JPH066066B2 - Heat treatment method for physiologically active substances produced by transgenic plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying a physiologically active substance produced from a recombinant. Specifically, in the process of isolating and purifying the physiologically active substance of the present invention from a culture solution of a microorganism (bacteria, yeast, etc.) artificially produced by a genetic recombination operation, or a disrupted extract of the cultured microorganism, crude purification is performed. The present invention relates to a purification method, which comprises subjecting an aqueous solution of the physiologically active substance to heat treatment in a state or in a state where purification has progressed.

Furthermore, the present invention relates to a purification method characterized in that the heat-treated aqueous solution is applied to a carrier column for gel filtration having an appropriate molecular weight fractionation ability to perform gel filtration.

The physiologically active substance in the present invention is a protein obtained from a gene recombinant, has a cytotoxic activity on LM cells as described below, and is BALB / c transplanted with mouse Meth A sarcoma cancer cells. When administered to mice, it has the property of causing a hemorrhagic necrotic reaction at the tumor site. In addition to the above properties, it also has a characteristic of having almost no harmful effect on normal cells in vitro. Further, as shown in the text, the physiologically active substance is derived from a human gene. Specifically, based on the amino acid sequence structure of rabbit tumor necrosis factor (hereinafter abbreviated as TNF), a human chromosomal gene was artificially used as a novel artificial DNA using DNA extracted by a conventional gene recombination procedure. It is a protein produced from a genetically engineered construct prepared by constructing DNA and incorporating the DNA. The physiologically active substance, which is considered to be a human TNF-like substance, is a substance that is expected to be a safe and highly effective anticancer agent considering that it is a protein originally possessed by humans.

In recent years, advances in biochemical research have been remarkable, and it has become possible to produce a large amount of various useful trace proteins existing in the natural world by utilizing the technology of gene recombination.
(Refer to Yasutaka Takagi, "Genetic Manipulation Manual", Kodansha, ibid., "Genetic Manipulation Experimental Method", Kodansha, etc.) At present, microorganisms such as Escherichia coli are most commonly used as genetic recombinants. There is. However, some problems still remain when using these microorganisms as genetic recombinants. One of them is a microbial-derived proteolytic enzyme. Also,
Pyrodiene (pyrogenic factor) derived from the microbial cell wall is also a problem. In other words, artificial proteins, which are originally regarded as foreign substances to microorganisms, are produced in recombinant microorganisms with high efficiency, but as the purification process progresses, a powerful proteolytic enzyme that is a mechanism for eliminating microorganisms is developed. Will be decomposed by. As a result, it has been extremely difficult to obtain the target protein with high yield and high purity.

In The Journal of Biological Chemistry 256, No. 189750-9740, 1981, when artificially producing and purifying interferon using recombinant Escherichia coli, the degradation of the interferon by a proteolytic enzyme derived from the bacterial cell was performed. There are reports on the use of various protease inhibitors for the purpose of prevention. However, the well-known proteolytic enzyme inhibitors are themselves highly toxic to humans. Therefore, it is not practical in terms of safety to use it during the manufacturing process of the drug, which is the object of the present invention, because complete removal is difficult.

In view of the above situation, the present inventors, as a result of intensive research, surprisingly, by carrying out a suitable heat treatment in the purification step, inactivation of the bacterial cell-derived proteolytic enzyme, We have found that it is extremely easy and safe to achieve.

Furthermore, surprisingly, nucleic acids, lipids, and proteins derived from cells are precipitated as an insoluble precipitate by heat treatment,
It was found that the degree of purification was dramatically improved when the gel filtration step was performed next, because aggregation and polymerization occurred. At the same time, it was surprisingly found that the lipopolysaccharide, pyrogen, can be easily removed, and the present invention has been completed.

The present invention relates to a purification method characterized in that a heat treatment is carried out when purifying a physiologically active substance produced from a genetically modified substance and having the following properties. That is, the property is L when measured by the method described in the text.
-Has cytotoxic activity on M cells. In addition, BAL transplanted with Meth A sarcoma cancer cells by the method described in the text
When administered to B / c mice, it causes hemorrhagic necrotic reaction at the tumor site.

The physiologically active substance used in the present invention is a protein containing at least 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 Gin Val Tyr Phe Gly Ile Ile Ala Leu The above sequence consists of L-amino acids represented by the following abbreviations,
The left to right orientation of the sequences indicates the orientation from the N-terminus to the C-terminus.

Cys: Cystine residue Gln: Glutamine residue Asp: Aspartic acid residue Pro: Proline residue Tyr: Tyrosine residue Val: Valine residue Glu: Glutamic acid residue Ala: Alanine residue Asn: Asparagine residue Leu: Leucine residue Group Phe: Phenylalanine residue Gly: Glycine residue His: Histidine residue Ser: Serine residue Thr: Threonine residue Ile: Isoleucine residue Trp: Tryptophan residue Arg: Arginine residue The recombinant gene used in the present invention is At least the following DN
It contains the A sequence.

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 GCA 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 CAC CCC TCC 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 CATT GTC GGG ATC ATT GCC CTG The above-mentioned DNA sequences use the following abbreviations, and the direction from left to right of the sequences indicates the direction from 5'to 3 '.

A: 2'-deoxyadenylic acid residue C: 2'-deoxycytidylic acid residue G: 2'-deoxyguanylic acid residue T: thymidylic acid residue Next, the physiologically active substance is as follows. Obtainable.

1. The bacteriophage λ / rabbit chromosomal gene library and the bacteriophage λ / human chromosomal gene library are available at the Department of Biochemistry and Molecular Biology of Harvard University (7 Divinity Avenue, Cambridge, Massachusetts 02).
138, U.S.S. S. A. ) From Professor T. Maniatis. These libraries can be created by the following method. [See Cell, 15 , p. 678 (1978)] (1) Rabbit or human tissue, for example, rabbit or human pancreas, is made into a frozen powder, and RNA and protein components are decomposed, and then precipitated by precipitation. Molecular DN
Get A

(2) This high-molecular-weight DNA is partially decomposed because the gene loci are randomly cut.

(3) 15-2 by molecular weight fractionation from the obtained DNA fragment
A fragment with a size of 0 kilobase pairs (kb) is obtained.

(4) The obtained DNA fragment is cloned using λCharon30 phage vector.

(5) The obtained vector is incorporated into infectious phage particles containing rDNA in vitro to obtain the above-mentioned rabbit or human chromosomal gene library.

2. The rabbit TNF cDNA obtained in Reference Example 1 is PWJRigby.
Nick translation method [J. Mol. Biol. 113 , p. 2
By 37 (1977)] ^and labeled with ³² P.

Rabbit TNF cDNA labeled with ³² P by inoculating each of the bacteriophage λ / rabbit chromosomal gene library and the bacteriophage λ / human chromosomal gene library so as to form plaques densely on a uniform layer of bacteria. Was screened by hybridizing with.

4 Isolate the corresponding DNA from an appropriate clone, create a restriction oxygen map, and use the Southern hybridization method [EMSouther
n, J. Mol. Biol., 98 , P. 503 (1957)].

A fragment of rabbit TNF and a gene of the physiologically active substance, which has been digested with a restriction enzyme, is introduced into a plasmid vector, and the nucleotide sequence is analyzed.

5. Compare the rabbit TNF cDNA and the nucleotide sequence of the rabbit TNF gene to determine the exon of the rabbit TNF gene (a nucleotide sequence that does not code for the amino acid sequence of rabbit TNF).

6. Then, the rabbit TNF gene is compared with the gene of the physiologically active substance to determine the exon and intron of the physiologically active substance.

7. Confirmed that the amino acid sequence of rabbit TNF determined by the nucleotide sequence obtained by combining exons in which the intron of the rabbit TNF gene has been deleted matches the amino acid sequence determined by the nucleotide sequence of the cDNA of rabbit TNF. To be done.

8. Next, the amino acid sequence of the physiologically active substance is determined from the nucleotide sequence obtained by deleting the intron of the physiologically active substance gene and connecting exons. It is confirmed that the amino acid sequence of the physiologically active substance partially matches the amino acid sequence of rabbit TNF.

9. After that, the DNA encoding the physiologically active substance is in vitro.
And then introduce into an appropriate expression vehicle to make a recombinant DNA containing the DNA.

10. The physiologically active substance thus obtained is in its mature form and has 155 amino acid residues starting from serine. On the other hand, it has a signal peptide as its preceding sequence.

Without changing the amino acid sequence due to reasons such as different usage frequencies of codons (genetic code) corresponding to each amino acid,
It is also possible to replace part or all of the base sequence with artificial DNA that is chemically synthesized.

It is also presumed that the physiologically active substance is produced in the cell in an immature form (pre- or prepropeptide), and becomes a matured physiologically active substance via an intermediate by a maturation process (processing). Such an immature form of the physiologically active substance can be deduced from the base sequence of the physiologically active substance gene. The physiologically active substance gene, including the immature form and the gene encoding the physiologically active substance in the intermediate, can also be recombined using natural or artificial DNA.

One of the applications of these methods is methionine codon (A
(TG) of mature or immature or intermediate TNF gene
It is to introduce at the 5'end. By doing so, mature or immature or intermediate physiologically active substance is produced from mRNA synthesized by an appropriate promoter. The methionine residue thus added to the N-terminus is naturally removed depending on the host.

As another form, by adding a sequence rich in hydrophobicity called a signal sequence, it can be secreted to a part called periplasma outside the host cell or in Gram-negative bacteria.

Also, in the case of a pecter incorporating a start codon, a fusion peptide of the peptide derived from the pecter and the physiologically active substance is formed, but in this case, the peptide is cleaved chemically or oxygenically, or If there is no change in the main activity of the substance, it can be used as it is.

The bioactive substance gene thus obtained is connected to a gene sequence in the 5'region such as a promoter in such a sequence that transcription and translation are correctly performed, and connected to a vector capable of replicating in bacteria or higher organism cells. The physiologically active substance can be obtained by obtaining the recombinant gene described above, transforming a bacterium or a higher organism cell with the recombinant gene, growing the transformant, and expressing the physiologically active substance gene.

