JPH06178687A - Dna coding human st2, expressed product of the same, method for producing expressed product by expressing the same - Google Patents

Abstract

PURPOSE:To isolate a genom DNA coding human ST2, a cDNA coding the human ST2 and the human ST2 for detecting cancer cells, preventing the multiplication of the cancer cells, etc. CONSTITUTION:A genom DNA fragment coding human ST2 is isolated from the library of human genom DNA with the cDNA of mouse ST2 as a probe. A cDNA fragment coding a part of the human ST2 is synthesized from various human cDNA libraries with a part of the DNA fragment as a PCR primer, and a cDNA coding the whole length of the human ST2 is isolated from the human cDNA library with the cDNA fragment as a probe. The cDNA is inserted into an expression vector, and the inserted vector is transduced into a culture cell. The human ST2 is massively expressed in the cultured cells.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding human ST2, a human ST having at least a part of the DNA.
DNA capable of selectively hybridizing with 2,
A vector containing the DNA, a transformant carrying the vector, human ST2, human ST by expressing the DNA
2 relates to a method of manufacturing 2.

[0002]

2. Description of the Related Art In cell division, DN called G1 phase
Preparatory period for A synthesis, DNA synthesizing period called S period, preparatory period called G2, mitotic period called M period and G0
It is known that a stationary phase called a phase appears. Here, a cell that repeats division repeats a growth cycle of G1 phase → S phase → G2 phase → M phase → G1 phase, but a cell transiting from M phase to G0 phase is in a quiescent state of division. However, cells in a quiescent state in the G0 phase can shift to the G1 phase by the growth stimulus or the like and enter the growth cycle again.

Cells of different cycles are present in the tissues of animals, but by extracting these cells, arresting their division at G0 phase, and giving artificial stimulus again, G0 to G1 phase can be obtained. As the technology for initiating the transfer of Escherichia coli has become known, studies have been actively conducted to elucidate genes (DNA) that are specifically expressed from the G0 phase to the G1 phase. If the mechanism by which cells in the G0 phase enter the G1 phase and enter the growth cycle is clarified, it can be applied to the control of cell division of cancer cells, etc., and this research is not limited to basic research. Absent.

Actually, an animal cell preparation in which division has been stopped at the G0 phase has been used conventionally, and artificial stimulation is applied to this to shift from the G0 phase to the G1 phase, at which time it is specifically expressed. Studies such as RNA extraction have been carried out (Linzer, D. I.H., et al., Proc. Nat.
l. Acad. Sci. USA, Vol. 30, pp. 4271 (1983), Linzer, D .; I. H. Et al., P
roc. Natl. Acad. Sci. USA, 81st
Volume, 4255 pages (1984), Hirschhor.
n, R.N. R. Et al., Proc. Natl. Acad. Sc
i. USA, Vol. 81, page 6004 (1984),
Lau, L .; F. EMBO J. et al. Volume 4, 3145
P. (1985), Lau, L .; F. Et al., Proc.
Natl. Acad. Sci. USA, Volume 84, 11
82 (1987), Chavrier, P .; ,
EMBO J.M. Volume 7, page 29 (1988)).

According to these studies, when cells move from G0 phase to G1 phase, for example, c-myc, c-
It has been revealed that an oncogene such as fos is specifically expressed.

However, these studies show that
Too much attention was paid to the expression of genes during the transition to the phase, and in order to eliminate non-specific DNA, the cells in the G0 phase were artificially stimulated in a very short time of 2-3 hours. Targeting only the specifically expressed DNA, there was no knowledge of what is expressed immediately before S phase.

[0007] The present inventor previously studied by paying attention to a gene that is specifically expressed after a relatively long time of about 10 hours after artificially stimulating cultured cells derived from a mouse in the G0 phase. As a result, a previously unknown DNA was isolated by synthesizing cDNA by using as a template an mRNA that is specifically expressed in the transitional period from the G0 phase to the G1 phase of mouse fibroblasts. We succeeded in expressing the encoded protein (Tominaga,
S. et al. , FEBS Lett. , 25
8 , 301-304 (1989), Tominaga.
S, et al. , Biochem Biophys.
Acta, 1090 , 1-8 (1991)), and this protein was named mouse ST2.

The characteristics of the DNA encoding this mouse ST2 are as follows.

