JPH0832239B2 - Recombinant DNA body containing a gene encoding a polypeptide having human interleukin-2 activity and prokaryotic cell transformed with the recombinant DNA body - Google Patents

Description

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

Interleukin 2 (hereinafter, abbreviated as “IL-2”) was previously called T cell growth factor, and is a soluble protein (generally, “T cell growth factor”) produced by T cells activated by lectins or antigens. (Known as the lymphokine)) (Morgan et al., Science, 193 , 1007-1008).
(1976), Gillis et al., J. Immunol., 120 , 2027-2033 (197
8)). IL-2 can regulate lymphocyte reactivity and antigen-specificity can enable long-term culture of effector T-lymphocytes in vitro (Gillis et al., Natur.
e, 26 8,154-156 (1977)). IL-2 also promotes thymic cell division (Chen et al., Cell Immunol., 22 , 221-224 (197
7), Shaw et al., J. Immunol., 120 , 1967-1973 (1978)), cytotoxic T lymphocyte activity in nude mouse base cell culture system (Wagner et al., Nature, 284 , 278-280, (1980)) and induction of anti-SRBC plaque forming cell reaction (Gillis et al., J. Exp. M.
ed. 149 , 1960-1968 (1979)) and other related biological activities. Therefore, this lymphocyte regulatory factor is useful for enhancing the liquid immunity and the cell-mediated immune response and for restoring the immunodeficiency state to the normal liquid or cell-mediated immunity. These immunological activities of IL-2 revealed that IL-2 is a malignant tumor, bacterial or viral infection, immunodeficiency, autoimmune disease, etc. (Papermaster et al., Ad
v.Immunopharm., 507, (1980)) and has been shown to be useful for medical immunotherapy. Similar to interferon, IL-2 has been shown to enhance natural killer cell activity, strongly suggesting its utility in treating malignant tumors. Furthermore, IL-2 enables retention of monoclonal activated T cells, which is useful for studying the molecular mechanism of T cell differentiation, the differentiation mechanism of T cell function, and the mechanism of T cell antigen receptor. It shows that it plays an important role. IL-2 can also be used to produce lymphokines derived from various T cells, which are useful in various other fields, by culturing monoclonal T cells for a long period of time.
Furthermore, the production of IL-2 and the responsiveness of lymphocytes to IL-2 are important parameters of immunological function and are useful for clinical diagnosis of immune disorders.

In the prior art, IL-2 has been produced by stimulating mouse, rat or human lymphocytes with mitogens (Gillis et al., Nature, 268 , 154-156, (197
7)), Farrar et al., J. Immunol., 121 , 1353-1360, (1978),
Gillis et al., J. Immunol., 120 , 2027-2033, (1978)). By stimulating human peripheral blood lymphocytes with mitogens (Gillis et al., J. Immunol., 124 , 1954-1962, (198
0)), Gillis et al. From mouse IL from a T lymphoma cell line.
-2 (Gillis et al., J. Immunol., 125 , 2570-2578 (19
80)) and the production of human IL-2 from a human leukemia cell line (Gi
Llis et al., J. Exp. Med., 152 , 1709-1719, (1980)).

The technique described by Gillis et al. Was performed on mitogen-activated T leukemia cell lines using a cell culture method to obtain human IL-
The present invention relates to a method for manufacturing 2. However,
This method has a drawback that only low concentrations of human IL-2 are produced, and in order to obtain a small amount of IL-2 from a large amount of culture solution,
Requires complex purification steps. Furthermore, since the human T leukemia cell line also produces a small amount of other physiologically active substances that closely resemble human IL-2, a cytotoxin that separates IL-2 from these other immunologically active molecules or sometimes coexists with them. Substance (toxi
It is quite difficult to separate it from lactic acid.

As another method for producing IL-2, a recombinant DNA (abbreviation of deoxyribonucleic acid) method used for producing other bioactive human-derived proteins such as interferon (G
ray et al., Nature 295 , 503〜508, (1981), Nagata et al., Nature
284 , 316〜320 (1980), Taniguchi et al., Gene 10 , 11〜15,
(1980)) seems preferred. However, prior to the present invention, attempts to produce IL-2 by recombinant DNA methods were unsuccessful. For example, an attempt to create an organism that produces IL-2 by recombinant DNA forms
It was reported in "Nikkei Biotechnology, No. 19, July 5, 1982" that the gene coding for the IL-2 polypeptide was not yet cloned and probably not successful.

In the meanwhile, there has been a longing for a cloned gene encoding IL-2 and a recombinant DNA body carrying the gene. Also,
Live cell line with recombinant DNA and I
There has been craving for a method of making L-2.

The subject matter of the present invention will be more readily apparent from the following description. It is an object of the present invention to create a recombinant DNA body containing a gene encoding a polypeptide having IL-2 activity, and of a prokaryotic organism transformed to produce IL-2 by the recombinant DNA body. It is about creating cells.

That is, the present invention is Having a human interleukin-2 activity having the amino acid sequence shown by or a human interleukin having an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted with respect to the amino acid sequence of the polypeptide. Recombinant DNA for transforming a prokaryote containing a gene encoding a polypeptide having 2 activity, a polypeptide having human interleukin 2 activity having the amino acid sequence represented by the above formula, or an amino acid of the polypeptide Recombination containing a gene encoding a polypeptide having human interleukin-2 activity having an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted to the sequence
The present invention provides a bacterium transformed with a DNA body. In the present specification, Gln and Asn of amino acids may be referred to as GIN and AsN, respectively.

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

In the above, specific examples of the polypeptide having human interleukin 2 activity include polypeptide (I) containing the following amino acid sequence in the molecule.

Specific examples of the polypeptide (I) include, for example, a polypeptide (II) having the following amino acid sequence; A polypeptide (III) having the following amino acid sequence; A polypeptide (IV) having the following amino acid sequence: Examples include a polypeptide (V) having the following amino acid sequence.

Specific examples of the recombinant DNA body containing the gene encoding the polypeptide having the human interleukin 2 activity include, for example, a recombinant DNA body having the following base sequence encoding the polypeptide (III); Alternatively, a recombinant DNA body having the following base sequence encoding the above-mentioned polypeptide (V) may be mentioned.

The polypeptide in the present invention includes a polypeptide lacking one or more amino acids in the above amino acid sequence; one or more amino acids in the above amino acid sequence are substituted with one or more amino acids. Polypeptide; A polypeptide in which one or more amino acids are added to the above amino acid sequence may be used.

As the bacterium in the present invention, those belonging to the genus Escherichia are preferable, and those belonging to Escherichia coli are more preferable.

By using the recombinant DNA body of the present invention, IL-
2 is modified by a recombinant method by insertion of a gene encoded to produce a polypeptide having IL-2 activity and insertion of a vector DNA that can be replicated in cells, and the coding sequence of the gene is a promoter sequence. It is produced by suspension culture (aerobic) of a bacterium of the present invention or Escherichia coli transformed to produce IL-2 with DNA located downstream of Escherichia coli.

A cloned gene encoding an IL-2 polypeptide is a messenger RNA (mRNA; mRNA) corresponding to IL-2 derived from a mammalian cell, which is characterized by the ability to produce a polypeptide having IL-2 activity. "RNA" is an abbreviation for ribonucleic acid, hereinafter referred to as "IL-2 mRNA") is complementary DNA
(CDNA) by reverse transcription. The obtained single-stranded cDNA (ss-cDNA) can be converted into double-stranded cDNA (ds-cDNA).

The mRNA used as a template for preparing cDNA is IL-
It can be isolated from mammalian cells which produce the 2 polypeptide. The isolated RNA is polyadenylated (GiL
Lis et al., Immunological Rev., 63 , 167-1209 (1982), polyadenylated RNA can be fractionated into 11-12S fractions by, for example, sucrose density gradient centrifugation. 13s
IL-2 mRNA activity may appear in mRNA, but in this case, it is considered to be an aggregate of 11 to 12S mRNA.

Mammalian cells capable of producing IL-2, which is a source of mRNA, are peripheral blood mononuclear cells that can be isolated from mammals,
T-lymphocytes such as tonsil cells and spleen cells may be used. Nylon column treatment, antiserum and scavenger treatment, in order to impart IL-2 producing ability to cells or enhance IL-2 activity
Pretreatment may be performed by a conventionally known method such as density gradient fractionation, combination treatment of neuraminidase and galactose oxidase, various enzyme treatments such as trypsin treatment, and X-ray irradiation. Cloned T lymphocytes obtained after culturing the above mammalian cells in the presence of T cell growth factor can also be used as a source of mRNA, which is a more preferred T-lymphocyte. Transformed lymphocyte cell lines or cloned transformed cell lines such as T lymphocytes themselves derived from leukemia or lymphoma cell lines or their derivatives pretreated or mutated by the above-mentioned method are also used as a source of mRNA. preferable. Apparently, the cloned cell line usually contains higher amounts of IL-2 mRNA compared to the parental line before cloning. T cell hybridomas obtained by fusing the above-mentioned lymphocyte-derived cells with tumor cell lines such as CEM, Molt 4F and BW5147 are also preferred mammalian cells for use in the present invention. The lymphocyte-derived cells in this case are
(1) IL-2 is produced only when the mitogen is introduced into the culture medium in the presence or absence of spontaneously producing cells for IL-2 and (2) other cells that support IL-2 production. Includes producing cells.

