JP2000004882A - Human mp52 gene promotor and screening of useful substance using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a promotor which consists of DNA which has human MP52 gene promotor domain having a specific base sequence, and is useful, for example, for screening of compounds which have bone-forming activity at a level similar to human BMP protein or inhibiting activity against it. SOLUTION: This promotor is new DNA which has a human MP52 gene promotor domain which has a base sequence of no. 1-3251 in the base sequence shown by the formula, or fragments of it, and is useful, for example, for screening of low molecular compounds which control, positively or negatively, expression of human MP52 using, as an index, a reporter activity of an animal cell in which a recombinant expression vector which is linked to this DNA and an appropriate reporter gene were introduced. Obtained low molecular compounds are useful, for example, for the treatment, for example, of skeletal diseases, chondropathy, tendon lesion, ligament lesion, and neurological disorders, and as anticancer agents. This promotor is obtained by screening a human genome library using a probe which was prepared by PCR of human embryo cDNA using degenerate primer(s).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a 5 'upstream sequence containing the promoter of the human MP52 gene. Also,
The present invention uses a cell population in which a 5 ′ upstream gene containing a human MP52 gene promoter and a recombinant expression vector incorporating an appropriate reporter gene have been introduced into animal cells, and corrects the expression of human MP52 using the reporter activity as an index. Alternatively, a method for searching for a low-molecular compound that is negatively controlled is provided.

[0002]

2. Description of the Related Art There are known a number of substances which promote bone regeneration and can be used for healing bone defects, fractures and the like. One of them, bone morphogenic protein (hereinafter referred to as BMP) is T
A protein belonging to the GF (transforming growth factor) -β superfamily, which was originally found as an ectopic ossification inducer present in bone demineralized matter (Tr
ends in Biotechnol. 11, 379-383, 1993). BMP
Is known to exist from BMP-1 to BMP-14, among which BMP-2 to BMP-14 have been confirmed to have an osteoinductive effect.

[0003] Human MP52 is a BMP-like protein isolated by PCR cloning (Biochem. Biophys.
Res. Comm. 204, 646-652, 1994), recombinant human M
P52 has been shown to be effective not only in inducing ectopic ossification but also in bone defects in animal models (Growth Factors 1
3, 65-74, 1996). These BMP proteins are thought to be effective in the treatment and prevention of various bone disorders and diseases, but are present only in trace amounts in nature, and in order to obtain them in large quantities for use in treatment, recombinant proteins must be used. Need to produce.

[0004] In general, the production of recombinant proteins involves:
It costs much more than the production of small molecules.
In addition, due to the property of being a protein, there are restrictions on the physical properties and administration method as a pharmaceutical. In view of these points, it is expected that the presence of a low molecular weight organic compound having the same activity as the BMP protein would be a very attractive drug.

[0005] The present invention relates to a human MP, a BMP-like protein.
A method for searching for a substance that induces 52 is provided. The useful substance obtained by this search method is human bone
Since it has the effect of inducing the expression of P52, it has the same effect as human MP52 despite being a small molecule, and is considered to be extremely useful. The present invention also provides a method for searching for a substance that suppresses the expression of human MP52. If human MP52 is involved in bone / cartilage hyperplasia, suppressing this expression can prevent hyperplasia.

[0006] Furthermore, it has recently been suggested that mutant human MP52 may not only reduce its own activity but also abolish its activity by forming a dimer with other BMPs. (Nature Genetics 17, 58-64, 1
997). It is considered that the human MP52 expression inhibitor is also effective in alleviating chondrodysplasia caused by mutant human MP52.

Further, human MP52 has tendon / ligament inducing ability (Toshihiro Tashiro et al., American Society of Bone and Minera
l Research, F336, 1997), neurogenesis / induction / protection effects (J. Neurosci. Res. 42, 724-732, 1995), and angiogenic activity (Exp. Cell Res. 235, 218-226, 1997). )
It is also known that there is. Therefore, by the search method presented by the present invention, it is also possible to obtain a tendon / ligament-inducing or inhibiting substance, a nerve-inducing or inhibiting substance, or an angiogenesis-inducing or inhibiting substance.

