JPS60185799A - Human cancer necrotic factor - Google Patents

Abstract

PURPOSE:To obtain the titled factor, by cloning cDNA coding human necrotic factor with a cloned DNA coding a rabbit cancer necrotic factor as a probe, integrating the resultant cloned cDNA in a plasmid, transforming a microorganism with the plasmid, and using the transformant microorganism. CONSTITUTION:A human macrophage is collected from an air cell, blood or abdominal cavity, etc. and cultivated in an inducer, e.g. endotoxin, to separate a fraction containing a human cancer nectrotic factor mRNA from the above-mentioned cell. A single-stranded cDNA is prepared from the above-mentioned mRNA with a reverse transcriptase, and converted into a double-stranded cDNA, which is inserted into a vector. The resultant vector is then transformed to a host to prepare a cDNA library. A cDNA coding a human cancer necrotic factor is cloned from the above-mentioned cDNA library with a DNA coding a rabbit cancer necrotic factor as a probe, and a microorganism transformed by a plasmid having the integrated cloned DNA is used to give the aimed factor, e.g. a polypeptide having amino acid sequence expressed by the formula.

Description

[Detailed description of the invention]

The present invention is directed to human cancer necrosis factor voripebutylation and its coating 1) NA, and their 11-
Transformation 1) Inject into vectors into which NA has been integrated and hosts transformed with these vectors. In this specification, the following approximate bow is used to abbreviate T of 1 Tsukuda. A adenino C cytonone G guanine r boomino Ala arani / A r sr Al1'! Nino Asn Asparagi / Asp γ Subashigi / Acid Cys Cystine Gin Glutami / Glu Glutamic acid G ] y Glyci / II i s Histidine lie Isoleucine L eur'in / 1,, y s Lysine Met Methioni / Gate 1C71 Nylalanine 1' r o P

[J Lin Scr Serino 1'hr Threoni 7 TrI) Tributov 7 Tyr Chi' J Gino Val Balino 1) NA 5' blade xyribonucleic acid cl) NA Complementary 1) N A 1 se NA Ribonucleic acid [1117 NA Messenger 1 se NA dATI) Deoxyadenone triphosphate d CT11
Deoxycytidine triphosphate/acid dC; T11 de"xyguanodinotriphosphate d'r Tl' deAxidemidine triphosphate/acid subsugo (dC) Ribo(dT) oligodeoxydemidylic acid, j1no(A)
Polyadenylic acid, 1'su (U), I! Rinawate lysolic acid poly(dA) rl! Lysooxyadenylic acid poly(
d C) ;I! Rideoxycytisolic acid, 1'su (d
G) ;l: Riboxyguanylic acid poly((IT) Bolide 4/cydimidylic acid E l) 1' A Ethylenediaminotetrachloroic acid bp salt vs. Carswell et al.
We found that the sera of mice infected with Caliettc-Gut immune 1 (IICG) and then treated with En/Todo-1-dino contained a substance that caused necrosis of transplanted IIIeLhΔ sarcoma vessels. This substance was named cancer necrosis factor [1'roe, NaL, ^cad].
, set, UsA, 72, 3U1i [1 (1975)]
. The cause of cancer cancer death is said to be a physiologically active substance released from macrophages, and its characteristics include (i) when administered to tumor-bearing animals, it causes certain cancers to be necrotized and eradicated (H) tn Certain types of cancer cells in vitro (e.g. mouse cancer cells, human cancer-derived 1) c-1
It is known that 1ti) its IL use is not species-specific; Because of these characteristics, cancer necrosis factor is highly anticipated for its clinical application as a new type of anticancer agent. However, the conventional method for producing cancer necrosis factor is to use I) CG or I'ropionibac in animals such as mice and rabbits.
Two shots of te-riu■acnes, then ethotoxin! After that, the photonecrosis factor is isolated and purified from the blood and body fluids, so it has been difficult to stably obtain a large amount of cancer necrosis factor at a low cost. Furthermore, in order to use cancer necrosis factor as a medicine, human-derived cancer necrosis factor is more preferable from the viewpoint of antigenicity, etc.
The above method cannot be applied to the production of human-derived cancer necrosis factor. Conventionally, as a cancer cytotoxic factor derived from human macrophages, a factor produced by peripheral monocytic cells or myeloid monocytic leukemia cells of normal humans (^nti-1umor cytotoxin) [li
munology. 44, H2S (1981) Koh and Reed et al. reported that the factor produced by culture vessel wall (=I adherent cell toxin) in human peripheral blood [
J, 1mmuno1. , υ5,395 (1975>). Also, ijlliamson et al.
H) A substance with a molecular weight of approximately 70,000 Daltons produced from 11 cell lines has been reported as a human cancer necrosis factor [Pr.
oc, Nat, 8cad, sci, UsA, 80.53
97 (Ij+83>]. However, in each case, the substance as a substance was unknown. The present inventors used the human-derived IF method to g+: iγ of human-derived cancerous cause of death -r. , 1st place in recombinant technology
1 and carried out the Eikura research, first to determine the cause of death of rabbit cancer:
We succeeded in isolating J-coated Ruri (1-Nation 1) NA (patent application 1982-228'10O': f Tani Hari). As a result of further research, we succeeded in cloning the cDNA encoding human cancer necrosis factor by using Tonori 1'l-7-conjugated 1)NA as a 1-bub, and furthermore, we succeeded in cloning the cDNA encoding human cancer necrosis factor. The present invention was completed by confirming that microorganisms transformed with the integrated plasmid produced human cancer necrosis factor. As used herein, human cancer necrosis factor is a protein produced by human-derived cells, and has the ability to injure at least I7 cells with 1lIVitrO, and the ability to necrotize cancer caused by at least transplanted MethA tissue in ViVO. The term "carcinogenic death factor" refers to the activity of a single substance. The present invention provides polypeptides comprising the amino acid sequence of human cancer necrosis factor or its active center portion, and cloned 1) NAs that coat them. In particular, the following amino acid sequence (1) SCr 5crSer Arz Thr Pro Sc
r Asp Lys Pr. ValAlaIljsValValAlaAsnPro
GinAlaGlu Gly Gln Leu Gin
TrP Lcu Asn Arg ArgAla A
sn Ala Leu Leu Ala Asn Gl
y Val GluLeu Arg Asp Asn
Gin Lcu Val Val Pro 5crGl
u Gly Leu Tyr Leu Ile Tyr
Scr Gln ValLcu l'llo Lys
Gly Gln Gly Cys l'ro Ser
Tl+r11is Val Leu Lau Thr
l1is Thr lle Ser Arff11c
AlaVatScrTyrGlnTbrLysValA
snLeu LCu Scr Ala Ilc Lys
Ser Pro Cys GlnArg Glu T
hr Pro Glu Gly Ala Glu Al
a LysPro Trp Tyr Glu l'ro
llc Tl'r 1. cu c, +y C:IVV
al PI+c Gln Leu Glu Lys G
ly AsP Arg LeuSCr Ala Glu
llc' ASn Arg I'ro ASp Ty
r LcuAsp Pbe Ala Glu Ser
Gly Gin Val Tyr I'be Gly I
lc Ilc Ala +, Cu (1) is a cause of death in human carcinogenesis T- [hereinafter referred to as polypeptide (1)], the following amino acid sequence (
1■) Met Scr TI+r Glu SCr Met
Ile Arc; AspVatGluLeuAl
a Glu Glu Ala Lcu I'ro l,
ys 1. ysT'l+r (ily Gly I'r
o (iln (ily Scr Lys Ar++
CysLCu PbC1, cu 'Scr Lcu I
'l+c SCr Pl+c teu l1cVal
Ala (ily Ala Thr Tbr 1.eu
Phc Cys 1. euLcuIlisPl+cG
lyValIlc(ilyI'roGlnArgGlu
Glu I'11! ! PrOArg Asp +,
, cu SCr Lr+u l1cSer Pro L
eu Ala Gln Ala Val 8rg (I
I) A polypeptide [hereinafter referred to as polypeptide (l→
-■)] and M at the N-terminus of polypeptide (1).
A polypeptide having Ct and the following salts J, 1. Sequence (1■) (5')-TCA TCT]'CT CGA ACC
CCGΔGT CACAAGCCT GTA GCCC
AT (iTT GTA GCA AACCCT CA
AGCT GAG GGG CA (i CTCCACT
GG CTG AACCGCCGG GCCAAT G
CCCTCCTG GCCAAT GGCGTGGAG
CT (i AGA GAT A to cc G CTG
GTG GTG CGATC8 GAG GGCCTG
TACCTCATCTACTCCCAGG1'CCT
CTTCAAG GGCCAA (iGc TGCCC
CTCCACCCAT (iTG CTCCTCACC
CACACCATCAGCCGCATCGCCGTCT
CCTACCAG ACCAAG GTCAAACCTC
CTCTCT GCCATCAAG 8C; CCCCT
GCCAG A (iG GAG ACCCGA CAG
G (iG GCT GAG GCCAAG CCCT
GG TAT GAG CCCATCTAT CTG
GGAGGG G'l'CTTCCCACCTG GAG
AACGGT GACCGACTCAGCGCT G
AG ATCAAT CG (i CCCGACTATC
TCGACTTT GCCGAG TCT GGG C
AG GTCTACTTT GGG ATCAT c
Cloning of cc crc-(3') (Ill) to coat polypeptide (I) 1) NA[
Hereinafter referred to as 1) NA (III)] and [) NA (II
At the 5' end of I), the following base sequence (IV) (5')
-ATG AGCACT GAA AGCATG AT
CCGG GACGTG GAG CTG GCCGA
G GAG GCG CTCCCCAAGAAG AC
A GGG GGG CCCCAG GGCTCCAA
G CGGTGCTTG TTCCTCAGCCTCT
TCTCCTTCCTGATCGTG GCA GGC
GCCACCACCGCTCTTCTGCCTGCT
G CACTTT GGA GTG ATCGGCCC
CCAGAGG GAA GAG TTCCCCAGG
GACCTCTCCTCTAATCAGCCCTC
TG GCCCAG GCA GTC8GA-(H1'
) (IV) Coating the polypeptide (1+II) with
], and I) AT at the 5' end of NA (Ill).
It is the one that provides the I)NA that owns the G. In this specification, 1F, a polypeptide containing the amino acid sequence of human cancer cause r is a polypeptide in which another 71! repeptide is bound to the N-terminus or C-terminus of polypeptide (1). This means a polypeptide that exhibits the same human cancer necrosis factor activity as polypeptide (1).In addition, a polypeptide that includes the active center portion of the amino acid sequence of human cancer necrosis factor is a polypeptide that exhibits the same human cancer necrosis factor activity as polypeptide (1). Of these, it refers to a polypeptide containing an essential amino acid sequence for expressing human cancer necrosis factor activity, which exhibits the same human cancer necrosis factor activity as polypeptide (1').The present invention includes: Polypeptides containing the amino acid sequence of human sweetfish necrosis factor or its active center portion, DNA containing them, vectors incorporating these I)NAs, and hosts transformed with these vectors. No?
