JPH0398587A - Human tnf-alpha gene, gene coding new tnf derivative and peptide obtained by same gene - Google Patents

Abstract

PURPOSE:To improve the efficiency of production by culturing a transformed colon bacillus, etc., transformed using a specified vector in a culture medium. CONSTITUTION:A prescribed length of a gene fragment produced using the amidite solid phase synthesis method, etc., is annealed to obtain a human TNF-alpha gene (A) containing a base sequence, etc., of formula I. The resultant component (A) is then integrated to a plasmid capable of propagation in a colon bacillus to obtain a mutagenic plasmid (B) as a plasmid for peptide manifestation. The resultant component (B) is then introduced into a colon bacillus strain (e.g. colon bacillus JM 101) to obtain a transformant (C). The obtained component (C) is subsequently cultured in L-medium, M9 medium, etc., to obtain a cultured material (D). Colon bacillus cell bodys are then separated from the component (D) and the resultant cell bodys are subjected to ultrasonic crushing to obtain a supernatant (E) as a water soluble fraction. The obtained component (E) is then purified by the ammonium sulfate precipitation method, dialysis, the anion-exchange chromatography method, etc., to produce the objective antitumor peptide of formula II (A1 is Asp or Arg; A2 is His or nothing; A3 is Gln or nothing; A4 is Glu or His).

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides a human gene for efficiently expressing human TNF'-α, which has antitumor activity, in bacterial cells such as Escherichia coli, and a fragment of this gene. The present invention relates to a gene with a mutated region and a peptide having antitumor activity obtained from this gene.

[Prior Art and Issues] Currently, multifaceted research on cancer treatment is being conducted, and the development of drugs with higher antitumor activity is desired.

In particular, there are many reports on gene cloning and expression in E. coli as a means of mass production of human, which is expected to be applied to cancer treatment as an antitumor peptide. No detailed study has been conducted on improving the manufacturing method for this purpose.

Specifically, there are significant differences in the codons used between prokaryotes such as E. coli and eukaryotes such as humans, so for example, by genetically transferring the gene encoding the amino acid sequence of human TNP-α to E. coli. When it is incorporated into a protein, the problem arises that the expression level becomes very low.

Therefore, there is a need for a human TNF-α gene that can express human TNF-α more efficiently in bacterial cells such as E. coli, and it is also desired to develop a peptide that has a higher antitumor effect than human TNF-α. Ta.

