JPH1175871A - Cell calcification suppressing active protein and gene coding for the same protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain both a protein C-11 protein which is a cell calcification suppressant useful as a medicine or the like and a gene coding for the C-11 protein. SOLUTION: This C-11 protein is composed of an amino acid sequence represented by the formula or an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added in the amino acid sequence represented by the formula and has cell calcification suppressing activities. The C-11 protein can be produced by finding a new isoform gene (C-11 gene) of a c-erg gene which is an erg gene derived from a chicken, determining the base sequence of the gene and recombining the resultant C-11 pretein encoded by the gene. Concretely, the total RNA is extracted from an embryo on the 4 to 10 days of the chicken and reversely transcribed. The whole translation region of the c-erg and C-11 gene can be amplified by a polymerase chain reactional(PCR) method (using 5'-ATCTTGATCACATTATGGCAAGC 3'- and 5'-CACTTAGTAGTAGGTGCCAAG.ATGG-3' or the like as a primer).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a protein for inhibiting cell calcification and a gene encoding the protein.

[0002]

2. Description of the Related Art Ets was initially avian acute leukemia vi.
It was found as an oncogene of rusE26. Recently, a group of Ets-related genes ranging from humans to Drosophila have been discovered,
These are assumed to be transcription factors that play a fundamental role in regulating cell growth and differentiation. Meanwhile, Ets
Little is known about the biological effects of gene superfamily.

[0003]

[Problems to be solved by the invention] Recently, Ets related ge
It was reported that ne (erg) was specifically expressed at the site of chondrogenesis, indicating that erg may play some role in early skeletal formation. The present inventors based on such knowledge,
Chicken-derived erg gene (hereinafter, chickin-erg or c-
erg gene) was introduced into osteoblasts, and the function of c-erg was successfully elucidated, thereby completing the present invention.

[0004]

Means for Solving the Problems The present inventors have found a novel isoform gene of c-erg gene (hereinafter referred to as C-11 gene or C11 gene), which is a chicken-derived erg gene, and have determined the nucleotide sequence thereof. It was determined. Further, the expression of a protein encoded by the gene (referred to as C-11 protein or C11 protein) was confirmed.

[0005] In addition, the above-mentioned c-erg or C-11 may
It has been found that calcification of the cells is suppressed by introducing the gene.

[0006] More specifically, the present invention provides the following C-11 protein (a) or (b).

(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence (a)
A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having a cell calcification inhibitory activity.

[0008] The present invention also provides a gene encoding the above-described protein.

Further, the present invention provides a medicine containing the above-mentioned protein, and also provides a medicine which is a cell calcification inhibitor.

Further, the present invention provides the following (a) or (b)
The present invention provides a cell calcification inhibitor containing c-erg protein.

(A) a protein comprising the amino acid sequence of SEQ ID NO: 4 in the sequence listing, (b) an amino acid sequence (a)
A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having a cell calcification inhibitory activity.

[0012] The present invention further provides an antibody against the C-11 protein, and further provides an antibody characterized in that the antibody is a monoclonal antibody.

Further, the present invention provides a method for measuring the expression of C-11 gene or c-erg gene in cells,
It is intended to provide a method for measuring calcification of cells.

[0014] The present invention also relates to the present invention, wherein the expression of the above gene is determined by using a DN specific to the C-11 gene or c-erg gene.
An object of the present invention is to provide the above-mentioned method for measuring the amount of C-11 mRNA or c-erg mRNA expressed in a cell by using a probe for the A sequence.

[0015] The expression of the gene may be determined by
Another object of the present invention is to provide the above-mentioned method for measuring the expression level of c-erg protein in cells.

[0016] Further, the antibody against C-11 or c-erg in a cell is obtained by using an antibody against the above-mentioned C-11 or c-erg protein.
The present invention provides the above method for measuring the expression of -erg protein.

The present invention also provides a method for diagnosing a disease causing pathological calcification or ossification, such as ossification of the spinal ligament (OPLL) or osteoarthritis, using the above-described method for measuring calcification of cells. Things.

The present invention also provides a kit for measuring cell calcification, which comprises an antibody against C-11 protein or c-erg protein or both.

Further, the present invention provides a cell calcification inhibitory effect using a cell transformed with a gene encoding a protein described in any one of the following (a) to (d): It is intended to provide a method for screening a substance having an inhibitory activity.

(A) a protein consisting of the amino acid sequence of SEQ ID NO: 2 in the sequence listing, (b) an amino acid sequence (a)
A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added, and having a cell calcification inhibitory activity, (c) SEQ ID NO: 4 in the sequence listing
And (d) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence (c), and having a cell calcification inhibitory activity.

Furthermore, the present invention provides a medicine containing erg protein.

The present invention also provides a medicine containing the erg gene.

Further, the present invention provides a medicine containing C-11 protein or c-erg protein.

The present invention also relates to the C-11 gene or c-
The present invention provides a medicine containing the erg gene.

Further, the present invention provides a drug containing a protein having an amino acid sequence common to the c-erg protein and the C-11 protein.

In the present specification and drawings, when abbreviations such as bases and amino acids are used, IUPAC-I
UB abbreviations or conventional abbreviations in the art were used.

(Nucleic acid) DNA Deoxyribonucleic acid cDNA Complementary DNA RNA Ribonucleic acid mRNA Messenger RNA A Adenine C Cytosine G Guanine T Thymine.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Isolation and Confirmation of C-11 Gene A novel gene according to the present invention is c-erg derived from chicken.
It is an isoform of a gene. As shown in FIG. 1, it has a nucleotide sequence in which 81 nucleotides from the 655th nucleotide to the 735th nucleotide are deleted as compared to the nucleotide sequence of the c-erg gene (SEQ ID NO: 1 in the sequence listing). The novel gene, C-11 gene according to the present invention can be isolated from various cells by a generally known method.

