JP3422963B2 - Peptides and their uses - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide that inactivates human T cells that specifically react with cedar pollen allergen, and an immunotherapeutic agent containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art In recent years, in Japan, the number of people complaining of rhinitis and conjunctivitis due to cedar pollinosis has been increasing in Japan in the early spring. Cedar pollinosis is a type of allergic disease, and it is said that the main cause thereof is an antigenic substance in cedar pollen, that is, a cedar pollen allergen. When cedar pollen scattered in the air enters the human body, an immunoglobulin E antibody against the cedar pollen allergen is produced. When cedar pollen invades next in this state, the allergen in the pollen and the immunoglobulin E antibody cause an immune reaction, and allergic symptoms are exhibited.

It is known to date that there are at least two types of allergens having different antigenicities in cedar pollen. One of them is the article by Jasueda [J. Allergy.
Clin. Immunol. 71, 77-86 (1983)], which is a protein called "Cryj1", and the other is the protein of Taniyai et al. [FEBS Letters, 239, 329-332 (19).
88)] and Sakaguchi et al. [Allergy, No. 45, 309].
-312 (1990)], it is a protein called "Cryj2". Cryj1 and Cry
To date, the entire amino acid sequence of both j2 has been determined [WO93 / 01213 and JP-A-8-5052].
No. 84].

In Japanese cedar pollen, Cryj1 and Cry are usually used.
j2 is present in a ratio of about 50: 1 to 5: 1, and most of the sera collected from hay fever patients have Cr and j.
It is said to react to yj2. Sawaya et al. [Allergy, Vol. 42, pp. 738-747 (1993)] describes Cryj1 in the intradermal reaction test and RAST test.
And Cryj2 are reported to exhibit similar antigenicity.

Since several cedar pollen allergens have already been isolated and their properties and properties have been elucidated to some extent, the purified cedar pollen allergens were administered to humans to desensitize them to obtain cedar pollinosis. The prospect of being able to treat and prevent Recently, some desensitizing agents for that purpose have been devised. For example, JP-A-1-156926 and JP-A-3-93730 disclose a complex produced by covalently binding a sugar to a cedar pollen allergen. Has been proposed as a desensitizing agent for humans.

However, a large amount of high-purity allergen is usually required for the diagnosis of allergic disease and desensitization therapy, and the allergen in cedar pollen is scarce and its stability is low, so that the diagnosis of cedar pollinosis is made. There is a great deal of difficulty in trying to cover the agents and desensitizing agents with cedar pollen alone. Therefore, in the recent treatment / prevention of allergic diseases, instead of administering the whole allergen to the patient as in the past, the minimum region specifically recognized by T cells in the allergen, that is, Immunotherapy in which a low-molecular peptide consisting essentially of a T cell epitope is administered has attracted attention.

[0007] Generally, when an allergen is taken up by antigen-presenting cells such as macrophages, it is digested there,
The digested fragments are HLA (Human Leukocyt
e Antigen) will bind to the protein and be presented as an antigen. The fragment presented as an antigen is limited to a part of a specific region in the allergen due to affinity for the HLA protein and the like, and the region specifically recognized by T cells is generally called "T cell epitope". .
For immunotherapy in which a peptide consisting essentially of a T cell epitope is administered, (i) the peptide lacks a B cell epitope, that is, an immunoglobulin E antibody specific for the allergen does not react, and thus the conventional crude Or side effects such as anaphylaxis that frequently occur with purified allergens cannot occur; (ii) The time period from starting with a small amount to reaching an effective dose can be significantly shortened compared to conventional desensitizing agents; (Iii) It has advantages such as inducing oral tolerance and reducing an allergic reaction to an allergen. However, all cedar pollinosis patients do not respond uniformly to a common T cell epitope, and in some cases administration of only one type of T cell epitope may have no effect. A reliable effect cannot be expected unless it is administered after investigating whether it reacts with the epitope or if multiple T cell epitopes are prepared and administered simultaneously. However, in the former method, it is necessary to previously check which T cell epitope the patient responds to, and in the latter method, a plurality of T cell epitopes must be prepared separately.

Therefore, in recent years, attempts have been made to use peptides in which a plurality of different T cell epitopes are artificially linked [see, for example, Japanese Patent Publication No. 7-503362]. In addition, such a linked T cell epitope has also been prepared in a cedar pollen allergen [JP-A-10-259].
No. 198].

[0009]

The first object of the present invention is to provide a prophylactic or therapeutic effect against cedar pollinosis to a wide range of people at a lower dose than conventionally known, which is excellent in cedar pollen. It is intended to provide a peptide having a continuous amino acid sequence of a plurality of human T cell epitopes derived from different allergens.

The second object of the present invention is to provide a DNA encoding the above peptide. A third object of the present invention is to provide a method for producing the above peptide. A fourth object of the present invention is to provide an anti-cedar pollinosis agent containing the above peptide as an active ingredient.

[0011]

[Means for Solving the Problems] The present inventors newly defined the range of T cell epitopes which are particularly excellent in the effect as a desensitizing therapeutic agent for cedar pollinosis. Furthermore, using these T cell epitopes, peptides were produced that could exert an excellent preventive or therapeutic effect on cedar pollinosis in a wider range of people than previously known. On the other hand, the present inventors have found that the peptides of the present invention in which irrelevant amino acids do not enter between the linked T cell epitopes have lower doses of cedar pollinosis than those obtained by simply mixing the respective T cell epitopes. It was revealed that immunotherapy has a therapeutic or preventive effect. The present inventors have completed the present invention by providing a preventive or therapeutic agent for cedar pollinosis containing these peptides as an active ingredient.

That is, the present invention provides the following formula (I): α ₁ -α ₂ -α ₃ -α ₄ -α ₅ -α ₆ -α ₇ (I) (wherein α _{1 to} α ₇ are different from each other). SEQ ID NO: 16 or SEQ ID NO: 7 in the sequence listing, SEQ ID NO: 17 in the sequence listing,
SEQ ID NO: 18 or SEQ ID NO: 8 of the sequence listing, SEQ ID NO: 19 of the sequence listing, SEQ ID NO: 20 or SEQ ID NO: 21 of the sequence listing,
Which represents an amino acid sequence selected from the group consisting of SEQ ID NO: 22 in the sequence listing and SEQ ID NO: 23 in the sequence listing), a peptide containing the amino acid sequence, a complex thereof, a derivative thereof or a polymer thereof; II): α ₁ -α ₂ -α ₃ -α ₄ -α ₅ -α ₆ (II) (wherein α _{1 to} α ₆ are different from each other, SEQ ID NO: 16 in the sequence listing and SEQ ID NO: in the sequence listing) 18, an amino acid sequence selected from the group consisting of SEQ ID NO: 19 of the sequence listing, SEQ ID NO: 20 or SEQ ID NO: 21 of the sequence listing, SEQ ID NO: 22 of the sequence listing and SEQ ID NO: 23 of the sequence listing). A peptide comprising a sequence, a complex thereof, a derivative thereof or a polymer thereof; a peptide consisting of the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing; and a peptide comprising the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing .

The present invention will be described in detail below. The present invention has 6 to 7 selected one from each of the amino acid sequence groups A, B, C, D, E, F and G defined below.
Kind, more preferably seven kinds of amino acid sequences linked in any order, a complex thereof, a derivative thereof or a polymer thereof is provided: Group A: the amino acid sequence shown in SEQ ID NO: 16 of the sequence listing (hereinafter "A") or the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing (hereinafter referred to as "A '"); B: the amino acid sequence represented by SEQ ID NO: 17 in the sequence listing; C group: represented by SEQ ID NO: 18 in the sequence listing The amino acid sequence shown (hereinafter referred to as "C") or the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing (hereinafter referred to as "C '"); D: the amino acid sequence shown in SEQ ID NO: 19 in the sequence listing; E group: Amino acid sequence represented by SEQ ID NO: 20 in the sequence listing (hereinafter referred to as "E") or amino acid sequence represented by SEQ ID NO: 21 in the sequence listing (hereinafter referred to as "E '"); F: In SEQ ID NO: 22 in the sequence listing Amino acid sequences; G: amino acid sequence shown in SEQ ID NO: 23.

Further, the present invention has an amino acid sequence which constitutes a novel human T cell epitope, and has the above amino acid sequence A'or C'which is useful as an active ingredient of a hyposensitizing therapeutic agent for cedar pollinosis. Provide a peptide. Here, "amino acid sequence constituting a human T cell epitope"
And, when the peripheral blood mononuclear cell group derived from a Japanese cedar pollinosis patient was cultured in the presence of a peptide having the amino acid sequence,
Activity for increasing the DNA synthesis rate of the peripheral blood mononuclear cell group to more than 2 times, more preferably 5 times or more, that of the peripheral blood mononuclear cell group cultured in the absence of the peptide having the amino acid sequence Having an amino acid sequence of In the above, E group, F and G are cedar pollen allergen Cr
It is a group of amino acid sequences constituting a human major T cell epitope derived from yj1, and all others are a group of amino acid sequences constituting a human major T cell epitope derived from Cryj2.

Further, as preferred peptides in the present invention, the following examples can be mentioned: Peptide 1 (SEQ ID NO: 1 in Sequence Listing); Peptide 2 (SEQ ID NO: 2 in Sequence Listing); Peptide 3 (Sequence in Sequence Listing) No. 3); Peptide 4 (SEQ ID NO: 4 of Sequence Listing); Peptide 5 (SEQ ID NO: 5 of Sequence Listing); Peptide 6 (SEQ ID NO: 6 of Sequence Listing); Peptide 7 (SEQ ID NO: 7 of Sequence Listing); Peptide 8 (SEQ ID NO: 8 in the sequence listing); peptide 9 (amino acid numbers 1 to 185 in SEQ ID NO: 10 in the sequence listing); peptide 10 (amino acid number 1 in SEQ ID NO: 12 in the sequence listing)
To 209); peptide 11 (SEQ ID NO: 13 of the sequence listing); and peptide 12 (amino acid number 1 of SEQ ID NO: 15 of the sequence listing).
To 95).

The above-mentioned peptide 1 has an amino acid sequence which constitutes the human major T cell epitope of the cedar pollen allergen protein, in which the amino acid sequence is linked from the N-terminus in the order of A-E-C-D-G-F. . Similarly, Peptides 2-6 have the following structures: Peptide 2: AE'-C-D-F-G; Peptide 3: A-E'-C-D-G-F; Peptide 4 : B-A-E-C-D-F-G; Peptide 5: B-A'-E-C'-D-F-G.

Peptide 6 is at the N-terminal side of peptide 5,
It has an amino acid sequence to which a sequence Gly-Asp-Pro-Arg consisting of 4 amino acid residues is added. Peptide 7 and peptide 8
Is the peptide itself having the amino acid sequences of A ′ and C ′ above, respectively.

Peptide 9 is a unit having a structure represented by AECDFG (hereinafter referred to as "H6-1").
Amino acid Nos. 4 to 84 of SEQ ID No. 25 in the sequence listing),
A linker peptide having the following amino acid sequence: Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser-Val-Pro-Gly-Asp-Pr
o-Arg (L1, SEQ ID NO: 26 in the sequence listing); Arg-Ala-Asp-Pro-Ar
g (L2, SEQ ID NO: 27 of the sequence listing); and Arg-Ala-As
p-Leu (L3, SEQ ID NO: 80 in the sequence listing) is (L1)-(H6-1)-(L2)-(H6-1)-
(L3) has a linked polymer structure. These linkers are unrelated to the cedar pollen allergen, and were added or inserted to easily produce a polymer of H6-1 in E. coli using recombinant DNA technology. It does not affect the activity itself. Similarly, peptide 10 is AECCDGF
A unit having a structure represented by -B (hereinafter referred to as "H7-1". SEQ ID NO: 29 in the sequence listing) and the linker peptide are (L1)-(H7-1)-(L2)-(H7 -1)-
(L3) has a linked structure.

Peptides having these polymer structures, which have a short linker intervening, have the same effects as the monomers as described above, and can be produced simply and in large quantities by recombinant DNA technology. Has the advantage. As will be described in Examples below, this polymer can be used as it is, but can also be decomposed and used as a monomer.
Moreover, the peptide 12 is A'-EC'-DFG-
It has a structure represented by B, and peptide 11 is peptide 12
Has a structure in which Arg is added to the C terminus of.

Further, permutations of T cell epitopes other than those listed above, for example, A'-BC'-D-E-F-G.
And G-F-D-D-C'-B-A 'are also suitable as the peptide of the present invention. Furthermore, A ', B, C', D,
The permutation of 7 kinds of T cell epitopes of E, F and G is 7 × 6 × 5 × 4 × 3 × 2 × 1 = 5040 kinds including the above, and the number of peptides connecting these T cell epitopes is 5040 kinds. The peptide of the present invention has a positive T cell epitope activity when examined by a method for measuring uptake of tritiated thymidine by T cells specific to cedar pollen allergen, which will be described below. Since the permutation itself is not particularly limited, those peptides are also included in the present invention.

Of the above peptides, more preferred in the present invention are peptides selected from the group consisting of peptides 4, 5, 6, 10, 11 and 12, and particularly preferred are peptides 5, 6, 11 And a peptide selected from the group consisting of 12 and 12, most preferably peptide 11 or 12.

Further, the peptide of the present invention has, in addition to the above-mentioned constitution, N of T-cell epitope linked
It also includes the presence of an additional amino acid sequence unrelated to the constitution of T cell epitope at the terminal and / or C terminal (for example, Gly-Asp-Pro-A at the N-terminal of peptide 6).
a sequence represented by rg, a linker for peptide 9 or 10,
Arg at the C-terminus of peptide 11). The structure of such an additional amino acid sequence is such that it impairs the function of the peptide of the present invention as a T cell epitope donor, or has some harmful activity such as toxicity when administered to humans. Unless otherwise, there is no particular limitation.

In particular, peptide 11 is obtained by the production method according to the present invention described later, and Arg
Has the same effect as peptide 12. Further, the present inventors have conducted various studies on methods for obtaining the peptides of the present invention, and have found suitable DNAs, recombinant vectors, host cells, and methods for producing peptides, in order to achieve the object of the present invention. Furthermore, the present inventors have found that an anti-cedar pollinosis agent containing the peptide according to the present invention as an active ingredient is effective for a wider range of cedar pollinosis patients, is active at a low dose, and has a small dose. I found that I can do it.

[0024]

The peptides described in the present invention can be prepared by peptide synthesis methods known in the art known as "solid phase method" or "liquid phase method". For example, "Renewal Chemistry Experiment Course", Volume 1, "Protein VI", edited by The Society of Biochemistry, Japan, pp. 3 to 44, 1992.
The details of peptide synthesis are described in, for example, published by Tokyo Kagaku Dojin. In addition, the peptide used was Fmoc (9-fluorenyl methyloxycarbonyl) using a multi-peptide synthesizer (Symphony, manufactured by Protein Technology).
It can be synthesized by the solid phase synthesis method according to the protocol of the same apparatus. That is, Fmoc-L-amino acid W into which an amino acid corresponding to the C-terminal of each peptide to be synthesized has been introduced
Set the ang resin (or Cl-Trt resin) in the reaction vessel of the peptide synthesizer, and use the deprotection solution.
Excluding Fmoc. Further, react an amino acid solution corresponding to the second amino acid from the C-terminal with an activator solution,
After the reaction, the Fmoc group is deprotected again, and the same procedure is repeated to synthesize the desired peptide. In the above method, the yield tends to decrease as the peptide to be synthesized generally becomes longer. It is possible to maintain a high yield by activating and reducing the number of C-terminal amino acids capable of subsequent reactions.

The peptides of the present invention are not limited to those prepared by chemical synthesis, and may be those prepared by recombinant DNA technology. For example, the peptides 1 to 1 above.
The DNA encoding any one of 2 was prepared and inserted into a vector capable of self-sustaining growth, and E. coli, Bacillus subtilis,
The peptide of the present invention may be collected from the culture by introducing it into a host such as actinomycete or yeast to form a transformant.

Accordingly, the present invention provides a DNA containing a nucleotide sequence encoding the above-mentioned peptide of the present invention. In the present invention, those incorporated in the plasmid vector retained in the transformed Escherichia coli described below can be preferably used. In another aspect, the present invention provides the above D.
A recombinant vector containing NA is provided. In the present invention, as the recombinant vector, those incorporated in a vector capable of expressing a peptide consisting of the amino acid sequence encoded by the above DNA can be preferably used.

A method for preparing a DNA having a desired nucleotide sequence is, for example, chemically synthesizing sense and antisense nucleotides which are partial sequence nucleotides of the desired DNA and have overlapping ends. Chain reaction [Saiki, RK et
al (1988) Science 239, 487-491] and the like, and a method for obtaining a product in which those partial sequences are linked by utilizing a DNA polymerase reaction or a ligase reaction. For example, as a microorganism transformed with a vector into which a DNA encoding the peptide of the present invention artificially synthesized in this manner is incorporated, D encoding peptide 9 is used.
Vector incorporating NA, vector incorporating DNA encoding peptide 10, and peptide 1
1 or Escherichia coli strain E. coli transformed with the vector into which the DNA encoding peptide 12 is incorporated. co
li pBR (h6-1) SANK 70199,
E. coli pBR (h7-1) SANK 702
99 and E.I. coli pBR (h7-3) SAN
K 70399 has been internationally deposited with the Institute of Biotechnology, Institute of Biotechnology, February 9, 1999, with deposit numbers FERM BP-6642 and FERM BP-, respectively.
6643 and FERM BP-6644 are attached. The most preferable DNA encoding the peptide of the present invention can be obtained from the above-mentioned deposited strain by a general genetic engineering technique, but the DNA encoding the peptide of the present invention is not limited thereto.

A prokaryotic or eukaryotic host cell can be transformed by incorporating the DNA encoding the amino acid sequence of the peptide of the present invention obtained as described above into a suitable vector. Furthermore, by introducing an appropriate promoter and a sequence involved in transformation into these vectors, the DNA can be expressed in each host cell. That is, the present invention also relates to a host cell carrying a recombinant vector in which the DNA of the present invention is incorporated in a vector that enables the expression of the peptide of the present invention.

Examples of prokaryotic cells include Escherichia coli and Bacillus subtilis.
Etc. In order to express the gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences.
Further, the vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cell. For example, E. coli is E. coli K12 strain, J
The M109 strain and the like are often used, and pBR322 and pUC type plasmids are generally often used as the vector, but the vector is not limited to these, and various known strains and vectors can be used.

As a promoter, in E. coli, tryptophan (trp) promoter, lactose (lac) promoter, tryptophan-lactose (tac) promoter, lipoprotein (lpp) promoter, bacteriophage-derived lambda (λ) P.
L promoter, polypeptide chain elongation factor Tu (tuf
B) promoter, lacUV5 promoter and the like, and any promoter can be used for production of the peptide of the present invention.

As the Bacillus subtilis, for example, strain 207-25 is preferable, and as the vector, pTUB228 [Ohmura,
K., et al. (1984) J. Biochem. 95, 87-93], etc., but are not limited thereto. As a promoter for Bacillus subtilis, a regulatory sequence of the α-amylase gene of Bacillus subtilis is often used, and if necessary, a DNA sequence encoding a signal peptide sequence of α-amylase is ligated to thereby secrete outside the cell. Will also be possible.

Taking the case of using Escherichia coli as a host cell as an example, an expression vector having a pBR322 origin of replication, capable of autonomous growth in Escherichia coli, and further provided with a transcription promoter and a translation initiation signal is used. be able to. The expression vector is a calcium-chloride method [Mandel, M. and Higa, A. (1970) J. Mol.
Biol. 53, 154], Hanahan's method [Hanahan, D.
and Meselson, M. (1980) Gene 10, 63] and electroporation [Neumann, E., et al. (1982) EMBO J.
1, 841-845], etc., and incorporated into Escherichia coli, and thus cells transfected with the desired vector can be obtained.

Eukaryotic host cells include cells of vertebrates, insects, yeasts and the like. Examples of vertebrate cells are COS cells which are monkey cells [Gluzman, Y. (1981).
Cell23, 175-182] and Chinese hamster ovary cells (CHO) deficient in dihydrofolate reductase [Urlaub, G. and Chasin, LA (1980) Proc. Natl.
Acad. Sci. USA 77, 4216-4220], human Namalwa cells, hamster BHK cells, etc. are often used, but are not limited thereto.

As the expression vector for vertebrate cells, one having a promoter located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used, which is further required. May have a replication origin. As an example of the expression vector, pSV2dhfr having a SV40 early promoter [Subramani, S., et al. (1981) Mo
l. Cell. Biol. 1, 854-864], etc.
It is not limited to this.

Yeast is generally often used as a eukaryotic microorganism. Among them, yeast of the genus Saccharomyces, for example, Saccharomyces cerevisiae.
visiae) is preferred. Examples of eukaryotic expression vectors such as yeast include promoters of alcohol dehydrogenase gene [Bennetzen, JL and Hall, BD (1982)
J. Biol. Chem. 257, 3018-3025] and the promoter of the acid phosphatase gene [Miyanohara, A., et al.
(1983) Proc. Natl. Acad. Sci. USA 80, 1-5] and the like can be preferably used.

