JPH02195888A - Recombinant dna body containing gene coding polypeptide having human interleukin-2 and procaryote cell transformed by the same recombinant dna body - Google Patents

Abstract

PURPOSE:To obtain a gene useful for mass production of human interleukin-2 coding polypeptide having human interleukin-2 activity, containing special amino acid sequence. CONSTITUTION:A recombinant DNA is prepared by using gene coding polypeptide having human interleukin-2 activity, containing amino acid sequence expressed by the formula and a procaryote, preferably Escherichia coli is transformed by said recombinant DNA, then cultured to produce human interleukin-2.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant DNA body containing a gene encoding a polypeptide having human interleukin-2 activity,
and prokaryotic cells transformed with the recombinant DNA.

Interleukin 2 (hereinafter abbreviated as rlL-2J).

), formerly called T cell growth factors, are soluble proteins (commonly known as "lymphokines") produced by T cells activated by lectins or antigens (Morgan et al.

5science, 193.1(107-1(108(
1976), G11lis et al., J.

Jmmunol,, 120.2027-2033 (
197B)), IL-2 can modulate lymphocyte reactivity and inhibit antigen-specific effector knee lymphocytes in vitro.
long-term culture in vitro (Gillis et al., 11ature).

8+ 154-156 (1977)). Also IL-2
promotes thymocyte division (Chen et al., Ce1l
Immunol, +22+ 221-224 (1977
), 5ha et al., J. Tmmunol, 12
0.1967-1973 (1978)), cytotoxic T lymphocyte activity in nude mouse lung cell culture system (Wa
gner et al., Nature+284°278-280.
(1980)) and induction of anti-3RBC plaque-forming cellular responses (Gillis et al., J. Exp, Me.
It has been revealed that it has other related biological activities such as d, litan, 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 is involved in malignant tumors, bacterial or viral infections, immunodeficiency, autoimmune diseases, etc. (Papermaster et al., Adv, I
mmunopharm.

507, (1,980))). Like interferon, TL-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 receptor. It shows that they play an important role. IL-2 can also be used to produce a variety of T cell-derived lymphokines useful in a variety of other fields simply by culturing clonal T cells for long periods of time. 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 disorders.

IL-2 has traditionally been produced by stimulating mouse, rat or human lymphocytes with mitogens (Gillis et al. + Nature, 26
8+154~156. (1977)), Farra
r et al., J. Immunol, +12L1353~1
360. (1978), G11lis et al., J.
1Huno1. , monthly title.

2027-2033. (1978)). By stimulating human peripheral blood lymphocytes with mitogens (Gil
lis et al., J.

Immunol, 124.1954-1962.
(1980)), G11lis et al. production of murine IL-2 from a T lymphoma cell line (Gitlis et al., J
, Immunol, , 125.2570-2578
(1980)) and human IL-2 from human leukemia cell lines.
(Gillis et al., J. Exp. Med,
, 152.1709-1719. (1980))
is reported.

The technique described above by Gllis et al. relates to a method for producing human 1L-2 from a mitogen-activated T leukemia cell line using cell culture methods. However, this method has the disadvantage that only a low concentration of human IL-2 is produced, and a complicated purification process is required to obtain a trace amount of IL-2 from a large amount of culture fluid. Furthermore, H)T
Because 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 the cytotoxins that sometimes coexist. (toxic lectin) is quite difficult to separate.

Another method for producing IL-2 is recombinant DNA (abbreviation for deoxyliponucleic acid), which has been used to produce other bioactive human-derived proteins such as interferon.
(Gray et al., Nature 295.503-5
08 (1981), Nagata et al., Nature
284.316-320 (1980).

Taniguchi et al., cene dish, 11-15.
(1980)) is considered preferable. However, prior to the present invention, IL-2
For example, attempts to create an organism that produces IL-2 by recombinant DNA have not been successful. “Nikkei Biotechnology,
No. 19, July 5, 1982.

Therefore, a cloned gene encoding IL-2 and a recombinant DNA containing the gene have been desired. Furthermore, there has been a desire for a living cell line containing recombinant DNA and a method for producing IL-2 using the cell line.

The gist of the invention will become more readily apparent from the following description.

The object of the present invention is to create a recombinant DNA body containing a gene encoding a polypeptide having IL-2 activity, and to transform prokaryotes to produce IL-2 with the recombinant DNA body. The reason lies in the creation of cells.

That is, the present invention provides a recombinant DNN rod containing a gene encoding a polypeptide having human interleukin-2 activity for transforming prokaryotes containing a gene encoding a polypeptide having human interleukin-2 activity. Recombinant DNA for transforming Escherichia coli, recombinant DNA containing a gene encoding a polypeptide having human interleukin-2 activity
The present invention provides prokaryotic cells transformed by a prokaryotic cell and Escherichia coli transformed by a recombinant DNA body containing a gene encoding a polypeptide having human interleukin-2 activity.

The polypeptide having human interleukin-2 activity may be any polypeptide having human interleukin-2 activity.

In the above, a specific example of a polypeptide having human interleukin-2 activity includes polypeptide (1) containing the following amino acid sequence in its molecule.

Pro Thr Ser Ser Ser Thr L
ys Lys Thr GIN LeuGIN Leu
Glu 1lis Leu Leu Leu Asp
Leu GIN Metlie Leu AsNG
ly Tie AsN AsN Tyr Lys As
N Pr.

Lys Leu Thr Arg Met
Leu Thr Phe l,ys Phe
TyrMet Pro Lys Lys Ala T
hr GluLeuLysHisLeuGIN
Cys L,eu Glu Glu Glu Leu
Lys Pro Leu GluGlu Val,
Leu AsN Leu Ala GIN
Ser Lys AsN Phe Olds Leu
Arg Pro 8 arg Asp Leu
Ile Ser ASN l1eAsN Va
l Ile Val Leu Glu Leu Lys
Gly Ser GluThr Thr Phe M
et Cys Glu Tyr Ala Asp
Glu Thr Ala Thr Ile Va
l Glu Phe Leu AsN Ar
g Trp TieThr Phe Cys GI
N Ser Ile Monthe Ser Thr Leu T
Specific examples of the polypeptide (1) include:
Polypeptide (■) having the following amino acid sequence; 8Ia Pro Thr Ser Ser
Ser Thr Lys Lys Thr
GIN Leu GIN Leu Glu His Le
u Leu Leu Asp Leu GINMet
Ile Leu AsN Gly Ile
AsN AsN Tyr Lys AsNP
ro Lys Leu Thr Arg Met Le
u Thr Phe Lys PheTyr Met
Pro Lys Lys Ala Thr
Glu Leu Lys Old 5Leu G
IN Cys [+eu Glu Glu
GluLeuLysProLeuGl
u Glu Val Leu AsN Le
u Ala GIN Ser Lys As
NPhe tlis Leu Arg Pro
Arg Asp Leu lie Ser
AsN11e AsN Vat Ile Val
Leu Glu Leu Lys Gly 5erGl
u Thr Thr Phe Met Cy
s Glu Tyr Aha Asp Gl
uThr Ala Thr Ile Val
Glu Phe Leu AsN Arg
Trplie Thr Phe Cys GIN S
er Ile Ile Ser Thr Leuhr A polypeptide having the following amino acid sequence (I[[);
Met Ala Pro Thr Ser
Ser Ser ThrGIN Leu GI
N Leu Glu His Leu Le
uGIN Met Ile Leu AsN
Gly Ile AsN8s Pro L
ys Leu Thr Arg Met L
euPhe Tyr Met Pro Lys
Lys Ala Thrtlis Leu
GIN Cys Leu Glu Glu Glu
Leu Glu Glu Vat Leu
AsN Leu AlaAsN Phe 1l
is Leu Arg Pro Arg A
spAsN lie AsN Val lie
Val Leu GluSer Giu T
hr Thr Phe Met Cys GluGl
u Thr Ala Thr lie Va
l Glu PheTrp lie Thr
Phe Cys (+IN Ser l1e
Leu Thr Poly(■) having the following amino acid sequence; Pro Thr Ser Ser Ser Thr L
ys LysGIN Leu Glu His
Leu Leu Leu Asplie L
eu AsN Gly Ile AsN A
sN TyrLys Leu Thr Arg Me
t Leu Thr PheMet Pro Lys
Lys Ala Thr Glu Leuys Leu ASN Thr lut Leu GIN Leu Leu Tyr Leu Ile Lys Ala Thr Asp Leu Tyr Lys Phe Lys Leu Lys Lys Pr.

Ser　　I,ys 11e 5er Lys Gly Ala　Asp AsN　Arg Ser　Thr peptide Thr　GIN Leu GIN Lys　AsN Lys　Phe Lys His Leu Met Pr.

Tyr eu GIN Cys Leu Glu Glu Glu
Leu Lys Pro Leu GluGlu V
al Lea AsN Leu Ala GIN
Ser Lys AsN PheHis Leu
Arg Pro Arg Asp Leu lie
Ser AsN 1ieASN Val lie V
al Leu Glu Leu Lys Gly
Ser GluThr Thr Phe Met Cy
s Glu Tyr Ala Asp Glu Thr
Aha Thr Ice Val Glu
Phe Leu AsN Arg Trp l
1eThr Phe Cys GIN Ser Ile
Examples include polypeptide (V) having the following amino acid sequence.

