JPH089640B2 - Human interleukin-2 receptor - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human interleukin-2 receptor obtained by a gene recombination technique and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION Interleukin-2 (hereinafter sometimes abbreviated as IL-2) allows T cells to proliferate in vitro while retaining their functions and enables long-term passage and maintenance. It is known to have an action of promoting the mitogenic reaction of cells, restoring the ability of nude mouse splenocytes to produce antibodies against T cell-dependent antigens, and promoting the differentiation and proliferation of killer cells (The Journal Of Immunology, Volume 123, 2928-292
Page 9, 1979; Immunological Review, 51st
Vol. 257-278, 1980), but all these actions require the interaction of IL-2 with the cell surface IL-2 receptor (Journal of Experimental. Medicine, Volume 154, 1455
-1474 pages, 1981; ibid., Volume 158, 189.
Pp. 5-1911, 1983; Proceeding of National Academy of Science, 80th.
Vol., 6957-6961, 1983). Recently, an anti-Tac monoclonal antibody obtained by immunizing a mouse with an IL-2 dependent cell line established from the blood of a human T cell leukemia patient [The Journal of Immunology, No. 126
, 1393 (1981)], the biochemical analysis of the IL-2 receptor using anti-Tac antibody has been rapidly advanced. [Journal of Experimental Medicine, 158, 1332 (1983); Nature, 300, 267 (1982); Proceedings of the National Academy of Sciences. , 80, 6957 (1983)]. The binding site between the anti-Tac antibody and the Tac antigen is IL-2 and IL.
Although it remains unclear whether or not it is the same as the binding site for the -2 receptor, it has become clear that the Tac antigen and the IL-2 receptor share most of their properties. Utilizing such interaction between IL-2 and IL-2 receptor, cloning of T cells and natural killer cells has been successful so far (for example, Nature,
Volume 268, pp. 154-156, 1977; The Journal of Immunology, Volume 130, 981-9.
87, 1983). In addition, T cell leukemia and Ta
It has been suggested to be related to the abnormal expression of c antigen (Brad, 62, 509-510, 198).
3 years), on the other hand, the number of IL-2 receptors decreased by anti-Tac antibody in normal cells, whereas the number of IL-2 receptors decreased by anti-Tac antibody was not observed in leukemia cells (down).・ Regulation deficiency is known) (The Journal of Blood, No. 61)
Vol., 1014-1016, 1983).

[0003]

As described above, the IL-2 receptor which plays an important role in cells
The nature of the L-2 receptor protein as a protein and its structural gene are unknown, and there is no report that the IL-2 receptor was produced by gene recombination technology.

[0004]

The present inventors have found that human IL
The present invention has been completed by conducting research on a polypeptide of the -2 receptor and a gene encoding the same, cloning the gene for the first time, and establishing a method for producing the polypeptide of the IL-2 receptor by gene recombination technology. That is, the present invention provides a novel recombinant D encoding a polypeptide having interleukin-2 receptor activity.
The present invention provides an interleukin-2 receptor obtained by gene recombination technology using NA and a method for producing these. Regarding the gene encoding the above-mentioned IL-2 receptor polypeptide, for example, the first to the second in FIG.
A gene encoding a polypeptide consisting of the 52nd amino acid sequence can be mentioned, and a gene consisting of the nucleotide sequence shown by the 1st to 252nd codons in FIG. 2 is particularly preferable. The above gene may have a base sequence encoding a signal peptide consisting of ATG or the -21st to -1st codon in FIG. 2 at its 5 ′ end. Further, the 3'end of the gene may have TAA, TGA or TAG as a translation termination codon, and TAG is particularly preferable. The gene preferably has a promoter upstream thereof, and any promoter may be used as long as it is suitable for the host used for gene expression. Examples thereof include SV40-derived promoters and retrovirus promoters suitable for expression in animal cells (eg, monkey cell COS-7, Chinese hamster cell CHO, etc.), and SV40-derived promoters are particularly preferable.

