An immunogenic composition for preventing and treating Staphylococcus aureus infection

Abstract

The present invention relates to a new immunogenic composition, fusion protein, recombinant vaccine, and molecular architecture design and application for preventing and treating Staphylococcus aureus infection. The present invention provides a new fusion molecular architecture, which comprises a fusion protein encoded by three genes Tuf, SpxA, and Hu, or a fusion protein encoded by two genes Hla_H35L and EsxB. The present invention finds that the new fusion molecule has good immunogenicity, and can provide an effective immune protection effect, and can eliminate Staphylococcus aureus colonization in some individuals, and can be used for the research and development of nucleic acid vaccines or subunit vaccines. The new molecular architecture of the present invention can effectively increase the expression level of the fusion protein, thereby further improving the immune protection effect. The present invention can be applied to the production and development of Staphylococcus aureus nucleic acid vaccine immune drugs for humans and animals, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, in particular to the field of immune medicine technology, and specifically relates to an immunogenic composition of Staphylococcus aureus capable of inducing immune protection and its application. Background Art

[0002] Staphylococcus is a common Gram-positive coccus that is widely found on human skin and mucous membranes. Currently, pathogenic staphylococci mainly include: Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus epidermidis ( Staphylococcus epidermidis ), coagulase-negative staphylococci, enterococci, etc. Among them, Staphylococcus aureus, referred to as golden Staphylococcus, is the most common pathogenic staphylococcus, which can widely infect humans and a variety of animals, causing a variety of diseases such as mastitis, skin and soft tissue infections, pneumonia, osteomyelitis, endocarditis, etc., which not only endangers human health, but also brings serious economic losses to animal husbandry.

[0003] The pathogenicity of S. aureus comes from the various virulence factors produced during its replication, such as alpha-hemolysin (Hla), immunoglobulin G-binding protein A (SpA), clumping factor (Clf), etc., which can dissolve host cells, cause inflammation, tissue damage, and induce immune escape after infection. For individuals infected with S. aureus, the conventional method is to use antibiotics for treatment. However, S. aureus is prone to drug resistance. Methicillin-resistant S. aureus (MRSA) has already existed. Staphylococcus aureus , MRSA), multi-drug resistant (MDR) and other drug-resistant strains, which bring great challenges to clinical treatment.

[0004] Vaccination is a better solution. Compared with antibiotic treatment, vaccines can provide more lasting protection and prevent infection through active immunization, which enables the body to produce specific antibodies and immune cells against S. aureus. At the same time, protective vaccines can replace antibiotic treatment, significantly reduce the need for antibiotic use, help inhibit the emergence and further spread of new drug-resistant strains, and alleviate clinical treatment pressure. However, due to the powerful immune escape mechanism of S. aureus and its multiple serotypes, the protective efficacy of subunit vaccines is insufficient, and currently no vaccine has been successfully used in clinical treatment.

[0005] Nucleic acid vaccine, also known as genetic vaccine, refers to the introduction of a vector containing a nucleic acid sequence encoding an antigen protein into the host through intramuscular injection or other methods, so that the host cells express the antigen protein and induce the host cells to produce an immune response to the antigen protein, thereby achieving the purpose of preventing and treating diseases. Nucleic acid vaccines are developed using modern biotechnology immunology, biochemistry, molecular biology, etc., and are divided into two types: DNA vaccines and RNA vaccines. Compared with traditional inactivated vaccines, subunit vaccines and genetically engineered vaccines, nucleic acid vaccines have many advantages: (1) Nucleic acid vaccines do not carry virulent virus particles and are safer; (2) Nucleic acid vaccines can complete the expression of antigen proteins in cells, thereby inducing the body's humoral immunity and cellular immunity to the protein, which can enhance the immune effect and prolong the duration of effective protection; (3) After nucleic acid vaccine vaccination, the antigen protein is expressed in the cell and can directly bind to the tissue compatibility complex MHC class I or II molecules without interference from maternal antigens.

[0006] At present, there have been studies on vaccine development based on the main virulence factors and immunodominant proteins of S. aureus, mainly multivalent recombinant protein vaccines and polysaccharide vaccines. For example, SA4Ag is a 4-valent vaccine composed of staphylococcal capsular polysaccharide types 5 and 8 (CP5 / 8), clumping factor A (ClfA), and new recombinant P305A, where P305A is a protein from lipoprotein manganese transporter C (MntC). SA4Ag has been advanced to Phase III clinical trials. Although the vaccine induced the production of specific antibodies, these antibodies failed to show significant protective effects. For example, PentaStaph is a multivalent vaccine composed of CP5, CP8, α-toxin, Valentine Leukocidin S (LukSPV) and cell wall acid. The vaccine was taken over by GlaxoSmithKline (GSK) after Phase I clinical trials, and was later jointly developed with the Technical University of Denmark (DTU) and changed to Vaccia Ankara (MVA)-based immunity. In addition, Novartis (now GSK) has developed a multivalent vaccine based on the S. aureus surface proteins SdrE, IsdA, SdrD and IsdB, which is still in the animal research stage.

[0007] The above subunit vaccines can all induce humoral immune responses, but the latest studies have shown that the protection provided by humoral immunity is not enough to deal with S. aureus infection. In the long-term protection against S. aureus infection, T cell-mediated immune responses play a more important role. Subunit vaccines are limited by their own properties and immune principles, and it is difficult to induce cellular immunity, but nucleic acid vaccines can effectively solve this problem. The above subunit vaccines have achieved good results in the animal experiment stage, but all ended in failure in clinical trials. The reason is that humans and animals have non-protective humoral immune imprints against S. aureus infection, and the antibodies activated by subunit vaccines are non-protective antibodies that already exist in the human body. Non-protective humoral immune imprints can lead to falsely high neutralizing antibody titers and produce misleading results. In the design of S. aureus mRNA vaccines, the selection of toxin antigens and subdominant cell wall antigens (CWAs) antigen fusion expression can avoid the adverse effects of humoral immune imprints and is an effective way to develop S. aureus vaccines or pathogenic staphylococcal vaccines.