Met Pro Thr Ser Ser Ser T
hr Lys Lys Thr GINLeu GI
N Leu Glu Old s Leu Leu
Leu Asp Leu GINMet I
Le Leu AsN Gly Ile AsN As
N Tyr Lys AsNPro Lys Leu
Thr Arg Met Leu Thr Phe L
ys PheTyr Met Pro Lys I,y
s Ala Thr Glu Leu Lys His
Leu GIN Cys Leu Glu Glu G
lu Leu Lys Pro LeuGlu Glu
Val Leu AsN Leu Ala GIN
Ser Lys AsNPhe ) Its Leu A
rg Pro Arg Asp Leu Tie Se
r AsN11e AsN Vat Ile Vat
Leu Glu Leu Lys Gly 5erGl
u Thr Thr Phe Met Cys Glu
Tyr^la Asp GluThr Ala Th
r lie Val Glu Phe Leu AsN
Arg Trplle Thr Phe Cys
GIN Ser lie lie Ser Th
r Leuhr Specific examples of recombinant DNA bodies containing a gene encoding a polypeptide having human interleukin 2 activity include, for example, a recombinant DNA body having the following base sequence encoding the polypeptide (III): AAT ATCAACGTA ATA GTT
CTG GAA CTA AAG GGAC
TAACT.

Alternatively, a recombinant DNA body having the following nucleotide sequence encoding the above-mentioned polypeptide (V) can be mentioned.

8CT The polypeptide of the present invention includes a polypeptide lacking one or more amino acids in the above amino acid sequence; one or more amino acids in the above amino acid sequence is replaced with one or more amino acids. polypeptide; it may be a polypeptide in which one or more amino acids are added to the above amino acid sequence.

The prokaryotic cells in the present invention preferably belong to the genus Escherichia, and more preferably those belonging to the genus Escherichia coli.

By using the recombinant DNA of the present invention, IL
-2 was modified by recombinant methods by insertion of a gene encoded to produce a polypeptide having IL-2 activity and insertion of vector DNA capable of replicating in cells, and the coding sequence of the gene was modified by the promoter. It is produced by suspending (aerobically) culturing in a medium the prokaryotic cells of the present invention or Escherichia coli that have been transformed to produce IL-2 using DNA located downstream of the sequence.

The cloned gene encoding the IL-2 polypeptide is derived from a mesenger RNA (a+RN
A: "RNA" is an abbreviation for ribonucleic acid, hereinafter "IL-2mRN"
A'') is obtained by reverse transcription of complementary DNA (cDNA).The resulting single 1l cDNA (referred to as
ss-cDNA) is double-stranded cDNA (ds-cDNA)
) can be converted to

a+RN^ used as a template for preparing cDNA
can be isolated from mammalian cells that produce IL-2 polypeptides. The isolated RNA was polyadenylated (GiLLis et al., Immunologic
al Rev., 63+167~209 (1982
)) Polyadenylated RNA can be fractionated into 11-12S fractions, for example, by sucrose density gradient centrifugation. IL-2 mRNA activity may appear in 13s mRNA, but in this case, 11-12 Sm
It is thought that it is an aggregate of RNA.

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 lung cells that can be isolated from mammals.

To impart IL-2 production ability to cells or to enhance IL-2 activity, treatments such as nylon column treatment, antiserum and complement treatment, density gradient fractionation, combined treatment with neuraminidase and galactose oxidase, trypsin treatment, etc. Pretreatment may be performed by conventionally known methods such as various enzyme treatments 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, which is more preferably T-
They are lymphocytes. T derived from leukemia and lymphoma cell lines
Transformed lymphocyte cell lines or cloned transformed cell lines, such as lymphocytes themselves or their derivatives pretreated or mutated in the above-described manner, may also contain mRNA.
preferred as a source of Apparently, cloned cell lines usually contain higher amounts of IL than the pre-cloned parent line.
-2 mRNA is included. The above-mentioned lymphocyte-derived cells and CEM.

T cell hybridomas obtained by fusing tumor cell lines such as Molt 4F, BW5147 are also preferred mammalian cells for use in the present invention. In this case, the lymphocyte-derived cells are 0) cells that spontaneously produce IL-2 and (2) mitogens are introduced into the culture medium in the presence or absence of 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 cell culture art. If mRNA is to be produced in cells that produce IL-2 only in the presence of mitogen, the cultured cells should be thoroughly washed with culture medium and then incubated with Rosewell Park Memorial Institute 1640 with or without serum (hereinafter referred to as " RP old 1640
' is abbreviated as '. ), Dulbera Cola modified Eagle medium (hereinafter “
DMEM") or click medium. These cell culture media include penicillin, streptomycin or other antibiotics, L-glutamine.

Various additives such as Hepes buffer or sodium bicarbonate may be added at concentrations commonly used in the field of cell culture. The preferred cell concentration is 0.5-4X10" cells/d. Appropriate stimulants are added to induce mRNA activation and IL-2 production. Among these suitable stimulants are mitogens, , neuraminidase, galactose oxidase, zinc compounds such as zinc chloride or bacterially derived lymphocyte activating factors such as protein A, streptolysin-0.The stimulated cells are collected and washed. , macrophages or puntolytic cells may also activate mRNA or increase the yield of activated mRNA. Also Raji, Daudi.

K562. Even if cell lines derived from 8923 cells or B lymphocyte cell lines such as BALL-1 coexist, mRNA remains
can be activated or 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 subculturing in vitro to prepare a source of mRNA, these cells will grow in any medium conventionally known to promote T cell growth. .

These media may be supplemented with mammalian serum 5 serum components or serum albumin. The incubation time for mRNA activation corresponds to the time required for activation to produce mRNA.

This time corresponds to the time required until production of IL-2 into the medium begins. The preferred time is
This is 3 to 12 hours after adding a stimulant such as a mitogen. If the culture time is too long, the generated IL-2mR
NA may be degraded. When activating IL2-producing cells, the activation level can also be increased by adding 10 to 50 ng/ml of phorbol esters such as PMA or TPA.

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

A method for obtaining and culturing mammalian cells that produce IL-2 is described below.

(b) Obtaining a spontaneous IL-2 producing strain and Jurkat cells, which are leukemia cells derived from human T lymphocytes (Fred Hutchinson Cancer Institute/Seattle/USA, Sorta Institute/San Diego/USA, West German National Cancer Center/ Heidelberg/West Germany, etc.) were suspended in Crick medium at a cell density of 1 x 106 cells/ml and exposed at an irradiation rate of 150 Roentgen/min for a total of 8. (Execute X-ray irradiation of 100 Roentgen.

After this, the cells were added to a 96-well flat-bottom microtiter plate (Falcon 3072J) at a cell density of 0.1 cells/2 (10μ! medium) for 3 weeks in Click medium containing 5% tallow serum. Culture in a 5% COt incubator at °C (cloning by limiting dilution method).Cells in culture wells where cell growth was observed are
Before the cells reach a density covering the entire bottom surface, they are transferred to a 24-well Nunc culture plate and grown in Crick medium for 5 days. Furthermore, the present cells were incubated at 1-2×]
The cells were suspended in a serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 0'' cells/ail and cultured for 2 days, and the main culture supernatant was separated by centrifugation.
．． Debris removal and sterilization were performed using a 22μ Millipore filter. IL-2 of the culture supernatant thus obtained
X that spontaneously produces IL-2 by measuring its activity
Line-treated mutants were selected and cloned.

(11) Obtaining an L-2 producing strain from human peripheral blood mononuclear cells. Human peripheral blood was collected and peripheral blood lymphocytes (hereinafter abbreviated as "P") were collected using Ficoll-Heiberg density gradient centrifugation.
. Collect. This PBL was suspended in Click medium containing 5% FCS at a cell density of 1×106 cells/adult, and each
Inoculate a 4-well Nunc culture plate. Here, 5 μg/rt of phytohemagglutinin-M (manufactured by Gibco) was added.
1 (10 μ!) to give a final concentration of tfl, culture for 48 h under the conditions described above, then wash the cells with culture medium and again in 1 ml of click medium at a cell density of] × 10′ cells/rnfl. In addition, concanavalin A (hereinafter abbreviated as ConA) was inoculated at 2.571 g/m at 4
Human T lymphocytes from PBL are cultured long-term by adding 1 ml of conditioned medium prepared from human tile cells stimulated for 8 hours and replacing the medium containing 50% of the conditioned medium every 3 days. The T lymphocytes obtained by long-g subculture in this manner are cloned in the presence of human tile cells derived from a 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 transferred to a cell density of 1×10′ cells/ml at 10 μg/m.
fl phytohemagglutinin (hereinafter abbreviated as PHA)
RPMI in 24-well Nunc culture plates in the presence of
Inoculate 1 ml of 1640 medium and incubate for 24 hours at 37°C.
The cells were cultured in a 7.5% CO□ incubator. The main culture supernatant was separated by centrifugation, and then sterilized using a 0.22μ Millipore filter.
-2 activity assay was performed.

(c) Obtaining human lymphocyte-derived malignant cells that produce IL-2 upon mitogen stimulation Con in BPMI 1640 medium containing ~2%
A 10 u g/ml and PHA 2.5 u g
/ml when incubated for 24 hours, 10-4,
(Able to produce 100 units/lack of IL-2.

