JPH08272A - DNA fragment containing gene encoding mouse interleukin-2, recombinant vector having the DNA fragment, and transformant carrying the vector - Google Patents

Abstract

(57) [Summary] [Structure] A DNA fragment encoding a polypeptide having a mouse interleukin-2 activity and containing the amino acid sequence set forth in SEQ ID NO: 1 of the Sequence Listing, a prokaryote in which the DNA fragment is inserted, or A recombinant vector for transforming a eukaryotic cell, and a transformant carrying the recombinant vector. Effects According to the present invention, DN containing a gene encoding a polypeptide having mouse interleukin 2 activity
The A fragment was successfully isolated, paving the way for the production of a polypeptide having mouse interleukin 2 activity by a microorganism.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA fragment containing a gene encoding mouse interleukin-2, and the DNA fragment.
The present invention relates to a recombinant vector having a fragment and a transformant carrying the vector.

[0002]

2. Description of the Related Art Mouse interleukin-2 (MIL-
Regarding the production of 2), it has been reported that mouse lymphoma and hybridoma produce this (S. Gillis).
et al., J. Immunol., 125, 2570 (1980), I. Clark-Lew.
is et al., 128, 168 (1982)).

[0003]

However, the production of MIL-2 by these cells is very small, and the production requires a large amount of cells to be cultured, and there are many problems to be solved. .

[0004]

Means for Solving the Problems The present inventors have conducted MIL-2.
Have been studied repeatedly, but finally, DN containing a gene encoding mouse interleukin-2
The A fragment was successfully prepared, and the thus obtained DNA fragment was ligated to a vector DNA capable of replicating in prokaryotic or eukaryotic cells to obtain a recombinant vector. Furthermore, a polypeptide having mouse interleukin-2 activity was successfully produced by culturing prokaryotic or eukaryotic cells transformed with the obtained recombinant vector.

That is, the present invention firstly relates to a DNA encoding a polypeptide having mouse interleukin-2 activity and containing an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
Regarding the fragment, secondly, it relates to a recombinant vector for transforming a prokaryotic or eukaryotic cell into which the DNA fragment is inserted, and thirdly, to a transformant carrying the recombinant vector.

A DNA fragment containing a gene encoding mouse interleukin-2 is messenger RNA (m) derived from a mouse lymphocyte-derived cell capable of producing MIL-2.
RNA), and using this as a template, complementary DNA (cD
NA). The mRNA corresponding to MIL-2 was obtained from 12S by sucrose density gradient (SDG) centrifugation, gel filtration, or agarose gel electrophoresis.
It can be obtained as a fraction corresponding to the fraction. mRNA
Whether or not the fraction contains mRNA corresponding to MIL-2 can be examined by translating the mRNA into a protein using an in vivo or in vitro protein translation system. mRNA can be obtained as a fraction corresponding to the 12S fraction by a sucrose density gradient (SDG) centrifugation method, a gel filtration method, an agarose gel electrophoresis method, or the like.

Mouse lymphocyte-derived cells having MIL-2 producing ability include lymphocytes themselves obtained from peripheral blood, lymph nodes, spleen and the like. In addition, these lymphocytes may be subjected to mutation treatment by a conventionally known method such as nylon column treatment, antiserum and complement treatment, density gradient fractionation, enzyme treatment (neuraminidase, galactose oxidase, trypsin, etc.), and X-ray irradiation. included. Further, preferred lymphocyte-derived cells are those obtained by cloning these lymphocyte-derived cells, for example, like T lymphocytes cloned in the presence of T cell growth factor and the like. Furthermore, human lymphoid malignant cells such as leukemia cells and T lymphoma cells, those obtained by pretreatment or mutation treatment of these mouse lymphocyte malignant cells, or cloned malignant cells are more preferable human lymphoid cells. It can be used as a sphere-derived cell. In particular, cloned cell lines usually yield a large amount of mRNA. Furthermore, a hybridoma obtained by fusing the mouse lymphocyte-derived cells with a tumor cell line such as BW5147 can also be used as a suitable lymphocyte-derived cell.

[0008] These mouse lymphocyte-derived cells include:
There are (1) those that spontaneously produce MIL-2, and (2) those that produce MIL-2 by contacting with and stimulating with mitogen in the presence or absence of other cells. When MIL-2 spontaneous production strain is used, these cells may be cultured by a usual method.

When using cells that produce MIL-2 upon stimulation with mitogen, the cells are thoroughly washed and then washed with Rosewell Park Memorial Institute 16
Ordinary cell culture medium (with or without serum or serum components) such as 40 medium (hereinafter abbreviated as RPMI 1640), Dulbecco's modified Eagles medium (hereinafter abbreviated as DMEM) or Click medium. Additives such as penicillin, streptomycin or other antibiotics, L-glutamine, Hepes buffer or sodium bicarbonate may be added to the medium.) 0.5 to 4 × 10 ⁶ cells / ml. Suspension at a density, zinc compounds such as mitogen, neuraminidase, galactose oxidase, and zinc chloride; lymphocyte-activating components derived from bacteria such as protein A and streptolysin-O; phorbol myristic acid (PMA) and TPA After the addition of the phorbol esters, the cells are washed and the stimulant is removed.
Furthermore, coexistence of macrophages or dendritic cells upon mitogen stimulation can increase the yield of mRNA corresponding to MIL-2.

The lymphocyte-derived cells capable of producing IL-2 can be subcultured and proliferated in vitro or in animal species under ordinary conditions. When subcultured in a test tube to prepare an mRNA source, any conventional medium known to promote the growth of T cells can be used. In these media, mammal-derived serum, serum components, or serum albumin may be added.

