JPH0242986A - Dna coding novel polypeptide having physiological activity to human - Google Patents

Abstract

PURPOSE:To produce a polypeptide having human tumor necrosis activity by integrating a specific DNA coding a human tumor necrosis factor(TNF) into a replicative expression vector and transforming a host microorganism with the vector. CONSTITUTION:The structure of the human TNF of formula has been determined by the following findings. (1) A human polypeptide gene and a rabbit TNF gene can be cloned by using a rabbit cDNA as a probe. (2) A DNA coding human polypeptide can be skillfully separated and its structure can be determined by comparing the homology of the base sequence with that of rabbit TNF gene, human polypeptide gene and rabbit cDNA. (3) The structure of pure DNA coding human polypeptide can be also skillfully determined and the DNA can be obtained in pure state. (4) A human polypeptide produced by using a DNA coding a human polypeptide has cytotoxic activity against L-cell, etc.

Description

[Detailed description of the invention]

(Industrial Application Field 1 The present invention relates to a polydeoxyribonucleic acid (hereinafter abbreviated as DNA) having genetic information of a novel physiologically active polypeptide.
and a microorganism or cell transformed with said replicable set 144 DNA. More specifically, the present invention relates to human-derived tumor necrosis factor (TuIler Necros).
I) NΔ encoding isFactor) (hereinafter referred to as human TNF), a replicable recombinant DNA containing this DNA
Δ and microorganisms or cells transformed with the replicable recombinant DN8. In the present specification, amino acids and peptides are indicated by the abbreviations adopted by the IRB Biochemical Nomenclature Committee (IUr'-10BCI3N), and for example, the following abbreviations are used. In addition, when an optical isomer is possible for an amino acid or the like, the L-isomer is indicated unless otherwise specified. Gin: Glutamine residue ^S

[): Aspartic acid residue Pro Nibroline residue,! Tyr: Tyrosine residue Jlli Val: Valine residue S: Lysine residue Glu: Glutamic acid residue Ala: Alanine residue S11: Asparagine residue Leu: Leucine residue Phe: Phenylalanine residue cty Nigericine residue 11is: Histidine Residue Ser: Serine residue TI+r: Threonine residue 11e: Isoleucine residue Trp Nitributophane residue Δrg: Arginine Met: Methionine residue Cys Nidistine residue Also, polydeoxyribonucleotides and oligonucleotides have the following abbreviations. It is expressed by the deoxyribonucleotide sequence represented by 82 2'-deoxyadenylic acid residue C: 2°-deoxycytidylic acid residue G 2'-deoxyguanylic acid residue "Thymidylic acid residue Unless otherwise specified, the left end of the deoxyribonucleotide sequence is 5 [Problems to be solved by the prior art and the invention] Various substances that have a reticuloendothelial system activating effect, such as various Durum-positive bacteria and endotoxins, induce physiological effects such as antitumor cell ability. The existence of many active substances has been reported. For example, Carswell et al.
Calmette Guerin (Bacillus Calmet)
te Guerin (BCG)), and that the serum obtained from the mice by intravenous injection of endotoxin two weeks later has a cytocidal effect on cultured cells, and that the serum of the mice (f3ALIll/c (
:57B/6) F, discovered a phenomenon that caused hemorrhagic necrosis in female A sarcoma (Metl + Asarcol + a), and investigated tumor necrosis factor (TulIIo).
r Necrosis Factor) (hereinafter referred to as l”
(referred to as NF) [brok, nato. Acad, Sai, United States (Proc, Nat, A
cad. Sci, USA) 720 (ffi 9) 366
6-3670 (1975)), then Ru
``[]mo
uno1. ) Volume 125 (44) pp. 1671-1677 (1980) J and Matthews (MaLLhews
) et al. [lr. J. Cancer, Vol. 42, pp. 416-4122 (19
1980)) is the entry of Nii-ki-Swell arsw61
1) We attempted to purify TNF from rabbit serum prepared according to the method of et al., and obtained products that were about 2000 times and about 100 times more purified than the original serum, respectively. However, in any case, the antitumor effect of the purified product has not been confirmed in animal experiments. JP-A No. 57-140725 discloses that one or more substances having a reticuloendothelial system activation effect are administered to mammals (mouses, rabbits, guinea pigs, etc.), and then endotoxin derived from Gram-negative bacteria is injected. Discloses a method for isolating and purifying a proteinaceous physiologically active substance having an anticancer effect induced by endotoxin derived from Durham-negative bacteria or by adding endotoxin derived from Durham-negative bacteria to a tissue culture system containing activated macrophages derived from mammals. . Furthermore, the molecular weight of the substance is 39.000±5.000 by gel filtration method and 5DS-polyacrylamide gel electrophoresis method, and the isoelectric point is pl.
+3.9±0.3 (isoelectric focusing method), but its detailed structure is not shown. On the other hand, MaLthews [Brj, Cancer, Volume 44 (3)
[pp. 418-424 (1981)] obtained mononuclear phagocytes from various tissues of rabbits that had been injected with BCG for two weeks, and added endotoxin to the cell culture solution to detect cytotoxic substances. This shows that it is possible to obtain However, the detailed structure of this substance has not been shown,
Furthermore, there is no evidence that it is the same substance as T and NF found in serum. Furthermore, there are many reports on factors that have TNF-like biological activity, i.e., biological activity similar to that of TNF. For example, Reed et al. Found [J, Immunology, 115@39
5 (1975)]. Matthews
) found such factors in leukemic cells derived from human peripheral blood monocytes or from patients with myelomonocytic leukemia [
Matthews et al., Immunology 441J135 (1981)]. Also, Barbara (narl+ara)
) et al. Nibstein-Barr Wills (EpsLei
n-barr virus) [D., Barbara et al.
eLal) +Brock, Nato, Acad 0g, United States (Proc, NaL, ^cad, Sci, U
SA) Vol. 80, p. 5397 (1983)], and Bharat et al. found a physiologically active substance like ``l'NF'' in the lymphoblastoid 1788 cell line. The above-mentioned factors are also included in JP-A-58-15921.5.
8-21621.58-107197.58-2250
No. 24, UK Special Application Publication Nos. 2.106.117 and 2,117,385. However, cells or cell lines that efficiently produce such factors have not been found, and furthermore, little knowledge has been obtained regarding the structures and physical properties of these factors. Ito studied the structure and properties of rabbit TNF, and Ugi TN.
Conducted research on F-producing cells [Patent application 1982-25]
No. 1817], as a result, by administering a substance having a reticuloendothelial system activation effect to rabbits and then injecting bacterial endotoxin, cells capable of producing a substance having Lf14a cytotoxic activity were obtained; The material was obtained using L cells. Ito further determined the molecular weight and immunological properties of the substance with cytotoxic activity obtained using the cells obtained as described above and determined the molecular weight and immunological properties of the substance obtained from rabbit serum.
I confirmed that it matches the one. On the other hand, advances in genetic engineering technology have made it possible to determine the structure of a certain protein if DNA encoding it can be isolated. This is because the structure of isolated DNA can be determined and then the structure of the protein can be derived from the structure of the DNA. Furthermore, it has become possible to produce proteins from DNA encoding them using microorganisms or cultured cells. Ito applied the above genetic engineering technology to cells capable of producing a substance with L cell-toxic activity, and as a result, rabbits'
Ito isolated the DNA encoding l'NF, determined the structure of this DNA and rabbit TNF, and succeeded in producing rabbit TNF using the DNA. As mentioned above, Ito isolated the rabbit i'NF. Although they have had great success in producing it, anaphylactic shock can occur when TNF is administered to animals different from the animal from which it was derived, since 'l' NF is a polypeptide. This is because when 1'NF is administered to an animal that is not capable of producing TNF, TNF acts as an antigen and causes the production of antibodies.For this reason, 1'
N I? If one intends to administer TNF to humans, the use of human-made TNF is highly preferred; however,
The structural shift of human rNF has not been fully elucidated. Therefore, it has been strongly desired to determine the structure of the DNA encoding human TNF.

[Means and effects for resolving the problem 1 The inventor
et al. have studied the path of the DNA encoding human TNF.
conducted research. As a result, surprisingly, human polypeptide
The gene and rabbit TNF gene were cloned into rabbit cDNA.
Can be cloned by using it as a robe.
DNA encoding human polypeptides
can be isolated and whose structure is related to nucleotide sequence homology.
Rabbit TNF gene, human polypeptide gene and
that can be determined by comparing rabbit cDNA;
11ζ construction of pure NA encoding human polypeptides
can also be determined skillfully, and its pure NA can be obtained.
and encode human polypeptides.
Cell damage caused by human polypeptides obtained using DNA
The present inventors discovered that it has activity. The present invention was completed based on these new findings.6 That is, the object of the present invention is to
The aim is to provide the DNA that It is also an object of the present invention to encode human 1' NF
A replicable vector consisting of DNA and a replicable expression vector.
Our goal is to provide recombinant DNA. Furthermore, the object of the present invention is to use recombinant DNA as described above.
In providing transformed microorganisms or cells to
Ru. The foregoing and other objects, features and advantages of the present invention
The benefits can be found from the detailed description below with reference to the attached drawings.
It will become clear. In terms of wood quality, according to the present invention, the following formula (1)% formula% (wherein, Gln is a glutamine residue, and Δsp is an asparagine residue)
Pro is a proline residue, Tyr is a tyrosine residue
group, Val is a valine residue, Lys is a lysine residue, Gl+
is a glutamic acid residue, ^la is an alanine residue, ^sn is a
Asparagine residue, Leu is leucine residue, Pl+e is
Phenylalanine residue, Gzy is glycine residue, l1i
s is histidine residue, Ser is serine residue, TI+r is
Threonine residue, lie is isoleucine residue, Trp is
tryptophan residue, Arg is arginine residue, Met
represents a methionine residue and Cys represents a cysteine residue
) Human physiologically active polypeptide containing the amino acid sequence represented by
A deoxyribocontaining nucleotide sequence that encodes a peptide.
A nucleic acid is provided. Furthermore, according to the present invention, a salt represented by the following formula (II)
Base sequence TCA TCT TCT CGA ACCCC
G AGT GACAAG (:(:T GT
AGCCCAT GTT GTA GCA A
ACC(:T CAA GCT GAG GG
GGAG CT(: (:AG TGG CT
G AAG CGC (:GG GCCAAT G
CCCTCGTG GCCAAT GGCGTG GA
G CTG AGA GAT AACGAG CTG
GTG GTG (:(:A TCA G
AG GGCCTG TACCTCATCTACT
CCCACGTCCTC: TTCAAG GGC
CAA GG(:TG(: (:(:CTC(:
ACCCAT GTG CTCCTCAC:CC
ACACCATCAGC(:G(1: ATCGCCG
TCTC(: TACGAG ACC: AAGGT(
: AACCTCCT(: TCT GCCATCAA
G AGCCCCTG (:CAG AGG GAG
ACCCCA GAG GGG GCT
GAG GGC^^GCCCTGG TAT G
AG CCCATC Ding ATC Ding G GGA GG
G GTCTT (: CAG CTG GAG^AG
GGT GAC: CGA CTCAGCGCTGA
G ATCAAT (:GG CCCGACTA
T (:TCGACTTT GCCGAG TCT
GGG CAG GTCTACTTT GGG AT
CATT GC (1:CTC (where Δ is a deoxyadenylic acid residue, G is a deoxyglyceride residue,
anilic acid residue, C is deoxycytidylic acid residue and
represents a thymidylic acid residue, and the left and right ends of formula (u) are
Represents the 5′-hydroxyl group side and the 3′-hydroxyl group side, respectively.
) and a nucleotide sequence complementary to the nucleotide sequence.
a deoxyritic acid containing at least one base sequence that is
Bonucleic acids are provided. The DNA of the present invention can be matured into a human by microorganisms or cultured cells.
In order to produce TNF, the 5' end of base sequence 011
ATG (A, T, and G are the serial number months described in niJ are combined)
It includes DNA consisting of the base sequence. DN of the present invention
A also represents a portion of the signal peptide of human 1'NF or
contains the 5'-flanking DNA encoding the entire D
Also includes NA. Due to natural or artificial mutations, the main activity
tM structure of DNA and then
It is possible to mutate part of the structure of the polypeptide to be played.
is possible. Therefore, the DNA of the present invention is as described in n;f.
Has a structure that corresponds to a homologous variant of all polypeptides.
Contains a base sequence that encodes a polypeptide that
is also possible. Polypeps produced from genes according to the degeneracy of the genetic code
base of the gene without changing the amino acid sequence of
Replace at least one base in the sequence with another type of base
can be done. Therefore, the DNA of the present invention also includes genetic
The base sequence changed by substitution based on the degeneracy of the code.
It is also possible to contain. In this case, the above substitution will result in
The amino acid sequence derived from the obtained base sequence is n;1
It matches the amino acid sequence of formula (1) defined in . Furthermore, according to the present invention, nf deoxyribonucleic acid and
A replicable set jAD consisting of a replicable expression vector
NA is provided. The recombinant DNA. in microorganisms or cells transformed thereby.
