JPH0119879B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a gene encoding a polypeptide having interleukin-2 activity. 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. .,
152, p. 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 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. That is, the present invention provides a gene encoding a polypeptide having interleukin-2 activity that contains the following amino acid sequence in its molecule.

[Table] The electrophile of the present invention encoding the above polypeptide contains a portion in its DNA chain in which sites to be cleaved with restriction enzymes BstNI, XbaI, and BstNI are arranged in this order, and the following polypeptide or It encodes and has the following base sequence or.

【table】

[Table] Such DNA is obtained as the 11-12S fraction by fractionation using sucrose density gradient centrifugation, and is isolated from human or murine lymphocyte-derived cells as messenger RNA (hereinafter abbreviated as mRNA). prepared from That is, DNA prepared from mRNA of the 11-12S fraction by sucrose density gradient centrifugation obtained from lymphocyte-derived cells capable of producing interleukin 2 is ligated to, for example, a plasmid vector of Escherichia coli to generate Escherichia coli. - A clone having DNA that can be expanded in coli cells and capable of expressing interleukin-2 polypeptide is isolated, and the DNA inserted into the plasmid of the isolated clone is isolated. The DNA encoding the polypeptide having interleukin-2 activity of the present invention can be ligated to a replicon derived from a microorganism, and the obtained recombinant DNA can be introduced into host microorganism cells to induce the interleukin-2 producing ability of the microorganism. can be transformed to have As mentioned above, mRNA corresponds to interleukin 2 and can be obtained by fractionation by SDG centrifugation method, gel filtration method, etc. and agarose gel electrophoresis method.
It is obtained as the 11-12S fraction, and this
mRNA can be produced by extracting and separating from lymphocyte-derived cells. The human lymphocyte-derived cells capable of producing interleukin 2 used in the present invention include, for example, lymphocytes obtained from human peripheral blood, tonsils, spleen, etc. In addition, these lymphocytes were treated with nylon column, antiserum-complement treatment,
Products that have undergone pretreatment such as density gradient fractionation and enzyme (neuraminidase, galactose oxidase, etc.) treatment, as well as products that have been given the ability to produce interleukin 2 by mutation treatment using X-rays, etc., and enzyme treatment such as trypsin, etc. Included in the human lymphocyte-derived cells of the invention. 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 extract more mRNA than the parent line. Furthermore, the above human lymphocyte-derived cells and CEN,
So-called hybridomas 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. Interleukin in human lymphocyte-derived cells
When using an interleukin 2 spontaneous strain to produce 2mRNA, these cells may be cultured in a conventional manner. When using cells that produce interleukin 2 upon stimulation with mitogens, wash the cells thoroughly and then wash them with Rosewell®.
Park Memorial Institute 1640 (hereinafter abbreviated as RPMI 1640) medium, Dulbecco's modified
0.5 to 4 x 10 ⁶ cells per cell in a normal cell culture medium (which may or may not contain serum or serum components) such as Eagles' medium or Krick's medium, or a synthetic serum-free medium.
After suspending the cells at a density of 1 ml and adding mitogens; neuraminidase, galactose oxidase; zinc compounds such as zinc chloride; bacterial cell-derived lymphocyte activation components such as protein A and streptolysin-O, the cells are washed. , remove irritants. When macrophages and dendritic cells coexist during mitogen stimulation, cells that can produce interleukin 2, and when B lymphocytes and B lymphocyte-derived cell lines Raji, Daudi, K562, and BALL-1 cells coexist, interleukin 2 can be produced as well. There are cells that have the ability to produce interleukin 2, and when these cells are used to produce interleukin 2 mRNA, mitogen stimulation is performed in the presence of these cells. 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. Culture passage in vitro can be carried out using normal cell culture media, and mammalian-derived serum,
It has been found that these cell lines can be cultured and proliferated in a medium containing serum components or serum albumin, or in a synthetic serum-free medium that does not even contain serum albumin, and can be used as the cell material of the present invention. Ivy. 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 unnecessarily, the interleukin 2 mRNA produced will be degraded.
