HU197938B - Process for producing gene encoding interleukin-2 polypeptide, recombinant dna carrying the gene, living cell line having the recombinant dna and process for producing interleukin-2 with such cells - Google Patents

Abstract

A method of producing interleukin-2 by culturing an interleukin-2-forming, transformed eukaryotic or prokaryotic cell line is provided. This cell line is obtained by first isolating a gene encoded for a polypeptide having interleukin-2 activity and having a vector DNA capable of replicating in prokaryotic or eukaryotic cells at a position downstream of a promoter gene in the Vector is linked. With the recombinant DNA thus obtained, the prokaryotic or eukaryotic cells are transformed and thus capable of forming interleukin-2.

Description

The present invention relates to a gene encoding an interleukin-2 polypeptide, more particularly to a cloned gene, a recombinant DNA carrying the gene, a live cell line having recombinant DNA, and a method using the cell line to produce interleukin-2.

Interleukin-2 (hereinafter "IL-2"), previously known as T cell growth factor, is a soluble protein (also known as "lymphokine **") obtained from lectin or antigen-activated T cells (Morgan, DA and Science, 193: 1007-1008 (1976); Gillis, S., et al., J. Immunol. 120: 2027-2033 (1978). Interleukin-2 (IL-2) is capable of modifying lymphocyte reactivity and promoting long-term cultivation of antigen-specific effector T-lymphocytes in vitro (Gillis, S. et al., Natura 268, 154-156 (1977)). IL-2 is also known to express other important biological activities, e.g. enhancement of thymocyte mitogenesis (Chen, M.M. et al., Cell. Immunol. 22, 211-224 (1977), Shaw, J. et al., J. Immunol. 120, 1967-1973 (1978)), cytotoxic Induction of T cell reactivity (Wagner, H. et al., Naturre, 284, 278-280 (1980)), and anti-SRBC plaque-forming cell responses (Gillis, S. et al., J. Exp. Med. 149, 1960). —1968 (1979)) in nude mouse spleen cell cultures. Consequently, this lymphocyte regulator is useful in enabling humoral and cellular immune responses and in restoring immune deficient states to normal humoral and cellular immune states. These identified immunological activities of IL-2 strongly indicate that IL-2 is useful for medical immunotherapy against immunological disorders, including; neoplastic diseases, bacterial and viral infections, immune deficiency diseases, autoimmune diseases, etc. (Papermaster, B., et al., Adv. Immunopharm, 507 (1980)). Like interferons, IL-2 has been shown to enhance the activity of natural killer cells and thus its potential use in the treatment of neoplastic diseases. In addition, IL-2 is capable of maintaining functional monoclonal T cell culture and therefore appears to play a key role in studying the molecular nature of T cell differentiation, the mechanism of differentiated T cell function, and the mechanism of T cell antigen receptors. It can also be used to produce a variety of other T-cell-derived lymphokines by widespread cultivation of monoclonal T cells. In addition, IL-2 production and lymphocyte response to IL-2 may be important parameters of immunological functions that are useful in the clinical diagnosis of abnormal immunity.

IL-2 has been produced so far by the stimulation of mouse, rat, and human lymphocytes with a mitogen (Gillis, S. et al. (1977), Natur. 268, 154-156; Farrar, J., et al., J. Immunok. 121, 1353-1360 (1978); Gillis, S. et al., J. Immunol. 120, 2027-2033 (1978)), and stimulation of human peripheral blood mononuclear lymphocytes with a mitogen (Gillis, S. et al., J. Immunol. 124, 1954-1952 (1980). Gillis et al. Reported the preparation of murine IL-2 from a murine T-cell lymphoma cell line (Gillis, S. et al., J. Immunol. 125: 2570-2578 (1980)) and the production of human IL-2 from a human leukemia cell line.

The above-mentioned publications by Gillis et al. Discuss the human IL-2 production method from mitogen-stimulated human T cell leukemia cell line cell culture methods. However, this technique results in undesirable low concentrations of human IL-2 and also requires complex purification procedures to obtain a small amount of IL-2 from a high volume medium. In fact, since the human T cell leukemia cell line produces trace amounts of several other biologically active compounds that are analogs of human IL-2, there are significant difficulties in isolating IL-2 from these other immunologically active molecules or occasionally isolating IL-2 of toxic lectins present.

As another approach, it seems desirable to use recombinant DNA techniques for the production of IL-2 (DNA is an abbreviation for DNA), as they are already used in other biologically active human proteins, e.g. for the production of interferons (Gray, PW et al., 1981, Naturre, 295, 503-508; Nagata, S. et al., Naturre, 284, 316-320 (1980). Taniguchi, T. et al., Ge.ne, 10, 11- 15 (1980). So far, however, attempts to produce IL-2 by recombinant DNA techniques have failed. so e.g. NIKKEI BIOTECHNOLOGY (Japan), 19, July 5, 1982, reported that attempts to create a microorganism producing IL-2 with recombinant DNA were unsuccessful, probably due to the fact that the IL-2 polypeptide the coding machine has not yet been cloned.

Thus, there remains a need for an interleukin-2 cloning gene and a recombinantly engineered DNA that carries the gene. There is also a need for a live cell line incorporating DNA produced by recombinant techniques and for a method of producing interleukin-2 using the cell line.

The + trick is briefly summarized below as follows:

Cloned gene encoding a polypeptide with IL-2 activity:

DNA produced by a recombinant technique comprising a gene encoding a poly-2197938 peptide having IL-2 activity, and a vector DNA capable of replication in a prokaryotic or eukaryotic cell; the coding sequence of said gene is downstream of the promoter sequence.

The present invention also provides for prokaryotic or eukaryotic cell lines that have been transformed with the above-mentioned DNA, vector DNA and coding gene to produce IL-2. The vector DNA can replicate in the cell; the coding sequence of said gene is downstream of the promoter sequence.

According to the present invention, IL-2 can be produced by aerobically culturing a medium containing a eukaryotic or prokaryotic cell line that has been transformed to produce IL-2. Transformation is accomplished by DNA that has been modified by recombinant techniques with the gene encoding the production of the polypeptides having IL-2 activity, on the one hand, and by inserting DNA capable of replicating in the cell, respectively; the coding sequence of said gene is downstream of the promoter sequence.

A much more complete appreciation of the invention and its advantages will be made possible, and the entire invention will be better understood in the following detailed description, provided some explanations are given in connection with the accompanying drawings.

Figure 1 shows a restriction endonuclease cleavage map of a cloned gene encoding the production of a polypeptide having IL-2 activity (hereinafter referred to as "IL-2 polypeptide").

Figure 2 (a) shows the base sequence of the cloned gene. Figure 2 (b) shows the amino acid sequence I, II, polypeptides of IL-2 activity. Amino acid sequence and III. The amino acid sequence.

Figure 3 is a flowchart illustrating the construction of recombinant DNA (pCEIL-2), the coding gene inserted into recombinant DNA.

Figure 4 shows plasmid vector pTrs-3.

Figures 5 (a), 5 (b) and 5 (c) are flowcharts illustrating the construction of pTIL 2-22, pTIL 2-21, pTIL 2-14 and pTIL 2-15, as vector pTrs-3 using.

Figure 6 is a flowchart showing the construction of pKIL 2-21 recombinant DNA using pKT-218 as vector.

Figure 7 is a flowchart illustrating the construction of recombinant DNA from pTuIL 2-22.

Figure 8 shows DNAs capable of replication in a Saccharomyces cerevisiae cell.

In the figures, "A", "G", "C" and "T" represent deoxyadenylic acid, deoxyguanilic acid, deoxycytidylic acid and thymidylic acid.

The preferred embodiment of the invention will now be briefly described.

A cloned gene encoding an IL-2 polypeptide can be obtained by transcribing DNA complementary to IL-2 (IL-2 mRNA) and mammalian cell-derived messenger RNA (mRNA, "RNA" for ribonucleic acid abbreviation) capable of producing a polypeptide having IL-2 activity. (cDNA). The resulting single-stranded cDNA (ss-cDNA) can be converted to double-stranded cDNA (ds-cDNA).

The mRNA used as a template for the production of cDNA can be isolated in conventional manner from a mammalian cell capable of producing the IL-2 polypeptide. Isolated RNA is polyadenylated (Gillis et al., Immunological Rew., 63, 167-209 (1982)), and polyadenylated RNA can be fractionated e.g. sucrose density by gradient centrifugation as 11 - 12 S pellet. Occasional occurrence of 13S mRNA also shows mRNA activity, and in such cases it seems likely that this mRNA is an aggregated form of 11-12 S mRNA.

A mammalian cell capable of producing IL-2, which is a source of the mRNA of the present invention, may be a T lymphocyte such as a lymphocyte. peripheral blood mononuclear cells, tonsil cells, spleen cells and the like, which can be surgically recovered from mammals. The cells may be pretreated in a conventional manner, e.g. nylon column, antiserum complement, density gradient centrifugation, multiple enzymatic treatments, e.g. a combination of neuraminidase and galactose oxidase with X-ray irradiation or trypsin to confer cells with IL-2 productivity or to increase IL-2 activity. After culturing in the presence of T cell growth factor, cloned T lymphocytes obtained from said mammalian cells can also be used as mRNA sources and are preferred lymphocytes. Transformed lymphocyte cell lines, e.g. T lymphocytes derived from leukemia or lymphoma cell lines, or derivatives thereof by mutation or pretreatment by the above methods, or cloned transformed cell lines are preferred as mRNA sources. Obviously, cloned cell lines generally contain greater amounts of IL-2 mRNA than the original cell line mass. T-cell hybridomas obtained by combining lymphocytes from the aforementioned cells with tumor cell lines (e.g., CEM, Molt 4F and BW 5147) are also preferred cell lines for the method of the invention. The cell line lymphocytes may include those that are (1) constitutive IL-2 producing and (2) producing IL-2 only when there is exogenously added mitogen in the culture or if other cells affecting IL-2 production are present. are present or are away.

In order to induce IL-2 mRNA formation in constitutive IL-2 producing cells, constitutive IL-2 producing cells are cultured under conditions generally known in the field of cell culture. Cells producing IL-2 only in the presence of mitogen

-3197938, to induce mRNA formation, cultured cells should be washed with appropriate medium and resuspended in appropriate medium, e.g. Rosewell Park Memories Institute 1640 ("RPMI 1640"), Dulbecco Modified Eagle Medium ("DMEM"), or Click medium, whether or not containing serum.

