CN112851770B - Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof - Google Patents
Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof Download PDFInfo
- Publication number
- CN112851770B CN112851770B CN202110163653.1A CN202110163653A CN112851770B CN 112851770 B CN112851770 B CN 112851770B CN 202110163653 A CN202110163653 A CN 202110163653A CN 112851770 B CN112851770 B CN 112851770B
- Authority
- CN
- China
- Prior art keywords
- staphylococcus aureus
- epitope peptide
- antibody
- hemolysin
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/36—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino acids, polyamines and polycarboxylic acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The invention discloses an antigen epitope peptide for diagnosing or preventing staphylococcus aureus infection, which comprises a polypeptide with an amino acid sequence of SEQ ID NO. 1, and also provides application of the antigen epitope peptide in preparation of medicines for diagnosing, preventing or treating staphylococcus aureus infection. The antibody dominant epitope peptide of the staphylococcus aureus hemolysin alpha is determined by screening, has stronger immunogenicity, and meanwhile, the amino acid sequence of the dominant epitope peptide is conserved in a plurality of S.aureus strains, so the antibody dominant epitope peptide can also be used for preparing diagnostic reagents for staphylococcus aureus infection or preventing or treating other S.aureus infections.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to an epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof.
Background
Staphylococcus aureus (s.aureus), a representative of gram-positive bacteria, is a group of pathogenic cocci widely existing in nature, and is also an important pathogenic bacterium causing nosocomial infections and community infections. Research studies have shown that the most common pathogenic bacteria of community skin and soft tissue infections in the united states are s.aureus (about 75%); s. aureus-initiated bullous impetigo accounts for 92% of all cases in japan; 55-72% of the pathogens of pyomyositis tropicalis in africa; in china, the first pathogen of infective endocarditis is s.aureus (31-34%). Moreover, s.aureus infection is characterized by acute, suppurative, localized, persistent suppurative infection of skin and soft tissues; the whole body can cause serious infection and complication such as osteomyelitis, septic arthritis, endocarditis, pneumonia, sepsis and the like, and the death rate is up to 20%. Meanwhile, exotoxin of staphylococcus aureus can also cause systemic lethal infections such as food poisoning, scalded skin syndrome and toxic shock syndrome.
For the treatment of staphylococcus aureus infections, antibiotics such as erythromycin, neopenicillin, gentamicin, vancomycin, or cephalosporins VI are usually selected. But with the advent of multidrug resistant staphylococcus aureus, it has become increasingly difficult to treat staphylococcus aureus infections with a single antibiotic. Clinical data show that staphylococcus aureus with multiple antibiotic resistance accounts for 60% of community staphylococcus aureus infections, while in hospitals this proportion accounts for 80%. Of particular note are methicillin-resistant staphylococcus aureus strains (MRSA), which account for 25-50% of clinical staphylococcus aureus strains in 9 european countries in 2009. MRSA-caused infections are difficult to cure with antibiotic therapy and have a high mortality rate. The national average detection rate of the Chinese MRSA in 2018 is 35.0%, and the gram-positive drug-resistant bacteria are the first. The mortality rate of bacteremia caused by MRSA invasive infection is more than 50 percent within 90 days. Currently, the main clinical antibiotic treatment fails to control MRSA infection, and vaccine development is imminent.
The pathogenic strength of MRSA is mainly determined byThe ability of toxin and invasive enzyme, wherein Hemolysin alpha (Hla) is also an important exotoxin causing staphylococcus aureus infection, usually generated by pathogenic staphylococcus aureus, especially MRSA, hla can cause clinical symptoms such as bacteremia and the like by promoting neutrophil lysis and damaging epithelial cells, and Hla immunity can relieve skin and soft tissue necrosis and pneumonia caused by MRSA epidemic strains. Through Blast comparison analysis, the Hla gene sequence has high conservation in the published pathogenic staphylococcus aureus, and the gene homology is more than 90 percent, so the Hla gene sequence is an ideal vaccine candidate antigen. However, since the hemolytic activity of Hla is strong, there may be a safety problem directly as a vaccine component. Researches prove that histidine (His) at the 35 th position plays an important role in forming a perforation complex, and the hemolytic activity of the Hla can be greatly reduced after the amino acid is subjected to site-directed mutation to leucine (Leu). Therefore, the Hla component in the product is mutant Hla obtained by mutation H35L No other changes in biological functions other than reduced hemolytic activity were found.
