JPH10313876A - Antitumor protein and its gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a protein having antitumor activities, and useful as a polyclonal antibody and for diagnosis, treatment, etc., by containing a specific amino acid sequence, etc. SOLUTION: This protein contains an amino acid sequence consisting of (A) an amino acid sequence of the formula or (B) an amino acid sequence modified from that of the formula, in which one or more amino acids are added and/or inserted, and/or one or more amino acids are substituted and/or deleted, and having antitumor activities. Further, it is preferable that the protein is produced by culturing a host cell such as the colin bacillus or yeast which has been transformed with a vector containing a base sequence encoding the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to antitumor proteins and D-encoding proteins.
For the NA sequence.

[0002] and research on the anti-tumor substance of the background art edible mushrooms have been carried out much from before.

For example, JP-A-52-61214, JP-A-55-74797, JP-A-61-293923,
JP-B-61-47518, JP-B-61-47519
No. 3,26,172, JP-A-5-70362 and JP-A-6-80699, describe antitumor polysaccharides and glycoproteins derived from mushrooms. In addition, it has been reported that mushrooms were orally administered and antitumor activity was observed.

However, as far as the present inventors know, the amino acid sequence of the antitumor protein derived from Matsutake ( Tricholo ma matsutake ) which directly acts on cancer cells and the DNA sequence encoding the same have not been reported.

[0005]

SUMMARY OF THE INVENTION The present inventors have now purified an anti-tumor protein derived from Matsutake mushroom and purified c-protein encoding this protein.
DNA and its amino acid sequence were determined. Further, a cDNA encoding the protein is isolated, and the cDNA
Escherichia coli was transformed with the vector containing the sequence, and the antitumor protein was successfully expressed. The present invention is based on this finding.

Accordingly, the present invention provides an anti-tumor protein, a nucleotide sequence encoding the protein, a vector comprising the nucleotide sequence, a host cell transformed with the vector, a method for producing the protein, and the like. The purpose is to provide an antibody against

[0007] The protein according to the present invention is
(A) the amino acid sequence of SEQ ID NO: 1, or (b) the amino acid sequence of SEQ ID NO: 1 in which one or more amino acids have been added and / or inserted, and / or one or more amino acids have been substituted and / or deleted. And an amino acid sequence having antitumor activity.

[0008]

DETAILED DESCRIPTION OF THE INVENTION Protein The protein according to the present invention has the amino acid sequence of SEQ ID NO: 1. The protein consisting of the amino acid sequence of SEQ ID NO: 1 has an antitumor activity as described in Examples below.

The protein according to the present invention comprises the amino acid sequence of SEQ ID NO: 1, wherein one or more amino acids are added and / or inserted to the amino acid sequence of SEQ ID NO: 1, and / or 1
The above amino acids may be substituted and / or deleted, and the modified amino acid sequence may have antitumor activity. The term “addition, insertion, substitution, and deletion” as used herein refers to those that do not impair the antitumor activity of the protein consisting of the amino acid sequence of SEQ ID NO: 1. Add, insert, replace,
The number of deletions is not particularly limited as long as the antitumor activity is not impaired, but can be usually about 1 to 8.

[0010] Additions, insertions, substitutions, and deletions in the amino acid sequence can be performed, for example, by using Molecular Cloning (A lab).
oratory manual), second edition, Cold Spring Harbo
r Laboratory Press, Vol.2, Chap. 15 (1989); Botstei
n, D. et al., Science, 229: 1193 (1985); Craik, CS,
Bio Techniques, 3:12 (1985); Itakura, K. et al., A
nnu. Rev. Biochem. 53: 323 (1984); Shortle, D. et a
l., Annu. Rev. Genet. 15: 265 (1981); Smith, M. Ann.
u, Rev. Genet. 19: 423 (1985).

In the present invention, the term “protein having antitumor activity” refers to a protein which is evaluated by those skilled in the art to have antitumor activity.
It means a protein that is evaluated to have antitumor activity when tested under the same conditions as (3).

The molecular weight of the protein consisting of the amino acid sequence of SEQ ID NO: 1 is about 65 as measured by SDS-PAGE.
kDa.

The amino acid sequence of SEQ ID NO: 1 can be obtained, for example, by expressing the cDNA sequence (DNA sequence of SEQ ID NO: 2) in bacteria or the like in a conventional manner. The cDNA sequence was obtained by using an antibody against a protein having antitumor activity as a probe, and
It can be obtained by screening a DNA library (Example 2). The protein according to the invention has antitumor activity. Therefore, it can be used as a therapeutic agent for cervical cancer, endometrial cancer, and various cancers (skin cancer, lung cancer, liver cancer, kidney cancer, breast cancer, etc.) caused by abnormal expression of the tumor suppressor gene p53 or pBR.

The therapeutic agent according to the present invention can be administered orally or parenterally (eg, intramuscular, intravenous, subcutaneous, rectal, transdermal, nasal, etc.). It is used in humans and non-human animals in various dosage forms suitable for parenteral administration. It is considered that a method of directly reaching a diseased part as a drug (for example, locally dissolving tablet, application, injection, etc.) is effective.

Therapeutic agents include, for example, tablets, capsules, granules, powders, pills, fine granules, lozenges and the like oral preparations taking into account the stability of the protein and the drug delivery route. Injections such as intravenous and intramuscular injections,
The preparation can be prepared in any form of rectal administration, oily suppository, water-soluble suppository and the like. These various preparations, commonly used excipients, for example, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants,
It can be produced by a conventional method using a buffer, a preservative, a solubilizing agent, a preservative, a flavoring agent, a soothing agent, a stabilizer and the like.

The dosage for various treatments is appropriately determined in consideration of the usage, the age, sex, degree of symptoms, etc. of the patient.