In addition, these human malignant cells contain zinc chloride and protein A.
.

Even when cultured in the presence of picibanil, IL-2 is produced.

(d) Obtaining cells that produce IL-2 by stimulating them with mitogens in the presence of other cells or the factors that produce them One clone of Jurkat cells, the Jurkat 99 strain, does not produce IL-2 even when cultured for 24 to 72 hours with the addition of the above-mentioned lectins and mitogens in a wide range of concentrations. However, during this period, 5 to 10 u of interleukin l, a type of monokine, was used.
IL-2
It produces 10 to 1 (10 u/mR) of lids that can be confirmed.

TL-2mRN from cells activated in this way.
A can be extracted by any conventional method regardless of the type of cell. For example, NP40, SDS, T
riton-X1 (10. Partially or completely disrupt the cells by adding a detergent such as deoxycholic acid, or by using physical methods such as a homogenizer or freeze-thaw). In order to prevent RNA degradation by RNase, it is recommended to add an RNase inhibitor such as heparin, polyvinyl sulfate, bentonite, ncaroid, diethylpyrocarbonate, vanadium complex, etc. to the extract. Preferably. 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-TL-2 antibody, and mRNA is extracted from the polysomes using a surfactant or the like.

In addition, for purification of mRNA containing poliA, there are purification methods using affinity columns such as oligo dT-cellulose and polyU-Sepharose containing 78 units of Sepharose 2B, purification methods using batch methods, fractionation using SDG centrifugation, and agarose gel electrophoresis. It can be done by etc.

The mRNA obtained as above is IL-211+RN
In order to confirm that it has 8 activities, m
Methods such as translating RNA into protein and examining its physiological activity, or identifying the translated protein using an anti-IL-2 peptide or a cloned antibody may be used. For example, mRNA is typically isolated by microinjection into the eggs of the African frog (kμm daevis).
Don et al., Nature, 233.177-182
(1972)) or translated into the corresponding protein by using the reticulocyte or wheat embryo cell-free translation systems.

IL-2 activity was previously determined by Gllis et al. (Gillis et al., J.

Immunol, 120.2027-2033 (1
This can be confirmed by the microassay method basically described by 978)). In this assay, G11li
IL of cytotoxic T lymphocyte cell line (hereinafter abbreviated as CTLL) created according to the method established by S et al.
-2-dependent increase in cellular DNA synthesis (IL-2depe
ndent cellular proliferati
on) is used as an index. That is, 4X103 pieces 0CTL
L cells in RPMI-1640 medium 1 containing 2% PCS
(10 μl) and inoculate a 96-well flat-bottomed microplate with 10 μl of serially diluted translation product.

After culturing for 20 hours at 37°C under 5% CCh, the cells were labeled with 0.5 μCi/well of 3H-TdR for 4 hours, and the cells were harvested onto ribbon glass fibers using an automatic cell harvester. The radioactivity taken up by the sample is measured using liquid scintillation method.

By this assay, CTLL cultured in the presence of IL-2
It was found that cells take up 3H-TdR in a dose-dependent manner, and from this it is possible to clearly calculate the amount of IL-2 contained in the sample.

Since IL-2 has the activity of promoting proliferation of 1978 cells,
IL-2 activity can be measured using the proliferation of 1978 cells as an indicator. That is, 5 CTLL cells were treated with 2% Fe.
2 in DMEM 1 (10 μl),
Seed 96-well flat bottom microplates with μ2 serially diluted translation products. 72-96 hours, 37°C, 5
After culturing under %COZ, the number of activated and proliferated cells is counted under a microscope. 1 (10U/ff1) as a control group.
j! , IOU/- of IL-2 is used to determine the 1C2 activity of the sample in comparison with the number of proliferated viable cells in this control group.

In this way, the most highly active two components were obtained from 11,-
2111 RNA 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, 5s-c, which is complementary to mRNA, is produced by reverse transcriptase in the presence of dATP, dGTP, dCTP, and dTTP.
After synthesizing DNA and decomposing and removing template mRNA by alkaline treatment, ds-cDNA is synthesized using reverse transcriptase or DNA polymerase using single-stranded cDNA as a template.
Synthesize.

The recombinant DNA of the present invention is produced from the ds-cDNA thus obtained and a vector DNA containing a replicon that can be replicated in prokaryotes or Escherichia coli. This recombinant DNA body is then integrated into the host cell.

The ds-cDNA and the vector DNA that can be propagated in prokaryotes are treated with exonuclease, added with a chemically synthesized DNA fragment, and ds-cD before being ligated together.
It is modified by various treatments such as elongating the G and C chains in order to attach terminals that can be linked to the ends of NA and vector DNA. These ligatable NAs can be spliced together, for example, by DNA ligase of T4 phage in the presence of ATP.

By transforming prokaryotic cells or Escherichia coli in order to amplify the cloned cDNA or to produce IL-2 polypeptide using the recombinant DNA of the present invention thus prepared,
Prokaryotic cells or Escherichia coli transformed with the recombinant DNA of the present invention are obtained.

Suitable prokaryotic hosts for IL-2 production include Bacillus subtilis and the like or Escherichia coli. Escherichia coli can be used as a host for the expansion of N^ in a host cell, but other host cells can also be used.

A suitable vector for Escherichia coli is pscl as an EK type plasmid vector (stringent type).
ol, pRK353. pRK646. pRK24
8. EK type plasmid vector (relaxed type) such as pDF41: Co1E1. pV
H51, pAc105. R5F2124゜pcRl,
pM89. pBR313, pBR322, pBR3
24, pBR325゜pBR327, pBR328, p
KV2289. pKY27 (10. pKN80.
pKC? .

pKB158. pMK2 (104. pAcYcl,
pAcYc184. dul et al., λgt type phage vectors: λgt, λC9λgt, λB.

λWES, λC1λ-ES, λB, λZJvir, +
λBl, λALO, λB.

λ-ES, Ts622. λDam, etc. are included. In general, pBR322 has often been used as a vector for Escherichia coli, and in this case the cloning sites with good abundance are the Ps tl and EcoR1 sites.

The following methods are commonly used for transforming host cells using recombinant DNA. When Nisierhia coli is the host, competent cells capable of taking up this DNA can be transformed by the well-known CaClt method after harvesting cells in the logarithmic growth phase. Transformation efficiency is improved by coexisting MgClz 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: Partially purified IL-2 mRNA is prepared as a 1112s fraction by sucrose density gradient centrifugation from an antigen-stimulated mammalian cell extract, and this partially purified mRNA is used as a template to generate ffgp-radioactive 5s-
Synthesize cDNA. ζ template mRNA for alkali treatment
After removal of 1l-12SmR, the isolated cDNA was extracted from non-antigen-stimulated mammalian cells and partially purified 1l-12SmR.
Hybridize with NA. Subsequently, unhybridized cDNA and hybridized cDNA are fractionated by hydroxyapatite column chromatography. The unhybridized cDNA and the hybridized cDNA were labeled as probe A and probe B, respectively.
It is called. Both recombinants are grown on nitrocellulose filter paper using the same method. And the cell D
NA is fixed on the filter paper by alkali treatment. Probe A
and B, respectively, are hybridized with DNA on two different filter papers. After that, autoradiography was performed to find recombinants that reacted positively with probe A (plus).
, select recombinants (minus) that have little or no thermal reaction with probe B (Noguchi et al.; Proc.

Jap, Acad,, V155B 464~
469 (1979).

(2) The second method is, for example, 1 (100 to 10.
The recombinant clones of 0 were roughly divided into clone groups of 2 to 30 or 2 to 3 (10 clones), and each clone group was cultured using a conventional method to obtain plasmid D.
Prepare NA5. Next, these plasmid DNA5
The 5s-cDNA is immobilized on nitrocellulose filter paper by heat denaturation, for example, and hybridized complementary to mRNA prepared from mammalian cells containing activated IL-2-mRNA. Alternatively, IL-2mR
When mRNA (mixture) containing NA is hybridized with heat-denatured plasmid DNA (mixture), DNA
A-mRNA hybrids are immobilized on nitrocellulose filter paper. The filter paper was washed with a low salt buffer such as 1mM 1-IEPES or 10n+M saline, and the mRNA adsorbed on the filter paper was washed with 0.5mM EDT.
AHo, for example at 95°C11 with a solution containing 1χ SDS solution.
Process and extract for a minute. Purified mRNA is olig
.

By dT-cellulose column chromatography? Collect the goods. Next, the mRNA is microinjected into African frog oocytes and translated into protein to confirm IL-2 activity. Alternatively, this mRNA is translated into protein using an mRNA-dependent reticulocyte system or wheat embryo in vitro cell-free synthesis system, and anti-IL-2
Antibodies can be used to analyze IL-2 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 experiments are repeated until a single clone having IL-2ON^ 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 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 Pstl DNA insert encoding the Lou2 polypeptide from DNA recombined with pIL-2-50A was determined by Maxam and G11ber.
t method (Me th.

Enzy+n, 65.499-560. (1980)
); as well as the niboxynucleotide chain termination method (Smi
th, A, J, M, Meth.

Enzym, 65.560-580. (1980)
) was decided.

