JPH08322576A - Proteasome activator - Google Patents

Abstract

PURPOSE: To obtain a new polypeptide which is a human proteasome activator PA-28β containing a specific amino acid sequence, having multifunctional protease activities and useful for elucidating the functions thereof, diagnosis and treatment, etc., of various disease conditions such as immunopathy. CONSTITUTION: This new polypeptide of a human proteasome activator PA28β containing at least an amino acid sequence represented by the formula in the molecule. The polypeptide has multifunctional protease activities and useful for elucidating the functions of the proteasome activator and diagnosis, treatment, etc., of various disease conditions such as immunopathy. This polypeptide is obtained by separating the whole RNA from a bovine liver according to a guanidine thiocyanate method. treating the resultant RNA with an oligo(dT)- latex, isolating an mRNA, preparing a cDNA library with the prepared mRNA as a template. screening the cDNA library with a probe, selecting a strain containing a gene of the proteasome activator, recovering the selected gene, joining the recovered gene to a vector and expressing the gene in a host cell.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a proteasome activator, and more particularly to a polypeptide of the proteasome activator and a gene encoding the polypeptide.

[0002]

2. Description of the Related Art The proteasome, which is also called a multifunctional protease, is an inactive type, is localized in the cell, and is a major enzyme constituting an energy-dependent proteolytic system. This enzyme is widely present in eukaryotes from yeast to humans, and is present in all tissues, and in the liver, it accounts for 1% of the total soluble protein. The enzyme has a plurality of catalytically active sites in the same molecule, and is a synthetic peptide containing a basic amino acid that is a substrate of a trypsin type enzyme, a neutral amino acid that is a substrate of a chymotrypsin type enzyme, and an acidic amino acid that is rare as a protease. It has the activity of cleaving the peptide bond at the carboxy terminus. However, it is presumed that the enzyme has a catalytic active site different from that of known proteases, since none of various known inhibitors act specifically and effectively. Also, the sedimentation coefficient is 20
It is estimated that S and the molecular weight are about 750,000, which is exceptionally large as a protease.

The proteasome is a major enzyme in the non-lysosomal proteolytic pathway, and is presumed to have various physiological actions such as function control by protein modification and regulation of protein turnover. Furthermore, recently, the proteasome has been considered to be a processing enzyme of an endogenous antigen, and has attracted attention.

[0004]

[Problems to be Solved by the Invention] The proteasome has been found as such a 20S type complex. further,
The present inventor has recently found that a large number of regulatory factors reversibly bind to this 20S proteasome and form a huge complex having a molecular weight of about 2 million and a sedimentation coefficient of 26S, and named the 26S proteasome ( Japanese Patent Laid-Open No. 5-29296
4, JP-A-6-022759). It is also known that the binding of these numerous regulatory factors promotes the activity of the 20S proteasome. However, the regulatory factors that activate the proteasome are these 26S
Only the group of proteasome regulators has been clarified, and details of other activators, that is, proteasome activators have not been clarified, and the details are clarified to establish the diagnosis and treatment of various pathological conditions. Is strongly requested.

In view of the above situation, the inventor of the present application has proposed that the proteasome activator is:
As a result of intensive studies on the structure, function, etc. of the individual subunits which are the constituents thereof, a novel proteasome activator was elucidated and the present invention was completed. That is, the object of the present invention is to identify the components of the proteasome activator and clarify their functions, and further to provide the amino acid sequence of the polypeptide of the proteasome activator. Another object of the present invention is to provide a nucleotide sequence of a gene encoding a polypeptide which is a constituent component of the proteasome activator. Another object of the present invention is to provide a method for elucidating the function of the proteasome activator and providing a diagnostic treatment method for various pathological conditions including immune diseases.

[0006]

Means for Solving the Problems In order to achieve the above-mentioned object, the inventors of the present invention have conducted detailed research on the structure, function, etc. of the individual subunits of the proteasome, as a result of the research. , Clarified the proteasome activator, which is a constituent of the proteasome. Furthermore, based on the above findings, it has become possible to provide a diagnosis and treatment method for various pathological conditions. That is, the present invention comprises identifying the proteasome activator and determining the amino acid sequence of the polypeptide, and further determining the base sequence of the polynucleotide encoding the polypeptide.

Furthermore, the present invention enables the production of a complementary polynucleotide of a polynucleotide encoding the amino acid sequence of a proteasome activator polypeptide, and using this, the expression level of mRNA of proteasome activator in various human organs can be quantified. Therefore, it can be used for a diagnostic method for immune diseases and the like.

Further, in the present invention, it is possible to prepare antibodies against various proteasome activators, and it is possible to quantify the proteasome activators in various cells using these antibodies, which is used in a diagnostic method for immune diseases and the like. It is possible.

More specifically, the present invention comprises the human proteasome activator PA28β, which is characterized by containing at least the amino acid sequence set forth in SEQ ID NO: 1 in the molecule.
And a polynucleotide encoding the amino acid sequence thereof.

The present invention further relates to a polynucleotide of human proteasome activator PA28β, which comprises at least the nucleotide sequence of SEQ ID NO: 2 in the molecule.

The present invention also relates to a polypeptide of rat proteasome activator PA28β characterized by containing at least the amino acid sequence of SEQ ID NO: 3 in the molecule, and further, a polypeptide encoding the amino sequence thereof. It relates to nucleotides.

Further, the present invention relates to a polynucleotide of rat proteasome activator PA28β characterized by containing at least the nucleotide sequence of SEQ ID NO: 4 in the molecule.

The present invention also relates to an amino acid sequence of a mutated or mutated polypeptide of SEQ ID NO: 1 or SEQ ID NO: 3, which has an activity of substantially promoting proteasome protease activity. It relates to a peptide or a polynucleotide encoding the same.

The present invention further relates to a polypeptide having an amino acid sequence which is a homologue of the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 3 and has an activity of substantially promoting the protease activity of proteasome, or a polypeptide thereof. The present invention relates to a polynucleotide encoding

Further, the present invention has 80% or more homology with the amino acid sequence of the mutated or mutated polypeptide shown in SEQ ID NO: 1 or 3, and has an activity of substantially promoting protease activity of proteasome. The present invention relates to a polypeptide having an amino acid sequence or a polynucleotide encoding the same.

The present invention further relates to a polypeptide consisting of at least 4 consecutive amino acids, which is an amino acid sequence constituting the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 3.

The present invention also provides a mutated or mutated amino acid sequence of the polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 3, a homologue of the polypeptide shown in SEQ ID NO: 1 or 3, which is a proteasome. 80% or more homology with the amino acid sequence of the polypeptide of the amino acid sequence of SEQ ID NO: 1 or 3 which has the activity of substantially promoting the protease activity of SEQ ID NO: 1 or the mutated or mutated polypeptide of SEQ ID NO: 1 or 3, The present invention relates to a polypeptide having an amino acid sequence having an activity of substantially promoting the protease activity, and comprising at least 4 contiguous amino acids.

The present invention also relates to a polynucleotide comprising the polynucleotide shown in SEQ ID NO: 2 or 4 and having at least 12 consecutive bases or a complementary polynucleotide.