A polypeptide containing the amino acid sequence of l-'l'N['
can express peptides. A suitable vector is
, e.g. pHTNF-1acUV 5-1 and pH'
rNF~lac UV 5-2 fQ QL vector
Can be mentioned. Furthermore, the present invention also provides. Contains the amino acid sequence of human 'I''NF.
tsn capable recombinant DN capable of expressing a polypeptide with
It pertains to microorganisms or cells transformed with A. like this
Examples of microorganisms or cells include Escherichia coli, Bacillus subtilis, yeast 1u,
Examples include higher animal cells. DNA encoding human TNF was obtained as follows.
Ru. ■, Bacte 1 Phage λ/Rabbit Chromosome Gene Live
Larry and bacteriophage/human chromosome gene library
Braley is from the Department of Chemistry and Molecular Biology at the University of Barbados.
[Ken, Massachusetts 02138, USA
Bridge, Divinity Avenue 7, (7Div
inity Avenue, Cambridge,
Massachusetts 02138. U, S, Δ
)] Tea, Maniatis (TlManiaLiS)
Use the one prepared by Professor. These libraries
Lee can be made by the following method. 〔cell(
Cell), p. 151゜687 (1978)
] (1) Rabbit or human tissue, such as rabbit
The human pancreas is frozen into powder and the RNA and protein components are extracted.
Rabbit or human polymers are produced by decomposition treatment and precipitation.
Get DNA. (2) This polymeric DNA randomly cuts gene loci
Partially disassembled for this purpose. (3) ft) from the resulting DNA fragments by molecular weight fractionation.
Then, a fragment of 15 or 20 kilobase pairs (Kb) in size is
obtain. (4) ] The DNA fragment obtained in Step 3 is λ Sharon
Clone using the 4Δ phage vector. (5) Transfer the obtained vector to an infectious vector containing rDN8.
in vitro into the rabbit particles described above.
Obtain a human chromosomal gene library. 2. The rabbit rNF cDNAΔ obtained in Reference Example 3 was
Nick translation by P. W., J. Rigby, et al.
method [J9mol, Paiol, (J, Mo1.
Biol, ), p. 1139.237 (1977).
3. Bacteriophage/Rabbit Chromosome Gene Live
Larry and bacteriophage λ/human chromosome gene library
Place each brary tightly over an even layer of bacteria.
Plant them so that they form plaques and label them with '''P.
Scribed by hybridizing with rabbit TNF cDNA.
-ning. 4. Isolate the corresponding DNA from the compatible clone,
Create a restriction enzyme map and use Southern
Ibridization method [EoM.
, Mo1゜11io1. ), vol. 98, p. 503 (1
975)]. A control system containing the rabbit 1'NF and human 'I' NF genes.
Introducing the limited enzyme-digested fragment into a plasmid vector
After subcloning, the base sequence is analyzed. 5. Rabbit “l’NF cDNA and rabbit TNF gene
By comparing the nucleotide sequences of
(Base sequence encoding the amino acid sequence of rabbit TNF)
and an intron (encoding the amino acid sequence of rabbit TNF)
Determine the nucleotide sequence (no nucleotide sequence). 6. And the rabbit TNF gene and human TNF gene
By comparing the exons and infusions of the human “rNF gene”
Decide on Tron. 7. Delete intron and exon of rabbit TNF gene
determined from the base sequence obtained by combining
The amino acid sequence of rabbit TNF is the c of rabbit 1” NF.
Matches the amino acid sequence determined by the base sequence to DN.
It is confirmed that 8. Next, delete the intron of the human TNF gene and create
Based on the base sequence obtained by combining the
The amino acid sequence of TNF has been determined. human]'NF's
The amino acid sequence is rabbit TN F. It is recognized that #l partially matches the amino acid sequence of
. The human 'r N F thus obtained encodes
Manufacture human 'rNF from DNA as follows.
can do. 1. Introducing DNA encoding human I”NF in vttr
. and introduce the DNA into an appropriate expression vector.
0 Recombinant DNA is placed in a suitable host.
The desired human TN
Express F. 2. The human 'rNF obtained in this way is the mature form.
There are 155 amino acid residues starting with serine. It has a signal peptide in the presequence
If the signal peptide has a very hydrophobic character
. The above describes the human TNF gene and the code for human TNF.
Method for obtaining DNA and human TNF using the DNA
This shows the manufacturing method. However, the above description is not intended to limit the present invention;
Those skilled in the art without changing the basic characteristics and basic concept of the invention.
Obvious transformations can be made by a person. The frequency of use of codons (genetic code) corresponding to each amino acid is
Without changing the amino acid sequence due to differences etc.
A part of the base sequence of the DNA encoding human TNF or
All replaced with artificial DNA synthesized organically.
It is also possible to Presumably, human TNF is a prepeptide or prepropepeptide.
Produced intracellularly in immature form as a
It is processed in the processing stage and passes through intermediates to produce mature TNF'.
It is thought that it forms. The immature form of human I'NF is the salt of the human 'NF gene.
Entaku is performed from the basic arrangement. Coating immature or intermediate TNF
TNF DNA, which consists of DNA that encodes
Alternatively, it can be recombined with artificially synthesized DNA. One of the applications of these methods is the methionine codon (
ATG) as a mature or immature or intermediate TNF gene.
introduced into the 5' end of the gene, and 'rAΔ or
is called the stop codon of 'r A G or TGA
The purpose is to introduce at least one triplet. The presence of the methionine codon allows for suitable promotion.
Mature or immature +mRNA synthesized by
Mature or intermediate TNF is produced. Like this TN
The methionine residue added to the end of F is
are removed naturally. The purpose of inserting a stop codon is 'I
'N I = from mRNA Δ transcribed from DNA
translation at an appropriate position [C-terminus of the polypeptide of formula (1)]
] is to stop. Another form is called a ``signal sequence.''
By adding a highly hydrophobic sequence, the host cell
outside or in Durham-negative bacteria. secretion into a region called the ``periplasm''
is also possible. In addition, if the vector has an integrated start codon,
A fusion peptide of a peptide derived from a vector and TNF
form, but in this case chemical or enzymatic cleavage is required.
or if there is no change in the main activity of TNF,
It can be used as is. The human i' NF gene is transcribed correctly and thereby
The translation from +oflNA obtained by
In the sequence, the gene in the 5' region such as the promoter
The resulting TNF DNA-promoter
- vectors that can replicate sequences in bacteria or higher organism cells
- Obtain a recombinant gene connected to
Bacteria or higher organism cells are transformed as hosts, and this
The transformants were grown to express the i'NF gene.
By tightening! 'NF can be obtained. Escherichia coli [Escl+e] is used as a host.
-ricl+ia coli)] (hereinafter referred to as ``E. coli''
'') is preferably used, it is preferable to
Various mutant strains of two strains, such as HB I 01 (ATC
C33694), C600K (8r[;[;33055
), I) I 2 1 0, 1 or RI (A-cho
CC31343), MC1061, LE392
(8 TCC33572), J M I Ol (
ATC (: 33876), JM83, χI 776
(ATCC31244) etc. are used. When using E. coli as a host, the vector is 1 mouth R3
22, p+31?325, p[lR327, pUc8
, r+U(:9, pMB9(ATC[:3701
9), pJI! 8 (ATCC37074), pK
Plasmids such as C7 (AT(I; 37084) or
is λgt. λB, λ phages like Sharon 4A, MI3 phages
etc. are used. The physiologically active polypeptide is produced in the bacterial cells of the large intestine α1.
In order to
lomotor is used. As a promoter like this
Preferably, a promoter of lactose degrading enzyme (LΔC) is used.
ter and its UV5 mutation, penicillinase ([3LA)
, l-ributophane synthase (TRI) promoter
-, PL promoter or tryptophage of λ phage
Fusion promoter of AAN synthase and lactose degrading enzyme
The Lac promoter and the like are used. Bacillus subtilis [Bacillus subtilis (3a (illus 5)
ubtilis)] (hereinafter referred to as Bacillus subtilis)
strain BD170 (ATCC3) as a host.
3608), BR151 strain (ATCG 33677),
MI112 strain (ATCC337+2) etc. are used,
The vector is pcI04(AT(1:C37034
), pUBIIo (ATCC37015), pS
^2100 (ATC:G37014), 194 etc. on p
plasmids are used. When using Bacillus subtilis as a host, the promoter
Ramphenicol acetyltransferase ((:AT) and
Promoter of genes such as linase and erythromycin resistance
A tar is used. When using yeast as a host, Satucharomyces cerevisi
Saccharomyces cerevisea
e) rll+218 strain (ATCC44076), 5
IIY 1 share (ATC (: 44769), 5IIY
3 strains (^1'CG 44771), DI31A strain, 48
3 strains and 830 strains, etc., are used as vectors.
YIEp l 3 (ATCC37+15), YEp6
, YRp 7, Ylp 5, etc. are used.
. When using a 1 year old yeast as a host, an acidic phosphatide is used as a promoter.
Sphatase, alcohol dehydrogenase (ΔDl-1
1), 1-Liptophan synthesis enzyme fA (TRP), phospho
glycerate kinase (PGK), cytochrome B (
COB), actin, and other gene promoters are used.
It will be done. When using cultured cells of higher organisms as hosts, monkey kidney cells,
CO3 cells, mouse C127 cells (C to AT: +616
) etc. are used, and the vector is 5v40 Wii.
Rus et al. can be used. The molecular weight of the novel bioactive polypeptide obtained by the present invention is
Laemmli (Laen+m1i) method [Laemmli, Nike
, (Laemmli, O.,) (1970) Nature
- (Nature) (London) Volume 227, Page 680~
SDS polyacrylamide electrophoresis according to page 685]
When measured with (SDS r'AGE), it is approximately 17,000.
In this case, the marker used is phosphorylase.
b (molecular weight 94K), bovine serum albumin (molecular ff1
67K), ovalbumin (molecular weight 43K), carbonyl
anhydrase (molecular weight 30K), soybean trypsin
Inhibitor (molecular weight 20, IK) and α-lactalb
Min (molecule ji 14.4K) is used. Novel physiologically active polypeptide obtained from the DNA of the present invention
causes necrosis of the tumor without damaging the normal tissue of the body
. The active polypeptides of the invention are formulated by known methods.
to inhibit cell proliferation, such as malignant tumor cell proliferation.
6 Activities of the Invention That Can Prepare Effective Pharmaceutical Compositions
The polypeptide is mixed with a pharmaceutically acceptable carrier.
It is also possible to administer an effective amount of the active polypeptide of the present invention.
In order to effectively administer it to patients by mixing it with the appropriate amount of 71
Suitable pharmaceutical compositions can be prepared. The physiologically active polypeptide obtained from the DNA of the present invention is
Injectable for patients in need of anti-tumor or anti-viral treatment
, eye drops, nasal drops, inhalants, external preparations, drugs for oral administration,
It can be administered as a rectal or intravaginal drug.
can. The daily therapeutic dose for an adult of the polypeptide of the present invention is generally
50 units to too, 000.000 units, and
More preferably 50 to 500.00 for topical application.
For systemic injections such as 0 units, intravenous injection, and intramuscular injection,
, 1,000 to 1,000,000 units for oral administration.
10.000~too, 000.000 units
The dosage may be increased or decreased as appropriate depending on the usage or symptoms.
can. (Left below) The "l unit" used above is the LM cell (Δ1'C
:CCCL 1.2) l X I O'fli2/m
means the amount of the bioactive substance of the present invention that kills 50% of Q.
. The above-mentioned quantities are measured as follows. As a culture container
9G hole tissue culture microplate (Flow Lab
L-M cells were incubated at 1 v/v% with
Eagle's Minimum Essentials with Fetal Serum
Medium (its composition is, for example, "Tissue Culture" Chugai Qjl
Edited by Nosukeike, Breakfast Shoten, 1967)
Cultivate inside. The bioactive polypeptide was serially diluted in culture medium.
0.1 mQ of Tido sample and L- of concentration of lO@pcs/mQ
Add 0.1 mA of medium suspension of M cells into each formula of the plate.
Mix and place the microplate in air containing 5% carbon dioxide.
Incubate at 37°C for 48 hours. After culturing, add 20%
Fix the cells by adding 20 μC of rutaraldehyde aqueous solution.
. After fixation, wash the microplate with distilled water and dry it.
, add O.1mQ of 0.05% methylene blue solution,
Stain the remaining cells. Wash away excess methylene blue
After draining and drying, remove the remaining methylene blue with 0.36N salt.
Extract with acid solution and tie its absorbance at 665 nm.
Tartic Multiscan (Flow Laboratory)
, USA). This absorbance is determined by the number of surviving cells.
is proportional to. l of the above-mentioned physiologically active polypeptide of the present invention.
The amount that kills 50% of ×10° L-M cells is determined by the dilution rate and absorption rate.