[0008] The prior art discloses a variety of Staphylococcus aureus vaccines. Patent document 1 (CN115975865A, published on 2023-04-18) discloses an antigen carrier strain, a recombinant strain and its application in the preparation of an oral Staphylococcus aureus vaccine. The strain is LR076, which was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 27, 2022, with a deposit number of CGMCC No. 25845; the recombinant strain is formed by transforming a recombinant plasmid carrying an exogenous Staphylococcus aureus antigen fragment into the LR076 strain; the antigen fragment includes HlaH35L, IsdB, and MntC. The application of the recombinant strain in the preparation of an oral Staphylococcus aureus vaccine can effectively alleviate lung lesions after infection and reduce the fixed amount of Staphylococcus aureus in the lungs. Patent document 2 (CN117355328A, published on 2024-01-05) discloses an immunogenic composition for inducing an immune response in a subject to treat and / or prevent Staphylococcus aureus infection, the immunogenic composition comprising a Staphylococcus aureus protein A (SpA) polypeptide and a Staphylococcus aureus leukocidin A (LukA) and / or leukocidin B (LukB) variant polypeptide. Patent document 3 (WO2024040363A1, published on 2024-02-29) discloses a Staphylococcus aureus vaccine and a preparation method thereof, and the use of a PLGA-PEG copolymer in preparing a vaccine; the vaccine contains adjuvant nanoparticles, the adjuvant nanoparticles contain PLGA or PLGA-PEG copolymers, and Staphylococcus aureus antigens, and the Staphylococcus aureus antigens are covalently linked to the adjuvant nanoparticles. However, the above-mentioned Staphylococcus aureus vaccine still has defects such as insufficient immunogenicity and T cell immune response.

[0009] Although the above-mentioned subunit vaccines and Staphylococcus aureus vaccines disclosed in the prior art are available, there is still an urgent need in the art for a Staphylococcus aureus vaccine with better immunogenicity and capable of providing effective immune protection. Summary of the invention

[0010] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a new immunogenic composition, fusion protein, recombinant vaccine, and molecular architecture design and application for preventing and treating Staphylococcus aureus infection. The present invention provides a new fusion molecular architecture, which comprises a fusion protein encoded by three genes Tuf, SpxA, and Hu, or a fusion protein encoded by two genes Hla_H35L and EsxB, which can be used for the research and development of nucleic acid vaccines or subunit vaccines. The present invention finds that the new fusion molecule has good immunogenicity, and can provide an effective immune protection effect, and can eliminate Staphylococcus aureus colonization in some individuals. The present invention also finds that the new molecular architecture can effectively increase the expression level of the fusion protein, thereby further improving the immune protection effect. The present invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, uses, etc.

[0011] One aspect of the present invention provides a fusion protein, characterized in that it is selected from the following (1) or (2):

[0012] (1) Fusion protein A, wherein the fusion protein A comprises an elongation factor Tu (Tuf) antigen, a transcriptional regulator SpxA (SpxA) antigen, and a HU family DNA-binding protein (DNA-binding protein Hu) antigen;

[0013] (2) A fusion protein B, wherein the fusion protein B comprises an alpha toxin H35L mutant (Hla_H35L) antigen and an ESAT-6-like protein EsxB (EsxB) antigen.

[0014] Furthermore, it is characterized in that the antigen is derived from cocci; preferably, from Staphylococci; most preferably, from Staphylococcus aureus.

[0015] Furthermore, it is characterized in that the amino acid sequence of the elongation factor Tu antigen is shown in SEQ ID NO: 1, the amino acid sequence of the transcription regulatory factor SpxA antigen is shown in SEQ ID NO: 2, the amino acid sequence of the HU family DNA binding protein antigen is shown in SEQ ID NO: 3, the amino acid sequence of the α-toxin H35L mutant antigen is shown in SEQ ID NO: 4, and the amino acid sequence of the ESAT-6-like protein EsxB antigen is shown in SEQ ID NO: 5.

[0016] Furthermore, it is characterized in that the fusion protein A comprises, from the N-terminus to the C-terminus, an elongation factor Tu antigen, a transcription regulator SpxA antigen, and a HU family DNA binding protein antigen; and the fusion protein B comprises, from the N-terminus to the C-terminus, an alpha toxin H35L mutant antigen and an ESAT-6-like protein EsxB antigen.

[0017] Furthermore, it is characterized in that the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 12, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO: 13.

[0018] Furthermore, it is characterized in that the N-terminus of the fusion protein B also comprises a signal peptide and an Fc domain of an immunoglobulin heavy chain constant region γ1 protein (Immunoglobulin Heavy Constant Gamma 1, IGHG1) from any species; preferably, the signal peptide is derived from human Azurocidin protein or bovine IGHG1 protein, more preferably, the amino acid sequence of the signal peptide derived from human Azurocidin protein is as shown in SEQ ID NO: 6, and the amino acid sequence of the signal peptide derived from bovine immunoglobulin heavy chain constant region γ1 protein is as shown in SEQ ID NO: 7; preferably, the Fc domain of the immunoglobulin heavy chain constant region γ1 protein is derived from human or bovine, more preferably, the amino acid sequence of the Fc domain of the immunoglobulin heavy chain constant region γ1 protein derived from human is as shown in SEQ ID NO: 8, and the amino acid sequence of the Fc domain of the immunoglobulin heavy chain constant region γ1 protein derived from bovine is as shown in SEQ ID NO: 9.

[0019] Optionally, the various elements of the fusion protein are connected by a connecting peptide; preferably, the connecting peptide is a spacer sequence or a linker sequence; more preferably, the amino acid sequence of the spacer sequence is as shown in SEQ ID NO: 10, and the amino acid sequence of the linker sequence is as shown in SEQ ID NO: 11.

