JP2009201379A - Muscarine receptor-activating agent and peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide usable as a muscarine receptor agonist, and a muscarine receptor-activating agent containing the peptide. <P>SOLUTION: The peptide contains an amino acid sequence represented by the sequence: XXXP (wherein, the first X is W or R; the second X is Y, H or R; and the third X is T, R or I), or an amino acid sequence obtained by deleting, substituting and/or adding one or several amino acids from, with and/or to the amino acid sequence, and exhibiting muscarine receptor-activating actions. The muscarine receptor-activating agent contains the peptide or a derivative thereof. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a muscarinic receptor activator and a novel peptide that can be used as an active ingredient thereof.

  Muscarinic receptors are proteins belonging to the seven-transmembrane receptor protein (GPCR; G protein-coupled receptor) family. Muscarinic receptors are known to have subtypes M1 to M5. Since the structure of muscarinic receptors is very similar, there is a characteristic that all the subtypes bind to the same ligand, agonist or antagonist, and there is no binding specificity of acetylcholine which is a ligand, for example. Therefore, it is very difficult to give the specificity to therapeutic agents that act on muscarinic receptors.

  Muscarinic receptors have been confirmed to be expressed in a wide variety of organs such as brain, autonomic nervous system, heart, smooth muscle, vascular endothelium and exocrine glands. Muscarinic receptor agonists (agonists) are used as therapeutic agents for various diseases. For example, cevimeline (agonist for M3) is an agent for improving dry mouth symptoms in patients with Sjogren's syndrome, and carbachol or pilocarpine (agonist for M3) is used. Each is used as a glaucoma treatment.

  Examples of peptidic agonists for muscarinic receptors include muscarinic toxin (Muscarinic Toxin) 3 (agonist for M4) (non-patent document 1) consisting of the amino acid sequence represented by SEQ ID NO: 41, and muscarinic toxin α consisting of a similar sequence. (Non-patent document 2), muscarinic toxin 1 (non-patent document 3), and muscarinic toxin 7 (non-patent document 4) are known.

“FEBS letters” (Netherlands), 1994, 352, 91 “European journal of biochemistry” (UK), 1995, 234, 579. “Toxicon”, (UK), 1995, 33, 399. “Life sciences” (UK), 1997, 60, 1069

  An object of the present invention is to provide a novel peptide that can be used as a muscarinic receptor agonist.

  The subject is the amino acid represented by SEQ ID NO: 21 (XXXP; first X = W, R, second X = Y, H, R, third X = T, R, I) according to the present invention. A sequence, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and a peptide exhibiting a muscarinic receptor activating effect, or a derivative thereof as an active ingredient This can be solved by a muscarinic receptor activator.

According to a preferred embodiment of the muscarinic receptor activator of the present invention, the active ingredient is SEQ ID NO: 22 (XXXXP; first X = T, L, H, F, S, second X = W, R, 3rd X = Y, H, R, 4th X = T, R, I), or one or several amino acids in the amino acid sequence are deleted, substituted, and / or It is a peptide containing an added amino acid sequence and showing a muscarinic receptor activating action, or a derivative thereof.
According to another preferred embodiment of the muscarinic receptor activator of the present invention, the active ingredient is SEQ ID NO: 23 (XXXXXX; first X = Y, F, second X = T, L, H, F, S, the third X = W, R, the fourth X = Y, H, R, the fifth X = T, R, I), or one or several amino acids in the amino acid sequence Is a peptide that includes an amino acid sequence in which the amino acid is deleted, substituted, and / or added, and that exhibits a muscarinic receptor activation effect, or a derivative thereof.

According to still another preferred embodiment of the muscarinic receptor activator of the present invention, the amino acid sequence represented by SEQ ID NO: 22 or 23 is
YTWYTP (SEQ ID NO: 8),
YSWYTP (SEQ ID NO: 15),
HWHTP (SEQ ID NO: 19) or YHRHTP (SEQ ID NO: 20)
It is.
According to still another preferred embodiment of the muscarinic receptor activator of the present invention, the first amino acid X in SEQ ID NO: 21, the second amino acid X in SEQ ID NO: 22, or the third amino acid X in SEQ ID NO: 23 is: W.

