JPH0851978A - Tgf-beta-receptor type ii gene - Google Patents

Abstract

PURPOSE:To obtain the subject gene coding a transforming growth factor-beta (TGF-beta) receptor type II having a specific amino acid sequence, and giving a TGF-beta receptor protein useful for the therapy of cancer, etc. CONSTITUTION:The new TGF-beta receptor type II gene contains a base sequence coding a transforming growth factor-beta (TGF-beta) receptor type II containing an amino acid sequence of the formula, has a TGF-beta-inhibiting activity, and is useful for the therapy of various diseases including cancer, etc., by inserting the gene into a host cell for transforming the host cell and subsequently expressing the gene in the transformed host cell. The gene is obtained by synthesizing a cDNA in the presence of a template comprising a crude RNA extracted from a cell strain originated from human liver cancer, preparing the library of the cDNA, screening the library with a labeled probe, etc., extracting the DNA from the obtained positive clone, and subsequently treating the DNA with a restriction enzyme.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a TGF-β receptor type II gene, more specifically, human TGF containing a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 which is useful, for example, in suppressing the growth of cancer cells. -[Beta] receptor type II gene.

[0002]

2. Description of the Related Art TGF-β (Transforming gro
wth factor-β) was found as a substance that promotes the growth of non-neoplastic fibroblasts on soft agar medium, and was purified and purified from platelets in 1983 (Assoian RK et al., J. Bio).
l. Chem., 258 , 7155-7160 (1983)) Molecular weight about 25,000
Is a protein substance. Currently, five types of TGF-β, β ₁ to β _5, have been reported (Roberts, AB and Spor.
n, MB, Handbook of Experimental Pharmacology, part
I, Springer-Verlag, Berlin, pp419-427 (1990)), and it has been clarified that it has an action of positively or negatively regulating proliferation / differentiation of various cells (Massague, J., Annu.
Rev. Cell. Biol., 6, 67-641 (1990)). That is, TGF-
The functions of β are diverse such as induction of embryogenesis, cell proliferation, angiogenesis, wound healing, and production of extracellular matrix.

On the other hand, with respect to the TGF-β receptor, it is known that three types, type I, type II and type III, are expressed in many cells, and in recent years type II and type III receptors have been cloned one after another. (Lin, HY et a
l., Cell, 68 , 775-785 (1992); Lopez-Casillas, F. et
al., Cell, 67 , 785-795 (1991); Wang, X.-F. et al.,
Cell, 67 , 797-805 (1991)).

The growth of cancer cells is autocrine or paracrine regulated by various growth factors, and the above TGF-β is also produced in various cancer cells, but its growth is suppressed. It has been reported that some cancer cells induce apoptosis (Yanagihara, K. and Tsumuraya, M. Cance).
r Res., 52 , 4042-4045 (1992)). TGF-β produced from stromal cells is considered to be a protective reaction against cancer cells on the host side, but TGF-β, which is disadvantageous to proliferation, is seen from the cancer cell side.
It is irrational to produce -β, and it is considered that the TGF-β receptor is abnormal in order for cancer cells to continue to grow. From this point, clinical use of the TGF-β receptor, which is expected to have a cancer cell growth inhibitory action (anticancer action), is awaited in the art for its research and development.

[0005] Furthermore, TGF-β has been recognized as a multifunctional cell regulator (multifunctional egulator) that maintains its homeostasis not only in the above cancer cells but also in various cells. Against this, for example, it acts to suppress the proliferation and differentiation of hematopoietic cells (Keller
JR et al., J. Exp. Med., 168, 737-750 (1988); Kell
er JR et al., J. Cell Biochem., 39 , 175-184 (198
9)), exerting a growth inhibitory action that prevents abnormal expansion of immunocompetent cells such as IL-2-dependent T cells (Kehrl JH, eta.
L., J. Exp. Med., 163, 1037-1050 (1986)) and the like are known.

[0006] It has been suggested that TGF-β is also useful for treating heart disease, especially myocardial infarction (Tompson, et al.
al., Growth Factors, 1 , 91-99 (1988)) and is involved in the development of lung diseases, especially pulmonary fibrosis and aplastic anemia (Khal
il, et al., J. Exp.Med., 1 70, 727-737 (1989)), and also exerts an inhibitory effect on liver regeneration and liver fibrosis (Br).
aawn, et al., Proc.Natl.Acad.Sci., USA, 85, 1539-1
543 (1988)) and renal disease (glomerular proliferative disease), the proliferation of mesangial cells was suppressed, and the administration of a neutralizing antibody against TGF-β suppressed the development of acute glomerulonephritis (Broder, et al. al., Ciba Foundation Symposium No.157
(abstract)), and dramatically promotes insulin secretion in islet cells isolated from the pancreas (Totsuka, et al., Biochem.
iophys.Res.Commun., 158, 1060-1065 (1989)) and suppress proliferation of thyroid follicular cells (Grabeck, et al.,
J. Clin. Invest., 83 , 764-770 (1989)) is also known.

In addition, TGF-β has been proposed for surgical use such as fracture healing and osteosynthesis due to its immunosuppressive action and bone formation promoting action (Masaki Noda, Experimental Medicine 8 , 345 (1990);
Wang, EA, et al., Proc.Natl.Acad.Sci., USA, 87 , 22
20 (1990), and it was shown to be useful for wound treatment due to its high concentration in platelets (Mustoe, T. et a.
l., Science, 237, 1333 (1987)), which is also involved in proliferation of human dermal fibroblasts, and suppresses proliferation of epidermal keratinocytes (Shipley GD, et al., Cancer Res., 46 ,
2085-2091 (1986)) and the secretion promoting action of fibronectin (Wikner NE, et al., J. Invest. Dermatol., 91, 207).
-212 (1988)) has been reported, and clinical application in the dermatological field as a wound treatment promoting substance is being researched and developed.

