An immunogenic composition for preventing mycoplasma pneumonia infection and use thereof

Abstract

This invention relates to a novel fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention and treatment of Mycoplasma pneumoniae infection. The invention provides a novel fusion molecular architecture comprising fusion proteins encoded by the PstS and P37 genes, which can be used in the development of nucleic acid vaccines. The invention reveals that the novel fusion protein exhibits excellent immunogenicity, providing effective immune protection, significantly inhibiting Mycoplasma pneumoniae colonization, and, compared to nucleic acid vaccines encoding the P1-P30 fusion protein, which are known in the art to have excellent efficacy, demonstrates a more significant immune effect and protective effect against Mycoplasma pneumoniae infection, showing broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, particularly the field of immunopharmaceutical technology, and specifically relates to an immunogenic composition, fusion protein, recombinant vaccine and its application for the prevention and treatment of Mycoplasma pneumoniae infection. Background Technology

[0002] Mycoplasma pneumoniae (MP) is a cell-wall-less, minimal prokaryotic microorganism belonging to the order Mycoplasma in the class Mycoplasmata. It is widely distributed on the surface of the human respiratory mucosa and is an important atypical pathogen causing community-acquired pneumonia (CAP), bronchitis, otitis media, and other diseases, posing a serious threat, especially to children, adolescents, and immunocompromised individuals. Epidemiological statistics show that approximately 10%-40% of community-acquired pneumonia cases are caused by Mycoplasma pneumoniae infection, and its infection rate has been on the rise in recent years, even becoming the leading cause of pneumonia in children in some areas.

[0003] The pathogenic mechanism of Mycoplasma pneumoniae mainly relies on the specific binding of its surface adhesion proteins (such as P1, P30, and P116) to the ciliated epithelial cells of the host's respiratory tract. It then disrupts the mucosal barrier by secreting virulence factors such as community-acquired respiratory distress syndrome toxin (CARDSTX) and hydrogen peroxide, triggering local inflammatory responses and systemic immune damage. Clinical treatment primarily relies on antibiotics, with commonly used drugs including macrolides (such as azithromycin), tetracyclines (such as doxycycline), and fluoroquinolones (such as levofloxacin). However, with the widespread use of antibiotics, drug resistance in Mycoplasma pneumoniae is becoming increasingly prominent: in some regions of my country, the macrolide resistance rate has exceeded 80%, and some strains have also shown cross-resistance to tetracyclines. This has led to a decline in the efficacy of traditional treatment regimens and an increase in the proportion of severe infections and refractory cases, necessitating proactive prevention through vaccination.

[0004] Currently, no Mycoplasma pneumoniae vaccines have been approved for marketing globally, and related research and development is still in the exploratory stage. The main technical routes include inactivated vaccines, live attenuated vaccines, subunit vaccines, recombinant protein vaccines, and viral vector vaccines, but all of them have obvious limitations.

[0005] Inactivated / live attenuated vaccines: Early studies attempted to prepare vaccines by inactivation with formaldehyde or temperature-induced attenuation, but the immunogenicity was generally weak and could only induce a brief humoral immune response in animal models; although live attenuated vaccines have good immunization effects, there is a potential risk of virulence reversion (such as gene recombination between wild-type strains and attenuated strains), and safety is difficult to guarantee.

[0006] Subunit vaccines: These focus on key virulence factors or adhesion proteins of Mycoplasma pneumoniae (such as P1 adhesin, CARDS TX toxin for community-acquired respiratory distress syndrome, P30 protein, etc.). For example, the P1 protein is a core antigen mediating host cell adhesion, but its gene is highly variable (the coding region is prone to recombination), and the C-terminal domain of the P1 antigen can differ by more than 30% between different strains, which limits the broad-spectrum protection of vaccines with a single P1 antigen C-terminal domain. CARDS TX, as the main toxin, can induce neutralizing antibodies, but its residual toxicity may cause adverse reactions, requiring the removal of toxic sites (such as the ADP-ribosyltransferase active site) through mutation, increasing the complexity of vaccine design.

[0007] Multivalent vaccines attempt to expand the scope of protection by combining multiple serotype-specific antigens (such as covering different subtypes of P1), but the serotype classification of Mycoplasma pneumoniae is not yet unified (at least 13 serotypes have been reported so far), and the distribution of serotypes varies geographically, making it difficult to cover all circulating strains with a fixed combination.

[0008] It is worth noting that existing research has largely focused on optimizing single antigens or a few known virulence factors. However, the Mycoplasma pneumoniae genome contains a large number of surface or secretory proteins with unknown functions. Some of these conserved proteins may play key roles in host colonization and immune evasion, but they have not yet been fully explored as vaccine candidate antigens. P1 and P30 are classic antigens of Mycoplasma pneumoniae, and fusion proteins constructed from these two are known in the field to have good immunogenicity. For example, a journal article (Hausner M., Schamberger A., ​​Naumann W., Jacobs E., Dumke R. Development of protective anti-Mycoplasma pneumoniae antibodies after immunization of guineapigs with the combination of a P1-P30 chimeric recombinant protein and chitosan. Microb. Pathog. 2013;64:23–32. doi: 10.1016 / j.micpath.2013.07.004) discloses the construction of a fusion protein by combining a P30 truncation protein (amino acid positions 17 to 274) with the C-terminus of P1 adhesin (amino acid positions 1287-1518 of P1). Using a classic guinea pig model of Mycoplasma pneumoniae infection, protective anti-Mycoplasma pneumoniae antibodies were produced after immunization, which can resist Mycoplasma pneumoniae infection.

