Mycoplasma pneumoniae vaccine and application thereof

Abstract

The invention provides a mycoplasma pneumoniae vaccine and application thereof. Specifically, the invention relates to a mycoplasma pneumoniae protein, a vaccine composition composed of the mycoplasma pneumoniae protein, a preparation method and medical application of the mycoplasma pneumoniae protein, and the vaccine composition has a good protection effect on mycoplasma pneumoniae infection, and can remarkably relieve weight loss, reduce lung pathogen load and reduce lung pathological score. The mycoplasma pneumoniae vaccine has extremely high application value in mycoplasma pneumoniae vaccine development.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering vaccine technology, and specifically relates to a Mycoplasma pneumoniae vaccine and its pharmaceutical uses. Background Technology

[0002] Mycoplasma pneumoniae is a cell-wall-less microorganism and a major causative agent of atypical pneumonia, often causing tracheobronchitis and other upper and lower respiratory tract infections. Approximately 70% of patients experience symptoms such as fever, dry cough, rhinitis, sore throat, and shortness of breath. Severe infections can lead to respiratory diseases such as pulmonary edema, obstructive bronchiolitis, chronic interstitial fibrosis, and acute respiratory distress syndrome (ARDS), and may also cause extrapulmonary symptoms affecting the neurological, cardiovascular, skin, digestive, hematologic, and musculoskeletal systems. Susceptible populations include children under 5 years old, the elderly, and those with weakened immune systems, accounting for 20%-30% of community-acquired pneumonia cases. Macrolide antibiotics are first-line treatments, but over the past decade, macrolide-resistant Mycoplasma pneumoniae (MRMP) has become a global public health problem, particularly prevalent in Asia, with resistance rates ranging from 13.6% to 100%, exceeding 90% in some epidemic seasons in Japan and China. Therefore, there is an urgent need to strengthen research on new drugs and novel preventative measures, among which vaccination can effectively block transmission and reduce the risk of infection.

[0003] One of the major obstacles to the development of human Mycoplasma pneumoniae vaccines is the lack of clearly defined protective antigens or immune targets. Its infection and pathogenesis are complex, involving pathways such as adhesion to host cells, nutrient deprivation damage, invasion, toxin production, cytokine-mediated inflammatory damage, and immune escape. Extrapulmonary manifestations are related to direct damage mediated by invasion and inflammatory factors, indirect damage from the host immune response, and vascular obstruction. Despite extensive vaccine development efforts, no Mycoplasma pneumoniae vaccine has yet been approved for marketing due to the complex pathogenesis, unclear vaccine targets, and the risk of vaccine-induced disease enhancement. Summary of the Invention

[0004] To address the aforementioned technical challenges, this invention innovatively selects CARDS, P1, P40-90, P116, and MPN133 as templates for segmented expression and site-directed mutagenesis to screen for effective immunogenic epitopes. Simultaneously, based on a multi-antigen combined immunization design strategy for bacterial vaccines, combining proteins with strong immunogenicity and protective efficacy unexpectedly yielded better combined immunization results, opening up new avenues for the development of Mycoplasma pneumoniae vaccines.

[0005] In a first aspect, the present invention provides a vaccine composition.

[0006] In some embodiments, the vaccine composition comprises at least three proteins selected from the group shown below or variants thereof: CARDS, P1, P40-90, P116, and MPN133;

[0007] In some specific embodiments, the CARDS or its variants comprise an amino acid sequence as shown in any one of SEQ ID NO:1, 6-10; the P1 or its variants comprise an amino acid sequence as shown in any one of SEQ ID NO:2, 11-18; the P40-90 or its variants comprise an amino acid sequence as shown in any one of SEQ ID NO:3, 19-20; the P116 or its variants comprise an amino acid sequence as shown in any one of SEQ ID NO:4, 21-23; and the MPN133 or its variants comprise an amino acid sequence as shown in any one of SEQ ID NO:5 or 24.

[0008] In some embodiments, the vaccine composition includes CARDS, P116, and MPN133;

[0009] In some specific embodiments, the vaccine composition may also contain P1 and / or P40-90;

[0010] In some specific embodiments, the vaccine composition includes CARDS, P116, P1, and MPN133; in some specific embodiments, the composition includes CARDS, P116, P40-90, and MPN133; in some specific embodiments, the composition includes CARDS, P116, P1, P40-90, and MPN133.

[0011] In some specific embodiments, the CARDS or its variants comprise amino acid sequences as shown in SEQ ID NO:1, 6-10; the P1 or its variants comprise amino acid sequences as shown in SEQ ID NO:2, 11-18; the P40-90 or its variants comprise amino acid sequences as shown in SEQ ID NO:3, 19-20; the P116 or its variants comprise amino acid sequences as shown in SEQ ID NO:4, 21-23; and the MPN133 or its variants comprise amino acid sequences as shown in SEQ ID NO:5 and 24.

[0012] In some specific embodiments, the vaccine composition comprises any one of the following groups:

[0013] 1-1) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6, 8-10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14, 16-18, P40-90 with amino acid sequences as shown in SEQ ID NO:19, P116 with amino acid sequences as shown in SEQ ID NO:21-23, and MPN133 with amino acid sequences as shown in SEQ ID NO:24;

[0014] 1-2) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6-10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14 and 16-18, P40-90 with amino acid sequences as shown in SEQ ID NO:19-20, P116 with amino acid sequences as shown in SEQ ID NO:21-23, and MPN133 with amino acid sequences as shown in SEQ ID NO:24;

[0015] 1-3) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6-8 or 10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14, P40-90 with amino acid sequences as shown in SEQ ID NO:20, P116 with amino acid sequences as shown in SEQ ID NO:21 or 22, and MPN133 with amino acid sequences as shown in SEQ ID NO:24.

[0016] 1-4) CARDS with amino acid sequences as shown in any one of SEQ ID NO: 6, 7, or 10; P1 with amino acid sequences as shown in any one of SEQ ID NO: 12; P40-90 with amino acid sequences as shown in SEQ ID NO: 20; P116 with amino acid sequences as shown in SEQ ID NO: 22; or MPN133 with amino acid sequences as shown in SEQ ID NO: 24; or

[0017] 1-5) CARDS with amino acid sequences as shown in SEQ ID NO:10, P116 with amino acid sequences as shown in SEQ ID NO:22, and MPN133 with amino acid sequences as shown in SEQ ID NO:24.

[0018] In some specific embodiments, the vaccine composition comprises: CARDS with the amino acid sequence shown in SEQ ID NO:10, P116 with the amino acid sequence shown in SEQ ID NO:22, and MPN133 with the amino acid sequence shown in SEQ ID NO:24.

[0019] In some embodiments, the vaccine composition further includes a vaccinologically acceptable carrier;

[0020] In some specific embodiments, the vaccinologically acceptable carrier includes an adjuvant; in some specific embodiments, the adjuvant includes, but is not limited to, aluminum adjuvant, MF59, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvant, BCG ribonucleic acid, CpG ODN (unmethylated cytosine-guanine dinucleotide oligodeoxynucleotide), cholera toxin B subunit (CTB), Escherichia coli heat-labile enterotoxin B subunit (LTB), polyinosinic acid-polycytidylic acid (Poly I:C), or monophospholipase A.

