Recombinant human mycoplasma pneumoniae adhesion protein vaccine

Abstract

The invention provides a recombinant human mycoplasma pneumoniae adhesion protein vaccine. The vaccine contains human mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116 fusion proteins C1, C1-C4S, C2 and C2-G4S. The recombinant human mycoplasma pneumoniae adhesion protein vaccine has the advantages that the human mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116 fusion proteins C1, C1-G4S, C2 and C2-G4S; in the invention, after a mouse is immunized by the recombinant human mycoplasma pneumoniae adhesion protein vaccine which is expressed and purified by escherichia coli, a strong immune response can be induced in a living body, the generation of cytokines and the colonization of mycoplasma pneumoniae in lung tissues can be reduced, and the recombinant human mycoplasma pneumoniae adhesion protein vaccine can be used for preventing, detecting or treating 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 the prevention, monitoring or treatment of human Mycoplasma pneumoniae infection and other related fields. Background Art

[0002] Mycoplasma pneumoniae, which has no cell wall structure, is the smallest known pathogen capable of self-replication. It can cause mycoplasma pneumonia in humans of all ages, especially in older children and young adults, accounting for 20% of community-acquired pneumonia cases. Its infection usually manifests as trachea or bronchitis. In severe cases, it can cause chronic lung disease and extrapulmonary sequelae, and aggravate asthma attacks. Macrolide antibiotics are the first choice for the treatment of Mycoplasma pneumoniae infection. However, with the rapid increase in the infection rate of macrolide-resistant Mycoplasma pneumoniae, the demand for new drugs and new preventive measures is more urgent. Although veterinary Mycoplasma pneumoniae vaccines have achieved good application and protection effects at home and abroad, so far, there is no approved Mycoplasma pneumoniae vaccine for human use. At present, Mycoplasma pneumoniae vaccines in the research stage include inactivated vaccines, live attenuated vaccines, recombinant protein vaccines, DNA vaccines, etc. 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, causing 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 vivo 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 purpose, 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 by a rigid connecting peptide and / or a flexible connecting 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 The amino acid sequence of the fusion protein is as described in SEQ ID NO.11, and the fusion protein having the technical solution in the present invention is named "C1-G4S"; or The amino acid sequence of the fusion protein is as described in SEQ ID NO.13. In the present invention, the fusion protein having the technical solution is named "C2"; or The amino acid sequence of the fusion protein is as described in SEQ ID NO.15. In the present invention, the fusion protein having the technical solution is named "C2-G4S".

[0012] In the present invention, the structure of the C1 fusion protein is: P1 (1160-1521 aa in the full-length sequence of the P1 original protein)-EAAAK-P30 (100-274 aa in the full-length sequence of the P30 original protein)-AAY-P40 / 90 (456-754 aa in the full-length sequence of the P40 / 90 original protein)-GPGPG-P116 (145-347aa in the full-length sequence of the P116 original protein), wherein EAAAK, AAY, and GPGPG are the rigid connecting peptides described in the present invention.

[0013] 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 (145-347aa), wherein GGGGS is the flexible connecting peptide described in the present invention.

[0014] The structure of C2 fusion protein is: P1(1287–1518 aa)-EAAAK-P30(100-274 aa)-AAY- P40 / 90(456-754 aa)-GPGPG- P116(145-347aa).

[0015] The structure of C2-G4S fusion protein is: P1(1287–1518 aa)-GGGGS-P30(100-274 aa)-GGGGS-P40 / 90(456-754 aa)- GGGGS-P116(145-347aa).

[0016] Secondly, the present invention provides a polynucleotide encoding the above fusion protein, 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 polynucleotide provided by the present invention is an artificially optimized polynucleotide.

[0017] Third, the present invention provides a vector containing the above polynucleotide.

[0018] Fourthly, the present invention provides a host cell containing the above-mentioned vector.

[0019] In a specific embodiment of the present invention, the optimized nucleotide sequence expressing the above fragments is embedded in the pET29a plasmid cloning site (NdeI and XhoI), transformed into Escherichia coli BL21 and induced for expression by IPTG, and the expressed protein is purified by Ni column and gel filtration in two steps to obtain the corresponding recombinant protein.