When Escherichia coli is used as the host, various mutant strains of the E. coli K12 strain, such as HB101 (ATCC33694) and C600K (AT
CC339955), D1210, RRI (ATCC3I343), MC1061, LE392 (AT
CC33572), JM101 (ATCC33876), JM83, JM103, χ1776 (AT
CC31244) is used.

When using Escherichia coli as a host, pBR322,
pBR325, pBR327, pUC8, pUC9, pMB9 (ATCC37019), pJB8
(ATCC37074), pKC7 (ATCC37084), etc. or lambda phage such as λgt, λB, Sharon 4A, M / 3
Phage or the like is used.

In order to produce the physiologically active substance in the cells of Escherichia coli,
The E. coli gene or phage gene promoters are used. Such promoters, preferably the promoter and UV5 mutant lactose degradation enzyme (LAC), and penicillinase (BLA), a promoter of tryptophan synthetase (TRP), P of λ phage _L promoter or tryptophan synthase, lactose degradation The TAC promoter, which is a fusion promoter of the enzyme, is used.

BD170 strain (ATCC33608), BR15 when Bacillus subtilis is used as a host
1 strain (ATCC33677), MI112 strain (ATCC33712) etc. are used,
As a pector, pC194 (ATCC37034), pUB110 (ATCC3701)
5), pSA2100 (ATCC37014), pE194 and other plasmids are used.

When Bacillus subtilis is used as a host, promoters of genes such as chloramphenicol acetylase (CAT), penicillinase and erythromycin resistance are used.

When yeast is used as a host, Saccharomyces cereviseae RH218 strain (ATCC4407
6), SHY1 strain (ATCC44769), SHY3 strain (ATCC44771), D131A
Strains, 483 strains, 830 strains, etc. are used, and plasmids such as YEp13 (ATCC37115), YEp6, YRp7, and XIp5 are used as the vector.

When yeast is used as a host, promoters of genes such as acid phosphatase, alcohol dehydrogenase (ADHI), tryptophan synthase (TRP), phosphoglycerate kinase (PGK), cytochrome B (COB) and actin are used as promoters.

The genetically modified microorganism prepared as described above is cultured in a large amount by a usual method, the desired physiologically active substance is produced, and then the desired physiologically active substance is extracted and isolated and purified. The culture solution after removal of microbial cells or the disrupted extract of microbial cells is used as a crude stock solution for the purification method of the present invention. As the heating time of the present invention, in the state of the crude stock solution,
As it is, or after diluting with an appropriate dilution buffer to adjust pH, ionic strength, etc., or after appropriately concentrating using ultraconcentration, the crude stock solution is salted out with ammonium sulfate, etc. After further purification, after roughly purifying using an ion exchange resin, etc., after performing gel filtration or the like to increase the degree of purification, it is possible to carry out at any of the above steps. In order to eliminate the effect of the degrading enzyme in a short period of time, it is desirable to treat the treatment as early as possible.

It is preferable after the crude stock solution stage or after the crude purification step is completed.

The pH of the aqueous solution containing the physiologically active substance during the heat treatment is 4-
11, preferably 6-8 in the neutral region.

The temperature of the heat treatment is 50 ° -65 ° C. Fifty
At a temperature of not higher than 0 ° C, inactivation of the degrading enzyme is insufficient, and at a temperature of not lower than 65 ° C, the physiologically active substance starts deactivating, which is not preferable. 55 is more preferable
The angle is -65 ° C, and more preferably 58-62 ° C.

Next, the heat treatment time is 5 minutes to 180 minutes. Also in this case, if it is too short, inactivation of the degrading enzyme is insufficient, and if it is too long, the physiologically active substance begins to deactivate, which is not preferable. It is more preferably 10 minutes to 120 minutes, further preferably 30 minutes to 60 minutes.

Next, the solution after the above heat treatment is centrifuged or filtered to remove insoluble substances generated by the heat treatment, and then 30,000-70,000 in the molecular weight fractionation range.
The carrier is a gel filtration column. Specifically as a carrier for gel filtration, Sephadex G75, G100, G150, G
200, Cefacrylic S200, S300, (Pharmacia, Sweden) Biogel P30, 60, 100, 200, (Bio-Rad, USA), Toyopearl HW65, HW55 (Toyo Soda), Cellulofine GC-700-m, GC- Although 200-m (Seikagaku Corporation) can be mentioned, Cefacrylic S200, S300, Toyopearl HW55, HW65, and Cellulofine GC-700-m are preferable from the viewpoint of strength.

By carrying out this step after the heat treatment, a high degree of purification and a high pyrogen removal rate were obtained as compared with the case where this step was carried out in the non-heat treatment.

Although a buffer solution is used as the eluent in this step, it is not particularly limited as long as it is a common buffer solution such as a phosphate buffer solution and a Tris-HCl buffer solution. However, pH
Used in the range of 6-9. Various salts may be added, but the concentration is preferably 0.1-2.0M.

As described above, the present invention is characterized by a heat treatment, a method for purifying a physiologically active substance producing a genetically modified body,
Further, the present invention relates to a purification method characterized by performing heat treatment and appropriate gel filtration. INDUSTRIAL APPLICABILITY The purification method of the present invention is extremely safe as compared with conventional methods, and is excellent in that the desired physiologically active substance can be purified from a genetically modified substance in a large amount easily and inexpensively. It has features.

Approximately 300 units of the purified physiologically active substance obtained by the purification method of the present invention showed (+) activity in the physiological activity evaluation using Meth A sarcoma carrier cancer mice described below. Furthermore, BALB / c which was carried by colon colon mouse 26
When the purified product of the present physiologically active substance was administered to mice, cancer growth inhibitory and regression effects were observed with a significant difference compared to the control group (physiological saline administration group). In addition, culture 37
The activity against various human cancer cells was evaluated by the same experimental method as the cytotoxic activity evaluation against LM cells described below except that the test was carried out at 72 ° C. for 72 hours. The cells used are K
The cells were B cells (nasopharyngeal cancer), PC-8 cells (lung cancer), and normal cells (human fetal kidney cells, human fetal foreskin cells) as comparative control cells. As a result, normal cells showed almost no cytotoxic activity, but cancer cells showed strong cytotoxic activity.

That is, the purified product of the physiologically active substance of the present invention has an extremely excellent antitumor activity and is a human-derived protein, and therefore it can be expected as a useful antitumor agent in the future.

Next, the details of the evaluation method performed in carrying out the present invention will be described below.

1) In vivo activity measurement (activity measurement using Meth A sarcoma carrier mouse) As an in vivo method, for example, the method of Carswell et al., Proc.
Nat. Acad. Sci. USA, 72 (1975) 3666.

The method used by the present inventors is an improved version of this method, in which the effect of necrosis of the transplanted Meth A sarcoma tumor by the physiologically active substance is measured. That is, 2 × 10 ⁵ ) Meth in the lower skin of BALB / c mice.
Transplant A sarcoma cells. Seven days later, the size of the transplanted tumor was 7 to 8 mm in diameter, a mouse with good blood circulation without hemorrhagic necrosis was selected, and 0.5 m of the physiologically active substance sample diluted with physiological saline from the tail vein was selected. Inject
After 24 hours, the judgment is made according to the following judgment criteria.

(-): No change (+): Subtle hemorrhagic necrosis (): Moderate hemorrhagic necrosis (necrosis over 50% of the center of transplanted cancer surface) (): Significant hemorrhagic necrosis (of transplanted cancer) Severe necrosis in the center and a small amount of surrounding cancer tissue) 2) In vivo activity measurement (activity measurement using LM cells) The in vitro method for measuring the activity of the physiologically active substance is, for example, Ruff. (Lymphokines Vol.11, ed. By E. Pick, Academic
Press, NY (1980) 235], or [J. Immunol., 126 (1
981) 1279].

The method used by the present inventors is an improved version of these methods, in which the effect of the physiologically active substance to kill LM cells (American Type Culture Collection, CCL1.20) is measured. . That is, 0.1 m of the physiologically active substance sample diluted with a culture medium and L of a concentration of 10 ⁵ / m
Add 0.1 m of M cell culture medium suspension to a 96-well tissue culture microplate (Flow Laboratory). The medium used is Eagle's minimum essential medium containing 10 V / V% fetal bovine serum (the composition is described, for example, in "Tissue Culture" edited by Junnosuke Nakai et al., Asakura Shoten, 1967). The microplate is cultured for 48 hours at 37 ° C. in air containing 5% carbon dioxide. After the culture is completed, 20 μl of 20% glutaraldehyde aqueous solution is added to fix the cells. After fixing, wash and dry the microplate, add 0.1m of 0.05% methylene blue solution,
Stain the surviving cells. After washing away excess methylene blue and drying, the remaining methylene blue is extracted with a 3% hydrochloric acid solution, and the absorbance at 655 nm thereof is measured by Titer Tech Multiscan (Flow Laboratory). This absorbance is proportional to the number of surviving cells.
The dilution rate of the physiologically active substance corresponding to a value of 50% of the absorbance of the control to which the physiologically active substance sample is not added is determined by a graph or calculation, and the dilution rate is defined as a unit (U) / m. Hereinafter, in vitro of the physiologically active substance in the present invention
All activities are expressed in this unit except for Reference Examples 1 and 2.

The method for measuring L-cell damaging activity in Reference Examples 1 and 2 differs from the above method in the following points. That is, use L-929 cells instead of LM cells, and use 1 V / V% in the medium.
Fetal bovine serum and 5 μg / m actinomycin D
The difference is that an Eagle minimum essential medium containing spores is used, and the culture time is 21 hours instead of 48 hours.

3) Pyrogen measurement using PYRODIC PYRO-DIC (Seikagaku Corporation), a commercially available pyrogen measurement kit
Was measured according to the attached operation standard.

4) Protein quantification Protein was measured and calculated according to the method of Lowry et al. (J. Biol. Chem., 193p265 (1951)).

5) Measurement of proteolytic enzyme The measurement is performed by a method improved by the present inventors using a commercially available synthetic substrate S-2222 (Daiichi Pure Chemicals Co., Ltd.).

