JPH0773506B2 - Method for producing cancer necrosis factor, etc. - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently producing a substance such as a cancer necrosis factor, which comprises culturing a transformant with an advanced trait expression vector for producing the substance.

[0002]

2. Description of the Related Art The production of a useful substance in a microorganism by applying gene recombination technology is carried out by using a gene expression vector so that transcription and translation from the DNA encoding the target substance can be performed correctly. Integration, and then introducing the expression vector into a suitable host,
It is carried out by culturing the host. In order to efficiently produce a target substance in a microbial cell by a foreign gene, in constructing a trait expression vector of the foreign gene, increasing the transcription efficiency of the foreign gene, increasing the translation efficiency, and Increasing the copy number of the gene is an important requirement. To improve transfer efficiency,
Strong promoters such as lac , trp , tac , λ
PL, rec A, pho A promoters and the like are used. The Shine-Dalgarno (SD) sequence and the nucleotide sequence between the SD sequence and the translation initiation codon (ATG) have a great influence on the translation efficiency. In order to determine the nucleotide sequence with the highest translation efficiency in the region, downstream of the start codon, that is, N of the target substance is determined.
The nucleotide sequence encoding the terminal portion must be carefully considered [European Patent No. 111814 A2, Proc. Nat. A
cad. Sci. USA, 81 , 5403 (1984)]. That is, depending on the substance to be produced, the SD sequence and the translation initiation codon AT
The base sequence between G must be selected appropriately. The same applies to the SD sequence, and the appropriate sequence required to enhance translation efficiency is not necessarily uniquely determined. Therefore, in order to increase the translation efficiency of the target substance, the base sequence between the SD region and the initiation codon ATG from the SD region should be examined and an appropriate base sequence should be selected according to the structure of the gene encoding the target substance. I have to. In addition, the higher the copy number of the expression vector,
It is desirable for efficient production of the target substance.

[0003]

[Means for Solving the Problems] The inventors of the present invention have conducted intensive studies from the above viewpoints on the production of highly trait expression vectors for useful substances, in particular, cancer necrosis factor. The present invention has been completed by finding that a useful substance such as a cancer necrosis factor can be produced very efficiently in a transformant such as Escherichia coli.

More specifically, the present invention relates to a gene encoding a cancer necrosis factor or a polypeptide highly homologous to the N-terminal portion of the factor, at least as an expression control sequence, and as a promoter region of another gene. , A highly expressed recombinant vector having the following nucleotide sequence [I] in the sequence between the Shine-Dalgarno sequence (SD sequence) and the translation initiation codon, and a host transformed with the vector were cultured, and The present invention also relates to a method for producing the substance, which comprises efficiently producing a cancer necrosis factor or a polypeptide having high homology with the N-terminal portion of the factor.

(5 ′)-AAGGAGGTT-X-ATTATG- (3 ′) [I] (In the formula, X represents T, TY or AZ, where Y
Means A, AA, AAT, and Z means T and TCG. )

According to the present invention, the nucleotide sequence from the SD sequence region having the nucleotide sequence represented by the above formula [I] to the initiation codon ATG is set as a suitable promoter for another gene (eg,
trp , tac , λPL promoter, etc.) region, and a nucleotide sequence encoding a desired gene, and further a stop codon are added to a vector such as an Escherichia coli plasmid, for example, at an appropriate position of pBR322 or pBR322. By inserting the copy number restriction region near the origin of replication into an appropriate position of the plasmid from which the copy number restriction region has been deleted, the highly-expressed expression vector according to the present invention can be prepared. The target substance can be efficiently produced by inserting the transformant expression vector into a host for transformation according to a conventional method and culturing the transformant. The amount of the target substance produced by such a trait expression vector is extremely high as compared with the case where a natural base sequence is used.

[0007]

[Effects of the Invention] The present invention will be specifically described below by taking a recombinant plasmid for expression of human cancer necrosis factor production and E. coli which is a transformant thereof as an example. SD that affects expression efficiency
The effect of the nucleotide sequence between the sequence region and the SD sequence to the start codon ATG was evaluated by culturing the E. coli transformed with the obtained recombinant plasmid and observing the expression level of human cancer necrosis factor. did.

5'of DNA encoding human cancer necrosis factor
The end is the t of the tryptophan operon that exists in nature.
rp promoter, trp L following operator region
(Leader) SD of the nucleotide sequence encoding the protein
The base sequence from the sequence to the start codon ATG was used, and it was bound downstream of this ATG. Furthermore, it is incorporated into a large DNA fragment obtained by cleaving with restriction enzymes Pst I and Hin dIII of plasmid pBR322, were constructed recombinant plasmid (pHTP101). After introducing the recombinant plasmid into E. coli HB101, it was cultured, and the production amount of human cancer necrosis factor in the cell extract was measured for cytotoxic activity against mouse LM cells (ATCC, CCL1.2). It measured by the method shown in a test example mentioned later. as a result,
Human cancer necrosis factor obtained from the transformant (using a natural nucleotide sequence) is about 400 per 1 ml of the culture medium.
It was a unit.

To obtain higher expression efficiency, the effect on the expression level of human cancer necrosis factor by artificially converting the SD sequence and the base sequence between the SD sequence and the start codon ATG using chemically synthesized oligonucleotides investigated. The obtained recombinant plasmid was transformed into E. coli under the same conditions as above .
After introduction into Escherichia coli HB101, the cells were cultured and the expression level of human cancer necrosis factor in the cell extract was measured.

As a vector, the above-mentioned plasmid p
Copy number control region from BR322 [Twigg, AJ et a
l .: Nature, 283 , 216 (1980)] was deleted, and the effect of using a plasmid (named pBRS6) was also examined. The results are shown in Table 1.

[0011]

[Table 1]

As shown in the above results, the SD sequence and /
Alternatively, the base sequence between the SD sequence and the start codon ATG is shown in Table 1.
As shown in the above, the expression level of human cancer necrosis factor is increased by the artificial conversion. Particularly, in the above-mentioned formula [I], X is T,
Consists of base sequences such as TA, TAA, TAAT,
And by using the plasmid pBRS6 in which the copy number control region of pBR322 is deleted as a vector,
The expression level of human cancer necrosis factor increases dramatically.

Such an increase in expression efficiency is caused not only by substituting the consensus sequence for the SD sequence and increasing the copy number of the plasmid as a vector, but also particularly by S
By substituting the base sequence between the D sequence and the start codon ATG with a specific base sequence as shown in Table 1 above, the mRNA portion corresponding to the gene portion encoding the N-terminal region portion of human cancer necrosis factor and SD Sequence to start codon AT
This is probably because it became difficult to form a secondary structure or a three-dimensional structure that would reduce the translation efficiency with the mRNA portion corresponding to the nucleotide sequence between Gs.

