JP2000032987A - Dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new DNA which has a specific base sequence, consists of a DNA having a strong promotor activity in a specific organ or tissue, has a high transcription efficiency and an excellent gene expression ability, and allows mass and economic production of a peptide. SOLUTION: This is a new DNA, shown by the formula, which has a strong promotor activity in a specific organ or tissue such as brain and nerve cell, is effective for the treatment of genetic diseases caused by abnormal production of this DNA and of tumors and cancers, is expected to be effective in the gene therapy, has an excellent gene expression ability, and is useful for mass and economic production of a peptide. This DNA is obtained by extracting chromosomal DNA from mouse embryonic stem cells, sonicating the obtained DNA to fragmentate, followed by the treatment of the obtained DNA fragments with Klenow fragment DNA polymerase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gene having a high transcription efficiency (having a strong gene expression ability) for gene expression,
The present invention also relates to a DNA that selectively exhibits promoter activity in a specific organ or tissue.

[0002]

2. Description of the Related Art A promoter is known as a specific base sequence on DNA which starts transcription of mRNA using DNA as a template. In addition, RN which is an mRNA synthase
A polymerase recognizes a promoter and synthesizes mRNA. The promoter determines the efficiency of transcription and the mRNA of the gene downstream of the strong promoter
Is synthesized in large quantities, and as a result, gene products of the gene are also produced in large quantities.

[0003] Therefore, utilizing a promoter having a strong activity is suitable for producing peptides in a large amount and economically by a genetic engineering technique. Furthermore, by using a promoter that is selectively active in a specific organ or tissue, a peptide can be produced in a specific organ or tissue, which is economical.

[0004] In addition, a promoter that selectively exhibits activity in a specific organ or tissue is a gene in which no physiologically active substance is produced in a specific organ or tissue at all, or even if the bioactive substance is produced, the production is abnormally low. Gene therapy is expected to be effective because it can be effectively used for treating diseases, tumors, cancers and the like.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a DNA having a strong promoter activity selectively in a specific organ or tissue, which is useful for gene therapy in a large amount and economically producing a peptide. It is to provide.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a certain DNA selectively has a strong promoter activity in a specific organ or tissue.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a DNA represented by SEQ ID NO: 1 in the Sequence Listing. Hereinafter, the present invention will be described.

The DNA represented by SEQ ID NO: 1 in the sequence listing of the present invention has been isolated from the chromosome of mouse embryonic stem cells by genetic engineering. The present inventors have found that this DNA selectively has promoter activity in nerve cells and even in brain. Search for a DNA having a promoter activity is performed by a method of analyzing an appropriate region of a structural gene, a method using a transcription factor-binding DNA fragment as a probe, or a method of randomly analyzing a DNA fragment.

Generally, when a new DNA is found,
Those skilled in the art search for hybridizing DNA using various libraries, using a part or all of the DNA as a probe. DN represented by SEQ ID NO: 1 in the sequence listing
DNA that hybridizes to A or a portion thereof is also expected to have promoter activity.

Accordingly, the DNA of the present invention includes not only the DNA represented by SEQ ID NO: 1 in the sequence listing but also a part thereof, a DNA hybridizing to the DNA represented by SEQ ID NO: 1 in the sequence listing and a DNA hybridizing thereto. Some are included. In particular, those having promoter activity are included in the present invention.

[0011] The DNA to be hybridized is a DNA represented by SEQ ID NO: 1 in the sequence listing or a part thereof, and used as a probe.
-, second edition, Cold Spring Harbar Laboratory Pr
Under general conditions as described in ess 1989,
A substance that hybridizes. The conditions for hybridization may be appropriately examined. For example, 0.75M NaC
1, 50 mM Tris-HCl buffer (pH 8.0),
0.2 mM ethylenediaminetetraacetic acid (EDTA),
0.2% bovine serum albumin, 0.2% polyvinylpyrrolidone, 0.2% Ficoll 400 (Pharmacia), 0.1% sodium lauryl sulfate (SDS), 5%
Conditions such as 0 μg / ml denatured salmon sperm DNA and a reaction at 65 ° C. for 16 hours can be mentioned, but the conditions are not particularly limited. Under these conditions, DNA having about 70% sequence identity with the probe hybridizes. Further, when it is desired to obtain a DNA having a sequence identity of 70% or more with the probe, it can be obtained by washing after hybridization at 65 ° C. while lowering the salt concentration.