These media may be supplemented with a variety of additives, e.g. penicillin, streptomycin or other antibiotics, or fresh L-glutamine, HEPES buffer and sodium bicarbonate at concentrations commonly used in cell culture. A preferred cell density is 0.5-4-10 ⁶ cells / ml. Appropriate stimulants are added to induce the activation of mRNA and IL-2 production. Examples of suitable stimulants include mitogens, neuraminidase, galactose oxidase, zinc derivatives such as, for example. zinc chloride, or lymphocyte activating agents from microorganisms, e.g. Protein A, O-streptolysin. The stimulated cells are harvested and washed. Macrophages or dendritic cells also present during mitogen stimulation may also activate mRNA or increase the amount of mRNA activated. Similarly, B lymphocytes or B lymphocyte lines such as The simultaneous presence of Raji, Daudi, K 562 and BALL-1 derived cell lines may activate mRNA or increase the amount of mRNA activated.

In order to multiply mammalian cells, they must be maintained in vitro in cell culture or in histologically compatible animals under normal conditions. When in vitro culture maintenance is used to prepare the mRNA source, cells can be grown on any culture medium previously found to promote T cell growth. These media may be supplemented (or not supplemented) with mammalian serum, serum components or serum albumin.

The time required for mRNA activation corresponds to the time required for cell activation to induce mRNA formation. This period generally coincides with the time required for the start of IL-2 secretion in the medium. The preferred duration may be from 3 to 12 hours for the stimulant, e.g. after adding nitrogen. Unjustified prolongation of the culture period may occasionally result in the degradation of the formed mRNA. During the activation process of IL-2-producing cells, phorbol esters, e.g. PMA or TPA may preferably be used at a concentration of 10-50 ng / ml to increase the level of activation.

The above-described process for IL-2 mRNA activation can be carried out at 32-38 ° C in a humidified 4 atmosphere at a pH of about 7.0-7.4.

The procedures required for culturing and harvesting mammalian cells capable of producing IL-2 are explained in more detail below.

(1) Obtaining constitutive IL-2 producing cell lines

The human leukemia T-cell Jurkat cell line (freely available from Fred Hutchinson Cancer Institute, Seattle, United States, Salk Institute, San Diego, United States; German Cancer Center, Heidelberg, West Germany) is suspended in Click medium. irradiation with a 150 R / min irradiation speed IX10 ⁶ cells / ml and a cell density of 8X10 ³ R X-rays. Then, Click media containing 5% FCS in 0.1 cell / 200 μΙ medium was inoculated from the cells so irradiated in a 96-well flat bottom microplate (Falcon 3072) and cultured for 3 weeks at 37 ° C in a 5% carbon dioxide incubator (cloning limited). dilution method). The overgrown pre-cells are transferred to 24-well culture plates (Nunc) before the cell layer becomes confluent and cultured for an additional 5 days. The overgrown cells are further cultured in serum and serum albumin-free synthetic medium for an initial cell density of 1-2X10 ^B / ml for approximately two days. The culture supernatant was harvested by centrifugation and filtered through a 0.22 millipore filter paper to purify the pellet and sterilize the supernatant, and the constitutively IL-2-capable X-ray-treated mutants were selected and cloned in IL-2.

(2) Recovery of IL-2 producing cell from human peripheral blood mononuclear cells. Human peripheral blood is harvested and peripheral blood lymphocytes (hereinafter "PBL") are isolated by density gradient centrifugation on Ficoll-Hypaque. 2 ml of Click media containing 5% FCS in PB is inoculated with 1X10 ⁶ / ml cell density. wells with 100 μΐ of 5 μg / ml phytohemagglutinin M (Gibco) (pHA) in hollow Nunc culture plates and cultured for 48 hours under the conditions described above The cells are washed and re-inoculated in 1 ml Click medium with ⁵ ml cell density 1 ml of "conditioned medium", a medium prepared from human splenocytes stimulated with 2.5 pg / ml of concanavalin A ("Con A") for 48 hours, and the medium containing 50% "conditioned medium" was changed every three days to long-term cultures of human T lymphocytes are obtained from PBL. The thus prepared long-term cultured human T lymphocytes are cloned as above. by the limited dilution method described herein, in the presence of conditioned media from human splenocytes, and cell clones are similarly amplified. The cloned human T lim-4197938 foci are then inoculated with 1 ml RPMI 1640 at a cell density of 1X10 ⁶ / ml in 24 well Nunc culture plates in the presence of 10 pg / ml PHA and cultured for 24 hours at 37 ° C in 7.5% CO ₂ - incubator. The culture supernatant was harvested, centrifuged, filtered through a 0.22 μm micropore filter and assayed for IL-2 activity to accurately determine normal human T-lymphocyte clones producing IL-2.

(3) Obtaining a malignant cell line from human lymphocytes capable of producing IL-2 in the presence of mitogen

Jurkat cell lines, or cloned cell lines, e.g. Jurkat 111, obtained by the limited dilution method described above, is capable of producing 10-4,000 units / ml IL-2 when cultured for 24 hours in RPM1 containing 1-2% serum-free synthetic medium or 1-2% mammalian serum. 1640, a mitogen such as. 10 pg / ml Con A or 2.5 pg / ml PHA. These malignant human cell lines also produce IL-2 when cultured in the presence of zinc chloride, protein A or picibanil.

(4) Generation of cells capable of producing IL-2 in the presence of mitogen, other stimulatory cells, or stimulating soluble factors Molt 4 F human malignant cell line and certain cloned cell lines, e.g. Jurkat J 99, obtained by the limited dilution method, does not produce IL-2 even when cultured for 24-72 hours in the presence of any concentration of lectin or mitogens. However, these cells are able to produce significant amounts (10-100 U / ml) of IL-2 over a 24 hour culture at 37 ° C when co-cultured with 5 to 0 U / ml of interleukin 1 (monokines). 50% of K 562 or Raji cells.

Extraction of IL-2 mRNA from cells activated as above is performed by a conventional, well-known method, regardless of the different sources of the cells. so e.g. the cells are partially or completely comminuted with detergent, e.g. NP-40, SDS, Triton X and deoxycholic acid, or by mechanical homogenization or freeze-thaw method. In order to prevent degradation of RNA by ribonuclease during mRNA extraction, preferred RNase inhibitors, e.g. heparin, polyvinyl sulfate, bentonite, macaloid (available from National Lead Company, Houston, Texas), diethyl pyrocarbonate or vanadyl complex. IL-2 mRNA can be obtained from a polysome precipitated in IL-2 biosynthesis which is precipitated with an anti-IL-2 antibody and detergent extracted.

The poly A-containing mRNA can be fractionated or concentrated by any conventional method, e.g. affinity chromatography, or single-pass absorption on oligo-dT cellulose or sepha8 rose 2B poly-U-sepharose, sucrose density gradient centrifugation, or agarose gel electrophoresis.

The mRNA fractions were then assayed for IL-2 mRNA activity by testing the biological activity of the transcribed proteins from the mRNA fractions or by identifying the transcribed proteins using a monoclonal antibody against the IL-2 peptide. so e.g. the mRNA is usually transcribed into the appropriate protein by microinjection into the egg of a frog (Xenopus laevis) (Gurdon, JB et al., Natura 233, 177-182 (1972)), or by using a mRNA-dependent reticulolysate or cereal germ translational cell-free system. .

IL-2 activity can be demonstrated by a microassay developed in principle by Gillis et al. (1978), Gillis, S. et al., J. Immunol. 120,2027-2033. The assay monitors IL-2-dependent cellular proliferation of cytotoxic T lymphocyte cell lines (CTLLs), which are induced by the method described by Gillis et al. That is, 4X10 ³ CTLL cells are inoculated into 100 μΐ RPMI 1640 medium containing 2% FCS with 96 μl serially diluted transfected product in a 96-well flat bottom microplate. After incubation for 20 hours at 37 ° C in a 5% CO ₂ incubator, cells are shaken for 4 hours with 0.5 pCi of ³ H-TdR, harvested on glass fiber strips using an automatic cell harvester, and the incorporated activity is measured by liquid scintillation counting. With these assays, CTLL cells cultured in the presence of IL-2 are found to incorporate ³ H-TdR in a dose-dependent manner, allowing for a definite calculation of the amount of IL-2 in the test sample.

IL-2 has an activity that promotes T-lymphocyte proliferation, allowing measurement of IL-2 activity using a T-cell growth activity index. That is, 5 CTLL cells are transferred to 100 μΙ DMEM containing 2% FCS in 96-well flat bottom microplates with 100 μΐ serial dilution transcription product. After incubation for 72-96 hours at 37 ° C in a 5% CO ₂ incubator, the number of overgrown cells and activated cells is counted under a microscope. As a positive external control group, 100 units / ml and 10 units / ml IL-2 were added and the IL-2 activity of the test sample was calculated by comparing the number of live cells grown in these control groups.

The IL-2 mRNA thus obtained from the most active fractions serves as a template for the synthesis of ds-cDNA and the ds-cDNA is linked to a vector DNA. Synthesis of cDNA is accomplished by conventional methods.

First, ss-cDNA, which is complementary to mRNA, is prepared by dATP, dGTP, dCTP,

-5197938 in the presence of dTTP, using reverse transcriptase and using mRNA as a template, and oligo-dT as a reading template (primer). The template mRNA is then removed by alkaline treatment and the ds-cDNA is generated using reverse transcriptase or DNA polymerase using the ss-cDNA synthesized above.

DNA produced by recombinant technique is prepared from the resulting ds-cDNA and a replicon-containing vector DNA capable of replication in eukaryotic or prokaryotic systems. The recombinant DNA is then incorporated into the host cell.

The ds-cDNA and a vector DNA that is capable of replication in eukaryotic or prokaryotic cells have been modified prior to coupling by various methods, e.g. exonuclease treatment, addition of chemically synthetic DNA fragments as well as G, C terminus giving a ds-CDNS and vector DNA terminus. The linkage of linkable DNAs can be accomplished e.g. Using phage T ₄ DNA ligase in the presence of ATP.

The resulting recombinant DNA live cells can be transformed to amplify the cloned cDNA or to produce IL-2 polypeptide;

Suitable eukaryotic host organisms commonly used for the production of IL-2 include vertebrates, yeasts, and the like. so e.g. monkey cells (e.g., CV-I cell) transformed with the SV-40 origin-defective mutant and expressing the SV-40 large T antigen (COS cells) as described by Y. Gluzman (Cell, 23, 175 -182 (1981)); mouse cells described by Ohno, S. and Taniguchi, T. (Nucleic Acids Research, 10, 967-977 (1982)); and yeast host-vector systems used for expression of the IF'N gene, described by R. Hitzeman (Natura, 293, 717-722 (1981)). Suitable prokaryotic host organisms include Escherichia. coli, Bacillus subtilis, etc. In order to amplify the DNA in the host, it is preferable to use E. coli as the host, although other hosts may be used.