[0008] Therefore, if TGF-β and its receptor can be produced in large quantities by genetic engineering techniques, it will be useful for elucidating the pathogenic mechanism of various diseases and the like, and by extension, therapeutic methods for the various diseases and the like can be established. It is considered to be.

The inventors of the present invention have conducted extensive studies based on the above-mentioned object. As a result, previously, using the mouse TGFβ type I receptor cDNA as a probe, a commercially available PHA-stimulated T cell cDNA was prepared.
The TGF-β receptor type I gene was successfully cloned from the A library by the plaque hybridization method, the invention relating to this gene was completed, and a patent application was filed (Japanese Patent Application No. 5-316113).

Further, in the subsequent studies, the inventors of the present invention reported the human TGFβ receptor type II cDNA (Lin, HY et al., Cell, 68 , 77) previously reported by Lin et al.
5-785 (1992)) has led to the identification and elucidation of the same type II gene having a new sequence that is essentially different in several respects, and has completed the present invention.

[0011]

That is, according to the present invention,
Provided is a TGF-β receptor type II gene characterized by comprising a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, and a TGF-β receptor type II gene comprising the nucleotide sequence represented by SEQ ID NO: 2. To be done.

Hereinafter, the abbreviations for amino acids, peptides, base sequences, nucleic acids and the like in the present specification refer to IUPA.
The provisions of C and IUB, “Guidelines for preparing specifications including base sequences or amino acid sequences” (Edited by the Patent Office), and symbols conventionally used in the field shall be followed.

The above SEQ ID NO: 1 provided by the present invention
The nucleotide sequence encoding the amino acid sequence shown in, typically the nucleotide sequence shown in SEQ ID NO: 2, is compared with the cDNA of the same type II gene reported in Lin et al. Extracellular domain of (extracellar
domain) has an insertion of 75 bases, and Va is the 9th amino acid residue from the N-terminus of the domain.
The difference is that 1 disappears and, instead, a specific 26 amino acid residue is newly increased and a subtraction of 25 amino acid residues is recognized. This difference is due to the gene
Difference in splicing after transcription of NA (Alternativ
e splicing).

Second, in the gene of the present invention, the codon TTT encoding the 53rd amino acid, Phe, is Se.
It has been changed to TCT that encodes r, and thirdly, 43
The codon GCT encoding the 9th amino acid Ala is V
It has changed to a GTT that encodes al.

The gene of the present invention having the above-mentioned characteristics has its N
A nucleotide sequence encoding an amino acid sequence of a signal peptide consisting of 17 amino acid residues for the expression of TGF-β receptor gene starting from methionine at the end, and a more hydrophobic transmembrane region (hydrophobic transmem) at the C-terminal
brane region) and subsequent intracellular domain (cy
toplasmic domain). Therefore, in the gene of the present invention, not only the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1 above,
Along with this, those containing the above-mentioned signal sequence, transmembrane region, and base sequence of intracellular domain are also included.

Further, the gene of the present invention is represented by a single-stranded DNA sequence as shown in the above SEQ ID NO: 2, but the present invention is a DNA sequence complementary to such a single-stranded DNA sequence or the like. Components that include both are also included. The DNA sequence representing the gene of the present invention shown in SEQ ID NO: 2 is an example of one combination of codons encoding each amino acid residue according to the amino acid sequence shown in SEQ ID NO: 1, and the gene of the present invention has Of course, it is also possible to have a DNA base sequence in which an arbitrary codon is combined and selected for each amino acid residue without changing the above amino acid sequence. The selection of the codon can be carried out according to a conventional method considering the frequency of codon usage of the host used for gene recombination (Ncl. Acids Res., 9, 43-74 (1981)). It can be produced by synthesis or the like.

Further, in the gene of the present invention, a part of amino acids or amino acid sequences of the amino acid sequence shown above is deleted,
Also included is a DNA sequence that is modified by addition or the like and encodes the same drug having the same activity as the TGF-β receptor. The production, modification (mutation) and the like of these polypeptides may occur naturally, and they can be produced by post-translational modification. Furthermore, a natural gene (the gene of the present invention) is modified by a genetic engineering method, for example, by a method such as cytospecific mutagenesis, or a mutated DNA is synthesized by a chemical synthesis means such as a phosphite triester method, Both can be combined to synthesize a desired gene.

Utilizing the gene of the present invention, that is, for example, by incorporating the gene into a microbial vector and culturing a transformed microorganism, the TGF-β receptor can be easily expressed in a large amount, and the protein can be expressed. It can be isolated and provided.
Since it has TGF-β inhibitory activity, it is effective for various pharmacological applications including the above-mentioned suppression of cancer cell growth, and is also useful for elucidating the mechanism of occurrence of various diseases and pathological conditions. .

T obtained by using the gene of the present invention
The GF-β receptor can also be used to produce antibodies specific to the TGF-β receptor. The component used as an antigen here may be a protein produced in large quantities according to the above-mentioned genetic engineering method, and the antibody obtained may be either a polyclonal antibody or a monoclonal antibody.
It can be advantageously used for measurement and identification. Furthermore, according to various genetic engineering techniques using a part or all of the nucleotide sequence of the gene of the present invention as a labeling probe, human TGF having an amino acid sequence encoded by the human TGF-β receptor gene is used.
It is possible to easily produce and purify -β receptor or its equivalent, and to obtain an antibody against them.