Heretofore, as such a search method, there has been reported an example utilizing a mouse BMP-2 promoter (WO97).
/ 15308), and there is no example using the human MP52 gene promoter. Since all recombinant expression vectors in the search method presented by the present invention can be human-derived, the substances obtained by the search are effective in actual clinical practice.

[0009]

According to the present invention, there is provided a DNA sequence comprising a human MP52 gene promoter region having a nucleotide sequence from No. 1 to No. 3521 in SEQ ID NO: 1 in the Sequence Listing.
A, or fragments thereof. Sequence Listing SEQ ID NO: 1 shows the 5 'upstream sequence of the human MP52 gene. The present invention relates to a method for producing DNA of SEQ ID NO: 1 in the following order. (1) Preparation of a cDNA probe by PCR using degenerate primers, (2) Plaque hybridization screening of a human genomic phage library using the probe, and (3) Subcloning of a target fragment from the isolated phage. The plasmid vector used here is not limited, and a commercially available vector can be used. A preferred example is pUC18.

[0010] The present invention relates to the full length DNA of SEQ ID NO: 1,
Alternatively, the present invention relates to a recombinant expression vector characterized in that a part thereof is incorporated into a reporter gene. Specifically, a recombinant expression vector is constructed in which an appropriate region of the 5 'upstream gene of the human MP52 gene shown in SEQ ID NO: 1 is arranged upstream of the reporter gene. The reporter gene indicates the expression status instead of the original product, and includes, for example, a luciferase gene and a β-galactosidase gene. There is no particular limitation on the vector on which the recombinant expression vector is constructed, and a commercially available plasmid vector can be used. In the present invention, pGL3-basic is used as a preferred example. pGL3
-Basic, recombinant vector pMP containing human MP52 promoter and luciferase reporter gene
52 L (8085 bp) was obtained. In the present invention, this was used as a recombinant expression vector. This vector is transformed into a mammalian cell, preferably a human osteoblast-like cell,
For example, it needs to be introduced into SaOS-2 cells. Animal cells into which the recombinant expression vector has been introduced are selected so as to be stably expressed using a resistance marker or the like.

[0011] The present invention relates to a method for searching for a useful substance using a recombinant expression vector, wherein the full length or a part of the DNA of SEQ ID NO: 1 is incorporated into a reporter gene. 28 in Sequence Listing SEQ ID NO: 1
The nucleotide sequence from 80 to 3521 has already been reported (Bioche
m. Biophys. Res. Comm. 204, 646-652, 1994). The use of a recombinant expression vector incorporating a longer sequence enables a search method that is more natural.
The present invention relates to a search method in which the useful substance obtained by the search method is a bone-related substance. The present invention particularly relates to a search method in which the bone-related substance is an osteogenesis-inducing substance or an osteogenesis-inhibiting substance. Furthermore, the present invention relates to a search method in which the useful substance is a neurogenic substance or an angiogenic substance. Human MP
To obtain a low molecular compound that induces or suppresses the expression of 52, a promoter regulating this gene expression is isolated, ligated to an appropriate reporter gene, and the resulting construct is introduced into an appropriate mammalian cell. A method using a test system is conceivable. In this assay system, the substance that regulates the expression of human MP52 acts on the promoter to increase or decrease the expression of the reporter gene, so that the target substance can be searched for by simple and convenient measurement of reporter activity. .

Animal cells into which the above-described vector has been transfected can be subjected to high-throughput screening (high through p
ut screening, Nature, 384, supp, 14-16, 1996), etc., and can be used as a method for searching for active substances from natural products. The cells are treated with the compound for an appropriate time, the reporter activity is measured, and a substance that increases or decreases the activity is searched for. The expression of the compound thus obtained can be regulated, for example, by acting directly on the transcription factor or indirectly by participating in the signal transduction system to act on the promoter of human MP52. Therefore, these compounds are effective as a preventive / therapeutic agent for bone / cartilage disease, a bone metastasis inhibitor, or a preventive / therapeutic agent for hyperostosis. Alternatively, these compounds are also effective as agents for repairing damage to tendons and ligaments, for treating neurological diseases, and for inducing or inhibiting angiogenesis.

