JPH0142279B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to polypeptides having human interleukin-2 activity. Interleukin 2 (hereinafter abbreviated as "IL-2"), formerly called T cell growth factor, is a soluble protein (generally called "IL-2") produced by T cells activated by lectin or antigen. lymphokines) (Morgan et al., Science, 193 , 1007-1008 (1976),
Gillis et al., J. Immunol., 120 , 2027-2033
(1978)). IL-2 can modulate lymphocyte reactivity and induce antigen-specific effector T-lymphocytes.
long-term culture in vitro (Gillis et al., Nature, 268 , 154-156).
(1977)). IL-2 also promotes thymocyte division (Chen et al., Cell Immunol., 22 , 221-224).
(1977), Shaw et al., J. Immunol., 120 , 1967~
1973 (1978)), cytotoxic T lymphocyte activity in a culture system of nude mouse splenocytes (Wagner et al.
Nature, 284 , 278-280, (1980)) and anti-SRBC
Induction of plaque-forming cell responses (Gillis et al., J.Exp.
Med. 149 , 1960-1968 (1979)). Therefore, this lymphocyte regulatory factor is useful for enhancing fluid and cell-mediated immune responses and restoring immunodeficiency to normal fluid and cell-mediated immunity.
These revealed immunological activities of IL-2 indicate that IL-2 can be used in malignant tumors, bacterial or viral infections, immunodeficiency, autoimmune diseases, etc.
et al., Adv. Immunopharm., 507, (1980)).
Like interferon, IL-2 has been shown to enhance natural killer cell activity, strongly suggesting its utility in the treatment of malignant tumors. Furthermore, IL-2 enables the maintenance of monoclonal activated T cells, which is useful for studying the molecular mechanism of T cell differentiation, the differentiation mechanism of T cell function, and the mechanism of T cell antigen receptors. It shows that they play an important role. In addition, IL-2 can be obtained by long-term culture of monoclonal T cells.
It can also be used to produce a variety of T cell-derived lymphokines useful in a variety of other fields. Furthermore,
The production of IL-2 and the responsiveness of lymphocytes to IL-2 are important parameters of immunological function and are useful in the clinical diagnosis of immune abnormalities. IL-2 has traditionally been produced by stimulating mouse, rat, or human lymphocytes with mitogens (Gillis et al., Nature;
268, 154-156, (1977)), Farrar et al., J.
Immunol., 121 , 1353-1360, (1978), Gillis et al.
J. Immunol., 120 , 2027-2033, (1978)). By stimulating human peripheral blood lymphocytes with mitogens (Gillis et al., J. Immunol., 124 , 1954-
1962, (1980)), Gillis et al. Production of murine IL-2 from a T lymphoma cell line (Gillis et al., J.
Immunol., 125 , 2570-2578 (1980)) and production of human IL-2 from human leukemia cell lines (Gillis et al., J.
Exp.Med., 152 , 1709-1719, (1980)). The technique described above by Gillis et al. relates to a method for producing human IL-2 from mitogen-activated T leukemia cell lines using cell culture methods. However, with this method, low concentrations of human
The drawback is that only IL-2 is produced, and complex purification steps are required to obtain trace amounts of IL-2 from a large amount of culture fluid. Furthermore, since human leukemia cell lines also produce small amounts of other physiologically active substances that closely resemble human IL-2, it is necessary to separate IL-2 from these other immunologically active molecules, or to isolate cytotoxic substances that sometimes coexist. (toxic lectin) is quite difficult to separate. Other methods for producing IL-2 include recombinant DNA (abbreviation for deoxyribonucleic acid) methods, which have been used to produce other bioactive human-derived proteins such as interferon (Gray et al., Nature 295 ;
503-508, (1981), Nagata et al., Nature 284 ,
316–320 (1980), Taniguchi et al., Gene 10 , 11
~15, (1980)) is considered preferable. However, prior to the present invention, recombinant DNA methods
Attempts to produce IL-2 have been unsuccessful.
For example, attempts to create organisms that produce IL-2 by recombinant DNA bodies likely
The lack of success because the gene encoding the -2 polypeptide had not yet been cloned was reported in ``Nikkei Biotechnology, No. 19.''
Therefore, there has been a desire for a cloned gene encoding IL-2 and a recombinant DNA body containing that gene. There has been a need for a cell line and a method for producing IL-2 using the cell line. The gist of the present invention will become more readily apparent from the following description. That is, the present invention has created a polypeptide that is obtained by genetic recombination, has no sugars, does not substantially contain other proteins of human origin, and has the following amino acid sequence in its molecule. Pro Thr Ser Ser Ser Thr Lys Lys Thr
GlN Leu GlN Leu Glu His Leu Leu Leu
Asp Leu GlN Met Ile Leu AsN Gly Ile AsN
AsN Tyr Lys AsN Pro Lys Leu Try Arg
Met Leu Thr Phe Lys Phe Try Met Pro
Lys Lys Ala Thr Glu Leu Lys His Leu GlN
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr Specific examples of the polypeptide of the present invention include the following. Polypeptide having the following amino acid sequence Ala Pro Thr Ser Ser Ser Thr Lys Lys
Thr GlN Leu GlN Leu Glu His Leu Leu
Leu Asp Leu GlN Met Ile Leu AsN Gly Ile
AsN AsN Tyr Lys AsN Pro Lys Leu Try
Arg Met Leu Thr Phe Lys Phe Try Met
Pro Lys Lys Ala Thr Glu Leu Lys His Leu
GlN Cys Leu Glu Glu Glu Leu Lys Pro Leu
Glu Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr Polypeptide having the following amino acid sequence Pro Thr Ser Ser Ser Thr Lys Lys Thr
GlN Leu GlN Leu Glu His Leu Leu Leu
Asp Leu GlN Met Ile Leu AsN Gly Ile AsN
AsN Tyr Lys AsN Pro Lys Leu Try Arg
Met Leu Thr Phe Lys Phe Try Met Pro
Lys Lys Ala Thr Glu Leu Lys His Leu GlN
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr According to the present invention, IL-2 is obtained by inserting a gene encoded to produce a polypeptide having IL-2 activity and a vector capable of replicating in a cell.
Modified by recombinant methods by the insertion of DNA, the coding sequence of the gene is transformed into a prokaryotic cell line, in particular Escherichia coli, transformed to produce IL-2 by the DNA located downstream of the promoter sequence. A polypeptide having IL-2 activity is produced by suspension culture (aerobically) in a medium. The cloned gene encoding the IL-2 polypeptide is a messenger RAM (mRNA) corresponding to IL-2 derived from mammalian cells characterized by the ability to produce polypeptides with IL-2 activity. ; "RNA" is an abbreviation for ribonucleic acid; hereinafter referred to as "IL-2 mRNA") may be obtained by reverse transcription of complementary DNA (cDNA). The obtained single-stranded cDNA (ss-cDNA) can be converted into double-stranded cDNA (ds-cDNA). Use as a template to prepare cDNA
mRNA can be isolated from mammalian cells that produce IL-2 polypeptides. isolated
RNA is polyadenylated (Gillis et al., J.
Immunological Rev., 63 , 167-209 (1982)),
Polyadenylated RNA can be fractionated into 11-12S fractions, for example, by sucrose density gradient centrifugation. 13S mRNA may exhibit IL-2 mRNA activity, but in this case, 11-
It is thought that it is an aggregate of 12S mRNA. Mammalian cells capable of producing IL-2 and serving as a source of mRNA may be T-lymphocytes such as peripheral blood mononuclear cells, tonsil cells, and spleen cells that can be isolated from mammals. To impart IL-2 production ability to cells or enhance IL-2 activity,
Pretreatment may be performed by conventionally known methods such as nylon column treatment, antiserum and capture body treatment, density gradient fractionation, combination treatment with neuraminidase and galactose oxidase, various enzyme treatments such as trypsin treatment, and X-ray irradiation. . Cloned T-lymphocytes obtained after culturing the mammalian cells described above in the presence of T-cell growth factors can also be used as a source of mRNA, with T-lymphocytes being more preferred. Also transformed lymphocyte cell lines or cloned transformed cell lines, such as T lymphocytes themselves derived from leukemia or lymphoma cell lines or derivatives thereof pretreated or mutated by the methods described above.
Preferred as a source of mRNA. Apparently, cloned cell lines usually contain higher amounts of IL-2 mRNA compared to the parent line before cloning. Lymphocyte-derived cells described above and CEM, Molt 4F,
T cell hybridomas obtained by fusing tumor cell lines such as BW5147 are also preferred mammalian cells for use in the present invention. In this case, the lymphocyte-derived cells are cells in which mitogen is introduced into the culture medium in the presence or absence of (1) cells that spontaneously produce IL-2 and (2) other cells that assist in IL-2 production. Contains cells that produce IL-2 only when To induce IL-2 mRNA in the spontaneous IL-2 producing cells, the spontaneous IL-2 producing cells are cultured by methods well known in the field of cell culture. When producing mRNA in cells that produce IL-2 only in the presence of mitogens, the cultured cells should be thoroughly washed with culture medium and incubated at Rosewell Park Memorial Institute 1640 with or without serum (hereinafter referred to as “ RPMI 1640”), Darbetzkow’s Modified Eagle Medium (hereinafter “DMEM”)
It is abbreviated as ) or resuspend in a medium such as Krick's medium. These cell culture media contain penicillin, streptomycin or other antibiotics,
Various additives such as L-glutamine, Hepes buffer, or sodium bicarbonate may be added at concentrations commonly used in the cell culture field. The preferred cell concentration is 0.5-4×10 ⁶ cells/ml.