Reagent 1) 4mM S-2222 100μ 2) 1M Tris-HCl (pH8.3) 100μ 3) Sample 1000μ Method Reagents 1, 2 and 3 are put in a reaction vessel (2cc) and 37 ℃
After carrying out the reaction at
After fixing by inserting, the absorbance at 405 nm is measured.

Note that instead of the sample in 3), trypsin (B
oivine Pancreas Type / SIGMA NO.T-800) is used to obtain a standard curve. Using the above method, the concentration of proteolytic enzyme contained in each step is calculated in terms of tryptocin concentration.

Hereinafter, the present invention will be described in detail by reference examples and examples.

In carrying out the present invention, the production of recombinant DNA and the introduction of the recombinant into microorganisms were carried out according to the following experimental documents, unless otherwise specified.

(1) Yasutaka Takagi, Gene manipulation manual, Kodansha (2) Yasutaka Takagi, Gene manipulation experiment method, Kodansha (3) T. Maniatis, E, F, Fritsch, J. Sambrook Molecular Cloning, Cold Spring Harbor Laboratory (USA (4) Ray Wu et al., Method in Enzymology, Vol. 101, Academic Press (USA), Abbreviations Used in Reference Examples and Examples MOPS: Morpholinopropane Sulfate LB Medium: Luria-Bertani Medium DMSO: Dimethyl Sulfoxide PFU: Plaque Forming unit EDTA: Ethylenediaminetetraacetic acid SDS: Sodium dodecyl sulfate BRL: Bethesda Research Laboratory DMT: Dimethoxytrityl lac: Lactose Tris: Tris (hydroxymethyl) aminomethane XAR-5: X-ray film (Eastman Kodak) Company, USA) 1 x SSC: 0.15M sodium chloride + 0 0.015 M sodium citrate, pH7 2 x SSC: 0.30 M sodium chloride + 0.030 M sodium citrate, pH7 3 x SSC: 0.45 M sodium chloride + 0.045 M sodium citrate, pH75 x SSC: 0. 75M Sodium Chloride + 0.075M Sodium Citrate, pH 7 6 x SSC: 0.90 M Sodium Chloride + 0.090 M Sodium Citrate, pH 7 FDSS: 50% Deionized Film Amide + 5 x Denhardt'S + 5 x SSPE + 0.1% SDS + 100 μg / M Denatured calf thymus DNA Denhardt's solution: An aqueous solution containing 200 mg of Ficoll, 200 mg of polyvinylpyrrolidone and 200 mg of bovine serum albumin in 1 SSPE: 0.18 M sodium chloride + 10 mM sodium dihydrogen phosphate + 1 mM EDTA, pH 7. 4 SM: Sodium chloride 5.8 g, magnesium sulfate heptahydrate 2 g, 1M Tris · HC in 1 Phage preservation medium containing 50m of l (pH7.5) and 5m of 2% gelatin NZ broth: 10g of NZ amine (Fumco Siefield Chemical Division of Crafts, USA), 5g of sodium chloride And 2g of magnesium sulfate heptahydrate 2g IPTG: Isopropylthiogalactoside x-gal: 5-dibromo-4-chloro-3-indrylgalactoside TAE: 0.04MTris-acetic acid (pH8.0) -0.002MEDTA bp: Base pair Reference Example 1 Step 1 (acquisition of rabbit serum TNF) 50 mg of Propionibacterium acnes (Corynebacterium parvum, Welcome, UK), which had been killed with formalin, was injected into a female rabbit (body weight 2.5-3.0 kg) through an ear vein. did. After 8 days, 100 μg of endotoxin (lipopolysaccharide derived from Escherichia coli O26: B6, Difco, USA) was injected into the rabbit via the ear vein, and whole blood was collected from the heart 2 hours later. 100m for collected blood
After adding 100 units of heparin sodium per
Centrifugation was carried out at 00 rpm for 30 minutes to remove blood cells and insoluble solid matters and cool. Serum TNF from 40 rabbits
A plasma of 2.4 was obtained with a titer of 3 × 10 ⁴ units / m 2.

Step 2 (partial purification of serum THF) To the plasma 2.4 obtained in Step 1 was added 24 g of Celite, and the mixture was stirred for 1 hour and then passed. After adding 1.2 0.04 M Tris-HCl buffer (pH 7.8) to the solution, DEAE Sepharose CL fully equilibrated with 0.04 M Tris-HCl buffer (pH 7.8) containing 0.1 M sodium chloride. Added to a column of -6B (Pharmacia, Sweden). After washing with 0.04M Tris-HCl buffer, it contains 0.18M sodium chloride
Flow out using 0.04 M Tris-HCl buffer (pH 7.2). Fractions showing L-cytotoxic activity were concentrated by ultrafiltration. Then, the concentrate was added to a column of Cefacryl S-200 (Pharmacia, Sweden) equilibrated with 5 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride, and the gel was filtered with the same buffer. went. The active fraction was concentrated by ultrafiltration to recover 3.5 × 10 ⁶ units. The specific activity based on protein determination was 18 × 10 ⁶ units / mg.

Step 3 (anti-THF antibody) TNF obtained from serum was partially purified as in Step 2, and Freund's complete adjuvant was mixed at a ratio of 1: 1 to prepare 12-week-old male BALB.
/ c was injected subcutaneously in the back of the mouse. This operation was repeated after 2 weeks and 4 weeks, and one week later, whole blood was collected to obtain the serum.

This serum was added at a final concentration of 5 to the medium for measuring L cytotoxic activity.
TN was obtained from rabbit serum by adding it so that it would be diluted 00-fold.
When the L cytotoxic activity of F was measured, the L cytotoxic activity was not observed. It was concluded that the mouse serum obtained here contained an antibody against rabbit serum TNF (referred to as anti-TNF antibody).

Step 4 (obtaining TNF-producing cells) Propionibacter obtained by killing female rabbits with formalin
ium scnes (Corynebactriium parvum, Welcome, UK) was intravenously administered, and 7 days later, tracheostomy was performed, and the lungs were washed with physiological saline to obtain floating cells. After washing these cells with physiological saline, RPMI1640 (Flow Laboratories, USA) containing 10% fetal bovine serum (Flow Laboratories, USA) was used as a medium and carbon dioxide containing 5% carbon dioxide was used as an atmosphere. 37 in the incubator
Cultured at ° C. The incubator was divided into two, and one side contained 10 μg / m of E. coli-derived endotoxin (E. coli O26: B6-derived lipopolysaccharide, Difco, USA).
The same amount of sterilized water was added to one side. The maximum value was reached 7 hours after the appearance of L-cytotoxic activity in the culture supernatant containing endotoxin. This activity is
It was cleared by TNF antibody but not by normal mouse serum.

On the other hand, L cell cytotoxic activity was not observed in the cell culture supernatant without addition of endotoxin.

Step 5 (molecular weight of TNF) In the cell culture in Step 4, radioactive L- [ ³⁵ S] methionine (1300 Ci / mmo, together with endotoxin in the cell culture,
Culture was performed by adding Amersham (UK, UK) so as to be 1 mCi / m. The culture supernatant was added to the Laemmli method [Laemml
i, UK (1970) Nature (London) Vol. 227 680
Page 685] and analyzed by SDS polyacrylamide gel electrophoresis. The gel concentration was adjusted to 12.5%. After the electrophoresis, it was treated with enhance (New England Nuclear Co., Ltd., USA), dried, and then contact-exposed to an X-ray film (Fuji RX, Fuji Photo Film). The production of a substance having a molecular weight of about 17500 was observed in the culture supernatant cultivated in the presence of endotoxin.

In addition, the supernatant of the cell culture in step 4 was SD-treated as above.
2.5% after S polyacrylamide gel electrophoresis
NP40 (Calbiochem, USA) for 1 hour, 2 in water
After shaking for a period of time, each migration lane was cut and separated, and sliced in a width of 2 mm at right angles to the migration method. L-cytotoxicity was examined by culturing each slice fragment with L cells. In the lane in which the culture supernatant was developed in the presence of endotoxin, L cytotoxicity was observed at the position of molecular weight of about 17500, and no activity was observed at other positions.

Obtaining Step 6 (mRNA) After adding endotoxin in the same cell culture as in Step 4 and culturing for 2 hours, cells (rabbit lung lavage cells) were collected by centrifugation. Cytoplasmic RNA and mRNA from it
Is extracted by the conditions of Chirgwin et al. [Chirgwin, J, Me
al., Biochemistry 18: 5294 (1979)]. To cells 3 × 10 ⁸ cells, the 4M guanidine thiocyanate solution 4m added homogenizer (A
It was crushed at M-7, Nippon Seiki Seisakusho. After removing the residue by centrifugation, 2.4 g of cesium chloride was dissolved and 2.5
A 5.7 M cesium chloride and 0.1 M EDTA solution (pH 7.5) of m was gently overlaid on a polyallomer tube.

After ultracentrifugation using a Beckman SW41 rotor (Beckman, USA) at 20 ° C. and 30,000 rpm for 12 hours, the supernatant was removed, and the pellet was washed with 10 mM Tris-HCl buffer (5 mM EDTA, 1% SDS). Dissolved in 1 m). This solution was extracted with a mixed solution of 1 m of chloroform-1-butanol (4: 1), 0.05 volume of 2M sodium acetate and 2.5 volume of ethanol were added to the aqueous layer, and -20
The RNA was precipitated by leaving it at 2 ° C. for 2 hours or more. The precipitate was collected by centrifugation, dried, and then dissolved in 500 µ of sterilized water to obtain a cytoplasmic RNA solution.

The cytoplasmic RNA solution is heated at 68 ° C for 2 minutes and then rapidly cooled to 50
10 mM Tris EDTA buffer pH 7.4 with 2 times concentration of 0μ
(Containing 1 mM EDTA, 0.1% SDS and 0.5 M lithium chloride) was added, the mixture was developed on a 200 mg oligo (dT) -cellulose (BRL) column, washed with 1 m of the above buffer 10 m, an elution buffer (10 mM Tris). -Hydrochloric acid pH 7.4, 1 mM EDT
A, containing 0.1% SDS) 2 m. 0.0 after elution
5 volumes of sodium acetate and 2.5 volumes of ethanol were added, and the mixture was precipitated by cooling at -20 ° C. The precipitate was collected by centrifugation, and the fraction adsorbed on the oligo (dT) cellulose column was collected again. 85μ by UV absorption spectrum analysis
g mRNA was recovered.