Find the luminosity by plotting it on a graph
Can be done. The physiologically active polypeptide obtained from the DNA of the present invention is
Can be applied parenterally. Preparations for parenteral administration
For this purpose, albumin, gelatin, globulin, protein
Stabilizers such as rotamine and protamine salts, sucrose, and
serine, methylcellulose, carboxymethylcellulose
Addition of thickeners such as thickeners, PL+adjusters of various inorganic salts, etc.
It can be added as an agent. Moreover, the physiological activity of the present invention
Polypeptides can also be administered in tablet form. Examples of additives used in the preparation of tablets include the above-mentioned additives.
In addition to stabilizers, excipients such as starch and milk
can be given. Specifically, as a result of animal experiments, the majority of tumors are found in mice.
, can be completely cured with two injections. For example, mouse skin
An artificial tumor (female A cells) was implanted into the tumor to a diameter of 6 to 7 mm.
At the stage when the novel bioactive polypeptide of the present invention becomes
After injecting 0.6 micrograms, scabs occur after - weeks.
After two weeks, the hair started to grow again and the hair was completely healed.
After that, pregnancy and childbirth. The present invention will be described in detail by reference examples and examples below.
However, the present invention is not limited thereto. In carrying out the present invention, the production of recombinant DNA, microorganisms of recombinant
For introduction into living organisms, unless otherwise specified, use the experimental book (1) below.
- (4). (1) Gene manipulation manual, edited by Yasutaka Takagi, Kodansha (2) Gene manipulation experimental method, edited by Yasutaka Takagi, Kodansha (3)
Tee, Maniatis (70M6niatis), E
F, Fr1tscb: L (E, F, Fr1tscb
), J, Sa*brook
, Molecular Cloning (Molecular C
loning), Cold Spring Harbor Laboratories
Lee (Cold Spring 1larborLab
(1982 USA) (4) Lei Wu
(Ray Wu) et al., Method in Enzymology (
MeLl+od in lEnzymology), 1
010, Academic Press (^ academic press)
'ress) (U.S.) Used in Examples and Examples
Abbreviation MOPS: Molyfluorinobrobanesulfonic acid L8
Medium: Luria-Bertani medium DMSO Nidimethyl sulfoxide PFU ・Plaque forming unit El) T8
: Ethylenediaminetetraacetic acid 5t) S, sulfur dodecyl sulfate, lithium +11? L
: Bethesda Research Laboratory, Inc., DMF, USA Nidimethylformamide 1ilac : Lactotris (Tris) ・L・Lis (Hydroxymethyl
) Aminomethane XAR-5+ X-ray film (Eastman Kodak
Inc., USA) I X5SC: 0.15M Sodium Chloride + 0.0I
5M Sodium Citrate, p117 2 X5SC: 0.30M Sodium Chloride + 0.03
0M sodium citrate, p1173X5SC: : 0.45M sodium citrate
+0.045 Sodium enoate, pl (7 5X5S(: : 0.75M Sodium chloride +0.
075M Sodium Citrate, p117 6 x SSC: 0.90M Sodium Chloride + 0.09
0M Sodium Citrate, p117 FDSS: 50% deionized formamide+
5 x DenhardL's + 5
X 5SPE+
m Q-denatured bovine thymus DNA 5SPE: 0.18M NaCl') Lamb +
l O+mMIJ sodium dihydrogen phosphate+1 mM E
DT^, p117.4 SM: l 1) i'l: Sodium chloride 5
．． 8g, magnesium sulfate heptahydrate 2g, 1M Tris-CQ (pH 7.5) 50m
NZ amine (casein type Δ
Hydrolyzate, Humco Chef Yield Chemical Division of Craft, USA) log, medium containing 5 g of sodium chloride and 28% magnesium sulfate heptahydrate 1r'TG: Isoprobyl thiogalactoside X-
gal: 5-bromo-4-chloro-3-indolylgala
Toside TAE: 0.04M Tris-
Acetic acid (pl+8,0) - 0,002M IEDT^ 5x Denhardt's solution: Ficoll in lθ
l) 1000 mg, polyvinylpyrrolidone 1000 mg, and bovine serum albumin 1000 mg.
Aqueous solution containing bp: base pairs [Example 1 Reference Example IL Cytotoxic Activity Evaluation Activity 3 using human cells in the following Reference Examples and Examples
Monovalents are Rur (etc.) [Lymphokines (Lym
pbokines) 2 uE, big ([shi, Pi
ck) Edited by Akademi Tsuku Breath (Hachicademi
c I'ress) p. 235 (1980)) or
[Jiei, Imtsuru. (J, Immunol,) Vol. 126, p. 1279 (19
1981)] method of i! I. Biologically active substances according to the present invention
Measurement of the effect of quality on killing L929 cells (ATCCCCCL1)
It shall be determined. i.e., samples diluted in sequential media
Culture of cells with a concentration of 0.1mα and 10′ cells/mQ.
Earth! Turbid liquid 0. l mα with a 96-hole tissue culture microphone.
Add to Roplate (Flow Laboratory, USA). The medium contained lv/v% fetal bovine serum and a final concentration of 5 μg.
Eagle's minimum containing actinomycin D of /mQ
・Essential medium (the composition of which is e.g.
"Cultivation" edited by Nosuke Middle Ear 1iff, et al., Shokusho Shoten, 1967.
(described in ). 5% microplate
Culture in air containing carbon dioxide gas at 37°C for 21 hours. After culturing, add 20 I of 20% glutaraldehyde aqueous solution.
Add tQ to fix cells. After fixation, microplate
Wash, dry and add 0.05% methylene blue solution.
Add 0.1 m(L) and 6 extra to stain the surviving cells.
After washing away the methylene blue and drying, remove the remaining methylene blue.
The 6fb was extracted with 0.36N hydrochloric acid solution, and the 6fb was extracted with Titertic Multiscan (Flow Laboratories).
Ill'l determined by Su~sha, USA). This absorbance is
proportional to the number of remaining cells. 50% of L929 cells
The amount of physiological activity required to kill the is defined as I unit/mQ.
, corresponds to a value of 50% of the absorbance of the control without sample
Determine the dilution rate of the sample using a graph or 4 calculations, and then
The reciprocal of the dilution rate is the amount of biological activity of the sample (expressed in units/mα)
). “1 unit’° used in the reference example is
Rabbit T kills 50% of L929 cells at 10' cells/mα
NF means the amount of F. On the other hand, the protein amount was determined by Bradford
[Anal, Biochem, (Anal,
Bi. cbem, ) Vol. 72, pp. 248-254 (1976)
Coomassie brilliant blue 6250 by j
Calculated from the dye binding method using Reference Example 2 Step 1 (Obtaining rabbit blood γftTNF) Female rabbit (body
(weight 2.5-3.0 kg) was sterilized with formalin.
Propionibacterium acnes (Pro ioni)
bacLerium acnes)
Lerium or nebacLerium
150+ng into the ear vein), Wellcome, UK
injection. The rabbits were again injected with 100 μg of endotoxin 8 days later.
Enterobacteriaceae 026: B6 Yamakinori Boboli Saccharide, De
Ifco, Inc., USA] was injected into the ear vein, and 2 hours later, the heart
Whole blood is taken from the viscera. 10 per 100mQ in the collected blood
After adding 0 units of heparin sodium, 5. OOOr
Perform refrigerated centrifugation at pm for 30 minutes to remove blood cells and insoluble
Remove solids. Serum TNF3XIO' units/mα from 40 rabbits
Plasma 2.4α with an activity of 2.4α is obtained. Step 2 (Blood? ff TN F c7) Partial incontinence
Add 24g of Serai l-1 to 2.4C of plasma obtained in step 1;
After stirring for 1 hour, filter. 1.2α 0 in llx solution
．． 04M+-Lis-HCl buffer (pl+ 7, 8)
After adding 0.04M containing 0.1M thorium chloride
Fill with Tris-HCl solution (pl+ 7, 8).
1) [Mi^I Micepharose CL-6B
(Pharmacia, Sweetin) column.
. After washing with 0.04M Tris-HCl solution 6j, O.18
0.04 IVl Lis-HCl buffer containing M sodium chloride
Elute using solution (pl+7.2). L cell cytotoxic activity
Concentrate both fractions by ultrafiltration showing 0 then 5mM
Sephacryl S-200 equilibrated with phosphate buffer
The concentrated solution was added to a column of Pharmacia (Sweatin).
The active fraction is limited.
Concentrate by external filtration and collect 3.5 x 10 units. ratio
The activity is 18XIO@units/mg. Step 3 (anti-T N l? antibody) '[' N F obtained from blood γ11i was injected as in step 2.
Mix purified Freund's complete adjuvant at f, 1.
Subcutaneously on the back of 12-week-old tli BAL [l/c mice]
Inject. Repeat this operation after 2 weeks and 4 weeks to update
One week later, whole blood was collected and serum was obtained. This serum was added to the medium for measuring L cell cytotoxic activity at a final concentration.
Obtained from rabbit serum by adding it to a 500-fold dilution.
The cytotoxic activity of TNF was measured by the method shown in Reference Example 1.
When obtained, no L cell-toxic activity was observed.
Mouse serum was tested with antibodies against rabbit serum 'rNF (
It can be concluded that the virus contains anti-TNF antibodies (referred to as anti-TNF antibodies). Reference Example 3 Step + (Obtaining 1'NF-producing cells) Propionibacterium sterilized in female rabbits with formalin
Therium acnes (Pro ionibacteri)
uma Ahiro) [Corynebacterium pervum (Yori black)
υbacLerium), Wellcome, UK
J was treated intravenously and 7 days later, the tracheotomy was performed and the lungs were injected with physiological saline.
Obtain the transmigratory cells by washing with water. this cell
After washing with physiological saline, add 10% fetal bovine serum (Flow La
R1) MI 1640 (Flat.
Low Laboratories Inc., USA) was used as a medium, and carbon dioxide gas
%-containing air as an atmosphere in a carbon dioxide gas incubator
and culture at 37°C. Divide the incubator into two, and one side has
Endotoxin derived from Escherichia coli [Escherichia coli 026:136 origin]
11
01L/mα, and the same amount of sterilized
Add water. L to the culture supernatant supplemented with endotoxin.
Cytotoxic activity appears and reaches its maximum level in 7 hours. This activity is abolished by anti-TNF antibody, but in normal mice
It is not removed by serum. On the other hand, in cell culture supernatant without endotoxin, L
No cytotoxic activity was observed. Step 2 (Molecular weight of TNF) Together with endotoxin in cell culture in Step 1
Radioactive -[1”S]methionine (1300c:i/m
I mC1/
Add the culture supernatant so that m Q and culture.
Laemmli's method
mli) UK (1970) Nat JIF - (Nat
(London) Vol. 227, pp. 680-685)
Therefore, it can be resolved by SDs polyacrylamide gel electrophoresis.
analyze. lllllI is prepared so that the gel concentration is 12.5%. After electrophoresis, set [ENHANCE] to
processed by England Nuclear Co., USA) and dried.
After that, it is closely covered with X-ray film (Fuji RX, Fuji Photo Film).
Let the light shine on. Cultures grown in the presence of endotoxin
The formation of a substance with a molecular weight of 17,500 is observed in the supernatant.
. In addition, the cell culture supernatant in step 1 was SD in the same manner as above.
After being subjected to S polyacrylamide gel electrophoresis, 2.5
%NP40 (Calbiochem, USA) for 1 hour, water
After incubating for 2 hours in the chamber, separate each electrophoresis lane and run
Slice into 2IIIl perpendicular to the direction. each slice
By culturing the fragments with L cells, cytotoxic activity can be detected.
Find out the gender. Develop the culture supernatant in the presence of endotoxin.
Cytotoxic activity was determined at the molecular weight position of 17,500 in the lane.
was observed, and no cytotoxic activity was observed at that pond location.
do not have. Step 3 (obtaining mflNA) Add endotoxin in the same cell culture as in step 1.
After culturing for 2 hours, the cells (rabbit lung wash) were centrifuged.
Collect the purified cells). Cytoplasmic RNAΔ and m from within it
Extraction of RNΔ was performed using Cbirg as described below.
win) et al. [Charbwin, J.M., (
Chirgwin, J. M.) et al., Biochemist
Lee (13iocl+amistry) l 8052
94 (1979)]. cell 3XIO
4mfi of 4M guanidine thiocyanate per piece
Add the solution and use a homogenizer (AM-7, manufactured by Nippon Seiki)
Crush at After removing the residue by centrifugation, 2.4g of chloride
Dissolve cesium and add 2゜5mQ of 5.7M salt in advance.
Cesium chloride, 0. IM EDTA solution (pH 7, 5
) into the polyaromer tube quietly! ■Layer
Ru. Beckman 3W410-tar (Beckman, USA)
Ultracentrifuged for 12 hours at 20°C and 3ooooorpm using
After separation, remove the supernatant and place the pellet in lomMh
- Hydrochloric acid edge tfli? IJL (5mM EDTA, l
(containing w/V% SDS) to dissolve at 1 m+1
The solution is 1 mQ, a mixture of chloroform and 1-butanol.