! The polypeptide of the present invention or the polypeptide of the present invention (■
), as long as it has high homology with I' L , exhibits similar human cancer necrosis factor activity, and is immunologically intersecting.
Like that, J! Also included are polypeptides and the 1)NAs encoding them, vectors incorporating those I)NAs, and hosts transformed with those vectors. For example, allelic variants of human cancer necrosis factor 1) NA and derivatives of human cancer necrosis factor 1) NA, including allelic variants of NA and degeneracy or partial modifications of the genetic code, and polypeptides obtained therefrom are also included in the present invention. . Note that derivatives of human cancer necrosis factor 1) NA that include some modifications include derivatives of human cancer necrosis factor 1) NA in which a portion of NA has been deleted, and derivatives of human cancer necrosis factor 1) NA with other derivatives. I) Means a derivative in which the NA fragment has (-1 added to it.) 1) NA can be produced, for example, as follows. (I) Rabbit carcinogenesis cause factor r- = 1-dozuruk ml obtain. Konori + J
-/formation 1) NA salt L', the sequence is as shown in Table 1, and the region that coats the polypeptide of Uta cancer necrosis factor is No. 277-7 (No. 8). Death factor cl) NA of appropriate length I
) The NAflli fragment is treated with a restriction enzyme so that it can be digested, and 1) the NA fragment is excised and used as a solution for the NA glue-coating described below. Examples of restriction enzymes include - 8val, 1laall
, 1lsLNI. 1linclll, RsaI. DNA
It is preferable to use two or more types of fragments derived from the 5'C region. Table 1 lists eight restriction enzymes.
299 bll using l and 1laall respectively.
and the cleavage site for cutting out the 88 bp 1) NA fragment. (Ride margin) (2) Human) Preparation of 1'XI + necrosis and cause of death NA.
Fetus a11゜It'll be collected from the viscera and other organs. For example, from the alveoli, the method of S Ll II O et al. [J
, Iamunol, 12!1, 1:II: [1982
)]. 1111 From ilk, MaLthews method [11
r, J, Cancer+48+405 (1!+81)]
, fetus f11? From the method of WiISOTl et al. [J, 1m
11 old 101-o, 1cal Metl+ods, 5i
i, :105(018:])]. Approximately 2X 1 per culture lid surface 1c+i
Sow 0'~106 pieces, :l! i-38°C1 Preferably about 37°C1 About 5-10% carbon dioxide 6-1 In air, humidity about 90-100% about: 10 minutes to 2 hours before J〆'+6. Next, Ethotoxins extracted from Gushimin V1-1 ¥1, such as Escherichia coli, Pseudomonas aeruginosa + 7i, and Noriboborisasokarai 1 derived from Salmonella typhi, were added, and the history of Futani? (~8
/i to human necrotic cause r111RN over time; 4
At this time, by adding an agent that inhibits protein synthesis (e.g., 7ri11, 1-cymito), it is possible to induce human necrosis factor rnRNA to ifl441.1 .i Ototo Go S may be omitted. Addition of 1 corn todo 1. The corn is generally about 0.1 to 100
0/lar/1 (114 final concentration, same hereinafter), preferably about 1 to 100tt4;11. At this time, about 1 to 2000 ng/ml of phorbol esters such as phorbol-12-myrisdate = 1; (-acetate, phorbol-12,13-endecanoate; You may add A. The amount of protein synthesis inhibitor added varies depending on the type of compound, etc.; for example, in the case of cycloheximide, it is 0.1 to 50%
It is 1g7ml. As a medium, 1i of higher animal cells
Various synthetic 17j bases suitable for lj cultivation are used, such as R
I'MI-1 (+40. Eagle's MI et al.
Regarding the composition of the medium for MEM culture JLII (hereinafter referred to as '-) according to Dulbecco's modified method, see, for example, So I (Kosuke, ed., "Cell Culture Manual' + trlF'a Publishing, 1982).
) is mentioned. ) All terms j? Approximately 1-20% animal serum (e.g. fetal bovine 1
It is preferable to add 1117: + + -J"'l serum). After completion of the cultivation, the cells are prepared using a conventional method, such as CI +
The method of irgwin et al. [formerly ochcmisLry, I8
, 5294 (1≦170)],
Next, this is added to the oligo (dr) cell 11 according to the furun method.
-su or poly(U)cepha1''-su (fufulmanat)
) etc. to absorb 7 ml of poly(^) ml by the patch method.
Separate the A fraction. Otsu's p61y (8) mRN'A
Human cancer necrosis factor mRNA can be purified by Q-condensation and purification by subjecting 1 µl of human cancer necrosis factor mRNA to 1 h of acidic urea agarose gel electrophoresis or 7 g sugar density gradient centrifugation. In order to confirm that mRNA contains +n RNA that coats the target human cancer necrosis factor, it is sufficient to translate the mRNA into protein and examine its biological activity. For example, mRNA is injected or added to a suitable protein synthesis system such as Xanopus 1aevis eggs (superior cell, reticulocyte lysate, wheat embryo) to translate it into protein, and the white protein is extracted. This test is performed by confirming that the mouse L-929 cells do not exhibit cytotoxicity.
The h method using one follicle is carried out, for example, as follows: Approximately 50B of RN RNA is injected per one egg and one upper cell using the Mic I-1 ino/:rri/yo/ method. 10 pieces to 100μβ birth”?'7 itk [Gurdo
n, J, 11. , J., Embryo:
, Exp, lIIorpho l, . 24-7 at 22°C in 20.401 (Hl[;8)]
After culturing for 2 hours and homogenizing, the centrifuged supernatant (
10,000 rpm, 10 minutes) as a sample,
Measuring cancerous death factor activity using -929 cytotoxic activity as an indicator [1. The method for measuring -929 cytotoxic activity is Rul'
I,M,R,,et al,,J,1m1luno1.
. 125.1071 (1980)]. 3) Human cancer necrosis factor cl) NA paste Ll-ninog (2)
) was used as a template, and Gub.
ler et al. 2 [Gene, 25.20: [19
83)] using A ribo(c+T) as a primer, reverse transcriptase (e.g., avian i'1) in the presence of dATI'', dGTl', dcTI).
Reverse transcription enzyme derived from medullary white 1F11 disease virus:+i) synthesizes single-chain cl) NA complementary to mRNA, and then synthesizes double-stranded cl) NA complementary to mRNA using E. coli ribonuclease H and E. coli 1) NA polymerase I. cl) Synthesize NA. 1) The NA obtained here is 1. j! Su(dc)-;I! (C+C) or poly(dA)-poly(dr) homopolymer extension method [Noda, M', et al. Nature, 2!15,202 (1!182): N
e1son, T.S., “Methods +nEn
zymology,' [8,4+ (1979), Ac
Academic Prcss Inc. For example, it is inserted into the restriction enzyme Pstl cleavage site of plasmid p 11 R322 according to a conventional method such as [New, '10rk]. ? 11 recombinant plasmid,
For example, Cohe et al.'s h method [Proc, Nat, ^ca
d, sci, UsA, li9.2110 (1972)]
cl) NA library is prepared by introducing into a host such as E. coli χ1776 strain and transforming it, and selecting a putral italin-resistant strain. Regarding this cl) NA library, eJ was obtained in step (1). I) NA coating ship necrosis factor
A fragment of labeled with 3211 was used as a probe:
J I: Niichi High Predification Test [11an
ahan, D., at al. Gene, 10, 1i3 (1080)] to screen for clones that hybridize with rabbit islet necrosis factor cl) NA fragment J1. This J, a lot of glue [1-) obtained by sea urchin 1) NA
For example, the 61axam-Gi 1bcrt method [
Proc, flat, 8cad. St:i, USA, 74. ．． 51:0 (+9'77)]
According to the ground, (solve the array 4Ji, the already revealed one 5) -1! i', (salt Jl of necrosis r
l) Due to non-extrusion of NA in humans: caries necrosis J';l!Lipepdite; 1 - salt h (sequence 1 no / 1) NA? I can do one thing. In particular, the obtained clone/transformation 1) NA-integrated recombinant plasmid, for example, E. coli 1111101
After obtaining a transformant by introducing it into a host such as a strain, the human cancer death factor polypeptide is obtained by culturing the transformant and separating and purifying the protein using L-920 cytotoxic activity as an indicator. be able to. In order to further increase the expression efficiency, the glue 1-
1) After cutting out the region of NA that coats human tumor necrosis factor with an appropriate restriction enzyme, it is inserted into a gene expression vector containing an appropriate protein motor for production of human cancer necrosis factor polypeptide. All you have to do is create a vector. Examples of promoters include lac promoter, tr
pfromotor, υC promoter. 1'+, / [' motor, SV40 Hatsutomo-Hatsuko promoter. As the vector, any vector that proliferates in the host to be transformed can be used. For example, plasmid (1) + 3R322, p1 month ≧ 54
0, etc.), phages (lambda phage H'H body, etc.). Viruses (such as SV40) can be cited. These may be used alone or in combination, such as p II R322
- It may be used in the form of a SV40 hybrid plasmid or the like. Cloning 1) A host for expression of expression can be obtained by causing the vector into which the NA has been incorporated to act on a suitable host for transformation using the '16' method. Examples of hosts used for transformation include E. coli. Wither! , 1 bacteria, microorganisms such as yeast, CO5monke)'
Examples include cultured animal cells such as cells and cultured plant cells. The human cancer necrosis factor of the present invention has an amino acid sequence that is extremely homologous to Deutsche M. necrosis factor, and 1) high homology is also observed in the NA base sequence. Table 2 shows the homology of the amino acid sequences of the precursors of human cancer necrosis factor and rabbit cancer necrosis factor, and Table 3 shows the homology of the amino acid sequences of the precursors of human cancer necrosis factor and rabbit cancer necrosis factor.
) Shows the homology of the NA base sequence. Special application +1/158-
As explained in No. 228790, No. 2 Gigannecrosis Factor exists in the form of an aggregate in the absence of urea, starting from Scr at No. 82 and No. 236 No. 11 O shown in Table 2.
) A polypeptide consisting of 154 amino acids ending in L Cu is considered to be its basic structural unit. That is, it is thought that the N-terminal portion of the precursor polypeptide is cleaved and activated by the appropriate enzyme in vivo. In the case of human cancer necrosis factor, a polypeptide consisting of 155 amino acids starting from Set No. 82 [1] and ending with Lcu at No. 236 is considered to be the basic constituent unit of human cancer necrosis factor. As is clear from Tables 2 and 3, the amino acid sequence homology between human and rabbit cancer necrosis factor polypeptides is 81.