Nucleotide Sequence According to the present invention, a nucleotide sequence encoding the protein of the present invention is provided. A typical sequence of this nucleotide sequence is
It has part or all of the DNA sequence of SEQ ID NO: 2. A typical sequence of this nucleotide sequence has a part or all of the DNA sequence of SEQ ID NO: 2.

The DNA sequence of SEQ ID NO: 2 was obtained from a cDNA library derived from Matsutake as described above. This DNA sequence contains the open reading frame of the protein, the open reading frame starting from the ATG number 1-3 and from 1699-17.
The process ends with TAA No. 01.

Given the amino acid sequence of the protein according to the present invention, the nucleotide sequence encoding it is easily determined, and various nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO: 1 can be selected.

Accordingly, the nucleotide sequence encoding the protein according to the present invention is a DNA sequence encoding the same amino acid in addition to part or all of the DNA sequence shown in SEQ ID NO: 2.
It also means a sequence having a degenerate codon as a DNA sequence, which is an A sequence, and further includes an RNA sequence corresponding thereto.

The nucleotide sequence according to the present invention may be of natural origin or totally synthesized. In addition, a product synthesized using a part of a product derived from a natural product may be used. The nucleotide sequence is a chromosome library or c
It can be obtained from a DNA library by a method commonly used in the field of genetic engineering, for example, a method of screening using an appropriate DNA probe prepared based on partial amino acid sequence information. The base sequence according to the present invention can be obtained, for example, from a Matsutake cDNA library by using an oligonucleotide corresponding to the peptide shown in any of SEQ ID NOs: 3 to 18 as a probe in screening.

When the nucleotide sequence is of natural origin, its origin is not particularly limited, and may be derived from Matsutake or other.

According to vectors and transformation invention, a vector comprising a nucleotide sequence according to the invention described above, the vector is in replicable state in a host cell, and in a state capable of expressing a protein whose nucleotide sequence codes Is provided. Further, according to the present invention, there is provided a host cell transformed with the vector. The host-vector system is not particularly limited, and a fusion protein expression system with another protein can be used.

Examples of the fusion protein expression system include fusion proteins with β-galactosidase, glutathione-S-transferase, luciferase and the like.

As the vector, a plasmid vector (for example, pBluescript SK (-), pBluescript SK (+),
pGEX-4T, pGEX-5T, pRIT2T, pB
PV, pSVK3 (Pharmacia, etc.), Z
AP Express, pYEUra3, pMAM, and pOG (Toyobo, etc.), pET-11a-d,
pET20b, pET28a-c, and pET-32
ab (above Novagen), pQE-10, 16, 3
0, 40, 50, 60, and 70 (all Qiagen)
And liposome vectors (for example, cationic liposome vectors).

In order to express a desired protein by actually introducing the vector into a host cell, a vector according to the present invention may be used in addition to a base sequence according to the present invention, as well as a sequence controlling its expression (for example, a promoter). Sequences, terminator sequences, enhancer sequences) and genetic markers for selecting host cells (eg, neomycin resistance gene,
(A kanamycin resistance gene) and the like. In addition, this vector may contain the nucleotide sequence according to the present invention in a repeated form (for example, in tandem). These may be introduced into a vector according to a conventional method, and a method of transforming a host cell with this vector may be one commonly used in this field.

The procedure and method for constructing a vector according to the present invention may be those commonly used in the field of genetic engineering.

As the host cell, for example, Escherichia coli (eg, SOLR, JM109, XL1-Blue)
MRF ′, and BL21 (DE3)), yeast (eg, strain YRG-2), Bacillus subtilis, and animal-derived cells (CHO
Cells, COS cells, human keratinocytes, COP-5,
C127, mouse 3T3 cells, FR3T3, HB101
Etc.). The transformed host cell is cultured in an appropriate medium, and the protein according to the present invention can be obtained from the culture. Thus, according to another aspect of the present invention, there is provided a method for producing a protein according to the present invention. By this method, large quantities of antitumor proteins can be produced.

The culture of the transformed host cells and their conditions may be essentially the same as for the cells used. In addition, the protein of the present invention can be recovered and purified from the culture solution according to a conventional method (see Examples below). For example, chromatographic methods such as ion exchange chromatography, gel filtration chromatography, and immunoaffinity chromatography can be used.

Antibodies According to the present invention, there are provided antibodies against the proteins according to the present invention. In the present invention, antibodies include polyclonal antibodies and monoclonal antibodies.

The antibody according to the present invention can be produced by a method commonly used in the art. For example, the protein described in SEQ ID NO: 1 can be converted to any carrier (eg, Freund's complete and incomplete adjuvant)
With animal body (eg rabbit, rat, mouse)
And, after a period of time, purify the serum of the animal.

The specific response of the polyclonal antibody (ie, the immune response) is one indicator of the presence of an antitumor protein. Therefore, the antibodies according to the present invention can be used for purification and screening of antitumor proteins.

[0033]

The present invention will be described in detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Example 1 Purification of antitumor protein (1) Purification of protein Fresh matsutake commercially available (or native) was crushed by a conventional method, and subjected to column chromatography,
A protein having an antitumor effect was obtained by purification means using HPLC, electrophoresis and the like. Specifically, the procedure was as follows.

A solution containing NaCl and a protease inhibitor in a Tris buffer (50 mM Tris-HCl)
(PH 7.5), 150 mM NaCl, 1 mM PM
Crude protein extraction was performed with SF, 1 mM EDTA, 0.1 mM IAA (iodoacetamide), 1 μg / ml pepstatin A, and 1 μg / ml leupeptin, and ammonium sulfate precipitation treatment (90% saturated ammonium sulfate) was performed. Precipitation with ammonium sulfate was performed using 25 mM Tris-HCl.
(PH 7.5) (protease inhibitor (PI) was 1/10 of the above, the same applies hereinafter) and dialyzed and desalted. Subsequently, DEAE Toyopearl (ion exchange chromatography), concentration of the active fraction, purification by phenyl sepharose (hydrophobic chromatography), concentration of the active fraction, HPLC (TSK gel G3000S)
The protein was purified by gel filtration according to W).