Figures 1 and 2 (a) show diagrams of cDNA inserts cleaved with restriction endonucleases. Figure 2 (a
), these cDNAs are BstNl and BstNl, respectively.
It has a structure that can be cleaved with restriction endonucleases Xba I and BsLN I.

The DNA sequence of this cDNA insert contains one large open reading frame. The first ATG sequence (Ko
zak, M, Ce1l, ■, 1109-1123
(1978)) exists at 48-50 nucleotide positions from one end of 5', and 152 sequences are connected to this ATG from nucleotide positions 507-509 where the reading end sequence TGA exists. An A link corresponding to the 3'-poly(A) end of 1I RNA was found at the cDNA end,
Six nucleotides AATAAA (positions 771-776) usually found in most eukaryotic mRNAs
is located first.

(Proudfoot, N. J., & Brownl.
ee C,G,, Nature 263°211-2
14. (1976)) The amino acid sequence encoded by the cDNA is shown in Figure 2 (
b) (Amino acid sequence I) Throat As reported to be seen in most cases (BlobelG, et al.
, 5yni, Soc, Exp, Med,, '
4.9-36 (1979)), the N-terminal region of the above described IL-2 polypeptide is also hydrophobic. This region probably plays the role of a signal peptide that is cleaved during secretion of 7fill, -2. Cleavage occurs between Ser and Ala at positions 20-21 or between Ala- between positions 21-22.
It is cleaved between Pro to produce a polypeptide having the amino acid sequences ■ and ■. This is because similar cleavage positions 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 153.
It is calculated to be 49.4 Daltons. This molecular weight value is molecule 1 (1) of human IL-2 protein obtained from Jurkat cells.
5 (100 Daltons) (Gillis et al., Immunolo
logical Rev,, 63+ 67~209 (1
982) ). In addition, as shown in Example 3, base sequence 11
DNA fraction starting from the CCT sequence located at positions 1 to 113,
That is, it was confirmed that the code for the polypeptide starting from Pro located at position 22 (amino acid sequence ■ in Figure 2(b)) expressed a polypeptide having ■C2 activity. GCA located at positions 107 to 110 of the base sequence
The DNA fraction starting from , that is, the DNA fraction encoding the polypeptide starting from Ala located at position 21, as shown in the amino acid sequence ■ in Figure 2(b), has < TL-2 activity as shown in Example 6. It was confirmed that it expressed a polypeptide with

Genes of nucleated organisms are known to exhibit polymorphism, as is also known for the human interferon gene (Noriguchi et al., Gene JJl, 11-15 (1980)
, Ohno, Noguchi, , Proc, Natl, Acad
, Sci, USA+ 77+5305-5309 (
1981); Gray et al.
Kugiho, 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 base sequence but no change at all. In the case of human IL-2 cDNA,
A residue at position 503 of pIL-2-50A cDNA is G
Another cDNA clone (plL-
2-503) can also be detected. p I L2-50A c
The existence of other cDNA clones with different base sequences from the DNA can also be expected.

As is clear from the above explanation, the gene according to the present invention includes DNA having the base sequence shown in FIG.
DNA having a continuous base sequence starting from the ATG sequence at positions 8-50 and ending at at least the ACT sequence at positions 504-506.Starting from the GCA sequence at positions 108-110,
DNA with a continuous base sequence from the GCA sequence to at least A (, TIF self-published, and C at positions 111-1.13)
It includes DNA having a continuous base sequence from the CT sequence to at least the ACT sequence. The gene according to the present invention also includes
DNA ending with the ACT sequence at positions 504-506 and starting with A at position 1, DNA starting with ATG at positions 48-50, 1
DNA starting with the GCA sequence at position 0B-110 or 111
-Includes the DN^ starting with the CCT sequence at position 113. Furthermore, the gene according to the present invention ends with the TGA sequence at positions 507 to 509, begins with A at position 1, and A at positions 48 to 50.
It includes DNA starting with the TG sequence, DNA starting with the GC8 column at positions 108-110, or DNA starting with the CCT sequence at positions 111-113. Furthermore, the gene according to the present invention has a DNA that ends with C at position 801 and begins with A at position 1,
DNA starting with ATG at position 48-50, 108-11
It includes DNA starting with Ge8 at position 0 or DNA starting with 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, 10B-1
DNA starting with GCΔ sequence at position 10 or 111-11
Contains DNA starting with the CCT sequence at position 3. The present invention also provides amino acid sequences 1. T.I.

Contains a gene with a base sequence corresponding to m. A polypeptide lacking one or more amino acids in the amino acid sequence (III) or a polypeptide in which one or more amino acids in the amino acid sequence (I) is substituted 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, amino acid sequence 1. Even if the gene has one or more extra bases that can represent one or more amino acids relative to II or Unless otherwise specified, they are included within the present invention. 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 is true for amino acid sequence 1. Amino acid sequence I of the gene corresponding to II and ■
, ■ and D encoding an amino acid addition at the C-terminus of ■
This also applies to DNA in which NA is additionally attached to the 3'-end. Therefore, the use of genes encoding such polypeptides is included in the present invention.

Prokaryotic cells or Escherichia spp. transformed with recombinant DNA bodies that produce IL-2 in living host cells.
Koli is made in various ways: For example, IL-2・
A sequence encoding the cDNA is inserted into the expression vector downstream of the promoter sequence. Alternatively, a cDNA piece having a promoter sequence can be inserted upstream of the IL-2 encoding sequence before or after cDNA insertion into the expression vector.

A detailed description of the method for constructing a prokaryote or Escherichia coli that expresses IL-2-cDNA and produces IL-2-polypeptide is as follows.

(1) IL2-cD by Escherichia coli
Expression of NA To express IL-2-cDNA in Escherichia coli, the cDNA is first ligated to various bacterial promoters, and then a hybrid plasmid containing the cDNA downstream of the promoter is created. For example, by infecting Escherichia coli Hn101 with this plasmid,
A bacterium that biosynthesizes a protein with two activities is cloned. Basically, any bacterial promoter can be used as cD.
If properly connected to NA, it will express TL-2-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. The 20 amino acid N-terminal region of this polypeptide 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;
It is not desirable to express a portion corresponding to 2 signal sequences.

(i) Construction of plasmid vector pTrS-3 pT
rS-3 contains the Escherichia coli Trp promoter, and the liposome binding site (SO sequence) for the leader peptide of pTrS-3 has been previously 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.
etal, Biochemistry 54. No, 8.676 (19B
2)). This plasmid vector also contains one Sphr site downstream of the ATG initiation sequence (Figure 3).

To express IL-2 cDNA, the plasmid was first digested with sph I and treated with Escherichia coli DNA polymerase I (K1.enow fragment) or bacteriophage T4 DNA Δ polymerase ■ to obtain an overhanging end at the 3′-position. (4th
Figure (a)). Plasmid pIL-2-5OA was transformed into Pst
Digested twice with Hg1AI and Hg1AI to generate larger cDNA
Isolate fraction A.

The DNA is then treated with Escherichia coli DNA polymerase I (Flenow fragment) or bacteriophage T4 DNA polymerase to cut off the 3'-overhanging ends. This treated cDNA encodes a 132 amino acid IL-2 polypeptide (fourth
(a) Figure). This cDN^ was pretreated as described above.
TrS3 is ligated to plasmid DNA, splicing the ATG initiation codon to the CCT (Pro) sequence of IL-2 cDN^. Thus plasmid pr IL-2
-22 is obtained. Trp promoter sequence and pT
The binding of IL-2-22 to the IL-2 cDNA sequence is the fourth (
a) Shown in the figure.

Plasmid pT IL-2-22 directs the synthesis of the 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;
A plasmid that directs the synthesis of a 33 amino acid IL-2 polypeptide can be constructed as follows.

Plasmid pTrS-3 has one C1a I cleavage site between the SD and ATG sequences (Figure 3). This plasmid contains C1aT and Sal! will be cut off.

Plasmid pIL-2-5OA is partially digested with Pst I, treated with Escherichia coli DNA polymerase r, and the longest DNA is isolated. The DNA was then ligated with a synthetic NA linker containing a restriction enzyme Xho I cleavage site to create a D
Plasmid pIL-2-5OA introduced with NA linker
A clone containing (Xho) is isolated. plasmid p
If 1L-2-5OA (Xho) is first cut with fl1giAl, treated with Escherichia coli Flenou fragment or T4 DNA polymerase, and digested with Xho I, cDNA can be isolated into eight fragments.

This cDNA fragment was digested with C1a I and Sal
The fourth
(b) As shown in the figure (connect to synthetic NA. Thus, Ala
IL-2 consists of 133 amino acids starting from
Plasmid pTIL-2-21 is obtained which allows the polypeptide to be synthesized in Escherichia coli. (Figure 4(b)). The same thing can be done without using the Xhol linker.

(iii) Different sized IL-2 polypeptides with different N-terminal amino acids are also produced using pTrS-3 expression plasmid vectors. As shown below
, IL-2 cDNA cloned into pIL-2-5OA
A has only one Dd in the nucleotide binding site 81-85
e has 1 part. Plasmid pIL-2-508 (Xho) is cut with Dbe I and the DNA fraction containing the larger section of cDNA is isolated. This fraction contains DNA having 3 (100 base pairs) from pB12322 (Figure 4(c)).

