Fusion protein for preventing pathogenic enterococcus infection and application thereof

Abstract

The invention relates to a fusion protein for preventing pathogenic enterococcus infection, an immunogenic composition, a recombinant vaccine, application of the fusion protein, the immunogenic composition and the recombinant vaccine and the like. According to the invention, HlyC / CorC transporter antigens of enterococcus faecium and enterococcus faecalis are selected to construct a fusion protein A, and elongation factor Tu antigens and 50S ribosomal protein L7 / L12 (50S ribosomal protein L7 / L12, rplL) antigens of the enterococcus faecium and the enterococcus faecalis are selected to construct a fusion protein B. According to the present invention, the fusion protein A and the fusion protein B are selected, the vaccine based on the two fusion proteins can significantly weaken tissue infection caused by enterococcus faecium and enterococcus faecalis, has good immunogenicity, plays effective prevention and immune protection roles, can efficiently prevent pathogenic enterococcus infection, and has wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, in particular the field of immunopharmaceutical technology, and specifically relates to a fusion protein, a vaccine and applications thereof for preventing pathogenic enterococcal infection. Background Art

[0002] Pathogenic enterococci ( Enterococcus spp. ) are clinically important opportunistic pathogens, mainly including Enterococcus faecalis ( Enterococcus faecalis ) and Enterococcus faecium ( Enterococcus faecium ), infecting immunocompromised individuals, such as critically ill hospital patients, postoperative patients, and those receiving long-term antibiotics. Both can cause a variety of illnesses, including urinary tract infections, intraperitoneal infections, bacteremia, and endocarditis, with hospital-acquired infections accounting for over 80%. In recent years, the global prevalence of vancomycin-resistant enterococci (VRE) has posed significant challenges to the treatment of these infections. In particular, the resistance rate of Enterococcus faecium to multiple antibiotics has exceeded 60%. According to CHINET surveillance data, the prevalence of clinical isolates of Enterococcus faecium has increased from 5%-10% to 40%-50%, with drug resistance generally higher than that of Enterococcus faecalis, further complicating treatment. Enterococcal infections often synergize with other pathogens. For example, co-infection with Escherichia coli can exacerbate the formation of intra-abdominal abscesses, and their biofilm-forming ability enhances resistance to the host immune system and antibiotics. According to the World Health Organization (WHO), enterococcal infections cause over 100,000 deaths worldwide annually, with mortality rates reaching 20%-30% in newborns and elderly patients.

[0003] Safe and effective vaccines are considered the best approach for preventing and controlling pathogenic enterococcal infections. However, there are currently no commercially available vaccines targeting pathogenic enterococci. This is primarily due to the diverse serotypes of enterococci, the high plasticity of resistance genes (e.g., the absence of CRISPR-Cas systems leading to frequent horizontal gene transfer), the high diversity of surface virulence factors, and the weak immunogenicity of traditional polysaccharide vaccines against Gram-positive bacteria. Furthermore, similar to Streptococcus pneumoniae, enterococcal capsular polysaccharides and cell wall components vary significantly between strains, making it difficult to cover all major pathogenic strains with a single antigen. Therefore, vaccine design based on conserved protein antigens has become a breakthrough. Conserved antigens are highly homologous across strains, avoiding the problem of serotype substitution and stimulating synergistic protection from both cellular and humoral immunity.

[0004] To overcome the limitations of existing vaccines, researchers are actively developing novel pathogenic enterococcal vaccines. For example, prior art patent WO2023205627A1 (published on October 26, 2023) discloses an Enterococcus faecalis vaccine and its uses, which can reduce tissue bacterial loads to a certain extent. While this prior art demonstrates the efforts of researchers in developing pathogenic enterococcal vaccines, many challenges remain. Pathogenic enterococci are divided into Enterococcus faecalis and Enterococcus faecium. While existing technologies primarily target Enterococcus faecalis, insufficient attention has been paid to Enterococcus faecium, which exhibits increasing drug resistance and infection rates. Furthermore, further research is needed into the immune mechanisms of vaccines to better understand how they induce a long-lasting and effective immune response, including the synergistic effects of humoral and cellular immunity. During vaccine development, safety, production costs, and quality control must also be considered to ensure large-scale production and widespread application.

[0005] In summary, the development of a new generation of pathogenic enterococcal vaccines is urgent and will be key to resolving the various issues with current traditional vaccines. These new vaccines not only have the potential to provide broader protection but also reduce production costs, making them easier to deploy and administer globally. These studies are not only crucial for controlling pathogenic enterococcal infections but also provide new insights and approaches for the development of other bacterial vaccines. Therefore, there remains an urgent need for a vaccine that can effectively prevent and provide immunization against pathogenic enterococcal infections, particularly those with Enterococcus faecalis and Enterococcus faecium. Summary of the Invention

[0006] In response to the deficiencies of the prior art, the present invention provides a fusion protein, encoding nucleic acid, expression vector containing the same, recombinant cells, immunogenic composition, recombinant vaccine, treatment method, and medical use, etc. for preventing pathogenic enterococcal infection. The present invention selects HlyC / CorC transporter (HlyC / CorC family transporter) antigens of Enterococcus faecium and Enterococcus faecalis to construct fusion protein A, and selects Elongation factor Tu (Tuf) antigen and 50S ribosomal protein L7 / L12 (rplL) antigens of Enterococcus faecium and Enterococcus faecalis to construct fusion protein B. Vaccines based on the two fusion proteins can significantly reduce tissue infections caused by Enterococcus faecium and Enterococcus faecalis, have good immunogenicity, play an effective preventive and immune protective role, and effectively prevent pathogenic enterococcal infections, and have broad application prospects. One aspect of the present invention provides a fusion protein, characterized in that it is selected from any one of the following (1)-(3): (1) fusion protein A, wherein the fusion protein A comprises Enterococcus faecalis ( Enterococcus faecalis ) and / or Enterococcus faecium ( Enterococcus faecium ) derived from an HlyC / CorC transporter (HlyC / CorC family transporter) antigen; preferably, the HlyC / CorC transporter antigen is truncated from the C-terminal domain of the HlyC / CorC transporter; more preferably, the HlyC / CorC transporter antigen comprises a mutation targeting a glycosylation site; most preferably, the mutation in the glycosylation site comprises N68Q and / or T56A; (2) fusion protein B, wherein the fusion protein B comprises Enterococcus faecalis ( Enterococcus faecalis ) and / or Enterococcus faecium ( Enterococcus faecium ) derived from elongation factor Tu (Tuf) antigen and 50S ribosomal protein L7 / L12 (rplL) antigen; (3) A combination of the fusion protein A and the fusion protein B.

