KR20090053228A - Novel anthrax diagnostic system using polyvalent functional peptide polymers - Google Patents

Abstract

The present invention relates to peptides and multiple recognition functional peptide polymers for diagnosing anthrax infection.

More specifically, the identification of peptides (PA-binding peptides) that specifically binds to anthrax protective antigen (hereinafter referred to as 'PA') protein low molecular weight diagnosis of anthrax infection, anthrax test method using the same and the same It relates to a diagnostic kit. In addition, the present invention relates to an anthrax infection polymer in which the peptide for recognition of a diagnosis of anthrax infection is bound to a polymer chain, and according to the present invention, a peptide of another recognition site is also bound to a diagnosis object by binding of one peptide. By having a cooperative characteristic (coativity) has a binding force of 100-1000 times higher than the binding to the target protein using the existing antibody, it can increase the sensitivity and selectivity for the diagnosis of anthrax infection.

Anthrax, protective antigen, polyvalent functional peptide polymer, polyvalent detection peptide

Description

Novel anthrax diagnostic system using polyvalent functional peptide polymers

The present invention relates to peptides and multiple recognition functional peptide polymers for diagnosing anthrax infection. More specifically, the present invention discovers peptides (PA-binding peptides) that specifically bind to anthrax defense antigen protein having high specificity and affinity using phage display through screening, and using the anthrax infection diagnostic peptide The present invention relates to the development of polyvalent detection peptide through polymer chain reaction.

Anthrax is an infectious disease caused by Bacillus anthracis, which is a gram positive, rod. Anthrax is a common infectious disease that is transmitted to humans by eating infected meat or processed products (Mock, M., and Fouet, A., Annu. Rev. Microbiol. 55: 647-671, 2001; Turnbull, PC, Curr. Top. Microbiol. Immunol. 271: 1-19, 2002). Anthrax infects and germinates animals with spores, which causes the host to die from septicemia due to spleen enlargement, swelling of the subcutaneous mucosa and bleeding (Moutrez, M., Rev. Physiol Biochem.Pharmacol. 152: 135-164, 2004).

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 fatalities in eating livestock and lung meat, and the need for 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. Anthrax vaccines are also 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.

Anthrax spores enter macrophages by phagocytosis of macrophage but are not destroyed by unknown mechanisms and enter the body and blood of the host via macrophages (Guidi-Rontani, C., Trends Microbiol. 10: 405-409, 2002).

Proteins expressed in two large plasmids pXO1 (181,654 bp, NC_001496) and pXO2 (96,231 bp, NC_002146) play an important role in the mechanism of infection. Plasmid Plasmid plasmid pXO1 carries the exotoxin gene (Smith, H., Int. J. Med. Microbiol. 291: 441-417, 2002), and the toxin is a protective antigen (PA), edema factor (EF) and lethal factor (LF). Edema toxin (ETx), composed of protective antigens and edema elements, causes edema when injected into experimental animals and, depending on the concentration, causes the host to die (Firoved, AM, et al., Am. J. Pathol) 167: 1309-1320, 2005). Lethal toxin (LeTx), consisting of protective antigens and lethal elements, kills experimental animals (Hanna, P.C., et al., Mol. Biol. Cell. 3: 1269-1277, 1992). Plasmid pXO2 expresses the capsule (Makino, S., et al., J. Bacteriol. 171: 722-730, 1989), and Sterne (1937) isolated strains from which plasmid pXO2 was removed, The possibility of a vaccine using live Sterne strains has been shown. These anthrax toxins play an important role in the mechanism of anthrax development (Pezard et al., Infect. Immun. 59: 3472-3477, 1991).

Anthrax toxin is synthesized while the strain grows into feeder cells and released outside the cell. Anthrax toxins are included in the bacterial "AB" toxin family, in which the A moiety, which is enzymatic in the cytoplasm, binds the cell and enters the cell by the B part action. Lethal and edema are included in part A and individual toxins are carried by part B of protective antigens (Mourez, M., Rev. Physiol Biochem. Pharmacol. 152: 135-164, 2004).

