JPH1052286A - Human interleukin beta 2a gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene coding IFN-β2 A having a mature type biological activity obtained by measuring hydropathy along a specific amino acid sequence and useful for production, etc., of IFN-β2 A having antiulcer activity and antiviral activity. SOLUTION: This cDNA comprises a nucleotide sequence coding an amino acid sequence of interferon-β2 A (IFN-β2 A) having mature type biological activity and capable of obtaining by measuring hydropathy along a specific amino acid sequence represented by the formula and production of interferon β2 A having antiviral activity and antiulce activity and useful for uses, etc., as a medicine can efficiently be carried out by culturing a cell line transformed with a vector containing the cDNA. The cDNA is obtained by reacting a human fibroblast with an exogenous proper factor, extracting mRNA, preparing a gene library by using the mRNA as a template and screening the gene library with a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human interferon.
beta _2A and human interferon-beta _2B, vectors containing genes encoding them relates to the use thereof as the cell lines and their pharmaceutical produced.

[0002]

BACKGROUND OF THE INVENTION Interferons are important proteins produced by the human body having antiviral and antitumor activities. Because of its species specificity, clinical use requires human interferon. Interferon
Based on the reaction with the antibody, it is classified into three types. Interferon-α produced mainly by leukocytes, interferon-β produced mainly by fibroblasts, and interferon-γ produced mainly by T-lymphocytes.

[0003] Interferon produced by human fibroblasts in response to double-stranded (ds) RNA, is mainly glycoprotein IFN-beta ₁ a 20 Kd, which is 0.9k
b), which is obtained from the gene on chromosome 9 excluding the introns.
However, when introduced into frog oocytes by microinjection, another mRNA that produces IFN activity undergoes IFN induction by poly (γI) (γC) followed by cycloheximide (CHX) -actinomycin D treatment. Has been observed in human fibroblasts. Further, when the cloning of complementary (c) DNA of IFN-beta _1, and cDNA clones have been isolated that corresponds to another different RNA of 1.3kb derived together, the RNA is dissolved in reticulocytes 2 which is one of the things
It encodes a 3-26 Kd polypeptide and has been tested to produce human interferon antiviral activity in oocytes [J. Weisse
nbach et al., (1980) Proc. Natl. Ac
ad. Sci. U. S. A. , 77, 7152-715
6; 2,063,882].

[0004] The activity, since it is blocked by anti-IFN-beta ₁ antibody, the polypeptide IFN-beta ₂
is called. IFN-beta and protein and IFN-beta ₁ obtained from the corresponding RNA to _2, the cross-reaction with the same antibody, but their biological activity is prevented, both protein immunological distinction Is what is done. IFN-β
₂ mouse RNA was except chromosomes 9 - from being formed by the human hybrid, the IFN-beta ₂ 1.3k
b of RNA are apparently derived from other genes other than IFN-beta ₁ gene [U. Nir, (1984) P
h. D. Thesis, Weizmann Insti
Tute of Science, Rehovot, I
[srael].

[0005]

Problems to be Solved by the Invention West German Patent Application No. P
The 3,043,981, pure form preparation of interferon-beta ₂ of is described. According to this production method, the cultured human cells capable of producing interferon, and then isolating the DNA comprising the nucleotide sequence of human cells which encode interferon-beta ₂ that when stimulated by interferon inducer by a production method comprising, to obtain the interferon-beta _2. In this production method, mRNA is isolated and purified from the induced cells, and then DNA is obtained by reverse transcription of the mRNA.
NA has been cloned in an appropriate vector.

[0006]

Means for Solving the Problems Obtained as described above
Isolate the gene and transform it into hamster or mouse cells.
DsRNA and cyclohexene
Causing human-specific IFN activity after stimulation of the simide,
IFN-β_TwoOf another gene that encodes
Was proved. IFN produced here is IFN-β
_TwoHave characteristics that are considered to belong to
Gala, IFN-β_TwoHas the same amino acid sequence as
And these are IFN-β_2AAnd IFN-β_2BNamed
Was. Therefore, it is an object of the present invention to
IFN-β_2AAnd human IFN-β_2BTo isolate
You. Another object of the present invention is to provide a biologically active human IFN
−β_2AAnd human IFN-β _2BIs to manufacture.

[0007] Still another object of the present invention is to provide a human IFN-β.
CDNA encoding _2, more particularly human IFN-beta
It is to produce cDNA encoding _2A and cDNA encoding human IFN-β _2B . Still another object of the present invention is to identify genes cDNA hybridizing IFN-beta _2. Still another object of the present invention is to provide a cDNA of IFN-β _2A or a cDNA of IFN-β _2B .
The purpose is to identify a gene that hybridizes to DNA. Still another object of the present invention, genomic clones containing the IFN-beta ₂ gene, the more is to isolate genomic clones containing the genomic clone and IFN-beta _2B gene containing the IFN-beta _2A gene. Yet another object of the present invention is to provide IFN-β _2A and / or IFN-β
An object of the present invention is to provide a recombinant vector containing a _2B gene and a promoter sequence that controls transcription of an interferon gene. It is still another object of the present invention to provide cells that produce biologically active human IFN-β _2A and / or IFN-β _2B .

