JPH0560480B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a human interleukin-1 polypeptide and a method for producing the same. Gary et al.
In the microphage culture supernatant, he discovered a substance that promoted mitogen-induced mouse thymocyte division and named it lymphocyte activating factor (LAF). The name interleukin 1 (hereinafter abbreviated as IL-1) is used. Therefore, such a substance is also treated as interleukin-1 in this specification. IL-1 has the effect of promoting the proliferation and differentiation of T cells and B cells, and also acts on T cells to promote the production of lymphokines, especially interleukin 2 (T cell growth factor), which promotes antibody production and cell growth factor. It is considered to be one of the factors that plays an important role in regulating 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, et al., “ymphokines” vol.6,
p.47 (1982) Academic Press Inc.]. As described above, IL-1 is a biological substance that is involved not only in the immune response but also in the defense and repair of the body, and is expected to be clinically applied as a therapeutic agent for immunodeficiency diseases and an antitumor agent. Previous methods for obtaining IL-1 have mainly focused on microphage, peripheral mononuclear cells, microphage-like established cell lines (e.g. mouse P388D ₁ cells), monocytic or myeloid leukemia cells, etc. in the presence of an appropriate inducer. It is isolated from the culture supernatant. Human IL-1 was isolated and purified from the culture supernatant of human monocytic leukemia cell line U937 cells and human peripheral mononuclear cells, and its molecular weight was 11,300 and 15,000.
It is reported that Dalton. [Mizel, SB, et al., J. Immunol. 131 , 1834
(1983); Schmidt, J.A., J.Exp.Med. 160 , 772
(1984)]. Recently, mouse P388D ₁ cells were used to detect mouse IL-1.
It has been reported that a cDNA encoding a polypeptide was cloned and a polypeptide consisting of 156 amino acids with IL-1 activity was successfully produced in Escherichia coli [Lomedico PTet al.,
Nature, 312 , 458 (1984)]. Regarding human IL-1, cDNA encoding human IL-1 polypeptide has been cloned using human mononuclear cells [Auron, PE et al., Proc. Nat. Acad. Sci.
USA 81 , 7907 (1984)]. No homology was observed between the base sequences of the DNA encoding the mouse IL-1 polypeptide and the human IL-1 polypeptide, suggesting the possibility of the existence of multiple IL-1s. As a result of intensive research to produce human IL-1 polypeptide by applying gene recombination technology, the present inventors cloned cDNA encoding human IL-1 precursor polypeptide using human HL-60 cells.
The above-mentioned known human IL-1 was successfully isolated and transformed with a recombinant plasmid incorporating DNA derived from this cloned DNA.
succeeded in producing a new human IL-1 polypeptide whose amino acid sequence is completely different from that of the polypeptide, and also succeeded in isolating a human IL-1 polypeptide substantially free of impurities,