Taking the case of using COS cells as a host cell, the expression vector has an SV40 origin of replication and is capable of autonomous growth in COS cells. It is possible to use a device having a part. The expression vector is a DEAE-dextran method [Luth
man, H. and Magnusson, G. (1983) Nucleic Acids Re
s. 11, 1295-1308], calcium phosphate-DNA
Coprecipitation method [Graham, FL and van der Ed, AJ (197
3) Virology 52, 456-457] and electric pulse perforation [Neumann, E., et al. (1982) EMBO J. 1, 841-845.
Reference] etc., and can be incorporated into COS cells,
Thus, the desired transformed cell can be obtained. When CHO cells are used as the host cells, they function as a G418 resistance marker together with the expression vector.
A vector capable of expressing the eo gene, for example pRSVne
o [Sambrook, J., et al. (1989) "Molecular Clonin
g: A Laboratory Manual "Cold Spring Harbor Laborat
ory, NY reference] and pSV2neo [Southern, PJ and
Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341
[Reference] etc., and a G418-resistant colony is selected to obtain a transformed cell stably producing the peptide of the present invention.

As described above, in the present invention, the host cell may be any one so long as it has been transformed so as to produce the peptide of the present invention, and is not particularly limited. And a vector into which a DNA encoding peptide 9 is incorporated, a vector into which a DNA encoding peptide 10 is incorporated, and a vector into which a DNA encoding peptide 11 or peptide 12 is incorporated, respectively. Strain E. coli pBR (h6-1) SANK7019
9, E.I. coli pBR (h7-1) SANK 7
0299 and E.I. coli pBR (h7-3) S
ANK 70399 (consignment number FERMBP-664
2, FERM BP-6643 and FERM BP-
6644) has already been deposited internationally and can be preferably used in the practice of the present invention.

The desired transformant obtained above can be cultured according to a conventional method, and the polypeptide of the present invention is produced intracellularly or extracellularly by the culture. As the medium used for the culturing, various kinds of medium commonly used can be appropriately selected depending on the adopted host cell. For example, in the case of Escherichia coli, tryptone-yeast medium (bactotryptone 1.6%, yeast extract 1 0.0%, sodium chloride 0.5% (pH 7.0)), peptone medium (manufactured by Difco) and the like can be used. As the COS cells, RPMI1640 medium and Dulbecco's modified Eagle medium (DMEM) may be used, to which serum components such as fetal bovine serum (FBS) are added, if necessary.

As described above, the peptide of the present invention produced inside or outside the transformant cell can be isolated or separated by various known separation operation methods utilizing the physical and chemical properties of the protein. It can be purified. Specific examples of such a method include treatment with an ordinary protein precipitant, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC). ) Etc., various chromatographies, dialysis methods, and combinations thereof. When a foreign gene is introduced into Escherichia coli or the like and expressed in large amounts, the produced peptide may form a water-insoluble aggregate called an inclusion body. In such a case, the peptide can be solubilized by denaturing the peptide with a strong denaturing agent such as guanidine isothiocyanate.

Further, the peptide of the present invention is in the form of a complex obtained by adding a sugar or polyethylene glycol to the peptide thus obtained, and further by acetylation, amidation and / or polyfunctional reagent of the peptide. It may be in the form of a derivative or polymer obtained by cross-linking polymerization. In the production of such a complex, derivative or polymer of the peptide of the present invention, the addition of saccharides, etc., acetylation, amidation and / or cross-linking polymerization is carried out from the cedar pollen allergen protein in the peptide of the present invention. It is preferable to perform the above-mentioned additional peptide at the N-terminal and / or C-terminal of the peptide of the present invention, or a linker peptide so as not to impair the function of the amino acid sequence constituting the human T cell epitope.

The complex of the peptide of the present invention comprises, for example, polyethylene glycol, monomethoxy polyethylene glycol, dextran, and maltotriose such as pullulan and erucinan as repeating units at the N-terminal or amino group of Lys residue. Those with added polysaccharides,
Those known to those skilled in the technical field of the present invention can be mentioned, for example, "Newborn Chemistry Experimental Course", Vol. 1, "Protein IV", Vol. 23, edited by The Japan Biochemical Society.
It can be produced according to the description on pages 6 to 252 (1991, issued by Tokyo Kagaku Dojin) and the like.

Examples of the derivative of the peptide of the present invention include those known to those skilled in the technical field of the present invention, such as those having an N-terminal acetylation and those having a C-terminal Gly residue amidated. You can Such derivatives are obtained, for example, in the above-mentioned "Shinsei Kagaku Koza", Vol. 1, "Protein IV", pages 18-20 and Vol. 9, "Hormones I".
It can be manufactured according to the description on pages 290 to 298 (all published in 1991 by Tokyo Kagaku Dojin). further,
Examples of the polymer of the peptide of the present invention include those known to those skilled in the art of the present invention, such as those obtained by polymerizing two molecules of the peptide of the present invention with a divalent crosslinking reagent such as disuccinimidyl suberate. Can be mentioned. Such a polymer can be prepared, for example, according to the description in the above-mentioned "Shinsei Kagaku Koza", Vol. 1, "Protein IV", pages 207 to 226.

Further, the present invention provides a method for producing a peptide by a DNA recombination technique. In particular, a method for easily obtaining a monomer from DNA encoding a peptide having a polymer structure is provided. For example, a polymer in which a plurality of peptides of the present invention are linked via a linker peptide containing an amino acid sequence that can be specifically cleaved by protease is produced in a host such as E. coli, and The peptide of the present invention can also be obtained by digesting the combined product. At that time, the polymer may be either a homopolymer in which one of the peptides of the present invention is polymerized or a heteropolymer in which a plurality of types of peptides of the present invention are polymerized, but it is possible to produce a large amount of a single peptide. If desired, homopolymers are selected. As the protease, any known protease can be used as long as it recognizes and cleaves a specific amino acid sequence.
Preferred are trypsin, cathepsin B, cathepsin D, cathepsin E and the like.

The peptide of the present invention exerts a desired therapeutic / prophylactic effect even when administered in a relatively crude form, but it is usually purified before use. For purification, for example, filtration, concentration, centrifugation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography,
Methods commonly used in the art for purifying peptides or proteins such as affinity chromatography, gel electrophoresis, isoelectric focusing, etc. are used, and these methods may be appropriately combined as necessary. Then, the purified peptide may be concentrated and lyophilized into a liquid or solid form depending on the final use form. The activity of the peptide of the present invention as a T cell epitope can be confirmed by measuring the uptake of tritiated thymidine by T cells specific to cedar pollen allergen.
For this measurement, for example, the method described below can be used.

That is, a peripheral blood mononuclear cell group of a Japanese cedar pollinosis patient is separated by Ficoll-Hypaque gravity centrifuge or the like, and this cell group is suspended in a medium such as RPMI1640 and dispensed on a 96-well microplate. . Next, a peptide as a test substance is added and cultivated. Conditions such as temperature and time during this culture can be appropriately adjusted for each experiment, but 37 ° C. and 3 days are preferable. After that, tritiated thymidine was added to the medium, and the culture was continued for a certain period of time.
The activity of the peptide of the present invention as a T cell epitope can be calculated by measuring the uptake of tritiated thymidine in the mononuclear cell group. In the present invention, a system containing no peptide is provided at the same time as a negative control, and a system in which the tritiated thymidine uptake amount exceeds a value twice that of the negative control is “positive”, and a system in which the system does not reach the value is The result was "negative".

It can be confirmed, for example, by the following experiment that the peptide of the present invention has a therapeutic effect on cedar pollinosis. Since the peptide of the present invention has a human T cell-specific activity, its effect may not be exhibited even when an experiment is conducted in a non-human animal. For example, C3
Since the lymphocytes of H / HeN mice have reactivity with the peptides included in the above-mentioned group A, the effect of the peptide of the present invention, which necessarily contains the amino acid sequence of group A, can be obtained by the method described below. Can be evaluated.

The peptide of the present invention is previously administered to C3H / HeN mice to induce immune tolerance to the peptide. Cryj2 human major T cell epitope peptide having the amino acid sequence described below in the mouse after a certain period of time: Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp (Peptide 13 ; SEQ ID NO: 16) in the sequence listing is administered with an adjuvant such as Freund's complete adjuvant to immunize. After a certain period of time, lymph node cells are removed from the popliteal part of the mouse to prepare a cell suspension. By adding the above-mentioned peptide 13 or Cryj2 to this and continuing the culture, further adding tritiated thymidine, and measuring the uptake amount of tritiated thymidine,
Proliferation of T cells can be measured.

In mice not previously induced with the peptide of the present invention, the T cells specific to the peptide were activated by immunization with peptide 13, and the peptide presented by the antigen-presenting cells in vitro. 1
It proliferates in response to a partial peptide of 3 or Cryj2.
On the other hand, in the mouse in which the peptide of the present invention was used to induce immunological tolerance, subsequent immunization with peptide 13 did not activate the peptide-specific T cells, which were presented by the antigen-presenting cells in vitro. Peptide 1
It does not react with the partial peptide of 3 or Cryj2 and does not grow. By measuring the difference, the therapeutic effect of the peptide of the present invention on cedar pollinosis can be confirmed.

Since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, it is specific to the cedar pollen allergen without substantially causing anaphylaxis when administered to mammals including humans in general. T cells can be inactivated.

The anti-cedar pollinosis agent of the present invention containing the peptide of the present invention, its complex, its derivative or its polymer as an active ingredient substantially causes side effects such as anaphylaxis when administered to a patient suffering from cedar pollinosis. Can be treated without causing it. On the other hand, when the anti-cedar pollinosis agent of the present invention is administered to healthy individuals or individuals with potential cedar pollinosis before the cedar pollen begins to disperse, while exerting a remarkable preventive effect against cedar pollinosis , Remarkably effective in ameliorating allergic symptoms at onset.

The anti-sugi pollinating agent of the present invention will be described in more detail. The anti-sugi pollinating agent of the present invention is usually one or more of the peptide of the present invention, a complex thereof, a derivative thereof or a polymer thereof. 0.01 to 100% (w /
w), preferably 0.05 to 50% (w / w), more preferably 0.5 to 5.0% (w / w). The anti-cedar pollinosis agent of the present invention is not only in the form of the peptide alone, but is physiologically acceptable other than the peptide, for example, serum albumin, gelatin, glucose, sucrose, lactose, maltose, trehalose, sorbitol, maltitol. , Carriers such as lactitol, mannitol, pullulan, excipients, immunostimulants, stabilizers, and if necessary, anti-inflammatory agents and antihistamines such as steroid hormones and sodium cromoglycate,
Includes forms as compositions in combination with one or more other agents including anti-leukotriene agents, anti-tachykinin agents. Furthermore, the anti-cedar pollinosis agent of the present invention also includes a drug in a dosage unit form, and the drug in a dosage unit form means the peptide of the present invention, a complex thereof, a derivative thereof, or a polymer thereof, for example. Means a drug in a physically separate, unitary dosage form suitable for administration containing a daily dose or an integer multiple (up to 4) or equivalent thereof (up to 1/40) . Such dosage unit forms include powders, fine granules, granules, pills, tablets, capsules, troches, syrups, emulsions, ointments, plasters, poultices, suppositories, eye drops. , Nasal drops, sprays, injections and the like.

The anti-cedar pollinosis agent of the present invention is administered orally, dermally, nasally, by instillation or by injection for the purpose of treating or preventing cedar pollinosis. The dosage in humans varies depending on the purpose of administration, method of administration, and symptoms, but usually, the daily dose for adults is 0.0
The dosage is usually 1 to 1000 mg, preferably 1 to 10 mg, once a day or once a month for about 1 to 6 months, and is usually repeatedly administered while increasing the dose.

[0053]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited by these. [Example 1] Peptide 1 A peptide was chemically synthesized by a method in which one amino acid is bonded to a resin-fixed amino acid derivative from the carboxyl terminal side (solid phase synthesis method). Peptide 1 (SEQ ID NO: 1 in the sequence listing) was synthesized in 5 steps as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer.

1) It was considered that the synthesis of long-chain peptides would be possible by reducing the proportion of peptide chains synthesized on the resin. Therefore, the C-terminal amino acid α previously introduced into the resin used for solid phase synthesis
-The amino group was partially blocked with an acetyl group to reduce the proportion of peptides synthesized on the resin, and an attempt was made to synthesize a long-chain peptide.

The amino acid used in each cycle was a special amino acid derivative in which the reactive group in the α-amino group and the residue portion was blocked with a protecting group. For example, the α-amino group of each amino acid derivative is blocked by Fmoc (9-fluorenylmethyloxycarbonyl).
Peptide synthesis was carried out by sequentially repeating the reaction of deprotecting the Fmoc of the α-amino group of the amino acid bound to the resin and then binding the amino acid derivative having the activated carboxyl group (Fmoc method).

For the synthesis of a peptide having Pro at the C terminus,
It is known that the peptide chain is easily lost from the resin when the Wang resin commonly used in the Fmoc method is used. ) Resin (hereinafter referred to as “Cl-Trt resin”) was used.

100 μm of Pro-Cl-Trt resin into which an amino acid (Pro) corresponding to the C-terminal residue of peptide 1 has been introduced
Mol equivalent was placed in a 15 ml polypropylene conical tube, and 5 ml of CAP solution (dimethylformamide: N-methylmorpholine: acetic anhydride = 520: 1: 1 (volume ratio)) containing 96 μmol of acetic anhydride was added for 30 minutes at room temperature. I let it stand. By this operation, a part of the α-amino groups of many C-terminal amino acids introduced into the resin is blocked by the acetyl group, and the subsequent peptide synthesis proceeds only from the α-amino group that has escaped the block. The maximum number of peptides synthesized above is reduced.

This resin was placed in a disposable plastic reaction vessel for a peptide synthesizer Symphony (made by Aloka), the resin was washed with dichloromethane, and then used for a peptide synthesizer Model 430A (made by Applied Biosystems). Transfer to reaction vessel and use Fmoc
Solid-phase synthesis method (FastMoc Chem
wastry)). First from the C end 2
Fmoc-Asp (OtBu) 1mm which is the derivative of the th amino acid
1 ml of an activator solution {2M diisopropylethylamine (DIEA) / N-methylpyrrolidone (hereinafter referred to as "NMP") and 0.45M 2- (1H-
Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (H
OBt) / dimethylformamide 2.2 ml and NM
P 1 ml} activated (10 times equivalent to the bound amino acid) and the resin blocked with the above acetyl group are reacted at room temperature, but at that time, the purpose of sufficiently binding the amino acids Then, a double coupling method was used in which this reaction was repeated twice. Also at this time, Pro-Cl-Trt
Since the amino acid is difficult to bind to the resin, the reaction time is 120
Minutes (usually 30 minutes).

Fmoc-Asp (OtBu) -Pro-Cl-Tr produced here
After washing the t resin 7 times with 7 ml of NMP, 7 ml of the deprotection solution (piperidine / NMP) was reacted twice for 3 minutes and 12 minutes, and the amino acid Fmoc bound to the resin was removed.
The group was removed, and the plate was washed 5 times with 7 ml of NMP solution. Next, activated Fmoc-Tyr (t is a derivative of the third amino acid from the C-terminal side.
The Bu) solution was subjected to double coupling reaction. Hereinafter, the same operation was repeated. However, the piperidine concentration of the Fmoc group deprotection solution was gradually increased from 23% (first cycle) to 36% (20th cycle) according to the length of the peptide to be synthesized.

Peptide from the C-terminal to the 21st residue by the above method: Fmoc-Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe
-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -Gly-Leu-Val-His (T
rt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -As
When the p (OtBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. Take 1 mg of the above resin,
After hydrolysis with N hydrochloric acid at 110 ° C. for 24 hours, the released amino acid was analyzed by an amino acid analyzer (L-8500).
Type, manufactured by Hitachi, Ltd.). as a result,
It was found that the amount of peptide bound to the resin was about 20% of the resin substitution amount.

Further, part of the resin was sampled and
l clavage solution (trifluoroacetic acid (hereinafter "TFA"
: Ethanedithiol: water: triisopropylsilane = 92.5: 2.5: 2.5: 2.5), and the mixture is reacted for 1 hour, and ether is added to the obtained peptide solution to precipitate the peptide. [1997/98 Peptide Synthesis Handbook S48; Novabiochem]. The molecular weight of the obtained peptide was measured by a mass spectrometer (VG platform, manufactured by VG Biotech Co., Ltd.) by an electrospray ionization (hereinafter referred to as “ESI”) method, and a synthesized product was confirmed. .

2) Peptide synthesis was subsequently carried out using the remaining resin of 1) above, and the peptide from the C-terminal to the 31st residue: Fmoc-Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu). -Gly
-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (B
oc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Tyr (t
Bu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt)
When the -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was taken out, a cribage reaction was performed, and a synthetic product was confirmed by the same method as in 1) above.

3) Peptide synthesis was continued using the remaining resin from 2) above, and the peptide from the C-terminal to the 51st residue: Fmoc-Phe-His (Trt) -Leu-Gln (Trt) -Lys ( Boc)-
Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -G
ly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt)
-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-
Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -G
lu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -G
ly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn
When the (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was taken out, a cribage reaction was performed, and a synthetic product was confirmed by the same method as in 1) above.

4) Subsequent peptide synthesis was carried out using the remaining resin of 3) above, and the peptide from the C terminus to the 71st residue: Fmoc-Ser (tBu) -Trp (Boc) -Ser (tBu) -Met. -Lys
(Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -P
he-Gly-Pro-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-
His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -L
ys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala
-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Ser (tBu) -Gly-Lys (B
oc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tB
u) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-As
n (Trt) -Val-Glu (OtBu) -Tyr (tBu) -Gly-Leu-Val-His (Trt)
-Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (O
When the (tBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. In addition, Fm at the 69th residue from the C terminus
The reaction for binding oc-Ser (tBu), Fmoc-Trp (Boc) at residue 70 and Fmoc-Ser (tBu) at residue 71 was predicted to be poor in reaction efficiency, so deprotection The reaction with the solution was repeated three more times so that the Fmoc group bound to the peptide was sufficiently displaced, and then the reaction time for binding the amino acid was 120 minutes.
Part of the resin is collected and subjected to a cribage reaction, and the above 1)
The synthetic product was confirmed by the same method as described above.

5) Subsequent peptide synthesis was carried out using the remaining resin of 4) above, and the peptide from the C-terminal to the 81st residue: Fmoc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln. (Tr
t) -Asn (Trt) -Pro-Ala-Ser (tBu) -Trp (Boc) -Ser (tBu) -Met
-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Tr
t) -Phe-Gly-Pro-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt)-
Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tB
u) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile
-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Ser (tBu) -Gly-L
ys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Ty
r (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Ph
e-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -Gly-Leu-Val-His
(Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu)-
Asp (OtBu) -Pro-Cl-Trt resin was synthesized.

Here, the amino acid derivatives used in the synthesis are as follows: Fmoc-Ala, Fmoc-Arg (Pmc), Fmoc-.
Asn (Trt), Fmoc-Asp (OtBu), Fmoc-Cys (Trt), Fmoc-Gln
(Trt), Fmoc-Glu (OtBu), Fmoc-Gly, Fmoc-His (Trt), Fm
oc-Ile, Fmoc-Leu, Fmoc-Lys (Boc), Fmoc-Met, Fmoc-Ph
e, Fmoc-Pro, Fmoc-Ser (tBu), Fmoc-Thr (tBu), Fmoc-Tr
p (Boc), Fmoc-Tyr (tBu), Fmoc-Val (() represents a protecting group for protecting the reactive group in the residue portion; Perkin Elmer Japan Applied Biosystems Division).

The protected peptide resin obtained as described above was reacted with a deprotection solution to deprotect the N-terminal Fmoc group. Next, after washing 6 times with 7 ml of NMP, it was washed with dichloromethane 3 times, and further dried by blowing argon gas for 15 minutes. The resin was taken out, 30 ml of the cleaved solution was added, and the mixture was reacted at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution.

This peptide solution was applied to a polytetralfluoroethylene (hereinafter referred to as "PTFE") filter (3.
0 μm, manufactured by Advantech Toyo Co., Ltd.) and the filtrate was collected in a centrifuge tube. After concentrating this using an aspirator, 80 ml of cold ether was added to precipitate the peptide. After cooling for a while, this was centrifuged (3,000 rpm, 10 minutes) to recover the precipitate, and the operation of adding cold ether again to disperse and recover the precipitate was repeated three times to wash the precipitate. The obtained precipitate was dried to obtain 252 mg of a crude peptide.

75 mg of this crude peptide was added to 0.1% TFA.
After being dissolved in a 20% aqueous acetonitrile solution containing hexane, it was subjected to high performance liquid chromatography (hereinafter referred to as “HPLC”) under the following conditions: Column: ODS column (TSK-gel ODS-120)
T, particle size 5 μm, pore size 120 Å, column size φ21.
5 mm × 300 mm, manufactured by Tosoh Corporation) Mobile phase: 35-37% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 220 nm Fractions eluted at 23 to 25 minutes were collected, concentrated and lyophilized to give 11.5 mg of the desired peptide. Obtained.

Since the peptide obtained here still contained impurities, it was further purified by HPLC under the following conditions: Column: C22 column (docosyl-B, column size φ)
10 mm × 250 mm, Senshu Co., Ltd.) Mobile phase: 30-31% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 1.5 ml / min Detection wavelength: 220 nm Fractions eluted at 32-34 minutes were collected, concentrated and freeze-dried to obtain the desired peptide 2. 5 mg was obtained.