[0020] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it encodes the fusion protein of the present invention.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, 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; preferably, the immunogenic composition or pharmaceutical composition also comprises a pharmaceutically acceptable carrier.

[0025] Furthermore, it is characterized in that the immunogenic composition or pharmaceutical composition contains 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.

[0026] Another aspect of the present invention provides a recombinant vaccine, 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, and / or one or more immunogenic compositions or pharmaceutical compositions described in the present invention.

[0027] 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 biological immunity drugs for prevention, treatment and / or vaccination.

[0028] Furthermore, it is characterized in that the drug is used to prevent and / or treat tissue infection caused by Staphylococcus; preferably, the Staphylococcus is Staphylococcus aureus; preferably, the tissue infection is skin tissue infection, breast infection, abdominal cavity infection, etc.; more preferably, the tissue infection is breast infection.

[0029] Another aspect of the present invention provides a method for preventing and / or treating a disease, characterized in that the immunogenic composition or pharmaceutical composition of the present invention, or the recombinant vaccine of the present invention is inoculated into a subject.

[0030] Furthermore, it is characterized in that the disease is a tissue infection caused by Staphylococcus; preferably, the Staphylococcus is Staphylococcus aureus; preferably, the tissue infection is skin tissue infection, breast infection, abdominal cavity infection, etc.; more preferably, the tissue infection is breast infection.

[0031] The fusion protein, immunogenic composition, recombinant vaccine, etc. of the present invention have the following beneficial technical effects:

[0032] 1. The present invention finds that the new fusion molecule has good immunogenicity and can provide immune protection, and can be used in the development of nucleic acid vaccines or subunit vaccines. The immunogenic composition of the present invention can provide effective immune protection against infection with Staphylococcus aureus.

[0033] 2. The present invention also found that the new molecular architecture can effectively increase the expression level of the fusion protein, thereby further improving the immune protection effect. The Tuf-SpxA-Hu fusion protein involved in the vaccine A of the present invention and the Hla_H35L-EsxB fusion protein involved in the vaccine B, the molecular architecture of their secretory expression effectively increases the expression level of the fusion protein, thereby improving the immune effect, and provides new technology for the field of immune drug design of Staphylococcus aureus multivalent vaccine.

[0034] 3. Example 4 of the present invention shows that the vaccines designed based on the present invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to the presentation of immune epitopes.

[0035] 4. Example 5 of the present invention shows that in a mouse mastitis model, the vaccine A based on the present invention can provide a good immunotherapy effect and eliminate bacterial colonization in 33.3% of individuals. Detection by tissue section and HE staining method showed that the inflammatory cell infiltration in the vaccine A treatment group was significantly alleviated, and the tissue structure was clear and complete.

[0036] 5. Example 5 of the present invention also shows that the immune effect of vaccine C with a single antigen is significantly lower than that of vaccine A with multiple antigens. The reason may be that Staphylococcus aureus has multiple immune escape mechanisms and can escape the host's immune system through multiple virulence proteins secreted by itself, so a single antigen target cannot provide good immune protection. The vaccine based on the combination of multiple antigen targets of the present invention can provide more immune epitopes, which is conducive to improving the effect of immunotherapy and achieving the elimination of colonized bacteria in some individuals.

[0037] 6. Example 6 of the present invention shows that in the mouse abdominal infection model, both vaccine A and vaccine B according to the present invention have good preventive effects after immunization. Among them, vaccine A and vaccine B co-immunization has the best preventive effect: compared with the control group, the blood bacterial load (median) is reduced by about 18 times, the peritoneal fluid bacterial load (median) is reduced by about 20 times, and the spleen bacterial load (median) is reduced by about 7000 times; the preventive effect of vaccine B alone is second: compared with the control group, the blood bacterial load (median) is reduced by about 6 times, the peritoneal fluid bacterial load (median) is reduced by about 12 times, and the spleen bacterial load (median) is reduced by about 3500 times; the preventive effect of vaccine A alone is the third: compared with the control group, the blood bacterial load (median) is reduced by about 4 times, the peritoneal fluid bacterial load (median) is reduced by about 5 times, and the spleen bacterial load (median) is reduced by about 2300 times. However, whether it is vaccine A or vaccine B alone, it provides strong immune protection.

[0038] 7. Examples 4, 5 and 6 of the present invention collectively show that the vaccine based on the present invention can induce effective immunity in model animals and mice, and has therapeutic and preventive effects on Staphylococcus aureus infection. The present invention can be applied to the production and development of human and animal immune drugs, filling the gap in the current research and development of Staphylococcus aureus nucleic acid vaccines, and has extremely high commercial value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2A-2C The quality control peak graph and purity test results of nucleic acid vaccines A, B, and C containing the antigen sequence of the present invention; wherein Figure 2A This is the quality control peak diagram and purity test results of vaccine A. Figure 2B This is the quality control peak diagram and purity test results of vaccine B. Figure 2C This is the quality control peak diagram and purity test results of vaccine C.

[0041] Figure 3A-3C The expression effect of vaccines A, B and C of the present invention after in vitro transfection of HEK293T cells; wherein Figure 3A This is the in vitro expression result of vaccine A. Figure 3B is the in vitro expression result of vaccine B. Figure 3C This is the in vitro expression result of vaccine C.

[0042] Figure 4 The present invention shows the immunization and sampling process of vaccines A and C in a mouse mastitis model.

[0043] Figure 5A-Figure 5B is the bacterial load in the mammary glands of mice with mastitis after treatment with vaccine C and vaccine A of the present invention; Figure 5A is the mammary bacterial load of mice with mastitis after treatment with vaccine C, Figure 5B is the mammary bacterial load of mice with mastitis after treatment with vaccine A.

[0044] Figure 6 This is a HE staining image of mammary tissue sections of mastitis mice after treatment with the vaccine A of the present invention.

[0045] Figure 7 The present invention discloses the immunization and sampling process of vaccine A, vaccine B, and vaccine A and B co-immunization in a mouse abdominal infection model.

