KR100868248B1 - Novel peptide probes for diagnosing anthrax infection - Google Patents

Abstract

The present invention relates to a method for screening a peptide as an anthrax infection diagnostic probe, and to a peptide obtained therefrom. More specifically, the present invention is a method for screening peptides (PA-binding peptides) that specifically binds to an anthrax protective antigen (PA) protein having high specificity and affinity using phage display And a peptide as an anthrax infection diagnostic probe discovered therethrough.

According to the present invention, a screening method that can be differentiated from a conventional diagnosis method using an antibody or an aptamer, as well as linking with various nanobiosensors using a physicochemical stability of a peptide, and a novel anthrax infection Diagnostic probes may be provided.

Anthrax, protective antigen, peptide, phage display

Description

Novel peptide probes for diagnosing anthrax infection

Figure 1 (a) is an anthracite antigen protein produced by genetic recombination in Escherichia coli for screening peptides binding to the PA protein using a 6- histidine tag of the carboxy terminus of the recombinant protein in a 96-well plate bound to Ni It is a figure showing the state bonded to the plate bottom directionally.

In addition, Figure 1 (b) is a graph showing the results of ELISA experiments using a PA recognition antibody to confirm the binding, it was confirmed that the PA binding.

2 is a schematic of M13 phage screening for screening peptides that bind to PA.

FIG. 3 is a table summarizing five-step biopanning conditions for detecting phage expressing peptides having high specificity and binding ability to PA.

Figure 4 is amplification of phage binding to PA obtained through the five-step biopanning of Figure 3 to perform DNA sequencing after separation and purification by comparing the amino acid sequence of the peptide expressed on the phage surface and based on homology Figures showing the results of creating the pseudo-system diagram.

FIG. 5 is a diagram illustrating sequencing of a specific one group out of 19 groups classified as a result of homology analysis.

Figure 6 is a graph analyzing the binding force and PA by amplifying the representative peptide expression phage of 19 groups and then measuring the concentration.

7 is a schematic diagram for selecting binding sites of peptides that bind to PA.

FIG. 8 is a diagram showing the result of confirming the binding peptide by ELISA, leaving PA63 on the surface of the plate according to FIG.

9 is a view summarizing the binding position, peptide amino acid sequence and binding force of the peptide that binds to anthrax defense antigen (PA) obtained through the present invention.

The present invention relates to a method for screening a peptide as an anthrax infection diagnostic probe, and to a peptide obtained therefrom. More specifically, the present invention relates to a method for screening a peptide that specifically binds to anthrocyte defense antigen protein having high specificity and high affinity using phage display, and a peptide as an anthrax infection diagnostic probe discovered through the same.

In general, anthrax is a Gram-positive bacillus, Bacillus anthracis ) is a common infectious disease that is primarily infected by livestock but also by humans.

The anthrax has been developed as a biological weapon for a long time, and has recently been deployed in practice, and has been a major concern for the general public following the invasion of anthrax vaccine in the Gulf War in 1990 and the US military stationed in 1998.

Anthrax caused by anthrax infections is known to have a very high mortality rate. There are three approaches for treatment, like other bacterial infections.

The first is vaccination to prevent anthrax infections, the second is to neutralize the infected anthrax with antibiotics, and the third is to detox the major disease toxins secreted by the infected anthrax.

Once infected, antibiotics and antitoxins are best used to neutralize germs and toxins at the same time, but currently the development of antidotes is less than that of antibiotics. In Korea, this is classified as a staggering epidemic, and in Korea, there have been reports of the death of livestock and lung meat. The need for control of anthrax has emerged.

Anthrax bacteria Killing antibiotics have already been developed, but the drug has the disadvantage of having no effect on the already produced anthrax toxin. Have. In addition, anthrax vaccines are difficult to use extensively because of side effects. Moreover, anthrax with a high mortality rate is found at an early stage of disease development and public treatment is very important. In addition, a thorough quarantine system for livestock imports is urgently needed, since more than half of domestic meat consumption is a US-dependent import route with a high risk of biological terrorism. However, in the early and advanced stages of livestock products without disease symptoms, the concentration of disease-causing bacteria is very low, making it difficult to diagnose and large amount of quarantine.

