Immunogenic mycoplasma pneumoniae polypeptide epitope and recombinant protein containing same, and mycoplasma pneumonia vaccine

Abstract

Provided is an immunogenic Mycoplasma pneumoniae polypeptide epitope, which is a polypeptide epitope derived from the key adhesion proteins P1, P30, P40 / 90 and P116 of type I and type II Mycoplasma pneumoniae, and / or the CARDS toxin thereof. Further provided is a recombinant protein, which contains one or more of the immunogenic Mycoplasma pneumoniae polypeptide epitopes. Further provided is a vaccine for preventing Mycoplasma pneumonia infections, which vaccine provides protection against at least one of type I and / or type II Mycoplasma pneumonia infections, and is characterized in that the vaccine contains the recombinant protein and is a vaccine capable of providing safe and effective protection against Mycoplasma pneumonia infections.

Description

Immunogenic Mycoplasma pneumoniae polypeptide epitopes and recombinant proteins containing them, as well as Mycoplasma pneumoniae vaccines. Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the sequence design, screening, expression, purification preparation method of recombinant protein vaccine for the prevention of Mycoplasma pneumoniae infection and its application. Background Technology

[0002] Mycoplasma pneumoniae (MP) is a cell-wall-less prokaryote belonging to the class Mycohymenospora, order Mycoplasma, family Mycoplasma, and genus Mycoplasma. It is one of the major pathogens causing acute or chronic human respiratory infections. Mycoplasma pneumoniae is primarily transmitted through droplets, with an incubation period of 2 to 3 weeks. It commonly causes respiratory infections, most frequently upper respiratory tract infections and acute bronchitis, and also often causes interstitial pneumonia, affecting the bronchi, bronchioles, alveoli, and pulmonary interstitium. Mycoplasma pneumoniae infection is usually self-limiting and mild, with main clinical manifestations including fever, cough, and may be accompanied by headache, runny nose, sore throat, and earache. Fever is predominantly moderate to high, with persistent high fever indicating a more severe condition. More severe cases may present with serious community-acquired pneumonia or extrapulmonary manifestations. Common extrapulmonary complications include neurological involvement such as encephalitis, circulatory involvement such as thrombosis and myocarditis, hematologic involvement such as immune thrombocytopenic purpura, hemolysis, skin and mucous membrane damage, or other complications such as acute kidney injury, liver failure, and rhabdomyolysis. Mycoplasma pneumoniae infection is more common in school-aged children, but it can also occur in toddlers and adults.

[0003] In China, the infection rate of Mycoplasma pneumoniae peaked in 2007, 2011, 2014, 2017, and 2023. In the peak years, the number of pneumonia cases caused by this pathogen was more than double that of previous years. According to statistics from Guangdong Provincial Maternal and Child Health Hospital in 2023, the infection rate was 19.86% in June and rose to 27.16% in August, with school-aged children accounting for the majority of cases. Severe transmission can lead to endemic or pandemic outbreaks (spreading across provincial, national, or even continental borders to form a global pandemic within a short period). During endemic outbreaks, pneumonia caused by this pathogen may account for 4% to 8% of community-acquired pneumonia, while during pandemics, this proportion can reach as high as 20% to 40%, and even as high as 70% in closed populations. It is estimated that there are approximately 2 million cases of pneumonia caused by this pathogen in the United States each year, resulting in about 100,000 hospitalizations among American adults. Due to the low clinical detection rate of Mycoplasma pneumoniae, the actual number of infections may be even higher.

[0004] The pathogenesis of Mycoplasma pneumoniae infection is not fully understood, but it is currently believed to involve two main mechanisms: direct damage from Mycoplasma pneumoniae and secondary damage caused by abnormal immune responses in the host after infection. In the initial stage of infection, Mycoplasma pneumoniae adheres to the host's bronchial epithelium through its terminal structures, inducing metabolic and ultrastructural changes in infected cells. Simultaneously, it releases CARD toxins, hydrogen peroxide, and superoxide free radicals, leading to direct cell damage. Furthermore, components of Mycoplasma pneumoniae, such as membrane lipids, lipoproteins, HapE enzymes, nucleases, oxidase GlpO, and capsule materials, can further induce cytokines and inflammation, causing indirect damage. In addition, Mycoplasma pneumoniae can evade the host's immune system through its immune evasion mechanisms, enabling it to survive long-term in the body and cause more severe clinical symptoms and harm.

[0005] Currently, treatment options for Mycoplasma pneumoniae infection are still relatively limited, mainly including anti-Mycoplasma pneumoniae therapy and symptomatic treatment, as detailed below:

[0006] 1) Macrolide antibiotics are the first-line treatment option. These mainly include azithromycin, clarithromycin, erythromycin, roxithromycin, and acetyl tyrosine.

[0007] 2) Tetracycline antibiotics, mainly including doxycycline and minocycline, are used to treat drug-resistant Mycoplasma pneumoniae infections. Because they may cause yellowing of teeth and enamel hypoplasia, they are only suitable for children over 8 years of age.

[0008] 3) Quinolone antibiotics. Due to the risk of chondrodysplasia and tendon rupture in humans, they are only suitable for patients aged 18 and older.

[0009] 4) Severe and critically ill children require glucocorticoid therapy and symptomatic treatment for co-infections and other complications.