Appropriate stimulants are added to induce mRNA activation and IL-2 production. Among the suitable stimulants are mitogenes, neuraminidase, galactose oxidase, zinc compounds such as zinc chloride or protein A, streptolysin-O
These include lymphocyte activating factors derived from cells such as. Stimulated cells are collected and washed. During mitogen stimulation, if macrophages or dendritic cells coexist, mRNA
or increase the yield of activated mRNA. Similarly Raji, Daudi, K562, BALL
Co-existence of B lymphocytes such as -1 or cell lines derived from B lymphocyte cell lines can also activate mRNA and increase the yield of activated mRNA. To propagate mammalian cells, the cells are maintained in vitro under normal conditions in cell culture or in a histocompatible animal. When passaging by in vitro culture to prepare a source of mRNA, these cells can grow in any medium conventionally known to promote T cell growth. . Mammalian serum, serum components, or serum albumin may be added to these media. The culture time for mRNA activation is a time corresponding to the time required for activation to produce mRNA. This time is usually
This corresponds to the time required until production of IL-2 into the medium begins. The preferred time is 3 to 12 hours after adding the stimulant such as mitogen. If the culture time is too long, the generated IL-
2mRNA may be degraded. When activating IL-2 producing cells, phorbol esters such as PMA or TPA can be added at 10-50 ng/ml to increase the activation level. The above steps for IL-2 mRNA activation are PH7.0
~7.4, carried out in a saturated steam environment with a temperature range of 32-38°C. A method for obtaining and culturing IL-2-producing mammalian cells is described below. (b) Obtaining a spontaneous IL-2 producing strain Jyurkat cells, human T lymphocyte-derived leukemia cells (Fred Hutchinson Cancer Institute/Seattle/USA, Salk Institute/San Diego/USA, West German National Cancer Center/
Freely available in Heidelberg/West Germany, etc. ) were suspended in Krick medium at a cell density of 1×10 ⁶ cells/ml, and irradiated with X-rays for a total of 8000 roentgens at an irradiation rate of 150 roentgens/min.
After this, the cells were added to a 96-well flat-bottomed microtiter plate (Falcon 3072) at a cell density of 0.1 cells/200μ medium and incubated at 37°C for 3 weeks in Krick's medium containing 5% fetal bovine serum. %
Cultivate in a CO ₂ incubator (cloning by limiting dilution method). Cells in culture wells in which cell growth has been observed are transferred to a 24-well Nunc culture plate before the cells reach a density that covers the entire bottom surface, and the cells are grown in Krick medium for 5 days. Furthermore, add 1 to 2 cells of this cell
Cells were suspended in a serum-free synthetic medium containing neither serum nor serum-derived albumin at a density of × ¹⁰⁶ cells/ml and cultured for 2 days. The main culture supernatant was separated by centrifugation, and then passed through a 0.22μ Millipore filter. Debris removal and sterilization were performed at By measuring the IL-2 activity of the culture supernatant thus obtained, an X-ray treated mutant strain that spontaneously produces IL-2 was selected and cloned. (b) Obtaining an IL-2 producing strain from human peripheral blood mononuclear cells Human peripheral blood was collected and peripheral blood lymphocytes (hereinafter abbreviated as PBL) were obtained by Ficoll-Hypark density gradient centrifugation. Collect. This PBL was suspended in Krick's medium containing 5% FCS at a cell density of 1×10 ⁶ cells/ml, and 2 ml each was inoculated into 24-well Nunc culture plates. 100μ of Phytohemagglutinin-M (manufactured by Gibco) was added to the final concentration of 5μg/ml, cultured for 48 hours under the above conditions, and then the cells were washed with culture medium and cultured again at 1 × 10 Inoculate 1 ml of Krick's medium at a cell density of ⁵ cells/ml. Furthermore, concanavalin A (hereinafter abbreviated as ConA). Human T lymphocytes from PBL were added by adding 1 ml of conditioned medium prepared from human splenocytes stimulated with 2.5 μg/ml for 48 hours and replacing the medium containing 50% of the conditioned medium every 3 days. The spheres are subcultured long-term. T obtained through such long-term subculturing
Lymphocytes are cloned in the presence of human splenocytes derived from a culture medium conditioned by the same limiting dilution method as described above, and the cells are expanded in the same manner. The thus obtained cloned T lymphocytes were adjusted to a cell density of 1×10 ⁶ cells/ml by 10μ
g/ml of phytohemagglutinin (PHA)
It is abbreviated as ) in the presence of 1 ml of RPMI 1640 medium in a 24-well Nunc culture plate for 24 hours.
Cultured at 37°C in a 7.5% _CO2 incubator. The main culture supernatant was separated by centrifugation and then sterilized using a 0.22μ Millipore filter, and an IL-2 activity assay was performed to identify IL-2-producing human normal T lymphocyte clones. Summer. (c) Obtaining human lymphocyte-derived malignant cells that produce IL-2 upon mitogen stimulation BPMI including 2%
ConA 10μg/ml and PHA 2.5μ in 1640 medium
When cultured for 24 hours in the presence of 10-4000 g/ml
units/ml of IL-2 can be produced.
Furthermore, even when these human malignant cells were cultured in the presence of zinc chloride, protein A, and picibanil,
Produces IL-2. (d) By stimulating with mitogens in the presence of other cells or factors produced by those cells.
Obtaining cells that produce IL-2 Molt 4F, a human lymphocyte malignant cell, and the Juyukatsu 99 strain, a clone of the Juyukatsu cell cloned using the limiting dilution method described above, are used to treat the above-mentioned lectins and mitogens in a wide concentration range. In addition, IL-2 is not produced even after culturing for 24 to 72 hours. However, when we cultured interleukin-1, a type of monokine, at 37°C for 24 hours in the presence of 5-10 u/ml or 50% of K562 or Raji cells, a detectable amount of IL-2 ( 10-100u/ml). IL-
2mRNA can be extracted by a conventional method regardless of the type of cell. For example, NP-40,
Partially or completely disrupt cells by adding detergents such as SDS, Triton-X100, deoxycholic acid, or using physical methods such as a homogenizer or freeze-thaw. , solubilize. At that time, RNA by RNase
In order to prevent decomposition of the extract, it is preferable to add an RNase inhibitor such as heparin, polyvinyl sulfate, bentonite, macaroid, diethylpyrocarbonate, vanadium complex, etc. to the extract. Depending on the case, a method may also be used in which polysomes in the process of IL-2 synthesis are precipitated using an anti-IL-2 antibody, and mRNA is extracted from this using a surfactant or the like. In addition, for the purification of mRNA containing poliA, affinity polymerases such as oligo dT-cellulose and polyU-Sepharose with Sepharose 2B as carriers are used.
This can be carried out by purification using a column or batch method, fractionation using SDG centrifugation, agarose gel electrophoresis, or the like. The mRNA obtained as above was IL-
2 To confirm that it has mRNA activity, methods such as translating mRNA into protein and examining its physiological activity, or identifying the translated protein using an anti-IL-2 peptide monoclonal antibody, etc. Just do it. For example, mRNA is typically extracted by microinjection into Xenops laevis eggs (Gurdon et al.
Nature, 233 , 177-182 (1972)) or translated into the corresponding protein by using reticulocyte or wheat embryo cell-free translation systems. IL-2 activity was previously described by Gillis et al. (Gillis et al., J.
Immunol., 120 , 2027-2033 (1978)). In this assay, IL-2-dependent cell DNA synthesis (IL-2 dependent
The index is cellular proliferation. i.e. 4 x ¹⁰³ CTLL cells containing 2% FCS
Suspend in 100μ of RPMI-1640 medium and inoculate 96-well flat-bottom microplates with 100μ of serially diluted translation products. After 20 hours of culture at 37°C and 5% _CO2 , cells were incubated with 0.5 μCi/well of ^3H -TdR.
Cells are time-labeled and harvested onto ribbon glass fibers using an automatic cell harvester, and the radioactivity taken up by the cells is measured by liquid scintillation.
This assay shows that cells cultured in the presence of IL-2
It was found that CTLL cells take up ^3H -TdR in a dose-dependent manner, and this indicates that it is contained in the sample.