The DNA fraction is treated with exonuclease Bal 31 and then cut with Xhor. DN obtained here
A was ligated with pTrS-3 cut with sph I, treated with Kleno-fragment or T4 DNA polymerase, and digested with Sal I (fourth
c) Figure). Escherichia coli J
Infect IB 101 and search for clones expressing human IL-2. These clones should express the false color size of Hinoki L-2. Because human IL-2
This is because the DNA corresponding to the N-terminal region of is cleaved and removed in various ways. (pT containing IL2 cDNA)
IL-2-14 and 15 are obtained.

(iv) IL-2 cDNA is also pKT 218
(Courtesy of Talmage; Proc, N
ATL, Acad, Sci, USA.

? ? , P., 3369-3373 (1980)). Plasmid PKT 218 was cut with PstI and pIL2-50A was ll5iA.
I, l! : I obtained by cutting at Pst 1 (Fig. 5)
Connect with the L-2 cDNA insert. The resulting plasmid pKIL-2-21 has a sequence at the starting position of protein synthesis, as shown in FIG.

Therefore, this plasmid pKIL-2-21 is rL
It consists of a polypeptide that is a fusion of the 133 amino acids of -2 and the amino acids of β-lactamase, and it should be possible to synthesize this in Escherichia coli (the first methionine is cleaved and removed in Escherichia coli). ).

(v) Expression of plasmid pTuBIp-5 in which the promoter sequence for tufB was inserted into p-BR・322 has already been carried out (Noguchi et al., Biochemistry 53966 (
1981)).

This plasmid contains one C1a I cleavage site,
As shown in FIG. 6, this cleavage site is located two base pairs downstream of the SD sequence. pTrS-3 also has SO sequences and AT
Contains one C1a I cleavage site located between the G start sequence, and this C1a I site also cleaves pTrS-3 and IL
It is extremely easy to replace the bacterial Trp promoter with the tufB promoter since it is not destroyed during the process of creating an expression plasmid using the -2 cDNA. Human IL-2 cDNA is therefore expressed under the control of the tufB promoter. For example, pTIL-2-22 is C1a r
and Pvu I [to cut the DNA containing IL-2 cDNA.
Separate fraction A.

Then, these two parts were joined together with pTuBIP-5, and C
After pre-cleavage with 1a I and PvuII, plasmid pTuIL-2-22 is constructed as illustrated in FIG. IL-2 activity can be detected in extracts of Escherichia 31 IBIOI containing plasmid pTulL-2-22.

(vi) It can be similarly constructed using, for example, pTIL-2-21, or basically any expression plasmid achieved using pTrS-3.

Alternatively, for example, pTulL-2-22 is cut with C1a I and treated with Ba131 or SI or DN^ polymerase ■
(Escherichia coli), it is also possible to make the distance between the SD and ATG sequences an optimal length by removing or adding 2 to 3 base pairs of DNA and connecting the plasmid again.

Prokaryotic cells or Escherichia coli transformed with the recombinant DNA of the present invention can be cultured to amplify the recombinant DNA or produce IL-2 polypeptide. This culturing is carried out in a conventional manner.

IL-2 produced intracellularly or extracellularly is ammonium sulfate precipitated,
Dialysis (under normal pressure or reduced pressure) gel filtration for salt removal;
Chromatography, isoelectric focusing plate concentration, gel electrophoresis, high performance liquid chromatography (hereinafter abbreviated as HPLC)
(ion exchange, gel filtration, and reversed-phase chromatography), and affinity chromatography using a dye-binding carrier, activated Sepharose 4B conjugated with a monoclonal antibody against IL-2, or lectin-conjugated Sepharose 4B, etc. be able to.

The method for isolating and purifying IL-2 is described by Watson et al.
p.Med.

150, 849-8601979), G11lis
et al., J. Immunol.

124, 1954-1962 (1980), Moch
Izuki et al., J.

Immunol, Methods 39.185-2
01 (1980), Welte.

K et al+ J, Kxp, Med, 15
6.454-464 (1982)).

The polypeptide thus obtained exhibits biochemical and biological behavior and has IL-2 activity identical to that known for IL-2 produced from mammalian cells upon mitogen stimulation. The molecular weight is approximately 15 (100 Daltons) and the IL-2 activity is approximately 15 (100 Daltons).
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, after treatment with NAse and RNA5e, and after heat treatment at 56° C. for 130 minutes. Activity is stable at pH 2-9. I produced in this way
L-2 promotes the proliferation of T cells (cytotoxic T lymphocytes) with monoclonal functions, strengthens the division of thymocytes, and further develops anticancer-specific cytotoxic T lymphocytes from a memory state in the absence of antigen. cause differentiation into Also, YAC
Helps enhance natural killer cell activation against -1 cells and RL1 cells.

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

Example 1 (1) Jurkat cells (freely available in Japan, West Germany, and the United States), a leukemia cell line of the Hil-T cell lineage, were suspended in RP old 1640 medium containing 10% PCS, and X-ray Irradiation was performed using an irradiation device Exs 150/3 (10-4 (Toshiba, Japan) for 50 seconds at room temperature until reaching 10. The cells were cultured in an incubator at 37°C under 5% carbon dioxide gas 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.

The clones obtained from the growing wells were transferred to a new medium to increase the amount of clones, and the increased clones were grown at an initial cell density of lX in the presence of 50 μg/d of ConA.
Cultured for 24 hours at 10" cells/ml. Then, IL-2
Activity was measured according to the method described above.

As a result, Jurkat-111 (ATCCCCRL81
29) A human T cell line named ``J-111'' (hereinafter referred to as ``J-111'') was cloned from the shell cut of the parent line.
The IL-2 production capacity of these cells was 40 times higher than that of the parent strain.

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 (IXIO5 cells/d) were cultured in serum-free synthetic medium RIT C55-9 (Sato, T., et al.
al,, Exp.

Ce1l Res, 138.127-134. (
1982)) 101 (10O!) and cultured in a roller culture bottle (Falcon 3027) at 37°C for 14 days, and the proliferated cells were obtained by centrifugation. The above C
on^25 μg/Id-containing medium was inoculated. Culture solution 1 (10 d) inoculated with cells was placed in each of 4 batches of roller culture bottles (Falcon), and cultured by rotation for 6 hours.

(3) Jurkat cells (1,2 x 106) stimulated with ConA 25ug/1ail for 6 hours in this manner were treated with physiological saline-phosphate buffer (hereinafter abbreviated as PBS) 8. (
Suspended in 100 rrdl. The cells were washed twice by centrifugation, and added to an RSB solution (10 d Tris-HCl buffer, pH 7.5, L Om
M NaCl,1, 5mM MgCl2) 8(10
resuspended in m1. Then, add surfactant NP-40 to a final concentration of 0.05% and mix slowly.
3. Cell nucleus. (Separated by centrifugation at 100 rpm, 5 min, 4'C. 5OS (0.5%) and EDTA (5 m
M) 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.
Dissolved in 1I Tris-HCl. The amount of RNA obtained was 19
It was 6■.

Fractionation of mRNA was performed using oligo(dT)-cellulose (P,
L.

Biochemicals+ Type? ) using affinity chromatography. The adsorption liquid is 20+iM Tris-HCl, 0.5M NaCj! , 1 s
+ME! pH 7, containing DTA and 0.5% SOS,
5 solution, ? For output 8, the column was washed with buffer (20mMl).
-Lis-hydrochloric acid, pH 7, 5, 0, 5M NaC/! ,
After washing with 1mM EDTA), water and 10mM Tris-
Hydrochloric acid (pH 7,5) was used alternately. The amount of mRNA obtained by elution was 3.6. Then this obtained mR
2.4μ of NA was centrifuged on sucrose density gradient (50mM) in a pH 7.5 solution containing Lis-HCl, 1mM EDTA, 0.2M NaCf, 5-25%, 2
6, (100 rpn+ at 4°C for 24 hours). Fraction No. 11 to 123 of mRNA 12.
13.14, 59μg and 46μg160 respectively
It was μg.

(4) The mRNA obtained from the fractionation of Nα13 was injected into oocytes of Xenopus laevis and Xenopus laevis.
0 ng mRNA/oocyte), and the culture solution of this oocyte was measured for IL-2 activity. As shown in Table 1, an increase in the uptake of 3H-thymidine (CH-TdR) and an increase in the number of activated T lymphocytes were confirmed, and it was clear that mRNA in this fraction
It was demonstrated that A contains human IL 2 mRNA.

Table 1 (a) Translation product of fraction 13 × 8 × 32 14.683 10.165 (b) Translation product proboscis of fraction 13 × 2 * Units are “II” of standard IL-2 (10 units/form) -T
Calculated by comparing with dR uptake amount.

(5) Then 11-12S containing IL-2n+RNA
cDNA in vitro from Nα13 fraction of mRNA
A was synthesized.