Suitable vectors for E. coli include EC type plasmid vectors (strict type): pSC101, pRK353, pRK646, pRK248, pDF4I, etc .; EC type plasmid vectors (moderate type): ColE1, pVH51, pAC105, RSF 2124, pCR1, pMB9, pBR313, pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700, pKN80, pKC7, pKN80, pKC7 rabbit, etc; λ gt phage vectors λ gt.Ác, λ gt.XB, XWES, XC, λΒ. Xzj vir., ΛΒ ', XALO, λΒ, XWES.Ts622, XDam, etc. In general, pBR322 is most commonly used as a vector for F, coli and in this case the best cloning sites are PstI and EcoRI.

1o

Transformation of the host cell with recombinant DNA can be accomplished in conventional manner as follows.

Where the host organism is prokaryotic, e.g. E coli, suitable cells are prepared which are capable of capturing DNA. These cells are prepared from cells obtained after the exponential growth phase and subsequently treated with the CaCl ₂ method according to known techniques. When MgCl ₂ or RbCl is present in the transformation reaction medium, the transformation efficiency increases. Transformation can also be performed after protoplast formation from the host cell.

Where the host used is eukaryotic, the method of DNA transfection, such as, e.g. calcium phosphate precipitation; conventional mechanical methods such as microinjection, insertion of a plasmid encapsulated in the red blood cell host or liposomes; treatment of the cells with an agent, such as lysophosphatidylcholine, or the use of viral vectors and the like.

Isolation of cells with the IL-2 gene after transformation can be performed by one of the following two methods:

(1) In the plus-minus method, partially purified IL-2 mRNA is obtained by sucrose gradient centrifugation of mRNAs extracted from mitogen-activated mammalian cells as 11-12S pellets followed by synthesis of 32 P-radiolabeled ss-cDNA. using partially purified mRNA as a template. After removing the template mRNA by alkaline treatment, the isolated cDNA was fractionated by hydroxylapatite column chromatography. The non-hybridized cDNA and the hybridized cDNA are temporarily referred to as "A" and "B" samples respectively. The transformants grow in exactly the same way on the two nitrocellulose filters and the DNA of the cells is attached to the filter paper by alkaline treatment. Sample A and sample B are hybridized with DNA on two different filter papers and then autoradiographed to select transformants that react positively to sample A (plus) but show little or no reaction sample B (minus) (Taniguchi et al., Proc. Jpn. Acad., v. 155B 464-469 (1979)).

(2) The second method is eg. It consists of distributing 1000 to 10000 transformant clones into tens or hundreds of clones. The distributed clone groups are sequentially grown in conventional manner to obtain plasmid DNA. These plasmid DNAs are then converted to ss-cDNAs, e.g. heat denaturation, and the resulting ss cDNAs are fixed on a nitrocellulose filter paper to achieve hybridization of mRNA from mammalian cells complementary to the fixed DNA and activated IL-2 mRNA. Alternatively, mRNA containing IL-2 mRNA is ridged with Hib-6197938 and heat-denatured plasmid DNA and the DNA-mRNA hybrid is fixed on nitrocellulose filter paper. These filter papers are then used with low salt concentration buffers, e.g. 1 mM HEPES buffer or 10 mM NaCl was washed and mRNA adsorbed on filter paper was extracted with 0.5 mM EDTA, 0.1% SDS for about 1 min at 95 ° C. Purified mRNA is recovered by elution with oligo dT-cellulose column chromatography. The mRNA is then translated into protein. Microinjection into the egg of Xenopus laevis to determine IL-2 activity or translate the mRNA into a protein using an mRNA-dependent reticulocyte or wheat germ in vitro cell-free translation (anti-IL-2) assay to analyze IL-2 activity antibody. According to these methods, the group in which IL-2 activity can be detected can be repeatedly subdivided into groups containing a smaller number of transformant clones, so long as a single clone with IL-2 DNA remains.

To obtain cDNA encoding an IL-2 polypeptide from IL-2 producing transformants, the recombinant DNA in the transformant is isolated and cleaved with restriction endonuclease. The inserted cDNA fractions were separated from the DNA fragments formed by cleavage.

The complete nucleotide sequence of the Pst I DNA insert (insert) obtained from pIL2-50A recombinant DNA coding for the IL-2 polypeptide by the method of Maxam and Gilbert (Meth. Enzym. 65,499-560 (1980)) and the end of the dideoxynucleotide chain! (Smith, A.J.M., Meth. Enzym. 65, 560-580 (1980)).

The restriction endonuclease cleavage map of the cDNA insert and the base sequence of the insert are shown in Figures 1 and 2 (a), respectively, in which the cDNA has restriction endonuclease cleavage sites (BstNI, XbaI and BstNI, respectively).

The DNA sequence of the insert contains a unique large open reading frame. The first ATG sequence, which generally serves as a starter sequence in eukaryotes (Kozák, M .: Cell, 15, 1109-1123 (1978)), is located at nucleotide 48-50 at the 5 'end. This ATG is followed by codon 152 before we encounter the TGA termination triplet at nucleotides 507-509.

A region of the 3'-poly (A) terminus of the mRNA is found at the end of the cDNA and precedes the AATAAA hexanucleotide (positions 771-766), which is usually found in most eukaryotic mRNAs (Proudfoot, NJ and Brownlee). , CG: Natura, 263, 211-214 (1976)).

The amino acid sequence encoded by the cDNA can be deduced from Figure 2 (b) (Amino acid sequence I) and has a polypeptide of amino acid sequence I containing 153 amino acids and a molecular weight of 17631,7 daltons. As described for most of the secretory proteins known to date (Blobel, G. et al., Sym. Soc. Exp. Med., 33, 9-36 (1979)), the N-terminal region of the derived IL-2 polypeptide is also quite hydrophobic and this region is likely to serve as a signal peptide that is cleaved during the mature IL-2 selection process. Such cleavage occurs either between Ser and Ala at positions 20 and 21, or between Al and Pro at positions 21 and 22, forming a polypeptide having amino acid sequence II and III, since similar cleavage sites are often found in other secretory proteins (Blobel, G., et al., Symp. Soc. Exp. Med. 33, 9-36 (1979)). The mature IL-2 polypeptide then contains 132 or 133 amino acids with a calculated molecular weight of 15420.5 daltons or 15349.4 daltons. This value is then compared with that of human IL-2 protein obtained from Jurkal cells (15,000 daltons) (Gillis, S. et al., Immunological Rev., 63, 67-209 (1982)). In addition, a DNA fragment from the CCT codon at position 111-113 in the base sequence, which therefore encodes a polypeptide at position 22 in Prc (amino acid sequence III, Figure 2 (b)), has been shown to express such a polypeptide. which has IL-2 activity as shown in Example 5. It has also been shown that the sequence 107 to 110 in the base sequence. a DNA fragment with the GCA sequence at position 2, which therefore encodes a polypeptide starting at Ala at position 21 (amino acid sequence II in Figure 2 (b)), expresses a polypeptide having IL-2 activity as shown in Figs.

Example 8 will show.

It is known that genes of eukaryotes often exhibit polymorphism e.g. human interferon genes. (Taniguchi et al., Gene. 1980, 10, 11-15); Ohno and Taniguchi, Proc. Natl. Acad. Sci. USA, 77, 5305-5309 (1980); Gray et al., Natura, 295, 501-508. (1981)). In some cases, polymorphism is associated with the substitution of certain amino acids of the protein product, while in other cases, the structure of the protein remains unchanged. In the case of human IL-2 cDNA, another cDNA clone (pIL2-503) in which residue "A" is replaced by residue "G" at position 503 of pIL-5C'A cDNA (Figure 2). Other cDNA clones are also expected with certain base substitutions relative to the pIL2-50A cDNA.

As will be understood from the above, the genes of the present invention may be: DNA having the base sequence shown in Figure 2 (a); DNAs starting with the ATG sequence at position 48-50 which are the base followed by the ATG sequence at least at position 504-5067

-7197938; at positions 108-110, DNAs having a GCA sequence having a base sequence at least ATC codon following the GCA sequence; and, at positions 111-113, DNAs having a CCT sequence having a base sequence following the CCT sequence extending at least to the ACT sequence.

The genes of the present invention may also be DNAs that terminate with the ACT sequence of genes 504 to 506. start with positions A and A at position 1, ATG their sequence at positions 48-50. at position 108, their GCAs are sequences 108 to 110. or CCT have their sequence at positions 111-1133. position. The genes of the present invention may also be DNAs that terminate with the TGA sequence of genes 507-509. starting with position A and starting with position 1, ATG has the sequence at positions 48-50. at position 108, their GCAs are sequences 108 to 110. or CCT have their sequence in positions 111 to 113. position. The genes of the present invention are also DNAs that terminate with C at position 801 and start with A at position 1, ATG having the sequence at positions 48-50. at position 108, GCA at positions 108 to 110 or CCT at positions 111 to 113. In addition, the genes of the present invention may be DNAs terminated with poly (A) and initiated with the ATG codon at positions 48-50. position, the GCAs have the sequence 108-110. or CCT are their sequence 111-113. position.

The genes of the present invention may be those which have a base sequence corresponding to amino acids 1, II and III. Amino acid sequences. In addition, polypeptides may have IL-2 activity in which one or more amino acids of the amino acid sequence I are missing, or polypeptides in which one or more amino acids of the amino acid sequence I are replaced by one or more amino acids. Therefore, genes encoding such polypeptides are also genes of the present invention. Likewise, genes which have an additional linkage to one or more base sequences capable of expressing one or more additional amino acids in I, II or III. Amino acid sequences are also within the scope of this invention as long as the additional amino acids do not interfere with the activity of the polypeptides to elicit IL-2 activity. A modified linked excess amino acid region that interferes with polypeptide function with respect to IL-2 can also be used in the present invention to the extent that the linked excess region can be readily removed. This situation is the same for the complementary region of the linked DNA, at the 3 'terminal of the gene that is located in I, II, or II. III. Corresponds to amino acid sequence; this additional DNA region encodes I, II, and II respectively. III Additional amino acids at the C terminus of the Amino8 acid sequence. Therefore, the use of genes encoding such polypeptides is also within the scope of the present invention.

Recombinant DNAs that direct IL-2 production in living cells can be constructed by a variety of methods. so e.g. the IL-2 cDNA coding sequence may be inserted into an expression carrier downstream of the promoter sequence. Alternatively, a piece of cDNA carrying a promoter sequence may be inserted upstream of the IL-2 coding sequence before or after the cDNA is inserted into an expression-capable carrier.