In the present invention, the hybridizing DNA can also be expressed as a derivative of the DNA represented by SEQ ID NO: 1 in the sequence listing. That is, it has at least about 70% sequence identity to the DNA represented by SEQ ID NO: 1 in the sequence listing.
One or more nucleotides inserted into the DNA sequence, one or more nucleotides added to the DNA sequence, one or more nucleotides missing from the DNA sequence Examples include those that have been lost, those that have one or more nucleotides replaced in the DNA sequence, and those that have a combination thereof. In the present invention, as the DNA hybridizing to the DNA represented by SEQ ID NO: 1 in the sequence listing, 8
Preferably, it has 0% or more sequence identity, and more preferably 90% or more sequence identity.

The DNA of the present invention can also be produced by synthesizing by a known method.

Producing peptides in large quantities and economically;
Alternatively, to perform gene therapy, the DNA of the present invention
It is necessary to construct a recombinant DNA in which the structural gene encoding the peptide to be expressed is linked in the direction in which the structural gene is correctly transcribed.

[0015] To the recombinant DNA, a transcriptional regulatory region such as an enhancer or a terminator can be further linked to an appropriate site upstream or downstream of the DNA of the present invention. The recombinant DNA can be produced by a known production method.

The DNA of the present invention having a promoter activity
The structural gene encoding the peptide to be expressed and linked to is not particularly limited, for example,
Examples include a gene encoding a physiologically active substance and a gene used for functional analysis of gene transcription. One or more of these structural genes may be ligated in such a direction as to be correctly transcribed into the DNA of the present invention having a promoter activity.

Examples of genes encoding physiologically active substances include prostaglandin, prostacyclin, thromboxane, leukotriene, interferon, interleukin, tissue plasminogen activator (t-PA), and tumor necrosis factor (TNF). , Epidermal growth factor (EGF), nerve growth factor (NGF), cerebral diuretic peptide (BNP), brain-derived neurotrophic factor (BDNF)
And genes encoding erythropoietin and the like, but are not particularly limited thereto. Examples of genes used for gene transcription function analysis include, but are not limited to, chloramphenicol acetyltransferase (CAT) gene, alkaline phosphatase gene, β-galactosidase gene, luciferase gene and the like. It should not be done.

To obtain a peptide to be expressed, a recombinant DNA is introduced into a vector, and then the recombinant DNA
The host may be transformed with the vector into which the DNA has been introduced. Examples of the host include animal and insect cells such as human, cow, mouse, rat, and silkworm.

It is necessary to select a vector for transforming the host in consideration of the host-vector system. For example, the vector for animal cells is not particularly limited, but may be a retrovirus or pM
Plasmids such as AM (manufactured by Clontech) and the like can be mentioned. The vector into which the recombinant DNA has been introduced can be produced by a known production method.

By transforming the host with the vector into which the recombinant DNA has been introduced, the peptide encoded by the structural gene to be expressed can be expressed in the transformant.

Transformation of a host using a vector can be performed by a known method. For example, calcium phosphate method, electroporation method, DEAE-Dext
ran method, protoplast method (Molecular Cloning, -A
laboratory manual-, secondedition, Cold Spring Har
bar Laboratory Press 1989), but is not particularly limited. In order to transform a host, a known gene transfer reagent and recombinant DN are used.
A mixture with A can also be used.

In order to obtain a peptide to be expressed, the transformant may be cultured in a medium, and the peptide may be isolated from the culture and collected. In consideration of the peptide to be expressed, the culture method and conditions may be appropriately examined.
For example, MEM medium, DMEM medium, and RPMI1640 medium (GIBCO BRL) supplemented with about 5 to 20% fetal calf serum (FCS) can be exemplified.

Examples of the method for isolating and collecting the peptide include gel filtration, ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like, but are not particularly limited. .

Further, using a recombinant DNA or vector containing a structural gene encoding a peptide to be expressed, or a mixture of a gene introduction reagent and recombinant DNA,
Transformation of fertilized eggs or undifferentiated somatic cells can also produce transformed animals (transgenic animals). Transformed animals include animals such as cows, pigs, baboons, mice, rats, guinea pigs, and dogs.

Furthermore, recombinant DNA in which the DNA of the present invention and a structural gene encoding a peptide to be expressed are ligated in a direction in which it is correctly transcribed, and a vector containing the recombinant DNA are useful for gene therapy of humans and the like. Can be used.

