A subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and mucosal adjuvant

Abstract

This invention provides a subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and a mucosal adjuvant. The subunit vaccine comprises the Mycoplasma pneumoniae HP14 / 30 fusion antigen and a mucosal adjuvant in a mass ratio of 1-5:1. The HP14 / 30 fusion antigen is a recombinant protein co-expressed by chimeric fragments of the P1 and P30 proteins, with the amino acid sequence shown in SEQ ID NO:1. This vaccine is administered via mucosal routes such as nasal drops or oral inhalation. It efficiently induces respiratory mucosal-specific IgA and humoral IgG responses, significantly reduces pathogen load in the lungs, alleviates pathological damage to lung tissue, and does not induce enhanced respiratory disease (ERD). Its safety and protective efficacy are significantly superior to traditional inactivated vaccines and intramuscular vaccines. This invention fills the technological gap in human Mycoplasma pneumoniae subunit vaccines, is suitable for the prevention of Mycoplasma pneumoniae infection in children and adults, and has significant clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biological vaccine technology, and specifically relates to a subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and mucosal adjuvant. Background Technology

[0002] Mycoplasma pneumoniae (Mp) is one of the leading pathogens causing community-acquired pneumonia (CAP) in children and adults, with a particularly high incidence in children and adolescents. Although Mp infection is mostly self-limiting, it can lead to serious pneumonia, encephalitis, and neurological complications, and the risk is significantly increased in cases of respiratory co-infection.

[0003] Currently, macrolide antibiotics are the first-line treatment for Mycoplasma gondii (Mp) infection in clinical practice. However, drug resistance is becoming increasingly serious globally. In recent years, the resistance rate of macrolide antibiotics in my country has reached over 80%, leading to decreased efficacy of conventional treatments, prolonged disease course, and increased severity of illness. Therefore, safe and effective vaccination is the most economical and fundamental means of controlling Mp infection and its spread.

[0004] Currently, the development of Mp vaccines mainly focuses on inactivated vaccines, live attenuated vaccines, and subunit vaccines. Among these, inactivated vaccines are the most extensively researched. However, inactivated vaccines are mostly prepared by chemically or physically inactivating whole bacterial cells. Although they can induce a certain immune response, they have significant drawbacks: first, the complex composition of whole bacterial cells can easily trigger non-specific immune disorders; second, traditional formulations using aluminum adjuvants and other similar agents can easily induce enhanced respiratory disease (ERD), leading to post-immunization infection that actually worsens lung pathological damage. Although adding novel adjuvants (such as mucosal adjuvants) or optimizing the inactivation process can improve these problems to some extent, the complex antigenic composition of inactivated vaccines, containing multiple bacterial components, still poses potential interference with the body's immune system and long-term safety risks.

[0005] In summary, current technologies still lack a human Mycoplasma pneumoniae vaccine that is highly safe, does not induce ERD, can effectively activate respiratory mucosal immunity, and precisely targets key antigens of Mp infection. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and a mucosal adjuvant. By using the key pathogenic fusion antigen HP14 / 30 of Mycoplasma pneumoniae in combination with a special mucosal adjuvant, a vaccine regimen that can be administered via the respiratory mucosal route is constructed. This achieves the technical effects of strongly inducing specific mucosal IgA and humoral immune responses, significantly reducing the pathogen load in the lungs, effectively alleviating pathological damage to lung tissue, and not inducing ERD. Thus, it fills the gap in the field of human Mycoplasma pneumoniae subunit vaccines and meets the urgent clinical need for safe and effective preventive agents.

[0007] The objective of this invention is achieved through the following technical solution: A first aspect of the present invention provides a subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and a mucosal adjuvant, the vaccine comprising: (a) Mycoplasma pneumoniae HP14 / 30 fusion antigen, wherein the fusion antigen is a recombinant protein obtained by chimeric co-expression of immunogenic fragments of P1 and P30 proteins; and (b) Mucosal adjuvants; The mass ratio of Mycoplasma pneumoniae HP14 / 30 fusion antigen to mucosal adjuvant in the vaccine is 1~5:1.