It is still another object of the present invention to provide cells that produce substantial amounts of IFN-β _2A and / or IFN-β _2B . Still another object of the present invention is to provide a biologically active human IFN-β _2A by genetic engineering technology.
And / or a method for producing IFN-β _2B . Yet another object of the present invention is to provide human IFN-β _2A and / or human IFN-β _2A having a substantial amount of biological activity when fused to a strong transcription promoter of a virus and transfected into hamster or mouse cells. Human I
And to provide a gene that produces a FN-beta _2B. It is a further object of the present invention to provide IFNs having the desired effect on cell growth or differentiation, particularly on the differentiation of terminal cancer cells.
In use as a medicament-beta _2A and / or IFN-beta _2B. Yet another object of the present invention is the use of IFN-β _2A and / or IFN-β _2B to inhibit fibroblast differentiation or prevent post-infection sclerosis.

According to the present invention, the presence of biologically active interferon-β ₂ (IFN-β ₂ ) molecules has been identified, and these molecules are produced by recombinant DNA technology. Such interferon-beta ₂ is a glycoprotein secreted by human fibroblasts production of interferon is induced by dsRNA or virus. IF
N-beta ₂ corresponds to about 5% of the total IFN activity produced by fibroblasts. The amino acid sequence of IFN-β ₂ is,
The sequence is determined by a method using cDNA, and its sequence is determined by other IFNs produced by human cells (IFN-β ₁ , I
FN-α) is only about 20% homologous.

According to the present invention, the human cDNA or gene encoding IFN-β _2A is fused to a strong transcription promoter of the virus. Hamster cells transformed with the fusion cDNA or gene produce human-specific interferon activity, which blocks viral replication and cytopathic effects and is characteristic of the biological response of human cells to interferon. Protein is induced. When isolated and transfected into hamster or mouse cells, dsD
After induction with NA and cycloheximide, human specific I
Causing a FN activity, another alternative of the gene encoding IFN-beta ₂ were found. IFN produced by this gene has the characteristics found to belong to the IFN-beta _2, the amino acid sequence are not the same,
These were named IFN-β _2A and IFN-β _2B .

[0011] comprising a nucleotide sequence encoding an interferon, when the gene for human fibroblasts are isolated, the presence of interferon-beta ₂ was confirmed.
At this time, an appropriate exogenous factor was allowed to act on fibroblasts to induce production of interferon mRNA, and mRNA was extracted. At the same time, mRNA was also extracted from fibroblasts not subjected to the derivatization treatment. M extracted from derivatized and untreated cells
From RNA, using these as templates,
An NA probe was synthesized. Double-stranded cDNA was synthesized from mRNA obtained from the cells subjected to the derivatization treatment, then inserted into an appropriate vector, and transfected into a microorganism. Next, the transformant was cultured under conditions in which the transformant having the vector selectively proliferated, to obtain a first-generation colony. Then, from this colony,
To form colonies of two generations, DNA from both colonies
Was extracted. The DNA obtained from the second colony was
Hybridization was carried out with a cDNA probe synthesized from mRNA obtained from the cells subjected to the derivatization treatment described above.

On the other hand, DN obtained from the second generation colony
A was obtained from cells not subjected to the derivatization treatment described above.
It was also hybridized to a cDNA probe synthesized from RNA. A colony having DNA that hybridized to a cDNA probe obtained from the cells subjected to derivatization but did not hybridize to a cDNA probe obtained from cells not subjected to derivatization was selected, and DNA was extracted from the colony. This DNA is further examined and DNs that hybridize to mRNA translated into interferon in frog oocyte or reticulocyte lysates.
A was selected. Such DNA is essentially DNA that encodes interferon, or DNA that contains sufficient sequence to obtain DNA that encodes interferon.

In the course of such studies, it was found that two other other mRNAs encoding interferon were isolated from derivatized fibroblasts. These mR
By centrifuging NA with glycerol, a small amount of mRNA was precipitated in the 11S fraction. This was translated in a cell to obtain a protein. Such proteins have a molecular weight of 20, 20, which precipitates in response to antibodies to one of the interferons purified from fibroblasts.
000 proteins. This protein is human interferon-beta _1, is part of its amino acid sequence, Knigh
[(1980) Science, 207, 525-
526]. A large amount of mRNA precipitates in the 14S fraction, and the mRNA has a molecular weight of 2
3,000 proteins were obtained. This protein was precipitated by reaction with an antibody against low-purity interferon obtained from fibroblasts.

[0014] was named this protein and human interferon -β _2. MRN to be translated into interferon -β ₂
A cDNA clone that hybridizes with A was prepared in the same manner as described above, and IFN-
The beta ₂ cDNA probes were prepared. CD to hybridize with the mRNA is translated into a interferon -β ₂
It is NA clone, IFN-beta ₂ clones A341 and E474 has a sequence in common, respectively, by fusing them, rebuild the IFN-β ₂ AE20cDNA (see FIG. 1). It was determined the IFN-beta ₂ cDNA nucleotide sequence. This revealed that it had an open reading frame of 212 amino acids expected to produce a 23,500 dalton protein. A transcription-translation test confirmed that the cDNA codes for the protein as described above.