Specifically, total RNA (total RNA) was extracted from chicken embryos 4 to 10 days, and reversely transcribed.
-All the translation regions of the erg and C-11 genes can be amplified by the PCR method.

The primers usable at this time include, for example, the following C11B and C11C which amplify both c-erg and C-11.

C11B: 5′-CACATTATGGCAAGCACTATTAAGG-3 ′ C11C: 5′-CACTTAGTAGTAGGTGCCAAGATGG-3 ′ C11A: 5′-ATCTTGATCACATTATGGCAAGC-3 ′ When primers C11B and C11C are used,
Two bands of 46 bp and 1355 bp appeared, and 1446 bp was ce
In rg, 1355 bp is C-11. In addition, primer C
When 11A and C11C were used, 1454 bp (ce
rg) and 1373b (C-11) can be amplified.

In each case, the RT-PCR condition was 1 μm.
g of total RNA was reverse transcribed and then using 2 units of Taq polymerase for 1 minute at 61 ° C, 2 minutes at 72 ° C, 10 seconds at 95 ° C.
It can be detected after 30 cycles of amplification.

Further, it is easy to prepare an oligonucleotide probe complementary to an appropriate partial base sequence based on the determined base sequence, and using such a probe, various methods can be used to prepare the C-11 gene. It is also possible to confirm the existence.

C-11 Protein The C-11 protein deduced from the nucleotide sequence of the C-11 gene has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.

[0035] The C-11 protein also comprises the above C-1
One gene can be incorporated into a viral vector (adenovirus or chicken retrovirus) and expressed in appropriate cells (Gene Transfer and Expression: A
Laboratory Manual by Michael Kriegler, WHFreeman
and Company, New York (1991), p29-56.). further,
The amino acid sequence of the obtained protein can be directly determined by an ordinary amino acid sequence analyzer.

Furthermore, the C-C described in SEQ ID NO: 1 in the sequence listing
An arbitrary nucleotide sequence can be substituted, deleted, or inserted into the sequence of 11 genes using, for example, the site-directed mutagenesis method. By repeating the above-described site-directed mutagenesis method one or several times, it is possible to produce a c-11 protein mutant in which one or several amino acids have been substituted, deleted, or inserted, and a gene mutant encoding the same. is there. Such mutants are also within the scope of the present invention, as long as they have cell calcification inhibitory activity.

Further, C-1 expressed in specific cells
Various known methods can be used for detecting one protein. More specifically, immunostaining using an antibody against the C-11 protein or a partial protein thereof, a method for examining localization using a fluorescent antibody method, and a method for homogenizing cells to obtain C-11 using a radioimmunoassay or an ELISA method
A method for measuring the expression level of is mentioned.

The c-erg protein described in SEQ ID NO: 4 in the c-erg protein sequence listing is also
It can be expressed by a method similar to that for the C-11 protein.

Furthermore, a c-erg protein or a mutant of the gene can be prepared by the same method as that used for the C-11 protein or the gene, and the present invention is satisfactory as long as it has a cell calcification inhibitory activity. Can be used.

Antibodies against C-11 protein (Monoc
(Ronal Antibody ) The antibody against the C-11 protein according to the present invention may be against any or all of the above-described C-11 proteins. In addition, other substances (proteins, etc.) suitable for antibodies to impart specific properties
Can be used.

The immunization method using the above-mentioned protein or the like as an antigen is not particularly limited, and a generally known immunization method can be used. By such a method, a serum containing the polyclonal antibody is obtained. Furthermore, ammonium sulfate fractionation and Protein A Sepharose
It is also possible to obtain a specific fraction such as IgG by purifying with the use of

Further, it is also possible to prepare a monoclonal antibody by using the cell fusion method.

Cell calcification inhibitory activity Cell calcification inhibitory activity by introducing the C-11 gene or c-erg gene according to the present invention refers to the calcification of osteoblasts infected with a viral vector into which the above gene has been cloned. It means that the inducibility is suppressed. Here, as a method for measuring the amount of calcified deposits induced by cells, specifically, it is preferable to use the Alizarin Red method. Further, it can be easily discriminated by the von Kossa (alum carmine method) method.

Pharmaceuticals and Means of Administering Pharmaceuticals The pharmaceuticals according to the present invention also include erg protein, erg gene, C-
11 proteins, c-erg protein, C-11 gene or c-erg gene, and thus various diseases based on the suppression of cell calcification by these, specifically ossification of spinal ligament It can treat diseases in which pathological calcification causes ossification, such as osteoarthritis (OPLL) and osteoarthritis.

The medicament provided by the present invention is the aforementioned erg
Protein, erg gene, C-11 protein, c-
It is a medicine containing erg protein, C-11 gene or c-erg gene. The means of administering these medicaments is
There is no particular limitation, and ordinary administration means can be used. Specific examples include local injection, transdermal injection, and oral administration. Further examples include microinjection into cells.

More specifically, by binding the c-erg or C-11 protein to an appropriate hormone or the like, or introducing the fused protein into a cell, c-erg or C-11 is added to the hormone receptor. Can be combined and taken into cells together.

