JPH0157955B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human cancer necrosis factor polypeptides, cloned DNAs encoding them, vectors incorporating these cloned DNAs, and hosts transformed with these vectors. The following abbreviations are used herein to simplify the description. A Adenine C Cytosine G Guanine T Thymine Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid Cys Cysteine Gln Glutamine Glu Glutamate Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine Trp Tryptophan Tyr Tyr Tyr Shin Val Valine DNA Deoxyribonucleic acid cDNA Complementary DNA RNA Ribonucleic acid mRNA Messenger RNA dATP Deoxyadenosine triphosphate dCTP Deoxycytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate oligo (dC) Oligodeoxycytidylate oligo (dG) Oligodeoxyguanylate (dT) Oligodeoxythymidylate poly(A) Polyadenylate poly(U) Polyuridylate poly(dA) Polydeoxyadenylate poly(dC) Polydeoxycytidylate poly(dG) Polydeoxyguanylate poly(dG) (dT) polydeoxythymidylate EDTA ethylenediaminetetraacetic acid bp base pair Carswell et al.
- In the serum of mice infected with Gue'rin (BCG) and then treated with endotoxin,
discovered that it contained a substance that caused necrosis in cancer caused by Meth A sarcoma, and named this substance cancer necrosis factor [Proc.Nat.Acad.Sci.USA, 72 , 3666
(1975)]. Cancer necrosis factor is considered to be a physiologically active substance released from macrophages, and its characteristics include:
(i) When administered to tumor-bearing animals, it causes necrosis and cure of certain cancers. (ii) In vitro, certain cancer cells (e.g. mouse cancer L cells, human cancer-derived cancer cells)
It is known that (iii) its effects are 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, conventional methods for producing cancer necrosis factor require BCG or
Since the method involves injecting Propionibacterium acnes, then administering endotoxin, and then isolating and purifying cancer necrosis factor from the blood and body fluids, 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. However, the above method cannot be adopted for producing human-derived cancer necrosis factor. Anti-tumor cytotoxin, a factor produced by normal human peripheral monocytic cells or myeloid monocytic leukemia cells, was previously reported by Matthews as a cancer cytotoxic factor derived from human macrophages.
[Immunology, 44 , 135 (1981)] and a factor produced by culture vessel wall-adherent cells in human peripheral blood (Adherent cell toxin) reported by Reed et al. [J.
Immunol., 115 , 395 (1975)]. In addition, Williamson et al. reported a substance with a molecular weight of approximately 70,000 Daltons produced from an established human B cell line as a human cancer necrosis factor [Proc. Nat.
Acad.Sci.USA, 80 , 5397 (1983)]. However, in each case, the substance as a substance was unknown. In order to establish a method for mass production of human-derived cancer necrosis factor, the present inventors conducted intensive research focusing on genetic recombination technology, and first isolated cloned DNA encoding rabbit cancer necrosis factor. It was successful (see patent application No. 58-228790). As a result of further research,
Using this cloned DNA as a probe, we succeeded in cloning a cDNA encoding human cancer necrosis factor, and further demonstrated that microorganisms transformed with a plasmid incorporating this cloned DNA produced human cancer necrosis factor. After confirming this, the present invention was completed. As used herein, human cancer necrosis factor is a protein produced by human-derived cells, and has the ability to at least injure L cells in vitro, and at least the ability to cause necrosis in transplanted Meth A sarcoma cancers in vivo. Cancer necrosis factor activity means such biological activity. The present invention provides polypeptides comprising the amino acid sequence of human cancer necrosis factor or its active center portion, and cloned DNA encoding them. In particular, the following amino acid sequence () Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Ala His Val Val Ala Asn Pro Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg
Arg Ala Asn Ala Leu Leu Ala Asn Gly Val
Glu Leu Arg Asp Asn Gln Leu Val Val Pro
Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln
Val Leu Phe Lys Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Ile Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys
Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu Glu Lys Gly Asp Arg
Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr
Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr
Human cancer necrosis factor having Phe Gly Ile Ile Ala Leu () [hereinafter referred to as polypeptide ()], the N-terminus of the polypeptide () further has the following amino acid sequence () Met Ser Thr Glu Ser Met Ile Arg Asp
Val Glu Leu Ala Glu Glu Ala Leu Pro Lys
Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg
Cys Leu Phe Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe Cys
Leu Leu His Phe Gly Val Ile Gly Pro Gln
Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg
() [hereinafter referred to as polypeptide (+
)], and a polypeptide having Met at the N-terminus of the polypeptide (), and the following base sequence () (5')-TCA TCT TCT CGA ACC CCG
AGT GAC AAG CCT GTA GCC CAT GTT
GTA GCA AAC CCT CAA GCT GAG GGG
CAG CTC CAG TGG CTG AAC CGC CGG
GCC AAT GCC CTC CTG GCC AAT GGC
GTG GAG CTG AGA GAT AAC CAG
CTG GTG GTG CCA TCA GAG GGC CTG
TAC CTC ATC TAC TCC CAG GTC CTC
TTC AAG GGC CAA GGC TGC CCC TCC
ACC CAT GTG CTC CTC ACC CAC ACC
ATC AGC CGC ATC GCC GTC TCC TAC
CAG ACC AAG GTC AAC CTC CTC TCT
GCC ATC AAG AGC CCC TGC CAG AGG
GAG ACC CCA GAG GGG GCT GAG GCC
AAG CCC TGG TAT GAG CCC ATC TAT
CTG GGA GGG GTC TTC CAG CTG GAG
AAG GGT GAC CGA CTC AGC GCT GAG
ATC AAT CGG CCC GAC TAT CTC GAC
TTT GCC GAC TCT GGG CAG GTC TAC
TTT GGG ATC ATT GCC CTG−(3′)
A cloned DNA encoding polyppeptide () having () [hereinafter referred to as DNA ()] and
The following base sequence () (5')-ATG AGC ACT GAA AGC ATG at the 5' end of the DNA ()
ATC CGG GAC GTG GAG CTG GCC GAG
GAG GCG CTC CCC AAG AAG ACA GGG
GGG CCC CAG GGC TCC AGG CGG TGC
TTG TTC CTC AGC CTC TTC TCC TTC
CTG ATC GTG GCA GGC GCC ACC ACG
CTC TTC TGC CTG CTG CAC TTT GGA
GTG ATC GGC CCC CAG AGG GAA GAG
TTC CCC AGG GAC CTC TCT CTA ATC
AGC CCT CTG GCC CAG GCA GTC AGA
-(3′) (), a cloned DNA encoding a polypeptide (+) [hereinafter referred to as DNA (+)],
and a DNA having an initiation codon ATG at the 5' end of the DNA (). As used herein, a polypeptide comprising the amino acid sequence of human cancer necrosis factor is a polypeptide () or a polypeptide having an amino acid or peptide bound to its N-terminus or C-terminus, and