Recombinant DNA containing a gene encoding the IL-2 receptor polypeptide of the present invention is, for example,
(A) culturing cells producing interleukin-2 receptor, (b) separating a messenger RNA encoding a polypeptide of interleukin-2 receptor from the culture, and (c) a single chain from the messenger RNA. Of the complementary DNA (cDNA) of (4), (d) incorporating the complementary DNA into a plasmid,
(E) converting the plasmid into a complete double-stranded plasmid,
(F) transforming a host with the obtained recombinant plasmid, (g) culturing the obtained transformant, and isolating the plasmid containing the target DNA from the transformant,
(H) If desired, the desired cloned DNA can be excised from the plasmid, and (ri) if desired, the cloned DNA can be ligated downstream of the promoter in the vehicle.

The mRNA encoding the IL-2 receptor is usually human T cells such as MT-1 cells (Cancer, No. 7).
Volume 1, pp. 155-156, 1980) Poly ARN
A is prepared, a cDNA chain is synthesized by a method known per se using this as a template using a reverse transcriptase, and after being incorporated into a plasmid, the cDNA is converted into a double-stranded DNA (Molecular and Cellular Bio). Rosie, Volume 2, 161
-170, 1982). On the other hand, for example, 5 × 10 ⁹ MT-1 cells are solubilized in a buffer containing NP-40, an anti-Tac antibody or IL-2 is bound to a carrier, and the column is used to detect IL-2 receptor. To purify. This was analyzed by an automatic amino acid sequencer according to a method known per se (The Journal of Biological Chemistry, 256, 7990-7997, 1981).
Year), about 20 N-terminal amino acid sequences are determined. After chemically synthesizing an oligonucleotide having a nucleotide sequence corresponding to this part of the amino acid sequence, it was labeled with ³² P to form a probe, which was then subjected to a colony hybridization method (Gene, vol. 10, 63. -67 pages, 1
980), the desired clone is selected from the ampicillin-resistant transformants. The plasmid having the gene (DNA) encoding the cloned IL-2 receptor polypeptide can be cut out as it is or, if desired, with a restriction enzyme. Furthermore, the above genes can be produced by chemical synthesis.
The cloned gene can also be linked downstream of a promoter in a vehicle suitable for expression as described above.
The DNA of the present invention is, for example, E. coli (294, DH
1, HB101, etc.) to transform and proliferate and preserve them. As the IL-2 receptor obtained by the gene recombination technique of the present invention, for example, a protein containing the polypeptide shown by the amino acid sequence from the 1st to the 252nd amino acid in FIG. 2 can be mentioned. The polypeptide may have Met at its N-terminus. The polypeptide is preferably a glycoprotein having a sugar chain.

The IL-2 receptor of the present invention is, for example, IL-
It can be produced by culturing an animal cell having a recombinant DNA containing a gene encoding a bireceptor polypeptide, and producing and accumulating IL-2 receptor in the culture. The animal cell having the above-mentioned recombinant DNA is transfected with the DNA by transfecting a suitable animal cell, preferably a mammalian cell line such as COS-7, mouse L cell, human B cell, human FL cell.
section).
The animal cell having the DNA of the present invention can also be produced as a transformant. Culturing of animal cells is carried out in a known medium for animal cells, for example, DMEM medium containing 5 to 20% fetal bovine serum at 30 to 40 ° C. for 15 to 60 hours.
The ability of the thus-produced IL-2 receptor to bind to IL-2 is determined by the fluorescent antibody method using IL-2 (The Journal of Immunology, Vol. 126, 1393-1397).
P., 1981) or isotope labeled IL
It can be measured by the binding ability to -2.