[0047]

【Example】

(Example 1) Isolation of C-11 gene and c-erg gene The sternum was separated from an 18-day-old chicken embryo, and total RNA was extracted (Iwamoto-M et al., Microscopy Research and Tec).
hnique (1994) 28: 483-491). After reverse transcription of the obtained RNA, PCR to amplify the entire coding region of c-erg
Using primers (C11A and C11C or C11B and C11C) for 1 minute at 61 ° C., 2 minutes at 72 ° C., 10 minutes at 95 ° C.
When 30 cycles of amplification were performed under the condition of seconds, two bands were obtained using any of the primer pairs. Cut out each band and QiaexII gel ex
The DNA fragment was confirmed using a traction kit (Qiagen, Germany). After subcloning this into a PCRII vector (Invitrogen, CA, USA), the entire nucleotide sequence was determined by the Dideoxy method. As a result, the 1454 bp DNA fragment amplified with the primer pair C11A and C11C was c-erg (Me
chanism of Development (1995) 50, 17-28), but the nucleotide sequence of the 1373 bp DNA fragment was c-er
This sequence lacked 81 bases from positions 655 to 735 of g. The new sequence obtained was named C-11.

The DNA fragments amplified using the C11B and C11C primer pairs were of two types, 1446 bp and 1335 bp, the former being c-erg and the latter being C-erg.
-11. This result suggests that the obtained C-11 gene may be an isoform of the c-erg gene.

[0049] The above C-11 gene could be isolated using the usual subtraction method. Specifically, cDNAs specifically expressed in embryonic sternum were cloned by a generally known subtraction method using a chicken embryo sternum and fibroblast cDNA library. From the obtained candidate clones, the entire base sequence of the amplification product gene amplified by the PCR reaction using appropriate primers was determined by an ordinary method.

From the following (Example 2-1) to (Example 2-
8) shows the results obtained by using the above-obtained c-erg gene. Similarly, the results obtained by using the above-obtained C-11 gene are shown together with the results of c-erg. .

(Example 2-1) Preparation of vector having c-erg gene The cloned c-erg was placed at the ClaI site of an RCAS vector (see Journal of Virology, Oct. 1987, pp. 3004-3012). Subcloning was performed in the sense (c-erg) and antisense (AS-c-erg) directions. The above sense or antisense was confirmed based on the DNA sequence.

The RCAS vectors having the subcloned c-erg and AS-c-erg were respectively co-precipitated into chicken embryo fibroblasts by calcium phosphate co-precipitation (Che
n, Cand Okayama, H (1987), Mol. Cell Biol. 7,2745-
2752), and the transfected cells were treated with 10% calf embryo serum-containing DM.
48 at 37 ° C in EM medium (Nikken Institute of Biological Sciences, Kyoto)
Cultured for hours. Thereafter, the virus produced in the culture supernatant was concentrated by an ultrafiltration membrane having a molecular weight cut off of 30,000 (C
entriprep, Amicon, MA, USA) (hereinafter virus-C)
M).

In the same manner as described above, only the RCAS vector was introduced into chicken embryo fibroblasts, and the virus recovered from the introduced cells was used as a control.

The virus stock from the chicken embryo fibroblasts used in the present invention was prepared by the following method. That is, CEF is added to calcium phosphate by R
A DN containing a proviral structure having the LTR at both ends by substituting the v-src of SV (Rous Sarcoma Virus) with the target gene
A was transfected. Cells into which the gene has been introduced transiently release large amounts of virus. After that, transfection (transfec
to the culture used in the cultivation. At this time, the virus stock was recovered from the medium. More specifically, C grown to confluence
0.8 × 10 ⁶ cels of EF (cultured for 4-5 days from the time of preparation)
Dried ls in 60mm dishes. The next day, using the calcium phosphate coprecipitation method, 10 μg of DNA having a provirus structure
Fragments or plasmids are introduced by transfection. Medium was changed 1-2 hours before transfection. After 10 hours, the medium was washed three times with normal medium,
Propagation continued for 2 days. Transfected cultur
e The whole was subcultured to a 90 mm dish and continued to grow for another 2 days. The medium was replaced with fresh one, and the medium after 48 hours was collected as a virus stock. Add the medium again and add 1
Two hours later, a second viral stock was collected.

(Example 2-2) Introduction of c-erg Gene into Cells According to the method of Iwamoto et al., The c-erg gene was introduced into chicken osteoblasts, and the effect was observed (Iwamoto et al.
J. Biol. Chem., (1993) 268 (13): 9645-52)). The osteoblasts cut out 18-day-old parietal bones, and then, Louis.
Osteoblasts were separated and used according to a method such as CG (Dev
elopmental Biology, 122, p49-60 (1987)).

Virus-CM prepared in Example 2-1
(And the control virus) were added to the osteoblasts prepared above to effect virus infection.

The presence or absence of infection is confirmed by changes in the differentiation characteristics of the cells.
19 Antiviral antibody (Developmental Studies Hybridoma
(Potts.WM)
et al. J. Gen. Viol., 68, 3177-3182 (1982)).

Example 2-3 Morphological Observation of c-erg Gene Transformant The virus-infected osteoblasts obtained in Example 2-2 were observed using a phase contrast microscope (magnification: 10 ×). As a result, it was found that the virus-infected osteoblasts exhibited a small-sized polygonal morphology when compared to the control cells (not shown).

(Example 2-4) c-erg gene, C-
Confirmation of expression of 11 genes In order to confirm expression of the isoform C-11 gene, RT-PCR was performed according to the following method.