The pathogenicity of anthrax is known to be determined by toxin production and capsular formation.

Proteins that affect the toxin-producing ability of anthrax are protective antigen (PA) and edema factor (EF). It is known to be a lethal factor (LF), and research on them is ongoing. Each of them is not toxic, but defense antigen (PA) and edema factor (EF) combine to form edema toxin (EdTx, edema toxin), and defense antigen (PA) and lethal factor (LF) bind to lethal toxin (LeTx, ethal toxin).

Here, PA binds to receptor proteins (ATRs) on the surface of infected cell lines, such as macrophages, and is then cut into PA20 and PA63 by furin family proteases, of which PA63 binds to LF or EF as heptamers It has been widely reported to play the role of a toxin delivery channel to transport into.

These complexes enter into cells through the endocytosis mechanism, and when the pH of the endosomes decreases, structural changes of PA63 heptamers occur, forming pores in the endosomal membrane, and as a result, LF or EF is released into the cytoplasm and has a toxic effect. Therefore, PA is an interesting target for the prevention and treatment of anthrax. Thus, various approaches to inhibiting anthrax toxin are under study by the PA.

However, no diagnostic probes with high sensitivity, rapidity, field adaptability, or diagnostic probes with high physicochemical stability, sensitivity, specificity, and applicability have been proposed.

Meanwhile, the most common method for diagnosing a diagnosis subject is PCR (Polymerase Chain Reaction), which analyzes a unique gene of the subject and then in vitro ( in in vitro ) to analyze small amounts of diagnostic subjects.

In addition, by using the unique nucleotide sequence of the gene to target the unique nucleotide sequence of the diagnosis target only, and the scope of use can be expanded by securing a comprehensive genetic information database through the recent human genome project.

It is also useful in conjunction with SSCP (Single-strand conformation polymorphism), RFLP (Restriction fragment length polymorphism), and AFLP (Amplified fragment length polymorphism) methods to identify genetic variants of amplified genes with various probes. Information is provided.

The diagnostic method using the PCR has the advantage of having a high sensitivity in obtaining a small amount of unique nucleotide sequence only for the diagnosis subject, but problems such as difficulty in sample preparation and in-field adaptation have limitations as a diagnostic system. .

Furthermore, in the diagnosis of human disease, not the diagnosis of external factors such as pathogens and viruses, the importance of protein as a diagnostic subject is highlighted in view of the fact that the change in protein level is greater than the chromosomal modification of living organisms. Since the method is not suitable, it is necessary to combine it with related research that can overcome it.

In this regard, the recent Enzyme-Linked Immunosorption Assay (ELISA) method determines the increase and change in the expression level of a specific protein caused by disease. For the recognition of these specific proteins, antibodies that bind specifically to them are used as diagnostic probes to isolate diagnostic subjects from biological samples such as blood and urine, and to use enzyme-antibody reactions (ECLs).

However, despite the merits of targeting nanomolecular reactions in vivo, many related researches are underway to overcome this problem because they have lower sensitivity than PCR diagnostics.

Recently, surface-enhanced Raman spectroscopy (SERS) and bio-barcode techniques have been developed based on the current diagnostic methods (PCR, ELISA), demonstrating that femtomol levels can be analyzed experimentally. .

Both methods have femto signal amplification using metal nanoparticles with the signal characteristics of Raman spectroscopy (narrow Raman band; low sensitivity to moisture, oxygen and soak; low photobleaching) and high sensitivity corresponding to fluorescence analysis. By recognizing the mole (10 ^-15 M) level of the target object, an approach for prostate specific antigen (PSA) using easy specimens such as blood was proposed.

Existing methods are based on a system using antibodies that recognize biomarkers (diagnostics), which suggest that the physical and chemical structure of the antibody is limited in the number of bonds due to size limitations and instability due to structural denaturation under various chemical reactions. By showing, there is a limit to the integration with modern NT technology. Therefore, the development of small molecular probes having specificity and binding ability of antibodies is expected to be the basis of the next generation diagnostic material that overcomes these limitations.

It is well known that nucleotide eptamers have recently been studied as such antibody replacement diagnostic probes. The development of new low molecular diagnostic probes, which is differentiated from the existing diagnostic system technology using antibodies and epsilers, is not only a technical foundation for the development of the next generation of altitude systems but also very important in preempting new technologies.