The method of producing prokaryotic or eukaryotic cells that express IL-2 cDNA and IL-2 polypeptide is explained in more detail below;

(1) Expression of IL-2 cDNA in E. coli

For the purpose of expressing IL-2 cDNA in E. coli. cDNA is combined with various bacterial promoters and hybrid plasmids containing cDNA downstream of the promoters are obtained. The plasmids are introduced e.g. Bacteria synthesizing a protein product having E. coli strain HB101 and human IL-2 activity are cloned. Essentially, any bacterial promoter can direct expression of IL-2 cDNA when properly aligned with the cDNA. Examples of such cDNA expression are described herein.

The cDNA cloned for IL-2 encodes a 153 amino acid polypeptide as shown in Figure 2. The N-terminal region, which corresponds to about 20 amino acids of this polypeptide, is quite hydrophobic and is characteristic of most secretory proteins. Such a hydrophobic sequence, the so-called "signal sequence", is cleaved during the secretion process. Therefore, the mature IL-2 polypeptide should have less than 153 amino acids. Therefore, it is desirable to express the portion of the cDNA encoding the mature IL-2 polypeptide, but not the portion corresponding to the IL-2 "signal sequence.

(i) The construction of an expressable plasmid carrier, pTrs-3, which includes the E. coli trp promoter, its ribosome binding site (SD sequence) to the leader peptide, has been previously described (G. Miozzari and Yanofsky, C. J. Bacterial. 133, 1457-1466 (1978) and described an ATG codon located 13 bp (base pair) down the SD sequence (Nishi et al., SEIKAGAKU, 54, 8, 676 (1982)). )). The plasmid carrier also contains a unique SphI site just downstream of the ATG starter sequence (Figure 4).

To express IL-2 cDNA, the plasmid was first digested with SphI and treated with either E. coli DNA polymerase I (Klenow-8197938

fragment) or bacteriophage T4 polymerase I to remove the 3 'protruding ends (Fig. 5 (a)). Plasmid pIL-2-50A was digested twice with PstI and HgAIl and a larger cDNA fragment was isolated. The DNA is then treated with either E. coli DNA polymerase I (Klenow fragment) or bacteriophage T4 DNA polymerase such that the 3 'protruding ends are flush. The above treated cDNA encodes the 132 amino acids of the IL-2 polypeptide as shown in Figure 5 (a). This cDNA is then linked to the pTrs-3 pre-treated plasmid so that the ATG primer codon fits to IL-2. cDNA for the CCT (Pro) sequence, resulting in a plasmid pTIL -2-22 The junction between the pTIL-2-22 trp promoter sequence and the IL-2 cDNA sequence is also shown in Figure 5 (a).

Plasmid pTIL-22 should direct the synthesis of the proline-initiated IL-2 polypeptide containing 132 amino acids in E. coli.

(ii) Since it is also possible that mature IL-2 contains alanine (position 21) as the N-terminal amino acid instead of proline, the following plasmid is also directed to direct the synthesis of the 133 amino acid IL-2 polypeptide.

Plasmid pTrs-3 contains a unique Cla I site between the SD sequence and the ATG sequence (Figure 4). The plasmid was digested with Cla I and Sal I. Plasmid pIL 2-50A is partially digested with Pst I, treated with E. coli DNA polymerase I, and the largest linear DNA is isolated. The DNA is then linked to a synthetic DNA linker containing a restriction Xho I site and a clone containing the plasmid pIL2-50A (Xho) into which the linker DNA is inserted downstream of the 3 'end of the IL-2 coding sequence. isolated. Plasmid pIL2-50A (Xho) was first digested with HgI A1 and treated with either E. coli Klenow fragment or T4 DNA polymerase, digested with XhoI and the cDNA fragment was isolated. This cDNA fragment is then linked to pTrs-3 DNA pre-treated with Clal and SalI and a synthetic DNA, as shown in Fig. 5 (b), to form a plasmid pTIL2-21 that can direct a 133 synthesis of the amino acid IL-2 polypeptide starting with alanine in E. coli. as shown in Figure 5 (b). Similar constructions can be made without using the Xhol switch.

(iii) IL-2 polypeptides of different sizes with different N-terminal amino acids can be prepared with the plasmid carrier expressing pTrs-3 according to the following procedure. The cloning IL-2 cDNA contains a unique Dde I site in pIL2-50Ab at positions 81-85. nucleotide sites. Plasmid pIL2-50A (Xho) was digested with Dde I and the DNA fragment containing most of the cDNA was isolated. The fragment also contains about 3000 base pairs of pBR 322 (Figure 5 (c)). The DNA fragment was treated with Bal 31 exonuclease and then digested with Xho I. The above treated DNA was then ligated with pTrs-3 which was digested with Sph I or treated with either the Klenow fragment or T4 DNA polymerase and then digested with Sal I as shown in Figure 5 (c). The linked DNA is then transferred to E. coli HB101. and screening bacterial clones expressing human IL-2. These clones express human IL-2 of different sizes because the DNA corresponding to the N-terminus of human IL-2 is differently cleaved. Thus, pTIL2-14 and pTIL2-15 carrying IL-2 cDNA can be obtained.

(iv) IL-2 cDNA can be expressed using pKT 218 (gift from K. Tolmage) (Proc. Natl. Acad. Sci. USA, 77, 3369-3373 (1980)). Plasmid pKT218 was digested with Pst I and ligated with an IL-2 cDNA insert obtained by digesting pIL-2-50A DNA with HgAI1 and Pst I. (Figure 6). The resulting plasmid pKIL2-21 directs the synthesis of the fusion polypeptide in E. coli. which is a fusion polypeptide comprising 133 amino acids of IL-2 and amino acids of β-lactamase (the first methionine is cleaved in E. coli).

(v) A plasmid capable of expressing pTuBIP-5, in which the promoter sequence is inserted into pBR322, is inserted into tuf B (Taniguchi et al., SEIKAGAKU, 53, 966 (1981)). The plasmid contains a unique Cla 1 helix located 2 bp downstream of the SD sequence as shown in Figure 7.

Because pTrs-3 also contains a Cla I site between the SD sequence and the ATG start sequence, and since this Cla 1 site is not destroyed during the construction of an expressible plasmid, using pTrs-3 and IL-2 cDNA as as described above, it is very easy to replace the bacterial Trp promoter with the tuf B promoter such that the human IL-2 cDNA is expressed under the control of the tuf B promoter. so e.g. pTIL2-22 was digested with Cla I and Pvu II and the DNA fragment containing the IL-2 cDNA was isolated. This fragment is then linked to pTUB1P-5 DNA, pre-digested with Cla I and Pvu II, and a plasmid pTuI 12-22 is constructed as shown in Figure 7. IL-2 activity can be detected in extracts of E. coli HB101 containing pTuIL2-22.

(vi) A similar construction can be made e.g. pTIL2-21 and essentially any expressable plasmid constructed with pTRS-3. It is possible to optimize the distance between the SD and ATG sequences e.g. by digesting pTuIL2-22 with Cla I. some DNA base pairs were removed (or added) with Bal 31 or S1 or DNA polymerase I (E. coli) and then recombined into the plasmid.

-9197938 (2) Expression of IL-2 cDNA in yeast

IL-2 cDNA can also be expressed in yeast by inserting the cDNA into vectors that are capable of expression and introducing the product into the host cell. Various migratory vectors capable of expressing foreign genes in yeast have been described (Heitzman, R. et al., 1981, 293,717-722 Valenzuela, P, et al., Natura, 298, 347-350 (1982); Miyanohara Proc. Natl. Acad. Sci. USA 80: 1-5 (1983). The vectors are capable of replication in both E, coli and yeast hosts and contain yeast gene promoter sequences. Essentially any of these expressable vectors can be used to express IL-2 cDNA. Higher levels of IL-2 production can also be achieved by the use of yeast relative to animal cells or bacteria. An example of the expression of human IL-2 cDNA in yeast is provided herein.

pAT77 and pAM82 yeast-E. coli transmigrating ("commuting") vectors have been described by Miyanohara et al. (Proc. Natl. Acad. Sci. USA 80, 1-5 (1983)). PAM82 is a derivative of pAT77 and both carry ars 1 (Stinchcomb, DT, et al., Naturre, 282, 39-43 (1979)), 2 pm (Broach, JR et al., Gene 8, 121-133 (1979). )) and leu 2 íRatzkin. B. et al., Proc. Natl. Acad. Sci. USA 74: 474-491 (1979) and the promoter of the yeast acid phosphatase (APase) gene. They also carry a 3.7 kb DNA segment of pBR322 carrying an ampicillin resistance marker (Ap ') and the origin of replication (Figure 8). The APase promoter can be induced by changing high concentrations of phosphate to low concentrations in the medium. To express human IL-2 cDNA, pIL2-50A was digested with PstI after treatment with either E. coli Klenow fragment or T4 DNA polymerase, the cDNA was ligated with pAM82 previously digested with Xhol and incubated with E. coli Klenow fragment to fill ends. Hybrid plasmids having the cDNA coding sequence downstream of the yeast APase promoter sequence are selected by cloning in E. coli. The resulting plasmid, PYIL-2a, is introduced into yeast, and after induction of the APase promoter, IL-2 activity is measured in the yeast extract. Plasmid pYIL-2a contains a GC radical region between the yeast promoter and the IL-2 cDNA. It is possible that such a sequence may inhibit IL-2 cDNA expression. To overcome this problem, the following plasmid was constructed: PIL2-50A was digested with PstI and the cDNA insert was isolated. This cDNA is then treated with T4 DNA polymerase in the presence of dATP, dGTP, and dTTP so that the C-core regions are cut off at both ends of the cDNA and then treated with Nuclease S1.

To remove sections of the G-root. This DNA was ligated with the XhoI DNA linker and your plasmid pBR 322, which had its EcoRI site cleaved and aligned with EcoRI and the Klenow fragment, and the resulting plasmid, pIL2-XhoI was digested with XhoI and the cDNA insert isolated. The cDNA is then introduced into the unique XhoI site of pAM82 and a plasmid containing the IL-2 coding sequence correctly oriented for the yeast APase promoter is cloned in E. coli. This plasmid, pYIL-2b, was introduced into yeast and, after induction of the APase promoter, IL-2 activity was measured in the yeast extract.

(3) Expression of cDNA in a mammalian cell

A plasmid that directs the synthesis of human IL-2 in mammalian cells can be constructed as follows: pCE-1 is constructed from pKCR (O'Hare, K., et al., Proc. Natl. Acad. Sci. 1527-1531 (1981)) and pBR328 (Soberon, X., et al., Gene, 9, 287-305 (1980)), using a series of modification procedures as shown in Figure 3, and IL-2. gene ATG primer is linked downstream of the SV40 early gene promoter. This plasmid contains a unique PstI site just downstream of the SV 40 promoter and an intron upstream of the rabbit β-globin chromosomal gene. The plasmid also contains the SV 40 origin of replication as well as the early gene polyadenylation site. Thus, a plasmid pCEIL-2 is obtained in which the IL-2 structural gene can be transcribed from the SV 40 early promoter gene in the appropriate host cell (Figure 3).