[0021] Secondly, the present invention provides nucleic acid molecules.

[0022] In some implementations, the nucleic acid molecule is a single nucleic acid molecule or a group of nucleic acid molecules.

[0023] In some specific embodiments, the nucleic acid molecule encodes a recombinant molecular form of any of the proteins described above or variants thereof;

[0024] Thirdly, the present invention provides a recombinant expression vector.

[0025] In some implementations, the recombinant expression vector comprises the nucleic acid molecules described above.

[0026] Fourthly, the present invention provides a host cell.

[0027] In some embodiments, the host cell expresses a protein or a variant thereof as defined in any of the vaccine compositions described above; or contains a nucleic acid molecule as described above; or contains a recombinant expression vector as described above.

[0028] In some embodiments, the host cell is selected from the group consisting of bacteria and fungi, preferably, the host cell is *Escherichia coli* Pichia pastoris; more preferably, the host cell is *Escherichia coli*.

[0029] Fifthly, the present invention provides a method for preparing a vaccine composition.

[0030] In some embodiments, the method includes: culturing host cells as described above; and isolating the protein or a variant thereof from the culture product to prepare a vaccine composition.

[0031] In a sixth aspect, the present invention provides the use of any of the vaccine compositions, nucleic acid molecules, recombinant expression vectors or host cells described above in the preparation of pharmaceutical compositions;

[0032] In some embodiments, the pharmaceutical composition has the following characteristics:

[0033] 1) To prevent or reduce Mycoplasma pneumoniae infection in humans;

[0034] 2) To generate an immune response in the subject; and / or,

[0035] 3) Prevention and / or treatment of diseases or conditions caused by Mycoplasma pneumoniae infection in subjects.

[0036] The present invention has the following technical effects:

[0037] 1) This invention screens for optimal immune targets and, drawing on bacterial vaccine development strategies, employs mixed antigens to enhance vaccine efficacy. The prepared vaccine combination exhibits effective protection against Mycoplasma pneumoniae infection and provides dual protection against early bacterial growth and inflammation.

[0038] 2) The three modified antigens have unique immune properties. When mixed in equal proportions, they exhibit a synergistic effect and can effectively protect mice from pathological damage induced by Mycoplasma pneumoniae infection. This combination of vaccine antigens has extremely high application value. Attached Figure Description

[0039] Figure 1 This diagram illustrates the design and preparation of protein sequences. 1A is a schematic diagram of the CARDS histone sequence: CARDS1 and CARDS2 are different modified products of the KELED sequence; CARDS3, CARDS4, and CARDS5 correspond to different domains of the CARDS protein. 1B is a schematic diagram of the P1 histone sequence: P1-1 to P1-3 are different truncated designs of the P1 protein; P1-4 is the N-terminal fragment of the P1 protein; P1-5 to P1-8 are different truncated designs of the C-terminal fragment of the P1 protein. 1C is a schematic diagram of the P40-90 histone sequence: P40-90-1 is the N-terminal fragment of the P40-90 protein; P40-90-2 is the P40 fragment. 1D is a schematic diagram of the P116 histone sequence: P116-1 is a partially truncated design product of the P116 protein, and P116-2 and P116-3 are different truncated design products of the N-terminal fragment of the P116 protein. 1E is a schematic diagram of the MPN133 histone sequence: MPN133-1 is the MPN133 protein lacking the EKS region (amino acids 72-110).

[0040] Figure 2 SDS-PAGE electrophoresis images of 19 proteins.

[0041] Figure 3The table shows the animal immunization process and post-boost immunization test results. 3A is a schematic diagram of the animal immunization process; 3B-3G are the results of antigen-specific antibody testing after booster immunization (n=6); 3H is the result of Mycoplasma pneumoniae-specific antibody testing after booster immunization (CARDS group vs other groups, p<0.05, n=6); 3I is the result of Mycoplasma pneumoniae metabolic inhibition antibody testing after booster immunization (serum samples were equal volumes of serum from 6 mice in each group, and three independent replicate experiments were performed, P116 group vs other groups, p<0.0001, n=6).

[0042] Figure 4 This section presents the results of antigen-specific antibody testing after primary immunization. Specifically, sections 4A-4E show the results of antigen-specific antibodies after primary immunization, with the horizontal axis representing serum dilution and the vertical axis representing absorbance at 450 nm; section 4F compares the geometric mean titers of antigen-specific antibodies after primary immunization.

[0043] Figure 5 To enhance the comparison results of geometric mean titers of antigen-specific antibodies after immunization.

[0044] Figure 6 This section presents the results of infection detection following Mycoplasma pneumoniae challenge. Specifically, for infections 6A-6E, weight monitoring was performed at days 0, 1, 3, 5, and 7 (percentage change in weight; n=6, p>0.05); for 6F, Mycoplasma pneumoniae viral load was detected in lung tissue at day 7 post-infection; for 6G, pathological examination of lung tissue was performed at day 7 post-infection (CARDS5, P116-2, MPN133-1 group vs. PBS group, n=6, p<0.05); and for 6H, all test indicators were summarized and analyzed.

[0045] Figure 7 The results show the viral load of Mycoplasma pneumoniae in the upper respiratory tract. 7A-7D represent the Mycoplasma pneumoniae viral load results from mouse throat swabs on days 1, 3, 5, and 7 after infection, respectively.

[0046] Figure 8This section presents the mixed immunization protocols and post-immunization test results. Specifically: 8A. Mixed immunization flowchart; 8B. Antigen-specific binding antibody detection after the first immunization (in the high-dose mixed immunization group, "highmix-1" represents the test results with CARD5 as the coating antigen, "highmix-2" represents the test results with P116-2 as the coating antigen, and "highmix-3" represents the test results with MPN133-1 as the coating antigen; in the low-dose mixed immunization group, "lowmix-1 / -2 / -3" correspond to antibody responses against CARD5, P116-2, and MPN133-1, respectively); 8C. Antigen-specific binding antibody detection after the second immunization; 8D. Mycoplasma pneumoniae-specific antibody detection after booster immunization; 8E. Mycoplasma pneumoniae metabolic inhibition assay after booster immunization; 8F. Cytokine detection after booster immunization (ELISPOT: IFN-γ, n=3); 8G. Cytokine detection after booster immunization (ELISPOT: IL-2, n=3).

[0047] Figure 9 The results show the antibody subtype analysis (after booster immunization) in multi-antigen immunization. 9A represents the geometric mean titer of antigen-specific IgG1 antibodies after booster immunization in a mixed immunization experiment; 9B represents the geometric mean titer of antigen-specific IgG2a antibodies after booster immunization in a mixed immunization experiment; and 9C represents the ratio of antigen-specific antibodies IgG2a / IgG1 after booster immunization in a mixed immunization experiment (Note: the vertical axis represents the value of IgG2a / IgG1 magnified 100 times).

