JPS62263200A - Human interferon beta 2a and human interferon beta 2b, vector containing gene encoding said interferons, cell line producing the same and use thereof as drug - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention <Industrial Application Field> The present invention relates to vectors containing genes encoding human interferon-β2- and human interferon-β2B', cell lines producing them, and pharmaceuticals thereof. Concerning the use of.

Background Art Interferon is an important protein produced by the human body that has antiviral and antitumor activities. Because of its species specificity, human interferon is required for clinical use. Interferons are classified into three types based on their reaction with antibodies. That is, interferon-α, which is mainly produced by white blood cells,
, interferon-β, which is produced primarily by fibroblasts, and interferon-γ, which is produced primarily by T-lymphocytes.

The interferon produced by human fibroblasts in response to two gl(ds) RNAs is primarily the 20 Kd glycoprotein IFN-β1, which is a 0.9 kb RNA
This RNA is obtained from the gene on chromosome 9, excluding introns. However, other distinct mRNAs that produce IFN activity when introduced into frog oocytes by microinjection
A was immunized with poly(71) (7C) followed by IFN by cyclopeximide (CHX)-actinomycin D treatment.
observed in induced human fibroblasts. In addition, during the cloning of the complementary (C) DNA of IFN-β1, another 1 to 3 kb RNA was co-induced.
A cDNA clone corresponding to this R
NA encodes the polypeptide 23-26d in reticulocyte lysates and has been investigated to produce human interferon antiviral activity in oocytes [J, Weissenbach (1980
) Proc, Natl.

^cad, Sci, IJ, S, A,, 77, 715
2-7156: British Patent Order 2,063,882].

This activity is blocked by anti-IFN-β1 antibodies, so this polypeptide is called IFN-β2. The protein obtained from the RNA corresponding to IFN-β2 and IFN-β cross-react with the same antibody.
Although their biological activity is blocked, the two proteins are immunologically distinct. Since IFN-β2 RNA is formed by a mouse-human hybrid with chromosome 9 removed, the 1.3 kb RNA of IFN-β2
is clearly derived from a gene other than the IFN-β1 gene [0.1r, (1984) ph, Thesis, Weizgiann In.
5 posture of sctence, Rehovo
t, l5rael].

German Patent Application No. P3.043,981 describes a method for producing interferon-β2 in pure form.

According to this production method, human SM that can produce interferon when stimulated with an interferon inducer
Interferon-β2 was obtained by the In method, which involves culturing the cells and then isolating the DNA containing the nucleotide sequence in human 18 cells encoding interferon-β2.
I am getting . In this tJ production method, mRNA is tn* purified from the induced cells, then DNA is obtained by reverse transcription of the mRNA, and this DNA is cloned into an appropriate vector.

<Disclosure of the Invention> When the gene obtained as described above is isolated and transfected into hamster or mouse 1a follicles,
dsRNA and cycloheximide sting? The existence of another distinct gene encoding IFN-β2, which gives rise to human-specific IFN activity after liI, has been demonstrated. The IFN produced here has characteristics that are recognized as belonging to IFN-β2, however, it does not have the same amino acid sequence as IFN-β2, and these are IFN-β2.
-β and IFN-β2B.

A. Therefore, it is an object of the present invention to obtain biologically active human IF.
N-β and human IFN-β, 2B@11111A.

Another object of the invention is to obtain biologically active human IFN-β.
and to produce human IFN-β2B.

Yet another object of the present invention is to produce cDNA encoding human IFN-β2, more specifically, cDNA encoding human IFN-β and cDNA encoding human IFN-β2°A.

Yet another objective of the present invention is to identify a gene that hybridizes with the IFN-β2 cDNA.

Still another object of the present invention is to identify a gene that hybridizes to IFN-β2A cDNA or IFN-β2B cDNA.

Yet another object of the present invention is IFN-β23! A genomic clone containing the I gene, more specifically IFN-β2A3
The genomic sequence containing 11 genes and [FN-β2B
The objective is to isolate a genomic clone containing the II gene.

Still another object of the present invention is to
The object of the present invention is to provide a recombinant vector containing the FN-β2BII gene and a promoter sequence that controls transcription of the interferon gene.

Yet another object of the invention is to provide biologically active human IFN
The object of the present invention is to provide m cells that do not produce -β and/or IFN-β2B.

Yet another object of the present invention is to
The object is to provide cells that produce a substantial amount of N-β2B.

Still another object of the present invention is to
Biologically active human IFN-β2A and/or IFN
- To provide a method for producing β2B.

