Staphylococcus aureus toxin LukG epitope peptide and application thereof

Abstract

The invention provides a staphylococcus aureus toxin LukG antigen epitope peptide, which comprises a polypeptide with an amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 40 or SEQ ID NO: 42, and also provides an application of the antigen epitope peptide in preparation of drugs for diagnosing, preventing or treating staphylococcus aureus infection. The antibody dominant epitope peptide of the staphylococcus aureus toxin LukG is determined through screening, and the antibody dominant epitope peptide has high immunogenicity and can be used for diagnosis, prevention or treatment of staphylococcus aureus infection.

Description

Technical Field

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

[0002] Staphylococcus aureus (S. aureus), as a representative of Gram-positive bacteria, is a type of pathogenic cocci widely found in nature, and is also an important pathogen causing hospital and community infections. Surveys and studies have shown that in the United States, the most common pathogen for community skin and soft tissue infections is S. aureus (accounting for about 75%); in Japan, bullous impetigo caused by S. aureus accounts for 92% of impetigo; in Africa, S. aureus accounts for 55-72% of tropical pyomyositis pathogens; in China, S. aureus is the number one pathogen for infective endocarditis (accounting for 31-34%). In addition, S. aureus infection is characterized by acute and suppurative nature, which can cause suppurative infections of the skin and soft tissues locally and last for a long time; systemic infections can cause serious infections and complications such as osteomyelitis, septic arthritis, endocarditis, pneumonia, sepsis, etc., with a mortality rate of up to 20%. At the same time, the exotoxin of Staphylococcus aureus can also cause fatal systemic infections such as food poisoning, scalded skin syndrome and toxic shock syndrome.

[0003] For the treatment of Staphylococcus aureus infection, antibiotics such as erythromycin, new penicillin, gentamicin, vancomycin or vancomycin VI are usually used. However, due to the emergence of multidrug-resistant Staphylococcus aureus, it is becoming increasingly difficult to treat Staphylococcus aureus infection with a single antibiotic. Clinical data show that Staphylococcus aureus with multiple antibiotic resistance accounts for 60% of Staphylococcus aureus infections in the community and 80% in hospitals. Among them, methicillin-resistant Staphylococcus aureus strains (MRSA) are particularly prominent. In 2009, MRSA accounted for 25-50% of clinical Staphylococcus aureus strains in nine European countries. Infections caused by MRSA are difficult to cure with antibiotic therapy and have a high mortality rate. In 2018, the average detection rate of MRSA in China was 35.0%, ranking first among Gram-positive resistant bacteria. The mortality rate of bacteremia caused by MRSA invasive infection exceeds 50% within 90 days. At present, the main clinical antibiotic treatment is unable to control MRSA infection, and vaccine development is imminent.

[0004] The pathogenicity of MRSA depends mainly on the ability of the toxins and invasion enzymes it produces. It can produce a variety of exotoxins, including bicomponent leukotoxins (BCLs), such as γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. In recent years, a new BCL pair, LukGH (also known as LukAB), has been identified from Staphylococcus aureus. LukGH is different from other bicomponent leukocidins of Staphylococcus aureus in structure and function. Studies have shown that LukGH is more cytotoxic to human neutrophils (PMNs) than PVL. In addition, LukGH can also induce PMNs to produce IL-8, which is similar to PVL, but LukG and LukH alone cannot induce calcium influx or cell lysis. They can each induce high levels of IL-8 transcription and secretion, and this induction depends on NF-κB. In another study, a LukG gene knockout strain was constructed to explore the effect of LukG deficiency on the inflammatory response of macrophages stimulated by Staphylococcus aureus. The study found that LukG protein can affect 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 being phagocytosed by PMNs.

[0005] In the vaccine response of MRSA, the antibody response plays a major protective role. Studies have shown that people who cannot produce antitoxin neutralizing antibodies are more susceptible to toxic shock syndrome associated with staphylococcal infection. Since protein antigens exert their functions mainly through epitopes to reflect specificity. Therefore, the identification of protective epitopes of immunodominant responses in LukG antigens is an important prerequisite for improving and optimizing the design of Staphylococcus aureus vaccines based on LukG antigens. Screening for immunodominant epitopes of Staphylococcus aureus toxin LukG is a prerequisite for stimulating a more effective LukG immune response. The currently known B cell epitopes of LukG are either inferred by bioinformatics software, identified by monoclonal antibodies, or identified in human or animal immune models. This method cannot identify the immunodominant epitopes of the population under clinical MRSA infection status. There is no report on comprehensive screening of dominant epitopes of Staphylococcus aureus toxin LukG that participate in immune response in clinical MRSA infection status. Therefore, it is urgent to obtain an accurate and effective screening and identification of B cell dominant epitope peptides of LukG that participate in immune response under clinical MRSA infection status. Summary of the invention

[0006] In order to solve the above problems in the prior art, the present invention provides an antibody dominant epitope peptide of Staphylococcus aureus toxin LukG, and the use of the epitope peptide in the preparation of a drug 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, comprising a peptide segment with an amino acid sequence of SEQ ID NO:40.