Step 7 (size fractionation of mRNA) 880 μg of nRNA obtained in the same manner as in Step 6 was dissolved in 250 μm of water and layered on a 5-25% linear sucrose density gradient of 10 m. The sucrose density gradient is a Tris buffer (25 mM Tris-HCl pH7.2 pH, containing 5 and 25% sucrose, respectively).
2 mM EDTA, containing 1% SDS) was used and made by ISCO570 Gragenter (ISCO, USA).

Using Beckman SW41Ti, ultracentrifugation was performed at 4 ° C., 40,000 rpm for 12 hours, and then 400 μ of each fraction was collected by a fraction collection device (Beckman, USA). Each fraction was ethanol-precipitated, centrifuged and dissolved in sterilized water.

Step 8 (mRNA translation experiment) Translation of mRNA by Xenopus oocytes is described in experimental books (for example, Hiroshi Teraoka, Mikio Itsuki, Kotaro Tanaka, protein, nucleic acid, enzyme, extra number, gene manipulation, page 1981.
Year). The Xenopus laevis was obtained from the Hamamatsu Biological Teaching Material. The fractionated mRNA obtained in Example 5 was dissolved in sterilized water so as to have a concentration of 1 μg / μ, and 50 n per 1 oocyte was obtained.
Barth solution (7.5 mM Tris-HCl pH 7.6, 88 mM NaCl, containing 1 mg / m bovine serum albumin,
The cells were cultured for 24 hours in 1 mM potassium chloride, 0.33 mM calcium nitrate, 0.41 mM calcium chloride, 0.82 mM magnesium sulfate, 2. mM sodium bicarbonate, 18 U / m penicillin G, 18 μg / m streptomycin.
The oocyte was crushed as it was in the culture solution, centrifuged, and then the L cell cytotoxicity of the supernatant was measured. In the vicinity of the sedimentation constant 16S, the L cytotoxic activity gave the highest value. This activity was eliminated by the anti-TNF antibody obtained in step 3, but not by normal mouse serum.

Step 9 (acquisition of transformation) Using 5 μg of the fractionated mRNA obtained in Step 5, double-stranded DNA was prepared according to Experiment Manual (1), page 97 et seq. Reverse transcriptase used was from Life Science (USA). Double-stranded DNA
Was fractionated by polyacrylamide gel electrophoresis with a 3.5% gel concentration to obtain a fraction of about 1000 to 2000 base pairs (hereinafter, base pairs are abbreviated as bp) of 330 ng. This fraction 7ng
According to the above-mentioned experimental manual, the terminal deoxynucleotidyl transferase (BRL) was used to connect the deoxy C chain, and the plasmid was annealed with the plasmid pBR32256ng in which the deoxy G chain was also connected to the Pst 1 site. E. coli K-12 strain (HB
101, ATCC33694) to obtain a transformant of strain 12000.

Step 10 (amino acid sequence of TNF portion) The TNF partially purified in Step 2 is treated with S in the same manner as in Step 5.
Purified by DS polyacrylamide gel electrophoresis. Partly stained with Coomassie PRILIANT BLUE and the molecular weight is approx.
The band at 17000 was excised from the gel and extracted with 1% ammonium bicarbonate. About 180 μg of protein was recovered using 5 × 10 ⁷ units of TNF.

Of this, 150 μg is 1% ammonium bicarbonate 75 μg
3 μg of TPCK trypsin (Worthington Biochemical Co., USA) was added, and the mixture was incubated at 37 ° C. for 4 hours. The reaction solution was fractionated by high performance liquid chromatography using Cosmo Seal 5C8 (Hanui Chemical Co., Ltd.) as a carrier to obtain a trypsin-digested fragment.

The highly purified TNF and its tryptic digestion fragments as described above were then desalted on a column of Sephadex G25, lyophilized and then RMHewick using an amino acid sequence analyzer model 470A (Applied Biosystems, USA). Et al. (J. Biol. Chem. 256, 799
0-7997, 1981), and endoman degradation was performed from the N-terminal. The phenylthiohydantoin amino acid released in each step was analyzed using a high performance liquid chromatography model SP8100 (Spectra Physics, Inc., USA) using Zorbax ODS (Du Pont, USA).
Was used as a column and analyzed by a conventional method. As a result, TNF
The amino acid sequence from the N-terminal side of was as follows.

Ser Ala Ser Arg Ala Leu Ser Asp Lys Pro Leu Ala His Val Val Ala Asn Pro Gln Val Glu Gly Gln Leu Gln Also, one of the tryptic digests had the following amino acid sequence from its N terminus.

Glu Thr Pro Glu Glu Ala Glu Pro Met Ala Step 11 (Synthesis of oligo DNA probe) mRNA deduced from the amino acid sequence of TNF obtained in Step 10
Oligo DNA complementary to the nucleotide sequence of was synthesized.
The synthetic method is the modified phosphate triester method (H.Ito et al. Nueleic Acids Acids Res.
0, 1775-1769, 1982).

128 kinds of oligo DNAs deduced from the amino acid sequence are divided into 5 groups, each of which is 16, 16, 32, 32, 32
Synthesized as a mixture of types.

First, a part of the amino acid sequence of the novel physiologically active substance of the present invention and the nucleotide sequences of 5 types of synthetic oligo DNA probes based on the amino acid sequence are shown. Each was deprotected according to a conventional method, and G-50
Purification by column chromatography using (Pharmacia, Sweden), 20% polyacrylamide gel electrophoresis containing 7M urea and DE52 (Whatman, USA) column chromatography, and 0.1 mM Tris ED.
It was dialyzed against TA buffer.

Each purified oligo DNA was radiolabeled with T4 polynucleotide kinase (Bio Rat Laboratories, USA) and γ-32p-adenosine triphosphate by a conventional method, and purified with DE52 column. About 3 each
Radioactivity of × 10 ⁸ cpm / μg was introduced.

Step 12 (Assay of Oligonucleotide) The mRNA of TNF-producing cells obtained according to Step 6 was treated at 50 ° C. for 60 minutes in the presence of glyoxal and dimethyl sulfoxide, and then fractionated by 1.1% agarose gel electrophoresis. The fractionated mRNA was transferred using an electrophoretic transfer blotting device (Bio-Rad Laboratories, USA) according to the manufacturer's manual. Then, the membrane was treated with 5 × Denhardt's solution containing 5 × SSC and 150 μg / m of denatured salmon sperm DNA at 65 ° C. for 2 hours, and then radiolabeled oligo DNA was treated with 1 × 10 ⁷ cpm / m 2. 5 × Denhardt's solution containing 5 × SSC solution at 50 ° C. Treated for 2 hours. The membrane is then placed in 6 × SSC at room temperature, 40
X-ray film XAR-5 after sequentially washing at 50 ℃, 50 ℃ and 60 ℃
I exposed it to. As a result, it was revealed that the oligo DNA that most strongly hybridizes with mRNA is MJ, and that the oligo DNA having a sequence completely complementary to mRNA is contained in the MJ mixture.

Step 13 (Cloning of TNF gene) The transformant obtained in Step 9 was transferred onto a cellulose filter according to the method in Experimental Manual (2), page 162, and the DNA and the radiolabeled oligo DNA (MJ) selected in Step 12 were transferred. And were hybridized under the same conditions as in step 12 (colony hybridization). Ninety-nine strains were selected by selecting 49 strains that strongly hybridized, fixing them on the filter and performing colony hybridization again.

From each of these 9 strains, about 5 μg of plasmid was obtained according to the rapid plasmid isolation method on page 6 of Experimental Manual (1). This plasmid is used as a restriction enzyme PstI, TaqI, RsaI, PvuII.
(All of them were manufactured by BRL) and cut according to the manufacturer's manual, and the lengths of cut pieces by each enzyme were compared by 1% agarose gel electrophoresis.

As a result, it was suggested that all 9 strains had a fragment of about 50 bp with RvuII and RsaI, and 8 strains had a fragment of about 200 bp with RsaI and had a common sequence. Figure 1 shows the results of analysis with restriction enzymes.

In addition, 7 of these strains were cultured in 2 m of L medium containing 10 μg / m of tetracycline, and the cells collected by centrifugation were disrupted by ultrasonication in 2 m of physiological saline, and the L supernatant of the centrifuged supernatant was damaged. When the activity was measured, as shown in Table 2, L cell cytotoxic activity was shown.

This activity was abolished by anti-TNF antibody, but not by normal mouse serum. Therefore, all these 9 strains are TN
It was carried out to have a plasmid containing the F gene.

Step 14 (Determination of nucleotide sequence of TNF gene) An E. coli strain containing plasmids pB2-7 and pR18 was
After culturing in M9 medium [Experimental Manual (3) page 440] 1 containing 10 μg / m tetracycline, Experimental Manual (3)
Plasmids were isolated according to the method on page 90, each containing approximately 15
0 μg was obtained.

The respective nucleotide sequences are represented by the Maxam-Gilbert method (Maxam et.al. Method in Enzymology, 55, 490,
1980, Academic Press). Further, the agreement between this base sequence and the partial amino acid sequence determined in step 9 revealed the entire structure of the TNF protein.