Since the substance obtained in the present invention has bone or cartilage inducing activity, it can be used in the field of orthopedic surgery (osteoarthritis such as fractures, knee osteoarthritis, osteoarthritis, osteoarthritis, meniscal injury, etc.). Bone and cartilage defects due to cartilage damage, trauma, tumor removal, bone reconstruction such as spinal fusion, spinal canal enlargement, congenital cartilage such as osteogenesis imperfecta, achondroplasia, and bone diseases) Alternatively, it is effective as a prophylactic and therapeutic agent for dentistry (bone reconstruction such as cleft palate, mandibular reconstruction, alveolar plasty) and osteoporosis. Further, the substance of the present invention can be used for treating bone transplantation in cosmetic surgery. These treatments are effective as treatments in the field of veterinary surgery. On the other hand, it is also possible to obtain a bone or cartilage induction inhibitor according to the present invention. In this case, it is applied as a prophylactic / therapeutic agent for bone / chondrodysplasia.

Further, according to the present invention, a substance for inducing or inhibiting tendons and ligaments can be obtained. Therefore, the present invention also provides a method for searching for a therapeutic agent for tendon / ligament damage. Furthermore, human MP52
Has also neurogenic, inducing and protective activities, and thus the compounds obtained according to the present invention can also be applied as therapeutic agents for neurological diseases. Furthermore, an angiogenesis inducer or inhibitor can be obtained. The obtained compound can be applied as a therapeutic agent for damage, an anticancer agent, or a metastasis inhibitor.

[0015]

EXAMPLES Next, the effects of the present invention will be specifically described with reference to examples. Note that the present invention is not limited by these examples. Example 1 Cloning of 5 'upstream gene of human MP52 Primer OD of SEQ ID NO: 2 and SEQ ID NO: 3 of Sequence Listing
And PCR was performed for 40 cycles using 1.5 units of Taq polymerase (manufactured by PerkinElmer Cetus) using cDNA of human fetus (8-9 weeks old) as a template. Sp. Amplified DNA
After the treatment with hI and AlwNI, the process of further performing the PCR reaction 13 times was repeated twice. Finally EcoR
After treatment with I, the PCR product was ligated with pBluescript SK (Stratadyne). Using the cloned sequence containing the MP52 cDNA sequence, a human genomic phage library (Stratadin) was screened by plaque hybridization. The obtained 20 kb DNA was subcloned into a pBluescript SK vector, and unnecessary portions were cut and removed to obtain an 8.8 kb human MP52 gene D.
A pBluescript vector containing NA was obtained. This vector was named E. coli pMP52. E. coli pMP52 was deposited on March 30, 1998 with the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry under the deposit number FERM P-16734.

EXAMPLE 2 Gene sequencing of human MP52 gene DNA pBlues containing 8.8 kb of human MP52 gene DNA
After digesting the cript vector with XbaI, the remaining larger (7.6 kb) fragment was purified by agarose electrophoresis and self-ligated using a ligation kit manufactured by Takara Shuzo. This 4.7 kb human MP5
Using a vector containing the upstream portion of two genes as a template, AL
F The sequence of 3.5 kb at the 5 'end was determined using an automatic DNA sequence reader (Pharmacia Biotech) (Proc. Natl. Acad. Sci. USA 74, 5463-5467,
1977). The 5 'upstream of the human MP52 gene consisting of 3521 bases is shown in SEQ ID NO: 1 in the Sequence Listing. Read at least 3
The sequence was read from both sides after subcloning the site where reading was difficult. In this SEQ ID NO: 1, the nucleotide sequence from 2880 to 3521 has been reported (Biochem. Biophys. Res. Comm. 204, 646-652, 199).
4) thing.