The present invention also provides an amino acid sequence represented by any one of SEQ ID NOs: 21 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence. In addition, the present invention relates to a peptide having 100 or less constituent amino acids, or a derivative thereof.
Furthermore, the present invention relates to a polynucleotide encoding the peptide, and an expression vector comprising the polynucleotide.

  According to the present invention, a novel muscarinic receptor activator can be provided. The peptide that can be used as an active ingredient in the muscarinic receptor activator of the present invention or its derivative itself is also a novel compound and has an activating action on the muscarinic receptor. The muscarinic receptor activator of the present invention can be used for the treatment of glaucoma and Sjogren's syndrome, for example.

  The muscarinic receptor activator of the present invention is an amino acid sequence represented by SEQ ID NO: 21 as an active ingredient, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence. And a peptide exhibiting a muscarinic receptor activation effect, or a derivative thereof.

  Hereinafter, the peptides and their derivatives that can be used as active ingredients in the present invention are collectively referred to as “muscarinic receptor activating peptides”. The term “peptide” in the present specification includes both oligopeptides and polypeptides.

  As used herein, “muscarinic receptor activating effect” means an effect of binding to a muscarinic receptor and exhibiting agonist activity. The muscarinic receptors include muscarinic receptors derived from various mammals such as humans, horses, sheep, rabbits, guinea pigs, goats, cats, dogs, cows, pigs, mice, etc., and human muscarinic receptors are particularly preferred. . The muscarinic receptor includes various subtypes of muscarinic receptors, that is, M1 to M5, and particularly preferably M2.

  Whether or not a certain peptide has a muscarinic receptor activating effect can be easily determined by a known method. For example, a method for immobilizing a muscarinic receptor on an appropriate carrier (for example, an ELISA plate or a bead carrier) and analyzing the presence or absence of binding to the muscarinic receptor (for example, see Example 1 below), or a muscarinic receptor and Gi Whether a certain peptide has a muscarinic receptor activating effect by a method for preparing a fusion polypeptide and measuring the amount of labeled GTP incorporated into the fusion polypeptide (for example, see Example 2 or 3 below) Whether or not can be easily determined.

  In the present invention, the number of amino acid residues constituting the muscarinic receptor activating peptide is not particularly limited as long as it exhibits a muscarinic receptor activating action. For example, 4 to 100, preferably 4 to 50, More preferably, it is 4-30, More preferably, it is 4-20, More preferably, it is 4-12, Most preferably, it is 4-6. The amino acid sequence may be composed of only an amino acid sequence (basic sequence) that exhibits a muscarinic receptor activation effect alone, or may consist of a repeated sequence thereof. In the case of consisting of repetitive sequences, it may be a repetitive sequence of only one type of basic sequence or a combination of two or more types of basic sequences.

In the present specification, “the amino acid sequence represented by SEQ ID NO: X (for example, SEQ ID NO: 21 to 23), or an amino acid in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence” A peptide containing a sequence and exhibiting a muscarinic receptor activating effect ”includes, for example,
A peptide consisting of the amino acid sequence represented by SEQ ID NO: X;
A peptide consisting of an amino acid sequence to which an appropriate amino acid sequence is added to the N-terminus and / or C-terminus of the amino acid sequence represented by SEQ ID NO: X and having a muscarinic receptor activating effect; or represented by SEQ ID NO: X Peptides that include an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence and that exhibit a muscarinic receptor activating action are included.

The amino acid sequence represented by SEQ ID NO: 21 is
XXXP (SEQ ID NO: 21)
[In the sequence, the first amino acid X is W, R (preferably W), the second amino acid X is Y, H, R, and the third amino acid X is T, R, I (preferably T )]
It is.