Step 15 Using a recombinant form of plasmid pR / 2, a plasmid was constructed for the purpose of expressing TNF using lac as a promoter in E. coli. As shown in Fig. 2, 10 µg of plasmid pR / 2 was treated with 10 units of ApaI (BRL) at 37 ° C.
Digested for 2 hours and electrophoresed on a 4% polyacrylamide gel to isolate an approximately 630 bp fragment. About 1 μg of fragment was electroeluted from the gel. The two deoxyoligonucleotides shown in the figure in the same manner as in Step 10, namely,
5'-GATCCATGTCAGCTTCTCGGGCC-3 'and 5'-CGAGAAGCTG
ACATG-3 'was synthesized (second view), 5 of each oligodeoxynucleotide about 100pmole according to the experimental manual (3) 122 pp' end was phosphorylated using of T ₄ polynucleotide kinase. The reaction completed liquid is extracted with phenol,
After further chloroform extraction, 0.5μ of oligomer
Ethanol-precipitated together with g of the Apa1 fragment of about 630 base pairs. Ten units of T ₄ D according to the experiment book (1) page 37
The fragments were reacted with NA ligase at 4 ° C. overnight and ligated. After the reaction completion liquid was precipitated with ethanol, 20 units of Ba
It was digested with mHI for 3 hours at 37 ° C., subjected to electrophoresis on 4% polyacrylamide, and a fragment of about 670 bp was recovered by electrophoretic elution. 1 μg of commercially available plasmid pUC-8 (PL Biochemical, Catalog No. 4916, USA) was added to BamH
Digested with I, extracted with phenol, extracted with chloroform, and precipitated with ethanol.
A fragment containing the 0 bp TNF whole structural gene and having BamHI sites at both ends was ligated using T ₄ DNA ligase. Experiment book
According to (4), page 20, E. coli JM101 (ATCC33876) was transformed to obtain white colonies of about 200 agar medium containing IPTC and x-gal. When plasmid DNA was prepared from 100 of these transformants and digested with BamHI, 15 of them contained the desired 670 bp BamHI fragment. Furthermore, in order to check the direction of insertion, the above 1
Each of the 5 plasmids has only one recognition site
EcoRI (recognition site on pUS-8) and PvuII (about 670 bp)
(There is a recognition site on the fragment of 4) and was examined using 6% polyacrylamide gel electrophoresis.
From this plasmid, a fragment of about 140 bp of interest was confirmed, which was found to be the forward direction from the lac promoter on pUC-8.

Analysis of the nucleotide sequences confirmed that these 7 plasmids were identical and had the desired nucleotide sequence at the junction between the lac promoter, synthetic DNA and cDNA.

Step 16 Plasmid pR17 was used to construct a plasmid for the purpose of directly expressing TNF as a lac UV5 promoter in E. coli. As shown in FIG. 3, 10 μg of plasmid pR1710 unit was digested with Apa1 (BRL) at 37 ° C. for 2 hours, and then subjected to 4% polyacrylamide gel electrophoresis to give about 6
A 30 bp fragment was isolated. About 1 μg of fragment was electroeluted from the gel. In the same manner as in Step 10, the two deoxyoligonucleotides shown in FIG.
GTCAGCTTCTCGGGCC-3 'and 5'-CGAGAAGCTGACATG-3' were synthesized, and 5'ends of about 100 pmole of the above two types of deoxyoligonucleotides were synthesized with T ₄ according to the experiment manual (3) page 122.
Phosphorylated using polynucleotide kinase. The reaction-terminated solution was extracted with phenol and chloroform, and then the Apa1-digested fragment of pR17 (about 6
(30 bp) 0.5 μg and ethanol precipitated. 4 ° C with 10 units of T ₄ ligase according to page (37)
The reaction was allowed to occur overnight and the binding was allowed to proceed. After the reaction, the reaction solution was ethanol-precipitated, digested with 20 units of Ecpr1 for 3 hours at 37 ° C., and a fragment of about 670 bp was recovered by electrophoresis on a 4% polyacrylamide gel.

Plasmid pOP95-15 is a fuller's method (F. Fuller, Gene, 1
Vol. 9, pp. 42-54, 1982).

1 μg of pOP95-15 was digested with EcoRl and extracted with phenol,
Chloroform extraction, and vector 0.5μg prepared by the ethanol precipitation, the synthetic deoxy oligonucleotides as described above, a fragment of approximately 67-bp obtained by combining the TNF gene, linked with T ₄ DNA ligase. Experiment book (4), 20
E. coli JM101 (ATCC33876) was transformed according to the
About 150 white colonies were obtained on the agar medium containing PTG and x-gal.

Prepare 100 of these colonies or plasmid DNA and
When digested with coR 1, 12 of the desired 670 bp
It had the EcoR 1 fragment. Further, in order to examine the direction of insertion, the above 12 plasmids were digested with PvuII and Pst 1 and examined by using 1.5% agarose gel electrophoresis. As a result, 4 plasmids of desired 1280 bp and 2600 bp fragments were obtained. Was confirmed, and it was revealed that the TNF gene was connected in the forward direction from the lacUV ₅ promoter.

Analysis of the nucleotide sequence, the four plasmids are identical, lacUV ₅ promoter, the synthetic oligodeoxynucleotide and cDNA are (designated as pTNF-lacUV ₅ -1.) That have been confirmed to be correctly coupled step 17 (Purification of TNF produced by E. coli) 1 E. coli strain containing the plasmid obtained in step 16
50 m of L medium containing 00 μg / m of ampicillin
The cells were cultured overnight in 5 days, transferred to the same medium as in 5 and further cultured for 3 hours. Isopropyl β-D-thiogalactopyranoside (Sigma Chemical Co. USA) was added to a final concentration of 1 mM, the culture was further continued for 6 hours, then cooled, and the strain was collected by centrifugation. As in step 13,
Ultrasonication was carried out in 0.04 M Tris-hydrochloric acid buffer solution (pH 7.8) 5 to obtain a bacterial protein solution. This solution is 5 x ¹⁰⁷ units /
L cell cytotoxic activity was shown.

As a result of purifying this solution in the same manner as in step 2, 1.2 × 10 ⁶
Got the unit TNF. The specific activity of this compound 6.8 × 10 ⁷ units /
It was mg.

Step 18 (Evaluation of activity using Meth A sarcoma bearing cancer mouse) The activity of the TNF obtained in Step 17 was evaluated by the in vivo method described in the text.

In addition, on the 20th day after the sample administration, it was observed whether the cancer had completely regressed, and the complete cure rate was obtained.

It measured by the above method. The activity of TNF produced by Escherichia coli is shown in the third.

Reference Example 2 Step 1 (E. coli K12 of plasmid pR18, pB2-7, pB2-2,
Transformation into MC1061 strain) The above-mentioned three types of plasmids obtained in Reference Example 1 were subjected to a conventional method.
Using E. coli K12, MC1061 strain colonies in LB medium,
The cells were cultured until the absorbance at 550 nm became 0.3. The culture medium 50
10mM including 25m 10mM Rbcl after collecting m
Wash with MOPS (pH 7.0) solution, then 50 mM CaCl ₂ , 10 m
The cells were resuspended in 0.1 MMOPS (pH 6.5) containing M Rbcl.

The suspension was ice-cooled for 30 minutes, centrifuged, the supernatant was removed, and the suspension was suspended in a mixed solution of 30 mM DMSO and 0.1 mM OPS (pH 6.5) containing 50 mM Cacl ₂ and 10 mM Rbcl. The suspension was dispensed in 203 μm aliquots, and the above-mentioned plasmid DNA solution 1
0 μ was added to each.

The mixture was ice-cooled for 30 minutes, and then 60 ° C. at 44 ° C.
Give a second heat shock, immediately immediately in advance at 37 ℃
5 ml of warmed LB medium was added. After incubating this solution at 37 ° C for 1 hour, each solution was centrifuged,
The supernatant was removed and a cell pellet was obtained. LB medium was added to the cell pellet, stirred, and then made into a suspension. The suspension was spread on an LB agar plate containing 30 μg / m tetracycline and cultured overnight at 37 ° C. As a result, tetracycline-resistant transformant colonies were obtained from the plasmids pR18, pB2-7 and pB2-2, respectively.

Step 2 (Preparation of pB2-7 and pR18 plasmid DNA) The transformants of the plasmids pB2-7 and pR18 obtained in Step 1 were cultured according to the method described in the following report to amplify the plasmid. Then, the obtained transformant was harvested, crushed, and the plasmid DNA was purified. [Experiment 3, 88-9
6] That is, each LB medium was inoculated with the transformant,
Incubated at 37 ° C with vigorous shaking.

This process was then repeated to grow the transformants and further plasmids. Then, while cooling the resulting culture of transformants to 4 ° C.,
After centrifugation at G for 10 minutes, the supernatant was removed.

100 m of ice-cold STE [0.1 M sodium chloride, 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA] was washed, and then 20 mg / m in 10 mM Tris-HCl buffer [pH 8.0].
The cells were disrupted using an aqueous solution containing lysozyme.
Transfer the obtained viscous liquid to an ultracentrifuge tube and place it at 25,000 rp.
A DNA solution was obtained by centrifugation at 4 ° C. for 30 minutes.

After measuring the volume of the DNA solution, 1 g of solid cesium chloride was added to 1 m of the solution, and the mixture was slowly and carefully stirred until the cesium chloride was completely dissolved. For every 10 m of the cesium chloride aqueous solution, 0.8 m of 10 mg / m ethidium bromide aqueous solution was added. As a result, the final specific gravity of the solution was 1.55 g / m, which was about 600 μg / m of the final concentration of ethidium bromide.

Transfer the cesium chloride aqueous solution to an appropriate centrifuge tube, add light paraffin oil to the voids, and let stand at 20 ° C for 36 hours.
If you continue centrifugation at 5,000 rpm, linear microbial DNA will be present in the upper layer.
And the circular plasmid DNA is opened, and the closed circular plasmid DNA is in the lower layer. The lower band of DNA was collected by inserting an injection needle next to the tube and transferred to a glass tube. The ethidium bromide was removed and the water bath was dialyzed against TE buffer. The plasmid DNA solution was treated with RNase and extracted with an equal volume of saturated phenol solution. Water layer 0.1
It was applied to Biogel A-150 (Bio-Rad, USA) equilibrated with TE buffer (pH 8.0) containing% SDS. D
NA was washed in and eluted with TE buffer containing 0.1% SDS in order to obtain active fractions. The fractionated solution was precipitated with ethanol to obtain a purified plasmid DNA.

Purified pB2-7 plasmid DNA was 250μ by the above method.
g, pB18 plasmid DNA was obtained at 134 μg.

