JPH01165399A - Recombinant human interleukin 2 and production thereof - Google Patents

Abstract

PURPOSE:To produce a recombinant interleukin 2 without containing other human-derived proteins, by cultivating a transformant transformed with a vector containing a gene capable of coding human interleukin 2 in a culture medium. CONSTITUTION:A transformant containing a vector containing a gene capable of coding human interleukin 2 is cultivated in a culture medium to produce a recombinant human interleukin 2 substantially without containing other human- derived proteins. A vector containing a gene capable of coding interleukin 2 containing an amino acid sequence expressed by formula I in the molecule is cultivated in a culture medium to produce a recombinant human interleukin 2 containing an amino acid sequence expressed by formula I substantially without containing other human-derived proteins.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to recombinant human interleukin 2 and methods for producing the same.

Interleukin 2 has attracted attention for its application in medicine due to its unique immune response effect, and it has been reported that a human T leukemia cell line that produces interleukin 2 has been discovered (
Gillis et al., J. Exp. Med.

Volume 152, page 1709, 1980).

However, the amount of interleukin 2 produced by this human T leukemia cell line is minute, and its production requires culturing a large amount of cells, so difficult problems remain. Another possible method for producing interleukin 2 is to incorporate DNA corresponding to interleukin 2 into a microbial vector and have it replicated, transcribed, and translated within microbial cells, and then produced by the microorganism.

The present inventors succeeded in isolating DNA encoding interleukin-2 polypeptide necessary for production by microorganisms, and paved the way for interleukin-2 production by microorganisms.

The present invention relates to a recombinant human interleukin 2 that does not substantially contain other human-derived proteins and a method for producing the same, and furthermore, the present invention relates to a recombinant human interleukin 2 that does not substantially contain other human-derived proteins, and further relates to a recombinant human interleukin 2 that substantially contains other human-derived proteins that contain the following amino acid sequence in the molecule. The above-mentioned recombinant human interleukin'/2 is characterized in that a transformant having a vector containing a gene encoding interleukin 2 and a gene encoding interleukin 2 containing the following amino acid sequence is cultured in a medium. Concerning the manufacturing method.

Ala Pro Thr Ser Ser
Ser Thr Lys Lys Thr
GIN Leu GIN Leu Glu ) Its L
eu Leu Leu Asp Leu GINMet
Ile Leu AsN Gly Ile
AsN AsN Tyr Lys AsN
Pro Lys Leu Thr Arg
Met Leu Thr Phe Lys
PheTyr Met Pro Lys Ly
s Ala Thr Glu Leu Ly
s His Leu GIN Cys Leu
Glu Glu Glu Leu Lys
Pro LauGlu Glu Val L
eu AsN Leu Ala GIN S
er Lys AsNPhe His Lau
Arg Pro Arg Asp Leu
Ile Ser AsN11e AsN
Val lie Val Leu Glu
Leu Lys Gly 5erGlu Th
r Thr Phe Met Cys Gl
u Tyr Ala Asp GluThr
Ala Thr Ile Val Glu
Phe Leu AsN Arg Trpl
ie Thr Phe Cys GIN S
er Ile lie Ser Thr L
euhr The human interleukin 2 of the present invention has the following amino acid sequence I or 11.

Amino acid sequence I Met Tyr 8rg Met GIN
Leu Leu Ser Cys Ile
Ala Leu Ser Leu Ala Leu Va
l Thr AsN Ser Ala Pr.

Thr Ser Ser Ser Ser Thr Lys L
ys Thr GIN Leu GINLau Glu
His Leu Leu Leu Asp Leu
GIN Met IleLeu AsN Gly
Ile AsN AsN Tyr Lys A
sN Pro LysLeu Thr Arg
Met Leu Thr Phe Lys P
he Tyr MetPro Lys Lys Al
a Thr Glu Leu Lys )lis Le
u GINCys Leu Glu Glu
Glu Leu Lys Pro Leu
Glu GluVat Leu AsN Leu A
la GIN Ser Lys AsN Phe)I
isLeu Arg Pro Arg Asp
Leu Ile Ser AsN Ile
AsNVal Ile Val Leu
Glu Leu Lys Gly Ser
Glu Thr Thr Phe Met Cy
s Glu Tyr Ala Asp Gl
u Thr AlaThr Ile Val
Glu Phe Leu AsN Arg Tr
p Ile ThrPhe Cys GIN
Ser lie Ile Ser Thr
Leu Thr amino acid sequence! ! Ala Pro Thr Ser Ser Ser T
hr Lys Lys Thr GINLeu GIN
Leu Glu His Leu Leu Leu
Asp Leu GINMet Ile Leu As
N Gly Ile AsN AsN Tyr Lys
AsNPro Lys Leu Thr Arg M
et Leu Thr Phe Lys PheTyr
Met Pro Lys Lys Ala Thr
Glu Leu Lys HisLeu GIN Cy
s Leu Glu Glu Glu Leu L'y
s Pro LeuGlu Glu Val Leu
AsN Leu Ala GIN Ser Lys A
sNPhe ) Iis Leu Arg Pro Ar
g Asp Leu Ile Ser AsN11e
AsN Val Ile Val Leu Glu L
eu Lys Gly 5erGlu Thr T
hr Phe Met Cys Glu T
yr Ala Asp GluThr Ala
Thr Ile Vat Glu Phe
Leu AsN Arg TrpIIs
Thr Phe Cys GIN Ser
Ile Ile Ser Thr Leuhr The human interleukin 2 of the present invention is interleukin 2.
It can be obtained by culturing in a medium a transformant containing a vector containing a gene encoding an active polypeptide. This gene contains a portion in its DNA chain in which sites to be cut with the restriction enzymes BstNI, XbaI, and BstNI are arranged in this order, and the following polypeptide I or! ■It codes for the following base sequence! or II.

Polypeptide ■ Met Tyr Arg Met GIN Leu L
eu Ser Cys Ile AlaLeu Ser
Lau Ala Leu Val Thr AsN
Ser Ala Pr.