As a sequence containing the amino acid sequence represented by SEQ ID NO: 21, for example,
XXXXP (SEQ ID NO: 22)
[In the sequence, the first amino acid X is T, L, H, F, S, the second amino acid X is W, R (preferably W), and the third amino acid X is Y, H, R And the fourth amino acid X is T, R, I (preferably T)]
XXXXXX (SEQ ID NO: 23)
[In the sequence, the first amino acid X is Y, F (preferably Y), the second amino acid X is T, L, H, F, S, and the third amino acid X is W, R (preferably W), the fourth amino acid X is Y, H, R, and the fifth amino acid X is T, R, I (preferably T)]
Can be mentioned.

As the amino acid sequences represented by SEQ ID NOs: 21 to 23, for example,
YTWYTP (SEQ ID NO: 8; sequence consisting of amino acids 3 to 8 of SEQ ID NO: 1)
YLWHTP (SEQ ID NO: 9; sequence consisting of amino acids 3 to 8 of SEQ ID NO: 2)
YHWHTP (SEQ ID NO: 10; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 3)
YTWHTP (SEQ ID NO: 11; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 4)
YHWHRP (SEQ ID NO: 12; sequence consisting of amino acids 1 to 6 of SEQ ID NO: 5)
FFWHTP (SEQ ID NO: 13; sequence consisting of amino acids 7 to 12 of SEQ ID NO: 6)
FFWRTP (SEQ ID NO: 14; sequence consisting of amino acids 5 to 10 of SEQ ID NO: 7)
YSWYTP (SEQ ID NO: 15)
YLWHIP (SEQ ID NO: 17)
HWHTP (SEQ ID NO: 19)
YHRHTP (SEQ ID NO: 20)
Can be mentioned.

  “Peptide consisting of an amino acid sequence in which an appropriate amino acid sequence is added to the N-terminal and / or C-terminal (preferably C-terminal) of the amino acid sequence represented by SEQ ID NO: X and having a muscarinic receptor activating effect” Examples of the amino acid sequence that can be added to the N-terminus and / or C-terminus include a linker sequence, a marker sequence, a polypeptide sequence, or another muscarinic receptor activating peptide sequence.

  Examples of the linker sequence include a sequence for supporting a peptide on a carrier, for example, an amino acid having a thiol group [for example, cysteine (L-form cysteine or D-form cysteine) or homocysteine) or a functional group that does not react with an amino group A linker sequence consisting of one amino acid having (for example, a maleimide group) in the side chain, or a linker sequence in which at least one terminal is an amino acid having a thiol group or an amino acid having a functional group that does not react with an amino group Can do.

  As the marker sequence, for example, a sequence for easily confirming expression of a peptide, confirmation of intracellular localization, purification or the like can be used. For example, FLAG tag, hexa-histidine tag, hemagglutinin -A tag, a myc epitope, etc. can be mentioned.

  Examples of the polypeptide sequence include, for example, a purification polypeptide [for example, all or part of glutathione S-transferase (GST)], a detection polypeptide [for example, all of hemagglutinin or β-galactosidase α peptide (LacZ α). Or a part thereof], or a polypeptide for expression (for example, a signal sequence).

  In “a peptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: X and exhibiting a muscarinic receptor activating activity”, The number of amino acids that can be substituted and / or added is preferably 1 to 10, more preferably 1 to 8, more preferably 1 to 6, still more preferably 1 to 4, and still more preferably 1. ~ 3, more preferably 1 or 2, particularly preferably 1.