Yet another object of the present invention is to provide human IFN-β2A and/or IFN-β2A that has substantial amounts of biological activity when fused to a strong viral transcriptional promoter and transfected into hamsters or mice. Human I
The object of the present invention is to provide a gene that produces FN-β2.

Still another object of the present invention is to provide an IF that has a desirable effect on cell growth or differentiation, particularly on terminal stage cancer cell differentiation.
N-β and/or IFN-β2°A are used as pharmaceuticals.

Still another object of the present invention is to provide IFN-β2A and/or IFN-β2A.

According to the invention, biologically active interferon-
The existence of β2 (IFN-β2) molecules has been confirmed and these molecules are produced by recombinant DNA technology. Such interferon-β2 is a glycoprotein secreted from human fibroblasts whose production of interferon has been induced by dsRNA or a virus. IFN-β2 represents approximately 5% of the total IFN activity produced by fibroblasts. The amino acid sequence of IFN-β2 was determined by a method using CDN8, and the sequence is similar to that of other IFNs (IFN-β, IFN-α) produced by human cells.
It is only about 20% homologous to the sequence.

According to the present invention, a human cD encoding IFN-β2^
The NA or gene is fused to a strong transcriptional promoter of the virus. Hamster cells transformed with this fusion cDNA or gene develop human-specific interferon activity, which inhibits viral replication and cytopathic effects and is characteristic of the biological response of human cells to interferons. proteins are induced. Another distinct gene encoding IFN-β2 was found to produce human-specific [FN activity] after induction with dsDNA and Schif0 heximide when isolated and transfected into hamster or mouse cells. . IF produced by this gene
Although N has characteristics recognized as belonging to IFN-β2, its amino acid sequence is not the same, and these are I
They were named FN-β and IFN-β2B.

A DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION The existence of interferon-β2 was confirmed while isolating a gene in human fibroblasts containing the nucleotide sequence encoding interferon. At this time,
Creating appropriate exogenous factors to fibroblasts to induce the production of interferon mRNA,
mRNA was extracted. At the same time, mRNA was also extracted from fibroblasts that were not subjected to derivatization treatment. The cDNA block O-
synthesized. Two gic DNAs were synthesized from mRNA obtained from the derivatized cells, and then inserted into an appropriate vector and transfected into microorganisms. This transformant was then cultured under conditions in which transformants carrying the vector selectively proliferate to obtain first-generation colonies. Next, a second colony was further formed from this colony, and DNA was extracted from both colonies. The DNA obtained from the second generation colony was hybridized with a cDNA probe synthesized from the mRNA obtained from the derivatized 1III1 described above. On the other hand, the DNA obtained from the second generation colony was also hybridized with a cDNA probe synthesized from mRNA obtained from cells that were not subjected to the above-described derivatization treatment. Colonies containing DNA that hybridized to cDNA probes obtained from derivatized cells but did not hybridize to cDNA probes obtained from non-derivatized cells were selected, and DNA was extracted from these colonies. Examine this DNA further and find frog eggsff1ll
DNA was selected that hybridized with the mRNA translated into interferon in lysates of ll or reticulocytes.

Such DNA is DNA that essentially encodes interferon, or DNA that encodes interferon.
This is DNA containing a sequence sufficient to obtain A.

During such studies, two other mRNAs encoding interferon were found to be isolated from derivatized aim blast cells. By centrifuging these mRNAs using glycerol, a small amount of mRNA was precipitated in both parts of 11S. The protein was obtained by translating this in cells. Such a protein is that of the molecule ff120.000, which is precipitated by reaction with an antibody against one of the interferons purified from fibroblast cells. This protein is human interferon-β1, and a portion of its amino acid sequence is described by Knight et al. [(
1980) 5science, 207, 525-
5261. A large amount of mRNA is precipitated on both sides of 14S, and from this mRNA, the molecule m23
．． 000 protein was obtained. This protein was precipitated in reaction with an antibody against impure interferon obtained from fibroblast cells.

This protein was named human interferon-β2. A cDNA clone that hybridized with the mRNA translated into interferon-β2 was subjected to IIJ in the same manner as described above, and the clone was used to prepare a cDNA probe for 'IFN-β2. Interferon-β2
IFN-β2 clones A341 and E4, which are cDNA clones that hybridize with mRNA translated into
74 each had a common sequence, and these were fused to reconstruct IFN-β2AE20 cDNA (see Figure 1). The nucleotide sequence of this IFN-β2 cDNA was determined. This revealed that it has an open reading frame of 212 amino acids expected to produce a 23,500 dalton protein. Transcription-translation tests confirmed that this cDNA encodes the protein described above.

After partial digestion of human adult blood cell DNA with ECORl, λcyt
On4A [V, Hory et al. (1981) Eur
.