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

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

[0010] The present invention also provides a fusion protein for preparing an anti-Staphylococcus aureus antibody, wherein the fusion protein 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 a polymeric antigen peptide.

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

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

[0013] In one embodiment of the present invention, in the diagnostic reagent, 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 microplate, a colloidal gold reagent strip, a magnetic bead and a microfluidic chip.

[0014] In one embodiment according to the present invention, it further comprises a second antibody that specifically recognizes human IgG antibodies;

[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 invention also provides the use of the above-mentioned Staphylococcus aureus toxin LukG antigen epitope peptide in the preparation of a drug for preventing and / or treating Staphylococcus aureus infection.

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

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

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

[0021] The beneficial effects of the above technical solution of the present invention are as follows:

[0022] The antibody dominant epitope peptides of Staphylococcus aureus toxin LukG provided by the present invention can induce high-level dominant epitope antiserum in animals, and can be used to prepare highly effective, low-toxic, and highly safe drugs based on Staphylococcus aureus toxin LukG for preventing Staphylococcus aureus infection, such as preventive vaccines.

[0023] The antibody dominant epitope peptide of Staphylococcus aureus toxin LukG provided by the present invention has strong immunogenicity. When animals are immunized with the dominant epitope, the immune preparation has no irrelevant or harmful components. The specific monoclonal antibody prepared with the dominant epitope peptide can better prevent Staphylococcus aureus infection.

[0024] Sequence comparison analysis shows that the antibody dominant epitope peptide of Staphylococcus aureus toxin LukG provided by the present invention has a conserved sequence in multiple S. aureus strains. Therefore, it can also be used as a diagnostic reagent for Staphylococcus aureus infection or for the prevention and treatment of other S. aureus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The result spectrum of ELISA detection of overlapping peptides screened by the present invention using MRSA positive antiserum of the population as a primary antibody;

[0026] Figure 2 The graph is the analysis result of the binding ability between the antiserum obtained by immunizing mice with the immunodominant epitope peptide screened by the present invention and the LukG epitope peptide;

[0027] Figure 3 The immunodominant epitope peptide LukG screened by the present invention 30-47、 LukG234-251 , LukG 246-263 The KLH fusion protein was tested for its ability 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 The results of the position distribution analysis 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 in Figure 2. DETAILED DESCRIPTION

[0030] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, 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 detected 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 expected length of the overlapping peptide, each time the number of amino acids less than the length of the overlapping peptide is moved downstream, another expected overlapping peptide is obtained again. Among them, the expected length of the overlapping peptide can be 15-30 amino acids, and the number of amino acids in each step can be 4-8. In this embodiment, starting from amino acid No. 1, 6 amino acids are stepped each time, and 18 amino acid polypeptides with overlapping steps are synthesized (Shanghai Jier Biochemical Co., Ltd.), and a total of 50 overlapping peptides are obtained. The amino acid sequence of the overlapping peptides is shown in Table 1. The purity is greater than 95%. The information of the synthesized step-shifted overlapping peptides is shown in Table 1. The synthetic peptide segment is dissolved in dimethyl sulfoxide (DMSO) to a storage concentration of 1 mg / mL, and frozen at -80°C after aliquoting, and diluted to 1mM with PBS before use.

[0034] Table 1 Overlapping peptides

[0035]

[0036]

[0037] Example 2 Collection and Storage of Clinical MRSA Positive Serum

[0038] For the clinical MRSA-positive serum population, the antiserum titer was measured using the sera of 25 patients with Staphylococcus aureus infection aged 18-64 years who signed the informed consent form, and the 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 the positive control), the well plate was coated-washed-blocked-washed again, and the clinical MRSA positive serum obtained in Example 2 was added at a dilution of 1:250. After incubation for 1.5 hours-washing, HRP-sheep 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 terminating 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 was a positive overlapping peptide. After Graphpad Prism 9.5 data inspection, a positive overlapping peptide with 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 has significant statistical significance with other dominant epitope peptide readings, is defined as an immunodominant epitope, and BSA is a negative control in this figure. This method not only screens out all B cell epitopes of the immune response of LukG, but also clarifies the B cell immunodominant epitope peptide of LukG.