%, and the homology of the 1) NA base sequences coating each is 85%. In addition, high homology was observed between the human necrosis factor precursor and No. is 84%. This -:1 homology between human and No. 5 Gigantosis death factors leads us to infer that they are derived from an intrinsic factor or a phylogenetically common molecule, and also indicates that the homology is extremely high. There is a region essential for the expression of the biological activity of the cancerous death factor in the high range region, suggesting that this is the central part of the active VL of the cancerous death factor. Table 2 Amino acid sequence of human cancer necrosis factor and rabbit cancer necrosis factor 1j11 precursor boribeptite 10 steps: Human cancer necrosis factor precursor means ゛Utagifu°-5 necrosis factor r-precursor flll fraud■
Seven sorrows: Idaha Minoh■ Masa? Missing':,: o px'r m = ,,;Rice;,
Is it easy...? [edited by 1] 太＋rp, 'Jōho ■light (-elbow) ◇ ni biriu Gly Ql'n 1. cu Qln
Trp1. co etc. 1;! Qrg20 Val l soil] +16 KKW (ily 3in (
ilV CVS I;, ;LA bsugo! ,!脣・・す” peeling 1-■” (”F・・Th・lA7 glue+゛170 董１昌: IF under U] Sweat 7;: 人性UTINGX■Asn 1
．． cu 1. cu S°'A1°11°1・Pa8°m)
1:?耶 [H Ding I was punished and attended a meeting on 31st; L calendar:
r, o, TrP 1'Yr (:NI I,'ro I
lc TVr 1. C! U Q11'10 GIV Val 1lle Gln Leu GIL
I 1. The part surrounded by ys Gly Asp Argon indicates a homologous region. (---) indicates deletion. Table 3 1) Nucleotide sequence of NA containing human cancer necrosis factor and 1111 precursor of rabbit cancerization factor r; 1) DNA encoding human cancer necrosis factor precursor; I) NA0 AT(i AGCACT GAA AGCAT(i A
TCCGG GACGTGATG AGCACT GA
G AGT ATG ATCCGG GACC;TCG
AG CTG GCCGAG GAG GCG CTC
CCCAAG AAG (iAG CTG (icG G
AG GGG CCG CTCCCCAAG AAG0 ACA GGG GGG CCCCAG GGCTCC
AAG CGG TGCGCA GGG GGG CC
CCAG GGCTCCAAG CGCTGCTTG
TTCCTCAGCCTCTTCTCCTTCCTG
ATCCTCTGCCTCAGCCTCTTCTCT
TTCCTG CTC50 ACCCΔT GTG CTCCTCACCCACAC
CATCAGC---TACGTG CTCCTCAC
c10 (---) in T CACACT GTCA indicates deletion. (Zero Zero *) indicates the end futon. As shown in the test examples below, human cancer necrosis factor is a polypeptide that has no immunological crosslinking with rabbit cancer necrosis factor and is immunologically distinguishable from chronic cancer necrosis factor. Thus, although human and Wu IJ1' cancer necrosis factors show high overall homology, there are clear differences in the sexes of their polypeptides. This means that the entire amino acid sequence of the cancerous death factor is not essential for its biological activity, and if the amino acid sequence of the part constituting the l, ηU center is the same, the amino acid sequence of the other parts may vary slightly. And, what is the nature of the polypeptide?
Even if it changes, ship necrosis cause r・1. 'irl is maintained (
This indicates that the Cause of death due to 1<rH necrosis revealed by the present invention
;l: Based on the knowledge about the amino acid sequence of lipeptate and the I) NA base sequence, a polypeptide containing the amino acid sequence of human cancer necrosis factor or the active center of human cancer necrosis factor was prepared according to the base method of the Shodenshi Kakkei technology. I) NA or partially modified variants of human cancer necrosis factor DNA can be obtained, and they can be used to easily produce polypeptides having human cancer necrosis factor activity. Something comes up. In addition, a part of the l) NΔ base sequence revealed in the present invention was chemically synthesized, and this was
- Human cancer necrosis factor 1) NA with allelic gene mutations or reduced gene coat can be easily produced by using it as a polypeptide with human cancer necrosis factor activity. I can do something. Furthermore, human ship necrosis factor, 1! Based on the amino acid sequence of Lipeptite, it is also possible to totally synthesize IUj, (I of the sequence) NA that coats it. All of these are included within the scope of this invention. The human cancer necrosis factor polypeptide of the present invention
14i It can be used for the treatment of li L (, H). Examples and reference examples are listed below in 1. The present invention will be explained more specifically, and the present invention is not limited to this example.Example: Preparation of probe for detection In accordance with the method disclosed in Japanese Patent Application No. 58-228790, Cloned 1) NA coated with rabbit cancer necrosis factor shown in Table 1 (previously listed) was obtained. This 1) Limit NA6
? It was excised using iAllae II and Ava I to obtain 1) NA fragment consisting of 88 bp from No. 3 to No. 120 and 1) NA fragment consisting of 299 bp from No. 285 to 583, respectively. The 88 bp 1)NAfli fragment excised with 11aell is the rabbit tumor necrosis factor precursor. This is the area corresponding to the note area. These two types of DNA fragments were labeled with 321) and used as a template for cloning human cancer necrosis factor cl)NA in section (8). (2) Human alveolar MacII phage G, 3X 107 alveolar macrophages were collected from the lungs of the mRNΔ fraction from the lungs using linic acid-buffered saline AI. Suspended in 1 inch of RP M 1-1040 medium containing '/? Preincubation was carried out in air containing gas at a humidity of 90 to 100%. After preincubation for 1IIlf, J-dotkin (Noriboli hydraccharide derived from Escherichia coli) was added. -13
- acetate) and quinamide to 7-11, respectively.
%, IO μlr/ml, 1011 g/ml, and 1 Jg/l were added and mixed, and the culture was continued. After 4 to 4.5 hours (5 to 5.5 hours after 3'lI), the culture solution was removed by suction, and the remaining macrophage in Di Noche-1-1 was distributed with 0.6% lau-11-ylsarcosinothorium. and 1 to 5 containing Orr+M thorium enoate
Guanisilchi: A/A Futose cutting 1B corner? ,L,
, I mosilyized. This 5 tomosine tube is 01M
El) cesium monochloride aqueous solution to 15.7 containing 1'A-
”-ifi layer and one ultracentrifugal machine (R1'527-2
1J-tar, +, +
i, centrifuged at 500 rpi for 20 hours, and all! ? The NA fraction was obtained as a pellet. This is 0. :] 55M NaCl
. 20mM Tris and 20mM +71)TA
It was dissolved in 1.1 volume of a 7M urea solution containing 1.1% of 7M urea solution, and recovered as an ethanol precipitate. 159+z+y was obtained for total RN. This total RNA fraction was dissolved in lO containing 1 mM El)TA.
mMT r i s ilc IIJ2 force i[+4 (
It was dissolved in pH 7.4) (hereinafter referred to as FTEilk) and heated at 65°C for 5 minutes. After adding NaCl solution to 0.5M, add 0.5M solution in advance.
A pre-war poly(8) m cell column equilibrated with a TE solution containing NaCl was applied to an A:I (6) cell I + - column.
RNA TIEilM is out! :
Ni, Ni, '81r poly(8) m RN A
I got it. This poly(8) m RNA A is 1.9
Dissolve 1B in distilled water at a temperature of 11%, add 5 Onl per 1:1 cell of African mega-T. Translated d1 white matter L-! l20 cytotoxicity was measured. As a result, the activity of 11 at position 611ψ was observed in the :1 modinate 011 of 10 eggs (Na cells), and this po
ly' (8) mRNA1. It was confirmed that the product contained human Ryo-1 caries necrosis r mRNA. (31c l) Synthesis of NA (21JJi) Poly(^)ml≧NA obtained by the method of Gublcr et al. [Ganc, 25.21
cl) NA was synthesized according to ;:] (198:l). Reaction 'Lvtht; 40u1 50mM Tris-11CI buffer (pH 8
, 3): 10mM MgCI2; 10mM DidiA threitol; 4mM Pyro; 1. 1.25mM dGTP, dAT
l', dTTI'; 0.5mM dcT+': 0. H
i7μM a-"I'-dcTI' (specific activity 3000C
i/ml1ole) ; 48g J Rigo (
dT)12-+8; Ei 8g Poly(A)m
17 NA; +20 units avian 1゛1 medullary white 1111 disease virus-derived inverted 7IIIF cord. After reacting at 43'C for 30 minutes, the reaction was stopped by adding EI)TA, extracted with a phenol-chloro, J, lum mixture (]:II, and ammonium acetate was added to the aqueous layer to a final concentration of 2. The reaction product (single chain cl) NA-RNA 'IJ
coalescence) was precipitated. This +11-strand cl)NA-RN
The A complex was dissolved in 100 μl of a slow reaction 8J solution containing δ11. Reaction 1A buffer: 20mM Tris-11CIIJ buffer (pH
7,5); 5mM M+rC+2; 10mM (
N114) 2504; 100mM KCI;
0. 15mM β-2: J tinamide adeni/dinucleotide; 50/lir/ml bovine blood i+'/albumin; 40μM dGTI', dATl', dTT
P. dCTP; 0.9 units Escherichia coli ribonuclease II
; 23 units of human coliform bacteria 1) NA polymerase 10 After reacting at 12°C for 60 minutes, then reacting at 22°C for 1) 0 minutes, add El) TA to stop the reaction, and proceed as in 1. The double-stranded cl)NA was extracted with a phenol-II:I form mixture and precipitated with ethanol.
1. (4) "ribo(dc)dale addition cl) 3 for preparation of NA"
Add 100μβ of the reaction buffer with the following composition to the double-stranded cDNA obtained in section ), incubate at 37°C for 00 minutes, and add the ribo(dc) tail to the double-stranded cDNA (=1 Reaction buffer; 2mM COCl2.
/r++ole) and lQ lj position terminal de A-
r-Synucle 1 Desiltra/Suf. I00mMh cosylate solution containing 1 ml (pl+7.2). The reaction was stopped by adding 1ε1) TA aqueous solution, and then removed. 7-Roof rJ+i ; l, Rum was extracted with tail liquid, and the oligo(ClC) tail (·1-added cDNA was precipitated and recovered with ethanol. This was added to 10 mM Tris −
11CI buffer (pl+7.4). 211 g of ``ribo (dc)
I8 was dissolved to contain 1 cl) NA. (5) Oligo (dc) Dale addition plasmid 1) 10≧
3221) Preparation of NA 20mM Tris-11CI buffer (pH 7,4),
10mM MgC12゜501n~+ (NIL)
0, I IIg of bovine II per 2SO4 and 11
1lrrtAk Fumi7'E: Contains water rBM&IoO
Dissolve 10 μg of 13R322, add 15 units of restriction enzyme 1'st I nuclease,
The reaction was carried out at 7°C for 1 hour. After the reaction was completed, the reaction solution was extracted with a mixture of phenol and rum, and NA was recovered from the aqueous layer by ethanol precipitation. obtained l)
NA was added to the oligo(aC) tail CI in an aqueous solution (however, '11-d CTI) was replaced with 'li'.
d containing G Tl') and using 80 units of terminal deoxynucle A desyltransferase: (incubated at 7°C for 20 minutes, approx.