This term refers to polypeptides that exhibit the same human cancer necrosis factor activity as the polypeptide (2012), either by themselves or by being cleaved with an enzyme or the like. Furthermore, a polypeptide () containing an essential amino acid sequence for expressing human cancer necrosis factor activity among the amino acid sequences of a polypeptide () containing an active center portion of the amino acid sequence of human cancer necrosis factor; means a substance that exhibits human cancer necrosis factor activity similar to that of . The present invention includes not only polypeptides containing the amino acid sequence of human cancer necrosis factor or its active center, DNA encoding them, vectors incorporating those DNAs, and hosts transformed with these vectors, but also As long as the resulting polypeptides have high homology with the polypeptides of the present invention (), exhibit similar human cancer necrosis factor activity, and are immunologically cross-reactive, such polypeptides and their Also included are DNAs encoding these, vectors incorporating those DNAs, and hosts transformed with these vectors. For example, allelic variants of human cancer necrosis factor DNA, derivatives of human cancer necrosis factor DNA containing degeneracy or partial modifications of the genetic code, and polypeptides obtained therefrom are also included in the present invention. Note that the derivative of human cancer necrosis factor DNA containing a partial modification refers to a derivative in which a portion of human cancer necrosis factor DNA is deleted and a derivative in which another DNA fragment is added to human cancer necrosis factor DNA. DNA having a base sequence encoding the human cancer necrosis factor of the present invention can be produced, for example, as follows. (1) Cloning encoding rabbit cancer necrosis factor
Preparation of DNA Rabbit cancer necrosis factor cDNA is obtained according to the method disclosed in Japanese Patent Application No. 58-228790 (for example, the method of Reference Example 2). This cloned DNA
The nucleotide sequence of is shown in Table 1, and the region encoding the polypeptide of rabbit cancer necrosis factor is
Numbers 277-738. This rabbit cancer necrosis factor cDNA is treated with a restriction enzyme to give a DNA fragment of an appropriate length, and the DNA fragment is cut out and used as a probe for cloning the human cancer necrosis factor cDNA described below. As a restriction enzyme, for example, Ava
, Hae, BstN, Hinc, and Rsa. DNA fragments originate from different regions2
It is preferable to use more than one species. Table 1 shows the cleavage sites for cutting out DNA fragments of 299 bp and 88 bp using Ava and Hae as restriction enzymes, respectively. [Table] ｜ ｜
｜ ｜
｜ ｜
AAGCCTCTAGCCCAC
GTAGTAGCAAACCCGCA
AGTGGAGGGCCAGCTC
CAGTGGCTGAGC
TTCGGAGATCGGGTG
CATCATCGTTTGGGCGT
TCACCTCCCGGTCGAG
GTCACCGACTCG
370 380
390 400
410 420
｜ ｜
｜ ｜
｜ ｜
CAGCGTGCGAACGCC
CTGCTGGCCAACGGCAT
GAAGCTCACGGACAAC
CAGCTGGTGGTG
GTCGCACGCTTGCGG
GACGACCGGTTGCCGTA
CTTCGAGTGCCTGTTG
GTCGACCACCAC
[Table] ｜ ｜
｜ ｜
｜ ｜
TTTGGGATCATTGCC
CTGTGAGGGGACTGACC
ACCACTCCTCCCCCTC
TCCCACCCCAGC
AAACCCTAGTAACGG
GACACTCCCCTGACTGG
TGGTGAGGAGGGGGAG
AGGGTGGGGTCG
790 800

｜ ｜


CCCCTCACTCTGGGC
GCCCTCAG



GGGGAGTGAGACCCG
CGGGAGTC



〓 〓 indicates the region encoding rabbit cancer necrosis factor.
(2) Preparation of human cancer necrosis factor mRNA Human macrophages were isolated from alveoli, blood, peritoneal cavity,
Taken from the placenta, spleen, and other tissues. For example, from the alveoli, the method of Sone et al. [J. Immunol.
129, 1313 (1982)], the method of Matthews [Br.J.Cancer, 48 , 405 (1983)] for blood, and the method of Wilson et al. [J.Immunological
Methods, 56 , 305 (1983)].
Approximately 2 macrophages per 1 cm ² of culture vessel surface
Sow ×10 ⁴ to 10 ⁶ pieces, 35 to 38℃, preferably about 37℃
°C, in air containing about 5-10% carbon dioxide, humidity about 90
Preincubate at ~100% for approximately 30 minutes to 2 hours. Next, endotoxin obtained from Gram-negative bacteria, such as lipopolysaccharide derived from Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi, is added, and the culture is continued for 3 to 8 hours to induce the production of human cancer necrosis factor mRNA. At this time, human cancer necrosis factor mRNA can be accumulated by adding a protein synthesis inhibitor (eg, cycloheximide). Note that the pre-culture may be omitted. The amount of endotoxin added is generally about 0.1 to 1000 μg/ml (final concentration, the same applies below), preferably about 1 to 100 μg/ml.
It is. At this time, phorbol esters such as phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-dibenzoate are added to
You may add 2000ng/ml. The amount of protein synthesis inhibitor added varies depending on the type of compound, etc.
For example, in the case of cycloheximide, 0.1 to 50 μg/
ml. As the medium, various synthetic media suitable for culturing higher animal cells are used, such as
RPMI-1640, Eagle's MEM medium, MEM medium by Dulbecco's modified method (compositions of the above media are described, for example, in "Cell Culture Manual" edited by Kosuke Munemura, Kodansha, 1982)
can be mentioned. The medium contains approximately 1 to 10% of the total culture solution volume.
It is preferable to add 20% animal serum (eg fetal bovine serum, calf serum). After culturing,
Cells were isolated using conventional methods, such as the method of Chirgwin et al. [Biochemistry, 18 , 5294 (1979)].
RNA is extracted and then subjected to adsorption column chromatography using oligo(dT) cellulose or poly(U) Sepharose (Pharmacia) according to a conventional method, or the poly(A) mRNA fraction is isolated using the batch method. Separate. this
Human cancer necrosis factor mRNA can be concentrated and purified by subjecting the poly(A) mRNA fraction to acid urea agarose gel electrophoresis or sucrose density gradient centrifugation. In order to confirm that the mRNA fraction obtained here contains mRNA encoding the desired human cancer necrosis factor, it is sufficient to translate the mRNA into protein and examine its biological activity.
For example, the African clawed frog (Xenopus laevis)
mRNA was injected or added to an appropriate protein synthesis system such as oocytes, reticulocyte lysate, or wheat germ to be translated into protein, and the protein showed cytotoxic activity against mouse L-929 cells. This is done by checking. The method using Xenopus oocytes is
For example, this is done as follows. Approximately 50 ng of mRNA per oocyte was injected using the microinjection method, and 10 of them were placed in 100 μ of Barth culture medium [Gurdon, JB, J. Embryol. Exp.
Morphol., 20 , 401 (1968)], 24-72 at 22°C.
After culturing for an hour and homogenizing, the centrifuged supernatant (10,000 rpm, 10 minutes) was used as a sample for L
-929 cytotoxic activity is used as an indicator to measure cancer necrosis factor activity [method for measuring L-929 cytotoxic activity is Ruff, MR, et al., J. Immunol., 125 ;
1671 (1980)]. (3) Cloning of human cancer necrosis factor cDNA Using the mRNA obtained in step (2) as a template,
Using oligo (dT) as a primer according to the method of Gubler et al. [Gene, 25 , 263 (1983)],
Single strands complementary to mRNA by reverse transcriptase (e.g. reverse transcriptase from avian myeloid leukemia virus) in the presence of dATP, dGTP, dCTP, dTTP
cDNA is synthesized, and then double-stranded cDNA is synthesized using E. coli ribonuclease H and E. coli DNA polymerase. DNA obtained here
, poly(dG) − poly(dC) or poly(dA) −
Poly(dT) homopolymer extension method [Noda, M.,
et al., Nature, 295 , 202 (1982); Nelson,
T.S., “Methods in Enzymology,” 68 , 41.
(1979), Academic Press Inc., New York]
For example, plasmid
Incorporate into the restriction enzyme Pst cleavage site of pBR322. The obtained recombinant plasmid, for example
Cohen et al.'s method [Proc.Nat.Acad.Sci.USA,
69, 2110 (1972)], for example, E. coli
Introduce it into a host such as the χ1776 strain, transform it, select a tetracycline-resistant strain, and extract the cDNA.
Create a library. Regarding this cDNA library, the DNA fragment encoding rabbit cancer necrosis factor obtained in step (1) labeled with ³² P was used as a probe.
