JPS6261585A - Polypeptide exhibiting interleukin 1 activity and dna coding said polypeptide - Google Patents

Abstract

PURPOSE:The titled polypeptide, having an amino acid sequence expressed by a specific formula, obtained by removing amino acid on the N terminal and C terminal sides respectively within a specific number range and suitable for use as a remedy for immunological insufficiency or antitumor agent. CONSTITUTION:A polypeptide exhibiting interleukin 1 (IL-1) activity i.e. polypeptide having an amino acid sequence expressed by formula I, that is amino acids are deficient as follows; 0-18, particularly 0-16 amino acids on the N terminal side and 1-10, particularly 1-7 amino acids on the C terminal side are respectively removed in the above-mentioned amino acid sequence. Derivatives of the above-mentioned polypeptide mean derivatives formed by utilizing side-chain functional groups on the above-mentioned polypeptide chain, amino group at the N terminal or carboxyl group at the C terminal. Examples thereof include esters of carboxyl groups with an aliphatic alcohol, etc.

Description

[Detailed description of the invention]

The present invention relates to DNA encoding a polypeptide exhibiting interleukin/1 activity, 1' ribeptide exhibiting interleuki/l activity, derivatives of the polypeptide thereof, and methods for producing them. Gerry et al. discovered a substance in cultured ln of human macrophages that promotes mitogen-induced mouse thymocyte division, and added this to lymphocyte activating factor (lymp).
hocyte activating factor. However, since 1979, the name Interleukin I (hereinafter abbreviated as IL-1) has been used. Therefore, in this specification, such a substance is also treated as interleukin I. IL-1 promotes the proliferation and differentiation of T cells and B cells, and also acts on T cells and has the effect of promoting the production of hokines, especially interleukin 2 (T cell growth factor), and has the effect of promoting the production of interleukin 2 (T cell growth factor). It is considered to be one of the factors that plays an important role in the production and regulation of cellular immunity [5 Taruch, M.
J., et al., J., IIIunol. 130.2191 (+983)]. In addition, it has been reported that it has the effect of promoting the production of prostaglansh E and collagenase, promoting the proliferation of fibroblasts, and enhancing the NK (natural killer) cell activation effect of interleukin 72 and interferon [Simon, P.
, L., et al., “Lymphokines
”vol, G, P, 47 (1982) ^cadem
+c Press lnc,]. As described above, IL-1 is a biological substance that is involved not only in the immune response but also in the defense of the living body and its repair, and is expected to find clinical application as a therapeutic agent for immunodeficiency diseases and an anti-ulcer agent. Until now, methods for obtaining IL-1 have mainly involved cultivating macrophages, peripheral mononuclear 4ff cells, or macrophage-like established cell lines (e.g., mouse P388D I cells), monocytic or myeloid leukemia cells, etc. in the presence of rapid inducers. Culture it with 1? (It is isolated from the inside.) IL-1 is isolated from the human monocytic leukemia cell line U9.
37 cells and human peripheral IIi nuclear scissors in culture, and their molecular weights were l I , 300 and 15
．． 000 Dalton [Mizel
,S.B., ,et al., ,J,Immunol,I.
31. +834 (+983); Schmidt, J, ^
,,J,Exp,Med, 100.7?2 (1984
>]. Recently, mouse P388D r cells were used to develop mouse IL-1
The cDNA encoding the polypeptide was cloned and showed IL-1 activity! It has been reported that a polypeptide consisting of 56 amino acid residues was successfully produced in the large intestine [Lo edico P, T, et al.
,, Nature, 312,458 (+984)
], also human IL-1
When it flashes, it's a human! cDN encoding human IL-1 polypeptide from fL karyocytes or human macrophages
It has been reported that A was cloned and successfully expressed.

[＾uron, P, E, et at,. r'roc, Nat, ^cad, sci, Us^81.
7907 (1984), March, C., Jet al.
,, Nature 315,041 (+985)] - The base sequences of the DNAs encoding the above-mentioned IL-1 polypeptides are not identical, indicating that a plurality of IL-1s exist. On the other hand, the present inventors used human IIL-GO cells to
We successfully isolated a cloned cDNA encoding a human H,-1ii'f precursor polypeptide consisting of one amino acid residue, and transformed it with a recombinant plasmid incorporating DNA derived from this cloned cDNA. In the microorganism in which the C-terminal side of the IL-1 precursor polypeptide
We succeeded in producing and isolating a polypeptide consisting of 9 amino acid residues, and have filed a patent application (Japanese Patent Application No. 112474/1980). The polypeptide, which is encoded as a precursor consisting of 271 amino acids on the gene, and is involved in the expression of its biological activities, such as the effect on T cell proliferation (LAF activity), is 271 a It was found that this is a polypeptide consisting of 159 amino acid residues on the C-terminal side of a precursor consisting of basic amino acids. As a result of further research, we found that the N-terminal amino acids of this polypeptide consisting of 15OIiII and amino acids are not necessarily essential for the expression of LAF activity, especially the N-terminal 1 amino acid.
