JPH0659220B2 - Gene encoding a polypeptide having human interleukin 2 activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a gene having a polypeptide having human interleukin 2 activity.

Interleukin 2 (hereinafter abbreviated as "IL-2")
Was formerly called T cell growth factor and is a soluble protein (commonly known as "lymphokine") produced by T cells activated by lectins or antigens (Morgan et al., Science, 193,1007-1008 (1976),
Gillis et al., J. Immunol., 120, 2027-2033 (1978)). IL-2
Can regulate lymphocyte reactivity and allow long-term culture of antigen-specific effector T-lymphocytes 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. Immu.
Nol., 120, 1967-1973 (1978)), cytotoxic T lymphocyte activity in the culture system of splenocytes of nude mice (Wagner et al., Natur.
e, 284, 278-280, (1980)) and induction of anti-SRBC plaque-forming cell response (Gillis et al., J. Exp. Med. 149, 1960-1968 (1979)).
Has been shown to have other related biological activities such as. Therefore, this lymphocyte regulatory factor is useful for enhancing the liquid immunity and the cell-mediated immune response and for restoring the immunodeficiency state to the normal liquid or cell-mediated immunity. These revealed immunological activities of IL-2 indicate that IL-2 is effective against malignant tumors, bacterial or viral infections, immunodeficiency, autoimmune diseases, etc. (Papermaster et al., Adv. Immunopharm., 507 (1980)). It has been shown to be useful for medical immunotherapy. Similar to interferon, IL-2 has been shown to enhance natural killer cell activity, strongly suggesting its utility in treating malignant tumors. Furthermore, IL-2 is capable of retaining monoclonal activated T cells, which means the molecular mechanism of T cell differentiation, the differentiation mechanism of T cell function, T
It shows that it plays an important role in studying the mechanism of the cellular antigen receptor. IL-2 can also be used to produce lymphokines derived from various T cells, which are useful in various other fields, by culturing monoclonal T cells for a long period of time. In addition, IL-2 production and lymphocyte
Responsiveness to IL-2 is an important parameter of immunological function and is useful for clinical diagnosis of immune disorders.

In the prior art, IL-2 has been produced by stimulating mouse, rat or human lymphocytes with mitogens (Gillis et al., Nature, 268,154-156, (1977)), Far.
rar 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 mouse IL-2 from T lymphoma cell line (Gillis
Et al., J. Immunol., 125, 2570-2578 (1980)) and production of human IL-2 from a human leukemia cell line (Gillis et al., J. Exp. Med., 15).
2, 1709 to 1719, (1980)).

The technique described by Gillis et al., Uses a cell culture method to transform human IL-2 from mitogen-activated T leukemia cell lines.
The present invention relates to a method of manufacturing. However, this method has a drawback in that only low concentrations of human IL-2 are produced, and a complicated purification step is required to obtain a small amount of IL-2 from a large amount of culture solution. Furthermore, since the human T leukemia cell line also produces a small amount of other physiologically active substances that closely resemble human IL-2, a cytotoxin that separates IL-2 from these other immunologically active molecules or sometimes coexists with them. Substance (toxic l
Separation from ectin) is quite difficult.

As another method for producing IL-2, a recombinant DNA (abbreviation of deoxyribonucleic acid) method (Graph) used for producing other bioactive human-derived proteins such as interferon is used.
y et al., Nature 295, 503-508, (1981), Nagata et al., Nature 2
84,316 ~ 320 (1980), Taniguchi et al., Gene 10, 11 ~ 15, (198
0)) seems preferred. However, prior to the present invention, attempts to produce IL-2 by recombinant DNA methods were unsuccessful. For example, with recombinant DNA bodies IL-
Attempts to create life forms that produce 2 are probably
The fact that the gene encoding the IL-2 polypeptide has not yet been cloned has made it unsuccessful.
"Nikkei Biotechnology, No. 19, July 5, 1982
The day was reported.

Therefore, a cloned gene encoding IL-2 and a recombinant DNA having the gene have been craved. In addition, using a live cell line containing a recombinant DNA and the cell line, IL
There has been much craving for a method of producing -2.

The subject matter of the present invention will be more readily apparent from the following description.
An object of the present invention is to create a gene encoding a polypeptide having IL-2 activity.

That is, the present invention provides a gene encoding a polypeptide having the following amino acid sequence having human interleukin 2 activity.

Met Pro Thr Ser Ser Ser Thr Lys Lys Thr GlN Leu Gl
N Leu Glu His Leu Leu Leu Asp Leu GlN Met Ile Leu
AsN Gly Ile AsN AsN Tyr Lys AsN Pro Lys Leu Thr Ar
g Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala
Thr Glu Leu Lys His Leu GlN Cys Leu Glu Glu GlN Le
u Lys Pro Leu Glu Gul Val Leu AsN Leu Ala GlN Ser
Lys AsN Phe His Leu Arg Pro Arg Asp Leu Ile Ser As
N Ile AsN Val Ile Val Leu Glu Leu Lys Gly Ser Glu
Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Th
r Ile Val Glu Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr The gene of the present invention has the following nucleotide sequence.

By utilizing the gene of the present invention, IL-2 becomes IL-
Insertion of a gene encoded to produce a polypeptide having two activities and vector DNA capable of being replicated in a cell
Modified by recombination with the insertion of the gene and the coding sequence of the gene is located downstream of the promoter sequence
It is produced by suspension culture (aerobic) of a prokaryotic cell line transformed with DNA to produce IL-2, particularly Escherichia coli in a medium.

The cloned gene encoding the IL-2 polypeptide of the present invention is a messenger RNA (IL-2) corresponding to IL-2 derived from mammalian cells, which is characterized by having the ability to produce a polypeptide having IL-2 activity. mRNA; "RNA"
Is obtained by reverse transcribing ribonucleic acid (hereinafter referred to as “IL-2 mRNA”) into complementary DNA (cDNA). The obtained 2 single-stranded cDNA (ss-cDNA) can be converted into single-stranded cDNA (ds-cDNA).

The mRNA used as a template for preparing cDNA is IL-2
It can be isolated from mammalian cells which produce the polypeptide. The isolated RNA is polyadenylated (GiLLi
S. et al., Immunolgical Rev., 63, 167-209 (1982)), polyadenylated RNA can be fractionated into 11-12S fractions by, for example, sucrose density gradient centrifugation. Thirteen
IL-2 mRNA activity may appear in S mRNA, but in this case, it is considered to be an aggregate of 11-12S mRNA.

Mammalian cells capable of producing IL-2, which is 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. Nylon column treatment, antiserum and trapping treatment, density gradient fractionation, combination treatment of neuraminidase and galactose oxidase, trypsin treatment, etc. in order to impart IL-2 producing ability to cells or enhance IL-2 activity. Various pretreatments such as various enzyme treatments and X-ray irradiation may be performed. Cloned T lymphocytes obtained after culturing the above mammalian cells in the presence of T cell growth factor can also be used as a source of mRNA, which is a more preferred T-lymphocyte. Transformed lymphocyte cell lines or cloned transformed cell lines such as T lymphocytes themselves derived from leukemia or lymphoma cell lines or their derivatives pretreated or mutated by the above-mentioned method are also used as a source of mRNA. preferable. Apparently, the cloned cell line usually contains higher amounts of IL-2 mRNA as compared to the parental line before cloning. T cell hybridomas obtained by fusing the above-mentioned lymphocyte-derived cells with tumor cell lines such as CEM, Molt 4F, BW5147 are also preferred mammalian cells for use. The lymphocyte-derived cells in this case are (1) IL-
2 spontaneously producing cells and (2) cells that produce IL-2 only when mitogen is introduced into the culture medium in the presence or absence of other cells that support IL-2 production .

To induce IL-2 mRNA in IL-2 spontaneous producer cells, IL-2 spontaneous producer cells are cultured by methods well known in the art of cell culture. IL- only in the presence of mitogen
When mRNA is produced in cells that produce 2, the cultured cells are thoroughly washed with a medium, and then Rosewell Park Memorial Institute 1640 (hereinafter abbreviated as “RPMI 1640”) with or without serum, Dulbecco's. Resuspend in a medium such as modified Eagle medium (hereinafter abbreviated as "DMEM") or click medium. To these cell culture media, various additives such as penicillin, streptomycin or other antibiotics, L-glutamine, hepes buffer, or sodium hydrogen carbonate may be added at a concentration commonly used in the field of cell culture. good. 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 mitogens, neuraminidase, galactose oxidase, zinc compounds such as zinc chloride or protein A, bacterial lymphocyte activating factors such as streptolysin-O. The stimulated cells are harvested and washed. Co-existence of macrophages or dendritic cells upon mitogen stimulation can also activate mRNA or increase the yield of activated mRNA. Similarly Raji, Daudi, K56
Co-existence of cell lines derived from B lymphocytes or B lymphocyte cell lines such as 2, BALL-1 can also activate mRNA or increase the yield of activated mRNA.