The amount of IL-2 can be calculated unambiguously. Since IL-2 has the activity of promoting proliferation of T lymphocytes, IL-2 activity can be measured using proliferation of T lymphocytes as an indicator. That is, 5
CTLL cells were suspended in 100 μl of DMEM containing 2% FCS and incubated with 100 μl of serially diluted translation products.
Inoculate a 96-well flat bottom microplate. 72～96
After culturing at 37° C. and 5% CO ₂ for hours, the number of activated and proliferating cells is counted under a microscope. Using 100U/ml and 10U/ml of IL-2 as control groups, the number of viable cells proliferated in the control group was compared with that of the sample.
Determine IL-2 activity. In this way, the most active fraction was obtained.
IL-2 mRNA is used as a template to synthesize ds-cDNA, and ds-cDNA is combined with vector DNA. cDNA synthesis is performed by conventional methods. First, using mRNA as a template and oligo dT as a primer, ss-
After cDNA is synthesized and the template mRNA is degraded and removed by alkaline treatment, the single-stranded cDNA is used as a template and reverse transcriptase or DNA polymerase is used.
Synthesize ds-cDNA. A recombinant DNA body is produced from the ds-cDNA thus obtained and a vector DNA containing a replicon that can be replicated in prokaryotes. This recombinant DNA body is then integrated into the host cell. This ds-cDNA and vector DNA that can be propagated in prokaryotes are processed by exonuclease treatment, addition of chemically synthesized DNA fragments, and ds-cDNA before ligation.
-It is modified by various treatments such as extending G and C chains in order to attach a terminal that can be linked to the end of cDNA or vector DNA. These connectable
For example, DNA can be extracted from T4 phage in the presence of ATP.
They can be spliced together using DNA ligase. The recombinant DNA body thus prepared is used to transform living cells in order to amplify the cloned cDNA or to produce IL-2 polypeptide. Suitable prokaryotic hosts for IL-2 production include E. coli, B. baltis 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. A suitable vector for Escherichia coli is
EK type plasmid vector (stringent type)
as pSC101, pRK353, pRK646, pRK248,
pDF41 etc. , EK type plasmid vector (relaxed type): ColE1, pVH51, pAC105,
RSF2124, pCR1, pMB9, pBR313, pBR322,
pBR324, pBR325, pBR327, pBR328,
pKY2289, pKY2700, pKN80, pKC7,
pKB158, pMK2004, pACYC1, pACYC184,
dul et al. , λgt type phage vector: λgt.λc,
λgt.λB, λWES, λC, λWES.λB, λZJvir.,
Includes λB′, λALO, λB, λWES.Ts622, λDam, etc. Generally, pBR322 is
In this case, the best cloning sites are the Pst1 and EcoR1 sites. The following methods are commonly used for transforming host cells using recombinant DNA. When the host is a prokaryote such as Escherichia coli, this
Competent cells capable of taking up DNA can be transformed by the well-known CaCl ₂ method after harvesting cells in the logarithmic growth phase. Transformation efficiency is improved by coexisting MgCl ₂ or RbCl in the transformation reaction solution. It is also possible to transform host cells after preparing protoplasts. Cells carrying the IL-2 gene can be isolated after transformation using either of the following two methods. (1) Plus-minus method: 11− by sucrose density gradient centrifugation from antigen-stimulated mammalian cell extract
Partially purified IL-2 mRNA was prepared as a 12S fraction, and this partially purified mRNA was used as a template for ^32P .
-Synthesize radioactive ss-cDNA. cDNA isolated after removing template mRNA with alkali treatment
is extracted from antigen-unstimulated mammalian cells and hybridized with partially purified 11-12S mRNA. did not subsequently hybridize
The cDNA hybridized with the cDNA is fractionated using hydroxyapatite column chromatography. The unhybridized cDNA and the hybridized cDNA are called probe A and probe B, respectively. Both recombinants are grown on nitrocellulose filter paper using the same method. Cell DNA is then fixed on filter paper by alkali treatment. Probe A
and B on two different filter papers, respectively.
Hybridize with DNA. After that, autoradiography was performed on recombinants that reacted positively with probe A (plus), and recombinants that reacted little or not at all with probe B (minus).
(Taniguchi et al.; Proc. Jap. Acad.,
V155B 464-469 (1979). (2) The second method is to roughly divide, for example, 1,000 to 10,000 recombinant clones into clone groups of 2 to 30 to 2 to 300 clones, and culture each clone group using a conventional method to generate plasmid DNA. Prepare. These plasmid DNAs are then heat-denatured to immobilize ss-cDNA on nitrocellulose filter paper and activated IL.
Hybridization is performed complementary to mRNA prepared from mammalian cells containing -2-mRNA. Alternatively, IL-2
When mRNA (mixture) containing mRNA is hybridized with heat-denatured plasmid DNA (mixture), the DNA-mRNA hybrid is immobilized on nitrocellulose filter paper. The filter paper is washed with a low salt buffer such as 1mM HEPES or 10mM saline, and the mRNA adsorbed on the filter paper is treated with a solution containing 0.5mM EDTA and 0.1% SDS at 95°C for 1 minute. Extract. The purified mRNA is eluted and recovered using oligo dT-cellulose column chromatography. Next, the mRNA is microinjected into Xenopus oocytes, translated into protein, and IL-2 activity is confirmed. or
mRNA-dependent reticulocyte system or wheat embryo
This mRNA is translated into protein using an in vitro cell-free synthesis system, and IL-2 is isolated using an anti-IL-2 antibody.
-Ability to analyze activity. Groups in which IL-2 activity was detected by these methods are further classified into groups containing a smaller number of recombinant clones, and the process is repeated until a single clone containing IL-2 DNA is finally obtained. . To obtain cDNA encoding an IL-2 polypeptide from a recombinant capable of producing IL-2, first, the recombinant DNA in the transformant is separated;
This is cut with a restriction enzyme endonuclease.
Incorporated from the DNA fraction obtained by cutting
Separate the cDNA fraction. The entire nucleotide sequence of the Pst1 DNA insert encoding the IL-2 polypeptide was determined from the DNA recombined with pIL-2-50A using the Maxam and Gilbert method (Meth. Enzym. 65 , 499-560, (1980)); Deoxynucleotide chain termination method (Smith, A.
JMMeth.Enzym. 65 , 560-580, (1980)). Diagrams of cDNA inserts cut with restriction endonucleases are shown in Figures 1 and 2a. As shown in Figure 2a, each of these cDNAs
It has a structure that can be cleaved by restriction enzyme endonucleases BstN1, Xba, and BstN. The DNA sequence of this cDNA insert possesses one large open reading frame. The first ATG sequence, which is often the read initiation sequence in eukaryotes (Kozak, M.Cell. 15 , 1109-1123
(1978)) is present at nucleotide positions 48-50 from the 5'-end, and 152 sequences from nucleotide positions 507-509, where the reading end sequence TGA exists, are connected to this ATG. An A link corresponding to the 3'-poly(A) end of mRNA is found at the end of the cDNA, and the six nucleotides AATAAA (positions 771-776), which are normally found in most eukaryotic mRNAs, are located first. do. (Proudfoot, N.J. & Brownlee C.
G., Nature 263 , 211-214, (1976)) The amino acid sequence encoded by cDNA can be expressed as shown in Figure 2b (amino acid sequence 1),
Moreover, the polypeptide amino acid sequence consists of 153 amino acids, and its molecular weight is calculated to be 17,631.7 daltons. As reported to be present in most of the secreted proteins known to date (Blobel G. et al, Sym.Soc.Exp.Med., 33 , 9-
36 (1979)), N of the above-mentioned IL-2 polypeptide
The terminal region is also hydrophobic. This field is mature
It probably plays the role of a signal peptide that is cleaved during secretion of IL-2. Cutting is 20-21st
Cleavage occurs between Ser and Ala or between Ala and Pro between positions 21 and 22, producing a polypeptide having the amino acid sequence and. This is because similar cleavage sites are often found in other secreted proteins known to date (Blobel, G. et al.
Symp.Soc.Exp.Med., 33 , 9-36 (1979)). Therefore, the mature IL-2 polypeptide consists of 133 to 132 amino acids and has a molecular weight of 15420.5 or
Calculated as 15349.4 Daltons. This molecular weight value is compared with that reported as the molecular weight (15,000 Daltons) of human IL-2 protein obtained from Jurkat cells (Gillis et al., Immunological
Rev., 63 , 67-209 (1982)). In addition, as shown in Example 3, the DNA fraction starting from the CCT sequence located at positions 111 to 113 of the base sequence, that is, the Pro
The code for the polypeptide starting with (second b)
It was confirmed that the amino acid sequence (in the figure) expressed a polypeptide having IL-2 activity.
