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  • C12N15/09—Recombinant DNA-technology
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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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  • C12N2830/00—Vector systems having a special element relevant for transcription
• • C—CHEMISTRY; METALLURGY
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  • C12N2830/00—Vector systems having a special element relevant for transcription
  • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
  • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

• the present invention relates to a 5 ' upstream region DNA sequence which contains a promoter of human P52 gene. Furthermore, the present invention provides a method for exploring low molecular weight compound regulating positively or negatively the expression of human MP52 by using a mass of animal cells transfected with a recombinant expression vector harboring the 5 ' upstream region DNA containing the human MP52 gene promoter in front of a suitable reporter gene, and by using a reporter activity as an indicator.
• BMP bone morphogenetic protein
• TGF transforming growth factor
• Human MP52 is a BMP-like protein isolated by PCR cloning (Biochem. Biophys. Res. Comm. 204, 646-652, 1994). Recombinant human MP52 has been reported as effective not only in inducing ectopic ossification, but also in curing bone deficit in an animal model (Growth Factors 13, 65-74, 1996) .
• BMP proteins are effective in treating, and preventing various bone disorders and bone diseases.
• the BMP proteins exist in very small quantity in nature, and for an available large quantity for therapeutic use, manufacturing a recombinant protein is necessary.
• the present invention provides a method for exploring a substance which induces human MP52, a BMP-like protein.
• a useful substance obtained by this exploring method can be effective in inducing expression of human MP52, a bone formation factor. Since it has a small molecular weight and the same activity as that of human MP52, it can have a very useful application.
• the present invention provides a method for exploring a substance which inhibits expression of human MP52. In case that human MP52 relates to bone and cartilage hyperplasia, it can prevent hyperplasia by inhibiting the expression of human MP52.
• Fig. 1 is a 3.5 kb of 5 ' upstream region of human MP52 gene and a restriction enzyme map.
• Fig. 2 is a recombinant expression vector (pMP52L) containing a 5' upstream region of human MP52 gene.
• Fig. 3 is a result of measuring a human MP52 promoter activity (transient expression) .
• pGL3B means pGL3 -basic in the figure.
• the present invention relates to a DNA whose nucleotide sequence is represented by the base sequence from No. 1 to No. 3521 in SEQ ID NO.: 1 of the Sequence Listing that encodes a human MP52 gene promoter region, or a fragment thereof.
• SEQ ID NO.: 1 of the Sequence Listing shows a 5' upstream region sequence of the human MP52 gene.
• the present invention relates to a method for preparing the DNA shown in SEQ ID NO.: 1 of the Sequence Listing having the steps of:
• the plasmid vector used here is not restricted and any commercialized vector can be used.
• a preferable example is pUC18.
• the present invention relates to a recombinant expression vector characterized by integration of the full length or a part of DNA shown in SEQ ID NO.: 1 of the Sequence Listing into a reporter gene.
• the recombinant expression vector is constructed to locate a suitable region of 5' upstream region of the human MP-52 gene, that is represented by SEQ ID No.: 1 of the Sequence Listing, in front of a reporter gene.
• the reporter gene such as luciferase or ⁇ -galactosidase gene shows an expressing status on behalf of an original product.
• the basic vector for constructing the recombination expression vector is not specially restricted to allow to use a plasmid vector commercialized.
• the present invention uses pGL3 -basic as a preferable example.
• the use of pGL3 -basic yielded a pMP52L (8085 bp) that is a recombination vector containing the human MP52 promoter and a luciferase reporter gene.
• the present invention assigned it to a recombination expression vector. It is necessary to introduce the vector, to mammalian cells, preferably a human osteoblast-like cells, such as SaOS-2 cells, with a liposome.
• the animal cells stably transfected with the recombinant expression vector are selected by using a resistance marker.
• the present invention relates to an exploring method for a useful substance by using a recombinant expression vector characterized by integrating the full length or a part of DNA shown in SEQ ID NO. : 1 of the Sequence Listing into a reporter gene.