The disease suitable for gene therapy is not particularly limited. For example, a genetic disease in which a specific physiologically active substance is not produced at all or is produced at an abnormally low level, and a tumor is , Cancer and the like.

Genetic diseases in which a specific physiologically active substance is not produced at all or whose production is at an abnormally low level include, for example, adenosine deaminase deficiency, hemophilia A, hemophilia B, phenyl ketone Urinary disease, growth hormone deficiency, Duchenne muscular dystrophy, ornithine transcarbamoylase deficiency, Peritzus-Melbacher disease, Gerstmann-Schetrausler disease, malignant hyperthermia, retinitis pigmentosa, hyperkalemia, periodic limb paralysis , Dutch cerebral amyloidosis, Alzheimer's disease, retinoblastoma, cystic fibrosis, type 1 neurofibromatosis, antithrombin III deficiency and the like.

In gene therapy for these diseases, a gene in which a gene that is genetically deficient or an antisense oligonucleotide that suppresses the expression of an oncogene is linked to the DNA of the present invention in a direction in which it is correctly transcribed. Replacement DNA
Is administered to humans or the like. The administration is preferably carried out by introducing the recombinant DNA into a vector, liposome, lymphocyte, or the like.

Therefore, the gene therapy composition of the present invention refers to a composition containing recombinant DNA or a vector, liposome, lymphocyte, etc., into which the recombinant DNA has been introduced.

As a method for administering the composition for gene therapy of the present invention to humans and the like, a method for oral or parenteral administration may be mentioned, but parenteral administration is preferred. The dose may be examined in consideration of a disease, a patient's condition, and the like.

Examples of preparations for oral administration include pills, powders, granules, fine granules, capsules, tablets, liquids, and dry syrups. Further, as a preparation for parenteral administration, for example,
Examples include injections, suppositories, inhalants, eye drops, ointments, patches and the like.

When administered to humans or the like, pharmacologically or pharmaceutically acceptable additives can be added to the composition for gene therapy of the present invention.

Examples of the additives include various things such as excipients, disintegrants, disintegration aids, binders, lubricants, isotonic agents, pH adjusters, stabilizers, colorants and the like. Can be.

Next, the present invention will be described with reference to examples.
The present invention is not limited to this.

[0035]

EXAMPLES [Examination of Promoter Activity] A plasmid was prepared in which a DNA having the nucleotide sequence shown in Sequence Listing 1 was connected upstream of the 5 ′ end of a chloramphenicol acetyltransferase (hereinafter abbreviated as CAT) gene.

When transfection is performed using this plasmid alone, the transfection efficiency cannot be corrected. Therefore, a plasmid (CLONTE) in which the β-galactosidase gene is linked to the cytomegalovirus promoter is used.
(CH Co., trade name: pCMVβ).

That is, the transfection efficiency was corrected by the β-galactosidase assay, and the promoter activity of the DNA having the nucleotide sequence shown in Sequence Listing 1 was examined by the CAT assay. A specific method will be described in detail below.

1. Preparation of DNA Chromosomal DNA was extracted from mouse embryonic stem cells by the method described in Analytical Biochemistry., 219 , 131 (1994), and then subjected to ultrasonic treatment to reduce the size of the DNA. Klenow Frag
Ment DNA polymerase (Toyobo) 37
The reaction was carried out at a temperature of 1 ° C. for 1 hour to blunt the DNA ends.

2. Preparation of Vector DNA The pCAT vector (promega) was reacted with Pst I (Toyobo) at 37 ° C. for 2 hours to cut the DNA. KlenowFrag
Ment DNA polymerase (Toyobo) 37
The reaction was carried out at 1 ° C. for 1 hour to blunt the ends. Furthermore, Alk
Aline Phosphatase (E. coli)
C75: manufactured by Takara Shuzo Co., Ltd.) at 65 ° C. for 1 hour, and DN
The phosphate at the 5 'end of A was removed.

3. Ligation of DNA (Construction of Recombinant DNA) DNA prepared in 1. and 2. Vector D prepared in
Both DNAs were ligated to NA using a DNA ligation kit (Takara Shuzo). That is, solution A (Reaction b) was dissolved four times with respect to one solution of the mixture of both DNAs
buffer) and a 1-fold solution B (Enzyme solution)
) was added thereto and reacted at 16 ° C for 16 hours to prepare a vector containing the recombinant DNA.

