JPS60262594A - Human interferon-gamma - Google Patents

Abstract

PURPOSE:A natural-type human interferon-gamma having high safety and obtained by culturing a specific transformed cultured animal cell. CONSTITUTION:A nucleus DNA coding human interferon-gamma and a DNA sequence connected with the above DNA and containing the promoter domain of other gene functioning in a cultured vertebrate cell, are prepared beforehand. A cultured animal cell is transformed with the nucleus DNA sequence, and the transformed cultured animal cell is proliferated in the body of an animal or in a culture liquid to produce human interferon-gamma.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides human interferon-γ()IuIFN-
A DNA sequence in which a nuclear DNA sequence encoding γ) and a DNA sequence of a promoter region that functions in animal cells are linked;
The present invention also relates to heterologous and homologous cells into which the DNA sequence thereof has been introduced to produce HuIFN-γ, and a method for producing Hu I FN-γ using the cells. The present invention also relates to the amino acid sequence of HuIFN-γ produced by the above production method.

(Prior Art) Interferon (IFN) was discovered as a substance having an antiviral effect, and is a protein that induces an antiviral state that is nonspecific to viruses at least in cells of the same type. Human interferon (HuIFN) is classified into three types depending on the physiological, biochemical, and immunological aspects of the protein, as well as the production cells and induction methods. β (IFN-β) and interferon-γ (I
FN-r) (Stewart ll
.

W, E, et al. (1980) Nature, vol. 286, p. 110). Recently, the complementary DNs of these three species
A (C+DNA) was cloned, and the base sequence of the CDNA and the amino acid sequence deduced from the base sequence were determined. Human interferon-α1 (HuIFN-α
1) is a protein with 166 amino acid residues, and has acid (PH2
, 0), heat (56°C), 0.1% SDS, etc. More than 10 nuclear genes for HuIFN-α have been identified, and it has been revealed that there are more than 10 subspecies of HuIFN-α (Goeddel, D, V, et al. (1981) Nature.

Volume 290, 2ON). Human interferon-β (Hu
IFN-β) is a glycoprotein with a molecular weight of 22,000 to 23,000, and is stable in acid (pH 2.0) but resistant to heat (pH 2.0).
It is unstable at 56°C). It has 166 amino acid residues (Taniguchi, T. et al. (1980) Gene
, vol. 10, p. 11), it is thought that there is only one type of gene in cells. HuIFN-7 is unstable to acid (pH 2.0), heat (56°C), or 0.1% SDS treatment.
FN with molecular weights of 20,000 and 25,000 has been found (Yip, Y., et al. (1982)).
proc, Natl, Acad, Sci.

USA, Vol. 79, p. 1820). It is a glycoprotein with 146 amino acid residues, and only one gene is known to exist (Gray, P., W. et al. (1982) N.
ature, vol. 295, p. 503). IIFN was discovered as an antiviral protein, but the antiviral effect of IFN is species-specific; in virus infection tests using human cells, HuIFN exhibits antiviral activity, but mouse IFN does not. However, the specificity of IFN for viruses is quite wide, and it shows activity against various viruses.

As the mechanism of action of the antiviral activity of IFN, ■ double-stranded R
Inhibition of protein synthesis associated with phosphorylation of protein synthesis initiation factor (eIF-2) by NA-dependent protein kinase (R
ober ts, W., et al. (1976) N.
ature, vol. 264, p. 477; Kinchi,
A.

(1979) Proc, Natl, Acad,
Sci.

USA, Vol. 76, p. 8208), ■Activation of RNA degrading enzyme by 2-5A produced by 2-5A synthetase (Hovaressian, A. G., et al. (1977)
(Nature, Vol. 268, p. 537), or (2) Inhibition of protein synthesis due to induction of phosphogesterase and cleavage of the CCA terminus of tRNA (Revel.

M (1979) Interferon 1979 (
ed, Gresaer, 1. ), 102 pages, Acade
micPress) etc. are being considered. IFN has already been used to treat or prevent viral infections, including hepatitis B virus, cytomegalovirus, varicella virus, herpes zoster virus, human papilloma virus,
Clinical trials are being conducted for diseases caused by various viruses such as rhinovirus and herpes simplex virus, and results have also been shown to be very effective against certain viral diseases. It is also expected to be used in the prevention and treatment of human adult leukemia, for which a relationship between viruses and disease onset has recently been revealed.

IFN was discovered as an antiviral substance, but later...
It has been shown that IFN is a substance with various biological and immunological activities. IFN has been known for a long time to have the effect of suppressing cell proliferation (Rubin, B.
Y. et al. (1980) Proc.

Natl, Acad, Sci, USA, 7
7, p. 5928), and with recent advances in immunology, IFN has been shown to activate natural killer cells and cells with antibody-dependent cytotoxic activity, which are thought to be involved in the so-called cancer immune surveillance mechanism. It has become known that it enhances the antitumor activity of these cells (C
atalona, W, J, et al. (198
1 year) Nature, vol. 291, p. 77). Also, enhancement of cytotoxic T cell activity (Lindahl, P., et al.
972) Proc, Natl, Acad, S
ci,, 69 volumes.

721) and that IFN has the effect of activating macrophages and turning them into antitumor activated macrophages (Lel J et al. (1988) J, Immun.
ol, vol. 181, p. 2821). These results showed the potential of IFN as an antitumor agent, but clinical trials of IFN are already underway for various tumors, including multiple myeloma, non-Hodgkin's lymphoma, renal cancer, and breast cancer. Other effective examples are also becoming known.

Among IFNs, IFN-γ is IFN-α.

It can suppress cell proliferation at much lower concentrations than IFN-β (Rubin, B, Y, et al. (1980) Proc, Natl, Acad, Sci, USA
.

(Vol. 77, p. 5928), it can also activate cell groups that function in the so-called cancer immune surveillance mechanism, such as natural killer cells, killer T cells, human cells, and macrophages, making it useful in clinical applications. Expectations are high.

HuIFN-γ is known to be induced in human lymphocytes upon stimulation with phytohemagglutinin, staphylococcal enterotoxin A, concanavalin A, or galactose oxidase (Wheelock).
, E.F. (1965) Science, vol. 149, p. 310: Langford.

M, P, et al. (1979) Infect, Immu.
n.

Vol. 26, p. 86 + de Lay + M, et al. (1980) Eur, J., Immunol.
Volume 10, page 877; Dianzani, F., et al.
979) Infect.

