Staphylococcus aureus toxin lukg antigen epitope peptide and application thereof

Abstract

The application provides a staphylococcus aureus toxin LukG antigen epitope peptide, comprising a polypeptide with an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 40 or SEQ ID NO: 42, and the application also provides application of the antigen epitope peptide in preparation of a staphylococcus aureus infection diagnostic, preventive or therapeutic product. The application determines a staphylococcus aureus toxin LukG antibody dominant epitope peptide through screening, and the staphylococcus aureus toxin LukG antibody dominant epitope peptide has strong immunogenicity and can be used for staphylococcus aureus infection diagnosis, prevention or treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis technology, and in particular to an antigenic epitope peptide for diagnosing or preventing and treating Staphylococcus aureus infection and an application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus), a representative of Gram-positive bacteria, is a widespread pathogenic coccus in nature and a major cause of both nosocomial and community-acquired infections. Studies have shown that in the United States, S. aureus is the most common pathogen causing community-acquired skin and soft tissue infections (approximately 75%). In Japan, S. aureus causes bullous impetigo in 92% of cases of impetigo. In Africa, S. aureus accounts for 55-72% of tropical pyomyositis cases. In China, S. aureus is the leading cause of infective endocarditis (31-34%). Furthermore, S. aureus infections are characterized by acute, suppurative symptoms, causing persistent, localized purulent infections of the skin and soft tissues. Systemic infections can lead to severe infections and complications, including osteomyelitis, septic arthritis, endocarditis, pneumonia, and sepsis, with a mortality rate as high as 20%. At the same time, the exotoxins of Staphylococcus aureus can also cause fatal systemic infections such as food poisoning, scalded skin syndrome and toxic shock syndrome.

[0003] Staphylococcus aureus infections are typically treated with antibiotics such as erythromycin, new penicillins, gentamicin, vancomycin, or cefotaxime VI. However, due to the emergence of multidrug-resistant S. aureus, treating these infections with a single antibiotic has become increasingly difficult. Clinical data show that multidrug-resistant S. aureus accounts for 60% of S. aureus infections in the community and 80% in hospitals. Methicillin-resistant S. aureus (MRSA) strains are particularly prominent. In 2009, MRSA accounted for 25-50% of clinical S. aureus isolates in nine European countries. MRSA infections are difficult to cure with antibiotics and have a high mortality rate. In 2018, the national average detection rate of MRSA in China was 35.0%, ranking first among Gram-positive drug-resistant bacteria. The 90-day mortality rate of bacteremia caused by MRSA invasive infections exceeds 50%. Current clinical antibiotic treatments are inadequate to control MRSA infections, making vaccine development urgent.

[0004] The pathogenicity of MRSA depends primarily on the toxins and invasive enzymes it produces. It produces a variety of exotoxins, including bicomponent leukotoxins (BCLs), such as γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. Recently, a new BCL pair, LukGH (also known as LukAB), has been identified from Staphylococcus aureus. LukGH differs structurally and functionally from other S. aureus bicomponent leukocidins. Studies have shown that LukGH is more cytotoxic to human neutrophils (PMNs) than PVL. Furthermore, LukGH, similar to PVL, can induce IL-8 production in PMNs. However, LukG and LukH alone do not induce calcium influx or cytolysis. However, each of them can induce high levels of IL-8 transcription and secretion, and this induction is dependent on NF-κB. In another study, a LukG knockout strain was constructed to investigate the effects of LukG deficiency on the inflammatory response of macrophages stimulated by Staphylococcus aureus. The study found that LukG protein can influence the inflammatory response of macrophages through the NF-κB pathway. LukGH is the only known leukotoxin that can enhance the survival of Staphylococcus aureus after phagocytosis by PMNs.