The separation and purification of IL-2 receptor from the above culture can be carried out, for example, by the following method.
That is, after collecting the cultured cells by a centrifugation method, a buffer solution containing a surfactant (for example, NP-40, Triton X-100, Tween-20 or SDS), a hypotonic or hypertonic buffer solution, or ultrasonic waves is used. Cell surface proteins including the IL-2 receptor are solubilized by treatment or enzyme treatment. Prior to solubilization, radioactive compounds (eg Na
¹²⁵ I, ¹⁴ C-labeled amino acid, ¹⁴ C-labeled carbohydrate, ³ H
-Labeled amino acid, ³ H-labeled carbohydrate, ³⁵ S-labeled amino acid, etc.) may be used to label the IL-2 receptor with an isotope in advance. IL-obtained in this way
2 The crude lysate containing the receptor is treated with a water-insoluble carrier on which various proteins are immobilized in advance to remove impurities slightly mixed in the final preparation as much as possible. Examples of proteins used for this purpose include bovine serum albumin, human immunoglobulin, mouse immunoglobulin, etc. Usually, the crude solubilized solution is passed through a column packed with cross-linked agarose or the like on which these proteins are immobilized. Impurities can be removed by collecting the passing liquid. The thus-obtained solution containing the IL-2 receptor was then added to immobilized IL-2 in which non-glycosylated IL-2 and a water-insoluble carrier were covalently bonded [a part of the present inventors The IL-2 receptor is adsorbed through an affinity column packed with the title of the invention “immobilized interleukin-2” by date]. The column is loaded with a surfactant (eg NP-40, Triton X-100,
Tween-20 or SDS) and high concentration (0.1
˜1.0 M) Washed thoroughly with a buffer solution (eg Tris-HCl buffer solution) containing salts (NaCl etc.), then washed with acetate buffer solution (pH 4.0), then IL-2
IL-2 is eluted by eluting the IL-2 receptor under conditions (eg, citrate buffer (pH 2.0), 3M NaSCN, etc.) that destroys the binding between the receptor and IL-2, and the solvent is distilled off if necessary. The receptor can be manufactured.

[0009]

The IL-2 receptor obtained by the present invention has substantially the same activity as known natural human IL-2 receptor and Tac antigen. Here, human IL-2 receptor and Tac
The activity substantially similar to that of the antigen refers to, for example, the following biological and immunological activities. That is, it has the activity of proliferating normal T cells and natural killer cells while retaining their functions by binding to IL-2. Therefore, it is possible to produce a IL-2 receptor by genetically infecting or transforming various animal cells (B cells etc.) that do not originally have an IL-2 receptor with the DNA used in the present invention. , T cells can be proliferated, subcultured, established, or cloned in vitro for a long period of time by culturing these cells in an IL-2-containing medium. By culturing a large amount of the above-mentioned animal cells, which can be propagated using the above-mentioned DNA, a large amount of various useful substances originally produced by these cells can be obtained. Further, as described above, it is known that there is a relationship between leukemic conversion of T cells and abnormal expression of Tac antigen, but since the IL-2 receptor of the present invention has almost the same properties as Tac antigen, For example, using the receptor as a substrate. In addition, anti-IL-2 produced by the receptor
Leukemia and immunodeficiency can be diagnosed by detecting the number of IL-2 receptors expressed in cells using the receptor antibody.

In the present specification and drawings, when abbreviations are used for bases, amino acids, etc., IUPAC-IUB
Commission on Biochemica
It is based on the abbreviations by Nomenelature or the abbreviations commonly used in this field, examples of which are given below. When amino acids may have optical isomers, the L-form is shown unless otherwise specified. DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A: adenine T: thymine G: guanine C: cytosine RNA: ribonucleic acid mRNA: messenger ribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidine triphosphate dGTP: deoxyguanosine tri Phosphoric acid dCTP: Deoxycytidine triphosphate ATP: Adenosine triphosphate EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate Gly: Glycine Ala: Alanine Val: Valine Leu: Leucine Ile: Isoleucine Ser: Serine Thr: Threonine: Threonine: Threonine: Threonine : Methionine Glu: Glutamic acid Asp: Aspartic acid Lys: Lysine Arg: Arginine His: Histidine Phe: Phenyla Nin Tyr: Tyrosine Trp: Tryptophan Pro: Proline Asn: Asparagine Gln: Glutamine

[0011]

INDUSTRIAL APPLICABILITY The recombinant DNA containing the gene encoding the IL-2 receptor polypeptide used in the present invention can be introduced into various animal cells which originally do not have IL-2 receptor. Since the cells can produce the IL-2 receptor, the cells can be proliferated and established as well as T cells, and therefore various useful substances produced by these cells can be obtained in large quantities. can do. Moreover, the IL-2 receptor of the present invention or the anti-IL-2 receptor antibody produced by the IL-2 receptor can detect an IL-2 receptor that is aberrantly expressed in leukemic transformation of cells, and thus leukemia or immunodeficiency It can be used as a diagnostic agent for.