At least not expressed in cells of brain tissue, heart tissue, lung tissue, liver tissue, spleen tissue, pancreas tissue, kidney tissue, muscle tissue or testis tissue of mouse CD-1, but at least BALB / c-3T
3 (mouse fibroblast) cells are expressed during cell proliferation starting from the G0 phase. That is, the DNA is not expressed in the cells in the G0 phase (quiescent phase), but is expressed as these cells move from the G0 phase to the G1 phase.

At least mouse BALB / c-3T3
It is also expressed in cells that have transited from the M phase to the S phase via the G1 phase without passing through the G0 phase,
The expression level is equal to or lower than the expression level in cell division that starts in the G0 phase of the cell. That is, for example, in cells derived from meristems, the cells may transit from the M phase to the next cell cycle immediately without passing through the G0 phase. The DNA is also expressed in such cells, but its expression level is small compared to the expression level at the time of transition from G0 phase to G1 phase.

Approximately 5 to 1 after the start of cell growth from the G0 phase
The expression reaches its maximum in 2 hours. That is, in the conventionally known DNA, 2-3 after the initiation of division from G0 phase.
Although it is specifically expressed in about time, the DNA has properties different from these.

By its expression, cytoplasmic RNA having a molecular weight of 18 to 28S is synthesized.

As described above, the DNA encoding mouse ST2 previously isolated by the present inventor had properties different from the conventionally known DNA. The present inventors further determined the nucleotide sequence of cDNA encoding mouse ST2. Then, based on the determined nucleotide sequence, 3 of mouse ST2
An amino acid sequence consisting of 37 residues was deduced (Tomin
aga, S.A. et al. , FEBS Let
t. , 258 , 301-304 (1989)). From this amino acid sequence, the properties of mouse ST2 were estimated as follows.

It is a protein belonging to the immunoglobulin superfamily and having a primary structure capable of forming three immunoglobulin-like domains. Here, the immunoglobulin superfamily is a protein having an immunoglobulin-like domain and involved in intercellular communication,
The immunoglobulin-like domain means a structure similar to the loop formed by the SS bond of cysteines existing in immunoglobulin. The ability to form three immunoglobulin-like domains means having at least 6 or more cysteine residues.

It is a protein having a site to which 9 sugar chains can bind. This is because the protein has 9 asparagines.

Mouse IL- whose amino acid sequence is known
Outer membrane position in 1 receptor, mouse neuronal cell adhesion protein, mouse basement membrane proteoglycan, HLA-6-2,
The amino acid sequence similarity (identity of amino acid sequence) with the constant site in the heavy chain constituting secretory chicken IgM was 25.1%, 22.7%, 19.0%, 20.8%, respectively.
It is 16.5%.

[0017]

[Problems to be Solved by the Invention]
The details of mouse ST2, which is expressed a relatively long time after the initiation of transition during the transition from 0 phase to G1 phase, have been clarified, but no findings have been found for proteins similar to mouse ST2, particularly human-derived proteins. There was no

[0018]

Means for Solving the Problems The present inventor
A genomic DNA library derived from human granulocytes was screened using a DNA probe derived from 2cDNA to obtain a clone that specifically hybridizes with the DNA probe. And the genomic DNA contained in this clone
The base sequence of the fragment was clarified, and the exon portion was estimated from the comparison with the base sequence of the genomic DNA encoding mouse ST2. PC synthesized based on the base sequence of this exon
DNAs in human cDNA libraries derived from various cells were amplified using the R primer, and a library in which the region sandwiched between the two PCR primers was amplified was selected. Using the human genomic DNA fragment that hybridizes with the previously isolated mouse ST2 cDNA fragment as a probe, the cDNA encoding human ST2 was successfully cloned.

Furthermore, the cloned cDNA was expressed in animal cells, and human ST2 was successfully obtained.

The DNA of the present invention is capable of expressing a protein having the amino acid sequence of SEQ ID NO: 4, for example.

An example of such DNA is one having the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

The protein of the present invention is the DNA of the present invention.
Coded by and there are no other restrictions. An example of the protein of the present invention is that having the amino acid sequence represented by SEQ ID NO: 4.

The protein of the present invention includes both "the one containing a signal peptide" and "the one not containing a signal peptide (mature protein)".

The DNA of the present invention can be obtained from human cells by the method described below. Further, the protein of the present invention can be prepared by genetic engineering using the DNA of the present invention ligated to an appropriate vector.