Furthermore, when activating lymphocytes, 10 to 50 ng/ml of whole ball esters such as PMA and TPA may be added. The culture temperature is preferably in the range of 32 to 37°C. Below, a method for culturing human lymphocyte-derived cells having the ability to produce interleukin-2 will be described in more detail. (b) Obtaining lymphokine spontaneously producing strains Juyukatsu cells, human T lymphocyte-derived leukemia cells (Fred Hutchinson Cancer Research Institute/
It is freely available at Seattle/Salk Institute/San Diego/USA, West German National Cancer Center/Heidelberg/West Germany, etc. ) to 1
Suspended in Krick medium at a cell density of × ¹⁰⁶ cells/ml, a total of 8000
Perform X-ray irradiation. After this, the cells were added to a 96-well flat-bottomed microtiter plate ("Falcon 3072") at a cell density of 0.1 cells/200 μl and kept at 37°C for 3 weeks in Krick's medium containing 5% fetal bovine serum. Cultivate in a CO ₂ incubator (cloning by limiting dilution method). Cells in culture wells in which cell growth has been observed are transferred to a 24-well Nunc culture plate before the cells reach a density that covers the entire bottom surface, and the cells are grown in 2 ml of Krick's medium for 5 days. . When a sufficient amount of cells is obtained, the cells are suspended in 2 ml of serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 2 × 10 ⁶ cells/ml, and cultured for 2 days. The culture supernatant is separated by centrifugation at 3000 rpm for 5 minutes, and then debris is removed and sterilized using a 0.22μ Millipore filter to obtain interleukin-2. 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-Hypark density gradient centrifugation. The peripheral lymphocytes are suspended in Krick's medium at a cell density of 1×10 ⁶ cells/ml, and each 2 ml is inoculated into a 24-well Nunc culture plate. Add 100 μl of phytohemagglutinin-M (manufactured by Gibco) to the final concentration of 5 μg/ml, and add it under the above conditions.
After culturing for 48 hours, the cells are washed with culture medium and plated again in 1 ml onto the original Nunc culture plate at a cell density of 1×10 ⁵ cells/ml. Add 1 ml of conditioned culture solution obtained by stimulating human spleen cells with 2.5 μg/ml of concanavalin A (hereinafter referred to as ConA) for 48 hours to each well, and repeat the same culture for 3 days. , T lymphocytes obtained from human peripheral blood are subcultured over a long period of 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 collected at 1×10 ⁶ cells/ml.
Here, suspend in RPMI1640 medium to a cell density of
Phytohemagglutinin (PHA) was added to a final concentration of 10 μg/ml and incubated at 37°C for 24 hours at 7.5 μg/ml.
%CO ₂ incubator, and the main culture supernatant was separated by centrifugation at 3000 rpm for 5 minutes.
Next, the mixture is sterilized using a 0.22μ Millipore filter to obtain interleukin-2. 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 The J-111 strain cloned by the above-mentioned Jyurukatsu cells and the above-mentioned limiting dilution method can be used in the above-mentioned serum-free medium or serum 1-1. In the presence of 10μg/ml of ConA and 2.5μg/ml of PHA in RPMI1640 medium containing 2%
It was found that 10 to 4000 units/ml of interleukin 2 was produced when cultured for 24 hours. 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 stimulating them with mitogen in the presence of other cells or factors produced by those cells Human lymphocyte malignant cells Molt4F and Diurukatsu cloned by the aforementioned limiting dilution method cell 1
One clone, the Jyurukatsu 99 strain, was prepared by adding the lectins and mitogens mentioned above over a wide range of concentrations.
Interleukin 2 is not produced even after culturing for 24 to 72 hours. However, during this period, interleukin 1, a type of monokine, was administered at 5 to 10 u/ml or K562.
or Raji cells at 0.5 per 1× ¹⁰⁶ cells/ml.
×10 ⁶ cells/ml coexist in Krick medium.
When incubated at 37°C for 24 hours, interleukin 2 is produced.
Produces 10-100u/ml. 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, using surfactants such as NP-40, SDS, Triton X 100 deoxycholic acid, or physical methods such as a homogenizer or freeze-thaw
A method is used to partially or completely destroy and solubilize cells. In order to prevent RNA degradation by RNase during extraction, it is preferable to add an RNase inhibitor such as heparin, polyvinyl sulfate, bendonite, macaroid, diethylpyrocarbonate, vanadium complex, etc. to the extraction solution. In addition, if necessary, interleukin 2 can be detected using an antibody corresponding to interleukin 2.
