Streptococcus pneumoniae PsaA protective epitope peptide, vaccine and preparation method and application thereof

Abstract

The invention discloses a streptococcus pneumoniae PsaA protective epitope peptide, a vaccine and a preparation method and application thereof, the nucleotide sequence of the epitope peptide is shown as SEQ ID NO.1, a novel epitope vaccine ZnuA-PsaA-Loop90 (for short, ZPL90) is successfully constructed based on the epitope, after the vaccine is inoculated, an anti-Loop90 antibody can be efficiently induced in a mouse, the mouse can be effectively protected from being infected with streptococcus pneumoniae, and the streptococcus pneumoniae PsaA protective epitope peptide can be used for preparing the vaccine. Good application prospects are realized.

Description

Technical Field

[0001] This invention relates to the field of vaccines, specifically to a protective epitope peptide of Streptococcus pneumoniae PsaA, and also to vaccines containing the epitope peptide, methods for preparing the vaccines, and their applications. Background Technology

[0002] Streptococcus pneumoniae (SP), a Gram-positive pathogen, colonizes the nasopharynx of 40% of healthy individuals, causing invasive diseases such as bacterial pneumonia, meningitis, and sepsis. Vaccines are a key means of combating Streptococcus pneumoniae infection. The two commercially available vaccines, polysaccharide vaccines (PPV) and conjugate vaccines (PCV), are effective but have limitations. PPV is ineffective in infants under 2 years old and lacks immunological memory; PCV is expensive, covers fewer serotypes, and is difficult to use against new strains. Currently, no SP protein vaccine has been successfully marketed, making the development of a broad-spectrum, highly effective, and low-cost novel protein vaccine a major clinical need.

[0003] Pneumococcus surface adhesin A (PsaA) is a highly conserved lipoprotein on the surface of Streptococcus pneumoniae, belonging to the extracellular substrate-bound lipoproteins of the manganese ion ABC-type transporter complex. As a key protein in the manganese transport system, PsaA is a major virulence factor that exacerbates Streptococcus pneumoniae colonization and pathogenicity. The conservation of PsaA makes it an ideal antigenic target for overcoming serotype limitations. Therefore, some vaccines have incorporated it as a component in Phase I human clinical trials. However, due to the relatively weak immunogenicity and limited protective efficacy of PsaA, no vaccine based on the PsaA antigen is currently on the market. Epitope vaccines are a proven and effective strategy for precisely inducing antibodies against specific epitopes. Identifying key protective epitopes of PsaA and constructing novel vaccines capable of inducing high levels of antibodies against these epitopes holds promise for overcoming this challenge. This invention discovers a novel vaccine constructed based on the ZnuA vector and the PsaA protective epitope Loop90 discovered by our team, which can efficiently induce anti-Loop90 antibodies and exhibits significant protective effects in animal challenge trials. Therefore, this invention provides a new design strategy for developing SP protein vaccines and may offer new ideas for promoting the clinical application of pneumococcal vaccines. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide a protective epitope peptide of Streptococcus pneumoniae PsaA; a second objective of the present invention is to provide a vaccine based on the protective epitope peptide of Streptococcus pneumoniae PsaA; a third objective of the present invention is to provide a recombinant expression vector; a fourth objective of the present invention is to provide a method for preparing a vaccine based on the protective epitope peptide of Streptococcus pneumoniae PsaA; a fifth objective of the present invention is to provide the use of the vaccine based on the protective epitope peptide of Streptococcus pneumoniae PsaA in medicaments for the prevention or treatment of Streptococcus pneumoniae infection; and a sixth objective of the present invention is to provide the use of the vaccine based on the protective epitope peptide of Streptococcus pneumoniae PsaA in inducing mammals to produce specific antibodies against the Loop90 epitope of the Streptococcus pneumoniae PsaA protein.