• the sequence from 2880 to 3521 in SEQ ID NO.: 1 of the Sequence Listing has been already reported (Biochem. Biophys. Res. Comm. , 204, 646-652, 1994).
• the longer sequence has been integrated in a recombinant expression vector, the more natural process of the exploring method can be used.
• the present invention relates to the method for exploring a useful substance which is a bone-related substance.
• the present invention relates to the method for exploring a useful substance which is an osteogenesis inducing substance or which is an osteogenesis inhibiting substance. Furthermore, the present invention relates to the method for exploring a useful substance which is a neurogenic substance or which is an angiogenic substance.
• a low molecular weight compound which induces or inhibits the expression of human MP52 can be obtained by isolating the promoter which regulates the expression of the gene, by ligating it to a suitable reporter gene and by introducing the gene structure into a suitable mammal cell to make an exploring system.
• the substance which regulates the expression of human MP52 in the exploring system works on the promoter to increase or decrease the expression level of the reporter gene. Therefore, a simple and easy measurement of the reporter activity makes an exploration of the aimed possible substance.
• the animal cell transfected with said vector can be used for a method for screening a chemical compound library by high throughput screening (Nature, 384, supp, 14-16, 1996) and finding an active substance from natural compounds.
• the substance which increases or decreases an activity is searched by treating the cell with a compound for an appropriate period of time and measuring a reporter activity.
• the compound obtained in such a way can regulate the expression by working directly on a transcription factor or indirectly on the promoter of human MP52 through regulating a signal transduction system. Therefore, these compounds are effective as a therapeutic agent for osteocartilaginous diseases, cancer metastasis to bone, or osteohyperplasia.
• these compounds are effective as a recovering agent for tendon and ligament injuries, a remedy for neural diseases, or an angiogenesis inducing or inhibiting agent.
• the substance obtained by the present invention has a bone or cartilage morphogenetic activity and is effective as an agent for therapeutic and preventive treatment in the field of orthopedic surgery (fracture, osteoarthritis such as joint osteoarthritis and hip joint osteoarthritis, arthrosteitis, damage of cartilage such as damage of meniscus, regeneration of bone and cartilage deficit caused by injury and tumor dissection, bone reconstruction such as spinal fusion and vertebral canal enlargement, and congenital cartilage and bone diseases such as dysosteogenesis and achondroplasia) , or dental fields (bone reconstruction such as palatoschisis, mandible reconstruction, and residual ridge construction), and osteoporosis.
• the substance of the present invention can be used for bone graft in aesthetic surgery. These therapeutic treatments are effective in treating the field of veterinary surgery.
• the present invention can provide a substance which inhibits bone or cartilage morphogenesis. In this case, the substance is applied as an agent to prevention and therapy of bone or cartilage hyperplasia.
• the substance which induces or inhibits tendon and ligament can be obtained according to the present invention. Therefore, the present invention provides a method for exploring medicinal drugs to treat damage of tendons and ligaments. Furthermore, since human MP52 has neural cells forming, inducing, and protecting effects, the compounds obtained from the present invention can be applied as therapeutic agents for neural diseases. In addition, it is possible to obtain a substance inducing or inhibiting angiogenesis. The compounds obtained can be used as remedy for injury, carcinostatic and metastasis inhibitor.
• low molecular weight compounds regulating positively or negatively the expression of human MP52 can be explored with reference to a reporter activity.
• Low molecular weight compounds and their derivatives have morphogenetic activity and inhibiting activity for bone and cartilage through the expression of human MP52 and are useful as preventive or therapeutic agents for cartilage and bone diseases, remedies for osteometastasis, or therapeutic and preventive agents for osteohyperplasia. Furthermore, these compounds are effective as repairing agent for injury of tendon and ligament, therapeutic agent for neural diseases, and inducing and inhibiting agent for angiogenesis.