Thr Ser Ser Ser Ser Thr Lys L
ys Thr GIN Leu GIN Leu Glu
His Leu Leu Leu Asp Leu
GIN Met l1eLeu AsN Gly
lie AsN AsN Tyr Lys
AsN Pro LysLeu Thr Arg
Met Leu Thr Phe Lys Phe
Tyr MetPro Lys Lys Ala
Thr Glu Leu Lys His
Leu GINCys Leu Glu
Glu Glu Leu Lys Pro
Leu Glu Glu Val Leu As
N Leu Ala GIN Ser Ly
s AsN Phe HisLeu Arg
Pro Arg Asp Leu Ile
Ser AsN lie AsNVal Il
e Vat Leu Glu Leu Ly
s Gly Ser Glu ThrThr
Phe Met Cys Glu Tyr
Ala Asp Glu Thr AlaT
hr Ile Val Glu Phe L
eu AsN Arg Trp Ile Th
rPhe Cys GIN Ser Ile
Ile Ser Thr Leu Thr Polypeptide II Ala Pro Thr Ser Ser Ser T
hr Lys Lys Thr GINLeu GIN
Leu Glu Olds Leu Leu Leu As
p Leu GINMet lie Leu A
sN Gly Ile AsN AsN T
yr Lys AsNPro Lys Leu T
hr Arg Met Leu Thr Phe Ly
s PheTyr Met Pro Lys Lys
Ala Thr Glu Leu Lys HisLe
u GIN Cys Leu Glu Glu Glu
Leu Lys Pro LeuGlu Glu V
at Leu AsN Leu Ala GIN Se
r Lys AsNPhe His Leu ArgP
ro Arg Asp Leu Ile Ser As
N11e AsN Val lie Val Leu
Glu Leu Lys Gly 5erGlu Th
r Thr Phe Met Cys Glu Tyr
Ala Asp GluThr Ala Thr I
le Val Glu Phe Leu AsN Ar
g Trplle Thr Phe Cys
GIN Ser Ile lie Ser
Thr Leuhr base sequence ■ ATG TACAGG ATG CAA CTCCTG
TCT TGCATT GCACTA AGT CT
T GCA CTT GTCACA AACAGT G
CA CCTACT TCA AGT TCT ACA
AAG AAA ACA CAG CTA (:AA
CTG GAG CAT TTA CTG CTG G
AT TTA CAG ATG ATTTTG AA
T GGA ATT AAT AAT TA
CAAG AAT CCCAAACTCACCAG
G ATG CTCACA TTT AAG
TTT TACATGCC (: AAG AA
G GCCACA GAA CTG AAA
(:8T CTT CAGTGT CTA GA
A GAA GAA CTCAAT [:CT CTG
GAG GAAGTG CTA AAT TTA G
CT CAAAGOAAA AACTTT CACTT
A AGA CCCAGG GACTTA ATCAG
CAAT ATCAACGTA ATA GTT CT
G GAACTA AAG GGA TCCGAA A
CAA(:A TTCATG TGT GAA TAT
GCT GAT GAG ACA GCAACCAT
T GTA GAA TTT CTG AACAGA
TGG ATT ACCTTT TGT CAA AG
CATCAT(: TCA A(:A CTA A(:
T base sequence n GCA CCT ACT TCA AGT
TCT 8CA AAG AAA ACA
CAGCTA CAA CTG GAG ni A
T TTA CTG (:TG GAT T
TA CAGATG ATT TTG AAT
GCA ATT AAT AAT TACAA
G AATCCCAAA CT (: ACCAG
G ATG CTCACA TTT AAG
TTTTAG ATG CCCAAG AAG
GCCACA GAA CTG AAA
CATCTT (:AG TGT CTA G
AA GAA GAA CTCAAT CCT
CTGGAG GAA GTG CTA
AAT TTA GCT CAA AGCAA
A AACTTT CACTTA AGA C
CCAGG GACTTA ATCAGCAAT^
TCAACGTA ATA GTT CTG
GAA CTA AAG GGA T (tl:
GAA ACA ACA TTCATG TG
T GAA TAT GCT GAT GA
GACA GCA ACCATT GTA G
AA TTT CTG AACAGA TGG
ATT ACCTTT TGT CAA AG
CATCATCTCA ACA CTACT Such NA is obtained as fractions 11 to 123 by fractionation by sucrose density gradient centrifugation, and is Messenger RNA isolated from human or murine lymphocyte-derived cells.
(hereinafter abbreviated as n+RNA). That is, 11-
The DNA prepared from the mRNA of the 123 fractions is ligated to, for example, an Escherichia coli plasmid vector and amplified in Escherichia coli cells, and a clone having DNA expressing the interleukin 2 polypeptide is isolated. The DNA inserted into the plasmid of the isolated clone may be isolated.

In the present invention, the DNA encoding a polypeptide having interleukin-2 activity can be linked to a replicon derived from a microorganism, and the obtained recombinant DNA can be introduced into a host microorganism cell to induce interleukin-2 producing ability in the microorganism. can be transformed to have

As mentioned above, mRNA corresponds to interleukin 2 and is obtained as fractions 11 to 123 by fractionation by SDG centrifugation, gel filtration, etc. and agarose gel electrophoresis, and this mRNA is found in lymphocytes. It can be produced by extracting and separating the derived cells.

To explain the human lymphocyte-derived cells capable of producing interleukin 2 used in the present invention as an example, human peripheral blood,
Tonsils, 1!1! It also includes the lymphocytes themselves obtained from organs, etc. In addition, these lymphocytes were treated with a nylon column and antiserum-complement treatment.

Those that have undergone pretreatment such as density gradient fractionation and enzyme (neuraminidase, galactose oxidase, etc.) treatment, and
Also included in the human lymphocyte-derived cells of the present invention are cells that have been given the ability to produce interleukin 2 by mutation treatment with rays or the like and treatment with enzymes such as trypsin. Further, cloned human lymphocyte-derived cells, such as T lymphocytes cloned in the presence of T lymphocyte growth factor, are also preferred human lymphocyte-derived cells.

Further, malignant human lymphocytes such as human leukemia cells and T lymphoma cells, human lymphocytes that have been pretreated or mutated as described above, or human lymphocytes that have been cloned from malignant cells are more preferable. It can be used as a derived cell. In particular, cloned cell lines usually have more mRNA extracted than the parent line.