[0046] Figure 8A-8C The preventive and protective effects of vaccine A, vaccine B, and vaccine A and B co-immunization in a mouse abdominal infection model; wherein Fig. 8A is the blood bacterial load of mice after immunization and challenge. Figure 8B is the bacterial load in the peritoneal fluid of mice after immunization and challenge. Figure 8C This is the bacterial load in the spleen of mice after immunization and challenge. DETAILED DESCRIPTION

[0047] Terms and Definitions

[0048] The term "S. aureus" refers to Staphylococcus aureus ( Staphylococcus aureus , S. aureus ), is a Gram-positive coccus belonging to the genus Staphylococcus. It is spherical and exists alone, in pairs, short chains or irregular agglomerations.

[0049] The term "Staphylococcal infection" is meant to include infections caused by S. aureus and / or S. epidermidis as well as other strains of Staphylococci that are capable of causing infection in a mammal, preferably a human host.

[0050] The term "Hla_H35L" refers to a mutant of an alpha toxin from Staphylococcus bacteria, i.e., the 35th amino acid of wild-type Hla is mutated from histidine (His) to leucine (Leu). Proteins comprising Hla_H35L polypeptides and segments thereof, and variants that can stimulate an immune response against Hla_H35L proteins of Staphylococcus bacteria. Preferably, the amino acid sequence of Hla_H35L is as shown in SEQ ID NO: 4.

[0051] The term "EsxB" refers to an ESAT-6-like protein EsxB from Staphylococcus bacteria. Proteins comprising isolated wild-type EsxB polypeptides and fragments thereof from bacteria and variants that can stimulate an immune response against EsxB proteins from Staphylococcus bacteria. Preferably, the amino acid sequence of the EsxB is as shown in SEQ ID NO: 5.

[0052] The term "Tuf" refers to the Elongation factor Tu protein from Staphylococcus bacteria. Proteins comprising isolated wild-type Tuf polypeptides and segments thereof from bacteria and variants that can stimulate an immune response against the Tuf protein from Staphylococcus bacteria. Preferably, the amino acid sequence of Tuf is shown in SEQ ID NO: 1.

[0053] The term "SpxA" refers to the global transcriptional regulator Spx protein from Staphylococcus bacteria. Proteins comprising isolated wild-type SpxA polypeptides and segments thereof from bacteria and variants that can stimulate an immune response against the SpxA protein of Staphylococcus bacteria. Preferably, the amino acid sequence of the SpxA is as shown in SEQ ID NO: 2.

[0054] The term "DNA-binding protein Hu" or "HU family DNA-binding protein" or "Hu" refers to a HU family DNA-binding protein protein from Staphylococcus bacteria. Proteins comprising isolated wild-type DNA-bindingprotein Hu polypeptides and segments thereof from bacteria and variants that can stimulate an immune response against the DNA-binding protein Hu protein of Staphylococcus bacteria. Preferably, the amino acid sequence of the DNA-bindingprotein Hu is as shown in SEQ ID NO: 3.

[0055] The term "PmtC" refers to a phenol-soluble modulin export ABC transporter ATP-binding protein PmtC (PmtC) from Staphylococcus bacteria. Proteins comprising isolated wild-type PmtC polypeptides and segments thereof from bacteria and variants that can stimulate an immune response against PmtC proteins from Staphylococcus bacteria. Preferably, the amino acid sequence of the PmtC is as shown in SEQ ID NO: 14.

[0056] The term "immune response" refers to a humoral response, a cellular response, or both a humoral and a 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 proteins or cell surface proteins, assays that measure T cell activation or proliferation, and / or assays that measure the modulation of one or more cytokine activity or expression.

[0057] The term "administration" or "vaccination" refers to administration based on the 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-injection administration), intralymph node, intradermal, intraperitoneal, rectal or vaginal administration, or by a combination of routes. Administration in the neck muscle of the animal is preferred. The administration regimen can be adjusted to provide optimal immunity using an accelerated regimen (boosting regimens).

[0058] 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.

[0059] 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", etc.

[0060] The term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. 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 start and stop sequences. The recombinant expression vector is 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 a combination of plasmids and phage DNA, DNA from viruses such as lentivirus, retrovirus, vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Both self-replicating and non-self-replicating vectors are included.

[0061] The term "mRNA" refers to messenger RNA, which is translated into Chinese as "messenger ribonucleic acid". It is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA as a template, carries genetic information and can guide protein synthesis.

[0062] The term "5'-UTR", which refers to the "5' untranslated region" or "5'UTR", is a portion of a gene that is transcribed into a primary RNA transcript (pre-mRNA) and is 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 physiologically active RNA. The processing to form mature mRNA includes modifying the ends, removing introns, capping and / or shearing off individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of an mRNA is a portion of the mRNA that is not translated into protein and is located upstream of the coding sequence. In a genomic sequence, the 5'UTR is usually defined as the region located between the transcription start site and the start codon. The 5' untranslated region (5'UTR) of vertebrate mRNA can be tens to hundreds of bases in length.

[0063] The term "3'-UTR", meaning "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, and which is transcribed but not translated into an amino acid sequence, or to the corresponding region in an RNA molecule. The 3'-untranslated region 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'-untranslated region of mammalian mRNA typically 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'-untranslated region may contain one or more inverted repeats and may fold to produce a stem-loop structure that acts as a barrier to exoribonucleases or interacts with proteins (e.g., RNA binding proteins) that are known to increase RNA stability.

[0064] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including 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, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells.

[0065] The terms "individual", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., pigs, cows, sheep, cats, dogs, and horses, etc.), primates (e.g., humans and non-human primates such as monkeys), and rodents (e.g., rabbits, mice, and rats, etc.).

[0066] 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.