J, Biochem, 120: 197-2021
From the human gene library obtained by cloning in
-11 was isolated. Clone IFA-2 is clone c
Contains a gene with at least four exons that hybridize with various segments of DNA (Figure 1)
. Because the Xh01 site is located near the 5'-end of the AE20 cDNA, genomic segment A132 (Figure 2)
We created a map of A132 contains the first exon of IFA-2 that ends 70 bp T downstream from this Xho1 site. Using a DNA probe labeled with the BStN1 site located 40 bp from the xh01 site,
By performing S1 nuclease analysis, derivatized human IiAM blast cells FS11 have a 5'-terminal S-1
Alternatively, it was found to contain two IFN-β2 RNAs having S-2 (Fig. 3). These two are a great starting point! It is present following the TATA box at position -30 (Figure 4). RNAs ending in S-2 can be compared with other longer RNAs under various derivatization treatment conditions.
It was more than A (Figure 3).

Two RNAs with different 5'-ends are located in the cytoplasmic R
It was also observed in the case of NA. It is therefore clear that two RNAs are formed and active in human cells. Both IFN-β2 RNAs were transformed into poly(γ!)(γ
C) in human fibroblast cells. This induction is
In the case of IFN-β1 RNA [0, Nir et al. (1984
) Nucl.

^cids, Res, 12: 6979-699
Similar to 31, this is facilitated by the addition of IFN itself. IFN-β2 RNA is also induced by long-term treatment (6,5 h) with CHX. Also c t
-+

When induced with CHX alone without the addition of dsRNA, IFN-β2 RNA is only slightly induced, which allows it to be distinguished from IFN-β1.

In order to express the IFA-2 gene, a DNA construct with the SV40 early promoter was created. IFA
A 4.8 kb segment of ρ5VE3 DNA [Y, Ch
ernajovsky 5. (1984) DNA,
3.294-3081, the ATG codon is 5
V40(7)6fj 150b from the initial RNA start point
pT flow (psVIFA2-Il, 5th
(see figure).

psV I FA2-1 [DNA, cos7 monkey II
Cell [Y, Gluzman, (198T) Cell
, 23: 175-1821 and IFN-C
By Northern blot method using 2CD NA, 1
．． Two RNAs of 35 and 2B2kb were detected. These two RNAs each have R
This corresponds to RNA whose transcription ends at the NA1 or SV40 polyadenylation site (Figure 5).

Regarding the culture medium of CO57 cells in which psV I FA2-II DNA was transiently expressed, human FS11 and Wick (14ish) cells [0, Novick et al.
(1983) J. Gen. Virol, 6'
4: IFN activity was measured based on the inhibitory t, lj effects on the cellular effects of vSv on 905-9101.

Antiviral activity was clearly detected 2 days after pSVIFA2-II DNA transfection (Table 1). The specific antiviral activity of IFN-C2 per unit is 1/30 to 1/100 of that of IFN-β1, and that of IFN-C1 ('5-10 x 106 units/unit).
It was close to q).

IFN of the medium in which the IFA-2 gene is expressed
The titer was, as expected, approximately 3% of the titer when the IFN-β1 gene integrated downstream of the same SV40 promoter was expressed (Table 1). Furthermore, psV
(2'-
5') oligomer synthetase [8, Revel et al. (
1981), Heth.

Enzymol, 79: 149-161], psv I FA2-I [DNA
It has been demonstrated that IFN-β2 activity occurs in cells transfected with (FIG. 6). (2'-5
') Induction of oligomer synthetase is dependent on Kawaishi and corresponds to antiviral activity.

In addition, the IFA-2 gene was fused so that the 5V40 promoter was located at the XhO1 site near S-2 (
psVIFA2-I DNA, Figure 5). Hamster CH
O-KIDHFR- cells were treated with psVI FA2-I and pSVDHFR [Y, Chernajovsky et al. (1984) DNA, 3:294-308] DN
Stable transformants 5 were co-transfected with A.
I-15 was isolated.

For this cell line, human FSII and IJ i
When measured using 5hlQ cells, 10-50 U/d of IFN antiviral activity was observed. On the other hand, psVDl
- In the case of CHO cells introduced with only IFR DNA,
No production of human IFN was observed (Table 1). By concentrating the medium of 5t-15 culture, approximately 3000U#! A γIFN-β2 solution is obtained with a titer of .