In the vaccine response of MRSA, the antibody response plays a major protective role. Studies have demonstrated that humans who fail to produce anti-toxin neutralizing antibodies are more susceptible to toxic shock syndrome associated with staphylococcal infection. Because the protein antigen plays its function, specificity is mainly embodied by epitope. Therefore, identification of protective epitopes for immunodominant responses in Hla antigens is an important prerequisite for the promotion and optimization of Hla antigen-based staphylococcus aureus vaccine design. Screening of immunodominant epitopes of Hla is a prerequisite for the stimulation of a more effective Hla immune response. The currently known B-cell epitopes of Hla cannot be identified by bioinformatics software speculation, monoclonal antibody identification, or use of human or animal immune models, and this method cannot identify immunodominant epitopes in humans in clinical MRSA-infected states. There is no report on comprehensive screening of the dominant epitopes of Hla involved in immune response in clinical MRSA infection status. Therefore, it is very important to establish a method for accurately and effectively screening and identifying the B cell dominant epitope of Hla participating in immune response under the clinical MRSA infection state.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides an antibody dominant epitope peptide of staphylococcus aureus hemolysin alpha and application of the epitope peptide in preparing a medicament for diagnosing, preventing and/or treating staphylococcus aureus infection.
The invention provides an alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection, which comprises amino acid sequence SEQ ID NO:1 (KVIFNNMVNQNW).
In one embodiment according to the present invention, the α hemolysin epitope peptide is a peptide having an amino acid sequence of SEQ ID NO:29 or SEQ ID NO: 30.
In one embodiment according to the invention, further comprising a polypeptide tag coupled to the N-terminus or C-terminus of said polypeptide.
In one embodiment according to the invention, the polypeptide label is a biotin label or a fluorescent label.
The invention also provides a fusion protein for preparing the antibody against staphylococcus aureus, which comprises a carrier protein and the alpha hemolysin epitope peptide; the carrier protein is selected from one of Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), thyroglobulin, fibrinogen, gelatin and multiple antigenic peptides; preferably, the multiple antigenic peptide is Polylysine (PLL).
The invention also provides application of the alpha hemolysin epitope peptide in preparing a medicament for diagnosing, preventing or treating staphylococcus aureus infection. The staphylococcus aureus can be any staphylococcus aureus capable of causing infection, particularly methicillin-resistant staphylococcus aureus strain with extremely strong drug resistance, and the methicillin-resistant staphylococcus aureus strain can be an MRSA252 standard strain.
The invention further provides a diagnostic reagent for staphylococcus aureus infection, which comprises the epitope peptide.
In one embodiment of the invention, the alpha hemolysin 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, a magnetic bead and a microfluidic chip.
In one embodiment according to the present invention, further comprising a second antibody specifically recognizing the human IgG antibody; for example, animals are immunized with immunodominant epitope peptide-KLH (keyhole limpet hemocyanin) conjugate fusion protein, and the immunogenicity and immunoreactivity of the dominant epitope peptide are analyzed.
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 to a coordinating group that activates or quenches the specific fluorophore.
In another aspect of the invention, the invention provides the application of the epitope peptide in preparing a medicament for preventing and/or treating staphylococcus aureus infection. The medicament may be formulated for administration by different routes in different dosage forms, preferably the vaccine is formulated for intravenous administration, e.g. as an injection, in order to avoid degradation of the antigenic component of the vaccine.
The invention further provides a medicament for preventing or treating staphylococcus aureus infection, which comprises the alpha hemolysin epitope peptide.
In one embodiment according to the invention, the adjuvant is selected from the group consisting of Quil-A adjuvant (available from InvivoGen corporation)
The beneficial effects of the invention are:
the antibody dominant epitope peptide of staphylococcus aureus hemolysin alpha provided by the invention can induce high-level dominant epitope antiserum in animals, and can be used for preparing high-efficiency, low-toxicity and high-safety Hla-based medicaments for preventing staphylococcus aureus infection, such as preventive vaccines.