[Means for Solving the Problems] As a result of extensive studies aimed at producing human TNF-α through genetic recombination with high production efficiency, the present inventors have determined that human TNF-α can be produced using gene codons suitable for E. coli. By constructing and creating genes, human TN can be produced by genetically recombining bacterial cells.
Mass production of F-α? Is this successful? This human TNF
The present invention was completed by discovering that a peptide having excellent antitumor activity can be obtained from a mutant gene in which the -α gene is mutated using site-specific mutagenesis. That is, the present invention provides: (1) The following base sequence MetValArgSerSerSerArgThrP
roSerAspLysPro5'-ATGGTTC
GTTCCTCTTCCCGTACTCCATCTGA
CAAACC^3' -TACC^^GCAAGGAG
AAGGGCATGAGGTAGACTGTTTGGT
GTAGCTCATGTTGTTGCTAACCCGC
AGGCAGAAGGCCAGTTACATCGAGT
ACAACAACGATTGGGCGTCCGTCTT
CCGGTCAATGInTrpLeuAsnArgA
rgAIaAsn^IaLeuLeuAlaAsnGl
yCAGTGGCTTAACCGTCGTGCAAAC
GCACTGCTGGCGAACGGCGTCACCG
AATTGGCAGCACGTTTGCGTGACGA
CCGCTTGCCCValGIuLeuArgAsp
AsnGInLeuValValProSerGIuG
IyGTAGAACTGCGTGATAACCAACT
CGTAGTTCCGTCCGAAGGTCATCT
GACGCACTATTGGTTGAGCATCAAG
GCAGGCTTCCALeuTyrLeu4eTyr
SerGInValLeuPheLysGIyGlnG
lyCTGTACCTGATCTACAGCCAGGT
TCTGTTCAAGGGTCAGGGTGACATG
GACTAGATGTCGGTCCAAGACAAGT
TCCCAGTCCCACysProSerThrHi
sValLeuLeuThrHisThr4eSerA
rgTGCCCGAGGCACTCACGTTTTGCT
GACTCATACTATCTCCCCGTACGGGC
TCGTG^GTGCAAAACGACTGAGTAT
GATAGAGGGCAValAIaHisValVa
lAlaAsnProGInAlaGluGIyG]n
Leu11eAlaValSerTyrGInThrL
ysValAsnLeuLeuSerAla^TCGC
AGTTTCTTACCAAAACTAAAGGTGAAC
CTGCTGTCTGCTTAGCGTCAAAGAA
TGGTTTGATTCCACTTGGACGACAG
ACGAGGTATCATCGCTCTGTAGTG
CCATAGTAGCGAGACATCACT11eL
ysSerProCysGInArgGluThrPr
oGluGlyAlaG! uATCAAATCCCCG
TGCCAACGCGAAAACCCCAGAAGGCG
Human TNF-α gene expressed as CTGAA. (below,
It is referred to as the human TNF-α gene of the present invention. )TAGTTTAGGGGCACGGTTGCGCdingTT
GGGGTCTTCCGCGACTT^1aLysPr
oTrpTyrGluPro4eTyrLeuGlyG
IyValPheGCTAAAACCGTGGTACGA
ACCGATCTATTCDINGCGGTGGTGTATTC
(2) The following base sequence 5゜-ATG-GTT-CGT-TCC-TCT-TC
C-CGT-ACT-CCA-TCT・3'-TAC
-CAA-GCA-AGG-AGA-AGG-GCA-
TGA-GGT-AGA・CGATTTGGCACCA
TGCTTGGCTAGATAGAGCCACCACA
TAAGGInLeuGluLysG]yAspArg
LeuSerAIaGIuIleAsnArgR1・A
^^・CCA-GTA-GCT-CAT-GTT-GT
T-GCT-AAC・R,・TTT-GGT-CAT-
CG^・GTA-CAA-CAA-CGA-TTG・C
AGCTCGAAAAGGGDINGGATCGTCTGTC
CGCAGAGATCAACCCGCGDGINGCGAGCTT
TTCCCACTAGCAGACAGGCGTCTCT
AGTTGGCGCCG-CAG-GCA-GAA-G
GC-CAG-TTA-CAG-TGG-CTT・GG
C-GTC-CGT-CTT-CCG-GTC-AAT
-GTC-ACC-GAA・ProAspTyrLeu
AJpPheAIaGluSerGlyGInValT
yrPheCCGGATTACCTGGACTTTGC
TGAGTCTGGTCAAGTTTAC DingTCGGC
CTAATGGACCTGAAACGACTCAGAC
CAGTTCAAATGAAGAAC-CGT-CGT
-GCA-AAC-GCA-CTG-CTG-GCG-
AAC・TTG-GCA-GCA-CGT・DingTG-C
GT-GAC-GAC-CGC-TTG・GIy4e4
eAlaLeu GGC-GTA-GAA-CTG-CGT-GAT-A
AC-CAA-CTC-GT A・CCG-CAT-CT
T-GAC-OCA-CT^・TTG-GTT-GAG
-CAT・GA^・GCT-AAA-CCG-TGG-
TAC-R,・CCG-ATC-TAT・CTT-C
G^・TTT-GGC-ACC-ATG-R.・GG
C-TAG-ATA/GTT-CCG-TCC-GA^
・GGT-CTG-TAC-CTG・^TC-TAC・
CAA-GGC-AGG-CTT-CCA-GAC-A
TG-GAC-TAG・^TG・CTC-GGT-GG
T-GTA-TTC-CAG-CTC-GAA-AAG
-GGT・GAG-CCA-CCA-CAT-AAG-
GTC-GAG-CTT-TTC-CCA・^GC-C
AG-GTT-CTG-TTC-AAG-GGT-CA
G-GGT-TGC・TCG-CTC-CAA-GAC
-AAG-TTC-CC^・CTC-CCA・^CG・
GAT-CGT-CTG-TCC-GCA-GAG-A
TC-AAC-CGC-CCG・CTA-GCA-GA
C-AGG-CGT-CTC-TAG-TTG-GCG
-GGC・CCG-AGC-ACT-R 3・GTT-
TTG-CTG-ACT-CAT-ACT・GGC-T
CG-TGA-R.・CAA-AAC-GAC-TG
A-GTA-TGA・GAT-TAC-CTG-GAC
-TTT-GCT-GAG-TCT-GGT-CAA・
CTA-ATG-GAC-CTG-AAA-CGA-C
TC-AGA-CCA-GTT・ATC・Ding CC-CG
T-ATC-GCA-GTT-TCT-TAC-CAA
・＾CT・TAG-AGG-GCA-TAG-CGT-
CA＾・AGA-ATG-GTT-TGA・＾AG-G
TG-AAC-CTG-CTG-TCT-GCT-AT
C・＾A＾・TCC・TTC-CAC-TTG-GAC
-GAC-AGA-CGA-TAG-T-T・8GG・
CCG-TGC-R 5・CGC-GAA・＾CC-C
CA-GAA-GGC-GCT・GGC-ACG-R
．．・GCG-CTT-TGG-GGT-CTT-CCG
-CGA・GTT-TAC-TTC-GGT-ATC-
ATC-GCT-CTG-TAG-TGACAA・^T
G-AAG-CCA-TAG-TAG-CGA-GAC
-ATC-ACT (However, RI is GAC or CGC
, R, is CTG (however, R1 indicates GAC or absent G, R4 indicates GTG or absent G, R, is CTG)
^^ or missing G, R6 shows GTT or missing G, R7 shows GAA or CAT, R6 shows CTT or GTA. ) A gene encoding bebutide. (Hereinafter referred to as the gene of the present invention.) (3) Following formula (A) Val-Arg-Ser-Set-Ser-Arg-T
hr-Pro-Ser-A. Shiys-Pro-Val
-Ala-4fis4a1-val-Ala-^sn-
ProGln-Ala-Glu-Gly-Gln-Le
u-Gln-Trp-Leu-Asn-Arg-Arg
-Ala-Asn-Ala-Leu-Leu-Ala-
^sn-Gly-Val-Glu-Leu-Arg-A
sp-Asn-Gln-Leu4al-ValPro-
Ser-Glu-Gly-Leu-Tyr-Leu-1
1e-Tyr-SerGln-ya1-1, eu-P
he-Lys-Gly-Gln-Gly-Cys-Pr
oSer-Thr-A t-val-Leu-Leu-
Thr-11is-Thr-11e-Ser-Arg-
11e-Ala-Val-Ser-Tyr-Gln-T
hr-LysVal-^sn-Leu-Leu-Ser
-Ala-11e-Lys-Ser-ProCys-A
*-Arg-Glu-Thr-Pro-Glu-Gl
y-Ala-GluAla-Lys-Pro-Trp-
Tyr-A a-Pro-11e-Tyr-LeuGl
y-Gly4al-Phe-Gln-Leu-(ilu
-Lys-Gly-Asp-Arg-Leu-Ser-
^1a-Glu-11e-Asn-Arg-Pro-A
spTyr-Leu-^sp-Phe-Ala-Glu
-Ser-Gly-Gln-valTyr-Phe-G
ly-11e-11e-Ala-Leu
(A) (A, represents Asp or Arg,
A1 indicates old S or missing G, A3 indicates Gln or missing G
A indicates Glu or old S. ) is represented by Bebjid. (Hereinafter, referred to as the bebutide of the present invention.) In this specification, the following symbols are used for simplicity.