[0009] In summary, developing a Mycoplasma pneumoniae vaccine that can cover multiple circulating strains, induce long-lasting mucosal and systemic immunity, and has controllable safety is of great significance for reducing infection rates, antibiotic overuse, and the incidence of severe cases. Existing technologies, due to limitations in antigen selection, insufficient immunogenicity, or safety concerns, are insufficient to meet clinical needs. Therefore, this invention focuses on conserved functional proteins or multi-antigen synergistic mechanisms in Mycoplasma pneumoniae, screening new vaccine candidate antigens, and proposing novel vaccine design schemes. The aim is to overcome the bottlenecks of existing technologies by optimizing antigen combinations and delivery methods, providing an efficient solution for the prevention of Mycoplasma pneumoniae infection. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a novel fusion protein, immunogenic composition, recombinant vaccine, molecular architecture design, and applications for the prevention and treatment of Mycoplasma pneumoniae infection. This invention provides a novel fusion molecular architecture comprising fusion proteins encoded by the PstS and P37 genes, which can be used in the development of nucleic acid vaccines, etc. This invention reveals that the novel fusion molecule possesses excellent immunogenicity and can provide effective immune protection, significantly inhibiting Mycoplasma pneumoniae colonization, and has broad application prospects. Furthermore, the nucleic acid vaccine A encoding the PstS-P37 fusion protein, constructed by screening for antigen and glycosylation site mutations, exhibits a more significant immunogenic effect and protective effect against Mycoplasma pneumoniae infection compared to the nucleic acid vaccine B encoding the P1-P30 fusion protein, which is known in the art to have excellent efficacy. This invention also provides corresponding recombinant nucleic acids, gene expression cassettes, vectors, host cells, pharmaceutical compositions, vaccines, and uses.

[0011] One aspect of the present invention provides a fusion protein, characterized in that it comprises the following two antigen proteins:

[0012] (1) Phosphate transport system substrate-binding protein (PstS) antigen or its antigen-selected fragment; and,

[0013] (2) High affinity transport system surface lipoprotein p37 (P37) antigen or its antigen-selected fragment.

[0014] Furthermore, the PstS and P37 antigens or their antigenic truncation fragments are derived from the genus Mycoplasma; preferably, they are derived from Mycoplasma pneumoniae.

[0015] Furthermore, the fusion protein further comprises a secretory-promoting element and / or an expression-promoting element; preferably, the secretory-promoting element is a signal peptide and / or the Fc domain of IGHG, and the expression-promoting element is the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit; more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of IGHG is as shown in SEQ ID NO: 4, and the STABILON sequence is as shown in SEQ ID NO: 5.

[0016] Further, the PstS antigen or its antigen-selected fragment, and the P37 antigen or its antigen-selected fragment contain glycosylation mutation sites; preferably, the PstS antigen or its antigen-selected fragment contains a glycosylation mutation site N97Q, and the P37 antigen or its antigen-selected fragment contains a glycosylation mutation site N38Q; more preferably, the amino acid sequence of the PstS antigen is as shown in SEQ ID NO: 1, and the amino acid sequence of the P37 antigen is as shown in SEQ ID NO: 2.

[0017] Furthermore, the antigen or its antigen-selected fragments and functional elements may optionally be linked by a linker sequence or a spacer sequence; preferably, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 6, and the amino acid sequence of the spacer sequence is shown in SEQ ID NO: 7.

[0018] Furthermore, the fusion protein comprises an amino acid sequence as shown in SEQ ID NO: 8.

[0019] Another aspect of the present invention provides a recombinant nucleic acid molecule, characterized in that it comprises a nucleic acid encoding a fusion protein according to any one of the present invention; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

[0020] Another aspect of the present invention provides a recombinant gene expression cassette, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention; preferably, the recombinant gene expression cassette further comprises one or more of a promoter, a terminator, and a regulatory sequence.

[0021] Another aspect of the present invention provides a recombinant vector, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention or the recombinant gene expression cassette described in the present invention.

[0022] Furthermore, the recombinant vector comprises a prokaryotic vector or a eukaryotic vector.

[0023] Furthermore, the prokaryotic vector includes, but is not limited to, Escherichia coli vectors.

[0024] Furthermore, the Escherichia coli vector includes, but is not limited to, pET vector, pGEX vector, pMAL vector, pBAD vector, pUC vector, and pBR vector.

[0025] Furthermore, the eukaryotic vector includes, but is not limited to, yeast expression vectors, insect expression vectors, and mammalian cell expression vectors.

[0026] Furthermore, the yeast expression vector includes, but is not limited to, pPICZ vector, pGAPZ vector, pYES vector, pGAP vector, pAO815 vector, and pPIC9 vector.

[0027] Another aspect of the present invention provides a recombinant host cell, characterized in that it comprises the recombinant nucleic acid molecule described in the present invention, or the recombinant gene expression cassette described in the present invention, or the recombinant vector described in the present invention.

[0028] Furthermore, the recombinant host cell comprises a eukaryotic cell or a prokaryotic cell.

[0029] Furthermore, the eukaryotic cells include mammalian cells, insect cells, and yeast cells.

[0030] Furthermore, the yeast cells include, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0031] Furthermore, the prokaryotic cells include, but are not limited to, Escherichia coli cells, Bacillus subtilis cells, and Pseudomonas cells.

[0032] Furthermore, the *E. coli* cells include, but are not limited to, BL21(DE3), DH5α, TOP10, and Rosetta.

[0033] Another aspect of the present invention provides an immunogenic composition or pharmaceutical composition, characterized in that it comprises one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention; preferably, the immunogenic composition or pharmaceutical composition further comprises a pharmaceutically acceptable vector.