In order to grow mammalian cells, the cells are maintained under normal conditions in vitro by cell culture or in histocompatible animals. in vit to prepare a source of mRNA
When the cells are subcultured by ro culture, these cells can be grown in any medium that is conventionally known to promote the growth of T cells. Mammalian serum, serum components or serum albumin may be added to these media. The culture time for activation of mRNA corresponds to the time required for activation to produce mRNA. This time usually corresponds to the time required to start the production of IL-2 in the medium. The preferred time is 3 to 12 hours after the addition of a stimulant such as mitogen. If the culture time is too long, the produced IL-2 mRNA may be degraded. Upon activation of IL-2 producing cells, phorbol esters such as PMA or TPA are added at 10 to 50 ng / m 2.
It can also be added to increase the activation level.

The above steps for IL-2 mRNA activation are carried out in an environment of pH 7.0-7.4, saturated steam at a temperature range of 32-38 ° C.

The method for obtaining and culturing IL-2 producing mammalian cells is described below.

(A) Acquisition of spontaneously producing IL-2 strain Jurkat cells, human leukocyte-derived leukemia cells (Fred Hutchinson Cancer Institute / Seattle / USA, Salk Institute / San Diego / USA, West German National Cancer Center / Heidelberg) / Freely available in West Germany, etc.) at a cell density of 1 × 10 ⁶ cells / ml in a click medium and subjected to X-ray irradiation of a total of 8,000 roentgens at an irradiation rate of 150 roentgens / minute. . After this, the cells were cultured at a cell density of 0.1 cells / 200 μl medium for 9
6-hole flat bottom microtiter plate ("Falcon 3
072 ") and cultured in Click medium containing 5% fetal bovine serum for 3 weeks at 37 ° C in a 5% CO ₂ incubator (cloning by limiting dilution). The cells in the culture wells in which the growth of cells are observed are transferred to a 24-well Nunc culture plate before the cells reach the density covering the entire bottom surface, and the cells are allowed to grow in Click medium for 5 days. Further, the present cells were suspended in a serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 1 to 2 × 10 ⁶ cells / ml, and cultured for 2 days, and the main culture supernatant was separated by centrifugation. Then, debris removal and sterilization were performed with a 0.22μ Millipore filter. By measuring the IL-2 activity of the culture supernatant thus obtained, IL-
An X-ray treated mutant strain that spontaneously produced 2 was selected and cloned.

(B) Acquisition of IL-2 Producing Strain from Human Peripheral Blood Mononuclear Cells Peripheral blood lymphocytes (hereinafter abbreviated as PBL) were collected from human peripheral blood and subjected to FICOL HYPERK density gradient centrifugation. To collect. This PBL is suspended in a click medium containing 5% FCS at a cell density of 1 × 10 ⁶ cells / ml and inoculated into a 24-well Nunc culture plate containing 2 ml each. To this, 100 μl of phytohemagglutinin-M (manufactured by Gibco) was added so as to have a final concentration of 5 μg / ml, and the cells were cultured under the above-mentioned conditions for 48 hours, and then the cells were washed with the culture medium and again
Inoculate 1 ml of Click medium at a cell density of × 10 ⁵ cells / ml. Furthermore, concanavalin A (hereinafter abbreviated as ConA)
Human T lymphocytes from PBLs were long-term subcultured 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. To do.
The T lymphocytes thus obtained by long-term subculture are cloned in the presence of human splenocytes derived from a medium conditioned by the same limiting dilution method as described above, and the cells are similarly proliferated. The cloned T lymphocytes thus obtained were added to a cell density of 1 × 10 ⁶ cells / ml at 10 μg / ml.
1 ml of RPMI 1640 medium in a 24-well Nunc culture plate was inoculated in the presence of ml of phytohemagglutinin (hereinafter abbreviated as PHA), and cultured at 37 ° C. for 24 hours in a 7.5% CO ₂ incubator. The main culture supernatant was separated by centrifugation and then sterilized with a 0.22 μm Millipore filter, and then an IL-2 activity assay was performed to identify an IL-2 producing human normal T lymphocyte clone. .

(C) Acquisition of human lymphocyte-derived malignant cells that produce IL-2 by mitogen stimulation The aforementioned Jurkat cells and the J-111 strain cloned by the above-mentioned limiting dilution method are serum-free medium and serum 1 ConA 10 μg in BPMI 1640 medium containing ~ 2%
When cultured for 24 hours in the presence of 1 / ml or 2.5 μg / ml of PHA, it is possible to produce 10 to 4,000 units / ml of IL-2. Further, these human malignant cells produce IL-2 even when cultured in the presence of zinc chloride, protein A and picibanil.

(D) Acquisition of IL-2 producing cells by stimulating with mitogen in the presence of other cells or factors produced by the cells Cloned by human lymphocyte malignant cells Molt 4F or the above-mentioned limiting dilution method One clone of Jurkat cells, the Jurkat 99 strain, does not produce IL-2 even when it is cultured for 24 to 72 hours by adding lectin and mitogen in a wide concentration range as described above. However, during this period, when interleukin 1 which is one of monokines is co-cultured with 5 to 10 μ / ml or 50% of K562 or Raji cells at 37 ° C. for 24 hours, the amount of IL-2 that can be confirmed ( 10-100 μ / ml).

IL-2 mRNA can be extracted from cells activated in this manner by a conventional method regardless of cell type. For example, the cells are partially or completely decomposed by adding a surfactant such as NP-40, SDS, Triton-X100, and eoxycholic acid, or the cells are partially decomposed by a physical method such as a homogenizer or freeze-thawing. Destructively or completely solubilized. At this time, in order to prevent RNA degradation by RNase, it is preferable to add an RNase inhibitor such as heparin, polyvinyl sulfate, bentonite, macaloid, diethylpyrocarbonate, vanadium complex, etc. to the extract. In some cases, anti-IL-
A method of precipitating a polysome in the process of IL-2 synthesis using 2 antibody and extracting the mRNA with a detergent or the like can also be performed.

For purification of mRNA containing poliA, oligo dT-cellulose, poly U-sepharose with Sepharose 2B as a carrier, or an affinity column or batch purification method, fractionation by SDG centrifugation method, agarose gel electrophoresis method, etc. Can be done by.

In order to confirm that the mRNA obtained as described above has IL-2 mRNA activity, the mRNA is translated into a protein and its physiological activity is examined, or an anti-IL-2 peptide monoclonal antibody is used. May be used to identify the translated protein. For example, mRNA is usually Xenopus laevis (X
(Enopus laevis) eggs by microinjection (Gurdon et al., Nature, 233, 177-182 (1972)) or by using the reticulocyte or wheat embryo cell-free translation system.

IL-2 activity was previously reported by Gilis et al. (Gillis et al., J. Immunol., 12
0,2027 to 2033 (1978), which can be confirmed by the microassay method basically described. In this test,
IL- of a cytotoxic T lymphocyte cell line (hereinafter abbreviated as CTLL) prepared according to the method established by Gillis et al.
Increased DNA synthesis in IL-2 dependent cells
lar proliferarion) is used as an index. That is, 4 × 1
RPMI-1640 medium containing 0. ³ CTLL cells with 2% FCS 10
Suspend in 0 μl and inoculate 96-well flat bottom microplates with 100 μl of serially diluted translation products. 37
After culturing at ℃, 5% CO ₂ for 20 hours, the cells were added at 0.5 μCi /
The wells are labeled with ³ H-TdR for 4 hours, the cells are collected on a glass fiber band using an automatic cell harvester, and the radioactivity taken up by the cells is measured by a liquid scintillation method. This assay revealed that CTLL cells cultured in the presence of IL-2 uptake ³ H-TdR in a dose-dependent manner, which clearly calculated the amount of IL-2 contained in the sample. can do.

Since IL-2 has the activity of promoting the proliferation of T lymphocytes, IL-2
-2 activity can be measured using the proliferation of T lymphocytes as an index. That is, 5 CTLL cells were added to DME containing 2% FCS.
Suspend in 100 μl M and inoculate 96-well flat bottom microplates with 100 μl serially diluted translation products.
After culturing for 72 to 96 hours at 37 ° C. under 5% CO ₂ , the number of activated and proliferated cells is counted under a microscope. IL-2 at 100 U / ml and 10 U / ml is used as a control group, and the IL-2 activity of the sample is determined by comparing with the number of proliferated living cells in this control group.

IL-2mR thus obtained from the most active fraction
NA was used as a template for synthesizing ds-cDNA, and ds-cD
NA binds to vector DNA. cDNA synthesis is performed by conventional methods.