Therefore, the present inventors have identified a peptide as a new diagnostic material that can replace the antibody currently used for diagnosing anthrax, and have come to propose it as a new detection material.

That is, an object of the present invention is to provide a method for screening a peptide that recognizes and specifically binds anthrax toxin protein for diagnosing anthrax infection.

Another object of the present invention is to provide a peptide discovered by the method as a use of anthrax infection small molecule diagnostic probe.

In order to achieve the above object, the present invention is to select a peptide having a binding capacity corresponding to the antigen-antibody reaction in the diagnosis target toxin transport protein exhibiting anthrax toxicity, and then the binding region of the action of the PA protein Distinguish by site (PA20 and PA63).

In other words, phage display library screening is performed to screen peptides that bind to PA and then analyze their binding regions and binding forces to confirm their performance as probes, and phage can be used with bacteriophages such as phage λ or M13, which are commonly available. Among them, it is more preferable to use M13.

As a result, eight types of low molecular peptides could be identified as anthrax infection diagnostic probes. The amino acid sequences shown below are from the amino terminus, where L is leucine, S is serine, T is threonine, A is alanine, H is histidine, P is proline, F is phenylalanine, M is methionine, R is arginine, N is Asparagine, Y is tyrosine, V is valine, Q is glutamine, I is isoleucine, G is glycine, D is aspartic acid, K is lysine, W is tryptic acid, X is other unknown amino acids.

Specific amino acid sequences are as follows:

P1: LSHNQTIQQDSD

At this time, the second amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is characterized in that consisting of the amino acid substituted by T.

In addition, the fifth amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is characterized in that consisting of the amino acid substituted by L.

In addition, the seventh amino acid I of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is characterized in that consisting of the amino acid substituted by L.

In addition, the tenth amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is characterized in that consisting of the amino acid substituted by A.

Further, the second amino acid S in the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is T and / or the fifth amino acid Q is L and / or the seventh amino acid I is L and / or D is the tenth amino acid. Includes those consisting of amino acids substituted with A.

P2: HKHAHNYRLPXS

A seventh amino acid of the amino acid sequence HKHAHNYRLPXS represented by SEQ ID NO: 2 is characterized by consisting of an amino acid substituted by T.

P3: TPYYWHHHHIPP

P4: QSPVNHHYHYHI

P5: GHKPQVHRHTHI

At this time, the first amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized in that consisting of the amino acid substituted by A.

In addition, the second amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized in that consisting of the amino acid substituted by L.

Further, the twelfth amino acid I of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized by consisting of an amino acid substituted by L.

P6: LMPTPHHRLFPM

P7: ALHPHRTPHHHH

P8: NAYKHHHPPVFY

In addition, four of the above-mentioned eight peptides P1, P5, P7, P8 was confirmed that the binding to PA20, the remaining four species of P2, P3, P4, P6 was confirmed that the binding to PA63 ( 9).

Since such peptides are composed of amino acids, such as antibodies, unlike epsamers, they can exhibit the specificity of various combinations. In addition, the high physical and chemical stability due to the simple three-dimensional structure is expected to have great advantages in connection with various nano sensor chips and development of excellent kits in the field.

Hereinafter, one or more exemplary embodiments will be described in detail in order to show the structure and effects of the present invention.

<Example>

Example  One: PA  Mass production and isolation of proteins

After genomic DNA of Bacillus anthracis sterne is isolated, PA gene is amplified by PCR and inserted into pET22b, a bacterial expression vector, which is produced to suppress protein disappearance during mass expression of PA in bacteria. In order to transpose the PA into the periplasmic region of the bacterium, the PelB gene was inserted as a leader sequence at the amino terminal.

The cloned PA expression plasmid was transformed into BL21 (DE3), an expression host, to induce PA production under conditions of 0.5 mM IPTG and 18 ° C. And bacterial cells were harvested by centrifugation to examine the solubility of the protein was confirmed that most of the protein is insoluble.

