阅读说明：本技术 一种用于诊断或防治金黄色葡萄球菌感染的α溶血素抗原表位肽及其应用 (Alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection and application thereof ) 是由 赵�卓 章金勇 曾浩 邹全明 刘智勇 宋旭 陈致富 段连礼 苟强 熊青山 于 2021-02-05 设计创作，主要内容包括：本发明公开了本发明提供了一种用于诊断或防治金黄色葡萄球菌感染的抗原表位肽,包含氨基酸序列为SEQ ID NO:1的多肽,本发明还提供了该抗原表位肽在制备用于诊断、预防或治疗金黄色葡萄球菌感染中的应用。本发明通过筛选确定了金黄色葡萄球菌溶血素α的抗体优势表位肽,具有较强的免疫原性,同时该优势表位肽的氨基酸序列在多种S.aureus菌株中序列保守,因此,也可用于制备金黄色葡萄球菌感染的诊断试剂,或用于其他S.aureus感染的预防或治疗。(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.)

1. An alpha hemolysin epitope peptide for diagnosing or preventing staphylococcus aureus infection, which is characterized by comprising a polypeptide with an amino acid sequence of SEQ ID NO. 1.

2. The alpha hemolysin epitope peptide according to claim 1, wherein said epitope peptide is a polypeptide having the amino acid sequence of SEQ ID NO. 29 or SEQ ID NO. 30.

3. The α hemolysin epitope peptide according to claim 1 or 2, further comprising a polypeptide tag conjugated to the N-terminus or C-terminus of said polypeptide; preferably, the polypeptide label is a biotin label or a fluorescent label.

4. A fusion protein for use in the preparation of an antibody against staphylococcus aureus, comprising an α -hemolysin epitope peptide of 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 multiple antigenic peptides.

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, prevention or treatment of a staphylococcus aureus infection.

6. A diagnostic reagent for staphylococcus aureus infection comprising the α hemolysin epitope peptide according to any one of claims 1 to 4.

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;

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.

9. Use of an α -hemolysin epitope peptide according to claim 1 or 2 for the manufacture of a medicament for the prevention and/or treatment of a staphylococcus aureus infection.

10. A medicament for preventing or treating staphylococcus aureus infection, comprising the α -hemolysin epitope peptide of claim 1 or 2 and a pharmaceutically acceptable excipient.

11. The medicament of claim 10, further comprising a pharmaceutically acceptable adjuvant.

12. The medicament of claim 11, wherein the adjuvant is Quil-a adjuvant.

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 that occur widely in nature and is also an important pathogenic bacterium causing nosocomial 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 this proportion accounts for 80% in hospitals. Of particular note are methicillin-resistant staphylococcus aureus strains (MRSA), which in 2009 account for 25-50% of clinical staphylococcus aureus strains in 9 european countries. 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. Bacteremia caused by MRSA invasive infection has a mortality rate of more than 50% in 90 days. Currently, the main clinical antibiotic treatment is not capable of controlling MRSA infection, and vaccine development is imminent.

The pathogenicity of MRSA mainly depends on the abilities of toxin and invasive enzyme produced by MRSA, wherein Hemolysin alpha (alpha-haemolysin, Hla) is also an important exotoxin causing staphylococcus aureus infection, usually produced 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, because of the strong hemolytic activity of Hla, there may be safety issues as a vaccine component directly. 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_H35LNo other biological function changes were found except for the reduction of hemolytic activity.

In the vaccine response of MRSA, the antibody response plays a major protective role. Studies have demonstrated that humans who are unable 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, the 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 human immunodominant epitopes in clinical MRSA infection, either by bioinformatics software speculation, or by monoclonal antibody identification, or by use of human or animal immune models. 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 the Hla participating in immune response in 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 an amino acid sequence of 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 can be prepared into different dosage forms for administration by different routes, and preferably, in order to avoid the degradation of the antigen component in the vaccine, the vaccine is prepared into a dosage form for intravenous administration, for example, an injection.