First, using mRNA as a template and oligo dT as a primer, dA
MRNA by reverse transcriptase in the presence of TP, dGTP, dCTP, dTTP
Synthesize ss-cDNA complementary to
After decomposing and removing, ds-cDNA is synthesized using reverse transcriptase or DNA polymerase using the single-stranded cDNA as a template.

A recombinant DNA body is prepared from the ds-cDNA thus obtained and a vector DNA containing a replicon capable of replicating in prokaryotes. Thereafter, this recombinant DNA body is integrated into a host cell.

This ds-cDNA and a vector DNA that can grow in prokaryotes
Exonuclease treatment before binding these,
It is modified by various treatments such as addition of a chemically synthesized DNA fragment and extension of G and C-chains in order to attach a terminal capable of being ligated to the end of ds-cDNA or vector DNA. These connectable DNs
A can be patched by DNA ligase of T4 phage in the presence of ATP, for example.

Living cells are transformed with the thus prepared recombinant DNA bodies to amplify cloned cDNA or to produce IL-2 polypeptide.

Suitable prokaryotic hosts for IL-2 production 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.

As a suitable Escherichia coli vector, pSC101, p is used as an EK type plasmid vector (stringent type).
RK353, pRK646, pRK248, pDF41 etc. , EK type plasmid vector (relaxed type): ColE1, pVH51, pA
C105, RSF2124, pCR1, pMB9, pBR313, pBR322, pBR324, pBR32
5, pBR327, pBR328, pKY2289, pKY2700, pKN80, pKC7, pKB158,
pMK2004, pACYC1, pACYC184, dul, etc. , Λgt type phage vector: λgt. λc, λgt. λB, λWES, λC,
λWES. λB, λZJvir., λB ′, λALO, λB, λWES.T
s622, λDam, etc. are included. In general, pBR322 has often been used as a vector for Escherichia coli, in which case the best cloning site is Pst1 and
EcoR1 site.

For transformation of host cells using recombinant DNA bodies, the following methods are commonly used. 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 preparation of protoplasts.

Cells carrying the IL-2 gene can be isolated after transformation using either of the following two methods.

(1) Plus-minus method: IL-2 mRNA partially purified as a 11-12S fraction by sucrose density gradient centrifugation was prepared from an antigen-stimulated mammalian cell extract, and this partially purified mR was used.
³² P-radioactive ss-cDNA is synthesized using NA as a template. After removing the template mRNA by alkaline treatment, the isolated cDNA was
Extracted from non-stimulated mammalian cells and hybridized with partially purified 11-12S mRNA. CDNA that subsequently hybridized with unhybridized cDNA
Is fractionated by hydroxyapatite column chromatography. The cDNAs that did not hybridize and the cDNAs that did not hybridize are called probe A and probe B, respectively. Each recombinant is grown on nitrocellulose filter paper by the same method. And the DNA of the cell
Is fixed on the filter paper by alkali treatment. Probes A and B are each hybridized with DNA on two different filter papers. After that, autoradiography is performed, and a recombinant that positively reacts with probe A (plus) and a recombinant that slightly or does not react with probe B (minus)
(Taniguchi et al .; Proc. Jap. Acad., V155B 464-46)
9 (1979). (2) In the second method, for example, 1,000 to 10,000 recombinant clones are roughly divided into clone groups addressed to 2 to 30 to 2 to 300 clones, and each clone group is cultured by a conventional method to obtain plasmid DNAs. To prepare. Then, these plasmid DNAs are heat-denatured to fix the ss-cDNA on nitrocellulose filter paper, and activated.
MRNA prepared from mammalian cells containing IL-2 mRNA
Hybridization is performed in a complementary manner. Alternatively, when mRNA (mixture) containing IL-2 mRNA is hybridized with heat-denatured plasmid DNA (mixture), DNA
-MRNA hybrids are immobilized on nitrocellulose filter paper. The filter paper was washed with a low salt buffer solution such as 1 mM HEPES or 10 mM saline and adsorbed on the filter paper.
mRNA containing 0.5 mM EDTA; 0.1% SDS solution-containing solution, for example, 95
Extract for 1 minute at ℃. Purified mRNA is oligo
Elute and collect by dT-cellulose column chromatography. Then, the mRNA is microinjected into Xenopus oocytes and translated into a protein to confirm IL-2 activity. Alternatively, this mRNA can be translated into a protein using an mRNA-dependent reticulocyte system or an in vitro cell-free synthesis system of wheat embryo, and IL-2 activity can be analyzed using an anti-IL-2 antibody. The groups in which IL-2 activity was detected by these methods were categorized into a group containing a smaller number of recombinant clones, and finally repeated until a single clone having IL-2 DNA was obtained.

To obtain a cDNA encoding an IL-2 polypeptide from a recombinant capable of producing IL-2, first, the recombinant DNA in the transformant is isolated and cleaved with a restriction enzyme endonuclease. The integrated cDNA fraction is separated from the DNA fraction obtained by cleavage.

The entire nucleotide sequence of the Pst1 DNA insert encoding the IL-2 polypeptide from the recombinant pIL-2-50A DNA was determined by the Maxam and Gilbert method ((Meth.Enzym.65,499-560,
(1980)); and the two-deoxynucleotide chain-end method (S
mith, AjMMeth.Enzym.65,560-580, (1980)).

Cleavage diagrams of the cDNA insert with a restriction endonuclease are shown in FIGS. 1 and 2 (a). As shown in FIG. 2 (a), this cDNA has a structure that is cleaved by the restriction endonucleases BstN1, XbaI, and BstNI, respectively.

The DNA sequence of this cDNA insert carries one large open reading frame. The first ATG sequence (Kozak, M. Cell.
5,1109 to 1123 (1978)) exist at the 48'-50 nucleotide position from the 5'end, and 152 sequences up to nucleotide positions 507-509 where the end-of-read sequence TGA exists are linked to this ATG. A linkage corresponding to the 3'-poly (A) end of mRNA is found at the cDNA end, and is usually a eukaryotic mRNA.
6-nucleotide AATA found in most of the
AA (771-776) comes first. (Proudfoot, NJ & Bro
wnlee CG, Nature 263, 211-214, (1976)) The amino acid sequence encoded by the cDNA is shown in Fig. 2 (b).
(Amino acid sequence 1), and the polypeptide of amino acid sequence I consists of 153 amino acids and its molecular weight is calculated to be 17631.7 daltons.

As reported to be found in most secreted proteins known to date (Blobel G. et al, Sym.Soc.Exp.Med.,
33, 9-36 (1979)), N of the above-mentioned deductive IL-2 polypeptide.
The terminal region is also hydrophobic. This region may play the role of a signal peptide that is cleaved upon secretion of mature IL-2. The cleavage occurs between Ser and Ale at the 20-21 position or between Ala-Pro at the 21-22 position, and the amino acid sequence I
Produces a polypeptide having I and III. This is because similar cleavage sites are often found in other secreted proteins known so far (Blobel, G. et al, Sy.
mp.Soc.Exp.Med., 33, 9-36 (1979)). Therefore, mature IL-
The two polypeptides consist of 133 to 132 amino acids and have a calculated molecular weight of 15420.5 or 15349.4 daltons. This molecular weight value is for humans obtained from Jurkat cells
Contrast what was reported as the molecular weight of the IL-2 protein (15,000 daltons) (Gillis et al., Immunological Re
v., 63, 67-209 (1982)). In addition, as shown in Example 3, a DNA fraction starting from the CCT sequence at nucleotide positions 111 to 113, that is, a code for a polypeptide starting from Pro at the 22nd position (amino acid sequence III in FIG. 2 (b)) Is
It was confirmed to express a polypeptide having IL-2 activity. The DNA fraction starting from GCA at nucleotide positions 107 to 110, that is, the DNA fraction encoding the polypeptide starting from Ala at position 21 as shown in the amino acid sequence II of FIG. IL-2 as shown in 6
It was confirmed that it expresses an active polypeptide.

It is known that nucleated genes exhibit polymorphism as well as the human interferon gene (Taniguchi et al., Gene 10, 11-15 (1980), Ohno, Taniguchi., Proc. Nat).
1.Acad.Sci.USA, 77,5305-5309 (1981); Gray er al, Nat
ure 295,501-508 (1981)).

Due to this polymorphism, some of the amino acids in the protein product may be substituted, or there may be changes in the nucleotide sequence but no change at all. In the case of human IL-2 cDNA,
Another cDNA clone (pIL-2-503) in which the A residue at position 503 of the pIL-2-50A cDNA is replaced with a G residue can also be detected. pIL
The existence of other cDNA clones whose nucleotide sequence differs from that of the -2-50A cDNA can also be expected.