Ala ＾rg Asn Asp Cys Gln Glu Gly If is 11e Leu Lys Net L-Alanine L-Arginine L-Asparagine L-Aspartic acid L-Nstein L-Glutamine L~Glutamate Glycine L-Histidine -Isoleucine L-Leusone L- Lysine L-Methionine Phe Pro Ser Thr Trp Tyr Val A C G T DTT ATP SDS EDTA PEG dATP dGTP dCTP dTTP L-Phenylalanine - Proline L- Serine L- Threnin L- Tributophan L- Tyrosine - Valine Adenine tosinganine Thymine dithiothreitol Adenosine triphosphate Sodium dodecyl sulfate Ethylenenoamine Tetraacetic acid Polyethylene glycol Deoxyadenosyl triphosphate Deoxyguanosyl triphosphate Deoxycytidyl triphosphate Deoxythymidyl triphosphate FCS Fetal bovine serum MT T [3-(4.5-Dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide] PBS Phosphate buffered saline Human T of the present invention
The NF-α gene can be produced by gene synthesis using the branching method or solid-phase method, particularly preferably by amidite solid-phase synthesis, and by annealing a gene fragment of an appropriate length.

Below, the method for producing the human TNF-α gene of the present invention will be explained.

Gene Design The human TNF-α gene of the present invention is designed with the following points in mind.

■ Among the DNA sequences encoding the amino acids of human TNF-α, select sequences that are highly utilized by E. coli.

■A region rich in A-T base pairs is not continuous.

(2) Synthetic fragments that serve as parts for synthesizing the gene of the present invention do not have an intramolecular self-component base sequence.

■Do not have repeating arrays.

■It should be about 30 bases in size.

■3゛The end must not be A.

In order to express the human TNF-α gene of the present invention, for example, it may be linked downstream of the tac promoter, which is a synthetic promoter of E. coli.
It is preferable to connect two or more.

Synthesis To synthesize the human gene of the present invention designed as described above, each of the eleven chains is divided into several fragments and these are chemically synthesized. Any method of combining may be used. Each chain is 1
It is preferable to divide it into about 40 fragments, each consisting of 8 to 30 bases, each overlapping by 10 to 12 bases.

Delicious.

Methods for synthesizing each fragment include a solid phase method and a liquid phase method, but solid phase synthesis using a phosphoramidite method is most preferred.

When synthesizing purified fragments, the longer the chain length, the more impurities there are, which generally makes purification difficult, or the protection of the 5' hydroxyl group, which is an intermediate in oligonucleotide synthesis by the phosphoramidite method, is necessary for purification. The group-attached oligonucleotide was subjected to reversed-phase high performance liquid chromatography (reversed-phase HP
This can be achieved by fractionating by LC), removing the protecting group, further fractionating by using anion exchange column chromatography, and then desalting.

Phosphorylation and binding Each fragment except for the 5″ end fragment was phosphorylated with T4 kinase to inactivate the enzyme in each reaction solution, then mixed and desalted. The desalted fraction was concentrated and Add the 5° terminal fragment and add 90
Gene fragments can be obtained by heating to ~100° C., slowly cooling to anneal, and then reacting with T4 DNA ligase to bond.

The bebutide of the present invention can be easily mass-produced by genetic recombination using Escherichia coli or the like.

The method for producing the peptide of the present invention will be explained below.

Gene Design Since the bebutide of the present invention does not exist in nature, it can be constructed by mutating the human TNF-α gene of the present invention using site-directed mutagenesis.

Desirably, the human TNF-α gene of the present invention described above is subjected to, for example, Kunkel's site-directed mutagenesis method (P
ro. N. ^． S. 82.488 (1985)) to obtain the gene of the present invention.