The gene of the present invention can be isolated by a general genetic engineering method, for example, TGF according to the report of Lin et al.
cDNA of βII type receptor (total RNA was isolated from cells capable of expressing TGF-β receptor, and
CDN isolated from RNA, purified, and synthesized according to a conventional method
It can be carried out by selecting a positive clone from a commercially available PHA-stimulated T cell cDNA library according to the plaque hybridization method using A) as a probe, purifying the positive clone, and determining its nucleotide sequence according to a conventional method. . Thus, the gene of the present invention (TGF-βI
CDNA of type I receptor) can be obtained.

In the above-mentioned method, the origin cells used for the isolation of total RNA include cells of various animals known to have TGF-β receptors, soluble fractions of cultured cells derived from tissues, tissues and the like. It may be any, and it can be isolated and purified from the culture supernatant according to various chromatographic procedures.

The TGF-β receptor expression-originating cells can be cultured according to a conventional cell culture method using an appropriate cell culture medium. The medium used here includes
For example, RPMI-1640 medium, CEM medium, CMRL
-1066 medium, Dulbecco's modified Eagle's minimum essential medium (Eagle's MEM), Fisher's medium, F-10 medium and the like can be exemplified. If necessary, serum such as fetal calf serum (FCS) or serum component such as albumin can be appropriately added to the iso-medium. Culturing can be carried out by a conventional method, for example, a carbon dioxide gas culturing method or the like, and is generally about 30-40.
℃, preferably around 37 ℃, for about 5 to 17 days,
Preferably, it takes about 8 to 11 days.

Separation of total RNA from the above-mentioned cultured cells or tissues can be carried out according to a general extraction method. This extraction operation is the time when the most amount of TGF-β receptor is produced and accumulated in the culture supernatant by the above-mentioned culture. It should be done at.
In addition, when total RNA is extracted from the above-mentioned tissue of origin or cultured cells, for example, when tissue is used, EDTA, D
After crushing the tissue in a suitable buffer solution such as potassium phosphate buffer solution containing TT (dithiothreitol) and the like, a guanidine / isocyanate mixture solution and a suitable surfactant such as SDS, NP-40, Triton X100 , Or deoxycholic acid, or by a physical method such as a homogenizer or freeze-thaw, it is partially or completely destroyed and solubilized, and then the chromosomal DNA is polytron (POLYTRON, Kinema).
It may be carried out by shearing to some extent using a mixer or a syringe such as tica Switzerland), and then separating the protein from the nucleic acid fraction. For this operation, in particular, a cesium chloride multi-layer method (Chirgwin, J. et al.) Using phenol / chloroform extraction or ultracentrifugation of about 100,000 × g.
M., et al., Biochemistry, 18 , 5294 (1979)) and the like can be generally adopted. In each of the above methods, RNa
RNase inhibitors such as heparin, polyvinylsulfate, diethylpyrocarbonate, vanadium complexes, bentonites to prevent RNA degradation by se.
It is advisable to add macaroid, etc.

To separate and purify mRNA from RNA obtained according to the above extraction procedure, the extract is treated with, for example, oligo dT-
Cellulose (Collaborative Research Inc.), Poly U
-Sepharose (Pharmacia), Sepharose 2B
(Manufactured by Pharmacia) or by a batch method.

The purified mRNA obtained as described above is usually unstable and is replaced by a stable complementary DNA (cDNA) type, and is ligated to a replicons derived from a microorganism in order to enable amplification of a target gene. The above mR in vitro
Conversion of NA into cDNA, that is, synthesis of cDNA can be generally performed as follows.

That is, first, oligo dT was used as a primer (this primer may be either free oligo dT or oligo dT already added to the vector primer), and mRNA is used as a template for dNTP (dAT).
P, dGTP, dCTP or dTTP) in the presence of reverse transcriptase from mRNA to a complementary single strand c thereof
Synthesize DNA. The next steps differ as follows depending on whether the free oligo dT or the oligo dT added to the vector primer was used in the above.

In the former case, mRNA used as a template is decomposed and removed by alkali treatment or the like, and then double-stranded DNA is prepared by using reverse-transcriptase or DNA polymerase with single-stranded DNA as a template. Double-stranded DN obtained next
Both ends of A are treated with exonuclease, a plurality of bases in an appropriate linker DNA or an annealable combination is added to each, and this is incorporated into an appropriate vector.
This is a known method depending on the vector used,
For example, it can be performed according to the method of Gubler and Hoffman. For the synthesis of the above cDNA, commercially available cDNA
It is also possible to use the A synthesis kit, which has the advantage of easier operation. The vector used here is not particularly limited, but it is preferable to select a λgt-based phage vector, an EK-based plasmid vector or the like, alone or in combination, and appropriately select according to the host. Λ above
Specific examples of the gt-based phage vector include λgt1
0, λgt11, etc. can be exemplified. The method using the λgt-based phage vector can be based on the method of Young et al. (Young, RA, et al., In DNA Cloning, 1,
49 (1985)).

In the latter case, an open circular plasmid in which a plurality of bases in the same annealable combination as described above are added while leaving the mRNA used as the template, and linker D
NA (often an autonomously replicating region and mRN in animal cells)
A DNA fragment containing the transcriptional promoter region of A) is used to anneal the
In the presence of P, RNase H and DNA polymerase I are allowed to coexist, and mRNA is replaced with a DNA chain to prepare a complete plasmid DNA.

