JPH11146786A - h-Hyd protein, polynucleotide encoding the protein, antisense polynucleotide thereof, and antibody recognizing the protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To isolate a homologous gene of a Drosophila hyd gene (d-hyd gene) in a human, determine its base sequence, produce a homologous protein encoded by the gene, and further provide the protein. It is an object of the present invention to produce an antibody to recognize. SOLUTION: A d-hyd homolog protein (h-Hyd) in human, and a gene (h-HH) encoding the protein
yd gene). Also provided is a mutant of h-Hyd. Further, the present invention provides an antibody that recognizes the protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a homolog protein of a Drosophila hyperplastic disc (hyd) protein and a polynucleotide encoding the homolog protein. In addition, the present invention relates to an antisense polynucleotide of the polynucleotide. The present invention also relates to an antibody that recognizes the homolog protein.

[0002]

BACKGROUND ART Among vertebrates, humans are organisms having the most highly differentiated nervous system. However, analysis of the nervous system has been delayed due to its complex mechanism. In particular, the analysis of tumors occurring in the cerebral nervous system is delayed compared to other tumors. In the cranial nervous system, not only malignant tumors, but also benign tumors can be life-threatening for patients depending on the site of occurrence, and elucidation of the mechanism of the onset of cranial nerve tumors is awaited.

On the other hand, in Drosophila, many mutants have been obtained in the course of analysis of neurogenesis, and genetic analysis thereof is in progress. The homoplastic deletion of the Drosophila hyperplastic disc gene (hereinafter sometimes referred to as the d-hyd gene) is lethal, and in various temperature-sensitive mutants, hyperplasia of the adult primordium is generated. Is considered to be a controlling factor. The Drosophila hyperplastic disc protein (hereinafter sometimes referred to as d-hyd protein) encoded by this gene is a protein comprising 2897 amino acids, and has a poly (A) binding domain at its C-terminal side. In addition, in the intracellular protein degradation system, ubiquitin
The active site of ligase and the C-terminal homologous domain of E6AP, hect (homologous to E)
6AP) domain.

[0004] In addition, the nucleotide sequence of a homologous gene (homolog gene) of the d-hyd gene in rat and the amino acid sequence of the protein (homolog protein) encoded by the gene are known.

[0005]

DISCLOSURE OF THE INVENTION The present invention is to isolate a homologous gene (homolog gene) of the hyd gene in humans, determine its base sequence, prepare a protein encoded by the gene (homolog protein), It is an object to obtain an antibody that recognizes the protein.

[0006]

Means for Solving the Problems The present inventor searched an EST database based on the nucleotide sequence of the hyd gene, and selected a nucleotide sequence having homology with the hyd gene from the database. Then, a primer consisting of a part of the base sequence was prepared. Then, the human hyd (h-Hyd) gene, which is a homolog gene in human, is subjected to PCR by using the primers and a human fetal brain cDNA library and a chronic myelogenous leukemia cell line (K562) cDNA library as templates. It was isolated and its nucleotide sequence was determined.

[0007] The present invention also provides an antisense polynucleotide comprising a base sequence complementary to the above base sequence.

[0008] Further, the present application discloses a method for producing h-Hyd protein (hereinafter sometimes referred to as h-Hyd). More specifically, a method is disclosed in which a transformant into which the above-mentioned h-Hyd gene has been introduced produces h-Hyd protein. It also discloses a method for producing a mutant of h-Hyd in which one or more amino acids in the amino acid sequence of h-Hyd are substituted, deleted or added.

[0009] The present invention also provides an antibody that recognizes the h-Hyd. In the present application, the antibody was prepared, and h
It is disclosed that the antigenicity of -Hyd, ie, it is generally possible to generate antibodies from h-Hyd.

The present invention also provides a gene probe capable of detecting h-Hyd mRNA from human tissues and cell lines by analysis by Northern blot hybridization. With the gene probe of the present invention, it is confirmed that h-Hyd mRNA is naturally expressed in humans.

[0011] The present application also discloses the chromosomal location of the h-Hyd gene.

[0012]

DETAILED DESCRIPTION OF THE INVENTION Due to natural or artificial mutations (eg, Molecular Cloning 2).
nd Edition (Cold Spring Ha)
rbor Laboratory Press) 15.
1-15.113), the structure of the polynucleotide can be changed without changing the main function of the protein encoded by the polynucleotide. According to this method, the h-Hy of the present invention is used.
Also for d, a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing, ie, h-Hy
It is possible to create d variants. More specifically, for a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing obtained from a cDNA library or the like, po
Hect, the active site of ubiquitin ligase in the ly (A) binding domain and intracellular proteolytic system
A so-called point mutation for substituting, deleting or adding some bases other than the part encoding the (E6APC terminal homology) domain is performed to obtain a mutant of the DNA. The DNA mutant is introduced into an appropriate host such as Escherichia coli to prepare a transformant, and the transformant is allowed to produce a protein encoded by the introduced DNA mutant, thereby obtaining a h-Hyd mutant. Fabrication is possible.

The amino acid sequence of the h-Hyd mutant of the present invention can be determined from the nucleotide sequence of the gene encoding the mutant. For example, it is possible to use a commercially available program (for example, Genetyx-Mac (registered trademark, manufactured by Software Development Corporation)).

Due to the degeneracy of the genetic code, at least a portion 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 encoding h-Hyd of the present invention includes all patterns of degeneracy.

The DNA consisting of the nucleotide sequence of SEQ ID NO: 2 in the sequence listing is a naturally occurring h-Hyd gene.

The present invention includes an antisense polynucleotide comprising the nucleotide sequence of the antisense strand of the polynucleotide encoding h-Hyd and a derivative thereof. The antisense polynucleotide includes a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 2 in the sequence listing or a mRNA having a nucleotide sequence corresponding to the cDNA having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. RNA comprising a complementary base sequence is exemplified. The antisense polynucleotide of the present invention is capable of hybridizing to a polynucleotide encoding h-Hyd, and if the polynucleotide to which it hybridizes is a polynucleotide of a coding region, the polynucleotide is coded. It is possible to inhibit the biosynthesis of the polypeptide. The antisense polynucleotide and its derivative are included in the polynucleotide. In the present specification, the term “antisense polynucleotide” is used when it is specifically indicated that the antisense polynucleotide comprises the base sequence of the antisense strand.