[0008] Furthermore, the Mycoplasma pneumoniae HP14 / 30 fusion antigen has the amino acid sequence shown in SEQ ID NO:1: MHHHHHHSGSLKTTTPVFGTSSGNLSSVLSGGGAGGGSSGSGQSGVDLSPVEKVSGWLVGQLPSTSDGNTSSTNNLAPNTNTGNDVVGVGRLSESNAAKMNDDVDGIVRTPLAELLDGEGQTADTG PQSVKFKSPDQIDFNRLFTHPVTDLFDPVTMLVYDQYIPLFIDIPASVNPKMVRLKVLSFDTNEQSLGLRLEFFKPDQDTQPNNNVQVNPNNGDFLPLLTASSQGPQTLFSPFNQGGGSMLVLFSA LIVLATLILVQHNNTELTEVKSELSPLNVVLHAEEDTVQIQGKPITEQAWFIPTVAGCFGFSALAIILGLAIGLPIVKRKEKRLLEEKERQEQLAEQLQRISAQQEEQQALEQQAAAEAHAEAEVE PAPQPVPVPPQPQVQINFGPRTGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMPPHPGMAPRPGFPPQPGMAPRPGMQPPRPGMPPQPGFPPKR.

[0009] Furthermore, the Mycoplasma pneumoniae HP14 / 30 fusion antigen is encoded by the nucleotide sequence shown in SEQ ID NO:2:

[0010] Furthermore, the mucosal adjuvant includes bacterial flagellin, Escherichia coli heat-sensitive toxin LT, and cholera toxin CT.

[0011] Furthermore, the bacterial flagellin flagellin was isolated and purified from Salmonella typhimurium.

[0012] Furthermore, the mass ratio of Mycoplasma pneumoniae HP14 / 30 fusion antigen to mucosal adjuvant in the vaccine is 2:1.

[0013] Furthermore, each dose of the vaccine contains 10 μg of Mycoplasma pneumoniae HP14 / 30 fusion antigen and 5 μg of bacterial flagellin.

[0014] Furthermore, the subunit vaccine is in the form of mucosal immunization and can be administered via nasal drops, oral inhalation, or vaginal suppositories.

[0015] The second aspect of the present invention provides the use of the subunit vaccine described in the first aspect in the preparation of a medicament for the prevention of Mycoplasma pneumoniae infection.

[0016] Furthermore, the drug is one or more of the following: (a) The drug can induce the production of secretory IgA antibodies against Mycoplasma pneumoniae in the respiratory mucosa of the subject; (b) The drug can reduce the pathogen load in the lungs of subjects after infection with Mycoplasma pneumoniae; (c) The drug can reduce lung pathological damage in subjects after infection with Mycoplasma pneumoniae; (d) The drug can prevent the subject from developing enhanced respiratory disease (ERD).

[0017] The advantages of this invention compared to the prior art are as follows: 1. The subunit vaccine of the present invention uses the HP14 / 30 fusion antigen, which is related to the key pathogenicity of Mycoplasma pneumoniae, as the immune component, abandoning the complex components of whole bacteria, avoiding non-specific immune interference, reducing potential safety risks, and having stronger immune targeting; at the same time, it abandons aluminum adjuvants that are prone to causing enhanced respiratory disease (ERD), and selects a compatible and safe mucosal adjuvant, so that it does not aggravate lung pathological damage after immunization, while achieving efficient immune enhancement. 2. The subunit vaccine of the present invention, administered via mucosal routes such as nasal drops, can significantly induce the production of specific secretory IgA antibodies in bronchoalveolar lavage fluid, respiratory mucosa, and other sites, directly blocking the adhesion and invasion of Mycoplasma pneumoniae, resulting in a more targeted protective effect. 3. The subunit vaccine of the present invention can effectively reduce the pathogen load in the lungs after immunization, and alleviate the weight loss, lung tissue inflammation, hemorrhage, fibrosis and other damage caused by infection. The overall protective effect is significantly better than that of the muscle immunization group and the adjuvant-free group. At the same time, the subunit vaccine adopts non-invasive mucosal immunization methods such as nasal drops and oral inhalation, which is simple to operate and has high compliance. It is especially suitable for susceptible populations such as children and is easy to promote and apply on a large scale. Attached Figure Description