Immun, vol. 25, p. 879). However, the production method described above requires a large amount of fresh lymphocytes, making it difficult to mass produce I(uIFN-γ) as a therapeutic agent. In recent years, the CDNA of HuIFN-γ has been cloned, and HuIFN-γ has been cloned in Escherichia coli. It became possible to produce γ-like proteins (Gray, P., W., et al. (1982)).
ure, vol. 295, p. 503). However, since the protein synthesis mechanisms of IFN-γ produced by microorganisms are somewhat different between animal cells and microorganisms, the amino terminus of the produced protein often differs from that of the natural protein.

In fact, the amino terminus of recombinant HuIFN-γ produced in Escherichia coli is methionine, whereas that of natural HuIFN-γ is cysteine.

Furthermore, IFN-γ produced by microorganisms does not have sugar chains attached to it, whereas natural HuIFN-γ has sugar chains. In this way, IFN-γ produced by the protein synthesis system of microorganisms and natural HuIFN-γ are different substances, and when used for a long time or frequently as a therapeutic agent, the titer may decrease due to the antigen-antibody reaction. There are concerns about allergic reactions such as reduction and shock. Le et al.
It has been reported that the reactivity of the antigen-antibody reaction using a monoclonal antibody differs between γ and γ, and it was shown that this difference in reactivity is not due to the presence or absence of sugar chains (La et al. (1984) J. Immunol.

132 volumes, 1100 pages). On the other hand, established T cell clones (Nathan, H, et al. (1981))
e, vol. 292, p. 842), T cell hybrid-7 (L
ll et al. (1982) Proc, Natl.

Acad, Sci, USA, vol. 79, p. 7857), adult leukemia virus-transformed T cells (Su
gamur+a, K et al. (1988) J, Imm.
uno 1. .

131, p. 1611), it is thought that these cells contain human leukemia virus as a provirus or release virus particles to the outside of the cells. C8ugamura et al., where the issue of risk remains. (1988) J.
, Immunol, vol. 181.

1611 pages).

In addition, HuIFN-γ (DCDNA sequence NiSV 4
HuTFN-0 promoter in animal cells.
Attempts were made to produce γ (Gray, P, W, et al. (1982) Nature, vol. 295, p. 5o3; Haynes, J. et al. (1983) Nuc
leic Acids Rag, vol. 11, p. 687;
5cahi l l.

S, J, et al. (1988) Pro oc, Nat.
l, Acad.

Sat, USA, vol. 80, p. 4654; Devos.

R1 et al. (1982) Nucleic Ac1ds
Reg.

Volume 10, page 2487). Many proteins in higher organisms are 1i
It is known that the DNA sequence is encoded in several parts. A DNA sequence encoding the amino acid sequence of a protein is called an exon (eXOn) plus an intervening sequence called an intervening sequence or an intron. Although much of the biological significance and function of introns is currently unknown, ovalbumin (Wickens
, M. P. et al. (1980) Nature.
285, p. 628) and viral proteins (Lai, C-J
, et al. (1979) Proc.

Natl, Acad, Sci, USA, vol. 76, 71
It is known that gene sequences that do not contain introns (page) produce significantly less protein in animal cells into which they are introduced, compared to sequences that contain introns. Furthermore, it has been found that adding an intron of the β-globin gene to the SV40 gene lacking an intron results in stable accumulation of metsenger RNA (mRNA) (Hamer, D., Hl et al. (1979)). CeII, 1
Volume 8, page 1299).

(Problems to be solved by the invention) Splicing is the removal of intron sequences from initial RNA transcribed from a gene.
However, splicing is presumed to be an event necessary for the accumulation of stable mRNA or the translocation of mRNA from the nucleus to the cytoplasm. Therefore, some of the production methods using the HuIFN-γ intron-free cDNA sequence described above involve adding a viral intron to the HuIFN-γ cDNA sequence to generate intracellular mRNA.
It is estimated that this enables the accumulation of HuIFN
As shown in Figure 1, the region containing the -γ gene consists of four exons, three introns, the 5' flanking sequence, and the 3'
It is composed of flanking sequences (Gray, P
, W. et al. (1982) Nature, vol. 298,
859 pages; Taya, Y et al. (1982) EMBo
J, vol. 1, p. 953).

The present inventors demonstrated that HuIFN-γ with native introns
m that is transcribed from the nuclear DNA sequence and undergoes splicing.
We hypothesized that RNA may be more stable and accumulate in the cytoplasm at higher concentrations than mRNA transcribed from fCD DNA sequences. In the 5-side flanking sequence of the HuIFN-γ gene, there is a regulatory site that controls the expression of HuIFN-γ, and in particular, a region containing a DNA sequence to which RNA polymerase binds and initiates mRNA synthesis. is called a promoter region, but the present inventors
We have learned that even if the HuIFN-γ gene sequence containing this region is directly introduced into animal cells, the production of HuIFN-γ is very weak.

This result indicates that the promoter region on the 5 side of the HuIFN-γ gene is in a dormant state in most animal cells, and therefore, in order to observe effective production in animal cells, this promoter region is m R area with animal cultured cells
This indicates that it is necessary to replace the promoter region of another gene that can initiate NA synthesis.

Splicing of introns at the correct positions is essential for normal and functional protein expression, but the insulin gene and simian virus 40 (SV'40.M
) Aberrant splicing has been reported when the promoter region is introduced into CO5 cells (Lanb
, Oo et al. (1988) J. Biol.Chem.
Volume 258, page 604B).

It is also known that tissue-specific splicing exists in the expression of amylase, and salivary gland amylase and liver amylase are synthesized from the same gene through two different splicing processes (Young, R.
A. et al. (1981) Cell.

Volume 23, page 451). Also, 5V40 (Bark.

A, J., et al. (1978) Proc, Natl., A.
cad.

Sci, USA, vol. 75, p. 1274), adenovirus (Chow, L. T. (1977) Cell.

12, p. 1), multiple mRNAs and proteins are synthesized from the same gene through different splicing processes. Therefore, normal splicing must occur in order to introduce the HuIFN-γ gene containing an intron into cultured animal cells and produce HuIFN-γ.
A sequence that functions in animal culture cells, i.e. rn RN
DNA of the promoter region that can initiate A synthesis
It was found that when the A sequence was ligated and introduced into various animal cultured cells, splicing occurred normally and HuIFN-r was secreted into the culture medium in significant amounts. It is believed that the present invention makes it possible to supply a large amount of extremely safe natural HuIFN-γ. The present invention will be explained in more detail below.

(Structure of the invention; means for solving problems) HuIF
The gene region encoding N-γ is contained in a DNA fragment of about 8.6 kilobases (Kb) when human chromosomal DNA is cut with the restriction enzyme BamHI as shown in FIG. The sequence (exon) encoding the HuIFN-γ polypeptide is divided into four locations. The 1st exon, 2nd exon, 3rd exon, and 4th exon from the 5' side of the DNA sequence (indicated by thick lines in the figure).