As is clear from the above explanation, the gene of the present invention is a DNA having the nucleotide sequence shown in FIG. 2 (a), AT at the 48-50th position.
DNAs having a continuous base sequence starting from the G sequence and extending to at least the ACT sequence at positions 504 to 506, DNA having a continuous base sequence starting from the GCA sequence at the 108-110 positions and extending from the GCA sequence to at least the ACT sequence, It also contains a DNA having a continuous base sequence extending from the CCT sequence at positions 111-113 to at least the ACT sequence. The gene of the present invention also ends in the ACT sequence at positions 504 to 506, DNA starting at A at position 1 and DN beginning at ATG at positions 48-50.
A, DNA starting with GCA sequence at 108-110 or C at 111-113
Contains DNA beginning with the CT sequence. Furthermore, the gene of the present invention is a DN that ends at the TGA sequence at positions 507-509 and starts at A at position 1.
A, DNA starting with ATG at positions 48-50, DNA starting with GCA sequences at positions 108-110 or DNA starting with CCT sequences at positions 111-113. Furthermore, the gene of the present invention ends with a C at position 801 and
DNA starting with A at position 48, DNA starting with ATG at position 48-50, 108-
It includes DNA starting with GCA at position 110 or CCT sequence at positions 111-113. The gene of the present invention is also poly (A)
It includes a DNA starting with the ATG sequence at positions 48-50, a DNA starting with a GCA sequence at positions 108-110 or a DNA starting with a CCT sequence at positions 111-113. Further, the gene of the present invention includes a gene having a nucleotide sequence corresponding to the amino acid sequences I, II and III. A polypeptide lacking one or more amino acids in amino acid sequence I, or a polypeptide in which one or more amino acids in amino acid sequence I is substituted with one or more amino acids is IL-2. It may have activity, and thus the gene encoding such a polypeptide can be used as the gene of the present invention. Similarly, even in a gene in which one or more bases capable of expressing one or more amino acids with respect to the amino acid sequence I, II or III are additionally linked, the added amino acid is the IL of the polypeptide. -2 is included in the gene of the present invention unless it interferes with the expression of activity. Even a modified region having an additional amino acid sequence that inhibits the polypeptide function of IL-2 can be used as the gene of the present invention as long as the newly added region can be easily removed. The same thing can be said in the case where the DNA coding for the amino acid addition at the C-terminal of the amino acid sequences I, II and III of the gene corresponding to the amino acid sequences I, II and III is additionally linked at the 3'-terminal. In order to produce IL-2 using the gene of the present invention obtained as described above, first, a recombinant DNA body containing the gene of the present invention is prepared, and then a host cell is prepared with the recombinant DNA body. It may be transformed, and the transformed biological cells may be cultured in a medium.

Recombinant DNA bodies that produce IL-2 in living cells can be produced by the following various methods. For example, a sequence encoding IL-2.cDNA is inserted downstream of the promoter sequence of the expression vector.
Alternatively, a piece of cDNA having a promoter sequence can be inserted upstream of the IL-2 coding sequence before or after the insertion of the cDNA into the expression vector.

The method for constructing a prokaryote that expresses IL-2-cDNA and produces an IL-2-polypeptide will be described in detail below.

(1) Expression of IL-2-cDNA by Escherichia coli In order to express IL-2-cDNA in Escherichia coli, first the cDNA is bound to various bacterial promoters, and then the hybrid containing the cDNA downstream of the promoter. Make a plasmid. Bacteria that infect Escherichia coli HB101 with this plasmid and biosynthesize a protein having human IL-2 activity are cloned. Any naturally-occurring bacterial promoter will express IL-2-cDNA if properly connected to the cDNA. An example of expression of such a cDNA is as follows.

The cloned cDNA encoding IL-2 encodes a 153 amino acid polypeptide as shown in FIG. N corresponding to 20 amino acids of this polypeptide
The terminal region is extremely hydrophobic and is characteristic of most secreted proteins. Such a hydrophobic sequence is called a signal sequence and is cleaved during the secretory process. Therefore, the mature IL-2 polypeptide should have less than 153 amino acids. Therefore, it is desirable to express the cDNA portion encoding the mature IL-2 polypeptide and not the IL-2 signal sequence equivalent portion.

(i) Construction of plasmid vector pTrS-3 pTrS-3 contains the Escherichia coli Trp promoter, and the ribosome binding site (SD sequence) for the leader peptide of pTrS-3 has already been reported (G. Miozzari and
Yanofsky J. Bacteriol. 133, 1457-1466 (1978)). The presence of an ATG codon located 13 base pairs downstream of the SD sequence has also been reported (Nishi et al, Biochemistry 54, NO.8.676 (1982)). This plasmid vector also contains a single Sph I site downstream of the ATG initiation sequence (Figure 3).

To express IL-2 / cDNA, the plasmid was first transformed into Sph.
Escherichia coli DNA polymerase I digested with I
(Klenow fragment) or treatment with bacteriophage T4 DNA polymerase I to remove the 3'-position overhanging end (Fig. 4 (a)). Plasmid pIL-2-50A
Is digested twice with Pst I and Hgi AI and the larger cDNA fraction is isolated. The DNA is then treated with Escherichia coli DNA polymerase I (Klenow fragment) or bacteriophage T4 DNA polymerase to cut off the 3'-overhang. This treated cDNA encodes an IL-2 polypeptide of 132 amino acids (4 (a)
Figure). This cDNA was ligated to pTrS-3 plasmid DNA pretreated as described above and the ATG initiation codon was added to IL-2 cDNA.
Connect to CCT (Pro) array of. Thus the plasmid pT
IL-2-22 is obtained. Trp promoter sequence and pT IL-2-2
The binding of the two IL-2 cDNA sequences is shown in Figure 4 (a).

Plasmid pT IL-2-22 directs the synthesis of a 132 amino acid IL-2 polypeptide starting from proline by Escherichia coli.

(ii) Mature IL-2 may contain alanine (position 21) as the N-terminal amino acid instead of proline, and a plasmid directing IL-2 polypeptide synthesis consisting of 133 amino acids should be prepared as follows. You can

The plasmid pTrS-3 contains a single C between the SD and ATG sequences.
There is a la I cleavage site (Fig. 3). This plasmid is Cla
Cut with I and Sal I. Plasmid pIL-2-50A for Ps
Partially digested with tI, treated with Escherichia coli DNA polymerase I, and the longest DNA is isolated. Then, the DNA was ligated to a synthetic DNA linker containing a restriction enzyme Xho I cleavage site, and a clone containing a plasmid pIL-2-50A (Xho) into which a DNA linker was introduced 3'-downstream of the IL-2 coding sequence was constructed. To isolate. The plasmid pIL-2-50A (Xho) was first transformed into HgiAI.
The cDNA fraction can be isolated by digestion with E. coli, treatment with Escherichia coli Klenow fragment or T4 DNA polymerase, and digestion with Xho I. This cDNA fragment is Cl
It is ligated to pTrS-3 DNA pretreated with a I and Sal I and ligated to the synthetic DNA as shown in FIG. 4 (b). Thus, a plasmid which synthesizes an IL-2 polypeptide consisting of 133 amino acids starting from Ala in Escherichia coli
pTIL-2-21 is obtained. (Fig. 4 (b)). Similar thing is Xh
o Made without the I linker.

(iii) different size I with different N-terminal amino acids
L-2 polypeptides can also be made using the pTrS-3 expression plasmid vector. As shown below, the IL-2 cDNA cloned in pIL-2-50A had a nucleotide binding site 81-
85 has only one Dde part. Plasmid pIL-2-5
0A (Xho) is cut with Dbe I and the DNA fraction containing the larger section of cDNA isolated. This fraction contains DNA having 3,000 base pairs of pBR322 (Fig. 4 (c)). The DNA fraction was treated with exonuclease Bal 31 and then Xho
Cut with I. The DNA obtained here was cleaved with Sph I
Klenow fragment or T4 DNA bound to pTrS-3
Treat with polymerase and then digest with Sal I (4th
(Fig. (c)). Escherichia coli HB 101
To search for clones expressing human IL-2. These clones should express human IL-2 in different sizes. This is because the DNA corresponding to the N-terminal region of human IL-2 is cleaved off in various ways. Thus, pTIL-2-14 and 15 containing IL-2 cDNA are obtained.

(iv) IL-2 cDNA was also pKT218 (provided by Talmage; Proc. Natl. Acad. Sci. USA, 77, P. 3369-3373 (1980)).
Can also be expressed using. Plasmid pKT 218 is Pst
Cleavage with I, pIL-2-50A with HgiAI and Pst I (5th
(Figure) Connect with the IL-2 cDNA insert obtained. The resulting plasmid pKIL-2-21 has a sequence at the initiation position of initiation of protein synthesis, as shown in FIG. Therefore, this plasmid pKIL-2-21 consists of a polypeptide in which both 133 amino acids of IL-2 and the amino acid of β-lactamase are fused together.
It should be able to be synthesized in E. coli (the first methionine is cleaved off in Escherichia coli).