Example 3 Construction of Recombinant Expression Vector Containing Human MP52 Gene Promoter and Luciferase Reporter Gene FIG. 1 shows a restriction enzyme diagram of 3.5 kb 5 'upstream region of human MP52. ApaLI in the 5 'upstream region of human MP52
The -BglII fragment (0.6 kb) was purified and the ApalI side was blunted. Then, this fragment was first converted to Hind II
The plasmid was treated with I and BglII, and inserted into a pGL3-basic vector in which the HindIII side was blunted. The resulting vector was treated with BglII and KpnI restriction enzymes to obtain MP52.
5 'region was ligated to a BglII-KpnI (derived from pBluescript vector) fragment (2.7 kb). The finally obtained recombinant expression vector pMP52L (8085 bp)
Contains 3.3 kb of the human MP525 5 'upstream region. FIG. 2 shows a schematic diagram of the constructed recombinant expression vector.

Example 4 Measurement of Human MP52 Promoter Activity (Transformation of Recombinant Expression Vector into Human Cells and Transient Expression) In order to transiently express the human MP52 recombinant expression vector pMP52L, the above vector and gene were used. An equal amount of a vector pRL-SV40 (Promega) containing a seapan luciferase gene as an internal control for measuring the transfection efficiency was mixed. Further, the DNA lysate was mixed with the cationic liposome Lipofectamine (manufactured by Lifetech Oriental) and the mixture was mixed with human osteosarcoma cells HOS, MG63, and SaOS.
-2 and transfected. Firefly luciferase activity and sea pansy luciferase activity are measured by Picka Gene Dual Kit (Toyo Ink Products)
Was measured by The result is shown in FIG. Promoter activity was expressed as the relative ratio of firefly luciferase activity to sea pansy luciferase activity. From this result, it was found that the DNA of SEQ ID NO: 1 of the present invention has promoter activity.

Example 5 Establishment of cells stably transfected with a human MP52 recombinant expression vector and utilization as an active compound search system In order to stably express a human MP52 recombinant expression vector (pMP52L), a plasmid containing a puromycin resistance gene was used. The mixture was mixed with pPUR at a ratio of 10: 1, and the mixture was mixed with cationic liposome Lipofectamine.
(Lifetech Oriental) and mixed with SaO
Added to S-2 cells and transfected. Cells into which the target gene was introduced were selected in a medium containing puromycin (a product of Sigma).

Example 6 Screening for Active Low-Molecular Compounds Selected cells were seeded on a 96-well plate, treated with substances in various compound libraries for 1-3 days, and then the cells were lysed with a cell lysing agent (Promega). And the enzyme activity was measured using a luciferase assay kit (promega). In this manner, various substances that promote or suppress the expression of human MP52 can be searched.

[0021]

EFFECTS OF THE INVENTION Using animal cells into which a 5 'upstream gene containing a human MP52 gene promoter and a recombinant expression vector incorporated into an appropriate reporter gene have been introduced, the expression of human MP52 is positive or negative using the reporter activity as an index. It is possible to search for low-molecular compounds that are negatively controlled. These low molecular weight compounds and derivatives thereof have an activity of inducing or inhibiting bone / cartilage through expression of human MP52,
It is useful as a prophylactic / therapeutic agent for bone and cartilage diseases, a therapeutic agent for bone metastasis or a prophylactic / therapeutic agent for hyperostosis. Further, these compounds are also effective as agents for repairing damage to tendons and ligaments, agents for treating neurological diseases, and agents for inducing or inhibiting angiogenesis.