After partially digesting human adult blood cell DNA with EcoRI, λ Sharon 4A [Y. More et al. (1981).
Eur. J. Biochem. 120: 197-20
From human gene library obtained by cloning in 2], genomic clone IFA-2 and I hybridizes to the above-described IFN-beta ₂ cDNA probes
FA-11 was isolated. Clone IFA-2 contains a gene with at least four exons that hybridizes to various segments of the cloned cDNA (FIG. 1). Xho ₁ near the 5′-end of the AE20 cDNA
Since there is a site, genomic segment A132 (FIG. 2) was mapped. A132 is the Xho ₁
It has the first exon of IFA-2 ending 70 bp downstream from the site. Using a DNA probe labeled with BstN1 site positions 40bp from Xho ₁ site, by performing the S1 nuclease analysis, human fibroblasts FS11 that have undergone induction process is distinguished by the nucleotide sequence of 20 pieces of , 5'-terminal S-1
Alternatively, it was found to contain _two IFN-β ₂ RNAs having S-2 (FIG. 3). These two starting points follow the TATA box at position -30 upstream of it (FIG. 4). RNA ending in S-2 was more abundant than other longer RNAs under various derivatization conditions (FIG. 3).

Two RNAs with different 5'-ends
Was also observed with cytoplasmic RNA. Thus, it is clear that two RNAs are formed and show activity in human cells. Both IFN-β ₂ RNAs were induced in human fibroblasts by poly (γI) (γC). This induction occurs in the case of IFN-β ₁ RNA [U. Ni
r et al., (1984) Nucl. Acids. Res. ,
12: 6979-6993], which is facilitated by the addition of IFN itself. CHX for a long time (6.
5h) IFN-β ₂ RNA is also induced by the treatment. When the processing time of CHX is short (3.5 h), only poly (γI) (γC) is effective.
when induced only by CHX without the addition of dsRNA, IFN-β ₂ RNA is not little induction,
It can be distinguished from IFN-beta ₁ by this.

In order to express the IFA-2 gene,
A DNA construct with the SV40 early promoter was created. A 4.8 kb segment of IFA-2 DNA is
PSVE3 DNA via synthetic oligonucleotide
[Y. Chernajovsky 5, (1984) D
NA, 3, 294-308] and the ATG codon is 150 bp from the start of the immediate early RNA of SV40.
It was made downstream (pSVIFA2-II, see FIG. 5). pSVIFA2-II DNA was transferred to cos7 monkey cells [Y. Gluzman, (1981) Cell, 2
3: 175-182] and IFN-β ₂ cDNA
1.35 and 2.2 by Northern blotting using
Two kb of RNA were detected. These two RNAs
Corresponded to the transfer ends RNA in each IFN-beta ₂ by the transfer is complete RNA or SV40 polyadenylation site, (Fig. 5).

For the medium of Cos7 cells in which pSVIFA2-II DNA is transiently expressed, human F
S11 and Wish cells [D. Novic
(1983) J. K. et al. Gen. Virol. , 64:
905-910], IFN activity was measured based on the inhibitory effect on cell effects of VSV.
Antiviral activity was clearly detected 2 days after pSVIFA2-II DNA transfection (first
table). Antiviral specific activity of IFN-beta ₂ per unit is 1 / 30-1 / 100 of the IFN-beta _1,
IFN-alpha ₁ it (5-10 × 10 ⁶ units / m
g). The IFN titer of the medium in which the expression of the IFA-2 gene was performed was, as expected, the same SV4
0 If IFN-beta ₁ gene incorporated downstream of the promoter is expressed was about 3% of the titre of (Table 1).

Further, by exposing FS11 cells to the medium of Cos7 cells into which pSVIFA2-II DNA has been introduced, (2'-5 ') oligo A synthetase [M.
Revel et al., (1981), Meth. Enzymo
l. 79: 149-161] is induced.
SVIFA2-IIDNA is IFN-beta ₂ activity in cells transfected was demonstrated to occur (Figure 6). The induction of (2'-5 ') oligo A synthetase is dose-dependent and quantitatively corresponds to antiviral activity.

Further, the IFA-2 gene was fused so that the SV40 promoter was located at the Xho ₁ site near S-2 (pSVIFA2-I DNA, FIG. 5). Hamster CHO-K1DHFR ^- cells were treated with pSVIFA2
-I and pSVDHFR [Y. Chernajovsk
y et al., (1984) DNA, 3: 294-308] DN.
A together with the stable transformant S
I-15 was isolated. As for this cell line, when measured using human FS11 and Wish cells, 10-
An IFN antiviral activity of 50 U / ml was observed. On the other hand, in the case of CHO cells into which only pSVDHFR DNA was introduced, no production of human IFN was observed (Table 1). By concentrating the medium of the SI-15 culture, a γI with a titer of about 3000 U / ml
FN-beta ₂ solution is obtained.

[0021] The gamma IFN-beta ₂ has a characteristic with human IFN, and by gamma IFN-beta ₂ FS11
In a cell, (2'-5 ') oligo A synthetase contains 1 U
It is induced even at a low concentration of / ml (FIG. 6). CHO
The SI-15 cells, since the gamma IFN-beta ₂ is produced in the presence of cycloheximide, (2'-5 ')
It was demonstrated that the mRNA for producing oligo A synthetase C56 and HLA was specifically induced in humans. The γIFN-β ₂ anti-viral activity, anti--IF
Neutralized by N-beta ₁ polyclonal or monoclonal antibodies, anti -IFN-alpha or anti -IFN-gamma
It was found that the antibody was not neutralized. Only when the mouse-human hybridoma contains chromosome 21 carrying the type 1 IFN receptor gene,
FN-beta _2, the mice - were found to act against human hybridomas. From such biological characteristics of _{γIFN-β 2, γIFN-β} 2 is a than acting as IFN, proves not to have to act as an inducer of IFN-beta _1.