In this specification, it is preferable that the amino acid substituted in order to maintain the function of a peptide is an amino acid which has a property similar to the amino acid before substitution. For example, amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substituting these amino acids with other amino acids in the group often does not compromise the essential function of the protein. Such amino acid substitution is called conservative substitution and is known as a technique for converting an amino acid sequence while retaining the function of a polypeptide.
Nonpolar amino acids: Gly, Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp
Neutral amino acids: Ser, Thr, Cys, Tyr, Asn, and Gln
Acidic amino acids: Asp and Glu
Basic amino acids: Lys, Arg, and His

  The amino acid sequence represented by SEQ ID NOs: 21 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence can be used as an active ingredient in the present invention. The “derivative of a peptide including and exhibiting a muscarinic receptor activating action” is not particularly limited as long as it is a derivative of the peptide and exhibits a muscarinic receptor activating action. Examples of such derivatives include peptide derivatives that have been subjected to various modifications that improve peptide stability. Examples of the modification include D-formation of L-form amino acid (eg, D-formation of N-terminal amino acid, D-formation of C-terminal amino acid, D-formation of amino acids other than N-terminal and C-terminal), N-terminal amino acid Acetylation of the group, amidation of the C-terminal carboxyl group, substitution of natural amino acids with non-natural amino acids (similar in nature), or combinations thereof.

  Since the muscarinic receptor activating peptide, which is an active ingredient of the muscarinic receptor activator according to the present invention, has a muscarinic receptor activating action, the muscarinic receptor activator of the present invention is, for example, a pharmaceutical (for example, glaucoma, Sjogren's syndrome (Therapeutic agent) can be used.

  When the muscarinic receptor activator of the present invention is used as a medicine, for example, the peptide itself, or optionally with a conventional pharmaceutically or veterinary acceptable carrier or diluent. Can be administered to animals, preferably mammals (especially humans). In this case, a polynucleotide encoding the peptide (preferably, an expression vector containing the polynucleotide) can be used instead of the peptide [eg, Gene Ther., Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle., 2002 Mar; 9 (6): 372-80].

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: Screening of muscarinic receptor 2 (M2) binding peptide by phage display method >>
(1) Preparation of cell membrane fraction containing fusion polypeptide of M2 and Gi1a In this example, cell membrane fraction containing human hGPCR44-Giα fusion polypeptide described in Example 1 of JP-A-2005-325055 According to the preparation method, a muscarinic M2-Gi1a fusion polypeptide in which the C-terminus of human M2 (Gene Accession Number NM_000739) and the N-terminus of human Gi1a subunit (Gene Accession Number NM_002069) are linked is produced in insect cell Sf9. Thereafter, the cell membrane fraction containing the fusion polypeptide was prepared by collecting the cell membrane fraction.

  Specifically, a fusion gene (M2-Gi1a fusion gene) of a human M2 gene and a human Gi1a subunit gene is inserted into a multicloning site of a vector pFASTBac1 (manufactured by Invitrogen) for producing a recombinant baculovirus. Then, Escherichia coli DH10Bac (manufactured by Invitrogen) that retains the baculovirus genome as a bacmid was transformed, and the bacmid in which the M2-Gi1a fusion gene was inserted was isolated. The isolated recombinant bacmid (baculovirus genome) was transfected into insect cell Sf9 (manufactured by Invitrogen) to obtain a recombinant baculovirus having the M2-Gi1a fusion gene. A cell membrane fraction containing the M2-Gi1a fusion polypeptide was obtained by infecting this recombinant virus with fresh Sf9 cells, collecting the cells after culture, disrupting the cells, and further collecting the cell membrane fraction. I got it. The transformation, bacmid isolation, transfection, and infection were performed according to the attached protocol.

(2) Screening of M2-binding peptides from peptide-displaying phage library Subsequently, the obtained cell membrane fraction was used to screen for peptides having binding properties to human M2 by the phage display method.
The specific operation was performed according to the screening method described in Example 2 of JP-A-2005-325055. In addition, as a library used in the phage display method, a library in which peptides are randomly displayed at the N-terminus of the minor protein pIII on the surface of M13 phage (a peptide library having 7 or 12 random amino acids to be displayed) Was prepared based on the description of Smith, GP, Science, 288, 1315-1317 (1985), JK Scott and GP Smith, Science, 249, 386-390 (1990). The binding of the phage library to the target (M2) was determined by ELISA (enzyme-linked immunosorbent assay).