(V) Expression of the plasmid pTuBlp-5 in which a promoter sequence for tufB is inserted into p-BR.322 has already been performed (Taniguchi et al., Biochemistry 53 966 (1981)). This plasmid contains one Cl
It contains an aI cleavage site, and as shown in FIG. 6, this cleavage site is located 2 base pairs downstream of the SD sequence. pTrS-3 is also
It contains one Cla I cleavage site between the SD sequence and the ATG initiation sequence, and at the same time, this Cla I site contains pTrS-3 and IL-2 cDNA.
It is extremely easy to replace the bacterial Trp promoter with the tufB promoter, as it is not destroyed in the process of making an expression plasmid using. Therefore, human IL-2 cDNA is tufB
It is expressed under the control of a promoter. For example pTIL-2-22
Is cleaved with Cla I and Pvu II to separate the DNA fraction containing IL-2 cDNA. Next, this fraction is ligated with pTuBlP-5 and precut with Cla I and Pvu II, and then the plasmid pTuIL-2-22 is constructed as shown in FIG. IL-2 activity includes Escherichia coli HB10 containing plasmid pTuIL-2-22
Detectable in 1 extract.

(vi) For example, using pTIL-2-21, but basically pTrS-
It can be constructed in the same manner by using all the expression plasmids achieved by using 3. Also for example pTuIL-2-
22 is cleaved with Cla I, and the base pair of DNA is 2- with Bal31 or SI or DNA polymerase I (Escherichia coli).
It is also possible to optimize the distance between the SD and ATG sequences by removing or supplementing three and reconnecting the plasmids.

Then, Escherichia coli with the recombinant DNA inserted,
Transformed prokaryotic cells such as Bacillus subtilis are cultured to amplify recombinant DNA bodies or produce IL-2 polypeptides. This culture is performed by a usual method.

IL-2 produced intracellularly or extracellularly is subjected to ammonium sulfate precipitation, dialysis for salt removal (under normal pressure or reduced pressure), gel filtration,
Chromatography, isoelectric focusing, gel electrophoresis, high performance liquid chromatography (hereinafter abbreviated as HPLC),
(Ion exchange, gel filtration and reverse phase chromatography) and affinity chromatography with dye-bound carrier, activated Sepharose 4B bound to IL-2 monoclonal antibody or lectin-bound Sepharose 4B, etc. You can IL
-2 is isolated and purified by Watson et al. (J. Exp. Med., 150, 849-861).
(1979), Gillis et al, J. Immunol., 124, 1954-1962 (198
0), Mochizuki et al, J. Immunol. Methods 39, 185-201 (19
80), Welte, K et al, J. Kxp. Med., 156,454-464 (1982)).

The polypeptide thus obtained exhibits the same biochemical and biological behavior as that known for IL-2 made from mammalian cells by mitogen stimulation.
-2 activity. The molecular weight is about 15,000 daltons and IL
-2 activity was completely neutralized with or without immunosorbents such as Igsorb (Enzyme Center) or precipitated with monoclonal anti-IL-2 antibody. In immunoelectrophoresis,
The IL-2 polypeptide shows only one settling line for the corresponding anti-IL-2 antibody. The IL-2 activity is stable even after reduction with 2-mercaptoethanol, and is stable even after treatment with DNAse and RNAse or heat treatment at 56 ° C. for 30 minutes. The activity is stable at pH 2-9. IL- produced in this way
2 promotes the proliferation of T cells (cytotoxic T lymphocytes) having a monoclonal function, enhances thymocyte division,
Furthermore, in the absence of antigen, it induces differentiation from memory state into anti-cancer-specific cytotoxic T lymphocytes. It also helps enhance the activation of natural killer cells for YAC-1 cells and RL 1 cells.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 (1) A Jurkat cell, which is a human T-cell leukemia cell line (freely available in Japan, West Germany, and the United States), was suspended in RPMI 1640 medium containing 10% FCS, and X-ray irradiation apparatus Exs was used. Irradiation with 150 / 300-4 (Toshiba, Japan) for 50 seconds at room temperature until reaching 10,000 roentgens. The irradiated cells were then cultured in the above-mentioned medium at an initial cell density of 1 × 10 ⁵ cells / ml in 5% carbon dioxide in an incubator at 37 ° C.
Cultured for a day.

The mutant cells (0.2 cells / well) were seeded in 10 wells of a 96-well flat-bottomed microplate, and cultured for 21 days in a 5% carbon dioxide gas, 37 ° C incubator. The clones obtained from the growing holes were transferred to a new medium to increase the amount of clones, and the clones with increased ConA 50 μg / m
The cells were cultured in the presence of 1 at an initial cell density of 1 × 10 ⁶ cells / ml for 24 hours. Then, IL-2 activity was measured according to the method described above.

As a result, a human T cell line designated as Jurkat-111 (ATCC CRL 8129) (hereinafter referred to as "J-111") was cloned and selected from its parent strain, Jurkat, and the IL-2 production ability of this cell was selected. Was 40 times higher than the parent strain.

This cloned J-111 cell line grew under normal conditions and its growth rate was almost the same as that of normal Jurkat cells.

(2) J-111 cells (1 × 10 ⁵ cells / ml) were mixed with serum-free synthetic medium RIT C 55-9 (Sato, T. et al., Exp. Cell Res., 138, 127).
-134, (1982)) was inoculated into 1000 ml and cultured in a roller culture bottle (Falcon 3027) at 37 ° C. for 4 days, and the grown cells were collected by centrifugation. 4x this cell again
The above medium containing 25 μg / ml of ConA was inoculated so that the concentration was 10 ⁶ cells / ml. 100 ml of the culture solution inoculated with the cells was placed in each of 4 batches of roller culture bottles (Falcon), and the cells were subjected to rotary culture for 6 hours.

(3) The Jurkat cells (1.2 × 10 ⁶ ) thus stimulated with ConA 25 μg / ml for 6 hours were suspended in 8,000 ml of physiological saline-phosphate buffer (hereinafter abbreviated as PBS). The cells were washed twice by centrifugation and contained the nuclease inhibitor ribonucleoside-vanadale complex (10 mM).
RSB solution (10 ml Tris-HCl buffer, pH 7.5, 10 mM N
NaCl, and resuspended in 1.5mM MgCl ₂₎ 800ml. Thereafter, the surfactant NP-40 was added so that the final concentration was 0.05%, the mixture was slowly mixed, and the cell nuclei were separated by centrifugation at 3,000 rpm for 5 minutes at 4 ° C. SDS (0.5%) and EDTA (5 mM) were added to the supernatant, and cytoplasmic RNA was extracted by adding the same amount of phenol as the supernatant. After extraction with phenol three times, RN
A was precipitated with 2 volumes of ethanol, the precipitate was collected by centrifugation and dissolved in 10 mM Tris-hydrochloric acid at pH 7.5. The amount of RNA obtained was 196 mg.

The fraction of mRNA was oligo (dT) -cellulose (PL Bioche
micals, Type 7) affinity chromatography. Adsorption liquid is 20 mM Tris-HCl, 0.5MN
It is a solution of pH 7.5 containing aCl, 1 mM EDTA and 0.5% SDS, and elution was carried out with a buffer solution (20 mM Tris-HCl, pH 7.
After washing with 5, 0.5 M NaCl, 1 mM EDTA), water and 10 mM Tris-HCl (pH 7.5) were used alternately. The amount of mRNA obtained by elution was 3.6 mg. Next, 2.4 mg of the obtained mRNA was subjected to sucrose density gradient centrifugation (50 mM Tris-HCl, 1 mM EDT).
A, sucrose density gradient in a solution containing 0.2 M NaCl at pH 7.5
Fractionation was performed at 25% and 26,000 rpm at 4 ° C. for 24 hours). Fractions No. 12, 13, 14 from 11 to 12S of mRNA
Were fractionated into 59 μg, 46 μg and 60 μg, respectively.

(4) The mRNA obtained in the fraction of NO.13 was used as Xenopus laevis
(Xenopus Iaevis) injected into oocytes (50 ng mRNA
/ Oocyte). IL-2 activity of this oocyte culture medium was measured. As shown in Table 1, ³ H- thymidine rise and increase in activated T lymphocyte counts uptake of ⁽³ H-TdR) was confirmed, clearly mRNA in this fraction contains human IL-2 mRNA The thing was proved.

(5) Thereafter, NO.13 of 11-12S mRNA containing IL-2 mRNA
CDNA was synthesized from the fractions in vitro.

Recombinant DNA was constructed with plasmid vector pBR322.
Recombinant DNA was transformed into Escherichia coli, and IL-
The clones that obtained 2 cDNA clones were selected by the following method.