[0007] Furthermore, the fusion protein A comprises, from N-terminus to C-terminus, an HlyC / CorC transporter antigen derived from Enterococcus faecalis and an HlyC / CorC transporter antigen derived from Enterococcus faecium; the fusion protein B comprises, from N-terminus to C-terminus, an elongation factor Tu antigen derived from Enterococcus faecalis, a 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecalis, an elongation factor Tu antigen derived from Enterococcus faecium, and a 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecium.

[0008] Furthermore, the amino acid sequence of the HlyC / CorC transporter antigen derived from Enterococcus faecalis is shown as SEQ ID NO: 1, the amino acid sequence of the HlyC / CorC transporter antigen derived from Enterococcus faecium is shown as SEQ ID NO: 2, the amino acid sequence of the elongation factor Tu antigen derived from Enterococcus faecalis is shown as SEQ ID NO: 3, the amino acid sequence of the 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecalis is shown as SEQ ID NO: 4, the amino acid sequence of the elongation factor Tu antigen derived from Enterococcus faecium is shown as SEQ ID NO: 5, and the amino acid sequence of the 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecium is shown as SEQ ID NO: 6.

[0009] Furthermore, the antigens can be optionally connected through a linker sequence or a spacer sequence; preferably, the linker sequence is a GGS linker sequence; more preferably, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 11, and the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 12.

[0010] Furthermore, the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 7, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO: 8.

[0011] Furthermore, the N-terminus of the fusion protein A further comprises a signal peptide and / or any secretion-promoting element; preferably, the signal peptide is derived from human azurocidin protein, and the secretion-promoting element is derived from the Fc domain of immunoglobulin heavy chain constant region γ1 protein (Immunoglobulin Heavy Constant Gamma 1, IGHG1); more preferably, the amino acid sequence of the signal peptide derived from human azurocidin protein is as shown in SEQ ID NO: 9, and the amino acid sequence of the Fc domain derived from immunoglobulin heavy chain constant region γ1 protein is as shown in SEQ ID NO: 10.

[0012] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the fusion protein of the present invention; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

[0013] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule of the present invention; preferably, the recombinant gene expression cassette further comprises one or more regulatory sequences; more preferably, the regulatory sequences comprise a promoter, an enhancer, and a terminator.

[0014] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule of the present invention or the recombinant gene expression cassette of the present invention.

[0015] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0016] Furthermore, the prokaryotic vector includes but is not limited to an Escherichia coli vector.

[0017] Furthermore, the E. coli vector includes but is not limited to pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0018] Furthermore, the eukaryotic vector includes but is not limited to yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0019] Furthermore, the yeast expression vector includes but is not limited to pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0020] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule of the present invention, or the recombinant gene expression cassette of the present invention, or the recombinant vector of the present invention.

[0021] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0022] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0023] Furthermore, the yeast cells include but are not limited to Saccharomyces cerevisiae, Pichia pastoris, and Hansenula.

[0024] Furthermore, the prokaryotic cells include but are not limited to Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0025] Furthermore, the Escherichia coli cells include but are not limited to BL21 (DE3), DH5α, TOP10, and Rosetta.

[0026] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins described in the present invention, and / or one or more recombinant nucleic acid molecules described in the present invention, and / or one or more recombinant gene expression cassettes described in the present invention, and / or one or more recombinant vectors described in the present invention, and / or one or more recombinant host cells described in the present invention; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0027] Furthermore, the immunogenic composition or pharmaceutical composition contains the fusion protein A and the fusion protein B, or the immunogenic composition or pharmaceutical composition contains a recombinant nucleic acid molecule expressing the fusion protein A and the fusion protein B; preferably, the mass ratio of the fusion protein A and the fusion protein B in the immunogenic composition or pharmaceutical composition is 1:1.

[0028] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention; preferably, the recombinant vaccine comprises the fusion protein A and fusion protein B, or the recombinant vaccine comprises the recombinant nucleic acid molecules expressing the fusion protein A and fusion protein B; more preferably, the mass ratio of the fusion protein A and fusion protein B in the recombinant vaccine is 1:1.

[0029] Another aspect of the present invention provides the use of one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention in the preparation of vaccines or drugs for biological immunity for prevention, treatment and / or vaccination.

[0030] Furthermore, the drug is used to prevent and / or treat infections or diseases caused by enterococci; preferably, the enterococci are pathogenic and / or drug-resistant enterococci; preferably, the enterococci are Enterococcus faecalis and / or Enterococcus faecium; preferably, the infections or diseases caused by enterococci are urinary tract infections, intra-abdominal infections, wound infections, bacteremia, endocarditis, meningitis, and other types of infections; preferably, other types of infections include respiratory tract infections, skin and soft tissue infections, and gynecological infections.

[0031] Another aspect of the present invention provides a method for preventing and / or treating infections or diseases caused by enterococci, characterized in that it includes administering to a subject one or more fusion proteins of the present invention, and / or one or more recombinant nucleic acid molecules of the present invention, and / or one or more recombinant gene expression cassettes of the present invention, and / or one or more recombinant vectors of the present invention, and / or one or more recombinant host cells of the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions of the present invention, and / or one or more recombinant vaccines of the present invention; preferably, the enterococci are pathogenic and / or drug-resistant enterococci; preferably, the enterococci are Enterococcus faecalis and / or Enterococcus faecium; preferably, the infections or diseases caused by the enterococci are urinary tract infections, intra-abdominal infections, wound infections, bacteremia, endocarditis, meningitis, and other types of infections; preferably, other types of infections include respiratory tract infections, skin and soft tissue infections, and gynecological infections.

[0032] The fusion protein, recombinant vaccine, etc. of the present invention have the following beneficial technical effects: 1. The present invention uses the reverse vaccinology analysis method and combines previous research and development experience to select HlyC / CorC transporter antigen (Hly), elongation factor Tu antigen (Tuf), and 50S ribosomal protein L7 / L12 antigen (rplL) as candidate antigens for vaccine development.

[0033] 2. The present invention adopts the method of antigen truncation and fusion expression to design vaccines. For Hly antigens with larger molecular weight, domain and epitope truncation is required. Through a variety of truncation and a variety of epitope combinations, orthogonal, single factor and other experiments are performed for screening, and finally the truncation Hly selected by the present invention is obtained, and then the truncation Hly of Enterococcus faecium and Enterococcus faecalis is fused and expressed as fusion protein A. The Tuf and rplL antigens of Enterococcus faecium and Enterococcus faecalis are fused and expressed as fusion protein B. Both fusion protein molecules can significantly reduce tissue infections caused by Enterococcus faecium and Enterococcus faecalis, have good immunogenicity, play an effective preventive and immune protection role, and efficiently prevent pathogenic enterococcal infections.

[0034] 3. The present invention mutates the glycosylation site of the truncated Hly antigen to prevent glycosylation of the prokaryotic protein during expression in eukaryotic cells, which could affect the correct presentation of the epitope. The mutated truncated Hly antigen can improve the expression of the candidate antigen and enhance the immunogenicity of the antigen.