The sequence adjacent to the 5th side of the first ecun contains HuIFN-
There are regulatory sites that control the expression of γ. In addition, the sequence adjacent to the 3' side of the fourth exon contains the mRNA polyadenylation oxidation signal AATA following the stop codon.
AA exists. The first exon encodes a polypeptide consisting of 38 amino acid residues.
The terminal 20 amino acids are signal peptides found at the N-terminus of many secreted proteins, and HuIFN-r is synthesized.
It is cleaved during the process of being secreted outside the cell. HuIFN-γ
The K DNA sequence encoding this refers to a DNA sequence containing the four exons and three mentrons shown in FIG.

[DNA sequence is Hu) DN encoding HuIFN-γ
Cloned from A. Human DNA can be obtained using the method of Blin et al. (Blin, N. et al. (1976) Nucleic
Aclds Res, Vol. 8, p. 2303). Vectors used for cloning the HuIFN-γ gene include lambda phage vectors typified by Charon28, plasmid vectors typified by pBR822, and cosmid vectors typified by pHC79; A genetic engineering method is used in which a lambda phage vector, which can clone DNA fragments of strands, is used as a vector. That is, after cutting human high molecular DNA with an appropriate restriction enzyme, it is inserted in place of the replaceable region of a λ phage vector to generate recombinant phage DNA. Next, using an in vitro packaging method, infectious Phage particles are created. They are then plated with host E. coli to form recombinant phage plaques (
Enquist, L. et al. (1979) Methods
inEnzymology Vol. 68, p. 281; Hor
n, B. (1979) Methods' in Enzy.
(Vol. 68, p. 299). To detect plaques of recombinant phage containing a DNA fragment encoding HuIFN-7, a plaque hybridization method using CDNA or synthetic NA as a prologue (Woo, S.
, L.C. (1979) Methods in En.
Zymology, Volume 68, Page 389 + 5zosta
k.

J, W et al. (1979) Methods in E
nzy-mology, Vol. 68, p. 419). Furthermore, recombinant phages carrying the HuIFN-γ gene can be prepared in large quantities by recovering them from plaques selected by plaque hybridization and culturing them with host E. coli. In addition, recombinant phage D
NA can be prepared by the phenol method etc. (Maniat
is, T et al. (1982) Molecular Clo
ning a Laboratory Manual, C
o1d Spting Harbor Labo-ra
tory).

Although there are differences in transfection efficiency as a method for introducing DNA into cultured animal cells, the calcium phosphate method (Wi
glet, M. et al. (1977) Cell.

I volume 1, p. 228), microinjection method (
Ander aon, W, 'F-, et al. (198
0 years) Proc.

Natl, Acad, Set, USA, ? '1 volume,
p. 5899), ribosome method, DEAE-dextran method, or cell fusion method (Schoffner, W., et al.
980) Proc, Notl, Acad, Scf
, USA, Vol. 77, p. 2168), etc. are used. As the DNA material used in the calcium phosphate method, in addition to a DNA solution, microorganisms such as Escherichia coli, phages, etc. can be used. The cell fusion method uses microbial prodoplasts that carry the target DNA sequence as a plasmid.

HuIFN-1 is an inducible protein and is induced by stimulating human leukocytes with various mitogenes.

Currently, mitogen stimulation or HulF
It is unknown whether HuIFN-r is induced by acting on the N-γ gene, but Hul
Even if a gene that blocks the regulatory site of FN-γ is introduced, HuIFN
-γ production was weak. The present inventors connected the sequence of the promoter region of another gene that functions in cultured animal cells to the 5' side of the nuclear DNA sequence of HuIFN-γ and introduced it into the cells. We have discovered that producing cells can be transformed into high producing cells.

In this case, HuIFN-r mRNA synthesis is under the control of the connected promoter region (j7; for example, if the connected promoter region is the promoter region of a constitutive protein gene, HulFN-r m of γ
RNA is constantly synthesized, so cells have HulFN-
It becomes a constitutive producer cell of f. If the connecting promoter region is that of an inducible protein, the transformed cells
Produce ulFN-γ as an inducible protein.

The SV40 early promoter is known as a promoter that functions in cultured animal cells.

Conobromo 9- is 5V40 DNA H4ndlll-
Pvu]l fragment, approximately 840 hair pairs (bp)
SV40 T antigen and t antigen mRNA are synthesized from this promoter. The starting point for mRNA synthesis is around 260, as shown in the table below.
ATG of the first amino acid (methionine) codon of T antigen
339 (indicated by underline in the table below).

II) ■ CATAGTCCCGCCCCTAACTCCGCCC
ATCCCGCCCCTAACTCCGCCCAGTT
(Note →mRNA indicates the vicinity of the mRNA synthesis start site) The herpes simplex virus (H5V) type I thymidine kinase promoter is also a constitutive promoter like the jV40 early promoter, and the structure of the region was shown by Wagner et al. Although it is (W
agner, M. J., et al. (1981) Proc, Na.
tl, Acad, Set, USA, 79 volumes.

(page 1441), m RNA is 208 as shown in the table below.
It is synthesized from the 08th position, and polypeptide is synthesized from the 310th ATG. (In the table below, m RNA is m
The vicinity of the RNA synthesis initiation site is shown, and Δ<-> indicates the codon for methionine, an amino acid of thymidine kinase polypeptide. ) What is a functional promoter region?
Refers to the sequence of a promoter region that contains the initiation site for mRNA synthesis but does not contain the methionine codon, the first amino acid regulated by these promoters.

From the above points of view, the promoter region sequence and Hul
Example 4 shows the preparation of DNA in which the FN-γ wing DNA sequence is connected.

(2) The amino acid sequence of ulFN-γ can be deduced from the base sequence of the exon portion of the cloned HuIFN-γ gene. The base sequence was determined using the Maxam-Gilbert method (M
axam, A., M., et al. (1980) Methods
in Enzymology, vol. 65, p. 499)
It can be determined by

When a gene is introduced into a cell, the introduced gene may be stably integrated into the host chromosomal DNA. The location on the chromosome where the gene is integrated appears to be random, and the number of copies of the integrated DNA is also irregular. HuI
When FN-γ is introduced into cells, the location and number of copies of FN-γ integrated differs from cell to cell, and the amount of IFN produced by each cell differs.

Therefore, by cloning cells, cells having various production amounts can be obtained. Introduce the target gene,
In order to selectively proliferate only cells that stably express
D that has a functional promoter sequence, a sequence connecting the HuIFN-γ gene, and a selection marker on the same DNA sequence.
NA sequences are suitable. Ecogpt (Muligan, RlC,
(1980) Science, vol. 209, 142
2 pages), ne.