[0010] With the widespread use (and overuse) of antibiotics, drug-resistant mycoplasma strains have been gradually identified. Currently, macrolide-resistant mycoplasma pneumoniae (MRMP) pneumonia has become a very challenging clinical problem. The global MRMP infection rate rose from 18.2% in 2000 to 41.0% in 2010, and then to 76.5% in 2019. Especially in East Asian countries, the macrolide resistance rate has risen to 90%, posing a significant challenge to treatment.

[0011] Currently, there are no commercially available vaccines for preventing Mycoplasma pneumoniae infection for clinical use. Therefore, developing a Mycoplasma pneumoniae vaccine is of great significance for the prevention and control of Mycoplasma pneumoniae pneumonia. Although several research groups have attempted to develop Mycoplasma pneumoniae vaccines, research has been limited to inactivated vaccines, live attenuated vaccines, or single antigen protein subunit vaccines. These vaccines offer low protective efficacy, short duration of immunity, and can lead to excessive immune responses and severe illness after vaccination. The lipid-associated membrane protein (LAMP) on the Mycoplasma pneumoniae cell membrane of live attenuated or inactivated vaccines can cause an increase in IL-17A concentration and neutrophil count in vivo, leading to an excessive inflammatory response and damage to lung tissue. Animal experimental data indicate that removing the lipid portion of LAMP before vaccination can eliminate the aforementioned vaccine-enhanced disease (VED).

[0012] Summary of the Invention

[0013] The technical problem to be solved by the present invention

[0014] As mentioned above, the development of a Mycoplasma pneumoniae vaccine is of great significance for the prevention and control of Mycoplasma pneumoniae pneumonia. However, there is currently no vaccine available on the market for the prevention of Mycoplasma pneumoniae infection for clinical use. Therefore, the purpose of this invention is to provide a safe and effective vaccine for the prevention of Mycoplasma pneumoniae infection.

[0015] Technical solutions for solving technical problems

[0016] Since the first step in causing cell damage by Mycoplasma pneumoniae is adhesion to the host cell surface, the focus of vaccine research is on inhibiting pathogen adhesion. After in-depth analysis, the inventors discovered that proteins mainly associated with Mycoplasma pneumoniae adhesion include P1, P30, P40 / 90, and P116. These adhesion proteins play a crucial role in Mycoplasma pneumoniae adhesion and infection of respiratory mucosal epithelial cells. Furthermore, CARDS toxin (Community-Acquired Respiratory Distress Syndrome toxin), an exotoxin produced by Mycoplasma pneumoniae with ADP-ribosyltransferase and cellular vacuolizing activities, plays an important role in the pathogenesis of Mycoplasma pneumoniae. During the acute phase of Mycoplasma pneumoniae infection, the level of anti-CARDS toxin antibodies in patients is slightly elevated; however, the level of anti-CARDS toxin antibodies in patients during the recovery phase is significantly elevated, indicating that CARDS toxin has good immunogenicity. Based on the above infection mechanisms, this invention uses bioinformatics techniques to analyze the structure, properties, and amino acid sequences of Mycoplasma pneumoniae P1, P30, P40 / 90, P116, and CARD's toxin proteins. Simultaneously, BLAST analysis was used to remove amino acid sequences that might induce human autoimmunity, thereby screening 23 polypeptide epitopes with strong immunogenicity and high safety. Based on this, the inventors utilized these 23 selected polypeptides, supplemented with an immune adjuvant protein (CRM197), and linked them using a GGGGS (glycine-glycine-glycine-serine) flexible linker, expressing them tandemly into multiple recombinant proteins using genetic engineering techniques. The expressed recombinant proteins exhibit excellent immunogenicity and can be used for Mycoplasma pneumoniae vaccines. Furthermore, they can also be used for the detection of Mycoplasma pneumoniae antibodies and the preparation of Mycoplasma pneumoniae monoclonal and polyclonal antibodies.

[0017] This invention is based on the above research and specifically includes the following:

[0018] 1. An immunogenic Mycoplasma pneumoniae polypeptide epitope, characterized in that the polypeptide epitope is a polypeptide epitope derived from the adhesion key proteins P1, P30, P40 / 90, P116 and / or CARDS toxin of Mycoplasma pneumoniae type I and II.

[0019] 2. The polypeptide epitope as described in 1 above, characterized in that,

[0020] The amino acid sequence of the polypeptide epitope from the P1 protein is the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4.

[0021] The amino acid sequence of the polypeptide epitope from the P30 protein is the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8.

[0022] The amino acid sequence of the polypeptide epitope from the P40 / 90 protein is the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12, or it is an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12.

[0023] The amino acid sequence of the polypeptide epitope from the P116 protein is the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18, or 19, or is an amino acid sequence that has more than 90% homology with and is immunogenic to the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18, or 19 by substituting, deleting, or adding one or more amino acid residues.

[0024] The amino acid sequence of the polypeptide epitope from CARDS toxin is the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23.

[0025] 3. A nucleotide sequence encoding the antigenic epitope described in 1 or 2 above.

[0026] 4. A recombinant protein comprising one or more immunogenic Mycoplasma pneumoniae polypeptide epitopes, said polypeptide epitopes being polypeptide epitopes derived from adhesion key proteins P1, P30, P40 / 90, P116 and / or CARDS toxins of Mycoplasma pneumoniae types I and II.

[0027] 5. The recombinant protein as described in 4 above, characterized in that,

[0028] The amino acid sequence of the polypeptide epitope from the P1 protein is the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4.

[0029] The amino acid sequence of the polypeptide epitope from the P30 protein is the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8.