This plasmid was digested with Hhal. followed by introduction of DNA into COS7-transformed monkey cells, which allows DNA replication of the SV 40 starter sequence. It appears important to digest the plasmid with Hhal prior to transfer (transfection) for efficient expression of the cDNA, since sequences that inhibit replication of the transferred DNA in COS cells can be removed from a substantial portion of the plasmid required for cDNA expression by this method for 1-3 days. In cultures (from transfer of this vector to the cultured COS-7 monkey cell) under conventional culture conditions (Gluzman, Y .: Cell, 23, 175-182 (1981)), IL-2 is generally secreted and produced in the culture medium of the cultured cell. Similarly, for the purpose of inserting the expanded DNA into other eukaryotic cells, a vector suitable for the host is linked to a cDNA insert excised and isolated from a prokaryotic cell, and the vector synthesized in this way can be transferred and cultured.

Cells incorporating recombinant DNA are cultured either to amplify the recombinant DNA or to produce the IL-2 polypeptide. Breeding is the traditional way

-10197938 can be executed. so e.g. the transformed yeast is a carbon source, a nitrogen source, inorganic salts and, if necessary, other organic nutrients, e.g. in a medium containing vitamins and amino acids, at 20 ° C to 37 ° C and in a pH range of 4 to 7 aerobically. Transformed prokaryotic organisms, e.g. E. coli or B, subtilis can also be grown under conventional conditions.

Intracellularly or extracellularly produced IL-2 can be recovered by any known method, such as ammonium sulfate precipitation, dialysis to remove salts (at normal pressure or in vacuum), gel filtration, chromatography, preparative flat bed isoelectric focusing, high performance liquid chromatography (hereinafter HPLC), ion exchange, gel filtration and reverse phase chromatography, and affinity chromatography on dye-supported vehicle, activated Sepharose 4B coupled with anti-IL-2 monoclonal antibody, or lectin-bound Sepharose, etc. Methods for recovering and purifying IL-2 have been described in more detail (Watson et al., J. Exp. Med. 150, 849-861 (1979), Gillis et al., J. Immunol. Methods, 39, 185-201 (1980)), and Welte, K., et al. (1982) J. Exp. Med. 156: 454-464.

The resulting polypeptide exhibits the same biochemical and biological behaviors as known IL-2 produced by a mitogen-stimulated mammalian cell and also has IL-2 activity. The molecular weight is about 15,000 daltons and the IL-2 activity is completely neutralized or precipitated by monoclonal anti-IL-2 antibody, such as immunoadsorpts. In the presence or absence of Igsorb (Enzyme Center). In immunoelectrophoresis, the IL-2 polypeptide shows only a single precipitate against the corresponding anti-IL-2 antibody. IL-2 activity remains stable after reduction with 2-mercaptoethanol and is resistant to treatment with DNA and RNA and heat treatment at 56 ° C for 30 minutes. Activity is stable between pH 2 and pH 9. The produced IL-2 promotes the growth of monoclonal functional T cells (cytotoxic T-lymphocytes), increases thymocyte mitogenesis, raises the generation of antitumor-specific cytotoxic T lymphocytes from memory in the absence of antigen, and can be used to enhance natural killer cell activity. and RLo 1 cells.

The invention has already been described in general terms, and the same will become more apparent upon reference to certain specific examples, which, however, are intended to be illustrative only and are not intended to be limiting unless otherwise indicated.

Example 1 (1) Human T leukemia cell line, Jurkat cells (available in Japan, West Germany and the United States) are suspended in 10 volumes of RPMI 1640 medium containing FCS and irradiated with 10,000 X-rays. up to X-rays at room temperature for 50 seconds using an Exs 150 / 300-4 (Toshiba, Japan) X-ray irradiation apparatus, and the irradiated cells are then cultured for 5 days at 37 ° C in a 5% CO ₂ incubator with an initial cell density of 1X10 ⁵ cells / ml. in the above-mentioned medium. Mutated cells (0.2 cells / well) were placed in wells of 10 flat-bottomed microplates with 96 wells and cultured at 37 ° C in a 5% CO ₂ incubator for 21 days.

Clones from growth cavities were repeatedly transferred to fresh medium to proliferate the clone size and proliferating clones were cultured for 24 hours at an initial cell density of ¹ x ^{10 6} cells / ml at 50 pg / ml Con A and the IL-2 activity was measured as described previously. . As a result, a human T cell line, designated Jurkat-111 (hereinafter "J-111") (ATCC 2RL 8129), which was cloned from the parent Jurkat cell, had an IL-2 productivity increased by a factor of 40. relative to the productivity of the parent strain. The J-111 cloned cell line can grow under normal conditions and the growth rate is almost identical to that of a normal Jurkat cell.

(2) lXl0 ⁵ / ml J-111 cells were inoculated with 1000 ml of a serum-free synthetic tápközegber (RITC 55-9) (Sato, T., et al..., Exp Cell Res 138, 127-134 (1982) is cylindrical in culture flasks (Falcon 3027) and cultured for 4 days at 37 [deg.] C. and the cultured cells were harvested by centrifugation, and the harvested cells were again inoculated at 4x10 ⁶ cells / ml in the medium mentioned below, to which 25 [mu] g / ml ConA was added. Falcon) four pieces form one batch, 1000 mL of inoculated culture is placed in each batch.

(3) 1.2X10 ⁶ Jurkat cells stimulated with 25 pg / ml ConA for 6 hours were suspended in 8000 ml saline-equilibrated phosphate buffer (PBS). The cells were washed twice by centrifugation and resuspended in 800 ml of RSB solution (10 mM Tris / HCl, pH 7.5, 10 mmol / l NaCl, 1.5 mmol / l MgCl _2), which ribonucleoside-vanadyl complex (10 mM per liter), which is a nuclease inhibitor. MP-40 detergent was then added to give a final concentration of 0.05%, followed by gentle agitation and centrifugation of the nuclei for 5 minutes at 3000 rpm at 4 ° C. SDS (0.5%) and EDTA (5 mM) were added to the supernatant and the cytoplasmic RNA was extracted with an equal volume of phenol. Three times phenolic

After extraction of -11197938, RNA was precipitated with two volumes of ethanol and the precipitate was collected by centrifugation, which was solubilized in 10 mM Tris-HCl pH 7.5. The amount of RNA obtained was 196 mg.

Fractionation of mRNA was performed using affinity chromatography on oligo (dT) cellulose (P. L. Biochemicals, Type 7). The composition of the adsorption solution is 20 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA and 0.5% SDS, pH 7.5, and elution with water and 10 mM Tris -HClel. (pH 7.5) after washing the column with buffer. (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM Tris-HCl). The resulting eluted mRNA was 3.6 mg. Subsequently, 2.4 mg of the resulting mRNA is fractionated by sucrose density gradient centrifugation1.

huk (gradient of 5 to 2.5% sucrose in a pH 7.5 solution of 50 mM Tris-HCl, 1 mM EDTA and 0.2 M NaCl, centrifugation 26,000 rpm at 24 ° C for 24 hours) and fractionating the mRNA 11-12S into fractions 12, 13 and 14 at 59 pg. 46 pg and 60 pg, (4) The mRNA obtained in fraction 13 was microinjected into Xenopus laevis oocytes (50 ng mRNA / egg) and the culture supernatant serves to measure IL-2 activity. As shown in Table 1, an increase in ³ H TdR incorporation and an increase in the number of activated T lymphocytes can be demonstrated, clearly demonstrating that mRNA contains human .IL-2 mRNA in this fraction.

Spreadsheet /

Sample Dilution ³ H-TdR capture / cpm / TT o. . X Amount of IL-2 /egység/.ml/ Control I - 553 0 / medium for measurement / Control II X2 590 0 / untreated eggs- X3 572 culture supernatant ja / Fraction 13 X8 14.683 32 translation product X32 10.165 / B / Dilution T-lymphocyte ττ o. / x Quantity of IL-2 cell count / U / ml / / Db / well / Control I X2 0 0 / Medium for measurement / X16 0 Control II X2 0 0 / Untreated eggs- X16 0 culture supernatant ja / Fraction 13 X2 1 · 5 40 translation product X16 55 x We calculate your unit by to compare the built-in ³ H-TdR amount of standard IL-2/10 units / ml / probit analysis by

(5) Subsequently, cDNA was synthesized with 11-12S mRNA containing IL-2 mRNA.

Fraction 13 was constructed in vitro and recombinantly with plasmid vector pBR322. Escherichia coli was transformed with recombinant 12

Clones obtained from DNA and IL-2 cDNA were selected as follows:

(5-1) 50 mM Tris-HCl buffer (pH 7.5), 30 mM NaCl, 6 mM MgCl ₂ -t, 5 mM dithiothreitol eitolt ^r (hereinafter referred to as DTT), 0.5 mM

-12197938 dATP, dGTP, dCTP, dTTP (dCTP ³² P radiolabeled), 0.7 pg oligo (dT), ₀ et, 10 pg mRNA and 15 units AMV reverse transcriptase ( JW Beard) and stirred for 90 minutes at 41 ° C.

After the reaction was stopped, the DNA was recovered after phenol treatment as an ethanol precipitate and the DNA was dissolved in a pH 7.5 solution containing 20 mM Tris and 1 mM EDTA.

2.5 pg ss-cDNA was synthesized. To remove the mRNA present in the solution, the solution was adjusted to 0.33 N NaOH by addition of NaOH, allowed to stand at room temperature for 15 hours, and then neutralized with an equal volume of 1 M Tris-HCl pH 7.5 and pass through a Sephadex G-50 column.

The recovered cDNA was 1.8 pg.

(5-2) 50 mM phosphate buffer (pH 7.5), 10 mM MgCl ₂ , 10 mM DTT, 0.75 mM dATP, dGTP, dCTP and dTTP (a) dCTP ³ H (radiolabeled), 1.8 pg ss-cDNA and 8 units of polymerase I (BRL, United States) were mixed and allowed to react for 15 hours at 15 ° C. After the reaction was stopped, the DNA was recovered as phenol and chloroform as an efanol precipitate. 1.10 pg ds-cDNA was generated. 50 ml of sodium acetate (pH 4.5), 0.2 mol of NaCl, 1 mmol of ZnCl ₂ and 1.10 pg of ds-cDNA were incubated for 20 minutes at 37 ° C with 0.25 units of nuclease. S1 (Sankyo, Japan) was added and incubated for an additional 15 minutes.

. After the reaction was stopped, the reaction product, which was twice treated with phenol, was applied to Sephadex G-50 to obtain 0.55 pg of ds-cDNA.

