JPH0977794A - New protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein derived in an apoptosis-induced cell, having a specific mol.wt., a specific isoelectric point, and specific partial amino acid sequences, and useful for diagnosing, preventing and treating apoptosis-related diseases such as cancers, autoimmune diseases, virus infectious diseases, etc. SOLUTION: This new protein is expressed and derived in an apoptosis- induced cell, has a mol.wt. of 38kDa (SDS-PAGE), and has partial amino acid sequences represented by the formulas: EHELLEQ, IPQALEK, APV, LAQQQAAL, RAAVLLEQE, LAEIGAPI and AELQLPP. The new protein is a marker effective for the apoptosis, and is useful for diagnosing, preventing and treating apoptosis-related diseases such as cancers, autoimmune diseases and virus infectious diseases, etc. The protein is obtained by adding actinomycin D to a culturing cell, strain, incubating the mixture to induce the apoptosis, grinding the cells, extracting the ground cells with a solvent, separating the protein from the extract and subsequently purifying the separated protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein whose expression is induced in cells in which apoptosis is induced. More specifically, it relates to a novel protein whose expression is induced in cultured human cells in which apoptosis is induced by an anticancer agent.
Furthermore, the present invention relates to an antibody having an affinity for a partial amino acid sequence of the protein.

[0002]

2. Description of the Related Art In recent years, attention has been paid to apoptosis in cell tissue death. Unlike the necrosis, which is a pathological cell death, this apoptosis is considered to be a death that is originally integrated into the gene of the cell itself.
That is, it is considered to be an active cell death caused by activation of a gene that programs apoptosis and activation of the self-destruction program triggered by some external or internal factor.

Apoptosis is involved in numerous life phenomena. That is, not only morphogenesis during development, but also the replacement of normal cells such as epidermal cells of the skin, epithelial cells of the small intestine and stomach in mature solids (removal of old cells), and thymus of hormone-dependent tissues It has been suggested that atrophy caused by glucocorticoids, atrophy of the prostate due to castration, elimination of immunocompetent cells that react with self-components, and neuronal cell death due to removal of neurotrophic factors are also due to apoptosis.

Apoptosis is found not only in such physiological cell death, but also in cell death of cells irradiated with radiation or cells infected with a virus. Further AIDS
It has been reported that the reduction of T cells due to virus is also caused by cell death due to apoptosis, and has been drawing attention. In addition to this, apoptosis is also caused by the administration of drugs or poisons and chemical and physical stimuli such as heat. Furthermore, it has been clarified that apoptosis is involved in neuronal cell death in neurodegenerative diseases such as Alzheimer's disease, spontaneous disappearance of tumor cells in cancer lesions, and cell death by anticancer agents.

Therefore, elucidation of the molecular mechanism of apoptosis is important for understanding the biochemical significance and role of cell death in the control of ontogeny and carcinogenesis. Further, genes involved in apoptosis or translation products thereof are used for diagnosis, prevention or prevention of apoptosis-related diseases (eg, cancer, autoimmune disease, viral infection, neurodegenerative disease, endocrine disease, organ transplantation disorder, radiation disorder, etc.). It is useful as a means to develop a therapeutic drug.

[0006] Common characteristic phenomena in apoptosis are morphological changes such as cell shrinkage accompanied by changes in cell membrane (loss of microvilli), chromatin condensation fragmentation and subsequent cell fragmentation (apoptotic body formation). Changes and fragmentation of chromatin DNA in nucleosome units (biochemical changes).

The research on the molecular mechanism of apoptosis is roughly classified into the following (1) to (3). That is, (1) research on the mechanism of recognizing internal or external stimuli and transmitting it as a death signal (2) research on expression of genes involved in apoptosis execution and its control mechanism (3) gene of the above (2) It is a study of the apoptotic process that results in fragmentation of chromatin DNA mainly by the product. A particular problem with apoptosis studies is that apoptosis does not occur uniformly in all cells in a tissue, but is usually sporadic in only some cells. Therefore, it is quite difficult to detect a molecular change specifically occurring in apoptotic cells. Methods for solving this problem include a) selection of a system containing a high proportion of apoptotic cells, and b) identification or isolation of apoptotic cells prior to research. Examples of the method a) include a method of artificially inducing apoptosis using a free cell line as a material. On the other hand, regarding b), a method of detecting apoptotic cells histochemically is considered to be effective.

Identification of apoptosis currently in use
Examples of the detection method include morphological, biochemical or histochemical methods using the above-mentioned characteristic phenomenon of apoptosis as an index. (i) Morphological method 1. Optical or electron microscope observation: Observation of nuclear fragmentation, cell shrinkage, etc., with an optical or electron microscope. Further, by using a scanning electron microscope (SEM), changes in the surface layer structure such as disappearance of microvilli can be confirmed. In any case, it requires a high level of training and time and labor. 2. Staining method: A dye that stains only dead cells (trypan blu
After staining with e, erythrosin B, etc.), detect dead cells by light microscopy. The disadvantage is that it cannot be distinguished from necrotic cells. (ii) Biochemical method 1. DNA electrophoresis (ladder detection method): DNA is extracted from tissues or free cells, and then subjected to agarose gel electrophoresis, and apoptosis-specific DNA fragmentation (having a molecular weight that is an integral multiple of 180 base pairs by cleavage in nucleosome units) (Ladder migration image composed of DNA fragments) is detected. The disadvantage is the inability to confirm apoptosis of individual cells. (iii) Histochemical method 1. in situ end labeling method (TUNEL method):
The DNA cleavage end generated by apoptosis is 3'-OH / 5'-P type having a phosphate group on the 5'side. Therefore, terminal deoxynucleotidyl transferase (Td
T) is used to bind biotinylated deoxyuridine to the 3'-OH end and the biotin is detected immunohistochemically.
The cells with more 3'-OH ends are more strongly stained. But,
Since all 3'-OH stumps are detected, some necrotic cells are also detected. 2. Immunohistochemical method using antibody of apoptosis-related protein: Fluorescent antibody staining method or enzyme-linked immunosorbent assay (ELISA method) using monoclonal antibody of protein (p53, Bcl-2, anti-Fas protein) expressed during apoptosis ). However, the proteins currently used are translation products of proto-oncogenes and tumor suppressor genes, and are not necessarily apoptosis-specific proteins. Further, in cancer cells and the like, the gene is often mutated and not expressed. Therefore, the search for a novel protein specifically expressed in apoptosis is essential for the development of a method and a reagent for easily and accurately identifying apoptotic cells,
The present invention aims to provide novel proteins that are induced during apoptosis. Another object of the present invention is to provide an antibody that has affinity for a peptide having all or part of the partial amino acid sequence of the novel protein.

[0009]

Means for Solving the Problems The present inventor has examined the protein fate in cells that have been induced to undergo apoptosis using two-dimensional electrophoresis. Then, the protein that appeared was confirmed. The protein was subjected to limited decomposition to determine the partial amino acid sequence, and the protein was confirmed to be novel by analysis using a database, p3
The present invention was completed under the name of 8.

That is, the present invention is a novel protein whose expression is induced in apoptotic cells and which has the following properties (hereinafter, referred to as
(referred to as p38). a) Molecular weight: 38 kDa (SDS-PAGE) b) Isoelectric point (pI): 5.2 c) Substantially having the following partial amino acid sequences (I) to (VII). (I) EHELLEQ (II) IPQALEK (III) APV (IV) LAQQQAAL (V) RAABLELEQE (VI) LAEIGAPI (VII) AELQLPP (wherein E is glutamic acid, H is histidine,
L is leucine, Q is glutamine, I is isoleucine, P is proline, A is alanine, K is lysine,
V is valine, M is methionine, R is arginine,
G means glycine. )

More specifically, the present invention is a novel protein which can be obtained by subjecting an extract of cultured human cells in which apoptosis is induced by several anticancer agents to two-dimensional electrophoresis, and which is a) above. ~ C) is a novel protein p38 having the properties shown in c).

The present invention is also an antibody showing affinity for a peptide having all or part of each amino acid sequence shown in c) above.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail below. Apoptosis is inhibited not only by protein synthesis inhibitors such as cycloheximide but also by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), and thus it has become clear that it depends on protein synthesis and protein degradation. . Polyacrylamide gel electrophoresis (PAGE) is used as the most general method for detecting the fate of such proteins. Sodium dodecyl sulfate (SDS) for PAGE
SDS-PAGE for separation by difference in molecular weight in the presence
First, the separation based on the isoelectric point difference is performed, and then SDS-P
There is two-dimensional electrophoresis that performs AGE.

The p38 of the present invention is isolated by subjecting an extract of cells inducing apoptosis to two-dimensional electrophoresis.

Cellular material is typical (ie, cell shrinkage with loss of microvilli, nuclear fragmentation, chromatin condensation,
Subsequent morphological changes such as cell fragmentation and fragmentation of chromatin DNA in nucleosome units are observed)
The cell is not particularly limited as long as it is a cell derived from a eukaryotic multicellular organism in which an apoptosis phenomenon is observed, and may be a free cell or tissue. Preferably, human, rat, mouse,
Free cells of mammals such as bovine, and more preferably human cultured cells.

Apoptosis can be artificially induced by a known method. Examples include radiation / ultraviolet irradiation, thermal treatment, drug treatment of anticancer agents / poisons, infection of viruses / Fas antigens, removal of cytokines, and the like. Treatment with an anticancer agent such as actinomycin D or camptothecin is preferable. Alternatively, cells / tissues that physiologically undergo apoptosis may be used. Specific examples include limb blast cells, lens, chondrocytes, vascular endothelial cells, nerve cells, thymocytes and the like.

Specific examples of the method for preparing the cell extract are as follows. Apoptosis-induced cultured cells of humans, rats, etc., preferably cultured human cells inducing apoptosis by treatment with an anticancer agent, are added in the presence of a suitable reducing agent and urea (in a preferred embodiment, 100 m
M dithiothreitol and 8M urea are used.) Crush by sonication and obtain supernatant fraction by centrifugation. A sample obtained by adding a nonionic surfactant to the obtained supernatant fraction is used as a sample for two-dimensional electrophoresis.
Preferably, 1/100 (v / v) amount of nonidet P-
The use of 40 (NP-40) is illustrated.

The proteins contained in the obtained cell extract are separated by two-dimensional electrophoresis. First, the extract is applied to an electric field in a gel support having a pH gradient formed in advance, and isoelectric focusing is performed. Each protein migrates towards a pH portion in the pH gradient that is equal to the isoelectric pH (pH when it has no net charge), creating a sharp band. Subsequently, the gel subjected to isoelectric focusing is immersed in an appropriate buffer for equilibration, and then adhered onto an SDS-polyacrylamide gel to perform SDS-PAGE. Proteins have a large number of negative charges due to the binding of SDS ^- ions, and the migration rate in gel and the logarithm of the molecular weight show a linear relationship. A preferable mode of the two-dimensional electrophoresis will be described in detail in (4) of Example 1. The separated protein is detected by Coomassie Brilliant Blue (CBB) staining or silver staining with higher sensitivity.

The p38 of the present invention has a molecular weight of 38 kD on a two-dimensional electrophoresis gel of a protein extract from apoptotic cells.
a, detected as a spot at the isoelectric point 5.2.
After separating the spot portion and transferring it to a nylon or nitrocellulose membrane, the protein is enzymatically or chemically limited decomposed on the membrane by a chemical substance such as endopeptidase or bromocyan, and the resulting peptide is subjected to reverse phase The partial amino acid sequence of the protein can be determined by separating and purifying using HPLC and further analyzing the amino acid sequence of each peptide by a protein sequencer.

The p38 of the present invention comprises (I) to (VII) of c) above.
It has a partial amino acid sequence shown in. The present invention also provides
The present invention relates to an antibody having affinity for a peptide having all or part of each of these amino acid sequences. The antibody of the present invention includes both a polyclonal antibody and a monoclonal antibody having the above properties. Further, the monoclonal antibody also includes monoclonal antibodies belonging to any immunoglobulin class IgG, IgM, IgA, IgD and IgE, and IgG or IgM immunoglobulin class monoclonal antibody is preferable.

The antibody of the present invention can be obtained according to a conventional method (successive biochemistry experiment course 5, immunobiochemistry research method, edited by the Japanese Biochemical Society: published by Tokyo Kagaku Dojin, etc.). For example, the polyclonal antibody of the present invention can be produced by the following method. The peptide having all or part of each partial amino acid sequence described in c) above is bovine serum albumin,
Using a mixture of a complex cross-linked to a carrier protein such as Keyhole Limpets Hemocyanin (KLH) and complete (incomplete) Freund's adjuvant [FCA (FIA)] as an antigen, mammals such as rabbits, mice, rats, guinea pigs or hamsters Immunize animals (boost 1 to several times every 1 to 4 weeks after the initial immunization, and measure the antibody titer of the serum partially collected about 3 to 10 days after each booster using a conventionally known antigen-antibody reaction. Measurement, confirmation of the increase, and about 3 to 10 days after the final immunization, whole blood is collected to purify antiserum.

The p38 monoclonal antibody of the present invention can be produced from a hybridoma (fused cell) produced by so-called cell fusion. That is, a fused hybridoma is formed from antibody-producing cells and myeloma cells, and the hybridoma is cloned,
It is produced by selecting a clone that produces an antibody showing a specific affinity for a peptide having all or part of the partial amino acid sequence of p38 of the present invention as an antigen. For the manipulation, conventionally known means can be used except that a peptide having all or part of the partial amino acid sequence of p38 of the present invention is used as an immunogen.

The "monoclonal antibody" of the present invention may be obtained by any method. In addition, a “monoclonal antibody” usually has sugar chains having different structures depending on the type of mammal to be immunized, but the “monoclonal antibody” in the present invention is not limited by the structural difference of the sugar chains. It also includes monoclonal antibodies derived from all mammals. Furthermore, for example, a human monoclonal antibody obtained by using a transgenic mouse engineered to produce a human antibody by incorporating a human immunoglobulin gene, or a certain mammalian A chimeric monoclonal antibody in which the constant region (Fc region) of an animal-derived monoclonal antibody is recombined with the Fc region of a human monoclonal antibody, and further, a complementarity determining region (CDR: co
chimera monoclonal antibody of the present invention is also a chimeric monoclonal antibody in which the entire region other than the complementarity determining region) is recombined with the corresponding region of the human monoclonal antibody
Included in.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Cell culture 7.5% (v / v) fetal calf serum (FCS), 75 μg
/ Ml (w / v) kanamycin sulfate and 5 μM 2-mercaptoethanol in RPMI640 culture medium for HL
-60 cells (human promyelocytic leukemia cultured cells) are suspended,
CO ₂ incubator [na co (registered trademark) Model 510
0, NAPCO SCIENTIFIC COMPANY] at 37 ℃, 5% CO
Cell density 1.0 × 10 ^{5 to} 1.0 × 10 ⁶ under the condition of ₂
Subculture was performed so that cells / ml were obtained.

(2) Induction of apoptosis HL-60 cells prepared by the method of (1) above (1
To 10 × 10 ⁶ cells / ml), actinomycin D was added so that the final concentration was 1 μg / ml, and the mixture was allowed to stand in a CO ₂ incubator for 6 hours and then centrifuged at 200 × g for 5 minutes to collect the cells. The cells were treated with phosphate buffered saline (Na
Cl 8g, KCl 2g, Na ₂ HPO ₄・ 12H ₂
O 29 g and KH ₂ PO ₄ 2 g were dissolved in 1 liter of purified water), and the mixture was centrifuged at 200 × g for 5 minutes and the supernatant was removed.

(3) Preparation of cell extract All extraction operations below were carried out at 4 ° C. 8M urea, 10
The cells treated in the above (2) were suspended in a solution of 0 mM dithiothreitol at 1 × 10 ⁸ cells / ml. The suspension was sonicated at 20 W for 1/10 seconds 10 times, cooled for 20 seconds, and then similarly sonicated twice. The treated solution was centrifuged at 10400 × g for 1 hour, and the supernatant was mixed with 1/100 (v / v) amount of Nonidet P-40 (N
The one to which P-40) was added was subjected to two-dimensional electrophoresis.

(4) Two-dimensional electrophoresis First dimension (isoelectric focusing): Immobiline (registered trademark)
A gel having a pH gradient of DryPlate (pH 4.5-5.4, manufactured by Pharmacia Co.) was placed at a width of 1 perpendicular to the gradient.
Cut into cm and rehydrate the gel with rehydration buffer (8 M urea, 0.
5% NP-40, 0.2% Ampolyte pH
3.5-10) and swelling for 12 hours or more,
Isoelectric focusing was performed using a MultiphorII migration tank (Pharmacia). 200 μl (2 per gel)
× 10 ⁷ cells) were applied together with a marker dye, bromophenol blue solution. Electrophoresis condition is 500V
5 hours, 2000V for 5 hours, and 3500V for 10 hours, a total of 47500 Vhr was performed. Second dimension (SDS-PAG
E): Equilibration buffer (50 mM)
Tris-HCl pH 6.8, 6 M urea, 30% glycerol, 1% SDS, 162 μM dithiothreitol) was immersed for 10 minutes for equilibration. Separately, a separating gel and a concentrating gel were prepared as follows. Separation gel (final concentration of acrylamide: 10%) 30% acrylamide 15 ml 1.5M Tris-HCl (pH 8.8) 7.5 ml 10% SDS 0.3 ml 10% ammonium persulfate 150 μl pure water 12.04 ml N, N, N ', N'- Tetramethyl 20 μl Ethylenediamine (TEMED) Concentration gel (acrylamide final concentration: 4%) 30% acrylamide 1.33 ml 0.5M Tris-HCl (pH 6.8) 1.25 ml 10% SDS 0.1. 1 ml 10% ammonium persulfate 100 μl pure water 7.5 ml TEMED 10 μl Next, the separating gel was poured between two glass plates, and water-saturated butanol was overlaid. After the gel solidifies,
The butanol layer was removed and the gel was washed with pure water. The concentration gel was layered, and then water-saturated butanol was layered. The solidified gel is brought into close contact with the equilibrated first-dimensional gel,
Running buffer (25 mM Tris, 192 mM glycine, 0.1% S
It was set in the electrophoresis tank containing DS). After the addition of the molecular weight marker, the electrodes were connected and electrophoresis was started. First, run at 10 mA, and if the marker dye passes through the concentrated gel part,
In terms of mA, electrophoresis was performed until the marker dye reached 5 mm from the lower end of the gel. After completion of the electrophoresis, the gel was immersed in a 0.25% Coomassie Brilliant Blue (CBB) staining solution and stained for 2 hours with gentle shaking. The resulting electrophoretic image (Fig. 1, right) shows HL that does not induce apoptosis.
-60 cells were extracted in exactly the same manner and compared with the migration image obtained by two-dimensional electrophoresis (Fig. 1, left).
At a position of 8 kDa and an isoelectric point of 5.2, a spot of protein which was not found in cells in which apoptosis was not induced was observed.
The present inventor named the protein p38.

Example 2 The gel obtained in Example 1 was transferred to a PVDF membrane (manufactured by Millipore) by electroblotting using a transblotting apparatus (manufactured by Bio-Rad), and P
The VDF membrane was stained with CBB, and the spot site of p38 was collected. First, add excess dithiothreitol to reduce the disulfide bond in the protein to a sulfhydryl group,
The sulfhydryl group was alkylated with iodoacetic acid to give an S-carboxymethyl derivative. Then Ly
Buffer (20 mM Tris-HCl (pH 9.0), 10% acetonitrile, Lys-C endopeptidase containing s-C endopeptidase (enzyme that specifically cleaves the peptide bond on the carboxyl group side of the lysine residue of the peptide) PVDF membrane with p38 protein bound in 1 pmol)
Was reacted. After enzymatic digestion, each peptide fragment released from the membrane was separated and collected by reverse phase HPLC. Fractions containing the peptide were monitored at a wavelength of 214 nm (Figure 2). Each fraction of fragments detected as a peak is dried in a centrifugal evaporator and then SD
Dissolve in S solution and vapor phase protein sequencer (PS
Amino acid analysis was carried out to determine the partial amino acid sequence of each peptide fragment (SEQ ID NOS: 1 to 7 in FIG. 2).

Based on the partial amino acid sequence of p38 obtained by the above-mentioned method, a homology search with a known protein was performed using Genome Net which is a protein / peptide database, and as a result, the p38 of the present invention was found to be:
It was found to be a novel protein.

Example 3 Actinomycin D was prepared using the same materials and method as in Example 1.
By changing only the treatment time by 0 to 6 hours,
The expression of 38 was observed over time (FIG. 3). p38 was expressed 3 hours after the addition of actinomycin D, reached its peak 5 hours later, and decreased thereafter. The time when p38 was expressed at a high level coincided with the time when DNA fragmentation proceeded (electrophoresis image in FIG. 3), suggesting that p38 may be involved in the process of apoptosis.

Example 4 The expression of p38 was observed in the same manner as in Example 1 except that the combination of cell material and treatment for cells was changed as follows. a) HL-60 cells treated with camptothecin 1 μM for 6 hours b) Jurkat (human T cell lymphoma) cells treated with camptothecin 1 μM for 6 hours c) Rat thymocytes irradiated with 10 Gy X-rays d) HL-60 cells at 50 ° C. or After heat treatment at 60 ° C for 30 minutes, incubation at 37 ° C for 6 hours. As a result, in any of the cases a), b), and c), p38
Spots were detected. These results suggest that p38 is induced across species, generally in cells during apoptosis. On the other hand, in the case of d), the spot of p38 was not detected. d) The described treatment induces necrosis in the cells. From this result, it was confirmed that p38 was not specifically induced in cell death but specifically induced in apoptosis.

Example 4 Preparation of p38 Antibody Based on the partial amino acid sequence of p38 of the present invention determined in Example 2, the following peptide synthesized was used as an antigen for p3.
A polyclonal antibody against 8 was prepared.

EHELLEQqk (wherein E is glutamic acid, H is histidine,
L means leucine, Q and q means glutamine, and k means lysine. In addition, in the formula, the part shown in upper case is the partial amino acid sequence of p38, and the part shown in lower case is the sequence added for cross-linking with the carrier protein. )

The above synthetic peptide was converted into a carrier protein, Keyhole Limpets Hemocyanin, by the maleimide method.
(KLH) cross-linked complex and a mixture of complete Freund's adjuvant (FCA) as an antigen, and rabbit [K
bl: JW, 15 years old, male, weight 3-3.5 kg (at the time of initial immunization), 2 birds are used], and about 0.5 m by subcutaneous injection in the back
g immunized. Approximately three times every three weeks from the first immunization, about 0.5
Each mouse was boosted with 10 mg of blood, and the antibody titer was measured by partial blood sampling about 10 days after each boosting. Furthermore, about 10 days after the final immunization (3rd boosting), whole blood was collected to obtain antiserum. .

The antibody titer was measured by the following method. The antigen was immobilized on a microtiter plate at a concentration of 10 μg / ml, and after blocking, the partial blood of the sensitized rabbit was 10
^It was diluted to ¹ to 10 ⁸ and reacted with the antigen. After washing, an anti-rabbit IgG-peroxidase-labeled secondary antibody was reacted, and after washing, the antibody titer was measured by coloring the substrate solution ABTS. As a result, it was confirmed that the antibody titer increased with time in each case.

[0037]

INDUSTRIAL APPLICABILITY The p38 of the present invention is an effective marker protein for apoptosis because it is characterized by being specifically induced during apoptosis and not induced during necrosis. Therefore, a labeled antibody against the protein is histochemic for apoptosis.
al) Useful as a detection reagent. Further, clarifying the function of p38 has important significance in elucidating the molecular mechanism of apoptosis from a new perspective, such as identification of a novel protein that interacts with p38.

Since the expression of p38 of the present invention reaches its maximum at the time when the fragmentation of chromatin DNA becomes remarkable, it is predicted that p38 is a protein involved in the execution process of apoptosis including DNA fragmentation. It Therefore, the p38 of the present invention and its antibody are useful for diagnosing and preventing cancer, immunosuppressive diseases, autoimmune diseases, viral infections, neurodegenerative diseases, endocrine diseases, organ transplantation disorders, radiation disorders and the like, which are diseases involving apoptosis. Alternatively, it is expected to be used as a therapeutic drug.

[0039]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 3 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 4 Sequence length: 8 Sequence type: Amino acid Topology: Linear sequence type: Peptide

SEQ ID NO: 5 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 6 Sequence length: 8 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 7 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 8 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Brief description of drawings]

FIG. 1 is a two-dimensional electrophoretic image of a protein extracted from cells in which apoptosis was induced by treating with actinomycin D at 1 μg / ml for 6 hours. Fig. 1 right. Electrophoresis image of proteins extracted from apoptotic cells. Fig. 1 Left Electrophoresis image of proteins extracted from non-apoptotic cells.

FIG. 2 is an elution pattern of each peptide fragment when the peptide fragment generated by limited digestion of p38 with Lys-C endopeptidase was separated by reverse-phase HPLC. The amino acid sequence of the peptide corresponding to each peak is added.

FIG. 3 is a graph showing changes over time in the appearance of p38. The amount of protein was measured by measuring the intensity of CBB staining as the absorbance. The figure shows the numerical value in which uneven staining is corrected based on the protein that hardly changes.

Claims (3)

[Claims] 1. A protein characterized by being induced during apoptosis and having the following properties. a) Molecular weight: 38 kDa (SDS-PAGE) b) Isoelectric point (pI): 5.2 c) Substantially having the following partial amino acid sequences (I) to (VII). (I) EHELLEQ (II) IPQALEK (III) APV (IV) LAQQQAAL (V) RAABLELEQE (VI) LAEIGAPI (VII) AELQLPP (wherein E is glutamic acid, H is histidine,
L is leucine, Q is glutamine, I is isoleucine, P is proline, A is alanine, K is lysine,
V is valine, M is methionine, R is arginine,
G means glycine. ) 2. An extract of cultured human cells in which apoptosis is induced by the addition of an anticancer agent is subjected to isoelectric focusing and S
The protein according to claim 1, which can be obtained by subjecting it to two-dimensional electrophoresis with DS-PAGE. 3. An antibody showing affinity for a peptide having all or a part of the amino acid sequence according to claim 1.