In order to induce IL-2 mRNA in IL-2 spontaneous producer cells, IL-2 spontaneous producer cells are cultured by methods well known in the field of cell culture. IL only in the presence of mitogen
In the case of producing mRNA in a cell producing -2, the cultured cells are thoroughly washed with a medium, and then rose well park memorial institute 1640 (hereinafter abbreviated as "RPMI 1640") with or without serum. Resuspend in a medium such as Dulbecco's modified Eagle medium (hereinafter abbreviated as "DMEM") or click medium. These cell culture media may be supplemented with various additives such as penicillin, streptomycin or other antibiotics, L-glutamine, hepes buffer, or sodium bicarbonate at concentrations commonly used in the field of cell culture. good. The preferred cell concentration is 0.5-4 × 10 ⁶ cells / ml. mRNA activation and IL
Appropriate stimulants are added to induce the production of -2. Among these suitable stimulants are mitogens, neuraminidase, galactose oxidase, zinc compounds such as zinc chloride or protein A, streptolysin-O.
Bacterial-derived lymphocyte activating factors such as The stimulated cells are harvested and washed. Upon mitogen stimulation, coexistence of macrophages or dendritic cells also activates mRNA or activates mRNA.
Can increase the yield of Similarly Raji, Daudi, K562, BALL−
MRNA is activated or activated even in the coexistence of a B lymphocyte or a cell line derived from a B lymphocyte cell line such as 1.
It may increase the yield of mRNA.

In order to grow mammalian cells, the cells are maintained under normal conditions in vitro by cell culture or in histocompatible animals. in v to prepare the source of mRNA
When the cells are subcultured by itro culture, these cells can grow in any medium which is conventionally known to promote the growth of T cells. Mammalian serum, serum components or serum albumin may be added to these media. The culture time for activation of mRNA corresponds to the time required for activation to produce mRNA. This time usually corresponds to the time required to start the production of IL-2 in the medium. The preferred time is 3 to 12 hours after the addition of a stimulant such as mitogen. If the culture time is too long, the produced IL-2 mRNA may be degraded. Upon activation of IL-2 producing cells, PMA or T
Phorbol esters such as PA can also be added at 10-50 ng / ml to increase activation levels. The above steps for activating IL-2 mRNA have a pH of 7.0 to 7.4 and a temperature range of 32 to 38 ° C.
It is carried out in an environment of saturated steam.

The method for obtaining and culturing IL-2 producing mammalian cells is described below.

(A) Acquisition of spontaneously producing IL-2 strain Jurkat cells, which are human T lymphocyte-derived leukemia cells (Fred Hutchinson Cancer Institute / Seattle / USA, Salk Institute / San Diego / USA, West German National Cancer Center / Heidelberg) / Freely available in West Germany, etc.) at a cell density of 1 × 10 ⁶ cells / ml in a click medium and a total of 150 roentgen / min irradiation rate.
Perform X-ray irradiation of 8,000 roentgens. After that, the cells were added to a 96-well flat-bottomed microtiter plate ("Falcon 3072") at a cell density of 0.1 cells / 200μ medium,
The cells are cultured in a click medium containing 5% fetal bovine serum for 3 weeks at 37 ° C. in a 5% CO ₂ incubator (cloning by limiting dilution method). The cells in the culture well in which the growth of the cells is observed are transferred to a 24-well Nunc culture plate before the cells reach the density covering the entire bottom surface, and the cells are allowed to grow in Click medium for 5 days. Further, the present cells were suspended in a serum-free synthetic medium containing neither serum nor serum-derived albumin at a cell density of 1 to 2 × 10 ⁶ cells / ml, and cultured for 2 days, and the main culture supernatant was separated by centrifugation. Then 0.22μ
The debris removal was sterilized by using a Millipore filter. By measuring the IL-2 activity of the culture supernatant thus obtained, an X-ray-treated mutant strain that spontaneously produced IL-2 was selected and cloned.

(B) Acquisition of IL-2 Producing Strain from Human Peripheral Blood Mononuclear Cells Human peripheral blood is collected and peripheral blood lymphocytes (hereinafter abbreviated as PBL) are collected by the Ficoll-Hyperk density gradient centrifugation method. . This PBL is suspended in a click medium containing 5% FCS at a cell density of 1 × 10 ⁶ cells / ml, and inoculated into a 24-well Nunc culture plate containing 2 ml each. Here, hemagglutinin-
M (manufactured by Gibco) to a final concentration of 5 μg / ml 10
0 μl is added and the cells are cultured under the above-mentioned conditions for 48 hours, then the cells are washed with the culture medium and inoculated into 1 ml of Click medium again at a cell density of 1 × 10 ⁵ cells / ml. Furthermore, Concanavalin A
(Hereafter abbreviated as ConA.) 1 ml of conditioned medium prepared from human splenocytes stimulated with 2.5 μg / ml for 48 hours
Is added and the medium containing 50% of the conditioned medium is replaced every 3 days, and human T lymphocytes from PBL are subcultured for a long period of time. The T lymphocytes thus obtained by long-term subculture are cloned in the presence of human splenocytes derived from a medium conditioned by the limiting dilution method as described above, and the cells are similarly proliferated. The cloned T lymphocytes thus obtained were added to RPMI 1 in a 24-well Nunc culture plate at a cell density of 1 × 10 ⁶ cells / ml in the presence of 10 μg / ml of phytohemagglutinin (hereinafter abbreviated as PHA).
1 ml of 640 medium was inoculated and cultured for 24 hours at 37 ° C. in a 7.5% CO ₂ incubator. The main culture supernatant was separated by centrifugation and then sterilized with a 0.22 μm Millipore filter, and then an IL-2 activity assay was carried out to identify an IL-2 producing human normal T lymphocyte clone. .

(C) Acquisition of human lymphocyte-derived malignant cells that produce IL-2 by mitogen stimulation The aforementioned Jurkat cells and the J-111 strain cloned by the limiting dilution method described above are serum-free medium and serum. ConA 10 μg / ml in BPMI 1640 medium containing 1-2%
When cultured in the presence of 2.5 μg / ml PHA for 24 hours, 10-4,000
Units / ml of IL-2 can be produced. These human malignant cells also produce IL-2 when cultured in the presence of zinc chloride, protein A and picibanil.

(D) Acquisition of cells that produce IL-2 by stimulating with mitogen in the presence of other cells or factors produced by the cells Cloned by human lymphocyte malignant cells Molt 4F or the limiting dilution method described above. One clone of Jurkat cells, Jurkat 99 strain, can be cultured for 24 to 72 hours by adding lectin and mitogen as mentioned above in a wide concentration range.
Does not produce IL-2. However, during this time 1 of monokine
Seed interleukin-1 of 5-10u / ml or 50%
When cultured at 37 ° C. for 24 hours in the coexistence of K562 and Raji cells, a detectable amount of IL-2 (10 to 100 u / ml) is produced.

In order to extract IL-2 mRNA from the cells activated in this way, a conventional method may be used regardless of the cell type. For example, detergents such as NP-40, SDS, Triton-X100, and deoxycholic acid are added to partially or completely degrade the cells, or the cells are treated with a physical method such as a homogenizer or freeze-thawing. Partial or complete destruction,
Solubilize. At this time, in order to prevent RNA degradation by RNase, it is preferable to add an RNase inhibitor such as heparin, polyvinyl sulfate, bentonite, macaloid, diethylpyrocarbonate, vanadium complex to the extract. Also, in some cases, anti-IL-2
It is also possible to use a method of precipitating a polysome in the process of IL-2 synthesis using an antibody, and then extracting the mRNA with a detergent or the like.

For purification of mRNA containing poliA, oligo dT-
It can be carried out by an affinity column such as poly U-sepharose using cellulose or sepharose 2B as a carrier, a purification method by a batch method, a fractionation by an SDG centrifugation method, or an agarose gel electrophoresis method.

To confirm that the mRNA obtained as described above has an IL-2 mRNA activity, the mRNA is translated into a protein and its physiological activity is examined, or anti-IL-2 peptide monoclonality is determined. A method such as identifying the translated protein using an antibody may be performed. For example, mRNA is usually microinjected into Xenopus laevis eggs (Gurdon et al., Nature, 233 , 177-182 (197
2)) or translated into the corresponding protein by using the reticulocyte or wheat embryo cell-free translation system.

IL-2 activity was previously reported by Gillis et al. (Gillis et al., J. Immunol.,
120 , 2027 to 2033 (1978), which can be confirmed by the microassay method basically described. In this assay, a cytotoxic T lymphocyte cell line (hereinafter abbreviated as CTLL) prepared according to the method established by Gillis et al.
Increased DNA synthesis in cells dependent on IL-2 (IL-2 dependent cel
lular proliferation) is used as an index. That is, 4 × 1
RPMI-1640 medium containing 2% FCS in 100 μl of ³ CTLL cells
96 μl with 100 μl of serially diluted translation product.
Inoculate flat-bottomed microplates with wells. After culturing at 37 ℃, 5% CO ₂ for 20 hours, the cells were added with 0.5 μCi / well of ³ H-TdR.
Label for 4 hours, collect the cells on the glass ribbon using an automatic cell harvester, and measure the radioactivity incorporated by the cells by liquid scintillation method. This assay revealed that CTLL cells cultured in the presence of IL-2 uptake ³ H-TdR in a dose-dependent manner, which clearly calculated the amount of IL-2 contained in the sample. can do.