The DNA obtained as described above is used as a vector host, for example, Escherichia coli.
a coli), Bacillus subtilis
, Saccharomyces cer
evisiae) and the like, and can be transformed into by introducing into a suitable host. As a method for introducing this DNA into a host and transforming it, a generally used method is used, for example, cells in a logarithmic growth phase are mainly collected, treated with CaCl ₂ and naturally DN.
It is possible to adopt a method in which A is easily incorporated so that the plasmid is incorporated. In the above method, MgCl ₂ or RbCl can be further coexistent to further improve the efficiency of transformation, as is generally known.
A method in which microbial cells are transformed into spheroplasts or protoplasts and then transformed can also be used. For details of these methods, see Gubler and Hoffman's method (Gubler, U. an.
d Hoffman, BJ, Gene, 25 , 263 (1983)). When a λ phage, which is commonly used as a phage vector, is used as a vector, a cDNA library using λ phage can be prepared by in vitro packaging. As the cDNA library, commercially available cD
NA library, eg Clontech
It is also possible to use various cDNA libraries commercially available from the company.

Screening of the gene of the present invention from the thus obtained cDNA library can be carried out by an ordinary method. As the screening method,
For example, a method of selecting a corresponding cDNA clone by Western blotting using a TGF-β receptor-specific antibody against a protein produced by cDNA, a Southern blotting method using a probe that selectively binds to a target DNA sequence, The northern blotting method and the like and combinations thereof can be exemplified. As the probe used here, it is common to use a DNA sequence chemically synthesized based on the information on the target DNA or RNA sequence or the amino acid sequence encoded by the sequence,
DNA or RNA prepared from nature can also be used as such a probe.

More specifically, the above-mentioned probe is prepared as follows. That is, poly (A) ⁺ RNA is selected from RNA obtained from tissues or cultured cells containing TGF-β receptor type II protein by an oligo dT-cellulose column, and single-stranded cDNA is selected according to the above method.
After synthesizing A and terminating the reaction, a primer that is considered to correspond to part of the amino acid sequence information of TGF-β receptor type II was synthesized by an automatic oligonucleotide synthesizer, and this was used to perform PCR (Saiki, RK, et al., S
cience, 230 , 1350-1354 (1985))
A is amplified.

Next, the amplified cDNA fragment is isolated and purified by 1.0% agarose gel electrophoresis. The base sequence of the DNA fragment obtained above can be determined by a conventional method. For example, the obtained DNA fragment is digested with an appropriate restriction enzyme, and then the dideoxy method (Sanger F., Nickl
en S. and Coulson AR, DNA sequencing with chain-
terminating inhibitors, Proc.Natl.Acad.Sci., US
A., 74 , 5463-5467 (1977)) and Maxam-Gilbert method (M
axam, AM and Gilbert, W., Method in Enzymology, 65 ,
499 (1980)) and the like. Furthermore, the base sequence can be easily determined using a commercially available sequencing kit or the like.

Thus, the determined gene of the present invention (TG
The entire DNA base sequence including F-β receptor type II) is as shown in SEQ ID NO: 3.

In the present invention, a part of the DNA fragment sequenced above is used as a probe, and this is used, for example, in a random prime DNA labeling method (Feinberg, AP, et al.,
Anal.Biochem., 137, 266-267 (1984)) and labeled with a random prime DNA labeling kit (manufactured by Takara Shuzo, Amersham, etc.), and the labeled probe thus obtained is the target TGF-β. Receptor type I
It can also be used for I gene screening.

The target DNA is screened by the plaque hybridization method (Benton, W. an developed by Benton and Davis using the above-mentioned labeled probe and the like.
d Davis, R., Science, 196 , 383-394 (1977)).

The gene of the present invention obtained above can be cloned into various plasmids according to a conventional method. For example, after cutting with an appropriate restriction enzyme, the purified gene fragment of the present invention is
The recombinant plasmid can be obtained by inserting into the cleavage site of a cloning vector such as a plasmid purified by cutting with the same restriction enzyme. The recombinant is transduced into an appropriate host, for example, Escherichia coli, and the transformant is subjected to a generally known method such as Molecular Cloning (A Lab).
oratory Manual), T. Maniatis, EFFritsch, J. Sambro
ok, Cold Spring Harbor Laboratory (1982) p104-106
By following the method described in 1) or the like, a restriction enzyme map of a clone encoding the gene can be prepared. The nucleotide sequence of the clone can be determined by digesting it with an appropriate restriction enzyme and then using the dideoxy method or the Maxam-Gilbert method. Furthermore, the above-mentioned base sequence can be easily determined even by using a commercially available sequence kit or the like.

Thus determined human TGF of the present invention
-D of the extracellular domain of the β receptor type II gene
The NA base sequence and the corresponding amino acid sequence encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

As is clear from the above SEQ ID NO: 2 and SEQ ID NO: 3, the DNA sequence of the gene of the present invention (extracellular domain) follows the 5'38 untranslated region 17
It has a total of 1918 bases including the translated region of 79 bases (including the termination codon TGA) and the untranslated region on the 3'side (101 bases) following it. The translation region is 5
It corresponds to a protein of 92 amino acid residues.

Utilizing the above-mentioned gene (DNA) of the present invention,
Various publicly known gene recombination techniques [for example, Science, 224 , 143
1, (1984); Biochem.Biophys.Res.Comm., 130, 692 (19
85): Proc.Natl.Acad.Sci., USA, 80, 5990 (1983)
Etc.], recombinant TGF-β receptor type II can be obtained.