Furthermore, the above human lymphocyte-derived cells and CEM.

So-called bilidoma, which is obtained by cell fusion with human tumor cells such as Molt4F, can also be used as suitable human lymphocyte-derived cells.

These human lymphocyte-derived cells (1) spontaneously produce interleukin 2, and (2) produce interleukin 2 when stimulated by contact with mitogen in the presence or absence of other cells. There is something.

When using an interleukin 2 spontaneous strain to produce interleukin 2 mRNA in human lymphocyte-derived cells, these cells may be cultured by a conventional method. When using cells that produce interleukin 2 upon stimulation with mitogens, wash the cells thoroughly and incubate at Rosewell Park Memorial Institute 1.
840 (hereinafter abbreviated as RPMI 1640) medium, Dulbacco's Eagles modification (Dulbacco's
s modified Eagles) medium, click medium (which may or may not contain serum or serum components) or synthetic serum-free medium.
Suspend the cells at a density of 4 x 106 cells/mJ, and add mitogen; neuraminidase, galactose oxidase;
After adding a zinc compound such as zinc chloride, nibrotiin A, and a bacterial cell-derived lymphocyte activation component such as streptolysin-○, the cells are washed to remove the stimulant.

IIm, B lymphocytes, and B lymphocyte-derived cell line Raji, which can produce interleukin 2 when coexisting with macrophages and puntolytic cells during mitogen stimulation;
[1audi, K562. There are cells that have the same ability to produce interleukin 2 when coexisting with BALL-1 cells, and when using these cells to produce interleukin 2 mRNA, in the presence of these cells. Perform mitogenic stimulation. In this way, the yield of mRNA may increase.

Human lymphocyte-derived cells are passaged and propagated in vitro or in animal species under normal conditions. In vitro culture and subculture can be carried out using normal cell culture media, including mammalian-derived serum.

Whether it is a medium that contains serum components or serum albumin, or a synthetic serum-free medium that does not even contain serum albumin,
It has been found that these cell lines can be cultured and propagated, and can be used as cell materials for the present invention.

The culture time of lymphocyte-derived cells is the time during which lymphocytes are activated and interleukin-2 mRNA is produced, and this time corresponds to the time when interleukin-2 begins to be produced in the cell culture supernatant. .

Specifically, it is usually 3 to 12 hours after the addition of the stimulant. If the culture time is extended in vain, the produced interleukin 2
mRNA is degraded. Furthermore, when activating lymphocytes, 10 to 50 ng/mil of phorbol esters such as PM^ and TPA may be added. The culture temperature is preferably in the range of 32 to 37°C.

The method for culturing human lymphocyte-derived cells having the ability to produce interleukin-2 will be described in more detail below.

(a) Obtaining a strain that spontaneously produces lymphokines Jurkat cells, human T-lymphocyte-derived leukemia cells (Fred Hutchinson Cancer Research Institute/Seattle/USA, Sota Institute/San Diego/USA, West German National Cancer Center/Heidelberg/West Germany etc.) are suspended in Click medium at a cell density of 1X10' cells/mz, and irradiated with X-rays for a total of a, ooo roentgens at an irradiation rate of 150 roentgens/min. After this, the cells were grown at a cell density of 0.1 cells/200 μp at 96 μm.
Hole flat bottom microtiter plate (Falcon 30
72J) and cultured in Click medium containing 5% tallow serum for 3 weeks at 37°C in a 5% CO2 incubator (cloning by limiting dilution method). Cells in culture wells in which cell growth has been observed are transferred to a 24-well Tank culture plate before the cells reach a density that covers the entire bottom surface, and the cells are grown in 2 ml of Click medium for 5 days. .

If a sufficient amount of cells is obtained, divide the cells into 2 x 106 cells.
piece/+nI! The cells were suspended in 2 mA of a serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 0.2 mA, and cultured for 2 days. Interleukin 2 is obtained by removing debris and sterilizing with a 22μ Millipore filter. An interleukin 2-producing strain is obtained from the cloned cells thus obtained.

(b) Obtaining an interleukin 2-producing strain from human peripheral blood T lymphocytes Human peripheral blood is collected, and peripheral blood lymphocytes are collected by Ficoll-Hyberk density gradient centrifugation. The peripheral lymphocytes are suspended in Click medium at a cell density of 1X10' cells/mi+ and inoculated into culture plates of 24 tanks each containing 2 mβ. Add 100μ of phytohemagglutinin-M (manufactured by Gibco) to a final concentration of 5μg/mβ.
! ! and cultured under the conditions described above for 48 hours, then the cells were washed with culture medium and returned to lXl0' cells/mj! 1 mI to the culture plate in the original tank at a cell density of ! Address.

Human spleen cells were added to each well of concanavalin A (hereinafter referred to as
It is abbreviated as ConA. )2.5μg/mj! Conditioned culture medium obtained by stimulation for 48 hours with 1 mR
The same culture is repeated for 3 days, and T lymphocytes obtained from human peripheral blood are subcultured for a long time. The T lymphocytes obtained through long-term subculturing in this manner are subjected to cloning and cell proliferation using the same limiting dilution method as described above. The thus obtained cloned T lymphocytes were suspended in RPMI 1640 medium at a cell density of 1X10' cells/mβ, and
Phytohemagglutinin (PHA) was added to a final concentration of 104l7 and incubated at 7.5% C at 37°C for 24 hours.
02 incubator, and the main culture supernatant was
Interleukin 2 is obtained by centrifuging at 0.000 rpm for 5 minutes and then sterilizing with a 0.22μ Millipore filter. An interleukin 2-producing strain is obtained from the cloned cells thus obtained.

(c) Obtaining malignant cells derived from human lymphocytes that produce interleukin 2 upon stimulation with mitogens.
Strain 111 was prepared using the above-mentioned serum-free medium or R containing 1-2% serum.
ConA 1101t/r in PMI 1640 medium
nR and P)IA 2. 2 in the presence of jμg/mi'
It was found that when cultured for 4 hours, interleukin 2 was produced at 10 to 4,000 cells/mf. Furthermore, interleukin 2 is produced even when cultured in the presence of zinc chloride, protein A, and picibanil.