(volume ratio 4 L) and add 0.05 volume of 2M to the aqueous layer.
Add sodium acetate and 2.5 volumes of ethanol, -
Leave to stand at 20°C for 2 hours or more to precipitate RNA. centrifugal
After collecting the precipitate and drying it, it was dissolved in 500 μα of sterile water.
to obtain a cytoplasmic RNA solution. The above cytoplasmic RNArB solution was heated at 68°C for 2 minutes and then rapidly cooled.
, 500μQ 2x concentration of l-region 1 Tris EDTA mild tf
Li solution pH 7.4 (1mM EDTA, 0.1w/v
% SDS and 0.5M lithium chloride),
200 mg of oligo(dT)-cellulose (P.R.
Developed on a column (BRL, USA) at 1x concentration.
Wash the elution edge with 10 mA of the buffer described above, and remove the elution edge with 1# solution (10 mA of
Lis-HCl pa17, 4.1 mM EDTA, 0,
Elute at 2 mQ (containing 1 w/v% sDs). to the eluate
0.05 volumes of sodium acetate solution, 2.5 volumes of ether
Add ethanol and cool to -20°C to precipitate. precipitation
Collected by centrifugation, oligo (dT) cellulose was added again in the same manner.
Collect both parts adsorbed on the column. By ultraviolet absorption spectroscopic analysis, 85 ILg of mRN
Collect A. Step 4 <mRNA size fractionation) 880 μg of +5 RNA prepared in the same manner as step 3
Dissolved in 250μ0 water, 5-25% linear sucrose density
Layer on gradient position 0mA. The sucrose density gradient is
Tris buffer (25mM Tris) containing 25% sucrose and 25% sucrose, respectively.
Liss hydrochloric acid pl+ 7, 2.2 +wM EDTA, I
containing w/v% SDS), l5CO570
Produced by Gradientor (Isco, USA). Using Beckman SW410-tar, 4℃40000r
pm, and after 12 hours of ultracentrifugation, the fraction collection device (vehicle)
Each fraction of 400/l Q was obtained by
to recover. Each fraction was precipitated with ethanol, centrifuged, and then transferred to sterile water.
dissolve. Step 5 (mRNA translation experiment) mRNA translation by African frog oocytes is
Experiment 8 (e.g. Hiroshi Teraoka, Mikio Itsuki, Kanetabe Tanaka, protein
, Nucleic Acids, Enzymes, Special Issue, Genetic Engineering, 602 pages 198
1 year). The African frog is a Hamamatsu biological teaching material.
can be obtained. The fractionated mRNA obtained in step 4 was reduced to lμg/μC.
Dissolve in sterile water so as to give 50n per oocyte.
microinjected with 1 m g/m Q of cattle.
Barth solution containing serum albumin (
7.5mMh lys-hydrochloric acid pl+ 7, 6.88 chloride
Sodium, 1mM potassium chloride, 0.33taM potassium nitrate
sium, 0.41mM calcium chloride, 0.82mM sulfur
Magnesium acid, 2.411 M sodium bicarbonate, 1
80/mQ Penicillin G, ■8μg, /mQ Strept
(containing mycin) for 24 hours. of culture solution
Crush the intact oocytes with a glass rod, centrifuge, and drain the supernatant.
Measure cell-damaging activity. Sedimentation constant: 163
gives the highest value of L cell-toxic activity. Also, this activity
Although it is erased by the anti-TNF antibody obtained in Step 3 of Reference Example 2,
It is not eliminated by normal mouse blood γN. Step 6 (Obtaining transformants) Using 5 μg of fractionated nRNA obtained in Step 4, experiment
1(1) Prepare two mInDNA according to page 96 onwards.
Ru. The reverse transcription rIP element is also available from LifeKuens (USA).
to use. Double-stranded DNA was added to a polyamide gel with a gel concentration of 3° to 5%.
Fractionated by acrylamide gel electrophoresis, length approximately 100
330 g of fractions from 0 to 2000 bp are obtained. This 2 minutes 7
According to the same experimental group using ng, terminal deoxynuclear
Creotidyl transferase [PRL (B)
RL), USA] to connect deoxy CAJ1,
Similarly, a plasmid with a deoxy C chain connected to the Pst1 site
Anneal with p13R3225611g. Ani
Colon α1K-12 strain (II[3
101, C33694) was transformed into AT, 2000
Obtain transformants of the strain. Koniho 7 (Rabbit “NF partial amino acid sequence”) Reference example
Partially purify the NF in step 2.
Purified by SDS polyacrylamide gel electrophoresis
do. Some parts were stained Coomassie brilliant blue,
The band corresponding to the molecular weight position of 17,000 was extracted from the gel.
Cut out and extract with 1% ammonium bicarbonate. 5X
Using 1' NF of IO' unit, approximately 18
Of the 0μg recovered, 150μ section was treated with 1% ammonium bicarbonate.
Dissolved in 75 μC of TPCK trypsin
Add 3 μg of Nton Biochemical Co., USA) and
Incubate the reaction solution for 4 hours at 7°C.
High performance liquid chromatography using 5C8 (Gyudon Kagaku) as a carrier
Fractionation is performed by fractionation to obtain trypsin-digested fragments. TNF purified to high purity as above and its trypsin
The digested fragments were then separated on a column of Sephadex G25.
Desalt and lyophilize. R. M, Hlewick (R, M, llewick) et al.
method [J, Biol, Kem, (J, Biol,
CI+e+s,) Vol. 256, pp. 7990-7997, 1
[981], perform Edman decomposition starting from the N-terminus.
, phenylthiohydan liberated in each step
Toin amino acids are analyzed by high performance liquid chromatography, model
5P8100 (Spectra Physics, USA)
Colored with Solpax DS (DuPont, USA).
As a sample, analyze using conventional methods. As a result, TNI? of
The amino acid sequence from the N-terminal side is as follows. 5er-^1a-3er-^rg-A 1a-Leu-
5er-^5p-Lys-f'ro-Leu-^1a-
11is-Val-Val-^1a-^5n-Pro-
Gln-Val-Glu-Gly-Gln-Leu-G
One of the ln-trypsin digested fragments has its N-terminus
It has the following amino acid sequence. Glu Tbr Pro Glu Glu
Ala Glu Pro Met 81 Step 8 (Synthesis of oligo DNA probe) Reference example 3 Step 7
mRNA deduced from the amino acid sequence of "I'NF" obtained in
Synthesize oligo DNAΔ complementary to the base sequence of A.
The method for synthesizing 0 is based on improved phosphoric acid, which has already been published by Ito et al.
Triester method (H, Ito, et al.,
Nucleic Acidless, (Nucleic
Ac1ds Res, ) Volume 10 1755-1769
Page, 1982). 128 types of oligo DNA deduced from amino acid sequences
Divide into 5 groups, each with 16, ■6.32.32.3
Synthesize as a mixture of two types. Each was deprotected according to a conventional method, and Sephadex G-50 was added.
Column glo using (Pharmacia, Sweetin)
Matography, 20% polyacrylic resin containing 7M urea
Midgel electrophoresis and DE52 (Whatman, USA)
) Purified by column chromatography, O,ll1
Dialyze against 1M Tris-EDTA 1/2 solution. Each purified oligo DNA was converted into T4 polynucleotide using a standard method.
Tidokinase (Bethesda Research Laboratories, USA)
(Japan) and γ-12-adenosine triphosphate.
Radioactively labeled DE52 column (Whatman, USA)
Purify by approximately 3×lO°cpm/μg, respectively.
Radioactivity is introduced. Oligo DNA probes obtained in the form of mixtures of each group
Name the blocks as shown in Table 1. Table 1 shows part of the amino acid sequence of rabbit TNF and rabbit I
−mRNAΔ base sequence deduced from the amino acid sequence of NF
column, and synthetic oligo DNA of each group based on this column.
The base sequence of the probe is shown below. Reference Example 3 mRNA of TNF-producing cells obtained according to Step 3
1M glyoxal, 50% by volume dimethyl sulfoxy
50 in the presence of
After processing for 60 minutes at °C, 1.1 wt% agarose gel
The mRNA after the 0 fraction fractionated by electrophoresis is divided into f! air
Pneumophoretic transfer plotting device (Bio-Rad
(Company, USA) for transfer according to the manufacturer's instructions.
Next, the mRNA on this membrane and 5xSS
C: and 150 μg/m Q of denatured salmon sperm DNA.
After treatment at 65°C for 2 hours with 5x Denhardt's solution containing
, radiolabeled oligo DNA at I x 10'cpm
/mQ, 5X Denhardt solution containing 5X5SC solution
Treat with liquid at 50°C for 2 hours. This film was then subjected to 6xss
Washed at room temperature, 40°C, 50°C, and 60°C sequentially, and exposed to X-rays.
Film XAR-5 (Eastman Kodak Company, USA)
) is exposed to n light. As a result, the strongest link between mRNA and
The oligo DNA that hybridizes is MJ, and MJ
If the mixture contains a sequence completely complementary to +aRNA,
It turns out that it contains Rigo DNA. Step 9 (cloning of rabbit'rNF gene)
The transformant obtained in Example 3 step 6 was placed in the experimental port (2) 16
Transfer onto a cellulose filter according to the method on page 2, and
DNA and the radioactive label selected in Step 8 of Reference Example 3.
RigoDNA (MJ) under the same conditions as in Reference Example 3 Step 8.
Hybridize (colony hybridization)
tion) 0Select 49 strains that hybridize strongly and further
Fix on the filter and perform colony hybridization again.
tion to the labeled oligo DNAΔ (probe MJ).
Select nine that hybridize more strongly. From these nine strains, the rapid plasmid on page 6 of the experimental book (1)
Obtain approximately 5 μg of each plasmid according to the decoupling method.
. Use this plasmid with restriction enzyme map 1, T! AI, average next year
1. Using Kyunyo ■ (both [3RL, USA), mail
Cut 1 wt% agarose according to Kerr's manual.
Compare the length of fragments cut by each enzyme using gel electrophoresis
do. As a result, all nine strains had I'vu II of approximately 50 bp.
and Rsa I fragments, and most of the nine strains had Rs
It has a fragment of approximately 200 bp due to a I, and is partially common.
It is suggested that the sequence is as follows. Figure 1 shows restriction enzymes.
The results of the analysis are illustrated below. The contents of the disclosure will be explained in FIG. That is, expression is observed in both pB2-2 and pR12.
pB2-7. pR9゜pR17,p
No expression was observed for R18 and pR25.
do not have. A β-lactamase promoter is used to express the inserted gene.
a protein is used, and the protein produced during the expression is
Fusion protein with β-lactamase (fusion pro
It is teinl. By the way, the inserted gene is expressed correctly.
In order for the β-lactamase promoter to be
The photographic direction must be the same. Therefore, the transfer direction
pR17, pR18 and pR25, which are the opposite, are not expressed.
Ino is natural. In addition, β-lactamase promoter
Even if the transcription direction of the target and the reading direction of the inserted gene are the same,
β-lactamase frame and cDNA encoding
If the protein frames do not match, it will not be expressed correctly. By the way
In the case of pB2-7 and pR9, the β-lactamase protein
No expression was observed even though the motor transcription direction was the same.
It is determined that this is due to a frame mismatch.
Ru. Furthermore, from the nucleotide sequence, the coding region of TNF is identified as
135bp upstream and 330bp downstream of PvuII in Figure 1
It is known that there are Therefore, pB2-2 and pR1
In the case of 2, the β-lactamase promoter
The transcription direction is the same and the frame of β-lactamase is
Then, the frame of the protein encoded by the cDNA matches, and
Expression is observed because it contains the coding region of TNF.
It is something that Also, as is clear from the content shown in Figure 1,
The Pst I site used for cloning was excluded.
The restriction enzyme maps of all clones are identical and the base
Essentially, all clones encode the same protein. Therefore, even if it contains the TNF coding region,
Either clone can be used for expression construction.
In this example, pRt7 is used for expression construction. Cause
Furthermore, the nucleotide sequence of the coding region of pR17 is also similar to that of pR12.
and that of pR18. In addition, 7 of these strains were treated with 10 μg/m 12 tetra
In 2mj2 LB medium containing cyclin, the absorbance is the second highest.
Culture until the values shown in the table are reached, and collect the cells by centrifugation.
Crush mβ with physiological saline using ultrasonic waves, and centrifuge the upper groove.
When cytotoxic activity was measured, as shown in Table 2, L cell
Shows damaging activity. Plasmid pBR32 as a control experiment
The same operation is repeated using a strain containing 2. The results are shown in Table 2. (Margin below) Table 2 Furthermore, this activity was abolished by anti-TNF antibody, and normal mice
Therefore, all nine strains are not erased by TN serum.
It is shown that it has a plasmid containing the F gene.