Colony hybridization test [Hanahan, D., et al., Gene, 10 , 63
(1980)] to screen for clones that hybridize with the rabbit cancer necrosis factor cDNA fragment. Many clones obtained in this way
The nucleotide sequence of the DNA was analyzed, for example, according to the Maxam -Gilbert method [Proc. Nat. Acad. Sci. A cloned DNA having a base sequence encoding a human cancer necrosis factor polypeptide is obtained by searching for a base sequence with a specific character and finally selecting a cDNA containing a base sequence corresponding to the entire translated region of human cancer necrosis factor. be able to. The recombinant plasmid containing the cloned DNA thus obtained is introduced into a host such as E. coli strain HB101 to obtain a transformant, which is then cultured to determine the cytotoxic activity of L-929. Human cancer necrosis factor polypeptide can be obtained by separating and purifying the protein using this as an indicator. In order to further increase the expression efficiency, the human cancer necrosis factor-encoding region of the cloned DNA of the present invention is cut out using an appropriate restriction enzyme, and then inserted into a expression vector having an appropriate promoter to induce human cancer necrosis. A vector for producing a factor polypeptide may be created. For example, as shown in the attached drawing, the DNA encoding the human cancer necrosis factor cloned in the Examples is treated with the restriction enzyme Ava, and the nucleotide sequence in Table 4 is
Obtain the DNA fragment from the 250th position onwards, and insert the deleted part on the 5′ end (TCA-TCT-TCT-CGA-
A DNA in which the start codon ATG has been added to ACC) is chemically synthesized, this is ligated to the above-mentioned Ava cleavage site, and then the DNA is inserted into a promoter-containing expression vector. Examples of promoters include lac
promoter, trp promoter, tac promoter, _PL promoter, SV40 early promoter, etc. Any vector that can be used to propagate in the host to be transformed can be used. For example, plasmids (pBR322, pDR540
), phage (lambda phage derivatives, etc.),
Examples include viruses (SV40, etc.). These may be used alone or in combination, e.g. pBR322
- It may be used in the form of a SV40 hybrid plasmid or the like. A vector incorporating the cloned DNA thus obtained is allowed to act on a suitable host for transformation using a conventional method to obtain a host for expression.
Hosts used for transformation include microorganisms such as Escherichia coli, Bacillus subtilis, yeast, COS monkey, etc.
Examples include cultured animal cells such as cells and cultured plant cells. Human cancer necrosis factor polypeptide can be obtained by culturing the thus obtained transformant according to a conventional method and purifying the culture using TNF activity as an indicator. The human cancer necrosis factor of the present invention has an amino acid sequence with extremely high homology to rabbit cancer necrosis factor, and high homology is also observed in the DNA 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,
Table 3 shows the homology of the base sequences of the DNAs encoding the precursors of human cancer necrosis factor and rabbit cancer necrosis factor. As explained in Japanese Patent Application No. 58-228790, rabbit cancer necrosis factor exists in the form of an aggregate in the absence of urea, starting from Ser at position 82 and Ser at position 236 shown in Table 2. A polypeptide consisting of 154 amino acids ending with Leu is thought to be its basic building block. That is, it is thought that the N-terminal portion of the precursor polypeptide is cleaved and activated by an appropriate enzyme in vivo. In the case of human cancer necrosis factor, it also begins at Ser 82 and begins at Ser 236.
A polypeptide consisting of 155 amino acids ending with Leu is considered to be the basic building block of human cancer necrosis factor. As is clear from Tables 2 and 3, the homology between the amino acid sequences of the human and rabbit cancer necrosis factor polypeptides is 81%, and the homology between the DNA base sequences encoding them is 85%. In addition, high homology was observed between human cancer necrosis factor precursor and rabbit cancer necrosis factor precursor, with polypeptide amino acid sequence homology of 79% and base sequence homology of 84%. be. This high degree of homology between human and rabbit cancer necrosis factors suggests that both factors are phylogenetically derived from a common molecule, and that the highly homologous regions of cancer necrosis factors are associated with cancer necrosis factors. This suggests that there is a region essential for the expression of biological activity, and that this region is the active center of cancer necrosis factor. [Table] [Table] [Table] [Table] [Table] [Table] Human cancer necrosis factor, as shown in the test example below,
There is no immunological interaction with rabbit cancer necrosis factor,
It is a polypeptide that is immunologically distinguishable from rabbit cancer necrosis factor. Thus, although human and rabbit cancer necrosis factors show high overall homology, there are clear differences in the properties of their polypeptides. This means that the entire amino acid sequence of cancer necrosis factor is not essential for its biological activity, and if the amino acid sequence of the part that constitutes the active center is the same, the amino acid sequence of other parts will change slightly, and accordingly, the polypeptide This shows that the activity of cancer necrosis factor is maintained even if the properties of cancer necrosis factor change. Based on the knowledge regarding the amino acid sequence and DNA base sequence of the human cancer necrosis factor polypeptide revealed by the present invention, a polypeptide containing the amino acid sequence of the human cancer necrosis factor or human cancer necrosis It is possible to obtain a DNA encoding a polypeptide containing the active center portion of the factor or a partially modified variant of the human cancer necrosis factor DNA, and use them to easily produce a polypeptide having human cancer necrosis factor activity. can be produced. In addition, the DNA revealed by the present invention
By chemically synthesizing a part of the base sequence and using it as a probe, it is possible to easily obtain human cancer necrosis factor DNA, which has allelic variations and degenerate genetic code, and use it to detect human cancer necrosis factor activity. It is possible to produce a polypeptide having the following.
Furthermore, based on the amino acid sequence of human cancer necrosis factor polypeptide, the nucleotide sequence encoding it was determined.
It is also possible to totally synthesize DNA. All of these are included within the scope of this invention. Since the human cancer necrosis factor polypeptide of the present invention has excellent antitumor effects, it can be used as an antitumor agent in the treatment of malignant tumors. The present invention will be explained in more detail below with reference to Examples and Reference Examples, but the present invention is not limited to these Examples. Example (1) Cloning encoding human cancer necrosis factor
Preparation of probe for detecting DNA Clones encoding rabbit cancer necrosis factor shown in Table 1 (already mentioned) were prepared according to the method disclosed in Japanese Patent Application No. 58-228790 (for example, the method of Reference Example 2). DNA was obtained. Restrict this DNA with a restriction enzyme
DNA fragments consisting of 88 bp from 33rd to 120th and 285th bp were excised using Hae and Ava.
A DNA fragment consisting of 299 bp of ~583 was obtained. The 299bp DNA fragment excised with the restriction enzyme Ava is a region encoding rabbit cancer necrosis factor.
The 88 bp DNA fragment excised with Hae corresponds to the region encoding rabbit tumor necrosis factor precursor. These two types of DNA fragments were labeled with ³² P and used as probes for cloning human cancer necrosis factor cDNA in Section (8). (2) Isolation of mRNA fraction from human alveolar macrophages 6.3 x 10 ⁷ alveolar macrophages were collected from human lungs using phosphate buffered saline. This alveolar macrophage was treated with 10% fetal bovine serum.
Suspended in RPMI-1640 medium, seeded on Petri dishes (8 cm in diameter) at 9 x ¹⁰⁶ cells per plate, and incubated at 37°C in air containing 5% carbon dioxide.
Preculture was performed at a humidity of 90-100%. After 1 hour of pre-incubation, endotoxin (lipopolysaccharide from E. coli), TPA (phorbol-12-myristate-13-acetate) and cycloheximide were added to a final concentration of 10 μg/ml and 10n, respectively.
The mixture was added and mixed at a concentration of 1 g/ml and 1 μg/ml, and the culture was continued. After 4 to 4.5 hours (5 to 5.5 hours in total), the culture medium was removed by suction, and the macrophages remaining on the date were dissolved in a 5M guanidyl thiocyanate solution containing 0.6% sodium lauroyl sarcosinate and 6mM sodium citrate. Homogenized. This homogenate was layered on a 5.7M cesium chloride aqueous solution containing 0.1M EDTA, and then placed in an ultracentrifuge (RPS27
-26500rpm using 2 rotors (Hitachi, Ltd.)