[0022]

[Sequence list] SEQ ID NO: 1 Sequence length: 3521 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA Origin: Biological name: Human (Homo sapiens) Tissue type : Whole blood Sequence characteristics: 5 'upstream gene sequence of human MP52. The ATG at the translation start is located at the end of the sequence. SEQ: TCGACTCGAT CACTTTATGC AATTTAATTT TATTTATTTT TTTAGTAGAG ACAGGGTTTC 60 ACCATGTTAG CCAGGATGGT CTTGATCTCC TGACCTCGTG ATCCACCTGC CTCGGCCTCC 120 CAAAGTGCTG GGATTACAGG CGTGAGCACT GCGCCTGGCC GCAATTTACT TCATTGAATC 180 TCCAACAAGA GTCCTGTGAG GTAAGCACTA TCGTTATCAC TGTTTAGAGT TTCAGAGGGG 240 TTTAGAGGCT TCCCAAGATC ACACAATACA TAAATAGCAG AACCAAGCTT CAAAACCAGG 300 TCTGTTTGTC TCCAGAATCC TTGCTCTTTA TCAAGCCACG TACAGAGACA TTGTGGTTGT 360 TCAACTCATT CATTTATTCA CTCTCTGAGC TTACAAAATG CTTAAGAAGT GGCAAGACAA 420 TTCTTCCCTT CAAGAAACTT AGAGTCTAAT GGGAAAGGCA GGTTATGTCC ACAAATAACT 480 ACACCTCAAA GTAGAAAATG ATGATTTTTG TCAATAAGGG CCAGTTAGAG ATTAATTTAT 540 TCATATAACA CGTACTGAGA GCTGCTGTGG GCTGGGCCTC TGCCAAGCAC TGGGTATAAT 600 TCAAAGATAA ATATGGCACA GTCTAGTCAT AATCTAATGG GAGAGACAGG TATGTAAAGA 660 AATTATTATA GTTATAAGGA ATTAAGTTAT GATTTTAAAA TATGACAAAA TACAGAGTGG 720 AAGAAACAAA CTGTGTTGGG ATAGGGGGCA GAGGCAGAGA CTGAATAAAC TTTCAGCTGC 780 CAAAAAATGT AAAAGAGCAC AGAAAATGTG TGATGGCAGC TCAGGGGGAG TGTGCATTTT 840 CTGGAGGATG CAAGAAGGCT TCATGGAGGA GGTGGCACGT GGGTTGGCAC TTGTTTGATG 900 GGTAGCTTTT AGTAGAGGGT TGAACCAGAG AGAGCCTGGA GGCCTAGAAG CCAGTTATAG 960 GGGAGAATCA TAGCGAGGCA AGCAATGAGG AGGACAGGGC AGTGACAGAG GTGGTGAGGA 1020 GGGAGTGAAG GGAGGGATGC CAGAGAGGAC TGGTTCAGTG GGAACAAGTC TGTTTTTAAT 1080 TTTTTTATTT TATTAATATT ATTTATTTAC TTATTTTGTG TGTGTGTGAT GGACTCTAGC 1140 TCTGTCACCT AGGCTGGHGT GCAGTGGTGC GATCTCGGCT CACTGCAACC TTTGCCTCCA 1200 CACCCGGCTA ATTTTTTGTA TTTTTGGTAG AGATGCCTGC AATCCCAGCA CTTTGAGAGG 1260 CCAAGGTGGG TGGATCACTT GAGGTCAGGA GCTCGAGACC AGACTGGCCA ACATGGTGAA 1320 ACTCCGTCTC TACTAAAAAT ACAAAAAATA GATAGGCATG GTGGTGTGCA CCTGTAGTCC 1380 CAGCTACTCG AGAGGCTGAG GCAGGAGAAT CGTTTGAACC CAGGAGGTGG AGATTACAGT 1440 GAGCCAAGAT CGCACCACTG CACTCCAGCC TAGGTGACAG AGTGAGACTC TGTCTCAAAA 1500 AAAAAAAAAA AAAAAAAAAA AAAGAAGGAA GCCTATCTGA CTATTGCTTC TCCCCCGCCA 1560 TCCTTCTTAC AGCGTGAAAA GTGTTGTTGT AGAGAAATGT ACAGAAGGGG TAACTGGCTC 1620 TGTCTGGGGA