As a result of screening, seven types of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 7 shown below were obtained as amino acid sequences showing binding to human M2.
NA YTWYTP EVLS (SEQ ID NO: 1; 12 amino acids)
TS YLWHTP AEVP (SEQ ID NO: 2; 12 amino acids)
YHWHTP E (SEQ ID NO: 3; 7 amino acids)
YTWHTPV (SEQ ID NO: 4; 7 amino acids)
YHWHRPP (SEQ ID NO: 5; 7 amino acids)
HSTQNTFFWHTP (SEQ ID NO: 6; 12 amino acids)
SHSEFFWRTPLP (SEQ ID NO: 7; 12 amino acids)

A homology search by BLAST (Basic local alignment search tool) was performed on each of the above underlined sequences having high homology among these sequences. As a sequence having 70% or more homology, amino acids of known proteins were obtained. The following homologous sequences were obtained from the sequences. The underlined portion in the homologous sequence indicates a mismatched amino acid. Also, underlined space "" Means that the corresponding amino acid does not exist because it is an N-terminal or C-terminal sequence.
[1] YTWYTP (SEQ ID NO: 8; Nos. 3 to 8 of SEQ ID NO: 1)
83% Y S WYTP tomato (Lycopersicon esculentum) aldehyde oxidase [SEQ ID NO: 15]
83% YTWYT Arabidopsis thaliana CKI1 (CYTOKININ-INDEPENDENT 1) [SEQ ID NO: 16]
[2] YLWHTP (SEQ ID NO: 9; Nos. 3 to 8 of SEQ ID NO: 2)
83% YLWH I P Arabidopsis (Arabidopsis thaliana) exo strike Shin family proteins (exostosin family protein) [SEQ ID NO: 17]
83% YLWHT Rice (Oryza sativa) expressed protein [SEQ ID NO: 18]
[3] YHWHTP SEQ ID NO: 10; Nos. 1 to 6 of SEQ ID NO: 3)
83% HWHTP Nicotiana langsdorffii nectarin III etc. [SEQ ID NO: 19]
83% YH R HTP rice (Oryza sativa) homeodomain transcription factor (homeodomain transcription factor), etc. [SEQ ID NO: 20]

<< Example 2: Confirmation of agonist activity of 12AA peptide (SEQ ID NO: 1) >>
Of the seven types of amino acid sequences obtained in Example 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as 12AA peptide) was synthesized and the agonist activity against M2 was confirmed.
The agonist activity was evaluated by measuring the amount of europium-labeled GTP (GTP-Eu) incorporated into the M2-Gi1a fusion polypeptide under the reaction conditions shown in Table 1 by a time-resolved fluorescence measurement method. In addition, the concentration of 12AA peptide was implemented in four steps (1 nmol / L, 100 nmol / L, 10 μmol / L, 1 mmol / L).

[Table 1]
10 mmol / L MgCl ₂
10μmol / L GDP
1000 μg / mL saponin 50 mmol / L HEPES
M2-Gi1a fusion polypeptide-containing Sf9 cell membrane predetermined concentration (4 steps) 12AA peptide
100 nmol / L GTP-Eu
Total volume 110μL / well

The results are shown in FIG. The vertical axis in FIG. 1 is the fluorescence intensity (unit: count).
As is clear from FIG. 1, the amount of GTP-Eu incorporated into the M2-Gi1a fusion polypeptide increased with an increase in the concentration of the 12AA peptide, confirming that the 12AA peptide has agonist activity for M2. It was.

<< Example 3: Confirmation of agonist activity of 6AA peptide (No. 3 to 8 of SEQ ID NO: 1) >>
In this example, a peptide having a sequence consisting of amino acids 3 to 8 in SEQ ID NO: 1 (hereinafter referred to as 6AA peptide) was synthesized, and agonist activity against M2 was confirmed.
This example was performed according to the activity measurement method described in Example 3 of JP-A-2005-325055 except that the synthetic peptide was used instead of the peptide-displayed phage. Specifically, the reaction was performed at 30 ° C. for 30 minutes under the reaction conditions shown in Table 2, and then evaluated by measuring the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide. Carbachol is a known agonist of muscarinic receptor, and the concentration of 6AA peptide was 8 steps (10 ⁻² to 10 ⁻⁹ mol / L).