The culture time of the lymphocyte-derived cells is the time during which the lymphocytes are activated and the mRNA corresponding to MIL-2 is produced.
2 corresponds to the beginning of production. The preferred time is 3 to 12 hours after the addition of the stimulant such as mitogen. If the culture time is too long, the generated MIL-2
May be degraded. It is desirable that the culture is carried out in an environment of saturated steam having a pH of 7.0 to 7.4 and a temperature of 32 to 38 ° C.

The extraction of mRNA corresponding to IL-2 from lymphocyte-derived cells having MIL-2 producing ability may be carried out by a conventional method regardless of the type of cells. For example N
MRNA is separated after the cells are partially or completely destroyed by using a detergent such as P-40, SDS, Triton X100, deoxycholic acid, or a physical method such as a homogenizer or freeze-thawing. To prevent RNA degradation by RNase during extraction,
RNA degrading enzyme inhibitors such as heparin,
It is preferable to add polyvinyl sulfate, bentonite, macaroid, diethyl pyrocarbonate, vanadium complex, and the like. In some cases, an antibody corresponding to MIL-2 may be used to precipitate polysomes in the process of MIL-2 synthesis, and then mRNA may be extracted with a surfactant or the like.

For purification of mRNA containing polyA, oligo dT-cellulose, Sepharose 2B
Can be carried out by affinity column chromatography such as poly-U-sepharose or a purification method by a batch method, fractionation by SDG centrifugation, agarose gel electrophoresis or the like.

A commonly used system for translating mRNA into protein is Xenopus laevis.
evis) oocyte-transfected mRNA for translation (Gurdon et al., Nature, 233, 177 (`72)) or cell-free system such as rabbit reticulocyte lysate and wheat germ The way is done. Also,
Since genes that encode the same polypeptide have homology between species, methods such as selecting a mouse gene using cDNA or chromosomal DNA for mRNA that encodes a human polypeptide as a probe can be performed ( Higashi, Y. et al., J. Biol.Ch
em., 258 , 9522 (1983), Gray, PWet al., Proc. Natl.
Sci. USA, 80 , 5842 (1983) etc.).

M corresponding to MIL-2 thus obtained
CDNA is synthesized from the RNA-containing fraction in vitro.
To determine whether the obtained cDNA encodes MIL-2, the cDNA is connected to, for example, a vector DNA capable of replicating in Escherichia coli cells or COS cells, and introduced into the host cell. be able to.

To synthesize cDNA, first, mRNA
As a template, oligo dT as a primer, and dAT
Ss-cDNA complementary to mRNA is synthesized by reverse transcriptase in the presence of P, dGTP, dCTP, dTTP,
After decomposing and removing the template mRNA by alkali treatment, this time use reverse transcriptase or DNA
Ds-cDNA is synthesized using a polymerase.

A recombinant vector is prepared from the ds-cDNA thus obtained and a vector DNA containing a replicon capable of replicating in a eukaryote or a prokaryote. This ds-cDNA and vector DNA capable of growing in eukaryote or prokaryote are treated with exonuclease, added with chemically synthesized DNA fragment, ds-
It is modified by various treatments such as extending the G, C-chain in order to attach a terminal that can be linked to the end of cDNA or vector DNA. These ligable DNAs are, for example, ATP
Coexisting with DNA ligase of T4 phage in the coexistence. The obtained cDNA is MIL-2
Is confirmed, it is processed so that the genetic information is expressed by the host cell of the vector DNA, connected to the vector DNA, and introduced into the host cell.

Suitable host cells for producing a polypeptide having mouse interleukin-2 activity include eukaryotic cells such as vertebrates and yeast. For example, CV-1 cells, which are monkey cells, are used.
-40 origin-deficient mutant, expressing the SV-40 large T antigen (COS
Cells) (Gluzman: Cell, 23, 175-182 (1981)). Also, Ohno, Taniguchi (Nucleic acid Research 10 , 967-97
7 (1982)), and a yeast host vector system applied for IFN gene expression studied by Hitzeman et al. (Nature 293, 717-722 (1981)). Can be Suitable prokaryotic hosts include Escherichia coli, Bacillus subtilis, and the like. Escherichia coli can be used as a host for DNA amplification in the host cell, but other host cells can also be used.

To process the gene encoding MIL-2 so that it can be expressed in host cells, control DNA such as a promoter is inserted into MIL-2 when connected to vector DNA.
The MIL-2 gene may be placed at an appropriate position upstream of the MIL-2 gene so that the -2 gene is expressed.
Furthermore, by directly connecting the signal peptide base sequence of the host cell to be used to the 5′-end of the MIL-2 gene, a polypeptide having mouse interleukin-2 activity can be secreted outside the host cell or its periplasm. it can.

Examples of suitable Escherichia coli vectors include EK type plasmid vectors (stringent type).
As pSC101, pRK353, pRK646, p
EK type plasmid vector (relaxed type) such as RK248, pDF41: Co1E1, pVH51,
pAC105, RSF2124, pCR1, pMB9,
pBR313, pBR322, pBR324, pBR3
25, pBR327, pBR328, pKY2289,
pKY2700, pKN80, pKC7, pKB15
8, pMK2004, pACYC1, pACYC18
4, dul et al., Λgt phage vector: λgt · λ
c, λgt · λB, λWES · λC, λWES · λB,
λZJvir. , ΛB ', λALO, λB, λWES.
Ts622, λDam, and the like. Generally pBR
322 has often been used as a vector for Escherichia coli, in which case the best cloning sites are the PstI and EcoRI sites.