The mixture was centrifuged for 20 hours to obtain a total RNA fraction as a pellet. This was dissolved in a small amount of 7M urea solution containing 0.35M NaCl, 20mM Tris and 20mM EDTA, and collected as an ethanol precipitate. all
159 μg of RNA was obtained. This total RNA fraction was added to 10 μg containing 1 mM EDTA
It was dissolved in 1 ml of mM Tris-HCl buffer (PH7.4) (hereinafter referred to as TE solution) and heated at 65°C for 5 minutes. After adding NaCl solution to 0.5M, it is applied to an oligo(dT) cellulose column equilibrated with TE solution containing 0.5M NaCl, and the adsorbed poly(A) mRNA is eluted with TE solution. As a result, 8 μg of poly(A) mRNA was obtained. This poly(A) mRNA was dissolved in distilled water at a concentration of 1.9 ng/n, injected at a rate of 50 n per Xenopus oocyte, and translated in the oocytes according to the method described above. protein L
−929 cytotoxic activity was measured. As a result, 10
An activity of 6.6 units/ml was observed in 0.1 ml of a homogenate of 2000 oocytes, confirming that this poly(A) mRNA preparation contained human cancer necrosis factor mRNA. (3) Synthesis of cDNA using the poly(A) mRNA obtained in section (2) as a template.
cDNA was synthesized according to the method of Gubler et al. [Gene, 25 , 263 (1983)]. Reaction volume: 40μ 50mM Tris-HCl buffer (PH8.3): 10mM
MgCl ₂ ; 10mM dithiothreitol; 4mM
Sodium pyrophosphate; 1.25mM
dGTP, dATP, dTTP; 0.5mM dCTP;
0.167μM α- ³² P-dCTP (specific activity 3000Ci/m
mole); 4μg oligo(dT) _12-18 ; 6μg poly(A)
mRNA; 120 units avian myeloid leukemia virus-derived reverse transcriptase. After reacting at 43°C for 30 minutes, EDTA was added to stop the reaction, extracted with a phenol-chloroform mixture (1:1), and ammonium acetate was added to the aqueous layer to a final concentration of 2.5M.
Reaction product with ethanol (single-stranded cDNA-
RNA complexes) were precipitated. This single-stranded cDNA
- Transfer the RNA complex to 100 μl of reaction buffer with the following composition.
dissolved in Reaction buffer composition: 20mM Tris-HCl buffer (PH7.5); 5mM
MgCl ₂ ; 10mM (NH ₄ ) ₂ SO ₄ ; 100mM
KCl; 0.15mM β-nicotinamide adenine dinucleotide; 50μg/ml bovine serum albumin; 40μM dGTP, dATP, dTTP,
dCTP; 0.9 units Escherichia coli ribonuclease H; 23
Unit Escherichia coli DNA polymerase. After reacting at 12°C for 60 minutes and then at 22°C for 30 minutes, the reaction was stopped by adding EDTA, extracted with a phenol-chloroform mixture in the same manner as above, and precipitated with ethanol to obtain double-stranded cDNA.
I got it. (4) Preparation of oligo(dC) tailed cDNA Add 100μ of the reaction buffer with the following composition to the double-stranded cDNA obtained in section (3), react at 37°C for 30 minutes, and add the oligo(dC) tail to the double-stranded cDNA. (dC) A tail was added. Reaction buffer; 2mM _CoCl2 , 0.2mM dithiothreitol, 0.1mM α- ^32P -dCTP (specific activity 3Ci/m
100m containing 10 units of terminal deoxynucleotidyl transferase (mole) and 10 units of terminal deoxynucleotidyl transferase.
M Sodium cacodylate (PH7.2). The reaction was stopped by adding EDTA aqueous solution.
It was extracted with a phenol-chloroform mixture, and the oligo(dC) tailed cDNA was precipitated with ethanol and recovered. This was added to 10mM Tris-
HCl buffer (PH7.4), 1mM EDTA and 100
The oligo(dC)-tailed cDNA was dissolved in an aqueous solution containing mM NaCl at a concentration of 2 μg per ml. (5) Oligo (dG) tailed plasmid
Preparation of pBR322DNA 20mM Tris-HCl buffer (PH7.4), 10mM
Aqueous solution containing MgCl ₂ , 50 mM (NH ₄ ) ₂ SO ₄ and 0.1 mg bovine serum albumin per ml.
Dissolve 10μg of pBR322 in 100μ, add restriction enzyme Pst
Add 15 units of endonuclease and incubate at 37°C.
Allowed time to react. After the reaction was completed, the reaction solution was extracted with a phenol-chloroform mixture, and DNA was recovered from the aqueous layer by ethanol precipitation. The resulting DNA was added to an aqueous solution of the aforementioned oligo(dC) tail (however, ³² P−dCTP was used instead of
^3H -dGTP) and reacted with 80 units of terminal deoxynucleotidyl transferase at 37°C for 20 min.
~15 dG residues were incorporated. The reaction solution was extracted with a phenol-chloroform mixture, and the oligo(dG) tailed plasmid pBR322DNA was recovered from the aqueous layer by ethanol precipitation.
This was dissolved in the same buffer as for the oligo(dC)-tailed cDNA so that 20 μg of oligo(dG)-tailed plasmid pBR322DNA was contained per ml. (6) Preparation of recombinant plasmid Addition of oligo(dC) tail obtained in section (4)
Mix 120μ of the cDNA solution with 120μ of the oligo(dG)-tailed pBR322DNA solution obtained in section (5), and incubate at 65℃ for 5 minutes and 57℃ for 120 minutes to perform annealing. Prepared. (7) Selection of transformants E. coli χ1766 strain (ATCC No. 31244) was transformed using the recombinant plasmid solution obtained in section (6). That is, E. coli
Culture in 20 ml of L-broth supplemented with 1.5 ml of L-broth at 37°C until the absorbance (600 nm) becomes 0.5, and then incubate the cells at 4°C.
10mM containing 50mM _CaCl2 .
Disperse in 10ml of Tris-HCl buffer (PH7.3) and
The mixture was centrifuged again at ℃ to precipitate it. The collected bacterial cells were dispersed in 2 ml of the same buffer solution and allowed to stand at 0°C for 5 minutes. Add 0.1 ml of the recombinant plasmid solution obtained in section (6) to 0.2 ml of this dispersion, mix, and heat at 0°C.
After standing still for 15 minutes and further holding at 42℃ for 2 minutes,
L-broth with the same composition as used in the previous culture
0.5 ml was added and cultured with shaking for 1 hour. A portion of this culture solution was taken and spread on an L-broth agar plate containing 15 μg/ml of tetracycline in addition to the above-mentioned ingredients, and cultured at 37°C for about 12 hours to select tetracycline-resistant bacteria and create a cDNA library. did. (8) Cloning The cDNA library obtained in section (7) was used to screen for transformants carrying a plasmid containing cDNA encoding human cancer necrosis factor. A colony hybridization test was performed using a ³² P-labeled fragment (299 bp) of the DNA encoding the DNA cut out with the restriction enzyme Ava as a probe.
It was carried out according to the method of Hanahan et al. [Gene, 10 , 63 (1980)]. From about 20,000 colonies, 43 transformants were selected that had a recombinant plasmid containing a base sequence that strongly binds to this labeled probe. Furthermore, for these 43 colonies, a DNA fragment (88 bp) corresponding to the upstream region of the rabbit tumor necrosis factor coding region was excised using the restriction enzyme Hae.
Secondary screening was performed using a ³² P-labeled substance as a probe, and six colonies that strongly hybridized with this probe were selected. Through these two rounds of screening, DNA containing a DNA base sequence encoding rabbit cancer necrosis factor and a base sequence highly homologous to the upstream portion thereof was obtained. (9) Character expression Six clones selected in section (8) (Plasmid numbers pHTNF-1, -4, -5, -13, -22, -26)
The respective recombinant plasmids were extracted from the plasmids, and transformants were obtained using E. coli HB101 strain (ATCC No. 33694) as a host. For each transformant, the method of Nagata et al.
284, 316 (1980)].