In general, an antisense polynucleotide for inhibiting the biosynthesis of a polypeptide preferably comprises 15 or more bases. On the other hand, if the antisense polynucleotide is too long, it is difficult to incorporate the antisense polynucleotide into cells. Incorporating the antisense polynucleotide into cells, h-Hy
When inhibiting the biosynthesis of d, generally 12 bases or more and 3
It is good to use an antisense polynucleotide comprising bases of 0 bases or less, preferably 15 bases to 25 bases, more preferably 18 bases to 22 bases. Also in the antisense polynucleotide of the present invention, it is preferable to use one comprising 12 to 39 bases, preferably 15 to 25 bases, more preferably 18 to 22 bases.

The antisense polynucleotide of the present invention or a part thereof includes all of a plurality of nucleotides consisting of a base, a phosphate and a sugar, including non-naturally occurring nucleotides. The typical one is antisense D
NA and antisense RNA.

For the antisense polynucleotide of the present invention, the desired DN
Various antisense polynucleotide derivatives having high binding strength to A or mRNA, tissue selection, cell permeability, nuclease resistance and intracellular stability can be obtained.

From the viewpoint of ease of hybridization, it is generally considered to be desirable to design an antisense polynucleotide or a derivative thereof having a base sequence complementary to the base sequence of the region forming the stem loop. ing. The antisense polynucleotide of the present invention and a derivative thereof,
If necessary, a stem loop can be formed.

An antisense polynucleotide having a sequence complementary to the sequence near the translation initiation codon, ribosome binding site, capping site, or splice site can generally be expected to have a high expression suppressing effect. Therefore, the antisense polynucleotide of the present invention or a derivative thereof, wherein the gene or mRNA encoding h-Hyd is located near the translation initiation codon, a ribosome binding site,
Those containing a sequence complementary to the capping site and the splice site are expected to have a high expression suppressing effect.

The derivatives generally known at present are preferably derivatives having at least one of enhanced nuclease resistance, tissue selectivity, cell permeability and binding strength. Particularly preferred are derivatives having a phosphorothioate bond as a skeletal structure. The polynucleotide of the present invention and its derivatives also include derivatives having these functions or structures.

As long as it is a natural type antisense polynucleotide, it can be synthesized using a chemical synthesizer, or h-Hyd
The antisense polynucleotide of the present invention can be prepared by a PCR method using a gene encoding as a template. Some derivatives, such as a methylphosphonate type and a phosphorothioate type, can be chemically synthesized. In this case, the desired antisense polynucleotide can be obtained by performing the operation according to the instructions attached to the chemical synthesizer and purifying the obtained synthetic product by HPLC using reverse phase chromatography or the like. Alternatively, a derivative thereof can be obtained.

The polynucleotide encoding the h-Hyd of the present invention, its antisense polynucleotide or a part thereof (polynucleotide having a continuous base sequence of 12 or more bases) is cDN.
It can be used as a probe for screening the h-Hyd gene from the A library or the like. At this time, those having a GC content of 30 to 70% can be suitably used. Further, a polynucleotide consisting of a continuous base sequence of 15 or more bases is particularly preferable because the gene to which the polynucleotide hybridizes is stochastically unique. The polynucleotide used as a probe may be a derivative. Usually, a sequence having the number of bases or more is recognized as a sequence having specificity. As a cDNA library to be used in screening using the probe, one prepared from mRNA can be preferably used. A group of cDNAs selected from these cDNA libraries by random sampling can be used as a sample to be searched. Also, commercially available products can be used.

For example, a DNA consisting of a continuous base sequence of 12 or more bases in the base sequence shown in SEQ ID NO: 2 of the sequence listing or a polynucleotide (antisense polynucleotide) hybridizing to the DNA is cDNA
It can be used as a probe for screening the h-Hyd gene from a library or the like.

Further, Northern blot hybridization is carried out on mRNA derived from each tissue using the polynucleotide encoding the h-Hyd of the present invention, its antisense polynucleotide or a polynucleotide which is a part thereof as a probe. h-Hyd
It is possible to find a tissue in which gene-derived mRNA is expressed.

When chemically synthesizing DNA or RNA,
It is well known to chemically modify side chains by methylating or biotinylating them, or by substituting O for S in the phosphate group. For example, when chemically synthesizing the DNA described in SEQ ID NO: 2 in the sequence listing, the chemical modification can be performed to synthesize a DNA having a secondary structure different from the DNA itself shown in the sequence listing. Further, even a cDNA obtained from a cDNA library can be labeled with a radioisotope or the like. Therefore, the DNA and RNA of the present invention include the chemically modified DNA, RNA or antisense polynucleotide in the range. Chemically modified D
NA or RNA can exhibit both the function of encoding a protein or the function as a probe,
The chemically modified antisense polynucleotide can exhibit both the function of inhibiting the biosynthesis of a probe or a protein or the function as a probe.

It is possible to obtain a transformant by introducing the plasmid into an appropriate host such as Escherichia coli by a known method. Culturing a transformant into which the h-Hyd gene of the present invention has been introduced to allow the gene to be amplified or the protein to be expressed;
It is possible to produce h-Hyd. Similarly, it is possible to produce a h-Hyd mutant by culturing a transformant into which a DNA encoding the h-Hyd mutant has been introduced. Further, as shown in the examples, h-Hyd
It is possible to produce a part of the h-Hyd by culturing a transformant into which an arbitrary part of the cDNA encoding is introduced. By producing a part of h-Hyd, a low-molecular-weight protein or peptide having h-Hyd function can be produced. By recovering the above-mentioned product and performing operations such as concentration, solubilization, dialysis, and various kinds of chromatography as needed, the h-
It is possible to purify Hyd (including a part thereof, hereinafter, the notation h-Hyd is used as including the part thereof) or h-Hyd mutant unless otherwise specified herein.

There are various textbooks on culturing the transformants, and h-Hy based on the nucleotide sequence described in the present invention is used.
The d or h-Hyd mutant can be produced by a known method. At this time, as the host, any of bacteria such as Escherichia coli, yeast, and animal cells can be used, and animal cells are particularly preferable. To introduce a gene into a cell, a ribosome method, an electroporation method, or the like can be used.

From the obtained culture, h-Hyd or h-Hyd
Purification methods for purifying the Hyd mutant include immunoprecipitation, salting out, ultrafiltration, isoelectric point precipitation, gel filtration, electrophoresis, ion exchange chromatography, hydrophobic chromatography, and the like. There are various affinity chromatography methods such as an antibody chromatography method, a chromatofocusing method, an adsorption chromatography method, a reverse phase chromatography method and the like, which may be appropriately selected and performed.