From the above, among the various factors that influence the expression efficiency, the correlation between the base sequence between the SD sequence and the start codon ATG and the base sequence of the structural gene following the start codon is particularly large. I know there is. In other words, the structure of the hyperplasmic expression vector of the present invention can be used not only for human cancer necrosis factor but also for a gene encoding a polypeptide having high homology with the N-terminal portion of the factor. In addition, a useful substance other than human cancer necrosis factor is efficiently produced by linking a structural gene of another useful substance downstream of the nucleotide sequence encoding the N-terminal portion of the human cancer necrosis factor of the hyperplasm expression vector. Therefore, it can be used.

In the present specification, the following abbreviations are used for simplification of description. A: adenine, C: cytosine, G: guanine, T: thymine, DNA: deoxyribonucleic acid, cDNA: complementary DNA, RNA: ribonucleic acid, mRNA: messenger RNA, dATP: deoxyadenosine triphosphate, dCTP: deoxycytidine triphosphate. Acid, dGTP: deoxyguanosine triphosphate, dTTP: deoxythymidine triphosphate, oligo (dC): oligodeoxycytidylic acid, oligo (dG): oligodeoxyguanylic acid, oligo (dT): oligodeoxythymidylate, poly (A): polyadenylic acid, bp: base pair, kbp: kilo base pair.

[0016]

EXAMPLES The present invention will be described more specifically with reference to Examples, Test Examples and Reference Examples below.

Reference Example 1: Expression of human cancer necrosis factor expression using a natural nucleotide sequence
Preparation of Lasmid: -Recombinant plasmid pHTNF13DNA (5 μg) obtained in Reference Example 2 was mixed with 50 mM NaCl and 6 mM Mg.
10 mM with Cl ₂ and 6 mM _2- mercaptoethanol
Dissolve in 45 μl of ris-HCl buffer (pH 7.5), and use Av
In addition, each 15 units of a I and Hin dIII restriction enzyme, 37 ° C.
Digested for 60 minutes. The obtained DNA fragments were separated by 5% polyacrylamide gel electrophoresis and
A DNA fragment of 600 base pairs was extracted by electrophoresis, and a final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract to obtain 0.4 μg of the DNA fragment as a precipitate.

A chemically synthesized linker (synthetic DNA fragment of FIG. 1) containing a start codon ATG having sticky ends of Cla I and Ava I restriction enzymes at both ends was prepared by a phosphate triester method using a nucleic acid synthesizer manufactured by Biosearch. Synthesized. The synthetic linker and the DNA fragment obtained above were combined with a ligation reaction solution (5 mM MgCl ₂ , 5 mM dithiothreitol and 1 mM).
66mM Tris-HCl buffer (pH7.5) 40μ containing TP
lysate and dissolve in 10 units of T ₄ DNA ligase at 16 ° C
The reaction was carried out for 16 hours. The reaction solution 60 ° C., after heated 15 minutes, the NaCl concentration fit 50 mM, was Hin dIII restriction enzyme hydrolysis. The obtained DNA fragments were separated by 5% polyacrylamide gel electrophoresis, and about 60
A 0 base pair DNA fragment was extracted by electrophoresis. A final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract to obtain the DNA fragment as a precipitate. This DN
The A fragment is designated as DNA fragment I.

A plasmid pTrpLC-1 having a tryptophan operator, a promoter region and a region encoding a leader peptide [see FIG. 1]. This plasmid is Proc. Nat. Acad.Sc; Proceeding of the National Academy of Sciences of the United States of America;
i. USA, 81 , 5956 (1984), containing the attenuator of the plasmid described as pCT-1.
It is the same as Taq I to Cla I region deleted. ] 5 μg of DNA was added to 100 mM NaCl, 6 mM MgCl ₂
And 10 mM Tris containing 6 mM 2-mercaptoethanol
It was dissolved in 50 µl of -HCl buffer (pH 7.5), and 15 units of Cla I and Pst I restriction enzymes were added, respectively, and digested at 37 ° C for 60 minutes. The obtained DNA fragments were separated by 5% polyacrylamide gel electrophoresis, and a DNA fragment of about 1.1 kbp was extracted from the acrylamide gel by electrophoresis.
A final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract, and 1 μg of the DNA fragment was obtained as a precipitate.

This DNA fragment and the above DNA fragment I were dissolved in a binding reaction solution, and 10 units of T ₄ DNA ligase was used.
The reaction was carried out at 16 ° C for 16 hours. The reaction solution is heated at 60 ° C for 15 minutes, and a buffer solution is added so that the final concentration of 50 mM NaCl is obtained.
It was carried out Hin dIII and Pst I restriction enzyme water solution. The obtained DNA fragments were separated by 5% polyacrylamide gel electrophoresis and 1.7 kb from the polyacrylamide gel.
The DNA fragment of p was extracted by electrophoresis. A final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract to obtain the DNA fragment as a precipitate. This DNA fragment is called DNA fragment II.

4.8 μg of pBR322 DNA was added to 50 mM NaC
10 mM Tris-HCl buffer containing l and 6mMMgCl ₂ (pH7.5) was dissolved in 45 [mu] l, Pst I and Hin dIII
15 units of each restriction enzyme was added and digested at 37 ° C for 60 minutes.
The obtained DNA fragments were separated by 0.7% agarose gel electrophoresis, and the 3.6 kbp DNA was separated from the agarose gel.
The fragment was extracted by electrophoresis. Final concentration 0.3M in extract
Add NaCl and 2.5 volumes of cold ethanol and add DN
2.5 μg of A fragment was obtained as a precipitate. This DNA fragment and DNA fragment II were dissolved in 40 μl of the ligation reaction solution, and T ₄ DN
Human cancer necrosis factor expression plasmid (pHTP101) was constructed by ligation using 10 units of A ligase (Fig. 1).
reference).

This expression plasmid was prepared by the method of Cohen et al. [Proc. Nat. Acad. Sci. USA, 69 , 2110 (1972)].
It was introduced into E. coli HB101 to obtain a transformant. The transformant was selected by LB agar medium containing 25 μg / ml of ampicillin (composition: 1 L of purified water, 10 g of bactotryptone, 5 g of bacto yeast extract, 10 g of salt, agar15).
g), isolate the transformant resistant to ampicillin,
The cells were cultured by the method described in the test example, and then the cytotoxic activity was measured to select a transformant producing human cancer necrosis factor. The desired recombinant plasmid pHTP101 was extracted from one of such transformants by the method of Wilkie et al. [Nucl. Acid Res., 7 , 859 (1979)].

The dideoxy method [Sanger et al .: Science, 214 ,
1205 (1981), Messing: Methods in Enzymology, 101 , 2
0 (1983)], the plasmid was determined to have the tryptophan leader peptide promoter,
It was confirmed that the human cancer necrosis factor structural gene was directly linked to the initiation codon ATG via the ATCGATT downstream of the SD region.