(d) Obtaining cells that produce interleukin 2 by stimulation with mitogen in the presence of other cells or factors produced by those cells Human lymphocyte malignant cell Mo1
Gelcuff 899, a clone of Jurkat cells cloned using t4F and the aforementioned limiting dilution method, does not produce interleukin 2 even after being cultured for 24 to 72 hours with the addition of the lectins and mitogens mentioned above in a wide range of concentrations. No production occurs. However, during this period, interleukin 1, a type of monokine, was administered at 5 to 10 u/mβ or 562 and RaJi cells at 0 per lXl0' cells/vaR.
．． When cultured in Click medium at 37°C for 24 hours in coexistence with 5 x 106 cells/mR, interleukin 2
It produces 10-100u/mf.

To extract mRNA corresponding to interleukin 2 from the cells thus obtained, a conventional method may be used regardless of the type of cell. For example, NP-40,S
Cells are partially or completely disrupted and solubilized using a surfactant such as DS, Triton X 100 deoxycholic acid, or a physical method such as a homogenizer or freeze-thaw. During extraction, RNA
RNA was added to the extract to prevent RNA degradation by se.
ase inhibitors, such as heparin, polyvinyl sulfate, bentonite, fucaroids. It is preferable to add it to diethylpyrocarbonate, vanadium complex, etc. Furthermore, if necessary, polysomes in the process of synthesizing interleukin 2 are precipitated using an antibody corresponding to interleukin 2, and mRNA can be extracted from this using a surfactant or the like. a+RNA can be purified using adsorption columns such as oligo dT-cellulose, polysepharose, Sepharose 2B, batch method, or SDG.
It can be purified by fractionation using centrifugation or the like. Through such purification operations, mRNA corresponding to interleukin 2 is obtained as a 11-12S fraction.

In order to confirm that the mRNA obtained as described above corresponds to the target interleukin 2, one can either translate the mRNA into protein and examine its physiological activity, or use an antibody etc. to analyze the protein. What is necessary is to perform a method such as identifying the . For example, the system commonly used to translate mRNA into protein, Xenopus
Translation has been carried out by injecting mRNA into oocytes (1aev+s) or translating it into protein using a cell-free system using rabbit reticulocyte lysate, wheat germ, or the like.

The interleukin 2 activity assay method used here is as follows. That is, 100 μp of the sample was added to the first row of a 96-well microtiter plate, and the dilution was repeated 2-fold in RPMI 1640 medium containing 2% fetal bovine serum to make each 100 μ2 dilution series on the 96-well microplate. create. Gillis et al. (Nature,
268, p. 154, (1977)) was prepared according to the method taught by
100μρ as cell density of X 103 cells/100μρ
Add to each well. After static culture in a 5% carbon dioxide incubator at 37°C for 20 hours, 0.5 μCi of tritiated thymidine was added and pulsed for 4 hours, and the cells were harvested according to methods well known in this field. Measure the amount of radiation taken into the Since a culture supernatant with high interleukin-2 activity has a large amount of tritium thymidine incorporated into activated T lymphocytes, the amount of interleukin-2 contained in the sample can be easily determined.

Furthermore, interleukin 2 has the effect of causing T lymphocytes to divide and proliferate, and the method for assaying T lymphocyte proliferative activity based on this effect is as follows.

Transfer 100 μP of the sample to one of the 96-well microtiter plates.
DMEM medium containing 2% fetal bovine serum is added to the column and repeated 2-fold dilution to create a dilution series of 100 μp each on a 96-well microplate. Five activated T lymphocyte strains described above are added to each well at a cell density of 7100 μl to 100 μβ.

The cells are incubated at 37° C. in a 5% carbon dioxide incubator for 72 or 96 hours, and then the number of surviving activated T lymphocytes is counted using an inverted microscope.

At this time, 1QQu/mj! , 10u/m! Interleukin 2, which has an activity of

Complementary NA (cDNA) is synthesized in vitro from the thus obtained interleukin 2 mRNA and connected to a replicon derived from a microorganism.

cDNA synthesis can usually be carried out in vitro by the following method.

Using mRNA as a template and oligo dT as a primer,
Jl, 1Jl complementary to mRNA by reverse transcriptase in the presence of dATP, dGTP, dCTP, dTTP
After synthesizing cDNA^ and decomposing and removing the template mRNA with alkali treatment, we use $1ficDNA as a template,
Duplex 1 using reverse transcriptase or DNA polymerase
Synthesize JlcDNA. Both ends of the obtained DNA are treated with exonuclease if necessary, and an appropriate linker-DNA is connected to each end, or a plurality of bases in an annealing possible combination are polymerized. This is then integrated into a vector containing a replicon capable of autonomous replication in, for example, Escherichia coli. The method of integration involves cleaving the vector with an appropriate restriction enzyme and, if necessary, polymerizing an appropriate linker or a plurality of annealing-enabled combinations of bases. Double glDNA processed in this way and vector D
Mix the NAs and connect using ligase.

The obtained recombinant DNA is introduced into a vector host microorganism. Although Escherichia coli is used as the host microorganism in the present invention, Bacillus subtilis, Satucharomyces cerevisiae, etc. can also be used. Examples of vectors used in the case of Escherichia coli are shown below. (Refer to Protein Nucleic Acid Enzyme Vol. 26, No. 4 (1981)) EK-based plasmid vector (stringent type) p
si:101. p) 353. pRK646.
248 to pR. pDF41 etc.

Ca1 of EK-based plasmid vector (relaxed type)
E1. pVE51. pAc105. R5F
2124. pcRl, pMB9゜pBR313
, p8R322, pBR324, pBR325, pBR
327゜pBR328, pKY2289. pKY27
00. pKN80. pKC7゜pKB158. p
MK2004. pAcYcl, pAcYc184.
λdu1 etc.

λgt-based phage vector λgt・λC1λgt-1
゜λWES-λC1λWES-λB', λZJvir・
λB', λ^LO・λB.

λWES4s622. λDam et al.

Among these vectors, pBR322 is commonly used for Escherichia coli.

In the case of pBR322, the cDNA integration site is Pst
I site and EcoRI site are often used.