[0005] To achieve the above objectives, the present invention provides the following technical solution: 1. A protective epitope peptide of Streptococcus pneumoniae PsaA, the sequence of which is shown in SEQ ID NO.1.

[0006] 2. A vaccine based on the protective epitope peptide of Streptococcus pneumoniae PsaA, characterized in that: the vaccine comprises a carrier protein and the epitope peptide according to claim 1; the carrier protein is ZnuA, characterized in that: the amino acid sequence of the carrier protein is as shown in SEQ ID NO.2, and the nucleotide sequence is as shown in SEQ ID NO.3.

[0007] Preferably, the amino acid sequence of the vaccine is shown in SEQ ID NO.4.

[0008] Preferably, the nucleotide sequence of the vaccine is shown in SEQ ID NO.5.

[0009] 3. A recombinant expression vector, wherein the recombinant expression vector contains the nucleotide sequence described above.

[0010] 4. The method for preparing the vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA involves inserting the sequence shown in SEQ ID NO.5 into the Nco I and Xho I restriction sites of the pET32a vector, followed by expression in Escherichia coli and purification.

[0011] 5. Use of the aforementioned Streptococcus pneumoniae PsaA protective epitope peptide in vaccines for the prevention or treatment of Streptococcus pneumoniae infection.

[0012] 6. The application of the vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA in inducing mammals to produce specific antibodies against the Loop90 epitope of Streptococcus pneumoniae PsaA protein.

[0013] The beneficial effects of this invention are as follows: This invention obtains the protective B-cell epitope peptide Loop90 of Streptococcus pneumoniae PsaA antigen, and successfully constructs a novel epitope vaccine ZPL90 based on this epitope. After vaccination, this vaccine can efficiently induce anti-Loop90 antibodies in SPF mice and effectively protect SPF mice from Streptococcus pneumoniae infection, showing good application prospects. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The protein expression of PsaA protein and PsaA mutants (mutants of all negatively charged amino acids in Loop90 mutated to neutral amino acids) and their binding curves with manganese ions are shown (a: protein electrophoresis; b: affinity of PsaA for manganese ions; c: affinity of PsaA mutants for manganese ions). Figure 2 Design pattern diagram for ZPL90 protein; Figure 3 The results of ZnuA and ZPL90 protein purification; Figure 4 The results of PsaA and PsaAΔLoop protein purification; Figure 5 ZPL90 can efficiently induce anti-Loop90 antibodies; Figure 6 ZPL90 has a protective effect against Streptococcus pneumoniae infection. Detailed Implementation

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0016] Example 1 The sequence of the protective B-cell epitope peptide Loop90 of Streptococcus pneumoniae PsaA was analyzed, and the amino acid sequence is as follows: AVSDGVDVIYLEGQNEKGKEDPHA (SEQ ID NO.1).

[0017] Obtaining the protective epitope peptide: PsaA plays a major role in the attachment of Streptococcus pneumoniae to host cells and the uptake of manganese ions to resist oxidative stress. Experiments revealed that Loop90 is a key "catcher" for PsaA's uptake of manganese ions. Figure 1Isothermal titration calorimetry (ITC) analysis showed that wild-type PsaA still retains the ability to bind manganese ions, while the Loop90 mutant PsaA protein (a mutant in which all negatively charged amino acids in Loop90 are mutated to neutral amino acids) has lost its ability to bind manganese ions; simultaneously, as Figure 2 As shown, Loop90 was also found to be an epitope exposed on the PsaA surface.

[0018] The ZnuA-PsaA-Loop90 (ZPL90) epitope vaccine sequence was constructed based on the Loop90 epitope peptide sequence. The amino acid sequence of ZPL90 is shown in SEQ ID NO.4, and the encoded DNA sequence is shown in SEQ ID NO.5. The vaccine utilizes ZnuA, which has a very similar structure to PsaA but poor sequence similarity, as a vector and is fused with Loop90 for expression.