[0042] Example 4 Identification of the Immunogenicity and Immunoreactivity of the Predominant 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 by Shanghai Jier Biochemical Co., Ltd.) to obtain a fusion protein. The epitope peptide-KLH fusion protein was 100 μg / mouse, and the Quil-A adjuvant (purchased from InvivoGen) was 15 ug / mouse. After mixing, BALB / c mice (8 mice, 6-8 weeks old) were immunized by thigh intramuscular injection on days 0, 14, and 21. Seven days after the last immunization, the mouse eyeball blood was collected, the antiserum was separated, the antiserum titer was measured, and the antiserum was frozen at -80°C after aliquoting.

[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-sheep 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 terminating 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 dominant epitope peptides had an average titer of 1:128,000, and Anti-LukG 234-251 The average titers of the antiserum against the dominant epitope peptides of mice immunized with KLH were 1:128000 and Anti-LukG 246-263 -The average titer of the antiserum of the dominant epitope peptide of KLH-immunized mice was 1: 64000. With the antiserum dilution as the horizontal axis and the OD value at 450nm as the vertical axis, it can be seen that the LukG dominant epitope peptide-specific antiserum can strongly bind to the LukG epitope peptide at the corresponding titer.

[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). On day 7 after the last immunization, a sublethal dose of MRSA252 standard strain (purchased from ATCC American Type Culture Resource Library, 8×10 7CFU / ml) infected mice. At the 48th hour of the attack, 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 mice. At the same time, a PBS control group without active immunization was set up. Detection of Staphylococcus aureus colonization: BALB / c mice were killed by cervical dislocation, 75% alcohol disinfection, sterile tissue specimens were weighed, placed in 1ml sterile PBS, and homogenized in a clean grinding tube with grinding beads. 1ml of homogenate was taken and diluted according to the ratio of 1:5, 1:25, 1:125, 1:625, and 1:3125. 5μL of each dilution was gently spread on LB solid culture medium, placed at 37°C, cultured for 24h, and colony counts (CFU / ml) were performed, and Gram staining typical microscopic morphology and PCR detection were performed in parallel 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 significant results 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 public 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 three-dimensional crystal structure of LukG is conducive to the binding with antibodies. It can be seen that the 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 antibody immunodominant epitope peptide LukG 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 the amino acid sequences were compared and analyzed 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 the scope of protection of the present invention.

Claims

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

40.

2. The Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 1, comprising a peptide segment with an amino acid sequence of SEQ ID NO:

40.

3. The Staphylococcus aureus toxin LukG antigen epitope peptide according to claim 1 or 2, comprising a polypeptide label coupled to the N-terminus or C-terminus of the polypeptide; preferably, the polypeptide label is a biotin label or a fluorescent label.

4. 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 as claimed in 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.

5. Use of the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 3, or the fusion protein according to claim 4, in the preparation of a medicament for diagnosing, preventing or treating Staphylococcus aureus infection.

6. A diagnostic reagent for Staphylococcus aureus infection, characterized in that: It comprises the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 3, or the fusion protein according to claim 4.

7. The diagnostic reagent according to claim 6, characterized in that The Staphylococcus aureus toxin LukG antigen epitope peptide or integrin 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, a magnetic bead and a microfluidic chip.

8. The diagnostic reagent according to claim 7, characterized in that Also comprising a second antibody that specifically recognizes human IgG antibody; 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; Preferably, the second antibody is coupled with a coordination group that activates or quenches the specific fluorescent group.

9. Use of the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 3 or the fusion protein according to claim 4 in the preparation of a medicament for preventing and / or treating Staphylococcus aureus infection.

10. A drug for preventing or treating Staphylococcus aureus infection, characterized in that: The invention comprises the Staphylococcus aureus toxin LukG antigen epitope peptide according to any one of claims 1 to 3 or the fusion protein according to claim 4, and pharmaceutically acceptable excipients.

11. The drug according to claim 10, characterized in that A pharmaceutically acceptable adjuvant is also included.

12. The drug according to claim 11, characterized in that The adjuvant is Quil-A adjuvant.

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