The antibody dominant epitope peptide of staphylococcus aureus hemolysin alpha provided by the invention has stronger immunogenicity, no irrelevant components or harmful components exist in an immune preparation when the dominant epitope peptide is used for immunizing animals, and a specific monoclonal antibody prepared from the dominant epitope peptide can better prevent staphylococcus aureus infection.
Through sequence comparison and analysis, the antibody dominant epitope peptide of staphylococcus aureus alpha provided by the invention has conserved sequences in various S.aureus strains, so that the antibody dominant epitope peptide can also be used as a diagnostic reagent of staphylococcus aureus infection or used for preventing and treating other S.aureus infections.
Drawings
FIG. 1 is a graph showing the results of ELISA detection of overlapping peptides screened according to the present invention using MRSA positive antiserum of the population as a primary antibody;
FIG. 2 is a graph showing the results of analysis of the binding capacity of antiserum and Hla epitope peptide obtained by immunizing a mouse with the immunodominant epitope peptide selected according to the present invention;
FIG. 3 shows immunodominant epitope peptide Hla selected by the present invention 162 -179、Hla 168 -185 KLH fusion protein is mapped by the ability test of active immunization to mitigate colonization of mouse kidney and lung staphylococcus aureus;
FIG. 4 shows the antibody immunodominant epitope peptide Hla selected by the present invention 162 -179、Hla 168 -185 location distribution analysis result map in the Hla three-dimensional structure;
FIG. 5 shows the antibody immunodominant epitope peptide Hla selected by the present invention 162 -179、Hla 168 -185 amino acid sequence conservation analysis result map.
Detailed Description
The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention and to clearly define the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 acquisition of overlapping peptides
Based on the Sequence ID of the Hla protein (ADQ 77533.1), another expected overlapping peptide is again obtained by moving downstream a number of amino acids smaller than the length of the overlapping peptide each time relative to the length of the expected overlapping peptide. The length of the expected overlapping peptide can be 15-30 amino acids, the number of amino acids in each step can be 4-8, in this example, starting from amino acid No. 1, 6 amino acids in each step, 18 amino acid polypeptides (Shanghai Qianzhizi Biotech Co., ltd.) with overlapping steps are synthesized, and 47 overlapping peptides are obtained. The purities are all more than 95%. The synthetic walking overlapping peptide information is shown in table 1. The synthetic peptide fragments were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 1mg/mL, dispensed and frozen at-70 ℃ and diluted to 1mM with PBS at the time of use.
TABLE 1 walking overlapping peptides
Example 2 Collection and preservation of clinical MRSA-positive sera
The antiserum titer was determined by the MRSA positive serum population (25 persons infected with staphylococcus aureus between 18 and 64 years old) who signed an informed consent, and the Hla titer was selected to be greater than 1:64000 clinical MRSA positive sera were used for the subsequent detection.
Example 3 screening of B cell immunodominant epitopes of Hla
Adjusting the coating concentration of the overlapping peptide to be 15 mug/hole (taking holoprotein Hla (Sequence ID: ADQ 77533.1) as a positive control), coating, washing, sealing and washing the well plate again, adding the Hla immune antiserum obtained in example 2, diluting at 1: 300, incubating for 1.5h, washing, adding HRP-goat anti-mouse IgG (purchased from Enjingyuan organism, product number E1WP 319), diluting at 1: 3500, washing, adding TMB substrate developing solution (purchased from Beyotime/Biyun, product number P0209-100 ml), reading the OD value at 450nm after terminating the reaction, and taking the positive overlapping peptide as the formula 18 according to the detection value of the amino acid overlapping peptide-the blank control detection value)/(the negative peptide-the blank control detection value) to be not less than 2.1. Through SPSS16.0 data inspection, positive overlapping peptides which have significant statistical significance relative to other positive overlapping peptide readings are obtained and are defined as immunodominant epitope peptides of B cells, namely immunodominant epitope peptides.
The results are shown in FIG. 1: there were 2 positive overlapping peptides Hla 162-179 (SEQ ID NO：29 DKKVGWKVIFNNMVNQNW)、Hla 168-185 (SEQ ID NO:30 KVIFNNMVNQNWGPYDRD), which has significant statistical significance in comparison with other dominant epitope peptide reads, was defined as the immunodominant epitope, in which OVA 192 Negative irrelevant peptide at-201. The method not only screens out all B cell epitopes of the Hla in immune response, but also defines the B cell immunodominant epitope peptide of the Hla.