[0012]

The present invention will be described in more detail with reference to the following Reference Examples and Examples, but the present invention is not limited to these. The transformant Escherichia Col disclosed in (vii) of Reference Example was used.
i) HB101 / Tac-2 (IFO-14364)
Has received the contract number FERM P from August 21, 1984 to the Institute of Microbial Technology (FRI), Ministry of International Trade and Industry.
Deposited as -7796.

Reference Example DNA Containing IL-2 Receptor Gene
(I) mRN encoding a polypeptide of IL-2 receptor
Production of A. MT-1 which is a human T-cell leukemia cell was added to RPMI164
It was cultured at 37 ° C. in 0 medium (containing 10% fetal bovine serum). 1 × 10 ^{9 of the} cultured cells were treated with 5M guanidine thiocyanate, 5% mercaptoethanol, 50 mM.
Tris-HCl pH 7.6, 10 mM EDTA
After destructive denaturation with a Teflon homogenizer in the solution, sodium N-laurylyl sarcosinate was added to 4%, and the homogenized mixture was added to a 5.7M cesium chloride solution (5.7M cesium chloride, 0.1M EDTA).
Layered on 6 ml and centrifuged at 15 ° C. for 2 hours at 24,000 rpm using a Beckman SW28 rotor to obtain RNA precipitate. This RNA precipitate was added to 0.25% N
-Dissolved in sodium lauroyl sarcosinate solution and then precipitated with ethanol to give 10 mg of RNA. A high salt solution of this RNA [0.5M NaCl, 10 mM
Tris-HCl pH 7.6, 1 mM EDTA, 0.
3% SDS] was adsorbed on an oligo (dT) cellulose column, and mRNA containing poly (A) was added to a low salt solution (10
mM Tris-HCl pH 7.6, 1 mM EDT
30 μg of mRNA containing poly (A) was collected by elution with A, 0.3% SDS). (ii) Synthesis of single-stranded DNA Single-stranded DNA was synthesized by a method known per se using the mRNA and reverse transcriptase obtained above. That is, 20 μl of reaction solution (5 μg of mRNA, 1.4 μg of vector / primer DNA, 5 units of reverse transcriptase, 2 mM of d each)
ATP, dCTP, dGTP and dTTP, 8 mM
After incubating in MgCl ₂ , 30 mM KCl, 0.3 mM dithiothreitol, 50 mM Tris-HCl pH 8.3 for 20 minutes at 37 ° C., the reaction was stopped by adding 2 μl of 0.25 M EDTA and 1 μl of 10% SDS. Then, it was deproteinized with phenol and precipitated with ethanol (Molecular and Cellular Biology, Vol. 2, 161-170, 1982). (iii) Addition of dC chain 15 μl of reaction solution (1 mM CoCl ₂ , 30
mM Tris-HCl pH 6.8, 140 mM sodium cacodylate, 0.1 mM dithiothreitol,
This was dissolved in 0.2 μg poly (A), 66 μM dCTP), 18 units of terminal transferase was added, and the mixture was incubated at 37 ° C. for 5 minutes. Then 1 μl
0.25 M EDTA and 0.5 μl of 10% SDS
Was added to stop the reaction, deproteinized with phenol, and precipitated with ethanol (Molecular and Cellular Biology, Vol. 2, 161-170, 1982).
Year). (iv) Cleavage with HindIII The precipitate was dissolved in 10 μl of HindIII buffer,
2.5 units of HindIII was added and digested at 37 ° C for 1 hour. Then phenol treatment followed by precipitation with ethanol and 10 μl of 10 mM Tris-HCl pH 7.
It was dissolved in 3.1 mM EDTA and 3 μl of ethanol was added. (v) Circularization with linker DNA with dG chain 1 μl of the above DNA was added with 7 ng of linker D with oligo dG.
10 μl of 10 mM Tris-HCl, p with NA
6 in H7.5, 1 mM EDTA, 0.1 M NaCl
After incubating at 5 ° C for 5 minutes and then at 42 ° C for 30 minutes, the mixture was cooled to 0 ° C. 20 mM Tris-HCl
(PH 7.5) 4 mM MgCl ₂ , 10 mM (NH ₄ ) ₂ S
O ₄ , 0.1 M KCl, 0.1 mM β-NAD, 50
μg / ml BSA was added and incubated overnight at 12 ° C. with 0.6 μg E. coli DNA ligase. (vi) Synthesis of double-stranded DNA 40 μM dTTP, 40 μM dGTP,
40 μM dCTP, 40 μM dATP, 0.15 mM
Add to make β-NAD (final liquid volume 104μ
l), 0.4 μg of E. coli DNA ligase, 0.3 μg
E. coli DNA polymerase I, 1 unit E. coli RN
After incubation with aseH at 12 ° C. for 1 hour and then at 25 ° C. for 1 hour, they were used for transformation of E. coli. (vii) Isolation of cDNA-containing plasmid In this way, 4 × 10 ⁵ ampicillin-resistant strains were isolated, and the DNA of each of them was immobilized on a nitrocellulose filter. Then, based on the amino acid sequence of the IL-2 receptor protein purified from MT-1 cells,
Amino acid No. 4-8 (Asp-Asp-Asp-Pr
o-Pro) (SEQ ID NO: 2) corresponding nucleotide sequence ( ^{3 '}