. Step 10 (Determination of the base sequence of the rabbit TNFill gene)
am strain containing IO μg/mQ of tetracycline.
Cultured in M9 medium [Experiment #J (3) p. 440] IQ.
After that, prepare the plasmid according to the method on page 90 of Experimental Book (3).
isolated, yielding approximately 150 μg each. Each base sequence was determined by Maxam-Gilbert (Maxa*-
Gilbert method [Maxa+m]
Method in enzymology
1980, Spring 55, 490 pages.
Academic Press (^ academic r'ress)
)). Also, this base sequence and Reference Example 3
Based on the match of the partial amino acid sequence determined in step 7,
The entire structure of the heron TNF protein is elucidated. Step 11 lac in E. coli using recombinant plasmid pR12
The purpose is to express T'NF using the promoter
Construct the plasmid (a) as shown in Figure 2.
μg of plasmid pl? 12 to ±1 [pi] of lO unit.
- RL (I3RL)] at 37°C for 2 hours.
6% by electrophoresis on a 4% polyacrylamide gel.
A 30 bp fragment is isolated. A fragment of approximately lILg was removed from the gel.
Elute electrophoretically. By the same method as Reference Example 31 Step 8
The two deoxyoligonucleotides shown in FIG.
5'-GATCCATGTCAGGTTCTCGG
GCC-3' and 5'-CGAG^^G(:TGAC:^
Synthesize TG-3° and experiment 1J (3) l on page 22.
Therefore, approximately 100 μmole of each deoxyoligonucleotide
The 5' end of the
After the reaction is complete, the reaction mixture is phosphorylated with phenol.
Extract using chloroform and then chloroform.
After that, 0.5 μg of the oligomer was added to a string of about 630 base pairs.
■ Combine the fragments and precipitate with ethanol. Experimental old (1)
a 1' of 10 units according to page 7, D N A
The above fragments are reacted with ligase overnight at 4°C to join together. After the reaction completed solution was precipitated with ethanol, 20 units of vagina 11
! Digested at 37°C for 3 hours, then digested with 4% polyacrylamide gel.
electrophoresis, and a fragment of approximately 670 bp was electrophoretically eluted.
Collected by January yield plasmid pUG-8 (P-
L Biochemical Co., catalog number 4916. United States)l
μg was digested with Ila*III, phenol extracted, and cloned.
Berita prepared by loform extraction and ethanol precipitation
- Approximately 670 bl) of TNF structural gene in 0.5 μg
A fragment with 13an+III sites on both ends containing
' , join using DNA ligase. Experiment book (4
), p. 20, by transforming E. coli.
MIPTG and 0.004% (w/v) x-gal
Approximately 200 white colonies were produced by culturing on an agar medium containing
obtain. Plasmid DNA was obtained from 100 of these transformants.
When A was prepared and digested with Ba+all+, 15 pieces were obtained.
contains the desired Bam1l I fragment of approximately 670 bp.
Ru. Furthermore, in order to check the direction of insertion, the above 15
There is only one 8μ site on the plasmid pUc-8.
Ec. R1 and Pvu II (recognition region on fragment of approximately 670 bp)
6 wt% polyacrylamide
By investigating using dog-gel electrophoresis, seven plaques were identified.
The desired fragment of about 140 bp was confirmed from Sumid, and pu
The forward direction from the lac promoter on c-s
Prove. Base sequence analysis revealed that these seven plasmids are identical.
, lac promoter, synthetic NA and cDNA
It has been confirmed that the desired nucleotide sequence is present at the junction.
Ru. Using plasmid pH 17, 1 acU in the large bacteria
TNF can be directly expressed using V5 as a promoter.
As shown in Figure 3, a plasmid is constructed for the purpose of
Add 10 μg of plasmid pR17 to sub-M of the IO unit.
I [Extinguished at 37℃ for 2 hours in BRL J.
and approximately 63% by 4% polyacrylamide gel electrophoresis.
Isolate a 0 bp fragment. Approximately 1 μg of fragment electrophoretically elutes from the gel. Step 8 and
The two deoxyoligonucnes shown in Fig. 3 were prepared in a similar manner.
Leotide River 1st, 5'-8^TTCATGTCAGCT
TCT(:GGGC(: -3' and 5°-11; GAG
AAG (:TGAC, ATG-3') was synthesized and the experimental book
(3) Approximately 1100p according to 122 True. 5' of two types of deoxyoligonucleotides on the upper 5 base of le
Phosphate the ends using 1゛4 polynucleotide kinase
Extract the completed reaction solution with phenol and further
After chloroform extraction, the sample of p1-17 obtained earlier was extracted with chloroform.
■ Digested fragment (approximately 630bp) together with 0.51Ag
Tanol precipitates. 10 units according to page 37 of Experiment Book (I)
Nitto's T was reacted with ligase at 4°C overnight to bind.
After the 0 reaction, the reaction solution was precipitated with ethanol and 20 units
Digested with EcoRI for 3 hours at 37°C and digested with 4% polyacrylate.
Approximately 670 bp fragment was recovered by Lumid gel electrophoresis.
do. Plasmid pOP95-15 was generated by Fuller's method [F.
Fuller (1”, Fuller), Gene (Gene),
Prepared according to Vol. 19, pp. 42-54, 1982, 1.
. Iμg of pOP95-15 was digested with EcoRI.
Perform nol extraction, chloroform extraction, and ethanol precipitation.
Prepare the vector. 0.5 μg of vector is
Synthetic deoxyoligonucleotides and TNF-coding
The approximately 670 bp fragment obtained by joining the DNA
, T, are joined using DNA ligase. Experiment w (4
), p. 20, by transforming E. coli with InMI
I'TG and 0.004% (w/v) x -g a l
Approximately 150 white colonies are obtained on the agar medium containing. Prepare plasmid DNA from 100 of these colonies
However, when I digested it with the old IEco, the 12 pieces were about 6 of the target.
The old 70bp Eco has been replaced. Furthermore, in order to investigate the direction of insertion, the above 12 plasmids were used.
Digested with r'vu II and r'stl, 1.
When examined using 5% agarose gel electrophoresis, there were 4
About 1280bp and 2600b of interest from the plasmid of
A fragment of p was confirmed, starting from the 1acUV5 promoter.
It turns out that the TNF gene is connected in this direction. Base sequence analysis revealed that these four plasmids are identical.
, 1acUV5 promoter, synthetic deoxyoligonuc
Verify that leotide and cDNA are correctly combined.
It is confirmed. The resulting plasmid was transformed into pTNF-1acU.
Name it V5-1. Step 12 (Purification of 'rNF produced by E. coli) Step 11
The E. coli strain containing the plasmid obtained in
at 37°C in 50 mΩ in LB medium containing Cultivate at night
, 5Q, and cultured for an additional 3 hours. stomach
Sopropyl β-lothiogalactopyranoside (Sigma)
(US) was added to a final concentration of 1- and then incubated for 6 hours.
After cooling, the bacterial strains are collected by centrifugation. engineering
In the same way as in Step 11, the bacterial cells were treated with 0.04M Tris-HCl.
Bacteria were disrupted by ultrasonication in buffer solution (pH 7, 8) 5 Q.
Obtain body protein solution. This solution exhibits a cytotoxic activity of 5X10" units/Q.
. This solution was purified in the same manner as in Step 2 of Reference Example 2 to obtain a concentration of 1°2 x 10
Obtain TNF in °. The specific activity of this product is 6.8X1
0" unit/n+g. Step 13 [Mesh A sarcoma (Mesh A sarc
oma) Activity ff'l' titer J[IA
2XIO@M female A in the abdomen of LI3/C mouse
Sarcoma (Net, b^sarcoma) Transfer m cells.
to plant After 7 days, the size of the transplanted tumor was 7-81IIIM in diameter.
This means that the tumor is in good orthogonal condition without hemorrhagic necrosis.
Select a mouse and add 0.5 m of saline diluted from the tail vein.
Inject the NF sample obtained in Reference Example 3 Step 12 of A.
, 24 hours later, the following judgment criteria are used. (=): No change 1): Faint hemorrhagic necrosis (I): Moderate hemorrhagic necrosis (from the middle of the bottom surface of the graft
0% or more necrosis) (-1+L): Significant hemorrhagic necrosis (central part of transplanted cancer)
20 days after administering the sample, whether the cancer had completely regressed or not.
Observe and determine the complete cure rate. By the above method, 81g of TNF produced by E. coli was determined.
The activity of is shown in Table 3. Number of experimental mice Table 3 Step I (Plasmid order 8, I) B2-7,
p[+2-2 E. coli Kl 2. MC, to 1061 shares
Transformation) The above 3111 plasmid obtained in Reference Example 3
E. coli Kl 2MC:1061 strain [Ka
Casadaban et al., Journal of Ba
Chuteiology (Journal of' I3acL
eriology), +43 Spring, 971 pages (1980
(published in 2013). For details, 1 large to 12
Colonies of MC1061 strain were grown at 550% using LB medium.
Culture until the positive absorbance reaches 0.3. The culture 50
Collect m8 and contain 25 mα I OmM RbCQ.
l On+MMOPs (pl! 7, 0) in solution
Wash, then 50mM Ca11, 10mM Rb
O, IM MOr'S (r+I+6.5
). The suspension was cooled on ice for 30 minutes, centrifuged, the supernatant was removed, and
0μα DMSO and 50+mM Gael, and lO
0.0 containing mMRbcQ. I M M O P S (pl
+6.5). 200% of the suspension
Dispense 10 pQ of plasmid DNA solution as described in If.
Add μ sadness to each. After cooling each mixture on ice for 30 minutes, the mixture was incubated at 44°C for 60 minutes.
Heat shock for seconds and immediately warm to 37°C.
Add 5 mQ of I, [3 medium that has been warmed to 100 mL. This melt
After incubating the solution at 37°C for 1 hour, each solution was centrifuged.
Then remove the supernatant and obtain a cell pellet. The cell beret
Add LB medium to the plate, stir, and make a suspension. The concern
? n solution containing 30 μg/m Q of tetracycline
Spread on LB agar plates and culture overnight at 37°C.
As a result, plasmids pR18, p[32-7 and pH2
-2 and 2 to 2, respectively, of tetracycline-resistant transformed bacteria.
Roni is obtained. Step 2 (preparation of pr+2-7 and pR18 plasmid DNA)
of plasmids p[+2-7 and pRI8 obtained in step 1)
The transformants were cultured according to the method in the paper below, and
6. Then, collect the resulting transformants.
, and then the plasmid DNA is purified. 〔experiment
11F(3), pp. 88-96] i.e. 30 μg/m
Q containing tetracycline and B medium, respectively.
Inoculate these transformants and incubate at 37°C with vigorous shaking.
6 Next, repeat this process to transform
The cells are propagated and the plasmid is further amplified.
While cooling the culture solution of the transformant to 4°C,
Centrifuge at 00g for 10 minutes and remove the supernatant. Ice-cold STE [0.1M sodium chloride, 10+mM sodium chloride]
Lis-HCl buffer (pl+7,8) and 1mM E
DTA) followed by washing with 100 mA of 101
2 in 1M Tris-HCl (pH 8,0)
cells using an aqueous solution containing 0 mg/mQ, Norizozyme.
lyse by boiling. Transfer the resulting viscous liquid to an ultracentrifuge tube
Transfer and centrifuge at 25,00 Orp + m for 30 minutes at 4°C.
to obtain a DNA solution. After measuring the volume of the DNA solution, per 1 mQ of the solution,
Add solid cesium chloride Ig until cesium chloride is completely removed.
Stir slowly and carefully until dissolved. The cecichloride
For every I OmQ of an aqueous solution of 10 tmg/mQ of ethyl
Add 0.8 mQ of aqueous dium bromide fi. This conclusion
As a result, the final specific gravity of the solution was 1.55 g/mQ, ethidium
The final concentration of bromide will be approximately 600 μg/mα. Transfer the cesium chloride aqueous solution to a suitable centrifuge tube and empty it.
Add light paraffin oil to the gap and incubate at 20℃ for 36 hours.
5. When centrifugation at OOOrpm continues, chain-shaped microorganisms appear in the upper layer.
derived DNA and nicked circular plasmid DNA,
Closed circular plasmid DNA comes in the lower layer. DNA at the bottom
Insert the syringe needle into the side of the tube and collect the band.
Transfer to tube. Remove ethidium bromide and water
Dialyze the layer into TΔE buffer. Plasmid DNA solution
I? RNase treated and an equal amount of saturated phenol
Extract with rEI solution. Prepare the aqueous layer at 0.1% SD in advance.
Equilibrate with TAE buffer (pl+8,0) containing S.
attached to biogel ([tiogel) A-150]
0.1 to wash the DNA and obtain the active fraction.
Elute the 6 fractions eluted with TAE slow injectate containing %SDS.
Precipitate with ethanol to obtain purified plasmid DNA
. Purified pn2-7 plasmid DNA was obtained by the above method.