Transformation of host cells with the recombinant DNA includes the following commonly used methods. When a prokaryote such as Escherichia coli is the host, competent cells capable of incorporating this DNA can be transformed by the well-known CaCl ₂ method after recovering the cells in the logarithmic growth phase. The coexistence of MgCl ₂ or RbCl in the transformation reaction solution improves the transformation efficiency. It is also possible to transform host cells after preparing protoplasts.

When the host cell used is a eukaryote, DNA
Such as a method of infecting cells with calcium phosphate precipitate, a method of microinjection; a method of entrapping and inserting a plasmid into red blood cells or liposomes, a method of treating cells with a reagent such as lysophosphatidylcholine, or a method using a viral vector. It can be transmitted by any method.

The cells carrying the IL-2 gene can be used as a marker possessed by the vector DNA for traits and MIL.
-2 is obtained by isolating a strain having the ability to produce. In addition, since it is already known that the MIL-2 gene has complementarity with the human IL-2 gene, human IL-
By selecting cells having DNA that hybridizes with the two genes, cells having a DNA fragment containing the gene encoding mouse interleukin-2 can be obtained.

The entire nucleotide sequence of the DNA fragment containing the gene encoding mouse interleukin-2 isolated in the present invention is determined by the Maxam and Gilbert method (Meth. Enz.
ym. 65 , 499, (1980) and M13 phage (J. Messing et a
L., Gene, 19 , 269 (1982)), using the dideoxynucleotide chain termination method (F. Sanger, et al., Proc. Natl. Acad. Sc.
i. USA, 74 , 5463 (1979)). cDN
FIG. 1 shows a cut diagram of the A insert by the restriction enzyme endonuclease. As shown in FIG. 1, these cDNAs were PstI, PvuII, HindIII, Taq
The cut points by I and RsaI are arranged in this order. The entire DNA sequence of this cDNA insert is as described in SEQ ID NO: 3 in the sequence listing.

As described above, mouse interleukin-
The DNA fragment containing the gene encoding 2 has one large open reading frame.
The open reading frame is the 169th codon from the Ala codon, which is the 21st codon from the 48th to 50th ATG sequence counted from the 5'-end, which is considered to be the initiation codon, in the above cDNA base sequence. It is thought to correspond to the sequence up to the Gln codon. Thus, the MIL-2 polypeptide is considered to consist of 149 amino acids with Ala at the N-terminus and Gln at the C-terminus, with a calculated molecular weight of 172333.5 daltons. In some cases, the polypeptide having IL-2 activity may have N-terminal at Pro next to Ala. In addition, even if some amino acids in the amino acid sequence starting from Ala above are replaced with other amino acids or deleted, IL-2 activity may be obtained. Further, the above M
In the gene encoding IL-2, even if the amino acid codon is replaced with another triplet having the same amino acid, the same action as the above nucleotide sequence may be obtained.

The recombinant vector is amplified by culturing cells into which the recombinant vector has been inserted, or a polypeptide having mouse interleukin-2 activity is produced. This cultivation is performed by a usual method. For example, infected yeast can be grown at 27 ° C. to 37 ° C. in a medium containing a carbon source, a nitrogen source, inorganic salts, and if necessary, organic nutrients such as vitamins and amino acids.
It is carried out under aerobic conditions in the range of ° C and pH 4-7. Transformed prokaryotes such as Escherichia coli or Bacillus subtilis can also be cultured by conventional methods.

Polypeptide having mouse interleukin-2 activity produced intracellularly or extracellularly is precipitated by ammonium sulfate, dialysis for removing salts (under normal or reduced pressure), gel filtration, chromatography, isoelectric focusing. Concentration, gel electrophoresis, high performance liquid chromatography (hereinafter HPLC)
Abbreviations), (ion exchange, gel filtration and reverse phase chromatography), and activated Sepharose 4B conjugated with a dye-binding carrier, a monoclonal antibody against MIL-2.
Alternatively, it can be recovered by a known method such as affinity chromatography using lectin-bound Sepharose 4B or the like. The isolation and purification method of MIL-2 is described by Gillis et al.
unol. Rev. 63 , 167 (1982)), Watson et al. (J. Immunol., 122,
1633 (1979), Clark-Lewis et al. (J. Immunol., 128, 168 (198
2), J. Immunol. , 128 , 175 (1982)), Mochizuki et al. (J.
unol., 125, 2579 (1980). The polypeptide thus obtained exhibits the same biochemical and biological behavior as is known for IL-2 produced from mammalian cells by antigen stimulation, and IL-2
Have activity.

[0028]

EXAMPLES Next, the present invention will be described in detail with reference to Examples. Example 1 (1) Lymphoma LBRM-33 cells obtained by irradiating splenocytes of B10.BR mice with X-rays (S. Gillis et al.,
J.Immunol., 125, 2570 and ('80)), 4 × 10 4 cells / ml
Cells were suspended in 50 ml of RPMI1640 medium containing fetal bovine serum (FBS) at a cell density of 5% / volume%, placed in a Petri dish 1058 made by Falcon, cultured at 37 ° C. for 4 days, and cells were collected by centrifugation. . This cell is 2 ×
Suspended in the above medium at a cell density of 10 ⁶ cells / ml,
To this was added 1% of PHA, and the mixture was placed in a Petri dish 1058 manufactured by Falcon Co., Ltd. at 75 ml, and cultured at 37 ° C. for 6 hours.