Since IL-2 has the activity of promoting the proliferation of T lymphocytes,
IL-2 activity can be measured using T lymphocyte proliferation as an index. That is, 5 CTLL cells containing 2% FCS in D
Suspend in 100μ of MEM and inoculate 96-well and flat bottom microplates with 100μ of serially diluted translation products. 72 ~ 96
After culturing at 37 ° C. for 5 hours under 5% CO ₂ , the number of activated and proliferated cells is counted under a microscope. 100 U / ml, 10 as a control group
Using IL / U of U / ml, the IL-2 activity of the sample is determined by comparing with the number of proliferated live cells in this control group.

IL-2 thus obtained from the most active fraction
mRNA was used as a template for synthesizing ds-cDNA, and ds-
The cDNA is combined with the vector DNA. cDNA synthesis is performed by conventional methods.

First, using mRNA as a template and oligo dT as a primer
mRNA by reverse transcriptase in the presence of dATP, dGTP, dCTP, dTTP
Synthesize ss-cDNA complementary to
After decomposing and removing, ds-cDNA is synthesized using reverse transcriptase or DNA polymerase using the single-stranded cDNA as a template.

The recombinant DNA body of the present invention is prepared from the thus obtained ds-cDNA and a vector DNA containing a replicon capable of replicating in a prokaryote or Escherichia coli. Then, this recombinant DNA body is integrated into a host cell.

This ds-cDNA and a vector DNA that can grow in prokaryotes
Is treated with exonuclease before binding them,
It is modified by various treatments such as the addition of chemically synthesized DNA fragments and the extension of G and C-chains in order to attach terminals that can be ligated to the ends of ds-cDNA and vector DNA. These connectable DNA
Can be combined with, for example, DNA ligase of T4 phage in the presence of ATP.

In order to amplify the cDNA cloned by the recombinant DNA body of the present invention thus prepared or IL-2
By transforming a bacterium or Escherichia coli to produce a polypeptide, the recombinant DN of the present invention can be obtained.
Bacteria or Escherichia
The stiffness is obtained.

Suitable bacterial hosts for IL-2 production include P. subtilis and the like or Escherichia coli. For amplification of DNA in host cells, Escherichia
E. coli can be used as a host, but other host cells can also be used.

As an appropriate Escherichia coli vector, pSC101, p as an EK type plasmid vector (stringent type)
RK353, pRK646, pRK248, pDF41 etc., EK type plasmid vector (relaxed type): ColE1, pVH51, pAC105,
RSF2124, pCR1, pMB9, pBR313, pBR322, pBR324, pBR325, pBR3
27, pBR328, pKY2289, pKY2700, pKN80, pKC7, pKB158, pMK200
4, pACYC1, pACYC184, dul etc., λgt type phage vector: λgt.λc, λgt, λB, λWES, λC, λWES.λB, λZJvi
r., λB ′, λALO, λB, λWES.Ts622, λDam, etc. In general, pBR322 has often been used as a vector for Escherichia coli, in which case the best cloning sites are the Pst1 and EcoR1 sites.

For transformation of host cells using recombinant DNA bodies, the following methods are commonly used. When Escherichia coli is the host, competent cells capable of incorporating this DNA can be transformed by the well-known CaCl ₂ method after recovering the cells in the logarithmic growth phase. If MgCl ₂ or RbCl coexists in the transformation reaction solution, the transformation efficiency will be improved. It is also possible to transform host cells after preparation of protoplasts.

Cells carrying the IL-2 gene can be isolated after transformation using either of the following two methods.

(1) Plus-minus method: IL-2 mRNA partially purified as 11-12S fraction by sucrose density gradient centrifugation was prepared from antigen-stimulated mammalian cell extract, and this partially purified mR
³² P-radioactive ss-cDNA is synthesized using NA as a template. After removing the template mRNA by alkaline treatment, the isolated cDNA was extracted from mammalian cells that did not stimulate antigen and partially purified.
Hybridizes with 11-12S mRNA. The unhybridized cDNA and hybridized cDNA are subsequently fractionated by hydroxyapatite column chromatography. The cDNAs that did not hybridize and the cDNAs that did not hybridize are called probe A and probe B, respectively.
Each recombinant is grown on nitrocellulose filter paper by the same method. Then, the DNA of the cells is fixed on the filter paper by alkali treatment. Probes A and B are each hybridized with DNA on two different filter papers. After that, autoradiography is performed and a recombinant (plus) that reacts positively with probe A, probe B
Recombinant (minus) that does not react with or slightly reacts with (Taniguchi et al .; Proc. Jap. Acad., V155B 464-469 (197
9). (2) In the second method, for example, 1000 to 10,000 recombinant clones are roughly divided into clone groups addressed to 2 to 30 to 2 to 300 clones, and each clone group is individually cultured to prepare plasmid DNAs. To do. Then, these plasmid DNAs are heat-denatured to ss-cDNA, for example.
Immobilized on nitrocellulose filter paper and activated IL-2-mR
Hybridization is performed complementarily with mRNA prepared from mammalian cells containing NA. Alternatively, IL-
2 When mRNA (mixture) containing mRNA is hybridized with heat-denatured plasmid DNA (mixture), the DNA-mRNA hybrid is immobilized on nitrocellulose filter paper. This filter paper is washed with a low salt buffer such as 1 mM HEPES or 10 mM saline, and the mRNA adsorbed on the filter paper is 0.5 mM.
Extract with EDTA; 0.1% SDS solution-containing solution at 95 ° C. for 1 minute, for example. The purified mRNA is collected by elution with oligod T-cellulose column chromatography. Then mRNA
Is microinjected into Xenopus oocytes and translated into a protein to confirm IL-2 activity. Or mRNA-dependent reticulocyte or wheat embryo in vitro
This mRNA is translated into protein using a cell-free synthesis system, and anti-IL
-2 antibody can be used to analyze IL-2-activity. The groups in which IL-2 activity was detected by these methods were categorized into a group containing a smaller number of recombinant clones, and finally repeated until a single clone having IL-2 DNA was obtained.

In order to obtain a cDNA encoding an IL-2 polypeptide from a recombinant capable of producing IL-2, first, the recombinant DNA in the transformant is isolated and cleaved with a restriction enzyme endonuclease. The integrated cDNA fraction is separated from the DNA fraction obtained by cleavage.

The entire nucleotide sequence of the Pst1 DNA insert encoding the IL-2 polypeptide from the recombinant pIL-2-50A DNA was determined by the Maxam and Gilbert method (Meth. Enzym. 65 , 499-560,
(1980)); and the two-deoxynucleotide chain end method (Smith, AJMMeth. Enzym. 65 , 560 to 580, (1980)).

Cleavage diagrams of the cDNA insert with a restriction endonuclease are shown in FIGS. 1 and 2 (a). Fig. 2 (a)
As shown in, the cDNAs are BstN1, Xba I, and BstN I, respectively.
It has a structure that is cleaved by the restriction enzyme endonuclease.

The DNA sequence of this cDNA insert carries one large open reading frame. The first ATG sequence (Kozak, M.Cel), which often serves as the read start sequence for eukaryotes.
l. 15 , 1109 to 1123 (1978) exist at the 48 'to 50' nucleotide positions from the 5'-end, and 152 sequences from nucleotide positions 507 to 509 at which the read end sequence TGA exists are connected to this ATG. . A connection corresponding to the 3'-poly (A) end of mRNA is found at the end of the cDNA, and is usually composed of 6 nucleotides AATAAA (77) found in most eukaryotic mRNAs.
1-776) is first.

(Proudfoot, NJ & Brownlee CG, Nature 263 , 211-2
14, (1976)) The amino acid sequence encoded by the cDNA can be deduced as shown in FIG. 2 (b) (amino acid sequence I), and the polypeptide of amino acid sequence I consists of 153 amino acids and has a molecular weight of 17631.7. Calculated as Dalton. As reported to be found in most secreted proteins known to date (Blobel G. et al, Sym.Soc.Exp.Med., 3
3 , 9-36 (1979), the N-terminal region of the above deduced IL-2 polypeptide is also hydrophobic. This region is mature IL-2
It may play the role of a signal peptide that is cleaved upon secretion of Escherichia coli. Does the disconnect occur between Ser and Ala in the 20-21 position?
Cleavage between Ala-Pro between positions -22 produces a polypeptide having amino acid sequences II and III. Because similar cleavage sites are often found in other secreted proteins known so far (Blobel, G. et al, Symp.
Soc.Exp.Med., 33 , 9-36 (1979)). Therefore, mature IL-
2 polypeptides consist of 133 to 132 amino acids,
The molecular weight is calculated to be 15420.5 or 15349.4 daltons.
This molecular weight value is human IL-2 obtained from Jurkat fat.
Contrast what was reported as the molecular weight of the protein (15,000 daltons) (Gillis et al., Immunological Rev., 63 ,
67-209 (1982)). In addition, as shown in Example 3, a DNA fraction starting from the CCT sequence at the 111-113th nucleotide sequence, that is,
It was confirmed that the code for the polypeptide starting from Pro located at the 22nd position (amino acid sequence III in FIG. 2 (b)) expresses a polypeptide having IL-2 activity. A DNA fraction starting from GCA at nucleotide positions 107 to 110,
That is, as shown in the amino acid sequence II of FIG. 2 (b), the DNA encoding the polypeptide starting from Ala located at the 21st position.
It was confirmed that the fraction expressed a polypeptide having IL-2 activity as shown in Example 6.