More specifically, the production of the TGF-β receptor type II is carried out by preparing a recombinant DNA capable of expressing the gene of the present invention in a host cell, introducing the recombinant DNA into the host cell and transforming it. It is carried out by culturing the transformant. Here, as a host cell, either a eukaryote or a prokaryote can be used. As a vertebrate cell expression vector, a vector having a promoter located upstream of the gene to be expressed, an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used. May have. As the eukaryotic microorganism, yeast is generally often used, and among them, Saccharomyces yeast can be advantageously used. Examples of expression vectors for eukaryotic microorganisms such as the yeast include pAM82 [A. Miyanohara et al, Proc. Natl.
Acad. Sci., USA, 80 , 1-5 (1983)], etc. can be used. Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. In the present invention, a plasmid vector capable of replicating in the host bacterium is used, and a promoter and an SD (Shine-and-Synthesis) upstream of the gene are used so that the gene of the present invention can be expressed in the vector. It is preferable to use an expression plasmid having a Dalgarno) nucleotide sequence and a start codon (eg, ATG) necessary for initiation of protein synthesis. Examples of Escherichia coli as the above host include Escherichia coli K1.
Two strains are often used, and pBR is commonly used as a vector.
322 is often used, but it is not limited thereto, and various known strains and vectors can be used. As the promoter, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, P _L promoter and the like can be used.

Various general methods can be adopted as a method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming the same. The resulting transformant can be cultured by a conventional method, and the desired TGF-β receptor encoded by the gene of the present invention is produced and accumulated by the culture. The medium used for the culture,
Various kinds of conventional ones can be appropriately selected and used according to the adopted host cell. For example, LB medium, E medium, M9 medium, M63 medium, etc. can be used for culturing the transformant using Escherichia coli etc. as a host cell. , Nitrogen sources, inorganic salts, vitamins, natural product extracts, physiologically active substances, etc. can be added.
Cultivation of the above transformant can be carried out under conditions suitable for the growth of host cells. In the case of E. coli, for example, pH is about 5-8, preferably 7 or thereabout, temperature is about 20-43 ° C, preferably 37 ° C. Or the vicinity can be adopted. Based on the above, the target recombinant TG inside or outside the transformant
F-β receptor protein is produced, accumulated or secreted.

The target protein is subjected to various separation operations utilizing its physical and chemical properties ("Biochemical Data Book II", pages 1175-1259, 1st edition, 1st edition, June 23, 1980).
Published by Nihon Tokyo Kagaku Dojin; Biochemistry, vol.25,
No.25, 8274-8277 (1986); Eur. J. Biochem., 163, 313
-321 (1987) etc.). Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out method), centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration, molecular sieve chromatography (gel filtration). , Adsorption chromatography, ion exchange chromatography, affinity chromatography,
Various liquid chromatography such as high performance liquid chromatography (HPLC), a dialysis method, a combination of these and the like can be exemplified. As described above, the desired recombinant protein can be easily produced in high yield on an industrial scale.

[0043]

According to the present invention, a TGF-β receptor type II gene is provided. With the gene,
The TGF-β receptor type II protein can be easily produced in a large amount, and the TGF-β receptor type II
Has a TGF-β inhibitory activity, and can be used for the treatment of various diseases including cancer.

[0044]

EXAMPLES Examples will be given below to explain the present invention in more detail.

[0045]

[Example 1] Cloning of TGF-β receptor type II (1) cDNA library As a cDNA library, human Keratinocyte cDNA (epidermal primary culture, ad) manufactured by Clontech was used.
ult, oligo (dT) -primed, CLHL1045b, sold by Toyobo Co., Ltd.)
Was used. This is 0.5 to 3.8 in the vector λgt10.
A cDNA of kb (1.1 kb on average) was inserted, and C600 was used as a dedicated host Escherichia coli in the above commercial products.
The stock is attached.

(2) Preparation of TGF-β Receptor Type II Screening Probe Using crude RNA extracted from HepG2 cells (human hepatoma-derived cell line, Dainippon Pharmaceutical Co., Ltd.) as a template, the two RNAs shown in Table 1 below were prepared. A 0.5 Kb cDNA fragment was prepared by amplification with a primer and used as a probe. The probe is a human TGFβ receptor type II cDNA
Among them, a portion encoding the secretory signal sequence and the extracellular domain, and the sequence of its cDNA fragment is shown in SEQ ID NO: 4.

[0047]

[Table 1]

(3) Cloning by Plaque Hybridization Method Using the cDNA library of (1) above and the screening probe of (2) above, the plaque hybridization method developed by Benton and Davis (Science, 196 , 383-394 (1977)).

First, L medium containing 0.2% maltose (1
% Bactotryptone, 0.5% yeast extract and 0.5%
The cDNA library of (1) above was added to 1.5 ml of the host strain C600 strain cultured overnight at 37 ° C. in NaCl), and the mixture was added at 37 ° C. After incubating for 15 minutes at 50 ° C., this was mixed with top agar (25 ml) heated to 50 ° C., and the mixture was immediately added to L medium 2 containing 10 mM MgSO _4.
Plastic petri dish containing 50 ml (24.5 x 2
4.5 cm) and spread it to solidify. When the dish was cultured overnight at 37 ° C, many plaques were formed on the medium surface.

Next, a nylon membrane filter (Biodyne A, pore size 0.2 μm, 22
A λ phage on the surface of the medium was adsorbed to the filter by placing a 2 × 222 mm, Pall) and contacting for 1 minute. The filter was placed in a denaturing solution (0.
Contact with 5N NaOH + 1.5M NaCl), then neutralizing solution (0.5M Tris HCl (pH 7.
0) + 1.5M NaCl), and further 2 minutes for 2 minutes
SSC solution (3M NaCl 17.54 g / l + 0.
3M sodium citrate dihydrate 8.82 g / l)
Contacted with. After air-drying the obtained filter, 8
Heat treatment was performed at 0 ° C. for 2 hours.

Then, the filter was put in a hybridization bag, and the prehybridization solution (100 × Denhardt's solution 5 ml, 20 × SSC solution 2
5 ml, 1 M Tris HCl (pH 7.5) 5 ml, 10
% SDS 1 ml, sonicated salmon sperm DNA (sssD
NA, 50-100 μg / ml) 1 ml and dH ₂ O 6
(3 ml) was added to each filter in an amount of 50 ml, and the bag was sealed while being careful not to leave air bubbles in the bag, and left at 65 ° C. overnight.