The recombinant DNA was constructed into plasmid vector pHR322. The modified 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) Lis-HCl buffer (pH 7,
5), 30mM NaCj! , 6n+M MgCj2.
, 5iM dithiothreitol (hereinafter abbreviated as DTT),
0.5mM each dATP, dGTP, dCTP, d
TTP (dCTP includes zzp radiolabeled)
, 0.1μg oligo(dT) + o, 10dgmR
N^ and 15 units^MV reverse transcriptase (J, 'A, B
eard) was mixed and kept at 41°C for 90 minutes. After the reaction, the DNA was treated with phenol and recovered as an ethanol precipitate, and this DNA was treated with 2011 IM Tris and 1
It was dissolved in a pH 7.5 solution containing mM EDTA.

2.5 μg of 5s-cDNA was synthesized. To remove 5RNA from this reaction solution, add Na0Hi solution to the reaction solution.
The solution was made into a 0.33 N NaOH solution, left at room temperature for 15 hours, then neutralized by adding the same amount of LM) Lis-HCl buffer (pH 7.5), and passed through a Sephadex G-50 column. The amount of cDNA recovered was 1.8 μg.

(5-2) 50mM phosphate buffer (pH 7,5),
10mM MgCfz, 10mM DTT, 0.75
n+M each dATP, dGTP, dCTP, dTT
P (dCTP includes those labeled with H), 1.8μ
g 5s-cDN eight and eight unit polymerase l (B
RL, USA) and the reaction was carried out at 15°C for 15 hours. After the reaction was completed, the DNA was treated with phenol and chloroform and recovered as an ethanol precipitate. 1.10dg d
s-cDNA was generated. 50mM Sodium Acetate (p)
I4.5), 0.2M NaCf, 1mM Zn
Cfz and 1.10dg mixture into double stranded cDN
After incubation at 7°C for 30 minutes, 0.25 units of Nuclease S, (Mitsui, Japan) was added and incubated for an additional 15 minutes. After the reaction was completed, the reaction product, which had been treated with phenol twice, was subjected to Sephadex G-50 to obtain 55 μg of double-stranded cDN 80.

(5-3) 0.14M potassium cacodylate, 301 Tris base, 0.1+M DTT, 1mM CoCj
2z, 0.64mM “P-dCTP (specific activity 2.7
X 10' cpm/n mol), 0, 5511
g ds-cDNA and 5 units of terminal transferase (BRL) were mixed, incubated at 37 °C for 7 minutes, treated with phenol, and then applied to a Sephadex G-50 column to obtain 0.50 dg DNA as an ethanol precipitate. Ta. It was found that approximately 50 dCMP residues were added to both 3'' ends of the recovered DNA.

After cutting pBR322DNA I 08g with the restriction enzyme Masu I, both of the cut DNAs were cleaved using exactly the same conditions as those used when adding the dCMP strand to the ds-cDNA described above, except that dGTP was used instead of dCTP. A dGMP chain was added to the 3' end.

(5-4) 50IIIM tris-hydrochloric acid (pH 7, 5>0
, 1 M NaC! ! , 5mM DETA, 0.
05 u g dGMP residue addition pBR322 and 0
．． 01dg dCMP residue-added cDNA was first heated at 65°C.
for 2 hours at 46°C, then 120 minutes at 46°C, then at 37°C.
C for 60 minutes and at room temperature for 60 minutes.

Escherichia coli z 1776 (Curtiss m
, R,etal,, in Mo1ecula
r Cloning of Recombina
nt DNA.

(-68, 5cott & R6Werner
ed,) 8 Academic Press.

(1977)') was added to 50 d of culture medium (1 (10 g g
/mfl diaminopimelic acid, 50dg/1rdl
of thymidine, 1% tryptophan, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose)
and cultured with shaking at 37°C until the absorbance of the culture solution reached around 0.3 at 562 nm. After the completion of the culture, the culture solution was kept at O'C for 30 minutes, the bacterial cells were collected by centrifugation, and added with 5mM) Lis-HCl (pl+ 7.6), 0.1
M NaCl, 5mM MgCj! z and L OmM
Washed twice with 25 m2 of a solution containing Rh(,e).

The obtained bacterial cells were treated with 5raM tris-hydrochloric acid (pH 7.6),
0.25 M KCj2.5mM MgCfz, 0
．． Suspended in 20 ml of a solution containing 1 M CaClz and 10 mM RbCff1, and left at 0°C for 25 minutes.
Collect the bacterial cells and use 1 mi of the same solution as above! Resuspend the bacterial cells in
The above-mentioned recombinant DNA was added to 0.27 g of the obtained bacterial cell suspension and incubated at 0°C for 60 minutes. After that medium 0.1
ml was added and cultured with shaking at 37°C for 30 minutes. The culture solution (0.1 m) obtained in this way was
1 of the medium containing /lnl diaminopimelic acid, 50 μg/ml thymidine and 15 μg/ml tetracycline.
．． It was plated all over a 5% agar medium 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) 1 (containing 10 μg/− of diaminopimelic acid, 50 μs/d of thymidine, and 10 μg of tetracycline). L medium 2 (10 tnl inoculated, 37°
The cells were cultured with shaking at C for 5 to 7 hours. Next, the final concentration is 170μ
Fresh medium 2 (10-g/rtdl) containing chloramphenicol was added and cultured for an additional night.

The thus enhanced plasmid DNA was purified according to a conventional method.

Clones containing rL-2 cDNA were selected by IllRNA hybridization-translation assay (hereinafter abbreviated as HT assay).

The HT assay used here was performed as shown below.

The purified DNA (25 Hg) was cleaved with the restriction enzyme old ndII, treated with phenol three times, phenol-chloroform and chloroform each, precipitated with ethanol, washed with 80% ethanol, and precipitated with 80% formamide 4.
Dissolved in 0d.

After heating at 90 °C for 5 minutes to denature the reaction solution,
Diluted to 1.3 ml with X5SC (1,5M NaCl, 0,15M sodium citrate). After that, zero 〇N^
was fixed on nitrocellulose filter paper, which was dried at 80°C for 3 hours and treated with 50% formamide, 20mM Pi.
pes (pH 6,5), 0.75 M NaCl! ,,
511+M EDTA, 0.2% SDS and J-111
The DNA immobilized on the filter paper and IL-2 mRNA were hybridized by incubation at 37°C for 118 hours in a solution containing 250 μg of cell-derived poly(A) mRNA.

The filter paper was then heated 3 times at 65 °C with 10 mM of p) 16.5
Pipes+ 0.15 M NaCl solution, 1mM
Pipes, 10eiMNa (/!Clean with i-liquid and
．． 95° in 5 mM EDTA, 0.1% SO3 solution
mRNA hybridized from filter paper after C11 minute treatment
was recovered.

The thus extracted mRN^ was purified on an oligo dT-cellulose column according to a conventional method, injected into African frog oocytes, and the IL-2 activity of the translated protein was measured.

One group out of 18 groups each consisting of 24 clones was found to have 48
It showed positive IL-2 activity of 11 units/ll.

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 medium 2 (10+d) with the same composition as described above, and 37"
The cells were cultured aerobically for 5 to 7 hours at C, and L medium containing chloramphenicol was further added thereto.

After overnight incubation and enrichment of plasmid DNA, plasmid DNA was similarly purified according to standard methods. Approximately 5 μg of each plasmid DNA was cleaved with 1 ml, and each plasmid was fixed onto nitrocellulose filter paper in the same manner.

The filter paper was hybridized with IL-2n+RNA, and the hybridized mRNA was injected into Xenopus oocytes and collected to measure the IL-2 activity of the translated protein.

As shown in Table 2, only plasmid DNA purified from a single colony designated p3τ16 showed positive rL-2 activity. Therefore, this clone has IL-2 cDNA (coli z 1?76/p
3 16AJ11995 (FERM-BP-225)
) was identified. In this way, plasmid DNA, p3
It was confirmed that -16 does indeed have DNA (IL-2 gene) capable of forming a specific hybrid with IL-2 rsRNA.

Table 2 (a) Translation product of mRNA x 2 X32 20.961 (b) Translation of "mRNA" X2 8832 product
X32 42* Plasmid p3-1
The cDNA insert of plasmid p3-16 was digested at one site with restriction enzyme Xba I and at two sites with Bst NI.
It was characterized by being cleaved at sites (upstream and downstream of the Xba I cleavage site). However, plasmid p3
-16 contains a cDN^ insert consisting of approximately 650 base pairs, which apparently corresponds to a portion of the 11-12S-sized IL2 mRN^. Therefore, other cDNA libraries were prepared using the method of Land et al. (Land et al.) using TL-2mRN8 as a template.
et al, Nucleic Ac1ds Res
, + vol 9+ p2551. (1981)). -This IJ cDNA (1.6 μg),
It was synthesized using 4 μg of IL-2a+RNA with added dCMP residues, and ds-cDNA was synthesized by using oligo(dG) 12=18 as a primer with DNA polymerase ■ (Kleno-fragment).

680 base pair DNA size marker
ker) longer cDNA (0.6 μg) was obtained by sucrose density gradient centrifugation and inserted into the Pst I site of pBR322 by standard GC Tilling method.

Escherichia coli χ1776 by recombinant DNA
nick-translated p3-16 as a probe in situ
Grunstein-Ho using cDN^ insert
About 2 (1
00 colonies were selected and transformed clones containing the plasmid prt, 2-508 containing approximately 850 base pairs and transformed clones (Escherichia coli χ 17
76/plL 2-508, 8J11996 (FERM
-BP-226) ) was identified. A restriction enzyme cleavage diagram of the cDNA insert of prL2-5OA is shown in FIG.