Nucleate genes are known to exhibit polymorphism as is also known for the human interferon gene (Taniguchi et al., Gene 10 , 11-15 (1980), Ohno, Taniguchi., Proc.
Natl.Acad.Sci.USA, 77 , 5305-5309 (1981); Gray et a
l, Nature 295 , 501-508 (1981)). Depending on the polymorphism, some of the amino acids of the protein product may be substituted, or the nucleotide sequence may be changed but not changed at all. In the case of human IL-2 / cDNA, pIL-2-50A cDN
Another cDNA clone (pIL-2-503) in which the A residue at position 503 of A is replaced with a G residue can also be detected. pIL-2-50A c
The presence of other cDNA clones that differ in nucleotide sequence from DNA can also be expected.

As is clear from the above explanation, the gene according to the present invention is a DNA, 48-having the nucleotide sequence shown in Fig. 2 (a).
It starts with the ATG sequence at position 50 and is at least A at positions 504-506.
DNAs having a continuous base sequence extending to the CT sequence, G at 108-110 position
It includes a DNA having a continuous base sequence starting from the CA sequence and extending from the GCA sequence to at least the ACT sequence, and a DNA having a continuous base sequence extending from the CCT sequence at positions 111-113 to at least the ACT sequence. The gene according to the present invention also contains ACTs at positions 504 to 506.
End of sequence, DNA starting at A at position 1, DNA starting at ATG at positions 48-50, DNA starting at GCA sequence at positions 108-110 or 111-113
Includes DNA beginning with the CCT sequence at position. Furthermore, the gene according to the present invention is the DNA ending in the TGA sequence at positions 507 to 509, the DNA starting at A at position 1, the DNA starting at the ATG sequence at positions 48 to 50, the G at positions 108 to 110.
DNA starting with CA sequence or CCT sequence at positions 111-113
DNA. Further, the gene according to the present invention is C at position 801.
End with, DNA starting with A at 1st position, D starting at ATG at 48-50th position
NA, DNA starting with GCA at 108-110 or CCT at 111-113
Includes DNA that begins with a sequence. The gene according to the present invention also ends in poly (A) and begins with the ATG sequence at the 48-50 position.
A, DNA starting with GCA sequence at 108-110 or 111-113
Contains DNA beginning with the CCT sequence. The present invention also includes a gene having a nucleotide sequence corresponding to the amino acid sequences I, II and III. A polypeptide lacking one or more amino acids in amino acid sequence I, or a polypeptide in which one or more amino acids in amino acid sequence I is substituted with one or more amino acids is IL-2. It may have activity, and thus the gene encoding such a polypeptide can be used as the gene according to the present invention. Similarly, an amino acid added to the amino acid sequence I, II or III, even in a gene in which one or more bases capable of expressing one or more amino acids is additionally linked,
It is included in the present invention as long as it does not interfere with the expression of IL-2 activity of the polypeptide. Even a modified region having an additional amino acid sequence that inhibits the polypeptide function of IL-2 can be used as the gene according to the present invention as long as the newly added region can be easily removed. The same applies to the amino acid sequences of the genes corresponding to the amino acid sequences I, II and III.
D encoding an amino acid addition at the C-terminus of I, II and III
The same can be said for DNA in which NA is additionally bound to the 3'-end. The deletion, addition, or substitution of amino acids as described above is carried out by the site-specific mutation technique (Geneti
c Engineering, vol.3, pp1-32, Plenum Press, New York
(1981), Nucleic Acid Research, vol.10, pp6487-6500
(1982) etc., and the deletion, addition or substitution of one or several amino acids means the number of amino acids that can be deleted, added or substituted by the site-specific mutation technique. Accordingly, the use of genes encoding such polypeptides is encompassed by the present invention.

Bacteria or Escherichia coli transformed with a recombinant DNA body capable of producing IL-2 in host live fatty acids are produced by the following various methods. For example, a sequence encoding IL-2.cDNA is inserted downstream of the promoter sequence of the expression vector. Alternatively, a piece of cDNA having a promoter sequence can be inserted upstream of the IL-2 coding sequence before or after the insertion of the cDNA into the expression vector.

The method for constructing a bacterium or Escherichia coli that expresses IL-2-cDNA and produces an IL-2-polypeptide will be described in detail below.

(1) Expression of IL-2-cDNA by Escherichia coli In order to express IL-2-cDNA in Escherichia coli, first, the cDNA is bound to various bacterial promoters, and then the hybrid containing the cDNA downstream of the promoter. Make a plasmid. Bacteria that infect Escherichia coli HB101 with this plasmid and biosynthesize a protein having human IL-2 activity are cloned. Any naturally-occurring bacterial promoter will express IL-2-cDNA if properly connected to the cDNA. An example of expression of such a cDNA is as follows.

The cloned cDNA encoding IL-2 encodes a 153 amino acid polypeptide as shown in FIG. N-corresponding to the 20 amino acids of this polypeptide
The terminal region is extremely hydrophobic and is characteristic of most secreted proteins. Such a hydrophobic sequence is called a signal sequence and is cleaved during the secretory process. Therefore, the mature IL-2 polypeptide should have less than 153 amino acids. Therefore, it is desirable to express the cDNA portion encoding the mature IL-2 polypeptide and not the IL-2 signal sequence equivalent portion.

(I) Construction of plasmid vector pTrS-3 pTrS-3 contains the Escherichia coli Trp promoter, and the ribosome binding site (SD sequence) for the leader peptide of pTrS-3 has already been reported (G.Miozzari.
and Yanofsky J. Bacteriol. 133 , 1457 ~ 1466 (197
8)). The presence of an ATG codon located 13 base pairs downstream of the SD sequence has also been reported (Nishi et al, Biochemistry 54 , No.8.676 (1
982)). This plasmid vector also contains a single Sph I site downstream of the ATG initiation sequence (Figure 3).

To express IL-2 / cDNA, the plasmid is first sp
It is digested with h I and treated with Escherichia coli DNA polymerase I (Klenow fragment) or bacteriophage T4 DNA polymerase I to remove the 3′-protruding end (FIG. 4 (a)). Plasmid pIL-2
-50A was digested twice with Pst I and HgiA I resulting in a larger cD
The NA fraction is isolated. Next, replace the DNA with Escherichia coli DNA.
Treatment with polymerase I (Klenow fragment) or bacteriophage T4 DNA polymerase cleaves the 3'-overhang. This treated cDNA encodes an IL-2 polypeptide of 132 amino acids (fourth (a)
Figure). This cDNA was ligated to pTrS-3 plasmid DNA pretreated as described above and the ATG initiation codon was added to IL-2 cDNA.
Connect to the CCT (Pro) array of. Thus the plasmid
pT IL-2-22 is obtained. Trp promoter sequence and pT I
Binding of the IL-2 cDNA sequence of L-2-22 is shown in Figure 4 (a).

The plasmid pT IL-2-22 is an IL-2 consisting of 132 amino acids starting from proline by Escherichia coli.
Directs polypeptide synthesis.

(Ii) Mature IL-2 may contain alanine (position 21) as an N-terminal amino acid instead of proline, and a plasmid directing IL-2 polypeptide synthesis consisting of 133 amino acids should be prepared as follows. You can

The plasmid pTrS-3 has one Cla I cleavage site between the SD sequence and the ATG sequence (Fig. 3). This plasmid is Cla
Cut at I and Sa1 I. Plasmid pIL-2-50A was added to Pst
Partially digested with I, treated with Escherichia coli DNA polymerase I, and the longest DNA is isolated. The DNA is then ligated with a synthetic DNA linker containing a restriction enzyme Xho I cleavage site,
A clone containing the plasmid pIL-2-50A (Xho) in which a DNA linker was introduced 3'-downstream of the IL-2 coding sequence was isolated. The plasmid pIL-2-50A (Xho) is first cleaved with HgiA I, treated with Escherichia coli Klenow fragment or T4 DNA polymerase, and digested with Xho I to isolate the cDNA fraction. This cDNA fragment is ligated to pTrS-3 DNA pretreated with Cla I and Sal I and ligated to synthetic DNA as shown in FIG. 4 (b). Thus A
The plasmid pTIL-2-21 is obtained, in which the IL-2 polypeptide consisting of 133 amino acids starting from la is synthesized in Escherichia coli. (Fig. 4 (b)). The same thing can be done without using the Xho I linker.

(Iii) Different size IL-2 polypeptides with different N-terminal amino acids are also made using the pTrS-3 expression plasmid vector. As shown below, pIL-2-5
The IL-2 cDNA cloned in 0A has a nucleotide binding site.
81-85 has only one Dde I portion. Plasmid pIL
-2-50A (Xho) is cut with Dbe I and the DNA fraction containing the larger section of cDNA isolated. This fraction is pBR322
Contains DNA with more than 3000 base pairs (fourth (c)
Figure). Treating the DNA fraction with exonuclease Bal 31 and
Then cut with Xho I. The DNA obtained here is ligated with pTrS-3 cleaved with Sph I, treated with Klenow fragment or T4 DNA polymerase, and then digested with Sal I (FIG. 4 (c)). Escherichia coli HB 101 is infected with the ligated DNA, and a clone expressing human IL-2 is searched. These clones are human of various sizes
It should express IL-2. Because of human IL-2 N
This is because the DNA corresponding to the terminal region is variously cut and removed. Thus pTIL-2-14 and 15 containing the IL-2 cDNA
Is obtained.