4. Preparation of transformant Was introduced into competent cells (Escherichia coli: DH5, manufactured by BRL) and transformation was performed to obtain a transformant. Since this transformant had ampicillin resistance, it was confirmed that it was a vector containing the recombinant DNA.

5. Extraction of Plasmid DNA Plasmid DNA (a plasmid in which a DNA having the nucleotide sequence of Sequence Listing 1 was ligated upstream of the CAT gene) was extracted from each of the transformants, and Qiagen Qiagen was used.
The product purified by n tip (trade name) was used for the following operation.

6. Transfection L, a synthetic polycation reagent from GIBCO BRL
IPOFECTAMINE (trade name) and LIPOF
Transfection was performed using ECTIN (trade name) according to the manual attached to the reagent. (Method) Ethanol-precipitated DNA was dissolved in a serum-free medium in a clean bench, and the required concentration (0-5 μg DNA / 100 μl serum-free medium) of D was added.
An NA solution was prepared. 100 μl of a synthetic polycation reagent (6 μl / 1 μg DNA for LIPOFECTAMINE, 4 μl / 1 μg DNA for LIPOFECTIN) is placed in advance in a polystyrene tube according to the amount of DNA in the prepared DNA solution.
100 μl of the prepared DNA solution
1 were mixed. This was allowed to stand at room temperature for 30 minutes to form two kinds of complexes of DNA and cation.

PC12D which was newly inherited the day before
Cells (PC12, a brown blast cell type derived from rat adrenal gland)
Cell variants. Nerve-like cells. ) And L cells (from mouse subcutaneous tissue), each 7 × 10 ⁵ cells / 2 ml growth medium, 3.5 cm dishes were washed twice with 2 ml serum-free medium kept at 37 ° C. In 200 μl of the complex of DNA and cation, serum-free medium 800
μl was mixed to make a total of 1 ml, added to the washed cells, and incubated at 37 ° C. for 5 hours in the presence of 5% CO ₂ .

The cation is LIPOFECTAMI.
In the case of NE, PC12D cells are used as the cells, and LIPO
In the case of FECTIN, L cells were used.

After the incubation, the medium was replaced with a growth medium (with serum added), and the medium was replaced again the next day. 48 hours after the start of transfection, cells were collected using a cell scraper.

7. Preparation of cell extract Basically, Molecular Cloning, -A laboratory manual-,
second edition, ColdSpring Harbar Laboratory Press
Followed 1989. (Method) 48 hours after transfection, an ice-cooled phosphate buffer containing no Mg ²⁺ and Ca ²⁺ (hereinafter referred to as PB
Abbreviated as S (-). 2.) Wash cells with 2 ml. PBS
(-) 1 ml was added, the cells were detached from the Petri dish with a cell scraper, and the cells were transferred to an Eppendorf tube. Centrifuge at 1000 rpm for 5 minutes at 4 ° C.
After discarding the supernatant, the suspension was suspended in 100 μl of a 250 mM Tris-HCl (pH 8.0) buffer, and the ice box prepared in advance was filled with dry ice.
A freeze-thaw cycle of 3 minutes in a water bath at 37 ° C. for 3 minutes was repeated three times. 15000 rpm for 5 minutes at 4 ° C
The supernatant was transferred to a new Eppendorf tube to obtain a cell extract (stored at -80 ° C).

8. β-galactosidase assay Basically, Molecular Cloning, -A laboratory manual-,
second edition, ColdSpring Harbar Laboratory Press
Followed 1989. (Method) 2 μg of cell extract was adjusted to 50 μl with 250 mM Tris-HCl (pH 8.0) buffer, and then 150 μl
Β-gal buffer (10 mM NaHPO ₄ pH 7.0, 0.1 m
M MgCl ₂ , 100 mM NaCl, 0.1% BSA, 0.1% NaN ₃ , 0.3 mM 4-methy
lumbelliferyl-β-D-galactoside). After 1 hour incubation at 37 ° C., 600 μl of 0.1 M
Glycine-NaOH (pH 10.3) was added and excitation 36
Fluorescence was measured at 0 nm and fluorescence 450 nm.

