JP2732005B2 - DNA encoding human ST2, expression product of the DNA, and method for producing expression product by expressing the DNA - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA encoding human ST2, and a human ST2 having at least a portion of the DNA.
DNA that can selectively hybridize with 2,
A vector containing the DNA, a transformant carrying the vector, human ST2, and a human ST2 expressing the DNA.
2 relates to a method for producing the same.

[0002]

2. Description of the Related Art In cell division, a DN called G1 phase is used.
A synthesis preparation phase, DNA synthesis phase called S phase, mitotic preparation phase called G2, mitotic phase called M phase and G0
It is known that a stationary phase called a phase appears. Here, the cells that repeat division repeat the growth cycle of G1 → S → G2 → M → G1 phases, but the cells that have transitioned from the M phase to the G0 phase are in a quiescent state of division. However, cells in the resting state in the G0 phase can also shift to the G1 phase by stimulation of proliferation or the like, and can enter the proliferation cycle again.

[0003] In animal tissues, cells having different cycles are present. These cells are extracted, the division is stopped in the G0 phase, and artificial stimulation is again applied to change the G0 phase to the G1 phase. As the technology for initiating the transfer of E. coli has become known, studies for elucidating genes (DNA) that are specifically expressed from the G0 phase to the G1 phase have become active. If the mechanism by which cells in the G0 phase shift to the G1 phase and enter the proliferation cycle becomes clear, it can be applied to the control of cell division of cancer cells, etc., and this study is not limited to basic research. Absent.

[0004] In practice, an animal cell preparation in which division has been arrested in the G0 phase has been used, and artificially stimulated to cause a transition from the G0 phase to the G1 phase, where the specific expression occurs. Research has been carried out such as extracting RNA (Linzer, DIH, et al., Proc. Nat.
l. Acad. Sci. USA, 30, 4271 (1983); Linzer, D .; I. H. Et al., P
rc. Natl. Acad. Sci. USA, No. 81
Volume, 4255 (1984), Hirschhor
n, R. R. Proc. Natl. Acad. Sc
i. USA, 81, 6004 (1984),
Lau, L .; F. EMBO J. et al. Volume 4, 3145
Page (1985), Lau, L .; F. Proc.
Natl. Acad. Sci. USA, Vol. 84, 11
82 (1987), Chavrier, P .; Et al.,
EMBO J.C. 7, 29 (1988)).

[0005] According to these studies, when a cell shifts from the G0 phase to the G1 phase, for example, c-myc, c-myc,
It has been revealed that oncogenes such as fos are specifically expressed.

[0006] However, these studies were conducted from G0 phase to G1 phase.
Attempts to focus on the expression of the gene during the transition to the phase, so that the cells in the G0 phase are artificially stimulated in a very short time of 2 to 3 hours after artificial stimulation in order to eliminate non-specific DNA. Only the DNA that is expressed in a targeted manner was targeted, and there was no knowledge about the DNA that was expressed immediately before the S phase.

[0007] The present inventor has previously proposed a mouse-like system in the G0 phase.
About 10 hours after artificially stimulating the next cultured cells
Focus on genes that are specifically expressed after a relatively long time
As a result of the study, the G0 phase of mouse fibroblasts
Use mRNA expressed specifically in the transitional phase of G1 phase as a template
Known by synthesizing cDNA as
The missing DNA is isolated and further encoded by the DNA
Successfully expressed the protein (Tominaga,
S. et al. , FEBS Lett. ,25
8, 301-304 (1989), Tominaga.
S, et al. , Biochem Biophys.
 Acta,1090, 1-8 (1991)).
The white matter was named mouse ST2.

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

[0009] At least not expressed in cells prepared from brain tissue, heart tissue, lung tissue, liver tissue, spleen tissue, pancreatic tissue, kidney tissue, muscle tissue or testis tissue of mouse CD-1 species, but at least BALB / c-3T
3 (mouse fibroblasts) are expressed during cell proliferation starting from the G0 phase of the cells. That is, the DNA is not expressed in the cells in the G0 phase (quiescent phase), but is expressed as these cells shift from the G0 phase to the G1 phase.