(Iv) IL-2 cDNA was also provided by pKT218 (Talmage; Proc. Natl. Acad. Sci. USA, 77 , P. 3369-3373 (198).
It can also be expressed using (0)). The plasmid pKT218 is
It is cleaved with Pst I, and pIL-2-50A is ligated with the IL-2 cDNA insert obtained by cutting with HgiA I and Pst I (Fig. 5). The resulting plasmid pKIL-2-21 has a sequence at the initiation position of initiation of protein synthesis, as shown in FIG.
Therefore, this plasmid pKIL-2-21 contains 13 of IL-2.
It consists of a fusion of 3 amino acids and β-lactamase amino acid, and it should be able to be synthesized in Escherichia coli (the first methionine is cleaved off in Escherichia coli). .

(V) Expression of the plasmid pTuBlp-5 in which the promoter sequence for tufB is inserted into p-BR.322 has already been performed (Taniguchi et al., Biochemistry 53 966 (1981)). This plasmid contains one Cla I cleavage site, which is located 2 base pairs downstream of the SD sequence as shown in FIG. pT
rS-3 is also a Cla located between the SD sequence and the ATG initial sequence.
It contains an I cleavage site and at the same time this Cla I site
Replacing the bacterial Trp promoter with the tufB promoter is extremely simple because it is not destroyed during the process of making an expression plasmid using L-2 cDNA. Therefore human IL-2
The cDNA is expressed under the control of the tufB promoter. For example
pTIL-2-22 is cut with Cla I and Pvu II, and the DNA fraction containing the IL-2 cDNA is separated.

This fraction was then ligated with pTuBlP-5 and Cla I
And pvu II, the plasmid pTuIL-2-22 is constructed as shown in FIG. IL-2 activity can be detected in the extract of Escherichia coli HB101 containing the plasmid pTuIL-2-22.

(Vi) For example, using pTIL-2-21, but basically
The same construction can be carried out by using all the expression plasmids achieved by using pTrS-3. Also for example pT
uIL-2-22 is cleaved with Cla I and Bal 31 or SI or DN
It is also possible to remove or replenish 2-3 base pairs of DNA with A polymerase I (Escherichia coli) and connect the plasmids again to make the distance between the SD and ATG sequences optimal.

Recombinant by culturing a prokaryotic cell transformed with the recombinant DNA of the present invention or Escherichia coli
The DNA body can be amplified or an IL-2 polypeptide can be produced. This culturing is carried out by a usual method.

IL-2 produced intracellularly or extracellularly is ammonium sulfate precipitated,
Dialysis (normal pressure or reduced pressure) for removing salts, gel filtration, chromatography, isoelectric focusing, gel electrophoresis, high performance liquid chromatography (hereinafter abbreviated as HPLC), (ion exchange, gel filtration and reverse) Phase chromatography), and dye chromatography, and affinity chromatography using activated sepharose 4B bound with a monoclonal antibody against IL-2 or lectin-bound sepharose 4B. IL-2 was isolated and purified by Watson et al. (J. Exp. Med., 150 , 8
49-861 (1979), Gillis et al, J. Immunol., 124 , 1954-
1962 (1980), Mochizuki et al, J. Immunol. Methods 39 ,
185-201 (1980), Welte, K et al, J.Kxp.Med., 156, 454
-464 (1982)).

The polypeptide thus obtained exhibits the same biochemical and biological behavior as that known for IL-2 produced from mammalian cells by mitogen stimulation.
It has IL-2 activity. The molecular weight is about 15,000 daltons and I
L-2 activity was completely neutralized with or without immunosorbents such as Igsorb (Enzyme Center) or precipitated with monoclonal anti-IL-2 antibody. On immunoelectrophoresis, the IL-2 polypeptide shows only one settling line for the corresponding anti-IL-2 antibody. The IL-2 activity is stable even after reduction with 2-mercaptoethanol, and is stable even after treatment with DNAse and RNAse or heat treatment at 56 ° C. for 30 minutes. The activity is stable at pH 2-9. IL- produced in this way
2 promotes the proliferation of T cells (cytotoxic T lymphocytes) having a monoclonal function and enhances the division of thoracic cells,
Furthermore, in the absence of antigen, it induces differentiation from memory state into anti-cancer-specific cytotoxic T lymphocytes. It also helps enhance the activation of natural killer cells for YAC-1 cells and RL 1 cells.

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

Example 1 (1) A Jurkat cell, which is a human T-cell leukemia cell line (freely available in Japan, West Germany, and the United States), was suspended in RPMI 1640 medium containing 10% FCS, and X-ray irradiation apparatus Exs was used. 150 / 300-4 (Toshiba / Japan) for 50 seconds,
Irradiation at room temperature until reaching 10,000 roentgens. The cells that were then irradiated had an initial cell density of 1 × 10 ^{5 in the} above medium.
5% carbon dioxide gas at 5 cells / ml in an incubator at 37 ° C
Cultured for a day.

The mutant cells (0.2 cells / well) were seeded in 10 wells of a 96-well flat-bottomed microplate, and cultured for 21 days in a 5% carbon dioxide gas, 37 ° C. incubator. Clones obtained from the growing holes were transferred to a new medium to increase the amount of clones, and the increased clones were cultured in the presence of 50 μg / ml ConA at an initial cell density of 1 × 10 ⁶ cells / ml for 24 hours. And
IL-2 activity was measured according to the method described above.

As a result, a human T cell line designated as Jurkat-111 (ATCC CRL 8129) (hereinafter referred to as "J-111") was cloned and selected from its parent strain, Jurkat, and the IL-2 producing ability of this cell was selected. Was 40 times higher than the parent strain.

This cloned J-111 cell line grew under normal conditions and its growth rate was almost the same as that of normal Jurkat cells.

(2) J-111 cells (1 × 10 ⁵ cells / ml) were treated with serum-free synthetic medium RIT C 55-9 (Sato, T. et al., Exp. Cell Res., 138 , 12).
7-134, (1982)) was inoculated into 1000 ml and cultured in a roller culture bottle (Falcon 3027) at 37 ° C. for 4 days, and the grown cells were collected by centrifugation. 4x this cell again
The above medium containing 25 μg / ml of ConA was inoculated so that the concentration was 10 ⁶ cells / ml. 100 ml of the culture solution inoculated with the cells was placed in each of 4 batches of roller culture bottles (Falcon), and the cells were subjected to rotary culture for 6 hours.

(3) The Jurkat cells (1.2 × 10 ⁶ ) thus stimulated with 25 μg / ml of ConA for 6 hours were suspended in 8,000 ml of a physiological saline-phosphate buffer solution (hereinafter abbreviated as PBS). The cells were washed twice by centrifugation and RS containing the ribonucleoside-vanadale complex (10 mM), which is a nuclease inhibitor.
B solution (10 ml Tris-HCl buffer, pH 7.5, 10 mM NaCl,
Resuspended in 800 ml of 1.5 mM MgCl ₂ . Then the surfactant
NP-40 was added to a final concentration of 0.05%, mixed slowly, and the cell nuclei were separated by centrifugation at 3,000 rpm for 5 minutes at 4 ° C. SDS (0.5%) and EDTA (5 mM) were added to the supernatant, and cytoplasmic RNA was extracted by adding the same amount of phenol as the supernatant.
After extraction with phenol was repeated 3 times, RNA was precipitated with 2 volumes of ethanol, and this precipitate was collected by centrifugation and dissolved in 10 mM Tris-HCl, pH 7.5. Obtained RNA
The amount was 196 mg.

The fraction of mRNA is oligo (dT) -cellulose (PLBioche
micals, Type 7) affinity chromatography. Adsorption liquid is 20 mM Tris-HCl, 0.5 M NaC
pH 7.5 solution containing 1 mM EDTA and 0.5% SDS, elution was performed with a buffer (20 mM Tris-HCl, pH 7.5, 0.5 M).
After washing with NaCl and 1 mM EDTA), water and 10 mM Tris-HCl (pH
7.5) alternate. 3.6 mg of mRNA obtained by elution
Met. Next, 2.4 mg of the obtained mRNA was subjected to sucrose density gradient centrifugation (in a pH 7.5 solution containing 50 mM Tris-hydrochloric acid, 1 mM EDTA, 0.2 M NaCl) at a sucrose density gradient of 5-25% at 26,000 rpm.
Fractionation was performed at 24 ° C for 24 hours). Fraction N from 11 to 12S of mRNA
Fractionated into o.12, 13, 14 and 59μg, 46μg, 60μg respectively
Met.

(4) The mRNA obtained in the No. 13 fraction was injected into oocytes of Xenopus Iaevis (50 ng m
RNA / oocyte). IL-2 activity of this oocyte culture medium was measured. As shown in Table 1, ³ H- thymidine ⁽³ H-TdR) increase in the uptake of and an increase in activated T lymphocytes were confirmed, clearly mRNA in this fraction contains human IL-2 mRNA It was proved that

(5) Then, No. 13 of 11-12S mRNA containing IL-2 mRNA
CDNA was synthesized from the fractions in vitro.

Recombinant DNA was constructed with plasmid vector pBR322. Convert the recombinant DNA to Escherichia coli
The L-2 cDNA clone-acquired clone was selected by the following method.

(5-1) 50 mM Tris-HCl buffer (pH 7.5), 30 mM
NaCl, 6 mM MgCl ₂ , 5 mM dithiothreitol (hereinafter abbreviated as DTT), 0.5 mM each of dATP, dGTP, dCTP, dTTP (dCTP is ³² P
Radiolabeled), 0.7 μg oligo (dT) ₁₀ , 1
0 μg mRNA and 15 units AMV reverse transcriptase (JWBeard) were mixed and kept at 41 ° C. for 90 minutes. After the reaction was completed, the DNA was treated with phenol and recovered as an ethanol precipitate.
It was dissolved in a solution of pH 7.5 containing 0 mM Tris and 1 mM EDTA.