9. Assay of CAT 100 μg of cell extract was added to 250 mM Tris-HCl (p
H8.0) After making up to 80 μl with buffer, the mixture was incubated at 65 ° C. for 10 minutes. Then, at 4 ° C. for 2 minutes, 1500
Centrifuge at 0 rpm and add 10 mM acetyl-Co
A 10 μl, [ ¹⁴ C] chloramphenicol 0.2 μl
20 μl of Ci / 20 μl was added, mixed, and incubated at 37 ° C. for 2 hours. 10 mM acetyl-CoA10
Further, μl was added and reacted overnight at 37 ° C., 200 μl of ethyl acetate was added, and the mixture was stirred and centrifuged. After that, 180 μl of ethyl acetate was taken from the supernatant, and acetylated chloramphenicol was extracted. It was dried with an evaporator, dissolved in 20 μl of ethyl acetate, and developed by thin layer chromatography.

10. For the plasmid in which the SV40 promoter was ligated upstream of the control CAT gene, as described in 1. above. ~ 9. The same operation as that described above was carried out to serve as a control.

11. Results The results are shown in FIG. As is clear from the results, it was found that the DNA represented by SEQ ID NO: 1 in the sequence listing selectively had a strong promoter activity in PC12D cells.

[0052]

Industrial Applicability The DNA of the present invention selectively has a strong promoter activity in the brain, is useful for producing peptides in large amounts and economically, and is useful for gene therapy.

[Brief description of the drawings]

FIG. 1 shows that the DNA of the present invention selectively expresses PC12D.
It shows promoter activity in cells.

[Sequence List] SEQ ID NO: 1 Sequence length: 254 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Myomorpha Mus. Strain name: 129 cells Type: Embryonic stem cell Sequence characteristics Symbol indicating characteristics: promoter Method for determining characteristics: E sequence GAGAAAATGG AAGGCTCCACAAAAAAGCAGG AAG
AGCATCA CAGCCTGGCT TTAGGGAGAT 60 TCCCATGACC TGGGCAGCCCGTGCAGGGCT GGC
GAGGGGGA TTTTAGGACC CTGAGGCCCT 120 GGCGCGAGCG TGAGGCGCAG GGGAGGGCGCTCC
CGGAGCC TGCATCCCGGG AGGGCGGGGGT 180 CTCGGCCCCG GGCGCGCTT CTATTGGCGG CAG
CGCGCGG GTGACGATGA CGGCTGGCGC 240 GAGGCCCCGC CCCG
254

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/02 C 4C087 C12P 21/02 C12N 5/00 B // (C12N 15/09 ZNA (C12R 1:91) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) F term (reference) 4B024 AA01 BA10 CA03 DA02 DA06 EA02 EA04 FA02 GA11 GA14 HA17 4B064 AG01 CA10 CA19 CC24 DA01 4B065 AA91X AA91Y AB01 AC14 BA01 BA02 BA03 CA29 CA44 4C084 AA06 AA13 CA53 NA05 ZB212 ZB262 4C086 AA01 AA02 EA16 NA05 ZB21 ZB26 4C087 AA01 AA02 BB57 CA12 NA05 ZB21 ZB26

Claims (13)

[Claims] 1. The DN represented by SEQ ID NO: 1 in the sequence listing
A. 2. The DNA represented by SEQ ID NO: 1 in the sequence listing
DNA that hybridizes to DNA. 3. The DNA according to claim 1 or claim 2.
DNA that is part of 4. The DNA according to any one of claims 1 to 3, which has a promoter activity. 5. The DNA according to any one of claims 1 to 4, which selectively exhibits promoter activity in a nerve cell. 6. The DN according to claim 1, which selectively shows promoter activity in the brain.
A. 7. A recombinant DNA comprising a structural gene linked to the DNA according to any one of claims 1 to 6 so as to be correctly transcribed. 8. A vector comprising the recombinant DNA according to claim 7. 9. A transformant transformed with the recombinant DNA according to claim 7 or the vector according to claim 8. 10. An animal transformed with the recombinant DNA according to claim 7 or the vector according to claim 8. 11. A method for producing a peptide, comprising culturing the transformant according to claim 9, separating a peptide encoded by a structural gene from the culture, and collecting the peptide. An expression kit comprising the DNA according to any one of claims 1 to 4. 13. D according to claim 5 or claim 6.
A gene therapy composition comprising NA, the recombinant DNA of claim 7, or the vector of claim 8.