(5outhern, P. J. et al. (1982) J.
Mol.

Appl, Genet, vol. 1, p. 327), dhft (
Wigler, M., et al. (1980) Proc., N.
atl.

Acad, Set, USA, Volume 77, 8567
Genes such as (page) are used. Also, like that N
In order to prepare large quantities of A sequences, it is desirable that such NA sequences be in the form of plasmids or phages that can be replicated in E. coli and can be prepared in large quantities. The plasmids psVesmaIγ and pSV2pTKy shown in Example 4 are plasmids that meet the above objectives.

That is, the origin of DNA replication (o) that enables replication in E. coli.
ri) and the [D
This plasmid is characterized in that the NA sequences are present on the same DNA sequence.

Hu that connects the promoter regions of other functional genes
Cells introduced with the nuclear DNA sequence of IFN-γ are HulF.
In order to produce N-γ, the DNA sequence needs to be compatible with the functions of the cell-specific RNA synthesis system, RNA maturation, protein synthesis system, protein maturation, secretion, etc. mRNA is synthesized from the introduced DNA, but mRN
The 5' end of A requires addition of a cap structure, splicing at the normal position, and polyazonylation at the 3 end. In addition, the expression of active IFN requires the formation and maintenance of the normal higher-order structure of the synthesized IFN peptide.
Furthermore, cleavage of the signal peptide and secretion from cells must be performed accurately. The animal cultured cells used by the present inventors and colleagues were cells derived from hamsters, monkeys, chimpanzees, and humans available from the American Type Culture Collection (ATCC), but the cells used in the production of HuIFN-γ as described herein are By using this method, at least vertebrate-derived cultured cells, fused cells, normal and mutant cells,
HuI active in virus-transformed cells, etc.
It is possible to produce FN-γ.

For example, CHO (hamster ovary cells), ) (eLa
(human cervical cancer cells), FL (human amniotic cells), WI
SH (human amniotic cells), Chimp, Liver (chimpanzee hepatocytes), and WI-26VA4 (human lung cells transformed with SV40) can be used.

Currently, there are many unknowns about the structure of the sugar chain of HuIFN-γ. For example, T(ul) produced in hamster cells
It is unknown whether there is a difference in the sugar chain structure and antigenicity of FN-f and HulFN-γ produced in human cells. However, human cells produce natural HuIFN-γ, which would avoid problems such as allergic reactions and shock during long-term administration as a therapeutic agent. In addition, human cells were SV4
Using a cell line transformed with 0 as a production cell is expected to improve the safety of the product by taking appropriate measures compared to cells that have become cancerous or established due to unknown causes.

A Hu I FN-γ wing DNA sequence connected to a functional promoter is introduced into cultured animal cells using, for example, the calcium phosphate method, and the cells that begin to produce Hu INF-γ do not contain the serum normally used for cell culture. HuI not only in a serum-free medium but also in a serum-free medium that does not contain any serum.
It was discovered that FN-γ was produced. Using a serum-free medium for the production of HuIFN-γ makes it easier to recover and purify HuIFN-γ from the medium.

(Effects of the Invention) Based on current knowledge, cells that have become a HulFN-γ producing strain through gene introduction as described above can be grown in animals such as nude mice or hamsters. In addition, HuIFN-
It seems possible to recover γ. It is also possible to produce HuIFN-γ by growing cells in an animal body, collecting the cells, and culturing them in a culture medium.

(Example) Examples are shown below, and experiments related to the present invention were conducted in accordance with the "Recombinant DNA Experiment Guidelines" established by the Prime Minister. Further, for detailed operations involving phages, plasmids, DNA, various enzymes, Escherichia coli, etc. in the Examples, the following magazines and books were referred to.

1. Proteins, Nucleic Acids, Enzymes, Vol. 26, No. 4, (1981) Extra Issue Genetic Manipulation (Kyoritsu Shuppan) 2. Genetic Manipulation Experimental Methods, edited by Yasutaka Takagi (1980) Kodansha 3, Genetic Manipulation Manual, edited by Yasutaka Takagi (1982) ) Kodansha 4, Mo1ecular Cloning a 1ab
orat-ory manual, T, Mania
tis et al. (1982) Co1d Spring
Harbor Labora-ory 5, Methods in Enzymology,
Volume 65.

Edited by L. Grossman et al. (1980) Acade.
-mic Press 6, Methods in Enzymology,
Volume 68.

Edited by R1Wu (1979) Academic Pre
ss Example I Cloning of HuIFN-γ gene After collecting blood from multiple healthy adults and collecting buffy coats,
After hemolysis by adding about 10 times the amount of 0.83% NH4, white blood cells were obtained by washing with Eagle's MEM medium.

1010 white blood cells, 50 mJ (D 0.5 M
EDTA.

50ml solution of 0.5% Sarcosyl, iooμ, 9'/ml protease! The solution was dissolved by shaking at 50'C for 8 hours. Phenol extraction was performed three times, and the aqueous layer was reduced to 20mM.
Trig-HCJ (pH 8,0), 10mM EDTA
.

Dialyzed against 10mM NaCJ. The dialysate was heated to 37°C.
8.5 hours Ribonuc L/7-se (100ttll/ml
) and after phenol extraction, 20mM Tris
・HCl (pH 8,0), 1mM EDTA, 10m
Dialysis was performed against MNaCJ to obtain high molecular weight human DNA of approximately 88 m, p. After cutting human DNA with the restriction enzyme BamH1, a BamHI DNA fragment having a size of about 8 to 9 kilobases was prepared by sucrose density gradient centrifugation.

After cutting lambda phage vector harom 28 DNA with BamHl, Char
Collect both parts including the left end fragment and right end fragment of on 28,
It was recovered by ethanol precipitation.

After ligating the DNA fragment at both ends of Charom28 and the human 8-9 kilobase BamH1 fragment using T4 DNA ligase,
Enquist and Stambarb Method (L, Enquist
and Sternberg, N. (1979) Met
Hods Enzymol, Volume 68, Page 281)
In vitro packaging was performed using E. coli LE.
Plaques of recombinant phage were formed using 892 as a host. Next, the plaque hybridization method (
Benton, W. D., Davis.

R, W. (1977) 5science, vol. 196, 1
A recombinant phage clone containing the HuIFN-γ gene was selected using the following method (p. 80). As a probe, Hu
Oligonucleotide CTTGGCTGTTAC, CCTGGCAGT having a sequence present in the IFN-γ gene
AAC and GCTCTTCGACCTCG were synthesized using the phosphotriester method (Miyoshi, K etal, (198
0) Nucleic Ac1ds Rea.

8, p. 5507), and synthesized 5'-OH to [γ-P]
It was used after labeling with ATP and T4 polynucleotide kinase.