CTRCTRCTRGGNGG ^{5 '} ) (SEQ ID NO: 3,
R represents A or G and N represents any of A, G, C, and T) by the triester method (Proceedings of the National Academy of Science U)
SA, Vol. 75, pp. 5765-5769, 1978).
It was chemically synthesized by. A 50 μl reaction solution (oligonucleotide 0.20 μg, 50 mM T was added to this oligonucleotide using T4 polynucleotide kinase).
ris · HCl pH 8.0, 10 mM MgCl ₂ ,
10 mM mercaptoethanol, 50 μCir- ³² PA
The reaction was carried out for 1 hour at 37 ° C. in TP, 3 units T4 polynucleotide kinase), and the 5 ′ end was labeled with ³² P. Using this labeled oligonucleotide as a probe, the method of Hanahan et al. (Gene, Vol. 10, 63-
P. 67, 1981), two strains (E. Coli HB101 /) which associate with the above-mentioned DNA immobilized on the nitrocellulose filter and react with the above-mentioned oligonucleotide probe by autoradiography.
Tac-1 and E. Coli HB101 / Tac-
2) isolated. Plasmid DNA from each of these strains was analyzed by the alkaline method (Birnboim H.C.
Et al., Nucleic Acids Research, Volume 7, 15
13-1524, 1979). These plasmids were named Tac-1 and Tac-2. Tac-1 and Tac-2 were the same except that Tac-2 had a longer nucleotide sequence at the 5'end by 16 bases. A restriction enzyme map of Tac-2 is shown in FIG. Next, the primary structure (base sequence) of the cDNA sequence inserted into this Tac-2 plasmid was determined by the dideoxynucleotide method and the Maxam-Gilbert method. Its primary structure (SEQ ID NO: 4) is shown in FIG. The peptide defined by this base sequence has a synthesis initiation signal [N
o. 175-177 ATG (codon-21)] and consists of 272 amino acids. Of these, 21 amino acids (amino acids -21 to -1) from the N-terminal are considered to be signal peptides. From the above primary structure, it was revealed that this plasmid has the entire nucleotide sequence encoding the human IL-2 receptor polypeptide. The amino acid sequence of the IL-2 receptor deduced from the above base sequence (SEQ ID NO: 5) is shown in FIG. (viii) Production of expression-type plasmid The plasmid Tac-2 (20 μm) obtained in (vii) of Reference Example
g) from human IL-2 receptor gene part to the restriction enzyme Ps
After cleaving with tI (partial decomposition) and PvuII (complete decomposition), the DNA fragments were separated by 1% agarose gel electrophoresis to obtain 2 μg of DNA. The single-stranded portion of this DNA is
After filling with 4 DNA polymerase reaction, phenol treatment and ethanol precipitation were performed. This DNA was added to 20 μl of ligation buffer (66 mM Tris-HCl p
H7.6, 6.6 mM MgCl ₂ , 10 mM DTT,
0.5 μg of a synthetic oligonucleotide linker (Hin
dIII linker) ^{5 ′} pCAAGCTTG and 5 units of T4 DNA ligase were mixed and reacted at 14 ° C. for 17 hours to ligate the gene encoding the IL-2 receptor polypeptide and the oligonucleotide linker. After inactivating the ligase by heat treatment at 65 ° C. for 10 minutes,
A 3-fold amount of distilled water was added, and a buffer solution of the restriction enzyme HindIII (50 mM NaCl, 10 mM Tris-HC was added.
1, pH 7.9, 6 mM MgCl ₂ , 100 μg / ml
1 with 20 units of HindIII) in bovine serum albumin
Time processed. The linker portion cut with a Sepharose 4B column (0.5 cm diameter, 20 cm length) and the IL-2 receptor DNA bound with the linker were separated, and the IL-2 receptor polypeptide bound with the linker was separated by ethanol precipitation. The encoding gene-containing DNA was recovered. On the other hand, plasmid pKCRH2 (Nature 307
Vol., Pp. 604-608, 1984) with the restriction enzyme Hin.
Double-stranded DN cleaved with dIII and bound with the above linker
Expression plasmid pKCR / Tac-2 / A by ligating A to the downstream of the SV40 promoter with T4 DNA ligase
Was built. In this case, a gene in which the gene encoding the IL-2 receptor polypeptide was linked in the reverse direction to the promoter was also isolated, which was named pKCR.Tac-2.Ai. Similarly, Hi obtained from the plasmid Tac-2
An expression-type plasmid was also constructed in which a HindIII linker was ligated to the ndIII-PvuII DNA fragment (including a gene encoding a polypeptide of IL-2 receptor) and inserted into the HindIII site of pKCRH2 to obtain pKCR / Tac-
I named it 2.