[0035] 4. Example 5 shows that Vaccine A (based on fusion protein A) detected significant expression of the target protein in both the supernatant and cell lysate, and the molecular weight was consistent with expectations. This demonstrates that the fusion protein A based on the present invention can not only be smoothly translated and correctly folded in eukaryotic cells, but also has a stable structure and can be efficiently secreted extracellularly, and can be used in subsequent animal trials to verify the immune protection effect. Vaccine B (based on fusion protein B) detected significant expression of the target protein in the cell lysate, and the molecular weight was consistent with expectations. This demonstrates that the fusion protein B based on the present invention can be smoothly translated, correctly folded, and expressed in high abundance in eukaryotic cells, and can be used in subsequent animal trials to verify the immune protection effect.

[0036] 5. Examples 6 and 7 respectively demonstrate the preventive effects of the recombinant nucleic acid vaccine of the present invention in the mouse Enterococcus faecium and Enterococcus faecalis gavage infection models. The results show that the recombinant nucleic acid vaccine A and vaccine B based on the present invention can provide effective immune protection against Enterococcus faecium and Enterococcus faecalis in the mouse model, and the vaccinated mice can produce significant protective immunity to prevent Enterococcus faecium and Enterococcus faecalis infection. Moreover, after combining the recombinant nucleic acid vaccine A and vaccine B, a synergistic effect was achieved compared to the use of vaccine A and vaccine B alone, which significantly reduced the bacterial load of each tissue after immune challenge and effectively prevented Enterococcus faecium and Enterococcus faecalis infection.

[0037] 6. The recombinant vaccine provided by this invention can induce effective protective immunity in model mice, demonstrating a strong preventive effect against two major pathogenic enterococcal infections. The efficacy is optimal when the two fusion proteins are administered together, demonstrating a synergistic effect. Therefore, this invention can be applied to the production and development of immunotherapies, filling a gap in the current field of enterococcal vaccine research and development and possessing broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagrams of the protein structure of Hly antigen before and after truncation; wherein A exemplarily shows the full-length protein structure of Enterococcus Hly antigen, and B exemplarily shows the truncated Enterococcus Hly antigen protein structure.

[0039] Figure 2 Schematic diagram of the non-limiting molecular structure of the vaccine of the present invention; wherein A is a molecular schematic diagram of the protein expressed by the vaccine; B is a molecular schematic diagram of the nucleic acid vaccine.

[0040] Figure 3 Schematic diagram of the template plasmid comprising the present invention.

[0041] Figure 4A-4B They are respectively the quality control peak graphs and purity test results of vaccine A and vaccine B of the present invention.

[0042] Figure 5A-5B The results of in vitro expression WB (Western blot) of HEK293 cells transfected with vaccine A and vaccine B of the present invention are shown respectively.

[0043] Figure 6 The present invention is an immunization and sampling process for the vaccine of the present invention in a mouse Enterococcus faecium gavage infection model.

[0044] Figures 7A-7D The figures are the comparison of bacterial loads in the liver tissue, spleen tissue, jejunum tissue, and fecal tissue of mice after immunization with Enterococcus faecium.

[0045] Figure 8The present invention is used to administer the vaccine to mice for immunization and sampling in an enterococcus faecalis gavage infection model.

[0046] Figures 9A-9D The figures are the comparison of bacterial loads in the liver tissue, spleen tissue, jejunum tissue, and fecal tissue of mice after immunization with Enterococcus faecalis. DETAILED DESCRIPTION

[0047] Terms and Definitions The term "Enterococcus faecium" refers to Enterococcus faecium , is a Gram-positive bacterium belonging to the genus Enterococcus ( Enterococcus ), which is γ-hemolytic or non-hemolytic and resistant to many commonly used antibiotics, is one of the important pathogens causing hospital-acquired infections.

[0048] The term "Enterococcus faecalis" refers to Enterococcus faecalis , like Enterococcus faecium, belongs to the genus Enterococcus ( Enterococcus ), which is resistant to multiple commonly used antibiotics and is one of the important pathogens causing hospital-acquired infections.

[0049] The term "enterococcal infection" refers to various diseases caused by Enterococcus faecium and / or Enterococcus faecalis, including but not limited to urinary tract infection, bacteremia, endocarditis, intra-abdominal infection, wound infection, meningitis, and other types of infection, including respiratory tract infection, skin and soft tissue infection, gynecological infection, etc.

[0050] The term "HlyC / CorC family transporter," or HlyC / CorC transporter (abbreviated as Hly), refers to a family of membrane transporter proteins that are widely present in a variety of bacteria. Members of this family typically contain hemolysin C (HlyC) and cobalt resistance protein (CorC) transport domains, which are associated with the efflux of magnesium and cobalt ions. Proteins in this family may also be involved in the regulation of ion substrate transport, but the exact mechanism is not yet fully understood. The present invention performs domain and epitope truncation on Hly to obtain HlyC / CorC transporter antigens. The HlyC / CorC transporter antigens are derived from any species, preferably from Enterococcus faecalis or Enterococcus faecium. The amino acid sequence of the HlyC / CorC transporter antigen from Enterococcus faecalis is shown in SEQ ID NO: 1, and the amino acid sequence of the HlyC / CorC transporter antigen from Enterococcus faecium is shown in SEQ ID NO: 2.

[0051] The term "Tuf" refers to Elongation Factor Tu (EF-Tu or Tuf), a GTP-binding protein that plays a key role in protein synthesis. It is widely present in the cytoplasm, mitochondria, and chloroplasts of prokaryotes and eukaryotes. It is an essential factor in translation elongation, responsible for the correct transfer of aminoacyl-tRNA (aminoacylated transfer RNA) to the A site (aminoacyl-tRNA binding site) of the ribosome. Tuf is derived from any species, preferably from Enterococcus faecalis or Enterococcus faecium. The amino acid sequence of Tuf from Enterococcus faecalis is shown in SEQ ID NO:3, and the amino acid sequence of Tuf from Enterococcus faecium is shown in SEQ ID NO:5.

[0052] The term "rplL" refers to the 50S ribosomal protein L7 / L12, a crucial component of the ribosome. The rplL is derived from any species, preferably from Enterococcus faecalis or Enterococcus faecium. The amino acid sequence of the rplL from Enterococcus faecalis is shown in SEQ ID NO:4, and the amino acid sequence of the rplL from Enterococcus faecium is shown in SEQ ID NO:6.

[0053] The term "C-terminal domain" refers to a functional or structural unit near the carboxyl terminus (C-terminus) in a protein molecule. It is located at the other end of the protein, opposite to the N-terminal domain. It usually has an independent three-dimensional structure, or contains a specific amino acid sequence or conserved region, thus possessing a specific biological function.

[0054] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. The immune response can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and / or assays that measure the modulation of the activity or expression of one or more cytokines.