[0030] The amino acid sequence of the polypeptide epitope from the P40 / 90 protein is the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12, or it is an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12.

[0031] The amino acid sequence of the polypeptide epitope from the P116 protein is the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19, or is an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19.

[0032] The amino acid sequence of the polypeptide epitope from CARDS toxin is the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23.

[0033] 6. The recombinant protein as described in 4 or 5 above, characterized in that the recombinant protein has protective modifications at the N-terminus or C-terminus, or at both ends, the protective modifications including linked protective proteins.

[0034] 7. The recombinant protein as described in 6 above, characterized in that the protective protein comprises at least one protein tag.

[0035] 8. The recombinant protein as described in 7 above, wherein the protein tag is selected from one or more of the following: His tag, S-tag tag, TrxA tag, GST tag, MBP tag, SUMO tag, flag tag, and NusA tag.

[0036] 9. The recombinant protein as described in 4 or 5 above, characterized in that the one or more polypeptide epitopes contained in the recombinant protein are linked by linkers consisting of any amino acid sequence.

[0037] 10. The recombinant protein as described in 4 or 5 above, characterized in that the recombinant protein is linked to or mixed with an amino acid sequence having stabilizing and immunogenicity-enhancing effects.

[0038] 11. The recombinant protein as described in 10 above, characterized in that the amino acid sequence having stability and immunogenicity-enhancing effects is the amino acid sequence of diphtheria toxin CRM197 and / or cholera toxin B subunit CTB.

[0039] 12. The recombinant protein as described in 4 or 5 above, characterized in that,

[0040] The amino acid sequence of the recombinant protein is the amino acid sequence shown in Serial No. 24 or Serial No. 25, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in Serial No. 24 or Serial No. 25 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in Serial No. 24 or Serial No. 25.

[0041] 13. A nucleotide sequence encoding the recombinant protein described in any one of 4 to 12 above.

[0042] 14. The nucleotide sequence as described in 13 above, characterized in that the nucleotide sequence is as shown in sequence number 26 or sequence number 27.

[0043] 15. A vaccine for preventing Mycoplasma pneumoniae infection, which provides protection against infection with at least one of Mycoplasma pneumoniae type I and / or type II, characterized in that the vaccine comprises any one of the recombinant proteins described in any one of 4 to 12 above.

[0044] 16. The vaccine as described in 15 above, characterized in that the dosage form of the vaccine is nasal drops, emulsion, suspension, gel, powder or injection.

[0045] 17. The vaccine as described in 15 or 16 above, characterized in that the route of administration of the vaccine is intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal mucosal administration, oral administration, local transdermal administration, or systemic transdermal administration.

[0046] 18. The application of the polypeptide epitopes described in 1 or 2 above in the preparation of vaccines for the prevention of Mycoplasma pneumoniae infection.

[0047] 19. The use of any one of the recombinant proteins described in 4 to 12 above in the preparation of vaccines for the prevention of Mycoplasma pneumoniae infection.

[0048] The effects of the invention

[0049] According to the present invention, a safe and effective vaccine for preventing Mycoplasma pneumoniae infection can be provided.

[0050] The recombinant protein provided by this invention exhibits excellent immunogenicity and safety. Compared to existing inactivated or attenuated vaccines, the recombinant subunit vaccine designed in this invention does not contain the entire pathogen or its lipid fraction, but only the key sequence of the antigenic epitope. This avoids selecting sequences that could cause allergic reactions or excessive autoimmune responses, thus improving immunogenicity while reducing damage from nonspecific immune responses. Therefore, the recombinant protein vaccine described in this invention has a low risk of causing venereal disease (VED) and is safer, more stable, and more effective than vaccines containing the complete pathogen. Furthermore, compared to existing recombinant protein subunit vaccines, the vaccine of this invention targets a greater number of pathogen target proteins, providing a more comprehensive protective mechanism and achieving stronger immunogenicity and protective efficacy. In addition, the antigenic epitopes selected in this invention are more precise, with shorter peptide chains and greater specificity, improving immunoprotection while significantly reducing the possibility of adverse immune reactions or allergens in the vaccine, resulting in lower safety risks. Moreover, the shorter peptide epitopes selected in this invention can be expressed as recombinant proteins through simple linker modification. These recombinant proteins are stable, exist in a soluble form, are easy to purify, and do not require refolding treatment, thus reducing the technical difficulty and cost of industrial production. Attached Figure Description

[0051] Figure 1 is an SDS-PAGE image of the protein sample from Mycoplasma pneumoniae lysate (SDS concentration of 15%), with a loading volume of 1 μL.

[0052] Figure 2 shows the serum titer of New Zealand white rabbits detected by indirect ELISA.

[0053] Figure 3 shows the detection of positive polypeptide epitopes by serum titer using indirect ELISA.

[0054] Figure 4 is a flowchart of the construction process of recombinant plasmid for recombinant protein 001 expressing 23 polypeptide epitopes.

[0055] Figure 5 is a flowchart of the construction process of the recombinant plasmid expressing the fusion protein 002, which expresses 23 polypeptide epitopes and the CRM197 immune adjuvant protein.

[0056] Figure 6 shows the SDS-PAGE (left) and Western blot (right) analyses of the recombinant protein 001, cloned in pET30a and expressed in different strains. A: BL21(DE3) strain; B: Rosetta strain TM 2(DE3) strain.