In the manufacturing stage, the hH to be manufactured is
The yd or h-Hyd mutant may be produced in a transformant as a fusion peptide with another polypeptide. In this case, in the purification step, an operation of treating with a chemical substance such as bromocyan or an enzyme such as a prosthesis to cut out h-Hyd or an h-Hyd mutant is required.

The present invention relates to the antigenicity of h-Hyd
As exemplified in Examples 3 and 4, h-Hyd or an oligopeptide having an amino acid sequence specific to h-Hyd obtained by the above method is immunized to an animal derived from h-Hyd and an animal other than a human. This makes it clear that antibodies can be easily obtained. Therefore, the antibody that recognizes h-Hyd of the present invention (hereinafter referred to as h-Hyd)
Hyd antibody) is used to convert h-Hyd to hH
An antibody obtained by immunizing an animal from which yd is derived and a non-human animal, wherein the antibody recognizes the h-Hyd of the present invention.
Antibodies confirmed by the LISA method, immunostaining method (for example, measurement by FACS) and the like are included in the range.

It is a commonly used method to use, as an immunogen, one obtained by binding a part of the protein to another carrier protein such as bovine serum albumin even if it is a part of the protein. A part of the protein may be synthesized using, for example, a peptide synthesizer. In addition, as a part of protein, it is preferable that it is 8 or more amino acid residues. It is well known that, for a substance whose antigenicity has been revealed, if a polyclonal antibody can be obtained by immunization, a monoclonal antibody is produced by a hybridoma using the lymphocytes of the immunized animal ("" Antibodies A Laboratory
Manual "(Cold SpringHarbo)
r Laboratory Press, 1988) Chapter 6). Therefore, the h-Hyd antibody of the present invention includes a monoclonal antibody within its scope.

In the present invention, an antibody also includes an active fragment. An active fragment means a fragment of an antibody having antigen-antibody reaction activity,
Specifically, F (ab ') ₂ , Fab', Fab, Fv
And the like. For example, when the antibody of the present invention is digested with pepsin, F (ab ') ₂ is obtained, and when digested with papain, Fab is obtained. F (ab ') ₂ is 2-
Reduction with a reagent such as mercaptoethanol and alkylation with monoiodoacetic acid gives Fab '. Fv is a monovalent antibody active fragment in which a heavy chain variable region and a light chain variable region are linked by a linker. A chimeric antibody can be obtained by retaining these active fragments and substituting other portions with fragments of another animal.

The antibody of the present invention can be used to elucidate functions such as expression of h-Hyd and elucidate the mechanism of malignant tumor formation.

The detection of h-Hyd includes a method using an antibody and a method using an enzyme reaction. As a method using an antibody, specifically, a labeled h-Hy
Method for detecting h-Hyd using d antibody, h-Hy
h-Hyd using an antibody d and a labeled secondary antibody of the antibody
Is detected. Labels include, for example, radioisotopes (RI), enzymes, avidin or biotin,
Alternatively, a fluorescent substance (FITC, rhodamine, or the like) is used.

Examples of the method using an enzyme reaction include an ELISA method, an immunoagglutination method, a Western blot method, a method for identifying an immunoreactive molecule using flow cytometry, or a method similar thereto.

[0038]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples. <Example 1> Isolation of h-Hyd gene and determination of base sequence EST database search Internet EST database (database in which sequences of mRNA fragments are registered, http: // w
ww. ncbi. nlm. nih. gov. / DbES
From the sequence (EST) registered in T), a sequence having homology with the drosophila hyd (d-hyd) gene was searched. 26 of the amino acid sequence of the d-hyd protein
Blast search (BL
ASTsearch) found that human fetal heart c
The sequence registered as W95857 derived from DNA was hit.

2. Synthesis of Primers The following primers were synthesized from a cDNA library in order to obtain a DNA fragment consisting of a partial nucleotide sequence of the above-mentioned nucleotide sequence of W95857. AR primer 5'-TAGTAATATTACAC as antisense primer
AGCAATACAC-3 '(base sequence described in SEQ ID NO: 4 in the sequence listing) and End primer 5'-CTACACAAAACCAAAATTCT
TGGT-3 ′ (base sequence described in SEQ ID NO: 5 in the sequence listing) was designed and synthesized using a DNA synthesizer (ABI, model 392). Synthesized primer is 20 pmo with distilled water
1 / μl. Using this as a PCR primer, the following PCR operation was performed.

3. As the PCR cDNA library, a human fetal brain cDNA library (manufactured by Stratagene) and a chronic myelogenous leukemia cell line K562 cDNA library (manufactured by UKHGMP Resource Center) were used, and PCR was performed under the following conditions. These cDNA libraries have a T7 sequence at the 5 'end. First, PCR was performed using T7 primer (sense side) and AR primer (antisense side) as primers. P
In the CR operation, a liquid having the following composition was placed in a test tube, and 20 μl of a mineral oil was layered thereon, and left at 94 ° C. for 5 minutes. cDNA library (1 μg / μl) 5 μl dNTP mixture (each NTP dissolved in distilled water to 20 pmol / μl) 1 μl T7 sense primer (20 pmol / μl) 1 μl AR antisense primer (20 pmol / μl) 1 μl 10-fold concentration PCR Buffer (400 mM Tris-HCl (pH 8.3), 1 M KCl, 100 mM MgCl ₂ , 100 mM DTT) 2 μl Taq polymerase (5 units / μl) 0.5 μl distilled water 9.5 μl total 20 μl Then, “30 ° C. for 30 seconds Subsequently, a cycle of 72 ° C. for 1.5 minutes, followed by 94 ° C. for 30 seconds ”was repeated 50 times to carry out the reaction. Then at 55 ° C for 2 minutes, finally 7
The reaction was carried out at 2 ° C for 10 minutes to carry out a fragment extension reaction,
R operation completed.

