JPH0581236B2 - - Google Patents

Description

Detailed Description of the Invention [0001] [Industrial Application Field] The present invention relates to DNA encoding human interleukin-1 polypeptide. [0002] [Prior Art] Gary et al. found a substance in the culture supernatant of human macrophages that promotes the division of mouse thymocytes by mitogen, and discovered that this substance was used as a lymphocyte activating factor (lymphocyte activating factor).
factor (hereinafter abbreviated as LAF),
Since 1979, interleukin-1 (IL-1)
(abbreviated as ) is used. Therefore, such a substance is also treated as interleukin-1 in this specification. [0003] IL-1 promotes the proliferation and differentiation of T cells and B cells, and also acts on T cells to induce lymphokines, especially interleukin 2 (T cell growth factor).
It is thought to be one of the factors that plays an important role in regulating antibody production and cell-mediated immunity [Staruch, MJ, et al., J.
Immunol., 130 , 2191 (1983)]. In addition, it is reported to have the effect of promoting the production of prostaglandin E and collagenase, promoting the proliferation of fibroblasts, and enhancing the NK (natural killer) cell activation effect of interleukin 2 and interferon [Simon ,pL,etal.,
“Lymphokines” vol.6, p.47 (1982), Academic
Press Inc., New York]. [0004] As described above, IL-1 is a biological substance that is involved not only in immune responses but also in the defense and repair of the body, and is expected to have clinical application as a therapeutic agent for immunodeficiency diseases and an antitumor agent. There is. [0005] Up until now, methods for obtaining IL-1 have mainly been from macrophages, peripheral mononuclear cells, or macrophage-like established cell lines (for example, mouse P388D ₁ cells).
Monocytic or myeloid leukemia cells, etc., are cultured in the presence of an appropriate inducing agent, and isolated from the culture supernatant. [0006] Human IL-1 is isolated and purified from the culture supernatant of U937 cells, which are human monocytic leukemia cell lines, and human peripheral mononuclear cells, and its molecular weight is 11,300.
and 15,000 Daltons.
[Mizel, SB, et al., J. Immunol., 131 , 1834
(1983); Schmidt, JA, J.Exp. Med., 160 ,
772 (1984)]. [0007] Recently, it was reported that a cDNA encoding a mouse IL-1 polypeptide was cloned using mouse P388D ₁ cells, and a polypeptide consisting of 156 amino acids with IL-1 activity was successfully produced in Escherichia coli. has been done. [Lomedico,
PT, et al., Nature, 312 , 458 (1984)]. Also,
Regarding human IL-1, human mononuclear cells were used.
A cDNA encoding the IL-1 polypeptide has been cloned [Auron, PE, et al.
Proc. Nat. Acad. Sci. USA, 81 , 7907 (1984)]. The above mouse IL-1 polypeptide and human IL-1
There is no homology between the DNA base sequences encoding each polypeptide, and multiple IL-
The possibility of the existence of 1 was suggested. [0008] [Object of the invention] The present inventors succeeded in isolating a cloned cDNA encoding human IL-1 precursor polypeptide using human HL-60 cells, and In a microorganism transformed with a recombinant plasmid incorporating DNA, we succeeded in producing a new human IL-1 polypeptide whose amino acid sequence is completely different from that of the above-mentioned known human IL-1 polypeptide. [0009] The present invention relates to a DNA encoding a novel human IL-1 polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1. [0010] [Configuration and effects of the invention] Represented by SEQ ID NO: 1
A typical base sequence of DNA encoding the IL-1 polypeptide itself is shown in SEQ ID NO:2. [0011] The method for producing DNA of the present invention will be described below. [0012] DNA having a base sequence encoding a human IL-1 precursor polypeptide can be isolated, for example, by the method shown in Reference Examples below. [0013] After cutting this DNA with an appropriate restriction enzyme, it was synthesized using conventional methods as needed.