(2) The PHA thus obtained is 6% with 1% of PHA.
Time-induced LBRM-33 cells (1.8 × 10 ⁹ cells) are suspended in 800 ml of PBS solution, and the cells are washed twice by centrifugation, and then ribonuclease inhibitor Ribo
R containing nucleosides-Vanadyl complex (10 mM)
SB solution (10 mM Tris-HCl, pH 7.5,
10mM NaCl, 1.5mM MgCl ₂₎ 800m
suspended in 1 l. Next, NP-40 was added so as to be 0.05%, and after gently stirring, 3000 rp
Nuclei were removed by centrifugation at m for 5 minutes and the supernatant was mixed with SDS (0.
5%) and EDTA (5 mM), and immediately, an equal amount of phenol was added to extract cytoplasmic RNA. The phenol extraction was repeated a total of three times, and then R
NA was precipitated and the precipitate was collected by centrifugation.
It was dissolved in is-HCl, pH 7.5. The amount of RNA thus obtained from LBRM-33 cells was 30 mg.

Next, to obtain mRNA from this RNA, oligo (dT) -cellulose (PL Biochemi
column chromatography was performed using cals, Type 7). Adsorption is 20 mM Tris-HCl, pH7.
5,0.5 M NaCl, 1 mM EDTA, 0.5% S
The elution is performed by dissolving the RNA in a DS solution, and elution is performed using a buffer solution (2
0 mM Tris-HCl, pH 7.5, 0.5 M Na
Cl, 1 mM EDTA), then water and 10 mM T
It was performed by alternately eluting mRNA with ris-HCl (pH 7.5). As a result, the eluted mRN
The amount of A was 576 μg.

Further, this mRNA was subjected to SDG centrifugation (50
mM Tris-HCl, pH 7.5, 1 mM EDT
A, 5-25% sucrose gradient containing 0.2 M NaCl, 27,000 rpm, 1 with Hitachi RPS28 rotor.
(8 hours, 4 ° C) and fractionated into 13 fractions.

(3) The fraction numbers 3 to 10 obtained here
From 1 μg of each of the mRNA fractions of 50 mM Tris-
HCl buffer (pH 7.5), 30 mM NaCl, 6
mMMgCl ₂ , 5 mM dithiothreitol (DT
T), 0.5 mM of each dATP, dGTP, dCTP, d
TTP (including dCTP labeled with ³² P), 0.1
₁₀ μg of oligo (dT) and 16 units of AMV reverse transcriptase (JW Beard) were mixed and kept at 41 ° C. for 90 minutes to synthesize single-stranded cDNA. After the reaction was completed, the protein was removed with phenol, and the DNA was recovered by ethanol precipitation. The obtained DNA is dissolved in distilled water, and
To remove RNA, add NaOH solution to add 0.33N Na
Place at 37 ° C. for 2 hours as OH, then neutralize the solution with equimolar hydrochloric acid.

Using the obtained single-stranded cDNA solution,
1% agarose gel electrophoresis was performed, and the cDNA was transferred to a nitrocellulose filter by Southern blotting according to a conventional method.
Niguchi, T et al., Nature, 302, 5906 ('83)) was digested with a restriction enzyme HinfI (New England Bio.Lab., USA) (450 base pairs) to perform hybridization. That is, HfI digested product of human IL-2 cDNA (450 base pairs) was introduced with ³² P by a nick translation method and hybridized at 65 ° C. for 24 hours using this as a probe.

Next, the nitrocellulose filter was immersed in 3 × SSC (1 × SSC is an aqueous solution containing 150 mM NaCl and 15 mM sodium citrate), and then immersed in 3 × SSC containing 0.1% SDS at 65 ° C. Was washed twice for 30 minutes, immersed in 3 × SSC, and air-dried. This was spent all day and night on autoradiography.

(4) cDNA from the mRNA fraction corresponding to the cDNA hybridized with human IL-2 cDNA
Was synthesized in a large amount to prepare a plasmid vector pBR322 and a recombinant vector, which were introduced into Escherichia coli, and cells having the MIL-2 cDNA clone were selected as follows. (4-1) 50 mM Tris-HCl, pH 7.5,
30 mM NaCl, 6 mM MgCl ₂ , 5 mM dithiothreitol (DTT), 0.5 mM dATP,
dGTP, dCTP, dTTP (dCTP includes those labeled with ³² P), 0.7 μg oligo (dT) ₁₀ , 13 μg
g mRNA and 16 units AMV reverse transcriptase (J.
W. Beard) was mixed and kept at 41 ° C. for 90 minutes. Then
Protein was removed from the reaction solution with phenol, and DNA was recovered by ethanol precipitation from the reaction solution.
It was dissolved in a 0 mM Tris, 1 mM EDTA, pH 7.5 solution. To remove the mRNA from the DNA solution, add 0.33N NaO
H and the solution was left at room temperature for 15 hours. Next, this solution was neutralized with an equal amount of 1 M Tris-HCl (pH 7.5) and passed through a "Sephadex G-50 column". As a result, 5 μg of single-stranded cDNA was recovered.

(4-2) From the single-stranded cDNA thus prepared, the method of Land et al. (Nucleic Acids Res., 9 , 25)
51 ('81)), and the cDNA mixture was fractionated by sucrose density gradient centrifugation. This gives 5
1.5 µg of a double-stranded cDNA of 00 base pairs or more was obtained.