[0057] Figure 7 shows the SDS-PAGE (left) and Western blot (right) analyses of the recombinant protein 002, cloned in pET30a and expressed in different strains. A: BL21(DE3) strain; B: Rosetta strain TM 2(DE3) strain.

[0058] In Figure 8, A is the SDS-PAGE image of recombinant protein 001 after purification; B is the SDS-PAGE image of recombinant protein 002 after purification.

[0059] Figure 9 is a schematic diagram of the results of detecting rabbit immune antiserum using recombinant proteins 001 and 002 by ELISA. Detailed Implementation

[0060] The present invention will now be described in detail.

[0061] As described above, based on the infection mechanism of Mycoplasma pneumoniae, the inventors analyzed the structure, properties, and amino acid sequences of the P1, P30, P40 / 90, P116, and CARDS toxin proteins of Mycoplasma pneumoniae (types I and II) using bioinformatics techniques. They screened out 23 polypeptide epitopes containing strong immunogenicity, including 4 polypeptides of the P1 protein (serial numbers 1-4), 4 polypeptides of the P30 protein (serial numbers 5-8), 4 polypeptides of the P40 / 90 protein (serial numbers 9-12), 7 polypeptides of the P116 protein (serial numbers 13-19), and 4 polypeptides of the CARDS toxin protein (serial numbers 20-23). ​​Details are shown in Table 1.

[0062] Table 1. Relevant information of 23 peptide epitopes obtained through bioinformatics

[0063] The immunogenic Mycoplasma pneumoniae polypeptide epitopes of this invention are polypeptide epitopes derived from the adhesion key proteins P1, P30, P40 / 90, P116 and / or CARDS toxins of Mycoplasma pneumoniae types I and II. Specifically, they include:

[0064] The P1 protein consists of amino acids 1222 to 1236 (Peptide-1), 1246 to 1260 (Peptide-2), 1478 to 1492 (Peptide-3), and 144 to 158 (Peptide-4).

[0065] The 37th to 51st amino acids of the P30 protein (Peptide-1), the 57th to 71st amino acids (Peptide-2), the 101st to 115th amino acids (Peptide-3), and the 123rd to 137th amino acids (Peptide-4);

[0066] The 827th to 841st amino acids (Peptide-1), 919th to 933rd amino acids (Peptide-2), 261st to 275th amino acids (Peptide-3), and 582nd to 596th amino acids (Peptide-4) of the P40 / 90 protein;

[0067] The following amino acids are present in the P116 protein: amino acids 895 to 909 (Peptide-1), 780 to 794 (Peptide-2), 599 to 613 (Peptide-3), 34 to 48 (Peptide-4), 68 to 82 (Peptide-5), 135 to 148 (Peptide-6), and 181 to 195 (Peptide-7);

[0068] The CARDS toxin protein consists of amino acids 506 to 521 (Peptide-1), 567 to 581 (Peptide-2), 175 to 189 (Peptide-3), and 68 to 82 (Peptide-4).

[0069] Based on this, the inventors immunized rabbits with Mycoplasma pneumoniae lysate to obtain antiserum, and verified through ELISA experiments that the above-mentioned polypeptide epitopes all exhibited varying degrees of immunogenicity. Using these 23 polypeptide epitopes, or further supplemented with an immune adjuvant protein (CRM197), multiple recombinant proteins were tandemly expressed using genetic engineering techniques via flexible linkers represented by GGGGS (glycine-glycine-glycine-serine). The expressed recombinant proteins can be used for the detection of Mycoplasma pneumoniae vaccines and antibodies, as well as for the preparation of Mycoplasma pneumoniae monoclonal and polyclonal antibodies.

[0070] The recombinant protein of the present invention comprises one or more polypeptide epitopes selected from the above-mentioned 23 polypeptide epitopes, wherein the polypeptide epitopes are polypeptide epitopes derived from the adhesion key proteins P1, P30, P40 / 90, P116 of Mycoplasma pneumoniae type I and II and / or CARDS toxin. Provided that immunogenicity is not affected, the amino acid sequences of the above-mentioned 23 polypeptide epitopes may also be substituted, deleted, or added with one or more amino acid residues, such that the modified amino acid sequence has more than 90% homology with the amino acid sequences shown in sequence numbers 1 to 23, preferably more than 95% homology, more preferably more than 96% homology, more preferably more than 97% homology, more preferably more than 98% homology, more preferably more than 99% homology, and most preferably 100% homology. The number and arrangement of the above-mentioned polypeptide epitopes in the recombinant protein are arbitrary. The recombinant protein of the present invention preferably comprises a recombinant protein consisting of at least one of the four polypeptide epitopes of the P1 protein, at least one of the four polypeptide epitopes of the P30 protein, at least one of the four polypeptide epitopes of the P40 / 90 protein, at least one of the seven polypeptide epitopes of the P116 protein, and at least one of the four polypeptide epitopes of the CARDS toxin protein, and more preferably a recombinant protein consisting of all of the above 23 polypeptide epitopes.

[0071] For example, the recombinant proteins of the present invention include recombinant protein 001 (serial number 24) and recombinant protein 002 (serial number 25). The nucleotide sequence encoding recombinant protein 001 is shown in serial number 26, and the nucleotide sequence encoding recombinant protein 002 is shown in serial number 27.

[0072] Recombinant protein 001 consists of 455 amino acids and is obtained by linking all 23 peptide epitopes mentioned above using a GGGGS flexible linker. The sequence is as follows:

[0073] Recombinant protein 002 consists of 995 amino acids in total. It is a recombinant protein obtained by linking all 23 polypeptide epitopes mentioned above using a GGGGS flexible linker and then linking the amino acid sequence of CRM197. The sequence is as follows:

[0074] in

[0075] The amino acid sequence is CRM197.

