JPH104968A - DNA - Google Patents

Abstract

(57) [Summary] [Object] To provide a DNA having a strong promoter activity, which is useful for producing a peptide in a large amount and economically. A DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, a part thereof, a DNA hybridizing to a DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, and a part of DNA hybridizing thereto.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA having a strong (high transcription efficiency) promoter activity for gene expression.

[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.

Therefore, in order to produce a peptide in a large amount and economically by a genetic engineering technique, it is necessary to use a strong promoter.

[0004] Currently, as a strong promoter, the promoter of the lactose operon, tryptophan promoter of, P _L promoter of lambda phage, SV40
Promoters, metallothionein promoters and cytomegalovirus promoters are known.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a DNA having a strong promoter activity, which is useful for producing a peptide in a large amount and economically.

[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 certain DNAs have strong promoter activity.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS That is, the present invention provides the following invention 1.
To invention 17. About.

Invention 1 DNA represented by SEQ ID NO: 1 in the sequence listing.

Invention 2 DNA represented by SEQ ID NO: 2 in the sequence listing.

Invention 3 DN hybridizing to DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing
A.

Invention 4. Invention 1 ~ Invention 3. A DNA which is a part of the DNA according to any one of the inventions.

Invention 5 Invention Having Promoter Activity ~ Invention 4. The DNA according to any one of the inventions.

Invention 6 Invention 1 ~ Invention 5. A recombinant DNA comprising the DNA according to any one of the inventions.

Invention 7 Invention 6 The recombinant DNA according to the above.
Vector containing.

Invention 8 Invention which is a plasmid7. The vector according to claim 1.

Invention 9 Invention 7. Or invention 8. A transformant transformed by the vector according to 1.

Invention 10 Invention 1 ~ Invention 5. A recombinant DNA comprising the DNA according to any one of the inventions and a structural gene linked downstream of the DNA.

Invention 11 Invention 10. Recombinant D described in
Vector containing NA.

Invention 12 Invention which is a plasmid 11.
The vector according to claim 1.

Invention 13 Invention 11. Or invention 12.
A transformant transformed by the vector according to 1.

Invention 14 Invention 10. Recombinant D described in
NA or invention 11. Or invention 12. An animal transformed with the vector according to 1.

Invention 15 Invention 13. A method for producing a peptide, comprising separating and collecting a peptide encoded by a structural gene from a culture obtained by culturing the transformant described in 1 above.

Invention 16 Invention 1 ~ Invention 5. An expression kit comprising the DNA according to any one of the inventions.

Invention 17 Invention 10. Recombinant D described in
NA or invention 11. Or invention 12. A composition for gene therapy, comprising the vector according to item 1.

Hereinafter, the present invention will be described.

The DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing of the present invention has been extracted from rat brain chromosomes. The present inventors have found that this DNA has promoter activity.

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. SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing
It is predicted that the DNA or a part thereof that hybridizes to the DNA represented by has promoter activity.

Therefore, the DNA of the present invention includes not only the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing, but also a part thereof, which hybridizes to the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. And a part of the hybridizing DNA are also included. In particular, those having promoter activity are included in the present invention.

The term "hybridizing DNA" refers to a DNA which hybridizes under a general condition using a DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 or a part thereof as a probe. Hybridization conditions may be appropriately examined, for example, 0.75 M 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 reaction at 65 ° C. for 16 hours can be mentioned, but the conditions are not particularly limited to these.

[0030] In the present invention, the hybridizing DNA can also be expressed as a derivative of the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. That is, it has at least about 70% sequence identity to the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing.
With one or more nucleotides inserted, D
One or two or more nucleotides added to either end of the NA sequence, one or more nucleotides at either end or inside the DNA sequence deleted, There can be mentioned a combination of these.

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

The recombinant DNA of the present invention is a DNA in which a structural gene encoding a peptide to be expressed is ligated downstream of the DNA of the present invention having a promoter activity in a direction in which the structural gene is correctly transcribed. is there.

Furthermore, in the recombinant DNA of the present invention, a transcription regulatory region such as an enhancer or a terminator can be linked to an appropriate site upstream or downstream of the DNA of the present invention having a promoter activity.
The recombinant DNA can be produced by a known production method.