Therefore, in order to separate and purify the PA, only the insoluble protein in the cell was obtained and dissolved in denaturing conditions (6M guanidine-HCl), and passed through a Ni-Sepharose column to bind the PA protein, followed by refolding the denatured PA. The PA was refolded and eluted under a 500 mM imidazole linear gradient while slowly denatured through a linear gradient (reduced concentration of urea) while bound to the column.

The obtained protein was dialyzed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4) solution to remove excess imidazole, and the degree of purification of the obtained protein was determined by SDS. It was confirmed by / PAGE and 3 mg / L protein was obtained by using the Bradford reaction solution to confirm the amount.

Then, trypsin treatment was performed to check whether the PA protein was folded back. In the case of (-) PA, trypsin treatment resulted in two specific fragments of 20 kDa and 63 kDa. Denatured by treating the PA protein purified by the above method with trypsin to see if the same result is obtained. After purification, it was checked whether the structure of the protein was normally refolded.

Trypsin treatment resulted in two protein fragments, PA20 and PA63.

Example  2: M13  scrap paper Peptide  Library Screening-Phage Display

First, in order to fix a recombinant protein to a 96-well plate, a Ni-Sorb plate in which Ni ions are fixed by coordination bonds is cloned so that it can be expressed with 6 histidine tags bound to the end of the PA gene carboxy. Using (Qiagen) to fix the PA protein with specific orientation by binding the carboxy terminus toward the bottom of the plate, facilitating higher specificity and binding site analysis when screening with M13 phage random peptide library (12 amino acids) Designed to be. The binding was analyzed in FIG. 1.

Specifically, in Figure 1 (a) to screen the peptides that bind to the PA protein, anthrax defense antigen protein produced by genetic recombination in Escherichia coli in the carboxy terminus of the recombinant protein in a Ni-coupled 96 well plate (Qiagen) 6 histidine tag was used to directionally bind to the bottom plate.

As a result of ELISA experiment using PA recognition antibody to confirm the binding, as shown in FIG.

The isolated and purified PA protein was bound to the surface of the plate, and then incorporated into the M13 phage peptide library (a library consisting of 12 amino acids having about 2.7 billion different amino acid sequences fused to M13 phage g3 minor coat protein). Peptide expression phages were then selected that bind with high affinity through various binding times and washing conditions. Screening schemes and respective reaction conditions are shown in FIGS. 2 and 3, respectively.

 Specifically, FIG. 2 is a schematic diagram of M13 phage screening for peptide binding to PA. The phage library expressing a peptide library consisting of 12 amino acids on the M13 phage surface was expressed in a PA immobilized on a 96 well plate surface. Put and washed under various conditions to obtain the bound phage eluted.

The resulting phages were infected with E. coli, amplified and combined with PA again, and the process was repeated to find strong binding phages under conditions such as higher washing conditions and shorter reaction times (biopanning).

In addition, FIG. 3 is a table of five-step biopanning conditions for detecting phages expressing peptides having high specificity and binding ability to PA. Each step was changed to stronger washing conditions and shorter binding time. Differently, biopanning was performed five times to obtain phage having peptides that bind to PA.

After amplifying the phages thus obtained, 80 phage plaques were selected, M13 phage genomic DNA (single stranded circular DNA) was isolated and purified, the gene sequence was confirmed and expressed on the phage surface protein (gp3 minor coat protein). The amino acid displayed peptide amino acid sequence of the peptide bound to PA was deduced.

Through this Clustal X sequencing program, a similar system based on mutual homology was created and all 19 groups were arranged and the results are summarized in FIG. 4.

In addition, as shown in FIG. 5 showing the sequence analysis of a specific one group among 19 groups classified as a result of amino acid homology analysis of each group of peptides, high similarity can be observed in the sequences of peptides belonging to the same group. The colored amino acids were found to be conserved in all peptides in the same group. This suggests that these amino acids PA correspond to residues that play an important role in the binding of the peptides.

Example  3: PA Combined with Peptide  Binding force analysis

For analyzing the binding capacity of the screened peptides, amplify the phage expressing each peptide, determine the concentration of phage solution through titering, and put the concentration into the PA-bound plate by measuring the amount of bound phage. Was deduced.

Phage was put on a plate to which PA was bound, followed by washing to perform ELISA using an antibody that recognizes phage surface protein. The ELISA signal intensity according to the concentration was indicated and the apparent Kd value was determined according to the saturation curve.