Preparation of expression vectors In the present invention, it is possible to use various plasmids that have an expression stem such as a promoter for expression and can be propagated in E. coli. It can be carried out according to the method, but it is also possible to use commercially available expression plasmids.

A plasmid for expressing the bebutide of the present invention (hereinafter referred to as a mutant plasmid) is constructed by integrating the gene of the present invention into these plasmids by a general genetic recombination operation.

Transformation A suitable E. coli strain is transformed using this mutant plasmid according to a conventional method to obtain a transformant.

For culturing the bebutide producing transformant, L1 medium, M9 medium,
M9-casamino acid medium, high phosphate medium, high phosphate-casamino acid medium, etc. can be used, and the peptide of the present invention can be mass-produced by culturing in these.

Purification of peptides Peptides expressed in E. coli are purified by ultrasonic disruption of E. coli.
By centrifugation, a water-soluble supernatant was obtained, and polyethylenimine, etc. was added to this to separate DNA and RNA.
A. Liposomes are precipitated and removed and centrifuged to obtain a supernatant. From this supernatant, crude fractions are collected using ammonium sulfate precipitation, and further purified by dialysis, anion exchange chromatography, etc.

In order to further increase the purity, it is also preferable to perform affinity stomatography using a specific antibody.

Add 100 passages of 0.01N hydrochloric acid and further boil at 37°C.I.8.
Cells were lysed by incubation for an hour. After dissolving 540～
Absorbance at 690 nm was measured using Titer Check Multiscan (Flow Laboratory) to confirm the survival of L929 cells.

The amount of biological activity required to kill 50% of L929 cells was defined as 1 unit, and the antitumor activity of the bebutide of the present invention was determined. The results are shown in Table I.

Next, the fact that the bebutide of the present invention has excellent antitumor activity will be explained by giving experimental examples.

Experimental Example L 9 2 9@cells were added to MEM medium containing 10% FCS at a concentration of 2XIO'/1! This was dispensed into a 96-well plate in loads, cultured at 37°C for 5 hours, and the peptide 10A and actinomain D of the present invention (2.5 l!9/
10 samples were added and cultured at 37°C for 18 hours. After culturing, 20 compositions of MTT (5 cells/d) were added and cultured at 37°C for 5 hours. The results of 10% SDS or higher confirmed the antitumor effect of the bebutide of the present invention. In addition, the acute toxicity (LDs) of the peptide of the present invention was 1 for both oral and intravenous administration in mice.
0, O Q Q I. U. /k9 or higher, 5,0001 for both oral and intravenous injections in rats. U. It has a rating of 7k9 or higher, making it extremely safe.

That is, the peptide of the present invention is a safe drug with few side effects and is useful for cancer treatment.

Next, the dosage and formulation of bebutide of the present invention will be explained.

The bebutide of the present invention can be administered to animals and humans as is or together with conventional pharmaceutical carriers. The dosage form is selected as appropriate and used, including injections,
Examples include parenteral preparations such as suppositories and preparations for mucosal administration.

In order to exert the desired effect as a parenteral agent, the potency of the bebutide of the present invention in a regular adult is 10 to 200 1 per day, although it varies depending on the age, weight, and severity of the disease of the patient.
．． U. Intravenous injections, intravenous drips, subcutaneous injections, intramuscular injections, suppositories, and preparations for mucosal administration are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and generally uses distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. be able to. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation is frozen after filling vials, etc., and the moisture is removed using normal freeze-drying technology, and immediately before use, the parenteral preparation is frozen. !
It is also possible to reconstitute a liquid formulation from the dried mixture. moreover,
If necessary, an isotonic agent, a preservative, an antiseptic, a soothing agent, a dispersing agent, an antioxidant, etc. may be added.

Other parenteral preparations include suppositories for rectal administration;
Examples include preparations for mucosal administration and liniments such as ointments, which are manufactured according to conventional methods.

Next, the gene of the present invention and the method for producing Bevudide of the present invention will be explained with reference to Examples, but the present invention is not limited thereto in any way.

Example 1 Synthesis of fragments The structure of the human gene of the present invention consists of 42 fragments shown below. Solid-phase synthesis was performed using the rumidite method.