[0035] Ion exchange chromatography and hydrophobic chromatography were performed using 25 mM Tris-HCl (p
H7.5) (including PI). The linear concentration gradient used was NaCl and (NH ₄ ) ₂ SO ₄ , respectively.
Gel filtration was performed using 0.1 M sodium phosphate (pH 7.0).
2) 0.1 M Na ₂ SO ₄ in buffer and PI
Eluted with a solution containing

The sample subjected to gel filtration by HPLC was subjected to SDS-PAGE. After transfer to the protein PVDF membrane on the gel, a single band (about 65 kDa) was detected by CBB staining.

It has been confirmed by the present invention that when matsutake mushrooms that are not fresh are used or when a protease inhibitor is not used in the purification step, the yield decreases and the antitumor activity described later also decreases.

For some samples, SDS
-After PAGE, the gel was stained with CBB, the gel was cut out, and a sample was collected by an electric extraction method. This sample was used for determining the amino acid sequence (Example 2).

Further, the antibody (2) described below was used for CNBr-activated
Affinity chromatography was performed using a column bound to Sepharose 6MB resin (Pharmacia) to confirm that the protein could be purified.

(2) Polyclonal Antibody A rabbit purified was immunized with the protein purified by (1) to prepare an antiserum. Specifically, the procedure was as follows.

15 μg of the purified protein was mixed with the complete adjuvant on the front, and was vigorously stirred to form an emulsion, which was then injected subcutaneously into the back of the rabbit. Three weeks later, 150 µg of the protein was mixed with Freund's incomplete adjuvant to form an emulsion, and boosting was performed.
Two weeks later, booster immunization was directly performed using 50 μg of the antigen, and one week later, blood was collected from the ear.

Then, 5 ml of antiserum was treated at 56 ° C. for 30 minutes, 5 ml of PBS (−) was added, and the mixture was saturated (N
An equal amount of H ₄ ) ₂ SO ₄ was added and left standing in ice water. After centrifugation, the precipitate was re-dissolved in a buffered sodium phosphate solution, and 20% (N
Saturated (NH ₄ ) ₂ SO ₄ was added to make H ₄ ) ₂ SO ₄ . After centrifugation, the supernatant was collected and 33% (NH ₄ ) ₂ SO
Saturated (NH ₄ ) ₂ SO ₄ was added so as to be _4, and after centrifugation, the precipitate was collected and redissolved. After dialysis and desalting, it was subjected to ion exchange chromatography (DE52 resin) to obtain an IgG fraction.

(3) Anti-tumor activity test The lethal effect on cells cancerated by simian virus 40 (SV40) and human papilloma virus (HPV), which are known to cause cancer in mouse and human cells, was tested. Examined. Specifically, antitumor activity was measured based on cell death. When the protein purified in the above (1) is given to cells, the amount of the protein that causes 50% of cells to die is 1 in SVT2 cells (SV40 transformed cells).
0 ng / ml, 1 for A31 (SV40 transformed cells)
00 ng / ml, human foreskin cells (HPV16 transformation)
Was 15-20 ng / ml.

Example 2 cDNA Cloning and Distribution
The N-terminal amino acid sequence of the protein purified in Example 1 (SEQ ID NOS: 3 and 4) was determined using a sequence determination protein sequencer (Hewlett-Packard).

Further, the protein obtained in Example 1 was digested with a lysylend enzyme (or further a post-prolinkage enzyme) to prepare many peptide fragments, and the amino acid sequence of 14 peptide fragments among them was prepared. Was determined (SEQ ID NOS: 5 to 18).

On the other hand, the mRNA of Matsutake was transformed into oligo-dT
Purified using Latex (oligo-dT particles) (Takara), and STRATAGENE ZAP-cDN.
Purification was performed using ASynthesis Kit (sold by Toyobo) to synthesize cDNA. After cDNA synthesis, the cDNA was converted to lambda phage in vitro using Gigapack III Gold (Stratagene, sales agency: Toyobo).
Packaging was performed to create a phage library.

Using the antibody obtained in Example 1 (2) as a probe, phage having an antitumor protein gene was screened from a phage library to obtain 21 positive phages. Specifically, the procedure was performed as follows.

To determine the library concentration, titers were measured. A plate of 150 mm NZYM medium contains about 2,000 to 20,000 phages and 60
0 ul of E. Coli (XL1-Blue) host 6m
Plated with 1 NZYM Top Agar (0.7%). Plaque 1m at 42 ° C for 3-4 hours
The cells were cultured until they reached an appropriate size of about m. 10 mM I
A 130-140 mm nitrocellulose membrane impregnated with PTG was placed on the plate and cultured at 37 ° C for 3 hours.
After the plate was cooled at 4 ° C. for 1 hour or more, the nitrocellulose filter was peeled off the plate, and the plate was washed with TBS-T buffer containing 3% skim milk.

Next, the filter was immersed in the buffer solution of the primary antibody ((2) of Example 1), and the mixture was gently shaken with a TBS-T buffer solution containing 3% skim milk. The filter was immersed in a buffer solution of an alkaline phosphatase (AP) -bound secondary antibody, and then washed with a TBS-T buffer solution. After washing with a buffer for alkaline phosphatase (AP),
Positive phages were detected.

Using the obtained positive phage, E. coli SOL
The R strain (Stratagene) was transformed by the in vivo excision method. Transformation was carried out by Stratagene ZA.
P-cDNA Synthesis Kit (Distributor:
(Toyobo Co., Ltd.) according to the instruction manual.

From this transformant, the plasmid pTS18 shown in FIG. 1 was obtained. The plasmid pTS18
(Including the cDNA sequence of SEQ ID NO: 1) was used as an expression vector in Example 3.