By binding with a DNA adapter, a DNA fragment encoding the IL-1 polypeptide represented by SEQ ID NO: 1, that is, a 5' of the DNA base sequence encoding the human IL-1 polypeptide represented by SEQ ID NO: 1. A DNA fragment containing a base sequence having a start codon ATG at the end and a stop codon at the 3' end can be produced. [0014] This is subsequently ligated to an appropriate promoter and Shein-Dalgarno (SD) sequence,
By integrating it into a vector, an expression vector for producing the polypeptide represented by SEQ ID NO: 1 can be obtained. [0015] Examples of promoters include lac ,
Examples include trp, tac , phoS , phoA , PL, and SV40 early promoter. Any vector that can be used to propagate in the host to be transformed can be used. For example, plasmid (pBR322
etc.), phages (λ phage derivatives, etc.), and viruses (SV40, etc.). Also, runaway
Plasmids are also useful. [0016] These expression vectors are transferred to a suitable host, such as Escherichia coli, using the method of Cohen et al. [Proc.Nat.Acad.Sci.USA, 69 , 2110 (1972)].
A transformant is obtained by introducing SEQ ID NO: 1 by culturing the transformant.
It is possible to produce a polypeptide represented by or a polypeptide having methionine added to its N-terminus. The product can be accumulated either within or outside the cytoplasm of the host depending on the promoter used and the construction method of the expression vector. For secretion to the outside of the cytoplasm, for example, the structural gene for alkaline phosphatase (phoA) or the structural gene for phosphate binding protein (phoS) is used, and the region encoding the signal peptide is followed by the sequence represented by SEQ ID NO: 1. encodes a polypeptide that
What is necessary is to construct a gene expression vector to which DNA is linked. [0017] After culturing the transformants thus obtained under appropriate culture conditions depending on each transformant until the desired polypeptide is sufficiently produced, the polypeptide is extracted from the culture. Extract. If the produced polypeptide is accumulated in cell materials, the host cells are destroyed by, for example, lysozyme digestion, freeze-thawing, ultrasonic disruption, French press, etc., and then the extract is collected by centrifugation or filtration. In addition, if it is accumulated in the periplasm, for example, the method of Virski et al. [J. Bacteriol.
127, 595 (1976)]. [0018] The extracted polypeptide is purified according to general protein purification methods (ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, etc.) to substantially eliminate the It can be obtained as a pure product. [0019] Next, human IL represented by SEQ ID NO: 1
The characteristics of the -1 polypeptide (hereinafter sometimes abbreviated as polypeptide 1) will be described in detail below. (1) Physicochemical and chemical properties Molecular weight Molecular weight is determined by polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) (gel concentration
12.5%) determined by electrophoretic analysis. [0021] Equal volume of 4% polypeptide 1 solution
mixed with 0.125M Tris-HCl buffer (PH6.8) containing SDS, 10% 2-mercaptoethanol, 20% glycerol and 0.02% bromophenol blue;
After being left at room temperature for 30 minutes, it was subjected to SDS-polyacrylamide gel electrophoresis analysis. As a running solvent
Electrophoresis was performed at 200 volts for 3 hours using a 25mM Tris-0.2M glycine solution containing 0.1% SDS. After the electrophoresis is finished, stain the protein using Coomassie Brilliant Blue G250 to determine the electrophoresis position of the protein, and separately clean the gel.
Each gel piece cut into mm width was immersed in tissue culture medium RPMI-1640 (Flow Labs.) containing 5% fetal bovine serum to extract the proteins in the gel. IL-1 activity was measured for each extract obtained from each gel piece using LAF activity as described later as an index. the result,
IL-1 activity was observed consistent with a single protein band at a molecular weight of approximately 18,000±500. [0022] Isoelectric point The isoelectric point is Pharmalite (Falmacia, Inc.,
It was measured by isoelectric focusing using a flat gel containing 5% polyacrylamide and a pH range of 4 to 6.5). As a result, the isoelectric point of polypeptide 1 is approximately 5.3±
It was 0.3. [0023] The electrophoresis was carried out at 5 Watts for 3 hours. The protein position was determined by staining with Coomassie brilliant blue G250, and the gel was cut into 2 mm width pieces.