Four strains of phage clones 88-11, 518-6, 519-5 and 520-1 were obtained from approximately 2 million recombinant phage clones that hybridized with all three types of synthetic NA probes used.

As a result of preparing DNA from each phage clone and cutting it with the restriction enzyme BamHI, all phage clones contained a DNA fragment of about 8.6 kilobases, and the size of 4 pieces cut with various restriction enzymes was Four recombinant phages selected from the analysis and DNA sequence analysis described below were identified as phages containing the BamHl 8.6 kilobase fragment of HuIFN-γ.

Example 2 pBRγ8.6-1, psV2r 8.6-1 and p
Construction of sV2r8.6-2 Phage clone S8- containing HuIFN-γ gene
After cutting the DNA of No. 11 with the restriction enzyme BamHI, the same (B
Plasmid pBR322 (Bolivar,
F. et al. (1977) Gene, 2 vols.

95) and transformed E. coli C6C600r. Among the transformants with ampicillin-resistant and tetracycline-sensitive traits, we selected a transformant with a plasmid in which the HuIFN-γ gene of approximately 8°6 kilobases was inserted into the BamH1 site of pBR822, and transformed the plasmid into pBRγ86-1. And so.

Figure 2 shows the structure of pBRγ86-1 (in the figure, B,
E and M are restriction enzymes BamHI, respectively.

Recognition sites for EcoRI and MstII are shown. Ampγ
is ampicillin resistance gene, HuIFN-γ is HuI
FN-γ gene is shown). pBRγ86-1 with BamH
A DNA fragment of about 8.6 kilobases was prepared by agarose gel electrophoresis and the same (BamHI-opened and bacterial alkaline phosphatase treated plasmid psV2gpt (Mulligan, R.C.).

and Berg, P. (1980) 5science,
209, p. 1422) using T4 DNA ligase, and E. coli C600rrn- was transformed. Plasmid psV2r, the structure of which is shown in Figure 3, was selected from among the transformed strains.
8.6-1, pS, and V218.6-2 (in the figure, B, E, M, and P are restriction enzymes BamHI, EcoRI, Mstl, respectively) and P
The recognition site of vul is shown. Ampγ is an ampicillin resistance gene, Ecogpt is an E. coli guanine phosphoribosyltransferase gene, and HulFN-γ is an H
The uIFN-γ gene is shown. ) psV2γ86-1 and pSV2γ86-2 are pSV2g
This plasmid contains a DNA fragment of approximately 8.6 kilobases containing the HuIFN-γ gene at the Bam)1.1 site of pt in the clockwise and counterclockwise directions, respectively. Each plasmid DNA was prepared by cesium chloride equilibrium density gradient centrifugation.

In addition, plasmid DNA can be extracted from Escherichia coli ()cM as necessary.
aa dam- was prepared as a host strain.

Example 4 Production of psVesmalγ and pSV2pTKγ SV4
Sequence of promoter region of 0 and HuIFN-γ nuclear DNA
psVes, a plasmid with connected sequences
maIγ is I) BR8,6-1, psv2gpt and p
sV3gpt (Muligan, R.C., and Berg.

P. (1980) 5 etence, vol. 209, 1
Figure 4 - (al, -) with starting material amount (page 422)
(Produced by the method shown in bl (In the figure, B, E, H
,M.

P, Sal and Smh are restriction enzymes BamT (I,
EcoRI.

Hind Ill, Mi+t■, Pvull
, shows the recognition site by Sal I and Sma I. A
mpr, T-a g, Ecogp□.

SVe and HuIFN-γ are ampicillin-reducing genes;
The T-antigen gene, E. coli guanine phospholipon transferase gene, SV40 promoter measurement, and HuIFN-γ gene are shown. ori indicates the origin of replication of the plasmid in E. coli).

In other words, psV8gpt was cut with Hindll, and the largest DNA fragment was converted into T4 DNA! It was circularized with J gauze to prepare pH1. Next, add the PvuII site at pH 1 to 5
Changed to 5al1 site using alIIJ linker, pH 2
was created. Furthermore, the Hindl1 site at pH 2 is Hind
A SmaJ site was introduced using the lTI SmaI adapter to create pH5mal. pH5maI Sa
I, BamH of pSV2gpt cut with EcoRI
After cutting the I site with BamHI, DNA polymerase I (
The same psi (Sali, EcoR
The DNA fragment containing the ampicillin surface I gene was ligated to pSVeSma'■ using T4 DNA calase.
I made it. Next, I) BRr8.6-1 was cut with Mstn, the ends were made blunt with DNA polymerase I (Klenow), then cut with BamHI, and HuIFN-
A DNA fragment having the γ sequence was obtained and was injected with SmaI.

psV was introduced into BamHI cut pSVeSma I.
esmal, r was created.

pSV2pT is a plasmid with a sequence connecting the promoter region of herpes simplex virus type 1 thymidine kinase and the HuIFN-γ gene.
Kγ is pBRγ8.6-1゜pH5V106 (Mc
Knight, S. L., and Gabis.

E, R. (1980) Nucleic Ac1ds
Res.

Volume 8, page 5931) and psV2gpt were used as the starting materials and produced by the method shown in Figure 5 (in the figure, B, B
cl ■, Bgl, E, and Sal are each restriction enzyme B
amH), Bcl■, BglII, EcoRI.

5al) is shown. A m pγ, pTK
.

TK, HuIFN-f, and Ecogpt represent ampicillin resistance gene, thymidine kinase promoter region, thymidine kinase gene, HuIFN-γ gene, and Escherichia coli phosphoribosyltransferase gene. or
i indicates the origin of replication of the plasmid in E. coli).

The 5alI linker and SmaI adapter used were d(pGGTCGACC) and d(pAGCTC), respectively.
CCGGG) was used. Also DNA
Klsnow fragment was used as polymerase ■.

Example 5 pSVe Sma I? ' and psVpTKr into cultured animal cells and production of HuTFN-γ Hu contained in psVemaIγ and psVpTKr
In order to examine the expression of the IFN-r gene, we applied the method of Wigler et al.
Plasmids were introduced according to Er et al. (1977) Cell, vol. 11, p. 223). Add the plasmid-calcium phosphate co-precipitate to cells (8 x 10 cells/8.6 ml medium/6 cm diameter culture dish) that were previously grown in Eagle's MEM medium containing 10% neonatal bovine serum.
After 15 hours, the medium was renewed, and the culture was continued. After 48 hours, the IFN activity contained in the medium was measured between FL cells and Vascular stomatitis virus (VasculaγStom).
Anti-CPE method using Sindobis Virus or Sindbis Virus Philip, C0 et al. (-1981) Met
Hodsin Enzymology, Volume 78, 38
(page 7). As shown in the table, psVesmaI
IFN in all cells transfected with γ and psV2pTKr.
expression of activity was observed, whereas pSV2γ8.6-1
．． .