Polypeptide with 6 amino acid residues deleted has strong LA
It was found that it exhibits F activity (Patent application 17149/1980)
(See No. 3). As a result of further research based on the above findings, the present inventors found that
The present invention, detailed below, was completed based on the new structural finding that even when the C-terminal amino acid of the above polypeptide is deleted, the polypeptide still retains LAF activity. It is something. The present invention provides a DNA encoding a polypeptide exhibiting IL-1 activity, that is, an amino acid sequence represented by the following formula [1], in which 0 to 18 amino acids on the N-terminal fi side are removed, and the C-terminal Side 77) A novel DNA having or containing a base sequence corresponding to an amino acid sequence from which 1 to 10 amino acids have been removed, and a method for producing the same. Ser Ser Pro Phe Ser Phe L
eu Ser Asn ValLye Tyr Asn
Phe Met Arg lie Ile Lys
TyrGlu Phe Ile Leu Asn As
1) Alm Leu Asn G1n5er IIs
lie Arg Ala Asn Asp Gln
Tyr LeuThr Alm Ala Ala Le
u His Asn Lau Asp G+uAla
Val Lye Phe AsP Met Gly A
la Tyr LysSer Ser Lys
Asp Asp Ala Lys Ile
Thr Valle Leu Arg li
e Sar Lye Thr Gln Le
uTyrVatThrAlaGlnALPGluA
spGinProVatLeuLeuLys
Glu Met Pro Glu IIs
Pro LyeThr IIs Thr G
ly Ser Glu Thr Asn L
eu LauPhe Phe TrP Glu
Thr Hls Gly Thr Lys
AmnTyr Phe Thr Ser
Val ALa Hls Pro Asn.
LeuPha IIs Ala Thr L
ys Gin Asp Tyr Trp Va
tCy* Leu Ala Gly Gly
Pro Pro Ser lie ThrA
SP Phe Gln lie Leu G
lu Asn Gln AIJL [I] The DNA according to the present invention has a base sequence represented by the following formula [11], in which 0 to 18 codes/ on the 5' end side are removed, and the 3' end An example is a DNA having or containing a base sequence from which 1 to 10 codons have been removed. (S')TCA TCA CCT TTT AGCTT
CCTG AGCAAT GTGAAA TACAAC
TTT ATG AGG ATCATCAAA TAC
GAA TTCATCCTG AAT GACGCCC
TCAAT CAAAGT ATA ATT CGA
GCCAAT GAT CAG TACCTCACG
GCT GCT GCA TTA CAT AAT C
TG CAT GAAGCA GTG AAA
TTT GACATG GGT GCT TA
T AAGTCA TCA AAG GAT
GAT GCT AAA ATT ACCG
TGATT CTA AGA ATCTCA
AAA ACT CAA TTG TATGT
G ACT GCCCAA GAT GAA
GACCAA CCA GTGCTG CTG
AAG GAG ATG CCT GAG
ATA CCCAAAACCCATCACAG
GT AGT GAG ACCAACCTCCT
CTTCTTCTGG GAA ACT CAC
GGCACT AAG AACTAT TTCA
CA TCA GTT GCCCAT CCA
AACTTTGTTT ATT GCCACA
AAG CAA GACTACTGG GTG
TGCTTG GCA GGG GGG CC
A CCCTCT ATCACTGACTTT
CAG ATA CTG GAA AACCA
G GCG(3')[I[] Preferred DNA is the amino acid sequence represented by the above formula CI], in which 0 to 16 amino acids on the N-terminal side are removed, and 1 to 7 amino acids on the C-terminal side are removed. DNA that has or contains a base sequence corresponding to the amino acid sequence from which the amino acid of Examples include DNA that exists or includes a base sequence in which 1 to 7 codons have been removed. Particularly preferred DNA
Examples include a DNA having or including a base sequence in which 0 to 18 codons at the 3' end of the base sequence ν11 shown in the above formula [11] have been removed, or a degeneracy thereof. The present invention also includes: In a polypeptide exhibiting IL-1 activity, that is, a tripeptide having the amino acid sequence represented by the above formula [11], 0 to 18 amino acids on the N-terminal side are
lvI is removed and its C-terminal Q (1111-1~
Polypeptides with 10 amino acids removed and their derivatives and their! I was inspired by the 2 construction method. Preferred polypeptides include polypeptides in which 0 to 16 amino acids at the N-terminus and 1 to 7 7mi/V at the C-terminus are removed from the amino acid sequence represented by formula [I] above, particularly Preferred examples include polypeptides in which 0 to 18 amino acids on the C-terminal side of the amino acid sequence represented by the above formula [11] are removed. The derivative of the polypeptide mentioned above means a derivative formed using a side chain functional group on the chain of the polypeptide, an N-terminal amine 7 group, or a C-terminal carboxyl group, for example, a carboxyl group and an aliphatic group. Esters with alcohol, No.
Alternatively, acid amides with secondary amines, N-acyl derivatives of amide 7 groups or O-acyl derivatives of hydroxyl groups, and salts formed with carboxyl groups or amino groups of the polypeptide, such as sodium hydroxide. , potassium hydroxide, arginine, caffeine, procaine, hydrochloric acid, gluconic acid and the like. 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 preparation and manufacturing method of the DNA and polypeptide of the present invention will be described below. h) DNA having a base sequence encoding an IL-1 precursor polypeptide can be isolated, for example, by the method shown in Reference Example 1 below. After cutting this DNA with an appropriate restriction enzyme, this DNA is ligated with a DNA adapter synthesized by a conventional method as necessary, thereby creating a start codon at the 5' end of the DNA base sequence encoding the polypeptide of the present invention. DNA containing a base sequence that hits a stop codon at the 13' end of ATG llr
pieces can be made. A suitable promoter and Shine War Darga 7 (
By subsequently ligating to the SD) sequence and incorporating it into a vector, an expression vector for producing the polypeptide of the present invention can be obtained. For example, Iac, trp
, tac. Examples include phoS, pho^, pt, SV40 early promoter, and the like. Any vector that can be used to propagate in the host to be transformed can be used. For example, plasmids (p13R322 etc.), phages (
λ phage derivatives, etc.), and viruses (SV40, etc.). Runaway plasmids are also useful. These expression vectors are transferred to a suitable host, such as E. coli, using the method of Cohen et al. [Proc. Nat, 8 cad, sci, Us^, 69.2110 (
+972>1 to obtain a transformant, and then by culturing the transformant, the polypeptide of the present invention or a polypeptide having methionine added to its N-terminus can be produced. The product depends on the promoter used and the construction method of the expression vector.
It can be accumulated in the cytoplasm or nematode of the host. To secrete it outside the cytoplasm, for example, the alkaline phosphatase ma gene (pho^) or the phosphate binding protein structural gene (phoS) are used, and the polypeptide is hooded following the region encoding these signal peptides. The expression vector ligated with the DNA for
do it. The transformants obtained in this manner 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 medium. Extract. When the PM-generated 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, when it is accumulated in the veriplasm, for example, the method of Wilsky et al. [J, Bacter+o1. ．． 127,
595 (1970)]. 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 desired polypeptide can be obtained in substantially pure form. The mIJs form of the polypeptide of the present invention is obtained by using the polypeptide obtained as described above as a raw material, and by treating the polypeptide with an aliphatic alcohol to form an ester form of the polypeptide, and by treating the polypeptide with a reactive derivative of carboxylic acid. N of the polypeptide
The -acyl or 0-acyl form can be reacted with a primary amine or fs2 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.The polypeptide of the present invention and its Is4 form can be used as a therapeutic agent for immunodeficiency or an anti-inflammatory agent. Pharmaceutical preparations of the polypeptide of the present invention and its derivatives include liquid preparations, lyophilized preparations, etc. When formulating, it is recommended to add a stabilizer. Preferred stabilizers include, for example, albumin, globulin, gelatin, protamine, protamine salt, glucose, galactose, xylose, manifol, curculo/fi, etc.