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 The results show the lung viral load of Mp in mice from different vaccine groups 4 days after infection with Mycoplasma pneumoniae. Data from seven groups—HP14 / 30IM, HP14 / 30+AlumIM, AlumIM, HP14 / 30IN, HP14 / 30+flagellinIN, flagellinIN, and the challenge group—were analyzed using one-way ANOVA to determine significant differences. The challenge group served as the control group. This represents a difference value p < 0.05; Figure 2 The changes in body weight of mice in different vaccine groups over 5 consecutive days after challenge with Mycoplasma pneumoniae are shown. The data at 4 dpi between the HP14 / 30+flagellin IN group and the challenge group were analyzed using a t-test to determine the statistical significance. This represents a difference value p < 0.05; Figure 3 The results of HP14 / 30 antigen-specific IgG antibody titers in the serum of mice in different vaccine groups after immunization and before challenge are shown. Data from the six groups—HP14 / 30 IM, HP14 / 30+Alum IM, Alum IM, HP14 / 30 IN, HP14 / 30+flagellinIN, and flagellinIN—were analyzed using one-way ANOVA to determine significant differences between groups. The mean values ​​for each group are listed above the bars in the graph, and the graph indicates… This represents a difference value p < 0.01. This represents a difference value p < 0.001; Figure 4 The results of HP14 / 30 antigen-specific IgA antibody titers in BALF of mice from different vaccine groups 4 days after immunization and challenge with the pathogen are shown. Data from the six groups—HP14 / 30 IM, HP14 / 30+Alum IM, Alum IM, HP14 / 30 IN, HP14 / 30+flagellin IN, and flagelin IN—were analyzed using one-way ANOVA to determine significant differences between groups. The mean values ​​for each group are listed above the bars in the figure. This represents a difference value p < 0.05; Figure 5 Representative images of lung pathological sections from mice in different vaccine groups 5 days after immunization and challenge with the pathogen are shown; the sections were photographed under 2x and 20x light microscopes, and the standard lines represent 1 mm and 100 μm, respectively. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0022] Example 1: Cultivation and Counting of Mp Take one vial (1 mL) of Mp stock solution and rapidly dissolve it in a 37°C water bath. Add 9 mL of preheated PPLO medium and 1 mL of Mp stock solution to a 15 mL centrifuge tube. Perform aseptic procedures throughout the process. After adding the stock solution, vortex to mix thoroughly. Incubate the centrifuge tube at 37°C in a 5% CO2 incubator for 3-5 days, observing the color change of the medium daily. Based on the physicochemical properties of Mp, during its metabolism, it can convert the sugars in the PPLO liquid medium into acids, thus lowering the pH of the PPLO medium from 7.8 to 6.8. At this time, the color of the PPLO liquid medium will change from red to orange-yellow. Typically, this color change occurs on the third day after thawing, when Mp enters the logarithmic growth phase.

[0023] Mp culture medium was serially diluted 10×, 100×, and 1000×, and 100 μL of each dilution was evenly spread onto PPLO solid medium plates. The solid medium was incubated at 37°C in a 5% CO2 incubator for 5-7 days, with daily observation of colony growth. After seven days of incubation, the plates were placed under an inverted microscope to count the number of colonies and calculate the CFU value using the following formula: .

[0024] Example 2: Preparation, Immunization, and Related Detection of Mp Subunit Vaccine The Mycoplasma pneumoniae HP14 / 30 fusion antigen (amino acid sequence shown in SEQ ID NO:1) expressed by Pujian Biotechnology was used, 1 mg / tube lyophilized, and reconstituted with 1 mL of sterile water to a stock solution concentration of 1 mg / mL; the mucosal adjuvant, bacterial flagellin (purchased from Invivogen), 100 μg / tube was added to 200 μL of sterile water to a stock solution concentration of 0.5 mg / mL. Vaccine preparation and immunization were performed according to the groupings in Table 1.

[0025] Experimental animals: 6-8 week old pathogen-free female BALB / c mice provided by the Experimental Center of the Institute of Medical Biology, Chinese Academy of Medical Sciences were used. The mice were randomly divided into 5 groups of 6 mice each and housed under SPF conditions with free access to food and water.

[0026] Immunization doses: Mice were immunized via intramuscular injection / nasal drop, 50 μL / mouse, as set in Table 1. The control group was injected with an equal volume of PBS. The second dose of vaccine was administered 4 weeks later using the same method.

[0027]

[0028] Two weeks after the second immunization, each group of mice was infected with either Mp or PBS, respectively. The mice were anesthetized with isoflurane gas, and a concentration of [missing information] was applied. Each mouse was challenged with 50 μL of Mp bacterial suspension at a concentration of CFU / mL. The control group received 50 μL of PBS intranasally. Day 0 of the challenge was recorded as day 0 (dpi). All mice were weighed daily after challenge, and weight changes were recorded for a total of 5 days (dpi, 1 dpi, 2 dpi, 3 dpi, 4 dpi).