! )5 2)3' 3)5 4)3 5)5 6)3 7)5゜8)3 9)5 1 0) I1) 12) AATTCATGGT CGTTCCTCTTCCCG
TACTGTACCAAAGCAAGGAGAAGGCC
ATCTGACAAAACCAGTAGCTGCATGA
GGTAGACTGTTTGGTCATGTTGTTG
CTAACCCGCAG-CATCGAGTAC^^C
AACGA DINGTGGCAGAAGGCCAGTTACA
GTGGGGCGTCCGTCTTCCGGTCAAT
CTTAACCG DingCGTGCAACGCACDingG3
゜-GTCACCGAATTGGCAGCACGTTT
G5'-CTGGCGAACGGCGTAGAACT
G3 -CGTGACGACCGCTTGCCGCA
T+3)5'-CGTGATAACCAACTC
GTAGTT1 4) 3゜-CTTGACGCACTA
TTGGTTGAG1 5) 5゜-CCGTCCGAA
GGTCTGTACCTGATC1 6) 3"-CAT
CAAGGCAGGCTTCCAGACATG1 7
)5”-TACAGCCAGGTTCTGTTCA
AGGGT1 8 ) 3'-GACTAGATGT
CGGTCCAAGACAAG19)5'-CA
GGGTTGCCCGAGCACTCAC2 0) 3゜-TTCCCAGTCCCAACGGGCTCG2
1) 5'-GTTTTGCTGACTCATACTA
TC2 2 )3'-TGAGTGCAAACG
ACTGAGTA23)5゜-TCCCGTATCGC
AGTTTCTTACCAAAACT2 4)3'-
TGATAGAGGGCATAGCGTCAAAGAA
TG2 5 ) 5'-AAGGTGAACCTGC
TGTCTGCTATC26)3'-GTTTG
ATTCCACTTGGACGACAGA2 7) 5゜-AAATCCCCGTGCCCAACGCGAAACC
2 8) 3'-CGATAGTTTAGGGGGCA
CGGTTGCG2 9')5'-CCAGAA
GGCGCTGAAGCTAAAACCGko0)3゜-C
TTTGGGGTCTTCCGCGACTTCGA3
l) 5゜-TGGTACGAACCGATCTATCT
CGGT3 2 ) 3'-TTTGGCAC
CA Ding GCTTGGCTAGATA3 3) 5'-
GGTGTATTCCAGCTCGAAAAGGGGT3
4) 3'-GAGCCACCACATAAGGT
CGAGCTT3 5 ) 5'-GATCGTCT
GTCCGCAGAGATCAAC3 6 ) 3'-
TTCCCACTAGCAGACAGGCGTCTC3
7) 5'-CGCCCGGATTACCTGG
ACTTT3 8 ) 3'-TAGTTGGCGGGG
CCTAATGGAC3 9) 5゜-GCTGAGTC
TGGTCAAGTTTAC4 0) 3°-CTGAA
ACGACTCAGACCAGTT4 1) 5゜-
TTCGGTATCA dingCGCTCTGTAGTGAA
4 2) 3'-CAAATGAAGCCATAGT
For purification of fragments of AGCGAGACATCACTTTCGA and above, after solid phase synthesis using a DNA synthesizer, an ammonia solution containing the obtained base and oligonucleotide with a 5° hydroxyl protecting group was heated at 55°C. The base was heated for 5 hours to remove the base protecting group, and after the reaction, ammonia was distilled off under reduced pressure, and then subjected to reverse phase HPLC to fractionate oligonucleotides with only the 5' hydroxyl protecting group attached. The obtained fraction was treated with 80% acetic acid at room temperature for 15 to 30 minutes to remove the 5° hydroxyl protecting group, and after evaporation under reduced pressure, an anion exchange force column (DEAE-
2 sw) to obtain an oligonucleotide fraction. This was desalted using a gel filtration column (Sephadex G25) to obtain each fragment.

i. Phosphorylated 800p each of the above 2) to 4N fragments in 60zM Lis-HCl buffer (p48.0), 10KM magnesium chloride, 15zM2-mercaptoethanol,
In addition to the mixed solution consisting of 0.25i MATP, T4
Add 10 units of kinase (+3 manufactured by R.L.),
The reaction was carried out at 7°C for 5 minutes. After the reaction, the enzymes were inactivated by heating at 65° C. for 15 minutes, and each reaction branch was desalted using a gel filtration ram (Sephadex G50).

iii. After concentrating the fraction after binding and desalting the fragments using a centrifugal concentrator,
) ~ 10), II) ~ 22), 23) ~ 32), 33)
~42) oligonucleotide fragments each 100p
Tris-HCl buffer (p47.6), I
O. In addition to the reaction arm consisting of M magnesium chloride, at 65°C
, After heating for 5 minutes, it was slowly cooled and annealed. After cooling, DTT was added to the reaction solution to bring it to a concentration of IOHM, ATP was further added to give a concentration of 1 mM, and the mixture was reacted at 16°C for 18 hours using 200 units of T4 DNA ligase (manufactured by NEB). After that, it was subjected to 5% polyacrylamide electrophoresis (PAGE), and the target band was cut out. The cut out gel was eluted with a solution consisting of 0.5M ammonium acetate and IRl4EDTA to obtain one block.

Each block thus obtained is 0. 2# further,
50xM} Lis-HCl buffer (pl!7.6), I
〇200 units of T4 DNA ligase (
(manufactured by NEB) at 16°C for 18 hours.

iv. Purified reaction limbs were subjected to 5% polyacrylamide electrophoresis (PAGE)
The desired band was cut out. The cut out gel was eluted with a solution consisting of 0.5M ammonium acetate, I, and 1% EDTA. After removing impurities from this solution with phenol and ether, 2.5 times the amount of ethanol was added to perform ethanol precipitation to obtain the human TNF-α gene of the present invention. This gene sequence was confirmed by restriction enzyme fragment length and dideoxy DNA sequencing.

As a result, the human TNF-α gene of the present invention shown below was confirmed. Note that l) to 42) correspond to the numbers of the above-mentioned chemically synthesized fragments.