DNA starting from GCA at base sequence 107-110
The fraction, ie, the DNA fraction encoding a polypeptide starting from Ala at position 21 as shown in the amino acid sequence in Figure 2b, expresses a polypeptide having IL-2 activity as shown in Example 6. It was confirmed that there is. Genes of nucleated organisms are known to exhibit polymorphism, as is also known for the human interferon gene (Taniguchi et al., Gene 10 , 11-15 (1980),
Ohno, Taniguchi. , Proc.Natl.Acad.Sci.USA, 77 ,
5305-5309 (1981); Gray et al, Nature 295 ,
507-508 (1981)). Due to this polymorphism, some of the amino acids in the protein product may be substituted, or there may be changes in the base sequence but no change at all. In the case of human IL-2 cDNA, pIL-
Another cDNA clone in which the A residue at position 503 of the 2-50A cDNA was replaced with a G residue (pIL-2-503)
can also be detected. The existence of other cDNA clones with different base sequences from pIL-2-50A cDNA can also be expected. As is clear from the above explanation, the gene for obtaining the polypeptide of the present invention (hereinafter referred to as the gene according to the present invention) is a DNA having the base sequence shown in Figure 2a, DNAs that have a continuous base sequence starting from the ATG sequence and ending at least to the ACT sequence at positions 504-506, and at positions 108-110.
Starting from the GCA sequence and starting from the GCA sequence at least
It includes DNA having a continuous base sequence leading to the ACT sequence, and DNA having a continuous base sequence from the CCT sequence at positions 111-113 to at least the ACT sequence.
The gene according to the present invention also contains ACT at positions 504 to 506
It includes DNA starting with A at position 1, DNA starting with ATG at position 48-50, DNA starting with GCA sequence at position 108-110, or DNA starting with CCT sequence at position 111-113. Furthermore, the gene according to the present invention is
DNA that ends with the TGA sequence at positions 507-509 and begins with A at position 1, DNA that begins with the ATG sequence at positions 48-50,
DNA starting with the GCA sequence at positions 108-110 or 111-
Includes DNA starting with the CCT sequence at position 113. Furthermore, the gene according to the present invention ends with C at position 801 and has 1
DNA starting with A at position 48-50, starting with ATG at position 48-50
DNA, DNA starting with GCA at position 108-110 or
Includes DNA starting with the CCT sequence at positions 111-113. The gene according to the present invention also ends with poly(A) and has 48
DNA starting from the ATG sequence at position -50, DNA starting from the GCA sequence at positions 108-110, or DNA starting from the GCA sequence at positions 111-113.
Contains DNA that begins with the CCT sequence. Moreover, the gene according to the present invention includes a gene having a base sequence corresponding to the amino acid sequence. A polypeptide lacking one or more amino acids in its amino acid sequence or a polypeptide in which one or more amino acids in its amino acid sequence has been replaced with one or more amino acids is called IL-2.
It may have activity, and therefore genes encoding such polypeptides can be used as genes according to the present invention. Similarly, even in the case of an amino acid sequence or a gene in which one or more bases that can represent one or more amino acids are added, the added amino acid may cause the IL-2 activity of the polypeptide. It is included in the present invention as long as it does not interfere with expression. Even a modified region having an additional amino acid sequence that inhibits the polypeptide function as IL-2 can be used as a gene according to the present invention if the newly added region can be easily removed. The same applies to the amino acid sequence, and the amino acid sequence of the gene corresponding to, and which encodes an additional amino acid at the C-terminus of
This also applies to DNA in which additional DNA is attached to the 3'-end. Therefore, the use of genes encoding such polypeptides is included in the present invention. Recombinant DNA bodies that produce IL-2 in living cells can be produced by the following various methods. For example, IL-
2. Insert the sequence encoding the cDNA downstream of the promoter sequence of the expression vector. Alternatively, insert a cDNA fragment with a promoter sequence into an expression vector.
It can be inserted upstream of the IL-2 encoding sequence before or after cDNA insertion. The detailed method for constructing a prokaryote that expresses IL-2-cDNA and produces IL-2-polypeptide is as follows. (1) IL-2-cDNA from Escherichia coli
Expression To express IL-2-cDNA in Escherichia coli, first the cDNA is linked to various bacterial promoters, and then
Create a hybrid plasmid containing the cDNA. This plasmid can be used for example in E.
E. coli HB101 is infected, and a bacterium that biosynthesizes a protein having human IL-2 activity is cloned.
Any bacterial promoter can be used as long as it is properly connected to cDNA.
Express cDNA. Examples of expression of such cDNA are as follows. The cloned cDNA encoding IL-2 encodes a polypeptide consisting of 153 amino acids as shown in FIG. of this polypeptide
The N-terminal region, corresponding to 20 amino acids, is extremely hydrophobic, which is also a characteristic of most secreted proteins. Such hydrophobic sequences are called signal sequences and are cleaved during the secretion process. Therefore, mature IL-2
A polypeptide should be less than 153 amino acids. For this reason, it is desirable to express the cDNA portion encoding the mature IL-2 polypeptide, and it is not desirable to express the portion corresponding to the IL-2 signal sequence. (i) Construction of plasmid vector pTrS-3 pTrS-3 contains the E. coli Trp promoter, and the ribosome binding site (SD sequence) for the leader peptide of pTrS-3 has been previously reported (G. Miozzari
and Yanofsky J.Bacteriol. 133 , 1457~
1466 (1978)). Located 13 base pairs downstream of the SD sequence
The existence of an ATG codon has also been reported (Nishi et al., Biochemistry 54 , No. 8.676 (1982)).
This plasmid vector also contains one Sph site downstream of the ATG initiation sequence (Figure 3). To express IL-2 cDNA, the plasmid is first digested with Sph and treated with E. coli DNA polymerase (Klenow fragment) or bacteriophage T4 DNA polymerase to remove the 3′-overhanging ends ( Figure 4 a). Plasmid pIL-2-50A was digested twice with Pst and HgiA and the larger
Isolate the cDNA fraction. The DNA is then treated with E. coli DNA polymerase (Klenow fragment) or bacteriophage.
Cut off the 3'-overhanging ends by treating with T4 DNA polymerase. I did this process
The cDNA encodes a 132 amino acid IL-2 polypeptide (Figure 4a). This cDNA
is ligated to pTrS-3 plasmid DNA pretreated as described above, joining the ATG initiation codon to the CCT(Pro) sequence of IL-2 cDNA. Plasmid pT IL-2-22 is thus obtained. Trp promoter sequence and pT IL−
The binding of the IL-2 cDNA sequence of 2-22 is the fourth
Shown in Figure a. Plasmid pT IL-2-22 directs the synthesis of the 132 amino acid IL-2 polypeptide starting from proline by Escherichia coli. (ii) Since mature IL-2 may contain alanine (position 21) as the N-terminal amino acid instead of proline, a plasmid that directs the synthesis of an IL-2 polypeptide consisting of 133 amino acids was constructed as follows. Can be done. Plasmid pTrS-3 has one Cla cleavage site between the SD and ATG sequences (Figure 3). This plasmid contains Cla and Sal
will be cut off. Plasmid pIL-2-50A
was partially digested with Pst, treated with Escherichia coli DNA polymerase, and the longest
Isolate the DNA. The DNA is then treated with restriction enzymes
A synthetic DNA linker containing an Xho cleavage site is attached to
A clone containing plasmid pIL-2-50A (Xho) with a DNA linker introduced downstream is isolated. Plasmid pIL-2-50A (Xho) was first cut with HgiA and then extracted with E. coli Klenow fragment or T4 DNA.
The cDNA fraction can be isolated by treatment with polymerase and digestion with Xho. This cDNA fragment is ligated to pTrS-3 DNA pretreated with Cla and Sal and ligated to synthetic DNA as shown in Figure 4b. Thus, an IL consisting of 133 amino acids starting from Ala
Plasmid pTIL-2-21 is obtained which allows the -2 polypeptide to be synthesized in E. coli. (Figure 4b). The same thing can be done without using the Xho linker. (iii) Different sizes of IL-2 polypeptides with different N-terminal amino acids can also be produced using pTrS-3 expression plasmid vectors. As shown below, the IL-2 cDNA cloned into pIL-2-50A has only one Dde moiety at nucleotide binding site 81-85. Plasmid pIL-2-50A (Xho)
Cut with Dbe and isolate the DNA fraction containing the larger section of cDNA. This fraction is
It contains DNA having 3000 base pairs from pBR322 (Figure 4c). The DNA fraction was treated with exonuclease Bal 31 and then Xho
Cut with. The DNA obtained here is Sph
Combined with pTrS-3 cut with
Treat with Klenow fragment or T4 DNA polymerase and then digest with Sal (Figure 4c). The spliced DNA was infected with E. coli HB 101, and human IL-2
Search for clones expressing . These clones should express human IL-2 in a variety of sizes. This is because the N of human IL-2
-This is because the DNA corresponding to the terminal region is cleaved and removed in various ways. Thus IL-2
pTIL-2-14 and 15 containing cDNA are obtained. (iv) IL-2 cDNA is also pKT 218 (Talmage
Provided by; Proc.Natl.Acad.Sci.