(4-3) 0.14 M potassium cacodylate, 30 mM Tris base, 0.1 mM DTT, 1 mM
CoCl ₂ , 0.64 mM ³² P-dCTP (specific activity 2.
7 × 10 ⁶ cpm / nmol), 1.5 μg double-stranded cD
NA and 5 units of terminal transferase (Bethesda Research Laboratories) were mixed and kept at 37 ° C. for 7 minutes, and the protein was removed from the reaction solution by the phenol method. This was passed through a "Sephadex G-50" column, and the DNA was recovered from the eluate by ethanol precipitation. As a result, about 50 dCMPs were added to the 3 'ends of the obtained cDNA.

(4-4) 50 mM Tris-HCl
(PH 7.5), 0.1 M NaCl, 5 mM EDT
A, pBR322 to which 0.10 μg of dGMP was added was cleaved with restriction enzyme PstI (BRL) and
An aqueous solution containing 2 μg of dCMP-added cDNA was kept at 65 ° C. for 2 minutes, then at 45 ° C. for 60 minutes, at 37 ° C. for 60 minutes, and at room temperature for 60 minutes.

On the other hand, 5 Escherichia coli HB101
0 ml of tryptone medium (1% tryptone, 0.5%
Yeast extract and 0.5% NaCl) were inoculated, and the culture was shake-cultured at 31 ° C. until the absorbance at 650 mμ of the culture reached about 0.6 to 0.7. After completion of the culture, the culture was left at 0 ° C. for 20 minutes, then the cells were collected by centrifugation, suspended in 25 ml of a 50 mM CaCl ₂ solution, and
After 1 hour, the cells were collected by centrifugation and 50 mM
The cells were resuspended in 5 ml of a CaCl ₂ , 20% glycerol solution, and the obtained cell suspension was placed in an Eppendorf tube in 0.5 ml portions and stored frozen at -70 ° C.

The above recombinant DNA was added to 0.1 ml of the frozen cell suspension, and the mixture was allowed to stand at 0 ° C. for 20 minutes. In addition, 42
After keeping it at ℃ for 1 minute, put it at 20 ℃ for 10 minutes.
Medium (1% "Bactopeptone", 0.5% yeast extract,
1 ml containing 0.5% NaCl and 0.1% glucose)
And shaking culture was performed at 37 ° C. for 1 hour. This culture solution 1
50 to 200 μl was spread on a 1.5% agar medium of L medium containing 25 μg / ml streptomycin and 10 μg / ml tetracycline, and kept at 37 ° C. for 1 day.

(4-5) Human IL-2c labeled with ³² P for about 15,800 colonies that appeared above
The method of Grunsteiu-Hogness (Proc. Natl. Acad. Sc) using a HinfI degradation product (450 base pairs) of DNA as a probe
Colony hybridization was performed using iUSA, 70, 2330 (1975)) to obtain a transformant containing plasmid pMIL-2-14 having a cDNA insert of about 900 base pairs. Therefore, this base sequence was changed to Maxam and Gilb
ert's chemical method (Meth. Enzym., 65 , 499, (1980)) and M1
Dideoxynucleotide chain termination method (F. Sanger, et al.) Using 3 phage (J. Messing et al., Gene, 19 , 269 (1982)).
al., Proc. Natl. Acad. Sci. USA, 74, 5463 (1979))
As a result of comparison with human IL-2 cDNA and a gene encoding a secreted protein, a nucleotide sequence encoding an initiation codon ATG at the 5′-upstream site and a subsequent hydrophobic peptide was found. Was not done. That is, this cDNA contains about 12S of IL.
-2 was found to correspond to part of the mRNA.

Then, again, in the same manner as described above, all mR
NA was size-fractionated by sucrose density gradient centrifugation, cDNA was synthesized from those having 500 base pairs or more, and plasmid pB was synthesized.
It was inserted into R322. This plasmid was used to transform E. coli. Co
lHB101 was transformed, and a cDNA clone having the same sequence as the plasmid pMIL-2-14 was cloned from 32,500 transformed strains into pMIL-2-labeled with ³² P.
Plasmid pMIL-, which had a cDNA insert of about 1,000 base pairs, was selected by the above colony hybridization method using the degradation products (585 base pairs) of the 14 insert restriction enzymes PvuII and AccI (Takara Shuzo Co., Ltd.) as probes. Escherichia coli HB101 / MIL2-45 AJ1215 containing 2-45
5 (FERM-P7758) was obtained.

The base sequence of the obtained cDNA is the above-mentioned Maxam
It was found to have the above-mentioned nucleotide sequence when examined according to and Gilbert's chemical method and dideoxynucleotide chain termination method. FIG. 1 shows a restriction enzyme cleavage map of this cDNA.

On the other hand, a pKCR vector (Proc. Natl. Aca. Sci. USA, 7
8 , 1527, ('81)), a pCE-1 vector having a region containing the promoter of the early gene of SV40 virus was constructed (see European Patent Application Publication No. 91539). Then, the cDNA was cut out from the plasmid pMIL2-45 clone with the restriction enzyme PstI to obtain pCE-1.
It was inserted into the PstI site of the vector (plasmid pC
EMIL-2-45).