This pTS18 was used for Exo / Mung DNA Sequenci
After dilation using ng System (Stratagene), both ends were blunt-ended and ligated to self-DNA (FIG. 2). Then E. coli JM109
(Toyobo) was transformed with the plasmid DNA thus obtained. ABI PRISM Cycle Sequencing
The entire nucleotide sequence of the sense strand and the antisense strand was decoded using Kit (Perkin Elmer).

The amino acid sequence and the cDNA sequence of the antitumor protein were obtained by joining the information of the decoded base sequences (SEQ ID NO: 2). The estimated molecular weight was about 62 kDa.

The N-terminal amino acid sequences (SEQ ID NOS: 3 and 4) corresponded to the amino acid sequences Nos. 2 to 30 and SEQ ID Nos. 2 to 58 of SEQ ID NO: 1, respectively.

Further, the sequence of the peptide fragment (SEQ ID NOS: 5 to 18) was identical to the amino acid sequence shown in SEQ ID NO: 1, respectively. The correspondence was as follows.

SEQ ID NO: 5: SEQ ID NO: 59-77,
SEQ ID NO: 6: 89 to 149 of SEQ ID NO: 1, SEQ ID NO: 150 to 178 of SEQ ID NO: 1, SEQ ID NO: 179 to 209 of SEQ ID NO: 1, SEQ ID NO: 210 to 267 of SEQ ID NO: 1 No., SEQ ID NO: 10: 268 to SEQ ID NO: 1
No. 297, SEQ ID NO: 11: 298 to 355 of SEQ ID NO: 1
No., SEQ ID No. 12: Nos. 356 to 406 of SEQ ID No. 1, SEQ ID No. 13: Nos. 407 to 436 of SEQ ID No. 1, SEQ ID No. 14:
SEQ ID NO: 1 at positions 437 to 486, SEQ ID NO: 15: SEQ ID NO: 1 at positions 487 to 521, SEQ ID NO: 16: SEQ ID NO: 1 at 52
Nos. 2 to 554, SEQ ID NO: 17: 555 to 56 of SEQ ID NO: 1
No. 6, SEQ ID NO: 18: Nos. 78 to 99 of SEQ ID NO: 1. These peptides are useful as antigens for obtaining antibodies against the anti-tumor protein that can be used for screening and purifying the anti-tumor protein.

Example 3 Production of Antitumor Protein (1) Thaw competent cells (strain JM109: Toyobo Co., Ltd. ) at −80 ° C. and mix them in a Falcon tube (code 205).
100 μl was transferred to 9). A pTS18 deletion clone (Example 2) was added and left on ice for 30 minutes. Heat shock (42 ° C) for 30 seconds
Cool for minutes. 900 μl of an SOC medium was added, and the cells were cultured with shaking at 37 ° C. for 1 hour. An appropriate amount was seeded on an LB / Amp plate and cultured at 37 ° C. overnight. Next, a platinum loop was scraped off from the colonies that appeared on the plate, and a liquid LB medium (Amp
) And absorbance at 660 nm (Abs) at 37 ° C.
660) was about 0.2. IPTG was added to a final concentration of 10 mM, and cells were collected by culture until Abs660 = about 1.

The cells were added to the extract containing PI used in (1) of Example 1 (50 mM Tris-HCl, pH
7.5) Suspended and sonicated. Extract liquid (50m
M Tris-HCl), and the supernatant was collected.
The antibody was subjected to affinity chromatography (CNBr-activated Sepharose 6MB resin, Pharmacia) to which the antibody of (2) was bound, and the eluate was collected.

The eluate was subjected to SDS-PAGE, and Western blot analysis was performed using the antibody of Example 1 (2).
As a result, it was confirmed that the protein according to the present invention was expressed.

Example 4 Production of Antitumor Protein (2) (1) Preparation of Expression Vector pET-28a A DNA fragment encoding an antitumor protein was prepared by using plasmid pTS18 (10 ng) (Example 2) as a template DNA. Obtained by the polymerase chain reaction (PCR). The PCR reaction was carried out using reagents of a commercial kit (Takara) and the following primers 1 and 2 (each 5 pmole)
Was performed based on the instruction manual. Primer 1: GAGAGACCATGGGGTATCGTCTTTCC (SEQ ID NO: 1
9) Primer 2: GAGAGAGGATCCGGAGACGCCAAGGAT (SEQ ID NO:
20) After the PCR reaction, the product was cut with NcoI and BamHI. The resulting fragment (0.1 μg) was pET-2
N of the 8a vector (0.5 μg) (Novagen)
Ligation to the coI / BamI site. The resulting DNA construct is transformed into competent cells (E. coli, DH5α and JM10).
9 stocks; Toyobo). Plasmid DNA collected from these transformed cells was further introduced into competent cells (BL21 (DE3) strain; Novagen).

(2) Preparation of Expression Vector pET-28b A DNA fragment encoding an antitumor protein was ligated to plasmid pTS18 (Example 2) using EcoRI and XhoI.
Prepared by collecting the EcoRI / XhoI fragment. The resulting fragment (0.1 μg) was pET-2
E of the 8b vector (0.5 μg) (Novagen)
Ligation to the coRI / XhoI site. The resulting DNA construct is transformed into competent cells (E. coli, DH5α and JM1).
09 (Toyobo). Plasmid DNA collected from these transformed cells was further introduced into competent cells (BL21 (DE3) strain; Novagen).

(3) Expression of anti-tumor protein gene One loopful of the transformed cell (pET-28a) obtained as described in Examples 4 (1) and (2) above
BL21 (DE3) strain having pET-28b and BL21 (DE3) strain were inoculated into NZYM medium (1 ml) containing 50 μg / ml kanamycin.
Precultured overnight at 7 ° C. 100 μl was taken out of the culture medium, inoculated into an NZYM medium (10 ml) containing 50 μg / ml kanamycin, and cultured at 25 ° C. until the Abs600 became about 0.4. After IPTG was added to a final concentration of 1.0 mM, the cells were cultured for 24 hours. Cells were collected from the culture medium, and the extract containing PI used in Example 1 (1) (25 mM Tris-HCl, pH
7.0) and sonicated.