Extract with 20mM Tris-HCl buffer (PH7.8) and IL
-1 activity was measured. Strong IL-1 activity was observed consistent with the protein location determined by staining. [0024] N-terminal partial amino acid sequence The amino acid sequence of the N-terminal portion was determined by the Edman degradation method [Arch.Biochem.Biophys., 22 , 475 (1949)]. [0025] Polypeptide 1 was subjected to a coupling reaction with phenyl isothiocyanate, and then the N-terminal amino acid was cleaved as a thiazolinone derivative, and after further conversion to phenylthiohydantoin amino acid, this was applied to a TSK-gel ODS-120A column (Toyo Soda Co., Ltd.). Identification was achieved by high performance liquid chromatography using a commercially available commercial laboratory. By sequentially repeating this operation, the amino acid sequence of the N-terminal portion was determined. [0026] As a result, the amino acid sequence of the N-terminal portion of polypeptide 1 is Ser-Ser-Pro-Phe...
It was hot. [0027] C-terminal partial amino acid sequence The C-terminal amino acid sequence was determined by an enzymatic method using carboxypeptidase. [0028] Carboxypeptidase A (Sigma Co., Ltd.) and carboxypeptidase Y (Oriental Yeast Co., Ltd.) are applied to polypeptide 1, and over a period of 2 hours from the start of the reaction, the amino acids liberated over time are analyzed using a trace amino acid analysis system. (Shimadzu Corporation). [0029] As a result, the amino acid sequence of the C-terminal portion of polypeptide 1 was Asx-Glx-Ala. Glx means Glu or Gln, and Asx means Asp or Asn. (2) IL-1 activity IL-1 activity was evaluated by biological activity that promotes mitogen-induced mouse thymocyte division, ie, lymphocyte activating factor (LAF) activity. [0031] Approximately 40 μg/ml solution of polypeptide 1 was added to tissue culture medium RPMI-containing 5% fetal bovine serum.
Diluted at 1640. Add 50 μl of each dilution to a 96-well flat bottom plate (Flow Labs.) and
Phytohemagglutinin-P at a concentration of 50μg/ml
(Difco) and 100 μl of a thymocyte (1 x ¹⁰ cells/ml) solution collected from C3H/He mice were added, and the mixture was heated at 37°C in air containing 5% carbon dioxide at a humidity of 90 to 100. % for 3 days. Then,
After adding 1 μCi of ³ H-thymidine and culturing for an additional 18 hours, LAF activity was measured by measuring the amount of ³ H-thymidine incorporated into the cells. The results are shown in Table 1. [0032] [Table 1] ■■■ Turtle shell [0049] ■■■ [0033] The human IL-1 polypeptide represented by SEQ ID NO: 1 can be used as an immunodeficiency therapeutic agent or an antitumor agent. [0034] The following abbreviations are used herein to simplify the description. A Adenine C Cytosine G Guanine T Thymine Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid Cys Cysteine Gln Glytamine Glu Glytamic acid Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine Trp Tryptophan Tyr Tyr Tyrosine Val Valine DNA Deoxyribonucleic acid cDNA Complementary DNA sscDNA Single-stranded cDNA dscDNA Double-stranded cDNA RNA Ribonucleic acid mRNA Messenger RNA Poly(A) mRNA
Polyadenylic acid-containing messenger RNA dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate Oligo(dC) Oligodeoxycytidylic acid Oligo(dG) Oligodeoxyguanylic acid Oligo(dT) Oligodeoxythymidic acid Poly(A) Polyadenylic acid Poly(U) Polyuridylic acid Poly(dA) Polydeoxyadenylic acid Poly(dC) Polydeoxycytidylic acid Poly(dG) Polydeoxyguanylic acid Poly(dT) Polydeoxythymidylic acid ATP Adenosine triphosphate EDTA ethylenediaminetetraacetic acid kb kilobase kbp kilobase pair bp base pair The present invention is not limited to this embodiment. [0036] FIG. 1 is also shown to facilitate understanding of the following embodiments. Example 1 Preparation of a transformant for producing human IL-1 polypeptide An expression plasmid for producing human IL-1 polypeptide (pHLP383) having the amino acid sequence shown in SEQ ID NO: 1 was transformed as shown in FIG. It was constructed. [0038] That is, the cloned cDNA portion was excised from the recombinant plasmid pHL4 obtained in Reference Example 1 using the restriction enzyme PstI, and the restriction enzyme AluI was further applied to the base sequence shown in SEQ ID NO: 3.