This vector was used for COS-7 of monkey cultured cells.
Cells (Gluzman, Y. Cell , 23, 175 (1981)) (McCutchan et al J, Natl. Cancer Inst., 41 , 351).
(1968)). That is, 6 × 10 ⁴ COS-7 cells / m
was suspended in DMEM containing 5% FBS.
After spreading on a 24-well Nunc culture plate and culturing at 37 ° C. for 4 hours, 1 mM T cell containing 0.1 mM EDTA was added.
ris-HCl, pH 7.5, 17.6 μl, 2M C
2.4 μl of aCl ₂ solution and 2 × concentrated Hanks balanced saline (2 × HBS) (50 mM Pipes, 2
_{80mM NaCl, 1.5mM Na 2 HPO 4} · 1
Contains 2H ₂ O. pH 7.12 20 μl mixed solution (4
0 μl) to the above plasmid pCEMIL-2-451.
What melt | dissolved μg was added. The mixture was allowed to stand at 37 ° C. for 4 hours, water was removed by suction from the resulting culture, the cells were washed with 1 ml of PBS, and 0.5 ml of PBS containing 20% glycerol was added to the plate. After the plate was left at room temperature for 3 minutes, water was removed by suction. The cells are washed with an additional 1 ml of PBS and 5% FBS
Culture at 37 ° C. in 1 ml of DMEM containing
At time, the medium is aspirated off and then 2-10% FBS
0.5 ml of DMEM containing was added and cultured for 2 days.

The IL in the culture supernatant thus obtained was
-2 activity was determined by the method of Gillis et al. (J. Immunol., 120, 2027 ('7
8)). As a control, the vector plasmid p
An experiment was conducted in the same manner using CE-1, IL-2
The activity was examined. From the results shown in Table 1, it was illuminated that the cDNA insert was a DNA fragment containing a gene encoding mouse interleukin-2. pCEM
Escherichia coli HB101 having IL2-45 (AJ 12156) has been deposited as FERM-P7759.

[0047]

[Table 1]

Example 2 (1) A recombinant vector used to express the MIL-2 gene was constructed as follows (FIG. 3). The fragments shown below were synthesized by the solid phase modified triester method. As shown in FIG. 2, P-1 and P-1
Except for T-12, T4 polynucleotide kinase and ATP
Was used to phosphorylate the 5'-end. Next, P-
1, P-2, P-3, P-4, P-5 are mixed, and T4
The DNA was converted to double-stranded DNA using DNA ligase, subjected to polyacrylamide gel electrophoresis, and purified. Similarly, P-
6, P-7, P-8, P-9, P-10, P-11, P
A double-stranded DNA was formed from -12. In addition, T
4 Ligation using DNA ligase, P-1 to P-1
2 isolated the correctly aligned double-stranded synthetic DNA (A) as shown in FIG.

On the other hand, pBR322 was replaced with the restriction enzyme EcoRI.
And a large DNA fragment (4.0K) digested with BamHI.
b) was isolated. Next, the obtained pBR322 fragment was mixed with three types of DNA fragments of a synthetic DNA (A) (FIG. 2) and a synthetic DNA (B) (described in SEQ ID NO: 4 in the sequence listing).
4 Ligation was performed using DNA ligase. The obtained recombinant vector was introduced into Escherichia coli HB101 strain, and a strain having ampicillin resistance was selected. From the isolated strain, a plasmid DNA was obtained, and a cutting test using a restriction enzyme was performed to select a bacterium holding pAJ-2.

50 μg of pAJ-2 was added to the restriction enzyme Bgt.
After cutting with II and XhoI, the fragment having the -35 region was isolated and purified by agarose gel electrophoresis according to a conventional method to obtain 2 µg. Next, 0.5 μg of this fragment and 20 pmol of each of the synthetic oligonucleotides (C) described in SEQ ID NOs: 5 and 6 in the sequence listing (this is the
ox sequence and lac operator sequence. ), Both of which are phosphorylated at the 5′-end, are treated with 6.6 mM MgCl ₂ , 1 mM ATP and 10 mM.
66 mM Tris-HCl (pH 7.
In 5), the mixture was mixed with 0.8 unit of T4 DNA ligase, and the mixture was reacted at 4 ° C. overnight to obtain pAJ20. P-1 to P-12 in FIG. 2 correspond to SEQ ID NOs: 7, 8, 9, 10, 11, 12, 1 in the sequence listing, respectively.
It is represented by 3, 14, 15, 16, 17, and 18.

(2) Using the obtained plasmid pAJ20, a recombinant vector expressing MIL-2 was constructed as follows (Fig. 4). Plasmid pAJ20 (10μ
g) is cleaved with restriction enzymes XhoI and PvuII, and a larger fragment is isolated and purified by agarose gel electrophoresis.
3 μg of DNA was obtained. On the other hand, 9 μg of the cDNA insert obtained by PstI digestion of pMIL2-45 obtained by the method shown in Example 1 was digested with the restriction enzyme HhaI, treated with T4 DNA polymerase, and the largest fragment 5 μg was isolated by agarose gel electrophoresis. Purified.

Next, 0.45 μg of the fragment containing the inducible synthetic promoter (described above) and the Hh containing the IL-2 cDNA were used.
0.5 μg of the aI fragment and 20 pmol each of the synthetic DNA (D) shown below (see SEQ ID NOs: 19 and 20 in the sequence listing)
Is phosphorylated at both the 5'-end, 6.6 mM MgCl ₂ so like, 1 mM ATP and 1m
66 mM Tris-H at pH 7.5 containing MDTT
In Cl, mixed with 0.8 units of T4 DNA ligase and kept the mixture at 4 ° C overnight.

The obtained plasmid was introduced into Escherichia coli HB101 strain according to a conventional method, and a strain which formed a colony on an L medium agar plate containing ampicillin was isolated. From the obtained strains, a strain having a recombinant vector that hybridizes with the HhaI fragment was selected by the colony hybridization method. The colonies thus selected were cultured again (10 ml) using the L medium, and the obtained cells were treated with lysozyme, frozen and thawed to obtain plasmid DNA. This plasmid DNA is
The resulting product was subjected to agarose gel electrophoresis to obtain pAJMIL2-21 in which the cDNA was ligated in the correct direction after the inducible promoter ATG sequence.