[0048] Figure 10 This study evaluates the protective effect against Mycoplasma pneumoniae challenge after multi-antigen mixed immunization. The results include: 10A. Body weight monitoring 0-4 days post-infection (high-dose mixed group vs. PBS group, p<0.001, n=7); 10B. Mycoplasma pneumoniae load in lung tissue 4 days post-infection (n=3); 10C. Lung tissue pathological score 4 days post-infection (n=3); and 10D. Lung tissue pathological sections 4 days post-infection in CARDS5, P116-2, MPN133-1, high-dose mixed group, low-dose mixed group, and PBS group. Detailed Implementation

[0049] The present invention will now be described in detail with reference to embodiments, but the embodiments provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0051] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Nevertheless, this invention still intends to provide a more detailed description and explanation of these terms and phrases. In the event of any inconsistency between the terms and phrases mentioned and their known meanings, the meanings expressed in this invention shall prevail.

[0052] Example 1: Design and preparation of recombinant proteins

[0053] The standard strain M129 of *Mycoplasma pneumoniae* was selected as the research object. Sequences encoding key antigen proteins of *Mycoplasma pneumoniae* were obtained from the NCBI database, including CARDSS (accession number: 4TLW_A), P1 (accession number: WP_010874498.1), P40-P90 (accession number: WP_010874499.1), P116 (accession number: GAB1890787.1), and MPN133 (accession number: WP_010874490.1), with specific sequences shown in SEQ ID NO: 1-5. Through amino acid mutation and truncation design, 19 protein sequences were finally obtained from 5 *Mycoplasma pneumoniae* proteins. Detailed information is shown in Table 1, with specific sequences shown in SEQ ID NO: 6-24.

[0054] Table 1. Detailed information on 19 proteins

[0055]

[0056] The CARDS group contains five proteins: CARDS1 and CARDS2 target the key amino acid signature sequence KELED (which plays a crucial role in toxin transport to target cells and subsequent toxin-mediated cytotoxicity); CARDS3, CARDS4, and CARDS5 target different domains of the CARDS protein ( Figure 1 A). The P1 protein contains 1530 amino acids, has a molecular weight of 167 kDa, and a complex spatial conformation. Based on the design principles of immunofocusing and maintaining the native spatial conformation, the full-length P1 protein was partially truncated to obtain P1-1, P1-2, and P1-3. Simultaneously, literature reports that antibodies recognizing the C-terminal domain of the P1 protein are more effective in inducing immunoprotection than antibodies recognizing the N-terminal variable region. Therefore, the N-terminus and C-terminus of the P1 protein were truncated and immunofocused to obtain P1-4 and P1-5 to P1-8 (…). Figure 1 B). In the P40-90 group, since the genetic variation of Mycoplasma pneumoniae is mainly concentrated in the N-terminal domains of P1 and P40 / P90, the N-terminus of P40-90 was truncated to obtain P40 / P90 N (P40-90-1) and P40 (P40-90-2). Figure 1 C). In the P116 group, partially truncated P116 protein (P116-1) and several truncated products of its N-terminal fragment (P116-2 / P116-3) were designed; studies have confirmed that the N-terminal fragment of P116 serves as a specific immunogen during Mycoplasma pneumoniae infection and can be used for the serological diagnosis of Mycoplasma pneumoniae infection. Figure 1 D). MPN133 is a calcium-dependent cytotoxic nuclease from Mycoplasma pneumoniae, containing a unique glutamate-lysine-serine enriched region (EKS region, amino acids 72-110). The deletion of this EKS peptide does not alter its nuclease activity. Based on this, MPN133 Δ72-110 (MPN133-1) was designed. Figure 1 E).

[0057] After systematic codon optimization of the above sequences in *E. coli*, they were cloned into the prokaryotic expression vector pET-30a to construct recombinant plasmids encoding 19 target proteins. These recombinant plasmids were transformed into the prokaryotic expression strain BL21(DE3). Following positive clone selection, large-scale culture, induced expression, cell lysis, and purification, SDS-PAGE analysis showed that all proteins were expressed in soluble form and exhibited a single band at the expected molecular weight, with purity meeting animal immunization requirements. Figure 2 ).

[0058] Example 2: Immunogenicity assessment of antigens in mice

[0059] 2.1 Antibody Detection

[0060] One hundred and twenty 6-8 week old female BALB / c mice were randomly divided into 20 groups of six each. Groups 1-19 were the experimental groups, and group 20 was the control group. All mice were immunized by intramuscular injection. Each mouse in the experimental group received 10 μg of antigen and 50 μg of aluminum hydroxide adjuvant per injection, for a total injection volume of 100 μL. The control group received an equal volume of PBS. The immunization schedule consisted of two injections, 21 days apart. Blood samples were collected from the inner canthal vein of the mice 21 days after the initial immunization and 21 days after the booster immunization to detect antigen-specific antibodies, Mycoplasma pneumoniae-specific antibodies, and metabolic inhibitory antibodies.

[0061] The antigen-specific antibody detection procedure is as follows: Five template proteins (CARDS, P1, P40-90, P116, and MPN133) were diluted to 2 μg / mL with coating buffer (sodium carbonate 1.59 g / L, sodium bicarbonate 2.93 g / L), and 100 μL was added to each well of a 96-well polypropylene microplate. The plate was incubated overnight at 4°C. 300 μL of 1% bovine serum albumin (BSA) was added to each well, and the plate was blocked at 37°C for 2 hours to block non-specific binding. Subsequently, mouse serum samples were added (1:100 dilution in the first well, 1:1000 dilution in the second well, and subsequent serial dilutions were performed), 100 μL per well, and incubated at 37°C for 1 hour. The plate was washed four times with PBST (PBS, pH 7.4, containing 0.05% Tween 20), 300 μL per well each time. Add 100 μL of horseradish peroxidase (HRP)-labeled anti-mouse IgG (1:10000 dilution) to each well and incubate at 37°C for 1 hour. Wash four times with PBST, 300 μL / well each time. Add 100 μL of tetramethylbenzidine (TMB) substrate to each well and incubate at room temperature for 5–8 minutes. Stop the reaction by adding 50 μL of 2 mol / L sulfuric acid and measure the absorbance at 450 nm using a microplate reader.

[0062] The steps for detecting Mycoplasma pneumoniae-specific antibodies are as follows: Mycoplasma pneumoniae was centrifuged at 10,000 rpm for 50 minutes. The precipitate was resuspended in PBS and washed three times. Subsequently, it was sonicated (350 W power, 3-second sonication followed by 5-second intervals, total sonication time 30 minutes). The protein concentration of the sonicated products was determined using the BCA method. The Mycoplasma pneumoniae lysate was diluted to 10 μg / mL with coating buffer, and 50 μL was added to each well of a 96-well polyacrylamide microplate. The plate was incubated overnight at 4°C. The next day, 300 μL of 1% BSA was added to each well, and the plate was blocked at 37°C for 2 hours. Mouse serum samples were added (1:100 dilution in the first well, subsequent serial dilutions), 100 μL per well, and incubated at 37°C for 1 hour. After washing four times with PBST, HRP-labeled anti-mouse IgG (1:10,000 dilution) was added to each well, 100 μL, and the plate was incubated at 37°C for 1 hour. After washing again, TMB substrate was added for color development, the reaction was terminated with sulfuric acid, and the absorbance at 450 nm was measured.

