JPH0832236B2 - Recombinant human interleukin 2 and method for producing the same - Google Patents

Description

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

It has been reported that interleukin-2 has been found to be a human T leukemia cell line that produces interleukin-2 due to its unique immune response and application to medicines (Gillis et al., J. Exp. Med. ., 152, 1709, 1980).

However, the amount of interleukin 2 produced by this human T leukemia cell line is very small, and the production thereof requires a large amount of cells to be cultured, which poses a difficult problem. As another method for producing interleukin 2, it may be considered that a DNA corresponding to interleukin 2 is incorporated into a vector of a microorganism, replicated, transcribed and translated in the cells of the microorganism to be produced by the microorganism.

The present inventors have succeeded in isolating a DNA encoding an interleukin 2 polypeptide necessary for production by a microorganism, and have opened the way for the production of interleukin 2 by a microorganism.

The present invention has the formula: Human interleukin 2 containing the amino acid sequence shown by
A polypeptide having activity or a deletion of one or several amino acid residues with respect to the amino acid sequence of the polypeptide,
A polypeptide having a human interleukin-2 activity having an added or substituted amino acid sequence and substantially free of other human-derived proteins with a sugar chain, and a polypeptide having these human interleukin-2 activities are encoded. A polypeptide having human interleukin-2 activity, which is associated with a sugar chain and is substantially free of other human-derived proteins, characterized by culturing a transformant into which a vector containing a gene for Manufacturing method.

A representative example of the polypeptide having human interleukin 2 activity of the present invention is one having the following amino acid sequence II. The following amino acid sequence I has Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Se at the N-terminal of the amino acid sequence II.
r Leu Ala Leu Val Thr AsN Ser is added and has human interleukin 2 activity.

Amino acid sequence I Amino acid sequence II The polypeptide having human interleukin 2 activity of the present invention can be obtained by culturing a eukaryotic cell into which a vector containing a gene encoding the polypeptide has been introduced in a medium. Specific examples of this gene include a portion of the DNA chain in which the sites cleaved by restriction enzymes BstNI, XbaI and BstNI are arranged in this order, and encode the following polypeptide I or II. , Having the following base sequence I or II.

Polypeptide I Polypeptide II Nucleotide sequence I Base sequence II Such DNA is obtained as a 11-12S fraction by fractionation by sucrose density gradient centrifugation, and is prepared from messenger RNA (hereinafter abbreviated as mRNA) separated from human or murine lymphocyte-derived cells. It That is, DNA prepared from mRNA of 11 to 12S fraction by sucrose density gradient centrifugation obtained from lymphocyte-derived cells having interleukin-2 producing ability is ligated to, for example, Escherichia coli plasmid vector. -A clone having a DNA capable of expressing the interleukin-2 polypeptide can be isolated by amplifying it in E. coli cells, and the DNA inserted into the plasmid of the isolated clone can be separated.

In the present invention, a DNA encoding a polypeptide having interleukin-2 activity is capable of producing an interleukin-2 producing ability of a microorganism by introducing a recombinant DNA obtained by connecting to a replicon derived from the microorganism into a host microbial cell. Can be transformed to have.

As described above, mRNA corresponds to interleukin 2 and is obtained as a fraction of 11-12S by fractionation by SDG centrifugation or gel filtration and agarose gel electrophoresis. The mRNA is lymphocytes. Extracted from origin cells,
It can be manufactured by separating.

When the human lymphocyte-derived cell having the ability to produce interleukin 2 used in the present invention is described as an example, lymphocytes themselves obtained from human peripheral blood, tonsils, spleen, etc. are also included. In addition, these lymphocytes were subjected to pretreatment such as nylon column treatment, antiserum-complement treatment, density gradient fractionation and enzyme (neuraminidase, galactose oxidase, etc.) treatment, mutation treatment by X-rays, trypsin, etc. The human lymphocyte-derived cells of the present invention include those to which the interleukin-2 producing ability has been imparted by the oxygen treatment and the like. Further, those obtained by cloning these human lymphocyte-derived cells, such as those obtained by cloning T lymphocytes in the presence of T lymphocyte growth factor and the like, are also preferred human lymphocyte-derived cells.

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

Furthermore, so-called hybridomas obtained by cell fusion of the above human lymphocyte-derived cells with human tumor cells such as CEM and 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 by stimulating them by contact with mitogen in the presence or absence of other cells. There is something.

When interleukin-2 mRNA is produced in human lymphocyte-derived cells, if a spontaneous interleukin-2 producing strain is used, these cells may be cultured by a usual method. When using cells that produce interleukin-2 by mitogen stimulation, wash the cells thoroughly and then use Rosewell Park Memorial Institute 1640.
(Hereinafter abbreviated as RPMI 1640.) Medium for normal cells such as medium, Dulbecco's modified Eagles medium and click medium (with or without serum or serum components) ) Or synthetic serum-free medium at a cell density of 0.5 to 4 × 10 ⁶ cells / ml, and mitogen; neuraminidase, galactose oxidase;
Zinc compounds such as zinc chloride; after addition of cell-derived lymphocyte activating components such as protein A and streptolysin-O,
Wash cells and remove stimulant.