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 invention has the beneficial effects that:

the antibody dominant epitope peptide of staphylococcus aureus hemolysin alpha provided by the invention can induce high-level dominant epitope antiserum in an animal body, 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 invention₁₆₂-179、Hla₁₆₈Active immunization of KLH fusion protein of-185 for reducing staphylococcus aureus in kidney and lung of miceDetecting the map of the planting ability;

FIG. 4 shows the antibody immunodominant epitope peptide Hla selected by the present invention₁₆₂-179、Hla₁₆₈-185 location distribution analysis result map in the Hla three-dimensional structure;

FIG. 5 shows the antibody immunodominant epitope peptide Hla selected by the present invention₁₆₂-179、Hla₁₆₈-185 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 was 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 15 mu g/well (taking holoprotein Hla (Sequence ID: ADQ77533.1) as a positive control), coating, washing, sealing, washing 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 Enjingyo organisms, product number E1WP319), diluting at 1: 3500, washing, adding TMB substrate developing solution (purchased from Beyotime/Biyun day, product number P0209-100ml), reading the OD value at 450nm after terminating the reaction, and taking the amino acid overlapping peptide OD-blank control detection value)/(the anionic peptide OD detection value-blank control detection value) as the formula 18 as the positive overlapping peptide, wherein the Hla immune antiserum obtained in example 2 is added, the dilution is 1: 300, and the washing is carried out for 1.5 h. 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), statistically significant for other dominant epitope peptide reads, defined as immunodominant epitopes, in which OVA₁₉₂Negative 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 B cell immunodominant epitope peptide of Hla identified in example 3 was coupled to KLH (hemocyanin) at equal concentrations (manufactured by GmbH, Shanghai Jier Biochemical Co., Ltd.) to obtain a fusion protein, the epitope peptide-KLH fusion protein was administered at 60. mu.g/mouse, the dosage of Quil-A adjuvant (purchased from InvivoGen) was administered at 10 ug/mouse, after mixing, the mice were immunized by injecting the thigh of BALB/c mice (10 mice, 6-8 weeks old) for 0, 14, 21 days, after the last immunization for 7 days, the mouse eye blood was taken, antiserum was isolated, the antiserum titer was measured, and the mice were frozen 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, coating, washing, sealing, washing again, adding antiserum with the dilution degree of 1: 50, 1: 100, 1: 200, 1: 400, 1: 800, 1: 1600, 1: 3200, 1: 6400, 1: 12800, 1: 25600 and 1: 51200 (normal mouse serum is used as negative control, PBS is blank control), incubating for 1.5h, washing, adding HRP-goat anti-mouse IgG (purchased from Enjingzi organism, product number E1WP319) with the dilution degree of 1: 3500, adding TMB substrate developing solution purchased from Beyotime/Biyun day, product number P0209-100ml after washing, 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 dominant epitope peptide from KLH immunized mice is 1: 22400, Anti-Hla_168-185-the mean antiserum titers of dominant epitope peptides of KLH immunized mice were 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 by intramuscular injection of BALB/c mice for 6-8 weeks three times on days 0, 14, 21 with the aid of Qui-A adjuvant (purchased from Inc.) and the sublethal dose of MRSA252 standard strain (purchased from ATCC American type culture Strain resource Bank, 6X 10, 7 days after the last immunization) was injected into tail vein⁸CFU/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 is set. Detecting the colonization amount of staphylococcus aureus: the BALB/c mice were sacrificed by cervical dislocation, sterilized with 75% alcohol, aseptically weighed tissue specimens, placed in 2ml of sterile PBS, homogenized in a clean glass homogenizer, diluted with 1ml of homogenate at a ratio 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 subjected to colony counting (CFU/ml), and parallel gram staining typical under-mirror morphology and PCR detection to confirm that Staphylococcus aureus.

The results are shown in FIG. 3, which shows: hla in contrast to PBS group_168-185Active 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_162-179Active KLH immunization did not achieve significant effect in eliminating visceral bacteria. Confirmation, Hla_168-185Is a protective immunodominant epitope against infection by the standard strain of MRSA 252.

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 reported 3D structure diagram of the 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_168-185The 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_168-185The result of amino acid sequence conservation analysis of (1).

The amino acid sequence of the Hla protein of 36 s.aureus strains was 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_168-185The 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 may be made by those skilled in the art within the spirit and principles of the invention. The scope of the invention is to be determined by the appended claims.

Sequence listing

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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