Thereafter, a second PCR operation was performed using the PCR product obtained above as a template and a T7 primer (sense side) and an End primer (antisense side). In the second PCR operation, a solution having the following composition was placed in a test tube, and 20 μl of mineral oil was layered thereon, and left at 94 ° C. for 5 minutes. PCR product (1 μg / μl) obtained above 5 μl dNTP mixture 1 μl T7 primer (20 pmol / μl) 1 μl End primer (20 pmol / μl) 1 μl 10-fold concentration of PCR buffer (400 mM Tris-HCl (pH 8.3), 1 M KCl, 100 mM MgCl ₂ , 100 mM DTT) 2 μl distilled water 9.5 μl Taq polymerase (5 units / μl) 0.5 μl total 20 μl Then, “30 ° C. for 30 seconds, followed by 1 minute at 72 ° C.
Subsequently, the cycle of 94 ° C. for 30 seconds ”was repeated 40 times to carry out the reaction. Then at 55 ° C for 2 minutes and finally at 72 ° C
For 10 minutes to perform a fragment extension reaction to complete the PCR operation.

4. Preparation of Transformant The h-Hyd DNA obtained above was subjected to mini-gel electrophoresis (0.75% agarose gel), and a band (2.7 kbp) of the DNA was cut out from the gel. GeneC
DNA was collected using lean (registered trademark, manufactured by Bio 101), and bands were checked by minigel electrophoresis.

1 μl of DNA was taken and diluted with 99 μl of TE. The absorbance at 260 nm (A260) was measured, and the DNA concentration was calculated (the DNA concentration when A260 was 1.0 was 50 μl / ml). The DNA was diluted with TE so that the DNA concentration was 1 μg / μl.

The full-length h-Hyd consisting of the above base sequence
The gene was inserted into a pCGN vector, and the vector was introduced into Escherichia coli MN522 to prepare a transformant. This transformant was named h-Hyd, and deposited on October 1, 1997 with the Institute of Biotechnology and Industrial Science and Technology (Accession No. FERM P-16453).

5. Determination of base sequence From the transformant, an alkali method (“Molecula”) was used.
r CloningSecond Edition "
The plasmid (pCRII vector) was recovered according to the method described on pages 1.33-1.43), and the chiawell (registered trademark) 8 plasmid kit (OIAweii 8) was collected.
Plasmid was purified using a plasmid kit (Qiagen). This plasmid was subjected to DNA sequencing using a T7 primer, M13 reverse primer, the AR primer and the End primer, and an autosequencer (ABI model 373A) by a dye terminator method, and h
-The nucleotide sequence of the Hyd gene was determined. This base sequence is shown as SEQ ID NO: 2 in the sequence listing.

The AR primer used for PCR was
A primer consisting of a nucleotide sequence complementary to the nucleotide sequence of the 3 ′ non-coding region of the h-Hyd gene, and the End primer is a nucleotide complementary to 24 nucleotides at the 3 ′ end of the coding region including the termination codon of the h-Hyd gene. It is a primer consisting of a sequence.

6 Determination of Amino Acid Sequence From the above base sequence, the amino acid sequence of h-Hyd was determined. This amino acid sequence is shown as SEQ ID NO: 1 in the sequence listing.
Further, h-Hyd (human-Hyd), d-hyd
FIG. 1 shows the results of comparison of the hect (E6APC terminal homologous) domain, which is the active site of ubiquitin ligase, in the intracellular proteolytic system of the hyd protein r-hyd (rat-Hyd) in rats (Drosophila-Hyd) and rat. . In FIG. 1, each letter h is an outline character.
It is an amino acid portion common to yd. h-Hyd's he
The ct (E6APC terminal homology) domain is a portion from Gln at position 672 to Val at position 890 of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing.

<Example 2> Expression of h-Hyd in each tissue 2 μg of each of poly A + RNA (mRNA) of human tissues and poly A + RNA (mRNA) of various human cells was blotted on a membrane (Human).
Multiple Tissue Northern
Blot, II, III and Human C
canceler Cell Line Multiple
Tissue Northern Blot (manufactured by Clontech)) was labeled with Molecu under the conditions of high stringency under the conditions of high stringency.
lar Cloning A Laboratory
Manual Second Edition 7.3
Hybridization was performed as described on pages 9 to 7.52, and analysis was performed by Northern blot hybridization.

The labeling of the h-Hyd gene was performed by the following operation. 1) Labeling was performed using Takara MEGA LABEL kit (registered trademark, manufactured by Takara Shuzo Co., Ltd.) according to the instruction manual. 2) Next, a DNA reaction solution having the following composition was prepared, and this solution was incubated at 37 ° C for 30 minutes, and then incubated at 70 ° C for 1 minute.
Heat treatment was performed for 0 minutes to inactivate the enzyme. DNA probe (2 pmol / μl) 2 μl 10-fold phosphorylation buffer 2 μl [γ-32P] ATP (370 MBq / ml) (Amersham) 5 μl T4 polynucleotide kinase 1 μl Total 10 μl

3) TE50 (50 mM Tris-Cl
Se equilibrated with pH 8.0, 1 mM EDTA)
phadex G-25 (registered trademark, manufactured by Pharmacia)
To a 1.5 ml polyprep column (Bio-Rad)
Then, the DNA reaction solution that had been heat-treated in 2) was packed on the column so that the bed volume became 1 ml. 4) After that, 200 μl TE50 was flown through the column four times,
A labeled DNA probe was obtained from the fraction eluted with 200 μl of the second time.

Photographs of the results for each tissue are shown in FIGS.
And 4. The photograph shows that the mRNA of h-Hyd (8
kb band) was found to be expressed in all tissues. In particular, it was found that the expression was strong in the heart and testis.

FIG. 5 shows photographs of the results for the various cell lines. From the photograph, Burkitt's lymphoma cells (Raji)
And K562 were found to be strongly expressed.

<Example 3> Preparation of antibody recognizing h-Hyd (1) Preparation of Antigen Amino acid sequence A which is the amino acid sequence of the amino acid sequence of h-Hyd described in SEQ ID NO: 1 from the 453rd Arg to the 503rd Phe with cysteine (Cys) added to the N-terminal. A peptide consisting of (the sequence described in SEQ ID NO: 3 in the sequence listing) was synthesized. Cysteine was added to bind to keyhole limpet hemocyanin (KHL). The synthesis was outsourced to Iwaki Glass Co., Ltd. 2 mg of the synthesized peptide was bound to 2 mg of maleimidated KLH (Pierce). The reaction was performed according to the method described in the instructions of the Pierce kit.