The present invention further relates to a mutated or mutated amino acid sequence of the polypeptide shown in SEQ ID NO: 1 or 3, a homologue of the polypeptide shown in SEQ ID NO: 1 or 3, which is a protease of proteasome. Proteasome protease activity having 80% or more homology with the amino acid sequence of the polypeptide having the activity of substantially promoting the activity, the amino acid sequence of the mutated or mutated polypeptide of SEQ ID NO: 1 or 3. The present invention relates to a polynucleotide comprising a polynucleotide encoding an amino acid sequence of a polypeptide having the activity of substantially promoting the activity of at least 12 consecutive bases.

Further, the present invention relates to a disease diagnostic kit characterized by utilizing the reaction specificity of proteasome activator with an antibody.

Further, the present invention relates to a disease diagnostic kit characterized by measuring the mRNA of the proteasome activator by using the reaction specificity of the polynucleotide complementary to the proteasome activator.

The present invention also relates to a recombinant microbial cell transformed with a recombinant plasmid containing the polynucleotide of human proteasome activator PA28β, or Escherichia coli.

The present invention also relates to a recombinant microbial cell transformed with a recombinant plasmid containing a polynucleotide of rat proteasome activator PA28β,
Or it is related to Escherichia coli. In this specification and the drawings, when abbreviations such as bases and amino acids are used, abbreviations according to IUPAC-IUB or abbreviations commonly used in the art are used. The amino acids are described in three letters or one letter. Ala (A) Alanine Arg (R) Arginine Asn (N) Asparagine Asp (D) Aspartic acid Cys (C) Cysteine Gln (Q) Glutamine Glu (E) Glutamic acid Gly (G) Glycine His (H) Histidine Ile (I) Isoleucine Leu (L) Leucine Lys (K) Lysine Met (M) Methionine Phe (F) Phenylalanine Pro (P) Proline Ser (S) Serine Thr (T) Threonine Trp (W) Tryptophan Tyr (Y) Tyrosine Val (V) Valine

The present invention will be described in detail below. (Polypeptide of human proteasome activator PA28β) Amino acid of the polypeptide of human proteasome activator PA28β deduced from the polynucleotide encoding the polypeptide of human proteasome activator PA28β whose nucleotide sequence is determined by the method described below. The sequence is as set forth in SEQ ID NO: 1. Further, the polypeptide of human proteasome activator PA28β according to the present invention includes a polypeptide in which methionine is not bound to the N-terminal of the amino acid sequence of SEQ ID NO: 1, and a human proteasome activator PA28β to the N-terminal of the amino acid sequence. It also includes an intermediate in which part or all of the signal peptide for is bound or deleted.

Further, it is possible to change a part of the structure of the DNA encoding the polypeptide without changing the main activity of the polypeptide by natural mutation or artificial mutation. The polypeptide of human proteasome activator PA28β of the present invention also includes a polypeptide having a structure corresponding to a homologous variant having the above amino acid sequence.

(Rat Proteasome Activator P
A28β polypeptide) The amino acid sequence of the rat proteasome activator PA28β polypeptide deduced from the polynucleotide encoding the rat proteasome activator PA28β polypeptide whose nucleotide sequence was determined by the method described below is SEQ ID NO: 3 As described in. The polypeptide of rat proteasome activator PA28β according to the present invention is a polypeptide in which methionine is not bound to the N-terminal of the amino acid sequence of SEQ ID NO: 3, and rat proteasome activator PA2 to the N-terminal of the amino acid sequence.
It also includes intermediates in which part or all of the signal peptide for 8β is bound or deleted.

Further, it is possible to change a part of the structure of the DNA encoding the polypeptide by natural mutation or artificial mutation without changing the main activity of the polypeptide. The rat proteasome activator PA28β polypeptide of the present invention also includes a polypeptide having a structure corresponding to a homologous variant having the amino acid sequence.

(Purification of Proteasome Activator)
The proteasome activator of the present invention can be purified by combining various chromatographies. For example, from bovine red blood cells, J. Biol. Chem. 267,1
According to the method described in 0515-10523,1992, after precipitation with ammonium sulfate, Sephacryl S-300, DEAE-
Sephacel, Hydroxyapatite column, Phenyl-Sep
Separation and purification with harose CL-4B is possible. In addition,
An assay method for proceeding the purification can be carried out by examining whether or not there is an activity of activating the protease activity of the 20S proteasome.

The purified proteasome activator was subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE, Antibodies A Laboratory Manual, Harlow D).
avid lane, Cold Spring Harbor laboratory, 636-
640, 1988) and O'Farrell's two-dimensional electrophoresis (J.Biol.Chem.250,4007-4021,1975), and then the gel was stained with Coomassie and observed as a band with a molecular weight of 28 kDa. .

(Isolation of Proteasome Activator)
To isolate the proteasome activator from the bands obtained by SDS-PAGE or two-dimensional electrophoresis, it is possible to select a band having a molecular weight of about 28 kDa. Furthermore, the proteasome activator separated and identified by the electrophoresis method was added to polyvinylidene difluorodide (PVD).
It is possible to transfer to F) or the like.

(Preparation of Probe) After the above treatment, a mixture of polypeptide fragments is obtained by treatment with various enzymes such as lysyl endopeptidase, and the obtained mixture of peptide fragments is separated by high performance liquid chromatography. The amino acid sequences of some of the polypeptide fragments obtained above can be determined using an automatic amino acid sequencer or the like.
The probe required for the search of the polynucleotide based on a part of the information of the amino acid sequence obtained by the above analysis,
It is possible to select a polypeptide fragment having a low degeneracy frequency from the obtained polypeptide fragments and synthesize it as a complementary polynucleotide corresponding thereto to prepare it.

When a plurality of amino acid sequences are obtained, the corresponding complementary polynucleotides are combined,
PCR using human cDNA as a template (eg, Michael
l A.Innis et al., ed., translated by Takashi Saito, PCR Experiment Manual, HBJ Publishing Bureau, 1991), it is possible to obtain a DNA probe of a longer fragment. As described above, the DNA probe for screening the proteasome activator is obtained.

(Preparation and Screening of Complementary Strand DNA Library) cD for screening a polynucleotide encoding a polypeptide in the present invention
A general method can be used to prepare the NA library, and for example, the following steps are possible.

That is, (i) messenger RNA (mRNA) is separated from proteasome-producing cells, and (i)
i) synthesizing a single-stranded complementary strand polynucleotide (cDNA) and then a double-stranded polynucleotide from the mRNA, (iii) incorporating the double-stranded polynucleotide into a plasmid or phage, and (iv) the resulting set After transforming an appropriate host cell with the recombinant plasmid or phage and (v) culturing the obtained transformant, the transformant is subjected to an appropriate method such as colony hybridization or plaque hybridization. A plasmid or phage containing the nucleotides is isolated, (vi) the polynucleotide of interest is excised from the plasmid or phage, and (vi)
i) subcloning the cloned polynucleotide into an appropriate plasmid.