[0020] Fifth, the present invention provides a composition comprising one or more of the above-mentioned fusion proteins.

[0021] 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.

[0022] Sixth, the present invention provides the use of the above-mentioned composition in the preparation of a recombinant protein vaccine of human Mycoplasma pneumoniae.

[0023] The fusion protein and fusion protein composition provided by the present invention were mixed evenly (recombinant protein quadruple preparation) and immunized mice by nasal drops, and immunized three times continuously on day 0, day 14 and day 28. After immunization, the mice were challenged with 1E+07 CCU / ml for three consecutive days. On the fourth day after 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 sera on days D21 and D42 were used for the detection of IgG antibodies against the corresponding antigens, respectively. Compared with the rP1C + rP30C immunization group, the fusion protein composition provided by the present invention (recombinant protein quadruple preparation) showed strong IgG antibody detection against rP1C, rP30, C2-G4S and iMP (human Mycoplasma pneumoniae inactivated by polyformaldehyde, 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 increasing to 8.15-fold, 10.79-fold, 35.64-fold and 3.56-fold on D42, respectively. Compared with the second immunization, the IgG antibodies 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.

[0024] The results of the mouse challenge experiment showed that after the quadruple immunization of the human pneumoniae candidate vaccine constructed by the present invention, compared with the rP1C + rP30C immunization group, the fusion protein composition (recombinant protein quadruple preparation) provided by the present invention also significantly increased the IgA antibody levels against rP1C, rP30, C2-G4S and iMP. Both rP1C + rP30C and quadruple immunization significantly reduced the content of proinflammatory cytokines IL-1β, IL-6 and TNF-α in alveolar lavage fluid, and significantly reduced the load of Mycoplasma pneumoniae in lung tissue, indicating that the human pneumoniae candidate vaccine constructed in this study has played a good protective effect.

[0025] In specific applications, the vaccine can be prepared into a variety of preparations, such as injections, oral preparations, and preferably, the recombinant vaccine can be prepared as an inhalation vaccine.

[0026] Finally, the present invention provides the use of the above-mentioned composition in preparing a human pneumonia Mycoplasma detection kit.

[0027] The present invention provides a human Mycoplasma pneumoniae recombinant protein composition that can be used for the detection of human Mycoplasma pneumoniae serum antibodies. Compared with the control group, in the serum of mice infected with Mycoplasma pneumoniae, antibodies binding to four human Mycoplasma pneumoniae recombinant proteins C1, C1-G4S, C2, and C2-G4S can be detected by ELISA. If specific antibodies against the recombinant proteins can be detected in human serum, it indicates the occurrence of historical or acute infection.

[0028] Preferably, the method can be used for the development of a human Mycoplasma pneumoniae serum antibody detection kit, and the method or kit may be used for human Mycoplasma pneumoniae serum epidemiological monitoring, human Mycoplasma pneumoniae vaccine humoral immune response evaluation, etc.

[0029] In summary, the present invention relates to four constructed human Mycoplasma pneumoniae recombinant protein compositions, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of SDS-PAGE of C1, C1-G4S, C2 and C2-G4S proteins are shown in Figure 2. The target proteins were obtained after two steps of affinity purification and gel filtration chromatography after the C1, C1-G4S, C2 and C2-G4S proteins induced and expressed by E. coli. The purity of the proteins was ≥80% as determined by SDS-PAGE. Figure 2 This is a schematic diagram of mouse immunization and grouping; Figure 3is the level of specific IgG antibodies against rP1C in the serum of mice after immunization; Figure 4 is the level of specific IgG antibodies against rP30C in the serum of mice after immunization; Figure 5 is the level of specific IgG antibody against C2-G4S in the serum of mice after immunization; Figure 6 is the level of specific IgG antibodies against iMP in the serum of mice after immunization; Figure 7 This is a schematic diagram of mouse immunization and grouping; Figure 8 The specific IgA antibody levels against different antigens in the bronchoalveolar lavage fluid of mice; Fig. 9 Cytokine levels in bronchoalveolar lavage fluid of mice; Fig.10 is the MP replication level in mouse lung tissue. DETAILED DESCRIPTION

[0031] The present invention is 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 are purchased from conventional biochemical reagent stores unless otherwise specified.