[0029] like Figure 1 The results showed that Mp challenge (model group) significantly increased the pathogen load in the lungs of animals to 28.61 copies / mg. The three intranasal immunization groups (HP14 / 30 IN, HP14 / 30+flagellin IN, and flagelin IN) reduced the lung load to 1.1, 0.6, and 1 copies / mg, respectively, showing significant differences compared to the challenge group. The three intramuscular immunization groups (HP14 / 30 IM, HP14 / 30+Alum IM, and Alum IM) reduced the lung load to 1.6, 3.7, and 28.35 copies / mg, respectively, showing no significant differences compared to the challenge group.

[0030] Figure 2The results showed that animals challenged with Mp (model group) reached their lowest weight on the second day after challenge (2 dpi), at 86.17% ± 1.96% of their initial weight. Compared to the model group, the three nasal immunization groups (HP14 / 30 IN, HP14 / 30+flagellin IN, and flagellin IN) showed weight recovery at 1 dpi, with the lowest weight loss points being 89.71% ± 1.33%, 89.18% ± 2.80%, and 91.34% ± 1.48%, respectively. There were no significant differences among the three nasal immunization groups. At 4 dpi, the weights of the HP14 / 30 IN, HP14 / 30+flagellin IN, and flagellin IN groups recovered to 96.85% ± 2.01%, 97.45% ± 2.00%, and 97.23% ± 3.37%, respectively, with no significant differences among the three groups. The HP14 / 30+flagellin IN group showed a significant difference compared to the challenge group.

[0031] Obtaining and testing immune serum: Two weeks after the final immunization and before challenge, venous blood was collected from mice. The blood was incubated overnight at 4°C and then centrifuged at 3000 rpm for 20 minutes to obtain serum. The titer of Mp antigen-specific antibodies was detected by indirect enzyme-linked immunosorbent assay (ELISA).

[0032] The specific implementation method is as follows: HP14 / 30 antigen was plated at a concentration of 4 μg / mL, with a volume of 50 μL per well. After incubation at 4°C overnight, the plate was washed twice with PBST. 50 μL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the plate was washed three times, and serially diluted serum samples were added to each well. The plate was incubated at 37°C for another hour. After incubation, the plate was washed four times, and HRP-labeled IgG antibody (diluted 10,000 times with 1% skim milk) was added to each well. After incubation for 30 minutes, the plate was washed five times, the liquid inside was blotted dry, and 50 μL of TMB chromogenic solution was added to each well. After 5 minutes, 50 μL of 2M sulfuric acid was added to each well to stop the chromogenic reaction. The absorbance value was read at 450 nm. The average of the readings from the blank control group was multiplied by 2.1 to obtain the cutoff value. The serum dilution factor in which the reading of other groups is higher than the cutoff value is defined as the IgG antibody titer of that group's serum.

[0033] like Figure 3As shown, muscle immunization with two groups of HP14 / 30 IM and HP14 / 30+Alum IM containing the HP14 / 30 antigen increased IgG antibody titers to 174,080 and 184,320, respectively. In contrast, nasal immunization with two groups of HP14 / 30 IN and HP14 / 30+flagellin IN induced lower IgG antibody titers, at 33,280 and 56,320, respectively.

[0034] Obtaining and testing mucosal samples: Four days after challenge (4 dpi), mice were euthanized by injecting an excessive amount of tribromoethanol (100 mg / kg). The trachea was exposed by dissecting the neck of the mice, and an endotracheal tube was inserted. 1 mL of PBS was injected to rinse the lungs, and the fluid was aspirated and stored as bronchoalveolar lavage fluid (BALF). The BALF was centrifuged at 1000 rpm for 10 min, and the supernatant was stored for IgA antibody titer detection.