[0076] The recombinant protein of the present invention can be recombinantly expressed and prepared using gene recombination techniques known and commonly used in the art, utilizing bacteria, yeast cells, insect cells, mammalian cells, and transgenic plants and animals.

[0077] The recombinant protein of this invention has excellent immunogenicity and can be used in Mycoplasma pneumoniae vaccines. In addition, it can also be used for the detection of Mycoplasma pneumoniae antibodies and the preparation of Mycoplasma pneumoniae monoclonal and polyclonal antibodies.

[0078] The recombinant protein of the present invention has protective modifications at at least its N-terminus or C-terminus, or both ends, preferably including a linked protective protein. The protective protein includes at least one protein tag, or a protein that has the same protective function after amino acid sequence modification. Amino acid sequence modification includes substitution, deletion, or addition of one or more amino acid residues in the amino acid sequence of the protein tag, such that the modified amino acid sequence has more than 90% homology with the amino acid sequence of the protein tag, preferably more than 95% homology, more preferably more than 96% homology, more preferably more than 97% homology, more preferably more than 98% homology, more preferably more than 99% homology, and most preferably 100% homology. The protein linked to the above protein fragment includes one or more of the following tags: His tag, S-tag tag, TrxA tag, GST tag, MBP tag, SUMO tag, flag tag, or NusA tag.

[0079] The amino acid sequence used for linking peptide epitopes in the recombinant protein of the present invention is not limited, but is preferably linked by a GGGGS flexible linker.

[0080] The recombinant protein of the present invention can be linked to or recombined with an amino acid sequence having stability and immunogenicity-enhancing effects. This amino acid sequence having stability and immunogenicity-enhancing effects is not particularly limited; for example, it can be linked to or mixed with the amino acid sequence of diphtheria-like toxin (CRM197) in any proportion, or linked to or mixed with the amino acid sequence of cholera toxin B subunit (CTB) in any proportion.

[0081] The recombinant protein of this invention has good immunogenicity and can be made into a safe and effective vaccine to block Mycoplasma pneumoniae infection.

[0082] The vaccine for preventing Mycoplasma pneumoniae infection of the present invention comprises the recombinant protein of the present invention, which provides protection against infection with at least one of Mycoplasma pneumoniae type I and / or type II. Compared with existing inactivated or attenuated vaccines, the recombinant subunit vaccine of the present invention does not contain the entire pathogen or its lipid fraction, but only the key sequence of the antigenic epitope, avoiding the selection of sequences that may cause allergic reactions or excessive autoimmune responses, thus improving immunogenicity while reducing damage from nonspecific immune responses. Therefore, the recombinant protein vaccine of the present invention has a low risk of causing venereal disease (VED) and is safer, more stable, and more effective than vaccines containing the complete pathogen. Furthermore, compared with existing recombinant protein subunit vaccines, the vaccine of the present invention targets more pathogen target proteins, providing a more comprehensive protective mechanism and achieving stronger immunogenicity and protective efficacy. In addition, the antigenic epitopes selected in the present invention are more precise, with shorter peptide chains and greater specificity, improving immunoprotection while significantly reducing the possibility of adverse immune reactions or allergens in the vaccine, resulting in lower safety risks. In addition, the peptide epitopes selected in this invention are relatively short and can be expressed as recombinant proteins through simple linker modification. The recombinant proteins are stable, exist in soluble form, are easy to purify, and do not require refolding treatment. Therefore, industrial production technology is less difficult and the cost is lower.

[0083] The recombinant protein provided by this invention can also be linked to protein subunits to form self-assembled nanoparticles, or provide multivalent antigen presentation through co-assembly with nanoparticles, covalent or non-covalent chemical linkage, or co-delivered with TLR2 agonists such as Pam2 Cys (P2C), TLR4 agonists such as monophospholipid A (MPLA), or TLR5 agonists such as FliC, thereby improving protein stability, immunogenicity, and the titer of protective antibodies after immunization. This allows for the development of safe and effective vaccines that can block Mycoplasma pneumoniae infection, demonstrating promising development and application prospects.

[0084] The dosage form of the vaccine of the present invention is not particularly limited and may be nasal drops, emulsion, suspension, gel, powder, or injection. The route of administration of the vaccine may be intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal mucosal administration, oral administration, local transdermal administration, or systemic transdermal administration. Nasal mucosal immunization via the nasal cavity is preferred, or immunization via subcutaneous or intramuscular injection.

[0085] Example

[0086] The present invention will now be described in further detail with reference to the embodiments. It should be understood that the specific embodiments described below are only for explaining the present invention and are not intended to limit the present invention. Furthermore, the experimental methods in the following embodiments, unless otherwise specified, are all performed according to conventional methods known in the art.

[0087] Example 1: Synthesis and purity determination of 23 polypeptide epitopes

[0088] The peptides were synthesized using the Fmoc solid-phase synthesis method. In addition, cysteine ​​(Cys) was added during the peptide synthesis process to facilitate subsequent coupling with the carrier protein bovine serum albumin (BSA) for plate testing of serum titers.