A DNA construct obtained by fusing the IFN-β _2A cDNA sequence to the SV40 early promoter (pSV
CIFβ ₂ , FIG. 5) was transfected into CHO cells together with pSVDHFR as described above,
ΓIFN-β ₂ was obtained in a high yield. And by selecting the resistance of cells to high concentrations of methotrexate, the yield of gamma IFN-beta ₂ was amplified. Even if not amplified, transformed CHO cells pSVCIFβ ₂ (e.g. clone B-132) is, SI-1
5 shows a similar IFN-beta ₂ activity and cell (Table 2).
However, if you select the cells resistant to methotrexate showed high 800U / ml IFN-β ₂ activity (Table 2).

[0023] Using such selected clones, the IFN-beta ₂ protein is secreted into the medium can be precipitated immunologically, also in the case where the cells are labeled with ³⁵ S- methionine, the IFN-beta ₂ protein can also be analyzed by dodecyl sulfate polyacrylamide gel electrophoresis. The size of γIFN-β ₂ is 21,0
00 daltons. According to actual experiments, the translation of IFN-β ₂ RNA in lysates of reticulocytes yielded a product of 23-26 Kd, which was shorter in vitro with a shorter 21 Kd of canine pancreatin membrane. It was found to be converted to protein. The derivatized human fibroblasts also produce a 21 Kd protein, which is immunologically identical to the 26 Kd product, which is a product from IFN-β ₂ RNA; Was different from _one . Mature IFN
N- terminus of 21Kd protein-beta ₂ were not identified. I
The FN-beta ₂ sequence, there are two glycosylation sites (Fig. 1), for this purpose, is to calculate the size of the region removed by processing has become difficult.

However, the area to be removed is I
Longer than in the FN-beta _1, its size, maturation [W. Degrave et al., (1981) Gene, 1
0: 11-15]. I
The FN-beta ₂ of 26Kd polypeptide of hydropathy plots (hydropathy plot), I
Mature FN-beta ₂ of hydrophilic and hydrophobic regions IFN-beta ₁
It has been found that there is remarkable commonality between the two proteins when they are aligned with respect to their C-terminus.
Moreover, IFN-beta ₂ amino acid sequence with other type I human IF
It was also found that the amino acid sequence of N had a consensus sequence (FIG. 7). All known IFN-α and β sequences have 38 common amino acid sequences (starred in FIG. 7). When arranged IFN-beta ₂ with respect to the same manner C- terminus, to all type I human IFN of common 38 amino acids, 18 amino acids are also found in IFN-beta ₂ (FIG. 7 ). IFN-beta ₂ and other type I I
There is about 20% homology with FN.

[0025] IFN-β ₁ amino acid sequence and the IFN-β ₂
Although homology lower between the amino acid sequence, the antiviral activity of IFN-beta ₁ and IFN-beta _2, for example anti -IFN-
β ₁ monoclonal antibody [D. Novick et al., (19
83) J. Gen. Virol. , 64: 905-91.
0] to be neutralized by cross reaction with the same antibody, such as, the active sites of these IFN-beta ₁ and IFN-beta ₂ it can be considered that there is some homology. IFN
Activity-beta _2, since it not neutralized by anti -IFN-alpha or -γ antibodies, this idea is supported. IF
Gene encoding N-beta ₂ is chromosome 9 [U. Ni
r, (1984) Ph. D. Thesis, Weizm
ann Institute of Science,
Rehovot, Israel], and is different from the type I IFN gene of chromosome 9 which has a cluster structure due to the presence of introns. The IFA-2 gene is located on chromosome 7 [P. B. Sehgal et al., (1986) Pro.
c. Natl. Acad. Su. , USA]. Since the homology in the amino acid sequence is detected, IFN-beta ₂ gene is believed to be associated with genes underlying the type I IFN genes to take cluster structure.

Another other genomic DNA clone IFA- hybridizing with IFN-β ₂ cDNA
11 has a different restriction enzyme map from IFA-2 (FIG. 8). From the transcription map and its sequence, the exon at the 3'-end of IFA-2 and IFA-11 (Hbal-H
ind ₁₁₁ segment) (99% homologous sequence), while no region cross-hybridizes with the exon at the 5 'end. The IFA-11 genomic clone (19 kb) was cloned from the HSV-TK gene [F.
Colberg-Garapin et al., (1981) J. Am.
Mol. Biol. , 150: 1-14] and L-
TK-cells were transfected to obtain stable transformants. As shown in Table 1, the L cell clone LI-39 containing the human IFA-11 gene was poly (γI)
When derivatized with (γC) and cycloheximide, human IFN activity was shown, whereas L-TK ⁺ cells did not show such activity.

CHO clone IC40 containing the IFA-11 gene also showed human IFN activity (Table 1). LI-39 or IC40 cells without derivatization did not show any activity. IF after derivation
The IFN activity of the A-11 transformant was IFA-2
It was significantly higher than that of the gene. IFA-11
ΓIFN-β _2B produced by the expression of the gene
As with the product of A-2 gene, its activity by anti -IFN-beta ₁ antibody is neutralized. The DNA probe specific to IFA-11 is, like the IFN-2 DNA probe, RN obtained from derivatized human fibroblasts.
A hybridized with A, indicating that both genes show activity in human cells.