This γIFN-β2 has the characteristics of human IFN, and is also caused by γIFN-β2 in FS11 cells (2
'-5') Oligom synthetase is induced even at a low concentration of 1 Ll/d (Fig. 6), CHO3I-
In the presence of cycloheximide, (2'-5')
It was demonstrated that the mRNA for producing oligomer synthetase 056 and HLA is specifically induced in humans. In addition, γIFN-β2 antiviral activity is associated with anti-IF
Neutralized by N-β1 polyclonal or monoclonal antibody, anti-IFN-α or anti-IFN-γ
It was found that some antibodies did not neutralize it. It was also found that mouse-human hybridomas act against mouse-human hybridoma 1. γIFN-β
From these biological characteristics of 2, γIFN-β2 is
It is proven that it acts as an FN and not as an inducer of IFN-β.

A DNA construct obtained by fusing the IFN-C2Ac cDNA sequence to the SV40 early promoter (DSVCIFβ
, Fig. 5) to CHo cells in the same manner as described above.
γIFN-β2 was obtained in high yield when transfected with HFR. The yield of γIFN-β2 was amplified by selecting IIIVll resistant to methotrexate in tbna. Even without amplification, CI-10 cells transformed with DSVCI Fβ2 (e.g. clone B-132) showed similar IFN-β2 activity as 81-15 cells (
Table 2). However, when cells resistant to methotrexate were selected, a high IFN-β2 of 800 U/−
showed activity (Table 2).

Using such selected clones, I
FN-β2 protein can be immunologically precipitated and, if the cells are labeled with 35s-methionine, IFN-β2 protein can also be analyzed by dodecyl sulfate polyacrylamide gel electrophoresis. . The size of γl FN-β2 was found to be 21.000 Daltons. Actual experiments have shown that in reticulocyte lysates, translation of IFN-β2 RNA yields a 23-26 Kd product, which can be expressed in VitrO by canine vancreatin membranes and the shorter 21
was found to be converted into the d protein. The derivatized human fibroblasts also produce protein 21d, which is immunologically identical to the product 26d, which is a product from IFN-β2 RNA, but is -It was different from β1. mature IFN-β2
21, the N-terminus of the d protein was not identified. IFN-
There are two glycosylation sites in the β2 sequence (Figure 1), which makes it difficult to calculate the size of the region removed by processing. However, this region of removal is longer than that of IFN-β1, and its size has been shown to increase upon maturation [14° Oegrave et al. (1981) Gane, 10: 11-151. d to 26 of IFN-β2
polypeptide hydride (hvdrOD)
8thV DIOt> shows that there is significant commonality between the hydrophilic and hydrophobic regions of IFN-β2 and those of mature IFN-β1 when the two proteins are aligned based on their C-termini. I understand. In addition, IFN-β2
It was also found that there is a common sequence between the amino acid sequence of this type and that of other type I human IFNs (Figure 7). All known IFN-α and β sequences have 38 common amino acid sequences (marked with an asterisk in Figure 7).

When IFN-β2 is similarly arranged based on the C-terminus, 18 of the 38 amino acids common to all type I human IFNs are also found in IFN-β2 (Figure 7). . IFN-β2 and other I'S! There is approximately 20% homology with IFN.

Although the homology between the IFN-β1 and IFN-β2 amino acid sequences is low, IFN-β1 and IFN-β2
The antiviral activity of, for example, anti-IFN-β1 monoclonal antibody [0, Novick et al. (1983) J, G
En, Virol, 64: 905-9101 and other antibodies are neutralized by cross-reaction, suggesting that there is some homology between the active sites of IFN-β1 and IFN-β2. Conceivable. IFN-β
This idea is supported by the fact that the activity of 2 was not neutralized by anti-IFN-α or -γ antibodies. IFN-
The gene encoding β2 is located on chromosome 9 [11, Nir
, (1984) Ph, D., Thesis,
Weizmann In5titude of Science
nce, Rehovot, and l5raell, and also differs from the ■-type IFN gene on chromosome 9, which has a cluster structure due to the presence of an intron. IFA-
211 Buddha has chromosome 7 [P, B, Sehgal et al. (1986) Proc, Watt, Acad,
Su5. I found out that it is in the USA. Since homology was detected in the amino acid sequence, IFN-β
23! It is thought that I Buddha is related to the gene underlying the type I IFN gene, which has a cluster structure.

Another genomic DNA gene, IFA-11, that hybridizes with IFN-β2 cDNA is
It has a different restriction enzyme map from A-2 (Figure 8)
. From the transcription map and its sequence, the 3'-terminal exon of IFA-2 and IFA-11 (Hbal-Hind
segment) (99% homologous sequence)
On the other hand, there is no region that cross-hybridizes with the 5' exon. IFA-11 Genomics 0-
(19Kb> wo, H3V-TK gene [F, Co
1bcre-Garapin et al. (1981) J, H
ot.