[0089] Table 2. Synthesis and purity detection of 23 polypeptide epitopes

[0090] Example 2: Preparation of Mycoplasma pneumoniae lysate

[0091] Mycoplasma pneumoniae NCTC 10119 was revived in ATCC2611 medium or other suitable liquid media. After one subculture, the Mycoplasma pneumoniae were collected by centrifugation (15000 g / min, 5–10 min), the supernatant was discarded, and the cells were washed three times with cold PBS. The lysate was obtained by sonication with PBS, inactivated at 56 °C for 30 min, and the protein concentration in the lysate was determined using the BCA method. The lysate was then frozen at -80 °C for later use. Simultaneously, a portion of the inactivated lysate was spread onto solid culture medium and cultured for 7–14 days to ensure that no surviving Mycoplasma pneumoniae remained in the lysate. The experimental results are shown in Table 3 and Figure 1.

[0092] Table 3. Information related to Mycoplasma pneumoniae lysis buffer

[0093] Example 3: Immunizing New Zealand rabbits with Mycoplasma pneumoniae lysate to obtain antiserum

[0094] The experimental procedure is as follows:

[0095] (1) Two rabbits were selected as immunized animals according to experimental requirements. They were injected intramuscularly with Mycoplasma pneumoniae lysis solution and immunized three times. The immunization information is shown in Table 4, where the adjuvant was RTIterFast adjuvant (rabbit rapid adjuvant), catalog number ATO00071.

[0096] Table 4

[0097] (2) Seven days after the completion of the three-immunization, 1-1.5 mL of blood was collected to detect the serum titer of the three-immunization. The collected blood was placed at 37℃ for 30 min to inactivate and then at 4℃ overnight to allow it to coagulate and release serum. The coagulated blood was centrifuged at 1000 g / min for 10 min to collect the supernatant, which is the serum, and used for ELISA experiments to detect serum titer.

[0098] (3) Coating the antigen onto the microplate: Dilute the antigen to 5 μg / mL in PBS. Transfer 100 μL of the antigen dilution to each well of the microtiter plate, coating the wells with the antigen. Cover the microtiter plate with an adhesive plastic film and incubate at 37°C for 2 hours. Discard the coating solution and add 300 μL of PBS to each well to wash the microtiter plate three times. Gently shake the microtiter plate over a water bath to remove the solution or washing liquid. Tap the microtiter plate on a paper towel to remove any remaining droplets.

[0099] (4) Blocking: Block the remaining protein binding sites in the wells by adding 300 μL of blocking buffer (3% BSA-PBS) to each well. Cover the microtiter plate with an adhesive plastic film and incubate overnight at 4°C. Wash the microtiter plate 2–3 times with PBST.

[0100] (5) Incubation with primary and secondary antibodies: Add serially diluted rabbit serum to each well, 100 μL / well, repeat twice. Cover the microtiter plate with adhesive plastic film and incubate at 37°C for 1 hour. Wash the microtiter plate 4 times with PBST. Add 100 μL / well of horseradish peroxidase (HRP)-conjugated secondary antibody, which has been diluted to the optimal concentration in blocking buffer before use (according to the manufacturer's instructions). Cover the microtiter plate with adhesive plastic film and incubate at room temperature for 30 minutes. Wash the microtiter plate 4 times with PBST.

[0101] (6) Detection: Add 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution to each well. Incubate at 37°C for 5 minutes (adjust as needed). Add 50 μL of stop solution (2M HCl) or an equal volume of stop solution (2M H2SO4), and then read the optical density at 450 nm to 630 nm.

[0102] The results are shown in Figure 2. The ELISA titer of the triple immunization serum was greater than 1:128000. The negative control was rabbit serum before immunization, and the blank control was PBS buffer.

[0103] Example 4: Screening of positive peptides using rabbit antiserum via indirect ELISA.

[0104] The experimental procedure is as follows:

[0105] (1) Antigen coating onto microplates: Dilute each antigen (peptide epitope) to 5 μg / mL in PBS, for a total of 23 antigens. Transfer 100 μL of the antigen dilution solution to each well of the microtiter plate, coating the wells with antigen. Cover the microtiter plate with an adhesive plastic film and incubate at 37°C for 2 hours. Discard the coating solution and add 300 μL of PBS to each well to wash the microtiter plate three times. Gently shake the microtiter plate over a water bath to remove the solution or washing solution. Tap the microtiter plate on a paper towel to remove any remaining droplets.

[0106] (2) Blocking: Block the remaining protein binding sites in the wells by adding 300 μL of blocking buffer (3% BSA-PBS) to each well. Cover the microtiter plate with an adhesive plastic film and incubate overnight at 4°C. Wash the microtiter plate 2–3 times with PBST.

[0107] (3) Incubation with primary and secondary antibodies: Rabbit serum obtained after immunization with serially diluted Mycoplasma pneumoniae lysate was added to each well, 100 μL / well. The microtiter plate was covered with adhesive plastic film and incubated at 37°C for 1 hour. The microtiter plate was washed 4 times with PBST. 100 μL / well of horseradish peroxidase (HRP)-conjugated secondary antibody was added, which had been diluted to the optimal concentration in blocking buffer before use (according to the manufacturer's instructions). The microtiter plate was covered with adhesive plastic film and incubated at room temperature for 30 minutes. The microtiter plate was washed 4 times with PBST.

[0108] (4) Detection: Add 100 μL of TMB (3,3',5,5'-tetramethylbenzidine) solution to each well. Incubate at 37°C for 5 minutes (adjust as needed). Add 50 μL of stop solution (2M HCl) or an equal volume of stop solution (2M H2SO4), and then read the optical density at 450 nm-630 nm.