IFN-β_TwoThe function of the gene is
IFN-β under conditions such that the gene is not expressed_TwoHeredity
It is considered that the offspring are expressed. we
Is tumor necrosis factor (TNF) or lymphokine I
When the fibroblasts were exposed to L-1, IFN-β_TwoRed rice
It was found that white was produced. In addition, I
FN-β is neutralized, thereby promoting cell growth
Have also been found by others [M. Koh
as et al., (1986) Posted in Cell: Resnit
Zky et al., (1986) Contributing Cell]. This thing
Et al., IFN-β _TwoIncreases cell growth in response to growth factors
It can be seen that it acts to suppress. This means that I
FN-γ mRNA and mitogen such as PHA
Therefore, IFN-induced peripheral blood mononuclear cells
N-β_TwoRNA that hybridizes with cDNA is detected
It is supported by the fact that it was.

In addition, lymphokines such as IL-1 are 2
In diploid fibroblasts and other human cell lines, IF
It has been reported that RNA that hybridizes with N-β ₂ cDNA is strongly induced [J. Content
Et al., (1985) Eur. J. Biochem. , 15
2: 253-257]. The promoter region of the IFA-2 gene contains two TATA boxes and two RNA start points (FIGS. 1 and 3). This IFN-β ₂
Promoters have been shown to respond to either poly (γI) (γC) or cycloheximide [Y. Chernajovsky et al. (1984), DN
A, 3: 294-308]. In an In vivo, 2 single IFN-beta ₂ gene promoter is different inducers (ds
Activated by RNA, protein synthesis inhibitor, IL-1, PHA). Various types of cells inhibit small amounts of autocrine in growth arrest or differentiation.
e) has been found to produce IFN-β [M.
Revel (1983) Interferon, 5, p.
p. 205-239, Acad. Press, London
on]. IFN-beta ₂ are those belonging to the small number of IFN species produced in accordance with the auto-regulated cell growth,
Type I IFN, on the other hand, is an IFN species that is specifically produced against viral infection.

[0030] IFN-β ₂ of biologically important point is, I
Conditions such as FN-beta ₁ is not induced, for example under conditions such as metabolic mechanism is inhibited [A. Zilberstei
n (1985) The Interferon S.
system, SeronoSymposia Rave
n Press, pp. 73-83] and IFN-β ₂
Is induced. And most importantly, TNF is, that induce IFN-beta ₂ in fibroblasts, fibroblast proliferation promoted by TNF is further enhanced by the addition of an anti -IFN-beta antibody That is. Many human cell lines undergo autocrine IFN-β during differentiation.
These IFNs induce HLA antigens in cells [A. Yarde
n et al. (1984) Embo. J. , 3; 969-97.
3].

In a mouse spinal cord leukemia cell line induced to differentiate by CSF-1, the growth arrest characteristic of terminal differentiation is abolished by anti-IFN-β antibodies. I
L-1 promotes the growth of some types of cells, and the growth factor PDGF has been shown to induce IFN-β RNA in mouse cells [J. N. Zullo et al. (1
985) Cell 43: 793-800]. Many I
Although there is FN-β, [P. B. Sehgal, (918
2), Interferon 4, Academic
Press, PP. 122, and M.P. Revel (19
83), Interferon 5, Academic
Press, PP. 205-239], IFN-β ₂
Is thought to be one of the autocrine autoregulators of cell growth that occurs in response to growth factors. Cells producing autocrine IFN-β show only low antiviral activity [M. Revel (1983), Interf
eron 5, Academic Press, pp. 147-146.
205-239].
₂ low specific activity (antiviral activity as a result of measurement by an immunoassay, IFN-beta ₁ of 1 / 50-1 / 100
).

The hamster cells expressing the IFN-β ₂ cDNA were replaced with hamster CHO cells carrying the human IFN-β ₁ gene [Y. Chernajovsky et al.
(1984), DNA 3: 297-308] can be explained by the above-mentioned concept. Interestingly, IFN-α1 (D)
Account for a large proportion of leukemia IFN [C. We
issmann (1981), Interferon
3, Academic Press, N.M. Y. pp. 1
01-134], despite IFN-α1 (D)
Also exhibit a specific activity as low as 1/100 of other IFN-α [T. Goren et al., (1983), Virology.
130: 273-280]. However, IFN-
With β ₂ , it should not be overlooked that some of the functions of many IFNs are more effectively expressed than in the case of anti-viral activity. Despite the low concentration, the growth regulator IFN-β is still HLA and (2'-
5 ') Induces oligo-A synthetases more strongly and also inhibits the growth of cells producing them longer and more strongly. IL-1 and from the fact that IFN-beta ₂ is induced by TNF, IFN-beta ₂ can not only regulate the growth of cells, plays a role in autocrine mediator of these cytokines in inflammation and acute-phase response It is suggested that

IFN-β₁IFN-β, not cDNA
_TwomRNA that hybridizes to a cDNA probe
Changed the human cell line to interleukin-1 (IL-
I) [J. Content et al. (1985), Eur.
J. Biochem. , 152: 253-257] and
Induction was observed when exposed to TNF-α.
Was. IFN-β using R-antibody_TwoImmune competition specific to
Combination assay (immuno-competition)
 assay) responds to these two cytokines.
In response, human fibroblast FS11 cells were actually IFN-β
_TwoIt was examined whether the protein secreted (FIG. 9). γIL-
α and TNF-α are both IFN-β_TwoProtein Synthesis and Minutes
Induces secretion and, by immunoassay, 10-20 U / m
l of IFN-β _TwoWas secreted. The concentration of γIL-1α is
At 4 U / ml (0.13 ng / ml), IFN-β
_TwoAn optimal induction of
E_Two, Collagenase or hyaluronic acid to human fiber
Concentration of IL-1 required for induction in blast cells [J.
H. Korn (1985), Arthritis Rh
um. , 28: 315-322].

