JPH0195784A - Novel dna, novel plasmid having said dna, novel polypeptide and production thereof and novel antitumor agent consisting of said polypeptide - Google Patents

Abstract

PURPOSE:To prepare an antitumor agent, capable of inhibiting propagation of cancerous cells, by cultivating a microorganism having a DNA capable of coding a specific amino acid sequence inserted into a plasmid propagable in host microbial cells. CONSTITUTION:A DNA capable of coding an amino acid sequence [amino acid sequence of the fourth exon part of tumor necrosis factor(TNF)] expressed by formula 1 (X represents Ser or Cys) is synthesized and integrated into a plasmid propagable in host microbial cells to form a recombinant DNA, which is then used to transform a host of a microorganism, such as animal cells, yeasts, Bacillus subtilis or Escherichia coli. The resultant transformed host microorganism is subsequently cultivated to produce a polypeptide having the amino acid sequence expressed by formula 1. An antitumor agent is prepared from the above-mentioned polypeptide having the amino acid sequence expressed by formula 1.

Description

Detailed Description of the Invention [Industrial Application Fields] The present invention relates to DNA, specifically, DNA that coats an antitumor polypeptide, a method for producing the same, a plasmid containing the same, a polypeptide, a method for producing the same, and a method for producing the same. , relates to an antitumor agent comprising the polypeptide.

[Prior Art] Antitumor polypeptides of human origin are described in The Journal of Biological Chemistry (TheJ
our own of Biol, Chem)! Danri, 234
TN obtained from the human cell line HL-60 (ATCC240) described on pages 5-2354 (1985).
A polypeptide named F is known. Its amino acid sequence is as follows.

Met-Val-Arg-Ser-Ser-Ser-A
rg-Thr-Pro-Ser-Asp-Lys-Pr
o-Va I-A Ia-Hi 5-Va 1-Va
l -A Ia-Asn-Pro-G l n-A I
a-Glu-Gly-Gln-Leu-G In-Tr
p-Leu-Asn-Arg-A rg-A Ia-A
sn-A Ia-Leu-Leu-A Ia-Asn-
G Iy-Va l -Gl u-Leu-Arg-A
sp-Asn-Gl n-Leu-Val -Val-
Pro-Ser-Glu-Gly-Leu-Tyr-L
eu-11e-Tyr-Ser-G In-Val
-Leu -Phe-Lys-Gly-Gln
-G I y-Cys-Pro-Ser-Thr-Hi
5-Val-Leu-Leu-Thr-H1s-
Thr-1l e-Ser-Arg-11e-A 1a
-Val -Ser-Tyr-G I n-Tyr-
Lys-Va I-Asn-Leu-Leu-5e
r-A l a-11e-Lys-Ser-Pro-
Cys-GIn-Arg-Glu-Thr-Pro-G
lu-Gly-A Ia-G Iu-A Ia-Ly
s-Pro-Trp-Tyr-G Iu-Pro-1
1e-Tyr-Leu-G I y-G ly-Va
1-Phe-G In-Leu-G 1u-Lys-
G I y-85p-A rg-Leu-Ser-A
l a-G l u-1l e-Asn-Arg-Pr
o-Asp-Tyr-Leu-Asp-Phe-A I
a-G Iu-Ser-G Iy-G In-Va I
-Tyr-Phe-Gly-11e-Ile-Ala
-Leu Furthermore, the polypeptide named TNF is
It is also known that it is produced by E. coli transformed with a recombinant plasmid [Nature
e), Tachijo ■, pp. 724-729, (1984, 12
Monthly 20th/27th issue), Nature + - (Nature),
Shutoyu, pp. 803-806, (February 28, 1985 issue), Science -LLL1249
'Page 154, (April 12, 1985)].

The polypeptides described in Nature, Uchiha ■, if inferred from the base sequence of the cloned DNA, are the same as those described in The Journal of Biological Chemistry (The Journal of Biol, C.
hem, ) is nearly identical to the TNF described in If-Standard, differing only in the absence of two N-terminal amino acids, namely valine and arginine. TNF lacking these two amino acids is hereinafter referred to as "conventional TNFJ" and will be used for comparison in the experimental examples described later. However, the cells in which these TNFs exhibit antitumor activity are extremely limited [Science (Science), l main, 94
3-945, 1985, it has also been reported to have so-called cachectin activity, which facilitates the catabolism of adipocytes [Therapeutic Research (Therapeutic Research)].
apeutic Research), Yu, No. 2,
pp. 184-190, 1987 Ko.

[Problems to be Solved by the Invention] Under the above-mentioned circumstances, the purpose of the present invention is to solve the problem of A349, which is reported to have no toxicity at all in conventional TNF.
It shows remarkable cytotoxicity to cells (lung carcinoma), and
A gene encoding a novel antitumor polypeptide that has no cachectin activity that conventional TNF has, and also suppresses the proliferation of cancer cells by suppressing their angiogenesis, and a novel plasmid containing the same; The object of the present invention is to provide the polypeptide, a method for producing the same, and a novel antitumor agent comprising the polypeptide.

[Means for Solving the Problems] The present invention provides DNA encoding the following amino acid sequence.