Almost no expression was observed in cells into which pBRγ8.6-1, psV5gpt, or calf thymus DNA was introduced.

ND indicates not detected, meaning 0.06 units or less.

Example 6 Production of HuIFN-γ in normal medium and serum-free medium The medium of CHO cells into which pSVeSmaIγ or psV2pTKγ was introduced in Example 5 was supplemented with 10% tallow serum, 25%
tllZ/ml mycophenolic acid, 250μg/ml
The medium was updated to MEM containing xanthine, and a mycophenolic acid-resistant strain was isolated. Mycophenolic acid part” sex strain 24
They were grown on the entire bottom surface of a hole multi-dish and cultured for 24 hours in MEM medium containing 5% tallow serum (Fe2) and MEM medium containing no fetal bovine serum, and the IFN activity contained in the medium was measured. As shown in the table, the isolated cell lines produced IFN in the presence or absence of serum.

Production of IFN by psVesmaIi-introduced CHO cells Production of IFN by psV2pTKr-introduced CHO cells Example 7 Purification and properties of uIFN-γ Mycophenolic acid-resistant CHO seeded in the same manner as in Example 6
Cells CHO-83 were cultured in MEM medium, and culture solution 120
I got mf. Add chondral pore glass (manufactured by Electronuclenics) cpG8 to 60 ml of culture solution.
50 (mesh size 120/200) to 1. After adding OI and stirring for 3 hours at 4°C, Chondral pore glass was packed in a column and 150 mM phosphate buffer (pH
7,4) After washing with 0.15MN a C73, 50%
Elution was performed with the same buffer containing ethylene glycol. Next, the active fraction was diluted 10 times with 20mM IJ phosphate buffer, passed through a 5ml ConA-5 epharose column to adsorb IFN, and then diluted with 0.15M NaC7 and 0.15M NaC7.
20 mM containing 15M α-methyl D-mannoside
! Elution was performed with J acid buffer.

The active fractions were collected and diluted with 0.2 M ammonia acetate (
Biogel P-100 (2.6 x 60 cm
) IFN samples were obtained.

The obtained sample was treated with acid (pH 2,0) or 0.1% S.
Activity was lost by DS treatment. Another purified IFN po! J
When we investigated the cytotoxic sensitization effect of I:C, we found that only human cells had a promoting effect.
Various cells were preliminarily formed in a monolayer in a 24-well multi-dish at 300 units/ml, treated with IFN for 20 hours, and then treated with ME containing poly I:C at 10 μg/ml.
When cultured in M medium for 24 hours, there was no change in CHO, BHK (hamster cells), but FL, WISH
.

In HeLa, circularization and detachment of cells were observed.

[Brief explanation of the drawing]