Reference Example Escherichia coli HB1 containing pAJMIL2-21
01 was cultured in the usual manner known for the growth of microorganisms. Cells were grown in medium containing 25 μg / ml streptomycin and 25 μg / ml ampicillin (2.5% “Bactotryptone”, 1% yeast extract, 0.
The cells were grown overnight at 37 ° C. in 10 ml of 1% glucose, 20 mM MgSO ₄ , 50 mM Tris-HCl buffer, pH 7.5. Next, 1 ml of the culture suspension was inoculated into the same χ medium (100 ml), and cultured at 37 ° C. When the absorbance of the culture at 650 nm reached approximately 0.5, 1 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium. One hour after the addition of IPTG, the cells were collected, and the obtained cells were treated with 20 mM Tr.
is-HCl buffer (pH 7.5, 30 mM NaCl
) And resuspended in 8 ml of the same buffer.

The protein thus produced in bacterial cells was treated with sonic (0 ° C., 30 seconds) and lysozyme (8 μm).
g) Following digestion (0 ° C, 20 minutes), freeze-thaw for 3 minutes.
It was extracted by performing it twice. The IL-2 activity of the extract thus obtained was determined by the method of Gillis et al. (J. Immunol.,
120, 2027 ('78)). An experiment was conducted in the same manner using the vector plasmid pAJ20 as a control, and the IL-2 activity was examined (Table 2). pAJMIL2
Escherichia coli HB101 (AJ
12162) has been deposited as FERM P-7821.

[0056]

[Table 2]

The host used in the above reference example, Escherichia coli HB101 (Boyer, HW et al., Mol. Bio
l., 41 , 459 (1969)) is readily available. In addition, pMIL2-45 is cultured at 37 ° C. in L medium to release the recombinant DNA in pMIL2-45, and the strain which has become sensitive to tetracycline and ampicillin is isolated. 45
HB101 is easily obtained from The plasmid vector pBR322 (for example, can be purchased from Bethesda Research Laboratory), pCE-1 is already known and readily available.

[0058]

Industrial Applicability According to the present invention, a DNA fragment containing a gene encoding a polypeptide having mouse interleukin 2 activity was successfully isolated. Opened.

[0059]

[Sequence list]

SEQ ID NO: 1 Sequence length: 148 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Polypeptide Origin Biological name: Mouse Strain name: Lymoma LBRM-33 Sequence Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln Gln 5 10 15 Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu Met 20 25 30 Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn Leu Lys 35 40 45 Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala Thr 50 55 60 Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu Arg 65 70 75 80 His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp Ala 85 90 95 Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys Gly 100 105 110 Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr Val 115 120 125 Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile Ser 130 135 140 Thr Ser Pro Gln 145

SEQ ID NO: 2 Sequence length: 444 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Mouse strain name: Lymphoma LBRM-33 sequence CCC ACT TCA AGC TCC ACT TCA AGC TCT ACA GCG GAA GCA CAG CAG CAG 48 CAG CAG CAG CAG CAG CAG CAG CAG CAG CAC CTG GAG CAG CTG TTG ATG 96 GAC CTA CAG GAG CTC CTG AGC AGG ATG GAG AAT TAC AGG AAC CTG AAA 144 CTC CCC AGG ATG CTC ACC TTC AAA TTT TAC TTG CCC AAG CAG GCC ACA 192 GAA TTG AAA GAT CTT CAG TGC CTA GAA GAT GAA CTT GGA CCT CTG CGG 240 CAT GTT CTG GAT TTG ACT CAA AGC AAA AGC TTT CAA TTG GAA GAT GCT 288 GAG AAT TTC ATC AGC AAT ATC AGA GTA ACT GTT GTA AAA CTA AAG GGC 336 TCT GAC AAC ACA TTT GAG TGC CAA TTC GAT GAT GAG TCA GCA ACT GTG 384 GTG GAC TTT CTG AGG AGA TGG ATA GCC TTC TGT CAAGC ATC TCA 432 ACA AGC CCT CAA 444