Each step will be described in more detail. Step (i): mRNA encoding a proteasome activator polypeptide is isolated from various animal tissues,
It can be obtained from organs and producing cells, more specifically from liver, kidney, heart, brain, lung, thymus, liver cancer cell line, kidney cancer cell line and the like. In addition, total RNA from the enzyme-producing cells
The method for preparing guanidinium / cesium chloride method (Guanidium / Cesium Chloride method, Maniat
is, T., Fritsch EF, and Sambrook, J., Molecula
r Cloning, Cold Spring Harbor Laboratory Press, 19
4-196, 1982) and the guanidinium thiocyanate method (Chomcz
ynski, P. et al., Analytical Biochemistry, 162, 156
-159, 1987) etc. are generally used.

M from total RNA obtained by the above operation
RNA separation and purification can be performed, for example, by oligo dT-cellulose (Collaborative Research).
(Tive Research)) and Oligotex dT30 (Takara), etc., and can be carried out by an adsorption column method or a batch method. Step (ii): Using the thus-obtained mRNA as a template and reverse transcriptase, for example, the Okayama, H. and Berg, P., Molecular and Cellu
lar Biology, 3, 280, 1983) and Gubler and Hoffman's method (Gubler, V. and Hoffman, BJ, Gene, 25, 263-
269, 1983) etc. to synthesize cDNA.

Step (iii): The obtained cDNA is incorporated into a plasmid or phage to prepare a cDNA library. Examples of plasmid vectors into which cDNA is incorporated include PBR322 (Gene, 2, 95, 1977), PB
R325 (Gene, 4, 121, 1978), PUC12 (Gene, 19, 259, 1
982), PUC13 (Gene, 19,259,1982), PUC18 (Gene, 33, 1
03, 1985), PUC19 (Gene, 33,103,1985), PUC118 (Meth
ods in Enzymology, 153, 3, 1987), PUC119 (Methods
inEnzymology, 153, 3, 1987), Bluescript II (Nuclei
C Acids. Res., 17,9494, 1989), etc., but any other one can be used as long as it is replication-retained in the host.

As a phage vector into which cDNA is incorporated, for example, λgt10 (Huynh, TV, Young,
RA and Davis, RW, DNA cloning, A Practical Appr
oach, IRL Press, Oxford, 1, 49, 1985), λgt11
(Proc. Natl. Acad. Sci., USA, 80, 1194, 1983) or λZAPII (Nucleic Acids. Res., 17,9494, 1989), etc. can be used, but even other vectors,
Any material can be used as long as it can grow in a suitable host. As a method for incorporating cDNA into a phage vector, for example, methods such as Hyunh, TV (DNA cloning, A Practic
al Approach, IRL Press, Oxford, 1, 49, 1985) can be used.

Step (iv): The plasmid or phage vector obtained by the above-mentioned method can be used in a suitable host, for example, Escherichia Coli,
It can be transformed by introducing it into Bacillus subtilis, Saccharomyces cerevisiae or the like. Examples of methods for transforming a host with a plasmid vector include, for example, literature (Mol
ecular Cloning, Cold Spring Harbor Laboratory Pres
s, 1.74-1.84, 1989) and the electroporation method or the calcium chloride method.
In addition, the phage vector can be introduced into, for example, Escherichia coli grown by the in vitro packaging method.

Step (v): cDNA according to the above method
To select the cDNA of the proteasome activator of interest from, for example, colony hybridization using a labeled probe or plaque hybridization (Molecular Cloning, Cold Spri
ng Harbor Laboratory Press, 1.85-1.104 or 2.112-2.
120, 1989) etc. can be used. The polynucleotide used as a probe in the above-mentioned hybridization may be any polynucleotide as long as it is a polynucleotide consisting of at least 12 bases that hybridize with the proteasome activator, and for example, an oligonucleotide chemically synthesized based on the amino acid sequence of the proteasome activator. , Or cDNA encoding other components of the proteasome, genomic DNA, chemically synthesized DNA, and partial DNAs thereof can be used.

In addition, cDNA encoding other components of the proteasome, genomic DNA, chemically synthesized D
NA and their partial DNAs can also be used as long as they can hybridize with the subunit. As described above, the polynucleotide encoding the proteasome activator can be prepared.

(Proteasome Activator PA28
Determination of the base sequence of β) The base sequence of the cDNA obtained as described above can be determined by, for example, Maxamu Gilbert (Maxia
m-Gilbert) method (Methods in Enzymology, 65, 499-560,
1980), dideoxy method (Messing, J. et al., Nucleic
Acids Research, 9, 309, 1981), Ta using fluorescent dye
q Cycle sequencing method, etc. (Biotechniques, 7,494
-499, 1989). The determined nucleotide sequence of the polynucleotide encoding the amino acid sequence of the polypeptide of human proteasome activator PA28β is represented, for example, as shown in SEQ ID NO: 2. The nucleotide sequence of a polynucleotide encoding the amino acid sequence of the rat proteasome activator PA28β polypeptide is represented by SEQ ID NO: 4, for example.

The above-mentioned polynucleotide according to the present invention includes a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 2 or 4 and having a nucleotide sequence in which ATG is not bound to the 5'end. The polynucleotides of the invention also include DNA containing a 5'flanking polynucleotide that encodes part or all of the signal peptide of the proteasome activator PA28β. Furthermore, it is possible to mutate the structure of the polynucleotide and part of the structure of the polypeptide deduced therefrom, either by natural mutations or by artificial mutations, without affecting the main activity. Therefore, the polynucleotide according to the present invention contains a nucleotide sequence encoding a polypeptide having a structure corresponding to homologues of all the above-mentioned polypeptides.

Further, according to the degeneracy of the genetic code, at least one base of the base sequence of the polynucleotide can be replaced with another type of base without changing the amino acid sequence of the polypeptide produced from the polynucleotide. . Therefore, the polynucleotide of the present invention also contains a base sequence converted by substitution based on the degeneracy of the genetic code. In this case, from the nucleotide sequence obtained by the above substitution, the deduced amino acid sequence matches the amino acid sequence of SEQ ID NO: 1 for human and the amino acid sequence of SEQ ID NO: 3 for rat. .

Proteasome Activator P of the Invention
A polynucleotide containing a cDNA encoding the A28β polypeptide can be prepared by the method described in
It can also be obtained by cloning genomic DNA from a mouse or the like library. CDNAs encoding these proteasome activators PA28β
The genomic DNA and the genomic DNA can be used as they are or after being cleaved with a restriction enzyme depending on the purpose.

The structure of the cDNA for the human proteasome activator PA28β obtained by the present invention is, as shown in FIG. 1, composed of 763 residues, the open reading frame is 717 residues, and encodes 239 amino acids. The calculated molecular weight is 27072 and the isoelectric point is 5.33.

As shown in FIG. 2, the structure of the rat proteasome activator PA28β cDNA obtained by the present invention is composed of 754 residues, the open reading frame is 714 residues and encodes 238 amino acids. The calculated molecular weight is 26791 and the isoelectric point is 5.28.