After the extract was centrifuged, the precipitate was recovered.
This precipitate was analyzed by SDS-PAGE. As a result, a single band was observed at a position of 65 kDa. The precipitate was also analyzed by Western blotting using the antibodies described in Example 1 (2). Immune response is S
It was observed at the same position as the protein band observed on the DS-PAGE gel. This result indicates that the anti-tumor gene was expressed in the host cell.

[0064]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 566 Sequence type: amino acid Number of chains: single-chain Topology: linear Sequence type: protein Sequence Met Pro Ile Arg Leu Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg 1 5 10 15 Ser Gln Gly Asp Leu Thr Ser Ser Gln His Glu Ile Val His Phe Thr 20 25 30 Asp Val Phe Ile Ala Gly Ser Gly Pro Ile Ser Cys Thr Tyr Ala Arg 35 40 45 His Ile Ile Asp Asn Thr Ser Thr Thr Lys Val Tyr Met Ala Glu Ile 50 55 60 Gly Ser Gln Asp Asn Pro Val Ile Gly Ala His His Lys Asn Ser Ile 65 70 75 80 Lys Phe Gln Lys Asp Ile Asp Lys Phe Val Asn Ile Ile Asn Gly Ala 85 90 95 Leu Gln Pro Ile Ser Ile Ser Pro Ser Asp Thr Tyr Gln Pro Thr Leu 100 105 110 Ala Val Ala Ala Trp Ala Pro Pro Ile Asp Pro Ala Glu Gly Gln Leu 115 120 125 Val Ile Met Gly His Asn Pro Asn Gln Glu Ala Gly Leu Asn Leu Pro 130 135 140 Gly Ser Ala Val Thr Arg Thr Val Gly Gly Met Ala Thr His Trp Thr 145 150 155 160 Cys Ala Cys Pro Thr Pro His Asp Glu Glu Arg Val Asn Asn Pro Val 165 170 175 Asp Lys Gln Glu Phe Asp Ala Leu Leu Glu Arg Ala Lys Thr Leu Leu 180 185 190 Asn Val His Ser Asp Gln Tyr Asp Asp Ser Ile Arg Gln Ile Val Val 195 200 205 Lys Glu Thr Leu Gln Gln Thr Leu Asp Ala Ser Arg Gly Val Thr Thr 210 215 220 Leu Pro Leu Gly Val Glu Arg Arg Thr Asp Asn Pro Ile Tyr Val Thr 225 230 235 240 Trp Thr Gly Ala Asp Thr Val Leu Gly Asp Val Pro Lys Ser Pro Arg 245 250 255 Phe Ala Leu Val Thr Glu Thr Arg Val Thr Lys Leu Ile Val Ser Glu 260 265 270 270 Thr Asn Pro Thr Gln Val Val Ala Ala Leu Leu Arg Asn Leu Asn Thr 275 280 285 Ser Asn Asp Glu Leu Val Val Ala Lys Ser Phe Val Ile Ala Cys Gly 290 295 300 Ala Val Cys Thr Pro Gln Ile Leu Trp Asn Ser Asn Ile Arg Pro Tyr 305 310 315 320 Ala Leu Gly Arg Tyr Leu Ser Glu Gln Ser Met Thr Phe Cys Gln Ile 325 330 335 Val Leu Lys Arg Gly Ile Val Asp Ala Ile Ala Thr Asp Pro Arg Phe 340 345 350 Ala Ala Lys Val Glu Ala His Lys Lys Lys His Pro Asp Asp Val Leu 355 360 365 Pro Ile Pro Phe His Glu Pro Glu Pro Gln Val Met Ile Pro Tyr Thr 370 375 380 Ser Asp Phe Pro Trp His Val Gln Val His Arg Asp Ala Phe Ser Tyr 385 390 395 400 400 Gly Asp Val Gly Pro Lys Ala Asp Pro Arg Val Val Val Asp Leu Arg 405 410 415 Phe Phe Gly Lys Ser Asp Ile Val Glu Glu Asn Arg Val Thr Phe Gly 420 425 430 Pro Asn Pro Lys Leu Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr 435 440 445 Gly Met Pro Gln Pro Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly 450 455 460 Asp Arg Asp Gln Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Met 465 470 475 480 Leu Gly Gly Tyr Leu Pro Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly 485 490 495 Leu Val Leu His Ile Thr Gly Thr Thr Arg Ile Gly Thr Asp Asp Gln 500 505 510 Thr Ser Val Ala Asp Pro Thr Ser Lys Val His Asn Phe Asn Asn Leu 515 520 525 Trp Val Gly Gly Asn Gly Cys Ile Pro Asp Ala Thr Ala Cys Asn Pro 530 535 540 540 Thr Arg Thr Ser Val Ala Tyr Ala Leu Lys Gly Ala Glu Ala Val Val 545 550 555 560 Asn Tyr Leu Gly Val Ser * 565