[0063] The steps for detecting the activity of metabolic inhibitory antibodies are as follows: In the experimental preparation stage, prepare Mycoplasma pneumoniae culture medium containing 56.4 μmol / L phenol red indicator (pH 7.9), collect serum from mice after booster immunization, and prepare sterile 96-well cell culture plates. Serum treatment and addition: The serum to be tested is serially diluted eight times in Mycoplasma pneumoniae culture medium. Add 50 μL of diluted serum to each well in columns 1-7 of the 96-well plate, and add 50 μL of culture medium to each well in column 8 as a Mycoplasma pneumoniae growth control. Simultaneously, Mycoplasma pneumoniae in the logarithmic growth phase is diluted 1:10 with culture medium, and 50 μL is added to all wells in columns 1-8. Standard curve setting: Using the 1:10 diluted Mycoplasma pneumoniae as the stock solution, prepare 10 gradient Mycoplasma pneumoniae solutions with concentrations ranging from 90% to 0%, and add 100 μL to each well in columns 9-10. Incubation and detection: The 96-well plate was placed in a 37℃, 5% CO2 incubator. The color change of the medium in column 8 was observed daily, and the incubation was stopped when it turned yellow. The absorbance (OD) at 560 nm of all wells was measured using a microplate reader. 560 ). Result determination: Using data from columns 9-10, fit the "Mycoplasma pneumoniae concentration-OD" data. 560 "Standard curve. Compare the OD values ​​of the experimental group and the control group." 560 Value: OD 560 The value was negatively correlated with the activity of inhibitory antibodies in serum; the inhibitory antibody titer was defined as the highest serum dilution that could inhibit 50% of Mycoplasma pneumoniae metabolism.

[0064] Antibody test results as follows Figure 3-5 As shown, the results indicated that antigen-specific antibody responses were generally low in all groups after the initial immunization. However, analysis of the initial immunization results revealed that the KELED sequence was crucial for the immunogenicity of the CARDS protein; in groups P1 and P116, antibody responses were positively correlated with protein sequence length; furthermore, the antibody levels induced by CARDS1 and CARDS5 in the CARDS group and by P116-1 and P116-3 in the P116 group were significantly higher than those induced by other antigens in the same group (…). Figure 4 21 days after the booster immunization, the levels of antigen-specific antibodies in all groups were significantly increased, and the OD450 value remained above 1 even after a 1000-fold dilution of serum. Figure 3B-3F). Results from booster immunization in the CARDS group further confirmed the crucial role of the KELED sequence in the immunogenicity of CARDS proteins, while CARDS5 still exhibited antibody response capabilities comparable to CARDS1. In the P1 group, the specific antibody responses induced by C-terminal truncated P1-5 to P1-8 were significantly lower than those induced by other antigens in the same group, indicating that excessive truncation of the P1 protein significantly weakens its immunogenicity. In the P116 group, booster immunization with all three antigens induced strong antibody responses with no significant differences within the group, indicating that the truncation strategy targeting the P116 protein successfully achieved the dual goals of immune focusing and immunogenicity preservation. The antibody response level after booster immunization in the MPN133-1 group was significantly enhanced compared to the initial immunization, suggesting that this antigen can effectively trigger humoral immune memory responses after booster immunization (…). Figure 5 ).

[0065] Results of Mycoplasma pneumoniae-specific antibody testing after booster immunization showed that antibodies induced by 19 recombinant proteins could bind to Mycoplasma pneumoniae, with geometric mean titers (GMT) ranging from 800 to 3200. Although there were no significant differences between groups using the same template design, intergroup comparisons showed that the antibody response in the CARDS group was significantly lower than that in other antigen groups (n=6, p<0.05). Figure 3 H).

[0066] The detection of Mycoplasma pneumoniae metabolic inhibitory antibodies showed that, compared with the Mycoplasma pneumoniae control group and the PBS immunization group, the serum of animals immunized with 19 recombinant proteins could partially inhibit the growth of Mycoplasma pneumoniae. Notably, the three antigens in the P116 group exhibited strong Mycoplasma pneumoniae metabolic inhibitory activity, significantly inhibiting Mycoplasma pneumoniae metabolism even at a serum dilution of 1:256 (p<0.0001). Figure 3 I).

[0067] Example 3: Evaluation of protective effect against Mycoplasma pneumoniae infection in mice

[0068] Twenty-two days after booster immunization, mice were challenged with Mycoplasma pneumoniae via intranasal inoculation. Body temperature and weight were monitored on days 1, 3, 5, and 7 post-infection, and pharyngeal swabs were collected to detect Mycoplasma pneumoniae load. Seven days post-infection, mice were sacrificed, and lung tissue was harvested for Mycoplasma pneumoniae load detection and histopathological examination.

[0069] 3.1 Mycoplasma pneumoniae viral load detection

[0070] The viral load of Mycoplasma pneumoniae in pharyngeal swabs and lung tissue was detected using real-time quantitative polymerase chain reaction (qPCR). Mice were anesthetized, and pharyngeal swabs were obtained by swabbing the pharynx with sterile swabs pre-soaked in physiological saline. The collected swabs were placed in pre-aliphatic 800 μL of sample preservation solution and vortexed to fully dissolve the contaminants. 200 μL of the sample solution was then used for nucleic acid extraction. After euthanizing the mice, lung tissue was dissected and separated. The right lung was weighed and homogenized in 800 μL of sample preservation solution. The homogenate was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and gently mixed by pipetting. 200 μL of the supernatant was used for nucleic acid extraction.

[0071] Primer and probe design (for the P1 gene) is shown below:

[0072] P1 upstream primer (F)

[0073] CCAACCAAACAACAACGTTCA

[0074] SEQ ID NO:25

[0075] P1 downstream primer (R)

[0076] ACCTTGACTGGAGGCCGTTA

[0077] SEQ ID NO:26

[0078] > P1 detection probe

[0079] FAM-TCAATCCGAATAACGGTGACTTCTTACCACTG-BHQ1

[0080] SEQ ID NO:27

[0081] qPCR reaction system composition and program parameters:

[0082] The reaction system (total volume 10 μL) consisted of: 2.5 μL sample, 0.5 μL upstream primer (concentration 20 μmol / L), 0.5 μL downstream primer (concentration 20 μmol / L), 0.5 μL probe (concentration 5 μmol / L), 2.5 μL 4×Master mix, and 3.5 μL enzyme-free water.

[0083] Reaction program: 95°C pre-denaturation for 1 minute; followed by 40 cycles (95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 40 seconds).

[0084] 3.2 Histopathological examination

[0085] Tissue was fixed in 10% formalin for 3 days. After paraffin embedding, it was cut into 5 μm thick sections and stained with hematoxylin and eosin (HE). The sections were scanned using CaseViewer 2.4 (3DHISTECH, Hungary). Scoring criteria included inflammatory cell infiltration, pulmonary hemorrhage, alveolar wall thickening, and consolidation.