The DNA may be isolated from the genome of human cells or from cDNA using mRNA derived from human cells as a template. Further, it can be produced by chemical synthesis. The cell from which the genomic DNA and mRNA are collected is not particularly limited as long as it is a human cell.
In the case of, it is desirable to obtain from cells expressing ST2. For example, after ligating an oligonucleotide to both ends of the DNA of the present invention, a DNA fragment having a restriction site formed by using an appropriate restriction enzyme is obtained, on the other hand, a selected host can be transformed and self-reacted in the host. A DNA fragment obtained by cleaving a sequence necessary for expression of a structural gene such as a promoter of a proliferative vector with the above-mentioned restriction enzyme is obtained, and an expression vector is obtained by ligating these fragments, which is transformed with the expression vector. It is possible to express the protein of the present invention in different host cells.

The DNA of the present invention can be expressed in a conventional host-vector system, but among them, an expression system using animal cells such as CHO and COS is preferably used.

With regard to the DNA of the present invention, it is also possible to prepare a DNA probe or the like by utilizing a sequence which is a part of the nucleotide sequence and is distinguishable from the conventionally known nucleotide sequence of DNA. By using such a probe, it is possible to detect, for example, a cell cluster or the like in a tissue where cell division is active.

At present, the known DNA can be partially or partially deleted, replaced, or inserted with another base to obtain the D
The protein expressed by NA expression is made smaller (sometimes solubilized) to enhance or eliminate the properties of the protein, and further facilitates expression in a certain host, for example. Is commonly done. Also in the present invention, there is no limitation in performing such an operation on the DNA of the present invention. On the other hand, regarding a known protein, a part of the protein is deleted, substituted or inserted with another amino acid residue to make the protein into a lower molecule (sometimes solubilized), The operation of enhancing or eliminating the property possessed or adding a new function is generally performed. Similar to the above-mentioned operation for DNA, there is no limitation in performing such operation for the protein of the present invention.

[0029]

EXAMPLES Examples will be described below to explain the present invention in more detail, but these examples do not limit the present invention.

Example 1 Genome encoding human ST2
Cloning of DNA A human genomic DNA library derived from granulocytes was cloned into mouse ST2 cDNA (Tominaga, S. et a.
l. , FEBS Lett. , 258 , 301-30
4 (1989)) (corresponding to SEQ ID NO: 1) as a probe (Maniatis, T. et.
al. , Molecular Cloning, Co
ld Spring Harbor Laborato
ry (1982)). Three positive clones were selected from approximately 2 × 10 ⁶ plaques and recombinant phage were purified. It was confirmed that the three clones contained the same genomic DNA fragment by mapping the recombinant phage by restriction enzyme cleavage, Southern hybridization analysis, and determination of the nucleotide sequence.

A part of the nucleotide sequence of the cloned genomic DNA fragment encoding human ST2 was cloned into mouse ST
2 nucleotide sequence of cDNA (Tominaga, S. e.
tal. , FEBS Lett. , 258 , 301-
304 (1989)). As shown in Figure 1,
A part of the nucleotide sequence of the genomic DNA encoding the human ST2 contained all of the 7th exon and a part of the 8th exon. In FIG. 1, the sequence surrounded by □ represents the exon portion, and the asterisk represents the stop codon.

Example 2 Human containing human ST2 cDNA
Selection of cDNA library Human ST2 genome D shown in Figure 1 (corresponding to SEQ ID NO: 2)
Based on the nucleotide sequence of NA, PCR primers used for selection of a human cDNA library containing human ST2 cDNA were designed. The PCR primers are two types of single-stranded DNA indicated by arrows in FIG.

As is apparent from FIG. 1, if human ST2 cDNA is present in the library, a DNA fragment of 305 bases is produced by amplification by the PCR method. If human ST2 genomic DNA is present in the library, DNA of 397 bp is obtained by amplification by PCR.
Fragments are produced.