[0109] As shown in Figure 3, all 23 polypeptide epitopes screened and synthesized in this invention showed positive results with an immunogenicity >1:2000, demonstrating significant immunogenicity. Among them, P1-Peptide-2, P30-Peptide-2, P40 / 90-Peptide-1, P40 / 90-Peptide-4, P116-Peptide-2, and P116-Peptide-6 showed strong positive results with an immunogenicity >1:4000. The negative control was rabbit serum before immunization, and the blank control was PBS buffer.

[0110] Example 5: Optimized synthesis of the target gene, construction of the recombinant vector, and expression and purification of the recombinant protein in this invention.

[0111] 1. Using codons readily accepted by both eukaryotes and prokaryotes, recombinant protein 001 gene sequence, consisting of 23 novel tandem polypeptide epitopes, and recombinant protein 002 gene sequence, consisting of 23 polypeptide epitopes tandemly with CRM197 immunoadjuvant, were constructed via chemical synthesis. The tandem gene fragments were then cloned into NdeⅠ and HindⅢ within plasmid pET30a(+) using genetic engineering techniques. The construction process is shown in Figures 4 and 5.

[0112] 2. Primers are synthesized using an automated synthesizer. After obtaining the target primers, PCR amplification is performed. The PCR product is then detected by electrophoresis. A PCR product of the expected size consistent with the target sequence is obtained. The band of the expected size consistent with the target sequence is cut, and the product is recovered for ligation and transformation.

[0113] 3. Ligation procedure. The amplified target gene PCR product was ligated to the linearized vector. The ligation product was then transformed into competent cells using a heat shock method. The transformed product was evenly spread on LB agar plates containing kanamycin resistance. The plates were then inverted and incubated overnight at 37°C.

[0114] 4. Colony screening. Using a sterile pipette tip, pick 3-6 single colonies and incubate them in LB liquid medium containing kanamycin resistance. After incubation, add nucleic acid dyes for electrophoresis detection. Select clones with bands consistent with the expected size for culture, extract plasmids, and sequence them.

[0115] 5. The recombinant plasmid was transformed into Escherichia coli strains BL21(DE3) and Rosetta. TM In step 2(DE3), single colonies were inoculated into LB medium containing the corresponding antibiotic and cultured. They were then induced with 0.5 mM IPTG at 15°C for 16 h, or at 37°C for 4 h. Expression was detected by SDS-PAGE and Western Blot. The results are shown in Figures 6 and 7.

[0116] 6. Scale-up culture. BL21(DE3) containing the recombinant expression plasmid was inoculated into TB medium containing the corresponding antibiotic for fermentation. When the OD600 reached approximately 1.2, 0.5 mM IPTG was added and expression was induced at 37°C for 4 h (recombinant protein 001) or 0.5 mM IPTG was added and expression was induced at 15°C for 16 h (recombinant protein 002). Finally, the bacterial cells were collected by centrifugation.

[0117] 7. Purification and Analysis. The cell pellet was resuspended in lysis buffer, then sonicated and centrifuged. The supernatant was retained for purification. One-step purification used a nickel column to obtain the target protein, which was then dialyzed to the final buffer. The target protein was sterilized by filtration through a 0.22 μm filter before aliquoting. Concentration was determined using the Bradford protein assay. The fusion protein was analyzed by standard SDS-PAGE, as shown in Figure 8.

[0118] The gene sequence of recombinant protein 001 (6624 bp) is shown in sequence number 26. The gene sequence of recombinant protein 002 (8244 bp) is shown in sequence number 27.

[0119] The amino acid sequence (602AA) of recombinant protein 001 is obtained by linking all 23 polypeptide epitopes using a GGGGS flexible linker and adding a protein tag, as shown in the following sequence number 28:

[0120] Serial number 28:

[0121] The amino acid sequence (1142AA) of recombinant protein 002 is obtained by linking all 23 polypeptide epitopes mentioned above with the amino acid sequence of CRM197 via a GGGGS flexible linker and adding a protein tag, as shown in the following sequence number 29:

[0122] Serial number 29:

[0123] The nucleotide sequence of recombinant protein 001 is shown in sequence number 30, and the nucleotide sequence of recombinant protein 002 is shown in sequence number 31.

[0124] Example 6: Immunogenicity assay of recombinant proteins 001 and 002

[0125] The experimental procedure is the same as in Example 4.

[0126] The experimental results are shown in Figure 9. The horizontal axis of Figure 9 represents the antiserum titer, and the vertical axis represents the absorbance at 450 nm-630 nm. The negative control (Negative) is rabbit serum before immunization, and the blank control (Blank) is PBS buffer. Compared to the individual peptide epitopes (Peptide 1-23), the recombinant proteins 001 and 002 showed significantly enhanced immunogenicity, with antiserum titers reaching 1:128000. Recombinant protein 002, obtained by tandem with the immunoadjuvant CRM197, showed even better results. This indicates that the recombinant proteins 001 and 002 prepared by tandem 23 peptide epitopes possess good immunogenicity.

[0127] The above results confirm that recombinant proteins 001 and 002 have excellent immunogenicity and can be used to produce vaccines to prevent Mycoplasma pneumoniae infection, thereby safely and effectively blocking the infection and spread of Mycoplasma pneumoniae.

[0128] After reading the above statement about the present invention, those skilled in the art can make various modifications or changes to the present invention, and these equivalent forms also fall within the scope defined in the appended claims.