[0086] 0 points: Normal organization;

[0087] 1 point: The change is slightly outside the normal range;

[0088] 2 points: Pathological changes are visible, but not serious;

[0089] 3 points: Significant change, possibly worsening;

[0090] 4 points: The changes are extremely serious and may affect the entire tissue or organ.

[0091] The scores of each item are added together to obtain the total lung pathology score.

[0092] Statistical analysis: Statistical analysis and data visualization were performed using Excel, GraphPad Prism 8, and Adobe Illustrator 2024. Differences among multiple groups were compared pairwise using one-way ANOVA combined with Tukey's post-hoc test. p < 0.05 was considered statistically significant.

[0093] 3.3 Comprehensive Comparative Indicators for Antigen Screening

[0094] The five core indicators and corresponding standards for screening superior antigens are as follows:

[0095] 1) Enhanced antigen-specific antibodies after immunization: when the antigen is diluted 8000 times, the absorbance value (OD450) is still ≥1;

[0096] 2) Enhanced post-immune metabolic inhibitory antibodies: When serum is diluted ≥4 times, it has significant inhibitory activity against Mycoplasma pneumoniae;

[0097] 3) Changes in body weight in mice after Mycoplasma pneumoniae challenge: The decrease in body weight was less than that in the PBS-immunized negative control group;

[0098] 4) Mycoplasma pneumoniae load after challenge: The Mycoplasma pneumoniae load in pharyngeal swabs (days 1, 3, 5, and 7) and lung tissue (day 7) was lower than that in the PBS immunization group;

[0099] 5) Lung tissue pathology score: The score was lower than that of the PBS immunization group (p<0.05);

[0100] 3.4 Experimental Results

[0101] The results showed no significant changes in body temperature among the groups of mice; however, one day after infection, all groups experienced a decrease in body weight, reaching 90%-95% of their original body weight; three days after infection, body weight continued to decrease, dropping to 85%-93% of the original body weight; five days after infection, body weight began to recover, and by seven days it had recovered to 90%-95% of the original body weight. Figure 6 A-6E). Pharyngeal swab tests collected at 1, 3, 5, and 7 days post-infection showed no significant difference in Mycoplasma pneumoniae load among the groups at each time point, suggesting that the 19 recombinant proteins, after intramuscular injection, failed to effectively activate local mucosal immunity and innate immune pathways in the respiratory tract, and could not form a targeted immune protective barrier on the mucosal surface. Figure 7 Seven days after infection, the viral load of Mycoplasma pneumoniae in lung tissue was approximately 10. 6 -10 7 The number of copies / g showed no statistically significant difference among the groups. Figure 6 F). Histopathological examination showed that mice in the PBS-immunized group had significant lung damage, manifested as inflammatory cell infiltration and alveolar wall thickening; pathological scoring results showed that the pathological scores of all protein-immunized groups were lower than those of the PBS-immunized group, and the pathological damage in the CARD5, P116-2, and MPN133-1 groups was significantly less than that in the PBS group (n=6, p<0.05). Figure 6 G).

[0102] By comprehensively evaluating multiple immunogenicity indicators, including the levels of antigen-specific antibodies and metabolic inhibitory antibodies after enhanced immunization, the viral load of Mycoplasma pneumoniae in lung tissue 7 days post-infection, and the pathological score of lung tissue, three highly effective candidate antigens were ultimately screened: CARDS5, P116-2, and MPN133-1. Figure 6 H). The list of candidate antigens obtained according to the screening criteria in Section 3.3 is detailed in Table 2.

[0103] Table 2. Comprehensive Analysis of Detection Indicators

[0104]

[0105] Example 4: Immunogenicity assessment of multiple antigens in mice

[0106] Based on preliminary antigen screening experiments, three different groups of antigens (CARDS5, P116-2, and MPN133-1) were selected for further evaluation. To investigate the immunogenicity and immunoprotective effects of single and combined antigens, six experimental groups were designed: three single antigen groups, two mixed antigen groups, and one PBS control group. Sixty SPF-grade female BALB / c mice (6-8 weeks old) were randomly divided into six groups, with 10 mice in each group. Groups 1-3 were immunized individually with CARD5, P116-2, or MPN133-1, with each mouse receiving 10 μg of antigen and 50 μg of aluminum hydroxide adjuvant per immunization. Group 4 (high-dose mixed group) received a high-dose mixture (each mouse receiving 10 μg of each antigen, 30 μg of total antigen, and 150 μg of aluminum adjuvant per immunization). Group 5 (low-dose mixed group) received a low-dose formulation (each mouse receiving 3.3 μg of each antigen, 10 μg of total antigen, and 50 μg of aluminum adjuvant per immunization). Group 6 received 100 μL of PBS per mouse via intramuscular injection. The immunization schedule consisted of two doses, 21 days apart. Blood samples were collected from the orbital venous plexus 21 days after the initial immunization and 21 days after the booster immunization to detect antigen-specific antibodies, Mycoplasma pneumoniae-specific antibodies, and Mycoplasma pneumoniae metabolic inhibitory antibodies. 21 days after the booster immunization, three mice from each group were sacrificed for ELISPOT assay to assess cellular immune response (see the mixed immunization protocol for details). Figure 8 A).

[0107] Antigen-specific antibody detection results showed that after the initial immunization, regardless of whether a single antigen, high-dose mixed, or low-dose mixed immunization regimen was used, the CARD5 antigen could induce a high level of antibody response in the host, which is consistent with previous antigen screening experimental data. Analysis of the CARD5-specific antibody levels after the initial immunization revealed that the antibody levels induced by the high-dose mixed immunization group were significantly higher than those induced by the CARD5 single antigen immunization group, indicating that the antibody responses induced by each antigen in the high-dose mixture did not interfere with each other, but rather promoted the antibody response to some extent; in addition, the CARD5-specific antibody levels induced by the high-dose mixed immunization group were significantly higher than those induced by the low-dose mixed immunization group, suggesting a dose-dependent effect. Figure 8 B).

[0108] Following booster immunization, antigen-specific antibody titers significantly increased in all experimental groups. Notably, there was no statistically significant difference in antibody levels against CARD5, P116-2, and MPN133-1 between the high-dose mixed immunization group and the single-antigen immunization group, further confirming that the antibody responses induced by each antigen in the high-dose mixture did not interfere with each other. It is noteworthy that the serum antibody binding capacity against CARD5, P116-2, and MPN133-1 in the low-dose mixed immunization group showed significant differences, with the binding capacity against CARD5 antigen remaining significantly stronger. Within the mixed immunization group, significant differences remained in antibody levels against P116-2 and MPN133-1 between the high-dose and low-dose groups, further confirming dose-dependent antibody responses. Figure 8 C).