The method for transforming a microbial host using a plasmid with cDNA integrated into a plasmid vector is mainly to collect cells in the logarithmic growth phase and treat them with CaCR2 to allow them to grow naturally, making it easier to take in NA and take in the plasmid. MgCl2 or Rb
It is also known that the efficiency of transformation is further increased by coexisting C1'. Alternatively, microbial cells may be transformed into sphnyloplasts or protoplasts before transformation.

The following methods can be used to select a strain in which the interleukin 2 gene has been integrated from transformed strains.

That is, this selection method is called the plus-minus method, and first, mRNA is extracted from Jurkat cells stimulated with ConA, and interleukin 2 mRNA is partially purified by SDG centrifugation.
2S mRNA), and this mRNA is used to synthesize one cDNA labeled with 32p. Then cD
After removing the mRNA that served as a template for N8 by alkaline treatment, this cDNA and 11-12s mRNA were extracted from Jurkat cells that had not been stimulated with ConA.
Hybridize with excess partially purified mRNA of A.

Then, c that hybridized with cDNA that did not hybridize to this ConA-unstimulated mRNA.
The DNA is fractionated using a hydroxyapatite column and designated as probe A and probe B, respectively.

Next, the transformed strain is grown in advance on two nitrocellulose filters in exactly the same manner, and the DNA is fixed on each filter by alkali treatment. Then, after hybridizing to the filter separately using probe A and probe B prepared earlier, autoradiography is performed, and the reaction is positive (positive) with probe A, but weak with probe B, which makes it difficult to react. searches for colonies that do not react at all (minus) (Taniguchi et aR, Paoc
, Jpn.

Acad, voR55B, 464-469 (19
79)).

Or a transformed strain, for example 1000 to 10,000
The clones are divided into clusters of several tens to hundreds of clones, the transformed strains of each cluster are mixed and cultured, and plasmid DNA is prepared (by a conventional method). Next, these DNAs are made into single-stranded DNA by heat denaturation, etc., fixed on a nitrocellulose filter, and then hybridized with mRNA prepared from human T leukocyte cells containing interleukin 2 mRNA as described above, which is complementary to these DNAs. Soybean. Alternatively, after heat denaturing the DNA, interleukin 2 is first added.
After hybridizing the mRNA containing mRNA, D
Another method is to immobilize the NA-mRNA hybrid on a nitrocellulose filter.

This filter was then treated with 1mM Bibes, 10mM N
aCJ! After thorough cleaning with a low salt solution such as
In a solution such as MEDTA, 0.1% SDS, for example at 95°C.

m that was adsorbed on the filter after heat treatment for about 1 minute.
Elute the RNA. Then, operations such as applying this to an oligo dT-cellulose column are performed to recover mRNA. Next, this 0I RNA is injected into African frog oocytes to be translated into protein, and interleukin 2 activity is measured. Alternatively, after translating into protein using a rabbit reticulocyte or wheat germ in vitro translation system, interleukin 2 is assayed using an interleukin 2 antibody.

Thus, once a population with detected interleukin-2 activity is found, the number of mixed clones in this population is subdivided into smaller populations, and the above-mentioned method is repeated to finally subdivide into single clones and interleukin-2 activity is detected. A clone containing DNA encoding Leukin 2 polypeptide! # Release.

In order to obtain DNA encoding interleukin-2 polypeptide from the obtained clone, a section corresponding to plasmid DNA is isolated from the microbial cell by a conventional method, and the DNA portion inserted into the vector used is isolated from the plasmid DNA. All you have to do is cut it out.

The structure of the DNA encoding the interleukin-2 polypeptide was determined by the chemical method of Maxam-Gi 1bert (Me
Th.

Enzym, 65.499-580. f980) and dideoxynucleotide termination method (Smith,
A., J., H., Meth.

Enzym, 65.560-580.1980) and the like.

The DNA isolated in the present invention and in which a polypeptide having interleukin-2 activity was expressed is shown below.

□-No-ku 20 zl-1/IF-12: 口ltηku L+ω Colo -Q ΦE--<<(C-!30 Colo-Q L-<2← L-<CIjE-t z< +e: w l/)< Koro -ω-ku ←
<<<+-<<mouth Σku L+! + Tameku ηRo 2← Seki C+50Ro -Ro ≧ku ηKoro ri<C5C5-<<< Kukutodo L+← Koro oI-) -Rho c5+e: ω ΦF-L, lj -ζRoto ←ku
−← −〇 〉Rozuku mouth t-(IJE--ro koku〈 -ω −← Φto< ku ← Ko(a)← ← ← ← C5C51-1<E-to ← ・ C5 ro ro, ← ku Ku ← Ku〉 Ro 〉l+5 ← ← Ro ←＜＜
<B)-1Ro Ku ← +-+<Fm<E-< 1m L) C
J/<<C5E-< Cj CJ Hee<<
Roku ← Ku ← <(j C50-ku ← ω Kukuku RoE-I Riku Ku Ro Kutoku ← =1-) ← Ku ← <<< b In the above base sequence, if the molecular weight is 15,000 Daltons. The only reported frame capable of encoding interleukin 2 is that shown in the above base sequence. In most cases, the initiation of protein synthesis begins with the first ATGT don on the mRNA, so the interleukin 2 polypeptide extends from the first initiation codon ATG to eight CT (threonine) before the termination codon TGA. It is thought that the base sequence corresponds to Furthermore, in secreted proteins such as interleukin 2, there is a so-called signal peptide rich in hydrophobic amino acids, and this peptide is cleaved during secretion to form a mature protein. Judging from the above base sequence, it is thought to correspond to an N-terminal signal peptide rich in hydrophobic amino acids. Therefore, even a peptide corresponding to, for example, from the A-D-G codon to the 21st codon GC^ (alanine) to ACT before the termination codon TGA is considered to have interleukin-2 activity. In addition, it is a polypeptide that retains the active site of the interleukin-2 protein even if it lacks some amino acids after the above-mentioned alanine or before the above-mentioned threonine and has multiple consecutive base sequences. If so, it is highly conceivable that it has interleukin-2 activity.

I) of the present invention At the 5'-end to N, interleukin 2
One or more bases that are not harmful to gene expression may be arranged. Furthermore, even if a base that can express one or more amino acids is arranged at the 5'-end, the added amino acid may not be harmful because the polypeptide has interleukin-2 activity, or
Furthermore, even if it is harmful, there is no problem as long as it can be easily removed.