Example 1: Conversion of base sequence between SD region and start codon ATG
Improvement of human cancer necrosis factor expression plasmid : -Reference Example 1
The plasmid pHTP101 DNA (4 μg) obtained in the above was added to 80 mM
40 μl of 10 mM Tris-HCl buffer (pH 7.5) containing aCl, 6 mM MgCl ₂ and 6 mM 2-mercaptoethanol
Dissolved in 15 units of Cla I and 1.5 units of Ava I
A restriction enzyme was added, digested at 37 ° C. for 60 minutes, separated by 0.7% agarose gel electrophoresis, and the target DNA fragment was extracted from the gel by electrophoresis. A final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract to obtain the DNA fragment as a precipitate.

A DNA fragment of 23 base pairs containing this DNA fragment and the initiation codon ATG chemically synthesized by the phosphotriester method, and corresponding to the downstream from the Ava I restriction enzyme cleavage recognition site of the human cancer necrosis factor structural gene. (Fig. 2
Synthetic DNA fragment) of T ₄ DN
Using 10 units of A ligase, the reaction was carried out at 16 ° C. for 16 hours (see FIG. 2). This reaction solution was introduced into E. coli HB101 to obtain a transformant. Selection of transformants was performed on LB agar medium containing 25 μg / ml of ampicillin, and ampicillin-resistant transformants were isolated. By the method described in Reference Example 1, a transformant producing human cancer necrosis factor was selected. By extracting the target recombinant plasmid pHTP102 and determining the nucleotide sequence by the dideoxy method, this plasmid pHTP102 was found to have 5 '... ATCGTC upstream of the initiation codon ATG of the human cancer necrosis factor structural gene.
It was confirmed that the nucleotide sequence is C-3 ', and only the 3 base pairs upstream of the initiation codon ATG differ from the plasmid pHTP101.

Example 2: (1) Construction of high copy number mutant plasmid vector :-
2.3 μg of pBR322 DNA was added to 50 mM NaCl, 6 mM Mg
10 mM with Cl ₂ and 6 mM _2- mercaptoethanol
Dissolve in 30 μl of ris-HCl buffer (pH 7.5), and use Av
Add 10 units each of a I and Pvu II restriction enzymes, and add at 37 ℃, 60
Digested for a minute. The obtained DNA fragments were separated by 0.7% agarose gel electrophoresis. About 3.7 kbp from the gel
DNA fragment was extracted by electrophoresis, and a final concentration of 0.3 M NaCl and 2.5 times the amount of cold ethanol were added to the extract to obtain 1.2 μg of the DNA fragment as a precipitate.

This precipitate was added to T ₄ DNA polymerase reaction buffer [50 mM Tris-HCl buffer (pH 7.5), 10 mM
MgSO ₄ , 0.1 mM dithiothreitol, 50 μg / ml bov
ine serum albumin] 30 μl, dATP, dG
TP, dTTP, dCTP 2nmole each and 1 unit D
Mixed with NA polymerase Iklenow fragment, 22
After incubation at 30 ° C. for 30 minutes, both ends of the DNA fragment were made blunt ends. Then, cold ethanol was added to obtain the desired DNA fragment as a precipitate. This precipitate was dissolved in the binding reaction solution, and using T ₄ DNA ligase at 16 ° C., 16
The reaction was carried out over time. 2.5 volumes of cold ethanol was added to the reaction solution to obtain DNA as a precipitate. This plasmid vector is called pBRS6 (Fig. 3). pBRS6 is pBR3
Compared with 22, the copy number per cell is 2 to 5 times.

(2) Human cancer necrosis factor having a high copy number
Preparation of expression plasmid : 2.5 μg of the plasmid pHTP102DNA obtained in Example 1 was added to 100 mM NaCl, 6 m
Contains MMgCl ₂ and 6 mM _2- mercaptoethanol 1
Dissolve in 30 μl of 0 mM Tris-HCl buffer (pH 7.5),
Sca I and Hin dIII restriction enzymes each 10 units added, 37 ° C., digested for 60 minutes. After 30 μl of water-saturated phenol was added for deproteinization, a final concentration of 0.3 M NaCl and twice the amount of cold ethanol were added to precipitate the DNA. This precipitate was added to T ₄ DNA polymerase reaction buffer 30
Dissolve in μl, dATP, dGTP, dTTP, dCT
Added P respectively 2nmole and DNA polymerase Iklenow fragment 1 unit, 22 ° C., by incubation for 30 minutes, filled with Hin dIII cleavage site, both ends were blunted. The DNA fragments were separated by 5% polyacrylamide gel electrophoresis, and the DNA fragment of about 1.7 kbp was eluted from the gel and precipitated with cold ethanol.
Recovered.

On the other hand, the plasmid vector pBRS6 prepared in (1) was added to 10 mM Tris-H containing 100 mM NaCl, 6 mM MgCl ₂ and 6 mM _2- mercaptoethanol.
Dissolved in Cl buffer (pH 7.5), Sca I and Eco R
Digested with I restriction enzyme, DNA polymerase Iklenow
The fragment was used to fill in the Eco RI cleavage site.
This DNA fragment was separated by 5% polyacrylamide gel electrophoresis, and a DNA fragment of about 3.8 kbp was eluted from the gel by electrophoresis and cold ethanol was added to obtain a precipitate.

This DNA fragment and the previously obtained 1.7 kbp DN
The A fragment was dissolved in 40 μl of the ligation reaction solution, and the reaction was carried out at 16 ° C. for 16 hours using 10 units of T ₄ DNA ligase. E. coli HB101 was transformed with this reaction solution. LB containing 25 μg / ml of ampicillin by the method described in Reference Example 1
The cells were cultured in an agar medium, ampicillin-resistant transformants were selected and isolated, and transformants producing human cancer necrosis factor were selected. The desired plasmid pHTP302 was extracted from one of such transformants (FIG. 3).

By determination of the nucleotide sequence by the dideoxy method, this plasmid shows the tryptophan promoter,
It was confirmed to be a high copy number mutant in which the human cancer necrosis factor structural gene was directly linked to the downstream of the SD region.

Example 3: SD region and nucleotide sequence between SD region and start codon ATG
Of human cancer necrosis factor expression plasmid by simultaneous conversion of
Improvement : -In the region from the Hpa I restriction enzyme recognition site in the operator region, the SD region downstream and the start codon ATG of the human cancer necrosis factor structural gene, to the Ava I restriction enzyme recognition site in the structural gene, DNA with variously converted corresponding 55 base pairs
The fragment was chemically synthesized by the phosphate triester method. The base sequences of these DNA fragments are as shown in FIG. These synthetic DNA fragments are obtained by converting the SD region of the promoter and operator of the tryptophan operon leader peptide into a consensus base sequence, and increase the distance between the SD region and the start codon ATG and its base sequence to enhance the expression efficiency. It is variously converted.