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. Furthermore, about 100 pmol of this peptide, an amino acid sequence analyzer (P
PSQ-10 type, manufactured by Shimadzu Corporation, using N
As a result of amino acid sequence analysis from the terminal to 10 residues, amino acid numbers 1 to 1 of SEQ ID NO: 1 in the sequence listing
It was confirmed to be in agreement with the amino acid sequence shown in 0.

Example 2 Peptide 2 Peptide 2 (SEQ ID NO: 2 in Sequence Listing) was synthesized as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer. The procedure was divided into 4 steps. 1) Amino acid corresponding to the C-terminal residue of peptide 2 (Gl
u) introduced Fmoc-Glu (OtBu) -Wang resin 100
Add 6 ml of 20% piperidine to 30 μmol
The Fmoc group was eliminated by reacting for minutes at room temperature. After washing the resin, 5 ml of the CAP solution was reacted at room temperature for 30 minutes. By this operation, as in Example 1, about 80% of the α-amino group of the C-terminal amino acid introduced into the resin was used.
Is blocked with an acetyl group and the subsequent peptide synthesis proceeds only from the unblocked α-amino group, thus reducing the maximum number of peptides synthesized on the resin.

After washing the resin, the resin was transferred to a reaction vessel. First, the peptide from the C-terminal to the 21st residue: Fmoc-Asp (O
tBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu)-
Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Ly
s (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wa
When the ng resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. Take out a part of the resin, carry out a cribage reaction,
The synthetic product was confirmed by the method described in 1) of Example 1.

2) Peptide synthesis was continued using the remaining resin from 1) above, and the peptide from the C terminus to the 41st residue: Fmoc-Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-. Ser (t
Bu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His
(Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu)-
Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (O
tBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Bo
c) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtB
When the u) -Wang resin was synthesized, the Fmoc reaction was removed and the synthesis was stopped. A part of the resin was taken out and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

3) Peptide synthesis was continued using the remaining resin from 2) above, and the peptide from the C-terminus to the 61st residue: Fmoc-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-. Phe-A
la-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln
(Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr
(tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (T
rt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val
-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu)
-Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-
Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Bo
When the c) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the de-Fmoc reaction. A part of the resin was taken out and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

4) Peptide synthesis was continued using the remaining resin from 3) above to synthesize the peptide from the C-terminal to the 79th residue. However, the 68th residue from the C terminus
The reaction for binding Fmoc-Trp (Boc) and Fmoc-Ser (tBu) at the 69th residue was predicted to have poor reaction efficiency, so the reaction with the deprotection solution was repeated three more times to bind to the peptide. After the Fmoc group is completely displaced, the reaction time for coupling amino acids is 120
It was postponed for a minute.

By the above method, Fmoc-Gly-Ile-Ile-Ala
-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -T
rp (Boc) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn
(Trt) -Gln (Trt) -Phe-Gly-Phe-Ala-Ser (tBu) -Lys (Boc) -A
sn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Tr
t) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys
(Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (t
Bu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt)
-Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys
(Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr
(tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe
-Asn (Trt) -Val-Glu (OtBu) -Wang resin was obtained. This was subjected to a Fmoc-removing reaction and a cleavage reaction to obtain 194 mg of a crude peptide.

97 mg of this crude peptide was added to 0.1%.
After dissolving in a 20% acetonitrile aqueous solution containing TFA,
HPLC purification was performed under the following conditions: Column: ODS column (TSK-gel ODS-120
T) Mobile phase: 35-38% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 220 nm Fractions eluted at 27 to 28 minutes were collected, concentrated and freeze-dried to obtain 7 mg of the desired peptide. .

Since the peptide obtained here still contained impurities, it was further purified by HPLC under the following conditions: Column: C22 column (docosyl-B) Mobile phase: 31-32% acetonitrile / 0.1 % T
FA, 30 minutes (linear concentration gradient) Flow rate: 1.5 ml / min Detection wavelength: 220 nm Fractions eluted at 20 to 22 minutes were collected, concentrated and lyophilized to obtain the desired peptide 2. 3 mg was obtained.

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. As a result of amino acid sequence analysis from the N-terminal to 10 residues of the purified peptide (100 pmol), it was confirmed that the amino acid sequence was identical to the amino acid sequences shown in amino acid numbers 1 to 10 of SEQ ID NO: 2 in the sequence listing. It was

Example 3 Peptide 3 Peptide 3 (SEQ ID NO: 3 in the Sequence Listing) was synthesized as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer. The procedure was divided into 5 steps. 1) First, using the method described in Example 1, 2 from the C terminus
Peptide up to the first residue: Fmoc-Lys (Boc) -Lys (Boc) -Glu
(OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -Gly
-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Tr
At the time when the t) -Tyr (tBu) -Asp (OtBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the de-Fmoc reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

2) Peptide synthesis was subsequently carried out using the remaining resin from 1) above, and the peptide from the C terminus to the 41st residue: Fmoc-Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-. Ser (t
Bu) -Cys (Trt) -Leu-Asn (Trt) -Ser (tBu) -Gly-Lys (Boc) -Ty
r (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr
(tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt)
-Val-Glu (OtBu) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-A
la-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -P
When the ro-Cl-Trt resin was synthesized, the Fmoc reaction was removed and the synthesis was stopped. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

3) Peptide synthesis was subsequently carried out using the remaining resin from 2) above, and the peptide from the C-terminal to the 51st residue: Fmoc-Phe-His (Trt) -Leu-Gln (Trt) -Lys ( Boc)-
Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -G
ly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt)
-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-
Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -G
lu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -G
ly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn
At the time when the (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Cl-Trt resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

4) Peptide synthesis was continued using the remaining resin from 3) above, and the peptide from the C-terminal to the 61st residue: Fmoc-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-. Phe-A
la-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln
(Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr
(tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (T
rt) -Leu-Asn (Trt) -Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Gl
u (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys
(Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu
(OtBu) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (T
rt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Cl-Tr
When the t resin was synthesized, the Fmoc reaction was removed and the synthesis was stopped. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

5) Peptide synthesis was subsequently carried out using the remaining resin from 4) above to synthesize the peptide from the C-terminal to the 79th residue. However, the 68th residue from the C terminus
The reaction for binding Fmoc-Trp (Boc) and Fmoc-Ser (tBu) at the 69th residue was predicted to have poor reaction efficiency, so the reaction with the deprotection solution was repeated three more times to bind to the peptide. After the Fmoc group is completely displaced, the reaction time for coupling amino acids is 120
It was postponed for a minute.

By the above method, Fmoc-Gly-Ile-Ile-Ala
-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -T
rp (Boc) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn
(Trt) -Gln (Trt) -Phe-Gly-Phe-Ala-Ser (tBu) -Lys (Boc) -A
sn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Tr
t) -Thr (tBu) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys
(Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Ser (t
Bu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Tr
t) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtB
u) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Tyr (tBu) -Gly-Leu
-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -T
An yr (tBu) -Asp (OtBu) -Pro-Cl-Trt resin was obtained. This was subjected to a Fmoc-removing reaction and a cribage reaction to obtain 380 mg of a crude peptide. Of this, 190 mg was
After dissolving in a 20% acetonitrile aqueous solution containing 1% TFA, HPLC purification was carried out under the following conditions: Column: ODS column (TSK-gel ODS-120
T) Mobile phase: 35-37% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 220 nm Fractions eluted at 25 nm to 27 minutes were collected, concentrated and lyophilized to give 15.6 mg of the desired peptide. Obtained.

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. As a result of amino acid sequence analysis from the N-terminal to 10 residues of 100 pmol of the purified peptide, it was confirmed that the amino acid sequence was identical to the amino acid sequences shown in amino acid numbers 1 to 10 of SEQ ID NO: 3 in the sequence listing. It was

Example 4 Peptide 4 Peptide 4 (SEQ ID NO: 4 in the Sequence Listing) was synthesized as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer. The procedure was divided into 5 steps. 1) First, using the method described in Example 2, 2 from the C terminus
Peptide up to residue 1: Fmoc-Asp (OtBu) -Pro-Ser (tB
u) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Tr
t) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtB
When u) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc removal reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

2) Peptide synthesis was subsequently carried out using the remaining resin from 1) above, and the peptide from the C terminus to the 41st residue: Fmoc-Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-. Ser (t
Bu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His
(Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu)-
Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (O
tBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Bo
c) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtB
When the u) -Wang resin was synthesized, the Fmoc reaction was removed and the synthesis was stopped. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

3) Peptide synthesis was continued using the remaining resin from 2) above, and the peptide from the C terminus to the 61st residue: Fmoc-Asn (Trt) -Gln (Trt) -Phe-Gly-Pro- Phe-A
la-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln
(Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Boc) -Leu-Thr
(tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (T
rt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val
-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu)
-Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-
Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Bo
When the c) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the de-Fmoc reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

4) Subsequent peptide synthesis was carried out using the remaining resin of 3) above, and the peptide from the C terminus to the 81st residue: Fmoc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln. (Tr
t) -Asn (Trt) -Pro-Ala-Ser (tBu) -Trp (Boc) -Ser (tBu) -Met
-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Tr
t) -Phe-Gly-Pro-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt)-
Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tB
u) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile
-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-L
eu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt)
-Tyr (tBu) -Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr
(tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr
(tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt)
-When Fal-Glu (OtBu) -Wang resin was synthesized, Fmoc was removed.
After the reaction, the synthesis was stopped. However, the reaction for binding Fmoc-Ser (tBu) at the 69th residue, Fmoc-Trp (Boc) at the 70th residue and Fmoc-Ser (tBu) at the 71st residue from the C-terminus has good reaction efficiency. Since it was predicted that the amino acid was not present, the reaction with the deprotection solution was repeated three more times so that the Fmoc group bound to the peptide was sufficiently displaced, and then the reaction time for binding the amino acid was extended to 120 minutes. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1. 5) Peptide synthesis was subsequently carried out using the remaining resin of 4) above, and peptides from the C-terminal to the 93rd residue were synthesized.

By the above method, Fmoc-Gln (Trt) -Phe-Al
a-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met-G
ly-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt)-
Pro-Ala-Ser (tBu) -Trp (Boc) -Ser (tBu) -Met-Lys (Boc) -Va
l-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-P
ro-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt)
-Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Thr (tBu) -Lys (Boc)-
Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tB
u) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His
(Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu)-
Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (O
tBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Bo
c) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtB
u) -Wang resin was obtained. Fmoc reaction to this thing,
And a cribage reaction was performed to obtain 47.4 m of the crude peptide.
g was obtained. 20% containing 0.1% TFA
After dissolving in acetonitrile aqueous solution, HPLC under the following conditions
Purification was carried out: Column: ODS column (TSK-gel ODS-120
T) Mobile phase: 39-49% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 220 nm Fractions eluted at 22 to 25 minutes were collected, concentrated and freeze-dried to obtain 4.4 mg of the desired peptide. Obtained.

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. Further, as a result of amino acid sequence analysis from the N-terminal to 10 residues of 100 pmol of the purified peptide, it was confirmed that the amino acid sequence was identical to the amino acid sequences shown in amino acid numbers 1 to 10 of SEQ ID NO: 4 in the sequence listing. It was

Example 5 Peptide 5 Peptide 5 (SEQ ID NO: 5 in the Sequence Listing) was synthesized as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer. The procedure was divided into 4 steps. 1) First, according to the method described in Example 2, the peptide from the C-terminal to the 11th residue: Fmoc-Ile-Tyr (tBu) -Thr
(tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt)
When the -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

2) Peptide synthesis was continued using the remaining resin from 1) above, and the peptide from the C-terminal to the 31st residue: Fmoc-Gly-Leu-Val-His (Trt) -Val-Ala-Asn. (Tr
t) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser (tB
u) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Tr
t) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtB
When the u) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

3) Peptide synthesis was subsequently carried out using the remaining resin from 2) above, and the peptide from the C terminus to the 54th residue: Fmoc-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt). -Phe-
His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Lys (Boc) -L
eu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu)
-Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Tr
t) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp
(OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtB
u) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc)
-Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu)
-At the time when Wang resin was synthesized, the synthesis was interrupted after the Fmoc removal reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

4) Peptide synthesis was subsequently carried out using the remaining resin from the above 3), and peptides from the C-terminal to the 95th residue were synthesized. However, the 70th residue from the C terminus
Fmoc-Ser (tBu), 71st residue of Fmoc-Lys (Boc), 72nd residue of Fmoc-Trp (Boc) and 73rd residue of Fmoc-Ser (tBu) are bound by reaction efficiency. Since it was predicted that the reaction was not good, the reaction with the deprotection solution was repeated three more times to ensure that the Fmoc group bound to the peptide was sufficiently displaced, and then the reaction time for binding the amino acid was set to 1
It was extended to 20 minutes.

By the above method, Fmoc-Gln (Trt) -Phe-Al
a-Lys (Boc) -Leu-Thr (tBu) -Gly-Phe-Thr (tBu) -Leu-Met-G
ly-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln (Trt) -Asn (Trt)-
Pro-Ala-Ser (tBu) -Trp (Boc) -Lys (Boc) -Ser (tBu) -Met-Ly
s (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Trt) -Gln (Trt)-
Phe-Gly-Pro-Asp (OtBu) -Ile-Phe-Ala-Ser (tBu) -Lys (Bo
c) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn
(Trt) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc)-
Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gl
y-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (T
rt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -T
yr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Th
r (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Tr
t) -Val-Glu (OtBu) -Wang resin was obtained. Fm to this thing
The oc reaction and the cribage reaction were performed to obtain 134 mg of a crude peptide. After the whole amount was dissolved in a 20% acetonitrile aqueous solution containing 0.1% TFA, HPLC purification was carried out under the following conditions: Column: ODS column (TSK-gel ODS-120
T) Mobile phase: 38-50% acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 5 ml / min Detection wavelength: 230 nm Fractions eluted at 20 to 23 minutes were collected, concentrated and freeze-dried to obtain 14 mg of the desired peptide. .

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. As a result of amino acid sequence analysis from the N-terminal to 10 residues of 100 pmol of the purified peptide, it was confirmed that the amino acid sequence was identical to the amino acid sequences shown in amino acid numbers 1 to 10 of SEQ ID NO: 5 in the sequence listing. It was

Example 6 Peptide 6 Peptide 6 (SEQ ID NO: 6 in the Sequence Listing) was synthesized as described below for the purpose of confirming the synthesis status and adding a reagent to the synthesizer. The procedure was divided into 5 steps. 1) First, according to the method described in Example 2, the peptide from the C terminus to the 21st residue: Fmoc-Asp (OtBu) -Pro-Ser.
(tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn
(Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu
When the (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was collected and subjected to a cribage reaction to give 1) of Example 1.
The synthetic product was confirmed by the method described in 1.

2) Peptide synthesis was subsequently carried out using the remaining resin from 1) above, and the peptide from the C terminus to the 54th residue: Fmoc-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt). -Phe-
His (Trt) -Leu-Gln (Trt) -Lys (Boc) -Asn (Trt) -Lys (Boc) -L
eu-Thr (tBu) -Ser (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu)
-Cys (Trt) -Leu-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Tr
t) -Val-Ala-Asn (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp
(OtBu) -Pro-Ser (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtB
u) -Gly-Gly-Asn (Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc)
-Lys (Boc) -Glu (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu)
-At the time when Wang resin was synthesized, the synthesis was interrupted after the Fmoc removal reaction. A part of the resin was collected and subjected to a cribage reaction to confirm the synthetic product by the method described in 1) of Example 1.

3) Peptide synthesis was continued using the remaining resin from 2) above, and the peptide from the C terminus to the 74th residue: Fmoc-Ala-Ser (tBu) -Trp (Boc) -Lys (Boc). -Ser
(tBu) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-Phe-Asn (Tr
t) -Gln (Trt) -Phe-Gly-Pro-Asp (OtBu) -Ile-Phe-Ala-Ser
(tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-Gln (Trt)-
Lys (Boc) -Asn (Trt) -Lys (Boc) -Leu-Thr (tBu) -Ser (tBu) -G
ly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-Asn (Trt)
-Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn (Trt) -As
n (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser (tBu) -Gl
y-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn (Trt) -Ile
-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu (OtBu) -Ala
When the -Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. However, Fmoc-Ser (tBu) at the 70th residue and Fmoc-Lys at the 71st residue from the C-terminus
(Boc), Fmoc-Trp (Boc) at 72nd residue and 73rd residue
Since the reaction efficiency of Fmoc-Ser (tBu) binding was predicted to be poor, the reaction with the deprotection solution was repeated three more times, and Fm bound to the peptide
After the oc group was completely removed, the reaction time for binding an amino acid was extended to 120 minutes. A part of the resin was collected and subjected to a cribage reaction to give 1) of Example 1.
The synthetic product was confirmed by the method described in 1.

4) Peptide synthesis was continued using the remaining resin from 3) above, and the peptide from the C terminus to the 94th residue was: Fmoc-Phe-Ala-Lys (Boc) -Leu-Thr (tBu)- Gly-P
he-Thr (tBu) -Leu-Met-Gly-Gly-Ile-Ile-Ala-Ala-Tyr (tB
u) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Trp (Boc) -Lys
(Boc) -Ser (tBu) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-P
he-Asn (Trt) -Gln (Trt) -Phe-Gly-Pro-Asp (OtBu) -Ile-Phe
-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-G
ln (Trt) -Lys (Boc) -Asn (Trt) -Lys (Boc) -Leu-Thr (tBu) -Se
r (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu-
Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-Asn
(Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Ser
(tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn
(Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu
When the (OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was synthesized, the synthesis was interrupted after the Fmoc-removing reaction. A part of the resin was collected and subjected to a cribage reaction to give 1) of Example 1.
The synthetic product was confirmed by the method described in 1. 5) Peptide synthesis was subsequently carried out using the remaining resin of 4) above, and peptides from the C-terminal to the 99th residue were synthesized.

By the above method, Fmoc-Gly-Asp (OtBu) -P
ro-Arg-Gln (Trt) -Phe-Ala-Lys (Boc) -Leu-Thr (tBu) -Gly-
Phe-Thr (tBu) -Leu-Met-Gly-Gly-Ile-Ile-Ala-Ala-Tyr (t
Bu) -Gln (Trt) -Asn (Trt) -Pro-Ala-Ser (tBu) -Trp (Boc) -Ly
s (Boc) -Ser (tBu) -Met-Lys (Boc) -Val-Thr (tBu) -Val-Ala-
Phe-Asn (Trt) -Gln (Trt) -Phe-Gly-Pro-Asp (OtBu) -Ile-Ph
e-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt) -Phe-His (Trt) -Leu-
Gln (Trt) -Lys (Boc) -Asn (Trt) -Lys (Boc) -Leu-Thr (tBu) -S
er (tBu) -Gly-Lys (Boc) -Ile-Ala-Ser (tBu) -Cys (Trt) -Leu
-Asn (Trt) -Tyr (tBu) -Gly-Leu-Val-His (Trt) -Val-Ala-As
n (Trt) -Asn (Trt) -Asn (Trt) -Tyr (tBu) -Asp (OtBu) -Pro-Se
r (tBu) -Gly-Lys (Boc) -Tyr (tBu) -Glu (OtBu) -Gly-Gly-Asn
(Trt) -Ile-Tyr (tBu) -Thr (tBu) -Lys (Boc) -Lys (Boc) -Glu
(OtBu) -Ala-Phe-Asn (Trt) -Val-Glu (OtBu) -Wang resin was obtained. This product was subjected to a Fmoc-removing reaction and a cribage reaction to obtain 706 mg of a crude peptide. 43 of these
After dissolving mg in a 20% acetonitrile aqueous solution containing 0.1% TFA, HPLC purification was performed under the following conditions: Column: C22 column (docosyl-B) Mobile phase: 32-36% Acetonitrile / 0.1% T
FA, 30 minutes (linear concentration gradient) Flow rate: 1.5 ml / min Detection wavelength: 230 nm Fractions eluted at 23 to 25 minutes were collected, concentrated and lyophilized to give 3.2 mg of the desired peptide. Got

The molecular weight of this purified peptide was confirmed by the ESI method using a mass spectrometer. As a result of amino acid sequence analysis from the N-terminal to 10 residues of 100 pmol of the purified peptide, it was confirmed that the amino acid sequence was identical to the amino acid sequences shown in amino acid numbers 1 to 10 of SEQ ID NO: 6 in the sequence listing. It was

[Example 7] Peptide 7 Peptide 7 (SEQ ID NO: 7 in the sequence listing) was synthesized by a Fmoc solid phase synthesis method using a multi-peptide synthesizer (Symphony, manufactured by Protein Technology) according to the protocol of the same apparatus. It was

That is, Fmoc-Lys in which an amino acid (Lys) corresponding to the C-terminal residue of the peptide to be synthesized has been introduced.
25 μm of (Boc) -Wang resin (0.50 mmol / g)
ol equivalent is set in the reaction vessel of the above peptide synthesizer, 1.25 ml of deprotection solution (20% piperidine / dimethylformamide) is added, and the reaction is carried out twice for 5 minutes, and the Fmoc group of the amino acid bound to the resin is added. Excluded.
The resin was washed 6 times for 30 seconds with 1.25 ml of dimethylformamide solution, and the second amino acid derivative Fm from the C-terminal side was obtained.
To 1.25 ml of a 100 mM oc-Trp (Boc) solution (solvent is dimethylformamide), an activator solution for a multipeptide synthesizer (100 mM 2- (1H-benzotriazol-1-yl-)-1,1,3,3- Tetramethyluronium-hexafluorophosphate / 4
1.25 ml of 00 mM N-methylmorpholine / dimethylformamide) was added (5 times equivalent amount to the bound amino acid), and the mixture was reacted at room temperature for 20 minutes. Fm generated here
After washing the oc-Trp (Boc) -Lys (Boc) -Wang resin with 1.25 ml of dimethylformamide for 6 times for 30 seconds, the Fmoc group was removed again, and after washing with 1.25 ml of dimethylformamide for 6 times for 30 seconds, the Fmoc was removed. -The Ser (tBu) solution and the activator solution for the multi-peptide synthesizer were added and reacted.
Protected peptide resin: Fm by repeating the same procedure
oc-Gly-Ile-Ile-Ala-Ala-Tyr (tBu) -Gln ((Trt) -Asn (Trt)
-Pro-Ala-Ser (tBu) -Trp (Boc) -Lys (Boc) -Wang resin was synthesized.