USA, 77 , P. 3369-3373 (1980)). Plasmid pKT 218
Cut pIL-2-50A with Pst and HgiA
IL− obtained by cutting with and Pst (Fig. 5)
2 Connect with the inserted cDNA part. The resulting plasmid pKIL-2-21 has a sequence at the beginning of protein synthesis, as shown in FIG. Therefore, this plasmid
pKIL-2-21 consists of a polypeptide that is a fusion of the 133 amino acids of IL-2 and the amino acids of β-lactamase, and it should be possible to synthesize this in Escherichia coli (the first methionine is Esierihia
In the case of stiffness, it is cut and removed). (v) Expression of plasmid pTuBlp-5, in which the promoter sequence for tufB was inserted into p-BR・322, has already been carried out (Taniguchi et al., Biochemistry
53 966 (1981)). This plasmid is one
It contains a Cla cleavage site, and as shown in Figure 6, this cleavage site is located two base pairs downstream of the SD sequence. pTrS-3 also has an SD sequence.
Contains one Cal cleavage site between the ATG starting sequences, and this Cla site also
Bacterial Trp is not destroyed during the process of creating an expression plasmid using -3 and IL-2 cDNA.
Replacing the promoter with the tufB promoter is extremely easy. Therefore, human IL
-2 cDNA is expressed under the control of the tufB promoter. For example, pTIL-2-22 is Cal
and Pvu to separate the DNA fraction containing IL-2 cDNA. This fraction was then ligated with pTuBlP-5, and after pre-cleavage with Cla and Pvu, the plasmid pTuIL-2 was added as illustrated in Figure 6.
−22 is created. IL-2 activity was determined using Escherichia coli containing plasmid pTuIL-2-22.
It can be detected in the extract of HB101. (vi) It can be similarly constructed using, for example, pTIL-2-21, or basically any expression plasmid achieved using pTrS-3. For example, pTuIL-2-
The distance between the SD and ATG sequences was reduced by cutting 22 with Cla, removing or adding 2-3 base pairs of DNA with Bal 31 or SI or DNA polymerase (Escherichia coli), and connecting the plasmid again. It is also possible to make the length suitable. Escherichia with recombinant DNA inserted
Cultivating transformed prokaryotic cells such as E. coli or B. subtilis to recombine
Amplify DNA bodies or produce IL-2 polypeptides. This culturing is carried out in a conventional manner. IL-2 produced intracellularly or extracellularly
is ammonium sulfate precipitation, dialysis to remove salts (at normal pressure or reduced pressure), gel filtration, chromatography, isoelectric focusing plate concentration, gel electrophoresis, high performance liquid chromatography (hereinafter abbreviated as HPLC), (ion exchange, gel filtration and reversed phase chromatography), and dye-binding carriers, IL-
It can be recovered by a known method such as affinity chromatography using activated Sepharose 4B bound to a monoclonal antibody against 2 or lectin-bound Sepharose 4B. IL-2 isolation and purification method
Watson et al. (J.Exp.Med., 150 , 849-861
(1979), Gilliss et al, J. Immunol., 124 ,
1954−1962 (1980), Mochizuki et al, J.
Immunol.Methods 39 , 185−201 (1980),
Welte, K et al, J.Kxp.Med., 156 , 454
-464 (1982)). The polypeptide thus obtained can be produced from mammalian cells upon stimulation with mitogens.
IL-2 exhibits the same biochemical and biological behavior as known for IL-2.
Has activity. The molecular weight is approximately 15,000 Daltons, and the IL-2 activity is similar to that of Igsorb (Enzyme
completely neutralized with or without immunoadsorbents such as Center) or precipitated with monoclonal anti-IL-2 antibodies. In immunoelectrophoresis, IL-2 polypeptides show only one sedimentation line relative to the corresponding anti-IL-2 antibody. IL-2 activity is stable even after reduction with 2-mercaptoethanol, and DNAse and
It is stable even after RNAse treatment and heat treatment at 56°C for 30 minutes. Activity is stable at pH 2-9. IL-2 produced in this way promotes the proliferation of T cells with monoclonal functions (cytotoxic T lymphocytes), strengthens the division of thymocytes, and furthermore, in the absence of antigen, is anticancer from a memory state. Causes differentiation into specific cytotoxic T lymphocytes. In addition, YAC-1 cells and RL
Helps enhance the activation of natural killer cells against 1 cell. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 (1) Jurkat cells, a human T-cell leukemia cell line (freely available in Japan, West Germany, and the United States), were suspended in RPMI 1640 medium containing 10% FCS, and subjected to X-ray irradiation equipment Exs 150. /300−
4 (Toshiba, Japan) for 50 seconds at room temperature until reaching 10,000 Roentgens. The irradiated cells were then grown at an initial cell density of 1× in the medium described above.
The cells were cultured at 10 ⁵ cells/ml in an incubator with 5% carbon dioxide gas at 37°C for 5 days. These mutant cells (0.2 cells/well) were seeded in 10 wells of a 96-well flat-bottomed microplate and cultured for 21 days in an incubator at 37°C with 5% carbon dioxide gas. Clones obtained from growing holes are transferred to a new medium to increase the number of clones.
The increased clones were cultured for 24 hours at an initial cell density of 1×10 ⁶ cells/ml in the presence of 50 μg/ml ConA. IL-2 activity was then measured by the method described above. As a result, Juyukatsu-111 (ATCC CRL
A human T cell line named 8129) (hereinafter referred to as "J-111") was cloned and selected from the parent cell line J-111, and the IL-2 production capacity of this cell was increased 40 times that of the parent line. This cloned J-111 cell line proliferated under normal conditions, and its proliferation rate was almost the same as that of normal Jurkat cells. (2) J-111 cells (1×10 ⁵ cells/ml) were grown in serum-free synthetic medium RIT C 55-9 (Sato, T. et al., Exp.
Cell Res., 138 , 127-134, (1982)) was inoculated into 1000 ml of roller culture bottle (Falcon
3027) at 37°C for 4 days, and the proliferated cells were obtained by centrifugation. This cell again 4
25 μg/ml of ConA as above to give ×10 ⁶ cells/ml.
ml containing medium was inoculated. 100 ml of culture solution inoculated with cells was placed in each of four batches of roller culture bottles (Falcon), and cultured by rotation for 6 hours. (3) Juyukatsu cells (1.2 × 10 ⁶ ) stimulated with ConA 25 μg/ml for 6 hours in this way were treated with saline.
It was suspended in 8000 ml of phosphate buffer (hereinafter abbreviated as PBS). The cells were washed twice by centrifugation and treated with the nuclease inhibitor ribonucleoside-vanadyl complex (10mM).
RSB solution (10ml Tris-HCl buffer, PH7.5,
Resuspend in 800ml (10mM NaCl, 1.5mM MgCl2 ₎ . Thereafter, surfactant NP-40 was added to a final concentration of 0.05%, mixed gently, and cell nuclei were separated by centrifugation at 3000 rpm for 5 minutes at 4°C. SDS (0.5%) and EDTA (5mM)
was added to the supernatant, and cytoplasmic RNA was extracted by adding the same amount of phenol as the supernatant. After repeating the extraction with phenol three times, the RNA was precipitated with twice the amount of ethanol, the precipitate was collected by centrifugation, and dissolved in 10 mM Tris-HCl at pH 7.5. The amount of RNA obtained was 196 mg. Fractionation of mRNA was performed using oligo(dT)-cellulose (PLBiochemicals, Type 7) affinity chromatography. Adsorption solution is 20mM Tris-HCl, 0.5M NaCl, 1m
This is a pH 7.5 solution containing M EDTA and 0.5% SDS, and elution is performed by immersing the column in buffer solution (20mM Tris-
Hydrochloric acid, PH7.5, 0.5M NaCl, 1mM EDTA)
After washing with water and 10mM Tris-HCl (PH7.5)
We took turns. mRNA obtained by elution
was 3.6 mg. Then this obtained mRNA2.4
mg by sucrose density gradient centrifugation (PH7.5 containing 50mM Tris-HCl, 1mM EDTA, 0.2M NaCl)
Sucrose density gradient 5-25% in solution, 26000 rpm
(at 4°C for 24 hours). mRNA
11 to 12S are fractionated into fractions No. 12, 13, and 14,
The amounts were 59 μg, 46 μg, and 60 μg, respectively. (4) The mRNA obtained in fraction No. 13 was injected into oocytes of Xenopus laevis (50 ng mRNA/oocyte). The IL-2 activity of this oocyte culture solution was measured. Table 1
As shown in Figure 2, an increase in the uptake of ^3H -thymidine ( ^3H -TdR) and an increase in the number of activated T lymphocytes was confirmed, clearly indicating that the mRNA in this fraction contains human IL-2 mRNA. has been proven. [Table] Products
×32 10165
[Table] Products
×16 55
*Units are ^3H- of standard IL-2 (10 units/ml)
Calculated by comparing with TdR uptake amount. (5) 11-12S, which then contains IL-2 mRNA
cDNA was synthesized in vitro from the mRNA No. 13 fraction. Recombinant DNA is plasmid vector pBR
It was configured as 322. The recombinant DNA was transformed into Escherichia coli, and clones from which IL-2 cDNA clones were obtained were selected by the method shown below. (5-1) 50mM Tris-HCl buffer (PH7.5), 30m
M NaCl, 6mM MgCl ₂ , 5mM dithiothreitol (hereinafter abbreviated as DTT), 0.5mM
Each of dATP, dGTP, dCTP, dTTP (dCTP
(including ^32P radiolabeled), 0.7 μg oligo(dT) ₁₀ , 10 μg mRNA and 15 units
Mix AMV reverse transcriptase (JWBread),
It was kept at 41℃ for 90 minutes. After the reaction, the DNA was treated with phenol and recovered as an ethanol precipitate, and this DNA was added to 20mM Tris and 1mM Tris.