[0019] ZnuA vector amino acid sequence: AVVASLKPVGFIASAIADGVTETEVLLPDGASEHDYSLRPSDVKRLQNADLVVWVGPEMEAFMQKPVSKLPGAKQVTIAQLEDVKPLLMKSIVPEQPDEPGEIEPIPEDFNMHLWLSPEIARATAVAIHGKLVELM PQSRAKLDANLKDFEAQLASTETQVGNELAPLKGKGYFVFHDAYGYFEKQFGLTPLGHFTVNPEIQPGAQRLHEIRTQLVEQKATCVFAEPQFRPAVVESVARGTSVRMGTLDPLGTNIKLGKTSYSEFLSQLANQYASCLKGD (SEQ ID NO.2).

[0020] ZnuA vector DNA sequence: GGATCCGCAGTTGTTGCCAGCCTGAAACCGGTGGGTTTTATTGCCAGCGCAATTGCAGATGGCGTTACCGAAACCGAAGTTCTGCTGCCGGATGGCGCAAGCGAACATGATTATAGCCTGCGCCCGAGTGATGTGAAACGTCTGCAGAATGCCGATCT GGTTGTGTGGGTGGGTCCGGAAATGGAAGCCTTTATGCAGAAACCGGTTAGT AAACTGCCGGGTGCCAAACAGGTTACCATTGCCCAGCTGGAAGATGTGAAAC CGCTGCTGATGAAAAGTATTCATGGCGATGATGATGATCATGATCATGCAGAA AAAAGTGATGAAGATCATCATCATGGCGATTTTAATATGCATCTGTGGCTGAGT CCGGAAATTGCACGCGCCACCGCCGTGGCCATTCATGGCAAACTGGTTGAAC TGATGCCGCAGAGTCGTGCCAAACTGGATGCAAATCTGAAAGATTTTGAAGC ACAGCTGGCCAGTACCGAAACCCAGGTTGGCAATGAACTGGCCCCGCTGAAA GGCAAAGGCTATTTTGTGTTTCATGATGCATACGGTTATTTTGAAAAGCAGTTT GGTCTGACCCCGCTGGGTCATTTTACCGTGAATCCGGAAATTCAGCCGGGCGC ACAGCGCCTGCATGAAATTCGTACCCAGCTGGTTGAACAGAAAGCCACCTGT GTTTTTGCCGAACCGCAGTTTCGCCCGGCAGTTGTGGAAAGCGTTGCACGTG GTACAAGTGTTCGTATGGGCACCCTGGACCCTCTGGGTACAAATATTAAACTG GGCAAAACCAGCTATAGCGAATTTCTGAGTCAGCTGGCAAATCAGTATGCCAG CTGCCTGAAAGGCGATTAACTCGAG (SEQ ID NO.3). The underlines indicate the BamH I restriction site and the Xho I restriction site respectively.

[0021] ZPL90 amino acid sequence: AVVASLKPVGFIASAIADGVTETEVLLPDGASEHDYSLRPSDVKRLQNADLVVWVGPEMEAFMQKPVSKLPGAKQVTIAQLEDVKPLAVSDGVDVIYLEGQNEKGKEDPHAWLSPEIARATAVAIHGKLVELMPQSRAKLDANLKDFEAQLASTETQVGNELAPLKGKGYFVFHDAYGYFEKQFGLTPLGHFTVNPEIQPGAQRLHEIRTQLVEQKATCVFAEPQFRPAVVESVARGTSVRMGTLDPLGTNIKLGKTSYSEFLSQLANQYASCLKGD (SEQ ID NO.4).