The amino acid derivatives used for the synthesis of Peptide 7 and Peptide 8 (SEQ ID NO: 8 in the Sequence Listing) are as follows: Fmoc-Ala, Fmoc-Asn (Trt), Fmoc-Asp (OtB.
u), Fmoc-Cys (Trt), Fmoc-Gln (Trt), Fmoc-Glu (OtBu),
Fmoc-Gly, Fmoc-His (Trt), Fmoc-Ile, Fmoc-Leu, Fmoc-
Lys (Boc), Fmoc-Met, Fmoc-Phe, Fmoc-Pro, Fmoc-Ser (t
Bu), Fmoc-Thr (tBu), Fmoc-Trp (Boc), Fmoc-Tyr (tBu),
Fmoc-Val (() indicates a protecting group that protects the reactive group in the residue portion .; Perkin Elmer Japan Applied Biosystems Division).

The protected peptide resin synthesized as described above was reacted with 1.25 ml of the deprotection solution twice for 5 minutes to deprotect the Fmoc group at the N-terminal. Next, the resin was washed 6 times with 1.25 ml of dimethylformamide and then 9 times with dichloromethane, and further dried by blowing nitrogen gas for 20 minutes. The resin was recovered and the cleaved solution (TFA: ethanedithiol: water:
Triisopropylsilane = 92.5: 2.5: 2.5:
2.5 ml (volume ratio)) was added and reacted at room temperature for 2 hours to cleave the peptide from the resin and remove the amino acid side chain protecting group to obtain a peptide solution. This peptide solution was filtered using a PTFE filter, and the filtrate was collected in a centrifuge tube. 10 ml of cold ether was added to this filtrate to precipitate the peptide. After cooling for a while, this was centrifuged (3,000 rpm, 10 minutes) to recover the precipitate, and cold ether was added again to disperse the precipitate and the operation of recovering was repeated 4 times to wash the peptide. The obtained peptide was dried to obtain a crude peptide.

7.0 mg of the obtained crude peptide was
After dissolving in a 20% acetonitrile aqueous solution containing 0.1% TFA, HPLC purification was performed under the following conditions: Column: C22 column (docosyl-B) Mobile phase: 20-25% acetonitrile / 0.1% T
FA, 20 minutes (linear concentration gradient) Flow rate: 7 ml / min Detection wavelength: 220 nm Fractions eluted at 16 to 17 minutes were collected, concentrated and freeze-dried to obtain 2.0 mg of the desired peptide. Obtained.
As a result of amino acid sequence analysis of 100 pmol of this purified peptide, the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing was confirmed.

Example 8 Peptide 8 By the same procedure as in Example 7, the protected peptide resin: Fmoc
-Asp (OtBu) -Ile-Phe-Ala-Ser (tBu) -Lys (Boc) -Asn (Trt)-
Phe-His (Trt) -Leu-Gln (Ttrt) -Lys (Boc) -Asn (Trt) -Wang
The resin was synthesized and deprotected and cleaved to give the crude peptide.

3.1 mg of the obtained crude peptide was
After dissolving in a 20% acetonitrile aqueous solution containing 0.1% TFA, HPLC purification was performed under the following conditions: Column: ODS column (TSK gel ODS-120
T) Mobile phase: 22-27% acetonitrile / 0.1% T
FA, 20 minutes (linear concentration gradient) Flow rate: 2 ml / min Detection wavelength: 220 nm Fractions eluted at 14 to 15.5 minutes were collected and concentrated.
Lyophilization was performed to obtain 0.9 mg of the desired peptide. As a result of amino acid sequence analysis of 100 pmol of this purified peptide, the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing was confirmed.

[Example 9] Peptide 9 1) Synthetic peptide 9 of DNA encoding a basic unit (Amino acid Nos. 1 to 185 of SEQ ID NO: 10 in Sequence Listing) was used to bind six T cell epitopes of cedar pollen allergen to A -E-C-D-F-G (hereinafter referred to as "H6-1", SEQ ID NO: 31 in the sequence listing) and a linker peptide having the following amino acid sequence: Thr-Met-Ile-Thr -Asn-Ser-Ser-Ser-Val-Pro-Gly-Asp-Pr
o-Arg (L1, SEQ ID NO: 26 in Sequence Listing); Arg-Ala-Asp-Pro-Arg (L2, SEQ ID NO: 2 in Sequence Listing)
7); and Arg-Ala-Asp-Leu (L3, SEQ ID NO: 80 in the sequence listing), (L1)-(H6-1)-(L2)-(H6-1)-
(L3) has a linked structure. To produce peptide 9 in Escherichia coli using a gene recombination technique, as described in detail below, first, a DNA encoding H6-1 (hereinafter referred to as "h6-1") is prepared and then H
A method was used in which an expression plasmid vector for Escherichia coli containing the DNA encoding peptide 9 was constructed by polymerizing the region containing the DNA encoding 6-1 and E. coli was transformed with the plasmid vector.

In designing h6-1, the codon usage frequency of E. coli [Cranthan, R. et al. (1981) Nucleic A
cids Res. 9, 43], and the restriction enzyme recognition sequences necessary for subsequent polymerization of h6-1 are located at the 5 ′ end side (BamHI) and 3 ′ end side (BglII) of the coding region, respectively. It was added. 3 '
A restriction enzyme SalI recognition sequence was added to the 3′-terminal side of the terminal side BglII recognition sequence in consideration of insertion into an expression plasmid (SEQ ID NO: 30 in the sequence listing).

First, h6 added with a restriction enzyme recognition sequence
The following oligonucleotide, which is a partial sequence of the sense or antisense strand of -1: 5'- gatccgcgtg gtatcatcgc agcataccag aacccggcat ct
tgg-3 '(F1, SEQ ID NO: 32 in the sequence listing); 5'- catagaccaa gatgccgggt tctggtatgc tgcgatgata cc
acgcg -3 '(F2, SEQ ID NO: 33 in the sequence listing); 5'-tctatgaaag ttaccgttgc tttcaaccag ttcggtccg -3'
(F3, SEQ ID NO: 34 in the sequence listing); 5'-tgcgaacgga ccgaactggt tgaaagcaac ggtaacttt -3 '
(F4, SEQ ID No. 35 in the sequence listing); 5'-ttcgcatcta aaaacttcca tctgcagaaa -3 '(F5, SEQ ID No. 36 in the sequence listing); 5'-ggtgtttttc tgcagatgga agtttttaga -3' (F6, SEQ ID No. 37 in the sequence listing). 5'- aacaccaaac tgacctctgg taaaatcgca tcttgc -3 '
(F7, SEQ ID NO: 38 in the sequence listing); 5'-gttcaggcaa gatgcgattt taccagaggt cagttt -3 '
(F8, SEQ ID NO: 39 in the sequence listing); 5'-ctgaactacg gtctggttca tgttgcaaac aacaactacg a
-3 '(F9, SEQ ID NO: 40 in the sequence listing); 5'- gacgggtcgt agttgttgtt tgcaacatga accagaccgt a
-3 '(F10, SEQ ID NO: 41 in the sequence listing); 5'- cccgtctggt aaatacgaag gtggtaacat ctacaccaaa a
-3 '(F11, SEQ ID NO: 42 in the sequence listing); 5'- cttctttttt ggtgtagatg ttaccacctt cgtatttacc a
-3 '(F12, SEQ ID NO: 43 in the sequence listing); 5'- aagaagcatt caacgttgaa cgtgcagatc tgtaag -3'
(F13, SEQ ID NO: 44 in the sequence listing); and 5'-tcgacttaca gatctgcacg ttcaacgttg aatg -3 '(F
14, SEQ ID NO: 45 of the sequence listing) was synthesized with a DNA synthesizer (model 394; manufactured by Applied Biosystems). These are F1 and F
2, F3 and F4, F5 and F6, F7 and F8, F9 and F1
In the combination of 0, F11 and F12, F13 and F14, the nucleotide sequences from the 7th nucleotide to the 3'terminal side of the 5'end are complementary, and F3 and F2, F5 and F4, F7 and F6, F9 and F8, F
5'in combination of 11 and F10, F13 and F12
The 6 terminal nucleotide sequences are complementary to each other. Therefore, first, F1 and F2, F3 and F4, F5 and F6, F7 and F8, F9 and F10, F11 and F12, F
The complementary strands are annealed by the combination of 13 and F14 to form a double-stranded DNA, and then the respective fragments are ligated by utilizing the complementary strand at the 5′-protruding end generated in each double-stranded DNA. By doing so, h6-1 can be produced.

Each of the synthesized oligonucleotides was blown with nitrogen to remove ammonia, dried, dissolved in sterile distilled water, and 10 to 30 μg of each was collected, and 8% polyacrylamide gel containing 7 M urea was prepared. Electrophoresis was performed. The gel of the band portion containing each oligonucleotide was cut out, an elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate) was added, and the mixture was kept at 37 ° C. for 16 hours while shaking to elute the oligonucleotide. To this eluate was added 2.5 volumes of cold ethanol to precipitate the oligonucleotide, and
Xg, it collect | recovered by centrifuging for 15 minutes at 4 degreeC. Precipitation 7
After washing with 0% ethanol, it was dissolved in 20 μl of sterile distilled water to obtain a purified oligonucleotide.

Next, except for F1 and F14, F2 to F13
5'end of each oligonucleotide was phosphorylated (hereinafter referred to as "F2p" to "F13p"). The reaction solution composition was as described below: Oligonucleotide (F2-F13) 5-10 μl 10-fold concentration buffer solution for phosphoryl transfer reaction (T4 polynucleotide kinase (attached to Takara Shuzo Co., Ltd.) 5 μl 10 mM adenosine triphosphate (hereinafter referred to as “ATP”) 5 μl T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) 10 units sterilized distilled water to 50 μl Temperature conditions: 1 hour at 37 ° C., then 70 ° C. The temperature was kept warm for 5 minutes to inactivate the enzyme to stop the phosphorylation reaction.

After completion of the reaction, 1/10 volume of 3M sodium acetate solution was added to each reaction solution, and cold ethanol was added to 2.5 times.
After adding, the solution was centrifuged at 10,000 × g and 4 ° C. for 15 minutes to recover F2p to F13p as a precipitate (hereinafter, referred to as “precipitate”).
This operation is called "ethanol precipitation"). After washing with 70% ethanol, it was dissolved in 5 μl of sterile distilled water.

Then, F2p to F13p and F1 and F14 were put together in one tube and annealed. The composition of the reaction solution was as described below: Oligonucleotide fragment (F1, F2p to F13p, F14) 2.5 to 5 μl each 1M Tris-hydrochloric acid buffer solution (pH 7.5) 10 μl 1M magnesium chloride solution 1 μl Distillation sterilization The total volume was made up to 100 μl with water. Temperature conditions: Using a thermal cycler (Model 9600; manufactured by Perkin-Elmer Co., Ltd.), after heating at 95 ° C. for 5 minutes, it was cooled from 95 ° C. to 25 ° C. in 90 minutes.

After completion of the reaction, ethanol precipitation was carried out to collect the precipitate, and 10 μm of 10 mM Tris-hydrochloric acid buffer solution (pH 8.0: hereinafter referred to as “TE buffer solution”) containing 1 mM ethylenediaminetetraacetic acid (hereinafter referred to as “EDTA”).
It was dissolved in 1. 3 μl of the annealed oligonucleotide mixture was ligated with a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) by keeping it at 16 ° C. for 16 hours according to the method described in the instructions attached to the kit. After completion of the ligation reaction, ethanol precipitation was performed to collect the precipitate, which was washed with 70% ethanol and then dissolved in 6 μl of sterile distilled water.

On the other hand, the cloning vector pUC18
(Manufactured by Takara Shuzo Co., Ltd.) with restriction enzymes BamHI and Sa
After sequential digestion with II, ethanol precipitation was performed to collect the precipitate, which was washed with 70% ethanol and then dissolved in TE buffer. This was subjected to 0.8% low melting point agarose gel (FMC Bioproducts) electrophoresis. After electrophoresis, the D in the gel can be
The NA was visualized, the gel of the band portion corresponding to about 2.7 kbp was cut out, and an equal volume of TE buffer (pH 8.0) was added so that the final concentration of agarose was 0.5% or less. The gel piece was melted by incubating at 5 ° C for 5 minutes. 2 with an equal volume of phenol solution (saturated phenol with 8-quinolinol added to a final concentration of 0.1%)
Times, phenol-chloroform solution (phenol solution,
50: 4 chloroform and isoamyl alcohol
It was extracted once with a 9: 1 mixture) and once with a chloroform solution (a 49: 1 mixture of chloroform and isoamyl alcohol). Further, ethanol precipitation was performed to collect the precipitate, which was washed with 70% ethanol and then dissolved in 10 μl of sterile distilled water. 1 μl of this BamHI-SalI-digested pUC18 solution was added to the above F1 to F14.
2 μl of the ligated fragment was added and ligated using a DNA ligation kit.

This ligation reaction solution was subjected to the Hanahan method [see Hanahan, D. (1983) J. Mol. Biol. 166, 557-580].
100 μl of the competent Escherichia coli JM109 strain (manufactured by Takara Shuzo Co., Ltd.) prepared by (1) was added and ice-cooled for 30 minutes. After keeping the temperature at 42 ° C. for 45 seconds, it was immediately ice-cooled for 3 minutes. To this, 900 μl of SOC medium was added, and the mixture was shake-cultured at 37 ° C. for 1 hour. 50 μg / ml of this culture solution
It was spread on an L-broth agar medium plate containing ampicillin and cultured at 37 ° C. overnight. The composition of the medium used here is as described below: L medium Bact tryptone (manufactured by Difco) 10 g Bact yeast extract (manufactured by Difco) 5 g Sodium chloride 5 g Total amount of 1 liter with ion-exchanged water And

[0123]   L-broth agar     Bact Trypton 10g     Bact East Extract 5g     Sodium chloride 5g     Bact agar (manufactured by Difco) 15g     The total amount was adjusted to 1 liter with ion-exchanged water.

[0124]   2xTY medium     Bact Trypton 16g     Bact East Extract 10g     Sodium chloride 5g     The total amount was adjusted to 1 liter with ion-exchanged water.

[0125]   SOC medium     Bact Trypton 20g     Bact East Extract 5g     5M sodium chloride solution 2ml     2M potassium chloride solution 1.25ml     1M magnesium chloride solution 10ml     1M magnesium sulfate solution 10ml     2M glucose solution 10ml     The total amount was adjusted to 1 liter with ion-exchanged water.

Ampicillin-resistant colonies appearing under the above-mentioned culture conditions were isolated and inoculated into L-broth medium.
The cells were shaken and cultured at 0 ° C overnight. From 3 ml of the culture broth, Qiagen Plasmid Mini Kit (manufactured by Qiagen) was used according to the method described in the instructions attached to the kit, and h6
The cloning vector pUC18 (hereinafter referred to as "p
UC (h6-1) ”was extracted (FIG. 1) (it should be noted that the above kit was used for extraction of all plasmids in the examples described below). The plasmid extracted by the kit was precipitated with ethanol, washed with 70% ethanol, and dissolved in 50 μl of sterile distilled water.

Thus obtained pUC (h6-
1) The nucleotide sequence of the DNA inserted in
As a result of examination using a sequencer (310 Genetic Analyzer, manufactured by Applied Biosystems), the cloned h6-1 has a nucleotide sequence (SEQ ID NO: 30 in the Sequence Listing) c of nucleotide number 247 of the originally designed nucleotide sequence. The amino acid Asn (SEQ ID NO: 31 in the sequence listing) encoded by that position
No change was made to the codon of amino acid No. 79) of the above, and it was used as it is in the following Examples. H6 after replacement
The nucleotide sequence of -1 and the amino acid sequence encoded by the sequence are shown in SEQ ID NO: 24 (nucleotide numbers 11 to 253) and SEQ ID NO: 25 (amino acid number 4 to 84), respectively.

2) Construction of Peptide 9 Expression Plasmid In FIG.
The outline of the construction of the 6-1 polymerized protein expression plasmid is shown. The pUC (h6-1) obtained in 1) above was digested with restriction enzymes BamHI and SalI to give a small fragment (about 270 bp).
(Equivalent) was purified by electrophoresis using a 2.5% low melting point agarose gel (manufactured by FMC Bioproducts). Separately, pUC (h6-1) was added to BglII and SalI.
Digested with and the large fragment (corresponding to about 2.9 kbp) was purified by electrophoresis on 1% low melting point agarose gel (manufactured by FMC Bioproducts). Next, BglII of pUC (h6-1)
-To the large fragment digested with SalI, Bam of pUC (h6-1)
The HI-SalI digested small fragment was ligated using a DNA ligation kit to obtain plasmid pUC.
(H6-1) ₂ was obtained. BamHI digestion or BglI
Since the cohesive ends generated by I digestion are identical, Ba
The mHI-digested fragment and the BglII-digested fragment can be ligated, but the nucleotide sequence after the ligation is not re-cut by BamHI or BglII.

3) Expression in Escherichia coli The E. coli YA21 strain (JM109 strain may be used) was spread on an L-broth agar medium plate and cultured overnight at 37 ° C. The formed single colony was inoculated into a 3 liter Erlenmeyer flask containing 250 ml of SOB medium, and kept at 18 ° C for 200
The culture was performed with shaking at rpm. After 45 hours, 660 of culture
When the absorbance at nm (OD _660nm ) reached 1.19, the flask was immersed in ice water and cooled for 10 minutes. The culture was centrifuged at 1200 xg for 15 minutes at 4 ° C to collect the precipitated bacterial cells. 80 ml of this fungus body
The cells were suspended in the ice-cold transformation buffer (hereinafter referred to as "TB"), cooled for 10 minutes with ice, and then centrifuged at 1200 xg for 15 minutes at 4 ° C to recover the precipitated cells. 20 ml of cells
Was resuspended in ice-cold TB, and 1.5 ml of dimethyl sulfoxide (hereinafter referred to as "DMSO") (final concentration 7%) was added, followed by ice-cooling for 10 minutes. Dispense 0.5 ml each into a tube in a dry ice-ethanol bath,
Immediately freeze and use as a competent E. coli sample at -80 ° C.
Saved in. The composition of the buffer and medium used here is
It was as described below: SOB medium: Bact tryptone 20 g Bact yeast extract 5 g 5M sodium chloride solution 2 ml 2M potassium chloride solution 1.25 ml 1M magnesium chloride solution 10 ml 1M magnesium sulphate solution 10 ml Ion-exchanged water to make the total amount. It was set to 1 liter.

[0130]   Transformation buffer (TB):     PIPES (Nakarai Tesque) 3.0g     Calcium chloride dihydrate 2.2g     Potassium chloride 18.6g     Manganese chloride tetrahydrate 10.9g     Bring the total volume to 1 liter with distilled water and adjust the pH to 6.7 with 5N potassium hydroxide solution. Adjusted to ~ 6.8.

Plasmid pUC constructed in 2) above
(H6-1) ₂ is a lacZ gene under the control of the lac promoter of pUC18 and a DN encoding peptide 9.
Since A is ligated on the same reading frame, by introducing these plasmids into the host,
Expression of peptide 9 is possible (Fig. 2).

60 ng of pUC (h6-1) ₂ was added to 100 μl of competent Escherichia coli prepared as described above, ice-cooled for 30 minutes, kept at 42 ° C. for 45 seconds and immediately ice-cooled for 3 minutes. . To this, 900 μl of SOC medium was added, and the mixture was shake-cultured at 37 ° C. for 1 hour. This culture solution was spread on an L-broth agar medium plate containing 50 μg / ml of ampicillin and cultured at 37 ° C. overnight. The ampicillin-resistant colonies that appeared were 50 μg /
2 ml of 2xTY medium containing 3 ml of ampicillin
Shaking culture was performed at 7 ° C. After 8 hours, culture medium OD
_{When 660 nm} reached 1 to 3, a part of the culture solution was adjusted to 100 μg / so that the final OD _{660 nm} was 0.01.
It was transferred to 100 ml of 2 × TY medium containing ml of ampicillin, and shake-cultured at 37 ° C. for 14 hours. At the end of the culture, the polymerized protein formed an inclusion body in the cells of the Escherichia coli YA21 strain.