Step 3 (Nick trusslation of purified pB2-7 and pB18 plasmid DNA) 40 μg of the purified plasmid DNA obtained in step 2 was digested with the restriction enzyme Pst 1 and then subjected to electrophoresis using 4% acrylamide gel.

After electrophoresis, stain 2.6 μg (calculated value 6 μg of 4
The desired band (3%) was cut out. Nick translation was performed using 500 ng of the excised fragment in 2.5 μg according to the method of T. Maniatis et al. [Proc. Natl. Acad. Sci. USA, 72 1184 (1974)]. A commercially available kit (BRL, USA) was used for nick translation. 25
80 pmole of dCTP activated by the reaction of μ was used (40
0Ci / m mole).

First, the following mixed solution was prepared.

2.5 μ solution A (αNTP solution) 2.5 μ solution B (500 ng DNA, ie Pst 1 insert) 5 μ α- ³² P-dCTP (3200
Ci / m mole) 1.3μ dCTP (65pmole, 50 pmole/μg dCTP) 11.2μ Solution E (H ₂ O) 22.5μ Total 22.5μ Solution 2.5μ solution C (DNase 1,
DNA polymerase 1) was added and reacted at 15 ° C. for 60 minutes.

Then, the solution D (stopping buffer 9 was added to stop the reaction. Further, carrier tRNA was added to carry out ethanol precipitation twice, and then dissolved in 500 µ of water.

The specific activity was 9.3 × 10 ⁷ cpm / μg DNA.

Using the purified pR18 obtained in step 2, nick translation was performed in the same manner as described above. The specific activity was 7 × 10 ⁷ cpm / μg DNA.

Step 4 (obtaining Rsa 1 fragment of pR18) 80 μg of pR18 plasmid DNA was digested with restriction enzyme Rsa 1 and subjected to 4% acrylamide electrophoresis. The following insert band of interest was excised and purified using a BND column.

About 640 bp 3.77 μg (recovery rate 52%) About 175 bp 1.77 μg (recovery rate 50%) This about 640 bp insert was inserted into the 3 ′ fragment of pR18 (of pR18).
An insert of about 175 bp, which means the 3'untranslated portion), was named pR18cfr (coding portion of pR18).

Furthermore, in the above method, instead of Rsa 1, Pst 1 and Mst II
When digested with, about 450 bp 3.65 μg (recovery rate 60%) was obtained. This insert was named the 5'fragment of pR18.

Step 5 (isolation of the physiologically active substance gene in the chromosome) Using the ³² P-labeled plasmid pB2-7 insert obtained in Step 3 as a probe for hybridization, Char
on 4A EcoRl cleavage site [Blattner et al., Science 196
161 (1977)], a fragment of human DNA cleaved with EcoR 1 [Mani
The atis et.cl.Cell 15 687 (1978)] was incorporated bacteriophage created by Charon4A / human chromosomal gene library of 10 ⁶ U plaques were screened. As the method, the puller hybridizing method of Benton and Davis et al. [Benton and Davis, Science 196 180 (1977)] was used.

Since not all of the bacteriophages in the starting medium contained the genetic material necessary to make the physiologically active substance, a probe having a sequence complementary to the gene of rabbit TNF was used. .

The phage plaque containing the gene of interest can be found by measuring its radioactivity by hybridizing with a plaque having radioactivity. In this way 9 hybridizing plaques were obtained from the library.

The method and conditions are as follows: 1) Number of plaques: ~ 1 x 10 ⁶ plaques (~ 4 x 10 ⁴ plaques / φ150 mm plate x 25) 2) Transfer to nitrocellulose filter: [Benton and
Davis, Science, 196 , 180 (1977)] 3) Hybridization: 1.25 × 10 ⁵ cpm / m of Reference Example 1 The pB2-7 insert probe obtained in Step 3 was added, and the temperature was changed to 42 ° C.
19.5 hours 4) Washing: Washing with 2 x SSC-0.1% SDS for 10 minutes at room temperature 4 times, followed by 1 x SSC-0.1% SDS at 50 ° C for 30 hours.
5) Exposure: XAR-5, -80 ° C, 2 intensifying screens, 39 hours. 12 candidate strains were obtained by the above screening. As a result of the secondary screening, 9 contained positive clones, and 1 was still possibly positive.
The positive plaques and potential plaques were picked up from the secondary screening plate, and the third screening was performed. As a result, 9 were positive. A fourth screening was performed on these 9 plaques, and 9 bacteriophages containing the fragment of interest were designated HG1 to HG9, respectively.

Step 6 (isolation of rabbit chromosomal TNF gene) Essentially the same as the method described in Step 5. That is, Char of 10 ⁶ prepared by using DNA cut with EcoRl
The plaques of the on4A / rabbit chromosomal gene library bacteriophage were searched.

Two bacteriophage strains (RG-1, RG-2) containing the rabbit chromosomal gene were obtained.

Step 7 (Southern Blot Analysis of Human Clone) Using the HG-3, HG-6, and HG-7 bacteriophages obtained in Step 5, DNA was obtained according to the following methods.

6 × 10 ¹⁰ E. coli LE392 suspended in 18m SM,
Bacteriophage HG-3 (3 × 10 ⁹ PFU) was added thereto and adsorbed at 37 ° C. for 20 minutes. Then, the obtained mixed liquid was added to NZ broth of 3 and cultured at 37 ° C. for 23 hours with stirring. Next, 60 m of chloroform was added to the mixed solution and stirred for 30 minutes. Sodium chloride was added to the mixture so that the final concentration was 1 M, and the mixture was left for 15 minutes. Then, centrifugation was performed. Next, the molecular weight is about 60
Polyethylene glycol of 00 was added so that the concentration of polyethylene glycol would be 10% (W / V), 4 ° C, 22
Left for hours. The bacteriophage was then harvested by centrifugation. SM obtained bacteriophage
28 m, and then an equal volume of chloroform was added. After mixing with a Portex mixer for 30 seconds, the mixture was centrifuged to collect an aqueous layer, and the total amount was adjusted to 30 m with SM. After adding 26.4 g of cesium chloride and gently dissolving it, a phage band was collected by ultracentrifugation (45,000 rpm, 20 hours). After dialysis against 50 mM Tris buffer (pH 8.0) containing 10 mM sodium chloride and 10 mM magnesium chloride, the final concentration of each was 20 mM, 50 μg / m, 0.1.
EDTA, proteinase K and SDS were added so as to be 5%, and the mixture was treated at 65 ° C. for 1 hour. Then, the mixture was extracted once with each of phenol, phenol: chloroform = 1: 1 and chloroform, and the obtained aqueous layer was dialyzed with 10 mM Tris buffer (pH 8.0) containing 1 mM EDTA. As a result of measuring the ultraviolet absorption of this solution, it was confirmed that bacteriophage HG-3 was pure.
It was confirmed that DNA was obtained.

Bacteriophage HG-6 and HG-7 DNAs were obtained by applying essentially the same method used to prepare the bacteriophage HG-3 DNA.

In this manner, 2920 μg, 1100 μg and 819 μg of HG-3, HG-6 and HG-7 DNAs were obtained, respectively.
Then, the Southern method [EM Southern.J. Mol. Biol., 98 503 (19
75)], Southern blotting of these DNAs was performed under the following experimental conditions.

1) DNA: HG-3 825ng HG-6 935ng HG-7 685ng 2) Degradation by various restriction enzymes: BamHI 10 units, EcoRI 10 units, BamHI 10 units, EcoRI 10 units HindpIII 10 units HindpIII 10 units + EcoRI 10 units RvuII 10 Unit: 37 ° C, 3 hours 3) Electrophoresis: 0.8% agarose gel TAE 28V, 15.5 hours 4) Transfer to nitrocellulose filter: [EM Southern.J. Mol. Biol., 98 503 (1975).
5) Pre-hybridization: 30m FDSS 42 ° C for 6 hours 6) Hybridization: 30mFDSS containing 5'fragment of pR18 (1 x 10 ⁵ cpm / m, prepared in step 4) 14 ° C for 14 hours 7 ) Washing: Washing with 2 × SSC-0.1% SDS for 10 minutes at room temperature 4 times, followed by washing with 1 × SSC-0.1% SDS at 50 ° C. for 30 minutes 2 times 8) Exposure: XAR-5 The results of hybridization at -80 ° C, two intensifying screens, and 14 hours are shown in Table 4.

Step 8 (Southern Blot Analysis of Rabbit Clone) In step 7, Southern bacteriophages were replaced by the same procedure except that HG-1 and RG-2 bacteriophages were used instead of HG-3, HG-6 and HG-7. Blot analysis was performed. As a result, RG-1 and RG-2
The fragments obtained by digestion with BamHI, EcoRl, BglII, HindIII and BamHI + EcoRl, respectively, and the 5'fragment of pR18 and the pB2-7 insert hybridized to a single band. It hybridized to a single band with both the 5'fragment of pR18 and the pB2-7 insert.
This indicates that the 5'fragment of pR18 and both fragments hybridized with the pB2-7 insert contain the entire base sequence encoding TNF.

Step 9 (Construction of a bacterial clone containing the physiologically active substance of human chromosome) The DNA of HG-3 obtained in Step 5 was obtained by the method of Landy et al. [Biochemistry, 13 , p. 2134 (1974)]. 33 μg of this HG-3 DNA was digested with 80 units of EcoRl at 37 ° C. for 3 hours. The degradation product was electrophoresed on a 1% low melting point agaromers gel (conditions: 1 × TAE, 20V, 14.5 hours). From the agarose gel, a 2.9 kb band was obtained from T. Maniatis (Molecular Cloning, Cold Spring
Harbor Laboratory, p. 377 (1982)].
For details, use a gel with the 2.9 kb band site cut out.
Heat at 15 ° C for 15 minutes. Furthermore, this EcoRl-degraded HG-3 fragment having a length of 2.9 kb (hereinafter referred to as "HG-3 / E"
(coRl 2.9 kb fragment "is often abbreviated), and extracted from the melted gel 3 times with phenol and 3 times with ether, precipitated with ethanol containing ammonium acetate and recovered. Thus, 637 μg (30% yield) of HG-3 / EcoR12.
A 9 kb fragment was obtained.