Polysomes in the process of synthesis are allowed to settle, and then
mRNA can be extracted using a surfactant or the like. mRNA is oligo dT-cellulose, polyU-
It can be purified by an adsorption column such as Sepharose or Sepharose 2B, a purification method using a batch method, fractionation using an SDG centrifugation method, etc. Through such purification operations, mRNA corresponding to interleukin 2 is obtained as an 11-12S fraction. In order to confirm that the mRNA obtained as described above corresponds to the target interleukin 2, it is necessary to translate the mRNA into protein and examine its physiological activity, or use antibodies etc. What is necessary is to perform a method such as identifying the . for example
Xenopus laevis, a system commonly used to translate mRNA into protein
mRNA is injected into oocytes and translated. Alternatively, translation into protein has been carried out using cell-free systems such as rabbit reticulocyte lysate and wheat germ. The interleukin 2 activity assay method used here is as follows. That is, 100 μl of the sample was added to the first row of a 96-well microtiter plate, diluted 2-fold in RPMI 1640 medium containing 2% fetal bovine serum, and each 100 μl dilution series was placed on the 96-well microplate. create. There, an activated T lymphocyte line prepared according to the method taught by Gillis et al. (Nature, vol. 268, p. 154, (1977)) was added at a cell density of 4 x 10 ³ cells/100 μl.
Add 100 μl 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, pulsed for 4 hours, and cells were harvested according to methods well known in the field. , measure the amount of radiation taken into cells. Since the culture supernatant with higher interleukin-2 activity has a larger amount of tritiated 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. Add 100 μl of sample to the first row of a 96-well microtiter plate containing 2% fetal bovine serum.
Repeat the 2-fold dilution of the DMEM medium to create a dilution series of 100 μl each on a 96-well microplate. Add 5 of the above activated T lymphocyte lines/
Add 100 μl to each well as a cell density of 100 μl. The cells were left to stand for 72 or 96 hours at 37°C in a 5% carbon dioxide incubator, and then the number of surviving activated T lymphocytes was counted using an inverted microscope. At this time, interleukin 2 having an activity of 100 u/ml and 10 u/ml is used as a positive control, and the number of proliferating T lymphocytes in the sample addition group is compared to calculate the interleukin 2 activity of the sample. Complementary DNA (cDNA) is synthesized in vitro from the thus obtained interleukin 2 mRNA and connected to a microbial-derived replicon. cDNA synthesis can usually be carried out in vitro by the following method. A single strand complementary to mRNA is produced using reverse transcriptase in the presence of dATP, dGTP, dCTP, and dTTP using mRNA as a template and oligo DT as a primer.
After cDNA is synthesized and the template mRNA is degraded and removed by alkaline treatment, double-stranded cDNA is synthesized using reverse transcriptase or DNA polymerase using the single-stranded cDNA as a template. Both ends of the obtained DNA are treated with exonuclease if necessary,
An appropriate linker DNA is connected to each, or multiple bases in combination that can be annealed are polymerized. This is then incorporated into a vector containing a replicon capable of autonomous replication in, for example, Escherichia coli. The integration method 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-stranded DNA processed in this way
and vector DNA, and connect them using ligase. The obtained recombinant DNA is introduced into a vector host microorganism. In the present invention, Escherichia coli was used as the host microorganism, but Bacillus subtilis, Satucharomyces cerevisiae, etc. can also be used. Examples of vectors used in the case of Escherichia coli are shown below. (Protein Nucleic Acid Enzyme Volume 26 4
(1981)) EK-based plasmid vectors (stringy end type) pSC101, pRK353, pRK646, pRK248,
pDF41, etc., EK-based plasmid vectors (relaxed type) CalE1, pVH51, pAC105,
RSF2124, pCR1, pMB9, pBR313, pBR322,
pBR324, bBR325, pBR327, pBR328,
pKY2289, pKY2700, pKN80, pKC7,
pKB158, pMK2004, pACYC1, pACYC184,
λgt and λc of λgt-based phage vectors such as λdu1,
λgt・λB, λWES・λC, λWEG・λB′, λZJvir・
λB′, λALO・λB, λWES・Ts622, λDam, etc. Among these vectors, pBR322 is commonly used for Escherichia coli.
In the case of pBR322, the cDNA integration site is Pst I
The Eco RI site is frequently used. The method for transforming a microbial host using a plasmid containing cDNA in a plasmid vector is mainly to collect cells in the logarithmic growth phase.
The method used is to treat the DNA with CaCl ₂ to make it naturally easy to take in the DNA so that the plasmid can be taken in. It is also known that the efficiency of transformation can be further increased by coexisting MgCl ₂ or RbCl. Alternatively, microbial cells may be transformed into spheroplasts 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. In other words, this selection method is called the plus-minus method. First, mRNA is extracted from Juyukatsu cells stimulated with ConA, and interleukin 2 mRNA is partially purified by SDG centrifugation. ), this mRNA is used to synthesize ^32P -labeled single-stranded cDNA. Next, after removing the mRNA that served as a template for cDNA synthesis by alkaline treatment, this cDNA and
Hybridize with an excess of partially purified 11-12S mRNA extracted from Juyukatsu cells that have not been stimulated with ConA. And it did not hybridize to this ConA-unstimulated mRNA.