2. Immune solution (1 μg / ml)
100 μl, 0.5 ml of PBS and 0.5 ml of Freund's complete adjuvant (manufactured by Difco) were taken in a syringe, mixed and made into an emulsion, and subcutaneously inoculated in four parts on the back of a rabbit. One week later, a second immunization was performed. From the second time, immunization was performed by changing the adjuvant to Freund's complete adjuvant (manufactured by Difco). Other operations are the same as in the first operation. After the second time, immunization was performed 6 times in total at one week intervals.

3. Antibody Purification One week after the final immunization, blood was collected. This blood was allowed to stand at room temperature for 3 hours, and after sufficient blood coagulation, 3,000 rpm
After centrifugation at 5 m for 5 minutes, the supernatant (serum) was collected.
Saturated ammonium sulfate was added to the serum to a final concentration of 50% and salted out. This sample was centrifuged to precipitate a fraction containing the antibody. Thereafter, the precipitate was dissolved in PBS, and further subjected to salting-out for PBS. Thereafter, the antibody was affinity-purified using a Protein G Sepharose column (registered trademark, manufactured by Pharmacia). As a result, a total of 5 mg of the peptide-specific antibody was obtained.

<Example 4> Preparation of antibody recognizing h-Hyd (2) Preparation of antigen Vector pGEX-2TH incorporating GST gene
(Pharmacia) between BamHI and HindIII, from A at position 1357 to 267 in SEQ ID NO: 2 in the sequence listing.
The 3'-terminal portion of the h-Hyd gene containing up to the third G was inserted, and the vector was introduced into Escherichia coli DH5α to prepare a transformant. H-Hyd C
GST-linked GST-terminal to the N-terminus of the terminal protein (protein consisting of the amino acid sequence from Arg at position 453 to Val at position 890 in SEQ ID NO: 1).
h-Hyd was produced. 2 mg of the prepared peptide was bound to 2 mg of maleimidated KLH (Pierce).
The reaction was performed according to the method described in the instructions of the Pierce kit.

2. Immunization In the same manner as in Example 3, antigen solution (1 μg / ml) 100
μl, 0.5 ml of PBS and 0.5 ml of Freund's complete adjuvant (manufactured by Difco) were taken in a syringe, mixed to form an emulsion, and immunized on the back of a rabbit.

3. Purification of Antibody The antibody was purified from rabbit blood in the same manner as in Example 3. Thus, an antibody using h-Hyd as an antigen was prepared.

Example 5 Western Blot GST-h-Hyd prepared in Example 4 was digested with thrombin and cut into h-Hyd and GST. GST-h-
1 for Hyd, h-Hyd (partial protein from Arg at position 453 to Val at position 890) and GST
Electrophoresis was performed on a 0-20% gradient gel. After the electrophoresis, the DNA was transferred to a nitrocellulose membrane using a trans-blot system (manufactured by Marisol). Nitrocellulose membranes were coated with 10% skim milk / PBS, 0.1% Tween
20 and left for 1 hour to block. Thereafter, the plate was washed twice with 0.3% Tween 20 / PBS for 5 minutes each.

The anti-h-Hyd antibody prepared in Example 4 was
After dilution with PBS to μg / ml, the mixture was added to a nitrocellulose membrane and reacted at room temperature for 40 minutes. Then 0.3%
The wells were washed three times for 5 minutes each with Tween 20 / PBS. Next, a liquid obtained by diluting peroxidase-labeled anti-rabbit IgG (manufactured by Amersham) 20,000-fold with PBS was added to the nitrocellulose membrane, and further reacted at room temperature for 40 minutes. Thereafter, the plate was washed three times with 0.3% Tween 20 / PBS for 5 minutes each.

Next, an ECL kit (manufactured by Amersham)
Was added to the nitrocellulose membrane in an amount of 5 ml and allowed to react for 1 minute. Thereafter, the membrane was exposed to an X-ray film for 20 seconds, developed, and photographed.

As a control, the same operation was performed using an anti-GST antibody instead of an anti-h-Hyd antibody. FIG. 6 shows the results. Anti-h-Hyd antibody (α-GST-
hyd) is shown on the left and the GST antibody (shown as α-GST in the figure) is used on the right. Only when an anti-h-Hyd antibody is used, h-Hyd
A band (indicated by hyd in the figure) was observed, confirming that the antibody prepared in Example 4 recognized h-Hyd.

Example 6 Chromosome Mapping Chromosome mapping of the full-length h-Hyd gene was performed as follows. 1. PCR PCR was performed using the Genebridge 4 Radiation Hybrid Screening Panel (GENEBRIDGE 4
Rradiation Hybrid Screeneni
ng Panel (manufactured by Research Genetics) was used as a template, and a part of the h-Hyd gene was used as a primer. Primer is S1 primer (sense direction)
5′-AGAGAGAGAC CGGGAAAGGG AGAGAGAAAG G-3 ′ (base sequence described in SEQ ID NO: 6 in the sequence listing) and AS1 primer (antisense direction) 5′-TCTCCAAGAGCCTGCCGATG T-3 ′ (base described in SEQ ID NO: 7 in the sequence listing) Were synthesized using a DNA synthesizer and dissolved in distilled water at a concentration of 20 pmol / μl.

The PCR operation was performed as follows. A liquid having the following composition was placed in a test tube, and 15 μl of mineral oil was overlaid thereon, and left at 94 ° C. for 5 minutes. Kit panel 1 μl dNTP mixture 1 μl S1 primer 1 μl AS1 primer 1 μl 10-fold concentration of PCR buffer 1.5 μl distilled water 9 μl Taq polymerase 0.5 μl total 15 μl Then, “60 ° C. for 30 seconds, followed by 72 ° C. for 30 seconds Subsequently, a cycle of “at 95 ° C. for 30 seconds” was repeated 35 times to carry out the reaction. Subsequently, the reaction was carried out at 60 ° C. for 2 minutes and finally at 72 ° C. for 10 minutes to carry out an extension reaction of the fragment.
CR operation completed. Then, 5 μl of the PCR product was
It was electrophoresed on a 2.0% agarose gel.

2. Mapping As a result, the section number of the panel to which the primer (S1 or AS1) was hybridized
head Institute / MIT Center
for Genome Research (http
p: www-genome. wi. mit. edu / c
gi-bin / contig / rhmapper. p
l. ) By e-mail to obtain information on the chromosome position and nucleotide sequence of the panel with the number. As a result, h-Hyd
Was mapped to chromosome 8q24 (near the c-myc gene).