A DNA fragment of approximately 533 bp downstream from position 411 was obtained.
Furthermore, this was treated with the restriction enzyme BstNI to isolate a DNA fragment corresponding to the nucleotide sequence 411 to 868 of SEQ ID NO: 3. To this DNA fragment, the following synthetic oligonucleotide shown by SEQ ID NO: 4 was synthesized by a conventional method.Adapter 5' -CGATTATGTCATCACCTTTTAG
[III] 3′-TAATACAGTAGTGGAAAATC and the following synthetic oligonucleotide adapter 5′-AGGCGTGATGA [] 3′-CCGCACTACTTCGA shown in SEQ ID NO: 5 are sequentially linked using _T4 DNA ligase to obtain the oligonucleotide shown in SEQ ID NO: 1. A start codon ATG is added to the 5' end of the base sequence encoding the human IL-1 polypeptide, and a stop codon TGATGA is added to the 5' end of the base sequence encoding the human IL-1 polypeptide.
A DNA fragment with added was obtained. This DNA fragment
It is called HIL-1 fragment. On the other hand, plasmid pCT-1 [Ikehara,
M.et al., Proc.Nat.Acad.Sci.USA, 81 , 5956
(1984)] was treated with restriction enzymes HpaI and AatII to generate trp.
Approximately 380bp of DNA including part of the promoter region
The fragment was cut out, and the following synthetic oligonucleotide shown by SEQ ID NO: 6 synthesized by a conventional method was added to this DNA fragment.
] 3'-TTGATCATGCGTTCAAGTGCATTCCTCCAATAGC was ligated using _T4 DNA ligase. [0040] This bound DNA fragment was prepared previously.
The HIL-1 fragment was ligated using _T4 DNA ligase to obtain a DNA fragment. This DNA fragment is called a promoter HIL-1 fragment. [0041] Separately, plasmid pBR322 was treated with restriction enzymes AvaI and PvuII, and a large DNA fragment (approximately 3.7 kbp) was separated by 0.7% agarose gel electrophoresis. Both ends of this DNA fragment were treated with DNA polymerase I (Klenow fragment) and dGTP.
The ends were made blunt using dATP, dCTP, and dTTP, and both ends were ligated using T ₄ DNA ligase.
This plasmid vector is called pBRS6. Furthermore, restriction enzymes AatII and Hin were added to this pBRS6 vector.
A large DNA fragment (approximately 3.6 kbp) is produced by dIII.
was isolated and purified. [0042] By linking the previously prepared promoter HIL-1 fragment to this DNA fragment using T ₄ DNA ligase, an expression plasmid for producing human IL-1 polypeptide (SEQ ID NO: 1) was constructed. This expression plasmid was named pHLP383. [0043] This expression vector (pHLP383)
was introduced into E. coli HB101 by the method described below to obtain a transformant. That is, E. coli HB101 was added to 5 ml of L-broth (composition: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 1 g of glucose, PH 7.2 per 1000 ml).
and cultured overnight at 37°C. 1 ml of the cell suspension was inoculated into 100 ml of L-broth, and cultured at 37°C until the turbidity (absorbance at 650 nm) reached 0.6.
After standing in ice water for 30 minutes, the bacterial cells were collected by centrifugation, suspended in 50ml of _50mMCaCl2 , and incubated at 0°C.
It was left to stand for 60 minutes. The bacterial cells were then collected by centrifugation and added to ₁₀ ml of 50 mM CaCl containing 20% glycerin.
resuspended in. [0044] Add the above-mentioned expression vector to this suspension.