[0010] At least mouse BALB / c-3T3
It is also expressed in cells that have transitioned 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 starting from the G0 phase of the cell. That is, for example, cells derived from meristems may transition from the M phase to the next cell cycle immediately without going through the G0 phase. The DNA is also expressed in such cells, but the expression level is small compared to the expression level at the time of transition from the G0 phase to the G1 phase.

Approximately 5 to 1 after the start of cell proliferation from G0 phase
Its expression reaches its maximum in 2 hours. That is, in the conventionally known DNA, a few minutes after the start of division from the G0 phase.
Although it is specifically expressed in about a time, the DNA has different properties.

The expression results in the synthesis of a cytoplasmic RNA having a molecular weight of 18 to 28S.

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

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 a loop formed by SS bonds between cysteines, which is present in an immunoglobulin. To be able to form three immunoglobulin-like domains means to have at least six or more cysteine residues.

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

Mouse IL-, whose amino acid sequence is known
Extracellular membrane in one receptor, mouse neural cell adhesion protein, mouse basement membrane proteoglycan, HLA-6-2,
The amino acid sequence similarity (identity of the amino acid sequence) to the constant site in the heavy chain constituting secretory chicken IgM is 25.1%, 22.7%, 19.0%, 20.8%,
16.5%.

[0017]

As described above, cells are G
When transitioning from phase 0 to G1 phase, the details of mouse ST2 that is expressed after a relatively long time after the initiation of the transition have been elucidated, but there is no knowledge of proteins similar to mouse ST2, particularly human-derived proteins. There was no.

[0018]

The inventor of the present invention has proposed a mouse ST.
Using a DNA probe derived from 2cDNA, a genomic DNA library derived from human granulocytes was screened, and a clone specifically hybridizing with the DNA probe was obtained. And the genomic DNA contained in this clone
The nucleotide sequence of the fragment was clarified, and the exon portion was estimated from the comparison with the nucleotide sequence of the genomic DNA encoding mouse ST2. PC synthesized based on this exon base sequence
DNA in human cDNA libraries derived from various cells was amplified using the R primer, and a library was selected in which the region between the two PCR primers was amplified. Using the human genomic DNA fragment hybridized with the mouse ST2 cDNA fragment isolated above 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 can express, for example, a protein having the amino acid sequence of SEQ ID NO: 4.

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

The protein of the present invention is a DNA of the present invention.
And there are no other restrictions. One example of the protein of the present invention is a protein having the amino acid sequence represented by SEQ ID NO: 4.

Further, the protein of the present invention includes both "protein containing signal peptide" and "protein not containing signal peptide (mature protein)".

[0024] The DNA of the present invention can be obtained from human cells using a technique described below. In addition, the protein of the present invention can be prepared by ligating the DNA of the present invention to an appropriate vector and using the resulting vector by genetic engineering.

The DNA may be isolated from the genome of human cells or from cDNA using mRNA derived from human cells as a template. It can also be produced by chemical synthesis. Cells from which genomic DNA and mRNA are collected are not particularly limited as long as they are human cells.
In this case, it is desirable to obtain from cells expressing ST2. For example, after binding oligonucleotides to both ends of the DNA of the present invention, a DNA fragment in which a restriction site has been formed using an appropriate restriction enzyme is obtained. A DNA fragment obtained by cleaving the downstream of a sequence necessary for expression of a structural gene such as a promoter of a proliferable vector with a restriction enzyme is obtained, and an expression vector is obtained by ligating the DNA fragments. It is possible to make the host cell express the protein of the present invention.

The DNA of the present invention can be expressed in a conventional host / vector system. Among them, an expression system using animal cells such as CHO and COS is preferred.