Biol, 150: 1-141 with L-T.
K-cells were transfected and stable transformants were obtained. As shown in Table 1, the Ll vacuole containing the human IFA-11 gene, Ll-39, contains poly(γN(I
C) and cycloheximide derivatization showed human IFN activity, whereas L-TK+ cells showed no such activity. CHO clone IC40, which contains the IFA-1111 gene, also exhibits human IFN activity (Table 1). Ll-39 without derivatization treatment
Alternatively, IC4011 cells did not show any activity. The IFA-11 transformant shows I after derivatization treatment.
FN activity was significantly higher than that of the IFA-2 gene. γIFN-β2B produced by expression of the IFA-11 gene, like the product of the IFA-2 gene, has its activity neutralized by anti-IFN-β1 antibodies. The NA probe specific for IFA-11 was
Similar to the FN-2 DNA probe, it hybridizes with RNA obtained from derivatized human fibroblast arn,
This indicates that both genes exhibit activity in human cells.

The function of the IFN-β2 gene is that IFN-β2! under conditions in which other IFN311 genes are not expressed. ! This is thought to be due to the manifestation of Buddha. We tested tumor necrosis factor (TNF) or lymphin-IL-1.
It was discovered that IFN-β2 protein was produced when fibroblasts were exposed to. In addition, antibodies can induce IFN-
It has also been found by other adults that β is neutralized and, as a result, arm proliferation is promoted [8, Kohas et al.
e et al. (1986) Cell submission HRe5nitzk
V et al. (1986) Cell submission]. From this,
It has been shown that IFN-β2 acts to suppress cell growth in response to growth factors. This means that I F
In peripheral blood mononuclear cells derivatized with N-1 mRNA and mitogenes such as PHA, IFN
This is also supported by the fact that RNA hybridizing with -β2 CD N A was detected. It has also been reported that lymphocytes such as IL-1 strongly induce RNA that hybridizes with IFN-β2 cDNA in diploid fibroblasts and human cell lines [
J, Content et al. (1985) Eur, J.
Biochet, 152: 253-257]
. The bromotor region of the IFA-2 gene consists of two TAs.
It contains a TA box and two RNA starts (Figures 1 and 3). This IFN-β2 promoter is
It has been shown to respond to either poly(γl) (IC) or cycloheximide [Y, Cher
najovsky et al. (1984), DNA, 3:294
-3081° In vivo, two IFN-β2 gene promoters are activated by different inducing agents (dsRNA, protein synthesis inhibitor, IL-1, PHA). Various types of cells are inhibited from growth or differentiated.
Small amount of autocrine IF
It has been found that N-β is produced [8. Re
vel (1983) Interferon, 5.
DD, 205-239, Acad, Pres.
[S, London], IF N-β2 belongs to a small number of IFN species that are produced in response to autoregulated growth of l1lrf&, whereas type I IFNs specifically respond to viral infection. The IFN species produced.

[The biologically important point of FN-β2 is that IFN-β1 is not induced under conditions such as inhibition of metabolic machinery.
Under three conditions [A, 7i1ber
Stein et al.

(1985) The tnterreron sys
tem, 5eron.

Symposia Raven Press, I)I
), 73-83 ], IFN-β2 is induced. And most importantly, TNF induces IFN-β2 in fibroblasts, which is promoted by TNF! The proliferation of IN blast cells was further promoted by the addition of anti-IFN-β antibodies. Many human cell lines use the autocrine IF N during differentiation.
-β is known to be produced, and these IFNs induce HLA antigens in cells [^, Wa
rden et al. (1984) Esbo, J., 3;969
-9731°C3F-1 induced myeloid leukemia cell line to differentiate, and anti-IFN-β antibody abolished growth arrest of m-cells characteristic of terminal stage differentiation. I
L-1 has been shown to promote the growth of several cell types and the growth factor PDGF induces IFN-β RNA in mouse cells [J.

H,,? ullo et al. (1985) Cell 43 Near 93-8001° Although there are many IFN-β [P,
B.

5ehoal, (9182), Interfe
ron4.

Academic Press, op, 122
, and H, Revel (1983), Interf.
eron 5, Academic Press,
DI).

205-2391, IFN-β2 is considered to be one of the autocrine autoregulators of cell growth that occurs in response to growth factors. Cells producing autocrine IFN-β exhibit only low antiviral activity [8, Rev.
el (1983).

Interrcron 5, ACadelliC
press, pi), 205-239].
The above idea is based on the fact that IFN-β2 has a low specific activity (antiviral activity, as measured by immunoassay).
1150-17100). Also I
Hamster i follicles expressing FN-β2Cl)NA are compared with hamster CHOi follicles carrying the human IFN-β1 gene [Y, Chernajovsky et al. (1984).