Next, the prehybridization solution was removed from the bag, and 5 μl of the labeled probe was added to the hybridization solution (100 × Denhardt solution 1 m
1, 20 × SSC solution 25 ml, 1M Tris HCl (p
H7.5) 5 ml, 10% SDS 1 ml, sssDN
A) 1 ml, dH ₂ O 67 ml and labeled probe 10 ⁶
cpm / bag) was added to each filter in an amount of 50 ml and left at 55 ° C. overnight. The labeled probe was prepared by using the 0.5 kb probe DNA shown in (2) above with a random primer DNA labeling kit (Takara Shuzo) according to the manufacturer's instruction manual.

Thereafter, the filter taken out from the bag was washed with a cleaning solution (2 × SSC + 0.1% S) heated to 55 ° C.
20 minutes in DS). This washing operation was repeated 4 times, and finally, washing was carried out in a washing solution of 0.2 × SSC + 0.1% SDS for 20 minutes, followed by air drying. The air-dried filter was wrapped in Saran wrap, brought into contact with the X-ray film, and allowed to stand at -80 ° C for 2 days, and then the X-ray film was developed.

As a result of the above operation, 6 positive signals which could be confirmed as black spots on the X-ray film were obtained. Compare the X-ray film with the original petri dish,
The plaque corresponding to the position where the above signal was found is sucked up together with the agar medium with the tip of a Pasteur pipette, and the phage particles contained therein are mixed with 1 ml of λdi solution (1
00 mM NaCl, 10 mM MgSO ₄ , 35 mM
It was extracted with Tris HCl (pH 7.5), 0.1% gelatin).

For each of the phage particle extracts corresponding to the positive signals thus obtained, λdi
A solution diluted to 500, 5,000 and 50,000 times with 1 l solution was prepared, and 0.2% for each 100 μl
C60 cultured overnight at 37 ° C in L medium supplemented with maltose
After adding 200 μl of 0 strain solution and incubating at 37 ° C for 15 minutes, 2.5m of top agar heated to 50 ° C
The mixture was mixed with 1 and immediately spread on a plastic petri dish having a diameter of 9 cm containing 25 ml of L agar medium containing 10 mM MgSO ₄ and spread to solidify.

After culturing at 37 ° C. overnight, 10 per dish.
Select a Petri dish in which about 0 plaques have been formed, and use this to repeat the procedures of filter adsorption, hybridization, washing, and autoradiography in the same manner as described above to detect recombinant phages showing a positive signal. The strain was isolated. Each of them is referred to as “λKβR
II-1 ”and“ λKβRII-6 ”.

(4) Preparation of recombinant phage DNA Escherichia coli LE392 strain (F ⁻ , hsdR514 (r _k ⁻ ,
m _k ⁺ ), supE44, supF58, lacY1,
galK2, galT22, metB1, trpR5
5, λ ⁻ , macA ⁻ , mcrB ⁺ , manufactured by Toyobo Co., Ltd.
-Mal medium (1% bactotryptone, 0.25% Na
Cl and 0.2% maltose) were cultured overnight at 37 ° C. in 5 ml, and the obtained cells were suspended in 1 ml of a λdi solution. To 70 μl of this suspension, 10 μl of the λdi solution of the recombinant phage isolated above was added, and the mixture was incubated at 37 ° C. for 15 minutes, and then L-MgSO ₄ medium (10 mM MgSO ₄
5 ml of added L medium) was added, and the mixture was cultured at 37 ° C. for about 6 hours. When the once turbid liquid became transparent again, 70 μl of chloroform was added and the mixture was further incubated for 15 minutes at 37 ° C.
The supernatant was obtained by centrifugation (10000 rpm, 10 minutes, 4 ° C.).

To the above supernatant, 5 ml of a PEG solution (λdi medium containing 10% polyethylene glycol and 11.7% NaCl) was added, mixed well and allowed to stand overnight at 4 ° C.
This mixture is centrifuged (12000 rpm, 10 minutes,
The precipitate was collected at 4 ° C). The precipitate was dissolved in 375 μl of L medium, and an L medium suspension (20 to 30 w / w%) of diethylaminoethyl cellulose (manufactured by Whatman, DE52) was dissolved.
After adding 375 μl and mixing well, centrifugation (120
(00 rpm, 5 minutes, room temperature) to obtain a supernatant. The supernatant was extracted with phenol and then with phenol / chloroform. Finally, 75 μl of 3M sodium acetate (pH 5) and 500 μl of isopropanol were added, and the mixture was allowed to stand at −20 ° C. for 90 minutes or longer, and the resulting precipitate was centrifuged (12000 rp).
m, 5 minutes, 4 ° C.) and dried, then TE (10
mM Tris HCl (pH 8.0) +1 mM EDTA)
It was dissolved in 50 μl.

(5) 2.8 kbE of λKβRII-6 strain
Subcloning of coRI fragment 10 μl of the prepared λDNA solution was added to 40 units of restriction enzyme EcoRI (Takara Shuzo) in 200 μl of the reaction solution.
The reaction was carried out at 37 ° C. for 4.5 hours for digestion, 1% agarose gel electrophoresis, and ethidium bromide staining. As a result, both λKβRII-1 strain and λKβRII-6 strain produced a DNA fragment of about 2.8 kb. Was confirmed.

From the above gel, 2.8 of strain λKβRII-6 was obtained.
The kb DNA fragment is cut out and DN is applied to a DNA collection filter-attached centrifugal tube Suprec-01 (Takara Shuzo).
A was collected.