With respect to the DNA of the present invention, a DNA probe or the like can be prepared using a part of the base sequence that can be distinguished from a conventionally known base sequence of DNA. The use of such a probe makes it possible to detect, for example, a cell mass in a tissue where cell division is active.

At present, known DNAs may be partially deleted, replaced or inserted with other bases to remove the DNA.
The protein expressed by the expression of NA is made smaller (sometimes solubilized) to enhance or eliminate the properties of the protein, and further to make it easier to express in a certain host, for example. This is commonly done. In the present invention, there is no limitation on performing such an operation on the DNA of the present invention. On the other hand, known proteins are also made smaller (sometimes solubilized) by deleting some of them, substituting them, or inserting other amino acid residues, so that the protein becomes An operation for enhancing or eliminating the property or adding a new function is generally performed. Similar to the above-described operation on DNA, there is no restriction on performing such operation on the protein of the present invention.

[0029]

EXAMPLES The present invention will be described below in more detail with reference to Examples, 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 for screening (Maniatis, T. et.
al. , Molecular Cloning, Co
ld Spring Harbor Laborato
ry (1982)). From about 2 × 10 ⁶ plaques, three positive clones were selected and the recombinant phage was purified. Mapping of the recombinant phage by restriction enzyme digestion, Southern hybridization analysis, and nucleotide sequence determination confirmed that the three clones contained the same genomic DNA fragment.

A part of the base sequence of the cloned genomic DNA fragment encoding human ST2 was
Nucleotide sequence of the cDNA of No. 2 (Tominaga, S. e.)
tal. , FEBS Lett. , 258 , 301-
304 (1989)). As shown in FIG.
A part of the nucleotide sequence of the genomic DNA encoding the human ST2 contained all of exon 7 and part of exon 8. In FIG. 1, the sequence enclosed by □ represents an exon part, and the asterisk represents a stop codon.

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

As is clear 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. Further, if human ST2 genomic DNA is present in the library, a DNA of 397 bp is amplified by PCR.
Fragments are produced.

Using the PCR primers, cDNA libraries derived from various human cells are amplified by PCR.
Thereafter, 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 minute, 50 ° C for 2 minutes, 7 minutes.
Three cycles of 2 ° C for 3 minutes were repeated. A photograph of the gel stained with ethidium bromide is shown in FIG. The names of the clones of the human cells used in producing the cDNA library are shown on the lane, and the size of the DNA fragment is shown on the right side of the lane. In five of the 17 cDNA libraries, a DNA fragment of 305 bases was amplified, and it was found 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 performed normally.

Of the above five 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. S. A. , 84 , 7388-739
2 (1987); Lee, F .; et al. , Pro
c. Natl. Acad. Sci. , U.S. S. A. , 8
2 , 4360-4364 (1985), Yokota,
T. et al. Proc. Natl. Acad.
Sci. , U.S. S. A. , 83 , 5894-5898
(1986)).

Example 3 cDN encoding human ST2
Cloning of A The cDNA library derived from the 5C10 clone of Example 2 was cloned from the 305 cDNA obtained in Example 2 by PCR.
Screening was performed using a base DNA fragment as a probe (Maniatis T. et al., Molec).
ullar Cloning, Cold Spring
According to the method described in Harbor Laboratory (1982)). Two positive clones were obtained from approximately 7 × 10 ⁵ colonies. A cDNA was isolated from a clone having a long cDNA fragment, subcloned into pUC19, and the entire nucleotide sequence was determined.
The A fragment contained the entire open reading frame (ORF) of human ST2. Figure 3 shows the determined base 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 indicated by an arrow.

Embedded image The position of a cysteine presumed to be involved in the formation of an immunoglobulin-like domain is indicated by □. In addition, human ST
From the amino acid sequence of No. 2, a known method (Von Heij
ne, G .; , Nucleic Acids Res. ,
14 , 4683-4690 (1986)), it was estimated that the 17th amino acid from the N-terminal constitutes the signal peptide. The cleavage site of the signal peptide is indicated by an upward white arrow in FIG.

The DNA of the present invention is human ST2 shown below.
G and the amino acid sequence of other proteins
As a result of searching using the enBank DNA database and the NBRF protein database, human ST2
L1-R1 (23.7% of overlapping 299 amino acids),
Human IL1-R2 (22.9 out of 319 overlapping amino acids)
%), Vaccinia virus B16R protein (2 overlapping
24.3% of 92 amino acids), Drosophila Cek
2 protein (23.5% out of 187 overlapping amino acids), chicken klg protein (25 out of 148 overlapping amino acids).
0%). The amino acid sequences of these proteins are shown in FIG. In FIG. 4, “Hu” indicates a human, “Mu” indicates a mouse, and “Vaccinia” indicates
2 shows vaccinia virus B16R protein. Also":"
Indicates the same amino acid, “*” indicates an uncontrasted amino acid, “□” indicates a conserved amino acid of 6 types of amino acids, and “▽” or “☆” indicates a conserved amino acid of 6 types of amino acids. The cysteines presumed to be involved in the formation of the immunoglobulin-like domain are indicated 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 isolated from the 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). It was confirmed whether the cDNA was inserted in the correct direction by restriction enzyme mapping, and the correctly inserted plasmid was introduced into COS7 cells by the DEAE-dextran method (also performing chloroquine treatment) (Sambrook, J 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 expressed protein was radiolabeled in a medium containing ³⁵ S-methionine, and the cell supernatant was recovered 12 hours after the start of radiolabeling. The protein in the supernatant was separated by SDS-polyacrylamide gel electrophoresis, and the position of the protein in the gel was confirmed by fluorography. FIG. 5 shows the result. In FIG. 5, lane 1 is pEF in which human ST2 cDNA is not inserted.
-Culture supernatant of cells transformed with BOS (control)
Lane 2 is pE into which human ST2 cDNA is inserted.
1 shows a culture supernatant of a cell transformed with F-BOS. Since the protein band indicated by the 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, it can be determined that the band corresponds to human ST2. From this, human ST2c
It was confirmed that COS7 cells transformed with the expression vector pEF-BOS into which DNA had been inserted express a large amount of human ST2.

[0039]

Industrial Applicability The DNA of the present invention is specifically expressed when a cell shifts from G0 phase to G1 phase. Therefore, it is possible to obtain important knowledge on cell proliferation by using an antisense RNA or the like against RNA that appears upon expression of the DNA and specifically suppressing the expression of the DNA. Such effects are the same for the protein of the present invention. For example, by preparing an antibody recognizing the protein and using the same, it is possible to obtain important knowledge on cell proliferation. These facts are needed to develop drugs that inhibit the growth of, or even selectively attack, such proliferative cells for diseases for which no effective therapeutic agent is known, such as cancer. It means providing the necessary basic skills.

By using a diffusion probe or a labeled antibody against these DNAs or proteins, it is possible to detect proliferating cells such as cancer cells existing in the tissue.

[0040]

[Sequence list]

[Brief description of the drawings]

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

FIG. 2 shows that various cDNA libraries were compared with human S
D obtained by performing 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).

FIG. 3 shows the nucleotide sequence of human ST2 cDNA and the DNA.
FIG. 3 is a view showing a predicted amino acid sequence 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 (electrophoresis photograph).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (6)

(57) [Claims] 1. A protein comprising the amino acid sequence of SEQ ID NO: 4. 2. A DNA encoding the protein according to claim 1. 3. A vector containing the DNA according to claim 2. A transformant carrying the vector according to claim 3. 5. The transformant according to claim 4, which is an animal cell. 6. A method for producing recombinant human ST2, comprising culturing the transformant according to claim 4 or 5, and recovering the expressed human ST2.