(Iwamoto et al., Microscopy Resear
ch and Technique 28, 483-491 (1994)), the total RNA of the thoracic spinal tissue of the 18-day chicken embryo was prepared. This R
Using NA, random hexamer and superscript reverse transcriptase (both Gibco-BRL, MD, USA)
After the conversion, the primer C11 having the base sequence of the ETA region (erg / ets transcription activation region) and the NRT region (transcription activation suppression region) of the c-erg protein gene shown in FIG.
A, C11C (SEQ ID NOS: 5 and 7 in the sequence listing, respectively)
Was used to perform a PCR amplification reaction. In FIG. 2, ETA
The C-11 gene has a deletion of 81 bases between the NRT domain and the NRT domain. The results of electrophoresis of the obtained amplification product on a 2% agarose gel are shown in FIG. As a result, two types of mRNA were amplified, and the c-erg gene (473 bp) and the isoform C-11 gene were 392 bp.
Appeared as a band.

(Example 2-5) Measurement of DNA synthesis ability The DNA synthesis ability of the c-erg transformant cells was measured as follows.

After washing the cells three times with cold physiological saline, the cells were collected with a physiological saline containing 0.01N NaOH and 0.2% (v / v) Triton X-100. After the collection, the cells were disrupted by ultrasonic waves and centrifuged, and the supernatant was used as a sample for measurement. Supernatant 1
Add 0.1 g / ml of DABA (3,5-diaminobenzoic acid di
After adding 200 μl of hydrochloride) and incubating at 65 ° C. for 45 minutes in the dark, the reaction was stopped by adding 300 μl of 2N HCl. After stopping the reaction, the fluorescence at 420 nm and 510 nm was measured.

Here, when Rcas (or RCAS) is infected with the vector alone, c-erg-L14 is
The RCAS into which the sense strand of c-erg was introduced,
-Erg-L44 means RCAS into which the antisense strand of c-erg was introduced. Furthermore, C11-L14
Means RCAS into which the C-11 sense strand has been introduced.

FIG. 4 shows that c-er introduced c-erg.
Both g-L14 and c-erg-L44 are c-erg
It was found that the DNA synthesis ability did not show a significant difference from that of RCAS not introduced with DNA. On the other hand, as shown in FIG.
C11-L14 introduced with -11 has a significant increase in DNA synthesis ability as compared to RCAS itself (particularly until day 6).
Was found.

Example 2-6 Measurement of Alkaline Phosphatase Activity Alkaline phosphatase activity was measured according to the method of Kato et al. (Kato. Y. et al. Endocrimology, 127, p114-118).
(1990)).

Specifically, the cells were washed three times on ice with cold physiological saline, and then recovered with physiological saline containing 0.2% (v / v) Triton X-100. After the collection, the cells were disrupted by ultrasonic waves and centrifuged, and the supernatant was used as a sample for measurement.

The alkaline phosphatase activity of the supernatant was 0.1%.
The measurement was performed in a 0.5 M Tris / HCl buffer (pH 9.0) containing 5 mM pNP (para-nitrophenyl phosphate) and 0.5 mM MgCl ₂ . The mixture was incubated at 37 ° C for 30 minutes and then 0.25 volume of 1N
The reaction was stopped by adding NaOH. After stopping the reaction, the absorbance at 410 nm was measured.

FIG. 6 shows the results obtained by using the RCAS into which c-erg was introduced. Control RCAS and c-erg-
Although there was no significant difference between L44 (introduced antisense strand), it was found that alkaline phosphatase activity was significantly suppressed in c-erg-L14 (introduced sense strand).

Similarly, C11-L1 introduced with C-11
The result in the case of No. 4 (sense strand) is shown in FIG. It was found that the alkaline phosphatase activity was significantly suppressed as compared with the control RCAS.

(Example 2-7) Alizarin Red staining Cultured osteoblasts (12 days after seeding) were washed with PBS (phosp
After washing twice with hate buffer saline), the cells were fixed with 100% ethanol. 1% Alizarin solution after fixing (Alizarin Red S in distilled water (sodium alizarin sulfonate, was dissolved to a Wako Pure Chemical) 1%, adjusted to at 0.1NH ₃ pH6.3~6.4) cells by adding 2 Stained for minutes. After dyeing, it was washed with distilled water and air-dried. By the above operation, the calcified portion is stained orange-red.

FIG. 8 shows the results of measuring the amount of calcified deposits of the osteoblast culture system on Alizarin Red 19 days after seeding. Uninfected cells and RCA
Cells infected with the S vector itself showed the same degree of calcification. On the other hand, c-erg-L14 (ce
rCAS sense-introduced RCAS) is c-erg-L44
(C-erg antisense strand-introduced RCAS), about half.

Further, FIG. 9 shows that in the RCAS into which C11-L14 was introduced, almost no calcified precipitate was observed as a result of measurement with Alizarin Red.

FIG. 10 shows the results of staining with Alizarin Red as a comparison of the amount of calcified deposits. RC
When compared under the condition where calcification is clearly seen in the infected cells of AS itself, c-erg-L44 shows very slight suppression of the amount of calcification vesicle deposition, but c-erg-L44
It can be seen that calcification deposits are almost completely suppressed in L14, and that it is almost completely suppressed in C11-L14.

(Example 2-8) von Kossa staining Cultured osteoblasts (12 days after seeding) were washed twice with PBS and fixed with 100% ethanol. Then add 1
% Silver nitrate aqueous solution was added and exposed to direct sunlight for 30 minutes. Next, the cells are washed with distilled water, a 5% aqueous solution of sodium thiosulfate is added, and the mixture is left for 5 to 10 minutes, and then washed with water. Nuclear staining was performed after washing with water, adding an alum carmine solution, and allowing to stand for 24 hours. By the above operation, the calcified portion is dyed black, and the nucleus is dyed red-red.