Met-Val-Arg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-A-tl i 5-Val-Va
l -A Ia-Asn-Pro-Gln-A Ia-
G Iu-G I y-G I n-Leu-G I
n-Trp-Leu-Asn-Arg-Arg-Al
a-Asn-A Ia-Leu-Leu-A la-
Asn-Gly-Val-Glu-Leu-Arg
-Asp-Asn-G I n-Leu-Va I -
Va I-Pro-Ser-G I u-G ly
-Leu-Tyr-Leu -11e-Tyr-Ser
-G I n-Va I -Leu-Phe-Lys-
Gly-G I n-G Iy-Cys-Pro-S
er-Thr-If i 5-Val -Leu -L
eu -Thr-)11s-Thr-1le-Ser-
Arg-l 1e-A Ia-Val-Ser-Ty
r-G l n-Tyr-Lys-Va I-Asn
-Leu-Leu-Ser-A la-I 1 e-L
ys-Ser-Pro-Cys-G I n-Arg-
G Iu-Thr-Pro-G Iu-G Iy-A
Ia-G l u-A Ia-Lys-Pro-Tr
p-Tyr-G Iu-Pro-I 1e-Tyr-L
eu -Gly-Gly-Va l -Phe-
G I n-Leu-G I u -Lys-Gly-
Asp-Arg-Leu-Ser-AIa-Glu-I
1e-Asn-Arg-Pro-Asp-Tyr-L
eu-Asp-Phe-AIa-Glu-Ser-Gl
y-GIn-Val-Tyr-Phe-GIy-11e
-11e-Ala-Leu (However, X is Ser or Cys) In the above amino acid sequence, from Ala, which is the 19th amino acid from the beginning, to the last L eu is the conventionally U number of TNF. It is the same as the amino acid sequence formed when guanine (G) is added to the beginning of the base sequence of the 4 exon portion. Therefore, in other parts of the specification, the Ala-Leu moiety is defined as rT
The amino acid sequence of the fourth exon of NF.

The DNA of the present invention can be obtained from Nucleitsu Acids Research (
Nucleic Ac1ds Res, ) Retired Emperor,
7439-7448 (1981), Biochemistry, 12
It can be chemically synthesized according to the method described on pages 57-1267 (1978). - As an example, a method for obtaining the DNA of the present invention from the genomic DNA of human acute monocytic leukemia cell THP-1 will be described in detail in Examples. In addition, in the polypeptide coated by the DNA of the present invention, when the base sequence of alanine (Ala), which is the 19th amino acid from the beginning, is GCG, the fourth It is extremely useful because it can cut the base sequence before the same base sequence as the exon part and introduce any base sequence.

To obtain an antitumor polypeptide using the obtained DNA, this DNA is integrated into an appropriate vector DNA so that it can be expressed, and the obtained recombinant DNA is used to inject animal cells, yeast, Bacillus subtilis, A host such as a microorganism such as E. coli is transformed and expression is induced.

As is well known, in order to incorporate the DNA of the present invention into vector DNA so that it can be expressed, a promoter sequence (usually located downstream of an operator sequence) and a Shine-Dalgarno sequence ( Either the DNA of the present invention is linked downstream of the SD sequence of the vector DNA having the SD sequence (hereinafter referred to as the SD sequence), or the DNA of the present invention is inserted into the vector DNA.
After incorporating A, a promoter sequence (usually also an operator sequence) and an SD sequence may be inserted upstream thereof.

A method for expressing the genetic information of a foreign gene in microbial cells using transferable DNA technology is described in "Practical Gene Recombination Technology (4) J (1983) (Science Forum),""MolecularCloning"
ar Cloning) J (1982) [Cold Spring Harbor Laboratory (ColdSp
It is generally described in "Introduction and Expression of Recombinant Genes into Cells" J (1983) (Kyoritsu Shuppan Co., Ltd.) and others.

Example 1 shows an example using E. coli as a host.

Furthermore, when yeast is used as a host, the genetic information of the DNA of the present invention can be expressed as follows.

Plasmid vector pMA56 into which the promoter of alcohol dehydrogenase (ADHI) was inserted
Nature) m, pp. 347-350 (1982)] has an EcoR1 site downstream of its promoter. Collected as a fragment, Ec downstream of the ADIT promoter of pMA56.

EcoRr/BamHI linker-1Ps t at Rr site
If inserted using I/EcoRI linker, ADHI
It is under the control of a promoter and can be expressed in yeast.

In addition, pAM82, which has a repressed acid phosphatase (PHOδ) promoter [Procedure of National Academy of Sciences of Nini
roci Natl, Acad, Sci, U.

S, A.) u, pp. 1-5 (1983)] is PH05
Since it has an XhoI site downstream of the promoter, the DNA of the present invention is recovered as a BamHI-Pstl fragment from the recombinant described in Example 1, for example, and pA
BamHI/Xhol linker, Ps t T/Xho at the Xho1 site downstream of the PH05 promoter of M82
If inserted using an I linker, it will be under the control of the PH05 promoter and can be expressed in yeast.

Furthermore, even if Bacillus subtilis is used as a host, the genetic information of the DNA of the present invention can be expressed as follows.