Example 4 immunogenicity and immunoreactivity characterization of dominant epitope peptides of Hla
The epitope peptide-KLH fusion protein was prepared by coupling the B cell immunodominant epitope peptide of Hla identified in example 3 with KLH (hemocyanin) at equal concentrations (manufactured by Shanghai Jier Biochemical Co., ltd.) to obtain a fusion protein, wherein the epitope peptide-KLH fusion protein was administered at 60. Mu.g/mouse, quil-A adjuvant (purchased from InvivoGen) at 10 ug/mouse, mixing, immunizing by injecting the thigh of BALB/c mouse (10 mice, 6-8 weeks old) on days 0, 14, and 21, immunizing the mouse 7 days after the last immunization, separating antiserum, measuring the antiserum titer, and freezing at-70 ℃ after split charging.
Detection of binding of antisera to dominant epitope peptides: adjusting the coating concentration of the Hla dominant epitope peptide to be 15 mug/hole, after coating, washing, sealing and washing again, adding antiserum (normal mouse serum is used as a negative control and PBS is a blank control) with the dilution of 1: 50,1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800,1: 25600 and 1: 51200 respectively, incubating for 1.5h, after washing, adding HRP-goat anti-mouse IgG (obtained from Enjingze organism color development, product number E1WP 319) with the dilution of 1: 3500, adding TMB substrate solution obtained from Beyotime/Biyun day after washing, product number P0209-100 ml), and reading the OD value at 450nm after terminating the reaction.
The results are shown in FIG. 2: anti-Hla 162-179 -the mean antiserum titer of the dominant epitope peptide of KLH immunized mice is 1:22400，Anti-Hla 168-185 -the mean antiserum titers of dominant epitope peptides of KLH immunized mice are 1:12800. by taking the antiserum dilution as the abscissa and the OD value at 450nm as the ordinate, the antiserum with the specificity of the Hla dominant epitope peptide can be strongly combined with the Hla epitope peptide when the corresponding titer is reached.
Example 5 detection of the ability of immunodominant epitope peptide active immunization to mitigate colonization of Staphylococcus aureus in the kidney and lung of mice
Immunodominant epitope peptide-KLH fusion protein was immunized with BALB/c mice by intramuscular injection three times on days 0, 14, and 21 with the aid of Qui-A adjuvant (purchased from), 6-8 weeks, and sublethal dose of MRSA252 standard strain (purchased from ATCC American type culture strain resource Bank, 6X 10, and 6D) was injected into tail vein on day 7 after the last immunization 8 CFU/ml) infected mice. Collecting mouse kidney and lung tissues at 48 h of the challenge, detecting the bacterial colonization quantity of each mouse organ, and evaluating the active immune protection effect of the dominant epitope peptide specific monoclonal antibody on the mice. Meanwhile, PBS non-active immunization control group was set. Detecting the colonization amount of staphylococcus aureus: cervical spine was sacrificed, BALB/c mice were sterilized with 75% alcohol, tissue specimens were aseptically weighed, placed in 2ml of sterile PBS, homogenized in a clean glass homogenizer, 1ml of homogenate was diluted in the proportion of 1: 10,1: 100,1: 1000, 100. Mu.L of each dilution was gently spread on a solid medium, placed at 37 ℃ for 24h, and colony count (CFU/ml) was performed, and a typical under-the-lens morphology and PCR assay for gram staining was performed to confirm that Staphylococcus aureus was present.
The results are shown in FIG. 3, which shows: hla in contrast to PBS group 168-185 Active KLH immunization significantly reduced the number of MRSA252 colonizations in the kidneys and lungs of mice, with statistical differences in P values (P < 0.05, P < 0.001). And Hla 162-179 Active KLH immunization did not achieve significant effect in eliminating visceral bacteria. Confirmation, hla 168-185 Is a standard strain against MRSA252Protective immunodominant epitopes of infection.
Example 6
This example is used to demonstrate the results of analysis of the positional distribution of antibody immunodominant epitope peptides in the three-dimensional structure of the full protein of Hla
Downloading a 3D structure chart of a reported Hla protein from a public database of PubMed protein, and labeling the sequence position of the immunodominant epitope peptide screened in the experiment by PyMOL 1.1 program software.