LB broth was used as the medium. Absorbance (600nm)
The cells were cultured until the number of cells reached about 0.8, and about 3 to 5 x ¹⁰¹⁰ cells were collected. 50m containing 0.1% lysozyme
M Tris-HCl buffer (PH8.0) and 30mM
Suspend the bacterial cells in 1 ml of a solution consisting of NaCl,
After standing in ice water for 30 minutes, the cells were repeatedly frozen and thawed six times to destroy the cells, and the cells were centrifuged to obtain a cell extract. As a result of measuring the L-929 cytotoxic activity of the extract obtained from each transformant, an activity of 186.1 units/ml was observed in the extract of the transformant using plasmid pHTNF-13. (10) Determination of base sequence of cloned DNA Recombinant plasmid selected in section (9)
The pHTNF-13 transformant was cultured in LB broth to obtain bacterial cells. This bacterial cell was collected using the method of Wilkie et al. [Nucleic Acids Res., 7 , 859 (1979)]
Plasmid DNA was obtained. This plasmid DNA was digested with the restriction enzyme Pst,
Cloned DNA was obtained by separation and purification. This cloned DNA fragment is digested with various restriction enzymes,
For 16 types of restriction enzyme fragments, each base sequence was dephosphorylated using the Maxam-Gilbert method.
It was determined by terminal labeling with ³² P, base-specific chemical decomposition reaction, gel electrophoresis, and autoradiography. The figure shows the cleavage site of the restriction enzyme used to determine the base sequence and the direction and range (indicated by arrows) where the base sequence was determined. The portion indicates the region encoding human cancer necrosis factor precursor polypeptide. The base sequence and the amino acid sequence translated from this base sequence are shown in Table 4. The region encoding the polypeptide of human cancer necrosis factor is
Estimation was made based on homology with the nucleotide sequence encoding rabbit cancer necrosis factor shown in Table 1. That is, the human cancer necrosis factor polypeptide starts from codons 235 to 237 of TCA (corresponding to Ser) in Table 4, and CTG (corresponds to Leu) to codons 697 to 699.
It consists of 155 amino acid residues.
The C-terminal leucine codon is followed by a TGA stop codon. Base sequences 1 to 234 are considered to be sequences necessary to encode the precursor of human cancer necrosis factor. [Table] [Table] [Table] Reference Example 1 Preparation of anti-rabbit cancer necrosis factor antibody Purified rabbit cancer obtained according to the method disclosed in Japanese Patent Application No. 58-228790 (for example, the method of Reference Example 3) A solution containing 1.9 x 10 ⁵ units of necrosis factor was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at several points on the back of guinea pigs. after that,
Immunization was performed in the same manner 1, 3 and 6 weeks later. After a further 8 weeks, the same amount of purified rabbit cancer necrosis factor was injected intraperitoneally with aluminum hydroxide gel. Nine weeks after the first immunization, whole blood was collected from the heart, and the serum was separated by centrifugation to obtain antiserum containing anti-rabbit cancer necrosis factor antibody. This antiserum was repeatedly passed through a Sepharose 4B (Pharmacia) column coupled with normal rabbit serum components three times to completely remove antibodies against rabbit serum components and to remove only specific antibodies against rabbit cancer necrosis factor. A purified antiserum was obtained. This antiserum formed a single sedimentation line only with purified rabbit cancer necrosis factor by immunoelectrophoresis and in-gel double diffusion. An approximately 60,000-fold dilution of this purified antiserum has the ability to neutralize 50% of the 500 units of L-929 cytotoxic activity of rabbit cancer necrosis factor. Reference Example 2 (1) Isolation and purification of mRNA fraction from rabbit alveolar macrophages Propionibacterium acnes was injected into a rabbit (approximately 2.5 kg in weight).
Acne) dead bacteria were injected intravenously at a dose of 100 mg per bird, and the birds were sacrificed 8 days later. Immediately, a thoracic tracheotomy was performed, and lung lavage was repeated using phosphate-buffered saline through a tube inserted into the trachea, and alveolar macrophages were collected. Approximately 3×10 ⁹ alveolar macrophages were obtained from 12 rabbits. The alveolar macrophages were suspended in RPMI-1640 medium containing 10% fetal bovine serum and placed in a Petri dish (diameter 8 cm) at 2×
They were seeded at 10 ⁷ cells and precultured at 37° C. in air containing 5% carbon dioxide at a humidity of 90 to 100%. 1
After a preincubation period of 10 h, endotoxin (lipopolysaccharide from E. coli), TPA (phorbol-12-myristate-13-acetate) and cycloheximide were each added to a final concentration of 10 μl.
They were added and mixed so that the concentrations were 1 g/ml, 10 ng/ml, and 1 μg/ml, and the culture was continued. After 4 to 4.5 hours (5 to 5.5 hours in total), remove the culture medium by suction and remove the macrophages remaining on the date.
0.6% sodium lauroyl sarcosinate and 6
It was dissolved and homogenized in 5M guanidyl thiocyanate solution containing mM sodium citrate. This homogenate containing 0.1M EDTA
Layered on 5.7M cesium chloride aqueous solution and ultracentrifuged (RPS27-2 rotor, Hitachi, Ltd.)
The whole RNA fraction was centrifuged at 26,500 rpm for 20 hours to obtain a pellet. Add this to 0.35M NaCl, 20
It was dissolved in a small amount of 7M urea solution containing mM Tris and 20mM EDTA and collected as an ethanol precipitate. 5.2 mg of total RNA was obtained from 12 rabbits. This total RNA fraction was mixed with 10 μM EDTA.
It was dissolved in 2 ml of mM Tris-HCl buffer (PH7.4) (hereinafter referred to as TE solution) and heated at 65°C for 5 minutes. After adding NaCl solution to 0.5M, it is applied to an oligo(dT) cellulose column equilibrated with TE solution containing 0.5M NaCl, and the adsorbed poly(A) mRNA is eluted with TE solution. As a result, 314 μg of poly(A) mRNA was obtained.
220 μg of the poly(A) mRNA obtained here was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6M urea, PH4), and 7
Divide into two fractions and thaw the gel (70°C, 10 minutes)
After that, the mixture was extracted with water-saturated phenol and chloroform, and then precipitated with ethanol to recover poly(A) mRNA from each fraction. The amount of rabbit cancer necrosis factor mRNA was measured for poly(A) mRNA in each fraction by a method using Xenopus oocytes, and rabbit cancer necrosis factor mRNA was enriched in the fraction corresponding to a molecular size of 1.6 to 2.7 kb. Collected at a concentration. This purified poly
The specific activity of (A) mRNA is approximately 1230 units/μg RNA, and the specific activity of the final purified poly(A) mRNA preparation is approximately
It was about 4 times more concentrated than 320 units/μg RNA. The purified poly(A) mRNA obtained here was used in the following experiment. (2) Synthesis of cDNA Using 4 μg of purified poly(A) mRNA, cDNA was synthesized under the conditions shown below. Reaction solution volume: 100μ 50mM Tris-HCl buffer (PH8.3); 10mM
_MgCl2 ; 70mM KCl; 1mM dithiothreitol; 0.5mM dTTP, dCTP, dATP,
dGTP (however, dCTP is labeled with ³² P, specific activity 4.4×
¹⁰⁶ cpm/nmole); 3μg oligo(dT) _12-18 , 80
Unit avian myeloid leukemia virus-derived reverse transcriptase. After reacting at 43°C for 90 minutes, the reaction was stopped with an aqueous EDTA solution. The cDNA-mRNA complex was extracted with a phenol-chloroform mixture (1:1), precipitated with ethanol, and collected. Furthermore, by alkali heating treatment
Single chain synthesized after mRNA is degraded and removed
cDNA was recovered and precipitated with ethanol. Transfer this single-stranded cDNA to 40μ of reaction buffer with the following composition.