The plasmid pHTP302 obtained in Example 2
Is a 10 mM Tris-HCl buffer solution (pH: 100 mM NaCl, 6 mM MgCl ₂ and 6 mM _2- mercaptoethanol).
7.5), digest with Ava I and Hpa I restriction enzymes, separate by 0.7% agarose gel electrophoresis, extract the target DNA fragment from the gel by electrophoresis, and add 2.5 volumes of cold ethanol. , Was obtained as a precipitate.

This DNA fragment and the chemically synthesized D shown in FIG.
Each of the NA fragments was dissolved in the ligation reaction solution, and 10 units of T ₄ DNA ligase was used to carry out a reaction at 16 ° C. for 16 hours. E. coli HB101 was transformed with each reaction solution. According to the method described in Reference Example 1, transformants were selected by culturing on LB agar medium containing 25 μg / ml of ampicillin, separating ampicillin-resistant transformants, and selecting transformants producing human cancer necrosis factor. did. The target recombinant plasmid DNA was extracted from each of the transformants.

That is, the plasmid pHTP301 containing the DNA fragment 01 and the plasmid p containing the DNA fragment 10 were inserted.
Plasmid pH with HTP310 and DNA fragment 12 inserted
Plasmid pHT with TP312 and DNA fragment 14 inserted
P314, a plasmid pHTP having DNA fragment 16 inserted
316, a plasmid pHTP3 having a DNA fragment 17 inserted
Plasmid pHTP3 with 17 and DNA fragment 18 inserted
18 were obtained (see FIG. 5). These inserted DNA
The structures of the base sequences in and around were confirmed by the dideoxy method.

Test Example: Quantification of human cancer necrosis factor by measuring cytotoxic activity : The method for measuring cytotoxic activity using mouse LM cells is as follows. Test solution 0.1m with serial 2-fold dilution of sample
l and LM cells (ATCC, CCL1.2) suspension (1 x 10 ⁵ cells / ml) 0.1 ml was added to 96-well tissue culture microplate (Fl
ow Labs.). Eagle's MEM medium containing 1% fetal bovine serum is used as the medium. The microplate is cultured in air containing 5% carbon dioxide at 37 ° C. for 48 hours.
After culturing, add 20 μl of glutaraldehyde to fix the surviving cells. After fixation, the microplate is washed and dried, and 0.1 ml of 0.05% methylene blue solution is added to stain the fixed cells. After washing away excess methylene blue and drying, methylene blue adhering to the fixed cells was extracted with 0.36 N hydrochloric acid, and the absorbance at 665 nm was measured by Titertec Multiscan (Flow Lab
s.). This absorbance is proportional to the number of cells that survived. The active concentration required to kill 50% of LM cells was defined as 1 unit / ml.

LB broth containing 25 μg / ml of ampicillin was transformed into E. coli HB101 transformed with the expression plasmid encoding human cancer necrosis factor (composition in 1 L: bactotryptone 10 g, bacto yeast extract 5).
5 g of sodium chloride (10 g of sodium chloride, 10 g of sodium chloride) and cultured overnight at 37 ° C., 0.1 ml of each culture solution was added to 25 μg / ml ampicillin, 20 ml each.
Modified M-9 synthetic medium containing μg / ml indolylacrylic acid and 1 μg / ml vitamin B ₁ (composition in 1 L: Na ₂ H
PO ₄ 6g, KH ₂ PO ₄ 3g, NaCl 0.5 g, NH ₄ C
1g, 1MMgSO ₄ 2ml, 20% glucose 10ml, 1M
CaCl _{2 (} 0.1 ml) and casamino acid (5 g) (10 ml) were added, and the culture was continued at 37 ° C. for another 24 hours, and then the cells were collected by centrifugation. 1 ml of 0.1% lysozyme and 30 mM NaCl
After resuspending in 50 mM Tris-HCl (pH 8.0) buffer containing 30 mM, the mixture was allowed to stand at 0 ° C. for 30 minutes, then frozen in a dry ice / ethanol bath and thawed at 37 ° C. was repeated 6 times. Then, the bacterial cell residue was removed by centrifugation to obtain a clear supernatant liquid. The supernatant fraction was used as a measurement sample for cytotoxic activity.

Reference Example 2: (1) Cloned DNA encoding human cancer necrosis factor
Preparation of probe for detection : -Restriction enzyme for the cloned DNA encoding rabbit cancer necrosis factor obtained in Reference Example 3
It was cut out using Hae II and Ava I, and the
~ 120 DNA fragment consisting of 88 bp and 285 ~ 583
A DNA fragment consisting of 299 bp was obtained. The 299 bp DNA fragment cut out with the restriction enzyme Ava I is a region encoding a rabbit cancer necrosis factor, and the 88 bp DN cut out with Hae II.
The A fragment is a portion corresponding to the region encoding the rabbit cancer necrosis factor precursor. These two DNA fragments were labeled with ³² P and used as a probe for cloning of human cancer necrosis factor cDNA, which was used in (8).

(2) mR from human alveolar macrophages
Isolation of NA fraction : 6.3 × 10 ⁷ alveolar macrophages were collected from human lung using phosphate buffered saline.
This alveolar macrophage contains 10% fetal bovine serum RPM
Suspended in I-1640 medium and petri dish (diameter 8c
m) was sowed so as to be 9 × 10 ⁶ cells per sheet, and precultured at 37 ° C. in air containing 5% carbon dioxide at a humidity of 90 to 100%. After 1 hour of pre-incubation, endotoxin (lipopolysaccharide derived from E. coli), TPA (phorbol-12-myristate-13-acetate) and cycloheximide were added to final concentrations of 10 μg / ml, 10 ng / ml and 1 μg / ml, respectively. The mixture was added and mixed so that the culture was continued. After 4 to 4.5 hours (5 to 5.5 hours in total), the culture solution was removed by suction, and the macrophages remaining on the dish contained 0.6% sodium lauroyl sarcosinate and 6 mM sodium citrate.
It was dissolved in M guanidyl thiocyanate solution and homogenized. This homogenate was overlaid on a 5.7 M cesium chloride aqueous solution containing 0.1 M EDTA, and an ultracentrifuge (RPS27-
2 rotor, Hitachi, Ltd.) and centrifuged at 26,500 rpm for 20 hours to obtain a total RNA fraction as a pellet. This is 0.
It was dissolved in a small amount of a 7 M urea solution containing 35 M NaCl, 20 mM Tris and 20 mM EDTA, and recovered as an ethanol precipitate. 159 μg of total RNA was obtained.

This total RNA fraction contained 10 mM of 1 mM EDTA.
It was dissolved in 1 ml of mM Tris-HCl buffer (pH 7.4) (hereinafter referred to as TE solution) and heated at 65 ° C for 5 minutes. Na for this
After adding Cl solution to 0.5M, 0.5M in advance
The poly (A) mRNA adsorbed onto an oligo (dT) cellulose column equilibrated with a TE solution containing NaCl was adsorbed on TE.
By elution with a liquid, 8 μg of poly (A) mRNA was obtained. The poly (A) mRNA was dissolved in distilled water at a concentration of 1.9 ng / nl, and 50 nl was injected per oocyte of Xenopus laevis, and the protein translated in the oocyte was prepared according to the method described above. L-929 cytotoxic activity was measured.
As a result, 6 in 0.1 ml of 10 oocyte homogenates.
An activity of 6 units / ml was observed, and it was confirmed that the human cancer necrosis factor mRNA was contained in this poly (A) mRNA preparation.