IFN-β ₂ induction (400 U / ml;
The optimal concentration of TNF-α for 40 ng / nl) is the concentration required to lyse sensitive cells [0.1 ng / m
l; M. Wang et al. (1985) Science,
228: 149-154], and higher than the concentration (2 ng / ml) required for promoting optimal growth of diploid fibroblasts. Start of the secretion of IFN-beta ₂ can be earlier by TNF-alpha, reach half of the maximum value of the secretion in about 6 hours. On the other hand, in the case of IL-1, it reaches half the value in 8-12 hours. The documents cited in the preceding sections are hereby incorporated by reference in order to complete the description of the present invention. It will be apparent to those skilled in the art that modifications and variations can be made in the present invention without departing from the scope of the invention.
Further, the present invention is not limited to the description in the specification and the drawings.

[0035]

[Table 1]

[0036]

[Table 2]

FIG. 1 shows a restriction map and nucleotide sequence of IFN-β _2A cDNA. This nucleotide sequence was obtained from the E474 cDNA clone and the A341 cDNA clone [Weissenbach et al., (1980), P.
rc. Natl. Acad. Sci. USA, 77:
7152-7156] at the Xba-1 site. Its 5'-end sequence is the genomic clone IF shown in FIG.
A-2. 3 and 4
Starting from S-1 of 1. The amino acid sequence of IFN-beta ₂ was determined by deduction.

FIG. 2 shows the structure of the genomic clone IFA-2 containing the IFN-β _2A gene. A132 is I
This is a subclone obtained by cloning the EcoRI segment of FA-2 with pBR322. The portions with scattered dots are cDNA clones A341 and E474 [Weis
Senbach et al., (1980), Proc. Nat
l. Acad. Sci. U. S. A, 77: 7152-
7156] shows a hybridizing region. R
Two starting points of NA (caps 1 and 2) and TATA
A box is shown. In RNA, an open reading frame (O) of 212 amino acids (FIG. 1)
RF) is indicated by a thick black line. M. C. Is the second A
TG is shown, and in IFN-β _2A cDNA,
No other ORF was found.

FIG. 3 shows IFN-β_Two5'-end of RNA
Figure 5 shows a partial S1 nuclease analysis. IFA-2 gene
The fragment of subclone A132 of FIG.
And a DNA probe labeled with a BstN1 site,
Total amount obtained from diploid fibroblasts treated by the following method
Hybridized with RNA. The processing method is IFN-β
₁Pretreatment at 200 U / ml for 16 hours; similar pretreatment and
And poly (γI) (γC) (pIC) 50 μg / ml,
3.5 hours treatment; similar pretreatment and cycloheximide
(CHX) 50 μg / ml, treated for 3.5 hours;
Treatment and treatment with pIC and CHX for 3.5 hours; similar pretreatment
6.5 hours treatment with water and CHX; 3.5 hours at pIC
Only treatment; only 3.5 hours treatment with CHX; pIC and C
HX only for 3.5 hours. S-1 and S-
2 is a symbol indicating the starting point of the two RNAs shown in FIG.
You. FIG. 4 shows IFN-β_2AIFA-2 inheritance encoding
Shows the promoter region of the offspring. Subclone A132
The sequence of the part (FIG. 2) is IFN-β_TwoAs cDNA
Indicated. S-1 and S-2 are the two RNs shown in FIG.
A indicates the starting point of A. Initiator ATG is indicated by X
You. The first intron starts with intγl.

FIG. 5 shows IFN-β_2AcDNA and SV40
1 shows the fusion of the early promoters of FIG. IFA
5 ′ of the -2 gene (5.5 kb to 7.5 kb in FIG. 2)
-Terminal Xho₁-BamH₁(Xh-B) segment
To a 26 bp Cla₁-Xho₁Synthetic adapter
(Reconstructing the cDNA sequence at the 5 'end)
Cla₁And BamH₁To the pBR plasmid cut with
This was subcloned and the plasmid pS
VIFA-II was constructed. Hind₁₁₁And BamH
₁Subcloned in pBR plasmid cut with
BamH at the 3 'end ₁-Hind₁₁₁(BH) gene
Segments (7.5 kb to 10.5 kb in FIG. 2)
Hind of pBR322₁₁₁Cla adjacent to site₁Department
And ligated to the 5'-terminal segment,
The entire IFA-2 gene was obtained.