The reason why DNA3 shows lower IFN activity than 297-3081 can be explained by the above idea.

Interestingly, IFN-α1(D)• accounts for a large proportion of leukocyte IFN [C.

%4eissmann (1981), Inter
feron 3.

Academic Press, N, Y, op.
Despite 101-1341, IFN-α1(D)
also shows a specific activity 1/100 lower than other IFN-α [
T., Goren et al.

(1983), Viroloail 30: 273
-2801° However, it cannot be overlooked that some of the many IFN functions are expressed more effectively by IFN-β2 than in the case of antiviral activity. Despite its low concentration, the growth regulator IFN
-β induces HLA and (2'-5') oligo A synthetase more strongly and also suppresses the growth of cells that produce them more strongly for a longer period of time. IL-1 and TNF
Since IFN-β2 is induced by
It is suggested that -β2 not only regulates cell proliferation, but also plays a role as an autocrine mediator of these cytokinetics in inflammation and acute phase responses.

The mRNA that hybridizes to the probe for IFN-β2 cDNA, but not IFN-β1 cDNA, induces human cell lines to become interleukin-1 (IL-1) [J
, Contcnt et al. (1985).

Eur, J. Biochem, 152
: 253-2571 and TNF-α when exposed to Competition
Assav), human fibroblast FS11m cells actually produce IFN-β2 in response to these two cytokinetics.
We investigated whether the protein was secreted (Fig. 9).
Both α and TNF-α induce the synthesis and secretion of IFN-β2 protein, and immunoassays show that 1O-20U/d
of IFN-β2 was secreted. The concentration of γIL-1α is 4
U/j11! (0,13 nMd), IFN-β2
An optimal induction of IL-1 was observed, which is the same concentration of IL-1 [J, 1
1. Orn (1985), Arthritis
Rheum, 28: 315-3221.

In addition, IFN-β2 induction (400U/d: 40no/nJ
) is the concentration required to lyse sensitive [l cells [0,1n (II/d; A,
H, IJanQ et al. (1985) Science, 22
8:1 higher than 49-1541 and the concentration required to promote optimal growth of diploid fibroblasts (2 nMd)
higher than The 1m start of secretion of IFN-β2 is determined by TNF-
It is accelerated by α and reaches half the maximum value of secretion m in about 6 hours. On the other hand, IL-1 reaches half its value in 8-12 hours.

The documents cited in the foregoing portions of the description are incorporated into the book [+11] in order to complete the description of the wood invention.

It will be obvious to those skilled in the art that modifications and changes to the invention may be made without departing from the scope of the invention.

Further, the present invention is not limited to the description in the specification or the drawings.

Table 1 1, IFA-2 gene (control CI-IQ cells
:OB, Co57 [transie in one cell
nt) Expression C) SV[FA2-II DN
A: 15LI/d<3> Tor IF
N-β1 psVEIF DNA: 500tJ/d)
■, IFA-11 gene A, expression after derivatization treatment in CHO cells **9 clone 1C40: 400-500 U/ad (control CHO cells: 100 U/d)
”: Assay using vSv in FSll and WISH cells.

Harvested 28 hours later.

[Brief explanation of drawings]