[0055] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and cellular response in an organism. The immune response can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and / or assays that measure the modulation of the activity or expression of one or more cytokines.

[0056] The terms "administering" or "vaccinating" refer to administration of a nucleic acid vaccine or vaccine composition of the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration can also be used, for example, oral, intranasal (e.g., aerosol or other non-injectable administration), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or a combination thereof. Administration into the neck muscle of an animal is preferred. Boosting regimens can be used to adjust the dosing regimen to provide optimal immunity.

[0057] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0058] The term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0059] The term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as lentiviruses, retroviruses, vaccinia, adenoviruses, fowlpox, baculoviruses, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0060] The term "mRNA" refers to messenger RNA, which is translated into "messenger ribonucleic acid" in Chinese. It is a type of single-stranded ribonucleic acid that is transcribed from a chain of DNA as a template, carries genetic information and can guide protein synthesis.

[0061] The term "5'-UTR," referring to the "5' untranslated region" or "5'UTR," is a portion of a gene that is transcribed into the primary RNA transcript (pre-mRNA) and located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription from DNA. Many primary transcripts must undergo RNA processing to form biologically active RNA. Processing to form mature mRNA includes terminal modification, intron removal, capping, and / or splicing of individual rRNA molecules from the pre-RNA. Therefore, the 5'UTR of an mRNA is the portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNAs can range from tens to hundreds of bases in length.

[0062] The term "3'-UTR," referring to a "3'-untranslated region" or "3'UTR," refers to a region located at the 3' end of a gene, downstream of the stop codon of the protein-coding region, that is transcribed but not translated into an amino acid sequence, or to 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 is typically attached after the transcription process. The 3'-UTR of mammalian mRNA often has a homology 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 and may fold to create a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins known to enhance RNA stability (e.g., RNA-binding proteins).

[0063] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are 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, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells.

[0064] The terms "individual," "patient," or "subject" include mammals, including, but not limited to, domesticated animals (e.g., pigs, cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats).

[0065] The terms "transformation, transfection, and transduction" have the meanings generally understood by those skilled in the art, namely, the process of introducing exogenous DNA or RNA into a host.

[0066] The term "pharmaceutical combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of pharmaceutical production. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe, stable, and / or has specific functionality, and also to provide a method for effective absorption in a subject. A pharmaceutically acceptable carrier can be an inert filler or an active ingredient that provides a specific function to the pharmaceutical combination (for example, 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, glidants, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0067] The term "prevention" means that before a subject develops a disease, the symptoms after developing the disease are alleviated by exposing the subject to (e.g., administering) the recombinant vaccine, composition, etc. according to the present invention, compared to when the subject has not developed the disease. It does not necessarily mean that the disease must be completely suppressed.

[0068] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0069] The present invention discloses a novel vaccine, preparation method, and application for preventing pathogenic enterococcal infection. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0070] The fusion proteins and encoding nucleic acids and their components, as well as their preparation methods and applications, provided herein can all be commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression constructs, vaccine preparation, and immunization, those skilled in the art can implement the methods of the present invention.

[0071] The present invention is further illustrated below with reference to the following examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for the preparation of a recombinant vaccine.

[0072] Example 1 Design ideas for universal enterococcal vaccine In order to design a vaccine that can prevent both Enterococcus faecium and Enterococcus faecalis, the present invention uses a fusion protein approach to construct a molecule for vaccine production. The specific idea is as follows: First, using reverse vaccinology analysis and combining previous R&D experience, the HlyC / CorC transporter antigen (Hly), elongation factor Tu antigen (Tuf), and 50S ribosomal protein L7 / L12 antigen (rplL) were selected as candidate antigens for vaccine development. Due to the low sequence homology between the antigens of Enterococcus faecium and Enterococcus faecalis, the present invention utilizes antigen truncation and fusion expression for vaccine design. For the larger molecular weight Hly antigen, domain and epitope truncation were required. Through multiple truncation and epitope combinations, orthogonal and single-factor screening experiments were performed to ultimately obtain the truncation Hly selected in the present invention. The truncation Hly from the two enterococci was then fused and expressed as fusion protein A. The Tuf and rplL antigens from the two enterococci were fused and expressed as fusion protein B.

[0073] Figure 1 Schematic diagram showing the protein structure before and after Hly antigen selection. Figure 1 Figure A illustrates the full-length protein structure of the Enterococcus Hly antigen, which has three relatively independent domains from the N-terminus to the C-terminus. Considering the stability of the fusion protein and the inclusion of as many antigenic epitopes as possible, this example selected domains 2 and 3 as the Hly antigen sequence for constructing the fusion protein.

[0074] like Figure 1 As shown in B, the structure of the truncated Hly antigen protein is exemplified. The truncated antigen still retains the original conformation of domain 2 and domain 3, proving that these two domains are relatively stable and can be used in the design of fusion proteins.

[0075] According to the above method, the final design is as follows Figure 2 The two vaccine molecules shown are: A is a molecular schematic diagram of the protein expressed by the vaccine; B is a molecular schematic diagram of the nucleic acid vaccine. Since the N-terminal signal peptide element originally possessed by the Hly antigen is lost after truncation, an additional secretion element is added to the N-terminus to promote the correct expression of the antigen in the host cell. In addition, the present invention mutates the glycosylation site of the truncated Hly antigen to avoid glycosylation of the prokaryotic protein when expressed in eukaryotic cells, which affects the correct presentation of the epitope. The mutated truncated Hly antigen can improve the expression of the candidate antigen and enhance the immunogenicity of the antigen.

[0076] Example 2 Construction of the recombinant nucleic acid vaccine of the present invention In order to prepare a recombinant nucleic acid vaccine comprising the antigen of the present invention and verify whether the vaccine based on the present invention has a good in vitro expression effect, the vaccine molecular architecture involved in the embodiment is as follows Figure 2 In order to prepare a product that can produce Figure 2 To prepare a recombinant nucleic acid vaccine for the protein shown in B, first construct a gene expression cassette for expressing the antigen sequence of the present invention. The expression cassette comprises, from the 5' end to the 3' end, 5'UTR, the coding sequence (CDS) of the present invention, 3'UTR, and PolyA. Subsequently, the complete gene expression cassette sequence is optimized based on codon degeneracy, and the DNA sequence is directly obtained by gene synthesis (commissioned to GenScript Corporation for synthesis). Finally, the synthesized gene expression cassette DNA sequence is inserted into an expression vector that can be used for in vitro RNA transcription. The schematic diagram of the template plasmid of the present invention is shown in FIG. Figure 3 As shown, a vector plasmid for preparing a recombinant nucleic acid vaccine was obtained.