SEQ ID NO: 2 Sequence length: 1701 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: cDNA to mRNA sequence ATG CCG ATA CGT CTT TCC AAA GAA AAA ATC AAC GAC CTG CTG CAA CGT 48 Met Pro Ile Arg Leu Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg 1 5 10 15 TCT CAA GGG GAT CTT ACT TCC TCG CAA CAC GAA ATT GTA CAT TTC ACT 96 Ser Gln Gly Asp Leu Thr Ser Ser Gln His Glu Ile Val His Phe Thr 20 25 30 GAT GTT TTC ATT GCT GGC AGT GGT CCC ATT AGC TGT ACT TAC GCC CGC 144 Asp Val Phe Ile Ala Gly Ser Gly Pro Ile Ser Cys Thr Tyr Ala Arg 35 40 45 CAC ATC ATT GAC AAT ACC TCA ACT ACA AAG GTT TAC ATG GCC GAA ATA 192 His Ile Ile Asp Asn Thr Ser Thr Thr Lys Val Tyr Met Ala Glu Ile 50 55 60 GGT TCT CAA GAT AAC CCT GTC ATC GGG GCC CAT CAC AAG AAC TCC ATA 240 Gly Ser Gln Asp Asn Pro Val Ile Gly Ala His His Lys Asn Ser Ile 65 70 75 80 AAG TTT CAG AAA GAC ATT GAC AAG TTT GTG AAT ATC ATC AAC GGT GCC 288 Lys Phe Gln Lys Asp Ile As p Lys Phe Val Asn Ile Ile Asn Gly Ala 85 90 95 CTC CAG CCG ATT TCG ATT TCG CCA TCG GAC ACC TAC CAG CCC ACT CTC 336 Leu Gln Pro Ile Ser Ile Ser Pro Ser Asp Thr Tyr Gln Pro Thr Leu 100 105 110 GCT GTA GCA GCG TGG GCG CCG CCC ATC GAT CCT GCC GAA GGC CAG CTC 384 Ala Val Ala Ala Trp Ala Pro Pro Ile Asp Pro Ala Glu Gly Gln Leu 115 120 125 GTG ATT ATG GGA CAC AAT CCG AAT CAG GAG GCC GGC CTG AAC CTT CCC 432 Val Ile Met Gly His Asn Pro Asn Gln Glu Ala Gly Leu Asn Leu Pro 130 135 140 GGT AGC GCT GTC ACT AGG ACA GTC GGG GGG ATG GCG ACC CAC TGG ACT 480 Gly Ser Ala Val Thr Arg Thr Val Gly Gly Met Ala Thr His Trp Thr 145 150 155 160 TGC GCG TGT CCT ACT CCA CAT GAC GAA GAG AGG GTC AAC AAC CCA GTT 528 Cys Ala Cys Pro Thr Pro His Asp Glu Glu Arg Val Asn Asn Pro Val 165 170 175 GAC AAG CAG GAG TTC GAC GCA CTG CTC GAA CGT GCT AAA ACA TTG CTC 576 Asp Lys Gln Glu Phe Asp Ala Leu Leu Glu Arg Ala Lys Thr Leu Leu 180 185 190 AAC GTT CAC AGC GAC CAG TAC GAC GAT TCT ATC CGT CAG ATA GTT GTC 624 Asn Val His Ser A sp Gln Tyr Asp Asp Ser Ile Arg Gln Ile Val Val 195 200 205 AAA GAG ACT CTT CAG CAG ACC CTT GAT GCG TCG CGG GGT GTG ACC ACT 672 Lys Glu Thr Leu Gln Gln Thr Leu Asp Ala Ser Arg Gly Val Thr Thr 210 210 220 CTC CCG CTG GGG GTG GAG CGC CGT ACG GAC AAT CCT ATT TAT GTC ACC 720 Leu Pro Leu Gly Val Glu Arg Arg Thr Asp Asn Pro Ile Tyr Val Thr 225 230 235 240 TGG ACC GGT GCC GAT ACC GTC CTT GGT GAT GTG CCG AAG AGT CCC CGA 768 Trp Thr Gly Ala Asp Thr Val Leu Gly Asp Val Pro Lys Ser Pro Arg 245 250 255 TTC GCT TTG GTT ACA GAG ACG AGA GTG ACG AAG CTT ATT GTC AGT GAA 816 Phe Ala Leu Val Thr Glu Thr Arg Val Thr Lys Leu Ile Val Ser Glu 260 265 270 ACC AAT CCG ACG CAG GTT GTT GCT GCG TTG CTA CGT AAC TTG AAT ACA 864 Thr Asn Pro Thr Gln Val Val Ala Ala Leu Leu Arg Asn Leu Asn Thr 275 280 285 AGC AAC GAT GAA CTT GTC GTG GCC AAG AGT TTC GTC ATA GCT TGT GGA 912 Ser Asn Asp Glu Leu Val Val Ala Lys Ser Phe Val Ile Ala Cys Gly 290 295 300 GCA GTC TGC ACA CCG CAA ATC TTG TGG AAC AGC AAC ATC CGC CCA TAT 960 Ala V al Cys Thr Pro Gln Ile Leu Trp Asn Ser Asn Ile Arg Pro Tyr 305 310 315 320 GCG CTT GGT CGC TAC CTC AGC GAA CAG TCC ATG ACT TTT TGT CAG ATC 1008 Ala Leu Gly Arg Tyr Leu Ser Glu Gln Ser Met Thr Phe Cys Gln Ile 325 330 335 GTT CTC AAG AGG GGC ATA GTC GAT GCC ATC GCT ACT GAC CCT CGC TTC 1056 Val Leu Lys Arg Gly Ile Val Asp Ala Ile Ala Thr Asp Pro Arg Phe 340 345 350 GCT GCG AAG GTT GAG GCG CAC AAG AAG AAG CAC CCC GAT GAC GTG CTG 1104 Ala Ala Lys Val Glu Ala His Lys Lys Lys His Pro Asp Asp Val Leu 355 360 365 CCC ATT CCA TTC CAC GAG CCT GAA CCT CAA GTG ATG ATT CCG TAC ACG 1152 Pro Ile Pro Phe His Glu Pro Glu Pro Gln Val Met Ile Pro Tyr Thr 370 375 380 380 TCG GAC TTC CCT TGG CAT GTT CAG GTG CAT CGC GAT GCA TTC TCA TAT 1200 Ser Asp Phe Pro Trp His Val Gln Val His Arg Asp Ala Phe Ser Tyr 385 390 395 395 400 GGT GAT GTT GGA CCC AAG GCC GAC CCG CGT GTT GTC GTC GAT CTG AGG 1248 Gly Asp Val Gly Pro Lys Ala Asp Pro Arg Val Val Val Asp Leu Arg 405 410 415 TTT TTC GGC AAA TCA GAT ATT GTC GAA GAA AAT CGA GTG ACT TTC GGT 1296 Phe Phe Gly Lys Ser Asp Ile Val Glu Glu Asn Arg Val Thr Phe Gly 420 425 430 CCG AAC CCT AAG CTA CGC GAG TGG GAA GCG GGT GTT ACA GAC ACT TAT 1344 Pro Asn Pro Lys Leu Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr 435 440 445 GGA ATG CCA CAG CCG ACA TTC CAT GTC AAG CGG ACC AAC GCC GAT GGA 1392 Gly Met Pro Gln Pro Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly 450 455 460 GAC CGT GAC CAG AGG ATG ATG AAT GAT ATG ACC AAC GTC GCG AAC ATG 1440 Asp Arg Asp Gln Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Met 465 470 475 480 CTG GGT GGG TAC CTT CCT GGC TCC TAC CCT CAA TTT ATG GCA CCT GGT 1488 Leu Gly Gly Tyr Leu Pro Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly 485 490 495 CTC GTA CTG CAC ATC ACG GGA ACT ACT CGG ATC GGG ACA GAT GAT CAA 1536 Leu Val Leu His Ile Thr Gly Thr Thr Arg Ile Gly Thr Asp Asp Gln 500 505 510 ACT TCT GTT GCT GAT CCG ACA TCA AAG GTT CAT AAC TTC AAC AAT CTG 1584 Thr Ser Val Ala Asp Pro Thr Ser Lys Val His Asn Phe Asn Asn Leu 515 520 525 TGG GTC GGC GGG AAT GGG TGC ATT CCA GAT GCG ACT GCC TGC AAC CCG 1632 Trp Val Gly Gly Asn Gly Cys Ile Pro Asp Ala Thr Ala Cys Asn Pro 530 535 540 ACT CGT ACG AGC GTC GCG TAT GCG CTC AAG GGT GCT GAG GCT GTA GTC 1680 Thr Arg Thr Ser Val Ala Tyr Ala Leu Lys Gly Ala Glu Ala Val Val 545 550 555 560 AAT TAC CTT GGC GTC TCC TGA 1701 Asn Tyr Leu Gly Val Ser * 565