SEQ ID NO: 3 Sequence length: 1039 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Mouse Strain name: Lymphoma LBRM-33 sequence Characteristic of the characteristic: Symbol indicating the characteristic: CDS Location: 48..554 Method determining the characteristic: S Symbol indicating the characteristic: sig peptide Location: 48..111 Method determining the characteristic: S Symbol indicating the characteristic: mat peptide Location: 112..554 Method for determining characteristics: S sequence ATCACCCTTG CTAATCACTC CTCACAGTGA CCTCAAGTCC TGCAGGC ATG TAC AGC 56 Met Tyr Ser 1 ATG CAG CTC GCA TCC TGT GTC ACA TTG ACA CTT GTG CTC CTT GTC AAC 104 Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu Leu Val Asn 5 10 15 AGC GCA CCC ACT TCA AGC TCC ACT TCA AGC TCT ACA GCG GAA GCA CAG 152 Ser Ala Pro Thr Ser Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln 20 25 30 35 CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAC CTG GAG CAG CTG 200 Gln Gln Gln Gln Gln Gln G ln Gln Gln Gln Gln His Leu Glu Gln Leu 40 45 50 TTG ATG GAC CTA CAG GAG CTC CTG AGC AGG ATG GAG AAT TAC AGG AAC 248 Leu Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn 55 60 65 CTG AAA CTC CCC AGG ATG CTC ACC TTC AAA TTT TAC TTG CCC AAG CAG 296 Leu Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln 70 75 80 GCC ACA GAA TTG AAA GAT CTT CAG TGC CTA GAA GAT GAA CTT GGA CCT 344 Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro 85 90 95 CTG CGG CAT GTT CTG GAT TTG ACT CAA AGC AAA AGC TTT CAA TTG GAA 392 Leu Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu 100 105 110 115 GAT GCT GAG AAT TTC ATC AGC AAT ATC AGA GTA ACT GTT GTA AAA CTA 440 Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu 120 125 130 AAG GGC TCT GAC AAC ACA TTT GAG TGC CAA TTC GAT GAT GAG TCA GCA 488 Lys Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala 135 140 145 ACT GTG GTG GAC TTT CTG AGG AGA TGG ATA GCC TTC TGT CAA AGC ATC 536 Thr Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile 150 155 160 ATC TCA ACA AGC CCT CAA TAACTATGTA CCTCCTGCTT ACAACACATA 584 Ile Ser Thr Ser Pro Gln 165 AGGCTCTCTA TTTATTTAAA TATTTAACTT TAATTTATTT TTGGATGTAT TGTTTACTAT 744 CTTTTGTAAC TACTAGTCTT CAGATGATAA ATATGGATCT TTAAAGATTC TTTTTGTAAG 804 CCCCAAGGGC TCAAAAATGT TTTAAACTAT TTATCTGAAA TTATTTATTA TATTGAATTG 864 TTAAATATCA TGTGTAGGTA GACTCATTAA TAAAAGTATT TAGATGATTC AAATATAAAT 924 AAGCTCAGAT GTCTGTCATT TTTAGGACAG CACAAAGTAA GCGCTAAAAT AACTTCTCAG 984 TTATTCCTGT GAACTCTATG TTAATCAGTG TTTTCAAGCT CATAGAA ATAACT

SEQ ID NO: 4 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence AATTCTTGCGG GAACGCCAATG 22

SEQ ID NO: 5 Sequence length: 49 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence GATCTATAAA TTTGTTATAA TGTGTGGAAT TGTGAGCGGA TAACAATTC 49

SEQ ID NO: 6 Sequence length: 49 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence ATATTTAAAC AATATTACAC ACCTTAACAC TCGCCTATTG TTAAGAGCT 49

SEQ ID NO: 7 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence GTTACCTGCCGTGAG 15

SEQ ID NO: 8 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence TAAGTTACTCACGGCAG 17

SEQ ID NO: 9 Sequence Length: 15 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic Oligonucleotide Sequence TAACTTAATTTTTGT 15

SEQ ID NO: 10 Sequence length: 16 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence CTATGTCAACAAAAAT 16

SEQ ID NO: 11 Sequence length: 15 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence TGACATAGATCTCTA 15

SEQ ID NO: 12 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence AACAAATTTAGAGAT 15

SEQ ID NO: 13 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence AATTTGTTATAATATAG 17

SEQ ID NO: 14 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence GAGATGCCTATATTAT 14

SEQ ID NO: 15 Sequence length: 16 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence GCATCTCGAGTAGGAG 16

SEQ ID NO: 16 Sequence length: 15 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic oligonucleotide Sequence TGTTACCTCCTACTC 15

SEQ ID NO: 17 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence GTAACAATTTGACCATG 17

SEQ ID NO: 18 Sequence length: 15 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence GATCCATGGTCAAAT 18

SEQ ID NO: 19 Sequence length: 45 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic oligonucleotide sequence AACTAGTACG CAAGTTCACG TAAAAAGGGT ATCGACAGCT GATGG 45

SEQ ID NO: 20 Sequence length: 45 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic oligonucleotide sequence TTGATCATGC GTTCAAGTGC ATTTTTCCCA TAGCTGTCGA CTACC 45

[Brief description of drawings]

FIG. 1 is a restriction map of a DNA fragment containing a gene encoding mouse interleukin-2.

FIG. 2 is an explanatory diagram of the progress of construction of a plasmid vector pAJ-2.

FIG. 3 is an explanatory diagram showing the progress of construction of a plasmid vector pAJ20.

FIG. 4 is an explanatory diagram showing the construction process of pAJMIL2-21.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12N 5/10 C12P 21/02 K 9282-4B // A61K 35/12 7431-4C 35/74 G 7431-4C 38/00 C07K 14/55 8318-4H (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1:91) A61K 37/02 (72) Inventor Haruo Sugano 4-8-13 Minamiogikubo, Suginami-ku, Tokyo

Claims (6)

[Claims] 1. A DNA fragment having a mouse interleukin-2 activity and encoding a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. 2. The base sequence of the DNA fragment contains the base sequence shown in SEQ ID NO: 2 in the sequence listing.
The described DNA fragment. 3. Transformation of a prokaryotic or eukaryotic cell into which a DNA fragment having a mouse interleukin-2 activity and encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing has been inserted. Recombinant vector for 4. A recombination for transforming a prokaryotic or eukaryotic cell according to claim 3, wherein the nucleotide sequence of the DNA fragment to be inserted contains the nucleotide sequence set forth in SEQ ID NO: 2 in Sequence Listing. vector. 5. Transformation of a prokaryotic or eukaryotic cell into which a DNA fragment having a mouse interleukin-2 activity and encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing has been inserted. Transformant carrying the recombinant vector for the purpose. 6. The nucleotide sequence of a DNA fragment inserted into a recombinant vector for transforming a prokaryotic or eukaryotic cell contains the nucleotide sequence set forth in SEQ ID NO: 2 in the Sequence Listing. The transformant described.