[0077] According to the above method, the carrier used in the subsequent examples was prepared: (1) Preparation of vectors based on the recombinant nucleic acid vaccine A of the present invention Step a: Synthesize a "signal peptide-IGHG Fc domain-Enterococcus faecalis HlyC / CorC transporter antigen-Enterococcus faecium HlyC / CorC transporter antigen" fusion gene fragment, wherein the Fc domain of IGHG (i.e., the "Fc domain of the immunoglobulin heavy chain constant region γ1 protein") and the Enterococcus faecalis HlyC / CorC transporter antigen are connected by a GGS linker sequence as shown in SEQ ID NO: 11, and the Enterococcus faecalis HlyC / CorC transporter antigen and the Enterococcus faecium HlyC / CorC transporter antigen are connected by a spacer sequence as shown in SEQ ID NO: 12. The amino acid sequence of the signal peptide (i.e., the "signal peptide of human blue pyrimidine protein") is shown in SEQ ID NO: 9, the amino acid sequence of the Fc domain of IGHG is shown in SEQ ID NO: 10, the amino acid sequence of the Enterococcus faecalis HlyC / CorC transporter antigen is shown in SEQ ID NO: 1, and the amino acid sequence of the Enterococcus faecium HlyC / CorC transporter antigen is shown in SEQ ID NO: 2. NO: 2. The HlyC / CorC transporter antigen of Enterococcus faecalis contains a glycosylation mutation site of N68Q. The HlyC / CorC transporter antigen of Enterococcus faecium contains a glycosylation mutation site of T56A. The fusion protein A is a core element, i.e., composed of the Enterococcus faecalis HlyC / CorC transporter antigen-spacer sequence-Enterococcus faecium HlyC / CorC transporter antigen, and the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 7.

[0078] Step b: Construct a nucleic acid vaccine framework vector.

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

[0080] Step c: Preparation of recombinant plasmid.

[0081] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine A based on the present invention.

[0082] (2) Preparation of vectors based on the recombinant nucleic acid vaccine B of the present invention Step a: Synthesize a "Enterococcus faecalis elongation factor Tu antigen-Enterococcus faecalis 50S ribosomal protein L7 / L12 antigen-Enterococcus faecium elongation factor Tu antigen-Enterococcus faecium 50S ribosomal protein L7 / L12 antigen" fusion gene fragment, wherein the four antigens are connected by a spacer sequence with an amino acid sequence as shown in SEQ ID NO: 12, the amino acid sequence of the Enterococcus faecalis elongation factor Tu antigen is shown in SEQ ID NO: 3, the amino acid sequence of the Enterococcus faecalis 50S ribosomal protein L7 / L12 antigen is shown in SEQ ID NO: 4, the amino acid sequence of the Enterococcus faecalis elongation factor Tu antigen is shown in SEQ ID NO: 5, and the amino acid sequence of the Enterococcus faecium 50S ribosomal protein L7 / L12 antigen is shown in SEQ ID NO: 6. The fusion protein B is a core element, which is composed of Enterococcus faecalis elongation factor Tu antigen-spacer sequence-Enterococcus faecalis 50S ribosomal protein L7 / L12 antigen-spacer sequence-Enterococcus faecium elongation factor Tu antigen-spacer sequence-Enterococcus faecium 50S ribosomal protein L7 / L12 antigen. The amino acid sequence of the fusion protein B is shown in SEQ ID NO: 8.

[0083] Step b: Construct a nucleic acid vaccine framework vector.

[0084] The nucleic acid vaccine framework vector comprises a 5'-UTR and a 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing a DNA vaccine.

[0085] Step c: Preparation of recombinant plasmid.

[0086] The gene synthesized in step a is inserted into the vector structure of step b to obtain a vector for preparing the recombinant nucleic acid vaccine B based on the present invention.

[0087] Table 1 Amino acid sequences of proteins involved in the present invention Amino acid sequence and sequence number HlyC / CorC transporter antigen of Enterococcus faecalis DSKMTRDEMRYMLETEGVLENEELEMLQGVFSLDTKVAREVMVPRTDAFMVDIQDDVQENINLILGEQYSRIPVYSEDKDKIVGILHTKTLLKAARNLGFENIELGAIIQEPLFVPETIFIDDLLYELKRTQNQMAILLDEY GGVVGLATLEDLLEEIVGEIDDETDEVENLYTQVADNEYLVQGRMLIDEFNEVFETDLHMSDVDTMAGYLITALGTIPDEGEKPSFEVGNIKLTAEMEGTRLLVLRVHFYDEETVDEEPEENRRFFRKEMEDDEPRR (SEQ ID NO:1) HlyC / CorC transporter antigen of Enterococcus faecium DSKMTRDEMRYMLENEGVLNNEELEMLQGVFSLDTKVAREVMVPRTDAFMIDINDAVEENVNEVLSENYSRIPVYNEDKDKVVGILHTKNLLKAAHKFGFDNLDIKKIMQEPLFVPETVFIDDLLYEMKK TQNQMAILLDEYGGVVGLATLEDLLEEIVGEIDDESDEVENLYEKIDEHEYIIQGRMLIDEFNEAFDSDLHMSDVDTMAGYLITALGMIPDEGEKLSFDVDNITLVSEEMEGSRILKIRVIFHDPEE (SEQ ID NO:2) Enterococcus faecalis elongation factor Tu antigen DRSKSHVNIGTIGHVDHGKTTLTAAIATVLSKHGGGEAQSYDSIDNAPEEKERGITINTSHIEYETETRHYAHVDCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHILLSRNVGVPYIVVFLNKMDMVDDEELLELVEMEVRDLLSEYDFPGDDVPVIAGSALKALEGDESYEEKILELMAAVDEYIPTPE (SEQ ID NO: 3) Enterococcus faecalis 50S ribosomal protein L7 / L12 antigen MALNIENIVAELETATILELSELVKAIEEKFDVSAAAPVAVAGPAAGGAAEEQTEFTVELTAAGDQKVKVIKAVREATGLGLKEAKAVVDGAPAPVKEAVSKEEAEALKAALEEVGASVTVK (SEQ ID NO: 4) Enterococcus faecium elongation factor Tu antigen DRSKPHVNIGTIGHVDHGKTTLTAAITTVLSKKNGGQAMAYDQIDGAPEERERGITISTAHVEYETDTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHILLSRQVGVPYIVVFLNKVDMVDDEELLELVEMEVRDLLTEYEFPGDDVPVVAGSALKALEGDASYEEKILELMAAVDEYIPTPE (SEQ ID NO:5) Enterococcus faecium 50S ribosomal protein L7 / L12 antigen MALNIENIVAELKEATILELNDLVKAIEEEFGVSAAAPVAVAAAGGAAAAEEQTEFTVELTAAGDQKVKVIKAVREATGLGLKEAKAVVDGAPAPVKEGVSKEEAEELKAKLEEVGASVTVK (SEQ ID NO: 6) Fusion protein A (SEQ ID NO:7) fusion proteinB DRSKSHVNIGTIGHVDHGKTTLTAAIATVLSKHGGGEAQSYDSIDNAPEEKERGITINTSHIEYETETRHYAHVDCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHILLSRNVGVPYIVVFLNKMDMVDDEELLELVEMEVRDLLSEYDFPGDDVPVIAGSALKALEGDESYEEKILELMAAVDEYIPTPEGGSGGGGSGGMALNIENIVAELETATILELSELVKAIEEKFDVSAAAPVAVAGPAAGGAAEEQTEFTVELTAAGDQKVKVIKAVREATGLGLKEAKAVVDGAPAPVKEAVSKEEAEALKAALEEVGASVTVKGGSGGGGSGGDRSKPHVNIGTIGHVDHGKTTLTAAITTVLSKKNGGQAMAYDQIDGAPEERERGITISTAHVEYETDTRHYAHVDCPGHADYVKNMITGAAQMDGAILVVSAADGPMPQTREHILLSRQVGVPYIVVFLNKVDMVDDEELLELVEMEVRDLLTEYEFPGDDVPVVAGSALKALEGDASYEEKILELMAAVDEYIPTPEGGSGGGGSGGMALNIENIVAELKEATILELNDLVKAIEEEFGVSAAAPVAVAAAGGAAAAEEQTEFTVELTAAGDQKVKVIKAVREATGLGLKEAKAVVDGAPAPVKEGVSKEEAEELKAKLEEVGASVTVK (SEQ ID NO:8) Signal peptide of human lanthionin protein MTRLTVLALLAGLLASSRA (SEQ ID NO:9) Fc domain of immunoglobulin heavy chain constant region γ1 protein EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:10) GGS linker sequence GGSGGSGGSG (SEQ ID NO: 11) spacer sequence GGSGGGGSGG (SEQ ID NO: 12) Example 3 Preparation of the recombinant nucleic acid vaccine of the present invention (1) Preparation of capped mRNA vaccines Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 2 is enzymatically cut and linearized to obtain a linearized plasmid for in vitro transcription.

