Recombinant human Mycoplasma pneumoniae adhesion protein vaccine

Abstract

The present invention provides a recombinant human Mycoplasma pneumoniae adhesion protein vaccine comprising human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90, and P116 fusion proteins C1, C1-G4S, C2, and C2-G4S. Upon immunization of mice with the recombinant human Mycoplasma pneumoniae adhesion protein vaccine, purified and expressed in Escherichia coli, the vaccine induces a strong immune response in vivo, reduces cytokine production, and reduces Mycoplasma pneumoniae colonization in lung tissue. The vaccine can be used for the prevention, detection, or treatment of Mycoplasma pneumoniae infection.

Description

Technical Field

[0001] The present invention relates to four human Mycoplasma pneumoniae recombinant protein vaccines, which can induce a strong immune response in vivo after combined immunization, reduce the production of cytokines and the colonization of Mycoplasma pneumoniae in lung tissue, and can be used in related fields such as the prevention, monitoring or treatment of human Mycoplasma pneumoniae infection. Background Art

[0002] Mycoplasma pneumoniae, lacking a cell wall, is the smallest known self-replicating pathogen. It can cause mycoplasma pneumonia in humans of all ages and is particularly common in older children and young adults, accounting for 20% of community-acquired pneumonia cases. Infection typically manifests as tracheitis or bronchitis, but in severe cases can lead to chronic lung disease and extrapulmonary sequelae, exacerbating asthma attacks. Macrolide antibiotics are the preferred treatment for M. pneumoniae infections. However, with the rapid increase in the prevalence of macrolide-resistant M. pneumoniae infections, the need for new drugs and novel preventive measures has become more urgent. While veterinary M. pneumoniae vaccines have demonstrated excellent protection and efficacy both domestically and internationally, no approved human M. pneumoniae vaccine has been developed. Currently, research is underway for M. pneumoniae vaccines, including inactivated, live attenuated, recombinant protein, and DNA vaccines. At present, inactivated vaccines and live attenuated vaccines have achieved certain protective effects in animal experiments, but both inactivated vaccines and live attenuated vaccines can cause lipid-associated membrane protein-induced, IL-17A-dependent, neutrophil-driven vaccine-enhanced diseases, thus limiting their further research and development.

[0003] Mycoplasma pneumoniae adheres to epithelial cells on the mucosal surface to promote its parasitism and survival, triggering colonization and infection. One end of its cell membrane extends outward to form a special adhesion organelle, and a series of adhesion-related proteins are located on the surface, including P1 (170 kDa), P30 (30 kDa), P116 (116 kDa), etc., which play a key role in the adhesion and sliding of Mycoplasma pneumoniae. The loss and mutation of these proteins can lead to the loss of their adhesion function and reduced sliding ability, making them potential vaccine targets. For example, vaccine candidate antigens such as HP14 / 30 and MP 559 have made certain progress at the in vitro and animal levels, but there is no relevant experimental data in human body to support them, and further in-depth research is still needed.

[0004] The purpose of the present invention is to provide a recombinant human Mycoplasma pneumoniae adhesion protein vaccine. Summary of the Invention

[0005] Based on the above objectives, the present invention first provides a fusion protein, which contains human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116.

[0006] In a preferred embodiment, the amino acid sequence of the mycoplasma adhesion protein P1 is shown as SEQ ID NO.1, the amino acid sequence of P30 is shown as SEQ ID NO.3, the amino acid sequence of P40 / 90 is shown as SEQ ID NO.5, and the amino acid sequence of P116 is shown as SEQ ID NO.7.

[0007] In a more preferred embodiment, the gene coding sequence of the mycoplasma adhesion protein P1 is shown as SEQ ID NO.2, the gene coding sequence of P30 is shown as SEQ ID NO.4, the gene coding sequence of P40 / 90 is shown as SEQ ID NO.6, and the gene coding sequence of P116 is shown as SEQ ID NO.8.

[0008] In a preferred embodiment, in the fusion protein, the human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116 are connected via a rigid linker peptide and / or a flexible linker peptide.

[0009] In a more preferred embodiment, the rigid linker peptide includes one or more of EAAAK, AAY, and GPGPG.

[0010] In another more preferred embodiment, the flexible connecting peptide is (G4S)n, where n is a natural number of 1-6.

[0011] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is as described in SEQ ID NO.9, and the fusion protein having the technical solution in the present invention is named "C1"; or

[0012] The amino acid sequence of the fusion protein is as shown in SEQ ID NO. 11. In the present invention, the fusion protein having the technical solution is named "C1-G4S"; or

[0013] The amino acid sequence of the fusion protein is as shown in SEQ ID NO. 13. In the present invention, the fusion protein having the technical solution is named "C2"; or

[0014] The amino acid sequence of the fusion protein is as shown in SEQ ID NO. 15. In the present invention, the fusion protein having the technical solution is named "C2-G4S".

[0015] In the present invention, the structure of the C1 fusion protein is: P1 (1160-1521 aa in the full-length sequence of the P1 proprotein)-EAAAK-P30 (100-274 aa in the full-length sequence of the P30 proprotein)-AAY- P40 / 90 (456-754 aa in the full-length sequence of the P40 / 90 proprotein)-GPGPG- P116 (145-347 aa in the full-length sequence of the P116 proprotein), wherein EAAAK, AAY, and GPGPG are the rigid connecting peptides described in the present invention.

[0016] The structure of the C1-G4S fusion protein is: P1 (1160-1521 aa)-GGGGS-P30 (100-274 aa)-GGGGS-P40 / 90 (456-754 aa)- GGGGS-P116 (145-347 aa), wherein GGGGS is the flexible connecting peptide described in the present invention.

[0017] The structure of the C2 fusion protein is: P1(1287–1518 aa)-EAAAK-P30(100-274 aa)-AAY- P40 / 90(456-754 aa)-GPGPG- P116(145-347aa).

[0018] The structure of the C2-G4S fusion protein is: P1(1287–1518 aa)-GGGGS-P30(100-274 aa)-GGGGS-P40 / 90(456-754 aa)- GGGGS-P116(145-347 aa).

[0019] Secondly, the present invention provides a polynucleotide encoding the above-mentioned fusion protein, wherein the sequence of the polynucleotide is shown in SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, or SEQ ID NO. 16. The coding sequence of C1 is shown in SEQ ID NO. 10, the coding sequence of C1-G4S is shown in SEQ ID NO. 12, the coding sequence of C2 is shown in SEQ ID NO. 14, and the coding sequence of C2-G4S is shown in SEQ ID NO. 16. The above-mentioned polynucleotide provided by the present invention is an artificially optimized polynucleotide.

[0020] Third, the present invention provides a vector containing the above polynucleotide.

[0021] Fourthly, the present invention provides a host cell containing the above vector.

[0022] In a specific embodiment of the present invention, the optimized nucleotide sequence expressing the above fragment was embedded in the pET29a plasmid cloning sites (NdeI and XhoI), transformed into Escherichia coli BL21 and induced for expression by IPTG. The expressed protein was purified by Ni column and gel filtration in two steps to obtain the corresponding recombinant protein.

[0023] Fifth, the present invention provides a composition comprising one or more of the above-mentioned fusion proteins.

[0024] In a preferred embodiment, the composition contains four fusion proteins, namely, fusion proteins whose amino acid sequences are shown as SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.15, respectively.

[0025] Sixth, the present invention provides the use of the above-mentioned composition in the preparation of a recombinant protein vaccine of human Mycoplasma pneumoniae.

[0026] The fusion protein and fusion protein composition provided by the present invention were mixed evenly (recombinant protein quadruple preparation) and immunized into mice by nasal drops, and immunized three times continuously on day 0, day 14 and day 28. After immunization, the mice were challenged with the virus by nasal drops at a dose of 1E+07 CCU / ml for three consecutive days. On the fourth day after the challenge, i.e., day D45, serum, alveolar lavage fluid and lung tissue were taken for the detection of antibodies, cytokines and bacterial load. Among them, the serum on day D21 and day D42 was used for the detection of IgG antibodies against the corresponding antigens, respectively. Compared with the rP1C + rP30C immune group, the fusion protein composition provided by the present invention (recombinant protein quadruple preparation) showed that the IgG antibodies against rP1C, rP30, C2-G4S and iMP (human Mycoplasma pneumoniae inactivated by paraformaldehyde, i.e., inactivated Mycoplasma pneumoniae ) were significantly increased, increasing to 2.39-fold, 2.11-fold, 8.23-fold and 2.49-fold on D21, and to 8.15-fold, 10.79-fold, 35.64-fold and 3.56-fold on D42, respectively. Compared with the second immunization, the IgG antibody levels against rP1C (1287–1518 aa), rP30C (100-274 aa), C2-G4S and iMP increased by 19-fold, 16-fold, 218-fold and 322-fold after the third immunization, respectively.

[0027] Results from mouse challenge experiments showed that after quadruple immunization with the human M. pneumoniae candidate vaccine constructed in this study, compared to the rP1C + rP30C immunization group, immunization with the fusion protein composition (recombinant protein quadruple formulation) provided by the present invention also significantly increased IgA antibody levels against rP1C, rP30, C2-G4S, and iMP. Both rP1C + rP30C and the quadruple immunization significantly reduced the levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in bronchoalveolar lavage fluid and the M. pneumoniae load in lung tissue, demonstrating that the human M. pneumoniae candidate vaccine constructed in this study exerted a good protective effect.

[0028] In specific applications, the vaccine can be prepared into a variety of preparations, such as injections and oral preparations. Preferably, the recombinant vaccine can be prepared as an inhalation vaccine.

[0029] Finally, the present invention provides the use of the above composition in preparing a human Mycoplasma pneumoniae detection kit.

[0030] The present invention provides a human Mycoplasma pneumoniae recombinant protein composition that can be used to detect human Mycoplasma pneumoniae serum antibodies. Compared to a control group, antibodies binding to four human Mycoplasma pneumoniae recombinant proteins, C1, C1-G4S, C2, and C2-G4S, can be detected in the serum of mice infected with Mycoplasma pneumoniae using an ELISA method. The detection of specific antibodies against the recombinant proteins in human serum indicates the occurrence of historical or acute infection.

[0031] Preferably, the method can be used for the development of a human Mycoplasma pneumoniae serum antibody detection kit. The method or kit may be used for human Mycoplasma pneumoniae serum epidemiological monitoring, evaluation of humoral immune response to human Mycoplasma pneumoniae vaccines, etc.

[0032] In summary, the present invention relates to a composition of four human Mycoplasma pneumoniae recombinant proteins, which can induce a strong immune response in vivo after combined immunization, reduce cytokine production and Mycoplasma pneumoniae colonization in lung tissue, and can be used in related fields such as the prevention, monitoring or treatment of human Mycoplasma pneumoniae infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of SDS-PAGE of C1, C1-G4S, C2 and C2-G4S proteins are shown. The target proteins were obtained after affinity purification and gel filtration chromatography after the expression of C1, C1-G4S, C2 and C2-G4S proteins induced by E. coli. The purity of the proteins was ≥80% as determined by SDS-PAGE.

[0034] Figure 2 Schematic diagram of mouse immunization and grouping;

[0035] Figure 3 is the level of specific IgG antibodies against rP1C in the serum of mice after immunization;

[0036] Figure 4 is the level of specific IgG antibodies against rP30C in the serum of mice after immunization;

[0037] Figure 5 is the level of specific IgG antibodies against C2-G4S in the serum of mice after immunization;

[0038] Figure 6 is the level of specific IgG antibodies against iMP in the serum of mice after immunization;

[0039] Figure 7 This is a schematic diagram of mouse immunization and grouping;

[0040] Figure 8 The specific IgA antibody levels against different antigens in the bronchoalveolar lavage fluid of mice;

[0041] Figure 9 Cytokine levels in mouse bronchoalveolar lavage fluid;

[0042] Figure 10 is the MP replication level in mouse lung tissue. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific examples, but it should be understood by those skilled in the art that the present invention is not limited to these specific examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0044] Example 1. Construction of a human Mycoplasma pneumoniae recombinant protein vaccine

[0045] 1. Construction of rP1C, rP30C, rP40 / 90N, and rP116N Recombinant Proteins

[0046] The optimized nucleotide sequences expressing rP1C, rP30C, rP40 / 90N and rP116N were inserted into the pET29a plasmid cloning sites (NdeI and XhoI), transformed into Escherichia coli BL21 and induced with IPTG for expression. The expressed proteins were purified by Ni column and gel filtration to obtain the corresponding recombinant proteins.

[0047] 2. Construction of C1, C1-G4S, C2, and C2-G4S recombinant protein vaccines

[0048] The optimized nucleotide sequences expressing C1, C1-G4S, C2, and C2-G4S were inserted into the pET29a plasmid cloning sites (NdeI and XhoI), transformed into E. coli BL21 and induced with IPTG for expression. The expressed proteins were purified by Ni column and gel filtration to obtain the corresponding recombinant proteins. The expression of each protein is shown in Figure 1 .

[0049] Example 2. Evaluation of the immunogenicity of human Mycoplasma pneumoniae recombinant protein vaccine

[0050] In this example, the immunogenicity of the human Mycoplasma pneumoniae recombinant protein vaccine was determined by enzyme-linked immunosorbent assay (ELISA). 27 BalB / C mice were divided into 3 groups on average and all were nasally dripped three times with an interval of 14 days. The PBS group was nasally dripped with 50 μl PBS, the P1+P30 immunization group was nasally dripped with 10 μg each of rP1C and rP30C (40 μl in total), and the C immunization group was nasally dripped with 5 μg each of C1, C1-G4S, C2, and C2-G4S (40 μl). Both the P1+P30 and C immunization groups were supplemented with c-di-GMP (10 μl, 1 μg / μl) (Invivogen, tlrl-nacdg) as an adjuvant. Serum was collected on the 21st day (D21) and 42nd day (D42) after immunization to detect the corresponding specific antibodies. The immunization procedure and grouping are shown in Tables 1 and Figure 2 .

[0051] Table 1. Immunization schedule and grouping

[0052]

[0053] The specific grouping, vaccination, and testing procedures were as follows: 27 BalB / C mice were equally divided into three groups and administered intranasal instillations three times, 14 days apart. The PBS group received 50 μl of PBS; the P1+P30 immunization group received 10 μg each of rP1C and rP30C (40 μl total); and the C immunization group received 5 μg each of C1, C1-G4S, C2, and C2-G4S (40 μl). Both the P1+P30 and C immunization groups received c-di-GMP (10 μl, 1 μg / μl) as an adjuvant. Serum was collected on days 21 (D21) and 42 (D42) after immunization to detect specific antibodies.

[0054] Specific IgG antibodies against rP1C, rP30C, C protein and iMP were detected in both P1+P30 and C immunization groups, and the antibody level in C immunization group was significantly higher than that in P1+P30 immunization group. Compared with D21, the specific IgG antibody levels against rP1C, rP30C, C protein and iMP in P1+P30 and C immunization groups on D42 were increased. The specific IgG antibody levels against rP1C, rP30C, C protein and iMP were shown in Table 1. Figure 3-Figure 6 .

[0055] Figure 3 Specific IgG antibodies against rP1C were detected in the serum of immunized mice on days 21 and 42 after immunization. Antibody levels in the BalB / C group were higher than in the P1+P30 group, increasing by 2.39-fold on day 21 and 8.15-fold on day 42. Compared to day 21, levels of rP1C-specific IgG antibodies in the P1+P30 and C groups increased by 5.67-fold and 19.33-fold on day 42, respectively.

[0056] Figure 4 Specific IgG antibodies against rP30C were detected in the serum of immunized mice on days 21 and 42 after immunization. Antibody levels in the BalB / C group were significantly higher than those in the P1+P30 group, increasing by 2.11-fold on day 21 and 10.79-fold on day 42. Compared to day 21, levels of rP1C-specific IgG antibodies in the P1+P30 and C groups increased by 3.22-fold and 16.50-fold on day 42, respectively.

[0057] Figure 5 The level of specific IgG antibodies against C2-G4S in the serum of immunized mice was detected on days 21 and 42 after immunization. Specific IgG antibodies against protein C were detected in the serum of BalB / C mice in the immunized group. Antibody levels in the C-immunized group were significantly higher than those in the P1+P30-immunized group, increasing by 8.23-fold on day 21 and 35.64-fold on day 42. Compared to day 21, levels of specific IgG antibodies against protein C in the P1+P30 and C-immunized groups increased by 50.39-fold and 218.18-fold on day 42, respectively.

[0058] Figure 6IgG antibody levels against iMPs in the serum of immunized mice. IgG antibodies against iMPs were detected in the serum of BalB / C mice on both days 21 and 42 after immunization. Antibody levels in the C-immunized group were significantly higher than those in the P1+P30-immunized group, increasing by 2.49-fold on day 21 and 3.56-fold on day 42. Compared to day 21, levels of iMP-specific IgG antibodies in the P1+P30 and C-immunized groups increased by 224.96-fold and 321.99-fold on day 42, respectively.

[0059] Example 3. Evaluation of the protective effect of human Mycoplasma pneumoniae recombinant protein vaccine

[0060] Immunization challenge program Figure 7 Specifically, on day 42 after immunization with PBS or protein, BalB / C mice (PBS*, P1+P30*, and C* groups) were infected intranasally with 50 μl (1E+07 CCU / ml) of MP for three consecutive days. 50 μl of normal saline was used intranasally as a negative control (PBS group). On day 45, serum, lung tissue, and bronchoalveolar lavage fluid were collected from the mice for corresponding tests.

[0061] In this example, the fully automated microfluidic immunoassay analyzer Ella was used to measure the expression levels of proinflammatory cytokines in bronchoalveolar lavage fluid after challenge. The Ella multifactor cartridge was removed from a -20°C freezer 30 minutes in advance and allowed to return to room temperature. Serum was centrifuged at 12,000 g for 4 minutes. 30 μl of the supernatant was diluted 1:1 with sample diluent. The Ella multifactor cartridge QR code was scanned, and 50 μl of sample and 1 ml of wash solution were added to each well. The cartridge was run, and data was acquired after 1 hour for analysis.

[0062] Figure 8 Specific IgA antibody levels against different antigens in the bronchoalveolar lavage fluid (BALF) of BalB / C mice in both the P1+P30* and C* groups were detected on day 45 in the BALF. Specific IgA antibodies against rP1C, rP30C, protein C, and iMPs were also detected in the BALF of BalB / C mice in both the P1+P30* and C* groups. Antibody levels in the C* group were significantly higher than those in the P1+P30* group (increased by 12.61-fold, 4.37-fold, 4.74-fold, and 15.91-fold, respectively).

[0063] Cytokine levels in bronchoalveolar lavage fluid Figure 9 Compared with the PBS* group, the levels of cytokines IL-1β, IL-6, and TNF-α in BALF of mice in the P1+P30* and C* groups were significantly decreased, decreasing to 0.67 and 0.61 (IL-1β), 0.13 and 0.27 (IL-6), and 0.15 and 0.28 (TNF-α) in the PBS* group, respectively.

[0064] In this example, real-time quantitative PCR (RT-PCR) was used to detect the copy number of MP genome in lung tissue. The ThermoFisher prepSEQ™ 1-2-3 nucleic acid extraction kit was used to extract the mycoplasma genome from lung tissue, and the Mycoplasma pneumoniae load was detected by fluorescence quantitative PCR (Zhijiang Bio, Mycoplasma pneumoniae nucleic acid detection kit). The Mycoplasma pneumoniae load in lung tissue is shown in Figure 10 Compared with the PBS* group, the MP genome copy number in the lung tissues of mice in the P1+P30* and C* groups was significantly decreased, and the MP load in the C* group was significantly lower than that in the P1+P30* group, decreasing by 2.87 times.

Claims

1. A composition, characterized in that The composition contains fusion proteins whose amino acid sequences are shown as SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.15 respectively.

2. Use of the composition according to claim 1 in the preparation of a recombinant protein vaccine of human Mycoplasma pneumoniae.

3. Use of the composition according to claim 1 in preparing a kit for detecting human Mycoplasma pneumoniae.

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