[0067] The term "drug combination" or "pharmaceutical composition" refers to auxiliary materials widely used in the field of drug production. The main purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature and / or has specific functionality, and also to provide a method so that it can be effectively absorbed in the body of a subject. A pharmaceutically acceptable carrier can be an inert filler or an effective component that provides a certain function for the pharmaceutical combination (such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, adhesives, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, glidants, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0068] The term "treatment" means that after a subject has contracted a disease, the subject is exposed to (e.g., administered) a recombinant vaccine, composition, etc. according to the present invention, thereby alleviating the symptoms of the disease compared to when the subject has not contracted the disease, and does not necessarily mean that the symptoms of the disease are completely suppressed. Contracting a disease means that the body has symptoms of the disease.

[0069] 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.

[0070] 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.

[0071] The present invention discloses a fusion molecule architecture that effectively improves the antigen presentation efficiency of cells, a method for preparing a recombinant vaccine based on the architecture, and an application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are deemed 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.

[0072] In the fusion protein and encoding nucleic acid and elements thereof, preparation methods and applications provided by the present invention, the raw materials and reagents used can all be obtained commercially.

[0073] The present invention is further illustrated below in conjunction with the examples, wherein, as a preferred embodiment, a nucleic acid vaccine framework is selected for preparing a recombinant vaccine.

[0074] Example 1 Construction of the recombinant nucleic acid vaccine of the present invention

[0075] In order to prepare a recombinant nucleic acid vaccine comprising an antigen of the present invention, a non-limiting schematic diagram of the nucleic acid vaccine of the present invention is shown in FIG. Figure 1 As shown, exemplary Figure 1 The molecular structure diagram of the fusion protein expressed by vaccine A and vaccine B of the present invention is shown in FIG. Figure 1 The recombinant nucleic acid vaccine of the molecular structure shown first constructs a gene expression cassette for expressing the antigen sequence described in the present invention. The expression cassette comprises, from the 5' end to the 3' end, 5'UTR, CDS region, 3'UTR, PolyA, wherein the CDS region comprises the fusion molecule architecture described in the present invention. 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 KingScript 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 to obtain a vector plasmid for preparing a recombinant nucleic acid vaccine.

[0076] According to the above method, the carrier used in the subsequent examples was prepared:

[0077] (1) Preparation of vectors based on the recombinant nucleic acid vaccine A of the present invention

[0078] Step a: Synthesize the fusion fragment of the three genes "Tuf-SpxA-Hu", which are connected by the linker sequence shown in SEQ ID NO: 11, and the amino acid sequence of the fusion protein encoded by the Tuf-SpxA-Hu fusion gene is shown in SEQ ID NO: 12. Among them, the amino acid sequence of the Tuf antigen is shown in SEQ ID NO: 1, the amino acid of the SpxA antigen is shown in SEQ ID NO: 2, and the amino acid of the Hu (i.e., HU family DNA-binding protein, DNA-binding protein Hu) antigen is shown in SEQ ID NO: 3.

[0079] Step b: Construct a nucleic acid vaccine framework vector.

[0080] 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.

[0081] Step c: preparing recombinant plasmid.

[0082] 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 C based on the present invention.

[0083] (2) Preparation of vectors based on the recombinant nucleic acid vaccine B of the present invention

[0084] Step a: Synthesize the gene fragment of "human signal peptide-Fc domain of human IGHG1-Hla_H35L-EsxB". The human signal peptide is a signal peptide of human Azurocidin protein, and its amino acid sequence is shown in SEQ ID NO: 6, the amino acid of the Fc domain of human IGHG1 is shown in SEQ ID NO: 8, the amino acid of Hla_H35L antigen is shown in SEQ ID NO: 4, the amino acid of EsxB antigen is shown in SEQ ID NO: 5, the Fc domain of human IGHG1 and Hla_H35L are connected by a spacer sequence shown in SEQ ID NO: 10, Hla_H35L and EsxB are connected by a linker sequence shown in SEQ ID NO: 11, and the amino acid sequence of the fusion protein encoded by the Hla_H35L-EsxB fusion gene is shown in SEQ ID NO: 13.

[0085] Step b: Construct a nucleic acid vaccine framework vector.

[0086] 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.

[0087] Step c: preparing recombinant plasmid.

[0088] 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.

[0089] (3) Preparation of vector based on the recombinant nucleic acid vaccine C of the present invention

[0090] Step a: Synthesize the gene fragment of "bovine signal peptide-Fc domain of bovine IGHG1-PmtC". The bovine signal peptide is a bovine IGHG1 protein signal peptide, whose amino acid sequence is shown in SEQ ID NO: 7, the amino acid of the Fc domain of bovine IGHG1 is shown in SEQ ID NO: 9, and the amino acid of PmtC is shown in SEQ ID NO: 14.

[0091] Step b: Construct a nucleic acid vaccine framework vector.

[0092] 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.

[0093] Step c: preparing recombinant plasmid.

[0094] 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 C based on the present invention.

[0095] Table 1 Protein amino acid sequences of the structural elements involved in the present invention

