococcus suis 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.

[0119] 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 intraperitoneally injected with 500 μL of Streptococcus suis bacterial solution (2 × 10⁻⁶). 7 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, and lung tissue bacterial load was measured. Lung tissue pathological sections were prepared to evaluate the immunoprotective effect of the vaccine. The immunization, challenge, and sampling procedures are as follows: Figure 9 As shown; Lung tissue was taken from each mouse, and the same weight was taken. The tissues were ground and diluted 1000 times, spread and cultured in solid culture dishes, and the bacterial load 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 bacterial load of Streptococcus suis in the immunized group was significantly reduced per mg of lung tissue, with an average decrease of approximately 14-fold. This result demonstrates that the recombinant nucleic acid vaccine based on this invention can provide effective immune protection in a mouse model of Streptococcus suis infection, and that vaccinated mice can develop protective immunity, preventing Streptococcus suis infection.

[0120] 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 12BAs shown, the alveolar septa of the lung tissue of mice in the PBS group showed varying degrees of thickening, and the alveolar morphology and structure were basically destroyed, accompanied by inflammatory cell infiltration; the alveolar tissue of mice in the immunized group had the best integrity, with only a small amount of thickening of the alveolar septa; the lung tissue of mice in the negative control group showed normal characteristics, with no obvious lesions or inflammatory infiltration.

[0121] 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 Streptococcus suis infection and play a good preventive role; (3) combined with the experimental results of Examples 7 and 8, the recombinant nucleic acid vaccine of the present invention can provide cross-immune protection and prevent different types of streptococcal infection.

[0122] 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 in the genus Streptococcus: Streptococcus pneumoniae type 2, Streptococcus pneumoniae type 5, and Streptococcus suis. Although the vaccine sequence is derived from Streptococcus pneumoniae type 19F and Streptococcus agalactiae, it still exhibits good immunoprotective effects against Streptococcus suis, which has a homology range of 93%-99%. Furthermore, the results of challenge experiments with two different serotypes of Streptococcus pneumoniae demonstrate that the antigen combination provided by this invention is not limited by serotype differences in bacteria, possessing the potential to develop a broad-spectrum vaccine and promising to provide an efficient and broad-spectrum immune protection strategy for clinical use.

[0123] 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 broad-spectrum streptococcal 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.

[0124] 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 for the prevention and treatment of pathogenic bacterial infections, 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 Streptococcus bacteria; preferably, the Streptococcus bacteria are pathogenic Streptococcus bacteria; more preferably, the pathogenic Streptococcus bacteria are derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae ) and / or agalactiae (Streptococcus agalactiae) Streptococcus agalactiae Most preferably, the Tuf antigen or a selected fragment thereof is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The DnaK antigen or its antigen-selected fragment is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The fusA antigen or a selected fragment thereof is derived from Streptococcus agalactiae (Streptococcus agalactiae). Streptococcus agalactiae ).

3. The fusion protein according to claim 1 or 2, characterized in that, The amino acid sequence of antigen Tuf has at least 94% identity with the sequence of SEQ ID NO: 1, preferably 95%, 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of antigen DnaK has at least 93% identity with the sequence of SEQ ID NO: 2, preferably 94%, 95%, 96%, 97%, 98%, 99%, or 100%; the amino acid sequence of antigen fusA has at least 95% identity with the sequence of SEQ ID NO: 3, preferably 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 93% homology in Streptococcus spp.; preferably, the selected fragment of the Tuf antigen has at least 96% sequence identity with SEQ ID NO: 4, and more preferably, the sequence identity is 97%, 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 93% sequence identity with SEQ ID NO: 6, and more preferably, the sequence identity is 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 Streptococcus bacteria; preferably, the Streptococcus is a pathogenic Streptococcus.

14. The use according to claim 13, characterized in that, The bacteria of the genus Streptococcus include, but are not limited to, Streptococcus pneumoniae, group A Streptococcus, group B Streptococcus, and Streptococcus suis; preferably, the group A Streptococcus includes Streptococcus pyogenes, the group B Streptococcus includes Streptococcus agalactiae, and the Streptococcus pneumoniae includes Streptococcus pneumoniae type 2 and / or Streptococcus pneumoniae type 5.

15. The use according to claim 13 or 14, characterized in that, The diseases mentioned include purulent inflammation, urinary tract infection, skin infection, respiratory tract infection, abdominal infection, vaginal infection, wound infection, pneumonia, meningitis, otitis media, and sepsis.

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