FIG. 1 shows the restriction enzyme map and nucleotide sequence of IFN-β2 A CD N A. This nucleotide sequence was compared to the E474 and A341 cDNA clones [Wclssenbach et al. (1980). Proc, Natl, Acad, Sci, US
A, 77:7152-7156] and xba-1
was determined from clone AE20 formed by fusion at the site. Its 5'-terminal sequence was determined from the genomic clone IFA-2 shown in the second VA. Also, the numbers start with 8-1 in FIGS. 3 and 4 as 1. The amino acid sequence of IFN-β2 was determined by a deduction method. Figure 2 shows IFN-β2A3! The structure of genomic clone IFA-2 containing the I gene is shown. A132 is IFA
This is a subclone in which the EcoRI segment of -2 was cloned in pBR322. The scattered dots indicate cDNA clones A341 and E474 [Wc1
Ssenbach et al. (1980). Proc, Natl, Acad, Sci, u,
NIK hybridizing to s, A, 77:7152-71561 is shown. The two starting points of the RNA (caps 1 and 2) and the TATA box are shown. In the RNA, the 212 amino M (FIG. 1) open reading frame (ORF) is indicated by a thick black line. M, C, indicates the second ATG, and no other ORF was found in the IFN-β2Ac DNA. Figure 3 shows the 5'-terminal portion of 81
Nuclease analysis is shown. It is a 7-ragment of subclone A132 of IFA-231 gene (Fig. 2), and Bs
Diploid 11N'! was prepared by treating the DNA probe labeled with the tN1 site in the following manner. It was hybridized with total RNA obtained from Jll cells. The treatment method is IFN-β
Pretreatment at 1200tJ/ae for 16 hours; similar pretreatment and poly(γI)(γC)(plG) 50μg/I11
．． 3.5 hour treatment; same pretreatment and cycloheximide (CHx) 50 μg/M! , 3.511111 treatment: similar pretreatment and treatment with pIC and CHX for 3.5 hours: similar pretreatment and treatment with C)IX for 6.5 hours; plcr3.
Between 5R (7) and 11 (7): CHXt'3.5 o'clock 1m
Treatment only: 3.5 hours treatment with pIc and CHX,
It is. S-1 and S-2 are the two RNs shown in FIG.
This is a symbol indicating the starting point of A. Figure 4 shows the promoter region of the IFA-2 gene encoding ``IFN-β2A''. Subclone A132
The sequence of the portion (Fig. 2) is shown as IFN-β2 cDNA. S-1 and S-2 indicate the starting points of the two RNAs shown in FIG. The initiator-ATG is indicated by an X. The first intron starts at intγ1. FIG. 5 shows the fusion of the IFN-β2A cDNA and the 5v40 early promoter. IFA-231! Gene (from 5.5kb to 7.5kb in Figure 2)
Xhol-BamHl (Xh-8
) segment into a 26bp C1a1-xhO1 synthesis 7
pBR cut with C1a1 and BamHl using a dabter (restores the 5' end (7) cDNA sequence).
This was fused to a plasmid and subcloned to construct plasmid pSVIFA-IF. Hf n d
3'-end BamHl-Hind subcloned in DBR plasmid cut with tll and BamHl.
(B-H) Gene segment (7.5k in Figure 2)
10.5kb) from DBR322)-1ind
Cut at the Claal site adjacent to the site, and
'-terminal segment to obtain the complete IFA-2 gene. Hin dl, 1 t' truncated osVE3
Vector [Y, Chernajovsky et al. (198
4) Re-ligate DNA, 3:294-3081 using C1a1 linker to pSVCj! form a)
Then, the IFA-11 gene was transformed into Cla of pSVCl.
SV4 forming I) SV I FA2-■ at l site
The promoter was introduced in the same direction as the 0 early promoter. IFA-2
Plasmid E) SV'1FA2-I was constructed in the same manner, except that the xho3 site of E) was fused directly to the ClaI site of pBR before introduction into DSVE3. To obtain the pVCI Fβ2 plasmid containing IFN-β, cDNA fused to the SV40 promoter, the ps
VIFA2-II DNA was cut with xbl and then
After partial cleavage at mn, the IFN-β2CD N A sequence f) X h o 60 bl from position 1a) lower 11. −
A certain X m n 1 site (Figure 1) was opened, but the X m n 1 site in the amp gene of the vector was not opened. This construct contains IFN-β, cDNA
Xmn-Xba1 Sen'J) of AE20
92-566 in the figure) to restore the complete IFN-C2A cDNA sequence as shown in the figure. EE
S is 5V-40T-aa311 gene 17) RNA A
t; fl 'JR point, ATG is 1FN-C2 (7) t
The lil start codon, pA, represents the polyadenylation site for IFN-C2 and SV40. psVc I Fβ2DNA
into CHO-1D along with the pDHER R plasmid.
HFR-[1111 was transfected with [Y,
Ch(!rnajOVskV et al. (1984), DNA,
3:294-3081, regarding the obtained clones,
Vesicular Stomatitis Viru
Human IFN antiviral activity in r=siimn was investigated using s (V SV ). Gene amplification was achieved by selecting CHO clones resistant to methotrexate (MTX). Assay of culture medium of CHO-3VCI Fβ2B132-5M cells resistant to 250 nM MTX is shown below the figure. (2) After the cytopathic effect of Sv appeared, the wA vesicles in the microplate were stained with methylene blue. Series of media 2
The fold dilutions are listed from left to right. [300tJ/ad! by comparison with FN standard (ST) and non-introduced CHOIII cells! of γIFN
- The titer of β2 was calculated. Figure 6 shows poly(γI)(7C)-7 galose (8, Revel et al., (1981 ) Heth, Enz
ymol, 79: 149-161]. i.e. @pSV I FA2-x
() Co57 cells 2 days after introducing N8 (Figure 5) (The results of the assay at this time are shown in
), (c) DSVIFA2- in FIG.
I()NAI' stably transformed hamster Cl-
1081-15 cells (next 4 rows), CHOM EIF-511111 expressing the IFN-β1 gene
11 [Y, Chernajovsky et al., (198
4), DNA, 3:294-3081 (next 2 columns)
, (d) Cl-1 transformed with DHFRII gene
011111 (next row), and Φ) fresh medium only (
None). Measurement results of IFN antiviral activity per d of medium added to FSl 1 cells are shown. Phosphorylated 32P-α-ATP labeling (2'-5'
) Shown are the results of electrophoresis of oligo A products. FIG. 7 shows a comparison of the amino acid sequences of type I human IFN. IFN-β1 and various IFN-α (excluding α-E, IFN-α denoted by α-A, α-C, and IF
The amino acids common to N-α class ■) are IFN-β
1 is marked with an asterisk above the array. Also, IFN-β
The amino acid that is common to all type I IFs is
IFN-β marked with an asterisk if common with N, or in a square if common with several IFNs.
2A is a hydropurge plot (bytiropat
hy DIOtS) and numbered from the predicted buO processing site. FIG. 8 shows restriction enzyme maps of two different human IFN-β2 expressed in cells of the cervix. (FN-11 DNA (IFN-β2-gene) was transfected into either CHO or LII follicles and expressed IFN-β activity. (IL-I) and tumor necrosis factor (TN
It has been shown that IFN-β2 protein is secreted in response to F-α). The optimal induction of both side forces at low levels, i.e. 1O-20U/all IFN-β2, is at a concentration of γI and 1α of 4LI/ad (0,13 ng/ad).
me). Optimal concentration of TNF-α for IFN-β2 induction (40
0LI/d: 40 nMd) is higher than the concentration required for cytopathic effects in susceptible cells (0,11 g/-) and also in diploid mm buds [111! higher than the concentration required to promote optimal growth (2 na/d).