DNA of the present invention having promoter activity
The structural gene encoding the peptide to be expressed, which is linked downstream of, is not particularly limited, and examples thereof include a gene encoding a physiologically active substance, a gene used for functional analysis of gene transcription, and the like. Can be.
One or more structural genes can be linked to the DNA of the present invention having promoter activity.

Examples of the gene encoding a physiologically active substance include prostaglandin, prostacyclin, thromboxane, leutocollien, interferon, interleukin, tissue plasminogen activator (t-PA), and tumor necrosis factor (TNF). And genes encoding epidermal growth factor (EGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), atrial diuretic peptide (ANP), erythropoietin, and the like. It should not be done.

Examples of the gene used for gene transcription function analysis include chloramphenicol acetyltransferase (CAT) gene, alkaline phosphatase gene, β-galactosidase gene and luciferase gene. It should not be limited to things.

To obtain a peptide to be expressed, a recombinant DNA is introduced into a vector, and then the recombinant DNA
The host will be transformed with the vector into which is introduced. As the host, for example, human, cow, mouse,
Examples include animal cells such as 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 the 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 Harb
ar Laboratory Press 1989).
There is no particular limitation.

To obtain the 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, M containing about 5 to 20% bovine serum (FCS)
Examples include EM medium, DMEM medium, and RPMI1640 medium (manufactured by GIBCO BRL).

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. .

When a fertilized egg is transformed with a recombinant DNA or vector containing a structural gene encoding a peptide to be expressed, a transformed animal (transgenic animal) can also be prepared.
Transformed animals include animals such as cows, pigs, baboons, mice, rats, guinea pigs, and dogs.

Further, the vector of the present invention containing the DNA and the structural gene encoding the peptide to be expressed can be used for gene therapy of humans and the like.

The disease suitable for gene therapy is not particularly limited as long as it is suitable for application. For example, a specific physiologically active substance is not produced at all or its production is abnormally low. Genetic diseases, tumors, cancers and the like at the level can be mentioned.

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 Urine, growth hormone deficiency, Dechenne muscular dystrophy,
Examples include ornithine transcarbamoylase deficiency and antithrombin III deficiency.

In gene therapy for these diseases, an antisense oligonucleotide that suppresses the expression of a genetically deficient gene or an oncogene is ligated in a direction that is correctly transcribed downstream of the DNA of the present invention. The administration is performed by administering the recombinant DNA thus obtained to humans or the like. The administration is preferably carried out by introducing the recombinant DNA into a vector, liposome, lymphocyte, or the like.

Accordingly, the composition for gene therapy 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 gene therapy composition 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 additives such as excipients, disintegrants, disintegration aids, binders, lubricants, isotonic agents, pH adjusters, stabilizers, and colorants. Can be.

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

[0054]

【Example】

1. Preparation of DNA First, chromosomal DNA was extracted from the brain of an 8-week-old Wistar rat by the method described in Analytical Biochemistry., 219 , 131 (1994), and this was extracted with Mse I (NewEngl).
amd Biolab) and Bam HI (manufactured by Toyobo Co., Ltd.) were reacted with each of them at 37 ° C. for 2 hours to reduce the size of DNA. The depolymerized DNA fragment was reacted with Klenow Fragment DNA polymerase (manufactured by Toyobo Co., Ltd.) at 37 ° C. for 1 hour to blunt the 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 buoy), which is 4 times dissolved in 1 solution of the mixed solution of both DNAs
ffer) and 1-time solution B (Enzyme solution)
ion) was added 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 was extracted from each transformant.

6. Introduction of plasmid DNA into mouse fibroblasts (L cells) 20 μg of the plasmid DNA obtained in the above was introduced into about 10 ⁶ L cells by the calcium phosphate method,
Allowed to react for days. At this time, a pCAT promoter vector (promega) having a cytomegalovirus promoter was used as a control.

7. Chloramphenicol acetyltransferase (CAT) assay. After washing the cells created in the above with PBS, peel off,
The cells were collected by centrifugation in a Spitz tube at 2000 rpm for 5 minutes. The collected cells are washed with 100 mM of pH 7.8.
The cells were suspended in 100 μl of Tris-HCl buffer. The cell suspension was freeze-thawed (liquid nitrogen-water bath at 37 ° C.)-Stirred three times to break the cells. At 4 ° C, 15000r
The supernatant was obtained by centrifugation at pm for 5 minutes. The protein concentration of this solution was quantified, and the amount corresponding to 50 μg of protein (maximum volume of 50 μg) was placed in a scintillation counter tube.
1) and 1.25 mM chloramphenicol 20
0 μl and 10 μl of 14 C-butyryl coenzyme A (manufactured by NEN Research Products) are mixed, and the mixture is mixed with Econofluor (NEN Research).
5 ml) (products) and reacted at room temperature for 3 hours. Thereafter, the CAT activity was measured with a liquid scintillation counter (Beckman LS3801).