Cells capable of producing interleukin 2 when coexisting with macrophages or dendritic cells during mitogen stimulation, B lymphocytes, and B lymphocyte-derived cell lines Raji, Daud
There are cells that have the same ability to produce interleukin 2 when i, K562 and BALL-1 cells are coexistent. When these cells are used to produce interleukin 2 mRNA, these cells can be produced. Stimulate in the presence of. This may increase the yield of mRNA.

Human lymphocyte-derived cells are passaged and grown in vitro or in animal species under normal conditions. Culture subculture in vitro can be performed using a normal cell culture medium, and a synthetic serum-free medium that does not even contain serum albumin even if it contains mammalian-derived serum, serum components or serum albumin. It was found that these cell lines can be cultivated and proliferated even in a medium and can be used as the cell material of the present invention.

The culture time of lymphocyte-derived cells is the time when lymphocytes are activated and interleukin-2 mRNA is produced, and this time corresponds to the time when interleukin-2 is started to be produced in the cell culture supernatant. . Specifically, it is usually 3 to 12 hours after the addition of the stimulant. If you extend the culture time to the people,
The generated interleukin-2 mRNA is degraded. In addition, phorbol esters such as PMA and TPA may be added in an amount of 10 to 50 ng / ml at the time of lymphocyte activation.
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.

(B) Acquisition of lymphokine spontaneously-producing strain Jurkat cells, which are human T lymphocyte-derived leukemia cells (Fred Hutchinson Cancer Institute / Seattle / USA, Salk Institute / Sanjego / USA, West German National Cancer Center / Heidelberg / West Germany) Etc.) are suspended in Click medium at a cell density of 1 × 10 ⁶ cells / ml, and the total is obtained at an irradiation rate of 150 roentgen / min.
Perform X-ray irradiation of 8,000 roentgens. After this, the cells were added to a 96-well flat-bottomed microtiter plate (“Falcon 3072”) at a cell density of 0.1 cells / 200μ and added to 5%.
5% at 37 ° C for 3 weeks in Click medium containing fetal bovine serum
Culture in a CO ₂ incubator (cloning by limiting dilution method). The cells in the culture wells in which the growth of the cells are observed are transferred to a 24-well Nunc culture plate before the cells reach the density covering the entire bottom surface, and the cells are allowed to grow in 2 ml of click medium for 5 days. . When a sufficient amount of cells is obtained, the cells are serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 2 × 10 ⁶ cells / ml.
Suspend in 2 ml and cultivate for 2 days.
Separation is carried out by a centrifugal operation for 1 minute, and then debris removal and sterilization are carried out with a 0.22 μ millipore filter to obtain interleukin-2. An interleukin-2 producing strain can be obtained from the thus obtained clonal cells.

(B) Acquisition of interleukin-2 producing strain from human peripheral blood T lymphocytes Human peripheral blood is collected and peripheral blood lymphocytes are collected by Ficoll-Hypaque density gradient centrifugation. The peripheral lymphocytes are suspended in a click medium at a cell density of 1 × 10 ⁶ cells / ml and inoculated into a Nunc culture plate having 24 holes each containing 2 ml. 100 μl of phytohemagglutinin-M (manufactured by Gibco) was added thereto so that the final concentration was 5 μg / ml, and the cells were cultured under the above-mentioned conditions for 48 hours, and then the cells were washed with the culture medium and again 1 × 10 ⁵ Spread 1 ml onto the original Nunc culture plate at a cell density of 1 / ml. Human spleen cells were added to each well at a concentration of 2.5 μg / ml of concanavalin A (hereinafter referred to as ConA) 4.
1 ml of conditioned medium obtained by stimulation for 8 hours
After addition, 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 thus obtained by long-term subculture are cloned and cell-grown by the limiting dilution method as described above. The cloned T lymphocytes thus obtained were added to a cell density of 1 × 10 ⁶ cells / ml.
Suspend in RPMI 1640 medium and add phytohemagglutinin (PHA) to this to give a final concentration of 10 μg / ml.
Cultivate at 37 ℃ in 7.5% CO ₂ incubator for
The main culture supernatant is separated by centrifugation at 3,000 rpm for 5 minutes,
Then, sterilization is performed with a 0.22 μ Millipore filter to obtain Interleukin 2. An interleukin-2 producing strain can be obtained from the thus obtained cloned cells.

(C) Acquisition of malignant cells derived from human lymphocytes that produce interleukin 2 by mitogen stimulation The Jurkat cells described above and the J-111 strain cloned by the limiting dilution method described above are serum-free medium and serum 1 described above. ConA 10 μg / ml in RPMI 1640 medium containing ~ 2%
When cultured in the presence of 2.5 μg / ml PHA for 24 hours, 10-4,000
It was found to produce units / ml of interleukin 2. It also produces interleukin 2 when cultured in the presence of zinc chloride, protein A and picibanil.