Figure 1 shows BamHl 8. containing the HuIFN-7 gene.
A 6 kilobase DNA fragment is shown. FIG. 2 shows the structure of plasmid pBRγ8.6-1. Figure 8 shows plasmids pSV2γ8.6-1 and pS
The structure of V2γ8.6-2 is shown. FIG. 4-(a) shows the method for producing psVesmaI. Figure 40 - (bl shows the production method of pSveSmaIr. Figure 5 shows the production method of psV2pTKγ. Patent applicant Makoto Asano, agent of Kanebuchi Chemical Industry Co., Ltd. -: n 623- α ζ Continuing from page 1 ■ Int, C1, 4 identification code Office docket number 625 - Procedural amendment (self-motivated) 1959 q Temple Ma1 Application No. 119648 2 Title of the invention Human interferon-γ4, Agent 5 Date of amendment order (2) Detailed description of the invention column A, page 8 of the specification, line 15 “146 "Amino acid" is corrected to "1148 amino acids." (Page 15, line 1, Vol. 110, p. 2487) is corrected as follows. However, there have been no examples of using human cells as these animal cells, and HuIFN-γ made from non-human cells and ■ made from human-derived cells.
It is unknown whether there are any differences in the sugar chain structure and antigenicity of IFN-f. "C," on page 19, line 2, "We have discovered that it is secreted. According to the present invention," is corrected as follows. [I discovered that it is secreted.] Still generally HLIIFN
-γ is highly toxic to human-derived cells, so transformed cells die due to their own f (uIFN-f), making it extremely difficult to obtain transformed strains that can be subcultured ( 2) This seems to be the case.In other words, it is possible to obtain a transformed strain capable of producing Hu I FN-γ that can be subcultured by transforming hamster-derived cells, but it is possible to obtain a transformed strain capable of producing Hu I FN-γ that can be subcultured by transforming cells derived from hamsters. No possible transformed strain has been obtained.The present inventors have found a transformed strain that can be subcultured by using human-derived cultured cells that are resistant to 1u IFN-γ or a mutant strain resistant to Hu I FN-γ. This led to the completion of the present invention. According to the present invention, HuJFN-
2. Add the following sentence between lines 12 and 13 on page 29. [Furthermore, when producing Hu I FN-γ using human-derived cells, it is necessary to use a HuIFN-γ resistant strain, and the resistant strain can be obtained as follows. That is, from many human-derived cultured cell lines, HuIFN-r resistant strains or Hu
As a method for selecting IFN-f resistant mutants, HuI
Cultured cells may be subcultured in a medium containing 1 unit to tens of thousands of units of FN-γ. Human-derived cultured cells sensitive to Hu I FN-γ are deformed and even die due to the toxicity of Hu I FN-γ, but Hu I FN-γ resistant strains continue to grow without any changes and are easily destroyed. You can choose. In addition, the Hu I FN-γ sensitive strain
Poly■:C which is a double-stranded RNA when treated with N-γ
When cultured in a medium containing poly I:C, the cells significantly degenerate and die, and are therefore distinguished as HuIFN-gamma sensitive strains. ” E. Next to the bottom line of page 46, the following descriptions of Examples 8 to 18 are added. Example 8 Selection of HuIFN-γ resistant strains I (eLa, F, L, WISH, WI-26V
100 units of human-derived cultured cells such as A4) /
After culturing for 24 hours using a 24-well multi-dish plate in MEM medium containing 1 ml of Hul, FN-γ and 5% FO8, the culture medium was removed and washed once with PBS.
MEM containing ltl poly■:C was added and cultured at 37°C. After 16 to 24 hours, we observed the morphology of the cells and found that the cells of HeLa, FL, and WISH were circular and detached from the bottom of the plate, but in WI-26
No such cell degeneration was observed in VA4. This indicates that WI-26VA,4 is resistant to E(uIFN-r. Example 9 HuI FN-γ-resistant mutant FL cells isolated at 75
After culturing on the entire bottom surface of a culture flask with a bottom area of 100 units)/ml, HLIIFN-γ and 10
Renew MEM medium containing μf//ll of poly■:C.
Cultured for 8 hours. Although most of the FL cells were detached from the bottom, the culture medium was diluted with 100 units/ynl of HuIFN-
Change to MEM containing γ and continue culturing for about 1 month.
Colonies of uIFN-γ resistant FL cells appeared. Colonies were grown to obtain I(uIFN-γ resistant FL cells. Example i. Obtaining transformed strains I)SVeSmaIr, pSV2pTK7 or pSV2gpt, which does not contain the uIFN-r gene, was used to transform HeLa, FL, which is a cell sensitive to old FN-γ.
, WISH cells or HuIFN-7 resistant strain W
It was introduced into I-26VA4 cells and Hu I FN-γ resistant FL cells according to the calcium phosphate method (Wigler et al. (1977) Ce0 (vol. 1, n, p. 223). 1. Plasmid containing 44 μ20 plasmid-calcium phosphate co-precipitation The cells were added to the above cells (3 x 105 cells/8.6 ml medium/diameter 6 crn culture dish) grown in Eagle's MEM medium containing 10% neonatal bovine serum in advance, and after 15 hours, the medium was renewed and culture was continued for 48 hours. After 25 μ?Al mycophenolic acid, 250 μf/ml xanthine, 0.1 μf Al aminopterin, 25 μt/m
The medium was updated to a medium containing 1 adenine and 5 μl/yxl of thymidine, culture was continued for another 2 weeks, and the number of colonies of the transformed strain that appeared was measured. As shown in the table, I)S
When using V2gpt, transformants were obtained from all cells, but pSVe8 containing the HuIFN-γ gene
ma■1 or p8V21] When using TKr, H
u I Only a few transformed strains were obtained from FN-γ sensitive strains, and WI-26, which exhibits Hul and FN-γ resistance,
A large number of colonies of transformed strains appeared from the VA4 and HuIFNgo-resistant FL cells. Example 11 Production example 1 of HuIFN-7 using WI-26VA4
T)SVeSmaIr of WI-26VA4 isolated at 0
Alternatively, colonies of transformed strains of pSV2I)TKr were isolated and grown in MEM medium containing 5% FO8. The cells were allowed to proliferate on the entire bottom surface of a 24-well multi-plate, and the culture medium was refreshed and cultured for 24 hours to reduce the IFN activity contained in the culture medium.
L cells and Pascular stomatitis virus (ya
scular Stomatitis yirus)
Or the Sindbis virus
rus) was measured by the CPE inhibition method. the result,
32.3 from the psVe8ma'Iγ transformed strain.
Unit/24h/106 cells (7)■
FN activity, 1) SV21) IF of 524 units/24 h/106 cells from the TKr-transformed strain
Expression of N activity was observed. Example 12 pS■2γ8.6-1, I)8V as a plasmid
γ to 2p'I'. Example 11 using 1.44 μr each of pSVeSma■γ
WI-26VA4 strain was transformed in the same manner as above, and the following results were obtained. Example 13 ■ Production of old FN-γ using IFN-γ resistant FL cells 1) SV2T) TKγ transformed colonies of ■ old FN-γ resistant Ii' L cells isolated in Example 6 were isolated. , grown in MEM medium containing 5% FO8. After growing on the entire bottom of a 24-well multi-plate and renewing the medium,
After culturing for 4 hours, the 1, FN activity contained in the culture solution was
L#] Pascular stomatitis will, z,
(yascular Stomatitis vir.
us) or SiudObI
It was measured by CPE inhibition method using SViruS). As a result, the I of 3 units/24h/106 cells
Expression of FN activity was observed. (Attachment) Claims (1) Nuclear DNA encoding human interferon-γ
A DNA sequence connected to the A sequence and including the promoter region of another gene that functions in cultured animal cells (2) The promoter region is a promoter region of a gene that is constitutively expressed. DNA sequence according to item 1. (3) The 7' promoter region is the promoter region of herpes simplex virus thymidine kinase (T(SV-I)) or the promoter region of simian virus 40 (SV40), according to claim 1 or 2. DNA sequence. (4) The DNA sequence according to any one of claims 1 to 3, wherein a selection marker in animal cells is present in the same DNA sequence. (5) A selection marker in microorganisms is present in the DNA sequence. (6) The DNA sequence according to any one of claims 1 to 4, which is present in the same DNA sequence. A DNA sequence according to any one of claims 5. (7) A DNA sequence according to any one of claims 1 to 6, wherein the DNA sequence is pSVel; 1 maHγ.
NA sequence. (8) The DNA sequence according to any one of claims 1 to 6, wherein the DNA sequence is pSV2pTKγ. (9) The DNA sequence according to claim 5, wherein the DNA sequence is 1) B'RpTKγ. αQ The amino acid sequence of human interferon-γ is G
ln Asp pro Qln Asp pro 'I
'yr Val LysQlu Ala Qlu AS
n Leu LyS Lys Tyr pheASn
Ala GIY His 8er Asp yal A
la AST) Asn Gly Thr leu ph
e I, eu Gly Ile 1. euLyS AS
n Trp LyS Glu Qlu Ser ASp
Arglys Ile Met Qln Ser Q
ln Ile Val 5erphe Tyr phe
Lys Leu Phe Lys Asn rheL
yS Asp, Asp Qln Ser Ile Q
ln Lys 5erVal Glu 'I'hr l
ie Lys Glu Asp Met Asnyal
Lys Phe phe Asn ser Asn
LVS LysL7S Arg AST) ASpPh
e Qlu I,ys Leu ThrASn 'l'
yr 8er Val Thr A, ST) Leu
Asn VatGln Arg Lys Ala Il
e His Glu I, eu l1eGln yal
Met Ala Glu Leu Ber pro
AlaAla Lys Thr Gly LVs Ar
g Lys Arg 5erQln Met LeU
phe Arg GIY Arg Ajg Alaer
The DNA sequence according to any one of claims 1 to 9, which is G1n. 01) Nuclear DNA encoding human interferon-γ
The DNA sequence according to any one of claims 1 to 10, wherein the A sequence is derived from human leukocytes. αri Nuclear DNA encoding human interferon-γ
DNA with a DNA sequence connected to this sequence and containing the promoter region of another gene that functions in cultured animal cells
Cultured animal cells transformed by A. Claim 1 in which the origin of α-cells is an animal
The cultured animal cell according to item 2. 04) The cultured animal cell according to claim 12, wherein the cell is derived from a mammal. 13. The cultured animal cell according to claim 12, wherein the cell is derived from a human. 0e Cells are CHO, HeLa, FL1wI8HICh
The cultured animal cell according to claim 12, which is implLiVer or WI-26VA4. Q7) The cultured animal cell according to claim 12, wherein the cell is a cell transformed with simian virus 40. Animal cells described in any of Items 15 to 17 (a) Nuclear DNA encoding human interferon-γ
Prepare a DNA sequence containing the A sequence and the promoter region of another gene that functions in cultured animal cells, connected to this,
Human interferon-γ, in which cultured animal cells are transformed with the nuclear I) NA sequence, the transformed cultured animal cells are cultured to produce human interferon-γ, and the human interferon-γ is collected.
manufacturing method. (b) The production method according to claim 18, which comprises culturing cultured cells of a transformed animal in a culture solution and recovering human interferon-γ from the culture solution. (b) Claim 1 in which the culture medium is a serum-free medium.
The manufacturing method according to item 9. (c) The production method according to claim 18, wherein cultured cells of a transformed animal are grown in the animal body, and human interferon-γ is recovered from the animal. Claim 19: The transformed animal cultured cells are grown in the animal body, and then the cells are collected and cultured in a culture solution.
Manufacturing method described in section. (c) Transformed animal cultured cells are human interferosomes
The DNA sequence is 1) BRT) TKr, pBRγ8, 6
-1, 1) T3Rγ8゜6-2. Or I) Bit
DNA sequence that is γ8, 6-8. A DNA sequence in which human interferon-r is connected to the cord-through nuclear DNA sequence, which contains the promoter regions of other genes that function in cultured animal cells, and also contains the DNA replication initiation region that enables replication in microorganisms. A microorganism transformed by DNA containing The microorganism that undergoes transformation is B. coli C600r.
The microorganism according to claim 24 which is mc6. (7) The NA sequence capable of replicating in the microorganism is
maIr, p8V2I)TKr or T)BRT
8. The microorganism according to claim 24, which is 6-1.