[0066]

The h-Hyd of the present invention is expected to be involved in intracellular proteolysis and cell proliferation based on the structure (poly (A) binding domain and hect domain) predicted from its amino acid sequence. In particular, it is expected to be involved in intracellular proteolysis and cell proliferation in the heart and testis where expression of the mRNA is remarkable. Therefore, h-Hyd or h-Hyd of the present invention is effective for analyzing the mechanism of the occurrence of malignant tumor.

The polynucleotide encoding the h-Hyd of the present invention, a polynucleotide consisting of a continuous base sequence of 12 or more bases, or a polynucleotide (including an antisense polynucleotide) hybridizing to the polynucleotide, It can be used as a probe for screening the h-Hyd gene from a cDNA library or the like.

Further, the antisense polynucleotide of the present invention can inhibit h-Hyd biosynthesis.

The antibody of the present invention can be used for elucidating functions such as expression of h-Hyd and elucidating the mechanism of malignant tumor occurrence.

[0070]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 890 Sequence type: amino acid Topology: Linear Sequence type: Protein sequence Met Met Ser Ala Arg Gly Asp Phe Leu Asn Tyr Ala Leu Ser Leu Met 1 5 10 15 Arg Ser His Asn Asp Glu His Ser Asp Val Leu Pro Val Leu Asp Val 20 25 30 Cys Ser Leu Lys His Val Ala Tyr Val Phe Gln Ala Leu Ile Tyr Trp 35 40 45 Ile Lys Ala Met Asn Gln Gln Thr Ala Leu Asp Thr Pro Gln Leu Glu 50 55 60 Arg Lys Arg Thr Arg Gly Leu Leu Glu Leu Gly Ile Asp Asn Glu Asp 65 70 75 80 Ser Glu His Glu Asn Asp Asp Asp Thr Asn Gln Ser Ala Thr Leu Asn 85 90 95 Asp Lys Asp Asp Asp Ser Leu Pro Ala Glu Thr Gly Gln Asn His Pro 100 105 110 Phe Phe Arg Arg Ser Asp Ser Met Thr Phe Leu Gly Cys Ile Pro Pro 115 120 125 Asn Pro Phe Glu Val Pro Leu Ala Glu Ala Ile Pro Leu Ala Asp Gln 130 135 140 Pro His Leu Leu Gln Pro Asn Ala Arg Lys Glu Asp Leu Phe Gly Arg 145 150 155 160 Pro Ser Gln Gly Leu Tyr Ser Ser Ser Ala Ser Ser Gly Lys Cys Leu 165 170 175 Met Glu Val Thr Val Asp Arg Asn Cys Leu Glu Val Leu Pro Thr Lys 180 185 190 Met Ser Tyr Ala Ala Asn Leu Lys Asn Val Met Asn Met Gln Asn Arg 195 200 205 Gln Lys Lys Glu Gly Glu Glu Gln Pro Val Leu Pro Glu Glu Thr Glu 210 215 220 Ser Ser Lys Pro Gly Pro Ser Ala His Asp His Ala Ala Gln Leu Lys 225 230 235 240 Ser Ser Leu Leu Ala Glu Ile Gly Leu Thr Glu Ser Glu Gly Pro Pro 245 250 255 Leu Thr Ser Phe Arg Pro Gln Cys Ser Phe Met Gly Met Val Ile Ser 260 265 270 His Asp Met Leu Leu Gly Arg Trp Arg Leu Ser Leu Glu Leu Phe Gly 275 280 285 Arg Val Phe Met Glu Asp Val Gly Ala Glu Pro Gly Ser Ile Leu Thr 290 295 300 Glu Leu Gly Gly Phe Glu Val Lys Glu Ser Lys Phe Arg Arg Glu Met 305 310 315 320 Glu Lys Leu Arg Asn Gln Gln Ser Arg Asp Leu Ser Leu Glu Val Asp 325 330 335 Arg Asp Arg Asp Leu Leu Ile Gln Gln Thr Met Arg Gln Leu Asn Asn 340 345 350 His Phe Gly Arg Arg Cys Ala Thr Pro Met Ala Val His Arg Val 355 360 365 Lys Val Thr Phe Arg Asp Glu Pro Gly Glu Gly Ser Gly Val Ala Arg 370 375 380 Ser Phe Tyr Thr Ala Ile Ala Gln Ala Phe Leu Ser Asn Glu Lys Leu 385 390 395 400 400 Pro Asn Leu Glu Cys Ile Gln Asn Ala Asn Lys Gly Thr His Thr Ser 405 410 415 Leu Met Gln Arg Leu Arg Asn Arg Gly Glu Arg Asp Arg Glu Arg Glu 420 425 430 430 Arg Glu Arg Glu Met Arg Arg Ser Ser Gly Leu Arg Ala Gly Ser Arg 435 440 445 Arg Asp Arg Asp Arg Asp Phe Arg Arg Gln Leu Ser Ile Asp Thr Arg 450 455 460 Pro Phe Arg Pro Ala Ser Glu Gly Asn Pro Ser Asp Asp Pro Glu Pro 465 470 475 480 Leu Pro Ala His Arg Gln Ala Leu Gly Glu Arg Leu Tyr Pro Arg Val 485 490 495 Gln Ala Met Gln Pro Ala Phe Ala Ser Lys Ile Thr Gly Met Leu Leu 500 505 510 Glu Leu Ser Pro Ala Gln Leu Leu Leu Leu Leu Ala Ser Glu Asp Ser 515 520 525 Leu Arg Ala Arg Val Asp Glu Ala Met Glu Leu Ile Ile Ala His Gly 530 535 540 Arg Glu Asn Gly Ala Asp Ser Ile Leu Asp Leu Gly Leu Val Asp Ser 545 550 555 560 Ser Gln Lys Val Gln Gln Glu Asn Arg Lys Arg His Gly Ser Ser Arg 565 570 575 Ser Val Val Asn Met Asp Leu Asp Asp Thr Asp Asp Gly Asp Asp Asn 580 585 590 Ala Pro Leu Phe Tyr Gln Pro Gly Lys Arg Arg Phe Tyr Thr Pro Arg 595 600 605 Pro Gly Lys Asn Thr Glu Ala Arg Leu Asn Cys Phe Arg Asn Ile Gly 610 615 620 Arg Ile Leu Gly Leu Cys Leu Leu Gln Asn Glu Leu Cys Pro Ile Thr 625 630 635 640 640 Leu Asn Arg His Val Ile Lys Val Leu Leu Gly Arg Lys Val Asn Trp 645 650 655 His Asp Phe Ala Phe Phe Asp Pro Val Met Tyr Glu Ser Leu Arg Gln 660 665 670 Leu Ile Leu Ala Ser Gln Ser Ser Asp Ala Asp Ala Val Phe Ser Ala 675 680 685 Met Asp Leu Ala Phe Ala Ile Asp Leu Cys Lys Glu Glu Gly Gly Gly 690 695 700 Gln Val Glu Leu Ile Pro Asn Gly Val Asn Ile Pro Val Thr Pro Gln 705 710 710 720 Asn Val Tyr Glu Tyr Val Arg Lys Tyr Ala Glu His Arg Met Leu Val 725 730 735 735 Val Ala Glu Gln Pro Leu His Ala Met Arg Lys Gly Leu Leu Asp Val 740 745 750 Leu Pro Lys Asn Ser Leu Glu Asp Leu Thral Ala Glu Asp Phe Arg Leu 755 760 765 Leu Val Asn Gly Cys Gly Glu Val Asn Val Gln Met Leu Ile Ser Phe 770 775 780 Thr Thr Phe Asn Asp Glu Ser Gly Glu Asn Ala Glu Lys Leu Leu Gln 785 790 795 800 Phe Lys Arg Trp Phe Trp Ser Ile Val Glu Lys Met Ser Met Thr Glu 805 810 815 Arg Gln Asp Leu Val Tyr Phe Trp Thr Ser Ser Pro Ser Leu Pro Ala 820 825 830 Ser Glu Glu Gly Phe Gln Pro Met Pro Ser Ile Thr Ile Arg Pro Pro 835 840 845 Asp Asp Gln His Leu Pro Thr Ala Asn Thr Cys Ile Ser Arg Leu Tyr 850 855 860 Val Pro Leu Tyr Ser Ser Lys Gln Ile Leu Lys Gln Lys Leu Leu Leu 865 870 875 880 Ala Ile Lys Thr Lys Asn Phe Gly Phe Val 885 890