255 ng of the above fragment and EcoRl-degraded pUC13 [J. Messin
g, Methods in Enzymology, 1
01 , p. 20 (1983)] was ligated with 2.5 units of T ₄ ligase.

E. coli K12JM83 strain was transformed with the ligated DNA obtained above. Specifically, E. coli K12 strain JM83 was cultured in LB medium until the absorbance of the culture pros at 550 nm was 0.3. JM of 50m grown E. coli K12
Collected 83 strains, 25m 10mM MOPS (pH7.0) -10mM
Wash with RbCl, 25m 0.1MMOPS (pH6.5) -50m
Suspended in M CaCl ₂ -10 mM RbCl. This suspension 203
To μ, 10 μ of an aqueous solution containing 10 ng of the above bound DNA was added. The mixture is chilled in ice for 30 minutes, 40
Heat at 60 ° C. for 60 minutes. Immediately afterwards, 37
The heated mixture was added to 5 m of LB broth which had been kept at 0 ° C, and the mixture was incubated at 37 ° C for 1 hour. The obtained culture broth was centrifuged and the supernatant was removed. LB medium is added to the spun cells to loosen them, and 30 μg / m ampicillin and 40 μg / m X-ga are added.
The LB plate containing 1 was inoculated. The colonies of the E. coli K12 strain JM83 introduced with the plasmid containing the insert were white, while the colonies of the strain introduced with only the plasmid were green. The white colony obtained again,
30 μg / m ampicillin and 40 μg / m X-
The LB plate containing gal was inoculated for confirmation.

From the white colonies obtained above, 10 colonies were selected and Holmes and Quigley's rapid analysis method [Anal. Biochem., 11
4 , p. 193 (1981)].

Specifically, each colony is cultured overnight in LB medium containing 30 μg / m of ancipiline. Collect the grown cells, 2mg / m lysozyme 50mM glucose-10mM
Suspended in EDTA-25 mM TrisHCl (pH 8.0). The suspension was placed at room temperature for 5 minutes and 200μ of 0.2N NaOH-1%
SDS was added. After gently stirring, add this suspension to 2
Placed at room temperature for minutes. Subsequently, 150 μM of 3M sodium acetate (pH5.2) was added, and the mixture was kept at −20 ° C. for 10 minutes to give 1
The supernatant was obtained by centrifugation for 5 minutes. 900m in this supernatant
Cold ethanol was added and the precipitate was obtained by centrifugation for 5 minutes. The obtained precipitate was washed with 70% ethanol and dried to obtain a plasmid DNA. Using this method, 10 plasmids were obtained.

Each plasmid DNA was 10 mM Tris-0.1 mM EDTA.
It was dissolved in (pH8.0), digested with EcoRl, and subjected to electrophoresis for restriction enzyme analysis. The conditions for restriction enzyme decomposition and electrophoresis are as follows.

Restriction enzyme digestion: Plasmid DNA solution = one-fifth amount of that obtained above, 3 units of EcoRl, 37 ° C, 1.5 hours Electrophoresis: 1% agarose gel, 1xTAE, 120V, 2 hours The above restriction enzymes Analysis showed that 8 out of 10 clones were of interest. That is, these eight clones had a 2.9 kb fragment. E. coli K12J83 was selected from one of eight target clones.
(PHGE) strain (ATCC 39656).

Then, by the same operation as in step 2, except that E. coli K12 JM83 (pHGE) strain was used instead of pB2-7 and pR18, 1.89 mg of pHGE DNA was obtained.

Step 10 (Subcloning of EcoRl-degraded RG-1) 30 μg of RG-1 obtained in Step 6 was digested with EcoRl. From the resulting mixture of various fragments, a fragment waiting for a length of about 3.5 kb was prepared by the same operation as in step 9 (however, using the mixture of various fragments described above and a 0.8% low melting point agarose gel was used). PUS13 digested with this fragment and EcoRl. Same operation as step 9 (However, EcoRl decomposition HG
-3 fragment (2.9 kb) instead of the EcoRl digested fragment (3.5 k
b) was used).

Transformation of E. coli K12 into JM83 strain, screening of bacterial clones, digestion of cloned DNA and electrophoresis were carried out by the same procedure as in step 9 (however, the above-mentioned binding DNA fragment was used). The resulting clone is E. coli K1
2 JM83 (pRGE) strain (ATCC39655).

Then, the same operation as step 2 (however, E.coli K12JM83
(pRGE) strain was used instead of pB2-7 and pR-18),
1.70 mg of RGE DNA was prepared.

Step 11 (restriction enzyme map of pHGE plasmid DNA) The restriction enzyme map of pHGE DNA obtained in step 9 is used for Maniatis
Method (Molecular Cloning, Cold Spring Harbor Labor
atory, 98 (1982)].

The method and conditions are as follows.

1) Degradation of pHGE DNA by EcoRl: 18.6 μg pHGE, 6
4 units EcoRl, 37 ° C, 2 hours 2) Ethanol precipitation 3) Dissolution: EcoRl decomposition Distilled water is added so that the solution has a pHGE of 1 µg / m.

4) Degradation with various restriction enzymes: 1 μg of the above EcoRl-degraded pHG
E, restriction enzyme: 5 units RvuII, 5 units RvuII + 10 units Rsal, 10 units Rsa 1, 4 units MstII, 3 units
Aval, 9 units Pst1, 36 ° C, 2 hours 5) Electrophoresis: 2% agarose gel, 1xTAE, 28V, 1
4.5 hours 6) Transfer to nitrocellulose filter: EMSouther
n, J. Mol. Biol., 98, p. 503 (1975) 7) First prehybridization: 30 mEDSS, 42
C., 6 hours 8) First hybridization: 30 mED containing 5 ′ fragment (5 × 10 ⁴ cpm / m) of pR18 (obtained in step 4)
SS, 42 ° C., 6 hours 9) Washing: Washing with 2 × SSC-0.1% SDS at room temperature for 10 minutes four times, followed by 1 × SSC-0.1% SDS at 50 ° C. for 3 times.
Wash twice for 0 minutes 10) Exposure: XAR-5, -80 ° C, 2 intensifying screens, 17.5 hours 11) Wash: 0.5M NaOH-1.5M NaCl for 1 minute, 0.5M Tris-
1.5M NaCl for 1 minute, 3 × SSC for 1 minute 12) Exposure: The same operation as 10) above except that the exposure time was 19 hours 13) Second prehybridization: The same as 7) 14) Second Time hybridization: pB2-7 insert (obtained in step 3), 42 ° C, 16.5 hours 15) Washing: same as 9) 16) Exposure: Except that the exposure time was 19.5 hours Ten)
Same as 17) Washing: Same as 11) 18) Exposure: Same as 10) above except that the exposure time was 20 hours 19) Third pre-hybridization: Same as 7) 20) Third hybrid Soybean: 3'fragment of pR18 (obtained in step 4) (4.5 × 10 ⁵ cpm / m), 42 ° C., 15 hours 21) Wash: the same as 9) 22) Exposure: the same as 10) A restriction enzyme map was prepared by cutting with the restriction enzymes shown in FIG.

The results of restriction map analysis are shown in FIG.

Step 12 (restriction enzyme map of pRGE plasmid DNA) The same method as step 11 was used instead of pHGE plasmid DNA, pRG
Using E plasmid DNA, restriction enzyme analysis of pRGE obtained in step 11 was performed. The restriction map of the obtained pHGE DNA is shown in FIG.

Step 13 (Determination of Nucleotide Sequences of Rabbit TNF Gene and the Physiologically Active Substance Gene) Step 2. The same operation was performed. Then, 150 μg of each of pRGE plasmid DNA and pHGE plasmid DNA was obtained.

pHGE and pRGE plasmid DNA was obtained.

The base sequences of pRGE and pHGE are the Maxam-Gilbert method [Maxam et.a.
l., Methods in Enzymology, 55 , p.490 (1980) Academic Pr
ess].

PR-18 nucleotide sequence determined in Reference Example 1 and pRG determined above
The structure of the rabbit TNF gene (exon, intron, etc.) was elucidated by comparing the nucleotide sequences of E. The structure of the pHGE DNA insert is shown in FIG. Subsequently, the base sequences of pHGE and pRGE were compared to examine the homology and the homology and similarity of the sequences near the intron / exon boundary. As a result, the structure (exon and intron) of the bioactive substance gene was elucidated. The structure of the bioactive substance gene is shown in FIG.

The nucleotide sequences encoding rabbit TNF and the physiologically active substance thus obtained are shown below. In this base sequence, the upper line shows the base sequence (R) encoding rabbit TNF, and the lower line shows the base sequence (H) encoding the physiologically active substance.

Step 14 (Synthesis of Oligodeoquine Nucletide) 20 mg of polystyrene resin having deoxyoligonucleotide (about 2 μM) bound thereto via a succinic acid residue,
50 with stainless steel filters on top and bottom
It was loaded into a 0 μ volume reaction vessel. The resin is a 1M zinc bromide dichloromethane-isopropanol solution (85:15).
Was removed with a dimethoxytrityl (DMT) protecting group, and the mixture was washed with dichloromethane-isopropanol (85:15), dimethylformamide, pyridine, and acetonitrile, and dried with a nitrogen stream. Then 200 μ of a solution of protected scleotide diester (20 uM) and mesitylene sulfonyl nitrotriazole (60 uM) in dry pyridine was added. After reacting at 45 ° C. for 20 minutes, the reaction solution was removed, the resin was washed with dry pyridine, and unreacted residues were protected with acetic anhydride in pyridine. this,
The cycle of deprotection and condensation was repeated to synthesize the desired oligodeoxynucleotide. After completion of the synthesis, the resin was taken out, the protective group was removed, the reaction of cleavage from the resin was performed, and the separation treatment from the resin was performed, followed by purification. The above oligodeoxynucleotides were synthesized and purified by Ito et al. [Nuc.Ac.R.
es. 10, p. 1755 (1982)].

In this way, the following oligodeoxynucleotide was obtained.

1) 5'-ATTCATGTCATCTTCTCGAACCCCGAGTGACAA-3 '2) 3'-GTACAGTAGAAGAGCTTGGGGCTCACTGTTCGG-5' 3) 5'-GCCTGTAGCCCATGTTGTAGCAAACCCTCAAAG-3 '4) 3'-ACATCGGGTACAACATCGTTTGGGAGTTCGACT-15H activity 13M 13H (13)
Preparation of GE) The plasmid pHGE (10 μg) was digested with EcoRl (20u),
After 1% low melting point agarose gel electrophoresis, the 2.9 kb fragment was excised and eluted. This fragment is M1
3mp9 phage was inserted into the EcoRl fragment of the replicative form. The DNA with the EcoRl fragment inserted is described in the BRL manual (User Manual / M13mp7Cloning / 'D
E. coli JM103 according to idesxy'sequenucing, 1980). The product was named M13mp9-HGE.

Step 16 (removal of intron 3 using M13mp9-HGE-heavy chain DNA and data E3-4) M13mp9-HGE-heavy chain DNA was prepared by BRL user's guide (User Manu).
al / M13mp7 Cloning / 'Didesxy'sequenucing, 1980).

Oligodeoxynucleosides prepared in step 14)
3'-ACATCGGGTACAACATCGTTTGGGAGTTCGACT-5 'was used as a deleter for Intron 3. Intron 3
The deleter was named E-3-4.

The deleter E3-4 is in front of the intron 3 (that is, exon 3) and behind (that is, exon 4) of the intron 3 to be removed.
Has a sequence complementary to. Removal of intron 3 follows the method of Wallace et al. [Science 209 , 1396 (1980)]
I went as follows.

E3-4 (104ng, 15pmole) is phosphorylated using of _{T 4} kinase (108 units) and ATP (3 mM), it was added to the mold M13mp-HGE (1.65μg, 0.5pmole) . The reaction mixture was heated at 65 ° C. for 65 minutes, cooled to room temperature for 5 minutes,
Further cooled in ice water. 0.4mM dATP, dCTP, dGTP, d
For TTP and ATP solutions, Kleno
W flagment) 5 units, T ₄ ligase 10 units Hin buffer [Wallace et al., Nuc. Ac. Res, 9: 3647 (1981)],
A solution containing 10 mM Tris-hydrochloric acid (pH 7.2), 2 mM MgCl ₂ and 1 mM mercaptoethanol was added. The reaction mixture (final dissolution volume 50 μ) was incubated at 4 ° C. for 30 minutes and at room temperature for 30 minutes. Double-strand synthesized DNA using oligonucleotides as primers is a guide for BRL users.
(User Manual / M13mp7 Cloning / 'Dideoxy' sequencing, 1
980) and infected with E. coli JM103. The plaques obtained in this way are YT plates [JHMi
ller, Experiments in Molceular Genetics, Cold Spring
Habor Laboratory (1972) p.433]. The obtained colonies were hybridized with ³² P-labeled E3-4 at 55 ° C. for 2 hours. Here, Deleater E3-4 itself was used as a probe to obtain a DNA having a desired sequence from various products obtained as a result of the intron removal step. Thus, Deleter-E
Colonies with which 3-4 hybridized were obtained, and further phages were obtained.

The obtained phages were plated and the resulting plaques were transferred to YT plates. Here again, it was hybridized with E3-4 radiolabeled with ³² P for 2 hours at 55 ° C. From clones that hybridize strongly, phage DN
A was obtained, the nucleotide sequence was analyzed, and a phage in which intron 3 was completely removed was selected. One such phage was named mp9-HGEΔ3-1.

Step 17 (Construction of pHTNF-lacUV5-2) The replicative form of mp9-HGEΔ3-1 was cleaved with EcoRl and isolated by electrophoresis. This fragment was inserted into pBR327 digested with EcoRl to obtain plasmid pHGEΔ3-1.

The construction of a plasmid capable of directly expressing the physiologically active substance in Escherichia coli using lacUV5 as a promoter using the following plasmid pHGEΔ3-1 is shown below. This construction method is shown in FIG.

First, 10 μg of plasmid pHGEΔ3-1 units of Aval and Eco
The fragment of interest was isolated by polyacrylamide gel electrophoresis after digestion with Rl (BRL, USA) for 2 hours at 37 ° C.
About 1 μg of fragment was recovered from the gel. As in step 14, the two oligodeoxynucleotides shown in FIG. 7, namely 5'-AATTCATGTCATCTTCTCGAACC-3 'and 5-TCGG.
GGTTCGAGAAGATGACATG-3 ′ and synthesized. Then literature
(3) The 5'ends of these two oligonucleotides (about 100 pmole) were phosphorylated according to the method on page 122. After the reaction
It was extracted with phenol / chloroform. The synthetic oligomer thus obtained and Aval-of pHGEΔ3-1 taken above
After mixing with EcoRl fragment and precipitating ethanol, literature
(1) According to the method on page 37, 10 units of T ₄ ligase was used for ligation at 4 ° C. overnight. After completion of the reaction, the mixture was ethanol precipitated and the target fragment was isolated by polyacrylamide gel electrophoresis.

The plasmid pOP95-15 was prepared by the method of F. Fuller [Gene 19, vol. 4, 4
2-54 (1982)].

Dilute 1 μg of pPP95-15 with EcoRl and extract with phenol,
Chloroform extraction and ethanol precipitation were used to ligate 0.5 μg of the prepared vector and the fragment obtained as described above using T ₄ DNA ligase. E.col according to the experiment book (4), page 20
i JM101 (ATCC33876) was transformed with 1 mM IPTG and 0.
About 100 white colonies were obtained on the agar culture containing 004% (W / V) x-gal.

Plasmid DNA was prepared from these transformants and EcoRl
It was digested with and the plasmid having the desired EcoRl fragment was fixed. Further, these plasmids were digested with RvuII and Pst1 and subjected to 1.5% agarose gel electric EcoRl.
A plasmid having 0 bp and about 2600 bp fragments and having the physiologically active substance gene correctly connected downstream of the lac UV5 promoter was selected.

When the nucleotide sequences were determined, it was shown that the synthetic oligonucleotide and the DNA derived from the chromosome were correctly connected in the two plasmids. The resulting plasmid was named pHTNF-lacUV5-2.

E. coli containing pHTNF-lacUV5-2 were cultured in a normal medium. When the activity of the physiologically active substance in the product was measured, it was found to show the same activity as the E. coli product containing pTHF-lacUV5-1 (expressing rabbit TNF).

Reference Example 3 Using the plasmid pTHF and oligonucleotides 1-4 prepared according to steps 1 to 14 in Reference Example 2, pH
The method for preparing TNF-lacUV5-1 is shown in FIG.

Example 1 Escherichia coli containing the gene assembly pHTNF-lacUV5-1 prepared in Reference Example 3 was cultured by a usual method. Then, an induction operation is performed so that the desired physiologically active substance is produced,
Further culturing was performed to obtain Escherichia coli containing the physiologically active substance. Collect the bacterial cells by centrifugation and collect the bacterial cells at 0.04M.
The mixture was ultrasonically disrupted in Tris-HCl buffer (pH 7.8) 1 to obtain a cell extract solution (A) containing the physiologically active substance.

This solution had an activity of 4.5 × 10 ⁵ U / m and a specific activity of 3.0 × 10 ⁴ .

The extraction solution was then added to 0.04 M Tris-base buffer (pH
It was applied to a column of DEAE-Sepharose CL-6B (Pharmacia, Sweden) which was sufficiently equilibrated with 8.0). 0.
Elution was performed with 04M Tris-hydrochloric acid buffer (pH 8.0).
The active fraction was concentrated by gel filtration to give a specific activity of 4.0 × 10 ⁵
A crude purified solution (B) of U / mg was obtained.

20 mM of the above extraction solution (A) containing 0.15 M sodium chloride
Dilute 25-fold with phosphate buffer (pH 7.4) and 60 at 60 ℃
Heated in a hot water bath for up to a minute. 3 at 6000 rpm after heat treatment
After centrifugation for 0 minutes, the activity of the supernatant, the concentration of proteolytic enzyme, and the amount of protein were measured. The results are shown in Table 5.

Similarly, Table 6 shows the results of heat treatment of the crudely purified solution (B) at 60 ° C. for 240 minutes.

Table 7 shows the results of heat treatment of the crudely purified solution (B) at 4 to 80 ° C for 30 minutes.

Example 2 Cefacryl S-200 (Pharmacia, Sweden) obtained by equilibrating the crude solution (B) obtained in Example 1 with 5 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride was used.
Column, and gel filtration was performed using the same buffer solution.
The active fraction was concentrated by ultrafiltration to obtain a purified solution (untreated).

On the other hand, the crude solution (B) was heated at 60 ° C. for 30 minutes. After the heat treatment, centrifugation was performed at 6000 rpm for 30 minutes to obtain a liquid from which insoluble matter was removed. This solution was added to a column of Cefacryl S-200 (Pharmacia, Sweden) equilibrated with 5 mM phosphate buffer (pH 7.4) containing 0.15 M sodium chloride, and gel filtration was performed with the same buffer. It was The active fraction was concentrated by ultrafiltration to obtain a purified solution (treatment).

The above results are summarized in Table 8.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme digestion map of a plasmid containing the rabbit TNF gene. FIG. 2 shows pTNF-lac-11.
Fig. 3 is a flow sheet showing the preparation method of pTNF.
-A flow sheet showing the method for preparing lac UV5-11. FIG. 4 shows the structure of the bioactive substance gene of the present invention. FIG. 5 shows the structure of the rabbit TNF gene. FIG. 6 is a flow sheet showing a method for preparing pTNF-lac UV5-1, and FIG. 7 is a flow sheet showing a method for preparing pTNF-lac UV5-2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12P 21/00 B 8214-4B (C12P 21/02 C12R 1:19)

Claims (1)

[Claims] 1. A LM produced by a genetic recombinant.
It has cytotoxic activity on cells and Meth Asarc
A purification method, which comprises performing heat treatment when purifying a physiologically active substance having a property of causing a hemorrhagic necrotic reaction at the tumor site when administered to BALB / c mice transplanted with oma cancer cells.