(5-3) 0.14 M potassium cacodylate, 30 mM Tris base, 0.1 mM DTT, 1 mM CaCl ₂ , 0.64 mM ³² P-dCTP (specific activity 2.7 x ^{10 6} cpm / nmole), 0 A mixture of 55 µg of ds-cDNA and 5 units of terminal transferase (BRL) was incubated for 7 minutes at 37 ° C and, after phenol treatment, applied to a Sephadex G-50 column, yielding 0.5 µg of DNA as an ethanol precipitate. The recovered DNA was found to have about 50 dCMP residues at both 3 'terminals. 10 µg of pBR 322 DNA was cleaved with the restriction enzyme PstI and the 3 'terminus of the cleaved DNA was joined to the dGMP chain in the same manner as described previously for adding dCMP to ds-cDNA, except that dGTP was replaced with dCTP. employed.

(5-4) 50 mM Tris-HCl buffer (pH 7.5), 0.1 M NaCl, 5 mM EDTA, 0.05 pg pBR322 (extended with dGMP) and 0.01 pg cDNA (dCMP) The mixture was incubated first at 65 ° C for 2 minutes, then at 46 ° C for 120 minutes, then at 37 ° C for 60 minutes and finally at room temperature for 60 minutes. E. coli K 1776 {Curtiss

111, R. et al., Molecular Cloning of Recombinant DNA (edited by WA Scott and R. Werner), Academic Press (1977)) is inoculated in 50 ml of L medium containing 100 pg / ml diamineprimelic acid, 50 pg / ml thymidine, 1% tryptophan, 0.5% yeast extract, 0.5% NaCl, and 0.1% glucose, and inoculated at 37 ° C with shaking until the absorbance of the culture medium at 562 nm was approximately 0.3 OD (optical density). ). After culturing, the culture medium is kept at 0 ° C for 30 minutes, and the bacterial cells are harvested by centrifugation, washed twice with 25-25 ml of a solution containing 5 mM TrisHCl, pH 7.6, 0.1 mol. NaCl, 5 mM MgCl ₂ and 10 mM RbCl.

The resulting cells were treated with 20 ml of 5 mM Tris HCl pH 7.6, 0.25 M KCl, 5 mM MgCl ₂ , 0.1 M CaCl ₂ . and 10 mM RbCl in solution and allowed to stand at 0 ° C for 25 minutes, the cells were harvested and resuspended in 1 ml of the above solution, the recombinant DNA described above was added to 0.2 ml of cell suspension and the suspension was allowed to stand at 0 ° C for 60 minutes. Subsequently, 0.7 ml of L-medium was added to the culture and shaken for 30 minutes at 37 ° C. The resulting culture medium (0.1 ml) was extensively plated on a 1.5% agarose medium containing 100 µg / ml diaminopimelic acid, 50 µg / ml thymidine and 15 µg / ml tetracycline. and incubated at 37 ° C for two days.

(5-5) Four hundred thirty-two emerged colonies are divided into 18 groups, each containing 24 different bacterial clones, inoculated with 200 ml of L-medium containing 100 pg / ml diaminopimelic acid, 50 pg / ml thymidine and 10 pg / ml tetracycline and shaking. At 5 ° C for 5-7 hours. Then 200 ml fresh L-medium containing chloramphenicol was added to the culture at a final concentration of 170 pg / ml and further cultured overnight. The plasmid DNA thus amplified is purified by conventional means. Clones with IL-2 cDNA are screened by mRNA hybridization-translation assay (hereinafter "H-T assay"). The HT assay used here is performed as follows: 25 µg of purified DNA is digested with Hind III, treated three times with phenol and then treated with phenol-chloroform and chloroform, precipitated with ethanol, washed with 80 µl of 80% ethanol, os formamide.

The mixture was heated at 90 ° C for 5 minutes to denature and then diluted with 10XSSC of

1.5 M NaCl, 0.15 M sodium citrate. The DNA was then fixed on a nitrocellulose filter, which was dried at 80 ° C for 3 hours and then 18 hours.

-13197938 incubate at 37 ° C with 50% formamide, 20 mM Pipes buffer, pH 6.5, 0.75 M NaCl, 3 mM EDTA, 0.2% SDS and 250 µg of a solution containing poly (A) mRNA from induced J-III cells to hybridize the DNA fixed on the filter with IL-2 mRNA. The filter was then washed three times at 65 ° C with 10 mM Pipes, pH 6.5, 0.15 M NaCl, then 1 mM Pipes, 10 mM NaCl then treated with 0.5 mM EDTA and 0.1% SDS at 95 ° C for 1 minute to recover the hybridized mRNA from the filter. The mRNA thus extracted was purified on an oligo dT-cellulose column by conventional means and injected into Xenopus oocytes to determine the activity of the translationally transformed protein IL-2. One of the 18 groups (each group contains 24 clones) gives positive 48 units / ml IL-2 activity with ³ H-TdR incorporation! study previously reported, while the others are clearly negative. The 24 individual colonies of the positive group are then inoculated with 200 ml of L-medium of the same composition as described above, aerobically cultured for 5 to 7 hours at 37 ° C, and fresh L-medium26 containing chloramphenicol is added again. After overnight culture of the plasmid DNA supplementation, the plasmid DNA is purified according to the standard procedure. The individual plasmid

After cleavage of about 5 pg of DNA with Hind III, each plasmid DNA is similarly bound to nitrocellulose. The filters were hybridized with IL-2 mRNA and the hybridized mRNA was recovered to generate Xenopus

It may be injected into oocytes to determine IL-2 activity of the translated protein.

As shown in Table 2, only one colony purified plasmid DNA, designated p3-16, gives positive IL-2 activity. Therefore, this clone was identified as a clone with IL-2 cDNA (E. coli K 1776) p3-16 AJ 11995 (FERM-BP-225). Thus, plasmid p3-16 DNA has been shown to be part of the DNA (IL-2 gene) capable of forming a specific hybrid with IL-2 mRNA.

The insert of p3-16 cDNA exhibits features that it can be cleaved with XbaI at a single site and BstNI at two sites (in the same direction and in the opposite direction to the XbaI site). Plasmid p3-I6 contains a cDNA insert containing about 650 base pairs, which appears to correspond to part of the 11-12S IL-2 mRNA.

Table 2 / a

Sample Dilution ³ H-TdR uptake How much IL-2 / Cpm / is / units / ml / I. Control - 2,010 0 / Medium for Definition / II. Control X2 2,120 0

/ untreated egg tap medium a-

supernatant of X32 2.482 mRNA translation X2 20.453 58 products X32 20.961 / B / Sample Dilution T-lymphocyte cell How much IL-2 number / cell t / cavity / / ml / ml / I. Control __ 0 0 / Medium of Definition- application / II. control X2 0 0

/ untreated eggs X32

-14197938

Table 2 (cont'd)

Sample Dilution T-lymphocyte cell count / cell t / well / Amount of IL-2 / unit / ml / medium-folyadékjá- supernatant of mRNA translation X2 88 32 termeke x X32 42

x mRNA was hybridized with cDNA from plasmid p3-16.

Another cDNA "library" was prepared according to the method of Land et al. (Nucleic Acids Res. 9, 2551 (1981)) using IL-2 mRNA as a template. Single-stranded cDNA (1.6 pg) was synthesized using 4 pg IL-2 mRNA, which was extended with the dCMP residue; and ds-cDNA was synthesized using oligo (dG) 2-Ig as a reading template (primer) for DNA polymerase I (Klenow fragment). CDNA (0.6 pg) longer than 680 base pairs of DNA size was recovered by sucrose gradient centrifugation and inserted into the PstI site of pBR322 by standard G-C tailing. Following E. coli K 1776 transformation, approximately 2000 colonies were screened by Grunstein-Hogness in situ hybridization with a nick translated p3-16 cDNA insert as a probe and a pIL2-50A plasmid containing approximately 850 bp. the transformed clone (E. coli K 1776) is identified as pIL2-50A, AJ 11996 (FERM-BP-226). A restriction endonuclease cleavage map of pIL-2-50A cDNA is shown in Figure 1.

To isolate the gene encoding the IL-2 peptide from transformed E. coli K 1776 pIL2-50A, plasmid DNA was digested with the restriction enzyme PstI after isolation of the DNA region obtained from the cells. The smaller of the two DNA fragments thus generated is the DNA gene encoding the IL-2 peptide. The complete nucleotide sequence of the PstI insert from pIL2-50A was determined according to the procedure of Maxam and Gilbert (Maxam A.W. et al., Enzym. 65, 499-560 (1980)) and is shown in Figure 2.

Example 2

Plasmid pKCR (O'Hare et al., Proc. Natl. Acad. Sci. USA, 78, 3, 1527-1531) consists of: (i) an early gene promoter and a replication origin (0.725-0.648). Morgan unit) and an SV 40 DNA segment containing a polyadenylation site from an early gene (shown as a bar coded section in Figure 3); (ii) a portion of the rabbit β-globin gene (shown as an open region) (BamHI-PvuII fragment); (iii) a segment derived from pBR322 (EcoRHBamHI fragment) containing an origin of replication and an ampicillin resistance gene. This plasmid was cleaved with BamHI and after completion of both ends of the cleaved DNA with DNA polymerase I (Klenow fragment), a synthetic PstI binding DNA was introduced to generate pKCR (PstI). Plasmid pKCR (PstI) was cleaved with SalI, treated with a Klenow fragment to fill in the ends, and then partially cleaved with EcoRI to obtain an EcoRI-SalI fragment containing the SV40 total DNA and the globin gene. This fragment is then linked to a piece of pBR 328 DNA cleaved with the appropriate enzyme containing the tetracycline resistance gene and the origin of replication as outlined in Figure 3. The plasmid pCE-1 generated in this manner contains a unique PstI site just downstream of the SV40 early promoter.

The cDNA insert of pIL2-50A was cleaved by PstI cleavage and ligated with PstI-cleaved pCE-1 to create pCEIL-2, in which expression of the structural gene for IL-2 is under the control of the SV40 early promoter. Plasmid pCE-1 was originally designed for cloning of cDNA by the G-C tailing method (Chang, A.C. Y. et al., Natura, 275, 617-624 (1978)) in bacteria and for direct expression in mammalian cells.