[0088] Step b: The linearized plasmid is subjected to an in vitro co-transcription capping reaction to add a 7-methylated guanylate cap structure to the 5' end of the transcribed mRNA, and the template DNA is degraded.

[0089] (2) Preparation of non-capped mRNA vaccines Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 2 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0090] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction, and the template DNA is degraded.

[0091] (3) DNA vaccine preparation Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 2 to obtain a large amount of target plasmid for purification.

[0092] Step b: Use an endotoxin-free plasmid extraction and purification kit to extract and purify the target plasmid.

[0093] Example 4 Quality Control of In Vitro Transcription of Recombinant Nucleic Acids and Vaccine Preparation of the Present Invention Vaccine A (based on the recombinant nucleic acid vaccine A of the present invention) and Vaccine B (based on the recombinant nucleic acid vaccine B of the present invention) were prepared using the capped mRNA vaccine preparation method described in Example 3. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiment was greater than 85%. The quality control peaks of the recombinant nucleic acid vaccines A and B of the present invention are shown in Figure 2. Figure 4A 、 Figure 4B The specific description is as follows: (1) The recombinant nucleic acid vaccine A based on the present invention has a purity of 94%, and the quality control peak diagram and purity test results are as shown in FIG. Figure 4A ; (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 87.8%, and the quality control peak diagram and purity test results are as follows Figure 4B The above purity meets the quality requirements for cell transfection experiments and vaccine production.

[0094] Example 5 In vitro expression effect of the recombinant nucleic acid of the present invention The vaccine in Example 4 was transfected into HEK293T cells using a cell transfection reagent. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot. The estimated molecular weights of the proteins expressed by vaccine A and vaccine B were calculated, as shown in Table 2.

[0095] Table 2 Estimated molecular weight of proteins expressed by vaccine A and vaccine B vaccine Protein molecular weight (kDa) A 94 B 81 Figure 5A-5B The in vitro expression of vaccines A and B in HEK293 cells was detected by Western blot analysis, with an exposure time of 1 second. The antigen expressed by vaccine A contains a secretion-associated element, and theoretically, significant expression should be detected in the supernatant and lysate proteins. The antigen expressed by vaccine B does not contain a secretion-associated element, and theoretically, significant expression should be detected in the lysate proteins.

[0096] like Figure 5A As shown, vaccine A detected significant expression of the target protein in both the supernatant and cell lysate, and the molecular weight was in line with expectations. This proves that the fusion protein A based on the present invention can not only be smoothly translated and correctly folded in eukaryotic cells, but also has a stable structure and can be efficiently secreted outside the cell, and can be used in subsequent animal experiments to verify the immune protection effect.

[0097] like Figure 5B As shown, vaccine B detected significant expression of the target protein in cell lysates, and the molecular weight was in line with expectations. This proves that the fusion protein B based on the present invention can be smoothly translated, correctly folded, and expressed in high abundance in eukaryotic cells, and can be used in subsequent animal trials to verify the immune protection effect.

[0098] Example 6 Preventive Effect of the Recombinant Nucleic Acid Vaccine of the Present Invention in a Mouse Enterococcus Faecium Infection Model In order to verify whether the recombinant nucleic acid vaccine based on the present invention has an immune protection effect against Enterococcus faecium, this example conducted immunization and challenge comparative experiments on vaccine-immunized mice (experimental groups, i.e., vaccine A and vaccine B) and non-immunized mice (blank control group).

[0099] Twenty male BALB / C mice, 6-8 weeks old and weighing 18-25g, were housed in individual cages at a constant temperature and humidity. They were acclimated for seven days in the housing room. The temperature was 20-26°C, the humidity was 40-70%, and the mice were kept in a light-dark cycle (light on from 8:00 AM to 8:00 PM, and dark on from 8:00 PM to 8:00 AM). They were provided with a continuous supply of feed and ad libitum, along with sterile water via a continuous drinking bottle. After acclimation, the mice were randomly divided into four groups of five mice each, each with ear tags. The details are shown in Table 3. Doses in this table and below refer to the amount of active ingredient.

[0100] Table 3 Grouping and immunization process of mouse immunization experiment

[0101] Each group of mice was immunized twice according to the immunization schedule in Table 3. On days 32-34, mice were gavaged with 100 μl of a 50 mg / ml penicillin and streptomycin antibiotic mixture once a day for 3 days. On day 35, mice were fasted for 4 hours before bacterial challenge and immunized with 100 μl of Enterococcus faecium solution (BNCC336951, 1×10 10 CFU) were gavaged, and the mice were raised normally after gavage. The clinical manifestations of the mice were observed and recorded every day. The mice were killed on day 38 and samples were collected. The feces, jejunum, liver and spleen of the mice were collected to detect the bacterial load of each tissue and evaluate the immune protection effect of the vaccine. Figure 6 shown.