(D) Acquisition of cells that produce interleukin 2 by stimulating with mitogen in the presence of other cells or factors produced by the cells Human lymphocyte malignant cells Molt4F and clones cloned by the limiting dilution method described above One clone of cut cells,
The Jurkat 99 strain does not produce interleukin 2 even if it is cultivated for 24 to 72 hours by adding the above-mentioned lectin and mitogen in a wide concentration range. However, during this time, 5 to 10 u / ml of interleukin 1 which is one kind of monokine or
0.5 × 1 per 1 × 10 ⁶ cells / ml of K562 and Raji cells
0 ⁶ cells / ml corresponding coexisted allowed to 37 ° C. C. in Click medium, and cultured for 24 hours, the interleukin 2 10～100U / ml production.

Interleukin-2 from the cells thus obtained
The mRNA corresponding to can be extracted by a conventional method regardless of cell type. For example, NP-40, SDS, Trito
Use a detergent such as n X 100 deoxycholic acid, or use a physical method such as a homogenizer or freeze-thaw to partially or completely destroy and solubilize the cells. In order to prevent RNA degradation by RNase during extraction, it is preferable to add RNase inhibitors such as heparin, polyvinyl sulfate, bentonite, macaloid, diethylpyrocarbonate, vanadium complex, etc. to the extract. In addition, if necessary, an antibody corresponding to interleukin 2 is used to interleukin 2
Polysomes in the process of synthesis are allowed to settle, and mRNA can be extracted from them with a detergent or the like. mRNA is oligo dT
-Cellulose, poly U-Sepharose, Sephalol 2B
It can be purified by an adsorption column such as the above, a purification method by a batch method, a fractionation by an SDG centrifugation method, or the like. The mR corresponding to interleukin 2 was purified by such a procedure.
NA is obtained as the 11-12S fraction.

In order to confirm that the mRNA obtained as described above corresponds to the target interleukin-2, the mRNA is translated into a protein and its physiological activity is examined, or the protein is detected using an antibody or the like. May be identified. For example, mRNA is injected into oocytes of Xenopus laevis, which is a system often used for translating mRNA into protein, to cause translation, or it is converted into protein in cell-free systems such as rabbit reticulocyte lysate and wheat germ. It is being translated.

The interleukin 2 activity assay method used here is as follows. That is, 100 μl of a sample was added to the first row of a 96-well microtiter plate, and 2-fold dilution was repeated in RPMI 1640 medium containing 2% fetal bovine serum to prepare 100 μl of each dilution series on a 96-well microplate. create. Gillis et al. (Nature, 268, 154, (1977))
100 microns activated T lymphocytes lines created according to the methods taught, as cell density of 4 × 10 ³ cells / 100 microns by
Add to each depression. After static culture for 20 hours in 37 ° C, 5% carbon dioxide incubator, tritiated thymidine 0.5 μC
After adding i and pulsing for 4 hours, the cells are collected according to a method well known in the art, and the radiation dose taken into the cells is measured. Since the amount of tritium thymidine incorporated into activated T lymphocytes is larger in the culture supernatant having higher interleukin 2 activity, the amount of interleukin 2 contained in the sample can be easily known.

Interleukin 2 has an action of causing T lymphocytes to divide and proliferate, and the assay method for T lymphocyte proliferating activity by this action is as follows.

Add 100μ of sample to the first row of 96-well microtiter plate and add 2% DMEM medium containing 2% fetal bovine serum.
Repeat doubling and repeat each on a 96-well microplate.
Make a 100 μ dilution series. The above-mentioned activated T lymphocyte strain was added thereto at a cell density of 5 cells / 100μ to each indentation for 100μ. After static culture for 72 hours or 96 hours in a 37 ° C, 5% carbon dioxide incubator, the number of activated T lymphocytes present is counted with an inverted microscope. At this time, interleukin-2 having activity of 100 u / ml and 10 u / ml was used as a positive control, and the interleukin-2 activity of the sample was calculated by comparing with the proliferation number of T lymphocytes in the sample-added group.

In vitro complementary DNA (cDNA) is synthesized from the interleukin 2 mRNA thus obtained and ligated to a replicons derived from a microorganism.

The synthesis of cDNA can usually be performed in vitro by the following method.

Using mRNA as template and oligo dT as brimer, dAT
In the presence of P, dGTP, dCTP, dTTP, single-stranded cDNA complementary to mRNA was synthesized by reverse transcriptase, and the template mRNA was decomposed and removed by alkaline treatment. Synthesize double-stranded cDNA using transferase or DNA polymerase. If necessary, both ends of the obtained DNA are treated with exonuclease, an appropriate linker DNA is connected to each, or a plurality of annealable bases are polymerized. Then, for example, Escherichia
It is integrated into a vector containing a replicon capable of autonomous replication in E. coli. As a method for incorporating, a vector is cleaved with a suitable restriction enzyme, and if necessary, a plurality of bases of a suitable linker or a combination capable of annealing is polymerized. The double-stranded DNA thus processed and the vector DNA are mixed and ligated with each other using ligase.