dissolved in Reaction buffer; 0.5mM dATP, dTTP, dGTP, dCTP;
5mM _MgCl2 ; 70mM KCl; 1.5mM β
- Murcaptoethanol; 8 units E. coli DNA
0.1 M Hepes buffer (PH 6.9) containing polymerase (large fragment). Double-stranded cDNA was synthesized by reacting at 15°C for 20 hours. The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction solution, and the double-stranded cDNA was extracted with a phenol-chloroform mixture, precipitated with ethanol, and collected. The obtained double-stranded cDNA was precipitated with 50mM sodium acetate (PH4.5), 1mM, ZnSO ₄ , 200mM
Dissolved in 100μ of an aqueous solution containing M NaCl, 0.5% glycerol and 0.5 units of S1 nuclease,
The hairpin structure was cleaved by reacting at 37°C for 20 minutes. The reaction was stopped by adding an aqueous EDTA solution, extracted with a phenol-chloroform mixture, and
After further extraction with ether, cDNA was recovered by precipitation with ethanol. (3) Preparation of oligo(dC)-tailed cDNA Add 100μ of the reaction buffer with the following composition to the double-stranded cDNA obtained above, react at 37°C for 20 minutes, and add oligo(dC) tails to the double-stranded cDNA. was added. Reaction buffer: 1mM CoCl ₂ , 0.1mM dithiothreitol, 0.2 μg poly(A), 0.1mM ³ H-dCTP (specific activity 5400 cpm/pmole) and 130mM cacodyl containing 10 units of terminal deoxynucleotidyl transferase. acid sodium 630m
M Tris.HCl buffer (PH6.8). The reaction was stopped by adding EDTA aqueous solution.
After extraction with a phenol-chloroform mixture and further extraction with ether, the oligo(dC) tailed cDNA was precipitated with ethanol and recovered. This was mixed with 10mM Tris-HCl buffer (PH
7.4), the oligo(dC)-tailed cDNA was dissolved in an aqueous solution containing 1mM EDTA and 100mM NaCl to contain 2μg per ml. (4) Oligo (dG) tailed plasmid
Preparation of pBR322DNA 20mM Tris-HCl buffer (PH7.4), 10mM
MgCl ₂ , 50mM (NH ₄ ) ₂ SO ₄ and per ml
100μ of an aqueous solution containing 0.1mg of bovine serum albumin
10 μg of pBR322 was dissolved in the solution, 15 units of restriction enzyme Pst endonuclease was added, and the mixture was reacted at 37° C. for 1 hour. After the reaction is complete, the reaction solution is extracted with phenol, and the aqueous layer is precipitated with ethanol.
DNA was recovered. The obtained DNA was added to the aqueous solution ( ^3H
-contains ^3H -dGTP instead of dCTP) 200μ
and reacted with 80 units of terminal deoxynucleotidyl transferase at 37°C for 20 minutes to incorporate approximately 10-15 dG residues. The reaction solution was extracted with a water-saturated phenol-chloroform mixture, and the oligo(dG)-tailed plasmid was extracted from the aqueous layer by ethanol precipitation.
pBR322DNA was recovered. This is oligo (dC)
1 ml of the same buffer as for tailed cDNA
Each sample was lysed to contain 2 μg of oligo(dG) tailed plasmid pBR322 DNA. (5) Preparation of recombinant plasmid Add 50μ of oligo(dC) tailed cDNA solution to 50μ of oligo(dG) tailed pBR322DNA solution.
Mix with 65℃ for 10 minutes and 57℃ for 120 minutes.
Annealing was performed by incubating at 45°C for 60 minutes, at 35°C for 60 minutes, and at room temperature for 60 minutes to prepare a recombinant plasmid solution. (6) Selection of transformants E. coli χ1776 strain was transformed using the recombinant plasmid solution obtained above. That is,
E. coli χ1776 strain was treated with diaminopimelic acid 100μ
The cells were cultured in 20 ml of L-broth supplemented with 40 μg/ml and 40 μg/ml of thymidine at 37°C until the absorbance (600 nm) reached 0.5, and the cells were collected using a _centrifuge with a condenser. −HCl
It was dispersed in 10 ml of buffer solution (PH7.3) and centrifuged again at 0°C to precipitate it. The collected bacterial cells were added to the same buffer solution 2.
ml and allowed to stand at 0°C for 5 minutes. Add 0.1ml of the above recombinant plasmid solution to 0.2ml of this dispersion.
Add and mix, leave at 0°C for 15 minutes, and then heat to 42°C.
After holding for 2 minutes, 0.5 ml of L-broth having the same composition as used in the previous culture was added and cultured with shaking for 1 hour. A portion of this culture solution was spread on an L-broth agar plate containing 15 μg/ml of tetracycline in addition to the above ingredients, and cultured at 37°C for about 12 hours to select tetracycline-resistant bacteria.
A cDNA library was created. (7) Hybridization test To screen for transformants carrying a plasmid containing cDNA encoding rabbit cancer necrosis factor using the above cDNA library.
A colony hybridization test using a ^32P -labeled cDNA probe was performed using the method of Hanahan et al. [Gene, 10 , 63 (1980)].
I followed. ³² The PcDNA probe was prepared using mRNA obtained from induced plus and minus alveolar macrophages using the method described in (1) above as a template, (2)
It was synthesized using the method described in Section 2. However, ³² P-dCTP with high radioactivity specific activity was used and labeled at a high concentration. Through this test, transformants were selected that had a recombinant plasmid containing a base sequence that strongly bound to the induction-plus probe but did not hybridize to the induction-minus probe. Fifty colonies were selected from approximately 20,000 colonies. These selected strains are then subjected to hybridization and translation tests called “molecular cloning”.
[Maniatis, T., et al., (ed) “Molecular
Cloning”, 329 (1982), Cold Spring Harbor
Lab.]. Extract plasmid DNA from each transformant,
After heat denaturation and fixation on a nitrocellulose filter, the poly(A) mRNA fraction containing rabbit cancer necrosis factor mRNA obtained in section (1) above was added to this and reacted at 50°C for 180 minutes to perform hybridization. I went to a seminar. After elution and recovery of the bound mRNA, it was injected into Xenopus oocytes, and the recovered mRNA was expressed as rabbit cancer necrosis factor.
It was tested whether it was mRNA or not. Through this test, three strains were found out of the 20 transformants selected above that had plasmids containing cDNA that strongly hybridized with rabbit tumor necrosis factor mRNA. The longest cDNA (approx.
750bp) from a plasmid with restriction enzyme
A DNA fragment was excised with Dde and used as a probe for secondary screening. This DNA fragment
A colony hybridization test was performed again on the cDNA library labeled with ³² P and obtained in section (6) above, and transformants having a plasmid containing cDNA that strongly binds to the labeled probe were selected. Of approximately 60,000 colonies in the cDNA library, 98 were positive colonies. The cDNA was excised from this using the restriction enzyme Pst, and its size was examined by polyacrylamide gel electrophoresis, and 17 clones with a size of 1 kbp or more were selected. the largest of these
Transformants containing cDNA (cell number:
PTNF802, cloned DNA number:
pRTNF802), the cloned DNA was isolated and the nucleotide sequence was determined by the method described below. (8) Determination of base sequence of cloned DNA The bacterial strain (RTNF802) selected in section (7) was cultured in L-broth supplemented with diaminopimelic acid and thymidine to obtain bacterial cells. The bacterial cells were collected using the method of Wilkie et al. [Nucleic Acids].
Res., 7 , 859 (1979)] to obtain plasmid DNA. This plasmid DNA was digested with restriction enzyme Pst, separated and purified to obtain cloned DNA. This cloned DNA fragment was digested with various restriction enzymes, and each base sequence of the appropriate restriction enzyme fragment was dephosphorylated using the Maxam-Gilbert method, end-labeled with ³² P, and base-specific chemical degradation. It was determined by performing reaction, gel electrophoresis, and autoradiography. Its base sequence is shown in Table 1. Reference Example 3 Isolation and purification of rabbit cancer necrosis factor Propionibacterium acnes was applied to rabbits (body weight 2.5 to 3.0 kg).