[0102] Jejunum, liver, and spleen tissues were collected from each mouse, each weighed 10 mg, ground separately, and diluted with sterile PBS buffer. After mixing, 100 μl of the diluted solution was evenly spread on a BHI solid culture medium. Feces were collected aseptically on day 38 before the mice were sacrificed, weighed, added to sterile PBS, and ground until no precipitate was visible. The solution was diluted and spread on a BHI solid culture dish. The dish was then placed in a 37°C incubator for incubation. After 24 hours of anaerobic bacterial culture, the dish was removed, bacterial colonies were counted, and photographed to assess the protective effect of the vaccine on the mice.

[0103] The results are as follows Figures 7A-7D As shown, Figures 7A-7D The comparison of bacterial load in liver tissue, spleen tissue, jejunum tissue and fecal tissue after mice were immunized with Enterococcus faecium. Figure 7AThe results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 7.7 times, and the maximum difference in counts decreased by about 24 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 12.7 times, and the maximum difference in counts decreased by about 118 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 11 times, and the maximum difference in counts decreased by about 31 times.

[0104] Figure 7B The bacterial load in the spleen tissue of mice after immunization and challenge was compared. The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 4.5 times, and the maximum difference in counts decreased by about 41 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 11 times, and the maximum difference in counts decreased by about 35 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 9.2 times, and the maximum difference in counts decreased by about 42 times.

[0105] Figure 7C The comparison of bacterial loads in the jejunum tissue of mice after immunization and challenge showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 4 times, and the maximum difference in counts decreased by about 13.5 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 6.8 times, and the maximum difference in counts decreased by about 27.5 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 13.7 times, and the maximum difference in counts decreased by about 30.7 times.

[0106] Figure 7D The bacterial load in fecal tissue of mice after immunization and challenge was compared. The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue of mice immunized with vaccine A decreased by about 4.4 times, and the maximum difference in counts decreased by about 8.3 times; the average bacterial CFU count per mg of tissue of mice immunized with vaccine B decreased by about 10 times, and the maximum difference in counts decreased by about 72 times; the average bacterial CFU count per mg of tissue of mice immunized with a mixture of vaccines A and B decreased by about 16 times, and the maximum difference in counts decreased by about 173 times.

[0107] These results demonstrate that both recombinant nucleic acid vaccines A and B, based on the present invention, can provide effective immune protection against E. faecium in a mouse model. Vaccinated mice developed significant protective immunity, preventing E. faecium infection. Furthermore, the combination of recombinant nucleic acid vaccines A and B achieved a synergistic effect compared to using either vaccine alone, significantly reducing bacterial loads in various tissues after immune challenge and effectively preventing E. faecium infection.

[0108] Example 7 Preventive Effect of the Recombinant Nucleic Acid Vaccine of the Present Invention in a Mouse Enterococcus Faecalis Infection Model In order to verify whether the recombinant nucleic acid vaccine based on the present invention has an immune protection effect against Enterococcus faecalis, this example conducted immunization and challenge comparative experiments on vaccine-immunized mice (experimental groups, i.e., vaccine A and vaccine B) and non-immunized mice (blank control group).

[0109] Twenty male BALB / C mice, 6-8 weeks old and weighing 18-25 g, were housed in individual cages at a constant temperature and humidity. They were acclimated for seven days in advance. The room temperature was 20-26°C, the humidity was 40-70%, and the mice were kept in a light-dark cycle (light on from 8:00 AM to 8:00 PM, and dark on from 8:00 PM to 8:00 AM). They were provided with a continuous supply of feed and ad libitum. Sterile water was also available from a drinking bottle and was available ad libitum. After acclimation, the mice were randomly divided into four groups of five mice each. Each mouse was ear-tagged. The details are shown in Table 3.

[0110] Each group of mice was immunized twice according to the immunization schedule in Table 3. On days 32-34, mice were gavaged with 100 μl of a 50 mg / ml penicillin and streptomycin antibiotic mixture once a day for 3 days. On day 35, mice were fasted for 4 hours before bacterial challenge and immunized with 100 μl of Enterococcus faecalis (BNCC186300, 1×10 10 CFU) were gavaged, and the mice were raised normally after gavage. The clinical manifestations of the mice were observed and recorded every day. The mice were killed on day 38 and samples were collected. The feces, jejunum, liver and spleen of the mice were collected to detect the bacterial load of each tissue and evaluate the immune protection effect of the vaccine. Figure 8 shown.

[0111] Jejunum, liver, and spleen tissues were collected from each mouse, each weighed 10 mg, ground separately, and diluted with sterile PBS buffer. After mixing, 100 μl of the diluted solution was evenly spread on a BHI solid culture medium. Feces were collected aseptically on day 38 before the mice were sacrificed, weighed, added to sterile PBS, and ground until no precipitate was visible. The solution was diluted and spread on a BHI solid culture dish. The dish was then placed in a 37°C incubator for incubation. After 24 hours of anaerobic bacterial culture, the dish was removed, bacterial colonies were counted, and photographed to assess the protective effect of the vaccine on the mice.

[0112] The results are as follows Figures 9A-9D As shown, Figures 9A-9D The figures are the comparison of bacterial loads in the liver tissue, spleen tissue, jejunum tissue, and fecal tissue of mice after immunization with Enterococcus faecalis. Figure 9A The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 2.2 times, and the maximum difference in counts decreased by about 4.3 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 2 times, and the maximum difference in counts decreased by about 3.4 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 10 times, and the maximum difference in counts decreased by about 42 times.

[0113] Figure 9B The bacterial load in the spleen tissue of mice after immunization and challenge was compared. The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 2.7 times, and the maximum difference in counts decreased by about 6.4 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 2.2 times, and the maximum difference in counts decreased by about 11 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 19 times, and the maximum difference in counts decreased by about 100 times.

[0114] Figure 9CThe bacterial load in the jejunum tissue of mice after immunization and challenge was compared. The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue in mice immunized with vaccine A decreased by about 3.5 times, and the maximum difference in counts decreased by about 13.4 times; the average bacterial CFU count per mg of tissue in mice immunized with vaccine B decreased by about 3 times, and the maximum difference in counts decreased by about 6 times; the average bacterial CFU count per mg of tissue in mice immunized with a mixture of vaccines A and B decreased by about 16 times, and the maximum difference in counts decreased by about 78.7 times.

[0115] Figure 9D The bacterial load in fecal tissue of mice after immunization and challenge was compared. The results showed that the Enterococcus faecium load in mice immunized with the vaccine of the present invention was significantly lower than that in the control group (p<0.001): compared with the control group, the average bacterial CFU count per mg of tissue of mice immunized with vaccine A decreased by about 3.6 times, and the maximum difference in counts decreased by about 5 times; the average bacterial CFU count per mg of tissue of mice immunized with vaccine B decreased by about 3.4 times, and the maximum difference in counts decreased by about 4.8 times; the average bacterial CFU count per mg of tissue of mice immunized with a mixture of vaccines A and B decreased by about 13 times, and the maximum difference in counts decreased by about 41 times.