CGAGGAAAGG CTTCCCGAAG GAGGTAAATT TTCAGCTAAA CTTCTCAGGA 1680 TGTAGAGTGC TTCCTCCA GT TGTGGGAGAG AGAGAGGAGG ATATTTCAGG TGAAGCGAAC 1740 AGCTTTCCCC GCTGACCCTG TGTTAAGTTG CCCAGGGGCT CTACGGCGAA GGTTCCCCAG 1800 AGGAAGGGAT CTTCCATACT TCAATGAGGC ACTTTAAGAA ACCCTTGAGT TCAGCTGGAA 1860 TTCAGACCTT CCAGAGCTAC CAGAAAAACA TCATGTGGGA AATTGTGCCC GGCGCGGTGT 1920 CACGCCTGTA ATCCCAGCAC TTTGGGAGGC CGAGGCGGGC GGATCACGAG GTCAGGAGAT 1980 CGAGACCAAC CTGGCTAACA TGGTGAAACC CCATCTCTAC TACAAACGTA AAAAATCAGC 2040 CAGGTGTGGT GGCAGGGGCC TGTAGTCCCA GCTACTGGGG AGGCTGAGGC AGGGGAATGG 2100 CGTGAACCTG GGAGGCAGAG ATTGCAGTGA GCTGAGATCA TGCCACTGCA CTCGACAGAG 2160 GCGAACTCCG TCTCAAAAAA AAAAAAAAAA AAAGCGTCAT GTGGGAAATG GCTTTTCCAG 2220 CCTCCTGTAG GGGCCGCTGC TGCCCCAGAC TCAGCCAGTC TTGTCTAAGA AAACTCAGAG 2280 GACGTCTCTG CTGGGGTGGT GGTGGGGTAC ACCCTGGACC TGCCGCCATC CAAGGAGTGA 2340 ACTTAAGGGC GACAGTGCCC CCAAGCCTGA AGAATATGCA CTCAGTCAAT GGCATTTGGG 2400 GGTAGGGAGG GGGTGTAGTG GGCACTGATA TTTTTCAGTC TCTGGGTCAC CACAAGTTTA 2460 TTAAAATAAA AGAAAATGTG GGTAAATGCT GCCATCTGTG CTGTCCCTAC TCCCAATACA 2520 CACACACAGG CACACACACA CAC AGGCACA CACACACACA GGCACACATA TACACACACA 2580 CACACACACA CACACACACA CACACACACA CACACACACG CATAACAAAT CAGGTCCCAG 2640 ACTAGATGCA GGAGTTGAAA CTGCTTTAAA ACACTCTAAG AACTTAACTA CAAACCCACA 2700 CTCCCCATGC TTCTGGGGCT TAAGATCTTT GAGCTCATTC TTCAGCATCT CTCCACGAGA 2760 AAGTGGGGTG GGCTGCTCCA TGAGGTGGAG GTGAAGACCC CTGAGTCTGC CCCGTGGAGG 2820 GGGAGGCCCC CTAAGCCTAG AGTCCCGCTG CAGGGCTCTG TGCCAGGAGC CCCCGTGAGC 2880 CATGGCCTCG AAAGGGCAGC GGTGATTTTT TTCACATAAA TATATCGCAC TTAAATGAGT 2940 TTAGACAGCA TGACATCAGA GAGTAATTAA ATTGGTTTGG GTTGGAATTC CGTTTCCAAT 3000 TCCTGAGTTC AGGTTTGTAA AAGATTTTTC TGAGCACCTG CAGGCCTGTG AGTGTGTGTG 3060 TGTGTGTGTG TGTGTGTGTG TGTGTGTGAA GTATTTTCAC TGGAAAGGAT TCAAAACTAG 3120 GGGGAAAAAA AAACTGGAGC ACACAGGCAG CATTACGCCA TTCTTCCTTC TTGGAAAAAT 3180 CCCTCAGCCT TATACAAGCC TCCTTCAAGC CCTCAGTCAG TTGTGCAGGA GAAAGGGGGC 3240 GGTTGGCTTT CTCCTTTCAA GAACGAGTTA TTTTCAGCTG CTGACTGGAG ACGGTGCACG 3300 TCTGGATACG AGAGCATTTC CACTATGGGA CTGGATACAA ACACACACCC GGCAGACTTC 3360 AAGAGTCTCA GACTGAGGAG AAAGCCTTT C CTTCTGCTGC TACTGCTGCT GCCGCTGCTT 3420 TTGAAAGTCC ACTCCTTTCA TGGTTTTTCC TGCCAAACCA GAGGCACCTT TGCTGCTGCC 3480 GCTGTTCTCT TTGGTGTCAT TCAGCGGCTG GCCAGAGGAT G 3521