250 (tg, p1318 plasmid DNA is 13
4 μg is obtained. Step 3 (Sperm*p[32-7 and pR18 plasmid DNA
Nick translation of A) Restrict 40 μg of purified plasmid DNA obtained in step 2
Digested with enzyme PsLI and then 4% acrylamide
Apply to gel electrophoresis. After electrophoresis, stain and cut out the desired band.
Isolate the Pstl insert. 500 ng of the Pst
Tee, Maniatis (ToM)
[Brock, Natl. Acad, Sai (Proc, Natl, ^cad,
Sci, ), USA, 720.1184 pages (1
974) performed Nick I. Lancelegeon according to J.
became. Nick translation is a commercially available kit [P.R.
BflL Company J is used. Activated dCTP in a reaction system at 25 μC (801')
Use Ilole (for 400Ci/a+ mole)
). First, prepare the following mixed solution. 2.5 μα Solution A (dNTP solution) 2.5 μQ Solution B (500 ng of DNA, i.e.
PsLI insert) 5p Q Radioactive dCTP (3200Ci/m mole) 1.31tQ dc:TP (65p pure ole, 50p
mole/μm2 dCTP) Engineering LL]"-Solution E (t50) Total 22.5/LA To this 22.5μΩ solution, 2.5μμ Solution C (DN
DNase I, DNA polymerase I
) and react at 15°C for 60 minutes. Solution D (stop buffer) is then added to stop. Change
Add carrier LRNA and perform ethanol precipitation twice.
and then dissolved in water at 500 μC. Specific activity is 9.3 x 10'cpm/μg DNA.
Ru. t+'l obtained in step 2! Using 1ApRl 3
, similarly follow the above method for nick translation
Do this. Specific activity is 7 X I O'cpm/μgDN
It is A. Step 4 (I) Rsa I cleavage of RI8 plasmid DNA
80 μg of pRI8 plasmid DNA was digested with restriction enzyme Rsa.
Digested with I and subjected to 4% acrylamide gel electrophoresis.
, cut out the band of the insert for the following purpose [3N
Purify using D column. Approximately 640bp 3.77μg (recovery rate 52%) Approx.
175bp 1.77μg (recovery rate 50%)
The approximately 640 bp insert was inserted into the 3' fragment of pRlB (pr
(meaning the untranslated part on the 3' side of l18), about 1
The 75 bp insert was inserted into pRl 8-clr (pR18
code part). Furthermore, in the above, instead of Rsa I, PsLI and Ms
Digested using t
g (recovery rate 60%). Is this insert pl? 1
It is named 5' fragment of 8. Step 5 (Isolation of human chromosomal TNF gene) Example 1 Step
1P-labeled plasmid p132-fi obtained in 3.
Using the sert as a hybridization river probe,
Ron 4A EcoRI cleavage site [Blattner (B
The method of Science (LaLtner) et al.
e) Vol. 196, p. 161 (1977) J. Hill-D.
Fragments obtained by partially digesting and size-fractionating NA (maniatei)
ManiaLis et al., Cell, vol. 15
, p. 687 (1978) 1.
Liophage Charon 4A/Human Chromosome Gene Library
Screening for Lee's 10" plaque.
As a method [Bent, on and Davis]
(Davis), Science, 19
6, p. 180 (197'7)]. All of the bacteriophages in the starting culture medium are
Contains the genetic material needed to create sex substances
Since the sequence is complementary to the rabbit TNF gene,
Use a probe with Phage plaques containing the gene of interest are radioactively
hybridizes with a probe that has a radioactivity of
can be found by In this way, the nine ha
hybridized plaques are obtained from the library.
. The method and conditions are as follows. Number of plaques: 2 ~ 1xlO@plaque (~4xlO")
Lark/φ150mm plate x 25) 2) Nitro cell
Transfer to loin filter: [13ento
n) and Davis, Sci.
ence), Vol. 196, p. 180 (1977)) 3) Hybridization: 1.25X I Ocpm/m
pR2-Finsertpro obtained in Example 1 Step 3 of E+
Addition of tube, 42°C, 19.5 hours 4) Washing: at room temperature with 2XSSC-0.1% SDS
4 x 10 min washes followed by lX5sc-0.1% SD
Wash twice for 30 minutes at 50°C with S 5) n light: XAR-5, -80°C, 2 intensifying screens, 39
Twelve candidate strains were obtained through the above screening. Perform secondary screening in the same manner as above to find the desired
Nine strains with fragments are obtained. As described above using these strains
Perform tertiary screening in the same manner as
We obtain 9 strains with . Regarding these nine stocks,
A fourth screening was performed using the same method, and nine strains were found.
Make sure it contains the desired fragment. 9 containing the desired fragment
bacteriophage) -I G I~
Named I-I G9. Step 6 (Isolation of rabbit chromosomal TNF gene) Digested human D
Digested rabbit DNA [Maniatis] instead of NA
iaLis) et al., Cell ((:e I l ), l 59
6871'f (1978)]
Bacteriophage Sharon 4A/Rabbit Chromosome Remains
Example 1 except that plaques from the gene library were used.
Perform substantially the same operation as step 5, 6.7 x 10' pieces
bacteriophage Sharon 4A/rabbit chromosomal genetics
Add 10” bacterial plaques to the child library.
-Disharon4Δ/human chromosomal gene library
Used in place of lark. Two bacteriophages containing rabbit chromosomal genes (
RG-1, RG-2) are obtained. Step 7 (Southern blot analysis of human gene clones) HG-3, HG-6, and HG-3 obtained in Step 5 of Example 1
Using bacteriophage 1-I G-7,
Each DNA was obtained according to the following method. 6 × lO°″ E. coli LE392 (host) in 18 mQ
bacteriophage HG-
Add 3 x 10” PFU of 3 and infect for 20 min at 3°C.
Then add the resulting mixture to 3 portions of NZ broth,
Incubate with stirring at 37°C for 23 hours, then incubate at 60 mA.
Add roform to the mixture and stir for 30 minutes. final dark
Sodium chloride was added to the mixture to give a degree of IM.
After that, let it stand for 15 minutes. Then, perform a centrifugation operation and remove the supernatant.
The obtained supernatant was then treated with polyester having a molecular weight of 6000.
The concentration of ethylene glycol and polyethine glycol is 10
% (W/V), and left at 1℃ for 22 hours.
Place the bacteriophage at 0 and then centrifuge the bacteriophage.
Collect. The resulting bacteriophages were transformed into SM 28
Suspend in mQ and then add an equal volume of chloroform. Pol
30 seconds with a tex mixer? After IJ mixing, centrifuge
Collect the aqueous layer and bring the total volume to 30 mQ with SM. this
After adding 26.4g of cesium chloride and gently dissolving it,
, phage by ultracentrifugation (45,000 rp++, 20 hours)
Collect the band. 10mM sodium chloride and 10mM
50 nM Tris buffer containing M magnesium chloride (pH
8,0), the final concentration of each was 2011
IM, 50 μg/mQ, 0.5% EDTA
Add SDS to proteinase and treat at 65°C for 1 hour.
0-order phenol, phenol:chloroform=
i = 1 (volume ratio), extracted once each with chloroform,
The resulting aqueous layer was divided into 10 volumes containing I n + M ED TA.
Dialyze against Tris buffer (pH 8,0). The purple color of this solution
When external light absorbance was measured, bacteriophage IIG-
It is confirmed that a purity of NA of 3 is obtained. To prepare the DNA of bacteriophage HG-3
by applying substantially the same methods as those used in
From bacteriophage IG-6 and JIG-7,
Get NA. In this way II G-3, I(G-6, tl
2920 μg and 1100 μg of G-7 DNA, respectively.
Soutl+ern method (Soutl+ern) yielding 819 μg
E, M. Southern (EoM, 5outhern), Chie 49mol
, Biol, (J, Mo1. Biol,), 98 volumes,
503 (1975)] under the following experimental conditions.
Perform Southern blotting of these DNAs. 1) DNA: HG-3825ng HG-6935ng HG-7685ng 2) Digestion with various restriction enzymes: 10 units, 10 units + 1 mouth of Eco 10 units 3 hours llindl 1'vu U 37℃ for 11 inds a month 3)? l Air swimming vJJ: 0.8% agarose gel 1゛ΔEio small unit 28V, 15.5 hours 4) Transfer to nitrocellulose filter: [E, E
M, Southern (E, M, 5outl+ern, )
J, Mo1. Biol
) 980.503 (1975) Reference J 5) Brehybridization: 30 mQ FDSS 42°C 6 hours 6) Hybridization: pl? 5'-fragment of I8 (I x I O'cpm/m
Q, prepared in Example 1 Step 4) at 42°C
, 14 hours 7) Washing: Wash for 10 minutes at room temperature using 2XSSC-0,1% SDS.
4 times, followed by 1X5SC-0.1% SDS.
8) Tl light: XΔR-5 (Eastman Kodak, USA) -80, washed twice for 30 minutes at 50°C.
The results of hybridization of two intensifying screens for 14 hours at °C are shown in Table 4. Table 4 (margin below) shows. Step 8 (Southern blot analysis of rabbit gene clone) In Step 7 of Example 1, HG-3,8G-6,11G
-7 instead of RG-1, RG-2 bacteriophage
substantially similar operations, except using each of the
Therefore, Southern plot analysis will be performed. As a result, pR1
The 5' fragment of 8 connects RG-1 and RG-2 to
I, far R1, black [■, crab dm and Ilaml
The fragments obtained by decomposition with each of l I IEcoRI
It can be seen that the fragments hybridize into a single band. Step 9 (Transformation of human chromosome TNFifl (bacillus containing zi)
Construction of terrier clone) The HG-3 DNA obtained in step 5 was
Landy Method [Biochemistry (mouth)
1ocl+e-sho 1stry), Volume 13, 2 l 3
4 pages (1974)]. This NG-
DNA33/Ag of 3 was treated with 80 units of IEcoR+.
and decompose at 37°C for 3 hours. The decomposition product is 1% low melting point agaro
- gel (conditions: IXTAE, 20V, 14.5 hours)
Perform electrophoresis. 2.9kb band with agarose gel
T, Maniatis
) (Molecular Cloning
Gloning), Cold Spring Harbor Labora
Tory (Cold Spring 1larbor L
aborat, ory), 377 pages (1982)
Isolate by method. For more information. The gel from which the 2.9 kb band was excised was incubated at 65°C for 1
Heat for 5 minutes. Furthermore, with the length of this 2.9kb
IEcoRI-digested HG-3 fragment (hereinafter referred to as rHG
-3/EcoRI 2.9kb fragment"
) was extracted from the melted gel three times with phenol.
Extracted three times with medium and precipitated with ethanol containing ammonium acetate.
Let it settle and collect it. In this way, 637 μg (yield approx.
30%) l-I G-3/Strong 1?12.9kb fragment
get. 255 ng of the above fragment and EcoR1-degraded pucta
I, Messing (JoMessing), Methods In
MeLbods in IEnzy
+mology), vol. 101, p. 20 (1983)]
are ligated using 2.5 units of 'r, ligase. Using the conjugation product obtained above, E. coli K12JM83 strain was used.
and transform. For details, please refer to E. coli 12 JM8
3 strains in LI3 medium at 550 nm in culture broth.
Culture until the absorbance reaches 0.3. 50mQ growth
Collect 83 strains of E. coli K12JM, and
Wash with MM0PS (pH 7,0) -10mM RbC1
0.1 M MOP 5 (pH 6.5) with 25mα
Suspended in -50mM CaC1, -10mM RbC1.
become cloudy To 203 μQ of this suspension, 10 ng of the above binding
Add an aqueous solution of lOμα containing the product. this mixture
Cool in water for 30 minutes and heat at 40° C. for 60 seconds. Immediately thereafter, add the LI3 tube, which had been preheated to 37°C.
Add the heated mixture to the loss 5m (L) and heat at 37°C for 1 hour.
Incubate. Centrifuge the resulting culture broth and remove the supernatant.
Ru. Add LB medium to the centrifuged cells to give 301tg/
Contains mQ of ampicillin and 40 μg/mQ of x-gal.
[Inoculate 3 plates. Plastics including inserts
Colony of E. coli Kl 2JM83 strain introduced with Sumid
- is white, but this is a strain with only the plasmid introduced.
Ronnie is blue. The white colonies obtained are again 3
0 μg/mα of ampicillin and 40 μg/mQ, x-
Inoculate into LB plate containing gal for confirmation. From the white colonies obtained above, 10 colonies (bats)
Select a cuticular clone (cutillary clone) and screen using the rapid method.
ning. Specifically, each colony was incubated with 30 μg/mQ of amperage.
Culture overnight in LI3 medium containing picillin. The proliferated cells were collected and 2 + ng/mα lysozyme −
50mM glucose-10mM EDTA-25mM
+-Squirrel (Tris) -11c Q (pl!
8,0). This suspension
was left at room temperature for 5 minutes, and then 0.2 N of 200 pQ was added to it.
Add Na011-1% SDS. I stirred it slowly.
This suspension was left at room temperature for 2 minutes, and then 150μ
Add α 3M sodium acetate (pH 5, 2) and 10 minutes
The mixture was incubated at -20°C and centrifuged for 15 minutes to obtain the supernatant. Add 900 μα cold ethanol to this supernatant and centrifuge for 5 minutes.
Obtain the precipitate with care. The resulting precipitate was diluted with 70% ethanol.
Wash with water and dry to obtain plasmid DNA. This method
to obtain 10 plasmids. Each plasmid DNA was diluted with lOmM Tris.
-Dissolved in 0.1mM EDTA (pH 8.0),
Ec. Digested with R1 and subjected to electrophoresis for restriction enzyme digestion.
. Conditions for digestion and electrophoresis are as follows. digestive nibs
Lasmid DNA solution = 5th L of the one obtained above
3 units of EcoRI, 37°C, 1°5 hours electrophoresis: 1
% agarose gel, IXTAE, +20V, 2 hours Out of the 10 clones, the above restriction enzyme analysis revealed that
Eight species are shown to be of interest. In other words, this 8? ]Clone 1 is a 2.9kb fragment
have. Select one of the eight desired clones.
and Escherichia coli Kl 2JM83 (pHGE) strain (ATCC
39656). In addition, this E. coli Kl 2J
M83 (pHGE) strain is classified as A based on the Budapest Treaty.
Deposited with Δ′I″CC under accession number TCC39656.
ing. Next, E. coli cells containing p[32-7 and pR18 were
Using Escherichia coli KI2 JM83 (pHGE) strain
Except for this, 1.89 mg of p
Obtain I-ICE DNA. Step 10 (Subclone of Ecol order flG-1
30 μg of RG-1 obtained in step 6 was added to EcoR.
By Ill! Become I. The resulting mixture of various fragments
and a mixture of the above various fragments and 0.8% low melting point agarose.
Follow substantially the same procedure as step 9 except for using the gel.
A fragment with a length of approximately 3.5 kb was collected, and a fragment of 1.0 μ
The IEcoR1-degraded RG-1 fragment (3.5 kb) of
Ru. This fragment was digested with EcoRI or UCl3,
IEcol? I-cleaved 1-I G-3 fragment (2,9k
b) Instead of the above εcal? I decomposition piece (3,5k
By substantially the same operation as step 9 except using b)
,Link. Transformation into Escherichia coli KI2JM83 strain, bacterial cloning
clone screening, clonal DNA degradation and electrophoresis
The procedure is the same as step 9 above except for using the bound DNA fragments.
Performed by essentially the same operation, the resulting clone is
E. coli Kl 2lM83 (pRGE) strain (ATCC3
9655). In addition, this large Jl & 1 bacteria K12
JM83(I)RGE) strain is based on the Budapest Treaty.
Deposited with the ATCC under deposit number ATCC 39655.
There is. Subsequently, E. coli Kl 2lM83 (pRGE) strain was transformed into pB
Same as step 2 except for using instead of pH-2-7 and pH-18.
By qualitatively the same procedure, 1.70 m
g Prepare. Step +f (1-mer 1-limit fermentation of pi-ICE plasmid DNA
(elementary analysis) Restriction enzyme of pHG E DNA obtained in step 9
The analysis was performed using the Maniatis method [Model].
Recular Cloning (Molecular Glo)
ning), Cold Spring Harbor Laboratory
(ColdSpring 1larbor Lab
(1982), p. 98 (1982)
)j. The method and conditions are as follows. 1) Degradation of pHGEDNΔ by Eco1 order: 18
゜Gltg p I-I CE, 64 units [EC0I
? I, 37°C 2 hours 2) Ethanol precipitation: Precipitate 3) Dissolution: EcoRI digestion p HG E is jμg/m
Add distilled water to make a solution of g. 4) Digestion with various restriction enzymes: I μg of the above EcoR1
Digested p I (G E, restriction enzyme: 5 units of Pvu II
. 5 units of Pbull + 10 units of Rsal, 10 units of
Rsa I, 4 units MstI+, 3 units Ava+,
PsL at position 911j [, 37°C, 2 hours 5) Electrophoresis: 2% agarose gel,
V, 14.5 hours 6) Transfer to nitrocellulose filter: [I, M
, Southern (E, M, 5ouT, l+ern), Je
I, mole, paiol, (J, Mo1.l3io1.)
, Vol. 98, p. 503 (1975)] 7) First prehybridization: 30mQFDSS,
42℃, 6 hours 8) ff! - times hybridization: 5″ fragment of pR18 (
What is obtained in step 4, 5XIO"cpm/m8)42
℃, 14 hours 9) Washing: Room temperature using 2XSSC-0,1% SDS
Wash 4 times for 10 minutes with lX5sC-0.1% S
Wash twice for 30 minutes at 50°C using DS10) D light: XAR-5 (Eastman Kodak Company)
, USA), -80°C, 2 intensifying screens, 17° for 5 hours + 1) Washing: 0, 5M Na0Il -1, 5M
0.5M Tris for 1 minute with NaCl
1, 5M Na1l for 1 minute, 3XSSC for 1 minute 12) Exposure = 19 hours above except for exposure time
Same operation as 0) 13) fAZ times prehybridization: Same operation as 7)!
4) i2 hybridization: pB2-finsert (
(obtained in step 3), 42°C, 16° for 5 hours + 5) Washing: Same procedure as in 9) 16) Exposure: Same procedure as above except that the exposure time was 19.5 hours.
Same procedure as in item 10) (1'-17) Washing 2 It) Same procedure as in item 18) n light = Exposure time was 20 hours.
Same operation as 10) +9) ff! Same operation as 3 times prehybridization = 7)
20) Third hybridization: 3″ fragment of pR18 (engineered
What is obtained in step 4 is 4.5 X I O”cpm/
mα), 42° C., 15 hours 21) Washing 2. Same procedure as 20) 22) n*: Same procedure as 10) The results of restriction enzyme map analysis are shown in FIG. Step 12 (Restriction enzyme analysis of pRGE plasmid DNA
) p It GE pRGE instead of E plasmid DNA
Substantially the same as step 11 except using plasmid DNA
pRGE plasmid D obtained in step 10 by the same method.
The resulting pRGIEDN was subjected to restriction enzyme analysis of NA.
The restriction enzyme map of the A insert is shown in FIG. Step +3 (Salt of rabbit TNF gene and human TNF gene)
Determination of base sequence) Escherichia coli Kl 2JM83 (pHGE
) strain and E. coli Kl 2JM83 (
1) Inject ['(GE) strain into E. coli harboring pB2-712
12MC1 to E. coli harboring MC1061 strain and pR18.
Substantially the same as step 2 above except that it is used instead of the 061 strain.
Perform the same operation and add 150 μg of each pR
GE plasmid DNA and pHG E plasmid DNA
get. The base sequences of pRGE and pHGE are Maxam-Gilva
(Maxa+a-Gilbert,) method [Maxam
(Maxa+*) et al., Methods in Enzymology (
Methods in Enzymology), Volume 55
490 pages (1980) Academic Press (Acad)
e+sic Press)]. The base sequence of pat-18 determined in Reference Example 3 and the above determined
By comparing the base sequences of the determined pRGE, the rabbit TNF gene was determined.
Elucidating the structure of genes (including exons and introns)
. The structure of the pflGE DNA insert is shown in Figure 5.
. Next, by comparing the base sequences of pRGE and pHGE,
, similarity and homologous sequences near intron-exon boundaries
investigate. In this way, the human 'rNF gene's 411￥
elucidate the structure (including exons and introns). Human
The structure of the NF gene is shown in FIG. Copy the rabbit TNF and human TNF obtained in this way.
The coded base sequence is shown below. In this base sequence,
The upper row shows the nucleotide sequence (R) encoding rabbit TNF,
The bottom row shows the nucleotide sequence (■(
) is shown. 1? TCAGC Ding TCT GGG G(:(:
CTG AGT GA (: Heto G (:C
TIt TCA TCT T to T to GA A
CCCG AGT GACAAG CCT+(CT
A GCCC76: GTA CTA GCA
AACG(:G CAA GTGGTA GC(:
C:AT GTT GTA GCA AA
(: CCT CAA G(:T1? GAG
GGCCAG CTC (:AG TGG CTG
AGC (:AG CGTIt GAG GGG (:
AG CTCGAG TGG CTG AACGGC
CGGRGCG AACGCCCTG (:TG
CGCAACGGCATG 8＾G CTCII
GCCAAT GCCCT(: TG G(:C
AAT GGCGTG GAG C'rG17 AC
G GAG AA(: (:AG (:TG GTG
GTG CCG GCCGA(:It AGA
GAT AACGAG (1:TG GTG
GTG (:CA TCA GAGrl G
GG CTG Ding A (: CTC: ATCT
ACTI;CCAG GTT CTGII G(
i to TG TACCT(: ATCTA to
TC(: AG GTC: (:T(:+i
'rT(: AG to GGT CAA GG
(: 'rG (: CGG TCC...-TA
TTCAAG GGG +1:AA GGCTG
CCCCTC:CA(:C:CATRGTGCT
CCTCAGT CACACT GTCAGC to GCT
TCII GTG CTC(:TCAC:CGA
C: Ace AT(: AGCCGCAT(:
1? GCCGTCTC:CTACCCG AA(:
AAG GTCAAACCTCII GCCGT(
: rCCTAC: CAG A(:CAAG
0 (: ^^(: CTC1? (:T(:
TCT GCCAT(: AAG AGG C(:(
: To TG ( : To A CGCII CTCTCT
GCCATCAAGAGOCCCTGCCAG A
GGRGAG ACG CC (: GAG GAG
GCT GAG CC (: ATG GCC mouth GAGA
ce(:(:AGAGGGGGGCTGAGGCCAAG
CC (:1? TGG TAC: GAG (:CC
ATCTA(: CTG GGCGGCGTCll
Ding GG Ding^T GAG CC: CATCTA
T (:TG GGA GGG GTCRTT
CCAc TTG GAG AAG GGT GAC
:CGG CTCAG(:TTC(:AG (:TG
GAG AAG GGT GACCGA CTCA
GCRA(:CGAG GTC: 8ACGAG
CCT GAG TAC (:TG GACII
GCT GAG ATCAAT (:GG
CCCGA(: TAT CT(: GA
R(1:TT G(:(1: GAG TCCG
GG CAG GT(: TA(: TTT
GGGII TTT Gee GAG TCT G
GG CAG GTCTA (: TTT GGG1?
AT(: ATT G to CCTGII ATCAT
T GC: CCTC Note) Symbol ゛...Pa is rabbit TN
This part exists in the base sequence of the DNA encoding F.
means no, therefore the 2 adjacent to each end of this symbol
The two codons are directly connected. Step +4 (Synthesis of oligodeoxynucleotide) Amber
2.0 μM deoxynucleoside is linked via acid residues.
Place 20 pieces of Borisdylene resin on top and bottom of stainless steel.
500μμ capacity filter with steel filter
Load into the reaction container. The resin is 1M zinc bromide dichloromethane-isopropanol.
solution (85:15) to dimethoxytrityl (D
MT) Remove the protecting group and dichloromethane-isopropyl
dimethylformamide (85:15), then dimethylformamide,
Wash with lysine, then acetonitrile, and dry with nitrogen stream.
do. Then DMT-nucleotide (207zM) and
and mesitylenesulfonylnitrotriazole (60
Add 200 μC of dry pyridine solution (μM) at 645°C.
After reacting for 20 minutes, remove the reaction solution and dry the
After washing the resin with gin, unreacted resin was washed with acetic anhydride in pyridine.
Protect residues. This cycle of deprotection and condensation is repeated.
Repeat, the desired oligodeoxynucleotide is coated on the resin.
The zero-order resin synthesized in
The above-mentioned oligodehyde is separated from the resin and purified.
Synthesis and purification of oxynucleotides were performed by Ito et al. [Nuc,
Ac, Res, I vol. 0, p. 1755 (1982)
]. In this way, as below
Oligodeoxynucleotides are obtained. ■ )5゛-he＾DingTCATGTCAT(:TTCTC
GAACCCGGAGTGA (1:AA-3'2)
3'-GTACAGTAGAAGAGCTTGGG
GCT(:ACTGTTCGG-5'3) 5'
-GCCTGTAGC(1:GATGTTGTAG(:
^^AGCC: TCAAGC-3'4)3'-A
C:ATCGGGTA(:AA(:ATCGTTdingGG
GAGTTCGACT-5' step 15 (human 'r N
Ml3mp9-HGE containing the minigene of F.
(preparation of plasmid ptlGE (I Oμg)) into IEcoR
Digested with 1% low melting point agarose gel (20 units)
After electrophoresis, a 2.9kL1 fragment was excised and eluted.
put out This fragment is a duplicate of the M13mp9 phage.
Insert into the α-R■ fragment of the mold. EcoRl fragment inserted I) NA is B
RL company's guide [User Manual (User Ma)
nual)/MI3mp7 cloning (Clonin
g) / 'Dideoxy'
Sequencing, 1980]
According to Escherichia coli JM103 (-Nie England Bio
Loves (New England 13io1abs)
, NEW IENGLAND 13io1abs CA
TALOG +981-1982]
. The product is named M13mp9-HGE. Step +6 (M13mp9-HGE m chain DNA and protein
Removal of intron 3 using Leeter E3-4) Ml 3mp9-HGE-m chain DNA is according to BRL's manual
[User Manual]
7M l3m p7 cloning (Clonin
g) /'Dideoxy'
Sequencing, 1980].
It is prepared as follows. Oligodeoxynucleo-F-t prepared in step 14
4) 3'-ACATCGGGTACAACAT
CGTTTGGGAGTTCGACT5' is an intron
It is used as a pleater for 3. intron 3 pre
The motor is named "E3-4". Pleater E3-4 is the intron 3 to be removed.
01j side (i.e. exon 3), and rear (i.e. exon 3)
4) has a complementary sequence. Removal of intron 3 was performed by Wallace et al.
Method [Science vol. 209 13
96 (1980)) as follows. E3-4 (164ng115p*ole) is T, key
phosphorylated using Nasase and ATP (3mM).
and the reaction mixture added to the template M13mp9-HGE (1,65/jg. 0,5 pmole) is 6
Heat to 5°C, cool to room temperature for 5 minutes, and then cool in ice water.
do. 0.4mM each dATP, dC:TP%dGTP
, Klenow fragment for dTTP and ATP solutions.
Klenow fragment 5 units, T
Hin buffer containing lO units of I ligase
-Wallace et al., Nuku, Aku, Res. (Nuc, ^c, Res, ), vol. 9, p. 3647 (1
981, i.e. 10mM Tris-HCl (pH 7.2)
, 2mM magnesium chloride and ImM β-mercap
Add a solution containing ethanol to the reaction mixture (ffi
The final volume fit 50 It Q) was measured at 4°C for 30 minutes and
Incubate for 30 minutes at room temperature. oligonucleo
The synthesized DNA is
PRL (BRL) Company Manual [User Manual]
User Manual/M l 3 m
p 7 Cloning /'Deide
Dideoxy' Sequencing (Seq)
Escherichia coli J
Infect with MI03 strain. The output obtained in this way
Lark is YT Bray 1 [Jay, H, Mirror (
J, Il, Miller), Experimentinzinmore
Qular Genetics (Experi+*epts
xn Mo1eclar Genetics), Cole
Cold Spr Harbor Laboratory
ing 1 labor Laboratory) (1
972) page 433]. The colonies obtained are °
``Hybrid with P-labeled E3-4 at 55℃ for 2 hours.
Breeded. Written as a result of the intron removal process
Obtain DNA with desired sequence from various products
Here, we use pleater E3-4 as a probe for
make use of itself. Thus, the pleater E3-4 and
Obtaining colonies that hybridize and further acquiring phages
Ru. The resulting phages are plated and the resulting plaques are
Transfer to YT plate, here again radiolabel with °゛P
Hybridize with E3-4 at 55°C for 2 hours.
From strongly hybridized clones, fur
We obtained di-DNA, analyzed the base sequence, and found that intron 3
Select phages that have been completely removed. like this
One of the phages was designated mp9-HG EΔ3-1.
Name. Step 17 (Construction of pHTNF-QaclJV5-2) The replication form of mp9-HG EΔ3-1 was converted into EcoRl
and isolated by electrophoresis. Eco this fragment
plasmid pBR327 cut with Rl.
Gains 3-1 to HG IE. Next, using plasmid pHGEΔ3-1, QacUV5
]'NF as a promoter directly in E. coli.
Construct a plasmid capable of expression (1X!). This construction method is shown in FIG.
^val and Ecorl I (B RL
Digested at 37℃ for 2 hours at 4ffiffi%
Target fragments by rearacrylamide gel electrophoresis
isolate. Approximately 1 μg of fragments were separated from the gel by electrophoretic elution.
recovered from. Same as step 14 as shown in Figure 7.
Two types of oligodeoxynucleotides, namely 5゛-^^T
TC^TGTGATCTTCTC:GAACC knee 3
'and 5'-TGGGGGGTdingCGAG^^GATG
Synthesize ACATG-3'. Next, literature (3)
l 22 According to the true method, these two oligonucleotides
(approximately 100 pmole) 5゛ end is T, polynucleotide
phosphorylate using dokinase. After the reaction, it is extracted with phenol and then with chloroform. The synthetic oligomer thus obtained and the p obtained above
HG EΔ3-1^va I-EcoRI fragment 0
．． After mixing with 5 μg and precipitating with ethanol, the method described in the literature (1
) using ligase according to the method on page 37.
After the reaction was completed, the mixture was evaporated into ether.
Nol precipitation and 4 wt% polyacrylamide gel electrophoresis
Isolate the target fragment by motion. Plasmid pOP95-15 was prepared using the method of F. Fullor [Gene I 90
．． 4.2-54 (1982) j. The t/Ag of pOP95-15 was transformed with EcoRI.
Phenol extraction, chloroform extraction, ethanol precipitation
and #i) as above.
The fragments are ligated using T. DNA ligase. experiment
1F (4), p. 20, using the obtained vector to
The fungus JMIOI strain (ATC:C33876) was transformed.
1mM IPTO and 0°004% (w/v) x
Approximately 100 white colonies on agar medium containing -gal
get. Plasmid DNA was prepared from these 'transformants,
Digest with EcoRI and create a plate containing the desired 10 fragments.
Identify Sumid. Furthermore, to investigate the direction of DNA insertion.
These plasmids were digested with Pvu U and 1'stl to
The results of 1.5% by weight agarose gel electrophoresis
It has fragments of about 1280 bp and about 2600 bp, and 1a
Copy TNF correctly downstream of the cUV5 promoter.
Select the plasmid to which the DNA to be coded is connected.
. When the base sequences were determined, the two plasmids showed the same base sequence.
sequence, synthetic oligonucleotides and chromosome-derived D
It shows that the NA is connected correctly. 11) et al.
The resulting plasmid was named pHTNF-IacUV5-2.
do. Large+19 bacteria containing pNTNF-1actJV5-2
is cultured in regular Sakae 4A medium. Product TNT” activity
When measuring sex, it is controlled by the lac promoter.
pTNFlacLJV5 containing the rabbit rNF gene
It has almost the same activity as the E. coli product containing -1.
show. Example 2 FJ11 according to steps 1 to 14 in Example 1! difference
Plasmid pIICE and oligonucleotide 1)
-4), pH"I'N F-1acU V
Prepare 5-1. The preparation method is shown in FIG. Novel microorganisms and cultured cells forming part of the present invention
The cells are important because they have the ability to produce human TNF.
It will be understood that this is new, therefore, the following
Transformed microorganisms and cultured cells prepared as described in
In addition to cells, the present invention further provides
Also includes those mutant strains and mutants that show activity.
It is something to do. Microorganisms and new plasmids containing the plasmids
By depositing samples of microorganisms, the United States;
Rockville, Maryland
Maryland, U, S, A) American Thai
Culture Collection (America Mouth Ty)
peCulLure Co11ection) 198
It was deposited on April 6, 2004. Microorganism Escherichia coli
2JM83 (pRGE) strain has ATCC deposit number 39.
No. 655 was infected with E. coli -12JM83 (pHGE) strain
has been assigned ATCC deposit number 39656.
. As described above, the present invention can be applied to various methods.
can be achieved with. Such changes are within the scope of the present invention.
It should not be considered as something outside the scope of God;
All such changes that are obvious to a person
This is included in the scope of Patent R/j.

[Brief explanation of the drawing]

FIG. 1 is a restriction enzyme map of plasmid inserts each containing DNA encoding a conventional rabbit physiologically active polypeptide. Figure 2 shows a recombinant DNA (pi'NF-1ac-
1) is a flow sheet showing the preparation method. Figure m3 is a flow sheet showing a method for preparing another recombinant DNA (p-I"NF-1ac UV5-1) encoding a conventional rabbit physiologically active polypeptide. Figure 0m4 is a flow sheet showing a method for preparing another recombinant DNA (p-I"NF-1ac UV5-1) encoding a conventional rabbit physiologically active polypeptide. Figure 5 is a restriction enzyme map of a part of a plasmid containing a gene for a peptide. Figure 5 is a restriction enzyme map of a part of a plasmid containing a gene for a conventional rabbit physiologically active polypeptide. recombinant DNA encoding a human physiologically active polypeptide of
It is a flow sheet showing the preparation method of (pHTNF-1acUV5-1). FIG. 7 shows another set of tADNA (pHT
1 is a flow sheet showing a method for preparing NF-1acUV5-2). Patent applicant: Asahi Kasei Industries, Ltd.

Claims (6)

[Claims] (1) The following formula (I) [There is a gene sequence] (In the formula, Gln is a glutamine residue, Asp is an aspartic acid residue, Pro is a proline residue, Tyr is a tyrosine residue, Val is a valine residue, Lys is a lysine residue, Glu
is a glutamic acid residue, Ala is an alanine residue, Asn is an asparagine residue, Leu is a leucine residue, Phe is a phenylalanine residue, Gly is a glycine residue, His is a histidine residue, Ser is a serine residue, and Thr is a threonine residue. residue, Ile is an isoleucine residue, Trp is a tryptophan residue, Arg is an arginine residue, Met is a methionine residue, and Cys is a cysteine residue). Encoding deoxyribonucleic acid. (2) Base sequence represented by the following formula (II) [There is a gene sequence] (In the formula, A is a deoxyadenylate residue, G is a deoxyguanylate residue, C is a deoxycytidylate residue, and T represents a thymidylic acid residue, the left end and right end of formula (II) represent the 5'-hydroxyl group side and the 3'-hydroxyl group side, respectively) and at least one base sequence complementary to the base sequence. Claim 1 contains a base sequence obtained by substituting at least one base in at least one base sequence of the deoxyribonucleic acid based on the degeneracy of the genetic code. deoxyribonucleic acid. (3) The following formula (I) [There is a gene sequence] (In the formula, Gln is a glutamine residue, Asp is an aspartic acid residue, Pro is a proline residue, Tyr is a tyrosine residue, Val is a valine residue, Lys is a lysine residue, Glu
is a glutamic acid residue, Ala is an alanine residue, Asn is an asparagine residue, Leu is a leucine residue, Phe is a phenylalanine residue, Gly is a glycine residue, His is a histidine residue, Ser is a serine residue, and Thr is a threonine residue. residue, Ile is an isoleucine residue, Trp is a tryptophan residue, Arg is an arginine residue, Met is a methionine residue, and Cys is a cysteine residue). A replicable recombinant DNA containing an encoding deoxyribonucleic acid and a replicable expression vector. (4) Base sequence represented by the following formula (II) [There is a gene sequence] (In the formula, A is a deoxyadenylic acid residue, G is a deoxyguanylic acid residue, C is a deoxycytidylic acid residue, and T represents a thymidylic acid residue, the left end and right end of formula (II) represent the 5'-hydroxyl group side and the 3'-hydroxyl group side, respectively) and at least one base sequence complementary to the base sequence. A patent claim containing a deoxyribonucleic acid containing a nucleotide sequence obtained by substituting at least one base of the deoxyribonucleic acid based on the degeneracy of the genetic code, and a replicable expression vector. The replicable recombinant DNA according to item 3. (5) The following formula (I) [There is a gene sequence] (In the formula, Gln is a glutamine residue, Asp is an aspartic acid residue, Pro is a proline residue, Tyr is a tyrosine residue, Val is a valine residue, Lys is a lysine residue, Glu
is a glutamic acid residue, Ala is an alanine residue, Asn is an asparagine residue, Leu is a leucine residue, Phe is a phenylalanine residue, Gly is a glycine residue, His is a histidine residue, Ser is a serine residue, and Thr is a threonine residue. residue, Ile is an isoleucine residue, Trp is a tryptophan residue, Arg is an arginine residue, Met is a methionine residue, and Cys is a cysteine residue). Containing at least one base sequence selected from the group consisting of a coding base sequence and a base sequence complementary to the base sequence, or based on the degeneracy of the gene code,
A microorganism transformed with a replicable recombinant DNA containing a deoxyribonucleic acid containing a base sequence obtained by replacing at least one base of the deoxyribonucleic acid and a replicable expression vector. (6) Claim 5, wherein the base sequence encoding a human physiologically active polypeptide containing the amino acid sequence represented by the formula (I) is represented by the following formula (II).
Microorganisms or cells described in Section. Formula (II) [There is a gene sequence] (In the formula, A represents a deoxyadenylic acid residue, G represents a deoxyguanylic acid residue, C represents a deoxycytidylic acid residue, and T represents a thymidylic acid residue, and the formula ( The left end and right end of II) represent the 5'-hydroxyl group side and the 3'-hydroxyl group side, respectively)