SEQ ID NO: 2 Sequence length: 2672 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence ATG ATG TCT GCA CGA GGA GAC TTC CTA AAT TAT GCT CTG TCT CTA ATG 48 CGG TCT CAT AAT GAT GAG CAT TCT GAT GTT CTT CCA GTT TTG GAT GTT 96 TGC TCA TTG AAG CAT GTG GCA TAT GTT TTT CAA GCA CTT ATA TAC TGG 144 ATT AAG GCA ATG AAT CAG CAG ACA GCA TTG GAT ACA CCT CAA CTA GAA 192 CGC AAA AGG ACG CGA GGA CTC TTG GAA CTG GGT ATT GAT AAT GAA GAT 240 TCA GAA CAT GAA AAT GAT GAT GAC ACC AAT CAA AGT GCT ACT TTG AAT 288 GAT AAG GAT GAT GAC TCT CTT CCT GCA GA ACT GGC CAA AAC CAT CCA 336 TTT TTC CGA CGT TCA GAC TCC ATG ACA TTC CTT GGG TGT ATA CCC CCA 384 AAT CCA TTT GAA GTG CCT CTG GCT GAA GCC ATC CCC TTG GCT GAT CAG 432 CCA CAT CTG TTG CAG CGA AAT AAG GAG GAT CTT TTT GGC CGT 480 CCA AGT CAG GGT CTT TAT TCT TCA TCT GCC AGT AGT GGG AAA TGT TTA 528 ATG GAG GTT ACA GTG GAT AGA AAC TGC CTA GAG GTT CTT CCA ACA AAA 576 ATG TCT TAT GCT GCC AAT CTG AAA AAT GTA ATG AAC ATG CAA AAC CGG 624 CAA AAA AAA GAA GGG GAA GAA CAG CCC GTG CTG CCA GAA GAA ACT GAG 672 AGT TCA AAA CCA GGG CCA TCT GCT CAT GAT CAT GCT GCA CAA TTA AAA 720 AGT AGC TTA CTA GCA GAA ATA GGA CTT ACT GAA AGT GAA GGG CCA CCT 768 CTC ACA TCT TTC AGG CCA CAG TGT AGC TTT ATG GGA ATG GTT ATT TCC 816 CAT CAT GAT ATG CTG CTA GGA CGT TGG CGC CTT TTA GAA CTG TTC GGC 864 AGG GTA TTC ATG GAA GAT GTT GGA GCA GAA CCT GGA TCA ATC CTA ACT 912 GAA TTG GGT GGT TTT GAG GTA AAA GAA TCA AAA TTC CGC AGA GAA ATG 960 GAA AAA CTG AGA AAC CAG CAG TCA AGA GATTG TCA CTA GAG GTT GAT 1008 CGG GAT CGA GAT CTT CTC ATT CAG CAG ACT ATG AGG CAG CTT AAC AAT 1056 CAC TTT GGT CGA AGA TGT GCT ACT ACA CCA ATG GCT GTA CAC AGA GTA 1104 AAA GTC ACA TTT AGG GAT GAG CCA GGA GGC AGT GGT GTA GCA CGA 1152 AGT TTT TAT ACA GCC ATT GCA CAA GCA TTT TTA TCA AAT GAA AAA TTG 1200 CCA AAT CTA GAG TGT ATC CAA AAT GCC AAC AAA GGC ACC CAC ACA AGT 1248 TTA ATG CAG AGA TTA AGG AAC CG A GGA GAG AGA GAC CGG GAA AGG GAG 1296 AGA GAA AGG GAA ATG AGG AGG AGT AGT GGT TTG CGA GCA GGT TCT CGG 1344 AGG GAC CGG GAT AGA GAC TTT AGA AGA CAG CTT TCC ATC GAC ACT AGG 1392 CCC TTT AGA CCC TCC GAA GGG AAT CCT AGC GAT GAT CCT GAG CCT 1440 TTG CCA GCA CAT CGG CAG GCA CTT GGA GAG AGG CTT TAT CCT CGT GTA 1488 CAA GCA ATG CAA CCA GCA TTT GCA AGT AAA ATC ACT GGC ATG TTG TTG 1536 GAA TTA TCC CCA CAG CTG CTT CTC CTT CTA GCA AGT GAG GAT TCT 1584 CTG AGA GCA AGA GTG GAT GAG GCC ATG GAA CTC ATT ATT GCA CAT GGA 1632 CGG GAA AAT GGA GCT GAT AGT ATC TTG GAT CTT GGA TTA GTA GAC TCC 1680 GTA CAAAG CAG CAG GAA AAC CGA AAG CGC CAT GGC TCT AGT CGA 1728 AGT GTA GTA AAT ATG GAT TTA GAT GAT ACA GAT GAT GGT GAT GAC AAT 1776 GCC CCT TTG TTT TAC CAA CCT GGG AAA AGA AGA TTT TAT ACT CCA AGG 1824 CCT AGC AAC ACA GAA GCA AGG TTG AAT TGT TTC AGA AAC ATT GGC 1872 AGG ATT CTT GGA CTA TGT CTG TTA CAG AAT GAA CTA TGT CCT ATC ACA 1920 TTG AAT AGA CAT GTA ATT AAA GTA TTG CTT GGT AGA AAA GTC AAT TGG 1968 CAT GAT TTT GCT TTT TTT GAT CCT GTA ATG TAT GAG AGT TTG CGG CAA 2016 CTA ATC CTC GCG TCT CAG AGT TCA GAT GCT GAT GCT GTT TTC TCA GCA 2064 ATG GAT TTG GCA TTT GCA ATT GAC CTG TGT AAA GAA GGT G GGA 2112 CAG GTT GAA CTC ATT CCT AAT GGT GTA AAT ATA CCA GTC ACT CCA CAG 2160 AAT GTA TAT GAG TAT GTG CGG AAA TAC GCA GAA CAC AGA ATG TTG GTA 2208 GTT GCA GAA CAG CCC TTA CAT GCA ATG AGG AAA GGT CTA GAT GTG 2256 CTT CCA AAA AAT TCA TTA GAA GAT TTA ACG GCA GAA GAT TTT AGG CTT 2304 TTG GTA AAT GGC TGC GGT GAA GTC AAT GTG CAA ATG CTG ATC AGT TTT 2352 ACC TCT TTC AAT GAT GAA TCA GGA GAA AAT GAG AGA CTT CTG CAG 2400 TTC AAG CGT TGG TTC TGG TCA ATA GTA GAG AAG ATG AGC ATG ACA GAA 2448 CGA CAA GAT CTT GTT TAC TTT TGG ACA TCA AGC CCA TCA CTG CCA GCC 2496 AGT GAA GAA GGA TTC CAG CCT ATG CCA TTC ATA AGA CCA CCA 2544 GAT GAC CAA CAT CTT CCT ACT GCA AAT ACT TGC ATT TCT CGA CTT TAC 2592 GTC CCA CTC TAT TCC TCT AAA CAG ATT CTC AAA CAG AAA TTG TTA CTC 2640 GCC ATT AAG ACC AAG AAT TTT G GT TTT GTG TAG 2673