SEQ ID NO: 2 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Origin: None Organism name: None Tissue type: None Sequence Features: forward PCR primer OD corresponding to human MP52 cDNA sequence. Sequence: ATGAATTCCC ATGGACCTGG GCTGGMAKGA MTGGAT 36

SEQ ID NO: 3 Sequence length: 29 Sequence type: Nucleic acid number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Origin: None Organism name: None Tissue type: None Sequence Characteristic: Reverse PCR primer OID corresponding to human MP52 cDNA sequence. Sequence: ATGAATTCGA GCTGCGYSGG SRCACAGCA 29

[Brief description of the drawings]

FIG. 1 is a restriction map of 3.5 kb 5 ′ upstream gene of human MP52.

FIG. 2 shows a recombinant expression vector (pMP52L) containing a 5 ′ upstream gene of human MP52. The one obtained by inserting the 1st to 3227th base (Apal position 1) of SEQ ID NO: 1 into the restriction enzyme sites HindII and KpnI of pGL3-basic.

FIG. 3 shows the results of measurement (transient expression) of human MP52 promoter activity. In the figure, pGL3B is pGL3-
Basic.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) (72) Inventor Gerd Lud Hetten Germany Day-69245 Bamentar. Veinweisenweg 17 F term (reference) 4B024 AA01 AA11 BA21 BA80 CA01 CA04 CA07 CA09 CA20 DA03 EA03 EA04 FA01 FA02 FA10 GA11 GA13 HA11 4B063 QA01 QA05 QQ08 QQ10 QQ22 QQ42 QR32 QR33 QR52 QR56 QR60 QS32 QS80 4B065 AA93X AA93Y AB01 AC14 BA02 BA25 BB37 CA24 CA44 CA46 4C084 AA17 ZA012 ZA362 ZA962 ZB262

Claims (11)

[Claims] 1. The sequence listing SEQ ID NO: 1 from 1 to 3
DNA containing a human MP52 gene promoter region having a nucleotide sequence of up to 521, or a fragment thereof. 2. In the sequence listing SEQ ID NO.
DNA containing a human MP52 gene promoter region having a nucleotide sequence up to position 879, or a fragment thereof. 3. A method for producing DNA of SEQ ID NO: 1 in the sequence listing in the following order. (1) Preparation of a cDNA probe by PCR using degenerate primers; (2) Plaque hybridization screening of a human genomic phage library using the probe; and (3) Subcloning of a target fragment from an isolated phage. 4. A recombinant expression vector wherein the full length or a part of the DNA of SEQ ID NO: 1 is incorporated into a reporter gene. 5. A method for searching for a useful substance, which comprises using the recombinant expression vector according to claim 4. 6. The search method according to claim 5, wherein the useful substance is a bone-related substance. 7. The method according to claim 6, wherein the bone-related substance is an osteogenesis-inducing substance. 8. The method according to claim 6, wherein the bone-related substance is a bone formation inhibitor. 9. The useful substance is a neurogenic substance.
Search method. 10. The method according to claim 5, wherein the useful substance is an angiogenic substance. 11. The method according to claim 5, wherein the useful substance is an angiogenesis inhibitor.