[0022] ZPL90 DNA sequence: CCATGGCTGCAGTTGTGGCTAGTCTGAAACCGGTGGGCTTTATTGCCAGCGCCATTGCAGATGGTGTTACCGAAACCGAAGTTCTGCTGCCGGATGGCGCATCAGAACATGATTATAGTCTGCGCCCGAGTGATGTTAAACGCCTGCAGAATGCCGATCTGGTTGTGTGGGTTGGCCCGGAGATGGAAGCATTTATGCAGAAACCGGTTAGCAAACTGCCGGGTGCAAAACAGGTGACCATTGCACAGCTGGAAGATGTTAAACCGCTGGCCGTGAGCGATGGCGTTGATGTGATTTATCTGGAAGGTCAGAACGAAAAGGGTAAAGAAGATCCGCATGCATGGCTGAGTCCGGAAATTGCCCGCGCAACAGCTGTTGCTATTCATGGTAAACTGGTGGAACTGATGCCGCAGAGCCGCGCAAAACTGGATGCAAATCTGAAAGATTTCGAGGCACAGCTGGCCAGTACCGAAACCCAAGTTGGTAATGAACTGGCACCGCTGAAAGGTAAAGGTTATTTTGTTTTCCACGACGCCTACGGTTATTTTGAAAAACAGTTCGGTCTGACCCCGCTGGGTCATTTTACCGTGAATCCGGAAATTCAGCCGGGCGCACAGCGTTTACATGAAATTCGTACCCAGCTGGTTGAACAGAAAGCAACCTGCGTTTTTGCAGAACCGCAGTTTCGTCCGGCCGTTGTTGAAAGCGTGGCCCGTGGTACAAGTGTTCGCATGGGTACCCTGGATCCGCTGGGTACCAATATTAAACTGGGTAAAACCAGCTACAGCGAATTTCTGAGCCAGCTGGCCAATCAGTATGCCAGCTGTCTGAAAGGCGATCTCGAG (SEQ ID NO.5). The underlines indicate the NcoI restriction site and the XhoI restriction site respectively.

[0023] Example 2 Construction of ZnuA expression vector: The pET32a vector and the synthetic ZnuA DNA sequence were digested with restriction endonucleases BamHI and XhoⅠ to obtain the vector and the target gene fragment. The excised vector and the target gene fragment were ligated with DNA ligase. After selection for resistance to ampicillin (Amp) in E. coli, the E. coli containing the expression vector were amplified, and the expression vector was obtained by plasmid extraction. Construction of ZPL90 expression vector: The pET32a vector and the synthetic ZPL90 DNA sequence were digested with restriction endonucleases NcoI and XhoⅠ to obtain the vector and the target gene fragment. The excised vector and the target gene fragment were ligated with DNA ligase. After selection for resistance to ampicillin (Amp) in E. coli, the E. coli containing the expression vector were amplified, and the expression vector was obtained by plasmid extraction.

[0024] The carrier protein and vaccine preparation method are as follows: Expression vector transformation: Add 25 µL of BL21 competent cells to a sterile EP tube, and add 1 µL of dissolved plasmid aqueous solution. Gently mix with a pipette, and incubate the EP tube on ice for 30 min. Heat shock at 42°C for 90 s in a water bath, then immediately remove and incubate on ice again for 2 min 30 s to terminate the heat shock reaction. Then add 500 µL of LB medium to the EP tube, mix gently, and place the EP tube in a shaker at 37°C and shake at 220 rpm for 45 min. Take 20 µL of the cultured bacterial solution, spread it evenly on an LB agar plate containing a final concentration of 100 µg / mL Amp, and incubate overnight at 37°C.