(3) Synthesis of cDNA : Using the poly (A) mRNA obtained in (2) as a template, the method of Gubler et al. [Ge
ne, 25 , 263 (1983)]. Reaction volume: 40 μl 50 mM Tris-HCl buffer (pH 8.3); 10 mM MgCl
₂ ; 10 mM dithiothreitol; 4 mM sodium pyrophosphate; 1.25 mM dGTP, dATP, dTTP; 0.5 m
M dCTP; 0.167 μM α- ³² P-dCTP (specific activity 30
00Ci / mmole); 4 μg oligo (dT) _12-18 ; 6 μg po
ly (A) mRNA; 120 units Avian myeloid leukemia virus-derived reverse transcriptase.

After reacting at 43 ° C. for 30 minutes, EDTA was added to stop the reaction, extraction was performed with a phenol-chloroform mixed solution (1: 1), and ammonium acetate was added to the aqueous layer to a final concentration of 2.5M. In addition, the reaction product (single-stranded cDNA-RNA complex) was precipitated with ethanol. This single-stranded cDNA-RNA complex is used as a reaction buffer having the following composition
It was dissolved in 100 μl.

Reaction buffer composition: 20 mM Tris-HCl buffer (pH 7.5); 5 mM MgCl ₂ ; 10 mM (NH ₄ ) ₂ S
O ₄ ; 100 mM KC1; 0.15 mM β-nicotinamide adenine dinucleotide; 50 μg / ml bovine serum albumin; 40 μ
M dGTP, dATP, dTTP, dCTP; 0.9 units E. coli ribonuclease H; 23 units E. coli DNA polymerase I.

After reacting at 12 ° C. for 60 minutes and then at 22 ° C. for 60 minutes, EDTA was added to stop the reaction, and the mixture was extracted with a phenol-chloroform mixed solution in the same manner as above and precipitated with ethanol to give a double-stranded chain. A cDNA was obtained.

(4) Oligo (dC) tail-added cDNA
Preparation : -To the double-stranded cDNA obtained in (3), 100 µg of the reaction buffer having the following composition was added, and the mixture was reacted at 37 ° C for 30 minutes to add an oligo (dC) tail to the double-stranded cDNA. .

Reaction buffer: ₂ mM CoCl ₂ , 0.2 mM dithiothreitol, 0.1 mM α- ³² P-dCTP (specific activity 3
Ci / mmole) and 10 units of terminal deoxynucleotidyl transferase, 100 mM sodium cacodylate, pH 7.2.

The reaction was stopped by adding an EDTA aqueous solution, extracted with a phenol-chloroform mixed solution, and the oligo (dC) -tailed cDNA was precipitated by ethanol and recovered. This is a 10 mM Tris-HCl buffer solution (pH 7.
4), 2 μg oligo (dC) -tailed cDNA per ml in an aqueous solution containing 1 mM EDTA and 100 mM NaCl
Was dissolved so as to contain.

(5) Oligo (dG) tail-added plasmid
Preparation of DopBR322 DNA: -20 mM Tris-HCl
Buffer solution (pH 7.4), 10 mM MgCl ₂ , 50 mM (NH ₄ ) ₂ S
10 μg of pBR322 was dissolved in 100 μl of an aqueous solution containing O ₄ and 0.1 mg of bovine serum albumin per ml, and 15 units of the restriction enzyme Pst I endonuclease was added to the solution.
Reacted for hours. After completion of the reaction, the reaction solution was extracted with a phenol-chloroform mixed solution, and DNA was recovered from the aqueous layer by ethanol precipitation. The obtained DNA was used in the above-mentioned oligo (dC) tail addition (however, ³² P-dCT
(Containing ³ H-dGTP instead of P) 200 μl,
Terminal deoxynucleotidyl transferase
The reaction was carried out at 37 ° C for 20 minutes using 80 units, and about 10 to 15 d
The G residue was incorporated. The reaction solution was extracted with a phenol-chloroform mixed solution, and the oligo (dG) -tailed plasmid pBR322DNA was extracted from the aqueous layer by ethanol precipitation.
Was recovered. This is an oligo (dC) tailed cDNA
20 μg oligo (d) in the same buffer as in
G) Lysed to contain the tailed plasmid pBR322 DNA.

(6) Preparation of recombinant plasmid :-
Oligo (dC) tailed cDNA obtained in item (4)
120 μl of the solution was mixed with 120 μl of the oligo (dG) -tailed pBR322 DNA solution obtained in section (5),
Annealing was carried out by incubating at 57 ° C. for 5 minutes and at 57 ° C. for 120 minutes to prepare a recombinant plasmid solution.

(7) Selection of transformants : -Using the recombinant plasmid solution obtained in (6), E. coli χ1776
The strain was transformed. That is, the E. coli χ1776 strain was cultured in 20 ml of L-broth supplemented with 100 µg / ml of diaminopimelic acid and 40 µg / ml of thymidine at 37 ° C until the absorbance (600 nm) reached 0.5, and the cells were cultured at 4 ° C. Collected by centrifugation, 50mM CaC
l ₂ dispersed containing 10 mM Tris-HCl buffer (pH 7.3) 10 ml, and precipitated again centrifuged at 4 ° C.. The collected bacterial cells were dispersed in 2 ml of the same buffer and allowed to stand at 0 ° C for 5 minutes. To 0.2 ml of this dispersion, 0.1 ml of the recombinant plasmid solution obtained in (6) was added and mixed, and the mixture was allowed to stand at 0 ° C for 15 minutes, and further 4
After holding at 2 ° C for 2 minutes, 0.5 ml of L-broth having the same composition as that used in the previous culture was added and shake culture was carried out for 1 hour. A part of this culture solution is taken, spread on an L-broth agar plate containing 15 μg / ml of tetracycline in addition to the components described above, cultured at 37 ° C for about 12 hours, and tetracycline-resistant bacteria are selected to prepare a cDNA library. did.

(8) Cloning : In order to screen a transformant having a plasmid containing cDNA encoding human cancer necrosis factor for the cDNA library obtained in (7), obtained in (1). The colony hybridization test was performed by the method of Hanahan et al. [Gene, 10 , 63 (1980)] using a ³² P-labeled fragment of a fragment (299 bp) excised with the restriction enzyme AvaI of DNA encoding rabbit cancer necrosis factor as a probe. Went according to. From about 20,000 colonies, 43 transformants having a recombinant plasmid containing a nucleotide sequence that strongly binds to this labeled probe were selected. Furthermore, these 43 colonies were subjected to a secondary screening using a ³² P-labeled DNA fragment (88 bp) corresponding to the upstream of the rabbit cancer necrosis factor coding region excised with the restriction enzyme Hae II as a probe, Six colonies that strongly hybridized were selected. As a result of these two rounds of screening, the DNA sequence encoding the rabbit cancer necrosis factor and its homologous portion with the upstream sequence were identified.
A DNA containing a base sequence with high R> property was obtained.