After completion of the culturing, 90 ml of the culture solution was taken,
Centrifugation at 6000 × g and 4 ° C. for 20 minutes was performed to collect the precipitated bacterial cells. After suspending the cells in 4.5 ml of TES buffer, the cells were disrupted by using an ultrasonic homogenizer (Sonifier 250, manufactured by Branson), and then centrifuged at 13000 × g at 4 ° C. for 20 minutes. The inclusion body was obtained as a precipitate. Since this precipitate fraction still contained a large amount of bacterial cell fragments, 1% Triton X-100 was used.
The suspension was suspended in 4.5 ml of a phosphate buffer solution containing a., Shaken at room temperature for 30 minutes, and then centrifuged at 25,000 × g and 4 ° C. for 20 minutes to recover the precipitate. After repeating this operation 4 times, the precipitated fraction was washed twice with 9 ml of distilled water. 4.5 ml of 6M guanidine hydrochloric acid solution (pH 7.6) was added to half of the precipitate fraction, and the mixture was shaken at 37 ° C for 1 hour to solubilize the precipitate, and after centrifugation at 25,000 xg and 4 ° C for 20 minutes. The supernatant was collected to obtain a crude purified solution of peptide 9. The composition of the buffer used here was as described below: TES buffer (pH 7.6): 1M Tris-HCl buffer (pH 7.6) 10 ml 5M sodium chloride solution 30 ml 0.5M EDTA solution. Make up to 1 liter with 20 ml distilled water.

[0134]   Phosphate buffer (containing 1% Triton X-100):     Sodium chloride 8g     0.2 g of potassium chloride     Disodium hydrogen phosphate dodecahydrate 2.9 g     0.2 g of potassium dihydrogen phosphate     Triton X-100 (manufactured by Sigma) 10g     The total volume was adjusted to 1 liter with distilled water.

[0135]   6M guanidine hydrochloric acid solution (pH 7.6):     Guanidine hydrochloride 57.32g     5 ml of 1M Tris-HCl buffer (pH 7.6)     1M dithiothreitol (hereinafter referred to as "DTT") solution 1ml     The total volume was adjusted to 100 ml with distilled water.

75 μl of the crude purified solution of the peptide 9 was subjected to HPLC purification under the following conditions: Column: ODS column (TSK-gel ODS-120)
T, φ7.8 mm × 300 mm, manufactured by Tosoh Corporation) Mobile phase: 20% acetonitrile / 0.1% TFA
(0-5 minutes), 20-70% acetonitrile (5-3
5 minutes) /0.1% TFA (linear concentration gradient) Flow rate: 2 ml / min Detection wavelength: 220 nm Peak at around elution time of 24.3 minutes was collected, and the obtained substance was from the N-terminal to 25 residues. As a result of amino acid sequence analysis, the amino acid sequence was in agreement with the amino acid sequences represented by amino acid numbers 1 to 25 of SEQ ID NO: 10 in the sequence listing. In addition,
The methionine residue at the N-terminal of the amino acid sequence encoded by h6-1 was lost during the purification process. Further, the molecular weight of the obtained substance was measured by the ESI method, and the result was in agreement with the molecular weight predicted from the amino acid composition of peptide 9. As described above, 4.1 mg of Peptide 9 was obtained per 100 ml of the culture solution.

[Example 10] Peptide 10 1) Synthetic peptide 10 of DNA encoding a basic unit (amino acid number 1 of SEQ ID NO: 12 in the sequence listing)
No. 209) to No. 209) in which seven types of T cell epitopes of cedar pollen allergen are linked like AECDFGFGB (hereinafter referred to as “H7-1”. SEQ ID NO: 29 in the sequence listing). ) And the linker peptides L1, L2 and L3 described in Example 9 are (L1)-(H7-1)-(L2)-(H7-1)-
(L3) has a linked structure. When producing peptide 10 in Escherichia coli using a gene recombination technique, as described in detail below, first, a DNA encoding H7-1 (hereinafter referred to as "h7-1") is prepared and then h
The expression plasmid vector for Escherichia coli containing the DNA encoding peptide 10 was constructed by polymerizing the region containing the DNA encoding 7-1, and Escherichia coli was transformed with the plasmid vector.

In designing h7-1, the codon usage frequency of E. coli [Cranthan, R. et al. (1981) Nucleic A
cids Res. 9, 43], and the restriction enzyme recognition sequences necessary for subsequent polymerization of h7-1 are located at the 5 ′ end side (BamHI) and 3 ′ end side (BglII) of the coding region, respectively. It was added. 3 '
A restriction enzyme SalI recognition sequence was added to the 3'-terminal side of the BglII recognition sequence on the terminal side in consideration of insertion into an expression plasmid (SEQ ID NO: 28 in Sequence Listing).

First, h7 with a restriction enzyme recognition sequence added
In addition to F1 to F12 synthesized in Example 9 as a partial sequence of the sense strand or the antisense strand of -1, the following oligonucleotide: 5'-aagaagcatt caacgttgaa cagttcgcta aactg -3 '(F
15, SEQ ID NO: 46 of the sequence listing); 5'-accggtcagt ttagcgaact gttcaacgtt gaatg -3 '(F
16, SEQ ID No. 47 in the sequence listing); 5'- accggtttca ccctgatggg tcgtgcagat ctgtaag -3 '
(F17, SEQ ID NO: 48 in the sequence listing); and 5'-tcgacttaca gatctgcacg acccatcagg gtgaa -3 '(F
18, SEQ ID NO: 49) in the sequence listing was synthesized by a DNA synthesizer and purified by the method described in 1) of Example 9. In addition, F15, F16 and F17
Was phosphorylated at the 5'end by the method described in Example 9-1) (hereinafter "F15p", "F16p" and "F17p").
p ”).

Next, oligonucleotides F1, F18 and F2p to F12p, F15 having phosphorylated 5'ends were prepared.
2.5 μl of each of p to F17p was annealed under the composition and temperature conditions described in 1) of Example 9, precipitated with ethanol, and then dissolved in 10 μl of TE buffer. 3 μl of that
The DNA was ligated using a DNA ligation kit, precipitated with ethanol, and then dissolved in 5 μl of TE buffer. On the other hand, in order to perform PCR using this ligated fragment as a template, the following oligonucleotides were used as primers: 5'- ggatccgcgt ggtatcatcg ca -3 '(PRv, SEQ ID NO: 50 in the sequence listing); and 5'- aggtcgactt acagatctgc ac -3. '(PFw, SEQ ID NO: 51 in the sequence listing) was synthesized.

Next, PCR was performed under the following reaction solution composition and conditions: Template DNA: Synthetic fragment after ligation 2 μl 10-fold concentration Ex Taq PCR buffer (Ex Taq polymerase (Takara Shuzo Co., Ltd.)) Attached) 10 μl dNTP mixed solution (attached to Ex Taq polymerase) 8 μl 20 pmol / μl primer PRv 2.5 μl 20 pmol / μl primer PFw 2.5 μl Ex Taq polymerase (manufactured by Takara Shuzo Co., Ltd.) 2.5 units Total amount with sterile distilled water Was set to 100 μl (the dNTP mixture represents an equimolar mixture of dATP, dCTP, dGTP and dTTP. The same applies to the following description). Temperature condition: Heat treatment at 94 ° C for 3 minutes, and then at 94 ° C for 3 minutes
0 second, 30 seconds at 55 ℃, 1 minute cycle at 72 ℃ 30
After repeating the number of times, the temperature was kept at 72 ° C. for 10 minutes (all the PCR reaction temperatures in the examples described below were adjusted using Thermal Cycler Model 9600; manufactured by Perkin Elmer).

After the reaction was completed, 50 μl of a 10 M ammonium acetate solution and 150 μl of isopropanol were added to the mixture to give D
NA was precipitated, collected by centrifugation (hereinafter, this operation is referred to as “isopropanol precipitation”), washed with 70% ethanol, and dissolved in 20 μl of sterile distilled water.
This product (2 μl) was digested with a restriction enzyme BamHI and further digested with SalI, and then ethanol precipitation was performed to recover the precipitate. The precipitate was washed with 70% ethanol, and then 4 μl of TE was added.
Dissolved in buffer. This BamHI-SalI-digested DNA fragment solution (2 μl) was ligated with the BamHI-SalI-digested pUC18 vector solution (1 μl) prepared in 1) of Example 9 using a DNA ligation kit. Using this ligation reaction solution, the competent E. coli JM10 was prepared by the method described in 1) of Example 9.
Nine strains were transformed and cultured at 37 ° C. The ampicillin-resistant colonies that appeared were isolated, inoculated into L-broth medium, and shake-cultured overnight at 37 ° C. The plasmid pUC (h7-1) containing h7-1 was extracted from 3 ml of this culture solution (FIG. 1). The extracted plasmid is precipitated with ethanol, washed with 70% ethanol, and then 50 μl of sterile distilled water
Dissolved in. h7-1 inserted into pUC (h7-1)
As a result of examination of the nucleotide sequence of, the nucleotide sequence was confirmed to be the nucleotide sequence encoding H7-1 (SEQ ID NO: 29 in the sequence listing) (SEQ ID NO: 28 in the sequence listing).

2) Construction of Expression Plasmid Encoding Peptide 10 FIG. 1 shows the cedar allergen epitope 7 connecting peptide H.
1 shows an outline of the construction of a 7-1 polymerized protein expression plasmid. The pUC (h7-1) prepared in 1) above was digested with restriction enzymes BamHI and SalI and electrophoresed on a 2.5% low melting point agarose gel to give a small fragment (about 300 bp).
Was purified. Separately, pUC (h7-1) was digested with restriction enzymes BglII and SalI, and a large fragment (about 3.0 kbp) was obtained by 1.0% low melting point agarose gel electrophoresis.
Was purified. Next, these fragments were ligated using a DNA ligation kit to obtain a plasmid pUC (h7-1) ₂ into which the DNA encoding peptide 10 (SEQ ID NO: 11 in the sequence listing) was inserted.

3) Expression in E. coli In the same manner as 3) of Example 9, 10 ng of pUC (h7
-1) ₂ in competent E. coli YA21 strain (JM10
9 strains are also possible). This transformant was transformed into 5
5 m of L-broth medium containing 0 μg / ml of ampicillin
Incubation was carried out at 37 ° C. with shaking in 1. After 9 hours, when the OD _{660nm of} the culture reached 3 or more, a part of this culture was adjusted to 100 so that the final OD _660nm was 0.01.
1 liter of 2xTY containing μl / ml ampicillin
The medium was inoculated and shake-cultured at 37 ° C. for 14 hours.
At the end of the culture, the polymerized protein formed an inclusion body in the bacterial cells of the YA21 strain. Take 90 ml of this culture,
The microbial cells that precipitated were collected by centrifugation at 000 × g for 20 minutes at 4 ° C. Suspend the cells in 9 ml of TES buffer and crush with an ultrasonic homogenizer, then 13000 × g,
Inclusion bodies were obtained as precipitates by centrifugation at 4 ° C. for 20 minutes.
The precipitate was washed 4 times with 9 ml of phosphate buffer containing 1% Triton. Half the amount of the precipitate was added to 2M urea buffer (2M urea, 50 mM Tris-HCl (pH 7.6), 10 mM).
DTT) was washed twice with 4.5 ml of distilled water and twice with 4.5 ml of distilled water, solubilized with 4.5 ml of 6M guanidine hydrochloride solution, centrifuged at 25,000 × g, 4 ° C. for 20 minutes, and the supernatant was removed. This was a crude purified solution of peptide 10. This crude purified liquid 1m
HPLC purification was performed for 1 under the following conditions: Column: C18 column (capsule pack C18 SG3
00, φ10 mm × 250 mm, manufactured by Shiseido Co., Ltd.) Mobile phase: 37-42% acetonitrile / 0.1% TF
A, 30 minutes (linear concentration gradient) Flow rate: 3 ml / min Detection wavelength: 220 nm The peak near the elution time of 13.9 minutes was collected, and the obtained substance was analyzed for the amino acid sequence from the N-terminal to 38 residues. As a result, it agreed with the amino acid sequences represented by amino acid numbers 1 to 38 of SEQ ID NO: 12 in the sequence listing. The N-terminal methionine residue of the amino acid sequence encoded by h7-1 was lost during the purification process. Further, the molecular weight of the obtained substance was measured by the ESI method, and the result was in agreement with the molecular weight predicted from the amino acid composition of peptide 10. As described above, 27 mg of Peptide 10 was obtained per liter of the culture solution.

[Example 11] Peptides 11 and 121 1) Synthetic peptide 12 of DNA encoding a basic unit (amino acid number 1 of SEQ ID NO: 15 in the sequence listing)
To the peptide consisting of the amino acid sequence shown in 95)
Has a structure in which seven T cell epitopes of cedar pollen allergen are linked as in A'-EC'-DFGGB, and peptide 11 (SEQ ID NO: 13 in the sequence listing) is a peptide It has a structure in which Arg is added to the C-terminal of 12. In order to produce these peptides in Escherichia coli using a gene recombination technique, a DNA encoding peptide 12 (hereinafter referred to as “h7-3”) was first prepared as described below. In designing h7-3, the codon usage of E. coli was taken into consideration, and the restriction enzyme recognition sequence necessary for polymerizing h7-3 later was determined by the 5'end (BamHI) and 3'ends of the coding region. Each side was added (BglII). Further 3'of the BglII recognition sequence on the 3'terminal side
A restriction enzyme SalI recognition sequence was added to the terminal side in consideration of insertion into an expression plasmid (SEQ ID NO: 5 in the sequence listing).
2).

First, h7 with a restriction enzyme recognition sequence added
-3 as a partial sequence of the sense strand or antisense strand, F1 and F8 synthesized in Example 9 and Example 10 to
In addition to F12 and F15-F18, the following oligonucleotides: 5'-aaatctatga aagttaccgt tgctttcaac cagttcggtc cg
-3 '(F19, SEQ ID NO: 54 in the sequence listing); 5'- agatttccaa gatgccgggt tctggtatgc tgcgatgata cc
acgcg -3 '(F20, SEQ ID NO: 55 in the sequence listing); 5'- gacatcttcg catctaaaaa cttccatctg ca -3' (F2
1, SEQ ID NO: 56 of the sequence listing); 5'- gatgtccgga ccgaactggt tgaaagcaac ggtaactttc at
-3 '(F22, SEQ ID NO: 57 in the sequence listing); 5'- gaaaaacaaa ctgacctctg gtaaaatcgc atcttgc -3'
(F23, SEQ ID NO: 58 in the sequence listing); 5'-gtttttctgc agatggaagt ttttagatgc gaa -3 '(F2
4, SEQ ID NO: 59) in the sequence listing was synthesized and purified by the method described in 1) of Example 9.
Further, F19 to F24 equivalent to 50 pmol were used in Example 9.
5'-terminal was phosphorylated by the method described in 1) (hereinafter "F19p", "F20p", "F21p", "F22").
p "," F23p "and" F24p ").

Next, oligonucleotides F1, F18 and F8p to F12p, F15 having phosphorylated 5'ends were prepared.
p to F17p and F19p to F24p are each 10 pmo
Each mixture (1 to 2 μl) was annealed under the composition and temperature conditions described in 1) of Example 9, precipitated with ethanol, and then dissolved in 10 μl of TE buffer. 5 μl of that
It was ligated using NA ligation kit, precipitated with ethanol, and then dissolved in 10 μl of sterile distilled water.

Further, 5 μl of the ligated DNA fragment solution
PRv prepared in Example 10 using
And PFw were used to carry out PCR under the reaction solution composition and temperature conditions described in 1) of Example 10. After isopropanol precipitation of the reaction solution after PCR, the precipitate was recovered and subjected to 2.5% low melting point agarose gel electrophoresis. The gel of the band portion corresponding to the amplified DNA fragment was cut out, 4 volumes of TE buffer was added, and the gel was dissolved by incubating at 65 ° C. for 5 minutes. This product was extracted twice with an equal volume of a phenol solution, once with a phenol / chloroform solution and once with a chloroform solution, followed by ethanol precipitation, washing the precipitate with 70% ethanol, and sterilized distilled water 10
It was dissolved in μl. This (4 μl) was added to the restriction enzyme Bam
The DNA fragment digested with HI and further digested with SalI was ligated with the BamHI-SalI digested pUC18 vector prepared in 1) of Example 9 using a DNA ligation kit. The ligation reaction solution was used to transform competent Escherichia coli JM109 strain by the method described in 1) of Example 9 and cultured at 37 ° C. The ampicillin-resistant colonies that appeared were isolated, inoculated into L-broth medium, and shake-cultured overnight at 37 ° C. From 3 ml of this culture solution, a plasmid pUC (h
7-3) was extracted (FIG. 1). The extracted plasmid was precipitated with ethanol, washed with 70% ethanol, and then dissolved in 50 μl of sterile distilled water. As a result of examining the nucleotide sequence of h7-3 inserted into pUC (h7-3), the cloned h7-3 was found to have nucleotide number 1 of the originally designed nucleotide sequence (SEQ ID NO: 52 in the sequence listing).
The t of 54 was replaced with c, but it did not change the codon of amino acid Ser (amino acid No. 48 of SEQ ID NO: 53 in the sequence listing) encoded at that position, so that it was used as it is in the following Examples. did. The nucleotide sequence of h7-3 after substitution and the amino acid sequence encoded by the sequence are shown in SEQ ID NOs: 14 and 15 of the sequence listing.

2) Preparation of peptide 11 pUC (h7-3) prepared in 1) above was treated with restriction enzyme B.
A small fragment (about 310 bp) was purified by digestion with amHI and SalI and electrophoresis on a 2% low melting point agarose gel. Separately, pUC (h7-3) was digested with restriction enzymes BglII and SalI, and a large fragment (about 3.0 kbp) was purified by 1.0% low melting point agarose gel electrophoresis. Next, by ligating these fragments using a DNA ligation kit, a plasmid pUC (h7- containing a nucleotide sequence (SEQ ID NO: 60 in the sequence listing) encoding the amino acid sequence shown in SEQ ID NO: 61 in the sequence listing.
3) ₂ was obtained (Fig. 1).

In the same manner as in Example 9 3), pUC (h
7-3) ₂ was transformed into competent E. coli YA21 strain (JM109 strain is also possible). This transformant
A 2 liter Erlenmeyer flask containing 400 ml of 2 × TY medium was inoculated and cultivated at 37 ° C. for 8 hours with rotary shaking. The total amount of the obtained culture solution (OD _660nm = 2.5) was 1
The mixture was transferred to a 200-liter culture tank containing 20 liters of 2 × TY medium, and the culture was continued at 37 ° C. for 17 hours while changing aeration and stirring conditions so as to maintain the dissolved oxygen concentration at 5 ppm.

After completion of the culture, the culture solution (OD ₆₀₀ = 8.6)
Was adjusted to pH 3.0 with 15% (v / v) sulfuric acid and
After stirring for a minute, the pH was adjusted to 20% (w / v) sodium hydroxide.
Returned to 7.0. After obtaining a bacterial cell concentrated liquid by membrane filtration (membrane pore diameter: 0.2 μm), centrifugation was performed to collect the precipitate and obtain 580 g of wet bacterial cells. 20 mM TES buffer (20
5 liters of cell suspension (concentration of polymerized protein by SDS-PAGE analysis was about 20%) was prepared with mM Tris-hydrochloric acid (pH 7.6), 150 mM sodium chloride, 10 mM EDTA, and this was used as a cell homogenizer. (Passed by APV Gorin) 4 times (50Mpa)
After crushing the cells, 0.4% Triton X-10
0 mM (final concentration) in 50 mM Tris-HCl buffer (p
H7.6) and 50 mM Tris-HCl buffer and washed and centrifuged to give a slurry (SDS-PAG).
Concentration of polymerized protein by E analysis is about 33%) 2.5
Got liters. The total amount of this slurry is 5 liters of 10
While adjusting the pH to 8.7 with a 25% (w / v) sodium hydroxide aqueous solution,
4.8 ml citraconic anhydride was added 8 times every 15 minutes. This citraconylated solution was adjusted to 37 ° C. and pH 7.6, 7.5 liters of 20 mM calcium nitrate and 264 mg of trypsin were added, and the mixture was kept at 36 ° C. After 1 hour, the reaction was stopped by adding 150 ml of TFA. The reaction solution was centrifuged to obtain 272 g of a precipitate.

68 g of this precipitate was suspended in 2 liters of 100 mM sodium phosphate buffer (pH 6.0), 8 liters of 10 M urea solution and 0.5 liter of β-mercaptoethanol were added, and then 2 liters of cations were added. It was adsorbed on an exchange resin (SP-Sepharose FF, manufactured by Pharmacia). After washing the resin with 8 M urea / 20 mM sodium phosphate (pH 6.0) / 10 mM DTT, the eluent containing 0.05 M, 0.1 M, 0.2 M, 0.3 M and 2.0 M sodium chloride (8 M urea Two
0 mM sodium phosphate and 10 mM DTT, pH
It elutes one by one in 6). Since the target peptide was contained in the elution fraction at a concentration of 0.2M sodium chloride, after diluting this fraction twice with pure water, sodium chloride was added to a final concentration of 3M and centrifuged. Done and collect the precipitate. After deionized water is added to the precipitate to suspend it, centrifugation is performed to collect the supernatant, and acetonitrile and TFA are each added to a final concentration of 2
After adding 0% and 0.1%, reverse phase chromatography purification was performed under the following conditions: Column: ODS column (YMC-pack ODS, φ10)
cm × 50 cm, manufactured by YMC) Mobile phase: 20-60% acetonitrile / 0.1% T
FA, 65 minutes (linear concentration gradient) Flow rate: 260 ml / min Detection wavelength: 230 nm The peak near the elution time of 44.3 minutes was collected, and the obtained substance was analyzed for the amino acid sequence from the N-terminal to 20 residues. As a result, it was in agreement with the amino acid sequences represented by amino acid numbers 1 to 20 of SEQ ID NO: 13 in the sequence listing. As described above, 650 mg of peptide 11 was obtained.