Five dG residues were incorporated. The reaction solution was extracted with Pheno-Roof V1+, 1, Luno 1lls i&, and the oligo(dG) tailed plasmid p11R3221)NA was recovered from the aqueous layer by ethanol precipitation. This was reconstituted to contain 20 μg of oligo(dG) tail, 1 plus psimit I) +3 R3221) NA in the same buffer i1k as for oligo(dC) tail plus cl) NA. (6) Production of f'I of recombinant plasmid Oligo (dc) obtained in section (4);--L addition cl)
NA solubility 120μ! (5) Oligo obtained by JjI ((1G door addition 1) 13 R3221)N
Mix with A solution 11120μβ and incubate at 5°C for 5 minutes at 57°C.
Annealing was performed by incubating at 120° C. for 120 minutes to prepare recombinant Bushisumi F tri i+k. (7) Selection of transformants Using the silk recombinant plasmid solution obtained in section (6),
・,', L: oliχ177[iK was transformed. That is, L: coli χ 1776 strain was treated with 2 oml of L-brace containing cyamino pimelic acid + 00 ti sr/m + and thymine y 40 ttg/m +.
Incubate at 37°C until the absorbance (lioonlI) reaches 0.5, collect the cells by centrifugation at 4°C, and add 50mM
The mixture was dispersed in 10ml of CaCl221OmMTris-11CI buffer (pH 7,3) and centrifuged again at 4°C to precipitate. The collected bacterial cells were dispersed in 2 ml of the same buffer and allowed to stand at 0°C for 5 minutes. This dispersion 0.21
0.1 m of the recombinant plasmid solution obtained in section (6)
1, mixed, left at 0°C for 15 minutes, and further heated to 42°C.
After incubation for 2 minutes, add 0.51 l of Shiichi broth of the same composition as that used in the previous incubation.
Shake; Performed 1 pike. Take a portion of this culture solution and l!
Contains 15 Izg/ml of tetracycline in addition to the above-mentioned ingredients] - Spread on agar plate and heat at 37°C for about 12
Incubate for 1 hour, select tetracycline-resistant bacteria, and cl.
) NA library ('1) was prepared. (8) ri I+-2/g (cl) NA library obtained in section (7) 1.1
In order to screen for transformants carrying a plasmid containing cDNA that coats the human septosomal cause of death r-,
(1) A fragment (299
The 3′l) labeled product of bp) was used as a bu IJ-pump, and the colony was
If the 11th Eve 1 Noguisei J Examination
The method was carried out according to the method of Han et al. IB 'f'l k cell 431114 was selected.Furthermore, for these 43 colonies, 0, 1 and 1 limit^1'
Rabbit cancer death factor Co-F excised with ZAllaell
DNA fragment (88bP) corresponding to the upstream of the f+31 region
321) Use #'? as the labeled substance and perform secondary screening. y), we selected 61!4 colloids that strongly hybridized (/%) with this probe.Through these two rounds of screening, we selected 1, which encodes rabbit cancer necrosis factor.
) A highly homologous NAl-Jλ sequence 11 and its upstream portion (I) NA containing sequence 1j was obtained. (9) Form group [Expression (8) selected [Multilateral glue +: + -7 (plasmid number IITNI'-1, -4, -5, -13, -2
2, -2 (i) h) Take out the transformant plasmid from each of them and use the coli 11 old OIc+ as a host to generate the transformant. Regarding the transformant thereof, Nagata et al.'s jjib [Natur
e. 284,: 116 (1980), 1 i culture of 501 was carried out. L 13 broth was used for Knia Jljl. The cells were cultured until the absorbance (000 rv) hS reached approximately 0.8, and approximately 3 to 5×1010 single cells were collected. 0.
50mM Tris-1 with 1% Lysodeme
1c l buffered LyL (pH 8,0) and: lO + nM N
Suspend the bacterial cells in a solution consisting of aCI, let stand for 0 minutes, then freeze and thaw repeatedly to destroy the bacterial cells, and centrifuge to remove the bacteria. Extract ilk?II. Extract i1 obtained from each transformant.
As a result of measuring the L-ri2 cytotoxic activity H for F,
Plasmid 1) Activity at position 11 of 186.111 was observed in the extract of the transformant with IITNI''-13. Recombinant plasmid pIIT selected by
The transformant produced by NI''-13 was cultured in 1,13 broth to obtain 111 cells.The cells were cultured using the method of Wilkia et al. [Nuclcct^cids Rcs, 7,859 (
1! J7!1)] and plasmid 1) NA
of? 1 again. This plasmid l) restricts NA78
The product was digested with PsLI to obtain 1''-N-1) NA, which was prepared using PsLI 1+7. In this way, the nucleotide sequence of each of the restriction enzyme fragments of I'l' L, , and Hi species was determined using various restrictions for NΔ fragmentation. Dephosphorylation, end-nJ labeling with P, base-specific chemical decomposition reaction, gel electrophoresis, and autochromography were performed using the following methods. The figure shows the cleavage position of the restriction enzyme used in the nucleotide sequence method>H, and the direction and range (indicated by arrows) in which the nucleotide sequence was determined. The rho portion indicates the region that corresponds to:J-1' of the human vessel necrosis r precursor polypeptide. The IM jA sequence and the amino acid sequence translated from this j-base sequence are shown in Table 4. The human cancer necrosis factor polypeptide: 2-F region is shown in Table 1. %C
, 4 necrosis-causing r polypeptides are Nos. 235-2 of Table 4:
TC of 17 fragrances, A O) : r Starting from F 7 (corresponding to Scr), CTG of No. 1i 07-0419 (
1-, corresponding to cu) and consists of 155 amino acid residues, 2. ing. Following the C-terminal "1 isine", there is 8% of TGΔ +l-coto/.The 15-base sequence number 2
No. 34 is considered to be a sequence necessary to encode the 1111-terminus of human cancer necrosis factor. Table 4 GACCCACGG (Explanation above) Reference Example Preparation of anti-tsugi cancer necrosis factor antibody 11 Purified rabbit cancer necrosis factor 1.9X 10" according to the method disclosed in Japanese Patent Application No. 58-228790
The solution containing the jpg was emulsified with LL Freund's complete adjuvant and injected at several points on the back of the guinea pig at 11:00. Thereafter, immunization was performed in the same manner 1.3 and 6 weeks later. Another 81! ! Afterwards, the same amount of purified rabbit cancer necrosis factor was intraperitoneally injected 11 times along with aluminum hydroxide and gel. One week after the first immunization, whole blood was collected from the heart, and the serum was separated by centrifugation by IE to prepare an antiserum containing an anti-carcinogenic antibody. This anti-blood j+'i was passed three times through a Ceph y II-S 413 (Pharmacia) column containing normal rabbit blood and Ili components cassoprinogated, thereby completely removing the UL form against the rabbit serum components. , a purified antiserum containing only specific antibodies against rabbit cancer necrosis factor was obtained. This antiserum formed a jp-· precipitation line only between it and the lead cancer necrosis factor purified by immunoelectrophoresis and in-gel double-layer diffusion. About a lid of this purified anti-1]11 supernatant,
The one diluted 1,000 times is L-U of Utagi necrosis factor.
The ability to neutralize 50% of cell cytotoxic activity of 500 or more. Test Example H) l', 4 Immunological Properties of Necrotic Prisoners Example (9
) The frj prepared in the reference example was added to the extract of the form De1 transformant by Bushismit pH NF-13 obtained by JJI.
A 100-fold dilution of the anti-depressant cancer necrosis factor antiserum prepared by Ik-1 was added to the mixture (11).
1 was determined as δ111. 1.11 results are shown in Table 5. Fifth
As is clear from the table, human ship necrosis cause r is Cooley 1
- There is no immunological interaction with cancer necrosis cause r. (bottom margin) Table 5 J, 111 Application No. 58-2287 ≦) Prepared according to the h method developed in 0-C.

[Brief explanation of the drawing]

The figure shows the cleavage site of the human husband', the necrosis factor coated with 1J-nated 1) the restriction site used for the DNA sequencing of NA, and the cleavage site of 11w;! l', the direction in which the arrangement was determined and φi!
Show the surroundings. Special: 1 person: 1 person: 1 person: 1 person: Mr. Kojima-Akira Procedural Amendment (1/2011) Indication of Matter A 11 1981; Group Former "Proposed IJ-+ Particulars, 1. Name of the invention, human, necrotic prisoner-j'- 3. Person making the amendment (Relationship with 'l) Patent application Address 25 J-rl, 3 Doshomachi, Higashi-ku, Osaka Name 2+
11 people 11 books manufacturing company 4J: j5i cam, 21rf”) CI.1,1.□1
．． 1111' 33 Engineering'9 Blue...Pin [' Honyaku Co., Ltd. General Research Institute 5, "Claims" and "Detailed Description of the Invention" of the specification subject to amendment
Contents of column 6 capture 1) "Range of special 7"la+"r" in the specification (the leap line is set to 71 as shown in the attached sheet).2) rTI+rG1yGlyGlyProGinGlySerLy on page 13, line 16 of the specification.
sArzCysJ table is 1' Tier Gly Gly Pro In G
ly Scr Arg Arg Cys J and: J stop. ;3) r AAG ACA GGG GGG CCCCAG on line 15Jj of the specification
G is CTCCAAG ccc; J is 1' AAG ACA G is G acc CCCCAG
ccc rcc AGG CGGJ and:il' +I
do. 4) In the specification i'i, line 151', line 6, add ``At the end of 5', after J, l' lfl begins 7'' by 1Φ. 5) In the specification, +i in the first Tij'JI line, [,'' is followed by ``1 itself or the polypeptide produced by Ill Ifli with an enzyme etc.''.
There will be 11 people. 6) Stop the statement on page 29, lines 12-14 of the specification. 7) Stop y+ on page 31, lines 22-24 of the specification. 8) Page 47, lines 11 to 13 of the specification states that Jnu 1], Machi 1 Toku 1; I' Claims (1) Amino acid sequence of human cancer necrosis factor or its 11
j′lipeptites or similar organisms containing an r+ sex center (11 and immune-; bi-crossing 51! lipeptites. (2) Human necrosis and necrosis ) Amino acid sequence or Scr
Scr Ser ArgThr I'ro Scr
Asp 1. ys l'r. Val AIa 1lis Val Val Ala
Asn Pro Gln AlaGlu Gly Gl
n1. cu Gln Trl>1. eu Asn.