(9)Determination of base sequence of cloned DNA
Fixed: -The 6 clones selected here (plasmid number
pHTNF-1, -4, -5, -13, -22, -2
From 6), the plasmid pHTNF-13
The transformant was cultured in LB broth to obtain bacterial cells. This bacterium
The method of Wilkie et al. [Nucleic Acids Res.,7, 859 (1979)
 ], And plasmid DNA was obtained. This program
Restriction enzyme for rasmid DNAPstDecompose with I, separate and purify
To obtain cloned DNA. This cloned DNA fragment
The fragment was digested with various restriction enzymes to obtain 16 types of restriction enzyme fragments.
The Maxam-Gilbert method.
Phosphorylation,³²End labeling with P, base-specific chemical decomposition reaction
, Gel electrophoresis and autoradiography
Decided The nucleotide sequence encoding the human cancer necrosis factor precursor
It is shown in SEQ ID NO: 1. The part surrounded by [] is human cancer necrosis
This is the region that codes the factor. [Japanese Patent Application No. 59-43617 (JP
(See Sho 60-185799))

Reference Example 3: (1) Analysis of mRNA fraction from rabbit alveolar macrophages
Isolation and purification : -Propionibacte for rabbits (body weight: 2.5 kg)
Killed rium acnes cells were intravenously injected at a dose of 100 mg per bird, and sacrificed 8 days later. Immediately perform an open chest tracheotomy,
Lung lavage was repeated using phosphate buffered saline through a tube inserted into the trachea to collect alveolar macrophages. About 3 × 10 ⁹ alveolar macrophages were obtained from 12 rabbits. This alveolar macrophage was suspended in RPMI-1640 medium containing 10% fetal bovine serum and seeded on a Petri dish (8 cm in diameter) to give 2 x 10 ⁷ cells per plate, containing 5% carbon dioxide at 37 ° C. Humidity 90-100 in air
Pre-cultured in%. After 1 hour of pre-incubation, endotoxin, TPA and cycloheximide were each added to a final concentration of
The mixture was added and mixed at 10 μg / ml, 10 ng / ml and 1 μg / ml, and the culture was further continued. 4 to 4.5 hours later (total 5 to 5.
After 5 hours, the culture solution was removed by suction, and the macrophages remaining on the dish were dissolved and homogenized in a 5M guanidyl thiocyanate solution containing 0.6% sodium lauroyl sarcosinate and 6 mM sodium citrate. This homogenate was overlaid on a 5.7 M cesium chloride aqueous solution containing 0.1 M EDTA, and the ultracentrifuge (RPS27-2 rotor,
(Hitachi, Ltd.) and centrifuged at 26,500 rpm for 20 hours for total RNA
Fractions were obtained as pellets. This is 0.35M NaCl, 20
It was dissolved in a small amount of 7 M urea solution containing mM Tris and 20 mM EDTA, and collected as an ethanol precipitate. 5.2 mg of total RNA was obtained from 12 rabbits.

This total RNA fraction was dissolved in 2 ml of TE solution,
Heated at 65 ° C for 5 minutes. After adding NaCl solution to this to 0.5M, TE containing 0.5M NaCl in advance.
The solution was applied to an oligo (dT) cellulose column equilibrated with the solution, and the adsorbed poly (A) mRNA was eluted with TE solution to obtain 314 μg of poly (A) mRNA. 200 μg of the poly (A) mRNA obtained here was subjected to agarose gel electrophoresis (gel concentration 1%, in the presence of 6 M urea, pH 4), divided into 7 fractions according to their molecular size, and the gel was melted (70 After the reaction was carried out at 10 ° C. for 10 minutes), extraction with water-saturated phenol and extraction with chloroform were performed, followed by precipitation with ethanol to recover poly (A) mRNA from each fraction. For each fraction of poly (A) mRNA, the amount of rabbit cancer necrosis factor mRNA was measured by a method using Xenopus oocytes, and the fraction corresponding to the molecular size of 1.6 to 2.7 kb was enriched with rabbit cancer necrosis factor mRNA. Recovered to concentration. The purified poly (A) mRNA obtained here was used in the following experiments.

(2) Synthesis of cDNA : -Purified poly (A) m
Using 4 μg of RNA, cDNA was synthesized under the following conditions. Reaction volume: 100 μl 50 mM Tris-HCl buffer (pH 8.3); 10 mM MgCl
₂ ; 70 mM KCl; 1 mM dithiothreitol; 0.5 mM dT
TP, dCTP, dATP, dGTP (however, dCTP is
³² P-labeled, specific activity 4.4 × 10 cpm / nmole); 3 μg oligo (dT) _12-18 , 80 units Avian myeloid leukemia virus-derived reverse transcriptase.

After reacting at 43 ° C. for 90 minutes, the reaction was stopped with an aqueous EDTA solution. The cDNA-mRNA complex was extracted with a phenol-chloroform mixed solution (1: 1),
It was precipitated with ethanol and collected. Further, after the mRNA was decomposed and removed by heating with alkali, the synthesized single-stranded cDNA was precipitated by ethanol and recovered.

This single-stranded cDNA precipitate was dissolved in 40 μl of a reaction buffer solution having the following composition. Reaction buffer: 0.5 mM dATP, dTTP, dGTP, d
CTP; 5 mM MgCl ₂ ; 70 mM KCl; 1.5 mM β-mercaptoethanol; 8 units E. coli DNA polymerase I
0.1 M Hepes buffer (pH 6.9) containing (large fragment).

Double-stranded cDNA was synthesized by reacting at 15 ° C. for 20 hours. The reaction was stopped by adding an aqueous solution of sodium dodecyl sulfate to the reaction solution, and the double-stranded cDNA was extracted with a phenol-chloroform mixed solution and precipitated by ethanol and recovered. The resulting double-stranded cDNA was precipitated with 50 mM sodium acetate (pH 4.5), 1 mM ZnSO ₄ , 200 mM NaCl,
The hairpin structure was cleaved by dissolving in 100 μl of an aqueous solution containing 0.5% glycerol and 0.5 unit of S1 nuclease and reacting at 37 ° C. for 20 minutes. The reaction was stopped by adding an aqueous EDTA solution, extracted with a phenol-chloroform mixed solution, re-extracted with ether, and then precipitated with ethanol to recover cDNA.