(Measurement Method for Diagnostic of Immune Diseases etc.) As shown in Example 2, the proteasome activator PA28β is strongly induced by interferon-γ called immune interferon. A homology search of the human proteasome activator PA28β of the present invention revealed that the autoantibody antigen protein Ki-Antigen (Clin.exp.) Found in a patient with systemic lupus erythematosus (SLE), which is a typical autoimmune disease. Immunol. 79, 209-2
14, 1990) and found to have a homology of 40%.

Therefore, the use of the obtained polynucleotide as a probe provides an effective means for the diagnosis of various diseases including immune diseases. That is,
By using the complementary chain polynucleotide of the proteasome activator obtained in the present invention as a probe, the mRNA expression level of various human organs is analyzed, for example, by Northern blot analysis or RT-PCR analysis (for example, Michael A. Inni.
s et al., ed., Translated by Takashi Saito, PCR Experiment Manual, H
BJ Publishing Bureau, 1991) enables quantitative measurement and provides a diagnostic means for the above-mentioned various diseases. Furthermore, the proteasome activator PA28β
By investigating the genetic polymorphism of C. cerevisiae, the association with various diseases can be clarified, and it can be effectively used for polynucleotide diagnosis.

Further, it is possible to prepare a polyclonal or monoclonal antibody against the proteasome activator PA28β obtained in the present invention (for example, Antibodies, A Laboratory Manual, Ed., Ha.
It is possible according to the method described in rlow et.al., Cold Spring Harbor Laboratory, 1988). It should be noted that the polypeptide used as an antigen when preparing a monoclonal antibody may be composed of at least 4 amino acids. An immunoassay using the obtained antibody provides a means for quantitatively measuring the change in the proteasome activator in various cells.

[0051]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. (Example 1) Determination of gene sequence of proteasome activator Preparation of proteasome activator (1) Preparation of probe (1-1) Preparation of starting material From bovine blood or heart cells, proteasome activator was identified as J. Biol. It can be purified according to the method described in Chem.267, 10515-10523, 1992. However, in order to prevent protein degradation during the purification process, 10 mM leupeptin (SIGMA) in buffers as a protein degradation inhibitor, 2.5 mM 4-
(2-aminoethyl) benzene-sulfonyl fluoride (SIGMA)
Need to be added.

The outline of purification is as follows. Bovine blood was collected in the presence of heparin, centrifuged at 2000xg for 1 hour to collect red blood cells, and the resulting precipitate was suspended in 4 volumes of phosphate buffer and washed by reprecipitation to obtain DE52.
(DEAE cellulose, Whatman) was added to prepare a bound fraction, and ammonium sulfate precipitation was performed, followed by Sephacryl S-300 and DEA.
E Sephacel, hydroxyapatite, Phenyl-Sepha
Purify the rose CL-4B (10x25 cm) by column chromatography. (1-2) Method of measuring active fraction Proteasome activator activity can be measured by mixing 20S proteasome and the protein of each step of purification and measuring whether or not the 20S proteasome peptidase activity is promoted. is there. The reaction is Suc
-Leu-Leu-Val-Tyr-AMC (synthetic substrate for measuring chymotrypsin-like activity), 50 mM, 50 mM Tris-HCl (pH8.0), 1 m
M dithiothreitol (DTT), 20S proteasome and purified protein at each step were added to a final volume of 1000 μl.
The reaction was incubated at 37 ° C for 10 minutes,
The fluorescence was measured. If the regulatory factor group has an activating activity, the activity of degrading the synthetic substrate by the 20S proteasome is increased, so that the active fraction can be identified.

(2) Determination of amino acid sequence of proteasome activator (2-1) Isolation of proteasome activator The proteasome activator fraction having a molecular weight of about 28 kDa obtained by the treatment of (1) was subjected to reverse phase high performance liquid chromatography. (RP-300 C8 column 2.1x100mm, PERKIN ELMER, Mo
(del130A) was used for separation under the following conditions. Separation conditions: Flow rate 50 μl / min, solvent A, 0.06% trifluoroacetic acid (TFA); solvent B, 0.05% TFA / 70% acetonitrile
/ 30% water.

The column was equilibrated with a mixed solvent consisting of A (64%) and B (0%) and eluted with a gradient using the B solvent from 0 to 70%.

Detected at a wavelength of 214 nm, the peak fraction was detected by a speed back concentrator (Savant Instruments).
Dried in. As a result, since two peaks were observed, the following operation was performed on the fraction eluted in the latter half.

(2-2) Separation by electrophoresis The dried sample obtained above was dissolved in an SDS sample buffer and 10% SDS-PAGE (Antibodies A Labora
tory Manual, Harlow David lane, Cold Spring Ha
rbor laboratory, 636-640, 1988).
The gel after the electrophoresis was transferred to Immobilon-PVDF membrane (Millipore) using a semi-dry electroblotting apparatus. The membrane filter on which the protein was blotted was washed with distilled water and then washed with Coomassie blue staining solution (0.2% Coomassie Brilliant Blue R
After staining with 40% methanol containing 250 and 10% acetic acid solution), the sample was immersed in a decolorizing solution (60% methanol solution) and shaken to decolorize the background.

A spot at a position of about 28 Da on this membrane was cut out, and L-1-tosylamido-2-phenyl ch was measured in situ.
loromethyl ketone treated trypsin (Worthington Enzym
es) or Lys-C protease (Boehringer Mannheim). The generated peptide is HP
The product was separated and purified by LC (manufactured by Perkin Elmer) (flow rate 50 μl / min, gradient of 0-70% acetonitrile using 0.1% TFA as solvent, RP300 column (2.1 × 100 mm)). Some of the obtained polypeptide fragments were analyzed by a peptide sequencer (Prokinque Elmer Protein Sequencer 497 type analyzer).

By the above amino acid analysis, the amino acid sequences of the following two kinds of peptide fragments were determined. Fragment 1; QVEVFR Fragment 2; QNLFQEAEEFLYR Fragment 3; FLPQK Fragment 4; IIYLNQLLQEDSFNVTDLNSLR Fragment 5; APLDIPIPDPPPKDDEMETD Fragment 6; TKVEAFQTTISK Fragment 7; ALVHERDEAVYGDLR Fragment 8; Synthetic (Proteasome Activator PA28β) From the sequences of the above-obtained peptide fragments, the sequences of fragment 5 to PPKDDEME and fragment 6 to ALVHERDEAV were selected as the sequences with low degeneracy.

Next, 5'-CGCATAAGGA (T / C) GA (T / C) GA (A / G) ATGGA- was used as a forward primer as a complementary oligonucleotide corresponding to these selected polypeptides.
-3 ', 5'-ACTGCCTCGTCT as the reverse primer
C (G / T) CTCATGTGAGIAGIGC-3 'is a DNA synthesizer 380B
(Manufactured by Applied Biosystems). The obtained oligonucleotide was purified using an oligonucleotide cartridge (manufactured by Applied Biosystems).

(3-2) Preparation of mRNA for PCR Total RNA from 5 g of bovine liver was separated and purified by the guanidine thiocyanate method (Anal. Biochem., 162, 156-159, 1987) as follows. A 5 g sample was added to a guanidine thiocyanate solution (sol.D solution; 6 M guanidine thiocyanate, 0.5% sarcosyl, 10 mM sodium citrate (pH 7), 100 mM 2-mercaptoethanol, 0.1%).
Was sonicated in the antiform A), denatured and then centrifuged to obtain a supernatant. To this, (1/10) amount of 2M acetic acid, equal amount of ethanol, and (1/5) amount of chloroformamisoamyl alcohol (49: 1 vol / vol) were added, and the mixture was allowed to stand on ice for 15 minutes to obtain 10,000g. The precipitate was obtained by centrifugation for 15 minutes at 4 ° C.

The aqueous phase was transferred to another test tube, an equal amount of isopropanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour to obtain 10,000 g.
Centrifuge for 20 minutes at 4 ° C. The obtained precipitate is so
l. Dissolve in D solution and add an equal volume of isopropanol,
After centrifugation, the precipitate was washed with 75% ethanol. Next, this precipitate was suspended in a sol. D solution, an equal amount of isopropanol was added again, and the mixture was cooled at -20 ° C for 1 hour and then centrifuged. The precipitate was collected by centrifugation, washed with 75% ethanol, centrifuged, and the supernatant was removed. This was dissolved in Milli-Q treated with DEPC (diethylpyrocarbonate 0.1% for 5 hours and then autoclaved), 54 mg total m
RNA was obtained.

From the total RNA obtained by the above treatment,
Oligo (dT) -latex (Takara Co .; oligotex-dT30)
Was used to obtain mRNA. That is, a reaction buffer (10 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% SDS) was added to 1 mg of total RNA to make a total volume of 1 ml. Next, add 1 ml of Oligotex-dT30 to this and mix at 5 ° C for 5
It was heat-denatured for 1 minute and quenched on ice for 3 minutes. 5M NaCl 0.2ml
After adding and stirring, incubate at 37 ℃ for 10 minutes,
After centrifugation at 5,000 rpm for 10 minutes, the supernatant was removed. Wash the pellet with 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% SD
It was suspended in 2.5 ml of S, 100 mM NaCl) and left at 37 ° C. for 10 minutes. This was centrifuged at 15,000 rpm for 10 minutes.

The resulting pellet was mixed with TE buffer (10 mM Tris-
It was suspended in 1 ml of HCl (pH 8.0) and EDTA 1 mM), heated at 65 ° C. for 5 minutes, and mRNA was eluted from Oligotex-dT30. Centrifuge at 15,000 rpm for 10 minutes, collect the supernatant, and perform 3M NaOAc (sodium acetate, pH 5.6) for ethanol precipitation.
100 μl and 2 ml of cold ethanol were added, and the mixture was left at −80 ° C. for 30 minutes. Centrifuge at 15,000 rpm for 10 minutes, wash the resulting pellet with 70% ethanol, and add 50 μl TE buffer.
Dissolved in. According to this example, from 10 mg of total RNA, 45
It was possible to obtain μg of mRNA.

(3-3) Synthesis of cDNA for PCR A complementary strand DNA (cDNA) was synthesized using the obtained mRNA as a template and Oligo (dT) ₁₅ as a primer (Pharmacia, cDNA synthesis kit). (3-4) Preparation of cDNA probe by PCR Using forward primer obtained in (3-1) and reverse primer together with cDNA synthesized from bovine liver mRNA obtained in (3-3) as a template PCR
(For example, Michael A. Innis et al., Ed., Translation by Takashi Saito, PCR experiment manual, HBJ Publishing Bureau, 19
1991). Each primer concentration was 20 pm / μl, Taq DNA polymerase was 0.025 U / μl, and cDNA was 1 ng / μl for 30 cycles, and amplification was performed using a DNA thermal cycler manufactured by Perkin Elmer (Cetus) to obtain a cDNA probe of about 600 bp.

(4) Preparation of cDNA library (4-1) Preparation of cDNA From human hepatocellular carcinoma HEPG2 (available from ATCC),
The guanidinium thiocyanate method described in (3-2) (Ana
l. Biochem., 162, 156-159, 1987).
Separated. Using oligo (dT) latex (Takara; Oligotex-dT30) from total RNA, 10 μg of mRNA was obtained from HEPG2 cells in the same manner as in the method described in (3-2). From the poly (A) + RNA obtained by the above method, cDNA was obtained using a TimeSaver cDNA synthesis kit manufactured by Pharmacia.

More specifically, an oligo dT primer is annealed to the poly A portion of the mRNA, and a single-stranded DN is prepared by reverse transcriptase (Murine Reverse Transcriptase).
A was synthesized and double-stranded cDNA was synthesized by E. coli DNA polymerase 1. The obtained cd
To add Not I / EcoR I adapters to both ends of NA,
T4 DNA ligase treatment and polynucleotide kinase treatment were performed to obtain cDNA having EcoR I restriction enzyme cleavage sites on both ends.

(4-2) In vitro packaging reaction cDNA having EcoR I restriction enzyme cleavage sites on both ends obtained
ΛZAPII (Stratage
λZAPI including neRI, EcoRI / CIAP processing λZAPII
I cloning kit) EcoRI was inserted using T4 DNA ligase. Ligation reaction solution 1 ml
Gigapack II Gold packaging extract (Stratagene
Packaging).

The packaging solution containing the packaged recombinant bacteriophage and E. coli XL-1 Blue
Incubated at 7 ° C for 15 minutes. This was added to 2 to 3 ml of top agar (48 ° C), plated on an NZY agar plate, and incubated at 37 ° C overnight. Approximately 50,000 plaques were plated on a 150 mm NZY plate, spread over 10 plates, and approximately 5x10 ⁵ plaques were used for screening.

(5) Isolation of clones (5-1) Preparation of screen filter NZY plate was cooled at 4 ° C. for 2 hours, and a nylon filter (Amersham, Hibond N +) was placed on the plate and left for 2 minutes. did. This was peeled off, dried on filter paper and fixed by irradiation with ultraviolet rays to prepare a screening filter. The following hybridization was performed using this.

(5-2) As a probe for hybridization, the DNA probe obtained in (1) was subjected to ³² P-dCTP (Amersham) by the random priming labeling method (Takara Co .; Random Primer DNA Labeling Kit Ver.2). (Labeled company) was used. As a pre-hybridization solution, 5xSSC (NaCl 0.15M, sodium citrate (pH7.0) 0.015M), 50% formamide, 1xdenhardt (bovine serum albumin (Frac
tion V) 0.2%, polyvinylpyrrolidone 0.2%, Ficoll400
0.2%) solution, 0.1% SDS, 200 μg / ml salmon spar DNA.

The filter was incubated at 42 ° C. for 3 hours with the prehybridization solution, followed by the hybridization solution (10%) to which the labeled probe was added.
Hybridization was carried out by incubating at 42 ° C. for 16 hours in a prehybridization solution containing dextran sulphate). By the above operation, 9 positive clones were obtained.

(6) Preparation of recombinant Escherichia coli by in vivo excision Positive ZA in agar plate obtained in (5-2)
The center of each of the 9 plaques of the P phage clone was attached with a bamboo comb, and the mixture was eluted in a mixed solution of 500 μl of SM buffer and 20 μl of chloroform, vortexed, and left overnight. E. coli XL-1 Blue 200 μl, positive phage clone 200 μl (> 1x10 ⁵ phage particles), helper phage R408 1 μl (> 1x10 ⁶ pfu / m)
l) is mixed in a 50 ml tube, and at 37 ℃ for 15 minutes, Z
AP and helper phage were infected.

5 ml of 2x YT medium (10 g NaCl, 10 g Bacto
Yeast Extract, 16g Bactotryptone / 1l), and add 37
The mixture was cultured at 3 ° C. for 3 hours with shaking to secrete the phagemid from Escherichia coli. Heat treatment at 70 ℃ for 20 minutes, then at 4000g 5
The cells were killed by centrifugation for a minute. The supernatant phagemid was transferred to another tube. This supernatant contains pBluescript SK (-)
Particles are included, and 200 μl or 100
20 μl of diluted solution and 200 μl of XL-1 Blue (OD600 =
1.0) was mixed and mixed at 37 ° C. for 15 minutes to infect.

1 to 100 μl of the culture solution was plated on an LB / Amp plate and then cultured at 37 ° C. overnight. Colonies of E. coli (XL-1Blue) transformants with double-stranded pBluescript SK (-) containing the insert DNA appeared. After culturing E. coli of these positive clones, plasmid DNA was prepared using the QIAprepPlasmid kit (Quiagen) and cleaved with the restriction enzyme NotI.
The base sequence was determined.

(7) Determination of nucleotide sequence The nucleotide sequence of the clone obtained above was determined by the Taq cycle sequencing method using a DNA sequencer 373A manufactured by Perkin Elmer. The analysis result of the nucleotide sequence of the obtained human proteasome activator PA28β is shown in FIG. The determined number of bases is 763 residues and the open reading frame is 717 residues.
Amino acids are encoded. Calculated molecular weight is 27
072 and the isoelectric point is 5.33. The underlined portion in FIG.
(2-2) shows a portion having homology with the peptide sequence determined from the proteasome activator PA28β purified from bovine described in (2-2).

(8) Analysis of gene sequence of rat proteasome activator PA28β A cDNA library was prepared from rat hepatoma cell line H4TG (available from ATCC) in the same manner as described in (4), and the cDNA library was prepared at about 5 × 10 ⁵ / One plaque was used for screening. Similar to the method described in (5), five clones were isolated and E. coli recombinants were prepared by in vivo excision described in (6). Then, the base sequence was determined.

The results of analysis of the nucleotide sequence of the rat proteasome activator PA28β thus obtained are shown in FIG. The determined number of bases is 754 residues, the open reading frame is 714 residues, and 238 amino acids are encoded. The calculated molecular weight is 26791 and the isoelectric point is 5.28.

The underlined portion in FIG. 2 shows a portion having homology with the peptide sequence determined from the proteasome activator PA28β purified from cattle described in (2-2). The E. coli BL-21 transformant having the 0.75 kb insert of the rat proteasome activator PA28β was PRO / BL-rP.
Deposited on May 25, 1995, under the name of A28, at the Institute of Biotechnology, Institute of Biotechnology (accession number: FERM).
P-14938).

(9) Homology of the proteasome activator PA28β between rat and human As shown in the result of homology alignment in FIG. 3, the homology between human and rat was high, 89
There was a% homology. From these results, it was found that the proteasome activator PA28β is highly conserved among species.

Example 2 Polynucleotide Expression Analysis of Proteasome Activator PA28β (1) Northern blot analysis of mRNA expression level in various human organs was carried out using the complementary chain polynucleotide of proteasome activator PA28β as a probe. .
As the probe, the recombinant obtained in Example 1 (4) was used, and the plasmid DNA was prepared from E. coli XL-1Blue.
QIAwell Plasmid Purificat
Ion System (QIAGEN). This plasmid was treated with restriction enzyme Not1 (Takara) and separated by 2% agarose gel electrophoresis, and a probe of subunit P45 of 1.4 kbp was QIA quick.
GEI Extraction Kit (QIAGEN
Company).

This was labeled with 32 P using a multiprime DNA labeling system (Amersham) and used as a probe. Filters are used for various human organs (heart, brain, placenta,
MRNA extracted from lung, liver, skeletal muscle, kidney, pancreas)
A positively charged nylon filter (Human Multi Tissue Northern Blot, Clontech) on which (2 μg) was blotted was used. As a prehybridization solution, 5x SSC (NaCl 0.15M, sodium citrate (pH 7.0) 0.015M), 50% formamide, 1x
denhardt (bovine serum albumin (Fraction V)
0.2%, polyvinylpyrrolidone 0.2%, Ficoll400 0.2%) solution, 0.1% SDS, 200 μg / ml salmon spar DNA was used.

The filters were incubated at 42 ° C. for 3 hours with the prehybridization solution, followed by the hybridization solution containing labeled probe (10%
Hybridization was carried out by incubating at 42 ° C. for 16 hours in a prehybridization solution containing dextran sulphate). The filter was washed 4 times with 2xSSC and 0.1% SDS at 42 ° C for 15 minutes, and further washed once with 1xSSC and 0.1% SDS for 30 minutes.

Autoradiography is based on Kodak X
It was carried out at -70 ° C using AR-5 film. As a result of autoradiography, as shown in FIG. 4, the proteasome activator PA28β had a mR of about 0.9 kb.
It can be clearly quantified that it reacts with NA and its expression level is high in heart and skeletal muscle and low in kidney and lung, and mRNA expression level can be measured by using the probe of the present invention. Is possible.

(2) Under the same conditions as in (1), Northern blot analysis was performed on human kidney cancer-derived cell lines and KPK-1 cells by adding interferon-γ. KPK-1 cells were cultured in a 10 cm Petri dish. As the culture medium, DME culture medium containing 10% fetal calf serum was used, and human interferon-γ (500 U / ml) was added to the medium, and the time-course (0, 12,
After 24, 36 and 48 hours), the cells were collected to obtain total RNA. A nylon membrane filter (Amersham, Hybond N +) was prepared using 10 μg of each total RNA.
Northern blot analysis was performed using the same probe as in (1). As a result, as shown in FIG. 5, it was revealed that PA28β was strongly induced 12 hours after the addition of interferon-γ. Therefore, it is considered that the diagnosis can be made early by measuring PA28β when interferon-γ is induced in human due to a disease caused by abnormality of the immune system.

Example 3 Analysis of Expression of Proteasome Activator PA28β Polypeptide (1) Preparation of Polyclonal Antibody Reactive with Proteasome Activator PA28β Proteasome, which is common to human and rat and has no homology with other subunits Activator P
Near the N-terminal side of A28β, 15 amino acids 3 to 17 (KPCGVRLSGEARKQVE) were selected, and this peptide was analyzed by the F-Moc method (Solid Phase Peptide synthesis, Aprac).
tical approach, IRL Press, Oxford, 1989, Atherton,
E., Sheppard, RC) using a peptide synthesizer 433A manufactured by Perkin Elmer.

Deprotection and purification of the synthesized polypeptide were carried out according to the anti-peptide antibody experiment protocol, Shin Omi et al.,
It was carried out by the method described on page 25-46 of Shujunsha. The peptide after deprotection was purified by a C ₁₈ ODS column (Shimadzu S
yn ProPep column, 4.6x150 mm, 0.1% TFA, 0-80% acetonitrile gradient) was used. (2) Hemocyanin (KLH) was bound to the peptide using an Imject Activated Immunoglobin Conjugation Kit (PIERCE), and a complex of the peptide and KLH was prepared by the column in the kit. (3) Approximately 150 μg of the initial solution was made into an emulsion with Freund's complete adjuvant (FCA, Capel), and the back of Rabbit (Japanese White) was immunized. After that, the mice were similarly immunized with incomplete adjuvant (FIA) three times every two weeks. ELISA at 7, 8 and 9 weeks after the first immunization
The antibody titer was measured by the method, and after confirming the increase in the antibody titer, whole blood was collected. (4) Rabbit blood was allowed to stand at room temperature for 3 hours, then applied to a separapid tube (Sekisui Chemical Co., Ltd.) and rotated at 3000 rpm for 3 hours.
Proteasome activator PA by centrifugation for 0 minutes
Serum against 28β was obtained.

(5) Immunoblot analysis was performed on the human kidney cancer-derived cell line, KPK-1, in the same manner as in Example 2 (2), by adding interferon-γ. KPK-1
Human interferon-γ (500 U / ml) was added to the cells, and the cells were collected over time (0, 48 hours later) and sonicated to obtain cellular proteins. After electrophoresing with 50 μg of each protein, it was transferred to a PVDF membrane, the antibody (3) was diluted 200-fold, and immunoblot analysis was performed using ECL (Amersham). As a result, as shown in FIG. 6, it was revealed that the proteasome activator antibody recognized the 28 kDa band and reacted, and it was revealed that PA28β was strongly induced by the addition of interferon-γ. Therefore, in the case where interferon-γ is induced in humans due to diseases caused by abnormal immune system, PA28β
It is considered that the diagnosis can be made early by measuring the amount of protein.

[0088]

EFFECT OF THE INVENTION Amino acid sequence of the polypeptide of each subunit constituting the proteasome activator,
And by determining the nucleotide sequence of the polynucleotide encoding the amino acid sequence of each polypeptide, it is possible to diagnose and treat various diseases, etc. by a method by an immune reaction with an antibody and a method using a complementary polynucleotide. Become.

[Sequence list]

 SEQ ID NO: 1 Sequence length: 249 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Met Ala Lys Pro Cys Gly Val Arg Leu Ser Gly Glu Ala Arg Lys 5 10 15 Gln Val Glu Val Phe Arg Gln Asn Leu Phe Gln Glu Ala Glu Glu 20 25 30 Phe Leu Tyr Arg Phe Leu Pro Gln Lys Ile Ile Tyr Leu Asn Gln 35 40 45 Leu Leu Gln Glu Asp Ser Leu Asn Val Ala Asp Leu Thr Ser Leu 50 55 60 Arg Ala Pro Leu Asp Ile Pro Ile Pro Asp Pro Pro Pro Lys Asp 65 70 75 Asp Glu Met Glu Thr Asp Lys Gln Glu Lys Lys Glu Val Pro Lys 80 85 90 Cys Gly Phe Leu Pro Gly Asn Glu Lys Val Leu Ser Leu Leu Ala 95 100 105 Leu Val Lys Pro Glu Val Trp Thr Leu Lys Glu Lys Cys Ile Leu 110 115 120 Val Ile Thr Trp Ile Gln His Leu Ile Pro Lys Ile Glu Asp Gly 125 130 135 Asn Asp Phe Gly Val Ala Ile Gln Glu Lys Val Leu Glu Arg Val 140 145 150 Asn Ala Val Lys Thr Lys Val Glu Ala Phe Gln Thr Thr Ile Ser 155 160 165 Lys Tyr Phe Ser Glu Arg Gly Asp Ala Val Ala Lys Ala Ser Lys 170 175 180 Glu Thr His Val Met Asp Tyr Arg Ala Leu Val His Glu Arg Asp 185 190 195 Glu Ala Ala Thr Gly Glu Leu Arg Ala Met Val Leu Asp Leu Arg 200 205 210 Ala Phe Tyr Ala Glu Leu Tyr His Ile Ile Ser Ser Asn Leu Glu 215 220 225 Lys Ile Val Thr Pro Lys Gly Glu Glu Lys Pro Ser Met Tyr 230 235 239 SEQ ID NO: 2 Sequence Length: 717 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: DNA Sequence ATG GCC AAG CCG TGT GGG GTG CGC CTG AGC GGG GAA GCC CGC AAA 45 CAG GTG GAG GTC TTC AGG CAG AAT CTT TTC CAG GAG GCT GAG GAA 90 TTC CTC TAC AGA TTC TTG CCA CAG AAA ATC ATA TAC CTG AAT CAG 135 CTC TTG CAA GAG GAC TCC CTC AAT GTG GCT GAC TTG ACT TCC CTC 180 CGG GCC CCA CTG GAC ATC CCC ATC CCA GAC CCT CCA CCC AAG GAT 225 GAT GAG ATG GAA ACA GAT AAG CAG GAG AAG AAA GAA GTC CCT AAG 270 TGT GGA TTT CTCCT GGG AAT GAG AAA GTC CTG TCC CTG CTT GCC 315 CTG GTT AAG CCA GAA GTC TGG ACT CTC AAA GAG AAA TGC ATT CTG 360 GTG ATT ACA TGG ATC CAA CAC CTG ATC CCC AAG ATT GAA GAT GGA 405 AAT GAT TTT G GG GTA GCA ATC CAG GAG AAG GTG CTG GAG AGG GTG 450 AAT GCC GTC AAG ACC AAA GTG GAA GCT TTC CAG ACA ACC ATT TCC 495 AAG TAC TTC TCA GAA CGT GGG GAT GCT GTG GCC AAG GCC TCC AAG 540 GAG ACT CAT GTATG GAT TAC CGG GCC TTS GTG CAT GAG CGA GAT 585 GAG GCA GCC TAT GGG GAG CTC AGG GCC ATG GTG CTG GAC CTG AGG 630 GCC TTC TAT GCT GAG CTT TAT CAT ATC ATC AGC AGC AAC CTG GAG 675 AAA ATT GTC ACC CCA AAG GAA GAA AAG CCA TCT ATG TAC 717 SEQ ID NO: 3 Sequence length: 238 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Ala Lys Pro Cys Gly Val Arg Leu Ser Gly Glu Ala Arg Lys 5 10 15 Gln Val Asp Ala Phe Arg Gln Asn Leu Phe Gln Glu Ala Glu Asp 20 25 30 Phe Leu Cys Thr Phe Leu Pro Arg Lys Ile Ile Ser Leu Ser Gln 35 40 45 Leu Leu Gln Glu Asp Ser Leu Asn Val Ala Asp Leu Ser Ser Leu 50 55 60 Arg Ala Pro Leu Asp Ile Pro Ile Pro Asp Pro Pro Pro Lys Asp 65 70 75 Asp Glu Met Glu Thr Glu Gln Glu Lys Lys Glu Val Pro Lys Cys 80 85 90 Gly Phe Leu Pro Gly Asn Glu Lys Leu Leu Ala Leu Leu Ala Leu 95 100 105 Val Lys Pro Glu Val Trp Thr Leu Lys Glu Lys Cys Ile Leu Val 110 115 120 Ile Thr Trp Ile Gln His Leu Ile Pro Lys Ile Glu Asp Gly Asn 125 130 135 Asp Phe Gly Val Ala Ile Gln Glu Lys Val Leu Glu Arg Val Asn 140 145 150 Ala Val Lys Thr Lys Val Glu Ala Phe Gln Thr Ala Ile Ser Lys 155 160 165 Tyr Phe Ser Glu Arg Gly Asp Ala Val Ala Lys Ala Ser Lys Asp 170 175 180 Thr His Val Met Asp Tyr Arg Ala Leu Val His Glu Arg Asp Glu 185 190 195 Ala Ala Tyr Gly Ala Leu Arg Ala Met Val Leu Asp Leu Arg Ala 200 205 210 Phe Tyr Ala Glu Leu His His Ile Ile Ser Ser Asn Leu Glu Lys 215 220 225 Ile Val Asn Pro Lys Gly Glu Glu Lys Pro Ser Met Tyr 230 235 238 SEQ ID NO: 4 Sequence length: 714 Sequence type: Nucleic acid Strand number: Double stranded Topology: Linear sequence Type: DNA sequence ATG GCC AAG CCT TGT GGG GTT CGC TTG AGT GGA GAA GCC CGT AAA 45 CAG GTG GAT GCC TTC AGA CAA AAT CTT TTC CAG GAG GCT GAG GAC 90 TTC CTT TGC ACT TTC TTG CCA CGG AAA ATC ATA TCC CTG AGT CAG 135 CTC TTG CAA GAG GAT TCC CTC AAT GTG GCT GAC CTC TCC TCC CTC 180 CGG GCT CCT CTG GAC ATC CCT ATC CCA GAT CCC CCA CCC AAG GAT 225 GAT GAG ATG GAA ACC GAG CAG GAG AAG AAA GAA GTC CCT AAG TGT 270 GGC TTC CTC CCT GGG AAT GAG AAG CTT CTG GCC CTG CTT GCC TTG 315 GTT AAG CCA GAA GTC TGG ACT CTC AAA GAG AAG TGC ATT CTG GTA 360 ATC ACA TGG ATC CAG CAC CTG ATC CCC AAG ATT GAG GAC GGA AAT 405 GAT TTT GGG GTG GCA ATT CAG GAG AAG GTG CTG GAG AGA GTG AAT 450 GCT GTC AAG ACC AAA GTG GAA GCC TTC CAG ACA GCC ATC TCC AAG 495 TAT TTC TCA GAG CGT GGG GAT GCT GTA GCC AAG GCC TCC AAG GAC 540 ACC CAT GTA ATG GAT TAC CGG GCC TTG GTG CAC GAG CGA GAT GAA 585 GCA GCC TAT GGG GCG CTT AGG GCC ATG GTG CTG GAC CTG AGA GCC 630 TTC TAT GCT GAG CTT CAT CAC ATC ATC AGC AGC AAC CTA GAG AAA ATT GTC AAC CCA AAG GGT GAA GAA AAG CCA TCT ATG TAC 714