[0025] Protein expression: The next day, single colonies growing on the plate were picked and inoculated into 5 mL of LB liquid medium containing 100 µg / mL Amp, and incubated overnight at 37°C and 220 rpm. The following day, 200 µL of the bacterial culture was transferred to 20 mL of LB liquid medium containing 100 µg / mL Amp and incubated overnight at 37°C and 220 rpm with shaking. Then, 20 mL of the culture was transferred to 2 L of LB medium containing 100 µg / mL Amp and incubated at 37°C and 220 rpm for 3 h. When the OD600nm reached between 0.6 and 1.0, isopropyl-1thio-β-D-galactoside (IPTG) was added to a final concentration of 2 mM for induction. ZnuA protein was cultured at 30°C and 200 rpm for 3 h and then centrifuged to collect the bacteria. ZPL90 was cultured at 16°C and 120 rpm overnight, and the bacteria were collected by centrifugation the next day. Resuspend the bacterial pellet in 50 mL of NTA-0 solution. Then place the centrifuge tube on ice and sonicate until the bacterial culture becomes clear or transparent. The sonication parameters were set as follows: power ≤40%, sonication for 9 seconds, interval for 9 seconds, total sonication time 20 min. Aliquot the disrupted bacterial culture into specially designed high-speed centrifuge tubes, balance, and centrifuge at 12000 rpm for 20 min at 4°C. Filter the supernatant through a 0.45 µM filter and collect it in 50 mL centrifuge tubes for subsequent purification.

[0026] Protein purification: Affinity chromatography (GST label): ① Affinity chromatography column preparation: Add 3 mL of GST-labeled Beads to the affinity chromatography column, add 10 times the volume of PBS buffer, open the stopper and let it flow down naturally, repeat the washing 3 times to ensure that the Beads are fully equilibrated; ② Protein binding: The filtered protein supernatant was added to an affinity chromatography column, sealed with a sealing film to prevent leakage, and then placed on a rotary shaker at 4°C for 4 hours. Afterward, the column was removed, the stopper was opened to allow the binding solution to flow out, at which point the target protein was bound to the beads. The beads were washed with 10 volumes of PBS buffer, repeated three times. Then, 3 mL of PBS buffer and 2 mL of PreScission Protease were added to the column, the column was sealed, and the column was placed on a rotary shaker at 4°C for overnight incubation. The next day, the digested liquid was collected in a 50 mL centrifuge tube for subsequent purification.

[0027] (2) Affinity chromatography (His label): ① Affinity chromatography column preparation: Add 3 mL of Ni-NTA packing material to the affinity chromatography column, add 10 times the volume of NTA-0 solution to equilibrate the Ni column, and wash 3 times to ensure that the beads are fully equilibrated; ② Protein binding: The filtered protein supernatant was added to a pre-equilibrated Ni column and allowed to flow down naturally, collecting the flowthrough. Imidazole was first added to the NTA-0 solution, gradually increasing the final concentration from 0 mM to 500 mM. Optimal elution concentrations were determined using gradient elution. In this study, the elution concentration was 100 mM with a elution volume of 50 mL; the elution concentration was 500 mM with a elution volume of 10–20 mL.

[0028] ③ Replacement Buffer: Turn on the AKTA purifier and install the Sephadex G-25 column. First, rinse the column with 100 mL of deionized water at a flow rate of 15 mL / min to remove impurities. Then, maintaining a flow rate of 15 mL / min, equilibrate the G25 column with PBS buffer until the A280 curve reaches zero and equilibrium is achieved. The washing volume should be one column volume. Finally, load the eluted sample onto the G25 column, set the flow rate to 10 mL / min, and begin replacement. During replacement, monitor the A280 absorbance and collect the replacement buffer corresponding to the protein absorption peak for subsequent purification.

[0029] Protein identification: Protein gel preparation: ① Separating gel preparation: Prepare the gel according to the instructions in the Dakota Rapid Protein Gel Reagent Kit. First, mix the separating gel solution A and solution B in a 1:1 ratio. Add an appropriate amount of 10% APS solution, mix thoroughly, and then pour into the electrophoresis mold until the liquid level is about 1.5 cm from the top of the glass plate. ② Preparation of stacking gel: Mix stacking gel A and B in a 1:1 ratio, add an appropriate amount of 10% APS solution, mix well, and then add directly to the upper layer of the separating gel; ③ Gel solidification and fixation: Insert the comb into the upper layer of gel. After the gel has completely solidified, remove the glass plate and place it in the electrophoresis tank containing electrophoresis buffer. Remove the comb.