Claims (1)

[Claims] (1) Human interferon-γ is encoded in nuclear DNA
A DNA sequence (2) comprising the A sequence and the promoter region of another gene that functions in cultured animal cells connected thereto.Claim 1, wherein the promoter region is a promoter region of a gene that is constitutively expressed. DNA sequence according to item 1. (3) The DNA sequence according to claim 1 or 2, wherein the promoter region is the promoter region of herpes simplex virus thymidine kinase (H5V-I) or the promoter region of simian virus 40 (sv40). (4) The DNA sequence according to any one of claims 1 to 8, wherein a selection marker for animal cells is present in the same DNA sequence. (5) The DNA'A sequence according to any one of claims 1 to 4, wherein a selection marker for microorganisms is present in the same DNA sequence. +61 The DNA sequence according to any one of claims 1 to 5, which is capable of being replicated in microorganisms. (7) The DNA sequence according to any one of claims 1 to 6, wherein the DNA sequence is psVasmaIγ. 7. The DNA sequence according to any one of claims 1 to 6, wherein the +81 DNA sequence is pSV2pTKγte. +91 DNA sequence according to claim 5, wherein the DNA sequence is pBRpTK7. 00) The amino acid sequence of human interferon-γ is Cyg Tyr Cys Gln AlID Pro
Tyr Val LysGlu Ala Glu As
n Leu Lys Lys Tyr PheAsn
Ala Gly His Ser ADD Val A
la AspAsn Gly Thr Leu Phe
Leu Gly IIs LeuLys Asn T
rp LyIIGlu Glu Ser Asp Ar
gLys Ile Met Gln Set Gin
Ile Val 5erPhe Tyr Phe Ly
s Leu Phe Lys Asn PheLys
Asp Asp Gln Ser Ile Gin L
ys 5erVal Glu Thr Ile Lys
Glu Asp Met AsnVal Lys P
He Phe Asn Ser Asn Lys Lys
sLyg Arg Asp Asp Phs Glu
Lys Lsu Thr Asn Tyr Ser Va
l Thr Asp Leu Aan ValGin
Arg LySAla IIs His Glu Le
u l1eGln Val Met Ala Glu
Leu Ser Pro AlaAla Lys Th
r Gly Lys Arg Lys Arg 5er
Gln Met Leu Phe Arg Gly A
The DNA sequence according to any one of claims 1 to 9, which is rg Arg AlaSer Gln. 01) Nuclear DNA encoding human interferon-r
The DNA sequence according to any one of claims 1 to 10, wherein the A sequence is derived from human leukocytes. a2 Human interferon-γ coated with nuclear DNA
DNA with a DNA sequence connected to this sequence and containing the promoter region of another gene that functions in cultured animal cells
Cultured animal cells transformed by A. aa Claim 1 in which the cell is derived from an animal
The cultured animal cell according to item 2. α4) The cultured animal cell according to claim 12, wherein the cell is derived from a mammal. 09. The cultured animal cell according to claim 12, wherein the cell is of human origin. 0Q cells are CHO, HeLa, PL, WI
SH, Ch imp. The cultured animal cell according to claim 12, which is Liver or WT-26VA4. The cultured animal cell according to claim 12, wherein the Q71 cell is a cell transformed with simian virus 40. (18) Nuclear D encoding human interferon-γ
Prepare a DNA sequence containing the NA sequence and the promoter region of another gene that functions in cultured animal cells, transform cultured animal cells with the DNA sequence, and culture the transformed cultured animal cells. Human interferon-γ that produces human interferon-r and collects it
manufacturing method. 19. The production method according to claim 18, wherein the transformed animal cultured cells are cultured in a culture solution, and human interferon-γ is recovered from the culture solution. (b) Claim 1 in which the culture medium is a serum-free medium.
The manufacturing method according to item 9. al) Propagating the transformed animal cultured cells within the animal body;
19. The manufacturing method according to claim 18, wherein human interferon-γ is recovered from the animal. (a) The production method according to claim 19, wherein the transformed animal cultured cells are grown in the animal body, and then the cells are collected and cultured in a culture solution. ) DNA sequence is pBRpTKγ, pBRγ8.6
-1. pBRγ8.62. Alternatively, the DNA sequence is pBR18,6-8. (c) Nuclear DNA encoding human interferon-γ
Transformed with DNA having a DNA sequence that includes the A sequence and the promoter region of another gene that functions in cultured animal cells, and also includes a DNA replication initiation region that enables replication in microorganisms. microorganisms. (c) The microorganism undergoing transformation is E, colt CC
25. The microorganism according to claim 24, which is 600r-. (c) NA sequences that can be replicated in microorganisms are psVes
malγ, pSV2pTKγ or PBRγ8.6
-1, the microorganism according to claim 24.