[0005] Antibody responses play a major protective role in the MRSA vaccine response. Studies have confirmed that individuals who are unable to produce neutralizing antibodies against the toxin are more susceptible to toxic shock syndrome associated with staphylococcal infection. Since protein antigens primarily exert their function through epitope specificity, identifying immunodominant protective epitopes within the LukG antigen is a crucial prerequisite for improving and optimizing the design of LukG-based Staphylococcus aureus vaccines. Screening for immunodominant epitopes within the S. aureus toxin LukG is essential for stimulating a more effective LukG immune response. Currently known LukG B cell epitopes are either inferred through bioinformatics software, identified through monoclonal antibody analysis, or identified using human or animal immune models. These methods are incapable of identifying immunodominant epitopes in humans during clinical MRSA infection. There are no reports comprehensively screening for dominant epitopes of the S. aureus toxin LukG that participate in the immune response during clinical MRSA infection. Therefore, there is an urgent need for a method to accurately and effectively screen and identify B cell dominant epitope peptides of LukG that participate in the immune response during clinical MRSA infection. Summary of the Invention

[0006] The present application provides a Staphylococcus aureus toxin LukG antibody dominant epitope peptide and application of the epitope peptide in preparation of a medicine for diagnosing, preventing and / or treating Staphylococcus aureus infection.

[0007] A Staphylococcus aureus toxin LukG antigen epitope peptide, comprising a peptide segment with an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 40 or SEQ ID NO: 42; preferably, a peptide segment with an amino acid sequence of SEQ ID NO: 40.

[0008] In one embodiment according to the present application, it comprises a peptide segment with an amino acid sequence of SEQ ID NO: 40.

[0009] In one embodiment according to the present application, it comprises a polypeptide marker coupled to the N-terminus or C-terminus of the polypeptide; preferably, the polypeptide marker is a biotin marker or a fluorescent marker.

[0010] The present application also provides a fusion protein for preparing an antibody against Staphylococcus aureus, which comprises the Staphylococcus aureus toxin LukG antigen epitope peptide and a carrier protein; the carrier protein is selected from one of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), thyroglobulin, fibrinogen, gelatin or polyantigen peptide.

[0011] In one embodiment according to the present application, the Staphylococcus aureus toxin LukG antigen epitope peptide or the fusion protein is used in preparation of a medicine for diagnosing, preventing or treating Staphylococcus aureus infection.

[0012] The present application also provides a diagnostic reagent for Staphylococcus aureus infection, which comprises the Staphylococcus aureus toxin LukG antigen epitope peptide or the fusion protein.

[0013] In one embodiment according to the present application, in the diagnostic reagent, the Staphylococcus aureus toxin LukG antigen epitope peptide is coated on a detection carrier, which is selected from any one of polystyrene micro-reaction plates, colloidal gold reagent strips, magnetic beads and microfluidic chips.

[0014] In one embodiment according to the present application, it further comprises a second antibody specifically recognizing human IgG antibody;

[0015] Preferably, the second antibody is selected from one of rabbit anti-human monoclonal antibody, rabbit anti-human polyclonal antibody, mouse anti-human monoclonal antibody, mouse anti-human polyclonal antibody, goat anti-human polyclonal antibody or goat anti-human polyclonal antibody;

[0016] Preferably, the second antibody is coupled with a coordination group that activates or quenches the specific fluorescent group.

[0017] The present application also provides the use of the Staphylococcus aureus toxin LukG antigen epitope peptide as described above in the preparation of a medicament for preventing and / or treating Staphylococcus aureus infection.

[0018] The present application further provides a medicament for preventing or treating Staphylococcus aureus infection, comprising the Staphylococcus aureus toxin LukG antigen epitope peptide as described above and a pharmaceutically acceptable adjuvant.

[0019] In one embodiment according to the present application, it comprises a pharmaceutically acceptable adjuvant.

[0020] In one embodiment according to the present application, the adjuvant is a Quil-A adjuvant.

[0021] The beneficial effects of the above technical solutions of the present application are as follows:

[0022] The antibody dominant epitope peptides of Staphylococcus aureus toxin LukG provided by the present application can all induce high-level dominant epitope antisera in animals, and can be used to prepare high-efficiency, low-toxicity and high-safety Staphylococcus aureus toxin LukG-based drugs for preventing Staphylococcus aureus infection, such as prophylactic vaccines.

[0023] The antibody dominant epitope peptides of Staphylococcus aureus toxin LukG provided by the present application have strong immunogenicity, and the immune preparation has no irrelevant or harmful components. The specific monoclonal antibody prepared by the dominant epitope peptide can better prevent Staphylococcus aureus infection.