The obtained recombinant DNA is introduced into the host microorganism of the vector. Escherichia coli was used as the host microorganism in the present invention, but Bacillus subtilis, Saccharomyces cerevisiae, etc. can also be used. The vector used in the case of Escherichia coli is exemplified below. (See Protein Nucleic Acid Enzyme Vol. 26 No. 4 (1981)) EK plasmid vector (stringent type) pSC1
01, pRK353, pRK646, pRK248, pDF41 etc., EK plasmid vector (relaxed type) CalEl, pVH51, pAC105, RSF212
4, pCR1, pMB9, pBR313, pBR322, pBR324, pBR325, pBR327, pBR
328, pKY2289, pKY2700, pKN80, pKC7, pKB158, pMK2004, pACY
C1, pACYC184, λdu1, etc., λgt-based phage vector λgt
・ Λc, λgt ・ λB, λWES ・ λC, λWES ・ λB ', λZJvir
・ ΛB ', λALO ・ λB, λWES ・ Ts622, λDam, etc.

Of these vectors, pBR322 is commonly used for Escherichia coli. In the case of pBR322, the Pst I site and the Eco RI site are frequently used as the cDNA integration sites.

As a method of transforming a microbial host using a plasmid in which cDNA is incorporated into a plasmid vector, cells in the logarithmic growth phase are mainly collected and treated with CaCl ₂ so that the DNA is naturally incorporated and the plasmid is incorporated. It is also known that the method is adopted and the efficiency of transformation is further increased by further coexisting MgCl ₂ or RbCl. Alternatively, the microbial cells may be transformed into spheroplasts or protoplasts before transformation.

The following method can be used to select a strain in which the interleukin 2 gene is integrated from the transformants.

That is, this selection method is called the plus-minus method. First, mRNA was extracted from Jurkat cells stimulated by ConA, and the interleukin 2 mRNA was partially purified by SDG centrifugation (11-12S mRNA). mRN
A is used to synthesize single-stranded cDNA labeled with ³² P.
Then, the mRNA serving as the template for cDNA synthesis is removed by alkaline treatment, and then hybridized with this cDNA and an excess of partially purified mRNA of 11-12S mRNA extracted from Jurkat cells not stimulated with ConA. Then, the cDNA that did not hybridize to the mRNA that was not stimulated by ConA and the cDNA that hybridized were fractionated by a hydroxyabatite column to obtain probe A and probe B, respectively.

Then, in advance, transform the transformants in exactly the same way with each 2
Grow on a piece of nitrocellulose filter, treat with alkali and fix DNA on each filter. Then, using the probe A and the probe B, which were prepared previously, respectively, were hybridized to the filter separately, and then autoradiography was performed. The probe A reacts positively (plus) but is weak to the probe B, or not at all. Search for unresponsive (minus) colonies (Taniguchi et al., Paoc. Jpn. Acad., Vol 55B.46
4-469 (1979)).

Alternatively, a transformant, for example, 1000 to 10,000 clones is divided into a population of several tens to several hundreds of clones, and the transformants are mixed and cultured for each population, and plasmid DN is used (by a conventional method).
Prepare A. Next, these DNAs are single-stranded DN by heat denaturation, etc.
Interleukin 2mR complementary to these DNAs fixed on a nitrocellulose filter as A described above
MRNA prepared from human T white blood cells containing NA is hybridized. Alternatively, there is a method in which after heat denaturing DNA, mRNA containing interleukin 2 mRNA is first hybridized and then the DNA-mRNA hybrid is immobilized on a nitrocellulose filter.

Next, this filter is thoroughly washed with a low temperature solution such as 1 mM pipes and 10 mM NaCl, and then heat treated with a solution such as 0.5 mM EDTA and 0.1% SDS at, for example, 95 ° C for about 1 minute to adsorb to the filter. Elute the selected mRNA. Then, the mRNA is recovered by performing an operation such as applying this to an oligo dT-cellulose column. Next, this mRNA is injected into Xenopus oocytes and translated into a protein, and interleukin 2 activity is measured. Alternatively, interleukin 2 is assayed with an interleukin 2 antibody after translation into a protein by an in vitro translation system of rabbit reticulocytes or wheat germ.

Thus, if a population in which interleukin-2 activity is detected is found, the number of mixed clones in this population is subdivided into smaller populations, and the above method is repeated until it is finally subdivided into single clones. A clone containing the DNA encoding the leukin 2 polypeptide is isolated.

In order to obtain the DNA encoding the interleukin-2 polypeptide from the obtained clone, the section corresponding to the plasmid DNA is separated from the microbial cells by a conventional method, and the DNA portion inserted from the plasmid DNA into the used vector is isolated. Just cut out.

The structure of the DNA encoding the interleukin 2 polypeptide is described by Maxam-Gilbert's chemistry (Meth. Enzym. 65,499.
-560,1980) and dideoxynucleotide chain termination method (Smith, AJH, Meth. Enzym.65, 560-580, 1980) and the like.

The DNA isolated in the present invention and expressing the polypeptide having interleukin-2 activity is shown below.