Add pHLP383 and store this in ice water for 20 minutes at 42
After incubating for 1 minute at °C and 10 minutes at room temperature, LB-broth (composition: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, PH7.5 per 1000 ml) was added, and the mixture was shaken at 37 °C for 60 minutes. A portion of the bacterial cell suspension was plated on an LB agar plate containing 25 μg/ml ampicillin, cultured overnight at 37° C., and then ampicillin-resistant clones were selected to obtain transformants. This transformant was named HB101/pHLP383. Example 2 Production and purification of human IL-1 polypeptide Transformant HB101/pHLP383 obtained in Example 1
was incubated with shaking in LB-broth at 37°C overnight. 10 ml of the cell suspension was added to 1000 ml of improved M9 medium (composition: 1.5% NA ₂ HPO ₄ 12H ₂ O, 0.3% KH ₂ PO ₄ ,
0.05%NACl, 0.1% _NH4Cl , 2mg/l vitamins
_B1 , 0.5% casamino acids, _2mMgSO4 ,
0.1mMCaCl ₂ , 0.5glucose) and cultured at 37°C for 1 hour, then indole-3-acrylic acid was added to a final concentration of 20μg/ml.
After continuing the culture for 24 hours, the bacterial cells were collected by centrifugation. Add 100ml of bacterial cells to 0.1% lysozyme and
Resuspend in 50mM Tris-HCl (PH8.0) buffer containing 30mM NaCl, leave to stand at 0°C for 30 minutes, freeze in a dry ice/ethanol bath and thaw at 37°C, then add 2ml of 10% Polyethyleneimine was added and left to stand. Next, bacterial cell residue was removed by centrifugation to obtain a clear extract. [0046] An equal volume of saturated ammonium sulfate aqueous solution was added to this extract and left to stand, and then a precipitate fraction was collected by centrifugation. Approximately 100 ml of this precipitate fraction
After dissolving in 20mM Tris-HCl buffer (PH8.0) and dialyzing against the same buffer, it was loaded onto a DEAE-Sepharose CL-6B column equilibrated in advance with the same buffer. After thoroughly washing the column with the same buffer, it was eluted with a concentration gradient of 0 to 0.5M NACl. Elution fractions having IL-1 activity were collected, concentrated by ultrafiltration, and then subjected to gel filtration using Sephacryl S-200 to collect fractions having IL-1 activity. Furthermore, a purified product was obtained by repeating the above column chromatography using DEAE-Sepharose and gel filtration using Sephacryl S-200. [0047] Approximately 15 mg of purified human IL-1 polypeptide (SEQ ID NO: 1) was obtained from the bacterial cell extract obtained from 1000 ml of culture. In this preparation, only a single protein band having IL-1 activity was detected by the SDS-polyacrylamide gel electrophoresis analysis described above, and no impurity protein was observed. Reference Example 1 Encoding human IL-1 precursor polypeptide
Cloning of cDNA and determination of base sequence (1) Preparation of human IL-1 mRNA from acute myeloid leukemia cell line (HL-60 cells) [0049] HL-60 cells were placed in a Petri dish (8 cm in diameter) at 1×10 It was seeded at ⁷ pieces/10ml/dish. The culture medium contains RPMI- containing 10% fetal bovine serum.
Using 1640 medium, phorbol was used as a differentiation inducer.
12-myristate-13-acetate and vitamin A
All acids were added to a final concentration of 500 ng/ml. In air containing 5% carbon dioxide at 37℃,
After culturing for 2 days at a humidity of 90-100%, the culture medium and floating cells were removed by suction. Add endotoxin (lipopolysaccharide derived from Escherichia coli) as an inducer to the dates to which the differentiated cells have adhered in RPMI-1640 medium containing 10% fetal bovine serum at a concentration of 10 μg/ml, and cycloheximide as a protein synthesis inhibitor to 1 μg/ml.
10 ml of the medium added to the concentration was added and cultured for an additional 5 hours. After culturing, the culture medium was removed by suction, and the differentiated cells remaining on the dish were treated with 6M containing 0.5% sodium lauroyl sarcosinate, 5mM sodium citrate, and 0.1M 2-mercaptoethanol.