• Example 1 Cloning of 5 ' upstream region of human MP52 gene The 40 cycles of PCR reaction was carried out by using the primer OD of SEQ ID No.: 2 of the Sequence Listing and the primer OID of SEQ ID No.: 3 of the Sequence Listing, cDNA derived from a human fetus (8-9 week old) as a template, and 1.5 unit Taq polymerase (provided from Perkin Elmer Cetus) . The following processes were repeated twice: the DNA amplified was treated with Sphl and AIwNI and then the PCR reaction was carried out 13 times.
• coli pMP52 was deposited in National Institute of Bioscience and Human- Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry 1-3, Higashi 1- chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan, in March 30, 1998 with depository No. FERM P-16734 and transferred to the International Depository Authority under Budapest Treaty on April 1, 1999 (Deposit No. FERM BP-6688) .
• Example 2 Determination of DNA sequence of 5' upstream region of human MP52 gene
• the pBluescript vector containing a human MP52 DNA of 8.8 kb was digested by Xbal , a remained large fragment (7.6 kb) was purified by agarose electrophoresis to subject to self-ligation by using a ligation kit provided from Takara Shuzo Ltd.
• the 3.5 kb sequence of the 5' terminal was determined by using a vector containing the upstream region of the human MP52 gene of 4.7 kb as a template and by a ALF automated DNA sequencer (proviced from Amersham Pharmacia Biotech Ltd.)(Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977) .
• the 5' upstream region of the human MP52 gene consisting of 3521 bases is presented in SEQ ID No.: 1 of the Sequence Listing. Sequencing was done at least three times. A site difficult to sequence was subcloned to sequence starting from both terminals. The base sequence from 2880 to 3521 shown in SEQ ID No.: 1 is already reported (Biochem. Biophys. Res. Comm. 204, 646-652, 1994).
• Example 3 Construction of a recombinant vector containing the human MP52 gene promoter and a luciferase reporter gene
• the restriction enzyme map of the 5 ' upstream region 3.5 kb of human MP52 gene is shown in Fig. 1.
• An Apahl -Bgll fragment (0.6 kb) of the 5' upstream region of human MP52 gene was purified and the ApaLI end was blunted. Then, this fragment was treated with HindiII and Bglll and inserted into a pGL3 -basic vector of which HindiII end was blunted.
• the vector obtained was treated with Bglll and Kpn restriction enzymes to ligate to the Bglll- pnl fragment (derived from pBluescript vector) (2.7 kb) of the 5' upstream region of the human MP52 gene.
• the recombinant expression vector pMP52L (8085 bp) finally obtained contains 3.3 kb of 5' upstream region of human MP52 gene.
• Fig. 2 shows the constructed recombinant expression vector.
• Example 4 Measurement of activity of the human MP52 promoter (introduction of the recombinant expression vector into human cells and transient expression)
• recombinant expression vector pMP52L of human MP52 promoter said vector was mixed with a vector pRL-SV40 (provided from of Pro Mega Ltd.) containing a sea pansy luciferase gene as an internal control for measurement of introducing efficiency of a gene in an equal quantity.
• cationic liposome lipofectamine provided from Lifetech Oriental Co. was mixed with said DNA solution to add to human osteosarcoma cells;
• said vector was mixed with a plasmid pPUR containing a gene resistant to puromycin in a proportion of 10 : 1, mixed with cationic liposome lipofectamine (provided from Lifetech Oriental Co.) and added to SaOS-2 cells for transfection.
• the cells into which the target gene has been introduced were selected from a culture medium containing puromycin (provided from Sigma Ltd.).
• the cells selected were inoculated in a 96-well plate, treated with substances of various chemical compound libraries for 1 - 3 days, dissolved with cytolytic agent (provided from Pro Mega Ltd.), and measured for enzyme activity by employing a luciferase assay kit (provided from Pro Mega Ltd.) .
• cytolytic agent provided from Pro Mega Ltd.
• luciferase assay kit provided from Pro Mega Ltd.

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