Among the above base sequences, the only frame capable of encoding interleukin 2 reported to have a molecular weight of 15,000 daltons is the one shown in the above base sequence.
In most cases, the initiation of protein synthesis starts from the first ATG codon on mRNA, and from the first initiation codon ATG to ACT (threonine) before the termination codon TGA corresponds to the interleukin-2 polypeptide. It is considered to be a nucleotide sequence. Also, interleukin 2
In such secretory proteins, there is a so-called signal peptide rich in hydrophobic amino acid, and this peptide is known to be a mature protein which is cleaved during secretion. Judging from the above nucleotide sequence, it is considered to correspond to the N-terminal signal peptide rich in hydrophobic amino acids. Therefore, for example, even a peptide corresponding to the 21st codon GCA (alanine) from the ATG codon to the ACT before the termination codon TGA is considered to have interleukin 2 activity. In addition, it is a noua polypeptide which retains the active site of the interleukin 2 protein even if it has several amino acid residues after the above-mentioned alanine or before the above-mentioned threonine and has a plurality of consecutive nucleotide sequences. If so, it is fully considered that it has interleukin 2 activity.

One or more bases that are not harmful for the information expression of the interleukin 2 gene may be arranged at the 5'-end of the DNA of the base sequence II. Moreover, even if a base capable of expressing one or more amino acids is arranged at the 5′-terminal, the added amino acid is not harmful because the polypeptide has interleukin 2 activity. There is no problem as long as it is harmful and can be easily desorbed.

Similarly for the 3'-end, a base sequence such that an amino acid that is not harmful because the polypeptide has interleukin 2 activity is added to the C-terminal of the polypeptide is 3'-end.
-It may be added to the terminal, and even if it is harmful, there is no problem as long as it can be easily removed. It is also possible to modify part of the gene or part of the polypeptide deduced from the gene structure by chemical synthesis of DNA or the like. Deletion, addition or substitution of amino acid residues can be performed by the site-specific mutation technique (Geneti
c Engineering, vol.3, ppl−32, Plenum Press, New York
(1981) Nucleic Acid Research, Vol.10, pp6487-6500
(1982), etc., and the deletion, addition, or substitution of one or several amino acid residues means the number of amino acids that can be deleted, added, or substituted by a site-specific mutation technique. To do. Furthermore, a gene can be obtained by using mRNA obtained from cells other than human cells described in Examples described later. Therefore, the point is that any DNA encoding a polypeptide having interleukin 2 activity can be used in the present invention.

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

Example 1 (1) RPMI 164 containing 10 volume / volume% fetal bovine serum
Human T cultured in 0 medium by methods well known in the art
Leukemia cells Jurkat cells (freely available in Japan, USA, West Germany, etc.) were suspended in the above medium, and 1,000 X-rays were taken for 50 seconds at room temperature using a Toshiba X-ray irradiation system EXS 150 / 300-4. The total irradiation dose was applied. Then, the irradiated cells were cultured at a cell density of 1 × 10 ⁵ cells / ml in the above medium in 5% carbon dioxide at 37 ° C. for 5 days. Next, this mutant cell was inoculated into 10 96-well microplates at 0.2 cells / well, and cultured in 5% carbon dioxide gas at 37 ° C. for 21 days. Clones with observed cell proliferation were sequentially subcultured, and then ConA 50 μg / m 2 at a cell density of 1 × 10 ⁶ cells / ml.
l Mutation after culturing in the presence of l for 24 hours, measuring the amount of interleukin 2 released into the culture supernatant by the method described above, and improving the amount of production to 40 cultures or more compared to the original Jurkat cells A clone cell Jurkat 111 strain (ATCC CRL8129) was obtained. This strain proliferated by an ordinary culture method, and its proliferation rate was similar to that of Jurkat cells.

(2) The Jurkat 111 cell line was suspended in 1,000 ml of serum-free synthetic medium RITC 55-9 at a cell density of 1 × 10 ⁵ cells / ml, placed in a Falcon rotary culture bottle, and cultured at 37 ° C. for 4 days. Then
The cells were collected by centrifugation. 4 × 10 ⁶ cells / ml
Suspended in the above medium at a cell density of
Add μg / ml and add the above Falcon rotary culture bottle (4
1,000 ml) and spin-cultured for 6 hours.

(3) Jurkat cells (1.2 × 10 ¹⁰ cells) induced by ConA 25 μg / ml thus obtained for 6 hours were added to 800 m of PBS solution.
Resuspend in l, wash the cells twice by centrifugation, and then use the nuclease inhibitor Ribonucleosides-Vanadyl Com
RSB solution containing plex (10 mM) (10 mM Tris-HCl, pH 7.
It was suspended in 800 ml of 5,10 mM NaCl and 1.5 mM MgCl ₂ . Then NP
After adding -40 to 0.05%, gently stir
Centrifuge at 3,000 rpm for 5 minutes to remove nuclei, and add SDS to the supernatant.
(0.5%) and EDTA (5 mM) were added, and then an equal amount of phenol was immediately added to extract cytoplasmic RNA. After repeating phenol extraction three times in total, RNA was precipitated with 2 volumes of ethanol, and the precipitate was collected by centrifugation and dissolved with 10 mM Tris-HCl, pH 7.5. RN thus obtained from Jurkat cells
The amount of A was 196 mg.