[0109] To further investigate the immunomodulatory properties of three antigens—CARDS5, P116-2, and MPN133-1—the levels of IgG1 and IgG2a binding antibodies in the immune serum were measured. The results showed that IgG1 titers ranged from 3000 to 500000, while IgG2a titers were only 100 to 2000. All antigen immunization groups exhibited a significantly higher IgG1 titer than IgG2a titer (IgG2a / IgG1 ratio < 1). This antibody subtype distribution pattern is consistent with the inherent immunomodulatory properties of the aluminum adjuvant used in this experiment, indicating that all five immunization groups induced a Th2-biased immune response. Notably, analysis of the IgG2a / IgG1 ratio magnified 100-fold revealed that, in both single-antigen and mixed-antigen immunization groups, the IgG2a / IgG1 ratio induced by P116-2 antigen was significantly higher than that induced by CARD5 and MPN133-1 antigens. This suggests that P116-2, under a Th2-biased immune background, can induce a relatively stronger Th1-type immune response and has the potential to trigger a balanced Th1 / Th2 immune response. Figure 9 Post-boost immunization, Mycoplasma pneumoniae-specific antibody testing showed no significant difference between the single-antigen immunization group and the high-dose mixed-antigen immunization group, while the mixed-antigen immunization group exhibited a clear dose-dependent effect. Figure 8 D).

[0110] Twenty-one days after booster immunization, the levels of metabolic inhibitory antibodies in mouse serum were measured. The serum pools from each group of mice were used, and the results were obtained from three independent replicates. The results showed that in the single-antigen immunization groups, the titer of metabolic inhibitory antibodies in the P116-2 group was significantly higher than that in the CARD5 and MPN133-1 groups (n=3, p<0.001), consistent with the antigen screening data. Interestingly, in the mixed immunization groups, the levels of metabolic inhibitory antibodies in both the low-dose and high-dose mixed immunization groups were significantly higher than those in the P116-2 single-antigen immunization group (n=3, p<0.05), indicating a synergistic effect of the mixture of three antigens in inducing metabolic inhibitory antibodies. Furthermore, the titer of metabolic inhibitory antibodies in the high-dose mixed immunization group was significantly higher than that in the low-dose mixed immunization group, showing a dose-dependent relationship (n=3, p<0.0001). Figure 8 E).

[0111] Enzyme-linked immunospot (ELISpot) assays were performed on isolated mouse spleen lymphocytes. The results showed that both P116-2 and MPN133-1 antigens induced high levels of IFN-γ and IL-2 production, regardless of whether a single antigen or a mixture of high and low doses was used. In contrast, immunization with the CARDS5 antigen alone induced a lower level of cellular immune response. However, when CARDS5 was combined with the other two antigens (high and low doses), the cellular immune response was significantly enhanced, indicating a synergistic effect among the three antigens. Figure 8 F and 8G).

[0112] Example 5: Evaluation of the protective effect of multiple antigens against Mycoplasma pneumoniae infection in mice

[0113] Twenty-two days after booster immunization, mice were challenged with Mycoplasma pneumoniae via intranasal inoculation. Body temperature and weight were monitored daily for 1-7 days post-infection. Four days post-infection, the first three numbered mice from the remaining group were sacrificed, and lung tissue was harvested for Mycoplasma pneumoniae load detection and histopathological examination. Weight changes in these three mice were monitored from 0-4 days post-infection. Seven days post-Mycoplasma pneumoniae challenge, the remaining four mice from each group were sacrificed, and lung tissue was isolated for pathogen load detection and lung pathological scoring.

[0114] The results showed that mice in the PBS-immunized control group experienced weight loss 1-4 days post-infection, reaching 85% of their baseline weight on day 4, consistent with the preliminary screening results. In contrast, the experimental group showed less weight loss 1-4 days post-infection compared to the PBS-immunized control group. Notably, all single-component groups began to recover weight 3 days after Mycoplasma pneumoniae challenge, while the high-dose mixed group showed weight gain as early as 2 days post-challenge, with the overall weight loss remaining less than 5% throughout the observation period. Figure 10A). Four days post-infection, the viral load of Mycoplasma pneumoniae in the lung tissue of all experimental groups was lower than that in the PBS-immunized group, but there was no statistically significant difference among the protein-immunized groups. Figure 10 B). Pathological analysis of lung tissue 4 days post-infection showed that the pathological scores of all protein-immunized groups were lower than those of the PBS-immunized group, with the high-dose mixed group having the lowest pathological score. There were no obvious inflammatory infiltration or alveolar wall thickening, typical pathological changes associated with Mycoplasma pneumoniae infection. Figure 10 D).

[0115] The specific embodiments are merely illustrative of the present invention and are not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

[0116] The partial sequence of this invention is as follows:

[0117] > CARDS

[0118] MDHHHHHHHASENLYFQGHMPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQRE WFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKELEDTPVYLRGIKTQKT FMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAAS TFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF

[0119] SEQ ID NO:1

[0120] > P1

[0121]

[0122] SEQ ID NO:2

[0123] > P40-90

[0124]

[0125] SEQ ID NO:3

[0126] > P116

[0127] MNKTHQVEHESEQSDFQDIRFGLNSVKLPKAQPAAATRITVENGTDKLVNYKSSPQQLFLAKNALKDKLQGEFDKFLSDAKAFPALTADLQEWVDQQLFNPNQSFFDLSAPRSNFTLSSDDKKASLDFIFRFTNFTESVQLLKLPEGVSVVVDSKQSFDYYVNASAQKLLVLPLSLPDYTLGLNYMFDHITLNGKVVNKFSFNPFKTNLNLAFSNVYNGVDVFEAQKNLVGKGKYLNTHVKAEDVKKDVNANIKNQFDIAKIIAELMGKALKEFGNQQEGQPLSFLKVMDKVKEDFEKLFNLVRPGLGKFVKDLIQSSSQAENKITVYKLIFDNKKTILNLLKELSIPELNSSLGLVDVLFDGITDSDGLYERLQSFKDLIVPAVKTNEKTAALSPLIEELLTQKDTYVFDLIQKHKGILTNLLKNFLADFQKSTPFMADQVAIFTELFDNEGAFDLFGEADFVDKIAE LFLTKRTVKNGEKIETKDSLLVTSLKSLLGEKVAALGDLLDSYIFKNELLNRSEVAKAEAKDTKGATDYKKEQAKALKKLFKHIGENTLSKTNLDKITLKEVKNTENVELEETETTLKVKKLDVEYKVELGNFEIKNGLIKAMLEFLPDTKDLETTLDKLLFKGESYKAMKDKYIKEGFPGYGWAKGVVPGAFESIENTFKSAIDKTKSIRDLFGDMLFGNDLSSVKETDSFITLGGSFDIKYGGENLNVLPAYYSLINSEIGYQIIGVDTTIDATKVKVELKNKEYKGKSPAINGQVKLSQSFFNVWTNMFDSITKQIFQKKYEFKDNIQVFARNEDNTSRLELDISDPEQRVIPFAFVDGFGIQLKAVDKNITKEAGNTEPKSPVIQLYEALNKEKDQKQQSKQSPKQLDTKTQLGYLLKLDNWSKDDYKSLIDDTIINNNYLEASFNSKITVDGGHHHHHHHHH