SEQ ID NO: 3 Sequence length: 29 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Sequence Pro Ile Arg Leu Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg Ser 1 5 10 15 Gln Gly Asp Leu Thr Ser Ser Gln His Glu Ile Val His 20 25

SEQ ID NO: 4 Sequence length: 57 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide Sequence Pro Ile Arg Leu Ser Lys Glu Lys Ile Asn Asp Leu Leu Gln Arg Ser 1 5 10 15 Gln Gly Asp Leu Thr Ser Ser Gln His Glu Ile Val His Phe Thr Asp 20 25 30 Val Phe Ile Ala Gly Ser Gly Pro Ile Ser Cys Thr Tyr Ala Arg His 35 40 45 Ile Ile Asp Asn Thr Ser Thr Thr Lys 50 55

SEQ ID NO: 5 Sequence length: 19 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Val Tyr Met Ala Glu Ile Gly Ser Gln Asp Asn Pro Val Ile Gly Ala 1 5 10 15 His His Lys

SEQ ID NO: 6 Sequence length: 61 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Phe Val Asn Ile Ile Asn Gly Ala Leu Gln Pro Ile Ser Ile Ser Pro 1 5 10 15 Ser Asp Thr Tyr Gln Pro Thr Leu Ala Val Ala Ala Trp Ala Pro Pro 20 25 30 Ile Asp Pro Ala Glu Gly Gln Leu Val Ile Met Gly His Asn Pro Asn 35 40 45 Gln Glu Ala Gly Leu Asn Leu Pro Gly Ser Ala Val Thr 50 55 60

SEQ ID NO: 7 Sequence length: 29 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Arg Thr Val Gly Gly Met Ala Thr His Trp Thr Cys Ala Cys Pro Thr 1 5 10 15 Pro His Asp Glu Glu Arg Val Asn Asn Pro Val Asp Lys 20 25

SEQ ID NO: 8 Sequence length: 31 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Gln Glu Phe Asp Ala Leu Leu Glu Arg Ala Lys Thr Leu Leu Asn Val 1 5 10 15 His Ser Asp Gln Tyr Asp Asp Ser Ile Arg Gln Ile Val Val Lys 20 25 30

SEQ ID NO: 9 Sequence length: 58 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Glu Thr Leu Gln Gln Thr Leu Asp Ala Ser Arg Gly Val Thr Thr Leu 1 5 10 15 Pro Leu Gly Val Glu Arg Arg Thr Asp Asn Pro Ile Tyr Val Thr Trp 20 25 30 Thr Gly Ala Asp Thr Val Leu Gly Asp Val Pro Lys Ser Pro Arg Phe 35 40 45 Ala Leu Val Thr Glu Thr Arg Val Thr Lys 50 55

SEQ ID NO: 10 Sequence length: 30 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Leu Ile Val Ser Glu Thr Asn Pro Thr Gln Val Val Ala Ala Leu Leu 1 5 10 15 Arg Asn Leu Asn Thr Ser Asn Asp Glu Leu Val Val Ala Lys 20 25 30

SEQ ID NO: 11 Sequence length: 58 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Ser Phe Val Ile Ala Cys Gly Ala Val Cys Thr Pro Gln Ile Leu Trp 1 5 10 15 Asn Ser Asn Ile Arg Pro Tyr Ala Leu Gly Arg Tyr Leu Ser Glu Gln 20 25 30 Ser Met Thr Phe Cys Gln Ile Val Leu Lys Arg Gly Ile Val Asp Ala 35 40 45 Ile Ala Thr Asp Pro Arg Phe Ala Ala Lys 50 55