[Brief description of drawings]

FIG. 1 is a diagram showing the determined nucleotide sequence of human proteasome activator PA28β obtained in Example 1 and the amino acid sequence deduced from the nucleotide sequence (primary structure of human proteasome activator PA28β) (the underline indicates the peptide sequence). It shows a portion having homology with the amino acid sequence obtained as a result.).

FIG. 2 shows the determined nucleotide sequence of rat proteasome activator PA28β obtained in Example 1 and the amino acid sequence deduced from the nucleotide sequence (primary structure of human proteasome activator PA28β) (underlined is the peptide sequence). It shows a portion having homology with the amino acid sequence obtained as a result.).

FIG. 3 shows comparison results of homology alignment of rat and human proteasome activator PA28β.

FIG. 4 m in various human organs using a polynucleotide complementary to human proteasome activator as a probe
It is a figure which shows the result of Northern blot analysis of RNA expression level.

FIG. 5 shows the results of Northern blot analysis of mRNA expression levels induced by interferon-γ in KPK-1 cells using a polynucleotide complementary to human proteasome activator as a probe.

FIG. 6 shows the results of immunoblotting using an antibody for human proteasome activator.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/53 G01N 33/53 DM 33/566 33/566 // (C12N 1/21 C12R 1 : 19) (C12N 9/64 C12R 1:19)

Claims (19)

[Claims] 1. A polypeptide of human proteasome activator PA28β, which comprises at least the amino acid sequence set forth in SEQ ID NO: 1 in its molecule. 2. A polynucleotide encoding the amino acid sequence according to claim 1. 3. A polynucleotide of human proteasome activator PA28β, which comprises at least the nucleotide sequence of SEQ ID NO: 2 in the molecule. 4. A polypeptide of rat proteasome activator PA28β, which comprises at least the amino acid sequence of SEQ ID NO: 3 in its molecule. 5. A polynucleotide encoding the amino sequence of claim 4. 6. A polynucleotide of rat proteasome activator PA28β, which comprises at least the nucleotide sequence of SEQ ID NO: 4 in the molecule. 7. An amino acid sequence of the polypeptide according to claim 1 or 4, which is mutated or mutated,
A polypeptide having an amino acid sequence, wherein the polypeptide has an activity of substantially promoting proteasome protease activity. 8. A homologue of the polypeptide according to claim 1 or 4, wherein the polypeptide has an amino acid sequence having an activity of substantially promoting proteasome protease activity. 9. An amino acid having 80% or more homology with the amino acid sequence of the polypeptide according to claim 1 or 4, which is mutated or mutated, and has an activity of substantially promoting the protease activity of proteasome. Sequence polypeptide. 10. A polynucleotide encoding the amino acid sequence of the polypeptide according to claim 7. 11. A polypeptide comprising the polypeptide according to claim 1 or 4, wherein the polypeptide is composed of at least 4 consecutive amino acids. 12. A polypeptide comprising the polypeptide according to any one of claims 7 to 9, which is composed of at least 4 consecutive amino acids. 13. A nucleotide sequence constituting the polynucleotide according to claim 2, 5 or 10,
A polynucleotide consisting of at least 12 consecutive bases. 14. A kit for diagnosing a disease, which utilizes the reaction specificity of a proteasome activator with an antibody. 15. A kit for diagnosing a disease, which comprises measuring mRNA by using the reaction specificity of a polynucleotide complementary to a proteasome activator. 16. Human proteasome activator P
A recombinant microbial cell transformed with a recombinant plasmid containing a polynucleotide of A28β. 17. The microbial cell according to claim 16, wherein the recombinant microbial cell is Escherichia coli. 18. A rat proteasome activator P.
A recombinant microbial cell transformed with a recombinant plasmid containing a polynucleotide of A28β. 19. The microbial cell according to claim 18, wherein the recombinant microbial cell is Escherichia coli.