The results are shown in FIG. 4, where the dominant peptide, hla 162-179 、Hla 168-185 The epitope peptide sequence is positioned in a beta sheet layer and a loop region of an Hla three-dimensional crystal structure respectively, and therefore, the dominant epitope peptide sequence (antibody dominant epitope) is a reliable candidate molecule of an Hla epitope vaccine.
Example 7
This example is for the antibody immunodominant epitope peptide Hla screened by the present invention 162-179 、Hla 168-185 The result of amino acid sequence conservation analysis of (1).
The amino acid sequences of the Hla proteins of 36 s.aureus strains were searched in the Genbank database, analyzed by amino acid sequence Alignment using Basic Local Alignment Search Tool (BLAST) software from NCBI, and 36 strains were randomly selected for Multiple sequence Alignment (Multiple Alignment) with the website address https: gov/blast.
The results are shown in FIG. 5, where the dominant peptide, hla 162-179 、Hla 168-185 The amino acid sequence of the staphylococcus aureus is conserved in each strain of 36 staphylococcus aureus, so that the staphylococcus aureus has a good application prospect.
The above summary and the detailed description are intended to demonstrate the practical application of the technical solutions provided by the present invention, and should not be construed as limiting the scope of the present invention. Various modifications, equivalent substitutions, or improvements within the spirit and principles of the invention may occur to those skilled in the art. The scope of the invention is to be determined by the appended claims.
Sequence listing
<110> China people liberation army, military and medical university
<120> alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof
<141> 2021-02-05
<160> 48
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 1
Lys Val Ile Phe Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Asp
1 5 10 15
Arg Asp
<210> 2
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 2
Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser
1 5 10 15
Asn Thr
<210> 3
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 3
Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser Asn Thr Thr Val Lys
1 5 10 15
Thr Gly
<210> 4
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 4
Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr
1 5 10 15
Tyr Asp
<210> 5
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 5
Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn Gly
1 5 10 15
Met Leu
<210> 6
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 6
Asp Leu Val Thr Tyr Asp Lys Glu Asn Gly Met Leu Lys Lys Val Phe
1 5 10 15
Tyr Ser
<210> 7
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 7
Lys Glu Asn Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp
1 5 10 15
Lys Asn
<210> 8
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 8
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His Asn Lys Lys
1 5 10 15
Ile Leu
<210> 9
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 9
Phe Ile Asp Asp Lys Asn His Asn Lys Lys Ile Leu Val Ile Arg Thr
1 5 10 15
Lys Gly
<210> 10
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 10
His Asn Lys Lys Ile Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly
1 5 10 15
Gln Tyr
<210> 11
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 11
Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser
1 5 10 15
Glu Glu
<210> 12
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 12
Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys
1 5 10 15
Ser Gly
<210> 13
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 13
Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro
1 5 10 15
Ser Ala
<210> 14
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 14
Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val Gln
1 5 10 15
Leu Gln
<210> 15
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 15
Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn
1 5 10 15
Glu Val
<210> 16
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 16
Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser
1 5 10 15
Asp Tyr
<210> 17
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 17
Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn
1 5 10 15
Ser Ile
<210> 18
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 18
Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu
1 5 10 15
Tyr Met
<210> 19
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 19
Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr
1 5 10 15
Tyr Gly
<210> 20
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 20
Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn
1 5 10 15
Val Thr
<210> 21
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 21
Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp Ser
1 5 10 15
Gly Lys
<210> 22
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 22
Phe Asn Gly Asn Val Thr Gly Asp Asp Ser Gly Lys Ile Gly Gly Leu
1 5 10 15
Ile Gly
<210> 23
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 23
Gly Asp Asp Ser Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser
1 5 10 15
Ile Gly
<210> 24
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 24
Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His Thr Leu Lys
1 5 10 15
Tyr Val
<210> 25
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 25
Ala Asn Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe
1 5 10 15
Lys Thr
<210> 26
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 26
His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser
1 5 10 15
Pro Thr
<210> 27