Example IL-2 in COS-7 cells
Expression of gene encoding receptor polypeptide 5 × 10 ⁵ COS-7 cells (Cell Vol. 23, 175
-182, 1981) in a Dulbecco's Minimal Essential (DMEM) medium containing 5% fetal bovine serum and plated on a cell culture dish of 10 cm in diameter.
After 0 hours, the medium was replaced. After 4 hours, 40 μg
1/10 volume of transfection solution containing 1 / ml of plasmid DNA (Proceeding of National Academy of Science USA, Vol. 76,
(Pp. 1373-1376, 1979), the mixture was incubated for 12 hours and then treated with 2.5% glycerol for 1 minute. After washing the cells, D containing 8% fetal bovine serum
The cells were cultured in MEM at 37 ° C for 48 hours. Next, the cells were treated with mouse anti-Tac monoclonal antibody and FITC (fluorescent dye).
Stained with labeled goat anti-mouse antibody (Journal of Immunology 126, 1393-1397, 19
1981). In a control experiment, a murine myeloma protein X5563, which is a type of antibody, was used instead of the anti-Tac antibody. The expression of the gene encoding the IL-2 receptor polypeptide was measured by flow cytometry using Spectrum III. The results are shown in Table 1.

[0015]

[Table 1]

[0016]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 251 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys 5 10 15 Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30 Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45 Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60 Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln 65 70 75 80 Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95 Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110 Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125 Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140 Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln 145 150 155 160 Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu 165 170 175 Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys 180 185 190 Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr 195 200 205 Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln Val Ala Val Ala Gly 210 215 220 Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu Ser Gly Leu Thr Trp 225 230 235 240 Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 245 250.

SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence Length: 14 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GGNGGRTCRT CRTC 14.

SEQ ID NO: 4 Sequence Length: 1309 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Nucleic Acid Sequence AACTCCTGAC TCCGATAGAG ACTGGATGGA CCCGCAAGGG TGGCAGCCCA GGCGGACCGA 60 TCTTCCCATC CCACATCCTC CGGCGCGATGCCTTACAAGTG GCTG CTGCAGAGAA AGACCTCCGC TTCACTGCCC CGGCTGGTCC CAAGGGTCAG GAAGATGGAT 180 TCATACCTGC TGATGTGGGG ACTGCTCACG TTCATCATGG TGCCTGGCTG CCAGGCAGAG 240 CTCTGTGACG ATGACCCGCC AGAGATCCCA CACGCCACAT TCAAAGCCAT GGCCTACAAG 300 GAAGGAACCA TGTTGAACTG TGAATGCAAG AGAGGTTTCC GCAGAATAAA AAGCGGGTCA 360 CTCTATATGC TCTGTACAGG AAACTCTAGC CACTCGTCCT GGGACAACCA ATGTCAATGC 420 ACAAGCTCTG CCACTCGGAA CACAACGAAA CAAGTGACAC CTCAACCTGA AGAACAGAAA 480 GAAAGGAAAA CCACAGAAAT GCAAAGTCCA ATGCAGCCAG TGGACCAAGC GAGCCTTCCA 540 GGTCACTGCA GGGAACCTCC ACCATGGGAA AATGAAGCCA CAGAGAGAAT TTATCATTTC 600 GTGGTGGGGC AGATGGTTTA TTATCAGTGC GTCCAGGGAT ACAGGGCTCT ACACAGAGGT 660 CCTGCTGAGA GCGTCTGCAA AATGACCCAC GGGAAGACAA GGTGGACCCA GCCCCAGCTC 720 ATATGCACAG GTGAAATGGA GACCAGTCAG TTTCCAGGTG AAGAGAAGCC TCAGGCAAGC 780 CCCGAAGGCC GTCCTGAGAG TGAGACTTCC TGCCTCGTCA CAACAACAGA TTTTCAAATA 840 CAGACAGAAA TGGCTGCAAC CATGGAGACG TCCATATTTA CAACAGAGTA CCAGGTAGCA 900 GTGGCCGGCT GTGTTTTCCT GCTGATCAGC GTCCTCCTCC TGAGTGGGCT CACCTGGCAG 960 CGGAGACAGA GGAAGAGTAG AAGAACAATC TAGAAAACCA AAAGAACAAG AATTTCTTGG 1020 TAAGAAGCCG GGAACAGACA ACAGAAGTCA TGAAGCCCAA GTGAAATCAA AGGTGCTAAA 1080 TGGTCGCCCA GGAGACATCC GTTGTGCTTG CCTGCGTTTT GGAAGCTCTG AAGTCACATC 1140 ACAGGACACG GGGCAGTGGC AACCTTGTCT CTATGCCAGC TCAGTCCCAT CAGAGAGCGA 1200 GCGCTACCCA CTTCTAAATA GCAATTTCGC CGTTGAAGAG GAAGGGCAAA ACCACTAGAA 1260 CTCTCCATCT TATTTTCATG TATATGTGTT CATTAAAGCA TGAATGGTA 1309.

SEQ ID NO: 5 Sequence length: 272 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro G ln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255 Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 270.

[Brief description of drawings]

FIG. 1 is a plasmid Tac-2 obtained in (vii) of Reference Example.
Restriction map of the gene site encoding the polypeptide having IL-2 receptor activity (the spotted portion indicates the signal peptide-encoding portion, the black-painted portion and the shaded portion sandwiched between them indicate the IL-2 receptor activity). It represents a portion that encodes a polypeptide having).

FIG. 2-1 shows the primary structure (base sequence) of the above-mentioned polypeptide having IL-2 receptor activity and the first part of the amino acid sequence of IL-2 receptor.