[Table 2]
20 mmol / L HEPES buffer (pH 7.0)
1mmol / L EDTA
160mmol / L NaCl
1 mmol / L dithiothreitol (DTT)
10 mmol / L MgCl ₂
1μmol / L GDP
1 mmol / L carbachol 0.2 mg / mL M2-Gi1 fusion polypeptide-containing Sf9 cell membrane predetermined concentration (8 steps) 6AA peptide
100 pmol / L [ ³⁵ S] GTPγS
Total volume 100μL

The results are shown in FIG.
As is clear from FIG. 2, the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide increases with an increase in the concentration of 6AA peptide, and the 6AA peptide has an agonistic activity against M2. confirmed.

  The muscarinic receptor activator of the present invention can be applied to the treatment of glaucoma, Sjogren's syndrome and the like.

Each amino acid sequence represented by the sequences of SEQ ID NOs: 1 to 14 and 21 to 23 in the sequence listing is an M2-binding peptide.
In the amino acid sequence represented by SEQ ID NO: 21 in the Sequence Listing, the first amino acid “Xaa” is W, R, the second amino acid “Xaa” is Y, H, R, and the third amino acid “Xaa” is T, R, I.
In the amino acid sequence represented by SEQ ID NO: 22 in the Sequence Listing, the first amino acid “Xaa” is T, L, H, F, S, and the second amino acid “Xaa” is W, R. The third amino acid “Xaa” is Y, H, R, and the fourth amino acid “Xaa” is T, R, I.
In the amino acid sequence represented by the sequence number 23 in the sequence listing, the first amino acid “Xaa” is Y, F, and the second amino acid “Xaa” is T, L, H, F, S, The third amino acid “Xaa” is W, R, the fourth amino acid “Xaa” is Y, H, R, and the fifth amino acid “Xaa” is T, R, I.

It is a graph which shows the change of the uptake | capture amount of GTP-Eu to M2-Gi1a fusion polypeptide when the density | concentration of 12AA peptide (sequence number 1) is changed. Changes in the amount of [ ³⁵ S] GTPγS incorporated into the M2-Gi1a fusion polypeptide when the concentration of the 6AA peptide (Nos. 3 to 8 in SEQ ID NO: 1) is changed in the presence of a known agonist (carbacol). It is a graph to show.

Claims (8)

  A peptide comprising the amino acid sequence represented by SEQ ID NO: 21, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and exhibiting an activity of activating muscarinic receptors, Alternatively, a muscarinic receptor activator comprising a derivative thereof as an active ingredient.   The active ingredient contains the amino acid sequence represented by SEQ ID NO: 22, or the amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and the muscarinic receptor activation The muscarinic receptor activator according to claim 1, which is a peptide having an action or a derivative thereof.   The active ingredient includes the amino acid sequence represented by SEQ ID NO: 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and activated muscarinic receptor The muscarinic receptor activator according to claim 1, which is a peptide having an action or a derivative thereof.   The muscarinic receptor activator according to claim 2 or 3, wherein the amino acid sequence represented by SEQ ID NO: 22 or 23 is SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 19, or SEQ ID NO: 20.   The muscarinic according to any one of claims 1 to 3, wherein the first amino acid Xaa in SEQ ID NO: 21, the second amino Xaa in SEQ ID NO: 22, or the third amino acid Xaa in SEQ ID NO: 23 is W. Receptor activator.   Including the amino acid sequence represented by any one of SEQ ID NOs: 21 to 23, or an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence, and the number of constituent amino acids is The peptide or its derivative which is 100 or less.   A polynucleotide encoding the peptide according to claim 6.   An expression vector comprising the polynucleotide according to claim 7.

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