Arg ArgAla Asn Ala Leu Lc
u Ala Asn Gly Val Glul, (!
11 A r l! ΔspΔsn Gin tau
Vat Val I'ro 5er Glu Gly +
,,cu Tyr Lcu lie Tyr Scr
Gln Vall, eu PI+c 1. ys Gly
Gin Gly Cys Pro Scr Tl+r
11is Val 1. cu 1. Cu 1'l+r
1lis Thr Ilc Ser Ar+r11c
Ala Val Ser ]'yr Gln Thr
1. ys Val Asn1, cu l, eu Scr
Ala Ilc1. ys Sar Pro Cys
(ilnArc: Glu Tier I'ro G
lu Gly Ala Glu Ala Lys summer'r
ol'rp1'yrGluProIlcTyrLcuG
lyGlyVal' Phc filn 1. eu G
lu 1. ys Gly As+>ΔrgLeuScr
ΔIa (ilu Ilc Asn ArgPro A
sp Tyr LcuAsl) Phe Ala Gl
u Ser Gly Gln Val Tyr Pl+
When eGly Ilc Ilc Ala Lcu, the polypeptide according to claim 1 J+1. Peptide. (4) The polypeptide according to any one of claims 1 to 3, which has Met bonded to its N-terminus. (5) The following amino acid sequence Met Scr Tier Glu Scr Met
Ilc Arg Asp Vat'GluLeuAla
GluGluAlaLcuPro1. ysLysTl+
r Gly Gly I'ro Gln (ily S
cr Arg ArgCysLcu Pbc Lcu
Scr1. cu Pbc Scr I'l+c Lc
u 1lcVal Ala Gly Ala Thr
1'br Lcu l'he Cys LcuLcu
1lis PI+c Gly V4cl Ilc Gl
y Pro Gin ArgGlu Glu Pbc
I'ro ArgAgpLcu Scr Lcu l1
cScr Pro LCu Ala Gin Ala
Val Arg Scr 5erSer Arg Th
r I'ro Ser Asp Lys Pro Va
l Ala11is Val Val Ala Asn
l'ro Gln Ala Glu GlyGln
Leu Gln Trp Leu Asn Ar11
Arg Ala AsnAla Lcu 1. cu A
la Asn Gly Vat Glu Lcu Ar
gAsp A'sn Gin Lcu Val Vat
Pro Scr Glu GlyLcu Tyr 1
．． cu Ilc Tyr Ser Gln val L
cu l'l+eLys Gly Gin Gly C
ys Pro Scr Thr l1is Val1,
cu 1. cu Thr l1is Tl+r IIC
Scr Arg Ile Δ1aVal Ser Ty
r Gln Thr Lys Val Asn Leu
LeuScr Ala llc Lys Scr P
ro Cys Gin Arg GluTl+r Pr
o Glu Gly Ala Glu Ala Lys
Pro TrpTyr Glu Pro llc T
yr Lcu Gly Gly Val l'l+cb
in 1. cu Glu Lys Gly Asp A
rg Leu Scr AlaGlu Ilc Asn
ArIX Pro Asp Tyr Leu Δsl
) PhcAla Glu Scr GISI Gln
Val Tyr Pl+c Gly 1le11 e
Ala 1. 1: Lipeptide according to claim 3, having .eu and .gamma.I. (6) Characteristics produced by minute t1 substances or cultured cells: 1
? The polypeptide according to any one of items 1 to 5. (7) Generate the amino acid sequence of human cancer necrosis factor or a salt-lambda sequence encoding a polypeptide containing its active center 1) NA or allelic mutation, degeneracy of the genetic code, or partial 1) A variant of NA containing a modification. , - (8) The J group sequence encoding the amino acid sequence of human cancer necrosis factor is (5')-TCA TCT TCT CGA ACCC
CG AGT GACAAGCCT GTA GCCC
AT GTT GTA GCA AACCCT CAA
GCT GA (i GGG CAG CTCCAG T
GG GTCAACCGCCGG GCCAAT GC
CCTCCTG' GCCAAT GGCGTGGAG
CTG AGA GAT AACCAG CTG G
TG GTG CCATCA GAG GGCCTG
TACCTCATCTACTCCCAGGTCCTCT
TCAAG GGCCAA GGCTGCCCCTCC
ACCCAT GTG CTCCTCACCCACΔC
CATCAGCCGCATCGCCGTCTCCTAC
CAG ACCAACGTCAACCTCCTCTCT
GCCATCAAGAGCCCCTGCCAG A
GG GAG ACCCCA GAG GGG GCT
GAG GCCAAG CCCTGG TAT GA
G CCCATCTAT CTG GGAGGG GT
CTTCCAG CTG GAG AAG GGT G
ACCGACTCA(ic GCT CAG ATCA
AT CGG CCCGACTATCTCGACTTT
GCCGAG TCT GGG CAG GTCTΔ
CTTT GGG ATCATT GCCCTG-(3
') according to claim 7. f! 11 Special 1 of starting code 7ATG on the 5' end of the wood.
1) NA of Claim 7 or 23. (10) Next stone base arrangement (5')-ATG AGCACT CAA AGCAT
G ATCCGG GAC(iT(i GAG C1'
G (icc GAG GAG GCG CTCCCC
AΔGAAG ACA (iGG GGG CCCCA
G GGCTCCAGG CGGT(ic TTG T
TCCTCAGCCTCTTTCTCCTTCCTGAT
CGi'G GCA GGCGCCACCACG CT
CTTCTGCCTG CTG CACTTT GGA
CTGATCGGCCCCGAG G GΔAG
ΔG TTCCCCAGG GACCTCTCT CT
AATCAGCCCTCT<;(icc CAG (i
cA GTCAGA TCΔTC'r TCT CGA
ACCCCG AGT GACΔA(i CCT (
iTA(icc CAT GTT GTA GCA A
ACCCT CAA GCT GAGGLi(i CA
G C1'CCACTGG CTG AACCGCCG
G GCCΔAT GCCCTCCTG GCCAAT
GGCGTG GAG C1'GA (iA (iAT
AACCAG CTG GTG GTG CCA T
CA GAGGGCCTG TACCTCATCTAC
TCCCAG GTCCTCTTCAAG GGCCA
A GGCTGCCCCTCCACCCATGTG C
TCCTCACCACACCATCACCCGCAT
CGCCGTCTCCTACCAG ACCA71G
GTCAACCTCCTCTCTGCCATCAAG
AGCCOCTGCCAG AGGGAG ACCC
CA GAG GGG GCT GAG GCCAAG
CCCTGG TAT GAG CCCATCTAT
CTG GGA GGG GTCTTCCAG CT
G GAG AAG GGT GACCGA CTCA
GCGCT GAG ATCAAT CGG CCCC
ACTAT CTCGACTTT GCCGAG TC
T GGG CAG GTCTACTTT GGGAT
I) NA of claim 7 having CATT GCCCTG-(3''). (11) A vector incorporating the DNA according to any one of claims 7 to 10. 021 Claim 1I vector of the company, wherein the vector is a gene expression vector.
1. A host transformed with a vector from Kisha. 134. The host according to claim 133, wherein the 0Φ transformed host is a microorganism or a cultured cell. ” Procedural amendment stamp ^) June 1985 (;] 1 1.1.4 Agency L+'11'
, '11-11 Indication 1981 Special; 'l Application No. 4; l Old No. 7 2 Name of Komei (h, Human ship necrosis r-3 N Mountain Climber 11 ('l Relationship with Special) ,':I Applicant Ire gr 31 Doshomachi, Higashi-ku, Osaka City It 25 Name 2!11
11 people Pharmaceutical Co., Ltd. (-1 representative director +1 Kanade 11j
71'＋゛ 4th generation male, 11 people 〒5184 Address 381-94 Honmachi, 111 City, Osaka Prefecture, 11 people
The number of inventions that will increase due to the 5th finding in the Comprehensive Research IJI 3.6 "Claims" and [Detailed Description of the Invention] before the specification subject to amendment
((Yl 7. Contents of correction (1) The column “Special FF, scope of claims” in specification 11F is supplemented as shown in the attached sheet.・
1. In the method disclosed in...
The method used in ... (for example, the method in Reference Example 2)
ni...'' and vl' + L'Jru. (;)) In the specification; Method (for example, reference example 2)... J and if' + l (do. (4) Details, 1: 50th shell, 1st line 11, "Reference 5 examples"
Correct j1, saying 1゛该尤1. (5) Specification, 1; In page 50, No. 2111I, [
71 amend the statement "In the method disclosed in ..." to "In the method disclosed in ... (for example, the method of Reference Example 3)...". (6) In Table 5 of Part 11, No. 52J'↓, r
11 of the entry ``l1fK (prepared according to the method disclosed in No. 1982-228790'')
111/I 58-2287410 (method of Reference Example 3)" and il' + I (7) Specification 11F No. 51j' ↓ No. 41r and Line 5 In between, that is, in +iii of the test example, the following reference examples 2 and 3 were added to 1-
1 + people. Reference I-5 Example 2 (1) Tsu・su1! m RN from the alveolar Mac 11 F7-Fun
111 purified woolly in fraction A 1. (About 2.5 inches tall,)
rium acnes) dead bacteria per bird.
O) The animals were injected intravenously at the end of the day and sacrificed 811 days later. Chi immediately opened the chest and trachea, and 11 people were inside the air shield.
- Through the phosphorus 1νj′, the ir, li, and li prior offices are repeated using the phosphorus 1νj′, and the 1di cell mak−
J phage was eradicated. I 2 j: + Woori, 1!
Approximately 3 x IO'1 alveolar macrophages were obtained. This alveolar McII fu -ji is 10% beef tallow +1
u 7+'r 3i1 no 1'Ml-10401r'
Suspended in the S ground and placed in the Petridisonyu (confidence 8 m) 1
The seeds were sown at 2 x 107 cells per sheet, and precultured at 7°C in air containing 5% carbon dioxide at a humidity of 510 to 100%. After 1 hour of 1IIF culture,: I: 7F
) ton/(large intestine 17.f yume nori; I!: 1! liritsukashiid). 'l''I'A (, 1, Le Ball-12-Millisfoot-
111, respectively, to Schiff I+. ii;Q degree or 10115;/
ml, 1011g/1 and I II 47/m
l (!: Add and mix as shown, and add '81'A to tB Gi. After 4-45 hours ('S?: 5-45 hours)
5.5 hours), remove the culture medium i1k by suction and place it in the dish 1
- The Macll phage remaining in the solution was dissolved in 1 to 5 guanidyl thiocyanate with 0.11%, 11%, 60/M Cu[/acid, and 1 guanidyl thiocyanate, and homosylized. . This, 1 module; 6-to 0.1M ICI)
1. Liquid-1-A''VN, ultracentrifugation!'Ilj machine (
RI' S 27-21+-tar, 111γ f'IJ
The total RNA was centrifuged for 20 hours using a 21i, 500jl+ tube to obtain a pellet of total RNA.