Bacillus subtilis
pTU8285 [Gene, 1st issue, p. 148 (1985)], which has the α-amylase promoter of the S. subtilis Marburs strain, has Hin in the promoter and downstream of the signal peptide.
Since it has a cll site, for example, the DNA of the present invention
was obtained from the recombinant BamHI described in Example 1.
- Recovered as a Pstl fragment and Hin of pTUB285
HincII/BamHI linker-11 (inc
If inserted using the ll/Pstl linker, it will be under the control of the α-amylase promoter and can be expressed in Bacillus subtilis. The antitumor polypeptide produced by the transformed host cell can be isolated and purified as follows.

Host cells are collected by centrifugation and disrupted using ultrasound or lysozyme. At this time, a hypotonic solution is used, but better results may be obtained by coexisting a surfactant such as SDS or a protein denaturing agent such as guanidine hydrochloride. The cell disruption solution is centrifuged to obtain a supernatant.

The crushed supernatant containing the antitumor polypeptide thus obtained is purified according to a conventional protein purification method. That is, it is purified by sequentially or appropriately combining ion exchange chromatography using a basic anion exchanger, salting out method, dialysis method, gel filtration method, hydrophobic chromatography, high performance molecular sieve chromatography, electrophoresis method, etc. .

For example, DEAE-Sephadex A-25, A-
50, DEAE-Sepharose
) CL-68, DEAE-Sephamil
i l ) (manufactured by Pharmacia) is preferred, and anion exchangers containing diethylamino groups, aminoethyl groups, or quaternized aminoethyl groups are also used.

The buffer used is preferably Tris-HCl or phosphate buffer with a pH of 6.0 to 9.0;
A culture solution of the antitumor polypeptide is diluted with a dilute buffer solution of about 0.05M, and the solution is brought into contact with an anion exchanger to adsorb the antitumor polypeptide. Elution of antitumor polypeptide is 0.1-0.2M
This is done with a saline solution such as NaCff1 or KCI. Anti-tumor polypeptides are eluted at salt concentrations around 0.2M.

A column method is preferable for contacting with the anion exchanger, but a batch method can also be used in the case of large quantities.

Before performing anion exchange chromatography, it is desirable to remove low-molecular substances using an ultrafiltration membrane as a pretreatment, which can increase the purification effect.

After dialysis, the solution obtained by anion exchange chromatography is concentrated and subjected to gel filtration. Supports for gel filtration include Sephadex G-75, G-100 (manufactured by Pharmacia), and Sephacryl S.
-200 (manufactured by Pharmacia), Biogel (Biog
el) P-100 (manufactured by Biorat Co., Ltd.) and Toyo Bar HW-50, HW-55 (manufactured by Toyo Soda Kogyo Co., Ltd.)
etc. The buffer used for gel filtration has a pH of 6.0.
~9.0 Tris-HCQ or phosphate buffer, preferably with addition of 0.2 to 0.5 M salt such as NaCl for the purpose of preventing adsorption.

Further, the antitumor polypeptide active solution obtained by anion exchange chromatography can also be purified by hydrophobic chromatography. In this case, a carrier such as Butyruto Yopal 650 (manufactured by Toyo Soda Kogyo Co., Ltd.) is used, and ammonium sulfate, N
The antitumor polypeptide is eluted using a salt such as aC14.

The antitumor polypeptide-containing solution purified by gel filtration or hydrophobic chromatography is then purified by mono
)Q Pharmacia F P using HR515 column (manufactured by Pharmacia, high performance anion exchanger column)
L C (Fast Protein.

Peptide, Polynucleotide,
A purified sample is obtained by high performance anion exchanger chromatography using a Liquid Chromatograph system.

The conditions for this high performance anion exchange chromatography are the same as for the first anion exchange chromatography using a carrier such as DEAE-Sepharose.

The polypeptide of the present invention not only exhibits excellent cytotoxicity against A349 cells, to which sensitivity has not been observed with conventional TNF, but also exhibits almost no so-called cachectin activity, which catalyzes the catabolism of lipid cells, which conventional TNF has. Furthermore, by suppressing angiogenesis in cancer cells, their proliferation is suppressed. The polypeptide of the present invention can be analyzed qualitatively and quantitatively as follows.

A349 cells were grown in Eagle Minimum Essential Medium (hereinafter referred to as MEM) supplemented with 10% calf fat serum (hereinafter referred to as FC8), and 4 x 103 cells were grown at 10
0μ2 of the same medium as above, and bred in 96 large flat bottom plates.

Breeding conditions were 37℃, -night, 5% CO2, 100% H20.
Any method used for normal cell culture may be used. Then, double the normal concentration of MEM containing 1% FC9 (100
µV) and immediately add an equal volume of the sample appropriately diluted with a buffer containing 1% FC3 (at this time, the dilution rate can be adjusted appropriately to determine the ED50).

A349 cells are cultured under the above conditions for 72 hours. To measure cell necrotic activity, first remove all the medium, then 0.
Fix and stain by adding 1% methyl alcohol solution containing 1% crystal violet. Since crystal violet stains all nucleated cells and does not stain cells that are released from the bottom of the plate as a result of cell necrosis, cell necrosis activity can be directly measured.

The degree of staining is measured by absorption at 0D595, and cell necrosis activity is determined by comparing the degree of staining with that of the control group. The definition of activity is as follows.