MetValArgSerSerSerArgThrP
roSerAspLysProl) 3) AATTCATGGTTCGTTCCTCTTCCCG
TACTCCA DINGCTGACAAAACCAGTACCA
AGCAAGGAGAAGGGCATGAGGTAGA
CTGTTTTGGT2) 4
) Val^IaHisValValAIaAsnProG
lnAIaGIuGlyGlnLeu5)7) GTAGCTCATGTTGTTGCTAACCCGC
AGGCAGAAGGCCAGTTACATCGAGT
ACAACAACGATTGGGCGTCCGTCTT
CCGGTCAAT6)8) GInTrpLeuAsnArgArgAlaAsnA
laLeuLeuA]aAsnGIy9)
11) CAG Ding GGCTTAA
CCGTCGTGCAAACGCACTGCTGGCG
AACGGCGTCACCGAATTGGCAGCAC
GTTTGCGTGACGACCGCTTGCCG10
) 12) ValGl
uLeuArgAspAsnGlnLeuValVal
ProSerGluG]y13)
15) GTAGAACTGCGTGATAAC
CAACTCGTAGTTCCGTCCGAAGGTC
ATCTTGACGCACTATTGGTTGAGCA
DINGCAAGGCAGGCTTCCA14)
16) LeuTyrl, eu4eTy
rserGInVa4, euPheLysGIyGln
GIy1.7) 19
)CTG dingACCTGATCTACAGCCAGGTT
CTGTTCAAGGGTCAGGGTGACATGG
ACTAGA DingGTCGGTCCAAAGACAAGTT
CCCAGTCCCA18)
20) Cys ProSerTh rH i
s Va l LeuLeuTh rH i sTh
r l lesserA rg21)
23) TGCCCGAGCACTCACGTTTTGCTGA
CTCATACTATCTCCCCGT^CGGGCTC
GTGAGTGCAAAACGACTGAGTATGA
TAGAGGGCA22)
24) 11e^laValSerTyrGlnThr
LysVal^snLeuLeuSerAIa25) ATCGCAGTTTCTTACCAAAACTAAGG
TGAACCTGCTGTCTGCTTAGCGTCA
AAGAATGGTTTGATTCCACTTGGAC
GACAGACGA26)
2g) 11eLysSerProCysGlnA
rgGluThrProGluGlyAlaGlu27
) 29)^TCAA
ATCCCCGTGCCAACGCGAAACCCCA
GAAGGCGCTGAATAGTTTAGGGGGCA
CGGTTGCGCTTTGGGGTCdingTCCGCG
ACTT30) AIaLysProTrpTyrGluProlIeT
yrl, euGlyGIyValPhe31)
33) GCTAAACCGT
GGTACGAACCGATCTATCTCGGTGG
TGTATTCCGATTTGGCACCATGCTT
GGCTAGATAGAGCCACCACATAAG3
2) 34) GlnL
euGluLysGlyAspArgLeuserAI
aGluIIeAsnArg35)
37) CAGCTCGAAAAGGG
DINGGATCGTCTGTCCGCAGAGATCAAC
CGCGTCGAGCTTTTTCCCAC dingAGCAG
ACAGGCGTCTCTAGTTGGCG36)
38) ProAspTy
rLeuAspPhe^laGluSerG1yGln
ValTyrPhe39)
41) CCGGATTACCTGGACTTTGCT
GAG Ding CTGGTCAAAGTTTACTTCGGCC
TAATGGACCTGAAACGACTCAGACC
AGTTCAAATGAAG40)
42) Gly4e4cAlaLeu GGTATCATCGCTCTGding AGTGAAAACC
ATAGTAGCGAGACATCACTTTCGA Next, an example of the production of the gene of the present invention and the bebutide of the present invention using the human TNF-α gene of the present invention will be shown.

Example 2 Preparation of template DNA Human gene 0.6 skin obtained in Example 1, M1 3mp pre-digested with EcoRl and Hindu
I 9RF (manufactured by Farmana) 0.17.11? , 5
0xM Tris-HCl buffer (pH 8.0), 10% magnesium chloride, IOxMDTT, IiMATP were mixed, 400 units of T4 DNA ligase was added, and the mixture was allowed to react at 6°C for 8 hours.

This reaction solution was transformed into Escherichia coli JMIOI to obtain a transformant, which was then cultured to obtain a phage (mpl9
TNF-a), Escherichia coli BM3 13 strain (dut”
-, ung-) three times to prepare a phage in which thymidine in the DNA was replaced with uracil. The obtained phage was concentrated under the conditions of 4% PEG-6000 and 0.5M sodium chloride, and subjected to phenol extraction and ethanol precipitation to extract the template DNA (mpl9 TNF-a
DNA) was obtained.

i. Preparation of Brimer DNA Next, as a Brimer ■ 5゜-TCCTCTTCCCGTACTCCATCT
An oligodeokine nucleotide (hereinafter referred to as S2 primer) having the base sequence of CGCAAA was produced by the amidite method using a DNA synthesizer.

The final concentration of this S2 primer 100p was 10
0MM Tris-HCl buffer (p}18.0), 10% magnesium chloride, 5xH D T T SO . 5
HMA TP, solution 30d consisting of 5 units T4 kinase
The oligonucleotide was reacted for 45 minutes at 37°C, and further treated at 65°C for 10 minutes to inactivate the enzyme, thereby phosphorylating the oligonucleotide.

111 Annealing Template DNA obtained as above (mpl
9 TNF-a DNA)O. A solution I consisting of 20 Tris-HCl buffer (pli 7.4), 2 magnesium chloride, and 50 sodium chloride, with the final concentration of phosphorylated S2 brimer DNA 2 rl-
After heating at 55°C for 5 minutes in Oμg, the mixture was slowly cooled to 25°C.

IV, to the reaction solution annealed with Extenoyon 11, 20 zM}Lis-HCl buffer/& (pt17.4), 0, 4 dATI', 0, 4 dGTP, 0.4 dCTP, 0.4 mM dTTP10
．． 75 ATP, 5 states magnesium chloride, 25r+M
Buffer tiI 0 0 A and T4 consisting of D T T
Add 5 units of DNA ligase and 1 unit of T4 DNA polymerase, react at 25°C for 5 minutes, and then incubate at 37°C for 9
The reaction was allowed to proceed for 0 minutes.

V. Transformation was performed using the method of Maniat et al. (■ ocular Cloning (1982)).
convitent E. coli JM created using p252)
1v extension reaction solution at IOI 0.1-
After one round of treatment at 4°C for 30 minutes and then at 42°C for 2 minutes, the cells were spread on LB plates and cultured at 37°C overnight.

The resulting plaques were collected and added to 2-7 medium together with 20ufl of E. coli JMIOI cultured overnight.
The cells were cultured at ℃ for 6 hours.

Furthermore, this E. coli was centrifuged (8, O O O rpm,
4% PEG-6000,
Template DNA (m
pl9-10-ArgTNF-αDNA) was obtained. Sequencing was performed using this template DNA to confirm mutations. Mutant Furno as canine enterobacterium JM 10
1 t. :: Infection was performed using the method of Maniatis et al. (Molecular Cloning (1982)).
) p90) by R F (Replicative
Form) DNA was adjusted.

vi, Preparation of expression vector V R P DNA (n+pl9-10-Arg
A DNA fragment obtained by digesting TNF-αDNA) with EcoR and Hind4 (In-^rgTN
F-a DNA) 0. 6ul, EcoRI
and pKK2 pre-digested with Hind4.
23-3 (manufactured by Pharmacia) 0. +7#, 50j IM Tris-HCl buffer group (pH 8.0), 10. In addition to the mixture consisting of M magnesium chloride, 10zMDTT, IxMATP, 400 units of T4 DNA ligase was added,
The reaction was carried out at 16°C for 18 hours.

v11. Preparation of transformants Using the reaction solution of vi, the method of Hanahan (J. Mol. Biol. 166, 557 (+9
83)) to transform E. coli strain E392,
A vector (hereinafter referred to as phio-^rgT) in which the gene of the present invention is linked downstream of the tac promoter of pKK223-3
It is called NF-a. ) transformant L E 3 9
2 (phi(1-ArgTNF-a)) was obtained.

Suke. Peptide production transformant LE392 (phlo-Arg-NF-σ) was grown in M9 medium (0.2% glucose, 0.1%
%casamino acids, 51. (contains li/td thiamine)
The cells were cultured at 37°C for 5 hours, and the cells were collected from the culture solution by centrifugation, and the collected cells were added to a lysis buffer (62
．． 5xM} Lith-HCl (p46.8), 3% SDS, 5%
2-mercaptoethanol, containing 10% glycerol) and! After heating at 00°C for 3 minutes, the total protein of E. coli obtained was analyzed using the method of Laemmi (CNature, 22 U., 680 (1970)). As a result, 10% of the total protein was expressed by the formula A, where A is Ar.
The peptide was g.

ix. Purification of peptide transformant LE392 (phlo-ArgT
NF-a) was pre-cultured overnight in M9 medium containing ambicillin (50A6I/d), further cultured with shaking in M9 medium 3Q at 37°C for 15 hours, and this culture was incubated at 10,000 rpm for 4 hours. ℃, collect bacteria by centrifugation for 10 minutes,
The mixture was suspended in 50 μM Tris-HCl buffer (pH 8.0).

The suspension was crushed using an ultrasonic crusher at 200 W for 20 minutes under cooling, and then crushed at 4°C and 2
The mixture was centrifuged for 0 minutes to obtain a supernatant.

Polyethyleneimine was added to this supernatant to give a concentration of 0.0125%, and the mixture was centrifuged at IQ, 000 rpm for 20 minutes at 4°C to obtain a supernatant.

Ammonium sulfate was added to the above iN to achieve 35% saturation, and the mixture was centrifuged at 10,000 rpm for 10 minutes at 4° C. to obtain a supernatant.

Further, ammonium sulfate was added to the supernatant to achieve 45% saturation, and the mixture was centrifuged at 10.00 rpm at 4° C. for 10 minutes to obtain a precipitate.