: 15M NaCl, 20mM Tris and 20mM ICI) dissolved in a small volume of 7M urea solution containing TA. It was recovered as an ethanol precipitate. 12 birds - 1
! From this, 5', 24+ was obtained as RNΔ. The total RNA fraction of the genus was diluted with 1 mM ICI) I containing TA.
O+nMTris-11CI uopi1k (+)11
7.4) (hereinafter referred to as FTIC liquid) dissolved in 21 and 65
Heat at °C for 5 minutes. After adding NaCl12+ilk to this to a concentration of 0.5 M, 0.5 M
TE11 including MNaC1! "Equilibrated" rib (d
T) Hill + 1 - I, l to Scalum, suck Ia 1゜ po
By eluting In IshiNA with TE11&: (171! 0) 1101y(^)
mRNA was obtained. The poly(^) m obtained here
RNA A's 200μ! Aga [l-sgelumi pneumophoresis (
Gel D degree 1%, OM urine 18 present (Dro) + 14'), divided into 7 halves according to their molecular size, and the gel
After melting (70°C, 10 minutes), extraction with water-saturated Tuunol was performed.
．． Poly(^
) lnlseNA was collected. Regarding poly(8)1111<NA of each fraction, γ Sorika Megador eggs 1, horn method using J cells, 1! Cause of ship breakdown 1'
Measuring mRNA i,i, min(-t4;(tl,
teI, li ~2.7kb ni411 This thread 1 (!
Rabbit ship necrosis factor m RNA A at +i min, X:, to)
It was collected once. Kono purification poly(8) m RNA
The specific activity is approximately 1.2: +0 units/ug NA, and the specific activity of the poly(8) mRNA preparation made by Mio, +1 is approximately 320 units/μ [approximately 4 times as much as RNA. I was cantered. The purified poly(8) In RNA thus obtained was used in the following experiments. f21cl) Synthesis of NA NA was synthesized using Po1y (A>InRNA 41Nr, not shown below). Reaction i1> i+i, 100μ! 50mM Tris-11Cits if
(pH 8,:l); +1 to 10mM; CI znia 0mM KCl lI +nM dithi 1 threitol. 0.5InM dTTl', dcTl', dATl',
Labeled with clGTI' (fLLl, dCTl' is 32I), specific activity 4.4x 1106cp/nmola)
+ 37ziHA Rigo (dT) +2-18.80! l
! - place)') Bone fg sex self 111115j +i Irus Yamaki reversal'JJ-drunk chord. At 431℃! After reacting for 0 minutes, the reaction was stopped with an ICI) TΔ aqueous solution. Extract the cl)NA-RO11SENA complex by Noruk11=1rutomixture (1:1), 1. The fish was given birth to Tanol and recovered. To the history, Arno J Rikat, 11 processing to Riro + RN
After decomposing and removing A, the synthesized medium chain cl)NA is
The precipitate was recovered using an I-tank/oil. This ll' + strand cl) NA precipitation is the reaction of Doki 611 formation! D %t+11440/7pI knee tri8' (L Buko. Reaction j〆 dl!; 0.5mM dA1'l', d'"Tl', dGT
l', dcTl'; 5 m to 1 to 11flC+ 2
: 70mM KCI;
) NA Borime Seise■ (No/Fusogume/To) included 1
O, IM Hepes (ll(!p+・s) i;<)
廿1ill: (1)II li, 9). I! + 'C and react for 20 hours] Heavy chain c I)N
Synthesize A! , :. On the other hand - 1k to nosil l/,
+ Add acid trichloride and water-soluble Ilk to carry out the -C reaction; (
11 11 ゛, mountain chain cl) NA with phenol [+
11 l-l-1, extracted with 1.1-tanol and l'-4. Tonosashi did 11'l once. ? 11) Precipitate the single strand cl) NA with 5oIIIM
II11 acid. ) ') □n l, (+>II 4.5), I m
M ZnSO4, 200mM NaC+. The hairpino structure was dissolved in 100 ul of an aqueous solution containing 0.5% Glyl+I+-L and 0.5% s1 nucleogamma-ase and reacted at 37°C for 20 minutes to cleave the hairpino structure. The reaction was stopped by adding an aqueous solution of El)TA, extracted with a mixture of phenol, 1+j,+; was recovered. (31-A ribo(dc)dale addition cl) Preparation of NA The double strand cl) obtained above was reacted with the following composition.
Add 100 μl of A 8i solution: (react at 7°C for 20 minutes, double-stranded cl) NA to A ribo(dc) pool (=
I added it. Reaction buffer: lmM COCl2.0. ImMnch” Slay Thor. 0.2trg poly(^>, 0.1InM “If −
dcTl'(relative llr tl: 5400(:111/
11101) and 130m containing the terminyl deoxynuclease tisyltransferase at position 1
M11:I acid retrieval, -:
10mM Tris-11Cl r'JI,
(1) II li, 8). - reaction is ICl)
After adding the TA aqueous solution and stopping, the phenoroof 11
Extract with I+Forl-mixture rI&, and further extract with ether for 11
f After extraction, Meligo (CIC) Dale addition cl) NA
was precipitated and recovered with Cortanol. Add this to I (1'In to 4 Tri s -11CI
tA reaction ilk (+) II 7.4>, I mM
l) A ribo(c+c) boule-added cpNA was dissolved in an aqueous solution containing TA and 100mM NaCI to contain 0.2uHHo)A ribo(c+c) boule-added cpNA per 11 days. (4) Preparation of 20 mM Tris-11CI tA solution (+
)117.4), l0nA4hUrc I 2.
50mM (Nl+4) 2sOa and 0 per 11,
I Ilg bovine blood l,', water containing albumin R4
ilk 100μp and pimejin 322 1OμlH7$
Restriction fermentation/) l'sL l:+:tonucleγ
-Add about 15 jpg of water; (react at 7°C for 1 hour)
is. After the reaction was completed, the reaction solution was extracted with phenol, and 1) NA was recovered from the aqueous layer by ethanol precipitation. 1) Dissolve the NA in the aqueous solution 1Ik (however, it contains "l l d G T P instead of 'If - <IcTl') 200111, which was used in addition to the above-mentioned 'ribo(dC)t-lef-1'. 1. About 10-15 dG residues were incorporated by reacting with synucleotidyltransferase gQjl'4 at 37°C for 20 minutes. The reaction solution was extracted with a water-saturated phenol-chloroform mixture,
Ribo(da)dale-adducted boulasmid l) 11 R'3221) NA was recovered from the aqueous layer by ethanol precipitation. Answer this: / (dC) Daley 4' Jjl
13 R322I)NA was included in the same buffer i1k as in the case of lcl)NA. (51II'll filtered body Brasmil's 4'1-made Karirigo (dC) Daley j addition cl) NA soluble i1 * 50 t
t 1 to 1 Rigo (c + 6 > tail visit P 11 R32
21) Mix with NA solution 50/lp,
℃ for 10 min, 57°C for 120 min, 45°C (;
0 min; 60 min at 15°C and 60 min at room temperature;
1. Incubate and perform annealing. A tll recombinant psismite d) solution was prepared. (6) Selection of transformants Using the recombinant plasmid solution obtained in the manual, F
, , coli χ177 (8 strains were transformed, i.e.
E. coli χ1771H strain was treated with diaminopimelic acid 1
Absorbance ((ioonw
) or 0.5, collect the bacterial cells in a centrifugal tube with a shaker, and add 50mM CaC+2).
lornM Tris-11CI t5&iii'i
(1) 117, :1N0ml and centrifuged again at 0°C to precipitate. The collected bacterial cells were dispersed in the same buffer solution 21 and allowed to stand at 0°C for 5 minutes. This dispersion 11M0.2m
J to l. Add and mix 0.11 of the silk IJ recombinant busismid solution,
It was left at 0°C for 15 minutes, and then kept at 42°C for 2 minutes.
1 of 1. -) IIsu051 was added and shaken for 1 hour, followed by i-culture. Take part of this culture medium 1'; ink and add 1 containing futonlikurin 15111H/if in addition to the ingredients mentioned in 11'I.
．． -No. 1 cold person ゛l', spread to II zo: (about 7°C! Incubate for 2 hours, buttoshiribokuri/〆11 t'l
I Tatsumi QI the bacteria! L/tecl) NA series I'1
It's made 7. ('/) Heino' redi lane, / test + iif, i
own t2I) Regarding N A soino series, ・Nrido broiling,) necrotic cause J'=l-tcl) 111 types of punsmite containing NA 37 p- 21s, i cl) Nhezo l'-16:
I'll be there: I 11 Knee + High Shiridia''
-''/, non-test Harino h7 (1lanal+an
) et al.'s method [Gcnc, 10. line-3 according to li:[l980)]. 32 +1-labeled cl) NAzo [1-b is 1. The IJ method in section (1) (11) and assuming that m RNA is υ1' type, (2
) was synthesized using method h. However, "I'-dcTI'
uses the quotient radiation 1i'Q specific activity 1+1, and lX'f+
I felt a deep sense of +jil. In this test, a recombinant psismite containing a 1.7-group sequence that binds strongly to the 1-1 group of the induced Ωpsos and does not bind to the 1-1 group of the induced mylis. The body was sorted. Approximately 2:11 to 50:1 to 50 were selected. These selected i7. I 11, the hybridization-1 translane test was carried out with "Moreki, Sea Re-11-2/G" [1+Iania
tis. T,,(!La1..((!υ”Mo1ac2ular
CloniB”, Ko 12++ (1980).
) is written in 1. According to the power of 1, it is 111. Bushisumi FI from each form fi1 transformant I)NA'a: l
1 liter was taken out, heat denatured on Nido 11 Cell 1 North filter, and then fixed, and then the poly(^)m RN containing the rabbit malignant death factor mRNA obtained in section (1) was added to this.
Fraction A was added and reacted at 50°C for 180 minutes, followed by hybridization. After recovering the bound mRNA, it was injected into the upper cells of African megaleech eggs, and whether the recovered mRNA was the cause of necrosis or not. was tested. As a result of this test, 13 strains were found that had a plasmid containing cl)NA that highly hybridized with NA of UriF*;pa+necrosis rIII from the 20 form TI transformants selected above. The most outrageous cl) NA
(approximately 750 bp) was subjected to restriction restriction to cut out the NA fragment 1"1 and use it as a block IJ-node for secondary screening. This l)NAIA4'tJ'+ was labeled with 321), and about the cl) NA schizosily obtained in -1 section (6)l')
Once again, a hybridization test was conducted.