The protective antigen is a 735-amino acid long 83-kDa protein, which is the first step in anthrax toxin, which binds to cellular receptors. When the protective antigen binds to cellular receptors, the N-terminal region is cut by furin or a purine-like endoprotease (Gordon, VM, et al., Infect. Immun. 63). : 82-87, 1995). Purines show specificity for the R-X-K / R-R sequence at the N-terminal portion of the protective antigen. When the N-terminal 20 kDa fragment (PA20) is cleaved and released by purine, the remaining 63 kDa fragment (PA63) quickly forms a haptamer. When 20 kDa of the N-terminal remains, a haptam is formed. (Milne, JC, et al., J. Biol. Chem. 269: 20607-20612, 1994). The edema and lethal elements combine in PA63, which forms a haptam. N-terminal domains of edema and lethal elements bind to the protective antigen (Arora, N. and Leppla, S.H., J. Biol. Chem. 268: 3334-3341, 1993). Three molecules of lethal or edema per PA63 haptam bind, and they bind competitively to each other (Mogridge, J., et al., Biochemistry 41: 1079-1082, 2002).

PA63 haptammer, combined with lethal toxin and edema, enters the cell by receptor mediated endocytosis and forms an endosome. When the pH inside the endosome is lowered, the structure of the haptam is changed, and the edema and lethal elements move from the endosome to the cytoplasm (Beauregard, KE, et al., Cell Microbiol. 2: 251-). 258, 2000; Krantz, BA, et al., Science 309: 777-781, 2005). After being transported into cells, lethal toxins and edema elements show enzymatic activity. Edema causes calcium and calmodulin to act as adenylate cyclase, increasing the concentration of cyclic adenosine monophosphate (cAMP) inside the cell. The edema factor activity in microorganisms is more than 1,000 times higher than the adenylate cyclase activity activated by eukaryotic calmodulin, but is active only in the cytoplasm of eukaryotic cells because there is no calmodulin in microorganisms (Leppla, SH). Proc. Natl. Acad. Sci. USA 79: 3162-3166, 1982).

The lethal factor is zinc-dependent endoproteases (Klimpel, KR, et al., Mol. Microbiol. 13: 1093-1100, 1994; Kochi, SK, et al., FEMS Microbiol. Lett. 124: 343-348, 1994). The substrate of the lethal element was identified as mitogen-activated protein kinase kinases (MAPSKK) (Duesbery, NS, et al., Science 280: 734-737, 1998; Vitale, GR, et al., Biochem. Biophys.Res. Commun 248: 706-711, 1998), lethality cleaves all known MAPKKs (MAPKK1-MAPKK7) except MAPKK5 (Duesbery, NS, et al., Science 280: 734-737, 1998; Vitale, GR, et al., Biochem. Biophys. Res. Commun. 248: 706-711, 1998; Pellizzari, RC, FEBS Lett. 462: 199-204, 1999; Vitale, GL, et al., Biochem.J. 352: 739 -745, 2000). The cleavage site is an N-terminal proline-rich site, and when this site is cleaved, MAPKK cannot bind to MAPK, which is a substrate, and the activity of MAPKKs is reduced (Chopra, AP, et al., J. Biol. Chem. 278: 9402-9406, 2003). Cleavage of these MAPKKs inhibits the MAPK metabolic pathway (MAP kinase pathway), which inhibits phosphorylation of proteins such as p38, ERK, and JNK (Agrawal, A and Pulendran, B., Cell. Mol. Life Sci. 61: 2859- 2965, 2004).

Macrophages, like all infectious diseases, are important for anthrax infections. When anthrax spores enter the body by respiration, they are first infected with lung macrophages and dendritic cells (DCs) and germinate to produce nutrient cells (Friedlander, AM, Curr. Clin. Top. Infect. Dis. 20: 335-349, 2000). Anthrax germinates in macrophages followed by expression of toxin genes (Guidi-Rontani, C., et al., Cell Micriobiol. 2: 259-264, 2000). During infection, anthrax escapes the phagocytosis of macrophages, in which anthrax toxin plays an important role (Welkos, S., et al., Microbiology 147: 1677-1685, 2001).

Anthrax diagnosis is characterized by anthrax toxin production and pathogenicity in laboratory animals (Parry et al., Wolfe Medical Altas no.19, 1983), physiological and chemical properties (Little and Knudson, Infect. Immuno. 52: 509-512, 1986 ) And serological properties (Graham et al., Eur. J. Clin. Microbiol. 3: 210-212, 1984; Philips and Ezzell, J. appl. Bacteriol. 66: 419-432, 1989).