2.5 μg of ss-cDNA was synthesized. To remove mRNA from this reaction solution, add NaOH solution to the reaction solution to make a 0.33 N NaOH solution, leave it at room temperature for 15 hours, and then add 1 M Tris-HCl buffer solution of pH 7.5 to neutralize it to give a sepharose solution. The Dex G-50 column was passed through the column. The recovered cDNA was 1.8 μg.

(5-2) 50 mM phosphate buffer (pH 7.5), 10 mM MgC
l ₂ , 10 mM DTT, 0.75 mM each dATP, dGTP, dCTP, dTTP (dC
TP includes those labeled with ³ H), 1.8 μg ss-cDNA and 8 units of Polymerase I (BRL, USA) were mixed for 15 hours, 1
The reaction was carried out at 5 ° C. After the reaction was completed, the DNA was treated with phenol and chloroform and then recovered as an ethanol precipitate. 1.
10 μg of ds-cDNA was produced. 50 mM sodium acetate (pH 4.
5), 0.2M NaCl, 1 mM ZnCl ₂ and 1.10 μg double-stranded cDNA
The mixture was incubated at 37 ° C. for 30 minutes, 0.25 unit of nuclease S ₁ (Mitsui, Japan) was added, and the mixture was further incubated for 15 minutes. After completion of the reaction, treat with phenol 2
The reaction product thus obtained was subjected to Sephadex G-50 to obtain 0.55 μg of double-stranded cDNA.

(5-3) 0.14 M potassium cacodylate, 30 mM Tris base, 0.1 mM DTT, 1 mM CoCl ₂ , 0.64 mM ³² P-dCTP (specific activity
2.7 × 10 ⁶ cpm / n mol), 0.55 μg ds-cDNA and 5 units of terminal transferase (BRL), and
After incubating at ℃ for 7 minutes, treated with phenol, and then subjected to Sephadex G-50 column to obtain 0.50 µg DNA as an ethanol precipitate. About 50 of this recovered DNA
It was found that dCMP residues were added to both 3'ends.

After cutting 10 μg of pBR322 DNA with the restriction enzyme Pst I, d
The ds-cDNA described above was used except that dGTP was used instead of CTP.
The dGMP chain was added to both 3'ends of the cleaved DNA under exactly the same conditions as the method used when the dCMP chain was added to.

(5-4) 50 mM Tris-hydrochloric acid (pH 7.5) 0.1 M NaCl, 5 m
M DETA, 0.05 μg d GMP residue added pBR 322 and 0.01 μg d
The CMP residue-added cDNA was first incubated at 65 ° C for 2 hours, then at
For an additional 60 minutes at 37 ° C. and 60 minutes at room temperature.

Escherichia coli χ1776 (Curtiss III, R. et al., I
n Molecular Cloning of Recombinant DNA, (WAScott
& R. Werner ed.) Academic Press, (1977)) 50 ml of L medium (100 μg / ml of diaminopimelic acid, 50 μg / ml of thymidine, 1% tryptophan, 0.5% yeast extract, 0.5%).
% NaCl and 0.1% glucose) and cultured at 37 ° C. with shaking until the absorbance of the culture solution was around 0.3 at 562 nm. After completion of the culture, the culture solution was kept at 0 ° C. for 30 minutes, the bacterial cells were collected by centrifugation, and 5 mM Tris-hydrochloric acid (pH 7.6), 0.
It was washed twice with 25 ml of a solution containing 1 M NaCl, 5 mM MgCl ₂ and 10 mM RbCl.

The obtained bacterial cells were treated with 5 mM Tris-hydrochloric acid (pH 7.6), 0.25 M KC.
Solution containing l, 5 mM MgCl ₂ , 0.1 M CaCl ₂ and 10 mM RbCl 2
After suspending in 0 ml and standing at 0 ° C for 25 minutes, the bacterial cells were collected and resuspended in 1 ml of the same solution as above to obtain a bacterial cell suspension.
The recombinant DNA was placed in 0.2 ml and left at 0 ° C. for 60 minutes. Then, 0.7 ml of L medium was added, and the mixture was shake-cultured at 37 ° C for 30 minutes. The thus obtained culture solution (0.1 ml) was smeared on one side of a 1.5% agar medium of L medium containing 100 μg / ml diaminopimelic acid, 50 μg / ml thymidine and 15 μg / ml tetracycline, and incubated at 37 ° C. for 2 days.

(5-5) The resulting 432 colonies were divided into 18 groups, each group containing 24 different bacterial clones: 100 μg / ml diaminopimelic acid, 50 μg / ml
Was inoculated into 200 ml of L medium containing thymidine and 10 μg of tetracycline and shake-cultured at 37 ° C. for 5 to 7 hours.
Next, 200 ml of a new L medium containing chloramphenicol added to a final concentration of 170 μg / ml was added, and the cells were further cultured overnight.

The plasmid DNA thus enhanced was purified by a conventional method.

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

The HT assay used here was performed as follows.

The purified DNA (25 μg) was cleaved with the restriction enzyme Hind III, treated with phenol three times, phenol-chloroform and chloroform respectively, and precipitated with ethanol.
It was washed with% ethanol and dissolved in 40 ml of 80% formamide.

After heating at 90 ℃ for 5 minutes to denature the reaction solution, 10 × SSC
It was diluted to 1.3 ml with (1.5 M NaCl, 0.15 M sodium citrate). After that, the DNA was fixed on nitrocellulose filter paper, dried at 80 ° C for 3 hours, and added with 50% formamide,
20mM Pipes (pH6.5), 0.75M NaCl, 5mM EDTA, 0.2% SD
After incubating at 37 ° C. for 18 hours in a solution containing 250 μg of poly (A) mRNA derived from S and J-111 cells, DNA immobilized on filter paper and IL-2 mRNA were hybridized.

Then filter the filter paper 3 times at 65 ° C, pH 6.5, 10 mM Pipes, 0.15M
Wash with NaCl solution, 1 mM Pipes, 10 mM NaCl solution, 0.5 mM EDT
A, 0.1% SDS solution was treated at 95 ° C. for 1 minute, and hybridized mRNA was recovered from the filter paper.

The mRNA thus extracted was treated with oligo dT according to a conventional method.
-Purified on a cellulose column and injected into Xenopus oocytes and the IL-2 activity of the translated protein was measured.

One out of 18 groups of 24 clones each contained 48 units / ml in the assay with ³ H-TdR incorporation described above.
IL-2 activity was positive. On the other hand, the other groups were clearly negative.

Next, 24 single colonies belonging to the positive group were inoculated into 200 ml of L medium having the same composition as described above, and the mixture was incubated at 37 ° C for 5 to 7
The cells were cultured aerobically for a period of time, and L medium containing chloramphenicol was also added.

Incubate overnight to enhance plasmid DNA and then plasmid D
NA was similarly purified according to standard methods. After cleaving about 5 μg of each plasmid DNA with Hind III, each plasmid was similarly immobilized on nitrocellulose filter paper. Filter the filter paper with IL-2 mRNA
And the hybridized mRNA was injected into Xenopus oocytes and the translated protein IL-
2 Collected for measuring activity.

As shown in Table 2, only plasmid DNA purified from a single colony designated p3-16 showed positive IL-2 activity. Therefore, this clone is a clone carrying the IL-2 cDNA (E. coli χ 1776 / p3-16AJ11995 (FERM-BP-22
5)) was identified. Thus plasmid DNA, p3-16
Was confirmed to have DNA (IL-2 gene) capable of forming a specific hybrid with IL-2 mRNA.

The cDNA insert of plasmid p3-16 contains the restriction enzyme Xba I.
And Bst NI at two sites (upstream and downstream of the Xba I cleavage site).
However, plasmid p3-16 contains a cDNA insert composed of approximately 650 base pairs, which is clearly
It corresponds to a part of the IL-2 mRNA having a size of 11 to 12S. Therefore, using other cDNA libraries as templates
Using IL-2 mRNA, the method of Land et al. (Land et al., Nuclei
c Acids Res., vol 9, p2551, (1981)). IL with single-stranded cDNA (1.6 μg) added with dCMP residue
-2 mRNA was synthesized using 4 μg, and ds-cDNA was used as DN.
A polymerase I (Klenow fragment) was used to synthesize oligo (dG) 12 = 18 as a primer. 680
CDNAs longer than the base pair DNA size marker
(0.6 μg) was obtained by sucrose density gradient centrifugation and could be inserted into the Pst I site of pBR322 by standard GC tailing.

After transformation of Escherichia coli χ1776 with the recombinant DNA body, G using the nick-translated p3-16 cDNA insert as a probe in its place.
About 2000 colonies were selected by the runstein-Hogness hybridization method, and colonies containing the plasmid pIL2-50A containing approximately 850 base pairs and transformed clones (Escherichia coli χ1776 / pIL2-50A, AJ
11996 (FERM-BP-226)) was identified. pIL2-50A cDN
A restriction enzyme digestion diagram of the A insert is shown in FIG.

Transformed Escherichia coli χ1776 / pIL2-50A
In order to isolate the gene encoding the IL-2 peptide from Escherichia coli, plasmid DNA was isolated from the bacterial cells by a conventional method.
Was isolated and then cleaved with the restriction enzyme Pst I. The smaller of the two DNA fragments generated by this treatment is IL-
It was a DNA gene encoding 2 peptides. pIL
The complete nucleotide sequence of the Pst I insert from 2-50A is the method of Maxam and Gilbert (Maxam, AW et al., Enzy.
m. 65 , 499-560, 1980). The whole structure is second
It is shown in FIG.