[0032] Example 1. Construction of human Mycoplasma pneumoniae recombinant protein vaccine 1. Construction of rP1C, rP30C, rP40 / 90N and rP116N recombinant proteins The optimized nucleotide sequences expressing rP1C, rP30C, rP40 / 90N and rP116N were embedded in the pET29a plasmid cloning sites (NdeI and XhoI), transformed into Escherichia coli BL21 and induced for expression by IPTG. The expressed proteins were purified by Ni column and gel filtration to obtain the corresponding recombinant proteins.

[0033] 2. Construction of C1, C1-G4S, C2, and C2-G4S recombinant protein vaccines 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 by 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 .

[0034] Example 2. Evaluation of immunogenicity of human Mycoplasma pneumoniae recombinant protein vaccine 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 evenly divided into 3 groups, and all were nasally dripped three times continuously with an interval of 14 days. The PBS group: 50 μl PBS was nasally dripped, the P1+P30 immunization group: 10 μg of rP1C and rP30C were nasally dripped (40 μl in total), the C immunization group: 5 μg of C1, C1-G4S, C2 and C2-G4S were nasally dripped (40 μl), and 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 .

[0035] Table 1. Immunization schedule and grouping

[0036] The specific grouping, vaccination and detection procedures were as follows: 27 BalB / C mice were evenly divided into 3 groups, and all were intranasally dripped three times with an interval of 14 days. The PBS group was intranasally dripped with 50 μl PBS, the P1+P30 immunization group was intranasally dripped with 10 μg of rP1C and rP30C (40 μl in total), and the C immunization group was intranasally dripped with 5 μg of C1, C1-G4S, C2 and C2-G4S (40 μl) each. The P1+P30 and C immunization groups were both adjuvanted with c-di-GMP (10 μl, 1 μg / μl). Serum was collected on the 21st day (D21) and 42nd day (D42) after immunization to detect the corresponding specific antibodies.

[0037] 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 .

[0038] Figure 3is the level of specific IgG antibodies against rP1C in the serum of mice after immunization. On D21 and D42 days after immunization, specific IgG antibodies against rP1C could be detected in the serum of BalB / C mice in the immunized group, and the antibody level in the C immunized group was higher than that in the P1+P30 immunized group, increasing to 2.39 times on D21 and 8.15 times on D42. Compared with D21, the levels of specific IgG antibodies against rP1C in the P1+P30 and C immunized groups on D42 increased to 5.67 times and 19.33 times, respectively.

[0039] Figure 4 It is the level of specific IgG antibodies against rP30C in the serum of mice after immunization. On D21 and D42 days after immunization, specific IgG antibodies against rP30C can be detected in the serum of BalB / C mice in the immunized group, and the antibody level in the C immunized group is significantly higher than that in the P1+P30 immunized group, increasing to 2.11 times on D21 and 10.79 times on D42. Compared with D21, the levels of specific IgG antibodies against rP1C in the P1+P30 and C immunized groups on D42 increased to 3.22 times and 16.50 times, respectively.

[0040] Figure 5 It is the level of specific IgG antibodies against C2-G4S in the serum of mice after immunization. On D21 and D42 days after immunization, specific IgG antibodies against protein C can be detected in the serum of BalB / C mice in the immunized group, and the antibody level in the C immunized group is significantly higher than that in the P1+P30 immunized group, increasing to 8.23 ​​times on D21 and 35.64 on D42. Compared with D21, the levels of specific IgG antibodies against protein C in the P1+P30 and C immunized groups on D42 increased to 50.39 times and 218.18 times, respectively.