In the von Kossa staining, Alizarin Red
Similar to staining, the c-erg-14 group showed a decrease in staining properties as compared to the control group (RCAS), and the amount of calcified deposits was suppressed. Furthermore, it was completely suppressed in the C11-L14 group. Since there was no difference in the staining properties between alum carmines between the groups, this effect was not a difference in staining properties due to the difference in cell number, but c-erg-L14 and C11-L14 functionally calcified osteoblasts ( It can be seen that the amount of calcium deposition per cell) was suppressed.

[0076]

As described above, according to the present invention,
A novel isoform gene of c-erg gene, which is a chicken-derived erg gene (referred to as C-11 gene or C11 gene), was found in osteoblasts.
By introducing the gene, calcification of the cells can be suppressed.

Therefore, according to the present invention, a protein (referred to as C-11 protein or C11 protein) having the activity of inhibiting cell calcification, encoded by the above gene,
In addition, it is possible to provide a cell calcification inhibitor containing the protein. Further, according to the present invention, it is possible to measure the calcification of the cell by measuring the expression of the gene in the cell.

[0078]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1447 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: DNA sequence GAATTCCGCG AACGAATAAT TATTATTAGC AATTATTAGC GATCAATAAT CTTGATCACA TT 62 ATG GCA AGC ACT ATT AAG GAA GCA GTA TTA TCA GTG GTG AGT GAA GAC CAG 110 TCC TTG TTT GAG TGT GCC TAC GGA TCG CCC CAC CTT GCA AAG ACA GAA 158 ATG ACA GCC TCC TCT TCC AGT GAA TAT GGG CAA ACA TCA AAG ATG AGC 206 CCG CGC GTT CCC CAG CAG GGG TTA TCA CAG CCC CCG GCC AGA GTT 254 ACC ATT AAG ATG GAG TGT AAC CCA AAC CAG GTT AAT GGG TCA AGG AAT 302 TCA CCT GAT GAC TGC AGC GTG GCA AAA GGA GGG AAA ATG GTT AGC AGT 350 TCA GAC AAT GTT GGG ATG AAC TAT GGA AGC TAC ATG GAA GAG AAG CAT 398 ATT CCG CCT CCA AAT ATG ACA ACC AAT GAA CGA AGA GTT ATT GTG CCA 446 GCA GAT CCT ACG TTA TGG AGC ACA GAC CAT GTA CGG CAG TGG CTG GAG 494 TGG GCA GTG GAG GAG GGT CTT CCA GAC GTG GAC ATC TTG TTG TTC 542 CAG AAC ATT GAT GGG AAA GAG TTG TGT AAA ATG ACC AAA GAT GAC TTC 590 CAG AGA CTC ACG CCG AGC TAT AAC G CA GAT ATC CTC CTG TCA CAC CTA 638 CAC TAC CTC AGA GAG AGA GGA GCC ACT TTT ATT TTT CCA AAT ACA TCA 686 GTT TAC CCA GAA GCA ACG CAA AGA ATA ACA ACA AGG CCA GAT TTA CCT 734 TAT GAG CAA GCG AGA TGA GCG TGG ACG AGT CAC AGC CAT CCC ACT 782 CAG TCA AAA GCT ACC CAA CCA TCA TCT TCA ACA GTG CCC AAA ACA GAA 830 GAC CAG CGT CCT CAG TTA GAT CCT TAT CAG ATT CTT GGA CCG ACC AGC 878 AGC CGT CTT GCA ACA GGG AGT GGG CAG ATA CAG CTA TGG CAG TTC 926 CTA CTG GAG CTT CTG TCG GAC AGC TCC AAC TCC AAC TGC ATC ACC TGG 974 GAG GGC ACA AAT GGG GAG TTC AAG ATG ACA GAC CCT GAT GAA GTG GCT 1022 CGG CGT TGG AGG AAA AGC AAA CCT AAC ATG AAC TAT GAC AAA 1070 CTC AGC CGT GCA CTT CGC TAC TAC TAT GAC AAA AAT ATT ATG ACT AAA 1118 GTT CAT GGT AAA CGC TAT GCC TAC AAA TTT GAT TTC CAC GGA ATC GCT 1166 CAG GCC CTC CAG CCT CAC CCT CCA GAA TCA TCC ATG TAC AAA TAC CCA 1214 TCA GAC CTC CCC TAC ATG AGT TCC TAC CAT GCA CAC CCC CAG AAG ATG 1262 AAC TTT GTA GCT CCC CAT CCC CCT GCT TTG CCC GTA ACC TCA TCC AGC 1310 TTT TT T GCT GCC CCT AAT CCA TAC TGG AAT TCA CCA ACT GGA GGC ATC 1358 TAC CCC AAT ACC AGG CTG CCA GCT GCT CAT ATG CCT TCC CAT CTT GGC 1406 ACC TAC TAC TAA