That is, as described above, the protective antigen (PA) binds to receptor protein (ATR) on the surface of an infected cell line such as macrophages and is then cut into PA20 and PA63 by furin family protease, of which PA63 is heptamerized. Toxin delivery channel that binds to LF or EF and transports it into cells, and these complexes enter into cells through the endocytosis mechanism and the structural changes of PA63 heptamers when the pH of the endosomes decreases. It forms 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. Therefore, various approaches to inhibiting anthrax toxin are under study in the PA.

However, no diagnostic probes with high sensitivity, rapidity, and 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, and further expand the scope of use by securing a comprehensive genetic information database through recent human genome projects.

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 diseases of humans rather than 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 that of 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 suggests that the physical and chemical structure of the antibody is limited by 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 diagnosis probes having specificity and binding ability of antibodies is expected to be the basis of the next generation diagnostic material that overcomes these limitations. In particular, it is well known that nucleotide eptamers in recent years have been studied as such antibody replacement diagnostic probes (Hong, J., J. Microbiol. Biotechnol. 14: 628-630, 2004).

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

According to the present invention, the diagnostic method using an antibody or an aptamer is not only differentiated, but the existing method is based on a recognition system using an antibody for a diagnosis object, and the site recognized by the antibody is the diagnosis object itself. The binding site is limited, and the antibody itself has a disulfide bridge structure, which limits the use of chemicals such as reducing agents. However, in the case of the peptide for recognition of a diagnosis subject according to the present invention, there is no such restriction, and as with the antibody, it is possible to secure a peptide that recognizes several parts of the diagnosis subject, and when the obtained peptide for recognition of the recognition subject is combined with a polymer chain type, By combining one peptide, peptides of other recognition sites also have a cooperative characteristic of binding to a diagnosis object, thereby obtaining 100-1000 times higher binding strength than that of a target protein using an existing antibody. have.

An object of the present invention is to provide a peptide for diagnosing anthrax infection, which peptide recognizes and specifically binds anthrax toxin protein for diagnosing anthrax infection.

In addition, an object of the present invention is to provide a new system for diagnosing anthrax infection by providing a polyvalent detection peptide through a polymer chain reaction using a multi-peptide for recognition of a target and a high-efficiency signal detection peptide.

In order to achieve the above object, the present invention provides an anthrax infection diagnostic low molecular peptide that can specifically bind anthrax toxin protein protective antigen and a diagnostic kit comprising the same.

In addition, the present invention has a property of having a cooperation (coativity) to bind the peptide for recognition of the anthrax infection to the diagnostic target by binding the peptide of the recognition target to the polymer chain by binding of one peptide. It provides an anthrax infection polymer having an avidity of about 100-1000 times higher than binding to a target protein using an antibody and an anthrax infection diagnostic kit comprising the same.

The present invention also provides an anthrax test method using the anthrax infection diagnosis peptide and anthrax infection diagnosis polymer.

Hereinafter, the present invention will be described in detail.

The present invention selects peptides having a binding capacity corresponding to antigen-antibody reactions based on existing toxin-carrying proteins exhibiting anthrax toxicity, and then distinguishes their binding regions according to functional sites (PA20 and PA63) of PA proteins. do.

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. In this case, phages such as phage λ or M13, which are commonly available, can be used. Among them, it is more preferable to use M13.

As a result, eight low molecular peptides could be identified as a probe used to diagnose anthrax infection. The amino acid sequence is as follows:

P1: L (S / T) HN (Q / L) T (I / L) QQ (D / A) SD (SEQ ID NO: 1-LSHNQTIQQDSD)

P2: HKHAHN (Y / T) RLPXS (SEQ ID NO: 2-HKHAHNYRLPXS)

P3: TPYYWHHHHIPP (SEQ ID NO: 3)

P4: QSPVNHHYHYHI (SEQ ID NO: 4)

P5: (G / A) (H / L) KPQVHRHTH (I / L) (SEQ ID NO: 5-GHKPQVHRHTHI)

P6: LMPTPHHRLFPM (SEQ ID NO: 6)

P7: ALHPHRTPHHHH (SEQ ID NO: 7)

P8: NAYKHHHPPVFY (SEQ ID NO: 8)

(The indicated amino acid sequence is shown from the amino terminus.

Amino acid notation: L (Leucine) S (Serine) T (Threonine) A (Alanine) H (Histidine) P (Proline) F (Phenylalanine) M (Methionine) R (Araginine) N (Asparagine) Y (Tyrosine) V (Valine ) Q (Glutamine) I (Isoleucine) G (Glycine) D (Aspartic acid) X (Any amino acid).