[0096] Amino acid sequence and sequence number Tuf antigen make IPTPERDSDKPFMMPVEDVFSITGRGTVATGRVERGQIKVGEEVEIIGLHDTSKTTVTGVEMFRKLLDYAEAGDNIGALLRGVAREDVQRGQVLAAPGSITPHTEFKAEVYVLSKDEGGRHTPFFSNYRPQFYFRTTDVTGVVHLPEGTEMVMPGDNVEMTVELIAPIAIEDGTRFSIREGGRTVGSGVVTEIIK (SEQ ID NO: 1) SpxA antigen MVTLFTSPSCTSCRKAKAWLQEHDIPYTERNIFSEHLTIDEIKQILKMTEDGTDEIISTRSKTYQKLNVDIDSLPLQDLYSIIQDNPGLLRRPIILDNKRLQVGYNEDEIRRFLPRKVRTFQLQEAQRMVD (SEQ ID NO: 2) DNA-binding protein Hu antigen MNKTDLINAVAEQADLTKKEAGSAVDAVFESIQNSLAKGEKVQLIGFGNFEVRERAARKGRNPQTGKEIDIPASKVPAFKAGKALKDAVK (SEQ IDNO: 3) Alpha toxin H35L mutant antigen ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKILVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLSDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVAGDDSGKIGGLIGAQVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN (SEQ ID NO: 4) EsxB antigen MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPIADHQKAIDQLSQNLAKYDTLSIKQGLDRVNP (SEQ ID NO: 5) Signal peptide of human Azurocidin protein MTRLTVLALLAGLLASSRA (SEQ ID NO: 6) Signal peptide of bovine IGHG1 protein MNPLWTLLFVLSAPRGVLS (SEQ ID NO: 7) Fc domain of human IGHG1 protein EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) Fc domain of bovine IGHG1 protein DPRCKTTCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK (SEQ ID NO: 9) Spacer sequence GGSGGSGGSG (SEQ ID NO: 10) Linker sequence GGSGGGGSGG (SEQ ID NO: 11) Tuf-SpxA-Hu fusion protein MAKEKFDRSKEHANIGTIGHVDHGKTTLTAAIATVLAKNGDSVAQSYDMIDNAPEEKERGITINTSHIEYQTDKRHYAHVDCPGHADYVKNMITGAAQMDGGILVVSAADGPMPQTREHILLSRNVGVPALVVFLNKVDMVDDEELLELVEMEVRDLLSEYDFPGDDVPVIAGSALKALEGDAQYEEKILELMEAVDTYIPTPERDSDKPFMMPVEDVFSITGRGTVATGRVERGQIKVGEEVEIIGLHDTSKTTVTGVEMFRKLLDYAEAGDNIGALLRGVAREDVQRGQVLAAPGSITPHTEFKAEVYVLSKDEGGRHTPFFSNYRPQFYFRTTDVTGVVHLPEGTEMVMPGDNVEMTVELIAPIAIEDGTRFSIREGGRTVGSGVVTEIIKGGSGGGGSGGMVTLFTSPSCTSCRKAKAWLQEHDIPYTERNIFSEHLTIDEIKQILKMTEDGTDEIISTRSKTYQKLNVDIDSLPLQDLYSIIQDNPGLLRRPIILDNKRLQVGYNEDEIRRFLPRKVRTFQLQEAQRMVDGGSGGGGSGGMNKTDLINAVAEQADLTKKEAGSAVDAVFESIQNSLAKGEKVQLIGFGNFEVRERAARKGRNPQTGKEIDIPASKVPAFKAGKALKDAVK (SEQ ID NO: 12) Hla_H35L-EsxB fusion protein ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKILVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLSDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVAGDDSGKIGGLIGAQVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNGGSGGGGSGGMGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPIADHQKAIDQLSQNLAKYDTLSIKQGLDRVNP (SEQ ID NO: 13) PmtC antigen MKLEHITKKYGSNVVLNDIDFDFGDSRIVGLIGKNGVGKTTVMKVMNGNIIKFDGKVDIDNADNIGFLIEHPKLYDQKSGLYNLKLFAQVLGKGFDKAYTDKIIDAFGMRPYIKKKVKKYSMGMKQKLAIAVSLMNKPKFLILDEPTNGMDPDGSIDVLTTIKSLVNELDMRILISSHKLEDIELICDRAVFLRDGHFVQDVNMEEGVASDTTIVTVDHKDFDRTEKYLAEHFQLQNVDKADGHLMINAQKNYQVILKALSELDIYPKYIETRKSSLRDTYFNINQRGDK (SEQ IDNO: 14)

[0097] Example 2 Preparation of the recombinant nucleic acid vaccine of the present invention

[0098] (1) Preparation of capped mRNA vaccines

[0099] Step a: The vector plasmid used to produce the capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0100] 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.

[0101] (2) Preparation of non-capped mRNA vaccines

[0102] Step a: The vector plasmid used to produce the non-capped mRNA vaccine in Example 1 is linearized by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0103] Step b: The linearized plasmid is subjected to an in vitro non-capped transcription reaction and the template DNA is degraded.

[0104] (3) DNA vaccine preparation

[0105] Step a: Amplify the vector plasmid used to produce the DNA vaccine in Example 1 to obtain a large amount of target plasmid for purification.

[0106] Step b: Use an endotoxin-free plasmid extraction and purification kit to extract and purify the target plasmid.

[0107] Example 3 Quality Control of In Vitro Transcription of Recombinant Nucleic Acid and Vaccine Preparation of the Present Invention

[0108] Vaccine A (based on the recombinant nucleic acid vaccine A of the present invention), vaccine B (based on the recombinant nucleic acid vaccine B of the present invention), and vaccine C (based on the recombinant nucleic acid vaccine C of the present invention) were prepared by the method for preparing the capped mRNA vaccine in Example 2. The purity of the produced recombinant nucleic acid was tested, and the purity of the recombinant nucleic acid used in the experiment was greater than 85%. The quality control peak diagram of the recombinant nucleic acid of the present invention is as follows: Figure 2A , Figure 2B , Figure 2CAs shown. Specifically described as follows: (1) The recombinant nucleic acid vaccine A based on the present invention has a purity of 90.8%; (2) The recombinant nucleic acid vaccine B based on the present invention has a purity of 89.1%; (3) The recombinant nucleic acid vaccine C based on the present invention has a purity of 91.5%. The above purities all meet the quality requirements of cell transfection experiments and vaccine production.

[0109] Example 4 In vitro expression effect of the recombinant nucleic acid of the present invention

[0110] Vaccines A and B in Example 3 were transfected into HEK293T cells using a cell transfection reagent, and proteins were collected after 48 hours of in vitro culture for Western blot detection.