On the other hand, the plasmid pU19 was used as a vector.
5 μg (Takara Shuzo) was digested with EcoRI40 unit, 0.5 μg of which was mixed with the DNA fragment recovered above and ligated with T4 DNA ligase (Takara Shuzo) (reaction volume 40 μl). Escherichia coli JM107 strain was transformed with the ligated DNA, and this was ampicillin 10
L containing 0 μg / ml, 5-bromo-4-chloro-3-indolyl-β-D-galactoside 40 μg / ml and isopropyl-β-D-thiogalactopyranoside 1 mM
After inoculating to an agar medium and culturing at 37 ° C. overnight, white colonies were separated. A plasmid DNA was prepared from the obtained colony by the mini-preparation method, and it was confirmed that a cDNA fragment of about 2.8 kb was inserted into this recombinant plasmid.

(6) 2.8 kbE of λKβRII-6 strain
Construction of restriction enzyme map of coRI fragment The recombinant plasmid obtained in (5) above was treated with ApaLI,
AvaI, BglII, HincII, HpaI, Na
eI, NarI, NcoI, NspI, PstI, Sa
Digested with cI and SmaI restriction enzymes to give 1% or 1.8
The restriction enzyme map was prepared by analysis by% agarose gel electrophoresis.

As a result, in the plasmid cDNA,
Restriction enzymes BglII (1), HincII (2), Ps
It was found that the recognition sites for tI (1) and SacI (1) are present at least in the numbers in parentheses.

The length of the restriction enzyme digested fragment obtained above was determined by electrophoresis, and the results are shown in FIG.

(7) 2.8 kbE of λKβRII-6 strain
Determination of Nucleotide Sequence of CoRI Fragment A sample reacted with an autocycle sequence kit manufactured by Pharmacia was manufactured by Pharmacia LKB.
L. F. It was analyzed with a DNA sequencer. As for the part which could not be completely decoded by this analysis method, the same sample was used as Sequenase Version 2.
React with a 0DNA sequence kit, V from Takara Shuzo
It was analyzed and decoded using an E-4 type DNA sequence analysis electrophoresis apparatus.

The nucleotide sequence thus determined (the result of sequencing the DNA fragment indicated by the arrow in FIG. 1) is shown in SEQ ID NO: 3.

As described above, the TGF-β receptor type II gene of the present invention consists of a total of 1918 bases including the translation region and the 5'side and 3'side untranslated regions, and the translation region contains 592 amino acid residues. 39 to 1 corresponding to the base protein
It has a length of 1779 bases at position 817, of which 3
The 23 amino acid residues corresponding to positions 9 to 107 are signal peptides, the 161 amino acid residues corresponding to positions 108 to 590 are extracellular domains, and the 30 amino acid residues corresponding to positions 591 to 680 are transmembrane domains. And 68
The 378 amino acid residues corresponding to positions 1-1814 were found to be the intracellular domain.

[Sequence list]

SEQ ID NO: 1 Sequence length: 161 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence: Ile Pro Pro His Val Gln Lys Ser Asp Val Glu Met Glu Ala Gln Lys 5 10 15 Asp Glu Ile Ile Cys Pro Ser Cys Asn Arg Thr Ala His Pro Leu Arg 20 25 30 His Ile Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys 35 40 45 Phe Pro Gln Leu Cys Lys Ser Cys Asp Val Arg Phe Ser Thr Cys Asp 50 55 60 Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu 65 70 75 80 Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn 85 90 95 Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp 100 105 110 Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys 115 120 125 Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu 130 135 140 Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro 145 150 155 160 Asp

SEQ ID NO: 2 Sequence Length: 483 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA Sequence: ATCCCACCGC ACGTTCAGAA GTCGGATGTG GAAATGGAGG CCCAGAAAGA TGAAATCATC 60 TGCCCCAGCT GTAATAGGAC TGCCCATCCGACTACGAGACATAGA120GACATAGA 120 ACTGACAACA ACGGTGCAGT CAAGTTTCCA CAACTGTGTA AATCTTGTGA TGTGAGATTT 180 TCCACCTGTG ACAACCAGAA ATCCTGCATG AGCAACTGCA GCATCACCTC CATCTGTGAG 240 AAGCCACAGG AAGTCTGTGT GGCTGTATGG AGAAAGAATG ACGAGAACAT AACACTAGAG 300 ACAGTTTGCC ATGACCCCAA GCTCCCCTAC CATGACTTTA TTCTGGAAGA TGCTGCTTCT 360 CCAAAGTGCA TTATGAAGGA AAAAAAAAAG CCTGGTGAGA CTTTCTTCAT GTGTTCCTGT 420 AGCTCTGATG AGTGCAATGA CAACATCATC TTCTCAGAAG AATATAACAC CAGCAATCCT 480 GAC 483