With the PCR primers, cDNA libraries derived from various human cells were amplified by the PCR method,
Then, the length of the amplified DNA fragment was analyzed by polyacrylamide gel electrophoresis. The reaction was carried out in the presence of Taq polymerase at 94 ° C for 1 min, 50 ° C for 2 min, 7 min.
Repeated 30 cycles of 2 ° C for 3 minutes. A photograph of the gel stained with ethidium bromide is shown in FIG. The clone name of the human cell used when producing the cDNA library is shown on the lane, and the size of the DNA fragment is shown on the right side of the lane. In 5 of the 17 cDNA libraries, a DNA fragment of 305 bases was amplified, and it was revealed that human ST2 cDNA was present in these libraries. In the control human genomic DNA library (the rightmost lane in FIG. 2), a DNA fragment of 397 bases was amplified, and it was confirmed that the PCR reaction was normally performed.

Of the above 5 cDNA libraries,
2F1 and 5C10 are clones of helper T cells activated by concanavalin A (Con A) or phorbol myristate acetate (PMA) (Yoko).
ta, T .; et al. , Proc. Natl. Aca
d. Sci. , U. S. A. , 84 , 7388-739.
2 (1987), Lee, F .; et al. , Pro
c. Natl. Acad. Sci. , U. S. A. , 8
2 , 4360-4364 (1985), Yokota,
T. et al. , Proc. Natl. Acad.
Sci. , U. S. A. , 83 , 5894-5898.
(1986)).

Example 3 cDNA encoding human ST2
Cloning of A A cDNA library derived from the 5C10 clone of Example 2 was obtained by amplification by the PCR method in Example 305.
Screening was carried out using a base DNA fragment as a probe (Maniatis T. et al., Molec.
ural Cloning, Cold Spring
According to the method described in Harbor Laboratory (1982)). Two positive clones were obtained from about 7 × 10 ⁵ colonies. CDNA was isolated from a clone having a long cDNA fragment, subcloned into pUC19, and the entire base sequence was determined.
The A fragment contained the entire open reading frame (ORF) of human ST2. Figure 3 shows the determined nucleotide sequence.
Shown in. In FIG. 3, the upper part of the sequence shows the determined base sequence (corresponding to SEQ ID NO: 3), and the lower part shows the amino acid sequence deduced from the base sequence (corresponding to SEQ ID NO: 4). The putative cleavage site of the signal peptide is indicated by an arrow, and the putative binding site of the N-linked sugar chain is

[Chemical 1] The position of cysteine, which is presumed to be involved in the formation of immunoglobulin-like domain, is indicated by □. Human ST
From the amino acid sequence of 2, a known method (Von Heij
ne, G.N. , Nucleic Acids Res. ，
14 , 4683-4690 (1986)), the signal peptide region was searched, and it was estimated that the 17th amino acid from the N-terminal constitutes the signal peptide. The cleavage site of the signal peptide is shown by an upward white arrow in FIG.

The DNA of the present invention is a human ST2 shown below.
G and the amino acid sequences of other proteins
As a result of searching using the enBank DNA database and the NBRF protein database, human ST2 was found to be human I
L1-R1 (23.7% in 299 overlapping amino acids),
Human IL1-R2 (22.9 out of 319 overlapping amino acids)
%), Vaccinia virus B16R protein (redundant 2
24.3% out of 92 amino acids), Drosophila Cek
2 proteins (23.5% of overlapping 187 amino acids), chicken klg protein (25.
0%) and homology. The amino acid sequences of these proteins are shown in comparison with each other in FIG. In FIG. 4, “Hu” is human, “Mu” is mouse, and “Vaccinia” is
The vaccinia virus B16R protein is shown. Also":"
Is the same amino acid, “*” is an unmatched amino acid, “□” is an amino acid conserved with 6 types of amino acids, and “▽” or “☆” is a conserved amino acid with 6 types of amino acids. Cysteines putatively involved in the formation of immunoglobulin-like domains are shown, respectively.