[0041] Figure 6 It is the level of specific IgG antibodies against iMP in the serum of mice after immunization. On D21 and D42 days after immunization, specific IgG antibodies against iMP can be detected in the serum of BalB / C mice in the immunized group, and the antibody level of the C immunized group is significantly higher than that of the P1+P30 immunized group, increasing to 2.49 times on D21 and 3.56 times on D42. Compared with D21, the levels of specific IgG antibodies against iMP in the P1+P30 and C immunized groups on D42 increased to 224.96 times and 321.99 times, respectively.

[0042] Example 3. Evaluation of the protective effect of human Mycoplasma pneumoniae recombinant protein vaccine Immunization challenge procedure Figure 7Specifically, on D42 after PBS or protein immunization, BalB / C mice (PBS* group, P1+P30* group and C* group) were infected with 50 μl (1E+07 CCU / ml) MP intranasally for three consecutive days, and 50 μl saline intranasally was used as a negative control (PBS group). On D45, serum, lung tissue and bronchoalveolar lavage fluid of mice were collected for corresponding tests.

[0043] In this example, the expression level of pro-inflammatory cytokines in alveolar lavage fluid after challenge was determined using the fully automatic microfluidic immunoassay analyzer Ella. The Ella multifactor cartridge was taken out of the -20° refrigerator 30 minutes in advance to restore to room temperature, the serum was centrifuged at 12000 g for 4 minutes, 30 μl of the supernatant was taken and diluted 1:1 with the sample diluent, the Ella multifactor cartridge QR code was scanned, 50 μl of sample and 1 ml of wash solution were added to each well, the cartridge was clicked to run, data was obtained after 1 hour, and the results were analyzed.

[0044] Figure 8 The specific IgA antibody levels against different antigens in the bronchoalveolar lavage fluid of mice. On D45, specific IgA antibodies against rP1C, rP30C, C protein and iMP were detected in the BALF of BalB / C mice in both P1+P30* and C* groups, and the antibody level in the C* group was higher than that in the P1+P30* group (increased to 12.61 times, 4.37 times, 4.74 times and 15.91 times, respectively).

[0045] Cytokine levels in bronchoalveolar lavage fluid Fig. 9 Compared with the PBS* group, the contents of cytokines IL-1β, IL-6, and TNF-α in BALF of mice in the P1+P30* and C* groups were significantly decreased, which were reduced 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.

[0046] 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 of lung tissue, and the fluorescence quantitative PCR method (Zhijiang Bio, Mycoplasma pneumoniae nucleic acid determination kit) was used to detect the Mycoplasma pneumoniae bacterial load. The Mycoplasma pneumoniae load in lung tissue is shown in Fig.10 Compared with the PBS* group, the MP genome copy number in the lung tissues of mice in the P1+P30* and C* groups decreased significantly, 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 fusion protein, characterized in that The fusion protein contains human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116.

2. The fusion protein according to claim 1, characterized in that The amino acid sequences of the human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116 are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7, respectively.

3. The fusion protein according to claim 1, characterized in that In the fusion protein, the human Mycoplasma pneumoniae adhesion proteins P1, P30, P40 / 90 and P116 are connected via a rigid connecting peptide and / or a flexible connecting peptide.

4. The fusion protein according to claim 3, characterized in that The rigid linking peptide includes one or more of EAAAK, AAY, and GPGPG.

5. The fusion protein according to claim 3, characterized in that The flexible connecting peptide is (G4S)n, where n is a natural number of 1-6.

6. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 or SEQ ID NO.

15.

7. A polynucleotide encoding the fusion protein of claim 6, characterized in that: The sequence of the polynucleotide is shown as SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 or SEQ ID NO.

16. A vector comprising the polynucleotide according to claim 7.

9. A host cell containing the vector according to claim 8.

10. A composition, characterized in that The composition contains one or more fusion proteins according to any one of claims 1-6.

11. The composition according to claim 10, 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.

12. Use of the composition according to claim 10 in preparing a recombinant protein vaccine of human Mycoplasma pneumoniae.

13. Use of the composition according to claim 10 in preparing a kit for detecting human Mycoplasma pneumoniae.

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