The cDNA and the hybridized cDNA are fractionated using a hydroxyl abatite column and designated as probe A and probe B, respectively. Before the next step, each transformed strain is grown in exactly the same way on two nitrocellulose filters, and the DNA is fixed on each filter by alkali treatment. Then, using probe A and probe B prepared earlier, hybridize each to a filter separately, and then perform autoradiography. Search for colonies that do not react at all (minus) (Taniguchi et al., Paoc.jpn.
Acad., vol55B.464-469 (1979)). Alternatively, the transformed strain, for example, 1,000 to 10,000 clones, is divided into groups of several tens to hundreds of clones, the transformed strains are mixed and cultured for each group, and plasmid DNA is prepared (by a conventional method). Next, these DNAs are made into single-stranded DNA by heat denaturation etc. and fixed on a nitrocellulose filter.
The mRNA prepared from human T leukocyte cells containing interleukin 2 mRNA as described above, which is complementary to DNA, is hybridized. or
After heat denaturing the DNA, first interleukin
Another method is to hybridize mRNA containing 2 mRNA and then immobilize the DNA-mRNA hybrid on a nitrocellulose filter. Next, add this filter to a 1mM pipette,
After washing thoroughly with a low salt solution such as 10mM NaCl,
The mRNA adsorbed on the filter is eluted by heat treatment at 95°C for about 1 minute with a solution such as 0.5mM EDTA and 0.1% SDS. Then, perform operations such as applying this to an oligo dt-cellulose column to recover mRNA. Then this
Interleukin 2 activity is measured by injecting mRNA into Xenopus oocytes and translating it into protein. Alternatively, after translating into protein using a rabbit reticulocyte or wheat germ in vitro translation system,
Interleukin 2 is assayed using 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 is isolated. To obtain DNA encoding interleukin-2 polypeptide from the obtained clone, a segment corresponding to plasmid DNA is isolated from the microbial cell using 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. encodes interleukin 2 polypeptide
The structure of DNA is determined by the Maxam-Gilbert chemical method (Meth. Enzym. 65, 499-560, 1980) and the dideoxynucleotide chain termination method (Smith, AJH,
Meth. Enzym. 65, 560-580, 1980), etc.). Interleukin 2 isolated in the present invention
The DNA in which the active polypeptide was expressed is shown below.

[Table] Among the above base sequences, the frame shown in the above base sequence is the only one that can encode interleukin 2, which is reported to have a molecular weight of 15,000 Daltons. The initiation of protein synthesis begins with mRNA
Most cases start with the first ATG codon above, so the first initiation codon
Before the termination codon TGA than ATG
The base sequence up to ACT (threonine) is considered to correspond to interleukin-2 polypeptide. 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 ATG codon to the 21st codon GCA (alanine) to ACT before the termination codon TGA is considered to have interleukin-2 activity. Furthermore, it is a polypeptide that retains the active site of interleukin-2 protein even if it lacks some amino acids after the above-mentioned alanine or before the above-mentioned threonine and has a plurality of consecutive base sequences. If so, it is highly conceivable that it has interleukin-2 activity. One or more bases that are not harmful to the information expression of the interleukin 2 gene may be arranged at the 5'-end of the DNA of the present invention. Also, 5′-
Even if there is a sequence of bases that can express one or more amino acids at the end, the added amino acids may not be harmful because the polypeptide has interleukin-2 activity, or may be harmful. If there is something that can be easily removed, there is no problem. Similarly, for the 3'-terminus, a base sequence may be added to the 3'-terminus such that a non-harmful amino acid is added to the C-terminus of the polypeptide because the polypeptide has interleukin-2 activity. Even if it is harmful, there is no problem as long as it can be easily removed. Furthermore, it is also possible to modify a part of the gene of the present invention or a part of the polypeptide deduced from the gene structure of the present invention by chemical synthesis of DNA or the like. Furthermore, cells obtained from cells other than human cells described in the Examples below.
The gene of the present invention can also be obtained using mRNA. Therefore, in short, any DNA encoding a polypeptide having interleukin-2 activity is included in the genes of the present invention. Next, the present invention will be explained in detail with reference to examples. Example 1 (1) Contains 10% v/v fetal bovine serum
Human T leukemia cells (freely available in Japan, America, West Germany, etc.) cultured in RPMI 1640 medium by a method well known in the art were suspended in the above medium and incubated at room temperature for 50 seconds.
A total irradiation dose of 1000 roentgens was applied using a Toshiba X-ray irradiation device EXS 150/300-4 model.
The irradiated cells were then concentrated at 1×10 ⁵ cells/ml.
in 5% carbon dioxide in the medium described above at a cell density of 37
The cells were cultured at ℃ for 5 days. Next, this mutant cell was inoculated into 10 96-well microplates at a density of 0.2 cells/well, and cultured for 21 days at 37°C in 5% carbon dioxide gas. Clones observed for cell proliferation were sequentially subcultured, and then cultured for 24 hours in the presence of ConA 50 μg/ml at a cell density of 1×10 ⁶ cells/ml.
The production amount of interleukin 2 released into the culture supernatant was measured using the method described above, and the mutant clone cell Juyukatsu 111 strain (ATCC
CRL8129) was obtained. This strain proliferated using a conventional culture method, and its proliferation rate was comparable to that of Jurkat cells. (2) This Juyukatsu 111 cell line was grown in serum-free synthetic medium RITC 55-9 at a cell density of 1 x 10 ⁵ cells/ml.
The cells were suspended in 1000 ml, placed in a rotary culture bottle manufactured by Falcon, and cultured at 37°C for 4 days, and the cells were collected by centrifugation. These cells were suspended in the above-mentioned medium at a cell density of 4 × 10 ⁶ cells/ml, and ConA
25 μg/ml was added, and 1000 ml of the above-mentioned rotary culture bottles (4 bottles) manufactured by Falcon were filled and cultured in rotation for 6 hours. (3) Juyukatsu cells (1.2×10 ¹⁰ cells) induced with 25 μg/ml of Cona thus obtained for 6 hours
The cells were suspended in 800 ml of PBS solution, washed twice by centrifugation, and then treated with Ribonucleosides-Vanadyl Complex, a nuclease inhibitor.
RSB solution containing (10mM) (10mM Tris-
HCl, PH7.5, 10mM NaCl, 1.5mM _MgCl2 )
Suspended in 800ml. Next, add NP-40 to 0.05% and stir gently at 3000 rpm.
Centrifuge for 5 minutes to remove the nuclei, and add SDS to the supernatant.
(0.5%) and EDTA (5mM), immediately added an equal volume of phenol to extract cytoplasmic RNA. Repeat the phenol extraction a total of 3 times, then 2
The RNA was precipitated with a volume of ethanol, and the precipitate was collected by centrifugation and dissolved in 10mM Tris-HCl, pH 7.5. The amount of RNA thus obtained from Juyukat cells was 196 mg. Next, to obtain mRNA from this RNA, oligo(dT)-cellulose (PL)
Column chromatography was performed using Biochemicals, Type 7). Adsorption is 20mM
Tris-HCl, PH7.5, 0.5M NaCl, 1mM
RNA was dissolved in EDTA and 0.5% SDS solution, and elution was performed using buffer solution (20mM Tris-HCl, PH
After washing with 7.5, 0.5M NaCl, 1mM EDTA),
Alternately with water and 10mM Tris-HCl (PH7.5)
This was done by eluating the mRNA. As a result, the amount of eluted mRNA was 3.6 mg. Furthermore, a portion (2.4 mg) of this mRNA was transferred to SDG
Centrifugation (50mM Tris-HCl, PH7.5, 1mM
EDTA, 5-25% sucrose density gradient with 0.2M NaCl, in Hitachi RPS 28 rotor
26,000 rpm, 24 hours, 4℃), fractionated, 11-
12S mRNA fraction with fraction numbers 12, 13, 14
As a result, 59μg, 46μg, and 60μg were obtained, respectively. (4) Oocyte culture obtained by injecting 50 ng of the mRNA of fraction number 13 obtained here into Xenopus oocytes using the micron excision method according to the assay method described above. When the supernatant was subjected to interleukin 2 activity assay, an increase in the uptake of tritiated thymidine and the number of activated T lymphocytes as shown in the following table was observed.
The mRNA of these fractions was proven to contain the human interleukin 2 mRNA of the present invention.

[Table] Oocyte culture
×32 572
Kiyoshi)

Claims (1)

[Scope of Claims] 1. A gene encoding a polypeptide having interleukin-2 activity containing the following amino acid sequence in its molecule. 【table】