This precipitate was dissolved in IOxM Tris-HCl buffer 'K1 (pt
i7.0), filtered with 20 RVI sodium chloride solution,
The dialysate fluid was transferred to M. Anion exchange column chromatography was performed using an onoQ force column (manufactured by Farmana).
A crude bebudide active fraction was obtained.

This precursor peptide active fraction was concentrated using Centribrep (manufactured by Amicon), and the concentrate was purified using TSK-gel Ph.
The purified TNF active fraction was subjected to hydrophobic chromatography using an enyl-5PII column (manufactured by Toyo Soda Co., Ltd.) and dialyzed against PBS (pH 7.4). In formula A, A1 is concentrated.
A peptide in which is Arg was obtained.

Example 3 As a brimer ■ 5' 1-choGCCCGAGCACTGTTTTGCTG
A peptide in which A in formula A was deleted was obtained in the same manner as in Example 2, except that an oligodeoxynucleotide (M1 primer) having the base sequence of ACT was used.

Example 4 As a brimer■5°-CCGTGGTACCATCCGATCTAT
A bebutide in which A4 is old S in formula A was obtained in exactly the same manner as in Example 2, except that an oligodeoxynucleotide (M3 primer) having the base sequence CTCGGTGGT was used.

Example 5 As a brimer ■5'-AATCCCCGTGCCGCGAAAACC
Oligodeoxynucleotide having a base sequence of CC (
A bebutide in which A3 in formula A was deleted was obtained in exactly the same manner as in Example 2 except for using M4 primer).

By integrating the human TNF-α gene of the present invention into bacterial cells by a commonly used gene recombination method, human
can be expressed. This will be explained using a reference example.

Reference Example: Preparation of expression vector Human TNF'-α gene of the present invention, EcoR
1 and old ndlI [pK
K223-3 (manufactured by Pharmacia) 0.1 A6I, 50 units of Tris-HCl buffer (pl{8.0), 10 units of magnesium chloride, 1 unit of IO DTTS and 1 unit of ATP, and in addition, 400 units of T4 DNA ligase. In addition, the mixture was reacted at 16°C for 18 hours.

ii. Creation of recombinant This reaction solution was used according to the Hanahan method (J. Mol. Bio
l. 166, 557 (1983)), the gene of the present invention was transformed into pKK2.
A transformant LE392 (phTNF-a) having a vector (hereinafter referred to as phTNF-a) linked downstream of the tac promoter of 23-3 was obtained.

iii. Production of peptides The transformant L E 39 2 (phTNF-a) was grown at 37°C in M9 medium (containing 0.2% glucose, 0.1% casamino acids, 5/-thiamine). , 5
After culturing for an hour, the cells were collected from the culture solution by centrifugation, and the collected cells were added to a lysis buffer (6.2.5xM Tris-HCl (pH 6.8), 3% SDS, 5% 2-merkabutylene). The total protein of E. coli obtained was suspended in E. coli (containing ethanol, 10% glycerol) and heated at 100°C for 3 minutes using the method of Laemmli (Nat
ure, 227, 680 (1970)), 15% of the total protein was human TNF-α.

iv. Purified transformant L E392 (phTNF-a) of phTNF-a was cultured in 3 liters of M9 medium (containing 0.2% glucose, 0.1% casamino acids, 5/monothiamine) at 37% °C,
After culturing for 5 hours, bacterial cells were collected from the culture solution by centrifugation at 8.00 rpm for 5 minutes.
aM} After suspending in Liss-HCl buffer (pl{8.0),
The bacterial cells were destroyed by ultrasonic disruption for 10 minutes. This is 8,0
00 rpm for 15 minutes to obtain a supernatant,
A crude extract was obtained. The final concentration of this crude extract was 0.
8. Add 2% polyethyleneimine to precipitate and remove DNA%RNA1 lysosomes. 0 0 Orp
The mixture was centrifuged for 15 minutes to obtain a supernatant. The precipitate obtained by adding 40% saturated ammonium sulfate to this supernatant was removed by centrifugation at 8.0 O O rpm for 15 minutes.
．． Collected by centrifugation at 00 Orpm for 10 minutes and diluted with 10 JAM Tris-HCl buffer (pH 7.0).
) 101 l (J!!!) and dialyzed against lO Tris-HCl buffer (pH 7.0). After dialysis, anion exchange force chromatography using DEAE Sepharose CL-6B was performed to obtain a concentration of 0 to 500 HCl. Human TNF-α was purified by elution with sodium.

Claims (3)

[Claims] (1) The following base sequence [There is a gene sequence] Human TNF-α gene represented by (2) The following base sequence [gene sequence is included] (However, R_1 indicates GAC or CGC, and R_2
indicates CTG or GCG, R_3 indicates CAC or deletion, R_4 indicates GTG or deletion, R_5 indicates CAA or deletion, R_6 indicates GTT or deletion, R_7 indicates GAA or CAT. , R_8 is C
Indicates TT or GTA. ) A gene encoding a peptide represented by (3) Formula (A) below [There is a gene sequence] (However, A_1 indicates Asp or Arg, and A_2
indicates His or deletion, A_3 indicates Gln or deletion, and A_4 indicates Glu or His. ) Peptide represented by.