50 mg of killed bacteria was injected through the ear vein. Eight days later, 100 μg of endotoxin (lipopolysaccharide derived from Escherichia coli) was injected into the ear vein, and 2 hours later, whole blood was collected from the heart. 100 per 100ml of blood taken
5000rpm after adding unit heparin sodium
A refrigerated centrifugation operation was performed for 30 minutes to remove blood cells and insoluble solids. 3000 units/ml from 400 rabbits
24 plasma was obtained with a titer of . This plasma 24 has 24g of EDTA and 240g of Celite.
After adding and stirring for 1 hour, the pore sizes were 3μ, 1μ and 0.2μ.
The mixture was sequentially filtered through the following filters. Add 0.04M Tris-HCl buffer (PH7.8) to filtrate 24
0.04M Tris containing 0.1M NaCl after adding 12
- Thoroughly equilibrated with HCl buffer (PH7.8)
DEAE-Sepharose CL-6B (Pharmacia)
column (27 x 45 cm). Then column equilibration buffer containing 75% and 0.15M NaCl
After sequentially washing with 50 ml of 0.04M Tris-HCl buffer (PH7.8), elution was performed using 0.04M Tris-HCl buffer (PH7.2) containing 0.18M NaCl. The eluate was fractionated into eight fractions and active fractions were collected. This active fraction was diluted by adding the same volume of 0.04M Tris-HCl buffer (PH7.8), and then applied to a DEAE-Sepharose CL-6B column (10 x 13 cm). Then 0.1M
0.04M Tris-HCl buffer (PH7.8) containing NaCl 1
containing 0.18M NaCl after washing with
Elution was performed using 0.04M Tris-HCl buffer (PH7.2). The eluate was fractionated into 250 ml portions and the active fractions were collected. Next, this eluate was transferred to a container, immersed in a 70°C water bath, and heated while stirring until the temperature of the eluate reached 60°C. Thereafter, it was transferred to another water bath at 60°C, heated for 30 minutes, and then quickly cooled to 4°C. The heat-treated solution was concentrated by ultrafiltration. 0.005M phosphate buffer (PH
7.4) Cephacryl S-200 fully equilibrated with
The concentrated solution was applied to a column (5 x 80 cm) manufactured by Pharmacia Co., Ltd., and eluted with the same buffer. The active fraction was collected in 40 ml portions and concentrated by ultrafiltration. Concentrate the active fraction obtained by gel filtration.
It was applied to a Zn ²⁺ chelate sepharose column. The method of Porath et al. [Nature, 258 , 598
(1975)] column packed with chelated cepharose (iminodiacetic acid immobilized resin) (1.6
20 cm), 120 ml of a 1 mg/ml zinc chloride aqueous solution was poured into the tube. After sufficient equilibration with 0.05M phosphate buffer (PH7.4) containing 0.1M NaCl, the concentrated solution obtained in the previous step was applied, and the non-adsorbed fraction eluted with the same buffer was collected. . Most of the activity was recovered in this fraction. The active fraction obtained in the prepurification step was concentrated and added to 0.005M phosphate buffer (PH) containing 0.15M NaCl.
7.4) was applied to a Toyopearl HW-55 (Toyo Soda Co., Ltd.) column (1.5 x 90 cm). The active fraction was collected by elution with the same buffer. The recovery rate of activity throughout the entire purification process was 60%, and the degree of purification was 7.5×10 ⁴ times. The specific activity of the purified rabbit cancer necrosis factor thus obtained was 6.0×10 ⁶ units/
mg protein (the definition of activity unit is JP-A-58-
It is the same as that of No. 174330). Furthermore, in biological evaluation using Meth A sarcoma transplanted into mice, the activity was above (+) when 3000 to 5000 units per mouse was intravenously administered. Test example Immunological properties of human cancer necrosis factor Plasmid pHTNF obtained in section (9) of Example
An equal volume of a 100-fold dilution of the purified anti-rabbit cancer necrosis factor antiserum prepared in Reference Example 1 was added to the extract of the transformant obtained by -13. After incubating the mixture at 37°C for 2 hours, the cytotoxic activity against L-929 cells was measured. The results are shown in Table 5. As is clear from Table 5, human cancer necrosis factor does not immunologically interact with rabbit cancer necrosis factor at all. [Table] Fluid discharge

Claims (1)

[Claims] 1. Human macrophages are cultured with an inducing agent, a fraction containing human cancer necrosis factor mRNA is separated from the cells, a single-stranded cDNA is produced from the mRNA using reverse transcriptase, and this is After converting into double-stranded cDNA, the DNA is inserted into a vector, the vector is introduced into a host, transformation is performed, a cDNA library is prepared, and the DNA encoding rabbit cancer necrosis factor or a fragment thereof is used as a probe. 1. A method for producing a DNA encoding human cancer necrosis factor, which comprises cloning a cDNA encoding human cancer necrosis factor from the library. 2. The DNA according to claim 1, wherein the cDNA encoding human cancer necrosis factor is a DNA encoding human cancer necrosis factor consisting of an amino acid sequence represented by the following formula or a DNA comprising the base sequence.
Manufacturing method: Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Ala His Val Val Ala Asn Pro Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg
Arg Ala Asn Ala Leu Leu Ala Asn Gly Val
Glu Leu Arg Asp Asn Gln Leu Val Val Pro
Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln
Val Leu Phe Lys Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Ile Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys
Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu Glu Lys Gly Asp Arg
Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr
Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr
Phe Gly Ile Ile Ala Leu 3 The method for producing DNA according to claim 1, wherein the cDNA encoding human cancer necrosis factor is a DNA consisting of a base sequence represented by the following formula or a DNA comprising the base sequence. : (5′)−TCA TCT TCT CGA ACC CCG
AGT GAC AAG CCT GTA GCC CAT GTT
GTA GCA AAC CCT CAA GCT GAG GGG
CAG CTC CAG TGG CTG AAC CGC CGG
GCC AAT GCC CTC CTG GCC AAT GGC
GTG GAG CTG AGA GAT AAC CAG
CTG GTG GTG CCA TCA GAG GGC CTG
TAC CTC ATC TAC TCC CAG GTC CTC
TTC AAG GGC CAA GGC TGC CCC TCC
ACC CAT GTG CTC CTC ACC CAC ACC
ATC AGC CGC ATC GCC GTC TCC TAC
CAG ACC AAG GTC AAC CTC CTC TCT
GCC ATC AAG AGC CCC TGC CAG AGG