SEQ ID NO: 3 Sequence length: 52 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Arg Asp Phe Arg Arg Gln Leu Ser Ile Asp Thr Arg Pro Phe Arg Pro 1 5 10 15 Ala Ser Glu Gly Asn Pro Ser Asp Asp Pro Glu Pro Leu Pro Ala His 20 25 30 Arg Gln Ala Leu Gly Glu Arg Leu Tyr Pro Arg Val Gln Ala Met Gln 35 40 45 Pro Ala Phe Cys 50

SEQ ID NO: 4 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid sequence TAGTAATATT ACACAGCAAT ACAC 24

SEQ ID NO: 5 Sequence length: 24 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid sequence CTACACAAAA CCAAAATTCT TGGT 24

SEQ ID NO: 6 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid sequence AGAGAGAGAC CGGGAAAGGG AGAGAGAAAG G 31

SEQ ID NO: 7 Sequence length: 21 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Other nucleic acid sequence TCTCCAAGAG CCTGCCGATG T 21

[Brief description of the drawings]

FIG. 1. Hec of h-Hyd, d-hyd, r-hyd
It is the figure which compared t domain.

FIG. 2 is an electrophoretic photograph showing the result of Northern blot hybridization obtained by hybridizing h-Hyd DNA with mRNA of each human tissue.

FIG. 3 is an electrophoretic photograph showing the results of Northern blot hybridization obtained by hybridizing h-Hyd DNA with mRNA of human tissues.

FIG. 4 is an electrophoretic photograph showing the results of Northern blot hybridization obtained by hybridizing h-Hyd DNA with mRNA of human tissues.

FIG. 5 shows h-Hyd for mRNA of each human cell line.
5 is an electrophoretic photograph showing the results of Northern blot hybridization obtained by hybridizing the DNAs of Example 1.

FIG. 6. GST-h-Hyd, h-Hyd and GST
5 is an electrophoretic photograph showing the results of Western Blot analysis performed using anti-h-Hyd antibody and anti-GST antibody.

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[Procedure amendment]

[Submission date] November 18, 1997

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] All figures

[Correction method] Change

[Correction contents]

FIG.

FIG. 2

FIG. 3

FIG. 4

FIG. 5

FIG. 6

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91) (C12P 21/02 C12R 1:19) (72) Inventor Yoshiomi Honda 1-chome Minami Kumamoto, Kumamoto City, Kumamoto Prefecture 9-23 (72) Inventor Masayuki Ando 1-7-53, Saburo, Kumamoto City, Kumamoto Prefecture (72) Inventor Hideyuki Saya 1-16-6, 31 Wataka, Kumamoto City, Kumamoto Prefecture

Claims (7)

[Claims] 1. A protein comprising an amino acid sequence having at least the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 2. An amino acid sequence comprising one or more amino acids substituted, deleted or added in the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and
A protein having a hep domain, which is an active site of ubiquitin ligase in an intracellular proteolysis system and is a C-terminal homologous domain of E6AP. 3. A polynucleotide encoding the protein according to claim 1 or 2. 4. A polynucleotide which is a part of the polynucleotide according to claim 3, wherein the polynucleotide comprises 12 or more consecutive bases. 5. An antisense polynucleotide comprising the nucleotide sequence of the antisense strand of the polynucleotide according to claim 3, or a part of a derivative of the antisense polynucleotide, comprising at least 12 consecutive bases. Antisense polynucleotides or derivatives of antisense polynucleotides. 6. The polynucleotide according to claim 3, which is chemically modified. 7. An antibody that recognizes the protein according to claim 1 or 2.