8. Results When the CAT activity of a vector containing a large number of recombinant DNAs was analyzed, a DNA having a CAT activity equivalent to that of a pCAT promoter vector (promega) into which a control cytomegalovirus promoter was inserted was obtained.
A vector containing (SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing) was found. The results are shown in FIG. Note that SEQ ID NOS: 1 and 2 in the sequence listing are in a sense-antisense relationship.

[0062]

Industrial Applicability The DNA of the present invention has a strong promoter activity and is useful for producing a peptide in a large amount and economically.

Further, since the DNAs represented by SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing having the promoter activity are in a sense-antisense relationship, the transcription of the DNA is linked to the promoter by the structural gene of the peptide to be expressed. Construction of a recombinant DNA or the like can be performed without paying attention to the direction.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows D having promoter activity of the present invention.
1 shows CAT activity of a vector containing NA.

[Sequence list]

SEQ ID NO: 1 Sequence length: 226 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Rattes nervegicus
) Organization Type: Brain sequence TAATGGCAGA AAAAGTGAGA TGACACAGAA CCTTCCCCTA ATAAATAAAG ATTTCAAGGA 60 GCCAATCAGT GGGTGAGTAG GAGGGACTTC TGAGTTGGAA AGGGAAAGAG AGGAAGCAGG 120 AGAAACAATA GGTCATTTGG ACCAGAGACA GCATGGAGGC CAAAATGTAG GTAGCAGAGC 180 TGCCCCCTGC CAACCTCCTC CACTCCCCCA ATTCAGGTGG TGGATC 226 SEQ ID NO: 2 Length of sequence: 226 SEQ type: nucleic acid strands Number: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Rattus nervegicus
) Tissue type: Brain Sequence GATCCACCAC CTGAATTGGG GGAGTGGAGG AG
GTTGGCAG GGGGCAGCTC TGCTACCTAC 60 ATTTTGGCCT CCATGCTGTC TCTGGTCCAA AT
GACCTATT GTTTCTCCTG CTTCCTCTCT 120 TTCCCTTTCC AACTCGAAG TCCCTCCTAC TC
ACCCACTG ATTGGCTCCT TGAAATCTTT 180 ATTTTATTGG GGAAGGTTCT GTGTCATCTC AC
TTTTTCTG CCATTA 226

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12P 21/00 C12P 21/00 C C12Q 1/68 7823-4B C12Q 1/68 A // A61K 38 / 00 A61K 37/02 38/16 37/04 38/22 37/24 38/46 37/54 (C12N 1/21 C12R 1:19)

Claims (17)

[Claims] 1. The DN represented by SEQ ID NO: 1 in the sequence listing
A. 2. The DN represented by SEQ ID NO: 2 in the sequence listing
A. 3. A DNA that hybridizes to the DNA represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. 4. A DNA which is a part of the DNA according to any one of claims 1 to 3. 5. The DNA according to claim 1, which has a promoter activity. 6. A recombinant DNA comprising the DNA according to any one of claims 1 to 5. 7. A vector comprising the recombinant DNA according to claim 6. 8. The vector according to claim 7, which is a plasmid. 9. A transformant transformed by the vector according to claim 7 or 8. 10. A recombinant DNA comprising the DNA according to any one of claims 1 to 5 and a structural gene linked downstream of said DNA. 11. A vector comprising the recombinant DNA according to claim 10. 12. The vector according to claim 11, which is a plasmid. 13. A transformant transformed by the vector according to claim 11 or 12. 14. An animal transformed with the recombinant DNA according to claim 10 or the vector according to claim 11 or 12. 15. A method for producing a peptide, comprising separating and collecting a peptide encoded by a structural gene from a culture obtained by culturing the transformant according to claim 13. 16. An expression kit comprising the DNA according to any one of claims 1 to 5. 17. A gene therapy composition comprising the recombinant DNA according to claim 10 or the vector according to claim 11 or 12.