3) Preparation of Peptide 12 Direct expression vector pU for preparing Peptide 12
C18M5 was prepared by substituting g for nucleotide number 177 of the nucleotide sequence of the pUC18 vector (GenBank Accession No. L08752) with c to prepare a pUC18M vector having a cleavage site for the restriction enzyme MunI (FIG. 4). The lac promoter was changed to the lacUV5 promoter by substituting gt of 169 to 170 with aa. Introduction of these mutations was carried out by the RT-PCR method (Douglas, H. et al. BioTechnique.
s, 8, 178 (1990)) (Fig. 3).

First, as a primer, an oligonucleotide described below: 5'-tccggctcgt atgttgtgtg caattgtgag c -3 '(P1,
5'- agcataaagt gtaaagcctg gg -3 '(P2, SEQ ID No. 63 in the sequence listing); 5'- attgttatcc gctcacaatt gcacacaaca ta -3' (P
3, SEQ ID NO: 64 of the sequence listing) and 5′-ttcacacagg aaacagctat gacc -3 ′ (P4, SEQ ID NO: 65 of the sequence listing) were synthesized.

Next, PCR was carried out under the following conditions: PCR (1) Reaction solution composition: template DNA pUC18 2 ng 10 pmol / μl primer P1 2.5 μl 10 pmol / μl primer P2 2.5 μl 10-fold concentration Ex Taq PCR Buffer 5 μl dNTP mixed solution 4 μl Ex Taq polymerase 0.5 unit The total volume was adjusted to 50 μl with sterile distilled water. Temperature condition: After heating at 94 ° C for 1 minute, 30 at 94 ° C
After repeating the temperature cycle of 30 seconds at 50 ° C. for 30 seconds and 2.5 minutes at 72 ° C. 30 times, the temperature was kept at 72 ° C. for 7 minutes.

[0156]   PCR (2)   Reaction solution composition:     Template pUC18 2ng     10 pmol / μl primer P3 2.5 μl     10 pmol / μl primer P4 2.5 μl     10 times concentration Ex Taq PCR buffer 5 μl     dNTP mixed solution 4 μl     Ex Taq polymerase 0.5 unit     The total volume was adjusted to 50 μl with sterile distilled water. Temperature condition: Same as PCR (1) above.

The reaction products of PCR (1) and (2) were
Isopropanol precipitation, 20 μl of sterile distilled water
Dissolved in. Each product was mixed and annealed under the following conditions: Reaction solution composition Reaction product of PCR (1) 1 μl Reaction product of PCR (2) 1 μl 10 times concentration annealing buffer (0.1 M Tris-HCl buffer) (PH 8.0), 10 mM EDTA, 1 M sodium chloride) 5 μl The total volume was adjusted to 50 μl with sterile distilled water. Temperature condition: The mixture was heated at 94 ° C. for 3 minutes, kept at 50 ° C. for 2 hours, and then cooled to room temperature (25 ° C.).

Then, the competent E. coli JM109 strain was transformed with 5 μl of the reaction solution after annealing by the method described in 1) of Example 9 to obtain 200 μg of isopropylthiogalactopyranoside (hereinafter referred to as “IPTG”).
And 800 μg of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (hereinafter “X-ga
1 ") and spread on L-broth agar containing 3),
Cultured at 7 ° C. The blue colonies that appeared were isolated and
The cells were cultured overnight in an L-broth medium containing 0 μg / ml ampicillin, and the plasmid was extracted from the culture solution. Whether or not this plasmid had a MunI recognition site was confirmed by the appearance of one band at a position of about 2.8 kbp as a result of digestion of the plasmid with the restriction enzyme MunI and electrophoresis on 1% agarose gel. . In addition, the nucleotide sequence of the site where the mutation was introduced was also confirmed. Furthermore, the transformants carrying the plasmid were IPTG and X-gal.
A blue colony was formed on the L-broth agar medium containing the above, indicating that the LacZ gene after substitution was functioning normally.

Next, using the pUC18M vector as a template, as a primer for introducing a mutation that changes the lac promoter to the lacUV5 promoter, the following oligonucleotides: 5'-tttatgcttc cggctcgtat aatgtgtgca at -3 '(P2
1, SEQ ID NO: 66 of the sequence listing); 5'- gtgtaaagcc tggggtgcct aa -3 '(P22, SEQ ID NO: 67 of the sequence listing); 5'- tccgctcaca attgcacaca ttatacgagc cg -3' (P2
3, 5'-taacaatttc acacaggaaa cag -3 '(P24, SEQ ID NO: 69 in the sequence listing) were synthesized and PCR was performed under the following conditions: PCR
(3) Composition of reaction solution: template pUC18M 10 ng 10 pmol / μl primer P21 2.5 μl 10 pmol / μl primer P22 2.5 μl 10-fold concentration Ex Taq PCR buffer 5 μl dNTP mixture 4 μl Ex Taq polymerase 1.25 units sterile distilled water. The total volume was 50 μl. Temperature condition: after heating at 94 ° C for 3 minutes, 30 at 94 ° C
Seconds, 55 ° C for 30 seconds, 72 ° C for 3 minutes for 3 minutes
After repeating 0 times, the temperature was kept at 72 ° C. for 10 minutes.

[0160]   PCR (4)   Reaction solution composition:     Template pUC18M 10ng     10 pmol / μl primer P23 2.5 μl     10 pmol / μl primer P24 2.5 μl     10 times concentration Ex Taq PCR buffer 5 μl     dNTP mixed solution 4 μl     Ex Taq polymerase 1.25 units     The total volume was adjusted to 50 μl with sterile distilled water. Temperature conditions: Same as PCR (3) above.

The reaction product after each PCR was precipitated with isopropanol, washed with 70% ethanol, dissolved in 20 μl of sterile distilled water, and both were annealed under the following conditions: Reaction solution composition: 10 times concentration Annealing buffer 5 μl PCR (3) reaction product 1 μl PCR (4) reaction product 1 μl Sterile distilled water was added to bring the total volume to 50 μl. Temperature condition: The mixture was heated at 94 ° C. for 3 minutes, kept at 50 ° C. for 2 hours, and then cooled to room temperature (25 ° C.).

Then, 5 μl of the reaction solution after annealing was used to transform competent Escherichia coli JM109 strain by the method described in 1) of Example 9 to transform L-broth agar containing 200 μg of IPTG and 800 μg of X-gal. It was spread on a medium and cultured at 37 ° C. The blue colonies that appeared were isolated, and L-containing 50 μg / ml ampicillin was isolated.
The plasmid was extracted by culturing overnight in broth medium. The introduction of the desired mutation was confirmed by analyzing the nucleotide sequence of the obtained plasmid. In addition, since the transformant carrying the plasmid forms a blue colony on the L-broth agar medium containing IPTG and X-gal, the lacUV5 promoter newly created by mutagenesis functions normally. It was shown that By the above operation, a direct expression vector pUC18M5 (SEQ ID NO: 70 in the sequence listing) for preparing peptide 12 was obtained.

Then, h7-3 obtained in the above 1) is
As a PCR primer for ligating immediately after the start codon (atg) on the 3'end side of the lacUV5 promoter of pUC18M5 vector, the following oligonucleotides are used: 5'- ggtatcatcg cagcatacca gaa -3 '(P101, in the sequence listing) SEQ ID NO: 71); 5'- ttaacccatc agggtgaaac cg -3 '(P102, SEQ ID NO: 72 in the sequence listing); 5'- gataacaatt tcacacagga aacagctatg ggtatcatcg ca
gcatacca gaa -3 '(P103, SEQ ID NO: 7 in the sequence listing)
3); 5'-agaggatccc cgggtaccga gctcgaattc ttaacccatc ag
ggtgaaac cg -3 '(P104, SEQ ID NO: 7 in the sequence listing)
4); 5'-gaattcgagc tcggtacccg gg -3 '(P105, SEQ ID NO: 75 in the sequence listing); 5'-catagctgtt tcctgtgtga aa -3' (P106, SEQ ID NO: 76 in the sequence listing); 5'-agagtcgacc tgcaggcatg ca- 3 '(P107, SEQ ID NO: 77 in the sequence listing); and 5'-cgctcacaat tgcacacatt at -3' (P108, SEQ ID NO: 78 in the sequence listing) were synthesized and PCR was carried out under the following conditions: PCR (5) Reaction solution composition: template DNA pUC (h7-3) 200 ng 10 pmol / μl primer P101 2.5 μl 10 pmol / μl primer P102 2.5 μl 10-fold concentration Ex Taq PCR buffer 5 μl dNTP mixture 4 μl Ex Taq polymerase 1.25 unit sterilization The total volume was adjusted to 50 μl with distilled water. Temperature condition: after heating at 94 ° C for 3 minutes, 30 at 94 ° C
Seconds, 55 ° C for 30 seconds, 72 ° C for 3 minutes for 3 minutes
After repeating 0 times, the temperature was kept at 72 ° C. for 10 minutes.

[0164]   PCR (6)   Reaction solution composition:     Template DNA pUC (h7-3) 200 ng     10 pmol / μl primer P103 2.5 μl     10 pmol / μl primer P104 2.5 μl     10 times concentration Ex Taq PCR buffer 5 μl     dNTP mixed solution 4 μl     Ex Taq polymerase 1.25 units     The total volume was adjusted to 50 μl with sterile distilled water. Temperature conditions: Same as PCR (5) above.

[0165]   PCR (7)   Reaction solution composition:     Template DNA solution pUC18M5 10ng     10 pmol / μl primer P105 2.5 μl     10 pmol / μl primer P106 2.5 μl     10 times concentration Ex Taq PCR buffer 5 μl     dNTP mixed solution 4 μl     Ex Taq polymerase 1.25 units     The total volume was adjusted to 50 μl with sterile distilled water. Temperature conditions: Same as PCR (5) above.

[0166]   PCR (8)   Reaction solution composition:     Template DNA solution pUC18M5 10ng     10 pmol / μl primer P107 2.5 μl     10 pmol / μl primer P108 2.5 μl     10 times concentration Ex Taq PCR buffer 5 μl     dNTP mixed solution 4 μl     Ex Taq polymerase 1.25 units     The total volume was adjusted to 50 μl with sterile distilled water. Temperature conditions: Same as PCR (5) above.

The reaction solution after each PCR was precipitated with isopropanol, washed with 70% ethanol, and then 20 times each.
Dissolved in μl sterile distilled water. Take 5 μl of each and take 2
% Low melting point agarose gel electrophoresis was performed. Cut out the gel of the band corresponding to the amplified DNA fragment, and
A double volume of TE buffer was added, and the mixture was incubated at 65 ° C for 5 minutes to dissolve the gel. 2 this with an equal volume of phenol solution
10 times, extracted once with a phenol / chloroform solution and once with a chloroform solution, followed by ethanol precipitation, washing the precipitate with 70% ethanol, and sterilized distilled water 10 times each.
It was dissolved in μl. Furthermore, these were mixed and annealed under the following conditions: Reaction solution composition: each reaction product solution of PCR (5) to (8) 1 μl 10 times concentration annealing buffer 10 μl Total volume of 100 μl with sterile distilled water. did. Temperature condition: The mixture was heated at 94 ° C for 3 minutes, kept at 50 ° C for 2 hours, and then cooled to room temperature (25 ° C).

Then, the competent Escherichia coli JM109 strain was transformed with 10 μl of the reaction solution after annealing according to the method described in 1) of Example 9 to obtain 200 μg of IPTG.
And spread on L-broth agar medium containing 800 μg of X-gal and cultured at 37 ° C. The white colonies that appeared were isolated and L containing 50 μg / ml ampicillin was isolated.
-The plasmid was extracted by overnight culture in broth medium. Whether or not the desired plasmid was obtained was confirmed by examining the nucleotide sequence of the insert fragment. Thus, the plasmid pUCM5-h73 was obtained.

Next, this pUCM5-h73 was digested with the restriction enzymes MunI and SalI and subjected to low melting point agarose gel electrophoresis to obtain a small fragment (about 360b) from the gel.
p) was extracted. On the other hand, separately from this, pUCM5-h7
3 was digested with restriction enzymes EcoRI and SalI and subjected to low melting point agarose gel electrophoresis to extract a large fragment (about 2.9 kbp) from the gel. Ligation of these two fragments using a DNA ligation kit gave plasmid pUCM5-2h73 (FIG. 6).

Similarly, the Mu of this pUCM5-2h73
nI-SalI digested small fragment (about 700 bp) and Ec
By ligating the large fragment digested with oRI-SalI (about 3.3 kbp), plasmid pUCM5-4h73 was obtained, and MunI-SalI of pUCM5-2h73 was obtained.
Digested small fragment (about 690 bp) and pUCM5-4h73
EcoRI-SalI digested large fragment of (about 3.9 kbp)
By ligating the plasmid pUCM5-6h7
3 was obtained (FIG. 6). In this pUCM5-6h73 (SEQ ID NO: 79 in the sequence listing), the multicopy unit [ribosome binding region + h7-3] is lac derived from pUC18M5.
All are directly linked immediately after the start codon on the 3'end side of the UV5 promoter. By introducing these plasmids into E. coli, peptide 12 can be directly and efficiently produced.

In the same manner as in Example 9 3), 5 ng of p
UCM5-6h73 was transformed into competent E. coli YA21 strain (JM109 strain is also possible). This transformant was treated with 2 × T containing 100 μg / ml of ampicillin.
The Y medium was inoculated into 100 ml and shake-cultured at 37 ° C. for 14 hours. After completion of the culture, take 45 ml of the culture solution and
The precipitated bacterial cells were collected by centrifugation at 00 × g, 4 ° C. for 20 minutes, suspended in 4.5 ml of TES buffer solution,
The cells were disrupted with an ultrasonic homogenizer. This one
After centrifugation at 3000 xg and 4 ° C for 20 minutes to recover the precipitate, and further washing with TES buffer three times,
It was washed 4 times with 4.5 ml of 0.75% N-lauryl sarcosine solution and further twice with distilled water (centrifugation conditions:
25,000 × g, 4 ° C., 20 minutes). The obtained precipitate was suspended in 4.5 ml of a 6 M guanidine solution and solubilized while shaking at 37 ° C. 1 hour later, 25000x
After centrifugation at 4 ° C. for 20 minutes, the supernatant was collected and used as a crude purified solution of peptide 12.

1 ml of the above crude purified liquid was subjected to gel filtration chromatography purification under the following conditions: Column: TSK-Gel G-3000SW _XL (φ7.8 ×)
300 mm, manufactured by Tosoh Corporation) Solvent: 0.1% T
30% acetonitrile containing FA Flow rate: 0.5 ml / min Detection wavelength: 280 nm The peak at a retention time of about 20.2 minutes was collected and further subjected to reverse phase HPLC purification under the following conditions: Column: ODS column (TSK- Gel ODS-120
T) Mobile phase: 36-43% acetonitrile / 0.1% TF
A, 35 minutes (linear concentration gradient) Flow rate: 2 ml / min Detection wavelength: 220 nm Peak at around retention time 13.4 minutes was collected and N-terminal 10
As a result of amino acid sequence analysis up to the residues, the amino acid sequence was in agreement with the amino acid sequences represented by amino acid numbers 1 to 10 of SEQ ID NO: 15 in the sequence listing. In addition, as a result of measuring the molecular weight by the ESI method, the calculated molecular weight of peptide 12 and the molecular weight of the substance mainly contained in this peak matched. As described above, about 6 mg of Peptide 12 could be obtained per 100 ml of the culture solution.

[Reference Example] p prepared in Examples 9 to 11
UC (h6-1), pUC (h7-1) or pUC
(H7-3) was digested with restriction enzymes BamHI and SalI, and their small fragments (about 270 bp and about 3 respectively) were digested.
00 bp or approximately 310 bp) were purified by agarose gel electrophoresis. On the other hand, cloning vector pBR322
Was digested with the restriction enzymes BamHI and SalI, and the large fragment (about 4.1 kbp) was purified by agarose gel electrophoresis. Plasmid pBR (h6-1), pBR (h7-1) or pBR obtained by ligating these DNA fragments
(H7-3) was introduced into competent E. coli JM109 strain, and the transformed E. coli E. coli pBR (h
6-1) SANK 70199, E.I. coli pB
R (h7-1) SANK 70299 and E.I. co
li pBR (h7-3) SANK 70399 is
Deposited at the Institute of Biotechnology, Institute of Industrial Science on February 9, 1998, with deposit number FERM.
BP-6642, FERM BP-6643 and F
ERM BP-6644 is attached.

Example 12 Cedar Pollen Antigen T Cell Epitope Activity Using the T cells derived from a patient with cedar pollinosis, the peptide of the present invention was used.
The cedar pollen antigen T cell epitope activities of 1 to 12 were confirmed by the method described below. 50 ml of peripheral blood was collected from a patient showing the symptoms of Japanese cedar pollinosis. Peripheral blood is converted to Hanks' Balanced Salt Solution (HB).
After dilution with SS), the peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cel
ls, hereinafter referred to as "PBMC") was fractionated and suspended in a medium (RPMI1640 containing 5% human AB serum).

4 per well in 96-well flat bottom plates
× 10 ⁵ cells were dispensed. At the same time, peptides 1, 2, 3, 7 were added to each well so that the final concentration was 1 μg / ml
Or 8 or peptide 4, 5, 6, 11 or 12 was added to a final concentration of 100 nM, or peptide 9 or 10 was added to a final concentration of 50 nM. As a negative control, a group to which no peptide was added was prepared. Cells in 200 μl medium at 37 ° C, 5%
The cells were cultured under carbon dioxide gas for 72 hours. After that, 0.5 μCi
Tritiated thymidine was added and the cells were further cultured for 16 hours. The cells in each well were collected on a glass fiber filter using a cell harvester, and the amount of tritiated thymidine incorporated into the cells was measured by a liquid scintillation counter. A value obtained by dividing the amount of thymidine uptake by the amount of uptake in the negative control group was used as a T cell stimulating index, and a group in which this T cell stimulating index reached a value exceeding 2 was defined as "positive" T cell epitope activity. The results are shown in Table 1.

[0176]

[Table 1]

The peptides prepared in Examples 1 to 11 all showed "positive" T cell epitope activity. [Example 13] Synergistic effect by ligation It was shown by the following method that the T cell epitope activity of the ligated peptide exhibited synergistically enhanced activity by ligating the T cell epitope peptide.

First, the following peptide, which is used as a control and consists of a single T-cell epitope that is not linked: Gly-Ile-Ile-Ala-Ala-Tyr-Gln-Asn-Pro-Ala-Ser-Trp.
(Peptide 13, corresponding to T cell epitope "A", SEQ ID NO: 16 in the sequence listing); Gln-Phe-Ala-Lys-Leu-Thr-Gly-Phe-Thr-Leu-Met-Gly
(Peptide 14, corresponding to T cell epitope "B", SEQ ID NO: 17 in the sequence listing); Phe-Ala-Ser-Lys-Asn-Phe-His-Leu-Gln-Lys-Asn-Thr
(Peptide 15, corresponding to T cell epitope "C", SEQ ID NO: 18 in Sequence Listing); Lys-Leu-Thr-Ser-Gly-Lys-Ile-Ala-Ser-Cys-Leu-Asn
(Peptide 16, corresponding to T cell epitope “D”, SEQ ID NO: 19 in the sequence listing); Ser-Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly-Pr
o (Peptide 17, corresponding to T cell epitope "E", SEQ ID NO: 20 in Sequence Listing); Met-Lys-Val-Thr-Val-Ala-Phe-Asn-Gln-Phe-Gly (Peptide 18, T cell epitope Corresponding to "E '", SEQ ID NO: 21) in the sequence listing; Tyr-Gly-Leu-Val-His-Val-Ala-Asn-Asn-Asn-Tyr-Asp-Pr
o (Peptide 19, corresponding to T cell epitope "F", SEQ ID NO: 22 in Sequence Listing); and Ser-Gly-Lys-Tyr-Glu-Gly-Gly-Asn-Ile-Tyr-Thr-Lys-Ly
s-Glu-Ala-Phe-Asn-Val-Glu (peptide 20, corresponding to T cell epitope "G", SEQ ID NO: 23 in the sequence listing) was synthesized by the same method as described in Example 7.