Example 4 Expression of Human ST2 in Recombinant Cultured Cells The human ST2 cDNA having the nucleotide sequence shown in FIG. 3 obtained in Example 3 was used as an expression vector pEF-BOS (Mi.
zushima, S .; et al. , Nucleic
Acids Res. , 18, 5322 (199
0)) at the BstXI site (located downstream of the EF-1α promoter). Whether the insertion of the cDNA was carried out in the correct direction was confirmed by restriction enzyme mapping, and the correctly inserted plasmid was introduced into COS7 cells by the DEAE-dextran method (also chloroquine treatment) (Sambrook, Jet et al., Mole.
color Cloning, Cold Spring
Harbor Laboratory, 16.30
(1989)). Thirty hours after the introduction of the plasmid, the cells were washed, the protein expressed in the medium containing ³⁵ S-methionine was radiolabeled, and 12 hours after the start of radiolabeling, the cell supernatant was collected. The proteins in the supernatant were separated by SDS-polyacrylamide gel electrophoresis, and the position of the protein in the gel was confirmed by fluorography. The result is shown in FIG. In FIG. 5, lane 1 is pEF in which human ST2 cDNA is not inserted.
-The culture supernatant of cells transformed with BOS (control),
Lane 2 is pE in which human ST2 cDNA is inserted.
1 shows the culture supernatant of cells transformed with F-BOS. The protein band indicated by an arrow has a molecular weight calculated from the deduced amino acid sequence of human ST2 shown in FIG. 3 and appears only in lane 2, so it can be determined to be a band corresponding to human ST2. From this, human ST2c
It was confirmed that COS7 cells transformed with the expression vector pEF-BOS in which DNA was inserted expressed a large amount of human ST2.

[0039]

INDUSTRIAL APPLICABILITY The DNA of the present invention is specifically expressed when cells move from G0 phase to G1 phase. Therefore, it is possible to obtain important knowledge about cell growth by specifically suppressing the expression of the DNA by using an antisense RNA or the like against the RNA that appears when the DNA is expressed. Such effects are the same for the protein of the present invention. For example, by preparing an antibody that recognizes the protein and using the antibody, it is possible to obtain important knowledge about cell proliferation. These are for the purpose of developing a drug that suppresses the growth of diseases such as cancer for which no effective therapeutic drug has been conventionally known and, in the case of necessity, selectively attacks such proliferative cells. It means providing the necessary basic technology.

Further, it becomes possible to detect proliferative cells such as cancer cells existing in a tissue by using a diffusion probe or a labeled antibody for these DNAs and proteins.

[0040]

[Sequence list]

[Brief description of drawings]

FIG. 1 is a diagram showing a part of the nucleotide sequence of genomic DNA encoding human ST2 and PCR primers used for selecting a cDNA library containing human ST2 cDNA.

FIG. 2. Human S against various cDNA libraries
D obtained by carrying out amplification by PCR using primers contained in exons of genomic DNA encoding T2
It is a figure which shows the result of having analyzed the NA fragment by the polyacrylamide gel electrophoresis method.

FIG. 3 Base sequence of human ST2 cDNA and the DNA
It is a figure which shows the deduced amino acid sequence which is encoded by.

FIG. 4 is a diagram comparing the amino acid sequence of human ST2 with the amino acid sequence of a protein similar to human ST2.

FIG. 5 shows the expression of human ST2 in COS7 cells.

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[Procedure amendment]

[Submission date] July 19, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 2

[Correction method] Change

[Correction content]

FIG. 2. Human S against various cDNA libraries
D obtained by carrying out amplification by PCR using primers contained in exons of genomic DNA encoding T2
It is a figure which shows the result of having analyzed the NA fragment by the polyacrylamide gel electrophoresis method (electrophoresis photograph).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 5

[Correction method] Change

[Correction content]

FIG. 5 is a diagram showing expression of human ST2 in COS7 cells (electrophoresis photograph).

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Claims (12)

[Claims] 1. A mouse S having the nucleotide sequence of SEQ ID NO: 1.
DNA encoding human ST2 that hybridizes with T2 cDNA 2. c synthesized using human mRNA as a template
The DNA according to claim 1, which is DNA. 3. A DNA derived from the human genome.
The described DNA. 4. The DNA according to claim 1, which encodes a protein having the amino acid sequence of SEQ ID NO: 4. 5. The method according to claim 1, which has the nucleotide sequence of SEQ ID NO: 2.
The described DNA. 6. The method according to claim 1, which has the nucleotide sequence of SEQ ID NO: 3.
The described DNA. 7. D according to any one of claims 1 to 6.
A DNA fragment having at least a part of NA and selectively hybridizing with a DNA encoding human ST2. 8. D according to any one of claims 1 to 6.
A vector containing NA. 9. A transformant carrying the vector according to claim 8. 10. The transformant according to claim 9, which is an animal cell. 11. A human ST2 encoded by the DNA according to any one of claims 1 to 6. 12. A method for producing human ST2, which comprises culturing the transformant according to claim 7 and recovering expressed human ST2.