Example 2 Constitutive IL-2 producing cell line JA 18 cloned from Jurkat cells according to the method described in Example 1.
86 (ATCC CRL 8130) was similarly grown in roller culture bottles. The grown cells were resuspended in fresh synthetic medium RITC-55-9 at an initial cell density of 1 × 10 ⁶ cells / ml, and 8 hours after the start of culture, 3 × 10 ⁶ cells according to the steps detailed in Example 1.
It was used for the ^nine cells of IL-2 _m RNA extraction as 11~12S fraction.

The ds-cDNA was synthesized in the same manner as in Example 1, and cDNA (2.4 μg) longer than 600 base pairs was obtained by fractionation by sucrose density gradient centrifugation. Next, this cDNA was extended with dCMP residues using terminal deoxynucleotidyl transferase, and 50 ng of which was extended with dGMP Pst I-digested pBR322 2
Annealed with 50ng.

The resulting hybrid plasmid was transformed into Escherichia coli χ1776, and a transformant of about 4,000 clones was obtained.

According to the Grunstein-Hogness method, three clones complementary to the plasmid 3-16 cDNA used as a probe were selected. That is, the transformed clone thus selected is a clone having the human IL-2 gene.

Example 3 A plasmid directing the synthesis of human IL-2 in Escherichia coli cells was constructed by the following method.

Plasmid pT IL2-22 was transformed into pTrS-3 (Nishi T., T by a series of modifications as illustrated in Figure 4 (a).
aniguchi T. et al., SEIKAGAKU 53 , 967, (1981), 54
676 (1982)) and pIL2-50A containing the IL-2 cDNA. Plasmid pTrS-3 contains Trp promoter and pBR322
Between the EcoR I and Cla I sites of Shine Dalgarno (hereinafter S
Abbreviated as D).

This plasmid also contains a single Sph, as shown in FIG.
Like the I site, it contains the ATG initiation codon 13 bp downstream of the SD sequence.

When the DNA sequence corresponding to the mentioned protein is inserted just downstream of the ATG codon, the vector is very effective in producing this protein. This ATG codon is generated by Sph I digestion of pTrS-3 followed by treatment with T4 DNA polymerase. Therefore the plasmid pTrS-
3 (30 μg) was a restriction enzyme Sph I, which was digested by a conventional method, and subsequently recovered by a phenol / chloroform treatment and an ethanol precipitation method, and both ends were flushed by a T4 DNA polymerase treatment.

Next, DNA (21.4 μg) was recovered by the same method as the phenol / chloroform treatment and the ethanol precipitation method. On the other hand, 380 μg of pIL2-50A containing IL-2 cDNA was Pst I
And the IL-2 cDNA insert was isolated by agarose gel electrophoresis. cDNA insert (11μ
g) was cleaved with HgiA I, treated with T4 DNA polymerase and 10 μg of the larger part of the DNA was isolated by agarose gel electrophoresis. According to this method, a cDNA (7.2 μg) encoding 132 amino acids was obtained, and this DNA fragment had a blunt end (4th (a)).
Figure).

The cDNA fragment thus obtained was then ligated just downstream of the ATG sequence into the pTrS-3 vector which had been treated with T4 DNA polymerase previously digested with SpHI. The thus ligated plasmid was then transformed into Escherichia coli HB 101 according to a conventional method. This ligation was performed as follows. IL-2 cDNA (0.4 μg)
Of the above-mentioned larger fragment and pTrS-3 vector DNA0.2
μg containing 6.6 mM MgCl ₂ , 1 mM ATP and 10 mM DTT at pH 7.5
Of 66 mM Tris-HCl with 0.8 units of T4 DNA ligase and the mixture was reacted overnight at 4 ° C. Among the transformants appearing on the L medium agar plate containing ampicillin, IL-2c encoding 132 amino acids was used.
Colonies with plasmids containing the DNA portion were selected in situ by colony hybridization assay. Re-cultivate the selected colonies (10 ml),
Plasmid DNA was prepared by lysozyme treatment and freeze / thaw treatment. Use this plasmid DNA for Pst I and Xba
Cleavage with I and analysis of the resulting product by agarose gel electrophoresis identified pTIL-2-22 whose cDNA was frozen in the correct orientation after the ATG sequence of pTrS-3.

Escherichia coli HB 101 containing pTIL-2-22 was cultured under conventional methods known for microbial growth.
Cells were χ medium containing 25 μg / ml streptomycin and 25 μg / ml ampicillin (2.5% bactotryptone, 1%
Yeast extract, 0.1% glucose, 20 mM MgSO ₄ , 50 mM Tris-
It was grown overnight at 37 ° C. in 10 ml of hydrochloric acid, pH 7.5. Then, 1 ml of the culture suspension was inoculated into the same χ medium (100 ml) and cultured at 37 ° C. 3-indole acrylic acid (IAA) was added when the OD of 650 m reached approximately 1.5-2.0. 3 hours after the addition of the inducer, the cells were collected, washed with 20 mM Tris-hydrochloric acid (pH 7.5, containing 30 mM NaCl), and 8 ml of the same buffer solution was added.
Suspended again.

For effective functional expression of the Trp promoter, an inducer such as IAA was added to a final concentration of 50 μg / ml. The protein thus produced in bacterial cells was extracted by sonication (0 ° C., 2 minutes) or lysozyme (8 μg) digestion (0 ° C., 20 minutes) followed by freeze-thawing three times. By this method, IL-2 was generally extracted from cells. IL-2 activity extracted is 10,000 to 120,00
The range was 0 unit / ml.

Escherichia coli HB 101 (AJ1200 including pTIL2-22
9) has been deposited as FERM-BP245.

Example 4 The plasmid pTuIL2-22 containing the IL-2 cDNA is pTuBlP-
5 (Taniguchi, T. et al., Seikagaku, 53 , 966, 1981) and pTIL2-22 shown in Example 3 were constructed by the method illustrated in FIG. The plasmid pTuBlP-5 has the tufB promoter sequence inserted into pBR322. This plasmid also contains a single Cla I site, which is located 2 bp downstream of the SD sequence as shown in FIG. pTr
S-3 also contains a Cla I site between the SD sequence and the ATG initiation codon, which was expressed by using pTrS-3 and IL-2 cDNA as described in Example 3. Since it is not destroyed during plasmid construction, Trp
Replacing the promoter with the tufB promoter is quite straightforward, so that the IL-2 cDNA is expressed under the control of the tufB promoter.

Therefore, the plasmid pTIL2-22 (30 μg) contains the restriction enzyme C
Cleaved in the usual way with la I and Pvu II. IL-2 cD
The NA-containing fragment (about 2.2 kb) was isolated and purified by agarose gel electrophoresis, and 3 μg of DNA was recovered. On the other hand, pT
Similarly, 20 μg of uBlP-5 vector was cleaved with Cla I and Pvu II, and the larger fragment (about 3.4 kb) containing the ampicillin resistance gene was isolated and purified by agarose gel electrophoresis to recover 3.5 μg of DNA. The two fragments thus obtained then contained one containing the tufB promoter (about 3.4 kb) and the other containing the IL-2 cDNA (about 2.2 kB).
b) Connected as shown below.

A fragment containing IL-2 cDNA (1.2 μg) and a fragment containing tufB promoter (0.3 μg) were added to 6.6 mM MgCl ₂ , 1 mM ATP and
T4 DNA in 66 mM Tris-HCl, pH 7.5 containing 10 mM DTT.
It was mixed with 0.8 unit of ligase and reacted overnight at 4 ° C. Next, the plasmid thus ligated was transformed into Escherichia coli HB 101 according to a conventional method.

Among the transformants appearing on the L medium agar plate containing ampicillin, IL such as pTuIL2-22 in FIG.
Eight colonies with recombinant DNA containing the −2 cDNA portion were selected and plasmid DNA was prepared as described in Example 3.

Escherichia coli HB 101 containing pTuIL2-22 was cultured in L medium (100 ml) at 37 ° C. OD of 650mμ is approximately
When it reached 0.5-1.0, the cells were collected, and 20m containing 30mM NaCl was added.
It was washed with M Tris-HCl (pH 7.5) and resuspended in 2 ml of the same buffer. The protein produced in this manner was obtained in Example 3
As well as extracted. IL-2 activity in the extract ranged from 6,000 to 56,000 units / ml.

Escherichia coli HB 101 (AJ1201 containing pTuIL2-22
0) has been deposited as FERM-BP 246.

Example 5 The plasmid pGIL2-22 containing the IL-2 cDNA was cloned into gGL101 (R
oberts, TMand Laucer GD, Meth Enzym., 68 , 473−48
3, (1979), Gail Lauer, et al, J. Mol. Appl. Genet., 1 , N
o.2,139-147 (1981), T. Taniguchi, et al, Proc.Natl.A
cad.Sci.USA, 77 , No.9,5230〜5233 (1980), Egon Amann,
et al, Gene, 25 , 167-178 (1983)) and pTIL2-22 shown in Example 3.