(5-1) 50 mM Tris-HCl buffer (pH 7.5), 30 m
M NaCl, 6 mM MgCl ₂ , 5 mM dithiothreitol (hereinafter DT
Abbreviated as T), 0.5 mM dATP, dGTP, dCTP, dTTP (dCTP)
³² P includes radiolabeled), 0.7 μg oligo
(dT) ₁₀ , ₁₀ μg mRNA and 15 units AMV reverse transcriptase (J.
W. Beard) was mixed and kept at 41 ° C. for 90 minutes. After completion of the reaction, the DNA was treated with phenol and recovered as an ethanol precipitate, and the DNA was added to a pH containing 20 mM Tris and 1 mM EDTA.
Dissolved in solution 7.5.

2.5 μg of ss-cDNA was synthesized. To remove mRNA from this reaction solution, add NaOH solution to the reaction solution to make 0.33N NaOH solution, leave it at room temperature for 15 hours, and then add 1M Tris-HCl buffer of pH 7.5 to the same volume to neutralize. Sephadex G
The -50 column was passed through the column. The recovered cDNA is 1.8
It was μg.

(5-2) 50 mM phosphate buffer (pH 7.5), 10 mM Mg
C ₂ , 10 mM DTT, 0.75 mM of each dATP, dGTP, dCTP, dTTP
(DCTP includes those labeled with ³ H), 1.8 μg ss-cD
Mix NA and 8 units Polymerase I (BRL, USA) 1
The reaction was carried out at 15 ° C for 5 hours. After completion of the reaction, DNA phenol and chloroform treatments were performed, and the precipitates were collected as ethanol precipitates. 1.10 μg of ds-cDNA was produced. 50 mM sodium acetate (pH 4.5), 0.2 M NaCl, 1 mM ZnCl ₂ and 1.10
The mixture of μg Japanese chain cDNA was incubated at 37 ° C. for 30 minutes, then 0.25 unit of nuclease S ₁ (Mitsui, Japan) was added, and the mixture was further incubated for 15 minutes. After completion of the reaction, the reaction product obtained by performing phenol treatment twice was subjected to Sephadex G-50 to obtain 0.55 μg of double-stranded cDNA.

(5-3) 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 / n mol), 0.55 μg ds-cDNA and 5 units Terminal transferase (BRL) of Example 1 was mixed, incubated at 37 ° C. for 7 minutes, treated with phenol, and then subjected to a Sephadex G-50 column to obtain 0.50 μg of DNA as an ethanol precipitate. The recovered DNA was found to have about 50 dCMP residues added to both 3'ends.

ds-cD described above except that 10 μg of pBR322 DNA was cleaved with the restriction enzyme Pst I and then dGTP was used instead of dCTP.
The dGMP chain was added to both 3'ends of the cleaved DNA under exactly the same conditions as the method used when adding the dCMP chain to NA.

(5-4) 50 mM Tris-hydrochloric acid (pH 7.5) 0.1 M NaCl,
5 mM DETA, 0.05 μg dGMP residue added pBR 322 and 0.0
1 μg dCMP residue added cDNA was first incubated at 65 ° C. for 2 hours, then 46 ° C. for 120 minutes, further 37 ° C. for 60 minutes, and room temperature for 60 minutes.

Escherichia coli χ1776 (Curtiss III, R. et al., In M
olecular Cloning of Recombinant DNA, (WAScott &
R. Werner ed.) Academic Press, (1977)) with 50 ml of L medium (100 μg / ml of diaminopimelic acid, 50 μg / ml)
Thymidine, 1% tryptophan, 0.5% yeast extract,
0.5% NaCl and 0.1% contained glucose), and the culture was shake-cultured at 37 ° C. until the absorbance of the culture solution was around 0.3 at 562 nm. After the completion of the culture, the culture solution was kept at 0 ° C. for 30 minutes, the cells were collected by centrifugation, and 5 mM Tris-hydrochloric acid (pH 7.6), 0.1 M NaCl, 5 mM MgCl ₂ and 10 mM RbC was added.
It was washed twice with 25 ml of a solution containing 1 l.

The obtained cells were treated with 5 mM Tris-hydrochloric acid (pH 7.6) 0.25 M KC.
l, 5, mM MgCl ₂ , 0.1 M CaCl ₂ and 10 mM RbCl were suspended in 20 ml of the solution and allowed to stand at 0 ° C. for 25 minutes, and then the bacterial cells were collected and resuspended in 1 ml of the same solution as above to obtain The recombinant DNA was added to 0.2 ml of the obtained bacterial cell suspension, and the mixture was incubated at 0 ° C for 60 minutes.
Let stand for a minute. After that, add 0.7 ml of L medium to 30 at 37 ℃.
It was shake-cultured for a minute. The culture solution thus obtained (0.1 m
l) 100 μg / ml diaminopimelic acid, 50 μg / m
l L-medium containing thymidine and 15 µg / ml tetracycline was smeared on one side of 1.5% agar medium and incubated at 37 ° C for 2
Incubated for days.

(5-5) The emerged 432 colonies were divided into 18 groups, and 100 μg / ml of diaminopimelic acid (the name group includes 24 different bacterial clones),
200 ml of L medium containing 50 µg / ml of thymidine and 10 µg of tetracycline was inoculated and shake-cultured at 37 ° C for 5 to 7 hours. Then fresh L containing chloramphenicol added to a final concentration of 170 μg / ml.
200 ml of medium was added and the cells were further cultured overnight.

The plasmid DNA thus enhanced was purified by a conventional method.

Clones having IL-2 cDNA were selected by mRNA hybridization-translation assay (hereinafter abbreviated as HT assay).

The HT assay used here was performed as follows.

The purified DNA (25 μg) was cleaved with the restriction enzyme HindIII, treated with phenol three times, phenol-chloroform and chloroform respectively, and precipitated with ethanol.
It was washed with% ethanol and dissolved in 40 ml of 80% formamide.

After denaturing the reaction solution at 90 ° C for 5 minutes, 10 × SS
Diluted to 1.3 ml with C (1.5 M NaCl, 0.15 M sodium citrate). Then, this DNA was fixed on nitrocellulose filter paper, dried at 80 ° C. for 3 hours, and then 50% formamide, 20 mM Pipes (pH 6.5), 0.75 M NaCl, 5 mM ED.
Poly (A) mRNA2 derived from TA, 0.2% SDS and J-111 cells
Incubation was carried out at 37 ° C. for 18 hours in a solution containing 50 μg to hybridize the DNA immobilized on the filter paper with IL-2 mRNA.

Then filter the filter paper 3 times at 65 ° C. with 10 mM Pipes, pH 6.5.
It was washed with 5M NaCl solution, 1 mM Pipes, 10 mM NaCl solution, treated with 0.5 mM EDTA, 0.1% SDS solution at 95 ° C. for 1 minute, and hybridized mRNA was recovered from the filter paper.

The mRNA thus extracted was subjected to oligo dT-
After purification on a lulose column and injection into Xenopus oocytes, the IL-2 activity of the translated protein was measured.

48 in assay 1 group of each 18 group of 24 clones by ^{3 H-TdR} incorporation of the aforementioned
Unit / ml of IL-2 activity was positive. On the other hand, the other goops were clearly negative.

Then, each of the 24 single colonies belonging to the positive group was inoculated into 200 ml of L medium having the same composition as described above, and incubated at 37 ° C for 5
The culture was aerobically carried out for about 7 hours, and L medium containing chloramphenicol was also added.

Incubate overnight to enhance plasmid DNA, then plasmid DNA
Was similarly purified according to standard methods. After cleaving about 5 μg of each plasmid DNA with Hind III, each plasmid was similarly immobilized on nitrocellulose filter paper. The filter paper was hybridized with IL-2 mRNA, and the hybridized mRNA was injected into Xenopus oocytes, and the translated protein IL-2 was injected.
Recovered for activity determination.

As shown in Table 2, only plasmid DNA purified from a single colony designated p3-16 showed positive IL-2 activity. Therefore, this clone is a clone having IL-2 cDNA.
(E. coli x 1776 / p3-16AJ11995 (FERM-BP-225)). Thus, the plasmid DNA, p3-16, is IL-2mRN
DNA capable of forming a specific hybrid with A (IL-
It was confirmed that they have 2 genes).

The cDNA insert of plasmid p3-16 contains the restriction enzyme Xba I
Was characterized in that it was cleaved at 1 site by Bst NI and at 2 sites (upstream and downstream of the Xba I cleavage site) by Bst NI. However, plasmid p3-16 contains a cDNA insert composed of approximately 650 base pairs, which clearly corresponds to a portion of the IL-12 mRNA of size 11-12S. Therefore, other cDNA libraries
Using IL-2 mRNA as a template, the method of Land et al.
l., Nucleic Acids Res., vol 9, p2551, (1981)). Single-stranded cDNA (1.6 μg) was synthesized using 4 μg of IL-2 mRNA with dCMP residue added, and ds-cDNA
Was synthesized with DNA polymerase I (Klenow fragment) by using oligo (dG) 12 = 18 as a primer. A cDNA (0.6 μg) longer than a 680 base pair DNA size marker was obtained by sucrose density gradient centrifugation and the P of pBR322 by standard GC tailing.
We were able to insert it into the st I site.