This plasmid was digested with Hhal and introduced into DNA into COS-7 transformed monkey cell lines by DNA transfer (transfection) (McCutchan et al., J. Natl. Cancer Inst. 41, 351-357 (1968)). Replication of DNA containing 40 starter sequences available from Gluzman Y. (Cell, 23, 175-182 (1981)). It appears important to digest the Hhal plasmid prior to transfer (transfection) for efficient expression of cDNA, since sequences that may inhibit replication of the transferred DNA in COS cells may be removed from a significant portion of the plasmids required for cDNA expression by this method. COS-7 cells (6X10 ⁴ / ml)

-15197938 was suspended in 0.5 ml DMEM medium containing 5% FCS, in 24 well culture plates (Nunc) and incubated for 4 hours at 37 ° C. Then 1 µg of the above-described vector, 17.6 µl of 1 mM Tris-HCl containing 0.1 mM EDTA, 2.4 µl of CaCl ₂ and 120 µl of 2xHBS (containing 50 mM) A mixture of Pipes, 280 mM NaCl and 1.5 mM Na ₂ HPO ₄ pXl ₂ H ₂ O (pH 7.10) was added to the cultured cells. The cells were further incubated for 4 hours at 37 ° C, and the medium was aspirated, washed with 1 mL of PBS, and 0.5 mL of 20% glycerol in PBS was added at room temperature for 1 minute. The medium is aspirated again and the cells are washed with 1 ml PBS and cultured in 1 ml DMEM containing 5% FCS. Every 24 hours, 500 µl of medium is replaced with fresh medium. Each medium collected at appropriate intervals is stored at 4 ° C until use. Two to three days after the transfer (transfection), the cultured cell medium is assayed for IL-2 activity. As shown in Table 3, the culture supernatant of the resulting process, the culture being a COS-7 cell containing PCEIL-2, contains IL-2 activity. IL-2 activity could not be detected in the medium of cells containing the transferred pCE-1.

DBS, which has been transmitted / transfected / done

Table 3

Activity 11-2 ⁵ K-Tdfi. measured by intake / unit / ml /

T-lymph oc ita growing s p Cili-2 12 -ΗΉ pCi-1 1

IL-2 activity found in the culture medium can be neutralized after pCEIL-2 transfer (transfection) to COS-7 with 12 units / ml to 1 unit / ml mouse (BALB) c) anti-human IL-2 monoclonal antibody. The result that COS-7 cells secrete human IL-2 after transfer of pCEIL-2 clearly demonstrates that a recombinant gene containing a gene encoding an IL-2 polypeptide and a vector capable of replicating in said cell. A DNA-transformed eukaryotic cell is definitely useful for the production of IL-2.

Plasmid pCEIL-2 (AJ 12008) incorporated into E. coli HB101 was deposited under accession number FERM-BP244.

Example 3

The strain Escherichia coli K 1776 pIL-2-50A (AJ 11996 (FERM-BP-226)) prepared in Example 1 is inoculated with 250 ml of 100 pg / ml diaminopimelic acid, 50 pg / ml of thymidine, 1% tryptophan, Cultured in L medium containing 5% yeast extract, 0.5% NaCl, and 0.1% glucose and shaking at 37 ° C until the optical density of the culture medium at 562 nm is 0.5. After termination of the culture, the culture medium was allowed to stand at 0 ° C for 30 minutes and cells are recovered by centrifugation, washed once with 20 mM Tris / HCl (containing 30 mmol / l NaCl) and re-suspended in ¹⁶ Juk 1.8 ml in buffer of the same composition. A 0.2 µl / ml lysozyme solution and 20 µl of a 0.5 M EDTA solution are then added to the cells and the mixture is allowed to stand at 0 ° C for 20 minutes followed by three successive freezing - Thawing process. Cell extract (1.5 mL) was recovered after centrifugation at 40,000 rpm for 30 min. The extract. desalted with 85% ammonium sulfate, desalted with Sephadex G15, subjected to DEAE cellulose column chromatography and freeze-dried fractions eluted with 0.06 M Tris-HCl buffer, pH 7.6 and controlled in a porous glass bed (250 A, Funakoshi Pharmaceuticals, Japan) was subjected to chromatography to obtain the IL-2 activity in the fractions eluted with 0.3 M glycine-HCl buffer, the fraction containing IL-2 having 12 units / ml IL-2 activity. explains. The results clearly indicate that E. coli K 1776 pIL2-50A, AJ 11996 actually produces IL-2.

Example 4

The constitutive IL-2 producing cell line of JA 1886 (ATCC CRL 8130), cloned from Jurkat cells as described in Example 1, was similarly grown in a cylindrical culture flask. Growth cells were resuspended in RITC-55-9 fresh synthetic medium65 with an initial cell density of ¹⁰ x ⁶ cells / ml, and

-16197938 hours after initiation of the culture, the cells used for extraction of the IL-2 mRNA, by 11-12S 3pl0 ⁹ cells, as detailed in Example 1, the fraction of steps.

Double-stranded cDNA was synthesized in the same manner as in Example 1, and cDNA containing more than 600 base pairs (2.4 pg) was obtained after fractionation on a sucrose density gradient. The cDNA was then expanded with the dCMP residue using terminal deoxynucleotidyl transferase and a 50 ng aliquot was fused with 250 ng of dGMP-extended PstI-cleaved pBR 322. The resulting hybrid plasmids were used to transform E. coli K 1776 and about 4000 clone transformants were obtained. Three clones complementary to the 3-16 cDNA plasmids (used as probe) were selected by the Grunstein-Hogness method. The clone thus selected is a transformed clone with the human IL-2 gene.

Example 5

A plasmid that directs the synthesis of human IL-2 in an E. coli cell was constructed as follows: pTIL2-22 from pTrS-3 (Nishi, T., Taniguchi, T., et al., SEIKAGAKU 53,976 (1981)). ) and pIL2-50A containing the IL-2 cDNA by a series of modifications as shown in Figure 5 (a). Plasmid pTrS-3 involves the insertion of the Trp promoter and Shine Dalgarno (hereinafter SD) site between the EcoRI site and the ClaI site of pBR 322. The plasmid also contains an ATG start codon 13 bp downstream of the SD sequence and a unique SphI site as shown in this insert.

Figure 4 illustrates. The vector can efficiently produce the protein of interest when the DNA sequence corresponding to said protein is in the downstream phase of the ATG codon, which is triggered by digestion with SphI and subsequent treatment with T4 DNA polymerase. -in. Therefore, plasmid pTrS-3 (30 µg) was cleaved with restriction enzyme SphI in a conventional manner, and after successive treatment with phenol and chloroform, an ethanol precipitate was obtained, and both ends were straightened by T4 DNA polymerase treatment. The DNA (21.4 pg) is then recovered in a similar manner to the former by successive phenol treatment followed by chloroform treatment and ethanol precipitation. On the other hand, pIL2'50A containing 380 pg of IL-2 cDNA was cleaved with PstI and the IL-2 cDNA insert was isolated by agarose gel electrophoresis. 11 pg of cDNA insert was digested with HgiAI, treated with T4 DNA polymerase and 10 pg of larger DNA was isolated by agarose gel electrophoresis. According to the method, a cDNA encoding 132 amino acids (7.2 pg) is obtained and this DNA fragment has cut ends (Fig. 5 (a)). The resulting cDNA is then ligated to a pTrS-3 vector previously digested with SphI and treated with T4 DNA polymerase, just downstream of the ATG sequence. The plasmid thus linked was then used to transform E. coli HB101 according to conventional procedures. The coupling was performed as follows: IL-2 cDNA (0.4 pg) larger fragment and 0.2 pg pTrS-3 vector DNA were mixed with 0.8 units T4 DNA ligase in 66 mM Tris-HCl pH 7 , 5) containing 6.6 mM MgCl ₂ , 1 mM ATP and 10 mM DTT, and the mixture was allowed to react overnight at 4 ° C. Colonies containing 132 amino acids encoding an IL-2 cDNA fragment from L-nutrient agar containing ampicillin were selected by in situ colony hybridization assay. The colonies thus selected were cultured (10 ml) again to generate plasmid DNA by lysosine treatment or freeze-thawing. Plasmid DNA is cleaved with PstI and XbaI and the resulting products are analyzed by agarose gel electrophoresis to identify pTIL-2-22, in which the cDNA is in the correct orientation for the TrS-3 ATG sequence. is attached. E. coli HB101 containing pT1L2-22 is cultured under conventional conditions known for the growth of microorganisms. Cells were grown in 10 ml of X medium (2.5% Bactotropton, 1.0% yeast extract, 0.1% yeast extract, 0.1% glucose, 20 mM MgSO ₄ containing 25 pg / ml streptomycin). and 25 µg ampicillin) at 37 ° C overnight. One ml of medium suspension is inoculated into the same medium X (100 ml) and cultured at 37 ° C. When the OD at 650 seconds reaches about 1.5-2.0, 3-indole acylic acid (IAA) is added. Three hours after the addition of the inducer, the cells were harvested, washed with 20 mM Tris-HCl buffer containing 30 mM NaCl and resuspended in 8 ml of similar buffer. To make the Trp promoter more effective, inducers, e.g. IAA is added until a final concentration of 50 pg / ml is reached. Proteins thus produced in bacterial cells are recovered by sonication (0 ° C, 2 min) or by lysozyme digestion (8 µg lysozyme, 0 ° C, 20 min) followed by three freeze-thawing sequences. In the present procedure, IL-2 is generally extracted from organisms. The extracted IL-2 activity ranges from 10,000 to 12,000 units. E. coli strain HB101 (AJ 12004) containing PTIL2-22 was deposited under accession number FERM-BP 245.

Example

Plasmid pTuIL-2-22 carrying the IL-2 cDNA was constructed from pTuBIP-5 (Taniguchi, T., et al., SEIKAGAKU, 53, 966 (1981)) and pTIL2-22, shown in Example 5. 7 using the procedure shown in Figure 7. A plasmid pTuBIP-5 includes a promoter insert for tufB17

-17197938 in pBR 322. The plasmid also contains a unique ClaI site and is located 2 bp downstream of the SD sequence as shown in Figure 7. Because pTrS-3 also contains a Clal site between the SD sequence and the ATG primer codon, and whereas this Clal site is not degraded using pTrS-3 and IL-2 cDNA in the construction of an expression plasmid, it is also described in Example 5, it is very simple to replace the bacterial trp promoter with the tufB promoter so that the IL-2 cDNA is expressed under the control of the tufB promoter.

Therefore, plasmid pTIL2-22 (30 pg) is cleaved by restriction enzymes Clal and Pvull in a conventional manner. The IL-2 cDNA containing fragment (about 2.2 kb) was isolated and purified by agarose gel electrophoresis to obtain 3 pg of DNA. On the other hand, 20 pg of pTuBIP-5 vector were similarly cleaved with ClaI and PvuH, and the larger fragment (about 3.4 kb) containing the ampicillin resistance gene was isolated and purified by agarose gel electrophoresis to yield 3.5 pg of DNA. The two fragments thus obtained, i.e. the fragment containing the tufB promoter (approximately 3.4 kb) and the fragment containing the IL-2 cDNA (approx.