M was dissolved in a solution containing EDTA at pH 7.5. 2.5 μg of ss-cDNA was synthesized. To remove mRNA from this reaction solution, NaOH solution was added to the reaction solution to make a 0.33N NaOH solution, and the mixture was left at room temperature for 15 hours.Then, the same amount of 1M Tris-HCl buffer (PH7.5) was added to neutralize the Sephadex. A G-50 column was passed through the column. The amount of cDNA recovered was 1.8 μg. (5-2) 50mM phosphate buffer (PH7.5), 10mM
MgCl ₂ , 10mM DTT, 0.75mM each
dATP, dGTP, dCTP, dTTP (dCTP is
(including those labeled with ^3H ), 1.8μg ss-
cDNA and 8-unit polymerase (BRL,
(USA) and the reaction was carried out at 15°C for 15 hours.
After the reaction was completed, DNA was treated with phenol and chloroform and recovered as an ethanol precipitate. 1.10 μg of ds-cDNA was produced. 50m
M sodium acetate (PH4.5), 0.2M NaCl, 1mM
A mixture of ZnCl ₂ and 1.10 μg double-stranded cDNA was incubated at 37 °C for 30 min before adding 0.25 units of nuclease S ₁ (Mitsui, Japan).
Incubate for an additional 15 minutes. After the reaction was completed, the reaction product that had been treated with phenol twice was subjected to Sephadex G-50, and the double-stranded
0.55 μg of cDNA was obtained. (5‐3) 0.14M potassium cacodylate, 30mM Tris base, 0.1mM DTT, 1mM CoCl ₂ ,
0.64mM ^32P -dCTP (specific activity 2.7×10 ⁶ cpm/
n mol), 0.55 μg ds-cDNA and 5 units of terminal transferase (BRL)
The mixture was mixed, incubated at 37°C for 7 minutes, treated with phenol, and then applied to a Sephadex G-50 column to obtain 0.50 μg DNA as an ethanol precipitate. This recovered
It was found that approximately 50 dCMP residues were added to both 3' ends of the DNA. Add 10μg of pBR 322 DNA to restriction enzyme Pst
dGMP was added to both 3′ ends of the cut DNA using exactly the same conditions as those used when adding dGTP strands to ds-cDNA described above, except that dGTP was used instead of dCTP.
Added a chain. (5-4) 50mM Tris-HCl (PH7.5) 0.1M NaCl,
5mM DETA, 0.05μg dGMP residue addition
pBR 322 and 0.01μg dCMP residue addition
The cDNA was first incubated at 65°C for 2 hours and then at 46°C.
Incubate for 120 minutes, then 60 minutes at 37°C, and 60 minutes at room temperature. Escherichia coli χ1776 (Curtiss,
R. et al., in Molecular Cloning of
Reocmbinant DNA, (WAScott & R.
Werner ed.) Academic Press, (1977))
Inoculate 50 ml of L medium (containing 100 μg/ml diaminopimelic acid, 50 μg/ml thymidine, 1% tryptophan, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose) until the absorbance of the culture solution reaches around 0.3 at 562 nm. 37℃ until
Cultured with shaking. After culturing, add the culture solution to 30%
The cells were kept at 0°C for minutes, collected by centrifugation, and mixed with 5mM Tris-HCl (PH7.6) and 0.1M.
NaCl, 5mM _MgCl2 and 10mM RbCl
Washed twice with 25 ml of a solution containing The obtained bacterial cells were diluted with 5mM Tris-HCl (PH
7.6), 0.25M KCl, 5mM MgCl ₂ , 0.1M
Suspend in 20ml of a solution containing CaCl ₂ and 10mM RbCl, let stand at 0℃ for 25 minutes, collect the bacteria, resuspend in 1ml of the same solution as above, and use 0.2ml of the resulting bacterial suspension. The above-mentioned recombinant DNA was added to the tube and left at 0° C. for 60 minutes. Then L medium
0.7 ml was added and cultured with shaking at 37°C for 30 minutes.
The culture solution (0.1 ml) obtained in this way was added to 100μ
The cells were plated on a 1.5% agar medium in L medium 1 containing g/ml diaminopimelic acid, 50 μg/ml thymidine and 15 μg/ml tetracycline, and incubated at 37° C. for 2 days. (5-5) The 432 colonies that appeared were divided into 18 groups (each group containing 24 different bacterial clones) and treated with 100 μg/ml diaminopimelic acid, 50 μg/ml thymidine, and
L medium 200 containing 10 μg of tetracycline
ml, and cultured with shaking at 37°C for 5 to 7 hours. Next, 200 ml of fresh L medium containing chloramphenicol added to give a final concentration of 170 μg/ml was added, and the mixture was further cultured overnight. Plasmids enhanced in this way
DNA was purified according to standard methods. Clones containing IL-2 cDNA
Selection was made by mRNA hybridization-translation assay (hereinafter abbreviated as H-T assay). The H-T assay used here was performed as shown below. Purified DNA (25 μg) was treated with restriction enzyme Hind.
cleaved with phenol, treated three times with phenol, each with phenol-chloroform and chloroform, precipitated with ethanol, washed with 80% ethanol, and dissolved in 40 ml of 80% formamide. In order to denature the reaction solution, it was heated at 90° C. for 5 minutes and then diluted to 1.3 ml with 10×SSC (1.5 M NaCl, 0.15 M sodium citrate). Then the book DNA
is fixed on nitrocellulose filter paper, and this
Dry at 80℃ for 3 hours, 50% formamide,
20mM Pipes (PH6.5), 0.75M NaCl, 5m
37°C in a solution containing M EDTA, 0.2% SDS, and 250 μg of poly(A) mRNA derived from J-111 cells.
After incubation for 18 hours, the DNA immobilized on the filter paper and IL-2 mRNA were hybridized. Next, the filter paper was heated 3 times at 65°C with 10mM of pH6.5.
Pipes, 0.15M NaCl solution, 1mM
Pipes, washed with 10mM NaCl solution, 0.5m
M EDTA, hybridized mRNA from filter paper treated with 0.1% SDS solution at 95℃ for 1 minute
was recovered. The thus extracted mRNA was purified on an oligo dT-cellulose column according to a conventional method, injected into Xenopus oocytes, and the IL-2 activity of the translated protein was measured. One group out of 18 groups each consisting of 24 clones showed positive IL-2 activity of 48 units/ml in the above-mentioned ³ H-TdR incorporation assay. On the other hand, the other groups were clearly negative. Next, each of the 24 single colonies belonging to the positive group was inoculated into 200 ml of L medium with the same composition as described above, and cultured aerobically at 37°C for 5 to 7 hours.
Similarly, L medium containing chloramphenicol was further added. After culturing overnight to enhance plasmid DNA,
Plasmid DNA was similarly purified according to standard methods. Approximately 5μ of each plasmid DNA in Hind
After cleaving G, each plasmid was similarly immobilized on nitrocellulose filter paper. IL-
2 Hybridize with mRNA, inject the hybridized mRNA into Xenopus oocytes, and extract the translated protein IL-2.
Collected to measure activity. As shown in Table 2, only plasmid DNA purified from a single colony designated p3-16 showed positive IL-2 activity. Therefore,
This clone has IL-2 cDNA (E. coli χ 1766/p3-16 AJ11995
(FERM-BP-225)). In this way, plasmid DNA, p3-16, is IL-2
It was confirmed that it did indeed have DNA (IL-2 gene) capable of forming a specific hybrid with mRNA. [Table] Things
×32 20961
[Table] The cDNA insert of plasmid p3-16 was digested at one site with the restriction enzyme Xba, and with Bst.