After transformation of Escherichia coli x 1776 with the recombinant DNA, nick-translated as a probe in its place
Grunks using (nick-translated) p3-16 cDNA insert
About 2000 by tein-Hogness hybridization method
Colonies were selected and colonies containing the plasmid pIL 2-50A containing 850 base pairs and transformed clones (Escherichia coli x 1776 / pIL2-50A, AJ11).
996 (FERM-BP-226)) was identified. A restriction enzyme digestion diagram of the pIL2-50A cDNA insert is shown in FIG.

Transformed Escherichia coli x 1776 / pIL2-50
To isolate the gene encoding the IL-2 peptide from A, plasmid DNA was isolated from the bacterial cells by the usual method.
After the NA was isolated, it was cut with the restriction enzyme PstI. The smaller of the two DNA fragments generated by this treatment is I
It was a DNA gene encoding the L-2 peptide.
The complete nucleotide sequence of the Pst I insert from pIL2-50A is the method of Maxam and Gilbert Maxam, AW et al., En
zym.65, 499-560, 1980). The whole structure is second
It is shown in Figure (a).

Example 2 Constitutive IL-2 producing cell line JA 1886 (A cloned from Jurkat cells according to the method described in Example 1).
TCC CRL 8130) was similarly grown in roller culture bottles. The grown cells were resuspended in fresh synthetic medium RITC-55-9 at an initial cell density of 1 × 10 ⁶ cells / ml, and 8 hours after the start of the culture, 3 × 10 6 according to the steps detailed in Example 1.
It was used for the extraction of IL-2 mRNA as a 11-12S fraction from ⁹ cells.

The ds-cDNA was synthesized in the same manner as in Example 1, and cDNA (2.4 μg) longer than 600 base pairs was obtained by fractionation by sucrose density gradient centrifugation. Next, this cDNA was extended with dCMP residues using terminal deoxynucleotidyl transferase, and 50 ng of which was extended with dGMP, Pst I cleaved pBR 3
Annealed at 22 250 ng.

The resulting hybrid plasmid was transformed into Escherichia coli x 1776, and a transformant of about 4,000 clones was obtained.

According to the method of Grunstein-Hogness, three clones complementary to the plasmid 3-16 cDNA used as a probe were selected. That is, the transformed clone thus selected is a clone having the human IL-2 gene.

Example 3 A plasmid directing the synthesis of human IL-2 in Escherichia coli cells was constructed by the following method.

The plasmid pT IL2-22 is shown in Figure 4 (a) as pTrS-3 (Nishi
T., Taniguchi T. et al., SEIKAGAKU 53,967, (1981), ibid.
4,676 (1982)) and pIL2-50A containing the IL-2 cDNA. Plasmid pTrS-3 is the Trp promoter and Ec of pBR322.
Includes a region of Shine Dalgarno (hereinafter abbreviated as SD) between the oRI and ClaI sites.

This plasmid also contains a single Sph, as shown in FIG.
Similar to the I site, it contains the ATG initiation codon 13 bp downstream of the SD sequence.

When the DNA sequence corresponding to the mentioned protein is inserted just downstream of the ATG codon, the vector is very effective in producing this protein. This ATG cordon is p
Produced by Sph I digestion of TrS-3 followed by treatment with T4 DNA polymerase. Therefore the plasmid pTrS-3 (30
μg) was digested with a restriction enzyme Sph I by a conventional method, subsequently recovered by phenol / chloroform treatment and ethanol precipitation, and both ends were flushed by T4 DNA polymerase treatment.

Next, DNA (21.4 μg) was recovered by the same method as the phenol / chloroform treatment and the ethanol precipitation method. On the other hand, 380 μg of pIL2-50A containing IL-2 cDNA was Pst I
And the IL-2 cDNA insert isolated by agarose gel electrophoresis.
g) was cleaved with HgiAI, treated with T4 DNA polymerase and 10 μg of the larger part of the DNA was isolated by agarose gel electrophoresis. 13 according to this law
A cDNA (7.2 μg) encoding 2 amino acids was obtained, and this DNA fragment had a blunt end (4th
(a) Figure).

The cDNA fragment thus obtained was then ligated just downstream of the ATG sequence into the pTrS-3 vector which had been treated with T4 DNA polymerase previously digested with SpH I. The thus ligated plasmid was then transformed into Escherichia coli HB 101 according to a conventional method. This ligation was performed as follows. IL-2 cDNA (0.4 μg)
0.2 μ of the above-mentioned larger fragment and pTrS-3 vector DNA
g of 6.6 mM MgCl ₂ , 1 mM ATP and 10 mM DTT at pH 7.
5 of 66 mM Tris-HCl was mixed with 0.8 unit of T4 DNA ligase and the mixture was reacted at 4 ° C overnight. Among the transformants appearing on the L medium agar plate containing ampicillin, the IL encoding 132 amino acids
Colonies harboring the plasmid containing the -2 cDNA portion were selected in situ by a colony hybridization assay. Re-cultivate the selected colonies (10m
Then, a plasmid DNA was prepared by lysozyme treatment and freeze / thaw treatment. Ps this plasmid DNA
Cleavage with t I and Xba I and analysis of the resulting product by agarose gel electrophoresis identified pTIL-2-22 whose cDNA was frozen in the correct orientation after the ATG sequence of pTrS-3.

Escherichia coli HB 101 containing pTIL-2-22 was cultured under conventional methods known for the growth of microorganisms. Cells are 25 μg / ml streptomycin and 25 μg / ml
X-medium containing 2.5% ampicillin (2.5% bactotryptone, 1% yeast extract, 0.1% glucose, 20 mM MgS
It was grown overnight at 37 ° C. in 10 ml of O ₄ , 50 mM Tris-HCl, pH 7.5). Then, 1 ml of the culture suspension was inoculated into the same x medium (100 ml) and cultured at 37 ° C. 650mμ
3-Indole acrylic acid (IAA) was added when the OD of about 1.5-2.0 was reached. Three hours after the addition of the inducer, the cells were collected, and 20 mM Tris-HCl (pH 7.5, 3
Washed with 0 mM NaCl) and resuspended in 8 ml of the same buffer.

For effective functional expression of the Trp promoter, an inducer such as IAA was added to a final concentration of 50 μg / ml. The protein thus produced in bacterial cells is treated with sonic (0 ° C, 2 minutes) or lysozyme (8 μg)
Extraction was carried out by digestion (0 ° C., 20 minutes) followed by freeze-thawing three times. By this method, IL-2 was generally extracted from cells. IL-2 activity extracted from 10,000
The range was 120,000 units / ml.

Escherichia coli HB 101 (AJ12009) containing pTIL2-22 is F
Deposited as ERM-BP245.

Example 4 The plasmid pTuIL2-22 containing IL-2 cDNA was pTuBlP-5 (Ta
niguchi, T. et al., Seikagaku, 53, 966, 1981) and pTIL2-22 shown in Example 3 and constructed by the method illustrated in FIG. The plasmid pTuBlP-5 has the tufB promoter sequence inserted into pBR322. This plasmid also contains a single Cla I site, which is located 2 bp downstream of the SD sequence as shown in FIG. pTrS-3 also contains Cla I between the SD sequence and the ATG initiation codon.
It is very easy to replace the Trp promoter with the tufB promoter since it contains a site and this Cla I site is not destroyed during the construction of the expression plasmid by using pTrS-3 and IL-2 cDNA as described in Example 3. , So that the IL-2 cDNA is expressed under the control of the tufB promoter.

Therefore, the plasmid pTIL2-22 (30 μg) was digested with the restriction enzyme Cl.
Cleavage with a I and Pvu II in the usual manner. IL-2
The cDNA-containing fragment (about 2.2 kb) was isolated and purified by agarose gel electrophoresis, and 3 μg of DNA was recovered. On the other hand, 20 μg of pTuBlP-5 vector was also treated with Cla I and Pvu.
The larger fragment (about 3.4 kb) containing the ampicillin resistance gene, which had been cleaved with II, was isolated and purified by agarose gel electrophoresis to recover 3.5 μg of DNA. The two fragments thus obtained were then ligated as shown below, one containing the tufB promoter (about 3.4 kb) and the other containing the IL-2 cDNA (about 2.2 kb).

0.3 μg of the fragment containing IL-2 cDNA (1.2 μg) and the fragment containing the tufB promoter were added to T4 in 66 mM Tris-HCl at pH 7.5 containing 6.6 mM MgCl ₂ , 1 mM ATP and 10 mM DTT.
It was mixed with 0.8 unit of DNA ligase and reacted overnight at 4 ° C. Next, the plasmid thus ligated was transformed into Escherichia coli HB 101 according to a conventional method.