Similarly, for the 3'-terminus, the nucleotide sequence is such that a non-toxic amino acid is added to the C-terminus of the polypeptide in order for the polypeptide to have interleukin-2 activity.
It may be added to the end, and even if it is harmful, there is no problem as long as it can be easily removed. It is also possible to modify a part of a gene or a part of a polypeptide deduced from the gene structure by chemical synthesis of DNA or the like. Furthermore, genes can also be obtained using mRNA obtained from cells other than the human cells described in the Examples below. Therefore, in short, any I)N8 that encodes a polypeptide having interleukin-2 activity can be used in the present invention.

Next, the present invention will be explained in detail with reference to examples.

Example 1 (1) RP containing 10% v/v fetal bovine serum
Human T leukemia gel-cut cells (Japan) were cultured in M11640 medium by a method well known in the art.

(freely available in the United States, West Germany, etc.) was suspended in the above medium and heated for 50 seconds at room temperature using a Toshiba X-ray irradiation device EX.
A total exposure dose of 1,000 roentgens was delivered using a model S 150/300-4. The irradiated cells were then cultured at a cell density of 1X10' cells/In1 in the above-mentioned medium at 37° C. in 5% carbon dioxide gas for 5 days. Next, this mutant cell was inoculated into 10 96-well microplates at 0.2 cells/well, and incubated at 37°C in 5% carbon dioxide gas.
It was cultured for 1 day. Clones with observed cell proliferation were serially subcultured, and then ConA SOB/mj! was grown at a cell density of 1 x 106 cells/mJ! The production amount of interleukin 2 released into the culture supernatant was measured using the method described above.
A mutant clonal cell strain Jurkat 111 (ATC (: CRL8129)) which was improved by more than 0 times was obtained. The tree strain was grown by a normal culture method, and its proliferation rate was comparable to that of Jurkat cells.

(2) 1 x 105 cells/I of this Jurkat 111 cell line
I! Serum-free synthetic medium RITfl at a cell density of: 55-
The cells were suspended in a solution of 91,000++1, placed in a rotary culture bottle manufactured by Falcon, and cultured at 37°C for 4 days, and the cells were collected by centrifugation. The cells were suspended in the above-mentioned medium at a cell density of 4 x 106 cells/mJ, and injected with 25μ of ConA.
g/mp, and the above-mentioned Falcon rotary culture bottle (4
The cells were plated at 1.000 n+iV in a tube) and cultured with rotation for 6 hours.

(3) ConA 25ug/++l thus obtained
Jurkat cells (1,2x 1a1
0 cells) were suspended in 800 ml of PBS solution, the cells were washed twice by centrifugation, and then treated with Ribonucleosides-Vanadyl, a nuclease inhibitor.
(: R5B solution containing oplex (10mM) (1
0mM Tris-HCjl, pH 7, 5, 10mM
NaCR, 1,5mM MgCj'z) 800+u
suspended in il. Next, NP-40 was added to a concentration of 0.05%, stirred gently, and centrifuged at 3,000 rpm for 5 minutes to remove the nuclei.
After adding 5%) and EDTA (5mM), an equal amount of phenol was immediately added to extract cytoplasmic RNA. Repeat the phenol extraction a total of three times and then RN with 2 volumes of ethanol.
A was precipitated, the precipitate was collected by centrifugation, and 10n+M Tri
Dissolved in s-HCi', pH 7.5.

The amount of RNA thus obtained from Jurkat cells was 196 mg.

Next, oligo(dT)-cellulose (P, L, Biochemical
Column chromatography was carried out using the following column chromatography. Adsorption was performed using 20mM Tris-HCl,
p) I 7.5.0.5M NaCl+.

RNA in 1 mM EDTA, 0.5% SDS solution
The elution was performed using a buffer solution (20+nM Tri
s-HCj), p)I 7.5°0.5 M Na
C1+, 1mM EDTA), then water and 10mM
mRNA alternately with Tris-HCJ (p)I 7.5)
This was done by eluting A. As a result, the amount of eluted mRNA was 3.6 mg.

Furthermore, a portion (2.4 mg) of this mRNA was subjected to SDG centrifugation (50 mM Tris-HCj!, pH 7,5,1
5-25 containing mM EDTA, 0.2M NaCl+
% sucrose density gradient, Hitachi RPS 280-ter at 26,000 rpm, 24 hours, 4°C), and the mRNA fractions 11-123 were fractionated with fraction number 12°13.
, 14, 59 μg, 46 μg, and 60 μg were obtained, respectively.

(4) Oocytes obtained by microinjecting 50 ng of the mRNA of fraction number 13 obtained here into African frog oocytes according to the above-mentioned assay method. Interleukin 2 was added to the cell culture supernatant.
When subjected to an activity assay, the incorporation of tritiated thymidine and the increase in the number of activated T lymphocytes as shown in the following table were observed, proving that the mRNA of these fractions contains the human interleukin 2 mRNA of the present invention. It was done.

Table-1 (a) (b) - Amount of tritiated thymidine incorporated at each dilution (cp
m) Probit plot of standard interleukin 2
(10 units/1aR).

(5) Next, the interleukin 2mRN obtained here
cDNA 8 was synthesized in vitro from the 11-123 mRNA fraction 13 containing A, and the plasmid vector PBR32
2 and recombinant DNA was prepared, which was transformed into Escherichia coli, and cells having an interleukin 2 cDNA clone were selected by the following method.

(5-1) 50mM Tris-HC1' buffer (p
H7,5), 30+nM NaCN, 6mM Mg
Cl! 2. 5mM dithiothreitol (DTT),
0.5mM each dATP, dGTP, dCT
P, dTTP (dCTP includes 32p labeled), 0.7μg oligo (dT), 10g
g mRNA and 15 unit 8MV reverse transcriptase (J
, W., Beard) and kept at 41°C for 90 minutes. After the reaction was completed, phenol treatment was performed once, and DN8 was recovered as ethanol precipitate, followed by 20 mM Tris.

Dissolved in 1 mhl EDTA pH 7.5 solution. As a result, about 2.5 μg of single-stranded CDNA was synthesized.