Transformed Escherichia coli χ1776/pl
In order to isolate the gene encoding the IL-2 peptide from L2-5OA, plasmid DNA was isolated from the bacterial cells and then cleaved with the restriction enzyme Pst I according to a conventional method. The smaller of the two DNA fragments produced by this treatment was the DNA gene encoding IL-2 peptide. The complete nucleotide sequence of the Pst I insert from pIL2-5OA is M
axam and G11bert's method (Maxam
, A. W. etal, Enzym, 65.
, 499-560.1980). The entire structure is shown in Figure 2(a).

Example 2 Constitutive IL-2 producing cell line J-A cloned from Jurkat cells according to the method described in Example 1
1886 (ATCC CRL 8130) was similarly grown in roller culture bottles. The grown cells were grown in fresh synthetic medium RITC-55-55 at an initial cell density of 1 x IOb cells/d.
9 and 8 hours after the start of culture, 3×10′ cells were resuspended in 11 to
IL-2 as 123 fraction was used for RNA extraction.

ds-cDNA was synthesized in the same manner as in Example 1, and 6 (10
cDNAs longer than base pairs (2,4 II g ) were obtained by fractionation by sucrose density gradient centrifugation. Next, this cO
Pst I-cleaved pBR32225, in which NA was extended with dCMP residues using terminal deoxynucleotidyl transferase, and 50 ng of it was extended with dGMP.
It was annealed to 0ng.

The generated hybrid plasmid was transformed into Escherichia coli χ1776, and approximately 4. (100 chrome transformants were obtained.

Grunstein-! (Following the method of ogness,
Three clones were selected that were complementary to the plasmid 3-16 cDNA used as a probe. That is, the transformed clones selected in this way are human I
This is a clone containing the L-2 gene.

Example 3 A plasmid that directs the synthesis of human IL-2 in Escherichia coli cells was constructed as follows.

Plasmid pTIL2-22 was transformed into pTrS-3 (
Nishi T.

Taniguchi T, et al, 5EIK
AGAKU u, 967, (1981).

54.676 (1982)) and pTL2-5OA containing IL-2 cDNA. Plasmid pTr
S-3 is Trp promoter Eco of pBR322
5hine Dal between R1 site and C1a 1 site
garno (hereinafter abbreviated as SD) region.

This plasmid also contains a single Sph
1 site, it contains an ATG initiation codon 13 bp downstream of the SD sequence.

If the DNA sequence corresponding to the protein in question is inserted at a site just downstream of the ATG codon, the vector is very effective for producing this protein.

This ATG codon is generated by forceful digestion of pTrS-3 followed by treatment with T4 ONA polymerase. Therefore, plasmid pTrS-3 (30 .mu.g) was cut with restriction enzymes in a conventional manner, followed by treatment with phenol and chloroform, recovery with ethanol precipitation, and both ends flushed with 74 DNA polymerase treatment.

Next, in the same manner, [1NA (21.4 μg)
was recovered. On the other hand, pIL2-5 containing IL-2 cDNA
0^380 μg was cleaved by Pst I and IL-2
cDN^ inserts were isolated by agarose gel electrophoresis. The cDNA insert (11 μg) was
and treated with T4 ONA polymerase, and 10 μg of the larger portion of DNA was isolated by agarose gel electrophoresis. A cDNA (7.2 μg) encoding 132 amino acids was obtained according to its nature, and this DNA fragment had a plant end (
Figure 4(a)).

The cDNA fragment thus obtained was then inserted just downstream of the ATG sequence into T4, which had previously been digested with Hoshi1.
DN was ligated into pTrS-3 vector treated with polymerase. The ligated plasmid was then transformed into Escherichia coli 118101 according to standard methods. This connection was performed as follows. I
The larger fragment of L-2 cDNA (0.4 ug) and 0.2 ug of pTrS-3 vector DNA were added to 66 mM of pTrS-17.5 containing 6.6 mM MgClz, 1 mM ATP and 10 mM DTT. It was mixed with 0.8 units of T4 DNA ligase in hydrochloric acid and the mixture was reacted for 1-night at 4°C. Among the transformants containing ampicillin and appearing on medium agar plates, an IL encoding 132 amino acids
Colonies carrying the plasmid containing the -2 cDNA portion were selected by in situ colony hybridization assay. The colonies selected in this way are cultured again (1
0if)l, lysozyme treatment and freezing.

Plasmid DltA was prepared by processing by melting.

This plasmid DNA was cut with b↓■ and μra I, and the resulting products were analyzed by agarose gel electrophoresis, indicating that the cDNA was frozen in the correct orientation after the ^TG sequence of pTrS-3. 22 were identified.

Escherichia coli HB l containing pTIL-2-22
(H) were cultured under conventional methods known for the growth of microorganisms. Cells were grown in χ medium (2
, 5% Bactodributon, 1% yeast extract, 0.1% glucose, 20mM MgSO4, 50mM Tris-HCl, pH 7,5) overnight at 37°C. Then, 1 ml of the culture suspension was inoculated into the same χ medium (1 (10 mf)) and cultured at 37°C.

0.0 of 650 mμ. 3-indole acrylic acid (IAA) was added when the value reached approximately 1.5-2.0.

Three hours after addition of inducer, cells were collected and 20 m
M) Washed with Lis-HCl (pH 7.5, containing 30mM NaCl) and resuspended in 8ml of the same buffer.

For effective functional expression of Trp promoter, IA
Use an inducer like A at a final concentration of 50 μg/Il! It was added so that The proteins thus produced in the bacterial cells were extracted by sonication (0'C, 2 minutes) or lysozyme (8 μg) digestion (0°C12O) followed by three freeze-thaw cycles.

By this method, IL-2 was extracted from the cells. The extracted IL-2 activity was 10, (100 to 12
0, (100 units/lan).

Escherichia coli II81 containing pTIL2-22
01 (AJ12 (109)) has been deposited as FERM-BP245.

Example 4 Plasmid pTulL2-2 carrying IL-2 cDNA
2 is pTuBIP-5 (Taniguchi, T.
et al, Seikagaku, 53+966
, 1981) and pTIL2-22 shown in Example 3.
was constructed by the method illustrated in Figure 6. Plasmid pTuBIP-5 has the tufB promoter sequence inserted into pBR322. This plasmid also contains a single "■" site, located 2 bp downstream of the SD sequence as shown in FIG. pTrS-3
also contains a C1a 1 site between the SD sequence and the ATG initiation codon, and this Φ 1 site is linked to pTrS-3 and IL-2cD
It is quite easy to replace the Trp promoter with the tufB promoter since it is not destroyed during expression plasmid construction by using NA, resulting in I
L-2 cDNA is expressed under the control of the tufB promoter.

Therefore, plasmid pTIL2-22 (30 μg) was cleaved with the restriction enzymes C1a I and α■ in the usual manner. Fragment containing IL-2 cDNA (approximately 2.2 kb
) was isolated and purified by agarose gel electrophoresis, and
g DNA was recovered. On the other hand, 20 μg of pTuBIP-5 vector was similarly digested with C1a I and Hatg [, and the larger fragment (approximately 3.4 kb) containing the ampicillin resistance gene was purified into a single mill by agarose gel electrophoresis. .5 μg was recovered.

Next, of the two fragments obtained in this way, one is tuf
B promoter (approximately 3.4 kb), and the other is IL-
2 cDNA (approximately 2.2 kb) and was ligated as shown below.

A fragment containing IL-2 cDNA (1,2 ug) and 0.3 µg of a fragment containing tufB promoter were added to 6.6
mM MgCl! z.

pH 7,5 with 1mM ATP and 10mM DTT
T4 DNA ligase 0 in 66mM Tris-HCl
．． 8 units 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 containing ampicillin and appearing on the medium agar plate, eight colonies having recombinant DNA containing the <IL-2 cDNA part, such as pTulL222 in FIG. 6, were selected. It was prepared as described in .

Escherichia coli HB containing pTulL2-22
101 was cultured in L medium (10 mf) at 37°C.

650mg of 0. When D reaches approximately 0.5-1.0, collect the bacterial cells and add 20ff containing 30mM NaCj2.
It was washed with 1M Tris-HCl (pH 7.5) and resuspended in 2 ml of the same buffer. The protein thus produced was extracted in the same manner as in Example 3. The IL2 activity in the extract ranged from 6,100 to 56,100 units/ml.

Escherichia coli HB containing pTulL2-22
101 (AJ12010) has been deposited as FERM-BP 246.

Example 5 Plasmid pGIL2-22 carrying IL-2 cDNA
is pci.

101 (Roberts, T. M. and La.
ucer G, D,, Meth Enzym,.

68, 473-483. (1979), Ga1l La
uer, et al., J. Mol.

Appl, Genet, L No. 2.139
~147 (1981), T.

Taniguchi, et al., Proc., N.
ATL, Acad, Sci, USA.

77, Na 9.5230-5233 (1980),
Egon Amann, et al.

Gene, 25.167-178 (1983)) and pTIL2-22 shown in Example 3.