[0030] (2) Electrophoresis: Add an appropriate amount of electrophoresis buffer to the electrophoresis tank until the gel is completely covered; take 5 µL of Protein Marker and 10 µL of sample and add them to the gel wells respectively; turn on the power and adjust the voltage to 100 V. When the band reaches the interface between the stacking gel and the separating gel, adjust the voltage to 200 V. Stop electrophoresis when the band is about 1.5 cm away from the bottom of the gel.

[0031] (3) Staining and destaining: Remove the gel from the electrophoresis tank and place it in a box containing instant blue staining solution. Place the box on a horizontal shaker and stain at room temperature for 5 minutes. Then discard the staining solution, add an appropriate amount of pure water, and continue to destain on the shaker until the gel background becomes clear.

[0032] (4) Gel imaging: Gel images were captured using a gel imaging system, and the position and intensity of the protein bands were recorded. The results are as follows: Figure 3 As shown, the purity of the obtained protein can reach over 95%.

[0033] Example 3 ZPL90 efficiently induces anti-Loop90 antibodies in SPF mice.

[0034] Mouse grouping: Healthy male C57BL / 6 mice aged 6-8 weeks were selected and randomly divided into three groups of 8 mice each; Mouse immunization regimen: The mice were randomly divided into an experimental group and a control group, with eight mice in each group. In the experimental group, 50 µg of PsaA and ZPL90 antigens were mixed with aluminum phosphate adjuvant and incubated at 4°C for 1–2 h to ensure sufficient binding between the antigen and adjuvant. In the control group, an equal volume of PBS was mixed with aluminum phosphate adjuvant. The mice were then immunized via intraperitoneal injection (600 µL / mouse), ensuring accurate injection site to prevent extravasation. All mice were immunized three times, on days 0, 7, and 14. Serum was collected seven days after the third immunization for subsequent antibody detection.

[0035] The antigen used for detection employs PsaA wild-type protein and PsaA knockout loop (PsaAΔLoop) protein, expressed using the same method as the ZPL90 epitope vaccine preparation method. Purification was performed using affinity chromatography (GST tagging). The amino acid sequence of the PsaA wild-type protein is shown in SEQ ID NO.6, and the encoded nucleic acid sequence is shown in SEQ ID NO.7. Simultaneously, the PsaA knockout loop (PsaAΔLoop) protein sequence was expressed using the same method. The amino acid sequence of the PsaA knockout loop (PsaAΔLoop) protein is shown in SEQ ID NO.8, and the encoded nucleic acid sequence is shown in SEQ ID NO.9.