Hind₁₁₁PSVE3 vector cut with
[Y. Chernajovsky et al. (1984) D
NA, 3: 294-308] with Cla₁Use linker
And religated (to form pSVCla), then
The IFA-2 gene was replaced with pSVCla Cla.₁In the site, p
SV40 early promoter forming SVIFA2-II
-Introduced in the same direction. Xho of IFA-2₁Part
Before introduction into pSVE3, the Cla of pBR₁To the site
Except for direct fusion, plasmid pSVI
FA2-I was constructed. Fused to SV40 promoter
IFN-β_2ApVCIFβ containing cDNA_TwoPlasmi
To obtain the pSVIFA2-II DNA,
Xb₁And then Xmn₁With IF and IF
N-β_TwoXho of cDNA sequence₁60 bp downstream from the site
Xmn in₁Open the site (Figure 1) and
Xmn in the amp gene₁The site did not open. this
The construct contains IFN-β_TwoX of cDNA clone AE20
mn₁-Xba₁Segments (corresponding to 92-566 in FIG. 1)
Corresponding to the complete IFN-β as shown in the figure. _2A
The cDNA sequence was recovered.

EES is the RN of SV-40T-ag gene.
A starting point, ATG is IFN-β_TwoStart codon, pA
IFN-β_TwoAnd shows the polyadenylation site of SV40.
You. pSVCIFβ_TwoDNA is transferred to pDHER plasmid
With CHO-K₁DHFR ^-Transfect cells
[Y. Chenajovsky et al. (198
4), DNA, 3: 294-308], and the resulting claw
About Vesicular Stomati
s Virus in FS11 cells using VSV
Human IFN antiviral activity was examined. Methotrexe
(MTX) resistant CHO clones
Thus, gene amplification was achieved. 250nM MTX
Resistant CHO-SVCIFβ_TwoCulture of B132-5M cells
The culture medium assay is shown below the figure. VSV
After the cytopathic effect appears, the cells in the microplate
Was stained with methylene blue. A series of 2-fold dilutions of medium
They are arranged from left to right. IFN Standard (ST) and
And 300 U /
ml of γIFN-β_TwoWas calculated.

FIG. 6 shows that human FS11 fibroblasts previously exposed to the cell culture medium shown below for 16 hours
Poly (γI) (γC) -agarose bordering P40 extract [M. Revel et al., (1981) Meth.
Enzymol. 79: 149-161],
Fig. 3 shows the results of an assay for (2'-5 ') oligo A synthetase. That is, (a) pSVIFA2-II DNA
Cos7 cells 2 days after the introduction (FIG. 5) (the results of the assay are shown in the leftmost four columns in FIG. 6), and (b) stable with pSVIFA2-I DNA in FIG. transformed hamster CHOSi-15 cells (4 rows of the next), CHO expressing (c) IFN-β ₁ gene
MEIF-5 cells [Y. Chernajovsky
(1984), DNA, 3: 294-308] (next row), (d) Transformed C with DHFR gene.
HO cells (next row), and (e) fresh medium only (none). I / ml of medium added to FS11 cells
The results of measuring FN antiviral activity are shown. The results of electrophoresis of the phosphorylated 32P-α-ATP labeled (2′-5 ′) oligo A product are shown.

FIG. 7 shows a comparison of the amino acid sequences of human type I IFN. IFN-β ₁ and a variety of IFN-α
(IFN- represented by α-A and α-C, excluding α-E
alpha, and amino acids that are common to IFN-alpha Class II) and is marked with an asterisk on the IFN-beta ₁ sequence.
The amino acids common to IFN-beta ₂ is that if an asterisk in the case of common that it all type I IFN, IFN-beta, which is marked with squares in the case of common with some IFN if _2A is a hydropersplot (hyd
, and numbered from the predicted processing site.

FIG. 8 shows two types expressed in rodent cells.
Different human IFN-β_Two1 shows a restriction map of the present invention. IF
N-11 DNA (IFN-β_2BGene) is CHO or
Is transfected into any of the L cells and
Expressed N-β activity. FIG. 9 shows IF using R-antibody.
N-β_TwoShows a specific immunocompetition assay
Yes, human fibroblast FS11 cells have two cytochromes.
, Interleukin-1 (IL-I) and tumor necrosis cause
IFN-β in response to offspring (TNF-α) _TwoSecretes protein
It is shown to be. Both cytokines are low
Bell, ie, 10-20 U / ml IFN-β_TwoMost
Suitable induction is when the concentration of γIL-1α is 4 U / ml (0.13 n
g / ml). IFN-β_TwoFor guidance
Optimal concentration of TNF-α (400 U / ml; 40 ng / m
l) is the concentration required for the cytopathic effect of sensitive cells.
Degree (0.1 ng / ml) and diploid fibroblast
From the concentration (2 ng / ml) required to promote optimal cell growth
Is also expensive.

[0046]

[Sequence list]