It was dissolved in guanidine thiocyanate solution and homogenized. This homogenate contains 0.1 MEDTA
It was layered on a 5.7M cesium chloride aqueous solution and centrifuged at 26,500 rpm for 20 hours using an ultracentrifuge (RPS27-2 rotor, Hitachi Koki) to obtain a total RNA fraction as a pellet. Add this to 0.35MNACl, 20mMTris
and a small amount of 7M urea solution containing 20mM MEDTA and collected as ethanol precipitate. [0050] This total RNA fraction contains 1mMEDTA.
It was dissolved in 2 ml of 10mM Tris-HCl buffer (PH7.4) (hereinafter referred to as TE solution) and heated at 65°C for 5 minutes. After adding a NaCl solution to a concentration of 0.5M, the column was applied to an oligo(dT) cellulose column equilibrated with a TE solution containing 0.5M NaCl, and the adsorbed poly(A) mRNA was eluted with the TE solution. [0051] The poly(A) mRNA obtained here was used in the following experiment. (2) Synthesis of cDNA Using the poly(A) mRNA obtained in section (1) as a template, the method of Gubler and Hoffman [Gene, 25 , 263
(1983)]. The poly(A) mRNA (6 μg) was dissolved in 6 μl of distilled water,
To this was added 0.6 μl of 100 mM aqueous methylmercury hydroxide solution, and the mixture was left at room temperature for 10 minutes. Then 20 units of
RNA degrading enzyme inhibitor [RNasin (registered trademark),
1.7 μl of a 500 mM 2-mercaptoethanol solution containing [Promega Biotec product] was added. After leaving at room temperature for 5 minutes, add 10mMgCl ₂ ,
1.25mMdGTP, 1.25mMdATP, 1.25mMdTTP,
0.5mMdCTP, 0.17μMα- ^32P -dCTP (specific activity:
Add 32 μl of 50 mM Tris-HCl (PH8.3) buffer containing 750 Ci/mmole), 4 μg oligo(dT) _12-18 , 120 units of avian myeloid leukemia virus reverse transcriptase, and incubate at 42°C for 60 minutes. After that, EDTA was added to stop the reaction. It was extracted with a phenol/chloroform mixture (1:1), ammonium acetate was added to the aqueous layer to a final concentration of 2.5M, and the reaction product (sscDNA-mRNA complex) was precipitated with ethanol. This sscDNA-mRNA complex was dissolved in 100 μl of a reaction buffer having the following composition. [0053] Reaction buffer composition: 5mMgCl ₂ , 10mM (NH ₄ ) ₂ SO ₄ ,
100mM KCl, 0.15mM β-nicotinamide adenine dinucleotide, 40μM dGTP, 40μM
dATP, 40μM dTTP, 40μM dCTP, and 5μg
20mM Tris-HCl (PH7.5) buffer containing bovine serum albumin, 1.25 units E. coli ribonuclease H, 24 units E. coli DNA polymerase I. [0054] The lysate was reacted at 12°C for 60 minutes, 2.5 units of E. coli DNA ligase was added thereto, and further reacted at 22°C for 60 minutes. After stopping the reaction by adding EDTA, extraction was performed with a phenol/chloroform mixture in the same manner as above, and the reaction product (dscDNA) was precipitated with ethanol and collected. (3) Preparation of oligo(dC) tailed cDNA The dscDNA obtained in section (2) was dissolved in 100 μl of a reaction buffer with the following composition, and reacted at 37°C for 30 minutes.
An oligo (dC) tail was added to dscDNA. Reaction buffer composition: 2mM CoCl ₂ , 0.2mM dithiothreitol,
0.1mM α- ³² P-dCTP (specific activity: 1Ci/mmole)
and 100 mM sodium cacodylate (PH7.2) containing 10 units of terminal deoxynucleotidyl transferase. [0056] The reaction was stopped by adding an aqueous EDTA solution, extracted with a phenol/chloroform mixture, and
The oligo(dC) tailed dscDNA was precipitated with ethanol and recovered. Add this to 10mM Tris-HCl (PH7.4) containing 1mMEDTA and 100mM NaCl.
It was dissolved in a buffer solution to a concentration of 2 μg/ml. (4) Preparation of recombinant plasmid Oligo (dG) tailed pBR322 (Bethesda
(manufactured by Res.Labs.Inc.) and oligo (dC) obtained in section (3)
Add tailed dscDNA to 1.5ml of 1mMEDTA and
After dissolving and mixing in 10mM Tris-HCl (PH7.4) buffer containing 100mM NaCl to contain 1.5μg and 0.09μg, respectively, at 65℃ for 10 minutes and at 57℃ for 2 hours.
The mixture was further heated at 45°C for 2 hours for annealing, and a recombinant plasmid solution was prepared. (5) Selection of transformants E. coli χ1776 strain was transformed using the recombinant plasmid solution obtained in section (4). That is, E.
coli
5g, NaCl5g, glucose 1g, PH7.2) in 20ml, 37℃
The cells were cultured until the absorbance (600nm) reached 0.5, and the cells were centrifuged and added to 10mM Tris− containing _50mMCaCl2 .
Washed with 10 ml of HCl buffer (PH7.3). [0059] The collected bacterial cells were suspended in 2 ml of the same buffer solution and allowed to stand at 0°C for 5 minutes. Add and mix 0.1 ml of the above recombinant plasmid solution to 0.2 ml of this suspension, let it stand at 0°C for 15 minutes, and then keep it at 42°C for 2 minutes. −
0.5 ml of broth was added and cultured with shaking for 1 hour.
A portion of this culture solution was taken and spread on an L-broth agar plate supplemented with tetracycline (15 μg/ml) in addition to the above composition, and cultured at 37°C for about 12 hours. Tetracycline-resistant bacteria were selected and cDNA live. I made a rally. (6) Cloning From the cDNA library obtained in section (5), colony hybridization was performed using a cloned cDNA fragment encoding rabbit IL-1 from the recombinant plasmid pRL15 obtained in Reference Example 2 as a probe. Dyization test and hybridization translation test [Maniatis, T., et al.,
“Molecular Cloning” 329 (1980), Cold Spring
A transformant containing a plasmid containing a cDNA encoding human IL-1 polypeptide was selected by the following method. [0061] This recombinant plasmid was named pHL4. (7) Determination of the base sequence of the cloned cDNA The base sequence of the cloned cDNA was determined by the dideoxy method using M13 Phage. M13mp18 and M13mp19 (Pharmacia PL Biochemicals
Amersham International) was used as a cloning vector, and M13 sequencing kit (Amersham International) was used as a cloning vector.
``M13 Cloning and Sequencing Handbook'' (Amersham)
International plc). [0063] The base sequence and the amino acids deduced from the base sequence are as shown in SEQ ID NO: 3, and encodes the human IL-1 precursor polypeptide. [0064] Bases 61 to 63 are the start codon ATG
and bases 874 to 876 are the stop codon TAG
It is. Reference Example 2 Preparation of rabbit IL-1 cDNA (1) Preparation of rabbit IL-1 mRNA Killed Propionibacterium acnes cells were intravenously injected into rabbits at a dose of 100 mg per rabbit, and sacrificed 8 days later. did. Immediately perform open tracheostomy,
Lung lavage was repeated using phosphate buffered saline through a tube inserted into the trachea, and alveolar macrophages were collected. This alveolar macrophage
Suspended in RPMI-1640 medium containing 10% fetal bovine serum, plated on Petri dishes (8 cm in diameter) at 1 x 10 ⁷ cells per plate, and incubated at 37°C in air containing 5% carbon dioxide with humidity. Precultured at 90-100%. 1
After pre-incubation for an hour, endotoxin (lipopolysaccharide derived from E. coli), TPA (phorbol-
12-myristate-13-acetate) and cycloheximide at a final concentration of 10 μg/ml, respectively.