That is, the plasmid pGL 101 containing the lac promoter.
(20 μg) was digested with a restriction enzyme Pvu II by a conventional method, and then 17 μg of DNA was recovered by a phenol / chloroform treatment and an ethanol precipitation method. On the other hand, pTIL2-22
(75 μg) was cleaved with Cla I and Sal I, and 2.2 μg of DNA fragment contained in IL-2 cDNA by agarose gel electrophoresis.
Was recovered. This fragment was flushed by treating with DNA polymerase I (Klenow fragment). The two fragments thus obtained (0.25 μg and 0.66
μg) was ligated with 1.0 unit of T4 DNA ligase in the same manner as in Example 4. Thus, the ligated plasmid was transformed into Escherichia coli HB 101 according to a conventional method. Cla containing IL-2 cDNA in the transformant
A transformant ³² P-labeled IL-2 cDNA with the insertion of the I-Sal I fragment was selected as a probe. Next, these transformants were cultured in 10 ml of χ medium containing 25 μg / ml of ampicillin, and plasmid DNA was prepared by the method described in Example 3. Thus, a plasmid DNA having the IL-2 cDNA initiation sequence ATG just downstream of the lac promoter was obtained by assaying for cleavage sites at Pst I and Xba I. PGIL2-2 thus obtained
Escherichia coli HB 101 containing 2 was inoculated and cultured in 100 ml of L medium containing 25 μg / ml ampicillin and 25 μg / ml streptomycin. When the OD of 650mμ reached about 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a concentration of 1mM and the cells were collected 1 hour later,
A cell extract was prepared according to the method described in Example 4. The IL-2 activity of the extracts ranged from 6,000 to 80,000 units / ml.

Escherichia coli HB 101 (AJ 1201 containing pGIL2-22
1) has been deposited as FERM-BP 247.

Example 6 The plasmid pTrS-3 (10 μg) was first treated with the restriction enzyme Sal I.
Cleaved with and the Sal I site is DNA polymerase (Klenow fragment)
Alternatively, flash (fl
ush).

After cutting with Cla I, the larger fragment having the Trp promoter region was isolated and purified by agarose gel electrophoresis according to a conventional method to recover 3 μg of DNA.

On the other hand, 11 μg of the cDNA insert obtained by PstI digestion of pIL2-50A was digested with HgiAI, treated with T4 DNA polymerase, and the larger fragment was isolated and purified by agarose gel electrophoresis. In this way, 7.2 μg of a cDNA fragment encoding the 132 amino acids of IL-2 was obtained. Next, a fragment containing trP promoter (above) 0.45μ
0.5 μg of HgiAI-Pst I fragment containing g, IL-2 cDNA and synthetic oligonucleotide (5 ′) CGATAAGCTATGGCA
(3 ') and (3') TATTCGATACCGT (5 ') (20pmo each)
le) are both phosphorylated at the 5'end, but these were treated in the same manner as described in Example 3 with T4 DNA.
Ligase was ligated by 1 unit (Fig. 4 (b)). The thus ligated plasmid was transformed into Escherichia coli HB 101. Among the transformants that appeared,
The target transformants were selected as follows. First, a transformant capable of hybridizing with both the IL-2 cDNA and the synthetic oligonucleotide was selected by the colony hybridization method. Then, just downstream of the ATGGCA sequence, 111 in Fig. 2 (a).
To the DNA fragment (CCTACT ...
……) was selected by transforming the plasmid with the inserted plasmid DNA by assaying the Pst I and Xba I cleavage sites.

The above transformants containing pTIL2-21a or pTIL2-21b were cultured in L medium according to the method described in Example 3, and when analyzed by the method described in Example 3, transformant cell extracts contained High IL-2 activity was observed. Escherichia coli HB 101 with pTIL2-21a
(AJ 12013) and Escherichia with pTIL2-21b
Escherichia coli HB 101 with E. coli (AJ 12014) has been deposited as FERM-BP 248 and EFRM-BP 249, respectively.

The hosts used in the above examples, Escherichia coli χ 1776 and HB 101 (Boyer HW et al., J. Mol. Biol. 4
1 , 459, (1669)) is publicly known and is easily available. In addition, recombination in transformants
The host can be easily obtained from the deposited transformant by culturing the transformant in an L medium at 37 ° C. to release the DNA, and separating the cells that have become sensitive to tetracycline and ampicillin.

Plasmid vectors pBR322 (available from Bethesda Research Laboratory, for example), pCE-1, pTrS-3 and
pGL 101 is publicly known and easily available. Furthermore, by isolating the recombinant plasmid in the transformant by a conventional method and further isolating the plasmid vector as is apparent from the explanation in each example, the plasmid vector from the deposited transformant is obtained. Can be obtained. pTrS-3 and pTuBlP
-5 is Escherichia coli FERM-P6735 (BP 32
8) and Escherichia coli ATCC 31878.

[Brief description of drawings]

FIG. 1 shows a cleavage map of a cloned gene encoding a polypeptide having IL-2 activity by a restriction endonuclease, FIG. 2 (a) shows the nucleotide sequence of the cloned gene, and FIG. ) Shows the amino acid sequences I, II and III of the polypeptide having IL-2 activity. FIG. 3 shows the plasmid vector pTrS-3. Figures 4 (a), 4 (b) and 4 (c) show recombinant DNAs (pTIL2) using pTrS-3 as a vector.
-22, pTIL2-21, pTIL2-14 and pTIL2-15) are flow charts showing the constitution. Figure 5 shows pK as a vector
It is a flowchart which shows the structure of recombinant DNA (pKIL2-21) using T218. Figure 6 shows pT as a vector
It is a flowchart which shows the structure of the recombinant DNA (pTuIL2-22) which uses UB1P-5. In the figure, "A", "G", "C" and "T" represent deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid and thymidylic acid, respectively.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Yutaka Matsui 1-19-16-101 Namiki, Kanazawa-ku, Yokohama-shi, Kanagawa (72) Inventor Shin-ichi Kashima 2-21-603 Wakabadai, Asahi-ku, Yokohama-shi, Kanagawa (72) ) Inventor Atsushi Hamuro 241-232, Fukaya-cho, Totsuka-ku, Yokohama-shi, Kanagawa

Claims (32)

[Claims] 1. The following formula Having a human interleukin-2 activity having the amino acid sequence shown by or a human interleukin having an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted with respect to the amino acid sequence of the polypeptide. A recombinant DNA body for transforming a prokaryote, which contains a gene encoding a polypeptide having two activities. 2. A recombinant DNA body for transforming a prokaryote according to claim 1, wherein the polypeptide has the following amino acid sequence in its molecule. 3. A recombinant DNA body for transforming a prokaryote according to claim 2, wherein the polypeptide has the following amino acid sequence. 4. A recombinant DNA body for transforming a prokaryote according to claim 2, wherein the polypeptide has the following amino acid sequence. 5. A recombinant DNA body for transforming a prokaryote according to claim 2, wherein the polypeptide has the following amino acid sequence. 6. A recombinant DNA body for transforming a prokaryote according to claim 2, wherein the polypeptide has the following amino acid sequence. 7. A recombinant DNA body for transforming a prokaryote according to claim 4, wherein the gene encoding the polypeptide has the following base sequence. 8. A recombinant DNA body for transforming a prokaryote according to claim 6, wherein the gene encoding the polypeptide has the following base sequence. 9. The recombinant DNA body according to claim 1, wherein the prokaryote is Escherichia coli. 10. A recombinant DNA body for transforming Escherichia coli according to claim 9, wherein the polypeptide has the following amino acid sequence in the molecule. 11. A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. 12. A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. 13. A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. 14. A recombinant DNA body for transforming Escherichia coli according to claim 10, wherein the polypeptide has the following amino acid sequence. 15. A recombinant DNA for transforming Escherichia coli according to claim 12, wherein the gene encoding the polypeptide has the following base sequence.
body. 16. A recombinant DNA for transforming Escherichia coli according to claim 14, wherein the gene encoding the polypeptide has the following base sequence.
body. 17. The following formula Having a human interleukin-2 activity having the amino acid sequence shown by or a human interleukin having an amino acid sequence in which one or several amino acid residues have been deleted, added or substituted to the amino acid sequence of the polypeptide. A prokaryote transformed with a recombinant DNA body containing a gene encoding a polypeptide having two activities. 18. The prokaryote according to claim 17, wherein the polypeptide has the following amino acid sequence in the molecule. 19. The prokaryote according to claim 18, wherein the polypeptide has the following amino acid sequence. 20. The prokaryote according to claim 18, wherein the polypeptide has the following amino acid sequence. 21. The prokaryote according to claim 18, wherein the polypeptide has the following amino acid sequence. 22. The prokaryote according to claim 18, wherein the polypeptide has the following amino acid sequence. 23. The prokaryote according to claim 20, wherein the gene encoding the polypeptide has the following base sequence. 24. The prokaryote according to claim 22, wherein the gene encoding the polypeptide has the following base sequence. 25. The prokaryote according to claim 17, wherein the prokaryote is Escherichia coli. 26. The Escherichia coli according to claim 25, wherein the polypeptide contains the following amino acid sequence in the molecule. 27. The Escherichia coli according to claim 25, wherein the polypeptide has the following amino acid sequence in the molecule. 28. Escherichia coli according to claim 25, wherein the polypeptide has the following amino acid sequence in the molecule. 29. The Escherichia coli according to claim 25, wherein the polypeptide has the following amino acid sequence in the molecule. 30. The Escherichia coli according to claim 25, wherein the polypeptide has the following amino acid sequence in the molecule. 31. The Escherichia coli according to claim 28, wherein the gene encoding the polypeptide has the following base sequence. 32. The Escherichia coli according to claim 30, wherein the gene encoding the polypeptide has the following base sequence.