[0036] Antigen sequences for immune serum detection: Amino acid sequence of wild-type PsaA: TSGQKLKVVATNSIIADITKNIAGDKIDLHSIVPIGQDPHEYEPLPEDVKKTSEADLIFYNGINLETGGNAWFTKLVENAKKTENKDYFAVSDGVDVIYLEGQNEKGKEDPHAWLNLENGIIFAKNIAKQLSAKDPNNKEFYEKNLKEYTDKLDKLDKESKDKFNKIPAEKKLIVTSEGAFKYFSKAYGVPSAYIWEINTEEEGTPEQIKTLVEKLRQTKVPSLFVESSVDDRPMKTVSQDTNIPIYAQIFTDSIAEQGKEGDSYYSMMKYNLDKIAEGLAK (SEQ ID NO. 6); DNA sequence: (SEQ ID NO.7). GGATCCACCAGCGGTCAGAAACTGAAAGTGGTGGCAACCAATAGCATTATTGCCGATATTACCAAAAACATTGCAGGTGATAAAATCGATCTGCATAGCATTGTGCCGATTGGTCAGGACCCTCATGAATATGAACCGCTGCCGGAAGATGTGAAAAAAACCAGCGAAGCCGATCTGATTTTTTATAATGGTATTAACCTGGAGACCGGTGGTAATGCATGGTTTACCAAACTGGTTGAAAATGCAAAAAAGACCGAAAATAAGGATTACTTTGCCGTTAGTGATGGCGTTGATGTTATTTATCTGGAAGGTCAGAATGAAAAAGGCAAAGAAGATCCGCATGCATGGCTGAATCTGGAAAATGGCATTATTTTTGCAAAGAATATCGCAAAACAGCTGAGTGCAAAAGATCCGAATAATAAAGAATTTTACGAGAAGAACCTGAAGGAATATACCGATAAACTGGATAAACTGGACAAAGAAAGTAAAGATAAGTTCAATAAGATCCCGGCAGAAAAAAAACTGATTGTTACCAGTGAAGGTGCCTTTAAATATTTTAGCAAAGCCTATGGCGTGCCGAGTGCCTATATTTGGGAAATTAATACCGAAGAAGAGGGCACCCCGGAACAGATTAAAACCCTGGTGGAAAAACTGCGTCAGACCAAAGTGCCGAGTCTGTTTGTGGAAAGCAGTGTGGATGATCGTCCGATGAAAACCGTTAGTCAGGATACCAATATTCCGATTTATGCACAGATTTTTACCGATAGCATTGCCGAACAGGGCAAAGAAGGCGATAGTTATTATAGTATG ATGAAATACAACCTGGACAAAATTGCAGAAGGCCTGGCCAAATAACTCGAG. The underlines indicate the BamH I restriction site and the XhoI restriction site respectively.

[0037] Amino acid sequence of the PsaA knockout Loop protein: TSGQKLKVVATNSIIADITKNIAGDKIDLHSIVPIGQDPHEYEPLPEDVKKTSEADLIFYNGINLETGGNAWFTKLVENAKKTENKDYFWLNLENGIIFAKNIAKQLSAKDPNNKEFYEKNLKEYTDKLDKLDKESKDKFNKIPAEKKLIVTSEGAFKYFSKAYGVPSAYIWEINTEEEGTPEQIKTLVEKLRQTKVPSLFVESSVDDRPMKTVSQDTNIPIYAQIFTDSIAEQGKEGDSYYSMMKYNLDKIAEGLAK (SEQ ID NO. 8); DNA sequence: (SEQ ID NO.9). The underscores represent the BamHI restriction site and the XhoⅠ restriction site, respectively.

[0038] After purification, the purity of PsaA protein and PsaA knockout loop protein (abbreviated as PsaAΔLoop) was detected, and the results are as follows: Figure 4 As shown in the figure. The results show that the purity of PsaA protein and PsaA knockout Loop protein can reach over 95%.

[0039] ELISA method for detecting whether anti-Loop90 specific antibodies are produced in immune serum: (1) Antigen coating: Dilute PsaA and PsaAΔLoop antigens to 5 µg / mL with coating solution, add to 96 microplate, 100 μL / well, and coat overnight at 4°C.

[0040] (2) Washing: Turn on the plate washer, prepare PBST containing 0.5% Tween-20 in advance, and set the plate washer parameters as follows: wash 4 times with PBST, add 300µL each time, shake for 5 seconds, aspirate for 2.5 seconds, and pat the plate dry after washing. (3) Sealing: Add 100 µL of sealing solution to each well and seal at 37℃ for 1 h; (4) Primary antibody incubation: First, dilute the serum with sample diluent at a ratio of 1:12800, and then dilute it in eight gradients. Add 100 µL of diluted serum sample to each well and incubate at 37°C for 2 hours. (5) Wash the plate: Repeat step (2); (6) Secondary antibody incubation: Dilute goat anti-mouse IgG antibody 1:200 with sample dilution buffer, add 100 µL to each well, and incubate at 37℃ for 1 h; (7) Wash the plate: Repeat step (2); (8) Color development: Under light-protected conditions, add 100 µL of TMB color development solution to each well and incubate at 37°C for 5–30 min. Determine the time to terminate the reaction based on the color in the well; (9) Termination: Add 100 µL of termination solution to each well to terminate the reaction; (10) Plate reading: Within 10 min after the reaction is terminated, the absorbance at a wavelength of 450 nm is detected using a multi-functional microplate reader.