(3) Oligo (dC) tail-added cDNA
Preparation : -Add 100 μl of the reaction buffer having the following composition to the double-stranded cDNA obtained above and react at 37 ° C. for 20 minutes,
An oligo (dC) tail was added to the double-stranded cDNA. Reaction buffer: 130 mM sodium cacodylate- ³⁰ mM Tris containing ₁ mM CoCl ₂ , 0.1 mM dithiothreitol, 0.2 μg poly (A), 0.1 mM ³ H-dCTP (specific activity 5400 cpm / pmol) and 10 units terminal deoxynucleotidyl transferase. -HCl buffer (pH 6.8
).

The reaction was stopped by adding an aqueous solution of EDTA, extracted with a mixed solution of phenol-chloroform, reextracted with ether, and then oligo (dC) tail-added cDN.
A was precipitated by ethanol and collected. This is 10mMT
ris-HCl buffer (pH 7.4), 1 mM EDTA and 100
The solution was dissolved in an aqueous solution containing mM NaCl so as to contain 0.2 μg of oligo (dC) -tailed cDNA per ml.

(4) Oligo (dG) tail-added plasmid
Preparation of DopBR322 DNA: -20 mM Tris-HCl
Buffer solution (pH 7.4), 10 mM MgCl ₂ , 50 mM (NH ₄ ) ₂ SO
₄ µl of pBR322 was dissolved in 100 µl of an aqueous solution containing 0.1 mg of bovine serum albumin per ml, and the restriction enzyme was added.
15 units of Pst I endonuclease was added and reacted at 37 ° C. for 1 hour. After the reaction was completed, the reaction solution was extracted with phenol, and DNA was recovered from the aqueous layer by ethanol precipitation. The obtained DNA was used for the oligo (dC) tail addition described above (provided that ³ H-dCTP was used instead of ³ H-dCTP).
200 μl) (containing dGTP) and 80 units of terminal deoxynucleotidyl transferase
The reaction was carried out at 37 ° C for 20 minutes to incorporate about 10 to 15 dG residues. The reaction solution was extracted with a water-saturated phenol-chloroform mixed solution, and the oligo (d
G) The tailed plasmid pBR322 DNA was recovered. This was dissolved in the same buffer as in the case of oligo (dC) -tailed cDNA so as to contain 2 μg of oligo (dG) -tailed plasmid pBR322 DNA per ml.

(5) Preparation of recombinant plasmid : 50 μl of oligo (dC) -tailed cDNA solution was mixed with 50 μl of oligo (dG) -tailed pBR322 DNA solution, mixed at 65 ° C. for 10 minutes, 57 ° C. for 120 minutes, 45 ° C. For 60 minutes,
Annealing was performed by incubating at 35 ° C. for 60 minutes and room temperature for 60 minutes to prepare a recombinant plasmid solution.

(6) Selection of transformant : E. coli χ1776 strain was transformed with the recombinant plasmid solution obtained above. That is, E. coli χ1776 strain was supplemented with L- supplemented with 100 µg / ml of diaminopimelic acid and 40 µg / ml of thymidine.
Incubate in 20 ml of broth at 37 ° C until the absorbance (600 nm) becomes 0.5, collect the cells with a centrifuge with a condenser, and add 50 mM Ca
10 ml of Cl ₂ containing 10 mM Tris-HCl buffer (pH 7.3)
And was centrifuged again at 0 ° C. to precipitate. The collected bacterial cells were dispersed in 2 ml of the same buffer and allowed to stand at 0 ° C for 5 minutes. 0.1 ml of the above recombinant plasmid solution is added to 0.2 ml of this dispersion.
Was mixed and allowed to stand at 0 ° C for 15 minutes, and further held at 42 ° C for 2 minutes, then 0.5 ml of L-broth having the same composition as used in the previous culture was added, and shake culture was carried out for 1 hour. . Take a portion of this culture solution and add tetracycline 15μ in addition to the above components.
The cells were spread on L-broth agar plates containing g / ml and cultured at 37 ° C. for about 12 hours, and tetracycline-resistant bacteria were selected to prepare a cDNA library.

(7) Hybridization test : -A colony hybridization test using a ³² P-labeled cDNA probe for screening transformants having a plasmid containing a cDNA encoding rabbit cancer necrosis factor for the above-mentioned cDNA library. Hana
It was performed according to the method of Han et al. [Gene, 10 , 63 (1980)].
^{The 32} P-labeled cDNA probe was obtained from the induced plus and minus alveolar macrophages by the method of (1) above.
It was synthesized by the method of (2) using NA as a template. However,
^{As 32} P-dCTP, one having a high radioactivity specific activity was used and labeled at a high concentration. By this test, transformants having a recombinant plasmid containing a nucleotide sequence that strongly binds to the induction plus probe and does not hybridize with the induction minus probe were selected. Fifty colonies were selected from about 20,000 colonies.

Next, a hybridization / translation test was performed on these selected strains by Ma.
niatis, T., et al., (ed) "Molecular Cloning", 329 (1
980), Cold Spring Harbor Lab. Plasmid DNA was extracted from each transformant, heat-denatured and fixed on a nitrocellulose filter, and the poly (A) mRNA fraction containing the rabbit cancer necrosis factor mRNA obtained in (1) above was fixed to this. In addition, 50 ℃
The reaction was carried out for 3 hours to carry out hybridization.
The bound mRNA was collected by elution and then injected into Xenopus oocytes to test whether or not the collected mRNA was rabbit cancer necrosis factor mRNA. By this test, 3 strains having a plasmid containing a cDNA that strongly hybridizes with rabbit cancer necrosis factor mRNA were found from the 20 transformants selected above. From the plasmid with the longest cDNA (about 750 bp),
The DNA fragment was cut out with the restriction enzyme Dde I and used as a probe for secondary screening. This DNA fragment was labeled with ³² P, and the cDNA library obtained in the above (6) was subjected to a colony hybridization test again to select a transformant having a plasmid containing a cDNA that strongly binds to the labeled probe. Of the approximately 60,000 colonies in the cDNA library, 98 were positive colonies. From these, the cDNA was cut out with the restriction enzyme Pst I, the size was examined by polyacrylamide gel electrophoresis, and 17 clones having a size of 1 kbp or more were selected. A transformant containing the largest cDNA of these (cell number: RTNF802, cloned DNA number:
For pRTNF802), the cloned DNA was isolated and the nucleotide sequence was determined.

(8)Determination of base sequence of cloned DNA
Fixed: -The strain selected in (7) (RTNF802) is
Cultivated with L-broth containing minopimelic acid and thymidine
It was cultivated to obtain bacterial cells. This bacterium was applied to the method of Wilkie et al. [Nucleic
 Acids Res.,7, 859 (1979)]
The obtained DNA was obtained. This plasmid DNA is a restriction enzymePs
tIt was digested with I, purified and separated to obtain cloned DNA.
This cloned DNA fragment is digested with various restriction enzymes,
For each restriction enzyme fragment,
dephosphorylation by m-Gilbert method,³²End labeling with P,
Base-specific chemical decomposition reaction, gel electrophoresis and autoradiation
Determined from ography.