FIG. 2-2 shows the primary structure (base sequence) of the polypeptide having IL-2 receptor activity and the amino acid sequence of IL-2 receptor, which is a part following FIG. 2-1.

FIG. 2-3 shows the primary structure (base sequence) of the polypeptide having IL-2 receptor activity and the amino acid sequence of IL-2 receptor, which is a part following FIG. 2-2.

FIG. 2-4 shows the primary structure (base sequence) of the polypeptide having IL-2 receptor activity and the amino acid sequence of IL-2 receptor, which is a part following FIG. 2-3.

FIG. 3 shows the structure of the expression type plasmid pKCR.Tac-2.A described in (viii) of Reference Example.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 15/09 (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) ( C12N 5/00 B C12R 1:91)

Claims (2)

[Claims] 1. A formula was obtained by genetic engineering GluLeuCysAspAspAspProProGluIleProHisAlaThr PheLysAlaMetAlaTyrLysGluGlyThrMetLeuAsnCys GluCysLysArgGlyPheArgArgIleLysSerGlySerLeu TyrMetLeuCysThrGlyAsnSerSerHisSerSerTrpAsp AsnGlnCysGlnCysThrSerSerAlaThrArgAsnThrThr LysGlnValThrProGlnProGluGluGlnLysGluArgLys ThrThrGluMetGlnSerProMetGlnProValAspGlnAla SerLeuProGlyHisCysArgGluProProProTrpGluAsn GluAlaThrGluArgIleTyrHisPheValValGlyGlnMet ValTyrTyrGlnCysValGlnGlyTyrArgAlaLeuHisArg GlyProAlaGluSerValCysLysMetThrHisGlyLysThr ArgTrpThrGlnProGlnLeuIleCysThrGlyGluMetGlu ThrSerGlnPheProGlyGluGluLysProGlnAlaSerPro GluGlyArgProGluSerGluThrSerCysLeuValThrThr ThrAspPheGlnIleGlnThrGluMetAlaAlaThrMetGlu ThrSerIlePheThrThrGluTyrGlnValAlaValAlaGly CysValPheLeuLeuIleSerValLeuLeuLeuSerGlyLeu ThrTrpGlnArgArgGlnArgLysSerArgArgThrIle (SEQ ID NO: 1) Interleukin containing the amino acid sequence represented by -
2 receptors. Wherein formula GluLeuCysAspAspAspProProGluIleProHisAlaThr PheLysAlaMetAlaTyrLysGluGlyThrMetLeuAsnCys GluCysLysArgGlyPheArgArgIleLysSerGlySerLeu TyrMetLeuCysThrGlyAsnSerSerHisSerSerTrpAsp AsnGlnCysGlnCysThrSerSerAlaThrArgAsnThrThr LysGlnValThrProGlnProGluGluGlnLysGluArgLys ThrThrGluMetGlnSerProMetGlnProValAspGlnAla SerLeuProGlyHisCysArgGluProProProTrpGluAsn GluAlaThrGluArgIleTyrHisPheValValGlyGlnMet ValTyrTyrGlnCysValGlnGlyTyrArgAlaLeuHisArg GlyProAlaGluSerValCysLysMetThrHisGlyLysThr ArgTrpThrGlnProGlnLeuIleCysThrGlyGluMetGlu ThrSerGlnPheProGlyGluGluLysProGlnAlaSerPro GluGlyArgProGluSerGluThrSerCysLeuValThrThr ThrAspPheGlnIleGlnThrGluMetAlaAlaThrMetGlu ThrSerIlePheThrThrGluTyrGlnValAlaValAlaGly CysValPheLeuLeuIleSerValLeuLeuLeuSerGlyLeu ThrTrpGlnArgArgGlnArgLysSerArgArgThrIle (SEQ ID NO: 1) Interleukin containing the amino acid sequence represented by -
A method for producing a receptor, which comprises culturing an animal cell having a recombinant DNA encoding a polypeptide having a 2-receptor activity, and producing and accumulating interleukin-2 receptor in the culture.