) Herose, dextran, hydroxyethyl starch/, nonionic surfactants (polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether lukyl phenyl ether, polyoxyethylene rubitan fatty acid ester, polyoxyethylene glycerin/fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, polyoxyethylene polyoxypropylene/alkyl ether, polyoxyethylene polyoxypropylene block polymer, Nrvita 7 fatty acid ester, sucrose fatty acid ester, glycerin fatty acid ester) etc. Formulation is carried out by adding appropriate excipients in addition to the above-mentioned stabilizers, and according to conventional methods. At Honkawa SA+IT! Il! The following abbreviations are used to simplify the description. A Adeni/ C Cytosine G Guanine T Thymine Ala Alanine Arg Arginine Asn Asparagi/ As p Aspartic acid Cys Cystine Gln Glutami/ Glu Glutamic acid Gly Glycine His Histidine 11e Inleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Se t Seri/ Thr Threoni/ Trp ) Liputra1 Tyr Tyr Tyr Tyr Val Valine DNA Deoxyliponucleic acid c DNA Complementary DNA s sc DNA Single stranded cDNA Ad s c D
NA double gl cDNA RNA liponucleic acid mRNA messenger RNA poly(^)mRNA polyadenylic acid-containing messenger RNA AdA
TI' deokine adenosine triphosphate dCTP
Deokinshitiji/triphosphate dGTr'
Deoxyguanosine triphosphate dTTP Deoxythymidine phosphate oligo (dC) Oligodeoxycytidylate oligo (dG) Oligodeoxyguanylate oligo (dT) Oligodeoxythymidylate poly(A) Polyadenylate poly(U) Polyuridylate fS (dA), t! Lysoxyadenylic acid'I
'S(dC) Polydeoxythymylic acid poly(dG
) Polydeokyanilic acid poly(dT)
Polydeoxythymidylic acid A T P Adenosine triphosphate EDTA Ethylenediaminetetraacetic acid kb Kilo base kbp Kilo base pair bp Base pair The present invention will be explained in more detail by giving examples and reference examples below, but the present invention The present invention is not limited to this embodiment. Further, FIG. 1 is shown to facilitate understanding of the following embodiment. (Left below) Example 1 Polypeptide showing 11,-1 activity (L-1(+5
Amino acid numbers 113 to 267 in the production if table of 5-C))! Polypeptide (IL-1) having an amino acid sequence consisting of 55 residues and 1λ
A production expression plasmid (pHLP373) (abbreviated as +55-C) was constructed as shown in FIG. That is, cloning c from the recombinant plasmid pHL4 obtained in Reference Example 1-(61) using the restriction enzyme Pstl.
The DNA portion was excised and further treated with restriction enzyme ^lul to obtain a DNA fragment of about 533 bp downstream from position 351 of the base sequence shown in Table 1. The third base sequence in Table 1
The DNA fragment corresponding to No. 51 to No. 768 was isolated using a single DNA fragment.
did. To this DNA fragment, oligonucleotide adapters synthesized by the following formula % formula % [ and the following formula] were sequentially added to T4.
By linking using DNA ligase, the polypeptide (IL-
A start codon ATG is added upstream of the nucleotide sequence encoding 1 (155-C)), and a stop codon TG is added downstream.
A DNA fragment to which ATGA was added was obtained. This DNA fragment is called the old L-155 fragment. On the other hand, plasmid pCT-1 [Ikehara, M, e
tif,. Proc, Nat, Acad, Sci, USA 81,
5956 (+984)] -■ and Aatl
Approximately 3 ml containing part of the trp promoter region
Cut out an 80bp DNA fragment, and add to this DNA fragment,
The oligonucleotide adapter represented by the following formula 5 [%
The ligation was performed using A ligase. The old L-155 fragment, previously prepared using rA, was ligated to this DNA fragment using T4 DNA ligase to obtain a DNA fragment. This DNA fragment is attached to promoter +11L-+55
It's called a fragment. Separately, add restriction enzyme ^vaI to plasmid pIIR322.
A large NA fragment (approximately 3.7k
bp) were separated by 0.7% agarose gel electrophoresis. Both ends of this DNA fragment were treated with DNA polymerase Natsu (Cre/
-7lagmet7) and dGTP, dATP, dcT
I). Make blunt ends using dTTP, and connect both ends with T4 DNA.
The conjugation was performed using ligase. This plasmid vector is called pflR58, and this p[1R96 vector is further treated with restriction enzyme ^at[I and old ndll+,
A large NA fragment (approximately 3.6 kbp) was isolated and purified. This DNA fragment was coated with ProStorter 111L-
An expression plasmid for IL-1 (+55-C) production was constructed by ligating the 155 fragments using T4 DNA ligase. This expression plasmid is p
It was named HLP373. This expression plasmid (pHLP373) was introduced into E. coli IIIBIOI by the method described below to obtain transformants. That is, if E.coli
aC15g, grape*1g: I) H7,2
) and cultured overnight at 37°C. One part of the bacterial cell suspension was inoculated into 1001 broth, and the turbidity (
The cells were cultured at 37°C until the absorbance [f050n■) reached 0.6. After standing in ice water for 30 minutes, the bacterial cells were collected by centrifugation, and suspended in 50mMCa of 501 (suspended in
The mixture was allowed to stand at ℃ for several minutes. The bacterial cells were then collected by centrifugation and resuspended in 50 mM CaCla IOm+ containing 20% glycerin. The above expression vector pHLP373 was added to this suspension, and the mixture was incubated in ice water for 20 minutes and at 42°C for 1 minute. After inking and baking at room temperature for 10 minutes, LB broth (composition: 10 g of tryptone, 5 g of yeast extract, and 110 g of NaC, pH 7.5 per 11) was added and incubated at 37°C for 6 hours.
Shake for 0 minutes. A portion of the bacterial cell suspension was added at 25 gg/■
1. Sow on LI 3t% flat plate containing ampicillin and grow at 37°C.
After incubating overnight), ampicillin-resistant clone 7 was selected to obtain transformants. This transformant was cultured overnight at 37° C. in L.B broth with shaking. 101 of the Δ body suspension was added to 11 modified M9 medium (
Composition: 1.5% NazHPO4・l2HtO, 0.3%
KHzPOa. 0.05% NaCl, 0.1% NHaCl, 2B/l
Vitamin' at 10.5% Kazami/acid, 2mM
Mg5Oa, 0.1mM cac+2. 0.5% glucose) and incubated at 37°C for 1 hour, then indole-3-acrylic acid was added to 20μ
g/l, and after continuing the culture for an additional 24 hours, the bacterial cells were collected by centrifugation. 0 of 1001 bacterial cells
．． 50m containing 1% lysozyme and 30mM NaCI
MTris-HCI (+)H8,0) was resuspended in Ta street solution, allowed to stand at 0°C for 30 minutes, and then repeatedly frozen in a dry ice/ethanol bath and thawed at 37°C.
Cell residue was removed by centrifugation to obtain a clear extract. When the IL-1 activity of this extract was measured by the method below, it was found that 27,109 C
pl publication - thymidine incorporation was observed. This extract contains equal volumes of 4% SD3, 10% 2-mercaptoethanol, 20% glycerol and 0.02% bromophenol blue. +25M Tris
-HCI buffer (pHOJ), left at room temperature for 30 minutes, and subjected to 5DS-polyacrylamide gel electrophoresis analysis. 25mM Tris-0.2M glycyol/solution containing 0.1% SDS was used as the ItI medium for electrophoresis, and electrophoresis was performed at 200 volts for 3 hours. After the electrophoresis is completed, the electrophoresis position of the protein is determined by staining with Coomassie Brilliant Blue 6250.Separately, the gel is cut out into 21-width pieces and each gel piece is placed in tissue culture medium JIll containing 5% tallow serum.
The gel was immersed in RPMI-11340 (Flow Labs) to extract the protein in the gel. IL-1 activity was measured for each extract obtained from each gel piece. Phosphorylase b (
MW94,000), bovine serum albumin (MW
137.000), Opoalpmin (MW 43.0
00), Carbonic anhydrase;i (MW 30,000)
, soybean trypsin inhibitor (MW 20,100
) and α-lactalbumi 7 (MW 14,400) were used. As a result, the molecular weight was about I? , strong IL-1 activity was observed at the position corresponding to 50 (l). This result indicates that the target polypeptide was produced by culturing E. coli transformed with the expression plasmid p) ILP373. This shows that. The IL-1 activity was evaluated by the biological activity that promotes the division of mouse thymocytes by mitogen, that is, the lymphocyte activating factor (LAF'J activity). Medium RPM
Diluted with I-1f340. 50μ of each dilution! 9
Place in a 6-well flat-bottomed plate (Flow Labs, ).
50μ of phytohemagglutinin-P (DHco) at a concentration of 25μg/■1 for each! and further C311
/lle? Thoracic cyst collected from a mouse (I
07 pieces/1) 100μ of solution! was added and cultured for 2 days at 37°C in air containing 5% carbon dioxide with a humidity of 90 to 100%.Next, 1 μCi of Morchimiji was added and an additional 18
LAF activity was measured by measuring the amount of thymidine incorporated into the cells after culturing for a period of time. Example 2 Polypeptide exhibiting IL-1 activity (IL-1(+52-
C) Production of polypeptide (IL-1 (15
2-C")] Production expression plasmid (p
HL+'4158) according to the method described in Example 1.
I built M. However, by using a synthetic adapter represented by the following formula instead of the synthetic oligonucleotide adapter [l)], 11,
, -1 (+52-C) production expression plasmids were constructed. This expression plasmid was injected into Escherichia coli according to the method described in Example 1, and further cultured into E. coli containing IL-1 (+52-C).
Yamade, I got the night. The IL-1 activity of this extract was 3 times diluted with 1×102
Incorporation of 3.281 cpm of 'II-thymidine was observed. Furthermore, in 5DS-polyacrylamide gel electrophoresis analysis, IL-1 was found at a position corresponding to a molecular weight of approximately 17,000.
Activity was observed. (Left below) Reference Example 1 Cloning of human IL-11iii cl) NA and determination of base sequence 11L-
60 cells were treated with 10% beef tallow blood In and phorbol-12-myristate-13-acetate as a differentiation inducer and vitamin AI'l (both 500N (final +5 degrees)).
/ml) at 37 °C in RPMI-1640 medium containing
After culturing in air at a humidity of 90 to 100% for 2 days, the culture solution and floating cells were removed by suction, and the dish containing 0 differentiated cells was added to a dish containing 10% beef tallow blood.
Add 10μ of entodoxy/(f1τ resafcalide derived from large intestine obstruction) as an inducer to T RPMI-1640 medium.
g/-1 iQ degree, protein E' was added [101 μg/min of the medium to which cycloheximide was added as a harmful agent at 1 μg/min degree.
was added and cultured for an additional 5 hours. Culture f! ! After finishing, culture f!