As a result, as shown in FIG. 6, 11 peptides among 19 peptide groups showed a picomolar binding capacity, and some peptides showed excellent binding strength of 20 pM or less, corresponding to the binding capacity of the existing antibody-antigen response. The binding force could be confirmed.

Example  4: PA Combined with Peptide  Combined Area Analysis

In the role of toxin-carrying protein for showing anthrax toxicity, PA must be converted to the amino terminal region (PA20) and carboxy terminal (PA63) by proteolytic enzymes after binding to the cell line of infection. It is known that it can be done.

Therefore, in the present invention, by performing the binding region analysis according to the respective sites of the identified PA binding peptide based on the plan shown in Figure 7 to build a data that can be used for various purposes.

Specifically, in the case of (-) PA, it is known that two specific fragments of 20 kDa and 63 kDa are generated when trypsin treatment is performed. After fixing the purified PA protein as shown in FIG. Trypsin treatment and washing to remove the PA20 to determine the binding by inserting phages expressed in 19 groups of peptides in the state fixed only to the PA63 protein on the plate. In particular, as shown in FIG. 8, the peptide was bound to the PA63 region by using the ELISA described in FIG. 6 as a result of confirming the peptide binding to 19 groups of peptides while leaving PA63 on the plate surface as shown in FIG. 8. Can be deduced by binding to the PA20 region.

9 shows information about amino acid sequences and binding sites of eight peptides having excellent binding strengths in the range of ^picomolar (10-12 M) among the peptides binding to PA proteins obtained through the present invention.

Specifically, Figure 9 is a view of the binding position and binding capacity of the peptides that bind to the anthrax defense antigen (PA) obtained through the present invention, in the present invention using the M13 phage peptide is expressed with a surface protein binding to PA Their binding force was measured as shown in FIG. 6, and they were divided into PA20 and PA63 regions as shown in FIG. 7.

The amino acid sequence of the binding peptide was shown by analyzing the gene of the peptide expression phage. As a result, the amino acid sequence listing of the corresponding eight peptides is as follows:

P1: LSHNQTIQQDSD

At this time, the second amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 includes an amino acid substituted with T.

In addition, the fifth amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 includes Q is composed of amino acids substituted with L.

In addition, the seventh amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 includes the amino acid substituted with L.

In addition, the 10th amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 includes D is an amino acid substituted with A.

Further, the second amino acid S in the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is T and / or the fifth amino acid Q is L and / or the seventh amino acid I is L and / or D is the tenth amino acid. Includes those consisting of amino acids substituted with A.

P2: HKHAHNYRLPXS

A seventh amino acid of the amino acid sequence HKHAHNYRLPXS represented by SEQ ID NO: 2 is characterized by consisting of an amino acid substituted by T.

P3: TPYYWHHHHIPP

P4: QSPVNHHYHYHI

P5: GHKPQVHRHTHI

At this time, the first amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized in that consisting of the amino acid substituted by A.

In addition, the second amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized in that consisting of the amino acid substituted by L.

Furthermore, the twelfth amino acid of amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 is characterized by consisting of amino acids substituted with L.

P6: LMPTPHHRLFPM

P7: ALHPHRTPHHHH

P8: NAYKHHHPPVFY

In addition, four of the above-mentioned eight peptides P1, P5, P7, P8 was confirmed that the binding to PA20, the remaining four species of P2, P3, P4, P6 was confirmed that the binding to PA63 ( 9).

As a result, the anthrax-antigen protein produced in large quantities using genetic recombination was bound to a 96-well plate in which Ni ions were coordinated to the plate surface using 6 histidine tags at the carboxy terminus of the protein, followed by a random peptide library ( Peptides specifically binding to anthrax defense antigen were identified using M13 expressed on the phage surface.

This can be expected to be differentiated from the existing diagnostic methods using antibodies or epsamers, and can be linked to various nanobio sensors using the physical and chemical stability of peptides.

According to the present invention, as an anthrax diagnostic probe, peptides that specifically bind to anthrax toxin protein PA were identified and their data were obtained by analyzing their amino acid sequence and binding properties.