GTG GGGAAAGAAA GAAAGCGCCA AGAAAA 1447 Sequence number: 2 : 451 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Ala Ser Thr Ile Lys Glu Ala Leu Ser Val Val Ser Glu Asp Gln 16 Ser Leu Phe Glu Cys Ala Tyr Gly Ser Pro His Leu Ala Lys Thr Glu 32 Met Thr Ala Ser Ser Ser Ser Glu Tyr Gly Gln Thr Ser Lys Met Ser 48 Pro Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Val 64 Thr Ile Lys Met Glu Cys Asn Pro Asn Gln Val Asn Gly Ser Arg Asn 80 Ser Pro Asp Asp Cys Ser Val Ala Lys Gly Gly Lys Met Val Ser Ser 96 Ser Asp Asn Val Gly Met Asn Tyr Gly Ser Tyr Met Glu Glu Lys His 112 Ile Pro Pro Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro 128 Ala Asp Pro Thr Leu Trp Ser Thr Asp His Val Arg Gln Trp Leu Glu 144 Trp Ala Val Lys Glu Tyr Gly Leu Pro Asp Val Asp Ile Leu Leu Phe 160 Gln Asn Ile Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 176 Gln Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 192 His Tyr Leu Arg Glu Arg Gly Ala Thr Phe Ile Phe Pro Asn Thr Ser 208 Val Tyr Pro Glu Ala Thr Gln Arg Ile Thr Thr Arg Pro Asp Leu Pro 224 Tyr Glu Gln Ala Arg Arg Ser Ala Trp Thr Ser His Ser His Pro Thr 240 Gln Ser Lys Ala Thr Gln Pro Ser Ser Ser Thr Val Pro Lys Thr Glu 256 Asp Gln Arg Pro Gln Leu Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser 272 Ser Arg Leu Ala Asn Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe 288 Leu Leu Glu Leu Leu Ser Asp Ser Ser Asn Ser Asn Cys Ile Thr Trp 304 Glu Gly Thr Asn Gly Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala 320 Arg Arg Trp Gly Glu Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys 336 Leu Ser Arg Ala Leu Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys 352 Val His Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala 368 Gln Ala Leu Gln Pro His Pro Pro Glu Ser Ser Met Tyr Lys Tyr Pro 384 Ser Asp Leu Pro Tyr Met Ser Ser Tyr His Ala His Pro Gln Lys Met 400 Asn Phe Val Ala Pro His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser 416 Phe Phe Ala Ala Pro Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile 432 Tyr Pro Asn Thr Arg Leu Pro Ala Ala His Met Pro Ser His Leu Gly 448 Thr Tyr Tyr 451 SEQ ID NO: 3 Sequence length: 1528 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: DNA sequence GAATTCCGCG AACGAATAAT TATTATTAGC AATTATTAGC GATCAATAAT CTTGATCACA TT 62 ATG GCA AGC ACT ATT AAG GAA GCA TTA TCA GTG GTG AGT GAA GAC CAG 110 TCC TTG TTT GAG TGT GCC TAC GGA TCG CCC CAC CTT GCA AAG ACA GAA 158 ATG ACA GCC TCC TCT TCC AGT GAA TAT GGG CAA ACA TCA AAG ATG AGC 206 CCG CGC GTT CCC CAG CAG GAC TGG TTA TCA CAG CCC CCG GCC AGA GTT 254 ACC ATT AAG ATG GAG TGT AAC CCA AAC CAG GTT AAT GGG TCA AGG AAT 302 TCA CCT GAT GAC TGC AGC GTG GCA AAA GGA GGG AAA ATG GTT AGC 350 TCA GAC AAT GTT GGG ATG AAC TAT GGA AGC TAC ATG GAA GAG AAG CAT 398 ATT CCG CCT CCA AAT ATG ACA ACC AAT GAA CGA AGA GTT ATT GTG CCA 446 GCA GAT CCT ACG TTA TGG AGC ACA GAC CAT GTA CGG CAG TGG CTG GAG 494 TGG GCA GTG AAG GAG TAT GGT CTT CCA GAC GTG GAC ATC TTG TTG TTC 542 CAG AAC ATT GAT GGG AAA GAG TTG TGT AAA ATG ACC AAA GAT GAC TTC 590 CAG AGA CTC ACG CCG AGC TAT AAC GCA GAT ATC CTC CTG TCA CAC CTA 638 CAC TAC CTC AGA GAG ACT CCT CTT CCA CAT TTG ACT TCA GAT GAT GTT 686 GAT AAG GCC TTA CAA AAC TCT CCA CGG TTA ATG CAT GCA AGA AGA 734 GGA GGA GCC ACT TTT ATT TTT CCA AAT ACA TCA GTT TAC CCA GAA GCA 782 ACG CAA AGA ATA ACA ACA AGG CCA GAT TTA CCT TAT GAG CAA GCG AGG 830 AGA TCA GCG TGG ACG AGT CAC AGC CAT CCC GACT CAG TCA AAA ACC 878 CAA CCA