SEQ ID NO: 12 Sequence length: 51 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Val Glu Ala His Lys Lys Lys His Pro Asp Asp Val Leu Pro Ile Pro 1 5 10 15 Phe His Glu Pro Glu Pro Gln Val Met Ile Pro Tyr Thr Ser Asp Phe 20 25 30 Pro Trp His Val Gln Val His Arg Asp Ala Phe Ser Tyr Gly Asp Val 35 40 45 Gly Pro Lys 50

SEQ ID NO: 13 Sequence length: 30 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence Ala Asp Pro Arg Val Val Val Asp Leu Arg Phe Phe Gly Lys Ser Asp 1 5 10 15 Ile Val Glu Glu Asn Arg Val Thr Phe Gly Pro Asn Pro Lys 20 25 30

SEQ ID NO: 14 Sequence length: 50 Sequence type: number of amino acid chains: single chain Topology: linear Sequence type: peptide sequence Leu Arg Glu Trp Glu Ala Gly Val Thr Asp Thr Tyr Gly Met Pro Gln 1 5 10 15 Pro Thr Phe His Val Lys Arg Thr Asn Ala Asp Gly Asp Arg Asp Gln 20 25 30 Arg Met Met Asn Asp Met Thr Asn Val Ala Asn Met Leu Gly Gly Tyr 30 40 45 Leu Pro 50

SEQ ID NO: 15 Sequence length: 35 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence Gly Ser Tyr Pro Gln Phe Met Ala Pro Gly Leu Val Leu His Ile Thr 1 5 10 15 Gly Thr Thr Arg Ile Gly Thr Asp Asp Gln Thr Ser Val Ala Asp Pro 20 25 30 Thr Ser Lys 35

SEQ ID NO: 16 Sequence length: 33 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide sequence Val His Asn Phe Asn Asn Leu Trp Val Gly Gly Asn Gly Cys Ile Pro 1 5 10 15 Asp Ala Thr Ala Cys Asn Pro Thr Arg Thr Ser Val Ala Tyr Ala Leu 20 25 30 Lys

SEQ ID NO: 17 Sequence length: 12 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide

SEQ ID NO: 18 Sequence length: 22 Sequence type: amino acid Number of chains: single chain Topology: linear Sequence type: peptide Sequence Asn Ser Ile Lys Phe Gln Lys Asp Ile Asp Lys Phe Val Asn Ile Ile 1 5 10 15 Asn Gly Ala Leu Gln Pro 20

SEQ ID NO: 19 Sequence length: 26 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GAGAGACCAT GGGGTATCGT CTTTCC 26

SEQ ID NO: 20 Sequence length: 27 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GAGAGAGGAT CCGGAGACGC CAAGGAT 27

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of a plasmid vector pTS18.

FIG. 2 is a diagram showing the TTM gene duration. Dotted lines indicate the parts that have been dilated.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI C12N 1/19 C12N 1/21 1/21 C12P 21/02 C 5/10 G01N 33/53 D C12P 21/02 33/574 A G01N 33/53 33/577 B 33/574 A61K 35/76 33/577 C12P 21/08 // A61K 35/76 A61K 37/02 ADU C12P 21/08 C12N 5/00 B (C12N 15/09 ZNA C12R 1 : 645) (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1: 125) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:19) (72) Inventor Izumo Koji 2-1-15 Kasukabe Central Corp., 7-1-15 Chuo, Kasukabe-shi, Saitama (72) Inventor Tomohide Saka 2-23-4 Matsushiro, Tsukuba-shi, Ibaraki 601 Noble Nomura 601

Claims (16)

[Claims] 1. A protein comprising the following amino acid sequence: (a) the amino acid sequence of SEQ ID NO: 1, or (b) one or more amino acids are added and / or inserted, and / or one or more amino acids are substituted and / or Or a deleted amino acid sequence of SEQ ID NO: 1 having an antitumor activity. 2. A nucleotide sequence encoding the protein according to claim 1. 3. The nucleotide sequence according to claim 2, which is the nucleotide sequence of SEQ ID NO: 2. 4. The base sequence is derived from Matsutake mushroom.
The base sequence described in 1. 5. The peptide according to any one of SEQ ID NOs: 3 to 18. 6. A vector comprising the nucleotide sequence according to any one of claims 2 to 4. 7. A plasmid vector, a virus vector,
And a liposome vector.
The vector according to claim 6. (8) pBluescript SK (-), pBluescript SK (+)
, PGEX vector, pRIT2T, pBPV, pS
The vector according to claim 7, which is selected from the group consisting of VK3, pET vector, and pQE vector. A host cell transformed by the vector according to any one of claims 6 to 8. 10. Escherichia coli, yeast, Bacillus subtilis, CHO cells, C
OS cells, human keratinocytes, COP-5, C12
7, mouse 3T3 cells, FR3T3, and HB101
10. The host cell according to claim 9, wherein the host cell is selected from the group consisting of: 11. The method according to claim 11, wherein the Escherichia coli is a SOLR strain, a JM109 strain,
The host cell according to claim 10, which is selected from the group consisting of a SURE strain, a TOPP strain, and a BL21 strain. 12. The yeast according to claim 10, wherein the yeast is strain YRG-2.
The host cell according to item 1. 13. The method of claim 1, comprising culturing the host cell of any one of claims 10 to 12, and isolating the protein of claim 1 from the culture. A method for producing proteins. 14. An antitumor agent comprising the protein according to claim 1 together with a pharmaceutically acceptable carrier. (15) an antibody against the protein of (1); 16. The antibody according to claim 15, which is a polyclonal antibody.
The antibody according to 1.