The invention has been completed. The present invention relates to a human IL-1 polypeptide, that is, a human IL-1 polypeptide having the amino acid sequence shown below, and a method for producing the same. Ser Ser Pro Phe Ser Phe Leu Ser Asn
Val Lys Tyr Asn Phe Met Arg lle lle Lys
Tyr Glu Phe lle Leu Asn Asp Ala Leu
Asn Gln Ser lle lle Arg Ala Asn Asp Gln
Tyr Leu Thr Ala Ala Ala Leu His Asn
Leu Asp Glu Ala Val Lys Phe Asp Met
Gly Ala Tyr Lys Ser Ser Lys Asp Asp
Ala Lys lle Thr Val lle Leu Arg lle Ser
Lys Thr Gln Leu Tyr Val Thr Ala Gln
Asp Glu Asp Gln Pro Val Leu Leu Lys
Glu Met Pro Glu lle Pro Lys Thr lle Thr
Gly Ser Glu Thr Asn Leu Leu Phe Phe
Trp Glu Thr His Gly Thr Lys Asn Tyr
Phe Thr Ser Val Ala His Pro Asn Leu
Phe lle Ala Thr Lys Gln Asp Tyr Trp
Val Cys Leu Ala Gly Gly Pro Pro Ser lle
Thr Asp Phe Gln lle Leu Glu Asn Gln
Ala [] A typical base sequence of the DNA encoding the IL-1 polypeptide [] itself is as shown below. (5′) TCA TCA CCT TTT AGC TTC
CTG AGC AAT GTG AAA TAC AAC
TTT ATG AGG ATC ATC AAA TAC
GAA TTC ATC CTG AAT GAC GCC
CTC AAT CAA AGT ATA ATT CGA
GCC AAT GAT CAG TAC CTC ACG
GCT GCT GCA TTA CAT AAT CTG
GAT GAA GCA GTG AAA TTT GAC
ATG GGT GCT TAT AAG TCA TCA
AAG GAT GAT GCT AAA ATT ACC
GTG ATT CTA AGA ATC TCA AAA
ACT CAA TTG TAT GTG ACT GCC
CAA GAT GAA GAC CAA CCA GTG
CTG CTG AAG GAG ATG CCT GAG
ATA CCC AAA ACC ATC ACA GGT
AGT GAG ACC AAC CTC CTC TTC
TTC TGG GAA ACT CAC GGC ACT
AAG AAC TAT TTC ACA TCA GTT
GCC CAT CCA AAC TTG TTT ATT
GCC ACA AAG CAA GAC TAC TGG
GTG TGC TTG GCA GGG GGG CCA
CCC TCT ATC ACT GAC TTT CAG
ATA CTG GAA AAC CAG GCG (3′)
[] Salts of the polypeptide of the present invention include salts formed with the carboxyl group or amino group of the polypeptide, such as sodium hydroxide, potassium hydroxide, arginine, caffein, procaine, hydrochloric acid, gluconic acid, etc. Examples include salt. The polypeptide of the present invention may exist as an aggregate, and such aggregates are also included in the polypeptide of the present invention. The following encodes the polypeptide of the present invention:
The method for producing DNA and the polypeptide of the present invention will be described. 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. After cutting this DNA with an appropriate restriction enzyme,
By linking this with a DNA adapter synthesized by a conventional method as necessary, a DNA fragment encoding IL-1 polypeptide [], that is, encoding human IL-1 polypeptide []
A DNA fragment containing a base sequence having a start codon ATG at the 5' end and a stop codon at the 3' end of the DNA base sequence can be produced. By subsequently ligating this to an appropriate promoter and a Shein-Dalgarno (SD) sequence and incorporating it into a vector, an expression vector for producing a polypeptide can be obtained. Examples of promoters include lac, trp, tac,
Examples include 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, plasmids (pBR322, etc.), phages (λ phage derivatives, etc.), viruses (SV40, etc.)
can be mentioned. Also useful are runaway plasmids. Transformants are obtained by introducing these expression vectors into a suitable host, such as E. coli, by the method of Cohen et al. [Proc.Nat.Acad.Sci.USA, 69 , 2110 (1972)], and then By culturing the transformant, it is possible to produce polypeptide [ ] or a polypeptide with 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 outside the cytoplasm, construct expression vectors using, for example, the structural gene for alkaline phosphatase (phoA) or the structural gene for phosphate-binding protein (phoS), to which DNA encoding these signal peptides is linked. do it. The transformants thus obtained are cultured under appropriate culture conditions depending on each transformant until the desired polypeptide is sufficiently produced, and then the polypeptide is extracted from the culture. . If the produced polypeptide is accumulated in the cytoplasm, 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, in the case of accumulation in the periplasm, for example, the method of Virski et al. [J. Bacteriol., 127 , 595
(1976)]. The crude polypeptide thus obtained is purified according to general protein purification methods (ultrafiltration, dialysis, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography, etc.). , the polypeptide of interest can be obtained as a substantially pure product. The polypeptide derivative of the present invention is produced by using the polypeptide obtained as described above as a raw material, reacting it with a fatty alcohol to obtain an ester form of the polypeptide, and reacting with a reactive derivative of carboxylic acid to obtain the polypeptide. The N-acyl or O-acyl form of a peptide can be reacted with a primary amine or a secondary amine to form an amide form of the polypeptide, respectively. These reactions are carried out according to conventional methods. Furthermore, an acid or alkali salt of the polypeptide of the present invention can be formed by adding an organic acid, an inorganic acid, or a base to the polypeptide of the present invention using a conventional method. Next, the properties of the polypeptide of the present invention [ ] 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. Polypeptide[] solution in equal volume of 4% SDS,
Contains 10% 2-mercaptoethanol, 20% glycerol and 0.02% bromophenol blue
Mix with 0.125M Tris−HCl buffer (PH6.8) and incubate for 30
After being left at room temperature for a minute, it was subjected to SDS-polyacrylamide gel electrophoresis analysis. 0.1 as a running solvent
Electrophoresis was performed at 200 volts for 3 hours using a 25mM Tris-0.2M glycine solution containing %SDS. After the electrophoresis was completed, the electrophoresis position of the protein was determined by staining with Coomassie Brilliant Blue G250.Separately, the gel was cut out into 2 mm width and each gel piece was placed in tissue culture medium RPMI-1640 (Flow I.abs) containing 5% fetal bovine serum. ) to extract the proteins in the gel. LAF as described below for each extract obtained from each gel piece.
IL-1 activity was measured using the activity as an index. As a result, IL-1 activity was observed, consistent with a single protein band at a molecular weight of approximately 18,000±500. Isoelectric point Isoelectric point is Pharmalite (Falmacia,
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 the polypeptide [] is approximately
It was 5.3±0.3. 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 out into 2 mm width and 20mM
After extraction with Tris-HCl buffer (PH7.8), IL-1 activity was measured. Strong IL-1 activity was observed consistent with the protein location determined by staining. 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)]. The polypeptide [] 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 Kogyo). It was identified by high performance liquid chromatography using By sequentially repeating this operation, the amino acid sequence of the N-terminal portion was determined. As a result, the amino acid sequence of the N-terminal portion of the polypeptide [] was Ser-Ser-Pro-Pbe... C-terminal partial amino acid sequence The C-terminal amino acid sequence was determined by an enzymatic method using carboxypeptidase. Carboxypeptidase A (Sigma) and carboxypeptidase Y (Oriental Yeast Co., Ltd.) are applied to the polypeptide [], and amino acids liberated over time are analyzed over a period of 2 hours after the start of the reaction using a trace amount amino acid analysis system (Shimadzu Corporation). It was quantified using As a result, the amino acid sequence of the C-terminal portion of polypeptide [] was...Asx-Glx-Ala. Glx means Glu or Gln, and Asx means Asp or Asn. (2) IL-1 activity IL-1 activity was evaluated based on the biological activity of promoting mitogen-induced mouse thymocyte division, that is, lymphocyte activating factor (LAF) activity. Approximately 40 μg/ml solution of polypeptide [] at 5%
It was diluted in tissue culture medium RPMI-1640 containing fetal bovine serum. 50μ of each dilution was placed in a 96-well flat-bottomed plate (Flow Labs.), and 50μg of each
ml concentration of phytohemagglutinin-P (Difco)
Add 50 μ of a solution of thymocytes collected from a C3H/He mouse (1×10 ⁷ cells/ml), and heat at 37°C in air containing 5% carbon dioxide at a humidity of 90 to 90°C.