TCA TCT TCA ACA GTG CCC AAA ACA GAA GAC CAG CGT CCT CAG 926 TTA GAT CCT TAT CAG ATT CTT GGA CCG ACC AGC AGC CGT CTT GCA AAT 974 CCA GGG AGT GGG CAG ATA CAG CTA TGG CAG TTC CTA G CTT CTG 1022 TCG GAC AGC TCC AAC TCC AAC TGC ATC ACC TGG GAG GGC ACA AAT GGG 1070 GAG TTC AAG ATG ACA GAC CCT GAT GAA GTG GCT CGG CGT TGG GGA GAG 1118 AGG AAA AGC AAA CCT AAC ATG AAC TAT GAC AAA CTC A GC CGT GCA CTT 1166 CGC TAC TAC TAT GAC AAA AAT ATT ATG ACT AAA GTT CAT GGT AAA CGC 1214 TAT GCC TAC AAA TTT GAT TTC CAC GGA ATC GCT CAG GCC CTC CAG CCT 1262 CAC CCT CCA GAA TCA TCC ATG TAC AAA TAC CCA TCA GAC CTC CCC TAC 1310 ATG AGT TCC TAC CAT GCA CAC CCC CAG AAG ATG AAC TTT GTA GCT CCC 1358 CAT CCC CCT GCT TTG CCC GTA ACC TCA TCC AGC TTT TTT GCT GCC CCT 1406 AAT CCA TAC TGG AAT TCA CCA ACTGA ATC TAC CCC AAT ACC AGG 1454 CTG CCA GCT GCT CAT ATG CCT TCC CAT CTT GGC ACC TAC TAC TAA GTG 1502 GGGAAAGAAA GAAAGCGCCA AGAAAA 1528 Sequence length: 478 Sequence type: amino acid Topology: linear sequence Type: Peptide sequence Met Ala Ser Thr Ile Lys Glu Ala Leu Ser Val Val Ser Glu Asp Gln 16 Ser Leu Phe Glu Cys Ala Tyr Gly Ser Pro His Leu Ala Lys Thr Glu 32 Met Thr Ala Ser Ser Ser Ser Glu Tyr Gly Gln Thr Ser Lys Met Ser 48 Pro Arg Val Pro Gln Gln Asp Trp Leu Ser Gln Pro Pro Ala Arg Val 64 Thr Ile Lys Met Glu Cys Asn Pro Asn Gln Val Asn Gly Ser Arg As n 80 Ser Pro Asp Asp Cys Ser Val Ala Lys Gly Gly Lys Met Val Ser Ser 96 Ser Asp Asn Val Gly Met Asn Tyr Gly Ser Tyr Met Glu Glu Lys His 112 Ile Pro Pro Pro Asn Met Thr Thr Asn Glu Arg Arg Val Ile Val Pro 128 Ala Asp Pro Thr Leu Trp Ser Thr Asp His Val Arg Gln Trp Leu Glu 144 Trp Ala Val Lys Glu Tyr Gly Leu Pro Asp Val Asp Ile Leu Leu Phe 160 Gln Asn Ile Asp Gly Lys Glu Leu Cys Lys Met Thr Lys Asp Asp Phe 176 Gln Arg Leu Thr Pro Ser Tyr Asn Ala Asp Ile Leu Leu Ser His Leu 192 His Tyr Leu Arg Glu Thr Pro Leu Pro His Leu Thr Ser Asp Asp Val 208 Asp Lys Ala Leu Gln Asn Ser Pro Arg Leu Met His Ala Arg Asn Thr 224 Gly Gly Ala Thr Phe Ile Phe Pro Asn Thr Ser Val Tyr Pro Glu Ala 240 Thr Gln Arg Ile Thr Thr Arg Pro Asp Leu Pro Tyr Glu Gln Ala Arg 256 Arg Ser Ala Trp Thr Ser His Ser His Pro Thr Gln Ser Lys Ala Thr 272 Gln Pro Ser Ser Ser Thr Val Val Pro Lys Thr Glu Asp Gln Arg Pro Gln 288 Leu Asp Pro Tyr Gln Ile Leu Gly Pro Thr Ser Ser Arg Leu Ala Asn 304 Pro Gly Ser Gly Gln Ile Gln Leu Trp Gln Phe Leu Leu Glu Leu Leu 320 Ser Asp Ser Ser Asn Ser Asn Cys Ile Thr Trp Glu Gly Thr Asn Gly 336 Glu Phe Lys Met Thr Asp Pro Asp Glu Val Ala Arg Arg Trp Gly Glu 352 Arg Lys Ser Lys Pro Asn Met Asn Tyr Asp Lys Leu Ser Arg Ala Leu 368 Arg Tyr Tyr Tyr Asp Lys Asn Ile Met Thr Lys Val His Pro Pro Glu 384 Ser Ser Met Tyr Lys Tyr Pro Ser Asp Leu Pro Tyr Met Ser Ser Tyr 400 His Gly Lys Arg Tyr Ala Tyr Lys Phe Asp Phe His Gly Ile Ala Gln 416 Ala Leu Gln Pro His Ala His Pro Gln Lys Met Asn Phe Val Ala Pro 432 His Pro Pro Ala Leu Pro Val Thr Ser Ser Ser Phe Phe Ala Ala Pro 448 Asn Pro Tyr Trp Asn Ser Pro Thr Gly Gly Ile Tyr Pro Asn Thr Arg 464 Leu Pro Ala Ala His Met Pro Ser His Leu Gly Thr Tyr Tyr 478 SEQ ID NO: 5 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type Sequence type: DNA sequence ATCTTGATCA CATTATGGCA AGC 23 SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type DNA sequence CACATTATGG CAAGCACTAT TAAGG 25 SEQ ID NO: 7 SEQ Length: 25 Type of sequence: number of a nucleic acid strand: single strand Topology: linear sequence type: DNA sequence CACTTAGTAG TAGGTGCCAA GATGG 25