That is, the plasmid pGL t containing the l promoter
ol (20 μg) was cleaved with the restriction enzyme Pvu U using a conventional method, and then 17 μg of DNA was recovered by treatment with phenol and chloroform and ethanol precipitation. On the other hand, pTIL2-22 (75 μg)
2.2 μg of DNA fragment containing IL-2 cDNA was obtained by cutting with 1 and Sal 1 and performing agarose gel electrophoresis.
− was recovered. This fragment was flushed by treatment with DNA polymerase I (Flenow fragment). Next, the two fragments (0.25ag and 0.66ag) obtained in this way were treated with T4 DN in the same manner as in Example 4.
The ligation was performed using 1.0 units of A ligase. (Then, this ligated plasmid was transformed into Escherichia coli H.
B101. Among the transformants, C1a I-5at containing IL-2 cDNA
A transformant 3tP-labeled IL-2 cDNA with an insertion of the I fragment was selected as a probe. Next, these transformants were treated with 25μ of ampicillin.
Example 3
lasmid DN was prepared by the method described in .

Thus, just downstream of the Iac phyromotor, the IL-2c
Plasmid DNA with DNA start sequence ATG
This was obtained by assaying the cleavage sites with st I and Xba I. pclL2 obtained in this way
Escherichia coli HB 101 containing -22 is 25a
10 l of medium I containing g/ml ampicillin and 25 g/ran streptomycin was inoculated and cultured. When 0.0 of 650 mμ reached approximately 0.5, Side (IPT
G) was added at a concentration of 1 mM, and after 1 hour, the cells were collected and a cell extract was prepared according to the method described in Example 4.

The IL-2 activity of the extract was 6. (100 to 80.(10
It was in the range of 0 units/ml.

Contains pGIL2-22! '/s') hif・
+18101 (AJ12011) is FERM-
Deposited as BP 247.

Example 6 Plasmid pTrS-3 (10ag) was first treated with restriction enzyme S.
It was cut with al I and the Sal 1 site was flushed by treatment with DNA polymerase (Flenow fragment) or T4 ON8 helimerase.

After cleavage with C1a I, 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 DN^ was recovered.

On the other hand, 11 μg of cDNA insert obtained by Pst I digestion of pIL2-5OA was digested with HB4 AI, treated with T4 DNA polymerase, and 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, t
0.45 μg of fragment containing the rP promoter (above).

0.5 ug of Hg1AI-Pstr fragment containing IL-2 cDNA and synthetic oligonucleotide (5°)
CGATAAGCTATGGCA (3゛) and (3°)T
ATTCGATACCGT (5') (20 pmo each
le), both of which are phosphorylated at their 5' ends, were prepared using the same method described in Example 3 to prepare T4.
They were ligated using one unit of DNA ligase (Fig. 4(b)). The plasmid thus linked is Escherichia coli H
B101. Among the transformants that appeared, the target transformant was selected as follows. First, transformants capable of hybridizing with both the IL2 cDN^ and synthetic oligonucleotides were selected by colony hybridization methods. Next, just downstream of the ATGGCA sequence, there is a DNA fragment (CCTACT......
) was selected by examining the Pst T and Xba 1 cleavage sites.

The above transformant containing pTIL2-21a or pTIL2-21b was transformed into L by the method shown in Example 3.
When cultured in a medium and analyzed by the method shown in Example 3, the transformant cell extract had a high fL-
2 activities were observed. E. coli 118101 (AJ 12013) with pTIL2-21a and E. coli (AJ 12013) with pTIL2-21b.
Escherichia coli HB with AJ 12014)
101 are FERM-BP 248. FERM-
Deposited as BP 249.

The host used in the above examples, Escherichia coli
Z 1776 and l(B Lot(Boyer H,
W. et al.

J, Mo1. Biol, 41.459. (196
9)) is known and easily available. In addition, in order to release the recombinant DNA in the transformant, the transformant was cultured at 37°C in L medium, and if the bacterial cells that became sensitive to tetracytalline and ampicillin were isolated, they could be deposited. Hosts are easily obtained from transformants.

Plasmid vector pBR322 (e.g. available from Hesesda Research Laboratory), pcE-1,p
TrS-3 and pat, 101 are known and readily available. Furthermore, the plasmid vector can be isolated from the deposited transformant by isolating the recombinant plasmid in the transformant using a conventional method, and further isolating the plasmid vector as is obvious from the description in each example. You can get it. pTrS-3 and pTuBIP-5 were each derived from Escherichia coli FERM-P6735 (BP 32
8) and Escherichia coli has been deposited as ATCC 3187g.

[Brief explanation of the drawing]

Figure 1 shows a restriction enzyme endonuclease cleavage map of a cloned gene encoding a polypeptide with IL-2 activity, Figure 2 (a) shows the nucleotide sequence of the cloned gene, and Figure 2 (b) ) shows the amino acid sequences I, ■, and ■ of polypeptides having IL-2 activity. Figure 3 shows the plasmid vector pTrS-3.
Figures (a), 4(b) and 4(c) show recombinant DNA5 (pTrS-3) using pTrS-3 as a vector.
TIL2-22. pTIL2-21. pTIL2-
14 and ρTIL2-15). Figure 5 shows I) KT 21B as a vector.
1 is a flowchart showing the construction of recombinant DN^(pKIL2-21) using pKIL2-21. Figure 6 shows recombinant DNA (pTUBIP-5) using pTUBIP-5 as a vector.
12 is a flowchart showing the configuration of Tu IL2-22). In the figure, "A", "G", "C" and "T" represent deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid, respectively. Patent applicant Cancer Research Foundation Ajinomoto Co., Inc. Figure 3 mp2: + Shiri - Ri
- KO111QI bu aI Φ
Wound Φ -← -ψ -Top - 2 Middle - 10 l -← Ko ・ - Spring - - 1 -bekoψ koyamako] Totot top,! i bu old 2 shi 2 = to ji to C to -1J
− ふ oI z 子 Φ −Φ り alM − for
− − Buko Ln − to ljt
Jc51-1? Ward≦i Dimensions

Claims (1)

Claims: (1) A recombinant DNA for transforming prokaryotes containing a gene encoding a polypeptide having human interleukin-2 activity. (2) A recombinant DNA body for transforming prokaryotes according to claim 1, wherein the polypeptide contains the following amino acid sequence in its molecule. [There is a gene sequence] (3) A recombinant DNA body for transforming prokaryotes according to claim 2, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (4) A recombinant DNA body for transforming prokaryotes according to claim 2, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (5) A recombinant DNA body for transforming prokaryotes according to claim 2, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (6) A recombinant DNA body for transforming prokaryotes according to claim 2, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (7) A recombinant DNA body for transforming prokaryotes according to claim 4, wherein the gene encoding the polypeptide has the following base sequence. [There is a gene sequence] (8) A recombinant DNA body for transforming prokaryotes according to claim 6, wherein the gene encoding the polypeptide has the following base sequence. [Gene sequence is available] (9) A recombinant DNA for transforming Escherichia coli containing a gene encoding a polypeptide having human interleukin-2 activity. (10) A recombinant DNA for transforming Escherichia coli according to claim 9, wherein the polypeptide contains the following amino acid sequence in its molecule. [There is a gene sequence] (11) A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (12) A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (13) A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (14) A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (15) A recombinant DNA body for transforming Escherichia coli according to claim 12, wherein the gene encoding the polypeptide has the following base sequence. [There is a gene sequence] (15) A recombinant DNA body for transforming Escherichia coli according to claim 14, wherein the gene encoding the polypeptide has the following base sequence. [Gene sequence is available] (17) A prokaryotic cell transformed with a recombinant DNA body containing a gene encoding a polypeptide having human interleukin-2 activity. (18) The prokaryotic cell according to claim 17, wherein the polypeptide contains the following amino acid sequence in its molecule. [There is a gene sequence] (19) The prokaryotic cell according to claim 18, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (20) The prokaryotic cell according to claim 18, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (21) The prokaryotic cell according to claim 18, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (22) The prokaryotic cell according to claim 18, wherein the polypeptide has the following amino acid sequence. [There is a gene sequence] (23) The prokaryotic cell according to claim 20, wherein the gene encoding the polypeptide has the following base sequence. [There is a gene sequence] (24) The prokaryotic cell according to claim 22, wherein the gene encoding the polypeptide has the following base sequence. [Gene sequence is available] (25) Escherichia coli transformed with a recombinant DNA body containing a gene encoding a polypeptide having human interleukin-2 activity. (26) The Escherichia coli according to claim 25, wherein the polypeptide contains the following amino acid sequence in its molecule. [There is a gene sequence] (27) Escherichia coli according to claim 26, wherein the polypeptide has the following amino acid sequence in its molecule. [There is a gene sequence] (28) Escherichia coli according to claim 26, wherein the polypeptide has the following amino acid sequence in its molecule. [There is a gene sequence] (29) Escherichia coli according to claim 26, wherein the polypeptide has the following amino acid sequence in its molecule. [There is a gene sequence] (30) Escherichia coli according to claim 26, wherein the polypeptide has the following amino acid sequence in its molecule. [There is a gene sequence] (31) Escherichia coli according to claim 28, wherein the gene encoding the polypeptide has the following base sequence. [There is a gene sequence] (32) Escherichia coli according to claim 30, wherein the gene encoding the polypeptide has the following base sequence. [There is a gene sequence]