[0129] Industrial availability

[0130] The Mycoplasma pneumoniae polypeptide epitope and the recombinant protein containing the polypeptide epitope of the present invention can be used to produce a safe and effective vaccine to prevent Mycoplasma pneumoniae infection, and have high industrial applicability.

Claims

1. An immunogenic Mycoplasma pneumoniae polypeptide epitope, characterized in that, The polypeptide epitopes are polypeptide epitopes derived from the adhesion key proteins P1, P30, P40 / 90, P116 of Mycoplasma pneumoniae type I and II and / or CARDS toxin.

2. The polypeptide epitope as described in claim 1, characterized in that, The amino acid sequence of the polypeptide epitope from the P1 protein is the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4. The amino acid sequence of the polypeptide epitope from the P30 protein is the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8. The amino acid sequence of the polypeptide epitope from the P40 / 90 protein is the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12, or it is an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 9, sequence number 10, sequence number 11 or sequence number 12. The amino acid sequence of the polypeptide epitope from the P116 protein is the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18, or 19, or is obtained by substituting, deleting, or adding one or more amino acid residues to the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18, or 19. The amino acid sequence shown in 19 has more than 90% homology and is an immunogenic amino acid sequence; The amino acid sequence of the polypeptide epitope from CARDS toxin is the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23.

3. A nucleotide sequence, characterized in that, The nucleotide sequence encodes the polypeptide epitope as described in claim 1 or 2.

4. A recombinant protein, characterized in that, The recombinant protein contains one or more immunogenic Mycoplasma pneumoniae polypeptide epitopes, wherein the polypeptide epitopes are polypeptide epitopes derived from adhesion key proteins P1, P30, P40 / 90, P116 and / or CARDS toxins of Mycoplasma pneumoniae type I and II.

5. The recombinant protein as described in claim 4, characterized in that, The amino acid sequence of the polypeptide epitope from the P1 protein is the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4 and has immunogenicity, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 1, sequence number 2, sequence number 3 or sequence number 4. The amino acid sequence of the polypeptide epitope from the P30 protein is the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 5, sequence number 6, sequence number 7 or sequence number 8. The amino acid sequence of the polypeptide epitope from the P40 / 90 protein is Serial No. 9, Sequence No.

9. The amino acid sequence shown in serial number 10, serial number 11 or serial number 12, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in serial number 9, serial number 10, serial number 11 or serial number 12 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in serial number 9, serial number 10, serial number 11 or serial number 12. The amino acid sequence of the polypeptide epitope from the P116 protein is the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19, or is an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 13, 14, 15, 16, 17, 18 or 19. The amino acid sequence of the polypeptide epitope from CARDS toxin is the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in sequence number 20, sequence number 21, sequence number 22 or sequence number 23.

6. The recombinant protein as described in claim 4 or 5, characterized in that, The recombinant protein has protective modifications at the N-terminus or C-terminus, or at both ends, including linked protective proteins.

7. The recombinant protein as described in claim 6, characterized in that, The protective protein includes at least one protein tag.

8. The recombinant protein as described in claim 7, characterized in that, The protein tag is selected from one or more of the following: His tag, S-tag tag, TrxA tag, GST tag, MBP tag, SUMO tag, flag tag, and NusA tag.

9. The recombinant protein as described in claim 4 or 5, characterized in that, The recombinant protein contains one or more polypeptide epitopes linked by linkers consisting of any amino acid sequence.

10. The recombinant protein as described in claim 4 or 5, characterized in that, The recombinant protein is linked to or mixed with an amino acid sequence that has stability and immunogenicity-enhancing effects.

11. The recombinant protein according to claim 10, characterized in that, The amino acid sequence that enhances stability and immunogenicity is the amino acid sequence of diphtheria-like toxin CRM197 and / or cholera toxin B subunit CTB.

12. The recombinant protein as described in claim 4 or 5, characterized in that, The amino acid sequence of the recombinant protein is the amino acid sequence shown in Serial No. 24 or Serial No. 25, or an amino acid sequence that has more than 90% homology with the amino acid sequence shown in Serial No. 24 or Serial No. 25 and is immunogenic, obtained by substituting, deleting or adding one or more amino acid residues to the amino acid sequence shown in Serial No. 24 or Serial No.

25.

13. A nucleotide sequence, characterized in that, The nucleotide sequence encodes the recombinant protein according to any one of claims 4 to 12.

14. The nucleotide sequence as described in claim 13, characterized in that, The nucleotide sequence is shown in sequence number 26 or sequence number 27.

15. A vaccine for the prevention of Mycoplasma pneumoniae infection, which provides protection against infection with at least one of Mycoplasma pneumoniae type I and / or type II, characterized in that, The vaccine comprises the recombinant protein according to any one of claims 4 to 12.

16. The vaccine as described in claim 15, characterized in that, The vaccine may be in the form of nasal drops, emulsion, suspension, gel, powder, or injection. Agent.

17. The vaccine as described in claim 15 or 16, characterized in that, The vaccine can be administered via intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, nasal mucosal administration, oral administration, local transdermal administration, or systemic transdermal administration.

18. The use of the polypeptide epitope according to claim 1 or 2 in the preparation of a vaccine for the prevention of Mycoplasma pneumoniae infection.

19. The use of the recombinant protein according to any one of claims 4 to 12 in the preparation of a vaccine for the prevention of Mycoplasma pneumoniae infection.

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