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of c-erg cDNA and the deduced amino acid sequence of c-erg protein (the nucleotide sequence lacking the underlined part in the figure indicates that the C-11 gene according to the present invention is not present). Base sequence).

FIG. 2 shows the gene domain encoding the c-erg protein. Here, ETA is the erg / ets transcriptional activation domain, NRT is the transcriptional activity repression domain, and EDB is erg / etsDN.
The A-binding domain and also CTA indicate the carboxy-terminal transcriptional activation domain.

FIG. 3 shows RT-P obtained in (Example 2-4).
It is a photograph showing the result of electrophoresis showing a CR amplification product.
The 473 and 392 bp bands were c-erg and C-
Corresponds to 11.

FIG. 4 shows Rcas (osteoblasts infected only with RCAS), c-erg-L14 (osteoblasts infected with RCAS transfected with c-erg sense strand), and c-erg-
The result of having compared the DNA-synthesizing ability of L44 (osteoblast infected with the c-erg antisense strand introduction RCAS) is shown.

FIG. 5 shows Rcas (osteoblasts infected only with RCAS), C11-L14 (RC with the C-11 sense strand introduced).
2 shows the results of comparing the DNA synthesis ability of osteoblasts infected with AS).

FIG. 6 shows Rcas (osteoblasts infected only with RCAS), c-erg-L14 (osteoblasts infected with RCAS transfected with c-erg sense strand), and c-erg-
The result of having compared the alkaline phosphatase activity of L44 (osteoblast infected with the c-erg antisense strand introduction RCAS) is shown.

FIG. 7 shows Rcas (osteoblasts infected only with RCAS), C11-L14 (C-11 sense strand-introduced RC).
2 shows the results of comparison of alkaline phosphatase activity of AS-infected osteoblasts).

FIG. 8 shows osteoblasts without infection (upper half) and RC
It is a photograph which shows the result of alizarin red staining of the calcified cell deposit of the osteoblast (lower half) culture system infected only with AS.

FIG. 9 shows c-erg-L14 (sense strand) RC
Osteoblasts infected with AS (upper half) and c-erg-L
It is a photograph which shows the result of having measured the calcification cell deposition of the osteoblast (lower half) infected with 44 (antisense strand) RCAS by Alizarin red staining.

FIG. 10 shows osteoblasts infected with RCAS alone, osteoblasts infected with c-erg-L14 (sense strand) RCAS, and c-erg-L44 (antisense strand)
It is a photograph which shows the result of having measured the degree of calcification cell deposition of the osteoblast cell infected with RCAS and the osteoblast culture system infected with C11-L14 (sense strand) RCAS by alizarin red staining.

FIG. 11 shows osteoblasts infected only with RCAS, osteoblasts infected with c-erg-L14 (sense strand) RCAS, and c-erg-L44 (antisense strand).
It is a photograph showing the result of measuring the degree of calcified cell deposition on the nucleus of osteoblasts infected with RCAS and nuclei of osteoblasts infected with C11-L14 (sense strand) RCAS by von Kossa staining ( (Right column, left) and photographs showing the results of double staining of osteoblast nuclei with alum carmine (left column).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // A61K 38/00 C12P 21/02 C C12P 21/02 21/08 21/08 A61K 37/02 (72) Inventor Yoshinobu Higuchi 1-135, Komamon, Gotemba-shi, Shizuoka Chugai Pharmaceutical Co., Ltd. (72) Inventor Marizio Pacificiki United States, Pennsylvania, Philadelphia, Locust Street 4010, Levi Building, Pennsylvania University Dental Medicine School (72) Inventor Joel Rosenbloom, Pennsylvania, United States, Philadelphia, Locust Street 4010, Levi Building, Pennsylvania University Dental Medicine School ( 72) Inventor Helena E. United States, Pennsylvania, Philadelphia, Locust Street 4010, Levi Building, Pennsylvania University Dental Medicine School

Claims (19)

[Claims] 1. A C-11 protein of the following (a) or (b): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (b) a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in (a), and cell calcification A protein having inhibitory activity. 2. A gene encoding the protein according to claim 1. 3. A medicament comprising the protein according to claim 1. 4. The medicament according to claim 3, which is a cell calcification inhibitor. 5. A cell calcification inhibitor comprising the following (a) or (b) c-erg protein. (A) a protein comprising the amino acid sequence of SEQ ID NO: 4 in the sequence listing; (b) an amino acid sequence comprising the amino acid sequence of (a) wherein one or more amino acids are deleted, substituted or added, and cell calcification A protein having inhibitory activity. 6. An antibody against the C-11 protein according to claim 1. 7. The antibody according to claim 6, wherein said antibody is a monoclonal antibody. 8. The C-11 gene or c-erg in a cell
A method for measuring the calcification of the cells by measuring the expression of the gene. 9. The expression of C-11m expressed in said cells using a probe for a DNA sequence specific to said C-11 gene or c-erg gene.
The method according to claim 8, wherein the amount of RNA or c-erg mRNA is measured. 10. The method according to claim 8, wherein the expression of the gene is measured by measuring the expression level of the C-11 protein or c-erg protein in a cell. 11. The C-11 gene or c-gene in the cell.
A C-11 protein or c, which expresses the erg gene in the cells using the antibody according to claim 6 or 7.
The method according to claim 8, wherein the measurement is performed by measuring -erg protein. 12. A method for diagnosing osteoarthritis or ossification of the spinal ligament (OPLL) by measuring the calcification of cells using the method according to claim 8. 13. A C-11 protein or c-erg
A kit for measuring cell calcification, comprising an antibody against a protein or both. 14. Using a cell transformed with a gene encoding a protein according to any one of (a) to (d) below, a substance having an inhibitory activity on cell calcification inhibition How to screen. (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing; (b) a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in (a), and cell calcification A protein having an inhibitory activity, (c) a protein consisting of the amino acid sequence of SEQ ID NO: 4 in the sequence listing, (d) an amino acid sequence in which one or more amino acids are deleted, substituted or added in (c). And a protein having cell calcification inhibitory activity. 15. A medicament containing erg protein. 16. A medicine containing the erg gene. 17. A medicament containing a C-11 protein or a c-erg protein. 18. A medicament containing the C-11 gene or c-erg gene. 19. A medicine containing a protein having an amino acid sequence common to c-erg protein and C-11 protein.