A transformant containing a plasmid containing cl)NA, which strongly binds to 1-zo, was selected. cl) About the monthly j I: of the NA library! 18 positive: I
I had a knee. From these, cl)NA was extracted using the restriction enzyme 1'sLI, and its size was determined by polyacrylic)' midgel electrophoresis.
1',)-! I selected 17 pieces of glue that would make 1 item. The form containing the largest cpNA among these 4L, etc.
F 802, Ri +, + -7 conversion 1) NA number Shi piece 1)
R1''N P2O3), RI+'- 1
) NA at the 111th position (and (multiple h-method sites), (sequence determined.
i744'A, (old 'NF30) selected with 71 sn
2) to which pimelic acid and timidium were added
, -J7'1 was cultured with IJ solution to obtain bacterial cells. This 1η body is 1. - (Wilkie) et al.
leic 8cids Rcs, 7,8541 (19
79)] and process the plasmid l) NA?
1) It was. This /cysmid l) NA is limited; tlte u) is understood, II'I preparation 1: iE is cloned 1)
NA? −). 1) Incubate the NA fragment with various restriction enzymes. Then, use appropriate restriction enzymes (Maxam-Gi 1bcr) to mark the sequence.
L) Dephosphorylated by jJ-, terminal G77 by 32P!
Two secrets: 1i-based properties (targeted IT decomposition reaction, gel electrophoresis and salts thereof), (sequences are as shown in Table 1. Reference Example 3 Wu IJ1 'l:;4 Cause of necrosis 1' 0) 'lj
kit 1'+'i Woori 1. (body lI'i, 2.5
~Mouth 1. Larium aenas (Larium aenas)
) Dead bacterial bodies were 50 g'a: 11: injected from ll' II vein. After 811 hours, Ir was injected 11 degrees from the 11th vein, and after 2 +11f, the heart If!
Whole blood was collected. 10 per 1001 to i+k collected
After adding ＼varinoritrium of fI′I in 0, 5
．． OOOrpm: (perform centrifugation for 0 minutes,
11 balls and undissolved solids were removed. From 400 rabbits: 1,000 units of blood with a titer of 11 5I241
! was gotten. Then add 24g of TA (151) to 121+1" and 240+r of Ceciite and stir for 1 hour (after stirring for 1", the pore size will change to Iu).
, Ht and 02μ filters for d·3. a'j 1ffl 24 N to 0.04M Tri
5-11Cj buffer ink' (1)II 7.8)12
After adding /, 0. 0.04M containing IM NaCI
T r i 5-1Icllil', 廿+1lk(p
1) 1ζAE-77 which was fully 11ζ equilibrated with H7, 8)
71-S C1, -e11 (Pharmaciane 1) column (27
0.04M 'r r including I, is -11c I
FD column a (rllll 7.1t) After sequential washing with 50N, 0.04MT containing 0.18M NaCI
r i s -11CI buffer 1IlX (pH 7,2)
It was eluted using The eluate was fractionated by 8p and active fractions were collected. This active vI fraction contains 0.04M of the same volume ii1.
Tris-11CI tA reaction i & (1) II 7.
After diluting by adding 8>,
'l-s C1, -011 column (Iox Ill,,
, ) to (,1. Then, Q containing 0.IMNaCI
,04MTris-11cI
117.8) After washing with 1 &, 0.18
0.04 M T r i s −1 containing M NaCI
Elution was performed using 1Cl kA buffer (1)II 7.2)5N. For elution i+, 250 ml fractions were fractionated and active fractions were collected. Next, transfer this l Furukawa itk to Kurei J:i, soak it in a hot water bath at 70°C, and stir until it elutes in 1! The temperature of ++0
It was heated to ℃. followed by another 4S' at 0°C
Transfer to If+, heat treat for 10 minutes, and then immediately
Cooled to °C. The heat-treated solution was concentrated by ultra-A4 filtration. 0.005 M salt/acid buffer containing 0, IM NaCl (
7F 7Cryl S-2 fully equilibrated with pit 7.4)
00 (Pharmacia) column (5X 1loci)
The active fraction was collected in 401 portions and filtered into dark blue by ultraviolet a.2 filtration. It was obtained by 4 gel filtration. Zn2
+ chelate ceph 10-silicon (=I') + chelate ceph l', +- Kara” (1,1iX20(:I)
, IIIg/Il+ zinc chloride water + 8 i & 120
ml is 1jLL. Then O, 0 containing IMNaCI
The dark blue 11k obtained by +ii1:I''1''5 was eluted with the same buffer (=tL) after equilibration with 05M phosphoric acid i. The J1 absorption 4'i 1iii fraction was collected. Most of the activity was recovered in both of these fractions. The activity 1<
+ fraction 'd:', flii L, 0.15M Na
0.005 Mυ acid buffer containing C+ (pH 7,4)
The mixture was applied to a column (1.5 x 90 cm) of Toyopearl IIW-55 (Toyo Soda Co., Ltd. J1) which had been sufficiently equilibrated. The IN:, lt L > 1 fraction was collected using the same solution. The recovery of activity throughout the entire purification process is (1
0%, the degree of purification was 7.5X 104 times. Thus obtained fl'i-manufactured Wooli Tsuki ship necrosis r j upper V [U, OX 106 K/II1g
i myself was dialed (the definition of PI 1' is from Unexamined Patent Publication No. 5
8-174: Same as that of +410 shish). In addition, in biological experiments using M (・Ll + eight meat) li transplanted into mice, the 1′ value per mouse: 1,000 to 5
, 000 ++ The story of injecting i'j into a vein t'
l was (+) or -1. 1 Attachment 1'('r +71Claims (1) j'ripebudite or similar biological story V1 containing the amino acid sequence of human yeast YI41/mortality or its active te + center portion, and immunologically intersecting 1' lipeptidate. (2) The amino acid sequence of human necrosis r is Scr SC
r Scr Arc: 'lr l'ro ScrΔs
l) 1. l's l'r. ValΔla l1is V(tl V; tl Ala
Δs n P r (l GI n A I a(il
ll (j15' (ill 1.cu (iln
Trpl, cu Δs n A r It A r R
AlaAsnAlalcu summer, euAlaAsn(il
yVat(iluLCu Az!Asp'Asn, (
iln 1. cu Vat Val I'ro 5cr
Gllll (ily 1.Cu TVr 1.-(!L
I Ilc TVr Ser (iln Vall, C
u I'l+C1,ys Gly Gln Gly C
ys l'ro sct Tbr old s Val 1. c.
u1. cu TI+r l1is Thr Ilc
Scr Arl? +1a Ala Val Ser T
yr Gin Tl+r 1. ys Val A9n1
, euLcuScrA'lal summer cT, ysSerPr
++Cys(ilnΔr++ Glu Thr Pro
GILI Gly Ala Glu Ala 1. y
sI'roTrl)TyrGluProllcTyrl
, cu'GlyGlyVal phylum 1c Gin 1. c.
u Glu I,ys GIST Asp Ar11
1. cuScr Ala Glu Ilc Asn A
rFI I'ro AsII Tyr 1. Cu eight p
PbcΔIaGluScrGlyGIITValTyr
I'heGly Ilc I'lCAla 1. '1 The polypeptide according to claim 1. 1'r +r with another polypeptide attached to the N-terminus
1. The polypeptide according to claim 1. Particularly, γ1 has McL bound to the N-terminus, Claim 1
'J'i+it! Crosspiece polypeptide′. (5) The following amino acid sequence 8 letSerThrGluSerMcLIlcΔrg
AsllValGlu 1. eu Ala Glu G
lu Ala 1. Cu Pro 1. ys 1. ys
Tllr Gly (ily I'ro GILI ni IsI Scr Ar+!ΔrI+cys1, cu P
l+C1, cu, Scr 1. cu l'l+c S
cr I'bc 1. cu 1lcValΔIa (i
ly Ala Thr Thr Lcu I'l+c
Cys 1. cul, cu l1is PhCGly
V, tl 1iCGly Prr+ Gln ArgG
l u (i I u I' II c I' r
+)Δr! rAsp lcu Scr 1. cu l1
cScrI'ro1. cu81aGlnAlaνalA
rgSCrScrScr Arg Tbr I'ro
Sar As1) Lys l'ro Val AIa
llis Val Val Ala Asn l'ro
Gln 8Ia Glu GlyGln 1. cu
GIn Trp l, cu Asn Arg Arg
Ala AsnAla 1. cu I-, cu Ala
As n G ly V a l G l u +
,, cu ΔrgASI) Asn Gin Lc
u Val Val Pro Scr Glu Gly
LcuTyrLcuIICTyrScrGinV; [1
LcuI'bc1, ysGlyGlnGlyCysl'
roScrThrsummer1isVa11, Cu1. cuTh
rIlisThrllcScrArIHIN'cAla
Val Scr,' Tyr Gln TI+, r L
ys Val Asn 1. eu 1. Cu5cr A
la Ilc LyS Scr Pro Cys'! n Ari'G111'I'l+r l'ro'GI
u Gly 1Ala Glu Ala Lys I'
ro TrpTyr Glu Pro Ilc Tyr
1. cu Gly Gly Val Gate 1Cblll
lcu Glu Lys Gly As1) Arg
Lcu Ser Ala(+lu Ilc Asn
Arl + I'r! l Asp Tyr Lcu As
p I'll (!AI+t Glu Scr Gly
(iln Val Tyr Pl+c Gly lIC
11c Ala Leu 1! Ripe boot. (6) fi = 1 produced by microorganisms or cultured cells;
γ1 The polypeptide according to any one of claims 1 to 5. (7) J≦1, which coats a polypeptide containing the amino acid sequence of human islet necrosis factor or its active center portion;
1) NA or an allelically digested 5°C form of 1) NA, which has the sequence 1) NA or an allelically digested 5°C form of 1) NA, including reduction or partial modification of the transmission coat. l) NA. Arg Glu Tbr Pro Glu Gly A
la GluΔla 1. ys1'ro Trp Ty
r Glu I'ro Ilc Tyr Leu Gl
y Gly8S1 Asn Gln 1. cu Va
l V；Ll l'ro SCr Glu (ilyt
Cu Tyr Lcu Ilc Tyr Scr Gl
n Val 1. cu l'bc11, c Ala L
cu (101hi) ii++ necrotic acid salt coating amino acid sequence, 1. The sequence is (5')-TCA TCT TCT CGAΔCCCC
(i AGT GACAAGCCT C; TA GCC
CAT GTT GTA GCA AACCCT CA
AGCT GAG GGG CAG CTCCAG T
GG CTG AACCGCCGG GCCAAT G
CCCTCCTG GCCAAT GGCGTGGAG
CT(i AGA C1AT AACCAG CTG
GTG (iTG CCATCA GAG GGCC
TG TACCTCATCTACTCCCAGGTCC
TCTTCAAG GGCCAΔG (i CT G C
CCCT CCACCCAT GTG CTCCTCA
CCCACACCATCAGCC (ic ATCGCC
GTCTCCTACCAG
CTCCTCTCT GCCATCAAG AGCCC
CTGCAG AG (i GAG ACCCAG
AG GGG GCT GAG GCCAAG CCC
TGG TAT CAG CCCATCTAT CTG
GGA(iGG GTC'rTCCAG CTG C
AG AAG GGT GACCGAC'l' C8G
CGCT GAG ATCAAT CGG CCCGA
CTΔ1CTCGACTTT GCCGAG TCT
GGG CAG GTCTACr'l'T (IGG
ATCATT (icc CTG-(3') '1
11' + i"l request) ko) range 71st ri or 8th Kawara battle 1) NAo (IIJ:; 'Start at end'"/A'rG
Special +i'F request for 911! Box No. 7 l) NA as stated in JJI. +171 and (the horse) -5 self tari (fi') -
AT(JAGCACTGAAAGCATGATCCGG
GAC to T (1GAGC'r to GCCGAGGAGGC
GCTCCCCAAGΔ8 8 CA G G (i
G G GCCCCA (i et al GCTCC8GG C
GGTGCT'lG'I'TCCTCAGCCTCT
CTCCTTCCTGATCCTG CCA GGCG
CCACCACCG CTCTTCT (icCTG CT
G CACTTT GGA GTG ATCGGCCC
CCAGAGG GAA GAG TTCCCCAGG
GACCTCTCT CT 8 to TCACCCCCT C
TG GCCCAC GCA GTCA (iA TCAT
CT TCT CGA ACCCCG AGT CAC
AAG CCT (iTΔGCCCAT GTT GT
A GCA AACCCT CAA GCT GAGG
G(i CAG CTCCACTGG CTG 8AC
cce ccc ccc to 8A (icc CTCCT
G GCCAAT GGc GT(i CAG CT(
iAGA CAT AACCACCT (i GTG C
TG CCA TCA GAGGGCCTCTACCT
CATCTACTCCCA (i GTCCTCTTCA
ACGGCCAA G(ic TGCCCCTCCAC
CCATGTG CTCCTCACCCACACCAT
CA(ic CGCATCG CCG T C1'C
CT A CCA G ACCAACGTCΔA(:
CTCCTCTCT GCCATCAAG AGCCC
CT(lc CAG AGGGAG 8CCCCA G
AG GGG GCT GAG GCCAAG CCC
TGG TAT GAG CCCATC AT CTC
GGA GGG GTCTTCCA('; CTG G
AG AAG GGT CACCGA CTC8CCG
CT GAG ATCAAT CGG CCCGACT
AT CTCGACTTT GCCCAG TCT G
GG CACGTCACTTT GGGicCTGA
TT (icc CTG-(3')) Claim 7 or () of claim 1) 1. I) Vector containing NA δ・11. 0.11 vector or expression vector
' The vector according to item 13. ψL1 Claims No. 1 or 18th Source 1. C-type [Transformed disease.