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 27
Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro Thr Asp Lys Lys Val
1 5 10 15
Gly Trp
<210> 28
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 28
Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe
1 5 10 15
Asn Asn
<210> 29
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 29
Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn Gln
1 5 10 15
Asn Trp
<210> 30
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 30
Lys Val Ile Phe Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Asp
1 5 10 15
Arg Asp
<210> 31
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 31
Met Val Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro
1 5 10 15
Val Tyr
<210> 32
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 32
Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu
1 5 10 15
Phe Met
<210> 33
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 33
Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn
1 5 10 15
Gly Ser
<210> 34
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 34
Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala
1 5 10 15
Glu Asn
<210> 35
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 35
Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Glu Asn Phe Leu Asp Pro
1 5 10 15
Asn Lys
<210> 36
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 36
Met Lys Ala Ala Glu Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu
1 5 10 15
Leu Ser
<210> 37
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 37
Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser
1 5 10 15
Pro Asp
<210> 38
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 38
Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr Val
1 5 10 15
Ile Thr
<210> 39
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 39
Ser Gly Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys
1 5 10 15
Ala Thr
<210> 40
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 40
Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Thr Lys Gln Gln Thr
1 5 10 15
Asn Ile
<210> 41
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 41
Met Asp Arg Lys Ala Thr Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr
1 5 10 15
Glu Arg
<210> 42
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 42
Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp
1 5 10 15
Tyr Gln
<210> 43
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 43
Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln Leu His Trp Thr
1 5 10 15
Ser Thr
<210> 44
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 44
Val Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly
1 5 10 15
Thr Asn
<210> 45
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 45
Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys
1 5 10 15
Trp Thr
<210> 46
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 46
Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser Ser
1 5 10 15
Glu Arg
<210> 47
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 47
Thr Lys Asp Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp
1 5 10 15
Trp Glu
<210> 48
<211> 18
<212> PRT
<213> Staphylococcus aureus (Staphylococcus aureus)
<400> 48
Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys Glu Glu Met
1 5 10 15
Thr Asn
Claims (13)
1. An alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection is characterized by comprising a polypeptide with an amino acid sequence of SEQ ID NO. 1, wherein the epitope peptide is the polypeptide with the amino acid sequence of SEQ ID NO. 29 or SEQ ID NO. 30.
2. An α -hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection, wherein a polypeptide label is coupled to the N-terminus or C-terminus of the epitope peptide according to claim 1.
3. The α hemolysin epitope peptide according to claim 2, wherein said polypeptide label is a biotin label or a fluorescent label.
4. A fusion protein for the preparation of antibodies against staphylococcus aureus, consisting of the alpha hemolysin epitope peptide of claim 1 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 multiple antigenic peptide.
5. Use of an α -hemolysin epitope peptide according to any one of claims 1-3 for the manufacture of a medicament for the diagnosis of a staphylococcus aureus infection.
6. A diagnostic reagent for staphylococcus aureus infection, further comprising the α hemolysin epitope peptide according to any one of claims 1 to 3.
7. The diagnostic reagent of claim 6, wherein the α -hemolysin epitope peptide is coated on a detection carrier selected from any one of a polystyrene microplate, a colloidal gold reagent strip, a magnetic bead, and a microfluidic chip.
8. The diagnostic reagent of claim 7, further comprising a second antibody that specifically recognizes a human IgG antibody.
9. The diagnostic reagent of claim 8, wherein the second antibody is selected from the group consisting 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, and goat anti-human polyclonal antibody.
10. The diagnostic reagent of claim 8, wherein the second antibody is coupled to a coordinating group that activates or quenches a specific fluorophore.
11. The use of an α -hemolysin epitope peptide according to claim 1, which is a polypeptide having an amino acid sequence of SEQ ID NO:30, in the manufacture of a medicament for the prevention and/or treatment of a staphylococcus aureus infection.
12. A medicament for preventing or treating staphylococcus aureus infection, comprising the alpha hemolysin epitope peptide of claim 1, which is a polypeptide having an amino acid sequence of SEQ ID NO:30, and a pharmaceutically acceptable excipient.