SEQ ID NO: 3 Sequence length: 1918 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA Direct origin Library name: Human T-Keratinocyte cDNA library Clone name : CLHL1045b Sequence features: Characteristic symbols: 5'UTR Location: 1. ． 38 Method of determining features: S Features of sequence: Symbol representing features: CDS Location: 39. ． 1817 Method of determining the feature: S Sequence feature: Symbol representing the feature: 3'UTR Location: 1818. ． 1918 was determined characteristic way: S sequence: GCTGGGGGCT CGGTCTATGA CGAGCAGCGG GGTCTGCCAT GGGTCGGGGG CTGCTCAGGG 60 GCCTGTGGCC GCTGCACATC GTCCTGTGGA CGCGTATCGC CAGCACGATC CCACCGCACG 120 TTCAGAAGTC GGATGTGGAA ATGGAGGCCC AGAAAGATGA AATCATCTGC CCCAGCTGTA 180 ATAGGACTGC CCATCCACTG AGACATATTA ATAACGACAT GATAGTCACT GACAACAACG 240 GTGCAGTCAA GTTTCCACAA CTGTGTAAAT CTTGTGATGT GAGATTTTCC ACCTGTGACA 300 ACCAGAAATC CTGCATGAGC AACTGCAGCA TCACCTCCAT CTGTGAGAAG CCACAGGAAG 360 TCTGTGTGGC TGTATGGAGA AAGAATGACG AGAACATAAC ACTAGAGACA GTTTGCCATG 420 ACCCCAAGCT CCCCTACCAT GACTTTATTC TGGAAGATGC TGCTTCTCCA AAGTGCATTA 480 TGAAGGAAAA AAAAAAGCCT GGTGAGACTT TCTTCATGTG TTCCTGTAGC TCTGATGAGT 540 GCAATGACAA CATCATCTTC TCAGAAGAAT ATAACACCAG CAATCCTGAC TTGTTGCTAG 600 TCATATTTCA AGTGACAGGC ATCAGCCTCC TGCCACCACT GGGAGTTGCC ATATCTGTCA 660 TCATCATCTT CTACTGCTAC CGCGTTAACC GGCAGCAGAA GCTGAGTTCA ACCTGGGAAA 720 CCGGCAAGAC GCGGAAGCTC ATGGAGTTCA GCGAGCACTG TGCCATCATC CTGGAAGATG 780 ACCGCTCTGA CATCAGCTCC ACGTGTGCCA A CAACATCAA CCACAACACA GAGCTGCTGC 840 CCATTGAGCT GGACACCCTG GTGGGGAAAG GTCGCTTTGC TGAGGTCTAT AAGGCCAAGC 900 TGAAGCAGAA CACTTCAGAG CAGTTTGAGA CAGTGGCAGT CAAGATCTTT CCCTATGAGG 960 AGTATGCCTC TTGGAAGACA GAGAAGGACA TCTTCTCAGA CATCAATCTG AAGCATGAGA 1020 ACATACTCCA GTTCCTGACG GCTGAGGAGC GGAAGACGGA GTTGGGGAAA CAATACTGGC 1080 TGATCACCGC CTTCCACGCC AAGGGCAACC TACAGGAGTA CCTGACGCGG CATGTCATCA 1140 GCTGGGAGGA CCTGCGCAAG CTGGGCAGCT CCCTCGCCCG GGGGATTGCT CACCTCCACA 1200 GTGATCACAC TCCATGTGGG AGGCCCAAGA TGCCCATCGT GCACAGGGAC CTCAAGAGCT 1260 CCAATATCCT CGTGAAGAAC GACCTAACCT GCTGCCTGTG TGACTTTGGG CTTTCCCTGC 1320 GTCTGGACCC TACTCTGTCT GTGGATGACC TGGCTAACAG TGGGCAGGTG GGAACTGCAA 1380 GATACATGGC TCCAGAAGTC CTAGAATCCA GGATGAATTT GGAGAATGTT GAGTCCTTCA 1440 AGCAGACCGA TGTCTACTCC ATGGCTCTGG TGCTCTGGGA AATGACATCT CGCTGTAATG 1500 CAGTGGGAGA AGTAAAAGAT TATGAGCCTC CATTTGGTTC CAAGGTGCGG GAGCACCCCT 1560 GTGTCGAAAG CATGAAGGAC AACGTGTTGA GAGATCGAGG GCGACCAGAA ATTCCCAGCT 1620 TCTGGCTCAA CCACCAGGGC ATCCAGATGG TGTGTGAGAC GTTGACTGAG TGCTGGGACC 1680 ACGACCCAGA GGCCCGTCTCTC ACAGCCCAGT GTGTGGCAGA ACGCTTCAGT GAGCTGGAGC 1740 ATCTGGACAG GCTCTCGGGG AGGAGCCT CCGGACGACAGCCGCAGCAGCCAGCCAGCCAGCCAGCCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCCAG

SEQ ID NO: 4 Sequence length: 503 Sequence type: Nucleic acid Number of strands: Single-stranded topology: Linear Sequence type: cDNA sequence: TCTAGAGATG GGTCGGGGGC TGCTCAGGGG CCTGTGGCCG CTGCACATCG TCCTGTGGAC 60 GCGTATCGCC AGCACGATCC CACCGCACGT TCAGAGTCG GTCAAGAGTCG GTAG CACTGACAAC AACAACGGTG CAGTCAAGTT TCCACAACTG TGTAAATTTT GTGATGTGAG 180 ATTTTCCACC TGTGACAACC AGAAATCCTG CATGAGCAAC TGCAGCATCA CCTCCATCTG 240 TGAGAAGCCA CAGCCACAGG AAGTCTGTGT GGCTGTATGG AGAAAAGAAT GACGAGAACA 300 TAACACTAGA GACAGTTTGC CATGACCCCA AGCTCCCCTA CCATGACTTT ATTCTGGAAG 360 ATGCTGCTTC TCCAAAGTGC ATTATGAAGG AAAAAAAAAA GCCTGGTGAG ACTTTCTTCA 420 TGTGTTCCTG TAGCTCTGAT GAGTGCAATG ACAACATCAT CTTCTCAGAA GAATATAACA 480 CCAGCAATCC TGACTAGGGA TCC 503

[Brief description of drawings]

FIG. 1 shows a restriction map of plasmid λKβRII-6 obtained according to Example 1 (6).

Claims (2)

[Claims] 1. A TGF-β receptor type II gene comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1. 2. The TGF-β receptor type II gene according to claim 1, which comprises the nucleotide sequence represented by SEQ ID NO: 2.