Cultured at 100% for 3 days. Next, 1 μCi of ³ H-thymidine was added, and after further culturing for 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. Table 1 Dilution factor ³ H-thymidine uptake (cpm) 10 ⁵ 32746 10 ⁶ 14498 10 ⁷ 4206 10 ⁸ 2582 Negative control 3210 The polypeptide of the present invention and its derivatives are used as immunodeficiency therapeutic agents or antitumor agents. sell. When formulating the polypeptide and its derivatives according to the present invention, either a solution or a lyophilized product may be used, but a lyophilized product is preferable from the viewpoint of long-term stability. It is also preferable to add excipients and stabilizers. Examples of the stabilizer include albumin, globulin, gelatin, protamine, protamine salt, glucose, galactose, xylose, mannite, glucuronic acid, trehalose,
Dextran, hydroxyethyl starch, nonionic surfactants (polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene hardening Castor oil, polyoxyethylene castor oil, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene polyoxypropylene block polymer, sorbitan fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester), and the like. These preparations can be manufactured according to conventional methods. 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 Glutamine Glu Glutamic acid Gly Glycine His Histidine lle 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) Oligodeoxythymidylic 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 will be described in more detail with reference to Examples and Reference Examples below, but the present invention is limited to these Examples. It's not a thing. In addition, in order to facilitate understanding of the following example,
A diagram was shown. Example 1 Preparation of a transformant for producing human IL-1 polypeptide Human having the amino acid sequence represented by the formula []
An expression plasmid (PHLP383) for producing IL-1 polypeptide was constructed as shown in FIG. That is, cloned from the recombinant plasmid PHL4 obtained in Reference Example 1 using the restriction enzyme Pst.
The cDNA portion was excised and further treated with restriction enzyme Alu to obtain a DNA fragment of approximately 533 bp downstream from position 351 of the base sequence shown in Table 2. Further, restriction enzyme BstN was treated with this DNA fragment, and We isolated a DNA fragment corresponding to nucleotides 351 to 808 of the nucleotide sequence. This DNA fragment was synthesized using the following formula 5'-CGATTATGTCATCACCTTTTAG using a conventional method.
[ ] 3′-TAATACAGTAGTGGAAAATC and oligonucleotide adapters represented by the following formula 5′-AGGCGTGATGA [] 3′-CCGCACTACTTCGA are sequentially linked using T ₄ DNA ligase to encode human IL-1 polypeptide [] Add a start codon ATG to the 5′ end of the base sequence,
Furthermore, a DNA fragment with a stop codon TGATGA added was obtained. This DNA fragment is called a 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 Hpa and Aat to cut out a DNA fragment of approximately 380 bp containing part of the trp promoter region, and this DNA fragment was synthesized using the following formula 5′-
AACTAGTACGCAAGTTCACGTAAGGAGG
TTAT [] 3'-
TTGATCATGCGTTCAAGTGCATTCCTCC
The oligonucleotide adapter denoted by AATAGC
The ligation was performed using _T4 DNA ligase. The previously prepared HIL-1 was added to this bound DNA fragment.
The fragments are joined using _T4 DNA ligase,
A DNA fragment was obtained. Promoter this DNA fragment
It is called HIL-1 fragment. Separately, add the restriction enzyme Ava to plasmid pBR322.
and Pvu to generate large DNA fragments (approximately
3.7kbp) were separated by 0.7% agarose gel electrophoresis. Both ends of this DNA fragment are treated with DNA polymerase (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, this pBRS6 vector was added with the restriction enzyme Aat.
Hind is applied to large DNA fragments (approximately
3.6 kbp) was isolated and purified. The promoter previously adjusted to this DNA fragment
By linking HIL-1 fragments using _T4 DNA ligase, human IL-1 polypeptide []
A production expression plasmid was constructed. This expression plasmid was named pHLP383. 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 mixed with L broth (composition: 10 g of tryptone, 5 g of yeast extract)
g, NaCl 5 g, glucose 1 g; pH 7.2) and cultured overnight at 37°C. 1 ml of the cell suspension was inoculated into 100 ml of L broth, and the turbidity (absorbance
650nm) was 0.6 at 37°C. After standing in ice water for 30 minutes, the bacterial cells were collected by centrifugation.
This was suspended in 50 ml of 50 mM CaCl ₂ and left at 0° C. for 60 minutes. The bacterial cells were then collected by centrifugation and resuspended in 10 _ml of 50 mM CaCl2 containing 20% glycerin. Add the above expression vector to this suspension.
Add pHLP383 and store it in ice water for 20 minutes at 42
After incubating for 1 minute at ℃ and 10 minutes at room temperature, LB broth (composition:
10g, yeast extract 5g and NaCl 10g, PH7.5))
was added and 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. 10ml of the cell suspension was added to 1 part of the modified 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/vitamin _B1 , 0.5
%casamino acids, 2mM _MgSO4 , 0.1mM _CaCl2 ,
0.5% glucose) and cultured at 37℃ for 1 hour.
Next, add indole-3-acrylic acid to a final concentration of 20μ.
g/ml, and after continuing the culture for an additional 24 hours, the bacterial cells were collected by centrifugation. bacterial body
Contains 100ml of 0.1% lysozyme and 30mM NaCl
Resuspend in 50M Tris-HCl (PH8.0) buffer and
After standing at ℃ for 30 minutes, freeze in a dry ice/ethanol bath and thaw at 37℃, then add 2 ml.
10% polyethyleneimine was added and allowed to stand. Next, bacterial cell residue was removed by centrifugation to obtain a clear extract. An equal volume of saturated ammonium sulfate aqueous solution was added to this extract, and the mixture was allowed to stand, and then centrifuged to collect a precipitate fraction. Transfer this precipitate fraction to approximately 100ml of 20mM Tris.
After dissolving in -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,
Elution was performed using a NaCl concentration gradient of 0 to 0.5M. IL
The eluted fractions having IL-1 activity were collected, concentrated by ultrafiltration, and then subjected to gel filtration using Sephacryl S-200 to collect the fractions having IL-1 activity.
Furthermore, column chromatography using the above DEAE-Sepharose and Sephacryl S-
A purified product was obtained by repeating gel filtration with 200 ml. Approximately 15 mg from the bacterial cell extract obtained by culturing 1.
Purified human IL-1 polypeptide [ ] was obtained. 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 cDNA encoding human IL-1 polypeptide
Cloning and base sequence determination (1) Preparation of human IL-1 mRNA from acute myeloid leukemia cell lines (HL-60 cells) HL-60 cells were placed in a petri dish (diameter 8 cm)
The cells were seeded at 1×10 ⁷ cells/10 ml/dish. RPMI-1640 medium containing 10% fetal bovine serum was used as the culture medium, and phorbol-12-myristate-13-acetate and vitamin A acid were added as differentiation inducers to a final concentration of 500 ng/ml. did. At 37℃, in air containing 5% carbon dioxide, humidity 90~
After culturing at 100% for 2 days, the culture solution and floating cells were removed by suction. Add endotoxin (lipopolysaccharide derived from Escherichia coli) as an inducer to the dates to which 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 at a concentration of 1 μg/ml. 10 ml of the medium added to the culture medium 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 dates were dissolved in a 6M guanidylthiocyanate solution containing 0.5% sodium lauroyl sarcosinate, 5mM sodium citrate, and 0.1M 2-mercaptoethanol, and homogenized. . This homogenate containing 0.1M EDTA
Layered on 5.7M cesium chloride aqueous solution and using an ultracentrifuge (RPS27-2 rotor, Hitachi Koki)
Centrifugation was performed at 26,500 rpm for 20 hours to obtain a total RNA fraction as a pellet. This was mixed with 0.35M NaCl and 20mM Tris.
and a small amount of 7M urea solution containing 20mM EDTA and collected as ethanol precipitate. This total RNA fraction was diluted to 10mM containing 1mM EDTA.