Amino acids in the (/) symbol indicate the same binding force and binding area)

In the present invention, a fluorescent probe selected from a FITC probe, a fluorescein derivative or a rhodamine derivative is linked to the small molecule peptide.

In addition, the present invention provides a peptide that specifically binds to anthrax toxin protein protective antigen; and a poly-peptide chain coupled to a chemical linker; cross-linking through a polymer chain substitution reaction. The present invention relates to a multi-recognition peptide and a signal detection peptide polymerized chain type anthrax infection diagnosis polymer, characterized in that the polymerized peptide is linked (linked).

In the present invention, a chemical linker for connecting a peptide and a signal label to an alternative polymer based on a poly-peptide chain, which is a biopolymer, is connected.

As the biopolymer type polymer, poly-lysine may be preferably used. Poly-lysine is an amino acid polymer with a positively charged side chain, which has a good solubility in water, making it easy to mix with diagnostic samples. By using the D-stereoisomer of poly-lysine, it is possible to block the cleavage by protease by differentiating it from the L-stereoisomer in the body. (Figure 1a)

Also preferably, SPDP (N-succinimidyl3- (2-pyridyldithio) propionate) having the chemical formula of FIG. 1B may be used as a chemical linker for linking peptides and signal labels. SPDP is a heterobifunctional molecular which has the aromatic of N-hydroxysuccinimide ester and 2-pyridyl disulphide, and is characterized by being substituted with primary amine group and sulfhydryl group.

In the present invention, the substitution reaction occurs with the amine group of the lysine side chain of poly-lysine and NHS (N-hydroxysuccinimide ester) of SPDP, so that the succinimide is separated and the linker and polymer are combined.

In linking peptides that specifically bind to anthrax toxin protein protective antigens, when synthesizing the peptides, a cysteine with a sulfhydryl group is attached to the pyridyldithio of SPDP and the SH group of Cys, resulting in cross-linking. Peptides are linked to the backbone (Figure 2).

In addition, the present invention preferably comprises at least two peptides each having a different sequence, specifically binding to anthrax toxin protein protective antigen; And a poly-peptide chain coupled with a chemical linker; two or more polymer based peptides cross-linked through a polymer chain substitution reaction. It further comprises a multi-recognition peptide and signal detection peptide polymerized chain anthrax infection diagnostic polymer.

Also preferably, the polymer of the present invention further includes that the peptide specifically binding to the anthrax toxin protein protective antigen is a peptide having an amino acid sequence identified through screening as described above. In addition, the peptide specifically binding to the anthrax toxin protein antigen, characterized in that the fluorescent probe selected from the FITC probe, fluorescein derivative or rhodamine derivative is connected, it may further include an anthrax infection diagnostic polymer.

The present invention also relates to anthrax test method using the anthrax infection diagnosis peptide or anthrax infection diagnosis polymer.

In addition, the present invention relates to an anthrax infection diagnosis kit comprising the above anthrax infection diagnosis peptide or anthrax infection diagnosis polymer.

As described above, by identifying a low molecular peptide specifically binding to an anthrax toxin protein PA, the anthrax diagnostic peptide comprising a fluorescent probe connected to the peptide and a diagnostic kit comprising the same will be provided. Can be.

In addition, the peptide probe is composed of amino acids, such as antibodies, unlike nucleotide epsamers, and has a specific combination of more diverse combinations and can be of great help in the study of various signal amplification methods with high physical and chemical stability due to a simple three-dimensional structure. It is expected to be.

In addition, when the peptide for recognition of the diagnosis of anthrax infection is coupled to the polymer chain, the peptide having the coordination of the other recognition site is also coupled to the diagnosis by the binding of one peptide. It is possible to provide anthrax infection polymer having an avidity of about 100-1000 times higher than binding with a target protein using an antibody and anthrax infection diagnostic kit including the same. In particular, according to the present invention, the polyvalent detection peptide through the polymer chain reaction using the multi-peptide for detecting the target object and the high-efficiency signal detection peptide may be applicable to a kit having excellent linkage with various nano-sensor chips and excellent field applicability.

In addition, it can be applied to metabolic diseases, cancer, infectious diseases, etc. by establishing a system for early diagnosis and monitoring of diseases through a diagnostic method having a higher sensitivity and selectivity presented in the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to help the understanding of the present invention but are not intended to limit the scope of the present invention.

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, and produced to suppress the protein disappearance that occurs during the 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, two fragments of 20 kDa and 63 kDa were generated when trypsin treatment was performed. Thus, the protein after denaturation purification was confirmed by treating PA protein purified by the above method with trypsin to see the same result. It was confirmed that the structure of the (refolding) was returned.

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

Example  2: Poly - lysine backbone Exploration and Synthesis of New Functional Polymers Using

First, we tried to connect chemical linker to connect peptide and signal label to alternative polymer based on poly-peptide chain.

First, poly-lysine was selected as a biopolymer. Amino acid polymer with side chain with poly-lysine positive charge. Solubility in water is very good and it is easy to mix with diagnostic samples. In addition, by using the D-stereoisomer of poly-lysine, the cleavage by proteolytic enzymes was blocked by the difference from the L-stereoisomer in the body (FIG. 1A).

In addition, SPDP (N-succinimidyl3- (2-pyridyldithio) propionate) was selected as a chemical linker for linking peptides and signal labels. SPDP is a heterobifunctional molecular having N-hydroxysuccinimide ester and aromatic of 2-pyridyl disulphide, and has a characteristic of being substituted with primary amine group and sulfhydryl group (FIG. 1B).

Using this feature, a substitution reaction occurs with the amine group of the lysine side chain of poly-lysine and NHS (N-hydroxysuccinimide ester) of SPDP. Peptides can then be linked. When synthesizing peptides, Cystein with sulfhydryl groups can be attached to the pyridyldithio of SPDP and SH group of Cys, resulting in cross-linking, resulting in the formation of a peptide to the polymer backbone. (FIG. 2).

Specifically, in FIG. 3, 1.1 pmol pLys and 2.7 mmol SPDP were reacted in 75 mM sodium acetate buffer (pH 8.5) in order to connect pLys and SPDP. After 1 hour, gel-filtration was carried out to bind pLys to SPDP. Isolate and purify. When pLys + SPDP is connected, the first molecular weight is expected to be the highest molecular weight.

Then, to determine whether pLys and SPDP are properly connected, sulfhydryl-active and pyridine2-thione were dissociated by cutting the disulfide bond in the pyridyldithiol-activate part using DTT. Since pyridine 2-thione has an absorbance at 343 nm, the absorbance at 343 nm after DTT treatment shows that SPDP and pLys are linked at fraction 3.

As shown in the table of FIG. 4, the result of the DTT treatment of the blue arrow fraction obtained in FIG. 3 was confirmed that pLys and SPDP were connected to fraction No. 3. For peptide synthesis, a peptide sequence having strong binding strength was selected for PA, and a peptide was synthesized by directly attaching a fluorescent probe to it and binding to a polymer to measure its binding strength.

Peptide 1) Ac-HKHAHNYRLPASGCGK- FITC

Peptide 2) Ac-LMPTPHHRLFPMGCGK- FITC

All peptides were expected to diversify and increase detection signal by binding FITC fluorescent probe to the carboxy terminus and put Cys group for binding to activated pLys (SPDP coupled pLYS). In addition, the N-terminal was prepared by acetylation to remove the amino-terminal reactivity.

Example  3: PA Combined with Peptide  Binding force analysis

For the analysis of the binding capacity of the synthesized peptide, the binding capacity of the peptide was determined by measuring the fluorescence intensity emitted from the fluorescence probe (FITC) of the peptide by binding each peptide to the plate to which the PA was bound. .

After the peptide was put on the plate to which the PA was bound, washing was performed to determine the maximum wavelength of the FITC fluorescent probe connected to the peptide, and fluorescence intensity measurement was performed using the Fluorometer. The signal intensity of the FITC fluorescence probe according to the concentration was indicated and the Kd value of the peptide was determined according to the saturation curve.

As a result, as shown in Figure 5, the two peptides synthesized determined the binding force in the nanomolar range.

Example  4: PA Functional diagnostic polymers combined with polyvalent functional peptide polymer , PFP ) Peptide  Binding force analysis

The binding force was analyzed in the same manner as in Example 3 to determine the binding force of the functional diagnostic polymer binding to PA.