It was characterized by being cleaved by NI at two sites (upstream and downstream of the Xba cleavage site). However, plasmid p3-16 is approximately
It contains a cDNA insert consisting of 650 base pairs, which apparently corresponds to a portion of IL-2 mRNA of size 11-12S. Therefore, using other cDNA libraries with IL-2 mRNA as a template,
Land et al.'s method (Land et al., Nucleic
Acids Res., vol. 9 , p. 2551, (1981)). Single-stranded cDNA (1.6μg)
4μg IL-2 mRNA with dCMP residue added
and ds-cDNA,
It was synthesized by DNA polymerase (Klenow fragment) using oligo(dG) 12=18 as a primer. Longer than 680 base pair DNA size marker
cDNA (0.6 μg) was obtained by sucrose density gradient centrifugation and inserted into the Pst site of pBR 322 using standard GC tailing methods. After transformation of E. coli χ 1766 with the recombinant DNA, it was nick-translated (nick-
Approximately 2000 colonies were selected by the Grunstein-Hogness hybridization method using the p3-16 cDNA insert.
Plasmid pIL2- containing approximately 850 base pairs
Colonies and transformed clones containing 50A (E. coli χ 1776/
pIL2−50A, AJ11996 (FERM−BP−26))
was identified. A restriction enzyme cleavage diagram of the cDNA insert of pIL2-50A is shown in FIG. Transformed Escherichia coli χ
In order to isolate the gene encoding the IL-2 peptide from 1776/pIL2-50A, plasmid DNA was extracted from the bacterial cells using standard methods.
After isolation, it was cut with the restriction enzyme Pst.
The smaller of the two DNA fragments produced by this treatment was the DNA gene encoding the IL-2 peptide. pIL2
The complete nucleotide sequence of the Pst insert from −50A was obtained by the method of Maxam and Gilbert (Maxam, AWet al., Enzym. 65 ,
499-560, 1980). The entire structure is shown in Figure 2a. Example 2 The constitutive IL-2 producing cell line J-A 1886 (ATCC CRL 8130), cloned from Jurkat cells according to the method described in Example 1, was also grown in roller culture bottles. The grown cells were grown in fresh synthetic medium at an initial cell density of 1 x ¹⁰ cells/ml.
Resuspend in RITC-55-9 and 8 hours after the start of culture, incubate 3 times according to the steps detailed in Example 1.
IL-2 _n as 11-12S fraction from × ¹⁰⁹ cells
used for RNA extraction. ds-cDNA was synthesized in the same manner as in Example 1, and 600
cDNA (2.4 μg) longer than base pairs was obtained by fractionation using sucrose density gradient centrifugation. Then this
cDNA was extended with dCMP residues using terminal deoxynucleotidyl transferase, and 50 ng of the cDNA was extended with dGMP. Pst-cleaved pBR 322
Annealed to 250ng. The generated hybrid plasmid was transformed into Escherichia coli χ 1776, and about 4000 clones of transformants were obtained. Three clones complementary to the plasmid 3-16 cDNA used as a probe were selected according to the Grunstein-Hogness method. That is, the transformed clone selected in this way is a clone having the human IL-2 gene. Example 3 A plasmid that directs the synthesis of human IL-2 in Escherichia coli cells was constructed as follows. Plasmid pT IL2-22 was transformed into pTrS-3 by a series of modifications as illustrated in Figure 4a.
(Nishi T., Taniguchi T. et al., SEIKAGAKU
53 , 967, (1981), 54 , 676 (1982)) and IL
-2 Constructed from pIL2-50A containing cDNA. Plasmid pTrS-3 contains the Trp promoter and pBR
Shine between EcoR site and Cla site of 322
Contains the insertion of a region of Dalgarno (hereinafter abbreviated as SD). This plasmid also has a single Sph site as well as 13 bp downstream of the SD sequence, as shown in Figure 3.
Contains the ATG initiation cordon. The DNA sequence corresponding to the protein mentioned is ATG
The vector is very effective in producing this protein when inserted just downstream of the codon. This ATG codon is generated by Sph digestion of pTr-3 followed by treatment with T4 DNA polymerase. Therefore plasmid pTrS-3
(30 μg) was cleaved with the restriction enzyme Sph by a conventional method, and subsequently recovered by phenol and chloroform treatment and ethanol precipitation.
Fragmented by DNA polymerase treatment. Next, DNA was extracted using phenol and chloroform treatment and ethanol precipitation using the same method.
(21.4 μg) was recovered. On the other hand, 380 μg of pIL2-50A containing IL-2 cDNA was cleaved by Pst.
IL-2 cDNA inserts were isolated by agarose gel electrophoresis. cDNA insert (11 μg) was cleaved with HgiA and T4 DNA
After treatment with polymerase, 10 μg of DNA from the larger portion was isolated by agarose gel electrophoresis. According to this method, cDNA (7.2 μg) encoding 132 amino acids was obtained, and this DNA fragment had blunt ends (Figure 4a). Next, the cDNA fragment obtained in this way is
Just downstream of the ATG sequence, it was ligated into the pTrS-3 vector, which had been treated with T4 DNA polymerase and previously digested with SpH. The ligated plasmid was then transformed into E. coli HB 101 according to standard methods. This connection was made as follows. IL-2
The larger fragment of cDNA (0.4 μg) and
0.2μg of pTrS-3 vector DNA at 6.6mM
It was mixed with 0.8 units of T4 DNA ligase in 66mM Tris-HCl at pH 7.5 containing _MgCl2 , 1mM ATP and 10mM DTT, and the mixture was allowed to react overnight at 4<0>C. Among the transformants appearing on L medium agar plates containing ampicillin,
IL-2 encodes 132 amino acids
Colonies carrying plasmids containing cDNA portions were selected in situ by colony hybridization assay. The colonies thus selected were cultured again (10 ml), and plasmid DNA was prepared by treatment with lysozyme, freezing, and thawing. This plasmid DNA was transformed into Pst and Xba
The resulting product was analyzed by agarose gel electrophoresis, and the cDNA of pTrS-3 was analyzed by agarose gel electrophoresis.
pTIL frozen in correct orientation after ATG sequence
-2-22 was identified. E. coli HB containing pTIL-2-22
101 were cultured under conventional methods known for the growth of microorganisms. Cells were incubated with χ containing 25 μg/ml streptomycin and 25 μg/ml ampicillin.
Medium (2.5% bactotryptone, 1% yeast extract,
The cells were grown overnight at 37°C in 10ml of 0.1% glucose, 20mM _MgSO4 , 50mM Tris-HCl, PH7.5). Next, 1 ml of the culture suspension was added to the same χ medium (100 ml).
and cultured at 37°C. 3-indoleacrylic acid (IAA) was added when the OD at 650 mμ reached approximately 1.5-2.0. Three hours after addition of the inducer, cells were collected, washed with 20mM Tris-HCl (PH 7.5, containing 30mM NaCl) and resuspended in 8ml of the same buffer. For effective functional expression of the Trp promoter, add an inducer such as IAA to a final concentration of 50 μl.
g/ml. The proteins thus produced in the bacterial cells were treated with sonication (0°C, 2 min) or digested with lysozyme (8 μg) (0°C, 20 min).
The sample was extracted by freezing and thawing three times. By this method, IL-2 was generally extracted from cells. The extracted IL-2 activity is 10000
The range was from 120,000 units/ml to 120,000 units/ml. E. coli HB containing pTIL2−22
101 (AJ 12009) has been deposited as FERM-BP245. Example 4 Plasmid pTuIL2- carrying IL-2 cDNA
22 is pTuBlP-5 (Taniguchi, T. et al.,
Seikagaku, 53 , 966, 1981) and pTIL2-22 shown in Example 3 by the method illustrated in FIG. Plasmid pTuBlP-5 is
The tufB promoter sequence has been inserted into pBR322. This plasmid also contains a single Cla site, which corresponds to the SD
It is located 2 bp downstream of the sequence. pTrS-3 also contains a Cla site between the SD sequence and the ATG initiation codon, and this Cal site is connected to pTrS-3 and IL-
2 The Trp promoter is not destroyed during expression plasmid construction using cDNA.
It is very easy to replace the tufB promoter, so that the IL-2 cDNA is expressed under the control of the tufB promoter. Therefore, plasmid pTIL2-22 (30 μg) was cleaved with restriction enzymes Cla and Pvu in the usual manner. Fragment containing IL-2 cDNA (approximately 2.2kb)
was isolated and purified by agarose gel electrophoresis,
3 μg of DNA was recovered. On the other hand, pTuBlP-5
20 μg of the vector was similarly digested with Cal and Pvu, and the larger fragment (approximately 3.4 kb) containing the ampicillin resistance gene was isolated and purified by agarose gel electrophoresis, and 3.5 μg of DNA was recovered.
Next, the two fragments obtained in this way are one
Contains tufB promoter (approximately 3.4kb), and the other is IL
-2 cDNA (approximately 2.2 kb) and was ligated as shown below. Fragment containing IL-2 cDNA (1.2 μg) and
0.3μg of fragment containing tufB promoter at 6.6mM
It was mixed with 0.8 units of T4 DNA ligase in 66mM Tris-HCl at pH 7.5 containing _MgCl2 , 1mM ATP and 10mM DTT, and reacted overnight at 4°C.