The dilution rate (N
). Rabbit TNS was used as a control, and the activity n (units/mg) of this rabbit TNS was 2.4X10'
Unit/m g/m 9. Determined using human TNF. The dilution rate that gives the ED5° of this rabbit TNS (
Find 'C).

The activity (units/ml) of the sample stock solution is calculated as −×n.

ゝI The amount of fat accumulated in adipocytes is regulated by the amount of fatty acids supplied to the cells and the amount of decomposed fat that has already been accumulated. The function of reducing fat accumulation by suppressing fatty acid supply and increasing the decomposition of accumulated fat is called cachectin activity. It falls into a state of severe consumption called liquid quality (kakekushia). Therefore,
It is undesirable for a drug to have cachectin activity as a side effect.

Fatty acid is supplied from outside the cell by riboprotein lipase (LPL)
Glycerol is also released by the decomposition of stored fat. Therefore, cachectin activity is based on LPL
The activity can be evaluated by measuring the amount of glycerol released.

(1) Method for measuring LPL activity Proceedings of the National Academy of Sciences of the United States (Proc.
i Natl, Acad, Sci, U, S,
Kawakami M Equivalent "Riboprotein Lipase Suppression in 3T3-Lll" described in A.), E., pp. 912-916 (1982).
Cell Type Ian Endotoxin-Induced Mediator From Ecudate Cells (Lipop
rotein 1ipase 5uppression
in 3T3-Llcells by an end
otoxin-induced mediator f
6 by the method of romexudate cells)
3T3-Ll mouse pre-azoibocyte cells were seeded in DME containing 10% Fe2 [manufactured by Wako Pure Chemical Industries, Ltd.] in a 0 mm Petri dish, and 2 days after the cells were deposited on the surface of the Petri dish, 0.5 mM isobutyl methyl was added. Add xanthine, 1 mM dexamethasone, and 10 μg/ml insulin, and culture for an additional 48 hours. Then 10% Fe2,
The medium is changed to DME containing 50 μg/mu of insulin, each concentration of specimen is added, and culture is carried out for about 1 day (within about 24 hours). Thereafter, 35 units of heparin were added, and the LPL activity released in the medium for 1 hour was measured using Nilson-Ehle and Schotts (Sc.
hotz) is Jiei, Lippid Res (J, Li
pid Res, ), pp. 536-541 (19
It is measured by the method described in 1976).

(2) Method for measuring the amount of glycerol released Glycerol released into the medium in the same manner as in (1) above, as described in J. Biochem, J. Koto, pp. 331-338 (1987). The amount was determined using a glycerol analysis kit [“Glycerol, U
V method (Glycerol, UV-method)
” [Measurement is performed using an instrument manufactured by Heringer Mannheim, Mannheim, West Germany.

Hereinafter, the present invention will be explained in more detail with reference to Examples and Experimental Examples.

THP-1 cells [International Journal of Cancer (Int, J, of Cancer)
) ■ Dan, pp. 171-176 (1980)], 10%
In RPMI-1640 medium containing FC5 at 37°C,
% CO2, and the number of cells was 1 x 106 cells/m.
9. When the cell temperature reached 100 μg/mg, the cells were further cultured in the presence of 12-tetradecanoylfarbol-13-acetate (hereinafter referred to as "TPA"), and 8 hours and 70 hours after the addition of TPA, the cells were cultured. Collect this and make it mR
The cells were used for NA extraction. Extraction of mRNA from cells is
It was carried out according to the following method.

Collect cells by centrifugation and add PBS (-) (0.8%
NaCl! , 0.02% KCIu, 0.02% J (H2
Washed with PO4,0,115%N62HPO4). Sufficiently add the collected cells to 50ml RNA extraction buffer! After it becomes cloudy, "Nonident J (Nonident)-P2O
was added to a final concentration of 0.5%, and homogenized using a Teflon homogenizer for 10 strokes to destroy the cells. Then, the homogenate was heated at 4°C.
The mixture was centrifuged for 1 minute at 10,000×g, and the supernatant was used as a cell extract. Add an equal volume of buffer-saturated phenol/chloroform mixture to the cytoplasmic extract and
Mixed for more than a minute.

Then, centrifugation was performed at 3000×g for 10 minutes to remove the phenol/chloroform solvent layer, and the same phenol and chloroform extraction procedure was performed two more times.

Then chloroform/isoamyl alcohol (24:1)
An equal amount of the mixed solvent was added to the supernatant, and after mixing at room temperature for 10 minutes or more, centrifugation was performed to collect the supernatant. 2.5 times as much ethanol was added to the supernatant and allowed to stand overnight at -20°C to precipitate RNA, which was collected at 4°C, 110,000X for 10 minutes, and used as a cytoplasmic RNA preparation. 20 of the sediment
After suspending in mA sterile water, some samples were
[The degree was measured. After suspending the RNA, add a 5x concentration of RNA.
5 ml of A wash buffer was added, and the mixture was passed through a poly(U) Sepharose column that had been equilibrated with an RNA wash buffer. rRNA other than poly(A)RNA
and after washing away the tRNA with the above RNA wash buffer,
Pol V (A) RNA was eluted with 5 ml formamide, poly (A) RNA fractions were collected, and ethanol precipitation was performed twice, followed by po I y (A) RNA1.
The suspension was suspended in sterilized water at a temperature of 1°C g/10μ, divided into portions, and stored at -80°C.