To remove mRNA from this solution, a NaOH solution was added to make 0.33N NaOH for 15 hours at room temperature, and then the solution was mixed with IMTris-11cj), pi(7,5
The cDNA was neutralized with an equal volume of 100 μg of cDNA and passed through a Sephadex G-50J column. As a result, 1.8 μg of cDNA was recovered.

(5-2) 50 mM phosphate buffer (pH 7.5),
10mM MgCl12. lomM DTT, 0.
75mM each of cATP, dGTP, dCTP.

dTTP (dCTP includes those labeled with 3H).

1.8μg single-stranded cDNA, 8 units Polya+erase I (manufactured by BRL, USA)
were mixed and the reaction was carried out at 15°C for 15 hours. After the reaction was completed, phenol treatment was performed once and chloroform treatment was performed once, and the DNA was recovered as ethanol precipitate. This reaction yielded 1.10 Mg of double-stranded cDNA.

Then 50 mM sodium acetate (pH 4,5),
0.2M NaCR, 1mM ZnCR2,1,10Mg
After mixing the duplicate 1jl cDNA and incubating for 20 minutes at 37°C, 0.25 units of nuclease S+ (
(manufactured by Sankyo ■) was added, and the mixture was further incubated for 15 minutes. After the reaction was completed, the mixture was treated with phenol twice and passed through a "Sephadex G-50" column to recover 0.55 Mg of duplex 1jl cDNA.

(5-3) 0.14M potassium cacodylate, 30
mM Tris base, 0.1mM DTT, 1mM
C0Cj! i, 0.64mM “P-dCTP
(Specific activity 2.7X 10'cpm/nmojri, 0
．． 55 μs double-stranded cDNA and 5 units of terminal transferase (BRL) were mixed and incubated at 37°C for 7 minutes. After the reaction was completed, phenol treatment was performed once, and the DNA was passed through a Sephadex G-50J column as ethanol precipitate. When 50 Mg was recovered, about 50 dCMPs were added to both 3' ends.

pBR322DNA (Gene, L?tar j+
3 (l?77))', Ni□--10 μg was cut with the restriction enzyme Pst I, and then subjected to dCT under exactly the same conditions as when dCM P kl was added to the double-stranded cDNA described above.
dGMPfi at both 3' ends using dGTP instead of P
Added. Approximately 50 dGMPs were thus added to both 3' ends.

(5-4) 50IIIM Tris-IR (pH 7,
5), 0. IM NaCR.

pBR322 with 5 mM EDTA, 0.05 Mg of dGMP, and 0.019 g of dc&IP-loaded cDNA were incubated at 65°C for 2 min, then at 46°C for 120 min, then at 37°C for 60 min, and at room temperature. 6
It was held for 0 minutes.

50 mj of Escherichia coli χ177G! of L medium 100 μg 7 mR diaminopimelic acid and 50 Mg/
ml! of thymidine, 1% tryptone, 0.5% yeast extract.

(containing 0.5% NaCl and 0.1% glucose), and the absorbance at 562 mμ of the culture solution was approximately 0.
．． The cells were cultured with shaking at 37°C until the number of cells reached 3. After culturing,
The culture solution was left at 0°C for 30 minutes, and then the bacterial cells were collected by centrifugation and treated with 5 mM Tris-HC1! (pH7,6
).

0.1M NaCl, 5mM MgCRt, 10m
M RbCR solution 2Smj! Washed twice with

The obtained bacterial cells were injected with 5 mM Tris-)ICJI (p
) I 7.6).

0.25M KCJ! , 51MM MgCR2,
0,1M CaCRz and 10mM RbCj! 20mj of solution containing! After suspension at 0° C. for 25 minutes, the bacterial cells were collected by centrifugation. 1 ml of the same solution as above
Resuspend the bacterial cells in 0.2 m of the resulting bacterial cell suspension.
The above recombinant DNA was added to A+ and allowed to stand at 0°C for 40 minutes. Furthermore, after keeping it at 37℃ for 2 minutes, it was heated again to 0℃ for 6 minutes.
It was left standing for 0 minutes. Next, add 0.7 mj of medium as described above!
was added and incubated at 37°C for 30 minutes. Add 0.1 mR of this culture solution to 100 μg/mp diaminopimelic acid,
0Mg/ml! It was spread all over a 1.5% agar medium containing thymidine and 15Mg7mA tetracytalline, and incubated at 37°C for 2 days.

(5-5) Diaminopimelic acid at 100 μg/mx as a mixture of 18 groups each consisting of 24 colonies of 432 colonies that appeared above.

Inoculated into 200 ml of L medium containing 50 Mg, 7 mR of thymidine and 10 μz/ml of tetracycline, and incubated at 37°C.
After shaking culture for 5 to 7 hours, the final concentration of chloramphenicol was 170 gg/mj! Add 200 mJ of fresh above-mentioned medium to give a total volume of 200 mJ, and incubate again with overnight shaking.

The plasmid DNA was thus amplified and purified according to a conventional method. Using this DNA,
ybridizatfon-translation
Cucoons containing interleukin 2 cDNA were screened using an assay method. Hybri used here
dLzation-translation assa
y was carried out as follows.

25μs of purified DNA was treated with restriction enzyme HindI.
II, the DNA was treated with phenol three times, phenol-chloroform once and once with chloroform, the DNA was precipitated with ethanol, washed with 80% ethanol and collected, and this was added to 40μ of an 80% formamide solution.
! and heat denatured at 90°C for 5 minutes. After that, 1
0x SSC (1,5M NaCj, 0.15M:'
diluted to 1.3 mj with trisodium citrate). This was fixed on a nitrocellulose filter and dried by heating at 80° C. for 3 hours. Filter this with 50% formamide.

20mM Pipes, pH 1! , 5.0.75M NaC
j, 5mM EDT^.

The DNA on the filter was incubated with interleukin 2 mRNA for 18 hours at 37° C. in a solution containing 0.2% SOS and 250 μg mRNA to hybridize. Next, give this filter a 10! L
M pipes pH 6, 5, 0, 15M NaCj,
1 e+M EDT^, 0.2% SDS solution at 65°C
Wash three times with additional 1 mM Pipez.