The peptide probe is composed of amino acids such as antibodies, unlike nucleotide aptamers, and may be of great help in the study of various signal amplification methods due to its high physical and chemical stability due to its simple three-dimensional structure. It is expected.

It is expected that the early diagnosis and monitoring system of diseases will be established through the establishment of diagnostic methods with higher sensitivity, which will create high expected effects on metabolic diseases, cancers and infectious diseases.

Furthermore, this technology is expected to contribute to securing national technological competitiveness and creating next-generation future business through the assetization of diagnostic technology.

<110> Industry-University Cooperation Foundation Hanyang University (UCU-HYU) <120> A method for screening peptides as a diagnostic probe for anthrax          infection and the resulting novel peptides specifically          recognizing anthrax protective antigen <130> DP20070022 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide for act as a diagnostic probe <400> 1 Leu Ser His Asn Gln Thr Ile Gln Gln Asp Ser Asp   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide to act as a diagnostic probe <400> 2 His Lys His Ala His Asn Tyr Arg Leu Pro Xaa Ser   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screended peptide to act as a diagnostic probe <400> 3 Thr Pro Tyr Tyr Trp His His His His Ile Pro Pro   1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide to act as a diagnostic probe <400> 4 Gln Ser Pro Val Asn His His Tyr His Tyr His Ile   1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide to act as a diagnostic probe <400> 5 Gly His Lys Pro Gln Val His Arg His Thr His Ile   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide to act as a diagnostic probe <400> 6 Leu Met Pro Thr Pro His His Arg Leu Phe Pro Met   1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide to act as a diagnostic probe <400> 7 Ala Leu His Pro His Arg Thr Pro His His His His   1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Screened peptide tp act as a diagnostic probe <400> 8 Asn Ala Tyr Lys His His His Pro Pro Val Phe Tyr   1 5 10  

Claims (21)

delete delete delete In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and functions as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide consists of amino acids represented by SEQ ID NO: 1. . The diagnostic peptide probe according to claim 4, wherein S, the second amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1, consists of an amino acid substituted with T. The diagnostic peptide probe of claim 4, wherein Q, which is the fifth amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1, consists of an amino acid substituted by L. The diagnostic peptide probe of claim 4, wherein I, the seventh amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1, consists of an amino acid substituted by L. 5. The diagnostic peptide probe of claim 4, wherein D, the tenth amino acid of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1, consists of an amino acid substituted by A. 6. The amino acid sequence of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is S is T, the fifth amino acid Q is L, the seventh amino acid I is L, and the tenth amino acid D is A. Diagnostic peptide probe, characterized in that consisting of amino acids substituted by. In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and functions as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide consists of amino acids represented by SEQ ID NO: 2. . The diagnostic peptide probe according to claim 10, wherein Y, the seventh amino acid of the amino acid sequence HKHAHNYRLPXS represented by SEQ ID NO: 2 consists of an amino acid substituted with T. In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and acts as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide consists of amino acids represented by SEQ ID NO: 3. . In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and acts as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide consists of amino acids represented by SEQ ID NO: 4. . In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and acts as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide is a diagnostic peptide probe comprising an amino acid represented by SEQ ID NO: 5 . The diagnostic peptide probe according to claim 14, wherein G, which is the first amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5, consists of an amino acid substituted with A. 15. The diagnostic peptide probe according to claim 14, wherein H, the second amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 consists of an amino acid substituted by L. The diagnostic peptide probe according to claim 14, wherein I, the twelfth amino acid of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 consists of an amino acid substituted by L. The amino acid sequence of the amino acid sequence GHKPQVHRHTHI represented by SEQ ID NO: 5 consists of amino acids in which G is A, H is L, L is 12, and I is L. Diagnostic Peptide Probes. In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and acts as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide is a diagnostic peptide probe comprising an amino acid represented by SEQ ID NO: 6 . In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and functions as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide is a diagnostic peptide probe comprising an amino acid represented by SEQ ID NO: 7. . In a diagnostic peptide probe that specifically binds to anthrax toxin protein protective antigen and acts as a low molecular picomol probe for diagnosing anthrax infection, the amino acid sequence of the peptide consists of amino acids represented by SEQ ID NO: 8. .

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