[0041] Record and analyze the data to assess antibody production levels (the steps for detecting antibody levels by peptide coating are the same as above). Figure 5 As shown, PsaA specifically induced the production of anti-Loop90 antibodies in SPF-grade mice.

[0042] Example 4 ZPL90 protects SPF mice from Streptococcus pneumoniae infection: (1) Mouse grouping: Healthy male C57BL / 6 mice aged 6-8 weeks were randomly divided into three groups of 8 mice each. The immunization regimen was the same as above. The survival test immunization group was supplemented with PsaAΔLoop and ZnuA antigen, with 8 mice in each group.

[0043] (2) Culture and preparation of Streptococcus pneumoniae: Streptococcus pneumoniae strain 7F was taken from a -80℃ freezer and inoculated onto blood agar plates using the three-zone streak method. The plates were then incubated overnight at 37℃. The next day, 40-50 single colonies were picked and inoculated into 50 mL of THYB liquid medium. The medium was placed in a 37℃ water bath and incubated for 6-8 hours until the logarithmic growth phase. Subsequently, the cultured bacterial solution was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the bacteria were resuspended in sterile PBS. The OD600nm of the bacterial solution was adjusted to 1.05 using a UV spectrophotometer, at which point the bacterial concentration was 3 × 10⁻⁶. 8 CFU / mL, the bacterial count used in the survival experiment was 1.5 × 10⁻⁶. 8 CFUs / each.

[0044] On day 7 after three immunizations of mice, the survival experiment was conducted at a dose of 1.5 × 10⁻⁶. 8 Mice were injected with CFUs / mouse via endotracheal intubation with Streptococcus pneumoniae 7F, and the 7-day survival rate was observed. Subsequently, the protective effect of ZPL90 against other different serotypes of Streptococcus pneumoniae (18C, D39, 3, 5) was investigated using the same method.

[0045] like Figure 6 As shown, ZPL90 can effectively help SPF mice resist infection by Streptococcus pneumoniae and has a broad spectrum of activity.

[0046] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A protective epitope peptide of Streptococcus pneumoniae PsaA, characterized in that: The sequence of the epitope peptide is shown in SEQ ID NO.

1.

2. A vaccine based on the pneumococcal PsaA protective epitope peptide of claim 1, characterized in that: The vaccine comprises a carrier protein and the epitope peptide of claim 1; the carrier protein is ZnuA, characterized in that: the amino acid sequence of the carrier protein is as shown in SEQ ID NO.2, and the nucleotide sequence is as shown in SEQ ID NO.

3.

3. The vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA according to claim 2, characterized in that: The amino acid sequence of the vaccine is shown in SEQ ID NO.

4.

4. The vaccine containing the protective epitope peptide PsaA of Streptococcus pneumoniae according to claim 2, characterized in that: The nucleotide sequence of the vaccine is shown in SEQ ID NO.

5.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleotide sequence of claim 4.

6. A method for preparing a vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA as described in any one of claims 2 to 4, characterized in that: The sequence shown in SEQ ID NO.5 was ligated into the Nco I and Xho I restriction sites of the pET32a vector, then expressed in E. coli and purified.

7. Use of the vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA according to any one of claims 2 to 4 in the prevention or treatment of Streptococcus pneumoniae infection.

8. The use of the vaccine containing the protective epitope peptide of Streptococcus pneumoniae PsaA according to any one of claims 2 to 4 in inducing mammals to produce specific antibodies against the Loop90 epitope of Streptococcus pneumoniae PsaA protein.

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