[0128] SEQ ID NO:4

[0129] > MPN133

[0130] MTRDYTTKNEFQLTTAQQAKLKPATIEYWRDGDTPEINYASEERRKEAEQKSKENAKKEDKKEEKKTEDSQDSSSASTQVRSSKHGLRIYGIDTPEKHVSSKGDSTGDEKIEAEKASNYAEKLIPKGSTVWVWSLNTYSYDREVGALFFKSNPKQTFFQSFEVAMVEAGHAIPIAGTGLNLIADPELSADDPLSVIGLQLANAANKAYNAKINIWSHDTDGYRSLTAVYKLRGADISWTRFLDEANGYSSASAGTGASLYQLWDQRQAKLAQKGS

[0131] SEQ ID NO:5

[0132] > CARDS1

[0133] MDHHHHHHHHASENLYFQGHMPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVKALADTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF

[0134] SEQ ID NO:6

[0135] > CARDS2

[0136] MDHHHHHHHHASENLYFQGHMPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSVTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF

[0137] SEQ ID NO:7

[0138] > CARDS3

[0139] MDHHHHHHHHASENLYFQGHMPNPVRFVYRVDLRSPEEIFEHGFSTLGDVRNFFEHILSTNFGRSYFISTSETPTAAIRFFGSWLREYVPEHPRRAYLYEIRADQHFYNARATGENLLDLMRQRQVVFDSGDREMAQMGIRALRTSFAYQREWFTDGPIAAANVRSAWLVDAVPVEPGHAHHPAGRVVETTRINEPEMHNPHYQELQTQANDQPWLPTPGIATPVHLSIPQAASVADVSEGTSASLSFACPDWSPPSSNGENPLDKCIAEKIDNYNLQSLPQYASSV

[0140] SEQ ID NO:8

[0141] > CARDS4

[0142] MDHHHHHHHHASENLYFQGHTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAAST

[0143] SEQ ID NO:9

[0144] > CARDS5

[0145] MDHHHHHHHHASENLYFQGHTPVYLRGIKTQKTFMLQADPQNNNVFLVEVNPKQKSSFPQTIFFWDVYQRICLKDLTGAQISLSLTAFTTQYAGQLKVHLSVSAVNAVNQKWKMTPQDIAITQFRVSSELLGQTENGLFWNTKSGGSQHDLYVCPLKNPPSDLEELQIIVDECTTHAQFVTMRAASTFFVDVQLGWYWRGYYYTPQLSGWSYQMKTPDGQIFYDLKTSKIFFVQDNQNVFFLHNKLNKQTGYSWDWVEWLKHDMNEDKDENFKWYFSRDDLTIPSVEGLNFRHIRCYADNQQLKVIISGSRWGGWYSTYDKVESNVEDKILVKDGFDRF

[0146] SEQ ID NO:10

[0147] > P1-1

[0148]

[0149] SEQ ID NO:11

[0150] > P1-2

[0151]

[0152] SEQ ID NO:12

[0153] > P1-3

[0154]

[0155] SEQ ID NO:13

[0156] > P1-4

[0157]

[0158] SEQ ID NO:14

[0159] > P1-5

[0160] MDHHHHHHHHEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQWPDYVLPL

[0161] SEQ ID NO:15

[0162] > P1-6

[0163] MDHHHHHHHHSLKTTTPVFGTSSGNLSSVLSGGGAGGGSSGSGQSGVDLSPVEKVSGWLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQ

[0164] SEQ ID NO:16

[0165] > P1-7

[0166] MDHHHHHHHHSGGGAGGGSSGSGQSGVDLSPVEKVSGWLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQ

[0167] SEQ ID NO:17

[0168] > P1-8

[0169] MDHHHHHHHHQSGVDLSPVEKVSGWLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTGPQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQ

[0170] SEQ ID NO:18

[0171] > P40-90-1

[0172] *

[0173] SEQ ID NO:19

[0174] > P40-90-2

[0175] MNTYLLQDHNTLTPYTPFTTPLNGGLDVVRAAHLHPSYELVDWKRVGDTKLVALVRSALVRVKFQDTTSSDQSNTNQNALSFDTQESQKALNGSQSGSSDTSGSNSQDFASYVLIFKAAPRATWVFERKIKLALPYVKQESQGSGDQGSNGKGSLYKTLQDLLVEQPVTPYTPNAGLARVNGVAQDTVHFGSGQESSWNSQRSQKGLKNNPGPKAVTGFKLDKGRAYRKLNESWPVYEPLDSTKEGKGKDESSWKNSEKTTAENDAPLVGMVGSGAAGSASSLQGNGSNSSGLKSLLRSAPVSVPPSSTSNQTLSLSNPAPVGPQAVVSQPAGGATAAVSVNRTASDTATFSKYLNTAQALHQMGVIVPGLEKWGGNNGTGVVASRQDATSTNLPHAAGASQTGLGTGSPREPALTATSQRGGHHHHHHHH*

[0176] SEQ ID NO:20

[0177] > P116-1

[0178] MNKTHQVEHESEQSDFQDIRFGLNSVKLPKAQPAAATRITVENGTDKLVNYKSSPQQLFLAKNALKDKLQGEFDKFLSDAKAFPALTADLQEWVDQQLFNPNQSFFDLSAPRSNFTLSSDDKKASLDFIFRFTNFTESVQLLKLPEGVSVVVDSKQSFDYYVNASAQKLLVLPLSLPDYTLGLNYMFDHITLNGKVVNKFSFNPFKTNLNLAFSNVYNGVDVFEAQKNLVGKGKYLNTHVKAEDVKKDVNANIKNQFDIAKIIAELMGKALKEFGNQQEGQPLSFLKVMDKVKEDFEKLFNLVRPGLGKFVKDLIQSSSQAENKITVYKLIFDNKKTILNLLKELSIPELNSSLGLVDVLFDGITDSDGLYERLQSFKDLIVPAVKTNEKTAALSPLIEELLTQKDTYVFDLIQKHKGILT NLLKNFLADFQKSTPFMADQVAIFTELFDNEGAFDLFGEADFVDKIAELFLTKRTVKNGEKIETKDSLLVTSLKSLLGEKVAALGDLLDSYIFKNELLNRSEVAKAEAKDTKGATDYKKEQAKALKKLFKHIGENTLSKTNLDKITLKEVKNTENVELEETETTLKVKKLDVEYKVELGNFEIKNGLIKAMLEFLPDTKDLETTLDKLLFKGESYKAMKDKYIKEGFPGYGWAKGVVPGAFESIENTFKSAIDKTKSIRDLFGDMLFGNDLSSVKETDSFITLGGSFDIKYGGENLNVLPAYYSLINSEIGYQIIGVDTTIDATKVKVELKNKEYKGKSPAINGQVKLSQSFFNVWTNMFDSITKQIFQKKYEFKDNIQVFARNEDNTSRLELDISDPEQRVIPFAFVDGGHHHHHHHH