13. The medicament of claim 12, further comprising a pharmaceutically acceptable adjuvant, wherein the adjuvant is Quil-a adjuvant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110163653.1A CN112851770B (en) | 2021-02-05 | 2021-02-05 | Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110163653.1A CN112851770B (en) | 2021-02-05 | 2021-02-05 | Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112851770A CN112851770A (en) | 2021-05-28 |
| CN112851770B true CN112851770B (en) | 2022-10-04 |
Family
ID=75989294
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110163653.1A Active CN112851770B (en) | 2021-02-05 | 2021-02-05 | Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112851770B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114685628B (en) * | 2022-03-24 | 2023-06-06 | 中国人民解放军陆军军医大学 | Antigen epitope peptide of RBD of SARS-CoV-2 and its application |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9181329B2 (en) * | 2007-08-31 | 2015-11-10 | The University Of Chicago | Methods and compositions related to immunizing against Staphylococcal lung diseases and conditions |
| WO2009029831A1 (en) * | 2007-08-31 | 2009-03-05 | University Of Chicago | Methods and compositions related to immunizing against staphylococcal lung diseases and conditions |
| BRPI1013780B8 (en) * | 2009-04-14 | 2022-10-04 | Novartis Ag | IMMUNOGENIC COMPOSITION USEFUL FOR IMMUNIZATION AGAINST STAPHYLOCOCCUS AUREUS, ITS PREPARATION METHOD AND PHARMACEUTICAL COMPOSITION |
| ES2684088T3 (en) * | 2011-06-19 | 2018-10-01 | New York University | Methods of treatment and prevention of staphylococcus aureus infections and associated conditions |
| EP2771349B1 (en) * | 2011-09-16 | 2020-02-26 | Iogenetics, LLC. | Bioinformatic processes for determination of peptide binding |
| CN102977214B (en) * | 2012-09-29 | 2014-03-12 | 重庆原伦生物科技有限公司 | Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof |
| GB201219420D0 (en) * | 2012-10-29 | 2012-12-12 | Novartis Ag | Staphyloccal immunogens |
| CA3061709A1 (en) * | 2017-03-28 | 2018-10-04 | The Children's Medical Center Corporation | A multiple antigen presenting system (maps)-based staphylococcus aureus vaccine, immunogenic composition, and uses thereof |
-
2021
- 2021-02-05 CN CN202110163653.1A patent/CN112851770B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112851770A (en) | 2021-05-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2016201992B2 (en) | Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use | |
| JP2012515551A5 (en) | ||
| Li et al. | The non-conserved region of MRP is involved in the virulence of Streptococcus suis serotype 2 | |
| WO2007145760A2 (en) | Anthrax compositions and methods of use and production | |
| CN112851770B (en) | Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof | |
| US7169393B2 (en) | Antigenic peptide fragments of VapA protein, and uses thereof | |
| EP2262828B1 (en) | Compositions, methods and kits | |
| Li et al. | Characterization of novel Omp31 antigenic epitopes of Brucella melitensis by monoclonal antibodies | |
| CN113462675B (en) | ApuA protein antigen polypeptide and application thereof | |
| CN112920259A (en) | IsdB epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof | |
| CN110540599B (en) | Klebsiella pneumoniae Elisa detection kit based on Klebsiella pneumoniae surface protein antibody and preparation method thereof | |
| US20090162369A1 (en) | Synthetic chimeric peptides | |
| AU2010234193B2 (en) | Methods and compositions for treating and preventing Shiga toxin-producing Escherichia coli infection | |
| CN111763263A (en) | Preparation method and detection kit of clostridium difficile antigen and polyclonal antibody | |
| Ma et al. | A novel monoclonal antibody against FbaA can inhibit the binding of the complement regulatory protein factor H to group A streptococcus | |
| JP2015509083A (en) | Pseudomonas aeruginosa OprM epitope used for diagnosis and therapy | |
| CN111378015B (en) | Synthetic peptide for detecting S.suis 2 infection and application thereof | |
| CN115093468B (en) | Helicobacter pylori specific antigenic peptide | |
| CN115073566B (en) | Helicobacter pylori specific immunogenic peptide fragments | |
| US20220221455A1 (en) | Antigen binding proteins and assays | |
| Ding et al. | Immunogenicity of a divalent group A streptococcal vaccine | |
| AU2001252038B2 (en) | Antigenic peptide fragments of vapa protein, and uses thereof | |
| Chang et al. | Identification of B Cell Epitopes in the MSG1 Protein of Mycoplasma suis | |
| AU2010229498B2 (en) | Polypeptides and immunizing compositions containing gram positive polypeptides and methods of use | |
| CN115925906A (en) | Monoclonal antibody for resisting novel coronavirus nucleoprotein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |