JP4238557B2 - Peptides with affinity for high mobility group proteins - Google Patents

Description

【図面の簡単な説明】
【図１】本発明のペプチド提示ファージを用いたＨＭＧ−１の測定結果（実施例１〜４）である。
【図２】抗ＨＭＧ−１抗体を用いたＨＭＧ−１の測定結果（比較例１）である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a peptide having affinity for a high mobility group protein (hereinafter abbreviated as HMG protein), and more particularly, to a peptide used for the treatment of highly sensitive quantification of HMG protein, diseases involving HMG protein, and the like. .
[0002]
[Prior art]
HMG protein is a non-histone DNA-binding protein present in eukaryotic cells, and HMG-1, HMG-2, HMG14, HMG17, HMG-I (Y) and the like are known. HMG was originally thought to be involved in functions such as transcription promotion and cell growth by binding to DNA in the cell, but it was found as a factor that exists on the surface of nerve cells and extends neurites. It has been shown that amphoterin is HMG-1 which is one of the HMG proteins, and the possibility that the HMG protein has a wide range of actions has been shown.
[0003]
Recently, an interesting report has been made that this HMG-1 is secreted extracellularly and acts as a powerful mediator of septic shock (see, for example, Non-Patent Document 1). That is, when lipopolysaccharide (LPS) is administered to mice, the serum HMG-1 concentration is markedly increased 8-24 hours later and the mice die. When purified HMG-1 itself is administered to mice simultaneously with LPS, it acts synergistically to show lethal activity, and when anti-HMG-1 antibody is administered, lethal action by LPS is suppressed. Has been shown to be an important mediator of endotoxin shock. Also in humans, HMG-1 concentration was significantly increased in the blood of septic patients, and was shown to be particularly high in death cases. HMG-1 is also found to increase in blood concentration in hemorrhagic shock. It has also been reported that HMG protein is involved in cancer growth (see, for example, Non-Patent Document 2).
[0004]
[Non-Patent Document 1]
Wang H. et al., 18 HMG-1 as a late mediator of endotoxin invasion in mice (Science, USA), 1999. 285, p248-251
[0005]
[Non-Patent Document 2]
Taguchi A et al., 18 "Blockage of RAGE-amphoterin signaling suppressor tumor growth and metastases", Nature, UK, Nature 2000, 405, p354-360
[0006]
[Problems to be solved by the invention]
As described above, HMG protein in the blood has been shown to be closely related to the pathology of diseases such as sepsis, and the accurate and highly sensitive measurement of its blood concentration is very useful for disease diagnosis. It is considered important. Until now, the measurement of HMG protein has been performed by immunological detection methods using antibodies such as Western blotting and enzyme immunoassay, but the quantitativeness, measurement sensitivity, etc. are not always sufficient. This is probably because the HMG protein has a very high sequence similarity between animal species, and it is difficult to obtain an antibody with high affinity by ordinary immunization. Therefore, it is considered necessary to obtain an affinity substance other than an antibody in order to perform detection with higher sensitivity. Furthermore, a substance that binds to HMG protein and inhibits its action is considered to be very useful as a therapeutic agent. For this purpose, a substance having a higher affinity than an antibody and a smaller molecular weight than that of an antibody should be obtained. Was strongly desired.
[0007]
[Means for Solving the Problems]
In order to solve this problem, the present invention has the following configuration.
(1) A peptide having affinity for a high mobility group protein and comprising 30 or less amino acids.
(2) The following peptide (a) or (b).
(A) Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr A peptide consisting of an amino acid sequence.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having affinity for a high mobility group protein.
(3) The following peptide (a) or (b).
(A) A peptide consisting of the amino acid sequence of Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Leu Ala.
(B) A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a) and having affinity for a high mobility group protein.
(4) The following peptide (a) or (b).
(A) A peptide comprising the amino acid sequence of Val Trp Val Val Pro Tyr Thr Thr Ala Thr Leu.
(B) A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a), and having affinity for a high mobility group protein.
(5) The following peptide (a) or (b).
(A) A peptide comprising the amino acid sequence of Cys Cys Tyr Ala Ser Phe Pro Gly Met Gly Met.
(B) A peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (a), and having affinity for a high mobility group protein.
(6) A phage displaying the peptide according to any one of (1) to (5).
(7) A peptide comprising the peptide according to any one of (1) to (5) as a part thereof and having affinity for a high mobility group protein.
(8) A polynucleotide encoding the peptide according to any one of (1) to (5).
(9) The polynucleotide according to (8), which is DNA.
(10) A method for measuring a high mobility group protein using the peptide or phage according to any one of (1) to (6).
(11) A kit for measuring a high mobility group protein comprising the peptide or phage according to any one of (1) to (6).
(12) A therapeutic agent for sepsis comprising the peptide or phage according to any one of (1) to (6).
(13) A therapeutic drug for cancer comprising the peptide or phage according to any one of (1) to (6).
[0008]
DETAILED DESCRIPTION OF THE INVENTION
The HMG protein referred to in the present invention refers to an HMG protein in mammals, and includes human HMG-1, HMG-2, HMG14, HMG17, HMG-I (Y) and the like.
[0009]
The peptide having affinity for HMG protein in the present invention refers to a peptide that can bind to HMG protein by non-covalent bond, that is, ionic bond, hydrophobic bond, hydrogen bond and the like. To examine whether a peptide has an affinity for HMG, the immobilized HMG protein is reacted with a phage that displays the peptide or peptide, and after washing away unbound substances, the bound peptide or peptide is displayed. It is possible to confirm by the enzyme immunoassay (ELISA method) which detects the phage to do with an antibody etc.
[0010]
In the present invention, the peptide or phage having affinity for HMG protein is detected by immobilizing the HMG protein on a plate and reacting the peptide or phage with this to bind the peptide or phage using an enzyme-labeled secondary antibody or the like. In the ELISA method, it means one having an absorbance twice or more that of a control peptide or phage having an irrelevant sequence.
[0011]
The peptide of the present invention consists of a peptide having a length of 30 amino acid residues or less having affinity for the HMG protein. Also, Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr (SEQ ID NO: 1 in the sequence listing), or Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Le sequence No. It consists of a peptide identified by the amino acid sequence represented by Val Val Pro Tyr Thr ThrAla Thr Leu (SEQ ID NO: 3 in the sequence listing) or Cys Cys Tyr Ala Ser Phe Pro Gly Met Gly Met (SEQ ID NO: 4 in the sequence listing) (In the above sequences, the left end represents the N-terminus, the right end represents the C-terminus, and the constituent amino acid residues are expressed in ordinary three-letter code). The peptide of the present invention identified by the amino acid sequence is characterized by having affinity for HMG protein. The peptide of the present invention has substitution, insertion, and / or deletion with one or more amino acid residues in the amino acid sequence of the above-mentioned specific peptide, and has substantially affinity for HMG protein. Also included are peptides having The type of one or more amino acids to be substituted and / or inserted is not particularly limited, but is preferably an L-amino acid. A person skilled in the art can easily confirm whether or not a peptide having substitution, insertion, and / or deletion by one or more amino acid residues substantially has HMG protein affinity. It is.
[0012]
The present invention also includes a phage displaying the above peptide on the surface. In this case, the type of phage is not particularly limited, but filamentous phage is preferably used. Moreover, the peptide of the present invention includes a peptide containing each of the above peptides as a partial sequence and having substantially affinity for HMG protein. Techniques for modifying specific proteins / peptides by substitution, deletion or insertion of amino acids are known, for example, from European Patent No. 77109 or British Patent Application No. 2119884. For example, European Patent No. 77109 discloses a method of replacing a coding amino acid by making a vector single-stranded, annealing with a primer containing a single residue mutation, and mixing with E. coli polymerase.
[0013]
A peptide having affinity for the high mobility group of the present invention and comprising 30 or less amino acids can be produced by the following method using a technique using a random peptide library, for example. Can do.
[0014]
Although a phage random peptide library etc. can be used as a random peptide library, the kind and preparation method of a phage random peptide library are not specifically limited, You may purchase and use a commercially available library.
[0015]
The length of the peptide in the random peptide library should be selected according to the length of the peptide to be produced, and is preferably 30 residues or less, and the length of the peptide to be produced is short. If there are 20 or 15 residues can be used.
[0016]
Phage displaying a peptide having affinity for HMG protein is immobilized on HMG protein on a column or plate directly or via an antibody, brought into contact with the peptide library, washed away unbound phage by washing, and then bound. The selected phage can be selected by a method of eluting with an acid or the like. In order to obtain a phage with high affinity, it is desirable to repeat this selection operation twice or more. The obtained phage is single-cloned and the sequence of the displayed peptide can be determined by analyzing the DNA sequence.
[0017]
Peptides (including each peptide of SEQ ID NO: 1 to 4 of the present invention) sequenced by the above method and substitution, insertion, and / or deletion with one or more amino acid residues in the amino acid sequence of the peptide Can be synthesized by a chemical method such as a solid phase method and a liquid phase method used for peptide synthesis. In the solid phase method, various commercially available peptide synthesizers can be used.
[0018]
In addition, each peptide of SEQ ID NOs: 1 to 4 and the amino acid sequence of the peptide in which substitution, insertion, and / or deletion with one or more amino acid residues are present are biology such as normal gene expression manipulation. A recombinant vector containing a DNA sequence encoding the peptide of the present invention is prepared according to a typical technique, a microorganism (transformant) transformed with the vector is prepared, and a desired culture is obtained from the culture in which the transformant is cultured. It can also be produced by separating and purifying peptides. In this case, the method for introducing substitution, insertion and / or deletion with one or more amino acid residues in the amino acid sequence is as described above.
[0019]
The phage presenting the above-mentioned various peptides according to the present invention incorporates a polynucleotide encoding the above-mentioned various peptide amino acid sequences into a phage DNA cleaved with a restriction enzyme, infects Escherichia coli, and precipitates polyethylene glycol from the culture supernatant. Or the like.
[0020]
The polynucleotide encoding the peptide according to the present invention can be obtained by using the above-mentioned phage DNA as a template and using replication chain reaction (PCR) (for example, US Pat. No. 4,683,195 and US Pat. 683,202 specification). In essence, PCR allows the production of a selected DNA sequence when the two source parts of the sequence are known. Primers, ie oligonucleotide probes, corresponding to each end of the sequence in question are obtained. Using PCR, the central portion of the polynucleotide sequence is then produced synthetically. When the encoding polynucleotide is DNA, it is obtained by infecting Escherichia coli with the amplified phage clone, culturing and amplifying, and then extracting the gene.
[0021]
The method of measuring HMG protein using the peptide or phage according to the present invention can be performed by techniques such as ELISA and Western blot. For example, in the case of ELISA, an anti-HMG protein antibody or a peptide or phage of the present invention is immobilized on a plate, a sample containing the HMG protein is reacted with this, and the bound HMG protein is measured with an enzyme-labeled peptide of the present invention. can do. It is also possible to detect the peptide with biotin-labeled enzyme-labeled avidin. It is also possible to react with peptide-displayed phages and detect them using enzyme-labeled anti-phage antibodies. To measure HMG protein by Western blotting, the polyacrylamide gel electrophoresis gel is transferred to a membrane, and the HMG protein is measured by reacting the transferred HMG protein with the enzyme-labeled peptide of the present invention. be able to.
[0022]
Moreover, the measuring kit of HMG protein containing the peptide concerning this invention takes the following structures, for example. For example, in the case of a kit that measures the concentration of HMG in a body fluid by ELISA, an anti-HMG protein antibody-immobilized plate, the peptide of the present invention having an affinity for HMG protein labeled with an enzyme, and a known concentration of HMG It can be used by containing a protein standard solution as a constituent element, reacting the anti-HMG protein antibody-immobilized plate with the HMG protein, reacting it with an enzyme-labeled peptide, adding an enzyme substrate, and developing a color.
[0023]
The peptide or phage according to the present invention can be expected to bind to an HMG protein in the body and inhibit its action when administered to a living body, thereby being used in a pharmaceutical preparation or formulation for the treatment of sepsis or cancer. be able to. The present invention provides a therapeutic agent for the aforementioned symptoms. The therapeutic agent can be mixed with a pharmaceutically acceptable carrier and can be administered parenterally systemically. If necessary, the therapeutic agent can be administered subcutaneously, topically, eg, at the site of injury.
[0024]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the scope of the present invention is not limited to these examples.
(1) Production of HMG-1
HL60 cells were cultured in RPMI1640 medium, and the culture supernatant was purified by heparin column chromatography and CM-Sephadex column chromatography.
(2) Measurement of HMG-1 by ELISA using phage
5 μg / ml anti-HMG-1 monoclonal antibody PBS solution was placed in a well of a 96-well microplate for ELISA, and solidified overnight at 4 ° C., followed by antibody PBS (137 mM NaCl, 8.10 mM Na ₂ PO4 · 12H ₂ O, 2.68 mM KCl, 1.47 mM KH ₂ PO _Four ) After removing the solution, blocking was performed by adding PBS in which 0.5% BSA was dissolved. After washing the plate with PBS-T (PBS containing 0.05% Tween-20), the buffer (0.25% BSA, 0.05% Tween-20, 0.01% n-propyl parahydroxybenzoate ( Diluted solution (0.25% BSA, 0.01% n-propyl parahydroxybenzoate (PHB)) containing 50 μl of 0.1 M Tris-hydrochloric acid buffer (pH 8.0) containing PHB) and various concentrations of HMG-1 , PBS-T solution containing 0.001% phenol red) was added and reacted at 25 ° C. for 60 minutes. Each well was washed 3 times with 400 μl PBS-T and then 1.0 × 10 ¹² 100 μl of a diluted solution containing Tu / ml peptide-displayed phage was added and reacted at 25 ° C. for 60 minutes. After discarding the phage solution, each well was washed 3 times with 400 μl of PBS-T, and 100 μl of HRP-labeled anti-M13 phage monoclonal antibody (Amersham Biosciences) solution diluted 5000 times with a diluent was added and reacted at 25 ° C. for 30 minutes. I let you. After discarding the phage solution, each well was washed 3 times with 400 μl of PBS-T, and then a color developing solution (0.1 M acetic acid-citric acid containing 0.006% hydrogen peroxide, 0.2 mg / ml tetramethylbenzidine (TMB)) 100 μl of sodium buffer (pH 4.5) was dispensed and reacted for 15 minutes. After the reaction, 100 μl of 1N sulfuric acid was added to stop the reaction. Absorbance was measured at 450 nm using a microplate reader.
(3) Preparation of phage displaying HMG-1 affinity peptide
2 ml of PBS in which 0.5 μg / ml of HMG-1 was dissolved was added to the petri dish, and the mixture was allowed to stand overnight at 4 ° C. for immobilization. The solution was removed from the petri dish, and blocking was performed by adding 8 ml of PBS in which 0.5% BSA (bovine serum albumin) was dissolved. After washing the petri dish with PBS, a phage library expressing a 15-residue random sequence peptide was added and left at room temperature for 30 minutes. The solution was discarded, and the plate was washed with PBS-T three times for 10 seconds and three times for 5 minutes. 400 μl of 50 mM glycine-hydrochloric acid buffer (pH 2.4) was added and left for 5 minutes to dissociate and recover the bound phages, and neutralized by adding 80 μl of 1M Tris-hydrochloric acid buffer (pH 8.0). The recovered phage was infected with E. coli and cultured and propagated. The grown phages were purified by the PEG (polyethylene glycol) method and used again for the reaction with HMG-1. These operations were performed for 3 rounds. After 3 rounds of selection, E. coli infected with phage and cultured on LB plate, picking up single colonies, and collecting the phages produced in the culture supernatant grown overnight in LB medium using 140 PEG method did. The recovered phages were selected using the ELISA method of (2) above, which had an absorbance that was at least twice that when the anti-HMG-1 antibody was used, and the phage DNA was extracted and sequenced. As a result, DNA sequences encoding the following amino acid sequences were found.
SEQ ID NO: 1; Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr
SEQ ID NO: 2; Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Leu Ala
SEQ ID NO: 3; Val Trp Val Val Pro Tyr Thr Thr Ala Thr Leu
SEQ ID NO: 4; Cys Cys Tyr Ala Ser Phe Pro Gly Met Gly Met
(Example 1) Production of phage displaying SEQ ID NO: 1 and detection of HMG-1 using the same
(A) Preparation of peptide-displayed phage
A DNA encoding the peptide containing the amino acid sequence Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr was added to the terminal and a DNA complementary to the following primer was synthesized and amplified by the PCR method. PCR is 2.5 mM MgCl ₂ , 0.2 mM dNTP, 0.5 μM 5 ′ terminal biotinylated SENSE primer (5′-CATTTCTCACTCGGCCCAGCG-3 ′), 0.5 μM 5 ′ terminal biotinylated ANTIENSE primer (5′-TTCAACAGTTCGGCCCAGAG 3 ′), 50 pg of polynucleotide Performed under conditions. For PCR, Takara Taq polymerase and the attached PCR buffer were used. One cycle was 96 ° C. for 0.5 minutes, 58 ° C. for 1 minute, and 72 ° C. for 2 minutes, for a total of 35 cycles of amplification. This PCR product was ethanol precipitated, and the obtained DNA was treated with BglI at 37 ° C. for 1 hour. Thereafter, biotinylated DNA fragments were removed using avidinized agarose, the solution was extracted with phenol / chloroform, and then DNA was ethanol precipitated. The obtained DNA was dissolved in sterilized water to obtain insert DNA. Filamentous phage DNA (FUSE5) was reacted with SfiI at 50 ° C. for about 19 hours to cleave the phage vector DNA. The reaction solution was extracted with phenol / chloroform, and DNA was ethanol precipitated. The obtained DNA was dissolved in sterilized water and then purified using Chromaspin TE400 (CLONTECH). Ligation of phage vector DNA and insert DNA is 10 ^-13 pmol of SfiI cut FUSE5 DNA and 10 ^-12 Mol of the insert DNA was treated with Ligation High (manufactured by Toyobo Co., Ltd.) at 16 ° C. for 3.5 hours. After the reaction solution is extracted with phenol / chloroform and chloroform, DNA is ethanol precipitated. The obtained DNA was dissolved in sterilized water and then stored at -20 ° C. Escherichia coli was transformed by a heat shock method, and 100 μl of a diluted E. coli solution was plated on an LB agar plate containing 50 μg / ml tetracycline and 50 μg / ml kanamycin, and cultured at 37 ° C. overnight. The resulting single colony was picked up and the phage (Example 1) produced in the culture supernatant cultured overnight in LB medium containing 50 μg / ml tetracycline and 50 μg / ml kanamycin was purified and recovered using the PEG method. .
(B) Measurement of HMG-1 using peptide-displayed phage by ELISA method
Measurement of HMG-1 by the ELISA method was performed in the same manner as in (2) above.
(Example 2) Production of phage displaying SEQ ID NO: 2 by screening and detection of HMG-1 using the same
(A) Preparation of peptide-displayed phage
This was carried out in the same manner as in Example 1 using DNA encoding a peptide containing the amino acid sequence Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Leu Ala.
(B) Measurement of HMG-1 using peptide-displayed phage by ELISA method
Measurement of HMG-1 by the ELISA method was performed in the same manner as in (2) above.
(Example 3) Production of phage displaying SEQ ID NO: 3 by screening and detection of HMG-1 using the same
(A) Preparation of peptide-displayed phage
This was carried out in the same manner as in Example 1, using a DNA encoding a peptide containing the amino acid sequence Val Trp Val Val Pro Tyr Thr Thr Ala Thr Leu.
(B) Measurement of HMG-1 using peptide-displayed phage by ELISA method
Measurement of HMG-1 by the ELISA method was performed in the same manner as in (2) above.
(Example 4) Production of phage displaying SEQ ID NO: 4 by screening and detection of HMG-1 using the same
(A) Preparation of peptide-displayed phage
This was carried out in the same manner as in Example 1 using DNA encoding a peptide containing the amino acid sequence Cys Cys Tyr Ala Ser Phe Pro Gly Met Gly Met.
(B) Measurement of HMG-1 using peptide-displayed phage by ELISA method
Measurement of HMG-1 by the ELISA method was performed in the same manner as in (2) above.
[0025]
The results of the binding of the phages of Examples 1 to 4 to HMG-1 are shown in FIG.
[0026]
(Comparative Example 1) Using the HRP-labeled anti-HMG-1 monoclonal antibody, the above-mentioned (b) HMG-1 was detected by the ELISA method. In this case, an HRP-labeled anti-HMG-1 monoclonal antibody was used instead of the peptide-displayed phage and the HRP-labeled anti-M13 phage antibody. The results are shown in FIG.
[0027]
By using peptide-displayed phages (Examples 1 to 4), the detection sensitivity of HMG-1 was greatly improved over the conventional method using a monoclonal antibody (Comparative Example 1).
(Example 5) Production of peptide displaying SEQ ID NO: 1 and detection of HMG-1 using the same
(A) Peptide production method
A peptide having the amino acid sequence Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr is synthesized by a peptide synthesizer.
(B) Measurement of HMG-1 using peptide by ELISA method
Place the 5 μg / ml anti-HMG-1 monoclonal antibody PBS solution in the wells of a 96-well microplate for ELISA, solidify overnight at 4 ° C., remove the antibody PBS solution, and then dissolve 0.5% BSA. Put PBS and block. After washing the plate with PBS-T (PBS containing 0.05% Tween-20), the buffer (0.25% BSA, 0.05% Tween-20, 0.01% n-propyl parahydroxybenzoate ( Diluted solution (0.25% BSA, 0.01% n-propyl parahydroxybenzoate (PHB)) containing 50 μl of 0.1 M Tris-hydrochloric acid buffer (pH 8.0) containing PHB) and various concentrations of HMG-1 , PBS-T solution containing 0.001% phenol red) is added and reacted at 25 ° C. for 60 minutes. Each well is washed 3 times with 400 μl of PBS-T, and then 100 μl of a biotinylated peptide solution is added and reacted at 25 ° C. for 60 minutes. The peptide solution is discarded, each well is washed 3 times with 400 μl of PBS-T, and then an HRP-labeled avidin solution diluted with a diluent is added and reacted at 25 ° C. for 30 minutes. After discarding the solution, each well was washed three times with 400 μl of PBS-T, and then a color developing solution (0.1 M acetic acid-sodium citrate containing 0.006% hydrogen peroxide, 0.2 mg / ml tetramethylbenzidine (TMB)). 100 μl of buffer solution (pH 4.5) is dispensed and allowed to react for 15 minutes. After the reaction, 100 μl of 1N sulfuric acid is added to stop the reaction. Measure absorbance at 450 nm using a microplate reader.
(Example 6) Production of peptide of SEQ ID NO: 2 and detection of HMG-1 using ELISA using the peptide
A peptide having the amino acid sequence Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Leu Ala is synthesized with a peptide synthesizer and performed in the same manner as in Example 5.
(Example 7) Production of peptide of SEQ ID NO: 3 and detection of HMG-1 using the same by ELISA
A peptide having the amino acid sequence Val Trp Val Val Pro Tyr Thr Thr Ala Thr Leu was synthesized by a peptide synthesizer and performed in the same manner as in Example 5.
(Example 8) Production of peptide of SEQ ID NO: 4 and detection of HMG-1 using the same by ELISA
A peptide having the amino acid sequence Cys Cys Tyr Ala Ser Phe Pro Gly Me Gly Met was synthesized with a peptide synthesizer, and the same procedure as in Example 5 was performed.
[0028]
The ELISA of Examples 5 to 8 provides significantly higher color development than when the control peptide is used.
[0029]
【The invention's effect】
The peptide of the present invention or the phage displaying the peptide has an affinity for HMG protein, and the HMG protein can be quantified with high sensitivity by obtaining this peptide by synthesis or screening from a library. In addition, a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the peptide of the present invention or a phage displaying the peptide also has an affinity for the HMG protein. Can be quantified with high sensitivity. It is also possible to provide a kit for measuring HMG protein by using the peptide of the present invention or a phage displaying the peptide. Moreover, the peptide of the present invention or the phage displaying the peptide is useful as an active ingredient such as a therapeutic drug for sepsis. Moreover, the peptide of the present invention or the phage displaying the peptide is useful as an active ingredient such as a therapeutic agent for cancer.
[0030]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a measurement result (Examples 1 to 4) of HMG-1 using the peptide-displayed phage of the present invention.
FIG. 2 is a measurement result of HMG-1 using an anti-HMG-1 antibody (Comparative Example 1).

Claims (12)

The peptide of the following (a) or (b).
(A) Gly Trp Leu Pro Gln Gly Trp Ile Phe Gly Ala Val Thr A peptide consisting of an amino acid sequence.
(B) consists of one or several amino acids have been pressurized with the amino acid sequence of (a), and high mobility group protein -1 (HMG-1) and peptide and having an affinity. The peptide of the following (a) or (b).
(A) A peptide consisting of the amino acid sequence of Gly Tyr Leu Met Phe Ile His Leu Tyr Pro Glu Leu Ala.
(B) consists of one or several amino acids have been pressurized with the amino acid sequence of (a), and high mobility group protein -1 (HMG-1) and peptide and having an affinity. The peptide of the following (a) or (b).
(A) A peptide comprising the amino acid sequence of Val Trp Val Val Pro Tyr Thr Thr Ala Thr Leu.
(B) consists of one or several amino acids have been pressurized with the amino acid sequence of (a), and high mobility group protein -1 (HMG-1) affinity peptide having. The peptide of the following (a) or (b).
(A) A peptide comprising the amino acid sequence of Cys Cys Tyr Ala Ser Phe Pro Gly Met Gly Met.
(B) consists of one or several amino acids have been pressurized with the amino acid sequence of (a), and high mobility group protein -1 (HMG-1) affinity peptide having. The phage which displays the peptide in any one of Claims 1-4. A peptide comprising the peptide according to any one of claims 1 to 4 as a part thereof and having affinity for high mobility group protein- 1 (HMG-1) . The polynucleotide which codes the peptide of any one of Claims 1-4. The polynucleotide of claim 7 which is DNA. The method to measure high mobility group protein- 1 (HMG-1) using the peptide or phage of any one of Claims 1-5. The kit which measures the high mobility group protein- 1 (HMG-1) containing the peptide or phage of any one of Claims 1-5. A therapeutic agent for sepsis comprising the peptide or phage according to any one of claims 1 to 5. The therapeutic agent of the cancer containing the peptide or phage of any one of Claims 1-5.

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