[0179] SEQ ID NO:21

[0180] > P116-2

[0181] MNKTHQVEHESEQSDFQDIRFGLNSVKLPKAQPAAATRITVENGTDKLVNYKSSPQQLFLAKNALKDKLQGEFDKFLSDAKAFPALTADLQEWVDQQLFNPNQSFFDLSAPRSNFTLSSDKKASLDFIFRFTNFTESVQLLKLPEGVSVVVDSKQSFDYYVNASAQKLLVLPLSLPDYTLGLNYMFDHITLNGKVVNKFSFNPFKTNLNLAFSNVYNGGGHHHHHHHH

[0182] SEQ ID NO:22

[0183] > P116-3

[0184] MVDVFEAQKNLVGKGKYNLTHVKAEDVKKDVNANIKNQFDIAKIIAELMGKALKEFGNQQEGQPLSFLKVMDKVKEDFEKLFNLVRPGLGKFVKDLIQSSSQAENKITVYKLIFDNKKTILNLLKELSIPELNSSLGLVDVLFDGITDSDGLYERLQSFKDLIVPAVKTNEKTAALSPL IEELLTQKDTYVFDLIQKHKGILTNLLKNFLADFQKSTPFMADQVAIFTELFDNEGAFDLFGEADFVDKIAELFLTKRTVKNGEKIETKDSLVTSLKSLLGEKVAALGDLLDSYIFKNELLNRSVEKAEAKDTKGATDYKKEQAKALKKLFKHIGENTLSKTNLDKITLKEVKNTEN VELEETETTLKVKKLDVEYKVELGNFEIKNGLIKAMLEFLPDTKDLETTLDKLLFKGESYKAMKDKYIKEGFPGYGWAKGVVPGAFESIENTFKSAIDKTKSIRDLFGDMLFGNDLSSVKETDSFITLGGSFDIKYGGENLNVLPAYYSLINSEIGYQIIGVDTTIDATKVKVELKNKEYKGKSPAINGQVKLSQSFFNVWTNMFDSITKQIFQKKYEFKDNIQVFARNEDNTSRLELDISDPEQRVIPFAFVDGFGIQLKAVDKNITKEAGNTEPKSPVIQLYEALNKEKDQKQQSKQSPKQLDTKTQLGYLLKLDNWSKDDYKSLIDDTIINNNYLEASFNSKITVDGGHHHHHHHHH

[0185] SEQ ID NO:23

[0186] > MPN133-1

[0187] MTRDYTTKNEFQLTTAQQAKLKPATIEYWRDGDTPEINYASEERRHGLRIYGIDTPEKHVSSKGDSTGDEKIEAEKASNYAEKLIPKGSTVWVWSLNTYSYDREVGALFFKSNPKQTFFQSFEVAMVEAGHAIPIAGTGLNLIADPELSADDPLSVIGLQLANAANKAYNAKINIWSHDTDGYRSLTAVYKLRGADISWTRFLDEANGYSSASAGTGASLYQLWDQRQAKLAQKGSGGHHHHHHHH*

[0188] SEQ ID NO:24

Claims

1. A vaccine composition, characterized in that, The vaccine composition comprises at least three proteins selected from the group shown below or variants thereof: CARDS, P1, P40-90, P116 and MPN133; Wherein, the CARDS or its variants contain the amino acid sequences shown in any one of SEQ ID NO:1, 6-10; the P1 or its variants contain the amino acid sequences shown in any one of SEQ ID NO:2, 11-18; the P40-90 or their variants contain the amino acid sequences shown in any one of SEQ ID NO:3, 19-20; the P116 or its variants contain the amino acid sequences shown in any one of SEQ ID NO:4, 21-23; and the MPN133 or its variants contain the amino acid sequences shown in any one of SEQ ID NO:5 or 24.

2. The vaccine composition according to claim 1, characterized in that, The vaccine composition includes CARDS, P116, and MPN133.

3. The vaccine composition according to claim 2, characterized in that, The vaccine composition may also contain P1 and / or P40-90.

4. The vaccine composition according to any one of claims 1-3, characterized in that, The vaccine composition comprises any one of the following groups: 1-1) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6, 8-10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14, 16-18, P40-90 with amino acid sequences as shown in SEQ ID NO:19, P116 with amino acid sequences as shown in SEQ ID NO:21-23, and MPN133 with amino acid sequences as shown in SEQ ID NO:24; 1-2) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6-10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14 and 16-18, P40-90 with amino acid sequences as shown in SEQ ID NO:19-20, P116 with amino acid sequences as shown in SEQ ID NO:21-23, and MPN133 with amino acid sequences as shown in SEQ ID NO:24; 1-3) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6-8 or 10, P1 with amino acid sequences as shown in any one of SEQ ID NO:11-14, P40-90 with amino acid sequences as shown in SEQ ID NO:20, P116 with amino acid sequences as shown in SEQ ID NO:21 or 22, and MPN133 with amino acid sequences as shown in SEQ ID NO:

24. 1-4) CARDS with amino acid sequences as shown in any one of SEQ ID NO:6, 7, or 10; P1 with amino acid sequences as shown in any one of SEQ ID NO:12; P40-90 with amino acid sequences as shown in SEQ ID NO:20; P116 with amino acid sequences as shown in SEQ ID NO:22; or MPN133 with amino acid sequences as shown in SEQ ID NO:24; or 1-5) CARDS with amino acid sequences as shown in SEQ ID NO:10, P116 with amino acid sequences as shown in SEQ ID NO:22, and MPN133 with amino acid sequences as shown in SEQ ID NO:

24.

5. The vaccine composition according to claim 4, characterized in that, The vaccine composition comprises: CARDS with the amino acid sequence shown in SEQ ID NO:10, P116 with the amino acid sequence shown in SEQ ID NO:22, and MPN133 with the amino acid sequence shown in SEQ ID NO:

24.

6. The vaccine composition according to claim 5, characterized in that, The vaccine composition also includes a vaccinologically acceptable carrier; The vaccinologically acceptable carriers include, but are not limited to, aluminum adjuvants, MF59, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvants, BCG ribonucleic acid, CpG ODN (unmethylated cytosine-guanine dinucleotide oligodeoxynucleotide), cholera toxin B subunit (CTB), Escherichia coli heat-labile enterotoxin B subunit (LTB), polyinosinic acid-polycytidylic acid (Poly I:C), or monophospholipase A.

7. A nucleic acid molecule, characterized in that, Its encoding comprises a protein or a variant thereof as defined in the vaccine composition of any one of claims 1-6, wherein the encoding comprises encoding the protein or a variant thereof by a nucleic acid molecule or a group of nucleic acid molecules.

8. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 7.

9. A host cell, characterized in that, It expresses the protein or a variant thereof as defined in any one of the vaccine compositions of claims 1-6; or contains the nucleic acid molecule as described in claim 7; or contains the recombinant expression vector as described in claim 8.

10. A method for preparing a vaccine composition, characterized in that, The method includes: culturing the host cells as described in claim 9; and isolating the protein or a variant thereof from the culture product to prepare a vaccine composition.

Images