Claims (1)

[Scope of claims] Amino acid sequence of human 1<; necrosis
r Ser Artr Tbr Pro Scr As
1) Lys l'rnVatΔIa l1is Va
l Val Ala Asn l'ro Gln Al
aG l u G I y et al In Lcu Gln
Trl)1. cu Asn ΔrIHArlBAla
Hesn Ala 1. eu 1. eu Ala Asn
Gly Vat GluI Cu Artr As1
l Asn Gln +, Cu Val V, LI I
'ro 5crtiluGlylCul'yr1. eu
Summer 1cTyrScrGlnVaSummer 1, eul'hc1.
ys(ilyGlnGlyCysI'roS[)rTb
r11isVat'1. Cu1. cuTl+rIlis
TbrIlcScrAr+++1CAlaValSCr
'I'yrGln1'l+rl, ysVaIAsn■,
cu 1. Cu Ser, Ala”llc 1.ys
Scr Pro Cys GinArg Glu T
hr Pro Glu Gly Ala Glu Al
a LysPro Trp Tyr Glu Pro
lie Tyr Lcu Gly Gly Vat Ph
c Gin Leu Glu Lys Gly Asp
Arg LcuSer Ala Glu lle A
sn Arg l'ro ASI) Tyr LeuA
sp I'he Ala Glu Ser Gly G
in Vat Tyr pH (!Gly Ile Il
e The polypeptide of claim 1 which is Ala Leu. (3) A polypeptide according to claim 1 or 2, which has another polypeptide bonded to its N-terminus. (The polypeptide according to any one of Claims 1JI'1 to 3, in which McL is bound to the 41 N-terminus. (5) The following amino acid sequence McLSetTI+rGluScrMetIlcΔrg
Asl) ValGlu Leu Ala Glu Gl
u Ala 1. cu Pro Lys LysTl+
rGlyGlyProGinGlySerLysArg
Cys+, cu Phe Lcu Scr 1. eu
l'he Scr Pl+c Lcu 1lcVal
Ala Gly Ala Thr Tl+r Lcu
l'l+c Cys LcuLeu 1lis Phc
Gly Val lle Gly Pro Gln
ArgGlu Glu Pbe l'ro Arg A
sp Leu Scr Leu 1ieSer Pro
Leu Ala Gln Ala Val Arg
Scr 5crScr Ari+ Thr Pro S
et Asp Lys I'ro Val ΔIa11
is Val Val Ala Asn l'ro G
in Ala Glu GlyGln Leu Gln
Trp1. eu Asn Arg Arg Ala
AsnAla Lcu 1. cu Ala Asn
Gly Val Glu Leu Arg Asll A
sn Gln leu Val Val l'ro S
cr Glu GlyLcuTyrlieuIlcTy
rScrGinVal+, cuPl+c1, ys Gl
y Gln Gly Cys Pro Ser Tl+
r l1is Vall, cu Lcu Tbr 1l
is Thr Ile Scr Arg lie Al
aVal Scr Tyr Gin TI+r Lys
Val Asn Lcu LcuScr Ala I
lc1. ys Scr Pro Cys Gin Δ
rgGIt+TI+r Pro Glu Gly Al
a Glu Ala Lys Pro TrpTyr
Glu I'ro lie Tyr Lau Gly
Gly Val Phe(iln Lcu Glu L
ys Gly Asp Arg +, cu Scr A
la(:1ulle Asn Arg l'ro AS
I) Tyr LCu Asp I'bcA I a
(i I u S cr et Iy Gln Val
Tyr Pl+c Gly II (!+1 e Al;
The polypeptide according to No. 3 JII, wherein t LCu is 4-. (6) The polypeptide according to any one of claims 1 to 5]l'I, which is produced by a microorganism or cultured cells. (7) The amino acid sequence of human cancer necrosis factor or the nucleotide sequence that coats the polypeptide containing its active center portion 1) NA or allelic gene r- mutation, i mountain or part of the gene code 1) A variant of NA comprising the modification. (8) The nucleotide sequence that forms two nodes of the amino acid sequence of human cancer necrosis factor is (5')-TCA TCT TCT CGA 8CCC
CG AGT CACΔAGCCT GTA GCCC
AT GTT GTA GCA AACCCT CAA
GCT GAG GGG CAG CTCCAG TG
G CTG AACCGCCGG GCCAAT GC
CCTCCTG GCCAAT GGCGTGGAG
CTG AGA CAT AACCAG CTGGTG
GTG CCATCΔGAGGGCCTG TACC
TCATCTACTCCCCAGGTCCTCTTCAA
G GGCCAA GGCTGCCCCTCCACCC
AT GTG CTCCTCACCACACACCATC
AGCCGCATC(icc GTCTCCTACCA
G ACCAAG GTCAAACCTCCTCTCT
GCCATCAAGAGCCCCTGCCAGAG
G CAG ACCCCA GAG GGG GCT
GA (i GCCAAG CCCTGG TAT GA
G CCCATCTAT CTG GGAGGG GT
CTTCCAG CTG GAG AAG GGT C
ACCGACTCAGCGCT GAG ATC, AA
T CGG CCCGACTA'rCTCGACTTT
GCCGAG TCT GGG CAG GTCTA
CTTT GGG ATCATT GCCCTG-(3
”) 1) NA of Claim 7 JJ1
. (Start at the 5' end of the sword: A special u that makes Notono ATG
l' Claim 7JJ'I or 8th statement I)NA
o(10) The following salt, (sequence (5')-ATG AGCACT GAA AGCAT
G ATCC to G GACG1'G CAG CT(i
GCCGAG GAG GCG CTCCCCΔAG
ΔΔGACΔ GGG GGG CCCCAG GGC
TCC,'tAG CGGTGCTTG TTCCTC
AGCCTCTTCTCCT'rCCTGATCGTG
GCA GGCGCACCACCG CTCTTCT
GCCT (i CTG CACT'rT (iGA G
TG ATCGGc CCCCAGAGG GAA G
AG TTCCCCAGG GACCTCCT CT
AATCA(ic CCT CTG GCCCAG G
CA GTCAGA i' CATCT TCT C (
+8 ACCCCG AGT GACAAG CCT
GTAGCCCAT GTT GTA GCA AAC
CCT CAA GCT GAGGGCCAG CTC
CAG TGG CTG AACCGCCGG GCC
AAT GCCCTCCTG GCCAAT GGCG
TG GAG CT (iAGA GAT AACCAG
CTG GTG GTG CCA TCA GAGG
GCCTG TACCTCATCTACTCCCAG
GTCCTCTTCAAG GGCCAA ccc T
GCCCCTCCCACCCATGTG CTCCTCA
CCCACACCA1'CAGCCC; CATCGCC
G1'CTCCTACCAG ACCAAG GTCA
ACCTCCTCTCT GCCATCAAG AGC
CCCTGCCAG AGGGAG ACCCCA G
A(i GGG GCT GAG GCCAAG CC
CTGG TAT GAG CCCATCTAT CT
(i CGA (i (i G (i T CTTCC
AG CTG GAG AAG GGT CACCGA
CTCAGCGCT GAG ATCAAT CGG
CCCGACTAT CTCGACT'rT GCC
GAG TCT GGG CAG GTCTACTTT
GGGATCATT GCCcTc-(3')
J'l Claim No. 7 Section 1) NA. 01) 1) A vector incorporating NA according to any one of claims 7 to 10 JJ1. The vector according to claim 1, which is an expression vector or a gene expression vector. θ) Particularly ii'l' A host transformed with the vector according to claim 1f or 12. 14. The host according to claim 13, which is a round-transformed host, microorganism, or cultured cell.