As shown in FIG. 6, by using Cystein with sulfhydryl group of the peptide, a cross-linked polymer backbone peptide was added to the PA-bound plate by the substitution reaction with pyridyldithio of SPDP and SH group of Cys. The binding force of the peptide was determined by measuring the fluorescence intensity emitted from the FITC fluorescent probe.

After the peptide was put on the plate to which the PA was bound, washing was performed to determine the maximum wavelength of the FITC fluorescent probe connected to the peptide, and a fluorometer was performed using the peptide. The signal intensity of the FITC fluorescence probe according to the concentration was indicated and the Kd value of the peptide was determined according to the saturation curve.

As a result, as shown in FIG. 7, the synthesized two peptides had relatively weak binding strength while the binding strength of the peptide itself remained in the nanomolar range, whereas the polymer based peptide cross-linked through the substitution reaction had a picomolar range. It was confirmed that the binding force.

In addition, as shown in FIG. 8, polymer based peptide-1 + 2, that is, a functional diagnostic polymer (polyvalent functional peptide polymer) in which two peptides of different sequences are simultaneously linked to a polymer chain, rather than the binding force when one peptide is linked , PFP) can be confirmed to have a better binding force. It was confirmed that the combination of one peptide can increase the binding strength by using the characteristic of having a cooperative (cooperativity) in which the peptide of the other recognition site is also coupled to the diagnosis target.

As a result of testing the binding force with the PA by using an antibody that recognizes a known PA, as shown in FIG. 9, it was confirmed that the binding force was almost similar to that of the functional diagnostic polymer (PFP) developed by the present inventors.

According to the present invention, the anthogenic defense protein produced in large quantities using genetic recombination is bound to a 96-well white plate having Ni ions coordinated to the surface of the plate using a 6-histidine tag at the carboxy terminus of the protein. Using a multi-peptide substituted with a FITC-labeled peptide through a polymer chain reaction, a new functional diagnostic polymer peptide was found to secure the binding capacity of the peptide compared to the antibody. This is different from the existing diagnostic method using antibodies or epsamer, and shows that it can be linked with various nanobio sensors using the physical and chemical stability of peptides.

FIG. 1A is a diagram illustrating a biopolymer-type polymer poly-lysine structure to be used to connect a peptide to an alternative polymer based on a poly-peptide chain.

In addition, Figure 1b is a diagram showing the structure of the SPDP (N-succinimidyl3- (2-pyridyldithio) propionate) as a chemical linker for connecting the peptide and the signal label.

FIG. 2 is a diagram illustrating a reaction in which a biopolymer-type polymer poly-lysine and a peptide are linked using a heterobifunctional group of a linker SPDP using FIG. 1.

Figure 3 shows the results of separation and purification using gel-filtration after the combination of poly-lysine and SPDP using the characteristics of the reaction shown in FIG. The red arrow shows that including SPDP + pLys.

FIG. 4 is an experimental schematic diagram for confirming cross-linking of poly-lysine and SPDP with a fraction including SPDP + pLys (red arrow) in FIG. 3, and purified by gel-filtration after the actual SPDP and pLys reactions. The results of observing the dissociation of the functional moeity of SPDP by DTT treatment of each fraction.

Figure 5-1) is a graph analyzing the binding force and the PA for the representative peptide-2 synthesized by connecting the FITC fluorescent probes, Figure 5-2) PA for the representative peptide-1 synthesized by connecting the FITC fluorescent probes This is a graph analyzing the binding force.

Figure 6 is a graph showing the results confirmed using gel filtration and fluorometer cross-linking for all peptides are activated pLys (SPDP coupled pLYS).

FIG. 7 is a diagram illustrating peptide-1, peptide-2, and peptide-1 + 2 using a fluorometer to bind a peptide by immobilizing PA on the surface of a plate and inserting a peptide based on a polymer chain. It is a graph analyzing the binding force between PA and binding force of the peptide itself.

Figure 8 shows the peptide amino acid sequence and the binding force of peptide-1 and 2, the polymer-based peptide-1 and 2 and 1 + 2 peptide-cross-linking to the polymer chains to bind anthrax defense antigen (PA) obtained through the present invention This is a diagram summarized.

FIG. 9 is a diagram summarizing the binding force between the polyvalent detection peptide binding to anthrax defense antigen (PA) obtained through the present invention and an antibody that recognizes a known PA.

10 is a result of combining a plurality of various signal markers to the diagnostic polymer chain in order to secure the original region by using a polyvalent detection peptide for the diagnostic object deviated from the conventional antibody recognition method through the present invention. Figure is a diagram.