The nucleotide sequence is shown in SEQ ID NO: 2. First
Numbers -15 are G-linked tails added to incorporate the cDNA into the vector. Nos. 16 to 276 are nucleotide sequences presumed to encode the polypeptide necessary for constituting the rabbit cancer necrosis factor precursor. The nucleotides 277 to 291 encode the amino acid sequence corresponding to the N-terminus of rabbit cancer necrosis factor, and the nucleotides 715 to 738 encode the amino acid sequence of the C-terminal portion. [Japanese Patent Application No. 58-228790 (Japanese Patent Laid-Open No. 60-
120990))

[Sequence list]

SEQ ID NO: 1, sequence length: 702, sequence type: nucleic acid, number of strands: double strand, topology: linear, sequence: ATG AGC ACT GAA AGC ATG ATC CGG GAC GTG GAG CTG GCC GAG GAG GCG 48 CTC CCC AAG AAG ACA GGG GGG CCC CAG GGC TCC AGG CGG TGC TTG TTC 96 CTC AGC CTC TTC TCC TTC CTG ATC GTG GCA GGC GCC ACC ACG CTC TTC 144 TGC CTG CTG CAC TTT GGA GTG ATC GGC CCC CAG AGG GAA GAG TTC CCC 192 AGG GAC CTC TCT CTA ATC AGC CCT CTG GCC CAG GCA GTC AGA [TCA TCT 240 TCT CGA ACC CCG AGT GAC AAG CCT GTA GCC CAT GTT GTA GCA AAC CCT 288 ↑ Ava I CAA GCT GAG GGG CAG CTC CAG TGG CTG AAC CGC CGG GCC AAT GCC CTC 336 CTG GCC AAT GGC GTG GAG CTG AGA GAT AAC CAG CTG GTG GTG CCA TCA 384 GAG GGC CTG TAC CTC ATC TAC TCC CAG GTC CTC TTC AAG GGC CAA GGC 432 TGC CCC TCC ACC CAT CTC CTC ACC CAC ACC ATC AGC CGC ATC GCC 480 GTC TCC TAC CAG ACC AAG GTC AAC CTC CTC TCT GCC ATC AAG AGC CCC 528 TGC CAG AGG GAG ACC CCA GAG GGG GCT GAG GCC AAG CCC TGG TAT GAG 576 CCC ATC TAT CTG GGA GGG GTC TTC CAG CTG GAG AAG GGT GAC CGA CTC 624 AGC GCT GAG ATC AAT CGG CCC GAC TAT CTC GAC TTT GCC GAG TCT GGG 672 CAG GTC TAC TTT GGG ATC ATT GCC CTG] TGA 702

SEQ ID NO: 2, Sequence length: 803, Sequence type: Nucleic acid, Number of strands: Double stranded, Topology: Linear, Sequence: GGG GGG GGG GGG GGG CCC TCT GGA GAG AGC GCC ATG AGC ACT GAG AGT 48 ATG ATC CGG GAC GTC GAG CTG GCG GAG GGG CCG CTC CCC AAG AAG GCA 96 GGG GGG CCC CAG GGC TCC AAG CGC TGC CTC TGC CTC AGC CTC TTC TCT 144 TTC CTG CTC GTG GCT GGA GCC ACC ACG CTC TTC TGC CTG CTG CAC TTC 192 AGG GTG ATC GGC CCT CAG GAG GAA GAG CAG TCC CCA AAC AAC CTC CAT 240 CTA GTC AAC CCT GTG GCC CAG ATG GTC ACC CTC AGA TCA GCT TCT CGG 288 GCC CTG AGT GAC AAG CCT CTA GCC CAC GTA GCA AAC CCG CAA GTG 336 GAG GGC CAG CTC CAG TGG CTG AGC CAG CGT GCG AAC GCC CTG CTG GCC 384 AAC GGC ATG AAG CTC ACG GAC AAC CAG CTG GTG GTG CCG GCC GAC GGG 432 CTG TAC CTC ATC TAC TCC CAG GTT CTC AGC GGT CAA GGC TGC CGC 480 TCC TAC GTG CTC CTC ACT CAC ACT GTC AGC CGC TTC GCC GTC TCC TAC 528 CCG AAC AAG GTC AAC CTC CTC TCT GCC ATC AAG AGC CCC TGC CAC CGG 576 GAG ACC CCC GAG GAG GCT GAG CCC ATG GCC TGG TAC GAG CCC ATC TAC 624 CTG GGC GGC GTC TTC CAG TTG GAG AAG GGT GAC CGG CTC AGC ACC GAG 672 GTC AAC CAG CCT GAG TAC CTG GAC CTT GCC GAG TCC GGG CAG GTC TAC 720 TTT GCC AATT CTG TGA GGG GAC TGA CCA CCA CTC CTC CCC CTC 768 TCC CAC CCC AGC CCC CTC ACT CTG GGC GCC CTC AG 803

[Brief description of drawings]

FIG. 1 is a start codon ATG at the amino-terminal portion of the gene encoding human cancer necrosis factor in the tryptophan promoter.
The manufacturing method of the expression plasmid ligated via is shown.

FIG. 2 shows a method of converting the ligation region using a Cla I restriction enzyme cleavage site in the ligation region between the SD region and the start codon ATG using a tryptophan promoter.

FIG. 3 shows a method for producing a plasmid vector having a high copy number derived from pBR322 and a method for incorporating a gene encoding human cancer necrosis factor into the plasmid vector.

FIG. 4 shows Hpa I restriction enzyme sites containing a novel SD region capable of high-level expression of human cancer necrosis factor and a nucleotide sequence of a connecting region between the SD region and the initiation codon ATG.
1 shows the base sequence of a chemically synthesized DNA fragment up to the Ava I restriction enzyme site.

FIG. 5 shows a method for producing a high-level expression plasmid for human cancer necrosis factor in which the chemically synthesized DNA fragment shown in FIG. 4 is replaced.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (3)

[Claims] 1. A gene encoding a cancer necrosis factor or a polypeptide highly homologous to the N-terminal portion of the factor, at least as an expression control sequence, comprising a promoter region of another gene and a Shine-Dalgarno sequence. In the sequence between the translation initiation codons, a transformed host was cultured with a highly-expressed recombinant vector having the following base sequence, and the host was cultivated in the culture to show homology with the cancer necrosis factor or the N-terminal portion of the factor. A method for producing the substance, which comprises efficiently producing a high polypeptide. (5 ')-AAGGAGGTT-X-ATTATG- (3') [I] (In the formula, X represents T, TY or AZ.
Means A, AA, AAT, and Z means T and TCG. ) 2. The method for producing human cancer necrosis factor according to claim 1, wherein the encoded gene is a human cancer necrosis factor gene. 3. The cancer necrosis factor or N of the factor according to claim 1, wherein the transformed host is a microorganism.
A method for producing a polypeptide having high homology with the terminal portion.