On the other hand, from patients showing symptoms of Japanese cedar pollinosis, 50
After collecting ml of peripheral blood and diluting it with HBSS, PBMCs were fractionated by Ficoll-pack specific gravity centrifugation and suspended in a medium (RPMI1640 containing 5% human AB serum). 4 x 1 per well in 96-well flat bottom plate
0 ⁵ cells were dispensed, and at the same time, peptide 1,
2, 3, 4, 5, 6, 11, or 12 for a final concentration of 1
It was added at 0 pM to 100 nM. For peptide 9 and peptide 10, peptide 9 or 10 was added to each well to give a final concentration of 5 pM to 50 nM. Further, the following four groups were provided as a peptide mixed culture group. Ie 1 peptide per well
3, 15, 16, 17, 19 and 20 each of which was added to a final concentration of 10 pM to 100 nM, 1
Group in which each of peptides 13, 15, 16, 18, 19, and 20 was added at a final concentration of 10 pM to 100 nM per well, and peptide 13, 1 per well
4, 15, 16, 17, 19, and 20 were added to each to give a final concentration of 10 pM to 100 nM, and peptides 7, 8, 14, 16, 1 were added per well.
7, 19 and 20 each with a final concentration of 10 pM to 10
A group was added so that the concentration was 0 nM. The cells thus conditioned were cultured in 200 μl of medium at 37 ° C. under 5% carbon dioxide gas for 72 hours. Then, 0.5 μCi of tritiated thymidine was added, and the cells were further cultured for 16 hours. The cells in each well were collected on a glass fiber filter using a cell harvester, and the amount of tritiated thymidine incorporated into the cells was measured by a liquid scintillation counter. And peptide 1 or peptide 9
Regarding the added group, the T cell epitope activity was compared with the mixed culture group of peptides 13, 15, 16, 17, 19 and 20 in which the composition of T cell epitopes was common to these (FIG. 7). Regarding the peptide 2 or peptide 3 addition group, the T cell epitope activity was similarly compared with the mixed culture group of peptides 13, 15, 16, 18, 19 and 20 (FIG. 8). For the group to which peptide 4 or peptide 10 was added, peptides 13, 14, 15, 16, 17,
The T cell epitope activity was compared with the mixed culture groups of 19 and 20 (FIG. 9). Further, for the group to which peptide 5, peptide 6, peptide 11 or peptide 12 was added, peptides 7, 8, 14, 16, 17, 19, and 20 were similarly added.
T cell epitope activity was compared with that of the mixed culture group (Fig. 1).
0, FIG. 11). However, the results of the groups to which peptide 9 and peptide 10 were added were plotted at the position of twice the actual molar concentration in order to correct that each peptide contained two T cell epitopes.

As a result, even if the total amount of each T cell epitope added was the same, when they were linked,
It was shown that the activity as a T cell epitope was at least 10 times higher than when added simply as a mixture.

Example 14 Importance of T Cell Epitope Peptide For 114 cedar pollinosis patients whose T cell reactivity to either the cedar pollen allergen Cryj1 or Cryj2 reached a value exceeding the T cell stimulation index 2. Then, the T cell epitope activities of peptide 7 and peptide 8 were evaluated according to the method described in Example 12, and the importance index was obtained. The importance index (Positivity Index) is a value obtained by multiplying the average T cell stimulation index by the positive frequency, and is WO94 / 015.
No. 60 publication. The average T cell stimulation index represents the geometric mean value of the T cell stimulation index of the subjects who showed a positive reaction to the peptide, and the positive frequency represents the ratio (percentage) of the subjects who showed a positive reaction to the peptide. The results are shown in Table 2.

[0182]

[Table 2]

Thus, peptide 7 and peptide 8
All of them showed high importance index among cedar pollinosis patients, and were shown to be useful as peptides for hyposensitization therapy for cedar pollinosis.

[Formulation Example 1] The peptide obtained by the method described in Examples 1 to 11 was added to physiological saline containing 1% (w / v) human serum albumin as a stabilizer for the final concentration of 0.01,0. 1 or 1 mg / ml
The solution is dissolved in the following manner, sterilized by filtration, dispensed in 2 ml portions into sterile vials, lyophilized, and sealed. Prior to administration, this product should first contain 1 m of distilled water for injection in a vial.
1 and then the contents are uniformly dissolved before use. The product, which has excellent stability and contains the peptide according to the present invention as an active ingredient, is useful as a dry preparation for treating and preventing cedar pollinosis.

[Formulation Example 2] Syrup agent [0185] One of the peptides obtained by the method described in Examples 1 to 11 was added to 0.1 mg / ml, and trehalose powder (Trehaose, manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) was added. % (W /
v) is dissolved in distilled water and the solution is sterilized by a conventional method to obtain 12 kinds of syrups.
2 ml of each of these syrups is separately dispensed into a sterile vial bottle and sealed. This product, which is highly stable and easy to take, is useful as a syrup for treating and preventing cedar pollinosis.

[Acute Toxicity Test] The mice obtained on the 20th day of life are orally or intraperitoneally administered with the preparation obtained by the method described in Preparation Example 1 or 2. LD50 (as a peptide) was 2 in the test sample regardless of the route of administration.
It is more than 00 mg / kg of mouse body weight, which means that the peptide of the present invention can be safely compounded and used as an anti-cedar pollinosis agent to be administered to mammals including human.

[0187]

According to the present invention, T of cedar pollen allergen
It was possible to provide a peptide comprising a cell epitope and an anti-cedar pollinosis agent containing them as an active ingredient. Since the peptide of the present invention does not substantially react with the immunoglobulin E antibody specific to the cedar pollen allergen, when administered to mammals including humans in general, it does not cause anaphylaxis and is specific to the cedar pollen allergen. T cells can be inactivated. Moreover, the peptide of the present invention containing 6 or 7 different T cell epitopes in its molecule is active at a lower dose than administration of the respective epitopes in combination,
The dose to the patient can be reduced. Further, the anti-cedar pollinosis agent comprising the peptide of the present invention as an active ingredient is effective for a wider range of cedar pollinosis patients even when the dose is reduced.

[0188]

[Sequence list]                                SEQUENCE LISTING    <110> Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo       Sankyo Company, Limited    <120> Peptides and The Uses Thereof    <130> P00-0129    <150> JP99 / 68316 <151> 1999-3-15    <160> 80    <170> PatentIn Ver. 2.0    <210> 1 <211> 81 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 1 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val   1 5 10 15    Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His              20 25 30    Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu          35 40 45    Asn Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala      50 55 60    Phe Asn Val Glu Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp  65 70 75 80    Pro    <210> 2 <211> 79 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 2 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Met Lys Val Thr   1 5 10 15    Val Ala Phe Asn Gln Phe Gly Phe Ala Ser Lys Asn Phe His Leu Gln              20 25 30    Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Tyr          35 40 45    Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys Tyr      50 55 60    Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu  65 70 75    <210> 3 <211> 79 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 3 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Met Lys Val Thr   1 5 10 15    Val Ala Phe Asn Gln Phe Gly Phe Ala Ser Lys Asn Phe His Leu Gln              20 25 30    Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Ser          35 40 45    Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn      50 55 60    Val Glu Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro  65 70 75    <210> 4 <211> 93 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 4 Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Gly Ile Ile Ala   1 5 10 15    Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val Thr Val Ala Phe              20 25 30    Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn          35 40 45    Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Tyr Gly Leu      50 55 60    Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys Tyr Glu Gly  65 70 75 80    Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu                  85 90    <210> 5 <211> 95 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 5 Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Gly Ile Ile Ala   1 5 10 15    Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys Val Thr Val Ala              20 25 30    Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys Asn Phe His Leu          35 40 45    Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn Tyr      50 55 60    Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys Tyr  65 70 75 80    Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu                  85 90 95    <210> 6 <211> 99 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which concentrated T cell epitopes derived       from ceder pollen allergens    <400> 6 Gly Asp Pro Arg Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly   1 5 10 15    Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys              20 25 30    Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys          35 40 45    Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser      50 55 60    Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro  65 70 75 80    Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe                  85 90 95    Asn Val Glu    <210> 7 <211> 13 <212> PRT <213> Cryptomeria japonica    <400> 7 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys   1 5 10    <210> 8 <211> 13 <212> PRT <213> Cryptomeria japonica <400> 8 Asp Ile Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn   1 5 10    <210> 9 <211> 558 <212> DNA <213> Artificial Sequence    <220> <221> CDS <222> (1) .. (558)    <220> <221> mat # peptide <222> (4) .. (558)    <220> <223> Description of Artificial Sequence: Designed       sequence encoding a polypeptide which coordinate       T cell epitopes derived from ceder pollen allergens    <400> 9 atg acc atg att acg aat tcg agc tcg gta ccc ggg gat ccg cgt ggt 48 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    atc atc gca gca tac cag aac ccg gca tct tgg tct atg aaa gtt acc 96 Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val Thr                  20 25 30    gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa aac ttc cat ctg 144 Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His Leu              35 40 45    cag aaa aac acc aaa ctg acc tct ggt aaa atc gca tct tgc ctg aac 192 Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn          50 55 60    tac ggt ctg gtt cat gtt gca aac aac aac tac gac ccg tct ggt aaa 240 Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys      65 70 75    tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca ttc aat gtt gaa 288 Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu  80 85 90 95    cgt gca gat ccg cgt ggt atc atc gca gca tac cag aac ccg gca tct 336 Arg Ala Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser                 100 105 110    tgg tct atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg ttc gca 384 Trp Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala             115 120 125    tct aaa aac ttc cat ctg cag aaa aac acc aaa ctg acc tct ggt aaa 432 Ser Lys Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys         130 135 140    atc gca tct tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac 480 Ile Ala Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn     145 150 155    tac gac ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa 528 Tyr Asp Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys 160 165 170 175    gaa gca ttc aat gtt gaa cgt gca gat ctg 558 Glu Ala Phe Asn Val Glu Arg Ala Asp Leu                 180 185    <210> 10 <211> 186 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which concentrated T cell epitopes derived       from ceder pollen allergens    <400> 10 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val Thr                  20 25 30    Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His Leu              35 40 45    Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn          50 55 60    Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys      65 70 75    Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu  80 85 90 95    Arg Ala Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser                 100 105 110    Trp Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala             115 120 125    Ser Lys Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys         130 135 140    Ile Ala Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn     145 150 155    Tyr Asp Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys 160 165 170 175    Glu Ala Phe Asn Val Glu Arg Ala Asp Leu                 180 185    <210> 11 <211> 630 <212> DNA <213> Artificial Sequence    <220> <221> CDS <222> (1) .. (630)    <220> <221> mat # peptide <222> (4) .. (630)    <220> <223> Description of Artificial Sequence: Designed       sequence encoding a polypeptide which coordinate       T cell epitopes derived from ceder pollen allergens    <400> 11 atg acc atg att acg aat tcg agc tcg gta ccc ggg gat ccg cgt ggt 48 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    atc atc gca gca tac cag aac ccg gca tct tgg tct atg aaa gtt acc 96 Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val Thr                  20 25 30    gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa aac ttc cat ctg 144 Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His Leu              35 40 45    cag aaa aac acc aaa ctg acc tct ggt aaa atc gca tct tgc ctg aac 192 Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn          50 55 60    tac ggt ctg gtt cat gtt gca aac aac aac tac gac ccg tct ggt aaa 240 Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys      65 70 75    tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca ttc aac gtt gaa 288 Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu  80 85 90 95    cag ttc gct aaa ctg acc ggt ttc acc ctg atg ggt cgt gca gat ccg 336 Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala Asp Pro                 100 105 110    cgt ggt atc atc gca gca tac cag aac ccg gca tct tgg tct atg aaa 384 Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys             115 120 125    gtt acc gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa aac ttc 432 Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe         130 135 140    cat ctg cag aaa aac acc aaa ctg acc tct ggt aaa atc gca tct tgc 480 His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys     145 150 155    ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac ccg tct 528 Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser 160 165 170 175    ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca ttc aac 576 Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn                 180 185 190    gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg atg ggt cgt gca 624 Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala             195 200 205    gat ctg 630 Asp Leu    <210> 12 <211> 210 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which concentrated T cell epitopes derived       from ceder pollen allergens    <400> 12 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val Thr                  20 25 30    Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His Leu              35 40 45    Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn          50 55 60    Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly Lys      65 70 75    Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val Glu  80 85 90 95    Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala Asp Pro                 100 105 110    Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys             115 120 125    Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe         130 135 140    His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys     145 150 155    Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser 160 165 170 175    Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn                 180 185 190    Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala             195 200 205    Asp Leu    <210> 13 <211> 96 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       sequence of a polypeptide which aggregate T cell       epitopes derived from ceder pollen allergens    <400> 13 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys   1 5 10 15    Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys              20 25 30    Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser          35 40 45    Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro      50 55 60    Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe  65 70 75 80    Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg                  85 90 95    <210> 14 <211> 291 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       sequence encoding a polypeptide which consists of       T cell epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (1) .. (291)    <220> <221> mat # peptide <222> (4) .. (291)    <400> 14 atg ggt atc atc gca gca tac cag aac ccg gca tct tgg aaa tct atg 48 Met Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met  -1 1 5 10 15    aaa gtt acc gtt gct ttc aac cag ttc ggt ccg gac atc ttc gca tct 96 Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser                  20 25 30    aaa aac ttc cat ctg cag aaa aac aaa ctg acc tct ggt aaa atc gca 144 Lys Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala              35 40 45    tcc tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac 192 Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp          50 55 60    ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca 240 Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala      65 70 75    ttc aac gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg atg ggt 288 Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly  80 85 90 95    cgt 291 Arg    <210> 15 <211> 97 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 15 Met Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met  -1 1 5 10 15    Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser                  20 25 30    Lys Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala              35 40 45    Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp          50 55 60    Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala      65 70 75    Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly  80 85 90 95    Arg    <210> 16 <211> 12 <212> PRT <213> Cryptomeria japonica    <400> 16 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp   1 5 10    <210> 17 <211> 12 <212> PRT <213> Cryptomeria japonica    <400> 17 Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly   1 5 10    <210> 18 <211> 12 <212> PRT <213> Cryptomeria japonica    <400> 18 Phe Ala Ser Lys Asn Phe His Leu Gln Lys Asn Thr   1 5 10    <210> 19 <211> 12 <212> PRT <213> Cryptomeria japonica    <400> 19 Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu Asn   1 5 10    <210> 20 <211> 13 <212> PRT <213> Cryptomeria japonica    <400> 20 Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro   1 5 10    <210> 21 <211> 11 <212> PRT <213> Cryptomeria japonica    <400> 21 Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly   1 5 10    <210> 22 <211> 13 <212> PRT <213> Cryptomeria japonica    <400> 22 Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro   1 5 10    <210> 23 <211> 19 <212> PRT <213> Cryptomeria japonica    <400> 23 Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe   1 5 10 15    Asn Val Glu    <210> 24 <211> 274 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed DNA       encoding a polypeptide which consists of T cell       epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (2) .. (265)    <400> 24 g gat ccg cgt ggt atc atc gca gca tac cag aac ccg gca tct tgg tct 49   Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser     1 5 10 15    atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa 97 Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys              20 25 30    aac ttc cat ctg cag aaa aac acc aaa ctg acc tct ggt aaa atc gca 145 Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala          35 40 45    tct tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac 193 Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp      50 55 60    ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca 241 Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala  65 70 75 80    ttc aat gtt gaa cgt gca gat ctg taagtcgac 274 Phe Asn Val Glu Arg Ala Asp Leu                  85    <210> 25 <211> 88 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 25 Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser   1 5 10 15    Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys              20 25 30 Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala          35 40 45    Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp      50 55 60    Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala  65 70 75 80    Phe Asn Val Glu Arg Ala Asp Leu                  85    <210> 26 <211> 14 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Linker peptide    <400> 26 Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg   1 5 10    <210> 27 <211> 5 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Linker peptide    <400> 27 Arg Ala Asp Pro Arg   1 5    <210> 28 <211> 310 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed DNA       encoding a polypeptide which consists of T cell       epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (11) .. (289)    <400> 28 ggatccgcgt ggt atc atc gca gca tac cag aac ccg gca tct tgg tct 49            Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser              1 5 10    atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa 97 Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys      15 20 25    aac ttc cat ctg cag aaa aac acc aaa ctg acc tct ggt aaa atc gca 145 Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala  30 35 40 45    tct tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac 193 Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp                  50 55 60    ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca 241 Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala              65 70 75    ttc aac gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg atg ggt 289 Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly          80 85 90    cgtgcagatc tgtaagtcga c 310    <210> 29 <211> 93 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 29 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val   1 5 10 15    Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His              20 25 30    Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu          35 40 45    Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly      50 55 60    Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val  65 70 75 80    Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly                  85 90    <210> 30 <211> 274 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed DNA       encoding a polypeptide which consists of T cell       epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (11) .. (253)    <400> 30 ggatccgcgt ggt atc atc gca gca tac cag aac ccg gca tct tgg tct 49            Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser              1 5 10    atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg ttc gca tct aaa 97 Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys      15 20 25    aac ttc cat ctg cag aaa aac acc aaa ctg acc tct ggt aaa atc gca 145 Asn Phe His Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala  30 35 40 45    tct tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac 193 Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp                  50 55 60    ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca 241 Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala              65 70 75    ttc aac gtt gaa cgtgcagatc tgtaagtcga c 274 Phe Asn Val Glu          80    <210> 31 <211> 81 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 31 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Ser Met Lys Val   1 5 10 15    Thr Val Ala Phe Asn Gln Phe Gly Pro Phe Ala Ser Lys Asn Phe His              20 25 30    Leu Gln Lys Asn Thr Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys Leu          35 40 45    Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser Gly      50 55 60    Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn Val  65 70 75 80    Glu    <210> 32 <211> 45 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 32 gatccgcgtg gtatcatcgc agcataccag aacccggcat cttgg 45    <210> 33 <211> 47 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 33 catagaccaa gatgccgggt tctggtatgc tgcgatgata ccacgcg 47    <210> 34 <211> 39 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 34 tctatgaaag ttaccgttgc tttcaaccag ttcggtccg 39    <210> 35 <211> 39 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 35 tgcgaacgga ccgaactggt tgaaagcaac ggtaacttt 39    <210> 36 <211> 30 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 36 ttcgcatcta aaaacttcca tctgcagaaa 30    <210> 37 <211> 30 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 37 ggtgtttttc tgcagatgga agtttttaga 30    <210> 38 <211> 36 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 38 aacaccaaac tgacctctgg taaaatcgca tcttgc 36    <210> 39 <211> 36 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 39 gttcaggcaa gatgcgattt taccagaggt cagttt 36    <210> 40 <211> 41 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 40 ctgaactacg gtctggttca tgttgcaaac aacaactacg a 41    <210> 41 <211> 41 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 41 gacgggtcgt agttgttgtt tgcaacatga accagaccgt a 41    <210> 42 <211> 41 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 42 cccgtctggt aaatacgaag gtggtaacat ctacaccaaa a 41    <210> 43 <211> 41 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 43 cttctttttt ggtgtagatg ttaccacctt cgtatttacc a 41    <210> 44 <211> 36 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 44 aagaagcatt caacgttgaa cgtgcagatc tgtaag 36    <210> 45 <211> 34 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 45 tcgacttaca gatctgcacg ttcaacgttg aatg 34    <210> 46 <211> 35 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 46 aagaagcatt caacgttgaa cagttcgcta aactg 35    <210> 47 <211> 35 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 47 accggtcagt ttagcgaact gttcaacgtt gaatg 35    <210> 48 <211> 37 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 48 accggtttca ccctgatggg tcgtgcagat ctgtaag 37    <210> 49 <211> 35 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 49 tcgacttaca gatctgcacg acccatcagg gtgaa 35    <210> 50 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 50 ggatccgcgt ggtatcatcg ca 22    <210> 51 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 51 aggtcgactt acagatctgc ac 22    <210> 52 <211> 316 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed DNA       encoding a polypeptide which consists of T cell       epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (11) .. (298)    <400> 52 ggatccgcgt ggt atc atc gca gca tac cag aac ccg gca tct tgg aaa 49            Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys              1 5 10    tct atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg gac atc ttc 97 Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe      15 20 25    gca tct aaa aac ttc cat ctg cag aaa aac aaa ctg acc tct ggt aaa 145 Ala Ser Lys Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys  30 35 40 45    atc gca tct tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac 193 Ile Ala Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn                  50 55 60    tac gac ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa 241 Tyr Asp Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys              65 70 75    gaa gca ttc aac gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg 289 Glu Ala Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu          80 85 90    atg ggt cgt gcagatctgt aagtcgac 316 Met Gly Arg      95    <210> 53 <211> 96 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 53 Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys   1 5 10 15    Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys              20 25 30    Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser          35 40 45    Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro      50 55 60    Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe  65 70 75 80    Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg                  85 90 95    <210> 54 <211> 42 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 54 aaatctatga aagttaccgt tgctttcaac cagttcggtc cg 42    <210> 55 <211> 47 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 55 agatttccaa gatgccgggt tctggtatgc tgcgatgata ccacgcg 47    <210> 56 <211> 32 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 56 gacatcttcg catctaaaaa cttccatctg ca 32    <210> 57 <211> 42 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 57 gatgtccgga ccgaactggt tgaaagcaac ggtaactttc at 42    <210> 58 <211> 37 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 58 gaaaaacaaa ctgacctctg gtaaaatcgc atcttgc 37    <210> 59 <211> 33 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence:       Oligonucleotide to construct a DNA encoding a       polypeptide which consists of T cell epitopes       derived from ceder pollen allergens    <400> 59 gtttttctgc agatggaagt ttttagatgc gaa 33    <210> 60 <211> 651 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed DNA       encoding a polypeptide which consists of T cell       epitopes derived from ceder pollen allergens    <220> <221> CDS <222> (1) .. (642)    <220> <221> mat # peptide <222> (4) .. (642)    <400> 60 atg acc atg att acg aat tcg agc tcg gta ccc ggg gat ccg cgt ggt 48 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    atc atc gca gca tac cag aac ccg gca tct tgg aaa tct atg aaa gtt 96 Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys Val                  20 25 30    acc gtt gct ttc aac cag ttc ggt ccg gac atc ttc gca tct aaa aac 144 Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys Asn              35 40 45    ttc cat ctg cag aaa aac aaa ctg acc tct ggt aaa atc gca tcc tgc 192 Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys          50 55 60    ctg aac tac ggt ctg gtt cat gtt gca aac aac aac tac gac ccg tct 240 Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser      65 70 75    ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa gaa gca ttc aac 288 Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn  80 85 90 95    gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg atg ggt cgt gca 336 Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala                 100 105 110    gat ccg cgt ggt atc atc gca gca tac cag aac ccg gca tct tgg aaa 384 Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys             115 120 125    tct atg aaa gtt acc gtt gct ttc aac cag ttc ggt ccg gac atc ttc 432 Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe         130 135 140    gca tct aaa aac ttc cat ctg cag aaa aac aaa ctg acc tct ggt aaa 480 Ala Ser Lys Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys     145 150 155    atc gca tcc tgc ctg aac tac ggt ctg gtt cat gtt gca aac aac aac 528 Ile Ala Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn 160 165 170 175    tac gac ccg tct ggt aaa tac gaa ggt ggt aac atc tac acc aaa aaa 576 Tyr Asp Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys                 180 185 190    gaa gca ttc aac gtt gaa cag ttc gct aaa ctg acc ggt ttc acc ctg 624 Glu Ala Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu             195 200 205    atg ggt cgt gca gat ctg taagtcgac 651 Met Gly Arg Ala Asp Leu         210 <210> 61 <211> 214 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Designed       peptide which consists of T cell epitopes derived       from ceder pollen allergens    <400> 61 Met Thr Met Ile Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Arg Gly  -1 1 5 10 15    Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys Ser Met Lys Val                  20 25 30    Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe Ala Ser Lys Asn              35 40 45    Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys Ile Ala Ser Cys          50 55 60    Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn Tyr Asp Pro Ser      65 70 75    Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys Glu Ala Phe Asn  80 85 90 95    Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu Met Gly Arg Ala                 100 105 110    Asp Pro Arg Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser Trp Lys             115 120 125    Ser Met Lys Val Thr Val Ala Phe Asn Gln Phe Gly Pro Asp Ile Phe         130 135 140    Ala Ser Lys Asn Phe His Leu Gln Lys Asn Lys Leu Thr Ser Gly Lys     145 150 155    Ile Ala Ser Cys Leu Asn Tyr Gly Leu Val His Val Ala Asn Asn Asn 160 165 170 175    Tyr Asp Pro Ser Gly Lys Tyr Glu Gly Gly Asn Ile Tyr Thr Lys Lys                 180 185 190    Glu Ala Phe Asn Val Glu Gln Phe Ala Lys Leu Thr Gly Phe Thr Leu             195 200 205    Met Gly Arg Ala Asp Leu         210 <210> 62 <211> 31 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18 vector    <400> 62 tccggctcgt atgttgtgtg caattgtgag c 31    <210> 63 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18 vector    <400> 63 agcataaagt gtaaagcctg gg 22    <210> 64 <211> 32 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18 vector    <400> 64 attgttatcc gctcacaatt gcacacaaca ta 32    <210> 65 <211> 24 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18 vector    <400> 65 ttcacacagg aaacagctat gacc 24    <210> 66 <211> 32 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18M vector    <400> 66 tttatgcttc cggctcgtat aatgtgtgca at 32    <210> 67 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18M vector    <400> 67 gtgtaaagcc tggggtgcct aa 22    <210> 68 <211> 32 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18M vector    <400> 68 tccgctcaca attgcacaca ttatacgagc cg 32    <210> 69 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       introduce a mutation in the pUC18M vector    <400> 69 taacaatttc acacaggaaa cag 23    <210> 70 <211> 2686 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Plasmid vector       pUC18M5    <400> 70 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60    cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120    cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtgcaat 180    tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 240    cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 300    ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360    atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420    agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 480    gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 540    taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 600    cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 660    caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 720    ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 780    gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 840    aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 900    tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 960    aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1020    aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 1080    tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1140    aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1200    tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 1260    ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1320    taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1380    agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1440    caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1500    tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1560    gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1620    cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1680    caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1740    ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1800    aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1860    gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1920    atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1980    tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 2040    gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 2100    actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2160    gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2220    agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2280    ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2340    aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2400    cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2460    gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2520    cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2580    cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2640    gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga 2686    <210> 71 <211> 23 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 71 ggtatcatcg cagcatacca gaa 23    <210> 72 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 72 ttaacccatc agggtgaaac cg 22    <210> 73 <211> 53 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 73 gataacaatt tcacacagga aacagctatg ggtatcatcg cagcatacca gaa 53    <210> 74 <211> 52 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a DNA encoding a polypeptide which       consists of T cell epitopes derived from ceder       pollen allergens    <400> 74 agaggatccc cgggtaccga gctcgaattc ttaacccatc agggtgaaac cg 52    <210> 75 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a fragment in the pUC18M5 vector    <400> 75 gaattcgagc tcggtacccg gg 22    <210> 76 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a fragment in the pUC18M5 vector    <400> 76 catagctgtt tcctgtgtga aa 22    <210> 77 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a fragment in the pUC18M5 vector    <400> 77 agagtcgacc tgcaggcatg ca 22    <210> 78 <211> 22 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: PCR primer to       amplify a fragment in the pUC18M5 vector    <400> 78 cgctcacaat tgcacacatt at 22    <210> 79 <211> 4613 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Plasmid       pUCM5-6h73    <400> 79 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60    cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120    cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa tgtgtgcaat 180    tgtgagcgga taacaatttc acacaggaaa cagctatggg tatcatcgca gcataccaga 240    acccggcatc ttggaaatct atgaaagtta ccgttgcttt caaccagttc ggtccggaca 300    tcttcgcatc taaaaacttc catctgcaga aaaacaaact gacctctggt aaaatcgcat 360    cttgcctgaa ctacggtctg gttcatgttg caaacaacaa ctacgacccg tctggtaaat 420    acgaaggtgg taacatctac accaaaaaag aagcattcaa cgttgaacag ttcgctaaac 480    tgaccggttt caccctgatg ggttaagaat tgtgagcgga taacaatttc acacaggaaa 540    cagctatggg tatcatcgca gcataccaga acccggcatc ttggaaatct atgaaagtta 600    ccgttgcttt caaccagttc ggtccggaca tcttcgcatc taaaaacttc catctgcaga 660    aaaacaaact gacctctggt aaaatcgcat cttgcctgaa ctacggtctg gttcatgttg 720    caaacaacaa ctacgacccg tctggtaaat acgaaggtgg taacatctac accaaaaaag 780    aagcattcaa cgttgaacag ttcgctaaac tgaccggttt caccctgatg ggttaagaat 840    tgtgagcgga taacaatttc acacaggaaa cagctatggg tatcatcgca gcataccaga 900    acccggcatc ttggaaatct atgaaagtta ccgttgcttt caaccagttc ggtccggaca 960    tcttcgcatc taaaaacttc catctgcaga aaaacaaact gacctctggt aaaatcgcat 1020    cttgcctgaa ctacggtctg gttcatgttg caaacaacaa ctacgacccg tctggtaaat 1080    acgaaggtgg taacatctac accaaaaaag aagcattcaa cgttgaacag ttcgctaaac 1140    tgaccggttt caccctgatg ggttaagaat tgtgagcgga taacaatttc acacaggaaa 1200    cagctatggg tatcatcgca gcataccaga acccggcatc ttggaaatct atgaaagtta 1260    ccgttgcttt caaccagttc ggtccggaca tcttcgcatc taaaaacttc catctgcaga 1320    aaaacaaact gacctctggt aaaatcgcat cttgcctgaa ctacggtctg gttcatgttg 1380    caaacaacaa ctacgacccg tctggtaaat acgaaggtgg taacatctac accaaaaaag 1440    aagcattcaa cgttgaacag ttcgctaaac tgaccggttt caccctgatg ggttaagaat 1500    tgtgagcgga taacaatttc acacaggaaa cagctatggg tatcatcgca gcataccaga 1560    acccggcatc ttggaaatct atgaaagtta ccgttgcttt caaccagttc ggtccggaca 1620    tcttcgcatc taaaaacttc catctgcaga aaaacaaact gacctctggt aaaatcgcat 1680    cttgcctgaa ctacggtctg gttcatgttg caaacaacaa ctacgacccg tctggtaaat 1740    acgaaggtgg taacatctac accaaaaaag aagcattcaa cgttgaacag ttcgctaaac 1800    tgaccggttt caccctgatg ggttaagaat tgtgagcgga taacaatttc acacaggaaa 1860    cagctatggg tatcatcgca gcataccaga acccggcatc ttggaaatct atgaaagtta 1920    ccgttgcttt caaccagttc ggtccggaca tcttcgcatc taaaaacttc catctgcaga 1980    aaaacaaact gacctctggt aaaatcgcat cttgcctgaa ctacggtctg gttcatgttg 2040    caaacaacaa ctacgacccg tctggtaaat acgaaggtgg taacatctac accaaaaaag 2100    aagcattcaa cgttgaacag ttcgctaaac tgaccggttt caccctgatg ggttaagaat 2160    tcgagctcgg tacccgggga tcctctagag tcgacctgca ggcatgcaag cttggcactg 2220    gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 2280    gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 2340    tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg 2400    catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc 2460    gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 2520    ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 2580    aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 2640    ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 2700    aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 2760    atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 2820    caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 2880    cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 2940    tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 3000    tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 3060    gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 3120    tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 3180    gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 3240    aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 3300    gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 3360    atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 3420    caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 3480    ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 3540    attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 3600    agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 3660    aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 3720    catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 3780    ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 3840    tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 3900    ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 3960    ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 4020    ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4080    gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4140    aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 4200    acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 4260    gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 4320    gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 4380    cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 4440    aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 4500    gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 4560    cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aga 4613    <210> 80 <211> 4 <212> PRT <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: Linker peptide    <400> 80 Arg Ala Asp Leu   1