Among the transformants appearing on the L medium agar plate containing ampicillin, IL-like pTuIL2-22 in FIG.
Eight colonies with recombinant DNA containing the 2 cDNA portion were selected and plasmid DNA was prepared as described in Example 3.

Escherichia coli HB 101 containing pTuIL2-22 was cultured at 37 ° C. in L medium (100 ml). When the OD of 650 mμ reached about 0.5-1.0, the cells were collected and 30 mM NaCl was added.
The cells were washed with 20 mM Tris-hydrochloric acid (pH 7.5) containing 10 mM and suspended again in 2 ml of the same buffer. The protein thus produced was extracted in the same manner as in Example 3. IL-2 activity in the extract ranged from 6,000 to 56,000 units / ml.

Escherichia coli HB 101 (AJ12010) containing pTuIL2-22
Deposited as FERM-BP 246.

Example 5 Plasmid pGIL2-22 containing IL-2 cDNA is pGL101 (Robe
rts, TMand Laucer GD, Meth Enzym., 68,473-483, (19
79), Gail Lauer, et al, J. Mol. Appl. Genet., 1, NO.2,13
9-147 (1981), T. Taniguchi, et al, Proc.Natl.Acad.Sci.
USA, 77, NO.9,5230〜5233 (1980), Egon Amann, et al, Gen
e, 25,167-178 (1983)) and pTIL2-22 shown in Example 3.

That is, the plasmid pGL 101 containing the lac promoter.
(20 μg) was cleaved with the restriction enzyme Pvu II by a conventional method,
Subsequently, 17 μg of DNA was recovered by phenol / chloroform treatment and ethanol precipitation. On the other hand, pTIL2
-22 (75 μg) was digested with Cla I and Sal I, and the DN contained in IL-2 cDNA by agarose gel electrophoresis
2.2 μg of A fragment was recovered. This fragment was flushed by treating with DNA polymerase I (Klenow fragment). Next, the two fragments (0.25
μg and 0.66 μg) in the same manner as in Example 4.
Ligase was linked with 1.0 unit. Thus, this ligated plasmid was transformed into Escherichia coli HB 101 according to a conventional method.
Was transformed into. IL-2 in the transformant
A transformant ³² P-labeled IL-2 cDNA having an insertion of Cla I-Sal I fragment containing cDNA was selected as a probe. Then, these transformants were lysed in 10 ml of x medium containing 25 μg / ml of ampicillin, and plasmid DNA was prepared by the method described in Example 3. Thus, IL-2c is located just downstream of the lac promoter.
Plasmid DNA containing ATG as the starting sequence of DNA was obtained by assaying the cleavage sites at Pat I and Xba I. Escherichia coli HB 101 containing pGIL2-22 thus obtained was 25 μg / ml ampicillin and 25
100 ml of L medium containing μg / ml streptomycin
Were inoculated and cultured. Isopropyl-β-D-thiogalactopyranoside (I
PTG) was added at a concentration of 1 mM, and the cells were collected 1 hour later, and a cell extract was prepared according to the method described in Example 4. The IL-2 activity of the extracts ranged from 6,000 to 80,000 units / ml.

Escherichia coli HB 101 (AJ12011) containing pGIL2-22 is F
Deposited as ERM-BP 247.

Example 6 The plasmid pTrS-3 (10 μg) was first treated with the restriction enzyme Sal I.
Cleavage with and flush Sal I site with DNA polymerase (Klenow fragment) or T4 DNA polymerase treatment
set to (flush).

After cleaving with Cla I, the larger fragment having the Trp promoter region was isolated and purified by agarose gel electrophoresis according to a conventional method to recover 3 μg of DNA.

On the other hand, 11 μg of cDNA insert obtained by Pst I digestion of pIL2-50A was digested with HgiAl, treated with T4 DNA polymerase, and the larger fragment was isolated and purified by agarose gel electrophoresis. In this way, IL-2 13
7.2 μg of a cDNA fragment encoding 2 amino acids was obtained. Next, a fragment containing the trp promoter (above) 0.45μ
g, 0.5 μg of HgiAI-Pst I fragment containing IL-2 cDNA and synthetic oligonucleotide (5 ′) CGATAAGCTATGGCA (3 ′)
Both (3 ') TATTCGATACCGT (5') (20 pmole each) are phosphorylated at the 5'end.
Ligation was carried out with one unit of T4 DNA ligase in the same manner as described in (4 (b) of FIG. 4). The thus ligated plasmid was transformed into Escherichia coli HB 101. Among the transhomants that appeared, the target Tonlas homanto was selected as follows. First, transformants capable of hybridizing with both IL-2 cDNA and synthetic oligonucleotides were selected by the colony hybridization method. Then ATGG
A transformant having a plasmid DNA in which a DNA fragment (CCTACT .........) starting from the CTT sequence at positions 111 to 113 in Fig. 2 (a) was inserted just downstream of the CA sequence was designated as Pst I, Xba I. It was selected by examining the cleavage site.

The above transformants containing pTIL2-21a or pTIL2-21b were cultured in L medium by the method shown in Example 3 and analyzed by the method shown in Example 3. High IL-2 activity was observed.
Escherichia coli HB 101 (AJ 1201 with pTIL2-21a
3) and E. coli with pTIL2-21b (AJ 120
14) with Escherichia coli HB 101 are FERM-
Deposited as BP 248, FERM-BP 249.

The host used in the above example, Escherichia coli
x 1776 and HB 101 (Bpyer HW et al., J. Mol. Biol. 4
1,459, (1969)) is publicly known and is easily available.
In addition, recombinant DNA in transformants
The host can be easily obtained from the deposited transformant by culturing the transformant in L medium at 37 ° C. in order to liberate the body, and separating the cells that became sensitive to tetracycline and ampicillin.

Plasmid pector pBR322 (available from Bethesda Research Laboratory, for example), pCE-1, pTrS-3 and pGL
101 is publicly known and is easily available. Furthermore, by isolating the recombinant plasmid in the transformant by a conventional method and further isolating the plasmid vector as is apparent from the explanation in each example, the plasmid vector was isolated from the deposited transformant. You can get it. pTrS-3 and pTuBlP-5 have been deposited as Escherichia coli FERM-P6735 (BP 328) and Escherichia coli ATCC 31878, respectively.

[Brief description of drawings]

FIG. 1 shows a cleavage map of a cloned gene encoding a polypeptide having IL-2 activity by a restriction endonuclease, FIG. 2 (a) shows the nucleotide sequence of the cloned gene, and FIG. ) Indicates the amino acid sequences I, II and III of the polypeptide having IL-2 activity. FIG. 3 shows the plasmid vector pTrS-3. Fig. 4
(a), FIG. 4 (b) and FIG. 4 (c) show pTrS- as a vector.
Recombinant DNAs using 3 (pTIL2-22, pTIL2-21, pTIL
2-14 and pTIL2-15) is a flowchart showing the configuration. FIG. 5 is a flow chart showing the construction of recombinant DNA (pKIL2-21) using pKT218 as a vector. FIG. 6 is a flow chart showing the construction of recombinant DNA (pTuIL2-22) using pTUB1P-5 as a vector. In the figure, "A", "G", "C" and "T" represent deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid, respectively.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location (C12P 21/02 C12R 1:19) (72) Inventor Yu Matsui 1 Namiki, Kanazawa-ku, Yokohama Chome 19-16-101 (72) Inventor Shinichi Kashima 2-21-603 Wakabadai, Asahi-ku, Yokohama-shi, Kanagawa (72) Inventor Atsushi Hamuro 241-232, Fukaya-cho, Totsuka-ku, Yokohama-shi, Kanagawa (56) Reference Reference JP-A-59-140882 (JP, A)

Claims (2)

[Claims] 1. A gene encoding a polypeptide having the following amino acid sequence having human interleukin 2 activity. Met Pro Thr Ser Ser Ser Thr Lys Lys Thr GlN Leu Gl
N Leu Glu His Leu Leu Leu Asp Leu GlN Met Ile Leu
AsN Gly Ile AsN AsN Tyr Lys AsN Pro Lys Leu Thr Ar
g Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala
Thr Glu Leu Lys His Leu GlN Cys Leu Glu Glu Glu Le
u Lys Pro Leu Glu Glu Val Leu AsN Leu Ala GlN Ser
Lys AsN Phe His Leu Arg Pro Arg Asp Leu Ile Ser As
N Ile AsN Val Ile Val Leu Glu Leu Lys Gly Ser Glu
Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Th
r Ile Val Glu Phe Leu AsN Arg Trp Ile Thr Phe Cys
GlN Ser Ile Ile Ser Thr Leu Thr 2. A first claim having the following base sequence:
The gene according to the item.