[0034] Another aspect of the present invention provides a recombinant vaccine, characterized in that it comprises one or more fusion proteins as described in any one of the present inventions, and / or one or more recombinant nucleic acid molecules as described in the present inventions, and / or one or more recombinant gene expression cassettes as described in the present inventions, and / or one or more recombinant vectors as described in the present inventions, and / or one or more recombinant host cells as described in the present inventions, and / or one or more immunogenic compositions or pharmaceutical compositions as described in the present inventions; preferably, the recombinant vaccine is a nucleic acid vaccine; more preferably, the nucleic acid vaccine is an RNA vaccine or a DNA vaccine; most preferably, the RNA vaccine is an mRNA vaccine.

[0035] Another aspect of the present invention provides the use of one or more fusion proteins according to any one of the present invention, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention in the preparation of vaccines or medicaments for prevention, treatment and / or inoculation; preferably, the medicament is used for prevention and / or treatment of infections or diseases caused by mycoplasma; more preferably, the mycoplasma is Mycoplasma pneumoniae.

[0036] Another aspect of the present invention provides a method for preventing diseases caused by mycoplasma infection, characterized in that an effective amount of one or more fusion proteins according to any one of the present inventions, and / or one or more recombinant nucleic acid molecules according to the present invention, and / or one or more recombinant gene expression cassettes according to the present invention, and / or one or more recombinant vectors according to the present invention, and / or one or more recombinant host cells according to the present invention, and / or one or more immunogenic compositions or pharmaceutical compositions according to the present invention, and / or one or more recombinant vaccines according to the present invention are administered to the subject; preferably, the mycoplasma is Mycoplasma pneumoniae.

[0037] Furthermore, the infection or disease includes mycoplasma pneumonia, pharyngitis, laryngitis, tracheitis, bronchitis, other pulmonary complications, and extrapulmonary complications; preferably, the bronchitis includes bronchiolitis obliterans, the other pulmonary complications include pulmonary consolidation and necrosis, pulmonary embolism, and acute asthma exacerbation, and the extrapulmonary complications include encephalitis, hemolytic anemia, myocarditis, kidney and urinary system diseases, musculoskeletal system diseases, sensory organ diseases, and skin and mucous membrane diseases.

[0038] The fusion protein PstS-P37 of the present invention and the nucleic acid vaccines, immunogenic compositions, pharmaceutical compositions, recombinant vaccines, etc. encoding it have the following beneficial technical effects:

[0039] 1. Multiple Mycoplasma pneumoniae antigens were selected, and the antigens were truncated and designed for fusion expression using bioinformatics techniques such as protein three-dimensional structure prediction and immune epitope screening. This included N / C-terminal truncation, full-length truncation, and prediction of fusion protein stability, ultimately obtaining the PstS antigen and P37 antigen selected in this invention.

[0040] 2. This invention involves glycosylation site mutation N97Q in the PstS antigen and N38Q in the P37 antigen. The fusion proteins constructed from the two mutated antigens (the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, and the amino acid sequence of the P37 antigen is shown in SEQ ID NO: 2) exhibit high expression abundance and more accurate epitope presentation.

[0041] 3. This invention constructs a nucleic acid vaccine A encoding the PstS-P37 fusion protein and a control vaccine B. Specifically, the classic antigens P1 and P30 of Mycoplasma pneumoniae, known in the art, are selected, and the fusion protein constructed from these two antigens is known in the art to have good immunogenicity (see journal article 1 in the background art). Therefore, the P1-P30 fusion protein is constructed as the control nucleic acid vaccine B of this invention to compare and demonstrate the efficacy of vaccine A.

[0042] 4. Vaccine A can be significantly expressed in the supernatant, proving that the molecular architecture of adding an Fc domain to the N-terminus of the antigen provided by the present invention, and the use of mutations N97Q at the PstS antigen glycosylation site and N38Q at the P37 antigen glycosylation site in Vaccine A, can enable the bacterial fusion protein to be translated smoothly and folded correctly in eukaryotic cells, and promote the efficient secretion of the fusion protein into the extracellular space.

[0043] 5. Example 5 illustrates the preventive effect of the recombinant nucleic acid vaccine based on the present invention in a rabbit model of Mycoplasma pneumoniae infection. The bacterial load in the lung tissue of rabbits immunized with vaccine A was the lowest. Compared to the vaccine immunized group, the bacterial load in the lung tissue of the vaccine A immunized group was only half that of the vaccine B immunized group. Therefore, it can be concluded that both vaccine A and vaccine B can effectively prevent Mycoplasma pneumoniae infection, achieving the expected results. Furthermore, vaccine A has the best preventive effect, significantly superior to vaccine B constructed from the fusion protein of the classic Mycoplasma pneumoniae antigens P1 and P30.

[0044] 6. In summary, the nucleic acid vaccine A encoding the PstS-P37 fusion protein constructed by screening antigens and glycosylation site mutations of the present invention has a more significant immune effect and protective effect against Mycoplasma pneumoniae infection compared with the nucleic acid vaccine B encoding the P1-P30 fusion protein, which is known in the art to have excellent effects. Attached Figure Description

[0045] Figure 1This is a schematic diagram of the molecular structure of the fusion protein PstS-P37 expressed in the vaccine of this invention, and the fusion protein P1-P30 expressed in the comparative vaccine. Figure 1 In this paper, A represents the fusion protein expressed in the vaccine of this invention. Figure 1 In the figure, B represents the fusion protein expressed by the vaccine used for comparison.

[0046] Figure 2 This is a schematic diagram of a template plasmid containing the gene expression cassette of the present invention.

[0047] Figures 3A-3B The table shows the quality control peak diagrams and purity test results for nucleic acid vaccine A containing the antigen sequence of this invention and nucleic acid vaccine B containing the sequence of the prior art; wherein... Figure 3A The image shows the quality control peaks and purity test results for vaccine A. Figure 3B This is a peak diagram of the quality control for vaccine B and the results of its purity test.

[0048] Figures 4A-4B The results of Western blot analysis of the expression of vaccines A and B after in vitro transfection into HEK293T cells are shown. Figure 4A The results of WB detection for in vitro expression of vaccine A are as follows. Figure 4B The results of WB detection for in vitro expression of vaccine B.

[0049] Figure 5 The procedure for immunization and sampling of vaccines A and B in a rabbit model infected with Mycoplasma pneumoniae.

[0050] Figure 6 The vertical axis represents the preventive effect of changes in bacterial load in lung tissue during Mycoplasma pneumoniae infection experiments, with the vertical axis representing the quantitative results of bacterial load count in lung tissue after challenge. Detailed Implementation

[0051] Terms and Definitions

[0052] The term "Mycoplasma pneumoniae" refers to a tiny prokaryote that can cause respiratory infections in humans, especially atypical pneumonia. Infections or diseases caused by Mycoplasma pneumoniae as described in this invention include, but are not limited to, mycoplasma pneumoniae pneumonia, pharyngitis, laryngitis, tracheitis, bronchitis, obliterative bronchiolitis, other pulmonary complications (including pulmonary consolidation and necrosis, pulmonary embolism, and acute asthma exacerbations), and extrapulmonary complications (including encephalitis, hemolytic anemia, myocarditis, kidney and urinary system diseases, musculoskeletal system diseases, sensory organ diseases, and skin and mucous membrane diseases).

[0053] The term "PstS" in Mycoplasma pneumoniae refers to a phosphate transport system substrate-binding protein, an important component of the phosphate transport system. The PstS antigen of this invention comprises the full-length PstS antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the PstS antigen. Preferably, the amino acid sequence of PstS is shown in SEQ ID NO: 1.

[0054] The term "P37" in Mycoplasma pneumoniae refers to the high affinity transport system protein p37, with a molecular weight of approximately 37 kDa. The P37 antigen of this invention comprises the full-length P37 antigen and selected fragments thereof, such as N-terminal truncation, C-terminal truncation, or partial full-length selection, all of which can stimulate an immune response against the P37 antigen. Preferably, the amino acid sequence of the P37 antigen is shown in SEQ ID NO: 2.

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

[0056] The terms "administration" or "inoculation" refer to the administration of the nucleic acid vaccine or vaccine composition based on the present invention, preferably via intramuscular or subcutaneous routes, although other routes of administration may also be used, such as oral, intranasal (e.g., aerosol or other non-injectable), intralymphatic, intradermal, intraperitoneal, rectal or vaginal administration, or by combination of routes. Intramuscular administration in the neck muscles of animals is preferred. Boosting regimens can be used to adjust the administration regimen to provide optimal immunization.

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

[0058] The term "recombinant nucleic acid molecule" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can then be transformed into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," a "recombinant protein," or a "fusion protein."

[0059] The term "recombinant expression vector" refers to a DNA structure containing a polynucleotide encoding, for example, a desired polypeptide. A recombinant expression vector may include, for example, a set of genetic elements that regulate gene expression, such as promoters and enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into a protein; and (3) a transcriptional subunit containing appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner and any vector, including plasmids, viruses, bacteriophages, and transposons, may be used. Possible vectors used in this disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as viral plasmids, bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viruses such as lentiviruses, retroviruses, vaccinia virus, adenovirus, fowlpox virus, baculovirus, SV40, and pseudorabies virus. Self-replicating vectors and non-self-replicating vectors are included.

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

[0061] The term "5'-UTR" refers to the "5' untranslated region" or "5'UTR," which is a portion of a gene transcribed into a primary RNA transcript (precursor mRNA) and located upstream of the coding sequence. Primary transcripts are the initial RNA products, containing introns and exons, produced by DNA transcription. Many primary transcripts must undergo RNA processing to form physiologically active RNA. The processing to form mature mRNA includes end modification, intron removal, capping, and / or cleavage of individual rRNA molecules from the precursor RNA. Therefore, the 5'UTR of mRNA is a portion of mRNA that is not translated into protein and is located upstream of the coding sequence. In the genome sequence, the 5'UTR is generally defined as the region between the transcription start site and the start codon. The length of the 5' untranslated region (5'UTR) of vertebrate mRNA can range from tens to hundreds of bases.

[0062] The term "3'-UTR" refers to the "3'-untranslated region" or "3'UTR," which refers to the region located at the 3' end of a gene, downstream of the stop codon in a protein-coding region, and which is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule. The 3'-UTR typically extends from the stop codon of the translation product to a poly(A) sequence that usually attaches after transcription. The 3'-UTR of mammalian mRNA typically has a homologous region known as the AAUAAA hexanucleotide sequence. This sequence may be a poly(A) attachment signal and is often located 10 to 30 bases upstream of the poly(A) attachment site. The 3'-UTR may contain one or more inverted repeats that can fold to create stem-loop structures that act as barriers to ribonucleases or interact with proteins known to enhance RNA stability, such as RNA-binding proteins.

[0063] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce recombinant vaccines based on the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells.

[0064] The terms “individual,” “patient,” or “subject” include mammals and birds. Mammals include, but are not limited to, domesticated animals (e.g., pigs, cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), and rodents (e.g., rabbits, hares, and rats). Birds include, but are not limited to, birds and poultry (e.g., chickens, ducks, geese).

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

[0066] The term "pharmaceutical combination" or "pharmaceutical composition" refers to excipients widely used in the pharmaceutical manufacturing industry. The primary purpose of using a carrier is to provide a pharmaceutical composition that is safe to use, stable in nature, and / or has specific functionalities, and also to provide a method for its effective absorption in a subject. Pharmaceutically acceptable carriers can be inert fillers or active ingredients that provide a function to the pharmaceutical combination (e.g., stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition). Non-limiting examples of pharmaceutically acceptable carriers include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adhesives, flow aids, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0067] The term "treatment" refers to exposing a subject to (e.g., administering medication) a recombinant vaccine, composition, etc., based on the present invention after contracting a disease, thereby reducing the symptoms of the disease compared to when not exposed, and does not imply the necessity of completely suppressing the symptoms of the disease. Contracting a disease means that the body has developed symptoms of a disease.

[0068] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a recombinant vaccine, composition, etc. based on the present invention before contracting the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0069] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art described herein.

[0070] This invention discloses a fusion molecular architecture for the prevention and treatment of Mycoplasma pneumoniae infection, a method for preparing a recombinant vaccine based on this architecture, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0071] The fusion protein and encoding nucleic acid and their elements provided by this invention, as well as the preparation method and application, all utilize commercially available raw materials and reagents. Based on conventional knowledge in molecular cloning, expression construction, vaccine preparation, and immunization, those skilled in the art can implement the methods and embodiments of this invention.

[0072] The present invention will be further illustrated below with reference to the embodiments. Preferably, a nucleic acid vaccine architecture is selected for the preparation of the recombinant vaccine.

[0073] Example 1: Construction of the recombinant nucleic acid vaccine of the present invention and a control vaccine for comparison.

[0074] Antigen screening: Multiple Mycoplasma pneumoniae antigens were selected, and bioinformatics techniques such as protein three-dimensional structure prediction and immunoepitope screening were used to truncate and design fusion expression, including N / C-terminal truncation, full-length selection, and fusion protein stability prediction, ultimately obtaining the PstS antigen and P37 antigen selected in this invention. Furthermore, to avoid glycosylation of prokaryotic proteins during expression in eukaryotic cells, which would affect the correct presentation of epitopes, this invention mutated the PstS antigen at the N97Q glycosylation site and the P37 antigen at the N38Q glycosylation site. The fusion proteins constructed from the two mutated antigens (the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, and the amino acid sequence of the P37 antigen is shown in SEQ ID NO: 2) exhibited high expression abundance and more accurate epitope presentation.

[0075] To prepare a recombinant nucleic acid vaccine containing the antigen of the present invention, a non-limiting schematic diagram of the nucleic acid vaccine of the present invention is shown below. Figure 1 As shown in A, an exemplary example Figure 1 In the diagram, A represents the molecular structure of the fusion protein PstS-P37 expressed in the vaccine of this invention. Figure 1 B in the diagram represents the molecular structure of the fusion protein P1-P30 expressed by the vaccine used for comparison.

[0076] In order to prepare a product capable of producing such Figure 1 The recombinant nucleic acid vaccine with the molecular structure shown in Figure A, and its preparation Figure 1 The prior art vaccine shown in Figure B is used to compare the efficacy of the vaccine of the present invention. First, a gene expression cassette is constructed to express the antigen sequence. A schematic diagram of the template plasmid containing the gene expression cassette is shown below. Figure 2 As shown, from the 5' end to the 3' end, the sequence includes: 5'UTR, CDS region, 3'UTR, and PolyA, where the CDS region contains the fusion molecular structure of the antigen. Subsequently, the complete gene expression cassette sequence was optimized based on codon degeneracy, and the DNA sequence was directly obtained through gene synthesis (commissioned to GenScript). Finally, the synthesized gene expression cassette DNA sequence was inserted into an expression vector suitable for in vitro RNA transcription, yielding a vector plasmid for preparing recombinant nucleic acid vaccines.

[0077] According to the above method, a carrier for use in subsequent embodiments is prepared:

[0078] (1) Preparation vector of recombinant nucleic acid vaccine A based on the present invention

[0079] Step a: Synthesize the gene fragment “signal peptide-IGHG Fc domain-PstS antigen-P37 antigen-STABILON”, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of IGHG is shown in SEQ ID NO: 4, the amino acid sequence of the PstS antigen is shown in SEQ ID NO: 1, the amino acid sequence of the P37 antigen is shown in SEQ ID NO: 2, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5. The Fc domain of IGHG and the PstS antigen are linked by a spacer sequence as shown in SEQ ID NO: 7, and the PstS antigen and the P37 antigen, and the P37 antigen and STABILON are linked by a linker sequence as shown in SEQ ID NO: 6. The amino acid sequence of the fusion protein encoded by the PstS antigen-P37 antigen fusion gene is shown in SEQ ID NO: 8.

[0080] Step b: Construct a nucleic acid vaccine architecture carrier.

[0081] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0082] Step c: Prepare recombinant plasmids.

[0083] The gene synthesized in step a is inserted into the vector structure in step b to obtain the recombinant nucleic acid vaccine A preparation vector based on the present invention.

[0084] (2) Recombinant nucleic acid vaccine B preparation vector used for comparison

[0085] The classic antigens P1 and P30 of Mycoplasma pneumoniae, known in the art, were selected, and a fusion protein constructed from them was known in the art to have good immunogenicity (see journal article 1 in the background art). Therefore, a nucleic acid encoding the P1-P30 fusion protein was constructed as the control nucleic acid vaccine B of the present invention, to compare and demonstrate the effect of vaccine A of the present invention.

[0086] Step a: Synthesize the gene fragment “signal peptide-IGHG Fc domain-P1 antigen C-terminal domain-P30 antigen-STABILON”, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of IGHG is shown in SEQ ID NO: 4, the amino acid sequence of the C-terminal domain of the P1 antigen is shown in SEQ ID NO: 9, the amino acid sequence of the P30 antigen is shown in SEQ ID NO: 10, and the amino acid sequence of STABILON is shown in SEQ ID NO: 5. The Fc domain of IGHG and the C-terminal domain of the P1 antigen are linked by a spacer sequence as shown in SEQ ID NO: 7, and the C-terminal domain of the P1 antigen and the P30 antigen, and the P30 antigen and STABILON are linked by a linker sequence as shown in SEQ ID NO: 6. The amino acid sequence of the fusion protein encoded by the P1 antigen C-terminal domain-P30 antigen fusion gene is shown in SEQ ID NO: 11.

[0087] Step b: Construct a nucleic acid vaccine architecture carrier.

[0088] The nucleic acid vaccine architecture vector includes 5'-UTR and 3'-UTR, and can be a vector for producing any form of RNA vaccine or a vector for producing DNA vaccines.

[0089] Step c: Prepare recombinant plasmids.

[0090] The gene synthesized in step a is inserted into the vector architecture in step b to obtain the recombinant nucleic acid vaccine preparation vector B for comparison.

[0091] Table 1. Amino acid sequences of the architectural elements involved in this invention.

[0092] amino acid sequence and sequence number PstS antigen CANINLISAVGSSSVQPLLSKLSSHYVLNHNDKDNLVEISVQAGGSSAGVKAITKGLADIGQVSKNTKSYAENKQLWMDKKLKTITLGKDAIAVIYKAPSEFKGKLVLTKDNLNDLYDLFAGSKSVDINKFVENGQTTKNSNHNLIGFPRTGGAFASGTAEAFLKFSGLTQTKTLD KDSKEILEGQRNYGPNARPTSETNIEAFNTFVTTLRQPNLYGMVYLSLGFVNNNMNLIKSEGFEVLKVKYDNNAVTPSSQAVSSNTYKWVRPLNSVVSLLPKQKTLPSIQRFFNWLLFSNNSEIKKIYDDFGVLELTADEKKKMFKTGNAEMSNIANFWVDDYSLNNQTFGAL (SEQ ID NO: 1) P37 antigen ARRESNHLIFQISLDHDADASISKFFELYSNNLSKKLDKKVTVSFNIIDDSFTKISNIQTAKADFAFVNSQSIKDNGIEEFNLILQTQTDAFKEDTNLDYYSDGQLKSKAEKMTTLFSKTPYKDWEDTAQQWTGSRYNFLYETNKLINFYRGMILITGSEEEIKKIKEAWDQKKWSDF MNYGIGHGSSGSAGKFQLPDLLLRKHFGSSYPGLQNAINQNPDKFANVRGREIGRDNKIKIVFDDANSFAWTHNDKNATNHFYTPTENNGKGDSEKSNNKNNKVEILTYTDPMLYDIGIVSDTLSDRYQKAIAEVFVELAKTKQDIYGPSYGYNGYNLITDPNKEILDVIHKTYG (SEQ ID NO: 2) signal peptide MTRLTVLALLAGLLASSRA (SEQ ID NO: 3) IGHG's Fc domain EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4) STABILON KDGKKDKKEEDKK (SEQ ID NO: 5) linker sequence GGSGGGGSGG (SEQ ID NO: 6) spacer sequence GGSGGSGGSG (SEQ ID NO: 7) PstS antigen - P37 antigen fusion protein CANINLISAVGSSSVQPLLSKLSSHYVLNHNDKDNLVEISVQAGGSSAGVKAITKGLADIGQVSKNTKSYAEENKQLWMDKKLKTITLGKDAIAVIYKAPSEFKGKLVLTKDNLNDLYDLFAGSKSVDINKFVENGQTTKNSNHNLIGFPRTGGAFASGTAEAFLKFSGLTQTKTLDKDSKEILEGQRNYGPNARPTSETNIEAFNTFVTTLRQPNLYGMVYLSLGFVNNNMNLIKSEGFEVLKVKYDNNAVTPSSQAVSSNTYKWVRPLNSVVSLLPKQKTLPSIQRFFNWLLFSNNSEIKKIYDDFGVLELTADEKKKMFKTGNAEMSNIANFWVDDYSLNNQTFGALGGSGGGGSGGARRESNHLIFQISLDHDADASISKFFELYSNNLSKKLDKKVTVSFNIIDDSFTKISNIQTAKADFAFVNSQSIKDNGIEEFNLILQTQTDAFKEDTNLDYYSDGQLKSKAEKMTTLFSKTPYKDWEDTAQQWTGSRYNFLYETNKLINFYRGMILITGSEEEIKKIKEAWDQKKWSDFMNYGIGHGSSGSAGKFQLPDLLLRKHFGSSYPGLQNAINQNPDKFANVRGREIGRDNKIKIVFDDANSFAWTHNDKNATNHFYTPTENNGKGDSEKSNNKNNKVEILTYTDPMLYDIGIVSDTLSDRYQKAIAEVFVELAKTKQDIYGPSYGYNGYNLITDPNKEILDVIHKTYG (SEQ ID NO: 8) C-terminal domain of P1 antigen SDKLDDDLKNLLDPNQVRTKLRQSFGTDHSTQPQPQSLKTTTPVFGTSSGQLSSVLSGGGAGGGSSGSGQSGVDLSPVEKVSGWLVGQLPSTSDGQTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQ (SEQ ID NO: 9) P30 antigen KRKEKRLLEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVEPAPQPVPVPPQPQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR (SEQ ID NO: 10) P1 antigen C-terminal domain - P30 antigen fusion protein SDKLDDDLKNLLDPNQVRTKLRQSFGTDHSTQPQPQSLKTTTPVFGTSSGQLSSVLSGGGAGGGSSGSGQSGVDLSPVEKVSGWLVGQLPSTSDGQTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQGGSGGGGSGGKRKEKRLLEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVEPAPQPVPVPPQPQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR (SEQID NO: 11)

[0093] Example 2: Preparation of the recombinant nucleic acid vaccine of the present invention

[0094] (1) Preparation of capped mRNA vaccines

[0095] Step a: Linearize the vector plasmid used in Example 1 for producing capped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

[0096] Step b: The linearized plasmid was subjected to an in vitro co-transcriptional capping reaction to add a 7-methylguanylate cap structure to the 5' end of the transcribed mRNA and the template DNA was degraded.

[0097] (2) Preparation of uncapped mRNA vaccines

[0098] Step a: Linearize the vector plasmid used in Example 1 for producing uncapped mRNA vaccines by enzyme digestion to obtain a linearized plasmid for in vitro transcription.

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

[0100] (3) DNA vaccine preparation

[0101] Step a: Amplify the vector plasmid used in Example 1 for producing DNA vaccines to obtain a large number of target plasmids for purification.

[0102] Step b: Extract and purify the target plasmid using an endotoxin-free plasmid extraction and purification kit.

[0103] Example 3: Quality control of recombinant nucleic acid in vitro transcription and vaccine preparation according to the present invention

[0104] Vaccine A (recombinant nucleic acid vaccine A based on the present invention) and vaccine B (recombinant nucleic acid vaccine B for comparison) were prepared using the method for preparing capped mRNA vaccines in Example 2. The purity of the produced recombinant nucleic acids was tested, and the purity of the recombinant nucleic acids used in the experiments was greater than 85%. The quality control peak diagrams and purity test results of recombinant nucleic acid vaccines A and B are shown below. Figure 3A , Figure 3B As shown. Specifically, it is described as follows: (1) Recombinant nucleic acid vaccine A based on the present invention, with a purity of 88.4%; (2) Recombinant nucleic acid vaccine B used for comparison, with a purity of 88.7%. The above purities all meet the quality requirements for cell transfection experiments and vaccine production.

[0105] Example 4: In vitro expression effect of the recombinant nucleic acid of the present invention

[0106] Using cell transfection reagents, vaccines A and B from Example 3 were transfected into HEK293T cells. After 48 hours of in vitro culture, the proteins were collected and analyzed by Western blot.

[0107] Figure 4A , Figure 4B The in vitro expression (WB) results of vaccines A and B transfected into HEK293T cells are presented. The antigens expressed by vaccines A and B are humoral immune antigens, and theoretically, their expression should be detectable in the supernatant and cell lysate. The molecular weights of the proteins A and B are shown in Table 2.

[0108] Table 2. Protein molecular weight of vaccines A and B

[0109] Name Estimated protein molecular weight (kDa) Vaccine A 135 Vaccine B 104

[0110] The results showed that both vaccines A and B were significantly expressed in the supernatant, demonstrating that the molecular architecture provided by this invention, which adds an Fc domain to the N-terminus of the antigen, and the use of mutations N97Q at the PstS antigen glycosylation site and N38Q at the P37 antigen glycosylation site in vaccine A, enable the bacterial-derived fusion protein to be successfully translated and correctly folded in eukaryotic cells, and promote the efficient secretion of the fusion protein into the extracellular space. Therefore, vaccine A based on this invention can be correctly expressed in eukaryotic cells, and the expressed protein structure is correct and stable, which is beneficial for immunoepitaxy presentation, while vaccine B can be used for subsequent comparative verification experiments.

[0111] Example 5: Preventive effect of the recombinant nucleic acid vaccine based on the present invention in a rabbit model of Mycoplasma pneumoniae infection.

[0112] To verify whether the recombinant nucleic acid vaccine based on the present invention can produce an effective preventive and protective effect in model animals after immunization, and whether it has a better preventive effect compared with existing antigen combinations, this embodiment uses vaccine A and vaccine B to conduct immunization and challenge experiments in a rabbit model.

[0113] Sixteen SPF-grade New Zealand White rabbits, each weighing approximately 2 kg, were used in the experiment. The rabbits were similar in size and weight among themselves. After 7 days of acclimatization, the rabbits were divided into four groups: a vaccine A immunization group (n=5), a vaccine B immunization group (n=5), a model control group (using PBS solution instead of the vaccine, negative control, group C) (n=5), and a blank control group (n=1, without immunization or challenge, group D). Details are shown in Table 3. In this table and below, dosage refers to the amount of active ingredient.

[0114] Table 3. Immunization procedure for experimental animals in Example 5

[0115] Rabbits in each group were immunized twice, on day 0 and day 21, according to the immunization schedule in Table 3. Challenge infection: Animals were anesthetized, and the hair around the trachea in the neck was shaved short using an electric shaver. The surface was then disinfected with alcohol. Using a 1mL syringe, 0.2mL (1×10^9 CFU) of Mycoplasma pneumoniae (ATCC29342) was injected through the trachea after drawing up the injection solution. This challenge was repeated for 3 consecutive days (D35-D37). Afterwards, the rabbits were fed normally, and changes in body temperature, weight, and survival rate were observed and recorded. Fourteen days after the last challenge, the animals were euthanized, and lung samples were collected for mycoplasma counting. The immunization and sampling procedures are as follows: Figure 5 As shown.

[0116] Changes in bacterial load in lung tissue of rabbits from different groups after viral challenge are as follows: Figure 6 As shown. According to Figure 6The results showed that a large number of Mycoplasma pneumoniae were detected in the PBS group, while no Mycoplasma pneumoniae were detected in the blank control group, proving that the challenge model was successful. Compared with the PBS group, the Mycoplasma pneumoniae load in the lung tissue of rabbits immunized with vaccine A and vaccine B was significantly reduced, with statistically significant differences. Figure 6 (where p < 0.0001 is represented by ****). The bacterial load in the lung tissue of rabbits immunized with vaccine A was the lowest. Compared to the vaccine B immunization group, the bacterial load in the lung tissue of the vaccine A immunization group was only half that of the vaccine B immunization group. Therefore, it can be concluded that both vaccines A and B can effectively prevent Mycoplasma pneumoniae infection, achieving the expected results. Furthermore, vaccine A has the best preventive effect, significantly superior to vaccine B constructed from the classic Mycoplasma pneumoniae antigens P1 and P30 fusion protein. In summary, the nucleic acid vaccine A encoded by the PstS-P37 fusion protein constructed by screening for antigen and glycosylation site mutations in this invention has a more significant immunogenic effect and protective effect against Mycoplasma pneumoniae infection compared to the P1-P30 fusion protein-encoded nucleic acid vaccine B, which is known in the art to have excellent efficacy.

[0117] In summary, the antigen and fusion molecular architecture provided by this invention successfully induced an effective immune response in rabbits, a model animal, demonstrating a good preventive effect against Mycoplasma pneumoniae infection. This breakthrough further expands the current field of Mycoplasma pneumoniae vaccine development, providing a novel solution for the production and development of animal immunotherapies, and possesses extremely high commercial value and broad application prospects.

[0118] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A fusion protein, characterized in that, It contains the following two antigen proteins: (1) Phosphate transport system substrate-binding protein (PstS) antigen or its antigen-selected fragment; and, (2) High affinity transport system surface lipoprotein P37 (P37) antigen or its antigen-selected fragment.

2. The fusion protein according to claim 1, characterized in that, The PstS and P37 antigens, or selected fragments thereof, are derived from the genus Mycoplasma; preferably, they are derived from Mycoplasma pneumoniae (…). Mycoplasma pneumoniae ).

3. The fusion protein according to claim 1 or 2, characterized in that, The fusion protein further comprises a secretory element and / or an expression-promoting element; preferably, the secretory element is a signal peptide and / or the Fc domain of IGHG, and the expression-promoting element is the C-terminal polypeptide fragment STABILON of the human S5a / PSMD4 proteasome subunit; more preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 3, the amino acid sequence of the Fc domain of IGHG is as shown in SEQ ID NO: 4, and the STABILON sequence is as shown in SEQ ID NO:

5.

4. The fusion protein according to any one of claims 1-3, characterized in that, The PstS antigen or a selected fragment thereof, and the P37 antigen or a selected fragment thereof, contain glycosylation mutation sites; preferably, the PstS antigen or a selected fragment thereof contains a glycosylation mutation site N97Q, and the P37 antigen or a selected fragment thereof contains a glycosylation mutation site N38Q; more preferably, the amino acid sequence of the PstS antigen is as shown in SEQ ID NO: 1, and the amino acid sequence of the P37 antigen is as shown in SEQ ID NO:

2.

5. The fusion protein according to any one of claims 1-4, characterized in that, The antigen or its antigen-selected fragments and functional elements may optionally be linked by a linker sequence or a spacer sequence; preferably, the amino acid sequence of the linker sequence is shown in SEQ ID NO: 6, and the amino acid sequence of the spacer sequence is shown in SEQ ID NO:

7.

6. The fusion protein according to any one of claims 1-5, characterized in that, The fusion protein contains the amino acid sequence shown in SEQ ID NO:

8.

7. A recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule comprises a nucleic acid encoding the fusion protein of any one of claims 1-6; preferably, the recombinant nucleic acid molecule is mRNA or DNA.

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

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

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

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

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

13. Use of one or more fusion proteins according to any one of claims 1-6, and / or one or more recombinant nucleic acid molecules according to claim 7, and / or one or more recombinant gene expression cassettes according to claim 8, and / or one or more recombinant vectors according to claim 9, and / or one or more recombinant host cells according to claim 10, and / or one or more immunogenic compositions or pharmaceutical compositions according to claim 11, and / or one or more recombinant vaccines according to claim 12 in the preparation of a vaccine or medicament for prevention, treatment and / or inoculation; preferably, the medicament is used for prevention and / or treatment of infections or diseases caused by mycoplasma; more preferably, the mycoplasma is Mycoplasma pneumoniae.

14. The use according to claim 13, characterized in that, The infection or disease includes mycoplasma pneumonia, pharyngitis, laryngitis, tracheitis, bronchitis, other pulmonary complications, and extrapulmonary complications; preferably, the bronchitis includes bronchiolitis obliterans, the other pulmonary complications include pulmonary consolidation and necrosis, pulmonary embolism, and acute asthma exacerbation, and the extrapulmonary complications include encephalitis, hemolytic anemia, myocarditis, kidney and urinary system diseases, musculoskeletal system diseases, sensory organ diseases, and skin and mucous membrane diseases.

Images