Washed three times with 10mM NaCj solution, then 0.5mM
The mRNA adsorbed on the filter was eluted by treatment with MEDT^ and 0.1% SDS solution at 95°C for 1 minute. This was collected by applying it to an oligo dT-cellulose column according to a conventional method. The recovered mRNA was injected into African frog oocytes, translated into protein, and interleukin 2 activity was measured. As a result, an activity of 48 units/mR of interleukin 2 was detected in one of the 18 populations by the above-mentioned activity assay method based on the amount of tritiated thymidine taken up. Therefore, 24 colonies belonging to this group were individually separated into 100 μl cells as described above.
200 ml of L medium containing g/mR diaminopimelic acid, 50 Bg/mR thymidine and 10 Mg/mp tetracytalline! After shaking culture at 37°C for 5 to 7 hours, chloramphenicol was added at a final concentration of 170 μg/
200ml of fresh medium added to rtllll
a+R was added, and the plasmid DNA was amplified by overnight shaking culture.
was purified. Then, 5 μs of each DNA was
After cutting with II, it was fixed on a nitrocellulose filter as before and hybridized with interleukin 2 mRNA, and the mRNA was collected and injected into African frog oocytes to be translated into protein and interleukin 2. Activity was measured to determine which of the 24 colonies contained the interleukin 2 clone. Purified plasmid DNA was obtained from one colony, and mRNA that hybridized to plasmid p3-16 was translated into oocytes. Interleukin 2 activity was found in the clone that had undergone interleukin 2 (Table 2), and this clone showed interleukin 2 activity.
Clone with cDNA (Escherichia coli χ177
a/3-teA, +11995 (FERM-BP22
5)). i.e. plasmid p3-18
cDNA was shown to contain DNA (interleukin 2 gene) that specifically hybridizes with interleukin 2 mRNA.

Next, we examined the restriction enzyme cleavage sites of the cDNA of plasmid p3-1fi, and found that one site was cut with XbaI (aRL, USA), and one site was cut with BstNI (New England, USA).
dBio Lab, Inc.) at two locations (upstream and downstream of the Xbal cleavage site). However, this cDNA consists of approximately 650 base pairs and was found to correspond to part of the 11-12S interleukin 21 RNA.
Human interleukin 2 mR prepared in the same manner again
Land et al.'s method using NA as a template (Land et al.
aR, Nuclefi Ac1ds Res.

vol, 9. p2551 (1981)) in the same manner as described above, and the plasmid pBR322
inserted into. Using this plasmid, E-coli χ1
776 was transformed, and cDNA clones having the same sequence as the cDNA of plasmid p3-16 were selected from approximately 2,000 transformed strains by Grunstein-Hognes
A transformant strain (Escherichia coli
A AJ11996 (FERM-BP22B)) was obtained.

A restriction enzyme cleavage map of this pIL2-50A cDNA is shown in Figure 1.

Table 2 (A) (B) ◆l mRNA piperidized to cDNA of plasmid p3-16 Next, as shown in Figures 2a and b, pBR328 plasmid vector (Gene, !, 287-305 (19
80)) into pKCR vector (Proc, Natl,
Aca, Sci, USA, vol 78°No
, 3.1527-1531.1981) SV 40
A pCE-1 vector incorporating a region containing the promoter of the viral early gene was constructed. From the plasmid pIL-2-50A clone, Pst
The cDNA inserted with I was cut out and inserted into the PstI site of the pCE-1 vector (plasmid pcEo,
-2). In addition, Escherichia coli) IB 101 (AJ
12008) has been deposited under accession number FERMBP-244. As shown in Figure 2, the insertion mode at this time is that the initiation codon ATG of the interleukin 2 gene is connected downstream of the promoter of the SV40 early gene (I in the diagram). This vector was introduced into monkey cultured CO5-7 cells (Gluzman, Y.
, Ce1l vat, 231) 175-182 (
Mc Cutchan e (1981))
tajl J, Natl, Cancer In5t
, vol, 41 p351-357 (1968))
The interleukin-2 activity in the culture supernatant was investigated. As a control, an experiment was conducted in the same manner using vector plasmid pcE-1 to examine interleukin 2 activity. The results are shown in Table-3.

Table 3 Furthermore, this interleukin 2 activity was neutralized by mouse anti-human interleukin 2 monoclonal antibody.
Interleukin 2 activity is 1 u/mj! It became the following.

The DNA encoding the polypeptide with interleukin-2 activity is derived from Escherichia coli χ1778/IL.
-2-5Isolate the plasmid DNA portion from OA cells by a conventional method, and digest the obtained plasmid DNA with the restriction enzyme PstI.
After digestion, a fragment with a smaller molecular weight than the two generated DNA fragments was separated.

The base sequence of the obtained DNA was determined by the Maxam-Gil
When investigated according to the chemical method of Bert, it was found that the sequence was as described above.

[Brief explanation of the drawing]

Figure 1 is a restriction enzyme map of a vine gene encoding a polypeptide with interleukin-2 activity. FIG. 2 is an explanatory diagram of the construction progress of pCEIL-2. Patent applicant Cancer Research Foundation Ajinomoto Co., Inc. 1.

Claims (6)

[Claims] (1) Recombinant human interleukin 2 that does not substantially contain other human-derived proteins. (2) Recombinant human interleukin 2 that does not substantially contain other human-derived proteins containing the following amino acid sequence in its molecule. [There is a gene sequence. ] (3) Recombinant human interleukin 2 substantially free of other human-derived proteins obtained by a transformant having a vector containing a gene encoding human interleukin 2. (4) Recombinant human interleukin obtained by a transformant having a vector containing the gene encoding human interleukin 2, and which does not substantially contain other human-derived proteins containing the following amino acid sequence in the molecule: 2. [There is a gene sequence. ] (5) Production of recombinant human interleukin 2 substantially free of other human-derived proteins, characterized by culturing a transformant containing a vector containing the gene encoding human interleukin 2 in a medium. Law. (6) culturing in a medium a transformant having a vector containing a gene encoding interleukin 2 containing the following amino acid sequence in the molecule; A method for producing recombinant human interleukin 2 substantially free of other proteins. [There is a gene sequence. ]