Hereinafter, poIy (A) RNA will be referred to as mRNA.

(2) --1-' cDNA using mRNA obtained as described above
The A library was created in two ways.

(j) Gubler method [Gene
), 25, pp. 263-269 (1983) By an ordinary method, an oligo (dT) complementary to the 3' poly(A) sequence of the mRNA is annealed with the mRNA to generate a reverse transcriptase primer. Prepared. Next, dATP, dG
cDNA complementary to mRNA was synthesized by allowing reverse transcriptase to act in the presence of TP, dCTP, and dTTP. Therefore, the mRNA/cD obtained in this way
RNaseH on mRNA A side of NA hybrid
DNA polymerase 11 and Escherichia coli D
Replace mRNA with DNA using NA ligase,
Full-stranded DNA was synthesized. 3' of the double-stranded DNA obtained
10 to 20 dC tails are added to the ends by the action of terminal deoxynucleotidyl transferase, and then the plasmid vector p, which can be replicated in E. coli, is
BR322 was treated with restriction enzyme PstI to obtain linear plasmid DNA. Terminal deoxynucleotidyl transferase is applied to the 3' end and
Added 0 dC tails. Next, the dC tail plasmid Hector and the dC tail double-stranded DNA were annealed and transferred to E. coli by a transformation method using the calcium method, and the resulting transformant was used as a cDNA library.

(II) Okayama-Barg
) method [Molecular and Cellular Biology (Molecular and Cellular Biology)
Biology)-21, pp. 161-170 (1980
) Unlike Kogabler's method, mRNA poly(A)
Double-stranded DNA with an oligo (dT) tail complementary to the sequence is annealed and treated with dATP.

Complementary cDNA was synthesized by reacting reverse transcriptase in the presence of dGTP, dCTP, and dTTP.

Next, terminal nucleotidyl transferase was applied to the newly synthesized cDNA to add dG detail. This is annealed and ligated with a plasmid vector that has been dC-tailed in advance, and mR
A plasmid containing an NA/c DNA hybrid was obtained. Next, similar to Gabler's method, RNa
The mDNA was replaced with DNA by the action of seH, DNA polymerase I, and Escherichia coli DNA ligase. In this way, a plasmid containing double-stranded cDNA was obtained. A cDNA library was created by introducing this into E. coli cells.

(3) Mum ■-2 The cDNA library obtained as described above was grown on a nitrocellulose filter and grown in a medium containing chloramphenicol [Gene, Retired Emperor, pp. 63-67]. (1980) and increased the number of plasmids.

Next, the grown nitrocell mouth of the cDNA library:
Immerse the filter in 0.5N NaOH solution for 5 minutes at room temperature to destroy the E. coli cell wall, convert the plasmid DNA from double strands to single strands,
) H7, 5) After soaking in the solution and leaving it at room temperature for 10 minutes,
0.5M) Lis-HC1j (pH7,5)/1.5M
After soaking in NaCg solution for 100 minutes at room temperature, it was air-dried.

After sufficiently drying, the nitrocellulose filter was then treated at 80° C. for 2 hours. Synthesized 23 base DNA
The 5' end was labeled with FATPST4 DNA kinase, and this 5' end labeled DNA was hereinafter referred to as cDNA.
It was used as a DNA probe for A clone screening. 8
6X nitrocellulose filter treated at 0°C
NET (IXNET.

0.15M NaC1,0,015) Squirrel-HC1! (
pH 7.5), 1mM EDTA, 250μg/m
g, Escherichia coli tRNA, 0.5% NP-40) 4
After hybridization overnight at 2°C, 6x SSC (IX
After washing at 0°C (SSC, 0.15M NaCl, 0.015M sodium citrate), it was washed twice with 2XSSC for 5 minutes at 0°C, and then further washed with 2XSSC at 42°C for 2 minutes. After washing, the nitrocellulose filter was air-dried and subjected to autoradiography.

The clones detected as positive by autoradiography were further sequenced according to the Maxam-Gilbert nucleotide sequencing method to narrow down the positive clones.

As a result, among the cDNA clones that hybridized with the 23-base synthetic NA, there was a cDNA clone having the C-terminal side of the antitumor polypeptide. Obtained cD
The NA clone was found to have approximately 1000 base pairs.

(4) ", After culturing for 8 hours in a medium containing 100 ng/ml of TPA, 3 x 109 THP-1 cells were cultured at 100 ng/ml.
mV 150mM NaCl, 100mM EDT
It was suspended in solution A.

Furthermore, 10m1! Add 10M sodium perchlorate solution and add 10ml of 10% SDS! added.