[0189]

[Sequence list free text]

SEQ ID NO: 1 Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 2: Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 3: Peptide SEQ ID number consisting of a plurality of cedar pollen allergen-derived T cell epitopes 4: Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 5: Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes

SEQ ID NO: 6: Peptide containing a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 9: DNA encoding a peptide containing a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 10: Plural cedar pollen allergen-derived T Peptide containing a cell epitope SEQ ID NO: 11: DNA encoding a peptide containing a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 12: Peptide containing a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 13: a plurality of cedar pollen allergens Peptide Containing Derived T Cell Epitope SEQ ID NO: 14: DNA Encoding Peptide Comprising Multiple Cedar Pollen Allergen-Derived T Cell Epitopes SEQ ID NO: 15: Peptide Comprised Plural Cedar Pollen Allergen Derived T Cell Epitopes

SEQ ID NO: 24: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
NA SEQ ID NO: 25: Peptide consisting of multiple T cell epitopes derived from cedar pollen allergen SEQ ID NO: 26: Linker peptide SEQ ID NO: 27: Linker peptide SEQ ID NO: 28: DNA sequence encoding a peptide consisting of multiple T cell epitopes derived from cedar pollen allergen No. 29: Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 30: DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes

SEQ ID NO: 31: Peptide comprising a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 32: Oligonucleotide SEQ ID NO: 33 for constructing a DNA encoding a peptide comprising a plurality of cedar pollen allergen-derived T cell epitopes: Oligonucleotide for constructing a DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 34: Oligonucleotide for constructing a DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 35: Oligonucleotide for constructing DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes

SEQ ID NO: 36: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
Oligonucleotide SEQ ID NO: 37 for constructing NA: Oligonucleotide SEQ ID NO: 38 for constructing a peptide encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 38: consisting of a plurality of cedar pollen allergen-derived T cell epitopes Oligonucleotide SEQ ID NO: 39 for constructing peptide-encoding DNA: Oligonucleotide for constructing DNA encoding peptide consisting of plural cedar pollen allergen-derived T cell epitopes SEQ ID NO: 40: Plural cedar pollen allergen-derived T Oligonucleotide for constructing DNA encoding peptide consisting of cell epitope

SEQ ID NO: 41: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
Oligonucleotide SEQ ID NO: 42 for constructing NA: Oligonucleotide SEQ ID NO: 43 for constructing a DNA encoding a peptide comprising a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 43: comprising a plurality of cedar pollen allergen-derived T cell epitopes Oligonucleotide SEQ ID NO: 44 for constructing peptide-encoding DNA: Oligonucleotide SEQ ID NO: 45 for constructing peptide-encoding DNA consisting of plural cedar pollen allergen-derived T cell epitopes: Plural cedar pollen allergen-derived T Oligonucleotide for constructing DNA encoding peptide consisting of cell epitope

SEQ ID NO: 46: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
Oligonucleotide SEQ ID NO: 47 for constructing NA: Oligonucleotide SEQ ID NO: 48 for constructing a DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 48: consisting of a plurality of cedar pollen allergen-derived T cell epitopes Oligonucleotide SEQ ID NO: 49 for constructing peptide-encoding DNA: Oligonucleotide for constructing DNA encoding peptide consisting of plural cedar pollen allergen T cell epitopes SEQ ID NO: 50: Plural cedar pollen allergen-derived T PCR primer for amplifying DNA encoding peptide consisting of cell epitope

SEQ ID NO: 51: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
PCR primer for amplifying NA SEQ ID NO: 52: DNA encoding a peptide composed of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 53: Peptide composed of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 54: plural Oligonucleotide for constructing a DNA encoding a peptide consisting of a cedar pollen allergen-derived T cell epitope SEQ ID NO: 55: an oligonucleotide for constructing a DNA encoding a plurality of cedar pollen allergen-derived T cell epitope peptides

SEQ ID NO: 56: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
Oligonucleotide SEQ ID NO: 57 for constructing NA: Oligonucleotide SEQ ID NO: 58 for constructing a DNA encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 58: consisting of a plurality of cedar pollen allergen-derived T cell epitopes Oligonucleotide for constructing DNA encoding peptide SEQ ID NO: 59: Oligonucleotide for constructing DNA encoding peptide consisting of plural cedar pollen allergen T cell epitopes SEQ ID NO: 60: Plural cedar pollen allergen derived T DNA encoding a peptide consisting of a cell epitope

SEQ ID NO: 61: Peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 62: PCR primer for introducing a mutation into pUC18 vector SEQ ID NO: 63: PCR primer sequence for introducing a mutation into pUC18 vector No. 64: PCR primer for introducing mutation into pUC18 vector SEQ ID NO: 65: PCR primer for introducing mutation into pUC18 vector

SEQ ID NO: 66: PCR primer for introducing mutation into pUC18M vector SEQ ID NO: 67: PCR primer for introducing mutation into pUC18M vector SEQ ID NO: 68: PCR primer for introducing mutation into pUC18M vector 69: PCR primer for introducing mutation into pUC18M vector SEQ ID NO: 70: Plasmid vector pUC18M5

SEQ ID NO: 71: D encoding a peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes
PCR primer for amplifying NA SEQ ID NO: 72: PCR primer for amplifying DNA encoding peptide consisting of plural cedar pollen allergen-derived T cell epitopes SEQ ID NO: 73: consisting of plural cedar pollen allergen-derived T cell epitopes PCR primer for amplifying DNA encoding peptide SEQ ID NO: 74: PCR primer for amplifying DNA encoding peptide consisting of a plurality of cedar pollen allergen-derived T cell epitopes SEQ ID NO: 75: Amplifying fragment in pUC18M5 vector PCR primer for

SEQ ID NO: 76: PCR primers for amplifying the fragment in the pUC18M5 vector SEQ ID NO: 77: PCR primers for amplifying the fragment in the pUC18M5 vector SEQ ID NO: 78: PCR for amplifying the fragment in the pUC18M5 vector Primer SEQ ID NO: 79: Plasmid pUCM5-6h73 SEQ ID NO: 80: Linker peptide

[Brief description of drawings]

FIG. 1 is an outline of construction of an expression plasmid for polymerizing a peptide of a sugi allergen epitope / connecting peptide.

FIG. 2 shows pUC (h6-1) ₂ , pUC (h7-1) ₂ ,
And schematic diagram of polymerized protein expressed in pUC (h7-3) ₂ .

FIG. 3: pUC18M and pU by RC-PCR method
Overview of CM5 construction.

FIG. 4: Expression vectors pUC18M and pUCM5
Schematic diagram of the expression control region of E. coli.

FIG. 5: Outline of construction of pUCM5-h73 by RC-PCR method.

FIG. 6: Outline of multicopy of h7-3 gene containing ribosome binding region.

FIG. 7: Comparison of human T cell proliferation stimulating activity of cedar pollen allergen-derived T cell epitope connecting peptides (peptide 1 and peptide 9) and a mixture of T cell epitopes.

FIG. 8: Comparison of human T cell proliferation stimulating activity of cedar pollen allergen-derived T cell epitope connecting peptides (peptide 2 and peptide 3) and a mixture of T cell epitopes.

FIG. 9: Comparison of human T cell proliferation-stimulating activity of cedar pollen allergen-derived T cell epitope connecting peptides (peptide 4 and peptide 10) and a mixture of T cell epitopes.

FIG. 10: Comparison of human T cell proliferation-stimulating activity of cedar pollen allergen-derived T cell epitope connecting peptides (peptide 5 and peptide 6) and a mixture of T cell epitopes.

FIG. 11: Comparison of human T cell proliferation-stimulating activity of cedar pollen allergen-derived T cell epitope connecting peptides (peptide 11 and peptide 12) and T cell epitope mixture.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12N 15/09 C12N 15/00 A C12P 21/02 A61K 37/02 (72) Inventor Hideyo Kirinaka 1-chome, Hiromachi, Shinagawa-ku, Tokyo No. 2 58 Sankyo Co., Ltd. (72) Inventor Akio Shiraishi 1-258 Hiromachi, Shinagawa-ku, Tokyo Sankyo Co. (72) Inventor Shinki Serizawa 1-25-2 Hiromachi, Shinagawa-ku, Tokyo No. Sankyo Co., Ltd. (56) Reference JP-A-10-259198 (JP, A) Special table 8-505284 (JP, A) Special table 6-508994 (JP, A) (58) Fields investigated ( Int.Cl. ^7, DB name) C12N 15/09 C12N 15/29 C07K 7/08 C07K 14/415 BIOSIS / WPI (DIALOG) PubMed SwissProt / PIR / GeneS eq GenBank / EMBL / DDB / G eneSeq

Claims (18)

(57) [Claims] 1. The following formula (I): α ₁ -α ₂ -α ₃ -α ₄ -α ₅ -α ₆ -α ₇ (I) (wherein α _{1 to} α ₇ are different from each other; SEQ ID NO: 7 of the sequence table, SEQ ID NO: 17 in the sequence listing, SEQ ID NO: 8 in the sequence listing, SEQ ID NO: 19 in the sequence listing, SEQ ID NO: of the sequence Listing 20 also
Represents an amino acid sequence selected from the group consisting of SEQ ID NO: 21 , SEQ ID NO: 22 of the sequence listing, and SEQ ID NO: 23 of the sequence listing), a peptide containing the amino acid sequence represented by SEQ ID NO: 21 , a complex thereof, a derivative thereof or Polymer. 2. A peptide comprising the amino acid sequence represented by SEQ ID NO: 5 in the sequence listing. 3. A peptide comprising the amino acid sequence represented by SEQ ID NO: 6 in the sequence listing. 4. Amino acid No. 1 of SEQ ID No. 13 in the sequence listing
A peptide consisting of the amino acid sequence shown by 1 to 96. 5. Amino acid No. 1 of SEQ ID No. 15 in the sequence listing
A peptide consisting of the amino acid sequence shown by 1 to 95. 6. A peptide comprising the amino acid sequence represented by SEQ ID NO: 7 in the sequence listing. 7. A peptide comprising the amino acid sequence represented by SEQ ID NO: 8 in the sequence listing. 8. A DNA containing a nucleotide sequence encoding the peptide according to any one of claims 1 to 7 . 9. Transformed E. coli E. coli pBR
(H7-3) SANK 70399 (FERM BP
9. The DNA according to claim 8 , which is incorporated into a plasmid vector retained in 6644) . 10. D comprising the nucleotide sequence shown in nucleotide numbers 4 to 288 of SEQ ID NO: 14 in the sequence listing
NA. 11. A recombinant vector containing the DNA according to any one of claims 8 to 10 . 12. The DNA according to any one of claims 8 to 10 is incorporated into a vector capable of expressing a peptide comprising an amino acid sequence encoded by the DNA. The recombinant vector according to claim 11 . 13. A host cell carrying the recombinant vector according to claim 11 or 12 . 14. A form proteins E. coli E. coli pBR
(H7-3) SANK 70399 (FERM BP
Host cell according to claim 13 , characterized in that it is -6644). 15. A host cell carrying the recombinant vector according to claim 12 is a recombinant D integrated into the vector.
The method for producing a peptide according to any one of claims 1 to 7 , which comprises culturing under conditions capable of producing a peptide encoded by NA, and then recovering the peptide from the culture. 16. The method for producing a peptide according to claim 4 , wherein 1) a host cell transformed with a recombinant vector containing the nucleotide sequence represented by SEQ ID NO: 60 in the sequence listing is used. Culturing under conditions capable of expressing a peptide consisting of the amino acid sequence encoded by the nucleotide sequence represented by nucleotide numbers 1 to 642, 2) then recovering the peptide from the culture obtained in 1), And 3) further, a step of recovering the peptide according to claim 4 after digesting the peptide obtained in 2) with trypsin, and a method for producing the peptide . 17. An anti-cedar pollinosis agent comprising the peptide according to any one of claims 1 to 7, a complex thereof, a derivative thereof or a polymer thereof as an active ingredient. 18. Glucose, sucrose,
Lactose, maltose, trehalose, sorbitol
Le, maltitol, lactitol, mannitol and
18. An anti-cedar flower according to claim 17, comprising / or pullulan.
Powdery drug.