[0024] Through sequence alignment analysis, the antibody dominant epitope peptides of Staphylococcus aureus toxin LukG provided by the present application are sequence-conserved in various S.aureus strains, and thus can also be used as diagnostic reagents for Staphylococcus aureus infection or for the prevention and treatment of other S.aureus infections. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 A result map of ELISA detection of the overlapping peptides screened by the present application using MRSA positive antisera of the human population as the primary antibody;

[0026] Figure 2 A result map of analysis of the binding ability of the antisera obtained by immunizing mice with the immune dominant epitope peptides screened by the present application to LukG epitope peptides;

[0027] Figure 3 A result map of analysis of the binding ability of the antisera obtained by immunizing mice with the immune dominant epitope peptides screened by the present application to LukG epitope peptides; 30-47、 LukG234-251 , LukG 246-263 The ability of KLH fusion protein to reduce the colonization of Staphylococcus aureus in the kidneys and lungs of mice after active immunization;

[0028] Figure 4 The antibody immunodominant epitope peptide LukG screened by the present invention 162-179 , LukG 168-185 A map of the position distribution analysis results in the three-dimensional structure of LukG;

[0029] Figure 5 The antibody immunodominant epitope peptide LukG screened by the present invention 162-179 , LukG 168-185 The results of amino acid sequence conservation analysis are shown. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, all reagents used in this example were of analytical grade, and the progress of all chemical reactions was monitored by thin-layer chromatography.

[0032] Example 1 Obtaining overlapping peptides

[0033] Based on the LukG protein sequence (Sequence ID: WP_000595324.1), relative to the length of the expected overlapping peptide, the number of amino acids less than the length of the overlapping peptide is moved downstream each time to obtain another expected overlapping peptide. Among them, the expected overlapping peptide length can be 15-30 amino acids, and the number of amino acids stepped each time can be 4-8. In this embodiment, starting from amino acid No. 1, 6 amino acids are stepped each time to synthesize 18 amino acid polypeptides with overlapping steps (Shanghai Jier Biochemical Co., Ltd.), and a total of 50 overlapping peptides are obtained. The amino acid sequences of the overlapping peptides are shown in Table 1. The purity is greater than 95%. The information of the synthesized stepped overlapping peptides is shown in Table 1. The synthetic peptide segment was dissolved in dimethyl sulfoxide (DMSO) to a storage concentration of 1 mg / mL, frozen at -80°C after aliquoting, and diluted to 1 mM with PBS before use.

[0034] Table 1. Stepping overlapping peptides

[0035]

[0036]

[0037] Example 2 Collection and preservation of clinical MRSA positive serum

[0038] For the clinical MRSA-positive serum population, sera from 25 patients aged 18-64 years infected with Staphylococcus aureus who signed informed consent were used to measure the antiserum titer, and clinical MRSA-positive sera with LukG titer greater than 1:64000 were selected for subsequent testing.

[0039] Example 3 Screening of B cell immunodominant epitopes of LukG

[0040] The overlapping peptide coating concentration was adjusted to 10 μg / well (with the peptide library as a positive control), and the well plate was coated, washed, blocked, and washed again. The clinical MRSA positive serum obtained in Example 2 was added at a dilution of 1:250. After incubation for 1.5 hours and washing, HRP-goat anti-human IgG (purchased from SAB, item number L35004) was added at a dilution of 1:10000. After washing, TMB substrate colorimetric solution (purchased from Beyotime / Biyuntian, item number P0209-100ml) was added. After stopping the reaction, the OD value was read at 450nm. According to the formula (18 amino acid overlapping peptide OD detection value-blank control detection value) / (negative control OD detection value-blank control detection value) ≥2.1, it was considered a positive overlapping peptide. After Graphpad Prism 9.5 data verification, a positive overlapping peptide with statistically significant statistical significance relative to other positive overlapping peptide readings was obtained, which was defined as an immunodominant epitope peptide of B cells, i.e., an immunodominant epitope peptide.

[0041] The results are as follows Figure 1 Shown: There are 3 positive overlapping peptides LukG 30-47 (SEQ ID NO:6QKNITQSLQFNFLTEPNY)、LukG 234-251 (SEQ ID NO:40MSHDKKDKGKSQFVVHYK)、LukG 246-263 (SEQ ID NO: 42FVVHYKRSMDEFKIDWNR), which showed statistically significant differences with other dominant epitope peptide reads and was defined as an immunodominant epitope. BSA is a negative control in this figure. This method not only screened all B cell epitopes of LukG immune response, but also identified the B cell immunodominant epitope peptide of LukG.