Next, 12 ml of 5M NaCL was added, and the mixture was heated at 60°C for 15 minutes.
Warmed for minutes. Add equal amounts of chloroform and isoamyl alcohol (24:1) to this solution and mix gently.
Centrifugation was performed for 0 minutes to obtain a supernatant. An equal amount of isopropyl alcohol was added to this supernatant, and the resulting DNA precipitate was poured onto a Pasteur pipette. DN with 70% ethanol
After washing A, 100 m 1. 10mM) of Lis-H
CQ (pH 7,5) + 10mM NaCl! +0.1
It was dissolved in mMEDTA solution (TSE). then 1
0% SDS was added to a final concentration of 0.5%, and protease K was added to a final concentration of 1 mg/mQ, and the mixture was heated at 55°C overnight. At this time, after centrifugation at 10,000 revolutions, the DNA-protein complex that comes to the middle layer is fractionated, and by performing the operations after centrifugation, the DNA-protein complex is separately fractionated, and by performing the operations after centrifugation. Increased DNA yield. The DNA solution treated with protease was gently mixed with a 100:14:0.1 mixture of water-saturated phenol, metacresol, and isoamyl alcohol, and centrifuged at 300 rpm for 10 minutes at room temperature to collect the supernatant. An equal amount of isopropanol was added to this supernatant, and the resulting precipitate was poured onto a Pasteur pipette. 7
After washing the DNA with 0% ethanol, 100 m 9.
of TSE.

After adding TSE to the DNA solution in 4 ways to a final concentration of 800 μg/m1, CsC1 was added at a rate of 0.95 g/mQ.
! and 1/10th volume of 5 mg/ml ethidium bromide to make a homogeneous solution, which was then washed in a Beckman type 60 rotor for 2 hours.
DNA was purified by density gradient method by centrifugation at 0°C for 48 hours.

After centrifugation, DN is irradiated with 360 nm UV.
While looking directly at the A band, pipet the DNA with a Pasteur pipette.
was recovered. Ethidium bromide was then removed by adding CsCl saturated isopropyl alcohol to the DNA solution and mixing several times, which was repeated 10 times.

DNA was obtained by dialyzing the resulting DNA solution against 22 TSEs at 4°C day and night. The DNA concentration at this time was 650 μg/ml, and a total amount of 18 mg of DNA was obtained.

(5) 15 μg of the obtained DNA was aliquoted and completely digested with several restriction enzymes, and the length of the antitumor polypeptide gene fragment was analyzed by the Southern method using various restriction enzymes. Apal (GGGCCC), Xhol (CTC
GAG), BamHI (GGATCC), EcoR
I (GAATTC), 5stl (GAGCTC)
, Kpnl (GGTA=28-CC) were used, and the DNA was incubated at 37°C overnight in a solution with appropriate buffer and salt concentration. After the reaction was completed, 5M NaCl was added to I? 2.5 volumes of ethanol was added to precipitate the DNA. The degraded DNA was recovered as a precipitate by centrifugation, dissolved in 10 μt of water, spread on a 1.5% agar gel, and the DNA fragments were adsorbed on a nitrocellulose filter. The nitrocellulose filter adsorbed with DNA was air-dried and then treated under vacuum at 80° C. for 2 hours.

2. -ゝ -・ゝゝ 200 n g of cDNA obtained in (3) is a
oμi reaction solution (50mM) Lis-HC1 (pI (
7,5) 10mM MgC1e, 10mM DT
20 μC of α-32PdCTP: dAT
P.

Add dGTP and dTTP to 5 μM each, and
Nase12.5pg, DNA polymerase (Polymerase)
merase) was added and reacted at 20°C for 1 hour. After the reaction is complete, add an equal amount of water-saturated phenol:chloroform 1:1 mixture, stir vigorously, centrifuge to remove protein, add an equal amount of chloroform, stir vigorously, and centrifuge to remove the aqueous layer. was separated.

Add 1mM EDTA and 100μg to this aqueous layer in advance.
/ml! The DNA portion was layered on a Sephadex G-50 column equilibrated with a solution containing tRNA of 100 μg/mff1 and eluted with a solution containing 1 mM EDTA and 100 μg/mff1 of tRNA. Labeled DNA with radioactive activity IXIO of 8 cpm/μg DNA was obtained. This DNA was heated at 100° C. for 10 minutes to form single-stranded DNA, which was used as a probe.

DNA
The nitrocellulose filter adsorbed was treated with 1 ml of 50% formaldehyde, 5
aCl, 0.015M sodium citrate), 5XFBP,
1% glycine, 20mM phosphate buffer (pH 6,8)
, after being uniformly immersed in a solution containing 100 μg/mff1 calf thymus denatured DNA and sealed in a plastic bag.
It was incubated overnight at ℃. Next, 1m9. 50% formamide, 5xssc, IXFBP, 20mM phosphate buffer (pH 6,8), 100μg/ml calf thymus denatured DN
The filter was immersed in a solution containing A and 2 x 10' cpm of probe and kept at 42°C overnight. After incubation, the nitrocellulose filter was transferred to 2XSSC solution and washed at 68°C for 1 hour to remove non-specific and NA probe adsorption, and then washed in the same manner for 5 minutes with O, 1X5SC, and then the filter was washed. The DNA fragments that specifically hybridized with the probe were detected by air drying and exposing Kodak X-ray film overnight.

,t, In the above Southern hybridization, the probe DNA described in (3) and the genomic DNA restriction enzyme ApaI were used.
The 2 to 6 kb DNA fragments generated when cleaved with the above hybridized with each other with good reproducibility. There, THP-
1 using 1.3 mg of genomic DNA in a 5 mfi reaction solution.
By adding 500 units of Apa+ and heating for -night, T
A complete Apa I degradation product of the HP-131-1 genome was obtained.