Tris-HCl buffer (PH7.4) (hereinafter referred to as TE solution)
The solution was dissolved in 2 ml and heated at 65°C for 5 minutes. to this
After adding NaCl solution to 0.5M, it was applied to an oligo(dT) cellulose column equilibrated with TE solution containing 0.5M NaCl, and the adsorbed poly(A)
mRNA was eluted with TE solution. The poly(A) mRNA obtained here was used in the following experiments. (2) Synthesis of cDNA Using the poly(A) mRNA obtained in section (1) as a template, Gubler and Hoffman's method [Gene 25 , 263 (1983)]
cDNA was synthesized according to . The poly(A) mRNA (6μ
Dissolve g) in 6μ of distilled water, add 0.6μ of
Add 100mM methylmercury hydroxide solution at room temperature.
It was left for 10 minutes. Then, 20 units of RNAase inhibitor (RNasin, a product of Promega Biotec)
of 500mM 2-mercaptoethanol solution containing
1.7μ was added. After leaving it at room temperature for 5 minutes,
Additionally 10mM MgCl ₂ , 1.25mM dGTP, 1.25mM
dATP, 1.25mM dTTP, 0.5mM dCTP,
0.17μM α-〓P-dCTP (specific activity, 750Ci/
mmole), 4 μg oligo(dT) _12-18 , 32 μg containing 120 units of avian myeloid leukemia virus-derived reverse transcriptase
Add 50mM Tris-HCl (PH8.3) buffer,
After reacting at 42°C for 60 minutes, EDTA was added to stop the reaction. Extract with phenol/chloroform mixture (1:1), add ammonium acetate to the aqueous layer to a final concentration of 2.5M, and extract the reaction product (sscDNA-mRNA complex) with ethanol.
was precipitated. This sscDNA-mRNA complex was dissolved in 100μ of a reaction buffer having the following composition. Reaction buffer composition: 5mM MgCl ₂ , 10mM (NH ₄ ) ₂ SO ₄ , 100mM
KCl, 0.15mM β-nicotinamide adenine
dinucleotide, 40μM dGTP, 40μM dATP,
Contains 40 μM dTTP, 40 μM dCTP, and 5 μg bovine serum albumin, 1.25 units E. coli ribonuclease H, 24 units E. coli DNA polymerase I
20mM Tris-HCl (PH7.5) buffer. The lysate was reacted at 12°C for 60 minutes, and then 2.5
Add one unit of E. coli DNA ligase and further incubate at 22°C.
was allowed to react 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 Dissolve the dscDNA obtained in section (2) in 100μ of reaction buffer with the following composition, react at 37℃ for 30 minutes,
An oligo (dC) tail was added to dscDNA. Reaction buffer composition: 2mM CoCl ₂ , 0.2mM dithiothreitol,
0.1mM α- ^32P -dCTP (specific activity 1Ci/mmole)
and 100 mM sodium cacodylate (PH7.2) containing 10 units of terminal deoxynucleotidyl transferase. 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. This was mixed with 1mM EDTA and
10mM Tris−HCl (PH7.4) containing 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 the oligo (dC) obtained in section (3)
Tailed dscDNA was added to 1.5ml of 1mM EDTA and
10mM Tris−HCl (PH7.4) containing 100mM NaCl
After dissolving and mixing in buffer solution to contain 1.5 μg and 0.09 μg, respectively, the mixture was incubated at 65°C for 10 minutes and at 57°C for 2 hours.
The mixture was further heated at 45° C. for 2 hours to perform annealing, and a recombinant plasmid solution was prepared. (5) Selection of transformants E. coli x1776 strain was transformed using the recombinant plasmid solution obtained in section (4). That is, E.
colix1776 strain at 100 μg/ml of diaminopimelic acid.
and L-broth supplemented with 40 μg/ml of thymidine (composition: 10 g of tryptone, 5 g of yeast extract,
5g NaCl, 1g glucose, PH7.2) in 20ml, 37℃
The cells were cultured until the absorbance (600nm) was 0.5, centrifuged, and added to 10mM Tris containing 50mM _CaCl2 .
- Washed with 10 ml of HCl buffer (PH7.3). The collected bacterial cells were suspended in 2 ml of the same buffer and heated to 0°C.
It was left standing for 5 minutes. Add and mix 0.1 ml of the above recombinant plasmid solution to 0.2 ml of this suspension, and
After standing at 42℃ for 15 minutes and further holding at 42℃ for 2 minutes,
L-broth with the same composition as used in the above culture
0.5 ml was added and cultured with shaking for 1 hour. A portion of this culture solution was 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 a cDNA library was created. was created. (6) Cloning Colony hybridization using the cloned cDNA fragment encoding rabbit IL-1 from the recombinant plasmid pRL15 obtained in Reference Example 2 from the cDNA library obtained in Section (5) as a probe. Tests and Hybridization Translation Tests [Maniatis, T., et al.,
“Molecula Cloning” 329 (1980) Cold Spring
Transformants containing a plasmid containing a cDNA encoding human IL-1 polypeptide were selected using the plasmid (Harbour Lab.). This recombinant plasmid was named PHL4. (7) Determination of base sequence of cloned cDNA The base sequence of cloned cDNA was determined by the dideoxy method using M13 Phage. M 13mp18
and M13mp19 (Pharmacia P-L
Biochemicals) was used as a cloning vector, and an M13 sequencing kit (Amersham Biochemicals) was used as a cloning vector.
International plc) using “M13 Cloning and Sequencing Handbook”
(manufactured by Amersham International plc). The nucleotide sequence and the amino acids deduced from the nucleotide sequence are as shown in Table 2 below.
It encodes an IL-1 precursor polypeptide. The first to third bases are the start codon ATG,
Bases 814 to 816 are the stop codon TAG. [Table] [Table] [Table]
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. Immediately perform a thoracotomy and 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 at a humidity of 90°C. Preincubated at ~100%. After 1 hour of preincubation, endotoxin (lipopolysaccharide derived from Escherichia coli), TPA (phorbol-12
- myristate-13-acetate) and cycloheximide, each at a final concentration of 10 μg/ml, 500 n
Add and mix to give a concentration of g/ml and 1 μg/ml,
Further culture was continued. After 4 hours, the culture solution was removed by suction, and poly(A) mRNA was obtained from the macrophages remaining on the dish according to the method shown in Reference Example 1-(1). The poly(A) mRNA obtained here was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6M urea, PH4).
Poly(A) mRNA was recovered from a migration position corresponding to a 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 methods shown in Reference Example 1-(2) to (5),
A cDNA library was created. (3) Cloning For the above cDNA library, rabbit
To screen for transformants carrying a plasmid containing cDNA encoding IL-1, a colony hybridization test using a P-labeled cDNA probe was performed using the method of Hanahan et al. [Gene,
1063 (1980)]. endotoxin,
Culture with TPA and cycloheximide [(1) above
Using the poly(A) mRNA obtained from the alveolar macrophages obtained by the method described in section (1) above as templates, and from the alveolar macrophages obtained by omitting these induction procedures, as templates, the method described in Reference Example 1-(2) was used. Synthesized and ³² P-labeled cDNAs were 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. Next, a hybridization/translation test was conducted on these selected clones using the poly(A) mRNA obtained in section (1) above, and clones that strongly hybridized with rabbit IL-1 mRNA were selected. The recombinant plasmid of this clone
It was named pRL15.

[Brief explanation of drawings]

FIG. 1 shows the construction steps of the expression vector pHLP383. In addition, the formulas [ ], [ ], and [ ] in the figure mean the respective oligonucleotide adapters shown in Example 1.

Claims (1)

[Scope of Claims] 1. A human interleukin-1α polypeptide consisting of an amino acid sequence represented by the following formula []. Ser Ser Pro Phe Ser Phe Leu Ser Asn
Val Lys Tyr Asn Phe Met Arg lle lle Lys
Tyr Glu Phe lle Leu Asn Asp Ala Leu
Asn Gln Ser lle lle Arg Ala Asn Asp Gln
Tyr Leu Thr Ala Ala Ala Leu His Asn
Leu Asp Glu Ala Val Lys Phe Asp Met
Gly Ala Tyr Lys Ser Ser Lys Asp Asp
Ala Lys lle Thr Val lle Leu Arg lle Ser
Lys Thr Gln Leu Tyr Val Thr Ala Gln
Asp Glu Asp Gln Pro Val Leu Leu Lys
Glu Met Pro Glu lle Pro Lys Thr lle Thr
Gly Ser Glu Thr Asn Leu Leu Phe Phe
Trp Glu Thr His Gly Thr Lys Asn Tyr
Phe Thr Ser Val Ala His Pro Asn Leu
Phe lle Ala Thr Lys Gln Asp Tyr Trp
Val Cys Leu Ala Gly Gly Pro Pro Ser lle
Thr Asp Phe Gln lle Leu Glu Asn Gln
Ala [] 2 The DNA encoding the human interleukin-1α polypeptide consisting of the amino acid sequence shown by the following formula [] is inserted into a gene expression vector that can be propagated in microorganisms, a gene expression vector is constructed, and this is used to infect microorganisms. A method for producing a polypeptide, which comprises transforming the microorganism, culturing the microbial transformant, and producing a polypeptide having human interleukin-1 activity. Ser Ser Pro Phe Ser Phe Leu Ser Asn
Val Lys Tyr Asn Phe Met Arg lle lle Lys
Tyr Glu Phe lle Leu Asn Asp Ala Leu
Asn Gln Ser lle lle Arg Ala Asn Asp Gln
Tyr Leu Thr Ala Ala Ala Leu His Asn
Leu Asp Glu Ala Val Lys Phe Asp Met
Gly Ala Tyr Lys Ser Ser Lys Asp Asp
Ala Lys lle Thr Val lle Leu Arg lle Ser
Lys Thr Gln Leu Tyr Val Thr Ala Gln
Asp Glu Asp Gln Pro Val Leu Leu Lys
Glu Met Pro Glu lle Pro Lys Thr lle Thr
Gly Ser Glu Thr Asn Leu Leu Phe Phe
Trp Glu Thr His Gly Thr Lys Asn Tyr
Phe Thr Ser Val Ala His Pro Asn Leu
Phe lle Ala Thr Lys Gln Asp Tyr Trp
Val Cys Leu Ala Gly Gly Pro Pro Ser lle
Thr Asp Phe Gln lle Leu Glu Asn Gln
The method for producing a polypeptide according to claim 2, wherein the DNA encoding the Ala [] 3 human interleukin-1α polypeptide has a base sequence represented by the following formula []. (5′) TCA TCA CCT TTT AGC TTC
CTG AGC AAT GTG AAA TAC AAC
TTT ATG AGG ATC ATC AAA TAC
GAA TTC ATC CTG AAT GAC GCC
CTC AAT CAA AGT ATA ATT CGA
GCC AAT GAT CAG TAC CTC ACG
GCT GCT GCA TTA CAT AAT CTG
GAT GAA GCA GTG AAA TTT GAC
ATG GGT GCT TAT AAG TCA TCA
AAG GAT GAT GCT AAA ATT ACC
GTG ATT CTA AGA ATC TCA AAA
ACT CAA TTG TAT GTG ACT GCC
CAA GAT GAA GAC CAA CCA GTG
CTG CTG AAG GAG ATG CCT GAG
ATA CCC AAA ACC ATC ACA GGT
AGT GAG ACC AAC CTC CTC TTC
TTC TGG GAA ACT CAC GGC ACT
AAG AAC TAT TTC ACA TCA GTT
GCC CAT CCA AAC TTG TTT ATT
GCC ACA AAG CAA GAC TAC TGG
GTG TGC TTG GCA GGG GGG CCA
CCC TCT ATC ACT GAC TTT CAG
ATA CTG GAA AAC CAG GCG (3') [] 4 The method for producing a polypeptide according to claim 2, wherein the constructed expression vector is pHLP383.