Claims (29)

[Claims] (1) Pure form of human interferon-β_2_A obtained by genetic recombination technology. (2) Pure form of human interferon-β_2_B obtained by genetic recombination technology. (3) cDNA consisting essentially of cDNA encoding human interferon-β_2_A. (4) cDNA consisting essentially of cDNA encoding human interferon-β_2_B. (5) cDNA having the nucleotide sequence substantially as shown in FIG. (6) cD encoding human interferon-β_2_A and capable of expressing human interferon-β_2_B
Genomic clone that hybridizes with NA. (7) cD encoding human interferon-β_2_B and capable of expressing human interferon-β_2_B
Genomic clone that hybridizes with NA. (8) The genomic clone according to claim 6, which contains the IFA-2 gene. (9) The genomic clone according to claim 7, which contains the IFA-11 gene. (10) A vector obtained by genetic recombination technology having a DNA sequence encoding human interferon-β_2_A and containing a regulatory gene region at a position where human interferon-β_2_A can be expressed. (11) The vector according to claim 10, wherein the DNA sequence is a genomic DNA sequence. (12) The vector according to claim 10, wherein the DNA sequence is a cDNA sequence. (13) The vector according to claim 12, wherein the DNA sequence is the sequence shown in FIG. (14) A vector obtained by genetic recombination technology that encodes human interferon-β_2_B and has a DNA sequence containing a regulatory gene region at a position where human interferon-β_2_B can be expressed. (15) The vector according to claim 14, wherein the DNA sequence is a genomic DNA sequence. (16) The vector according to claim 14, wherein the DNA sequence is a cDNA sequence. (17) The regulatory gene region is a promoter, preferably S
17. The vector according to any one of claims 10 to 16, comprising the V40 early promoter. (18) The vector according to any one of claims 10 to 13, wherein the vector is pSVIFA2-I. (19) The vector according to any one of claims 10 to 13, wherein the vector is pSVIFA2-II. (20) The vector according to claim 13, wherein the vector is pSVCIFβ_2. (21) A cell line transformed with the vector according to any one of claims 10 to 20. (22) The cell line according to claim 21, wherein the cell line is a eukaryotic cell. (23) The cell line according to claim 22, wherein the cell line is a hamster cell line. (24) The cell line according to claim 22, wherein the cell line is a CHO cell line. (25) The cell line according to claim 22, wherein the cell line is an L-TK cell line. (26) The cell line according to any one of claims 21 to 25, which is capable of producing human interferon-β_2_A and/or human interferon-β_2_B. (27) Cell line is CHO-SVCIFβ2 B13
25. The cell line according to claim 24, which is 2-10M. (28) Claim 1 using the cell line according to any one of Claims 21 to 27.
Human interferon-β_ as described in Section 1 and/or Section 2
A method for producing 2_A and/or human interferoso β_2_B. (29) Pharmaceutical use of human interferon-β_2_A and/or human interferon-β_2_B for regulating inflammation, acute phase response, or cell differentiation.