Applicant name: Yeda Research and Development Company Limited Name of invention: Human interferon-β _2A gene Reference number: PA-19165 Application number: Filing date: Priority number: 860883 Priority date: May 8, 1986 Number: 1 SEQ ID NO: 1 Sequence length: 1145 Sequence type: Number of nucleic acid chains: Topology: Sequence: AGGACTGGAG ATGTCTGAGG CTCATTCTGC CCTCGAGAGCCC ACCGGGAACG AAAGAGAAGC 60 TCTATCTCCC CTCCAGGAGC CCAGCT ATG AAC TCC TTC TCC ACA AGC GTC TCC ACA AGC GTC Ast Ser Thr Ser Ala Phe 1 5 GGT CCA GTT GCC TTC TCC CTG GGG CTG CTC CTG GTG TTG CCT GCT GCC 161 Gly Pro Val Ala Phe Ser Leu Gly Leu Leu Leu Val Leu Pro Ala Ala 10 15 20 25 TTC CCT GCC CCA GTA CCC CCA GGA GAA GAT TCC AAA GAT GTA GCC GCC 209 Phe Pro Ala Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala 30 35 40 CCA CAC AGA CAG CCA CTC ACC TCT TCA GAA CGA ATT GAC AAA CAA ATT 257 Pro Hi s Arg Gln Pro Leu Thr Ser Ser Glu Arg Ile Asp Lys Gln Ile 45 50 55 CGG TAC ATC CTC GAC GGC ATC TCA GCC CTG AGA AAG GAG ACA TGT AAC 305 Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu Arg Lys Glu Thr Cys Asn 60 65 70 AAG AGT AAC ATG TGT GAA AGC AGC AAA GAG GCA CTG GCA GAA AAC AAC 353 Lys Ser Asn Met Cys Glu Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn 75 80 85 CTG AAC CTT CCA AAG ATG GCT GAA AAA GAT GGA TGC TTC CAA TCT GGA 401 Leu Asn Leu Pro Lys Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly 90 95 100 105 TTC AAT GAG GAG ACT TGC CTG GTG AAA ATC ATC ACT GGT CTT TTG GAG 449 Phe Asn Glu Glu Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu 110 115 120 TTT GAG GTA TAC CTA GAG TAC CTC CAG AAC AGA TTT GAG AGT AGT GAG 497 Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu 125 130 135 GAA CAA GCC AGA GCT GTC CAG ATG AGT ACA AAA GTC CTG ATC CAG TTC 545 Glu Gln Ala Arg Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe 140 145 150 CTG CAG AAA AAG GCA AAG AAT CTA GAT GCA ATA ACC ACC CCT GAC CCA 593 Leu G ln Lys Lys Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro Asp Pro 155 160 165 ACC ACA AAT GCC AGC CTG CTG ACG AAG CTG CAG GCA CAG AAC CAG TGG 641 Thr Thr Asn Ala Ser Leu Leu Thr Lys Leu Gln Ala Gln Asn Gln Trp 170 175 180 185 CTG CAG GAC ATG ACA ACT CAT CTC ATT CTG CGC AGC TTT AAG GAG TTC 689 Leu Gln Asp Met Thr Thr His Leu Ile Leu Arg Ser Phe Lys Glu Phe 190 195 200 CTG CAG TCC AGC CTG AGG GCT CTT CGG CAA ATG TAGCATGGGC ACCTCAGATT 742 Leu Gln Ser Ser Leu Arg Ala Leu Arg Gln Met 205 210 GTTGTTGTTA ATGGGCATTC CTTCTTCTGG TCAGAAACCT GTCCACTGGG CACAGAACTT 802 ATGTTGTTCT CTATGGAGAA CTAAAAGTAT GAGCGTTAGG ACACTATTTT AATTATTTTT 862 AATTTATTAA TATTTAAATA TGTGAAGCTG AGTTAATTTA TGTAAGTCAT ATTTTATATT 922 TTTAAGAAGT ACCACTTGAA ACATTTTATG TATTAGTTTT GAAATAATAA TGGAAAGTGG 982 CTATGCAGTT TGAATATCCT TTGTTTCAGA GCCAGATCAT TTCTTGGAAA GTGTAGGCTT 1042 ACCTCAAATA AATGGCTAAC TTTATACATA TTTTTAAAGA AATATTTATA TTGTATTTAT 1102 ATAATGTATA AATGGTTTTT ATACCAATAA ATGGCATTTT AAA 1145

[Brief description of the drawings]

FIG. 1A shows a restriction map of IFN-β _2A cDNA.

FIG. 1B shows the nucleotide sequence of IFN-β _2A cDNA.

FIG. 1C shows the nucleotide sequence of the IFN-β _2A cDNA.

FIG. 2 shows the structure of a genomic clone IFA-2 containing the IFN-β _2A gene.

FIG. 3 shows S1 nuclease analysis of the 5′-end portion of IFN-β ₂ RNA.

FIG. 4 shows the promoter region of the IFA-2 gene encoding IFN-β _2A .

FIG. 5 shows the fusion of the IFN-β _2A cDNA and the early promoter of SV40.

FIG. 6 shows the results of assays for (2′-5 ′) oligo A synthetase.

FIG. 7 is a comparison of the amino acid sequences of human type I IFN.

FIG. 8 shows restriction maps of _two different human IFN-β2.

9 is a photograph showing the results of electrophoresis, and more specifically, shows the immune competition assay IFN-beta ₂ with R- antibody.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12P 21/02 C12R 1:91)

Claims (4)

[Claims] 1. A cDNA consisting essentially of a nucleotide sequence encoding an amino acid sequence of a mature biologically active interferon-β _2A obtainable by measuring hydropathy according to the amino acid sequence shown below. . Embedded image 2. A recombinant vector comprising a cDNA sequence and a regulatory region placed further such as to enable biologically expression of active human interferon-beta _2A position of claim 1. 3. A host cell transformed with the recombinant vector according to claim 2, which is capable of producing biologically active human interferon-β _2A . 4. A consists of culturing the host cell of claim 3 and recovering the interferon-beta _2A, biologically method for producing active human interferon-beta _2A.