[0042] Example 4 Identification of the Immunogenicity and Immunoreactivity of the Dominant Epitope Peptide of LukG

[0043] The B cell immunodominant epitope peptide of LukG identified in Example 3 was coupled with KLH (keyhole limpet hemocyanin) at equal concentrations (commissioned to Shanghai Jier Biochemical Co., Ltd.) to obtain a fusion protein. The epitope peptide-KLH fusion protein was administered at a dose of 100 μg / mouse and Quil-A adjuvant (purchased from InvivoGen) at a dose of 15 μg / mouse. After mixing, BALB / c mice (8 mice, 6-8 weeks old) were immunized intramuscularly in the thigh on days 0, 14, and 21. Seven days after the last immunization, eye blood was collected from the mice, and antiserum was isolated. The antiserum titer was measured, and the mice were aliquoted and frozen at -80°C.

[0044] Detection of the binding of antiserum to dominant epitope peptide: The coating concentration of LukG dominant epitope peptide was adjusted to 10 μg / well. After coating-washing-blocking-washing again, antiserum with dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 and 1:256000 were added (normal mouse serum was used as negative control, and PBS was used as blank control). After incubation for 1.5 h and washing, HRP-goat anti-mouse IgG (purchased from Abcam, product number AB97265) was added at a dilution of 1:7500. After washing, TMB substrate colorimetric solution (purchased from Beyotime / Biyuntian, product number P0209-100 ml) was added. After stopping the reaction, the OD value was read at 450 nm.

[0045] The results are as follows Figure 2 Shown: Anti-LukG 30-47 -KLH immunized mice with the dominant epitope peptide antiserum average titer of 1:128000, Anti-LukG 234-251 The average titers of the antiserum against the dominant epitope peptides of KLH-immunized mice were 1:128000, Anti-LukG 246-263 The average titer of the antiserum against the dominant epitope peptide in mice immunized with KLH was 1:64,000. Using the antiserum dilution as the horizontal axis and the OD value at 450nm as the vertical axis, it can be seen that the antiserum specific for the dominant epitope peptide of LukG at the corresponding titer can strongly bind to the LukG epitope peptide.

[0046] Example 5: Detection of the ability of active immunization with immunodominant epitope peptides to reduce the colonization of S. aureus in the kidneys and lungs of mice

[0047] The immunodominant epitope peptide-KLH fusion protein was immunized three times intramuscularly on days 0, 14, and 21 in the presence of Qui-A adjuvant (purchased from InvivoGen). BALB / c mice aged 6-8 weeks were injected intramuscularly 7 days after the last immunization. A sublethal dose of MRSA252 standard strain (purchased from ATCC American Type Culture Collection, 8×10 7CFU / ml) infected mice. At 48 hours after the challenge, the kidney and lung tissues of the mice were collected, and the number of bacterial colonization in each mouse organ was detected to evaluate the active immune protection effect of the dominant epitope peptide-specific monoclonal antibody on the mice. At the same time, a PBS non-active immunization control group was set up. Detection of Staphylococcus aureus colonization: BALB / c mice were killed by cervical dislocation, disinfected with 75% alcohol, and tissue specimens were taken aseptically and weighed. They were placed in 1 ml of sterile PBS and homogenized in a clean grinding tube with grinding beads. 1 ml of homogenate was taken and diluted according to the ratio of 1:5, 1:25, 1:125, 1:625, and 1:3125. 5 μL of each dilution was gently spread on LB solid culture medium, placed at 37 ° C, and cultured for 24 hours. The colony count (CFU / ml) was performed, and Gram staining with typical microscopic morphology and PCR detection were performed to confirm that it was Staphylococcus aureus.

[0048]

[0049] The results are as follows Figure 3 The results showed that compared with the PBS group, LukG 234-251 -KLH active immunization can significantly reduce the number of MRSA252 colonization in the kidneys and lungs of mice, and the P values ​​are statistically significant (* indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, ns indicates P > 0.05). 30-47 -KLH and LukG 246-263 -KLH active immunization did not achieve a significant effect in clearing bacteria from organs. 234-251 It is a protective immunodominant epitope against infection by the MRSA252 standard strain.