Then oligo (dT) to obtain mRNA from RNA
-Column chromatography was performed using cellulose (PL Biochemicals. Type 7). Adsorption is 20 mM Tris-H
Cl, pH 7.5, 0.5M NaCl, 1mM EDTA, 0.5% SDS, and dissolve the RNA, and elution is performed with a buffer (20mM Tris-HCl, pH 7.5,
After washing with 0.5M NaCl, 1mM EDTA), water and 10mM Tris-HCl
This was performed by alternately eluting the mRNA at (pH 7.5). As a result, the amount of eluted mRNA was 3.6 mg.

Furthermore, part of this mRNA (2.4 mg) was subjected to SDG centrifugation (50 mM T
5-25% sucrose density gradient containing ris-HCl, pH 7.5, 1 mM EDTA, 0.2 M NaCl, Hitachi RPS 28 rotor at 26,000 rpm, 24
Fractionation of the 11-12S mRNA fraction for 1 hour at 4 ° C
59 μg, 46 μg and 60 μg were obtained as 2, 13 and 14, respectively.

(4) The mRNA of fraction No. 13 obtained here was added to the oocytes of Xenopus laevis in an amount of 50 ng per oocyte according to the above assay method.
When the oocyte culture supernatant obtained by injecting E. coli was subjected to the interleukin 2 activity assay, the uptake of tritiated thymidine and the increase in the number of activated T lymphocytes shown in the following table were observed. It was demonstrated that the mRNAs of these fractions contained the human interleukin 2 mRNA of the present invention.

(5) Next, cDNA was synthesized in vitro from the thus obtained 11-12S mRNA fraction 13 containing interleukin 2 mRNA, to prepare a plasmid vector PBR322 and recombinant DNA,
This was transformed into Escherichia coli and cells containing the interleukin 2 cDNA clone were selected by the following method.

(5-1) 50 mM Tris-HCl buffer (pH 7.5), 30 mM NaC
l, 6mM MgCl ₂ , 5mM dithiothreitol (DTT), 0.5mM
dATP, dGTP, dCTP, dTTP (dCTP includes those labeled with ³² P), 0.7 μg oligo (dT) ₁₀ , 10 μg mRNA and 15 units AMV reverse transcriptase (JWBeard) were mixed and kept at 41 ° C. for 90 minutes. After the reaction was completed, phenol treatment was carried out once, and DNA was collected as an ethanol precipitate, 20 mM Tris, 1 mM EDTA.
Dissolved in pH 7.5 solution. This gives approximately 2.5 μg of single-stranded cD
NA was synthesized. To remove mRNA from this solution, use NaOH
The solution was added to make 0.33 N NaOH and left at room temperature for 15 hours, then the solution was neutralized with an equal volume of 1 M Tris-HCl, pH 7.5 and passed through a "Sephadex G-50" column. This gives 1.8 μg of c
The DNA was recovered.

(5-2) 50 mM phosphate buffer (pH 7.5), 10 mM MgCl ₂ , 1
Mix 0 mM DTT, 0.75 mM each cATP, dGTP, dCTP, dTTP (including dCTP labeled with ³ H), 1.8 μg single-stranded cDNA, 8 units Polymerase I (manufactured by US BRL), 1
The reaction was carried out at 5 ° C for 15 hours. After the reaction was completed, phenol treatment was carried out once and chloroform treatment was carried out once, and DNA was recovered as an ethanol precipitate. By this reaction, 1.10 μg of double-stranded cDNA was obtained.

Next, 50mM sodium acetate (pH4.5), 0.2M NaCl, 1m
M ZnCl ₂ , 1.10 μg double-stranded cDNA was mixed and incubated at 37 ° C. for 20 minutes, 0.25 unit of nuclease S ₁ (manufactured by Sankyo Co., Ltd.) was added, and the mixture was further incubated for 15 minutes. After completion of the reaction, phenol treatment was performed twice, and the mixture was passed through a “Sephadex G-50” column to give 0.55 μg of double-stranded cD.
NA was recovered.

(5-3) 0.14 M cacodylate Karimuu, 30 mM Tris _{base, 0.1mM DTT, 1mM CoCl 2,} 0.64mM 32 P-dCTP ( specific activity 2.
7 × 10 ⁶ cpm / nmol), 0.55 μg double-stranded cDNA and 5 units of terminal transferase (BRL) are mixed at 37 ℃
After incubating for 7 minutes at room temperature, after completion of the reaction, phenol treatment was performed once, and 0.50 μg of DNA was recovered as an ethanol precipitate by passing through a “Sephadex G-50” column. About 50 dCMPs were added to both 3 ′ ends. It was

After cleaving 10 µg of pBR322 DNA (Gene, 2 , 95-113 (1977)) with the restriction enzyme Pst I, dCMP was added to the above double-stranded cDNA.
The dGMP chains were added to both 3'ends using dGTP instead of dCTP under exactly the same conditions as the chains were added. Thus about 50
dGMP was added to both 3'ends.

(5-4) 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 5 mM
EDTA, 0.05 μg dGMP-added pBR322, 0.01 μg d
CDNA with CMP added was first at 65 ° C for 2 minutes, then at 46 ° C
For 120 minutes, 37 ° C. for 60 minutes, and room temperature for 60 minutes.

Escherichia coli χ1776 50ml L medium 100μg / ml
Diaminopimelic acid with 50 μg / ml thymidine, 1% tryptone, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose) and shaken at 37 ° C until the absorbance at 562 mμ of the culture reaches approximately 0.3. Cultured. After the culture was completed, the culture solution was left at 0 ° C. for 30 minutes, and then the cells were collected by centrifugation, and 5 mM Tris-HCl (pH 7.6), 0.1 M NaCl, 5 mM M
It was washed twice with 25 ml of a solution of gCl ₂ , 10 mM RbCl.

The obtained bacterial cells were treated with 5 mM Tris-HCl (pH 7.6), 0.25 M KCl, 5
The cells were suspended in 20 ml of a solution containing mM MgCl ₂ , 0.1 M CaCl ₂ and 10 mM RbCl, allowed to stand at 0 ° C. for 25 minutes, and then collected by centrifugation. The cells were resuspended in 1 ml of the same solution as above, and 0.2 ml of the obtained cell suspension was added with the above recombinant DNA, and the mixture was incubated at 0 ° C.
Let stand for 40 minutes. Furthermore, after maintaining at 37 ° C. for 2 minutes, the mixture was allowed to stand again at 0 ° C. for 60 minutes. Next, add 0.7m of the L medium
l was added and the mixture was incubated at 37 ° C. for 30 minutes with shaking. This culture 0.1
ml with 100 μg / ml diaminopimelic acid, 50 μg / ml thymidine
The L medium containing 15 μg / ml tetracycline was smeared on one side of a 1.5% agar medium and incubated at 37 ° C. for 2 days.

(5-5) As a mixture of 18 groups, each of which consists of 432 colonies that appeared in the above, 24 colonies
100 μg / ml diaminopimelic acid, 50 μg / ml thymidine
200 ml of L medium containing 10 μg / ml tetracycline was inoculated and shake-cultured at 37 ° C. for 5 to 7 hours, and then 200 ml of the above fresh L medium added with chloramphenicol to a final concentration of 170 μg / ml. Add and further shake culture overnight. In this way, the plasmid DNA was amplified and purified by a conventional method. Hybrid using this DNA
Interleukin 2cDN by ization-translation assay
Clones with A were screened. H used here
The hybridization-translation assay was performed as follows.

25 μg of purified DNA was cleaved with restriction enzyme Hind III,
Phenol treatment 3 times, phenol-chloroform treatment 1
DNA was precipitated by ethanol, washed with 80% ethanol, recovered and dissolved in 40 µ of 80% formamide solution.
Heat denatured for minutes. After that, 10 × SSC (1.5M NaCl, 0.15M
Dilute to 1.3 ml with trisodium citrate). This was fixed on a nitrocellulose filter and dried by heating at 80 ° C. for 3 hours. The filter is treated with 50% formamide, 20 mM pipes, pH 6.5, 0.75M NaCl, 5 mM EDTA, 0.2% SDS and 250.
Incubation was performed at 37 ° C. for 18 hours in a solution containing μg mRNA to hybridize the DNA on the filter with interleukin 2 mRNA. Then, this filter was washed 3 times with 10 mM pipes pH6.5, 0.15M NaCl, 1 mM EDTA, 0.2% SDS solution at 65 ° C., and then further washed with 1 mM pipes, 10 mM NaCl solution.
Wash twice, then with 0.5 mM EDTA, 0.1% SDS solution at 95 ° C for 1
After treatment for minutes, the mRNA adsorbed on the filter was eluted. This was applied to an oligo dT-cellulose column according to a conventional method and recovered. The collected mRNA was injected into Xenopus oocytes and translated into a protein to measure the interleukin 2 activity. As a result, one group out of 18 groups was analyzed by the activity assay method using the amount of tritiated thymidine incorporated as described above.
48 units / ml of interleukin-2 activity was detected.
Therefore, 24 colonies belonging to this population were inoculated to 200 ml of L medium containing 100 μg / ml of diaminopimelic acid, 50 μg / ml of thymidine and 10 μg / ml of tetracycline, respectively, as described above. After shaking culture for ~ 7 hours, 170 μg of chloramphenicol at final concentration
200 ml of the above-mentioned fresh L medium added so that the amount became / ml was added, and the mixture was further cultured overnight with shaking to amplify the plasmid DNA, and then the plasmid DNA was purified according to a conventional method. Then, 5 μg of each DNA was cleaved with Hind III, fixed on a nitrocellulose filter and hybridized with interleukin 2 mRNA as in the previous time, and the mRNA was recovered and injected into Xenopus oocytes to be translated into a protein. Leucine 2 activity was measured and assayed in which of 24 colonies the interleukin 2 clone was present. Purified plasmid DNA obtained from 1 colony,
Interleukin-2 activity was found in the translation of mRNA hybridizing to plasmid p3-16 into oocytes (Table 2), and this clone was a clone with Escherichia coli χ1776 / 3- 16AJ 11995
(FERM-BP225)). That is, it was proved that the cDNA of the plasmid p3-16 has a DNA (interleukin 2 gene) which specifically hybridizes with interleukin 2 mRNA.