This DNA fragment was fractionated on a 1.5% agar gel, and
．． The agar gel part around 6 kb was cut out. DNA was recovered from agar as follows. First, agar was added to 15 ml of a solution prepared by adding 22.5 g of Kl to 15 ml of 10 mM phosphate buffer (+) H7, O), and the agar was dissolved by heating to 60°C. Thereafter, the solution containing the DNA was adsorbed onto Bio-Gel HTPJ manufactured by Bio-Rad, and after thoroughly washing with 10 mM phosphate buffer, the DNA was eluted with IM phosphate buffer and 0.5% SDS. Dialysis for TSE all day and night 4
The reaction was carried out at ℃ to obtain DNA.

(6) --1-' A dCTP tail was added to the obtained DNA using terminal deoxynucleotidyl transferase. On the other hand, pNF used as a vector was made into a single chain with KpnI, and a dCTP tail was added in the same manner, followed by annealing to produce a chimeric ring. This chimera was introduced into E. coli RRI to create a library consisting of 2 x 10' independent colonies.

As a result of selecting the above genomic library using the probe described above, one type of clone was obtained. The restriction enzyme map of the obtained clone is shown in FIG. 1, and its partial nucleotide sequence is shown in FIG.

(7) [I de.・ (1) The genomic gene shown in Figure 1 is digested with the restriction enzyme Xho.
15Ps t I and cut with Xho I/
The Ps t T fragment (811 base DNA fragment from 340 bases to 1150 bases of the partial base sequence of the aforementioned TNF genomic DNA) was digested with the restriction enzyme 5aij of plastic tarpUc540 having a tac promoter and an SD sequence.
Plasmid pUc540T was inserted into the l/Pst1 site.
NF x/p was produced.

This plasmid contains the restriction enzyme XhoI/P of the genomic gene.
It has a gene that coats a protein whose N-terminus is the first methionine present in the stl fragment. (Figure 4) Plasmid vector pUc540 is an Eco variant of plasmid vector pUC8 commercially available from Pharmacia.
Plasmid pDR540, which has a tac promoter in the RI/BamHI site and is also commercially available from the same company.
The Eco T/BamHI fragment of .

Plasmid pUc540TN'x/p has a restriction enzyme BamH1 site under the SD sequence.

Therefore, when a foreign gene is inserted into this BamH1 site, expression of the foreign gene becomes possible only by the addition of isopropyl β-lowthiogalactopyranoside (hereinafter referred to as IPTG).

(11) The genomic gene shown in Figure 1 is
I, Ps t I and cut XhoI- as shown in Figure 3
Pstl fragments were recovered. Next, add this fragment to H1nall
Cut the 294 base pair XhoI-HinclI fragment.
Fragment, 521 base pairs Hinc I I-Ps t I
The fragment was recovered, and the 294 base pair fragment was partially digested with DdeI to produce a 206 base pair Dde l-Hlnc.
The II fragment was recovered. The 521 base pair HincII-PstI fragment recovered in this way, the 206 base pair D
The deI-Hinclll fragment was obtained from AB1 (USA) at 38
After binding with any of the 72/73 base pair double-stranded DNAs shown in Table 1 synthesized with a 0A DNA synthesizer, the BamHI-
It was inserted into the Pst1 site.

In this way, the two new TNF recombinants of the present invention
TNF-3-AMI (X=in the above amino acid sequence
corresponding to Serin), rTNF-5-AM2 (corresponding to
(corresponding to X=Cys in the above amino acid sequence) was obtained. Since these silk recombinants are under the control of the lactose operon promoter, their genes can be expressed in E. coli.

1st Table Tsuba 1 72/73 base pair A (X = corresponds to Serin) 72/73 base pair B (X = corresponds to Cys)
The novel TNF silk recombinant rTNF-3- thus prepared
AMI, rTNF-9-AM2, is linked to the BamHIT stream of the TaC promoter, has an ATG as a start codon immediately after the BamHI restriction enzyme cut point, and furthermore, the first codon of the 2'# amino acid following this ATG. This is a TNFI recombinant whose codon is G. Also, the molecular weight is 17500
±500 (SDS polyacrylamide electrophoresis method).

The TNF of the present invention! All of the mutants are under the control of the lactose operon promoter, so their expression is controlled by IPTG.
It can be induced by

・rTNF-S-A M1 or rTNF-
E. H. JM103 harboring 5-AM2 was treated with ampicillin 5.
I
1XYT medium 1 containing 50 μg/ml ampicillin after preincubation at 37 °C with
00ml! 1 in a 500 ml slope flask containing
% seedlings were planted and similarly cultured at 37°C, and 0Deao = 0.
When the concentration reached 3, IPTG was added to a final concentration of 0.7 mM, and the culture was continued for an additional 24 hours. The Escherichia coli thus obtained was collected using a centrifuge and IXP
BS (NaC1! 0.8%, KCI! 0.02%
, KH2PO40.02%, Na2HP O40,
After washing with 115%), the cells were suspended again in 10 mfl of IxPBS, and the E. coli cells were disrupted using ultrasound.

A portion of it was used to measure antitumor activity. The results are shown in Figure 5. In FIG. 5, the lines connected by ○ indicate the rTNF-5-AMI of the present invention, and the lines connected by △ indicate the rTNF-5-AMI of the present invention.
The lines connected between TNF-5 and AM2 are conventional TNF.
The following data is shown.