[0116] These results demonstrate that both recombinant nucleic acid vaccines A and B, based on the present invention, can provide effective immune protection against E. faecalis in a mouse model. Vaccinated mice developed significant protective immunity, preventing E. faecalis infection. Furthermore, the combination of recombinant nucleic acid vaccines A and B achieved a synergistic effect compared to using either vaccine A or B alone, significantly reducing bacterial loads in various tissues after immune challenge and effectively preventing E. faecalis infection.

[0117] In summary, the fusion protein and immunogenic composition provided by the present invention can induce effective protective immunity in model mice, showing good preventive effects against two major pathogenic enterococcal infections. Furthermore, the two fusion proteins are most effective when used together for immunization, demonstrating a synergistic effect. Therefore, the present invention can be applied to the production and development of immunopharmaceuticals, filling a gap in the current field of enterococcal vaccine research and development, and possesses extremely high commercial value and broad application prospects.

[0118] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the embodiments of the present disclosure. For those skilled in the art, other variations or changes in different forms can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present disclosure shall be included in the scope of protection of the claims of the present disclosure.

Claims

1. A fusion protein, characterized in that Select any one of the following (1)-(3): Fusion protein A, the fusion protein A comprises Enterococcus faecalis ( Enterococcus faecalis ) and / or Enterococcus faecium ( Enterococcus faecium ) derived from an HlyC / CorC transporter (HlyC / CorC family transporter) antigen; preferably, the HlyC / CorC transporter antigen is truncated from the C-terminal domain of the HlyC / CorC transporter; more preferably, the HlyC / CorC transporter antigen comprises a mutation targeting a glycosylation site; most preferably, the mutation in the glycosylation site comprises N68Q and / or T56A; Fusion protein B, the fusion protein B comprises Enterococcus faecalis ( Enterococcus faecalis ) and / or Enterococcus faecium ( Enterococcus faecium Elongation factor Tu (Tuf) antigen and 50S ribosomal protein L7 / L12 (rplL) antigen derived from The combination of the fusion protein A and the fusion protein B.

2. The fusion protein according to claim 1, characterized in that The fusion protein A comprises, from N-terminus to C-terminus, an HlyC / CorC transporter antigen derived from Enterococcus faecalis and an HlyC / CorC transporter antigen derived from Enterococcus faecium; the fusion protein B comprises, from N-terminus to C-terminus, an elongation factor Tu antigen derived from Enterococcus faecalis, a 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecalis, an elongation factor Tu antigen derived from Enterococcus faecium, and a 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecium.

3. The fusion protein according to any one of claims 1 or 2, characterized in that The amino acid sequence of the HlyC / CorC transporter antigen derived from Enterococcus faecalis is shown in SEQ ID NO: 1, the amino acid sequence of the HlyC / CorC transporter antigen derived from Enterococcus faecium is shown in SEQ ID NO: 2, the amino acid sequence of the elongation factor Tu antigen derived from Enterococcus faecalis is shown in SEQ ID NO: 3, the amino acid sequence of the 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecalis is shown in SEQ ID NO: 4, the amino acid sequence of the elongation factor Tu antigen derived from Enterococcus faecium is shown in SEQ ID NO: 5, and the amino acid sequence of the 50S ribosomal protein L7 / L12 antigen derived from Enterococcus faecium is shown in SEQ ID NO:

6.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The antigens can optionally be connected via a linker sequence or a spacer sequence; preferably, the linker sequence is a GGS linker sequence; more preferably, the amino acid sequence of the GGS linker sequence is shown in SEQ ID NO: 11, and the amino acid sequence of the spacer sequence is shown in SEQ ID NO:

12.

5. The fusion protein according to any one of claims 1 to 4, characterized in that The amino acid sequence of the fusion protein A is shown in SEQ ID NO: 7, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO:

8.

6. The fusion protein according to any one of claims 1 to 5, characterized in that The N-terminus of the fusion protein A further comprises a signal peptide and / or any secretion-promoting element; preferably, the signal peptide is derived from human azurocidin protein, and the secretion-promoting element is derived from the Fc domain of immunoglobulin heavy chain constant region γ1 protein (Immunoglobulin Heavy Constant Gamma 1, IGHG1); more preferably, the amino acid sequence of the signal peptide derived from human azurocidin protein is as shown in SEQ ID NO: 9, and the amino acid sequence of the Fc domain derived from immunoglobulin heavy chain constant region γ1 protein is as shown in SEQ ID NO:

10.

7. A recombinant nucleic acid molecule, characterized in that Encoding the fusion protein according to any one of claims 1 to 6; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

8. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 7; preferably, the recombinant gene expression cassette further comprises one or more regulatory sequences; more preferably, the regulatory sequence comprises a promoter, an enhancer, and a terminator.

9. A recombinant vector, characterized in that Comprising the recombinant nucleic acid molecule according to claim 7 or the recombinant gene expression cassette according to claim 8.

10. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 7, or the recombinant gene expression cassette according to claim 8, or the recombinant vector according to claim 9.

11. An immunogenic composition or pharmaceutical composition, characterized in that Comprising one or more fusion proteins according to any one of claims 1 to 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; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

12. The immunogenic composition or pharmaceutical composition according to claim 11, characterized in that The immunogenic composition or pharmaceutical composition comprises the fusion protein A and the fusion protein B, or the immunogenic composition or pharmaceutical composition comprises a recombinant nucleic acid molecule expressing the fusion protein A and the fusion protein B; preferably, the mass ratio of the fusion protein A and the fusion protein B in the immunogenic composition or pharmaceutical composition is 1:

1.

13. A recombinant vaccine, characterized in that Comprising one or more fusion proteins according to any one of claims 1 to 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 or 12; preferably, the recombinant vaccine comprises the fusion protein A and the fusion protein B, or the recombinant vaccine comprises the recombinant nucleic acid molecules expressing the fusion protein A and the fusion protein B; more preferably, the mass ratio of the fusion protein A to the fusion protein B is 1:

1.

14. Use of one or more fusion proteins according to any one of claims 1 to 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 or 12, and / or one or more recombinant vaccines according to claim 13 in the preparation of vaccines or medicaments for prevention, treatment and / or vaccination.

15. The use according to claim 14, characterized in that The drug is used to prevent and / or treat infections or diseases caused by enterococci; preferably, the enterococci are pathogenic and / or drug-resistant enterococci; preferably, the enterococci are Enterococcus faecalis and / or Enterococcus faecium; preferably, the infections or diseases caused by enterococci are urinary tract infections, intra-abdominal infections, wound infections, bacteremia, endocarditis, meningitis, and other types of infections; preferably, other types of infections include respiratory tract infections, skin and soft tissue infections, and gynecological infections.

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