GAG ACC CCA GAG GGG GCT GAG GCC
AAG CCC TGG TAT GAG CCC ATC TAT
CTG GGA GGG GTC TTC CAG CTG GAG
AAG GGT GAC CGA CTC AGC GCT GAG
ATC AAT CGG CCC GAC TAT CTC GAC
TTT GCC GAG TCT GGG CAG GTC TAC
TTT GGG ATC ATT GCC CTG-(3') 4 Production of DNA according to claim 1, wherein the cDNA encoding human cancer necrosis factor is DNA encoding a polypeptide consisting of an amino acid sequence represented by the following formula: Method: Met Ser Thr Glu Ser Met Ile Arg Asp
Val Glu Leu Ala Glu Glu Ala Leu Pro Lys
Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg
Cys Leu Phe Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe Cys
Leu Leu His Phe Gly Val Ile Gly Pro Gln
Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg Ser
Ser Ser Arg Thr Pro Ser Asp Lys Pro Val
Ala His Val Val Ala Asn Pro Gln Ala Glu
Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala
Asn Ala Leu Leu Ala Asn Gly Val Glu Leu
Arg Asp Asn Gln Leu Val Val Pro Ser Gln
Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu
Phe Lys Gly Gln Gly Cys Pro Ser Thr His
Val Leu Leu Thr His Thr Ile Ser Arg Ile
Ala Val Ser Tyr Gln Thr Lys Val Asn Leu
Leu Scr Ala Ile Lys Ser Pro Cys Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro
Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp
Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly
Ile Ile Ala Leu 5 The method for producing DNA according to claim 1, wherein the cDNA encoding human cancer necrosis factor is a DNA consisting of a base sequence represented by the following formula: (5')-ATG AGC ACT GAA AGC ATG
ATC CGG GAC GTG GAG CTG GCC GAG
GAG GCG CTC CCC AAG AAG ACA GGG
GGG CCC CAG GGC TCC AGG CGG TGC
TTG TTC CTC AGC CTC TTC TCC TTC
CTG ATC GTG GCA GGC GCC ACC ACG
CTC TTC TGC CTG CTG CAC TTT GGA
GTG ATC GGC CCC CAG AGG GAA GAG
TTC CCC AGG GAC CTC TCT CTA ATC
AGC CCT CTG GCC CAG GCA GTC AGA
TCA TCT TCT CGA ACC CCG AGT GAC
AAG CCT GTA GCC CAT GTT GTA GCA
AAC CCT CAA GCT GAG GGG CAG CTC
CAG TGG CTG AAC CGC CGG GCC AAT
GCC CTC CTG GCC AAT GGC GTG GAG
CTG AGA GAT AAC CAG CTG GTG
GTG CCA TCA GAG GGC CTG TAC CTC
ATC TAC TCC CAG GTC CTC TTC AAG
GGC CAA GGC TGC CCC TCC ACC CAT
GTG CTC CTC ACC CAC ACC ATC AGC
CGC ATC GCC GTC TCC TAC CAG ACC
AAG GTC AAC CTC CTC TCT GCC ATC
AAG AGC CCC TGC CAG AGG GAG ACC
CCA GAG GGG GCT GAG GCC AAG CCC
TGG TAT GAG CCC ATC TAT CTG
GGA GGG GTC TTC CAG CTG GAG AAG
GGT GAC CGA CTC AGC GCT GAG ATC
AAT CGG CCC GAC TAT CTC GAC TTT
GCC GAC TCT GGG CAG GTC TAC TTT
GGG ATC ATT GCC CTG-(3') 6 DNA encoding human cancer necrosis factor consisting of the amino acid sequence shown by the following formula or DNA comprising the base sequence: Ser Ser Ser Arg Thr Pro Ser Asp Lys
Pro Val Ala His Val Val Ala Asn Pro Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg
Arg Ala Asn Ala Leu Leu Ala Asn Gly Val
Glu Leu Arg Asp Asn Gln Leu Val Val Pro
Ser Glu Gly Leu Tyr Leu Ile Tyr Ser Gln
Val Leu Phe Lys Gly Gln Gly Cys Pro Ser
Thr His Val Leu Leu Thr His Thr Ile Ser
Arg Ile Ala Val Ser Tyr Gln Thr Lys Val
Asn Leu Leu Ser Ala Ile Lys Ser Pro Cys
Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala
Lys Pro Trp Tyr Glu Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu Glu Lys Gly Asp Arg
Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr
Leu Asp Phe Ala Glu Ser Gly Gln Val Tyr
Phe Gly Ile Ile Ala Leu 7 The DNA according to claim 6, wherein the DNA encoding human cancer necrosis factor is a DNA consisting of a base sequence represented by the following formula or a DNA comprising the base sequence: (5 ′) −TCA TCT TCT CGA ACC CCG
AGT GAC AAG CCT GTA GCC CAT GTT
GTA GCA AAC CCT CAA GCT GAG GGG
CAG CTC CAG TGG CTG AAC CGC CGG
GCC AAT GCC CTC CTG GCC AAT GGC
GTG GAG CTG AGA GAT AAC CAG
CTG GTG GTG CCA TCA GAG GGC CTG
TAC CTC ATC TAC TCC CAG GTC CTC
TTC AAG GGC CAA GGC TGC CCC TCC
ACC CAT GTG CTC CTC ACC CAC ACC
ATC AGC CGC ATC GCC GTC TCC TAC
CAG ACC AAG GTC AAC CTC CTC TCT
GCC ATC AAG AGC CCC TGC CAG AGG
GAG ACC CCA GAG GGG GCT GAG GCC
AAG CCC TGG TAT GAG CCC ATC TAT
CTG GGA GGG GTC TTC CAG CTG GAG
AAG GGT GAC CGA CTC AGC GCT GAG
ATC AAT CGG CCC GAC TAT CTC GAC
TTT GCC GAG TCT GGG CAG GTC TAC
TTT GGG ATC ATT GCC CTG-(3') 8 DNA according to claim 6, wherein the DNA encoding human cancer necrosis factor is DNA encoding a polypeptide consisting of an amino acid sequence represented by the following formula: Met Ser Thr Glu Ser Met Ile Arg Asp
Val Glu Leu Ala Glu Glu Ala Leu Pro Lys
Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg
Cys Leu Phe Leu Ser Leu Phe Ser Phe Leu
Ile Val Ala Gly Ala Thr Thr Leu Phe Cys
Leu Leu His Phe Gly Val Ile Gly Pro Gln
Arg Glu Glu Phe Pro Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg Ser
Ser Ser Arg Thr Pro Ser Asp Lys Pro Val
Ala His Val Val Ala Asn Pro Gln Ala Glu
Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala
Asn Ala Leu Leu Ala Asn Gly Val Glu Leu
Arg Asp Asn Gln Leu Val Val Pro Ser Glu
Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu
Phe Lys Gly Gln Gly Cys Pro Ser Thr His
Val Leu Leu Thr His Thr Ile Ser Arg Ile
Ala Val Ser Tyr Gln Thr Lys Val Asn Leu
Leu Ser Ala Ile Lys Ser Pro Cys Gln Arg
Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro
Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val
Phe Gln Leu Glu Lys Gly Asp Arg Leu Ser
Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp
Phe Ala Glu Ser Gly Gln Val Tyr Phe Gly
Ile Ile Ala Leu 9 DNA according to claim 6, wherein the DNA encoding human cancer necrosis factor is a DNA consisting of a base sequence represented by the following formula: (5')-ATG AGC ACT GAA AGC ATG
ATC CGG GAC GTG GAG CTG GCC GAG
GAG GCG CTC CCC AAG AAG ACA GGG
GGG CCC CAG GGC TCC AGG CGG TGC
TTG TTC CTC AGC CTC TTC TCC TTC
CTG ATC GTG GCA GGC GCC ACC ACG
CTC TTC TGC CTG CTG CAC TTT GGA
GTG ATC GGC CCC CAG AGG GAA GAG
TTC CCC AGG GAC CTC TCT CTA ATC
AGC CCT CTG GCC CAG GCA GTC AGA
TCA TCT TCT CGA ACC CCG AGT GAC
AAG CCT GTA GCC CAT GTT GTA GCA
AAC CCT CAA GCT GAG GGG CAG CTC
CAG TGG CTG ACC CGC CGG GCC AAT
GCC CTC CTG GCC AAT GGC GTG GAG
CTG AGA GAT AAC CAG CTG GTG
GTG CCA TCA GAG GGC CTG TAC CTC
ATC TAC TCC CAG GTC CTC TTC AAG
GGC CAA GGC TGC CCC TCC ACC CAT
GTG CTC CTC ACC CAC ACC ATC AGC
CGC ATC GCC GTC TCC TAC CAG ACC
AAG GTC AAC CTC CTC TCT GCC ATC
AAG AGC CCC TGC CAG AGG GAG ACC
CCA GAG GGG GCT GAG GCC AAG CCC
TGG TAT GAG CCC ATC TAT CTG
GGA GGG GTC TTC CAG CTG GAG AAG
GGT GAC CGA CTC AGC GCT GAG ATC
AAT CGG CCC GAC TAT CTC GAC TTT
GCC GAC TCT GGG CAG GTC TAC TTT
GGG ATC ATT GCC CTG− (3′)