By the method described above, the TNF, I recombinant r of the present invention
T N F -S -A M 2 and conventional T
Cachectin activity of NF was measured.

(1) The LPL activity results are shown in FIG. In Figure 6, the vertical axis shows LPL activity, and the horizontal axis shows the corresponding sample amount (unit/m).
shows. The solid line shows data for rTNF-9-AM2 of the present invention, and the dashed line shows data for conventional TNF. According to the same figure, for example,
Conventional TNF reduces LPL activity to 30 units/mu at 8 units/mu.
Milliunits/m g - compared to ζ for inhibiting cellular proteins,
The amount of rTNF-3-AM2 of the present invention is about 10 times that amount.
It can be seen that the same degree of suppression is shown for the first time at 00 units/m Q.

(2) The results of glycerol release amount are shown in FIG. In FIG. 7, the vertical axis shows the amount of glycerol released (μg/mg/cell protein), and the horizontal axis shows the corresponding amount of sample (unit/mt) (the solid line is the rTNF-S- of the present invention). A M 2, dashed line shows conventional TNF data). From the same figure, for example, for conventional TNF, 75 μg/m at 4 units/ml
rTNF-3-AM2 of the present invention releases 10 times the amount of glycerol from cell proteins.
It can be seen that only 0 units/mfl gives a comparable release.

(3) Evaluation of Cachectin Activity From (1) and (2) above, it can be seen that the Cachectin activity of the novel TNF recombinant of the present invention is extremely low compared to the same activity of conventional TNF.

Goods.・Human umbilical vein cells coated with 0.1% gelatin2
Cells were seeded at 1.0 x 10' cells/cm2 in a 5-well plate and left for 2 hours to allow the cells to adhere to the plate. The medium contains 10
MCDB-104 containing % FC5 [KCBiological Inc.
), final volume 70 ng/ml ECGF (endothelial cell growth factor prepared from newborn bovine fetal brain) + 10
mg/ml EGF (epidermal growth factor), 100 μg/m
(Includes L heparin.

Then, various amounts of the novel rTNF-5AM2 of the present invention or conventional TNF were added and cultured at 37° C. for 5 days, and then the number of cells was counted.

Conventional TN when administered at 5 × 10 units/ml
In F, the number of cells increased to twice the initial value, but rTNF
-5-AM2 had no significant difference from the initial value.

[Effect of the invention] As described above, the novel DNA synthesized by the present invention can express a novel antitumor polypeptide, and this novel antitumor polypeptide is completely insensitive to conventional TNF. It exhibits excellent cytotoxicity against A349 cells, which are known to be anti-inflammatory agents, and has virtually no cachectin activity that conventional TNF has. Furthermore, by suppressing angiogenesis in cancer cells, their proliferation is suppressed.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme map of the genomic gene of an antitumor polypeptide purified from THP-1 cells. FIG. 2 shows a partial base sequence of the gene. FIG. 3 shows the Xho-Pstl fragment of the genomic gene. FIG. 4 shows the nucleotide sequence of the antitumor polypeptide gene possessed by pUc540TN' x/p and the amino acid sequence of the polypeptide encoded by it. FIG. 5 shows A of the TNF recombinant of the present invention and conventional TNF.
349 is a graph showing 349 cytotoxicity. The line connecting ○ is r
The line connecting Δ of TNF-S-AM1 is rTNF-S-AM2, and the line connecting . is conventional TNF.
The following data is shown. FIG. 6 shows the TNllfl recombinant rTNF-5- of the present invention.
The LPL activity inhibitory effects of AM2 (solid line) and conventional TNF (dashed line) are shown. FIG. 7 shows the TNF recombinant rTNF-3-AM of the present invention.
2 (solid line) and the glycerol release promoting effect of conventional TNF (dashed line). Patent applicant Genbetsu Yuzu (and 1 other person) Inguchi<<0
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Claims (5)

[Claims] (1) DNA encoding the following amino acid sequence. Met-Val-Arg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-Ala-His-Val-Val- (amino acid sequence of the 4th exon part of TNF) (X is Ser or Cys) (2) A plasmid containing DNA encoding the following amino acid sequence. Met-Val-Atg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-Ala-His-Val-Val- (amino acid sequence of the 4th exon part of TNF) (X is Ser or Cys) (3) A polypeptide represented by the amino acid sequence below. Met-Val-Arg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-Ala-His-Val-Val- (amino acid sequence of the 4th exon part of TNF) (X is Ser or Cys) (4) Production of a polypeptide represented by the following amino acid sequence, which is characterized by culturing a microorganism having DNA encoding the following amino acid sequence inserted into a plasmid that can proliferate within a host microorganism cell. Method. Met-Val-Arg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-Ala-His-Val-Val- (amino acid sequence of the 4th exon part of TNF) (X is Ser or Cys) (5) An antitumor agent consisting of a polypeptide represented by the following amino acid sequence. Met-Val-Arg-Ser-X-Thr-Arg
-Thr-Pro-Ser-Arg-Lys-Pro-
Val-Ala-His-Val-Val- (amino acid sequence of the 4th exon part of TNF) (X is Ser or Cys)