Aspirate the liquid and remove the differentiated cells remaining on the dish.
．． It was dissolved in a 6M di-729nium thiocyanate solution containing 5% sodium lauroyl sarcosinate, 5mM sodium citrate, and O,IM 2-mercaptoethanol, and then modified. 0.1M of this homogenate
I'': l) rJ15L, ili i core molecule lI machine (RPS
2[i, 500
The total RNA fraction was obtained as a pellet by centrifugation at rpm for 20 hours. This was mixed with 0.35M NaCl, 20mMTri
It was dissolved in a small amount of 7M urea solution containing S and 20mM EDTA and collected as an ethanol precipitate. This total RNA fraction was dissolved in IOmM containing 1mM EDTA.
Tris-)ICII buffer (PH7,4) (hereinafter TE
(referred to as liquid) 211 and heated at 65°C for 5 minutes. After adding NaC+ solution to this to make it 0.5M,
The adsorbed I
(A) mRNA was eluted with TE solution. The poly(A) mRNA obtained here was used in the following experiment. ■ Synthesis of c DNA Using the poly(A) mRNA obtained in section (1) as a template, the Gubler and Hoffman method [Gene 25.203 (1)
983)] cDNA was synthesized. The poly(A)
Dissolve mRNA (6 μg) in 6 μj of distilled water and add O,lliμ! A 100mM aqueous solution of methylmercury hydroxide was added and left at room temperature for 10 minutes.
500mM 2- containing ga [1iotec product]
1.7 μl of mercaptoethanol solution was added. After leaving for 5 minutes at room temperature, add 10mM MgCl2.1
．． 25mM dGTP, 1.25mM dATP. 1.25mM dTTP, 0.5mM dCTP, O
, +7μM a-"P-dCTI' (specific activity, 75
0Ci/wvole), 4uz oligo(dT)
rt~+i, 32ul of 50mM Tri containing 12Ot1 Trimyeloid Leukemia Virus Yamamai Reverse Transcriptase
5-11c I (pH 8,3) & 1 buffer solution was added, 4
After reacting at 2° C. for 0 minutes, EDTA was added to stop the reaction. Phenol/chloroform mixture (1:l)
and add ammonium acetate to the aqueous layer at Hiiragi C degree of 2.5.
M, and the reaction product (s
scDNA-mRNA 211 combination) was precipitated. This sscDNA-mRNA 61 combination was dissolved in 100 μl of a reaction buffer having the following composition. Reaction buffer composition: 5mM MgCIz, 10mM (NH4) 2s
O, 100mM KCI, O, 15mM β-di+thi/amyl'7denine nucleotide, 40μM dG
TP, 40 μM dATP, 40 μM dTTP,
40 μM dCTP, and 5 μg bovine serum albumin,
20mM Tris-containing 1.25 units Colon ribonuclease H, 24 units E. coli DNA polymerase I
HCI (PH7,5) II buffer. The M lysate was reacted at 12°C for 60 minutes, 2.5 units of E. coli:i DNA ligase was added, and the mixture was further incubated at 22°C.
The reaction was carried out for 60 minutes. After stopping the reaction by adding EDTA, extraction was carried out with a mixture of Fer/chloroform and the reaction product (dscDNA) was extracted with ethanol.
) was precipitated and collected. (3) Addition with dC tail Preparation of DNA The d sc DNA obtained in Section 2 was added to the reaction buffer with the following composition at 100%
Dissolve dscD in μl and react at 37°C for 30 minutes.
A dC tail was added to NA. Reaction! ! Solution composition: 2mM COCl2.0.2mM dithiothreitol. 0.1mM a-”P-dCTP (specific activity I H/
mmole) and 100 mM sodium cacodylate (pH 7.2) 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 dC-tailed dscD
NA was precipitated and collected with ethanol. This is 1
10m containing mM EDTA and 100mM NaCl
M Tris-HCI (pH 7,4)! ! In street liquid,
It was dissolved in 2 μg/■1 alf. (4) Preparation of recombinant plasmid dG-tailed pBR322 (manufactured by Bethesda Res, Labs. Inc.) and dC-tailed pBR322 (obtained in Section 31)
s c DNA in 1.51 mM EDTA and 1
10mM Tris-HCI with 00mM NaCl
(PH7,4) After dissolving and mixing in Ta solution to contain 1.5 μg and 0.09 μg, respectively, annealing was performed by heating at 65°C for 10 minutes, at 57°C for 2 hours, and then at 45°C for 2 hours. , a recombinant plasmid solution was prepared. 6) Selection of transformants Using the recombinant plasmid solution obtained in section (4),
E, col+χ1776 strain was transformed. That is, E
, coli χ1776 strain was treated with diaminopimelic acid 100
Shishi broth supplemented with μg/1 and 40 μg/■1 of thymidine (composition: 710 g of tryptic acid per 11 g, 5 g of yeast extract, 15 g of NaC, 1 g of glucose + p
H7,2) Absorption Dlj (80
0nm) was 0.5, the bacterial cells were centrifuged, and 10mM Tris-HCI containing 50mM CaCl* was added.
It was washed with 10 ml of 5lrII solution (PH7,3). The collected bacterial cells were suspended in the same buffer solution 21 and allowed to stand at 0°C for 5 minutes. Add and mix 0.11 of the above recombinant plasmid solution to 0.21 of this suspension, let it stand at 0°C for 15 minutes, and then keep it at 42°C for 2 minutes. Add Noshiichi Gloss 0.51 for 1 hour*i
Culture was performed. A portion of this culture solution was poured onto a Pross agar plate to which tetracycline (15 μg/1) was added 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. (6) Reference Example 2 from the cDNA library obtained in Section 2/G■
rabbit IL from the recombinant plasmid pRL+5 obtained in
Colony hybridization, 7 studies and hybridization ts7 trans XI/-V, 7 studies using fragments of cloned c DNA encoding ts7 as probes [111aniatis, T., et al. “Mo1ecularC1on+ng” 329(
1980) Co1d Springllarbo
Transformants containing a plasmid containing a cDNA encoding a human IL-1 polypeptide were selected by using the following method. This recombinant plasmid was named pHL number. (2) Determination of the base sequence of cloned cDNA The base sequence of the four-cloned cDNA was determined by the ditheokine method using M13 phage. M13■plB and M13mp
19 (Pharmacia P-L llioches
icals) as a cloning vector, M13
Sequen/Gukisoto (Amersham Inte
``M+3 Cloning and Sequencing Handbook'' (Am
It was carried out in accordance with the method (manufactured by Ersham International plc). The base sequence and the amino acids deduced from the base sequence 11 are as shown in Table 1 below, and encode the IL-1 precursor polypeptide. The 1st to 3rd bases are the start codon ATG, and the 814th base is the start codon ATG.
The base at positions 816 to 816 is the stop codon TAG. (Leaving space below) Table 1 (Sono) [Note] ※※※ indicates a stop codon. Numbers in parentheses indicate amino acid numbers. Reference Example 2 Preparation of rabbit IL-1 cDNA (11 Preparation of rabbit IL-1 mRNA) Inject 1 rabbit with killed Propionibacterium acnes cells.
Each animal was injected intravenously at a dose of 1001 g and sacrificed 8 days later. Immediately, a thoracic tracheotomy was performed, and lung lavage was repeated using phosphorus ff1ll saline through a tube inserted into the trachea, and alveolar macrophages were collected. RPM containing alveolar macrophages with 10% tallow serum
Suspend it in I-1t340 medium, plate it on a Vetri dish (diameter 8cm) at 1×10' pieces per plate, and incubate it at 37°C in air containing 5% carbon dioxide.
Preculture was carried out at 0-100%. After 1 hour pre-incubation, endotoxin (Nori boboli subcharide from Major IIg)
, TPA (phorbol-+2-myristate-13
- acetate) and cycloheximide each at maximum na.
m is 10μg/■l, 500ng/■1 and 1μg/
(2) The mixture was added and mixed so that the concentration was 1, and the culture was further continued. After 4 hours, the culture solution was removed by suction, and the sample remaining on the dish was used as a reference example! - [1) Poly(A) mRNA was obtained according to the method shown in m. The poly(A) mRNA obtained here was subjected to agarose gel electrophoresis vI (gel concentration 1%, in the presence of 6M urea, PH4), starting from the electrophoresis position corresponding to the molecular size of 2.6 to 3.7 kb. A) mRNA was collected. ■ Preparation of cDNA library Using the poly(A) mRNA obtained in section (1) as a template,
Reference Example 1 - A cDNA library was prepared using the methods shown in (■) to (■). (3) Cloni/G For the above cDNA library, rabbit IL-
To screen for transformants carrying a plasmid containing cDNA encoding 32 p-marked cDNA
Colony hybridization using probes was performed using the method of Yaschen et al. [Gene. 10.03 (+980)]. endotoxin. Culture f! with TPA and cycloheximide! 4 [Top! 2
Using the poly(A) mRNA obtained by the method described in Section 11 as a template, the poly(A) mRNA obtained by the method described in Section 11 above was used as a template, and the method described in Reference Example! The synthesized and 32p-52 cDNA was used as g#4 plus and induction minus probes, respectively.This test revealed that a recombinant plasmid containing a nucleotide sequence that binds to the induction plus probe but does not hybridize to the induction minus probe. These selected clones were then subjected to a hybridization and transrage test as described in (1) above.
Using the poly(^) mRNA obtained in Section 1, rabbit IL-1m
A clone that strongly hybridized with RNA was selected. The recombinant plasmid possessed by this clone was called pRL+
I named it 5.

[Brief explanation of drawings]

FIG. 1 shows the steps for constructing the expression plasmid pHLP373. The formulas [al, [bl, and [cl] in the figure mean the respective oligonucleotide adapters shown in Example 1. ↓AM

Claims (25)

[Claims] (1) A polypeptide or a derivative thereof that exhibits interleukin-1 activity. (2) In the amino acid sequence represented by the following formula [I], 0 to 18 amino acids at the N-terminus are removed, and 1 to 10 amino acids at the C-terminus are removed. A polypeptide or a derivative thereof according to claim (1). [There is an amino acid sequence] [I] (3) In the amino acid sequence represented by the following formula [I], 0 to 16 amino acids at the N-terminus are removed, and 1 to 7 amino acids at the C-terminus are removed. Claim (1) or (2)
) or a derivative thereof. [There is an amino acid sequence] [I] (4) Any one of claims (1) to (3), which has an amino acid sequence represented by the following formula [I], in which 4 to 7 amino acids on the C-terminal side are removed. The polypeptide described in. [There is an amino acid sequence] [I] (5) Any of claims (1) to (4) which has or includes an amino acid sequence represented by the following formula [I'] or an amino acid sequence in which three amino acids on the C-terminal side are removed. A polypeptide described in crab. [There is an amino acid sequence] [I ′] (6) The polypeptide according to claim (1), wherein Met is bound to the N-terminus of the polypeptide according to any one of claims (2) to (5). (7) DNA having or containing a base sequence encoding a polypeptide having interleukin-1 activity. (8) In the amino acid sequence represented by the following formula [I], 0 to 18 amino acids at the N-terminus are removed, and 1 to 10 amino acids at the C-terminus are removed. The DNA according to claim (7), which has or contains a corresponding base sequence. [There is an amino acid sequence] [I] (9) Corresponds to an amino acid sequence in which 0 to 16 amino acids from the N-terminus are removed and 1 to 7 amino acids from the C-terminus are removed from the amino acid sequence represented by the following formula [I] The DNA according to claim (7) or (8), which has or contains a base sequence. [There is an amino acid sequence] [I] (10) Claim No. 7 which has or contains a base sequence corresponding to the amino acid sequence represented by the following formula [I] from which 4 to 7 amino acids on the C-terminal side have been removed. - The DNA described in item (9). [There is an amino acid sequence] [I] (11) Claims Nos. (7) to (11) have or include a base sequence corresponding to the amino acid sequence represented by the following formula [I'] or an amino acid sequence in which three amino acids on the C-terminal side have been removed. 10) DNA described in section 10). [There is an amino acid sequence] [I ′] (12) In the base sequence represented by the following formula [II], 0 to 18 codons at the 5' end have been removed, and 1 to 10 codons at the 3' end have been removed. Claims (7) or (8) having or including
) The DNA described in the claims. [There is a base sequence] [II] (13) In the base sequence represented by the following formula [II], 0 to 16 codons at the 5' end are removed, and 1 to 7 codons at the 3' end are removed. Claims (7) or (9) having or including
DNA described in section. [There is a base sequence] [II] (14) Base sequence represented by the following formula [II'] or its 3
The DNA according to claim (7) or (11), which has or contains a base sequence in which three codons at the 'terminus are removed. [There is a base sequence] [II′] (15) It has or contains a base sequence in which a start codon and/or a stop codon are bound to the 5' end and/or the stop codon to the 3' end of the base sequence according to any one of claims (8) to (14). DNA according to claim (7). (16) A recombinant vector in which the DNA according to any one of claims (7) to (15) is incorporated into the vector. (17) The recombinant vector according to claim (16), wherein the vector is a gene expression vector. (18) The recombinant vector according to claim (16) or (17), wherein the vector is a vector that can be propagated in E. coli. (19) The recombinant vector according to any one of claims (16) to (18), wherein the vector is an E. coli plasmid. (20) The recombinant vector is pHLP373 or pH
The recombinant vector according to claim (19), which is LP358. (21) A host transformed with the recombinant vector according to any one of claims (16) to (20). (22) The host according to claim (21), wherein the transformed host is a microorganism. (23) The host according to claim (21) or (22), wherein the transformed host is E. coli. (24) The DNA according to any one of claims (7) to (15) is incorporated into a expression vector, a host transformed with the vector is cultured, and the produced interleukin-1 activity is obtained. A method for producing a polypeptide, which comprises collecting the polypeptide. (25) A pharmaceutical composition containing the polypeptide or derivative thereof according to any one of claims (1) to (6).