[0035] The specific implementation method is as follows: HP14 / 30 antigen was plated at a concentration of 4 μg / mL, with a volume of 50 μL per well. After incubation at 4°C overnight, the plate was washed twice with PBST. 50 μL of 5% skim milk was added to each well, and the plate was blocked at 37°C for 1 hour. After blocking, the plate was washed three times, and serially diluted serum samples were added to each well. The plate was incubated at 37°C for another hour. After incubation, the plate was washed four times, and HRP-labeled IgA antibody (diluted 200-fold with 1% skim milk) was added to each well. After incubation for 30 minutes, the plate was washed five times, the liquid inside was blotted dry, and 50 μL of TMB chromogenic solution was added to each well. After 5 minutes, 50 μL of 2M sulfuric acid was added to each well to stop the chromogenic reaction. The absorbance value was read at 450 nm. The average of the readings from the blank control group was multiplied by 2.1 to obtain the cutoff value. The serum dilution factor in which the readings of other groups are higher than the cutoff value is defined as the IgA antibody titer of the BALF in that group.

[0036] like Figure 4 As shown, in BALF, muscle immunization with either HP14 / 30 IM or HP14 / 30+Alum IM containing the HP14 / 30 antigen did not increase IgA antibody titers. In contrast, intranasal immunization with either HP14 / 30 IN or HP14 / 30+flagellin IN induced higher IgA antibody titers, at 20 and 24, respectively. Among all groups, HP14 / 30+flagellin IN induced the highest IgA antibody titer level.

[0037] Lung pathology examination: After euthanasia of the mice, the lung tissue was aseptically isolated and promptly fixed in 4% paraformaldehyde solution. It was then sent to Wuhan Saiwei Company for subsequent embedding, sectioning, and hematoxylin-Eosin (H&E) staining. The sections were scanned in their entirety and magnified in certain areas using a scanner.

[0038] like Figure 5 As shown, in the HP14 / 30 IM group, a very small number of lymphocytes were infiltrated on the surface of the lung tissue capsule (brown arrows); a small number of scattered macrophages were visible in the alveolar cavities (green arrows), a small number of alveolar stenosis and a large number of alveolar dilation (purple arrows), and the alveoli varied in size; lymphocytes were occasionally seen around the bronchioles (blue arrows); a small number of lymphocytes were seen around a small number of blood vessels (yellow arrows), and a small number of interstitial vascular congestions were observed (orange arrows).

[0039] In the HP14 / 30+Alum IM group, focal infiltration of lymphocytes was observed on the surface capsule of lung tissue (brown arrows); numerous scattered macrophages and lymphocytes were observed in the alveolar cavities (green arrows), with a small number of alveolar stenosis and a small number of alveolar dilation (purple arrows), and alveoli of varying sizes; a small amount of eosinophilic material and desquamated epithelial cells were observed in the lumen of the bronchioles (silver arrows), with occasional hemorrhage (pink arrows), and a small amount of lymphocyte infiltration was observed around the bronchioles (blue arrows); focal infiltration of lymphocytes was observed around numerous blood vessels (yellow arrows).

[0040] In the Alum IM group, focal lymphocyte infiltration was observed on the surface capsule of lung tissue (brown arrows); a small number of scattered macrophages were observed in the alveolar cavities (green arrows), a small number of alveolar stenosis and a small number of alveolar dilation (purple arrows), and alveoli of varying sizes; occasional lymphocyte infiltration was observed around the bronchioles (blue arrows); a small number of lymphocyte infiltrations were observed around a small number of blood vessels (yellow arrows), and a small amount of connective tissue hyperplasia was observed (cyan arrows).

[0041] In the HP14 / 30 IN group, focal lymphocytic infiltration was observed in several places on the surface of the lung tissue capsule (brown arrows); numerous scattered macrophages and lymphocytes were observed in the alveolar cavities (green arrows), with a small number of alveolar stenosis and a small number of alveolar dilation (purple arrows), and the alveoli varied in size; a small number of foam cells were observed in the lumen of the bronchioles (silver arrows), and a small number of lymphocytic infiltrations were observed around the bronchioles (blue arrows); focal lymphocytic infiltrations were observed around many blood vessels (yellow arrows).

[0042] In the HP14 / 30+flagellin IN group, focal lymphocyte infiltration was observed in multiple areas of the lung tissue capsule (brown arrows); a small number of scattered macrophages and lymphocytes were observed in the alveolar cavities (green arrows), with a small number of alveolar stenosis and a small number of alveolar dilation (purple arrows), and alveoli of varying sizes; occasional lymphocyte infiltration was observed around the bronchioles (blue arrows); and a small number of focal lymphocyte infiltrations were observed around blood vessels (yellow arrows).

[0043] In the flagellin IN group, small focal infiltrations of lymphocytes were observed on the surface of the lung tissue and within the parenchyma (brown arrows); a small number of scattered macrophages and lymphocytes were observed in the alveolar cavities (green arrows), with a small number of alveolar stenosis and a small number of alveolar dilation (purple arrows), and alveoli of varying sizes; occasional lymphocyte infiltration was observed around the bronchioles (blue arrows); a small number of lymphocyte infiltrations were observed around a small number of blood vessels (yellow arrows).

[0044] In the challenge group, focal lymphocytic infiltration was observed in several places on the surface of the lung tissue capsule (brown arrows); a small number of scattered macrophages were observed in the alveolar cavities (green arrows), many alveoli were narrowed, a small number of alveoli were dilated (purple arrows), the alveoli were of varying sizes, and small areas of alveolar hemorrhage were observed (pink arrows); occasionally detached cells were observed in the lumen of the bronchioles (silver arrows), and a small number of lymphocytic infiltrations were observed around the bronchioles (blue arrows); focal lymphocytic infiltrations were observed around many blood vessels (yellow arrows).

[0045] In the blank control group, the lung tissue surface membrane structure was clear; the lung parenchyma consisted of the various branches of the bronchi and their terminal alveoli, with occasional lymphocyte infiltration around the bronchioles (blue arrows); a very small number of scattered macrophages were visible in the alveolar cavities (green arrows), a small number of alveoli were narrowed, and a large number of alveoli were dilated (purple arrows), with alveoli of varying sizes.

[0046] In summary, the lung lesions in the challenge group mice were successfully established. Correspondingly, the nasal immunization groups showed varying degrees of reduction in lung lesions, with the HP14 / 30+flagellin IN group showing a significant reduction in lesions.

[0047] In summary, this invention represents a groundbreaking innovation in vaccine safety, understanding of immune mechanisms, and clinical applicability, providing an efficient, safe, and convenient solution for the prevention of Mycoplasma pneumoniae infection, and has significant scientific value and application prospects.

[0048] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A subunit vaccine containing Mycoplasma pneumoniae HP14 / 30 fusion antigen and mucosal adjuvant, characterized in that, The vaccine contains: (a) Mycoplasma pneumoniae HP14 / 30 fusion antigen, said fusion antigen being a recombinant protein obtained by chimeric co-expression of immunogenic fragments of P1 and P30 proteins; and (b) Mucosal adjuvants; The mass ratio of Mycoplasma pneumoniae HP14 / 30 fusion antigen to mucosal adjuvant in the vaccine is 1~5:

1.

2. The subunit vaccine according to claim 1, characterized in that, The Mycoplasma pneumoniae HP14 / 30 fusion antigen has the amino acid sequence shown in SEQ ID NO:

1.

3. The subunit vaccine according to claim 1, characterized in that, The Mycoplasma pneumoniae HP14 / 30 fusion antigen is encoded by the nucleotide sequence shown in SEQ ID NO:

2.

4. The subunit vaccine according to claim 1, characterized in that, The mucosal adjuvants include bacterial flagellin, Escherichia coli heat-sensitive toxin LT, and cholera toxin CT.

5. The subunit vaccine according to claim 4, characterized in that, The bacterial flagellin flagelin was isolated and purified from Salmonella typhimurium.

6. The subunit vaccine according to claim 1, characterized in that, The mass ratio of Mycoplasma pneumoniae HP14 / 30 fusion antigen to mucosal adjuvant in the vaccine is 2:

1.

7. The subunit vaccine according to claim 1, characterized in that, Each dose of the vaccine contains 10 μg of Mycoplasma pneumoniae HP14 / 30 fusion antigen and 5 μg of bacterial flagellin.

8. The subunit vaccine according to claim 1, characterized in that, The subunit vaccine is administered via mucosal immunization and can be given via nasal drops, oral inhalation, or vaginal suppositories.

9. Use of the subunit vaccine according to any one of claims 1 to 7 in the preparation of a medicament for the prevention of Mycoplasma pneumoniae infection.

10. The use according to claim 9, characterized in that, The drug is one or more of the following: (a) The drug can induce respiratory mucosa-specific IgA and humoral IgG responses in subjects; (b) The drug can reduce the pathogen load in the lungs of subjects after infection with Mycoplasma pneumoniae; (c) The drug can reduce lung pathological damage in subjects after infection with Mycoplasma pneumoniae; (d) The drug can prevent the subject from developing enhanced respiratory disease (ERD).

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