<110> Hanyang University <120> Novel anthrax diagnostic system using polyvalent functional          peptide polymers <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 for 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> Screened peptide for 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 for 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 for 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 for 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 for 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 for act as a diagnostic probe <400> 8 Asn Ala Tyr Lys His His His Pro Pro Val Phe Tyr   1 5 10  

Claims (14)

To a peptide having an amino acid sequence of any of the following amino acid sequences, characterized in that it binds specifically to anthrax toxin protein defense antigen, to which a fluorescent probe selected from a Fluorescein Isothiocynate (FITC) probe, a fluorescein derivative or a rhodamine derivative is linked. Anthrax infection diagnostic peptide. (1) the amino acid sequence of any one of SEQ ID NOs: 1 to 8, (2) The second amino acid S of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is T, the fifth amino acid Q is L, the seventh amino acid I is L, and the tenth amino acid sequence D is A. The amino acid sequence of any one of the substituted amino acid sequences, (3) an amino acid sequence of which Y, the seventh amino acid of amino acid sequence HKHAHNYRLPXS represented by SEQ ID NO: 2 is substituted with T, (4) The amino acid sequence of any one of the amino acid sequences of amino acid sequence GHKPQVHRHTHI, represented by SEQ ID NO: 5, wherein G is A, the second amino acid H is L, and the twelfth I is L. Anthrax test method using the anthrax infection diagnosis peptide according to claim 1. A peptide that specifically binds to anthrax toxin protein protective antigen; and Poly-peptide chains coupled with chemical linkers; multi-recognition, characterized in that they are polymer-based peptides that are cross-linked through a polymer chain substitution reaction Peptide and Signal Detection Peptide Polymerized Chain Anthrax Infection Diagnostic Polymer. At least two peptides each having a different sequence that specifically binds to anthrax toxin protein protective antigen; And Poly-peptide chains combined with chemical linkers; characterized by the coupling of two or more polymer based peptides cross-linked through a polymer chain substitution reaction Multi-recognition peptide and signal detection peptide polymerized chain anthrax infection diagnostic polymer. The method according to claim 3 or 4, Peptides specifically binding to the anthrax toxin protein protective antigen has an anthrax infection diagnostic polymer, characterized in that it has an amino acid sequence of any one of the following sequences. (1) the amino acid sequence of any one of SEQ ID NOs: 1 to 8, (2) The second amino acid S of the amino acid sequence LSHNQTIQQDSD represented by SEQ ID NO: 1 is T, the fifth amino acid Q is L, the seventh amino acid I is L, and the tenth amino acid sequence D is A. The amino acid sequence of any one of the substituted amino acid sequences, (3) an amino acid sequence of which Y, the seventh amino acid of amino acid sequence HKHAHNYRLPXS represented by SEQ ID NO: 2 is substituted with T, (4) The amino acid sequence of any one of the amino acid sequences of amino acid sequence GHKPQVHRHTHI, represented by SEQ ID NO: 5, wherein G is A, the second amino acid H is L, and the twelfth I is L. The method of claim 5, The anthrax toxin protein protective antigen is an anthrax infection diagnostic polymer, characterized in that PA20 or PA 63. The method according to claim 3 or 4, The polypeptide chain is an anthrax infection diagnostic polymer, characterized in that the D-stereoisomer of poly-lysine (poly-lysine) having the formula of Figure 1a. The method according to claim 3 or 4, The chemical linker is an anthrax infection diagnostic polymer, characterized in that the SPDP (N-succinimidyl3- (2-pyridyldithio) propionate) having the chemical formula of Figure 1b. The method according to claim 3 or 4, Anthracnose infection diagnostic polymer, characterized in that the fluorescent probe selected from the FITC probe, fluorescein derivative or rhodamine derivative is linked to the peptide specifically binding to the anthrax toxin protein antigen. Anthrax test method using the anthrax infection diagnosis polymer according to claim 3 or 4. Anthrax test method using the anthrax infection diagnosis polymer according to claim 5. Anthrax Infection Diagnostic Kit comprising the low molecular weight peptide according to claim 1. Anthrax infection diagnostic kit comprising anthrax infection diagnostic polymer according to claim 3. Anthrax infection diagnostic kit comprising anthrax infection diagnostic polymer according to claim 5.

Images