[0111] Figure 3A , Figure 3B , Figure 3C The results of in vitro expression of HEK293 cells transfected with vaccines A, B, and C are shown in the Western blot test. The antigen expressed by vaccine A is a cellular immune antigen, which can theoretically be detected in the cell lysate. The antigens expressed by vaccines B and C are humoral immune antigens, which can theoretically be detected in the supernatant. Vaccines containing multiple cellular immune antigens can detect significant protein expression in cell lysates; vaccines containing multiple humoral immune antigens can detect significant protein expression in the supernatant. The protein molecular weights of vaccines A, B, and C are shown in Table 2.

[0112] Among them, vaccine A can be significantly expressed in the cell, proving that the molecular architecture that promotes antigen fusion expression provided by the present invention can make multiple antigens smoothly translated and correctly folded in eukaryotic cells, and the structure is stable and the half-life is long. Vaccine B can be significantly expressed in the supernatant, proving that the molecular architecture that promotes the secretory expression of fusion proteins provided by the present invention can make multiple antigens smoothly translated, correctly folded, and secreted to the extracellular space in eukaryotic cells. Vaccine C can be significantly expressed in the supernatant, proving that the humoral immune antigen target provided by the present invention can be smoothly translated, correctly folded, and secreted to the extracellular space in eukaryotic cells. Therefore, the vaccine designed based on the present invention, whether it is a single antigen or a combination of antigens, can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is conducive to immune epitope presentation.

[0113] Table 2 Protein molecular weight of vaccines A, B, and C

[0114] Name Protein molecular weight (kDa) Vaccine A 82.9 Vaccine B 77.5 Vaccine C 65.2

[0115] Example 5 The therapeutic effect of the recombinant nucleic acid vaccine of the present invention in a mouse mastitis model

[0116] In order to verify whether the cellular immune antigen fusion molecule has an immune protection effect, this example selected vaccine A and vaccine C to conduct immunotherapy experiments.

[0117] The experiment used 16 8-week-old Balb / c female mice of similar size and weight. After 3-7 days of adaptive feeding, they were caged together for littering. S. aureus Mastitis model was established in female mice for immunotherapy experiments, as shown in Table 3.

[0118] Table 3 Modeling method of mouse mastitis model in Example 5

[0119]

[0120] The mouse mastitis model was established in the above manner, with a total of 3 groups. The immunization and treatment process is shown in Table 4, and the dosage in this table and below refers to the amount of active ingredient.

[0121] Table 4 Treatment schedule for animals in the immunization experiment of Example 5

[0122]

[0123] Note: PBS refers to the use of PBS solution instead of vaccine, which is the control group (non-treatment group) and serves as the control group for immunotherapy experiments.

[0124] The mice in the above groups were immunized five times according to the immunization process in Table 4. Samples were collected on Day 17 to detect the bacterial load in the tissues. Figure 4 shown.

[0125] The mammary bacterial load of mice with mastitis treated with vaccine C was as follows: Figure 5A As shown. The experimental results show that vaccine C has a therapeutic effect, but the effect is not significant (p>0.05). This proves that the recombinant vaccine containing a single antigen target based on the present invention can provide a certain degree of immunotherapy, but cannot eliminate bacterial colonization.

[0126] The mammary bacterial load of mice with mastitis treated with vaccine A was as follows: Figure 5B As shown, the results showed that the mammary bacterial load of mice treated with vaccine A was significantly reduced (p<0.05). Compared with the bacterial load of the control group (~10,000 CFU / g), 5 / 6 (83.3%) mice in the treatment group (vaccine A) had a bacterial load below 500 CFU / g, of which 2 mice (33.3%) had a bacterial load of 0 CFU / g, indicating that Staphylococcus aureus in their bodies had been completely eliminated. This proves that the cellular immune recombinant vaccine based on the present invention can provide excellent immunotherapy effects and can eliminate bacterial colonization in 33.3% of individuals.

[0127] In addition, the recovery of mammary gland tissue in the control group and the vaccine A treatment group was detected by tissue sectioning and HE staining ( Figure 6 ), in order to evaluate the therapeutic effect of vaccine A. As can be seen from the result figure, the mammary gland tissue alveolar structure of the control group was severely damaged, the alveolar wall was significantly thickened, and inflammatory cells gathered in the mammary gland alveolar cavity. In the vaccine A treatment group, the inflammatory cell infiltration was significantly alleviated, and the tissue structure was clear and complete. This result proves that vaccine A can provide a good therapeutic effect for mastitis caused by Staphylococcus aureus infection.

[0128] The reason for the above results is that Staphylococcus aureus has multiple immune escape mechanisms and can escape the host's immune system through multiple virulence proteins secreted by itself, so a single antigen target cannot provide good immune protection. The vaccine based on the combination of multiple antigen targets of the present invention can provide more immune epitopes, which is conducive to improving the effect of immunotherapy and achieving the elimination of colonized bacteria in some individuals.

[0129] Example 6 Preventive effect of the recombinant nucleic acid vaccine of the present invention in a mouse abdominal infection model

[0130] In order to verify whether the humoral and cellular immune recombinant nucleic acid vaccines of the present invention can prevent Staphylococcus aureus infection after immunization, this example uses vaccines A and B for experiments. A total of 20 7-week-old Balb / c male mice with similar body shape and weight were selected for the experiment. After adaptive feeding, the mice were randomly divided into 4 groups, 5 in each group, for immunotherapy experiments. The details are shown in Table 5.

[0131] Table 5 Immunization process of experimental animals in Example 6

[0132]

[0133] Note: PBS refers to the use of PBS solution instead of vaccine, which is the control group (non-immune group) and serves as the control group of the immunization experiment.

[0134] The mice in the above groups were immunized twice on day 0 (Day 0) and day 21 (Day 21) according to the immunization process in Table 5. On day 35 (Day 35), the mice were intraperitoneally injected with 500 μl of Staphylococcus aureus solution (1×10 8 CFU), and the mice were raised normally after injection, and the clinical manifestations of the mice were observed and monitored every day. The mice were killed on Day 38, and samples were collected to test the bacterial load in blood, peritoneal fluid, and spleen. The immunization and sampling process is as follows Figure 7 shown.