2.2 kb) connect as follows:

1.2 pg of IL-2 cDNA fragment and 0.3 pg of tufB promoter fragment were mixed with 0.8 units of T4 DNA ligase in 66 mM Tris-HCl, pH 7.5, 6.6 mM containing MgCl ₂ -t, 1 mmol / l ATP and 10 mmol / L of DTT, and the mixture was allowed to react overnight. The plasmid thus linked was then used to transform E. coli HB101 according to the conventional method. Of the transformants appearing on ampicillin-containing L-medium agar plates, 8 colonies containing the IL-2 cDNA moiety, such as lymphocytes, were isolated. a pTuIL2-22 a

In Figure 7, plasmid DNA was prepared from these as described in Example 5. E. coli strain HB101 containing pTuIL2-22 was grown in L medium (100 mL) at 37 ° C. When the O.D. at 650 seconds is about 0.5 to 1.0, the bacterial cells are harvested, washed with 20 mM Tris-HCl buffer containing 30 mM NaCl and resuspended in 2 ml of the same buffer. The resulting protein is extracted in the same manner as in Example 5. The extracted IL-2 activity is between 6,000 and 56,000 units / ml.

Escherichia coli strain HB 101 (AJ 12010) containing ApTuML2-22 was deposited under accession number FERM-BP 246.

Example 7

Plasmid pGIL2-22 carrying IL-2 cDNA was constructed from pGL 101 (Roberts, TM and Laucer, GD: Meth. Enzym. 68, 473-483 (1979)) and pTIL2-22 in Figure 5. according to a demeanor.

A plasmid pGL 101 (20 pg) containing the lac promoter was cleaved with restriction enzyme Pvull in a conventional manner, and 17 pg of DNA was recovered after phenol treatment, then chloroform treatment and finally ethanol precipitation. On the other hand, pTIL2-22 (75 pg) was cleaved with ClaI and SalI and a DNA fragment containing 2.2 pg IL-2 cDNA was obtained by agarose gel electrophoresis. The fragment was straightened by treatment with DNA polymerase I (Klenow fragment) and the resulting two fragments (0.25 pg and 0.66 pg) were ligated with 1 unit T4 DNA ligase in the same manner as in Example 6. . The plasmid thus linked was then used to transform E. coli HB 101 according to a conventional method. Among the transformants, the transformants with the Clal-SalI fragment insert containing the IL-2 cDNA are the samples to be tested. These transformants are then cultured in 10 ml of X medium containing 25 pg / ml ampicillin and plasmid DNA is added to

It is isolated as described in Example 5 to obtain a plasmid DNA having the ATG primer sequence of the IL-2 cDNA just downstream of the lac promoter by cleavage with PstI and Xba.

The thus prepared pGIL2-22 is inoculated and cultured in 100 ml of L medium containing 25 pg / ml ampicillin and 25 pg / ml streptomycin. To obtain an optical density of about 0.5 at 65 sec, isopropyl pD-thiogalactopyranoside (IPTG) is added at a concentration of 1 mM, and one hour later, the bacterial cells are harvested and the cell extract is prepared as described in Example 6. . Extracted IL-2 activity is between 6,000 and 80,000 units.

Escherichia coli strain HB 101 (AJ 12011) containing pGIL2-22 was deposited under accession number FERM-BP 247.

Example 8 Plasmid pTrS-3 (10 µg) was first cleaved with SalI restriction enzyme and the SalI site was straightened by treatment with DNA polymerase (Klenow fragment) or T4 DNA polymerase. After cleavage with Clal, the larger fragment containing the trp promoter region was isolated by agarose gel electrophoresis in a conventional manner to obtain 3 pg of DNA.

On the other hand, 11 pg of the cDNA insert obtained by PstI cleavage of pIL2-50A was cleaved with HgAII. It is digested with T4 DNA polymerase and the larger fragment is isolated and purified by agarose gel electrophoresis. Thus, a cDNA fragment encoding the 132 amino acids of IL-2 is recovered

7.2 pg. This was followed by a 0.45 pg trp promoter fragment (see above), a 0.5 pg IL-2 cDNA HgiAI-PstI fragment, and synthetic oligonucleotides (20-20 moles), such as (5 ') CGATAAGCTATGGCA (3'). ) and (3 *) TATTCGATACCGT (5 ') are both phosphorylated at the 5' terminal as described in Example 5 (see Figure 5 (b)).

-18197938

The plasmid thus linked was used to transform E. coli HB101. Among the transformants that appear, the target transformants are selected as follows.

Transformer candidates capable of hybridizing both the IL-2 cDNA and the synthetic oligonucleotides were first selected by colon hybridization followed by the CCT sequence at positions 111-113 of Figure 2 (a) (CCTACT ......). Transformants with DNA fragment insertion downstream of the ATGGCA sequence were selected by PstI, XbaI cleavage.

The above transformants containing pTIL2-21a or pTIL-2-21b were cultured in L-medium as described in Example 5, and high IL-2 activity was found in the cell extract of the transformants when Measured as in Example 5.

Escherichia coli strain HB 101 with pTIL2-2 / a (AJ 12013) and Escherichia coli strain HB 101 with pTIL2-2 / a (AJ 12014) are deposited under accession numbers FERM-BP 1248 and FERM BP 249, respectively. .

The E. coli K 1776 and HB 101 strains used in the above examples (Boyer, H.W. et al., J.Mol. Biol. 41, 459 (1969)) are known and available to anyone. In addition, the host organisms can be recovered from the deposited transformants by culturing the transformants with L-medium at 37 ° C to release the relevant recombinant DNA in the transformant and selecting strains that have become sensitive to ampicillin and tetracycline. as the host.

Plasmid pBR322 (commercially available, for example, from Bethesda Research Laboratory), as well as plasmids pCE-1, PtrS-3 and pGL101 are all known and available to anyone. In addition, plasmid vectors may be recovered from deposited transformants by conventional means of isolating recombinant plasmid DNA from the transformant, or by separating plasmid vectors in a manner obvious from the teachings of the corresponding examples. For example, pCE-1 can be recovered by digesting pCEIL-2 with PstI and isolating the resulting larger DNA fragment. In addition, pTrS-3 and pTuB1P-5 are deposited as E. coli FERM-P 6735 and E. coli ATCC 31878.

Those skilled in the art will, after understanding the present disclosure, understand that the same can be done in a wide and equivalent range of different conditions, parameters, without affecting the spirit and scope of the invention or any of their realization.

Claims (16)

A method for producing interleukin-2 (IL-2) mRNA, comprising producing RNA from human T-lymphocyte, transformed human T-lymphocyte or human T-cell hybridoma cells, or cultures thereof, from oligo (dT) cellulose. and sucrose density gradient centrifugation of mRNA (Priority: 15.12.1982) 2. A method of producing a gene encoding a polypeptide having interleukin-2 (IL-2) activity, comprising producing a single-stranded and then a double-stranded cDNA using the mRNA of the method of claim 1, which is inserted into an appropriate vector and transformed into E. coli. plasmids are prepared from the transformants, the plasmids bearing the IL-2 gene are selected by hybridization with IL-2 mRNA and the gene encoding the peptides having IL-2 activity is removed from the plasmids by appropriate restriction enzyme digestion. (Priority: 12/24/1982) The method of claim 2, with the base sequence shown in Figure 2 (a), but at least 111 to 113 thereof. from at least 504 to 506. characterized in that the plasmids selected after hybridization are digested with the appropriate restriction enzyme digestion. (Priority: 12/24/1982) The process according to claim I or 3, which is numbered 48-50 according to Figure 2 (a). starting with the ATG sequence at position 504-506. for the preparation of a gene terminating with an on-site ACT sequence, wherein the restriction enzyme digestion is used to cleave the gene of interest. (Priority: 12/24/1982.) The method of claim 2 or 3, which is numbered 108-110 according to Figure 2 (a). starting with the GCA sequence at position 504-506. (a), characterized in that the restriction enzyme digestion cuts the gene of interest. (Priority: 12/24/1982.) The method of claim 2 or claim 3, wherein the method according to FIG. starting with the CCT sequence at position 504-506. for the preparation of a gene terminating with an on-site ACT sequence, wherein the restriction enzyme digestion is used to cleave the gene of interest. (Priority: 12/24/1982.) The composition of claim 2 or claim 3. The method beginning with base A at position 2 in Figure 2 (a) and starting at 504-506. for the preparation of a gene terminating with an on-site ACT sequence, wherein the restriction enzyme digestion is used to cleave the gene of interest. (Priority: 12/24/1982) The method according to claim 2 or 3 at position 1 in the numbering of Figure 2 (a) -19197938 Beginning with the base and 507-509. a TGA sequence, characterized in that the restriction enzyme digestion cuts the gene of interest. (Priority: 12/24/1982) The method of claim 2 or 3 for producing the gene starting at base A at position 1 and ending at base C at position 801 of Figure 2 (a), characterized in that, by restriction enzyme digestion, gene. (Priority: December 24, 1982) The method of claim 2 or 3 for producing a gene having the poly (A) sequence of Figure 2 (a), wherein the restriction enzyme digestion is used to cleave the gene of interest. (Priority: December 24, 1982) 11. The method of claim 2 for producing a gene having the amino acid sequence I shown in Figure 2 (b), wherein the restriction enzyme digestion is used to cleave the gene of interest. (Priority: December 24, 1982) 12. The method of claim 2, wherein the method of FIG. characterized in that the restriction enzyme digestion deletes the gene of interest. (Priority: December 24, 1982) 13. The method of claim 2, which is a process according to claim 2, illustrated in Figure 2 (b). characterized in that the restriction enzyme digestion deletes the gene of interest. (Priority: December 24, 1982) 14. A method of producing recombinant DNA capable of expressing a gene encoding a polypeptide having interleukin (IL-2) activity, comprising the step of: The gene produced by the method of any one of claims 13 to 13 is inserted into vector DNA capable of replication in a strain or animal tissue culture of the genus Escherichia such that said gene is a vector. 1θ promoter downstream. (Priority: December 15, 1982) 15. A method of expressing an interleukin-2 (IL-2) gene, an Escherichia genus, or an animal tissue culture transformant cell. Characterized in that a strain or animal tissue culture of the genus Escherichia is transformed with a recombinant DNA vector according to claim 14. (Priority: ²⁰ 1983. 02. 03.) 16. A process for the production of interleukin-2-acting proteins, wherein a transformant of the genus Escherichia produced by the method of claim 15 is cultured in an appropriate medium and the resulting interleukin-2-acting proteins are isolated from the medium. (Priority: December 27, 1982) 17. A method of producing interleukin-2-acting proteins, comprising culturing an animal tissue culture transformant produced by the method of claim 15 in a suitable medium and isolating the generated interleukin-2-acting proteins from the medium. (Priority: 12/29/1982)