Next, when the restriction enzyme cleavage site of the cDNA of the plasmid p3-16 was examined, it was found that there were 1 site in Xba I (US BRL) and 2 sites in BstNI (New England Bio Lab. US) (upstream of Xba I site and Downstream) disconnected. However, this cDNA is about 65
It consisted of 0 base pairs and was found to correspond to a part of the 11-12S interleukin 2 mRNA, so the method of Land et al. (Land et al, Nucleii Acids Res., Vol.9, p2551
(1981)) and cDNA was synthesized in the same manner as described above and inserted into the plasmid pBR322. Using this plasmid, E · c
oliχ1776 was transformed, and from about 2000 transformants, a cDNA clone having the same sequence as the cDNA of the plasmid p3-16 was selected using the Grunstein-Hogness method,
Plasmid with 0 base pair cDNA insert, pIL2-50A
With a strain (Escherichia coli χ1776 / IL-2-50A
AJ11996 (FERM-BP226)) was obtained. This pIL2-50A cD
The restriction enzyme digestion map of NA is shown in Figure 1.

Next Figure -2a, as shown in b, pBR 328 plasmid vector (Gene, 9, 287-305 (1980 )) in pKCR vector (Pr
oc, Natl.Aca.Sci.USA, vol 78, No.3,1527-1531,1981)
A pCE-1 vector incorporating a region containing the promoter of the SV 40 virus early gene of Escherichia coli was constructed. From the plasmid of the plasmid pIL-2-50A clone, Pst I
The cDNA inserted in
It was inserted into the site (plasmid pCEIL-2). The Escherichia coli HB 101 (AJ 12008) in which the plasmid pCEIL-2 is incorporated has the accession number FERMBP-244.
Has been deposited as. The insertion pattern at this time is such that the initiation codon ATG of the interleukin 2 gene is connected downstream of the promoter of the SV40 early gene as shown in FIG. 2 (I in the figure). This vector was added to COS-7 cells (Gluzman, Y. Cell vol.23p1) of monkey culture cells.
75-182 (1981)) (Mc Cutchan et al J,
Natl. Cancer Inst. Vol.41 p351-357 (1968)), the interleukin 2 activity appearing in the culture supernatant was examined. As a control, the vector plasmid pGE-1 was used to carry out the same experiment to examine the interleukin 2 activity. The results are shown in Table-3.

Furthermore, this interleukin 2 activity was neutralized by a mouse anti-human interleukin 2 monoclonal antibody, and the interleukin 2 activity was 1 u / ml or less.

The DNA encoding the polypeptide having interleukin 2 activity is Escherichia coli χ1776 / IL-2-50.
The plasmid DNA portion was separated from A cells by a conventional method, the obtained plasmid DNA was digested with a restriction enzyme Pst I, and then the fragment having a smaller molecular weight than the two generated DNA fragments was separated.

When the base sequence of the obtained DNA was examined according to the Maxam-Gilbert chemical method, it was found to have the above-mentioned sequence.

[Brief description of drawings]

FIG. 1 is a restriction enzyme map of a gene capable of encoding a polypeptide having interleukin 2 activity. FIG. 2 is an explanatory view of the construction process of pCEIL-2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // A61K 38/00 ABA ADU (C12P 21/02 K C12R 1:91) A61K 37/02 ABA ( 72) Inventor Hiroshi Matsui 1-19-16-101 Namiki, Kanazawa-ku, Yokohama-shi, Kanagawa (72) Inventor Shin-ichi Kashima 2-21-603 Wakabadai, Asahi-ku, Yokohama-shi, Kanagawa (72) Inventor Junji Hamuro 241-32, Fukaya-cho, Totsuka-ku, Yokohama-shi, Japan

Claims (2)

[Claims] 1. A formula: A polypeptide having human interleukin-2 activity having the amino acid sequence shown by or an amino acid sequence in which one or several amino acid residues are deleted, added or substituted to the amino acid sequence of the polypeptide, Substantially free of other proteins of human origin with chains,
A polypeptide having human interleukin 2 activity obtained by a genetic engineering method. 2. The formula: Having a human interleukin-2 activity having the amino acid sequence shown by or a human interleukin having an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted with respect to the amino acid sequence of the polypeptide. A human, characterized by culturing in a medium, a transformant in which a vector containing a gene encoding a polypeptide having 2 activity is introduced, which is substantially free of other human-derived proteins with sugar chains A method for producing a polypeptide having interleukin 2 activity.