[0050] Example 6

[0051] This example is used to illustrate the analysis results of the positional distribution of the antibody immunodominant epitope peptides in the three-dimensional structure of the entire LukG protein.

[0052] The 3D structure of the reported LukG protein was downloaded from the PubMed protein database, and the sequence positions of the immunodominant epitope peptides screened in the experiment were annotated using PyMOL 1.1 program software.

[0053] The results are as follows Figure 4 As shown, the dominant peptide LukG 234-251 The β sheet located on the surface of the LukG three-dimensional crystal structure is conducive to binding with antibodies. It can be seen that this dominant epitope peptide sequence (antibody dominant epitope) is a reliable candidate molecule for LukG epitope vaccine.

[0054] Example 7

[0055] This example is used for the immunodominant epitope peptide LukG of the antibody screened by the present invention. 234-251 The results of amino acid sequence conservation analysis.

[0056] The amino acid sequences of LukG proteins of 38 strains of Staphylococcus aureus were retrieved from the Genbank database, and amino acid sequence alignment analysis was performed using the Basic Local Alignment Search Tool (BLAST) software of NCBI. 38 strains were randomly selected for multiple sequence alignment (Multiple Alignment). The website address is https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0057] The results are as follows Figure 5 As shown, the dominant peptide LukG 234-251 The amino acid sequence is conserved among 38 strains of Staphylococcus aureus, so it has good application prospects.

[0058] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A Staphylococcus aureus toxin LukG antigen epitope peptide, whose amino acid sequence is a peptide segment of SEQ ID NO: 6, SEQ ID NO: 40 or SEQ ID NO:

42.

2. The Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 1, wherein the amino acid sequence thereof is a peptide segment of SEQ ID NO:

40. 3 . The Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 1 , comprising a polypeptide tag coupled to the N-terminus or C-terminus of the Staphylococcus aureus toxin LukG antigen epitope peptide.

4. The Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 3, characterized in that: The polypeptide is labeled with biotin or fluorescence.

5. A fusion protein for preparing an anti-Staphylococcus aureus antibody, characterized in that: The fusion protein comprises the Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 1 or 2 and a carrier protein; the carrier protein is selected from one of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), thyroglobulin, fibrinogen, gelatin or a polymeric antigen peptide.

6. Use of the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 4, or the fusion protein according to claim 5, in the preparation of a medicament for diagnosing Staphylococcus aureus infection.

7. Use of the Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 2 in the preparation of a medicament for preventing or treating Staphylococcus aureus infection.

8. A diagnostic reagent for Staphylococcus aureus infection, characterized in that: The method comprises the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 4, or the fusion protein according to claim 5.

9. The diagnostic reagent according to claim 8, wherein The Staphylococcus aureus toxin LukG antigen epitope peptide is coated on a detection carrier, and the detection carrier is selected from any one of a polystyrene micro-reaction plate, a colloidal gold reagent strip, magnetic beads and a microfluidic chip.

10. The diagnostic reagent according to claim 9, characterized in that It also contains a second antibody that specifically recognizes human IgG antibodies.

11. The diagnostic reagent according to claim 10, characterized in that The second antibody is selected from one of rabbit anti-human monoclonal antibody, rabbit anti-human polyclonal antibody, mouse anti-human monoclonal antibody, mouse anti-human polyclonal antibody or goat anti-human polyclonal antibody.

12. The diagnostic reagent according to claim 11, wherein The second antibody is coupled with a coordination group that activates or quenches a specific fluorescent group.

13. Use of the Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 2 in the preparation of a medicament for preventing and / or treating Staphylococcus aureus infection.

14. A drug for preventing or treating Staphylococcus aureus infection, characterized in that: The invention comprises the Staphylococcus aureus toxin LukG antigen epitope peptide as claimed in claim 2, and pharmaceutically acceptable excipients.

15. The drug according to claim 14, wherein A pharmaceutically acceptable adjuvant is also included.

16. The drug according to claim 15, wherein The adjuvant is Quil-A adjuvant.

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