[0135] After infection, the blood bacterial load of mice in different groups was as follows: Figure 8A As shown in Figure 2, the bacterial load in peritoneal fluid is as follows:Figure 8B As shown, the bacterial load in the spleen is Figure 8C As shown in the experimental results, the bacterial load in the blood, peritoneal fluid and spleen of mice immunized with the vaccine was significantly lower than that of the control group (p<0.001). This proves that all three immunization schemes can induce protective immunity in mice, and both vaccine A and vaccine B have good preventive effects. Among them, the combined immunization of vaccine A and vaccine B has the best preventive effect: compared with the control group, the blood bacterial load (median) is reduced by about 18 times, the peritoneal fluid bacterial load (median) is reduced by about 20 times, and the spleen bacterial load (median) is reduced by about 7000 times; the preventive effect of vaccine B alone is second: compared with the control group, the blood bacterial load (median) is reduced by about 6 times, the peritoneal fluid bacterial load (median) is reduced by about 12 times, and the spleen bacterial load (median) is reduced by about 3500 times; the preventive effect of vaccine A alone is the third: compared with the control group, the blood bacterial load (median) is reduced by about 4 times, the peritoneal fluid bacterial load (median) is reduced by about 5 times, and the spleen bacterial load (median) is reduced by about 2300 times. However, whether it is vaccine A or vaccine B alone, it provides strong immune protection.

[0136] In summary, the antigen and fusion molecule architecture provided by the present invention can induce effective immunity in model animals and mice, and has therapeutic and preventive effects on Staphylococcus aureus infection. Therefore, the present invention can be applied to the production and development of animal immune drugs, filling the gap in the current research and development of Staphylococcus aureus nucleic acid vaccines, and has extremely high commercial value and broad application prospects.

[0137] The above embodiments of the present disclosure are merely examples for clearly illustrating the present disclosure, and are not intended to limit the implementation methods of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation methods here. Any modifications, equivalent substitutions, improvements, etc. 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 It is fusion protein A or fusion protein B, the amino acid sequence of the fusion protein A is shown in SEQ ID NO: 12, and the amino acid sequence of the fusion protein B is shown in SEQ ID NO:

13.

2. The fusion protein according to claim 1, characterized in that The fusion protein A comprises an elongation factor Tu antigen, a transcriptional regulator SpxA antigen and a HU family DNA binding protein antigen; the fusion protein B comprises an alpha toxin H35L mutant antigen and an ESAT-6-like protein EsxB antigen.

3. The fusion protein according to claim 1 or 2, characterized in that The N-terminus of the fusion protein B further comprises a signal peptide and / or an Fc domain of a γ1 protein of an immunoglobulin heavy chain constant region from any species.

4. The fusion protein according to claim 3, characterized in that The signal peptide is derived from human blue protein or bovine immunoglobulin heavy chain constant region γ1 protein, and the Fc domain of the immunoglobulin heavy chain constant region γ1 protein is derived from human or bovine.

5. The fusion protein according to claim 4, characterized in that The amino acid sequence of the signal peptide derived from human blue protein is shown in SEQ ID NO: 6, the amino acid sequence of the signal peptide derived from bovine immunoglobulin heavy chain constant region γ1 protein is shown in SEQ ID NO: 7, the amino acid sequence of the Fc domain of human immunoglobulin heavy chain constant region γ1 protein is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc domain of bovine immunoglobulin heavy chain constant region γ1 protein is shown in SEQ ID NO:

9.

6. A recombinant nucleic acid molecule, characterized in that Encodes the fusion protein according to any one of claims 1 to 5.

7. A recombinant gene expression cassette, characterized in that: Comprising the recombinant nucleic acid molecule of claim 6.

8. A recombinant vector, characterized in that: Comprising the recombinant nucleic acid molecule according to claim 6, or the recombinant gene expression cassette according to claim 7.

9. A recombinant host cell, characterized in that Comprising the recombinant nucleic acid molecule according to claim 6, or the recombinant gene expression cassette according to claim 7, or the recombinant vector according to claim 8.

10. An immunogenic composition or pharmaceutical composition, characterized in that: Comprising one or more fusion proteins according to any one of claims 1 to 5, and / or one or more recombinant nucleic acid molecules according to claim 6, and / or one or more recombinant gene expression cassettes according to claim 7, and / or one or more recombinant vectors according to claim 8, and / or one or more recombinant host cells according to claim 9.

11. The immunogenic composition or pharmaceutical composition according to claim 10, characterized in that 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.

13. The immunogenic composition or pharmaceutical composition according to claim 12, characterized in that The mass ratio of the fusion protein A to the fusion protein B in the immunogenic composition or the pharmaceutical composition is 1:

1.

14. A recombinant vaccine, characterized in that: Comprising one or more fusion proteins according to any one of claims 1 to 5, and / or one or more recombinant nucleic acid molecules according to claim 6, and / or one or more recombinant gene expression cassettes according to claim 7, and / or one or more recombinant vectors according to claim 8, and / or one or more recombinant host cells according to claim 9, and / or one or more immunogenic compositions or pharmaceutical compositions according to any one of claims 10 to 13.

15. Use of one or more fusion proteins according to any one of claims 1 to 5, and / or one or more recombinant nucleic acid molecules according to claim 6, and / or one or more recombinant gene expression cassettes according to claim 7, and / or one or more recombinant vectors according to claim 8, and / or one or more recombinant host cells according to claim 9, and / or one or more immunogenic compositions or pharmaceutical compositions according to any one of claims 10 to 13, and / or one or more recombinant vaccines according to claim 14 in the preparation of vaccines or biological immunization drugs for prevention, treatment and / or vaccination; the drugs are used to prevent and / or treat tissue infections caused by Staphylococcus aureus.

16. The use according to claim 15, characterized in that The tissue infection is skin tissue infection, breast infection, or abdominal cavity infection.

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