Add and mix to give 500ng/ml and 1μg/ml,
Further culture was continued. [0066] After 4 hours, the culture solution was removed by suction, and reference example 1-
Poly(A) mRNA was obtained according to the method shown in section (1). The poly(A) mRNA obtained here was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6M urea, PH
4), poly(A) mRNA was recovered from the migration position corresponding to the molecular size of 2.6 to 3.7 kb. (2) Preparation of cDNA library Using the poly(A) mRNA obtained in section (1) as a template, according to the method shown in Reference Example 1-(2) to (5),
A cDNA library was created. (3) Cloning Regarding the above cDNA library, rabbit
In order to screen transformants carrying a plasmid containing cDNA encoding IL-1, a colony hybridization test using a 32P-labeled cDNA probe was performed using the method of Hanahan et al. [Gene,
10, 63 (1980)]. Poly(A) mRNA obtained by the method described in item (1) above from alveolar macrophages cultured with endotoxin, TPA, and cycloheximide [see item (1) above] and from alveolar macrophages in which these induction procedures were omitted, respectively.
was synthesized by the method described in Reference Example 1-(2) using as a template,
cDNA labeled with 32P was used as induction plus and induction minus probes, respectively. Through this test, transformants were selected that had a recombinant plasmid containing a base sequence that bound to the induction-plus probe but did not hybridize to the induction-minus probe. [0069] Next, a hybridization translation test was performed on these selected clones using the poly(A) mRNA obtained in section (1) above.
Using this method, clones that strongly hybridized with rabbit IL-1 mRNA were selected. The recombinant plasmid of this clone
It was named pRL15. [0070] SEQ ID NO: 1 Sequence length: 159 Sequence type: Amino acids Topology: Linear Sequence type: Protein sequence ■■■ Tortoise shell [0050] ■■■ [0071] SEQ ID NO: 2 Sequence length :477 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear sequence ■■■ Tortoise shell [0051] ■■■ [0072] Sequence number: 3 Sequence length: 900 Sequence type: Nucleic acid strands Number: Double-stranded Topology: Linear Type of sequence: cDNA to mRNA Type of cell of origin: Human acute myeloid leukemia cell line: HL-60 Sequence characteristics Symbol representing characteristics: CDS Location: 61.. 876 How the feature was determined: E Symbol representing the feature: mat peptide Location: 397..873 How the feature was determined: E Sequence ■■■ Tortoise shell [0052] ■■■ [0073] Sequence number: 4 Sequence length S: 22 Type of sequence: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Other information: Complementary from 3 to 22. Sequence CGATTATGTC ATCACCTTTT AG [0074] Sequence number: 5 Sequence length: 15 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Other information: 2 to 11 complementary to the other strand, up to 12
TCGA exists from 15 to 15. Sequence AGGCGTGATG A [0075] Sequence number: 6 Sequence length: 34 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Other information: 1 to 32 Complementary, other strand has 33
There are GCs from 34 to 34. Array AACTAGTACG CAAGTTCACG
TAAGGAGGTT AT

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the construction steps of the expression vector pHLP383. In addition, [III] and [] in the figure
and [ ] mean the respective synthetic oligonucleotide adapters shown in Example 1

Claims (9)

[Claims] 1. A DNA encoding a human interleukin-1α polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 1. 2. The DNA according to claim 1, wherein the DNA encoding the human interleukin-1α polypeptide has the base sequence shown by SEQ ID NO: 2. 3. A vector capable of proliferating in microorganisms, into which DNA encoding a human interleukin-1α polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is incorporated. 4. The vector according to claim 3, wherein the vector is a gene expression vector. 5. The vector according to claim 3, wherein the vector is an E. coli plasmid. 6. The vector according to claim 3, wherein the vector is pHLP383. 7. A bacterial host transformed with a vector in which DNA encoding a human interleukin-1α polypeptide consisting of the amino acid sequence shown by SEQ ID NO: 1 has been integrated into a proliferative expression vector. 8. The bacterial host according to claim 7, wherein the transformed host is E. coli. [Claim 9] The transformed host is E. coli.
The microorganism host according to claim 7, which is HB101/pHLP383.