Next, the thus ligated plasmid was transformed into Escherichia coli HB 101 according to a conventional method. Among the transformants appearing on the L medium agar plate containing ampicillin, Fig.
Eight colonies with recombinant DNA containing the IL-2 cDNA portion, such as pTuIL2-22, were selected;
Plasmid DNA was prepared as described in Example 3. E. coli HB containing pTuIL2−22
101 was cultured in L medium (100 ml) at 37°C. 650
When the mμ OD reached approximately 0.5-1.0, the cells were collected, washed with 20mM Tris-HCl (PH7.5) containing 30mM NaCl, and resuspended in 2ml of the same buffer. The protein thus produced was extracted in the same manner as in Example 3. IL-2 activity in the extract is
It ranged from 6000 to 56000 units/ml. E. coli HB containing pTuIL2−22
101 (AJ 12010) has been deposited as FERM-BP 246. Example 5 Plasmid pGIL2-22 carrying IL-2 cDNA
is pGL 101 (Roberts, TMand Laucer GD,
Meth Enzym., 68 , 473−483, (1979), Gail
Lauer, et al, J. Mol. Appl. Genet., 1 , No. 2,
139-147 (1981), T. Taniguchi, et al, Proc.
Natl.Acad.Sci.USA, 77 , No.9, 5230-5233
(1980), Egon Amann et al, Gene, 25 , 167~
178 (1983)) and pTIL2-22 shown in Example 3. i.e. a plasmid containing the lac promoter
pGL 101 (20 μg) was cleaved with the restriction enzyme Pvu using a conventional method, and then 17 μg of DNA was extracted by phenol and chloroform treatment and ethanol precipitation.
was recovered. On the other hand, pTIl2-22 (75 μg)
After cutting with Cla and Sal, 2.2 μg of a DNA fragment containing IL-2 cDNA was recovered by agarose gel electrophoresis. This fragment was made into a flash by treatment with DNA polymerase (Klenow fragment). Next, the 2 obtained in this way
The two fragments (0.25 μg and 0.66 μg) were ligated in the same manner as in Example 4 using 1.0 unit of T4 DNA ligase. This ligated plasmid was then transformed into Escherichia coli HB 101 according to standard methods. Among the transformants, IL-2
Transformant ³² P-labeled IL-2 with insertion of Cla-Sal fragment containing cDNA
cDNA was selected as a probe. Next, these transformants were treated with ampicillin 25 μg/ml.
Plasmid DNA was prepared by the method described in Example 3. Thus, just downstream of the lac promoter, IL-2
Plasmid DNA with cDNA start sequence ATG
was obtained by assaying the cleavage sites at Pst and Xba. obtained in this way
E. coli HB 101 containing pGlL2−22
was inoculated into 100 ml of L medium containing 25 μg/ml ampicillin and 25 μg/ml streptomycin and cultured. When the OD of 650 mμ reached approximately 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a concentration of 1 mM, and after 1 hour, the bacterial cells were collected and cultured according to the method described in Example 4. A body extract was prepared. The IL-2 activity of the extract is 6000
The range was from 80,000 units/ml to 80,000 units/ml. E. coli HB containing pGIL2−22
101 (AJ 12011) has been deposited as FERM-BP 247. Example 6 Plasmid pTrS-3 (10 μg) was first digested with restriction enzymes.
After cutting with Sal, the Sal site was flushed by treatment with DNA polymerase (Klenow fragment) or T4 DNA polymerase. After cleavage with Cla, the larger fragment containing the Trp promoter region was isolated and purified by agarose gel electrophoresis according to a conventional method, and 3 μg of DNA was recovered. On the other hand, 11 μg of cDNA insert obtained by Pst cleavage of pIL2-50A was cleaved with HgiAI,
After treatment with T4 DNA polymerase, the larger fragment was isolated and purified by agarose gel electrophoresis. In this way, 7.2 μg of a cDNA fragment encoding 132 amino acids of IL-2 was obtained. Next, a 0.45μ fragment containing the trp promoter (above)
g, HgiAI-Pst fragment containing IL-2 cDNA
0.5μg and synthetic oligonucleotide (5')
CGATAAGCTATGGCA (3′) and (3′)
TATTCGATACCGT(5') (20 pmole each), both of which are phosphorylated below 5', were prepared from T4 by the same method as described in Example 3.
The ligation was performed using one unit of DNA ligase (Fig. 4b). The plasmid thus linked is E.
coli HB 101. Among the transformants that appeared, the target transformant was selected as follows. First of all,
Transformants capable of hybridizing with both IL-2 cDNA and synthetic oligonucleotides were selected by colony hybridization. Next, transformants containing plasmid DNA in which a DNA fragment (CCTACT...) starting from the CTT sequence at positions 111 to 113 in Figure 2a was inserted just downstream of the ATGGCA sequence were transformed into Pst.
, was selected by testing the Xba cleavage site. The above transformant containing pTIL2-21a or pTIL2-21b was L by the method shown in Example 3.
When cultured in a medium and analyzed by the method shown in Example 3, high IL-2 activity was observed in the transformant cell extract. E. coli HB101 with pTIL2-21a (AJ12013) and E. coli HB101 with E. coli (AJ12014) with pTIL2-21b
have been deposited as FERM-BP248 and FERM-BP249, respectively. The hosts used in the above examples, E. coli x1776 and HB101 (Boyer HWet al.,
J. Mol. Biol. 41 , 459, (1969) is well known and readily available. In addition, in order to release the recombinant DNA in the transformant, the transformant can be cultured in L medium at 37°C, and the cells that have become sensitive to tetracycline and ampicillin can be isolated. The host is easily obtained from. Plasmid vectors pBR322 (e.g. available from Bethesda Research Laboratory), pCE-1,
pTrS-3 and pGL101 are known and easily available. Furthermore, the deposited transformant can be isolated by isolating the recombinant plasmid in the transformant using conventional methods, and further by isolating the plasmid vector as is obvious from the description in each example. Plasmid vectors can be obtained from. pTrS-3 and pTuBIP-5 are Escherichia coli, respectively.
FERM−P6735 (BP328) and Escherichia
Cori ATCC31878.

[Brief explanation of drawings]

Figure 1 shows a restriction endonuclease cleavage map of a cloned gene encoding a polypeptide with IL-2 activity, Figure 2a shows the base sequence of the cloned gene, and Figure 2b shows
The amino acid sequence of a polypeptide having IL-2 activity is shown. Figure 3 shows plasmid vector pTrS-3. Figures 4a, 4b and 4c show recombinant DNAs (pTIL2-22, pTIL2-21, pTIL2-21,
Fig. 2 is a flowchart showing the construction of pTIL2-14 and pTIL2-15). Figure 5 is as a vector
Recombinant DNA using pKT218 (pKIL2−
This is a flowchart showing the configuration of 21). FIG. 6 is a flowchart showing the construction of recombinant DNA (pTuIL2-22) using pTUBIP-5 as a vector. In the figure, "A", "G", "C" and "T" represent deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid, respectively.

Claims (1)

[Scope of Claims] 1. Human interleukin 2 activity, which is obtained by genetic recombination, is free from sugars, and does not substantially contain other human-derived proteins, and contains the next amino acid sequence in the molecule. A polypeptide having Pro Thr Ser Ser Ser Thr Lys Lys Thr
GlN Leu GlN Leu Glu His Leu Leu Leu
Asp Leu GlN Met Ile Leu AsN Gly Ile AsN
AsN Tyr Lys AsN Pro Lys Leu Try Arg
Met Leu Thr Phe Lys Phe Try Met Pro
Lys Lys Ala Thr Glu Leu Lys His Leu GlN
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr. 2. The polypeptide according to claim 1, which has the following amino acid sequence. Ala Pro Thr Ser Ser Ser Thr Lys Lys
Thr GlN Leu GlN Leu Glu His Leu Leu
Leu Asp Leu GlN Met Ile Leu AsN Gly Ile
AsN AsN Tyr Lys AsN Pro Lys Leu Try
Arg Met Leu Thr Phe Lys Phe Try Met
Pro Lys Lys Ala Thr Glu Leu Lys His Leu
GlN Cys Leu Glu Glu Glu Leu Lys Pro Leu
Glu Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr. 3. The polypeptide according to claim 1, which has the following amino acid sequence. Pro Thr Ser Ser Ser Thr Lys Lys Thr
GlN Leu GlN Leu Glu His Leu Leu Leu
Asp Leu GlN Met Ile Leu AsN Gly Ile AsN
AsN Tyr Lys AsN Pro Lys Leu Try Arg
Met Leu Thr Phe Lys Phe Try Met Pro
Lys Lys Ala Thr Glu Leu Lys His Leu GlN
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu
Glu Val Leu AsN Leu Ala GlN Ser Lys
AsN Phe His Leu Arg Pro Arg Asp Leu Ile
Ser AsN Ile AsN Val Ile Val Leu Glu Leu
Lys Gly Ser Glu Thr Thr Phe Met Cys Glu
Tys Ala Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr.