DK175194B1 - Recombinant DNA molecules, interferon omega-type genes, transformed hosts containing the DNA information, expression plasmids for interferon-omega types, E. coli transformed therewith, human type I interferons, method ....... - Google Patents

Abstract

The type I interferon peptides encoded by the genetic DNA sequences are called omega-interferons and are produced using appropriate expression vehicles and organisms.

Description

in DK 175194 B1

This invention relates to recombinant DNA molecules containing a coding sequence for novel type I human interferon proteins containing 168-174 amino acids for interferon pseudo-o) 2 (see Figure 12), in-terferon pseudo -o3 (see Figure 13) or interferon-pseudo-u) 4 (see Figure 14). In addition, the invention relates to interferon-type genes, transformed hosts containing the DNA information, expression plasmids for such interferon ω types, E. coli transformed herein, methods of producing these proteins, medically useful interferon-containing mixtures , use of ω-interferon to prepare medically useful mixtures and method for producing said expression plasmids.

The term interferon was formed to describe a selection of proteins that are endogenous in human cells and characterized by exhibiting biological overlap and divergence. These proteins alter the body's immune system and are believed to participate in effective protection against viral diseases. For example, the interferons were divided into three classes, namely o-, 3- and γ-interferon.

At present, only one subtype of 3- and γ-interferon is known in the human organism (see, e.g., S. Ohno et al., Proc. Natl. Acad. Sci. 78, 5305-252530 (1981); Gray et al., Nature 295, 503-508 (1982);

Taya et al., EMBO Journal 1/8, 953-958 (1982)). In contrast, various subtypes of α-interferon have been described (see, e.g., Phil. Trans. R. Soc. Lond. 299, 7-28 (1982))

The fully developed α-interferons differ from each other by at most 23% divergence and are approx. 166 amino acids in length. Of particular interest is a report on a (?) Interferon having a surprisingly high molecular weight (26,000 determined by SDS polyacrylamide / gel electrophoresis) (Goren,

I DK 175194 B1 I

I 2 I

In P. et al. Virology 130, 273-280 (1983)). This inter- I

In feron, IFN-α is called 26K. It has been established that it

You exhibit the highest specific antiviral to date and I

In anticellular activity. 'IN

Apparently the known interferons are I

You have a lot of illnesses, but you have little

You have or have no effect on many other diseases

In me (e.g. Powledge, Bio / Technology, March 1984, 215 - I

In 228 "Interferon on Trial"). In addition, bi- I

In effects of interferons. For example, I was determined

I for samples of recombinant α-interferon side effect against I

In cancer, doses of about 50 million units that you

According to experience from Phase I tests, you should be safe

Caused acute cases of confusion, joint pain that

In the case of incapacity for work, very severe fatigue, I-

I ^ gel on appetite, loss of orientation, epileptic an- I

In the fall and poisoning of the liver. After death, I

In straight heart attacks when treating cancer patients with I

In IF-α, in 1982, the French government banned trials of it

I tea. At least two deaths were also reported in I

In 20 America by experiment recently performed there. It has become you

More and more clearly, at least part of the bi- I

The effects, such as fever and inappropriateness, are direct

You are caused by the interferon molecule itself and cannot reach you

You are brought to contaminants. IN

I ^ Due to the high hopes that interferon I

You had awakened and spurred on the desire to find so far

In unknown interferon-like molecules with minor side effects

In things, the object of this invention was to find and

You make such novel substances. IN

Thus, in the invention, certain novel ones relate to I

In 30 type I interferons which may contain a Leader peptide

In time, and their N-glycosylated derivatives (herein designated I

I as ω-interferon or IFN-ω) containing from 168 I

3 DK 175194 B1 to 174, suitably 172 amino acids, and exhibit a divergence of 30 - 50%, suitably 40-48% against the known subtypes of α-interferon and a divergence of 70% against β-interferon, and on on the one hand, 5 acts as α-interferons, but on the other hand do not possess the many therapeutic disadvantages of these known substances.

Thus, the object of the invention is novel interferons in completely pure form, their non-glycosylated and glycosylated forms, the gene sequences encoding 1Q, as well as recombinant molecules containing these sequences. Furthermore, as plasmids containing said gene sequences, various host organisms, such as microorganisms or tissue cultures, enable the production of these novel interferons by fermentation or by tissue culture methods.

These objects of the invention mentioned above are peculiar to the features of the main claims.

In particular, according to the invention, ω-interferons as well as the corresponding gene sequences of the following formula are highlighted:

I DK 175194 B1 I

I I

I 5 10 15 I

I Cys Asp Leu Pro Gin Asn To Gly Leu Leu Ser Arg Asn Thr Leu I

I TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG I

I 20 25 30 I

I 5 Val Leu Leu His Gin With Arg Arg Ile Ser Pro Phe Leu Cys Leu I

I GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC I

I 35 40 45 I

List Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu With Fall List Gly I

I AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG I

I 1Λ 50 55 60 I

AU Ser Gin Leu Gin Lys Ala His Fall With Ser Val Leu His Glu Met

I AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG I

I 65 70 75. IN

I Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala I

I CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT I

I 15 80 85 90 I

I Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His I

I GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT I

I 95 100 105 I

I Gin Gin Leu Gin His Leu Glu Thr Cys-Leu Leu Gin Val Val Gly I

I CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA I

I 20 110 115 120 I

I Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu Thr Leu I

I GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG I

I 125 130 135 I

Arg Arg Tyr Phe Gin Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys I

I AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA I

I 140 145 150 I

Tyr Ser Asp Cys Ala Trp Glu Val Val Arg With Glu Ile With List I

I TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA I

I 155 160 165 I

Ser Leu Phe Leu Ser Thr Asn With Gin Glu Arg Leu Arg Ser List I

I 30 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA I

I 170 I

I Asp Arg Asp Leu Gly Ser Ser I

I GAT AGA GAC CTG GGC TCA TCT I

I 35 I

In position 111, the sequence GGG (encoding Gly) can be replaced by GAG (encoding Glu). The preferred molecules also include derivatives which are N-glycosylated at amino acid position 78.

As an example, both of the aforementioned ω interferons may contain a Leader peptide of the formula:

With Ala Leu.Leu Phe Pro Leu Leu Ala Ala Leu Val With Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly 10

Explanation of the drawings:

FIG. 1: Restriction map for clone E76E9 Fig. 2: Restriction map for the clone P9A2 Fig. 3: DNA sequence of clone P9A2 FIG. 4: DNA sequence of clone E76E9

FIG. 5: Genomic Southern Blot analysis with DNA from clone P9A2 used as probe Fig. 6: Construction of the expression clone pRHW12 Fig. 7: Amino acid and nucleotide differences between type I interferons

FIG. 8a; List of singular nucleotide positions in the IFN-αA gene

FIG. 8b: List of singular nucleotide positions in the IFN-ωΐ gene.

FIG. 8c: List of singular nucleotide positions in the IFN-αD gene

FIG. 9: Schematic representation of the interferon subtype synthesis for specific probe

FIG. 10: Detection of interferon subtype from specific 30 rnRNA

FIG. 11: DNA sequence of the IFN-wl gene FIG. 12: DNA sequence of the IFN pseudo-β2 gene. 13: DNA sequence of the IFN pseudo-td3 gene FIG. 14: DNA sequence of the IFN pseudo-4 gene 4 FIG. 15: Corrected alignment of the sequences in IFN-ω-

I DK 175194 B1 I

I I

In the genes I

In FIG. 16: Homologies for the signal sequences I

In FIG. 17: Homologies for the "finished" protein sequences I

In FIG. 18: Homologies for the 4-DNA sequences against each other

In 5 second. IN

In accordance with the invention, they are heretofore provided

In unknown ω interferons and the DNA sequences encoding I

Hereby, as follows:

In A line of human B lymphoma cells, e.g. IN

In 10 Namalwa cells (see G. Klein et al., Int. J. Cancer 10, I

I 44 (1972)), when treated with a virus, e.g.

In Sendai virus, is caused to simultaneously produce α- and β-I

In interferon. One can isolate mRNA formed in the stimuli

In prepared Namalwa cells, this can be used as template I

I 15 by cDNA synthesis. In order to increase the yield of the inter-

In feron-specific sequences by the cloning method, I

You allow the preparation of mRNA gradient separators into one I

In sugar solution according to the individual mRNA mols

In different lengths of cooler. Intent is collected

In the 20 term, the mRNA molecules in the 12S region (about 800 to 1

In 1000 base length for mRNA). Here, mRNA-I secretes

In the molecules specific to α- and β-interphase I

In rones. mRNA from this gradient region is concentrated at I

In precipitation and redissolution in water. IN

Preparation of a library for cDNA follows in I

Essentially methods known from the literature (see f. I

In E. E. Dworkin-Rastl, M.B. Dworkin and P. Swetly, Jour- I

I nal of Interferon Research 2/4, 575-585 (1982)). Man I

In primer mRNA by the addition of oligo-dT. Then, I-

At 30, the four desoxynucleoside triphosphates (dATP, I) are added

In dGTP, dCTP, dTTP), and the enzyme reverse transcriptase, and I

In the synthesis of cDNA, a suitable buffer solution I is performed

You are at 45 ° C and lasts an hour. CDNA / mRNA hybrid I is purified

by chloroform extraction and chromatography on a gel column

In 35 le, e.g. Sephadex G50. RNA is hydrolyzed by alkali I

B1 action (0.3 mol NaOH at 50 ° C, one hour) and, after neutralization with acidic sodium acetate solution, cDNA is precipitated with ethanol.

Double strand synthesis is carried out in a suitable solution (six hours at 15 ° C) after the addition of the four deoxynucleoside triphosphates and E. coli DNA polymerase I, thereby forming the hairpin structure at the 3 'end cDNA simultaneously as a template and primer. (see A. Eftratiadis et al., Cell 7, 279, (1976)). After extraction with phenol, chromatography on Sephadex G50 and precipitation with ethanol, the DNA in appropriate solution is treated with the single-strand specific endonuclease Si.

This breaks down the hairpin structure of the cDNA that is not built into the double strand. It is extracted with chloroform and precipitated with ethanol, and gradient separates double-stranded DNA (dsDNA) in a sugar solution by size. In the further clone preparation, only dsDNA of size 600 bp or higher is suitably used to increase the probability of providing only 20 clones containing the complete coding sequence for the novel interferons. dsDNA of more than 600 bp in length is concentrated from the sugar gradient to precipitation with alcohol and solution in water.

The dsDNA molecules formed must be placed in a suitable vector for amplification, and then in the bacterium E. coli. As a vector, the plasmid pBR322 is conveniently used (F. Bolivar et al., Gene 2, 95 (1977)). This plasmid consists essentially of a replicon and two selection markers. These confer resistance to certain antibiotics, Ampicillin and tetracycline (Apr, Tcr). The gene for 6-lactamase (Apr) contains the sequence recognizing the restriction endonuclease PstI. pBR322 can be cut with Pstl. The hanging 3 'ends are extended in a suitable buffer solution 35 by the enzyme terminal deoxynucleotide transfer agent.

I DK 175194 B1 I

i 8 I

In see (TdT) using dGTP. At the same time you are being extended

I dsDNA also at the 3 'ends with the enzyme TdT, using I

In the dCTP. The homopolymer ends of plasmid and dsDNA are com

In plementary and will hybridize when plasmid DNA and dsDNA I

I 5 is mixed, taking into account appropriate concentration levels.

In hold and under suitable salt, buffer and temperature conditions

In gels (T. Nelson et al., Methods in Enzymology 68, 41 - I

In 50 (1980)). IN

In E. coli of strain HB 101 (Genotype P-, hsdS20 I

I (r-B, m-B) recAl3, ara-14, proA2, lacYl, galK2, rpsL20 I

I (Smr), xyl-5, mtl-1, supE44, λ-) prepared by washing with H

In a CaCl2 solution so that it is ready to absorb it

In combining vector dsDNA molecule. Prepared E.coli HB I

I 101 is mixed with DNA and, after incubation at 0 ° C, exposes I

In this, the plasmid DNA thus formed is transformed for a transformation I

I at a heat shock at 42 ° C for two minutes (M. Dagert et I

In al., Gene 6, 23-28 (1979)). The transformed bacteria I

You are then placed on agar plates containing tetra

In cyclin (10 µg per liter). On this agar, E. coli HB I

I 20 101 only grow if it has occupied a vector or re- I

In combinator vector molecule (Tcr). Recombinant vector I

In dsDNA molecules, the host transmits the genotype ApsTcr, since β-I

In the lactamase information by placing dsDNA in $ -lac- I

In the tama gene has been destroyed. The clones are transferred to I

In 25 agar plates containing 50 µg / ml ampicillin. Only approx.

In 3% of the clones grow here, which means that 97% of I

A dsDNA molecule has been inserted into the clones. From 0.5 l

In addition, dsDNA provides more than 30,000 clones. Here

In of, 28,600 clones are transferred individually to the holes in I

In 30 microtiter plates containing nutrient medium, 10 µg / ml I

In tetracycline and glycerine. After clone growth, plate I is stored

Therein at -70 ° C (cDNA library). IN

When to scan the cDNA library for clones, I

In containing genes for novel interferon, I let

After thawing, the clones are transferred to a nitrocellulose

9 DK 175194 B1 release filter. This filter is on growth agar containing tetracycline. The bacterial colonies are grown and then the bacteria's DNA is fixed to the filter.

As a probe, advantageously, the insert is inserted into the clone pER33 (E. Rastl et al., Gene 21, 237 -248 (1983)) - see also EP-A-0.115,613) containing the gene for IFNα2-Arg . This piece of DNA is radiolabeled by notch translation, using DNA polymerase I, dATP, dGTP, dTTP and α- β P-dCTP. The nitro-cellulose filter is pretreated under appropriate, but not stringent, hybridization conditions without the radioactive probe and then hybridized for approx. 16 hours in the presence of the radioactive probe. Then, the filter is washed under appropriate but not stringent conditions · Due to the lack of rigor during hybridization and washing, not only are interferon α2-Arg-containing clones captured, but also other interferon-containing clones whose gene sequence may differ significantly from the previously known interferon-α. After drying, the filter is exposed to an X-ray film. A dark staining that differs clearly from the background level indicates that a clone with interferon-specific sequences exists.

Since the radioactivity signals are of different quality, both the positive and the suspiciously positive reacting clones are allowed to propagate on a small scale.

The plasmid DNA molecules are isolated, allowed to digest by the restriction endonuclease PstI, and separated by size by electrophoresis on an agarose gel (Birnboim et al., Nucl. Acid. Res. 7, 1513 (1979)). DNA 30 in agarose gel is transferred by Southern's method (E.M. Southern, J. Mol. Biol. 98, 503-517 (1975)) to a nitrocellulose filter. The DNA on this filter is hybridized with the radioactive IFN-containing denatured probe. As a positive control, plasmid 1F7 (deposited with DSM 35 under DSM number 2362) which contains the gene for inter

I DK 175194 B1 I

in 10 I

In feron-o2-Arg. According to the autoradiogram, it is clear that I

In that clone E76E9 and clone P9A2 contain a sequence, I

You hybridize under non-stringent conditions with I

In the gene for interferon-α2-Arg. For more detailed information

In 5 characteristics of the dsDNA inserted into clones E76E9 I

I and P9A2. plasmids for these clones are obtained

In silence on a larger scale. The DNA is digested with difference I

In even restriction endonucleases, e.g. with Alul, Sau3A, I

In Bglll, Hinfl, PstI and Haelll. The fragments formed ad- I

I 10 is separated on an agarose gel. By comparison with the pass- I

In the size cursor, e.g. the fragments arising i

I by digesting pBR322 with the restriction endonuclease I

In Hinfl and Haelll, respectively, one can determine the size of I

fragments. Cards are drawn, as directed by Smith and I.

In Birnstiel (H. O. Smith et al. Nucl. Acid. Res. 3, 2387 - I

In 2398 (1967)), and the order of these fragments becomes I

Certainly. Using the thus provided I

Surprisingly, in restriction enzyme maps (Figures 1 and 2), I

You conclude that by inserting pieces into clones E76 I

I 20 is about novel interferon genes, namely I

Similar to the genes for the ω interferons. IN

I This information about the ω interferons is an I

Helps when digesting the inserted cDNA by suitable re- I

In stricture endonucleases. The resulting fragments are ligated I

In 25 with the dsDBA form (the replicative form) of bacterial suspension

In gene M13 mp9 (J. Messing et al., Gene 19, 269-276 (1982)) I

In and divided into sequences using Sanger's dideoxyme-I

In Tode (F. Sanger et al., Proc. Natl. Acad. Sci. 74, 5463-1)

I 5467 (1977)). Single strand DNA is isolated from the re- I

30 combined phages. These are bound to a synthetic oligo-I

In more, and then in four parallel experiments double

In belt string synthesis using the large fragment I

Intended by E. coli DNA polymerase I (Klenow fragment). IN

For each of the four partial reactions, one of the four I is added

In didesoxynucleoside triphosphates (ddATP, ddGTP, ddCTP, I

11 DK 175194 B1 DDTTP). This leads to statistically distributed chain breaks at the sites where the template DNA contains complementary bases for the didesoxynucleoside triphosphate. In addition, radiolabeled dATP is also used. When the synthesis reaction is complete, the products are denatured and the fragments of single-strand DNA divided by size on a denaturing polyacrylic amide gel (F. Sanger et al., FEBS Letters 87, 107-111 (1978)). Then the gel is exposed to an X-ray film.

The DNA sequence of the recombinant M13 phage can then be read from the autoradiogram. The sequences of the inserted pieces in different recombinant phages are further processed in appropriate computer programs (R. Staden,

Nuel. Acid. Res. 10, 4731-4751 (1982)).

FIG. 1 and 2 show the sequencing strategy. FIG.

Figure 3 shows the DNA sequence of the insert in clone P9A2; 4 for the clone E76E9. The non-coding DNA strand is shown in the direction 5 '+ 3' along with the corresponding amino acid sequence.

20 cDNA isolated from the clone E76E9 for β - (Glu) interferon has a length of 858 base pairs and contains a 3 'untranslated region. The region coding for complete β - (Glu) interferon extends from nucleotide 9 to nucleotide 524. cDNA isolated from the clone P9A2 for u> (Gly) 25 interferon has a length of 877 base pairs and the coding sequence for completed ω-interferon runs from nucleotide 8 to nucleotide 523. The 3 'untranslated region extends at P9A2 to the poly-A section.

The DNA sequences encoding complete ω interferon are completely found in clones E76E9 and P9A2. They begin at the N-terminal end with the amino acids cyst-in-aspartic acid leucine. Surprisingly, it has been found that both of the completed ω interferons have a length of 172 amino acids, which differs markedly from the length of hide to 35 known interferons, such as, for example, 166 (

I DK 175194 B1 I

I 12 I

In 165) amino acids by α-interferons. It has surprised- I

It has been found that both ω-interferons possess a po- I

At tentatively N-glycosylation site at amino acid position 78 I

I (asparagine-methionine-threonine). IN

I 5 A comparison of DNA from clones E76E9 and

In P9A2 shows a difference. The triplet encoding amino I

In acid 111 of clone E76E9 is GAG and codes for glutamic acid, I

In contrast, this triplet is GGG in clone P9A2 and encodes H

In glycine. Thus, the two ω interferons differ from H

In each other at an amino acid site and are designated u> - (Glu) -inter- I

In feron (E76E9) and ui (Gly) interferon (P9A2). IN

I Comparison of the ω-interferons with the hitherto I

In known human α-interferon subtypes, the following I

In picture: I

I 15 I

I ω ^ ι · -

In Protein Length I

In amino acids_172_166 _ I

I 20 I

I Potential N-glycosyl- I

In ring location, position_78_- (++) I

I +) Interferon o-A has only 165 amino acids I

In 25 ++) Interferon o-H has a potential N-glycosyl

In teaching site at position 75 (D. Goeddel et I.

In Nature, Nature 290, 20-26 (1981)). IN

In E. coli HB 101 with the plasmid E76E9 and E. coli 101 I

I 30 with the plasmid P9A2 has been deposited with Deutschen I

In the collection fur the Microorganism (DMS Gottingen) with the numbers I

In DSM 3003 and DSM 3004, respectively, on July 3, 1984. I

I As confirmation that the novel clones H

You produce an interferon-like activity, one can get

In 35 examples, cultivate the clone E76E9 and test the bacterial degradation

Post-Growth Inoculation Product by a Plaque Reduction Test

13 DK 175194 B1 ve. As expected, the bacterium produced interferon-like activity (see Example 3).

Furthermore, as confirmation that both of the novel interferons were members of a new interferon group, all DNA from Namalwa cells were isolated and digested with various restriction nucleases. This provides an estimate of how many genes are encoded by cDNA from clones P9A2 and E76E9.

The obtained DNA fragments were separated on agarose gel by Southern's method (EM Southern et al., J. Mol. Biol. 98, 503-517 (1975)), applied to nitrocellulose filters and hybridized under relatively strict conditions. with radiolabeled specific DNA from clone P9A2.

The results of the hybridization with DNA from plasmids P9A2 and pER33 are shown in FIG. 5a.

On this, the individual traces are denoted by letters that refer to the different DNA samples (E = EcoRl, H = HindiII, B = BamHI, S = SphI, P = PstI, C = Clal).

The left half of the filter was hybridized with the interferon-α gene probe ("A") and the right half with the cDNA inserted from clone P9A2 ("O"). The band group that either hybridizes with the α-interferon gene probe or with the novel interferon gene probe is different. The two different probes show no cross-hybridization, judged by the corresponding traces.

In FIG. 5b, cDNA from clone P9A2 is mapped and the fragment used for hybridization. The sites of attack for some restriction enzymes are shown (P - path, S =

Sau3A, A * Alul). The probe comprises only two of the three possible Pst I fragments. The hybridization sample shows only one hybridizing fragment of approximately 1300 base pairs (bp) belonging to the homologous gene. The smaller, 120 bp long fragment, had run through and out of the gel. The band,

I DK 175194 B1 I

I 14 I

In the 5 'portion of the gene cannot be detected because I

the probe does not contain this region. At least I can be seen

In six different bands in this Pstl track. This means, I

In that there must be some other genes present in the human I

In 5 genomes which are related to the novel sequences. IN

I If in these genes there should be one or more PstI- I

attack sites, then it is expected that one will be able to isolate

In clay at least three additional genes. IN

These genes may advantageously be isolated from an I

10 human gene library embedded in a plasmid tumor

In tor, a phage vector or cosmid vector (see Example 4e). IN

I At this point it must be mentioned that the ω-interferons I

According to the invention, not only do the two finished parts comprise I

In terferons, which are described in detail, but also about for- I

In 15 different modifications of these polypeptides, not I

You touch the IFN-io activity. These modifications may f. I

For example, truncation of the molecule by the N- or C-I

At the terminal end, the exchange of amino acids with other re- I

In steroids where the activity does not change significantly, chemistry

20 spoon or biochemical bonds of the molecule to others I

In molecules that can be inert or active. By the I

the latter modifications it may e.g. revolve around you

In hybrid molecules of one or more ω-interferons and / - I

In or known α or β interferons. IN

In order to find the differences in amino acids I

I and the nucleotide sequences of the novel interfero I

, especially io (Gly) - and u) (Glu) interferon, compared with I

In the already known α-interferons and the β-interferon, which I

You are published (C. Weissmann et al., Phil. Trans. R. Soc. I

I 30 London 299, 7-28 (1982); A. Ullrich et al., J. Molec. IN

In Biol. 156, 467-486 (1982); T. Taniguchi et al., Proc. IN

Night. Acad. Sci. 77, 4003-4006 (1980); K. Tokodoro et al

In al., EMBO J. 3, 669-670 (1984)), similarly I

In sequences in pairs, counting the differences of the individual I

In 35 positions. IN

15 DK 175194 B1

From the results depicted in FIG. 7, it is found that the DNA sequences in clones P9A2 and E76E9 are related to the sequences of type I interferons (e.g., α and β interferons). Elsewhere, the difference between the 5 amino acid sequences between the individual α-interferons and the novel sequences is shown to be greater than 41.6% and less than 47.0%. The differences between the novel sequences or sequences of the individual α-interferons and the sequences of the β-interferons are of the order of 10 70%. For the purposes of Example 4, demonstrating the existence of a whole group of related genes, and for the nomenclature proposed for the interferons {J. Vilceck et al., J. Gen. Virol.

65, 669-670 (1984)), it is believed that the inserted pieces of cDNA in clones P9A2 and E76E9 encode a novel class I type interferon, which may be termed interferon-ω.

Furthermore, it is shown that the gene expression of ω-interferon is analogous to the expression of an interferon-type gene. To this end, the transcription of the individual members of the multigen family o and ω interferons is investigated by the S1 mapping method (A.J.

Berk et al., Cell 12, 721 (1977)) (see Example 7), and it is shown that the expression ω-1 mRNA is virus inducible.

25 Since the transcripts of such a gene family differ only in some bases out of ca. 1000, hybridization alone is not a sufficiently sensitive means to determine differences between the various IFN mRNAs.

To this end, the mRNA sequences 30 of 9 α-interferons, of interferon-ω-Ι and of β-interferon are prepared. The bases specific to the upper sequence are denoted here in capital letters. Such specific places can easily be found using a trivial computer program. A hybridization probe based on a specific site can only hybridize perfectly with mRNA

I DK 175194 B1 I

I 16 I

In from a certain subtype. All other mRNAs cannot be hybridized

In bridging at the specific place of the subtype. When he- I

In the bridging probe is marked radioactive in the speci- I

At the 5 'end, only the radioactive labels I are protected

I against digestion of a single strand-specific nuclease I

I (preferably S1 nuclease) which has hybridized with the I

In the interferon subtype mRNA that the probe was constructed in

In to. IN

This principle applies not only to interferon but to

I 10 can be used for any group of known sequences which I

You show the specific sites described in FIG. 8. I

I The aforementioned specific positions in subtypes I

In most cases, they are not restricted places

In why restriction endonuc- cDNA cDNA intersection

15 leases are not suitable for producing subtype speci- I

In neat hybridization probes. Therefore, in this example, they are

pellets used probes constructed by extending one into one

The H 5 'end radiolabeled oligonucleotide which is I

In complementary to the mRNA of interferon-ω-Ι above it spe- I

In 20 specific place. IN

In Pra fig. 10, as expected, it appears that ω-1 mRNA can I

induced in Namalwa and NC37 cells. IN

In the invention, therefore, not only relates to gene sequences, I

specifically encoding the indicated ω interferons but I

25 also modifications that can be easily and routinely provided

You are brought about by mutation, degradation, transposition or I

In addition. All sequences encoding the described I

In human ω interferons (i.e., exhibiting the biological I

In the activity spectrum described herein) and which are degenerate

30 compared to the sequences shown, I

You are included, as those skilled in the art are able to:

You make the degenerate DNA sequences of the coding I

In regions. Furthermore, any sequence encoding an I

In IFN-β spectrum of polypeptide, and as hybridis I

35 with the sequences shown (or portions thereof), under I

B1 stringent conditions (for example, conditions which selectively yield more than 85%, preferably more than 90% homology) are included.

The hybridizations are performed in 6 x SSC / 5 x 5 Denhardt's solution / 0.1% SDS at 65 ° C. The degree of string is determined by the degree of leaching. For a selection of DNA sequences by ca. At 85% or greater homology, the conditions are 0.2 x SSC / 0.01%, SDS / 65 ° C, and for a selection, the selection of DNA sequences by ca.

In 90% or greater homology, the conditions 0.1 x SSC / - 0.01% SDS / 65 ° C are suitable.

Scanning a cosmid library under these conditions (0.2 x SSC) yields some cosmids that hybridize with an IFN-β probe. The sequence analysis of 15 restriction enzyme fragments, isolated therefrom, yields the authentic IFN-iu gene (see Fig. 11), as well as three additional related genes designated as IFN-pseu-do-2, IFN-pseudo-u ) 3 and IFN-pseudo-u> 4 (see Figs. 12-14).

These as well as the encoding peptides are also encompassed by the invention. They are characterized in claims 11 to 13 and 36 to 38.

From the DNA comparisons it is seen that there is an approximate 85% homology between the pseudogens and the IFN-ωΙ gene. (Interferon-w-a gene).

In addition, the IFN-ωΙ gene shows that mRNA, upon transcription, contains information for a functional interferon protein, i.e. encoding a 23 amino acid signal peptide of the formula: 30 Met Ala Leu leu Phe Pro Leu Leu Ala Ala Leu Val

Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly followed by the 172 amino acids, complete IFN-ωΙ.

Interferon-w genes can be placed in any organism that will yield high yields. Suitable hosts and growers

I DK 175194 B1 I

in 18 I

Tors are best known to one of ordinary skill in the art

Referring to EP-A-0.093.619. IN

For expression, prokaryotes are particularly useful

In fair. In particular, E. coli is K 12, strain 294 (ATCC No. I

I 5 31,446) suitable. Among other suitable strains, I

Mention is made of E. coli X1776 (ATCC No. 31,537). As well as you

In the above-mentioned strains, E. coli W 3110 (F ", λ", I

In prototroph, ATCC, No. 27325), bacilli like Bacillus I

In subtilis, and other Enterobacteriaceae, such as Salmonella I

In 10 typhimurium or Serratia marcensens, as well as I

In different Pseudomonads are used. IN

In general, plasmid vectors, I, can be used

I containing replicon and control sequences derived from I

From species compatible with the host cells together

In 15 with these hosts. In the vector, there is usually besides an I

In place of replication, recognition sequences that make it mu- I

It is easy to select the transformed cells by phenotype I

I pi. For example, E. coli is usually transformed with

In pBR322, a plasmid derived from E. coli cells I

I 20 (Bolivar, et al., Gene 2, 95 (1977)). pBR322 contains I

In genes for ampicillin and tetracycline resistance, and do I

In that, easily identifying transformed cells. IN

In addition, the plasmid pBR322 or other plasmids must contain I

Do you have promoters or must be modified to

I 25 will contain promoters that microbe I

The ganism can utilize to express its own protein

Iner. The promoters most often used during manufacture I

In the lining of recombinant DNA, β-rlactamase I contains

I (phenicillinase) and lactose promoter systems, (Chang et I

In al., Nature 275, 615 (1989); Itrakura et al., Science I

I 198, 1056 (1977); Goeddel et al., Nature 281, 544 I

I (1979)) and Tryptophan (trp) promoter sisters (Goeddel et I

In al., Nucleic Acids Res. 8, 4057 (1980); EP-A-0036776). IN

Although the above are the most commonly used promotions

In 35 tumors, other microbial I have also been developed and used

19 DK 175194 B1 le promoters. For example, the gene sequence of IFN-ω can be inserted under the control of the Leftward promoter belonging to the bacteriophage λ (P1). This promoter is one of the most powerful, controllable promoters known. It can be controlled by the λ repressor for which the restriction cut sites are known.

A temperature-sensitive allele of this repressor gene can be inserted into a vector containing a complete IFN-α sequence. If the temperature is raised to 42 ° C, 10 the repressor is inactivated and the promoter is expressed up to its maximum concentration. The sum of mRNAs produced under these conditions should be sufficient to provide a cell containing, among its new synthetic ribonucleic acids, about 10% derived from the P1 promoter. In this way, it is possible to establish a clone bank in which a functional IFN two sequence is located in the neighborhood of a ribosome binding site at varying distances from the X-P1 promoter. These clones can then be tested and 20 selected for the highest yield.

The expression and translation of an IFN-to-se sequence may also proceed under the control of other regulatory systems that may act as "homologous" to the organism in its untransformed form. Thus, e.g. chromosomal DNA from a lactose-dependent E. coli a lactose or lac operon that, by shaking off the enzyme β-galactosidase, enables the degradation of lactose.

The Lac controls can be obtained from the bacteriophage X-plac5 that can infect E. coli. The phage contains lac-30 operon, which may be derived from transduction of the same bacterial species. Regulatory systems that can be used according to the invention may be derived from plasmid DNA belonging to the organism. The system lac promoter operator can be induced by IPTG.

35 Other promoter-operator systems or parts thereof can be used to the same extent: arabinose opera-

I DK 175194 B1 I

I 20 I

In rator, colicin operator operator, galactose operator, alkaline I

In phosphatase operator, trp operator, xylose A operator ,, I

In tac promoter and others. IN

In addition to prokaryotes, eukaryotic micro- I

In 5 organisms are used, such as yeast cultures. The most used

reversed is Saccharomyces cerevisiae among the eukaryotic I

In microorganisms, although a number of other species can also

You are used. For expression in Saccharomyces, e.g. IN

In using the plasmid YRp7 (Stinchcomb et al. Nature 282, 39 I)

I 10 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper I

In et al. Gene 10, 157 (1980)) and the plasmid Yep 13 (Bwach I

In et al., Gene 8, 121-133 (1979)) is used normally. IN

In the plasmid YRp7, the gene contains TRP1, which confers a se I

In lesson marking available to a yeast mutant, I

In 15 that cannot grow in tryptophan-containing medium; eg. IN

In ATCC No. 44,076. IN

The defect in TRP1, which characterizes the genome of I

Thus, in the host organism, a useful aid is, I

By which the transformation can be detected by making I

In the 20 growth without tryptophan. In a very similar way

It is related to the plasmid YEpl3 containing I

the yeast gene LEU 2, which can be used to supplement an I

In LEU-2 minus mutant. Suitable promoter sequences for yeast I

In species vectors, the region is in the immediate vicinity of 51 in I

In the gene for ADH I (Animates G., Methods of Enzymology 101, I

In 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman et al

In al., J. Biol. Chem. 255, 2073 (1980)) or other glycols

In lytic enzymes (Kawaski and Fraenkel, BBRC 108, 1107 - I

(1112 (1982)) as enolase, glyceraldehyde-3-phosphate, de-I

Hydrogenase, hexokinase, pyruvate, decarboxylase, phosphol

In phofructokinase, glucose-6-phosphate isomerase, phospho-I

In glucose isomerase and glucokinase. By efforts of own- I

In the expression plasmids, termination sequences can be asso- I

In addition to these genes, they are additionally inserted into the expression I

In the 35 vector at the 3 'end of the sequence to be expressed

21 DK 175194 B1 is primed so that polyadenylation and termination of mRNA can be predetermined.

Other promoters possessing the advantage that transcription can be controlled by growth conditions are the promoter regions of genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase degrading enzymes coupled with the nitrogen metabolism, the above said glyceraldehyde-3-phosphate dehydrogenase and enzymes capable of degrading maltose and galactose. Promoters regulated by a yeast species "Mating type Locus", e.g. promoters for the genes BARI, MFol, STE2, STE3 and STE5 can be inserted into temperature-regulated systems using temperature-dependent seismic mutations. (Rhine PH. D. in Thesis, University of Ore-15 gon, Eugene, Oregon (1979); Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, Part I, 181-209 (1981), Cold Spring Harbor Laboratory) . These mutations affect the expression of dormant "Mating type cassettes" of yeast species and thereby indirectly on promoters that depend on "Mating type". In general, however, any plasmid vector is suitable that contains a yeast compatible promoter and appropriate replication and termination sequences.

Suitable host organisms besides microorganisms are also cultures of multicellular organisms. In principle, any such culture can be used, whether it comes from vertebrate or invertebrates. Cores of vertebrates are of greatest interest, so that growth of tissue cell cultures has become a routine method for the past 30 years (Tissue, Culture, Academic Press # Kruse and Patterson, Editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells, gold hamster egg (CHO) cells, and W138, BHJ, COS-7 and MDCK cell lines. Expression vectors from these cells also contain (if necessary) a site of replication,

I DK 175194 B1 I

In 22 in I motor placed before the gene to be expressed,

In conjunction with any necessary ribosome binding site, RNA-I

In Splicing site, polyadenylation site and transcriptional I

In termination sequences. IN

I 5 For use in mammalian cells one bases

In often the control functions of the expression vectors of vi- I

In substance. As an example, frequently used promo- I

In tumors from Polyoma Adenovirus 2, and especially often from Simian I

In virus 40 (SV 40). They first and foremost placed the final promo

In 10 SV 40 towers are particularly useful as both can easily be obtained

You are extracted from the virus as a fragment that also contains I

You hold the viral replication sites in SV 40. (Fiers et I

In al., Nature 273, 113 (1978)). Smaller or larger frag- I

Ments from SV 40 can also be used under condition I

In that they contain the approximately 250 bp sequence, I

In the running from the Hindlll incision site to the Bgl I incision site in I

In the viral domain of replication. In addition, so are you

Possible and often advisable to use promoter I

I or control sequences which have the usual form

In association with the desired gene sequences, assuming I

In that these control sequences are compatible with host I

In the cell systems. IN

An area of replication can either be prepared by an I

In appropriate vector construction, so that it can incorporate an exo-I

In 25 gent range, e.g. from SV 40 or another virus source I

I (e.g., Polyoma, Adeno, VSV, PBV, etc.) or can be prepared

You are generated by the chromosomal replica of the host cell

In tions mechanism. If the vector is to be integrated into the host I

In the chromosome of the cell, the latter method is usually added

In 30 extensible. IN

However, it is appropriate to integrate the genes into I

In an expression plasmid pER103 (E. Rastl-Dworkin et al., I

In Gene 21, 237-248 (1983) and EP-A-0,115,613, filed with I

In DSM under the number DSM 2773 on December 20, 1983), when I

In this vector, all control elements containing I

B1 leads to high expression of the cloned gene. Accordingly, according to the invention, the plasmid pBR322 replaces the contained EcoRl / BamHI fragment with a DNA sequence of formula 5

EcoRI Sau3A

10 qaattcacqctGATCGCTAAAACATTGTGCAÅAAAGAGGGTTGACTTTGCCTTCGCGA 59 mRNA Start With CAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RBS Hindlll

Zys Asp ^ rGT GAT C --- IFN-omeca-Gen->

Sau 3A

20 25 1 35

I DK 175194 B1 I

I 24 I

In Por to achieve the object of the invention, reference I can be made

1 to FIG. 6, for example, use the following procedure:

Preparation of the required single DNA fragments:

I 5 fragment and I

I For the preparation of fragment a), an I

In plasmid containing a gene for IFN-ω, e.g. plasmid I

In P9A2 digest with the restriction endonuclease Avail. After I

In chromatography and purification of the cDNA obtained

In 10 joints, this is allowed to be digested twice with re- I

In the restriction endonucleases Ncol and Alul and then iso- I

In clay using chromatography and electroelution. This I

In fragment, most of the corresponding I contains

In the ω-interferon gene. For example, u> (Gly) interfere

In the ring network of clone P9A2 the following structure:

I 10 15 I

In His Give Leu Leu Ser Arg Asn Thr Leu I

I CI CAT GGC CTA CTT AGC AGG AAC ACC TTG 2 £ I

In Ncol I

I 20 20 25 30 I

I Val Leu Leu His Gin With Arg Arg Ile Ser Pro Phe Leu Cys Leu I

I GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 I

I 35 40 45 I

I List Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu With Fall List Gly I

I AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 I

I 25 I

50 55 60

Ser Gin Leu Gin List Ala His Fall With Ser Val Leu His Glu With I

I AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 I

I 65 70 75 I

I Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala I

I 30 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG Ci3 <· 208 I

I 80 85 90 I

Ala Trp Asn With Thr Leu Leu Asp Gin Leu His Thr Gly Leu His I

I GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 I

I 95 100 105 I

In Gin Gir. Leu Gin His Leu Glu Thr Cys Leu Leu Gin Val Val Give I

I 35 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 I

25 DK 175194 B1 110 115 120

Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu 'Thr Leu 5 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA C TG ACC TTG 343 125 130 135

Arg Arg Tyr Phe Gin Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 140 145 150

Tyr Ser Asp Cys Ala Trp Glu Val Val Arg With Glu Ile With List 10 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 155 160 165

Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Arg Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 170

Asp Arg Asp Leu Gly Ser Ser GAT GAC CTG AGA GGC TCA TCT TGAAATGATTCTCATTGATTAÅTTTGCCATA 530 TAAC ACTTGC AC ATGTGAC TCTGGTCAATTC AAAAGAC TC TTATTTCGGC TTTAATC AC 589 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 648 707 20 TT TT AC ATTC ATTTT ATC ATATTTATAC TATTTATATTC TTATATAAC AAATGTTTGCC 766 802 TTTACATTGTATTAAGATAACAAAACATGTTCAGfct

Alul 25 1 35

I DK 175194 B1 I

I 26 I

In Fragment b I

I To produce fragment b), plasmid I is charged

In P9A2 digest with the restriction endonuclease Avail. After I

In chromatography and purification of the cDNA obtained

In 5 sentence pieces this is allowed to be digested with re- I

In the string endonuclease Sau3A and isolate the desired I

In 189 bp fragment by "chromatography and electr

In faith. This fragment has the following structure:

I 5 10 15 I

I Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr Leu I

I IgAT CTG CCT CAG AAC CAT GGC CTA C TT AGC AGG AAC ACC TTG 42 I

I s7u3a | NcoI I

I 20 25 '30 I

I 15 ^ eu ^ eu Gin ^ et Arg Arg Ile Ser Pro Phe Leu Cys Leu I

I GTG C TT CTG CAC C AA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 87 I

I 35 40 45 I

I List Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu With Fall List Gly I

I AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 132 I

I 20 I

I 50 55 60 I

Ser Gin Leu Gin List Ala His Fall With Ser Val Leu Claim Glu With I

I ACG CAG TTG CAG AAG GGC CAT GTC ATG TCT GTC CTC CAT GAG ATG 177 I

I 25 Leu Gin Gin Ile I

I CTG CAG CAjg atc 189 I

In Sau3A | IN

I 30 Fragment c I

To produce fragment c), plasmid I is allowed

In that pER33 (see E. Rastl-Dworkin et al./Gene 21, 237-248 I)

I (1983) and EP-A-0.115.613) digest twice with restriction I

In the tions enzymes EcoRI and PvuII. It after agarose gel- I

In 35 fractionation and purification, 390 bp long 27 DK 175194 B1 fragment containing Trp promoter, the ribosomal binding site and start codon was then digested with Sau3A. The desired 108 bp fragment is obtained by agarose gel electrophoresis, electroelution, and elutip column purification. It has the following structure:

EcoRI | Sau3A

qaattcacgctjGATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTCGCGA 5 9, JmRNA Start With 10 ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RBS Hindlll

Cys Asp TGT hole c 123

Sau 3A

15

Binding of fragments b and c: 20 Fragments b and c are joined by T4 ligase and cut after destruction of the enzyme by HindIII.

This ligated fragment has the following structure:

25 NcoI HindIII Sau3A alftGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 -] - ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 30 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAkqct t Sau3A HindIII! 35

I DK 175194 B1 I

I 28 I

I 5 I

I 10 I

Alternatively, this DNA fragment which is I

Necessary for the production of plasmid pRHW10, also I

I is provided by using two synthetic preparations

In direct oligonucleotides: I

In the oligonucleotide of formula I

I 5 '-AGCTTAAAGATGTGT-3' I

I 20 I

You should be dephosphorylated at its 5 'end. IN

In the oligonucleotide of formula I

I 5'-GATCACACATCTTTA-3 'I

I 25 I

I is treated with T4 polynucleotide kinase and ATP at 5 'one-I

In it. IN

By hybridizing the two oligonucleotides, you obtain

The following short DNA fragment is:

I 30 I

I 5'-AGCTTAAAGATGTGT 3 'I

I 3'- ATTTCTACACACTAGp 5 'I

At one end there is a 5 'overhanging I

In 35 pieces, typical of HindIII, and at the other end I

In a 5 'overhang typical of Sau3A. IN

29 DK 175194 B1

Fragment b) is dephosphorylated by calcium phosphatase. Fragment b) and the fragment described above are brought together and joined by T4 ligase.

Since the ligase requires a 5 'phosphate-containing end, the synthetic DNA piece at the Sau3A end can be linked to fragment b) or another synthetic DNA piece.

Since the two resulting fragments have different lengths, they can be separated by selective isopropanol precipitation. The fragment thus purified is phosphorylated by T4 polynucleotide kinase and ATP.

II. Preparation of Expression Plasmids 15 a) Preparation of the plasmid pRHW10:

The expression plasmid pER103 (E. Rastl-Dworkin et al. Gene 21, 237-284 (1983) and EP-A-0115,613, deposited by DSM with DSM number 22773) is linearized with HindiII and then treated with calf intestinal phosphatase.

After isolation and purification of the DNA obtained, this is dephosphorylated and then linked to the ligated fragments b and c (after their treatment with HindIII). Then E. coli HB 101 is transformed with the provided mixture and cultivated on 25 LB agar plus 50 µg / ml ampicillin. The plasmid exhibiting the desired structure is called pRHW 10 (see Fig. 6), which, after its replication, serves as an intermediate in the production of additional plasmids. b) Preparation of plasmid pRHW122 Plasmid pRHW10 is cut with BamHI, adds

The Klenow fragment from DNA polymerase I and the four deoxy nucleoside triphosphates. The linearized plasmid, which has blunt ends, is incubated after incubation, then purified and cut with NcoI. The large fragment obtainable after agarose gel electrophoresis, electrically

I DK 175194 B1 I

I 30 I

In faith elution and elutipuration, saramen is bound by fragment

Then, the mixture is transformed with E. coli HB 101 I

I and cultivated on LB agar plus 50 µg / ml ampicillin. Pias- I

In the middle exhibiting the desired structure, the designation I is given

In 5 pRHW12 (see Fig. 6) expressing the desired I

I w (Gly) interferon. IN

For example, 1 liter of it thus contains I

In bacterial culture provided (optical density: 0.6 at 600 L)

In nm) 1 x 10 5 IU interferon. IN

I 10 I

I c) Preparation of plasmid pRHWII: I Plasmid pRHW10 is cut up with BamHI and

You add the Klenow fragment from DNA polymerase I and the 4 I

In desoxynucleotide phosphates. It after incubation I

In 15 linear straight-ended plasmid generated, I

You then see and intersect with Ncol. The great frag- I

In ment obtained after agarose gel electrophoresis, electr

In elution and elutip purification, bond with fragment a I

In an analogous manner, derived from plasmid E79E9, in which, however, I

I 20 for the 111th amino acid (Gly) coding GGG codon is substituted I

Into the GAG codon (Glu). Then it is transformed

In admixture with E. coli HB 101 and cultured I

In res on LB agar. The plasmid exhibiting the desired

In structure, the designation is given pRHWII (see analogous to Figure 6) and I

In it it expresses the desired u> (Glu) interferon. IN

In Transformation of the cells with the vehicles, you can

You follow many approaches. One can, for example. use I

In calcium, either washing the cells in magnesium, I

You suspend them in calcium and add DNA, or add

In the 30 cells to a common precipitate of DNA and calcium phosphate

In phat · At the following gene expression, cells I are transferred

I to a medium selective to transformed I

In cells. IN

I After transformation of the host, expression of I

In the gene and fermentation or cell culture under betin I

It is usually possible to express IFN-ω expressions under which IFN-ω is expressed. extract the product by known chromatographic separation methods, thereby providing a material containing IFN-ω with or without Leader and 5 Tailing sequences · IFN-ω can be expressed with a Leader sequence at the N end (pre-IFN-ω ) that can be removed by certain host cells. Otherwise it is necessary to cleave off any Leader polypeptide to provide complete IFN-ω. It is also possible to modify the IFN-ω-10 clone so that the finished protein is produced directly in the microorganism instead of Pre-IFN-ω. In this case, the precursor sequence of the yeast-feeding pheromone MF-e-1 can be used to obtain a correct completion of the fused protein and to excrete the product in the growth medium or in the periplasm. The DNA sequence of functional or complete IFN-ω can be associated with MF-α-Ι at the putative cathepsin-like incision site (after Lys-Arg) at position 256, calculated from initiation codon ATG (Kurjan, Herskowitz Cell 30, 933-943 (1982)).

On the basis of their biological field of action, the novel interferons of the invention are applicable to any type of treatment to which known interferons are used. Here, for example, mentioned are Herpes, Rhino-25 virus, possible AIDS infections, various species of cancer and the like. The novel interferons can be used alone or in combination with other known interferons or biologically active products, e.g. with IFN-α, IL-2, other immune modulators and the like.

IFN-ω may be administered parenterally in cases where antitumor or antiviral therapy is desired and in cases where the immunosuppressive properties are to be utilized. Similar dosages and dosage rates can be used currently used in clinical studies with IFNα materials, e.g. ca. (1-10) x 10®

I DK 175194 B1 I

I 32 I

In units daily, and preparations that are more than 1% pure I

For up to e.g. 5 x 10 ^ units daily. IN

For example, as a suitable dose at one in I

Essentially bacterially produced IFN-ω to be used

In 5, parenterally, dissolve 3 mg of IFN-ω in 25 ml of 5% human I

In serum albumin. This solution is passed through a batch

In rheological filter and the filtered solution is partitioned

Aseptically on 100 small bottles, each containing 6 liters

In x 10 ^ units pure IFN-ω for parenteral use. Before applying- I

In the case, the small bottles are suitably stored cold

I (-20 ° C). The compounds of the invention may be Formula I

In known manner for pharmaceutically acceptable agents, I

In which the polypeptide of the invention is mixed with an I

In pharmaceutically acceptable carrier. Applicable carriers and

In their formulation, E.W. Martin in Remington's I

In Pharmaceutical Sciences, to which express reference is made

See you. IFN-ω is mixed with a measured amount of carrier to I

You provide a suitable pharmaceutical agent which, in effect

To be used effectively in the patient. The agent is used I

In 20 advantageously parenterally. IN

The following examples are not intended to limit op

In the finding, the invention describes in detail. IN

Example 1 I

I Selection of Clones Specific to IFN Sequence I

I a) Preparation of cDNA Library I

In mRNA from cells stimulated with Sendai virus, I

I 30 is used as starting material for producing a cDNA-I

In Library by Methods Known from Literature (E. I

In Dworkin-Rastl et al., Journal of Interferon Research Vol

I 2/4, 575-585 (1982)). They formed 30,000 clones transfer

You are individually resized for the indentations in microtiter plates. IN

I The following medium is used for growth and freezing of cow I

33 DK 175194 B1 lonies: 10 g Trypton 5 g yeast extract 5 5 g NaCl

0.51 g Na-citrate, 2 H 2 O

7.5 g K2HPO4 H2O 1.8 g KH2PO4 0.09 g MgSO4, 7 H2O

0.9 g (NH 4) 2 SO 4

44 g glycerin 0.01 g tetracycline, HCl ad 1 L H2O

The microtiter plates with the individual clones are incubated overnight at 37 ° C and then stored at -70 ° C.

b) Hybridization Probe 20 As the starting material for hybridization probes, the recombinant plasmid pER33 (E. Dworkin-Rastl et al. Gene 21, 237-248 (1983)) serves. This plasmid contains the coding region for the final interferon IFN-α2 arg plus 190 bases belonging to the 3 'nontranslating region. 20 µg pER33 is incubated for one hour at 37 ° C with 30 units of HindIII restriction endonuclease in 200 µl reaction solution (10 mM tris-HCl pH 7.5, 10 mM MgCl 2, 1 mM dithiotreitol (DTT), 50 mM NaCl). The reaction is quenched by the addition of 1/25 vol of 0.5 M ethylenedi-trinotetraacetic acid (EDTA) and heated to 70 ° C for ten minutes. After adding 1/4 vol of 5 x buffer (80% glycerine, 40 mM tris-acetic acid pH 7.8, 50 mM EDTA, 0.05% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue), the fragments formed are charged. separating by size 35 electrophoretically on a 1% agarose gel [gel and flow

I DK 175194 B1 I

I 34 I

In Buffer (TBE)? 10.8 g / l of trishydroxymethylaminomethane I

I (Trisbase), 5.5 g / L boric acid 0.93 g / L EDTA]. Man incu- I

Prepare the gel with a 0.5 µg / ml ethidium bromide solution, I

In which the DNA bands become visible in UV light and cut out

In the portion of the gel containing the IFN gel-containing DNA-I

In piece (about 800 bp length). You put a voltage and you

In electroeluted DNA in 1/10 x TBE buffer. The DNA solution I

Extract once with phenol and four times with ether, I

I and DNA are precipitated by the addition of 1/10 vol of 3 M sodium I

In 10 acetate (NaeAc) pH 5.8, and 2.5 vol EtOH from the aqueous I

In solution at -20 ° C. The DNA is centrifuged, washed

Once with 70% ethanol and dry for five minutes in vacuo. IN

In this, 50 μΐ water (about 50 µg / yl) is added. This DNA I

You are labeled radioactive by chin translation (modifi- I

In cited by T. Maniatis et al., Molecular Cloning, ed. IN

In CSH). 50 μΐ incubation solution contains: 50 mM I

In Tris pH 7.8, 5 mM MgCl 2 # 10 mM mercaptoethanol, 100 ng I

In insert DNA from pER33, 16 µg DNasel, 25 µMol dATP, I

I 25 µΜο dGTP, 25 µΜ dTTP, 20 µΟ a32P-dCTP (> 3000 I

In 20 Ci / mMol) as well as 3 units of DNA polymerase I (E. coli). IN

The incubation takes place at 14 ° C for 45 minutes. You stand- I

You see the reaction by adding 1 vol 50 mM EDTA, 2% SDS, I

In 10 mM Tris pH 7.6 solution and heat to 70 ° C for ten liters

In minutes. DNA is separated by chromatography over Sephadex I

In 25 G-100 in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) from

In no built-in radioactivity. The radiolabelled I

The probe exhibits a specific activity of approx. 4 x 10 ^ I

In cpm / yg. IN

I c) Selection of clones with IFN-containing insert I

In 30, the bacterial cultures that are frozen are thawed

I on the microtiter plates (a). A piece of nitrocellulose fil- I

Iters of appropriate size (Schleicher and Schiill, BA 85, I

In 0.45 µm pore size) is applied to LB agar (LB agar: 10 L

In g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15 g / l bacto I

In 35 agar, 20 mg / L tetracycline HCl). With a stamp which I

35 DK 175194 B1 fits the microtiter plate, the individual clones are transferred to the nitrocellulose filter. At night, the bacteria grow at 37 ° C to colonies of approx. 5 frame diameter.

To destroy the bacteria and denature the DNA, the nitrocellulose filter is applied sequentially to stacks of Whatman 3MM filter sprinkled with the following solutions: 1) 8 minutes on 0.5 M NaOH, 2) 2 minutes 1 M Tris pH 7.4, 3) 2 minutes 1 M Tris pH 7.4 and 4) 4 minutes 1.5 M NaCl, 0.5 M Tris pH 7.4. The filters are air dried and then kept at 80 ° C for two hours. The pretreatment of the filters lasted four hours at 65 ° C in the hybridization solution consisting of 6 x SSC (1 x SSC corresponds to 0.15 M NaCl; 0.015 M trisodium citrate; pH 7.0), 5 x

Denhardt's solution (1 x Denhardt's solution corresponds to 15% 0.02% PVP (polyvinylpyrrolidone)? 0.02% Ficoll (MG:

40,000 D); 0.02% BSM (Bovine serum albumin) and 0.1% SDS

(Sodium dodecyl sulfate). Ca. 1 x 10 ^ cpm per filter of the probe made in b) is denatured by boiling and added to the hybridization solution. The hybridization lasts for 16 hours at 65 ° C. The filters are washed four times an hour at 65 ° C with 3 x SSC / 0.1% SDS. The filters are air dried, protected with Saran Wrap and exposed to Kodak X-OmatS film. 1 2 3 4 5 6 7 8 9 10 11 2

Example 2 3

Control of IFN-containing Recombinant Plasmids by Southern Transfer_5

Of the positive or suspiciously positive-reacting 6 colonies, 5 ml cultures are grown in L-herb (10 g / l 7 tryptone, 5 g / l yeast extract, 5 g / l NaCl, 20 mg / l tetra-8 cyclin x HCl) overnight at 37 ° C. Plasmid DNA isolate 9 res modified after Birnboim and Doly (Nucl. Acid Res.

7, 1513 (1979)). Cells are centrifuged from 1.5 ml suspension (Eppendorf centrifuge) and resuspended at

I DK 175194 B1 I

I 36 I

In 0 ° C in 100 µl lysozyme solution consisting of 50 mM glu-I

In cure, 10 mM EDTA, 25 mM Tris-HCl pH 8.0 and 4 mg / ml lyso-I

I zym. After five minutes of incubation at room temperature I

2 volumes of ice-cold 0.2 M NaOH, 1% SDS-I are added

In 5 solution and incubate for another five minutes. Then you

You add 150 µl of ice-cold potassium acetate solution pH I

For 4.8 and incubate five minutes. The precipitated cell portions I

I centrifuge away. The DNA solution is extracted with 1 L

In the phenol / CHCl 3 (1: 1) portion, and DNA is precipitated at-I

In 10 statement of 2 volumes ethanol. After centrifugation I

You wash the precipitate once with 70% ethanol and dry

For five minutes in vacuum. DNA is dissolved in 50 µl (TE) buffer. IN

To 10 µl thereof is added to 50 µl reaction solution (10 mM I

In Tris-HCl pH 7.5, 10 mM MgCl 2> 50 mM NaCl, 1 mM DTT)

For 15 hours and digested for one hour at 37 ° C with 10 units of PstI-re I

In stricture endonuclease. 1/25 part volume is added

In 0.5 M EDTA and 1/4 volume 5 x buffer (see Example 1b)), I

You heat for ten minutes to 70 ° C and then separate DNA I

In electrophoretic on a 1% agarose gel (TBE buffer). DNA in I

In 20 agarose gel is transferred by Southern's method (E. I. I

In Southern, J. Mol. Biol. 98, 503-517 (1975)) to a Ni

In trocellulose filters. The gel is incubated for one hour with a 1.5 L

In M NaCl / 0.5 M NaOH solution, thereby denaturing DNA. IN

Then neutralize for 1 hour with a 1 M Tris x HCl I

In pH 8 / 1.5 M NaCl solution. DNA is transferred to nitrocel-I

In the lulose filter with 10 x SSC (1.5 M NaCl, 0.15 M sodium I)

In citrate, pH 7.0). When the transfer is complete (approx

For 16 hours) rinse the filter briefly in 6 x SSC buffer

In ferns, air dry and bake for two hours at 80 ° C. File I

For 30 hours four hours are pretreated with a 6 x SSC / 5 x I

In Denhardt's solution / 0.1% SDS (see Example 1c) at 65 ° C. IN

In Approx. 2 x 10 5 cpm of the hybridization probe (see Example 1b) I

I is denatured by heating to 100 ° C and placed in the hy

In the bridging solution. 16 hours are hybridized at 1

At 65 ° C. Then the filter is washed 4x1 hour at 65 ° C

37 DK 175194 B1 with a 3 x SSC / 0.1% SDS solution. After air drying, the filter is covered with Saran Wrap and exposed to Kodak X-Omat film.

5

Example 3 Detection of Interferon Activity in Clone E76E9

A 100 ml culture of clone E76E9 is grown in L-herb (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 5 g / l glucose, 20 mg tetracycline x HCl per 1) to Optical density Agoo = 0 * 8 * The bacteria are centrifuged for 10 minutes at 7000 rpm, washed once with a 50 mM Tris x HCl pH 8.0, 30 mM NaCl solution and suspended in 1.5 ml wash solution. Incubate for 1 hour at 0 ° C with 1 15 mg / ml lysozyme and freeze and then re-thaw the bacterial suspension five times. The cell debris is centrifuged at 40,000 rpm for one hour. The centrifugate is sterile filtered and tested for interferon activity.

The Plaque Reduction Test with 20 V3 Cells and the "Vesicular Stomatitis Virus" (G. R. Adolf et al., Arch. Virol. 72, 169-178 (1982)) is used. Unexpectedly, the clone produces up to 9000 IU of interferon per day. liter of starting culture.

Example 4

Genomic Southern Blot to Determine Number of Genes with New Sequences a) Isolating DNA from Namalwa Cells 400 Centrifuge 400 ml of a Namalwa cell culture in a JA21 centrifuge at 1000 rpm to precipitate the cells. The precipitate was washed gently, suspended in NP40 buffer (NP40 buffer: 140 mM NaCl, 1.5 mM MgCl 2, 10 mM Tris / Cl pH 7.4) and precipitated again at 35 1000 rpm. The new precipitate is suspended in 20 ml of NP40-

I DK 175194 B1 I

I 38 I

In buffer and add 1 ml of 10% NP40 solution, then cell I

In the walls can be destroyed. After five minutes in an ice bath you leave

In the intact cell nuclei, centrifuge at 1000 rpm

In and discard the centrifugate. Cell nuclei are suspended

In 5 ml of 10 ml of a solution consisting of 50 mM Tris / Cl I

At pH 8.0, 10 mM EDTA and 200 mM NaCl, adding 1 ml of I

In 20% SDS to remove proteins. It provided vi- I

In shoe solution, extract twice with the same amount of I

In phenol (saturated with 10 mM Tris / Cl pH 8.0) and twice with I

In chloroform. DNA is precipitated by ethanol addition, from I

You are separated by centrifugation, after which the DNA precipitate I

You wash once with 70% ethanol, dry for 5 minutes in 1

In vacuum and dissolve in 6 ml TE buffer (TE buffer: 10 mM I)

In Tris / Cl pH 8.0, 1 mM EDTA). The concentration of DNA is I

In 0.8 mg / ml. IN

I b) Restriction endonuclease digestion of Na-I DNA

I malwaceller__ I

In the restriction endonuclease digestion is performed at I

In 20 conditions specified by the manufacturer (New England I

In Biolabs). 1 µg DNA is digested with 2 units of suitable restriction

I thion endonuclease in a volume of 10 µl at 37 ° C for two liters

For hours or longer · EcoRI, Hindlll, BamHl, SphI, I

In PstI and Clal are used as restriction endonucleases. IN

I 25 Each time 20 µg of DNA is used. For digestion control I

In the course of the reaction the reaction is separated each time by reaction I

Initially 10 yl from (aliquots) and transitions

I with 0.4 µg Xphag DNA. These aliquots are checked

In quantities after two hours of incubation using agarose I

In 30 gel electrophoresis and assess the completeness of the test

In the breeding process using a swatch for λ-phage- I

In DNA fragments. IN

After the control, the reaction is stopped by:

You add EDTA to a final concentration of 20 mM and raise

For 35 minutes, ten minutes to 70 ° C. DNA is precipitated by addition I

Of 0.3 M NaAc pH 5.6, and 2.5 parts by volume of ethanol. After 30 minutes incubation at -70 ° C, DNA is precipitated in an Eppendorf centrifuge, washed once with 70% ethanol and dried. The DNA obtained is stored in 30 µl of TE buffer.

c) Gel electrophoresis and Southern Transfer

The digested DNA probes are separated by size of 0.8% agarose gel in TBE buffer (10.8 g / l Tris base, 5.5 10 g / l boric acid, 0.93 g / l EDTA). To this end, 15 µl of the DNA probe is mixed with 4 µl of buffer (0.02% SDS, 5 x TBE buffer, 50 mM EDTA, 50% glycerine, 0.1% Bromophenol blue), briefly heated to 70 ° C and place the solution in a well in the gel. λ DNA, cut with Eco RI and HindIII, is placed in a corresponding well and used as a marker for the DNA major pig. The gel electrophoresis is performed over 24 hours at about 1 V / cm. Then, by Southern's method, DNA is placed on a nitrocellulose filter (Schleicher and Schuell, BA 20 85), using as transfer buffer 10 x SSC (1 x SSC: 150 mM trisodium citrate, 15 mM NaCl, pH 7.0). The filter is dried at room temperature and then heated for two hours to 80 ° C to bind DNA to it. 1 2 3 4 5 6 7 8 9 10 11 d) Hybridization probe 2 20 µg plasmid P9A2 is cut with Avail to form 3 fragment of approximately 1100 pb in length, which 4 contains the entire cDNA insert. This piece of DNA up 5 is cut again with Sau3A and Alul, and the largest DNA piece 6 is isolated after agarose gel electrophoresis (see Fig. 5b) 7 by electroelution and elutip column chromatography.

8

The DNA (1.5 µg) thus obtained is dissolved in 9 15 μΐ water.

10 20 µg plasmid pER33 is cut with HindIII, which cuts this expression plasmid for IFN-α 2-Arg two genes.

I DK 175194 B1 I

I 40 I

(Ge Rastl-Dworkin et al. Gene 21, 237-248 (1983)). IN

In the small DNA fragment, the gene for interferon I contains

In ct2Arg and isolated in the same way as in plasmid P9A2. IN

I Both DNA pieces are chopped after a method I

In 5 de, proposed by P.W.J. Ribgy et al. (J. Mol. Biol. 113, I

In 237-251 (1977)). The chin translation is both performed i

In 0.2 µg DNA in a 50 µl solution consisting of I

I of 1 x notch buffer (1x notch buffer: 50 mM Tris / Cl pH 7.2, I

In 10 mM MgSO 4, 0.1 mM DTT, 50 pg / ml BSA), each time with I

In 10 100 μΜοΙ dATP, dGTP and dTTP, 150 pCi a-1P-dCTP I

I (Amersham, 3,000 Ci / mMol) and 5 units of DNA polymerase I I

I (Boehringer-Mannheim, hook translation quality). After I

For two hours at 14 ° C, the reaction is stopped by addition I

I of the same amount of an EDTA solution (40 mmol) and I

In 15, the unreacted radioactive material is removed by means of I

I of G50 column chromatography with TE buffer. The future I

In specific radioactivity, about 100 x 10 5 cpm / pg I

In DNA. IN

(E) Hybridization and autoradiography

In 20 The nitrocellulose filter is cut in two halves.

In the two halves contain identical additions of Na-I

In walwa DNA treated with the restriction enzymes, I

Referred to in Example 4a. The filters are prehybridized to two

For hours at 65 ° C in a solution containing 6 x SSC, 5 L

I 25 x Denhardt's (1 x Denhardt's 0.02% bovine serum albumin I

I (BSA), 0.02% polyvinylpyrrolidone (PVP), 0.02% Ficoll I

I 400), 0.5% SDS, 0.1 mg / ml denatured calf DNA I

I and 10 mM EDTA. The hybridization is carried out for 16 hours I

I at 65 ° C in a solution containing 6 x SSC, 5 x 1

In 30 Denhardt's, 10 mM EDTA, 0.5% SDS and about 10 x 106 cpm I

In notch-translated DNA. One half of the filter is hybrid

You interfere with interferon-α2-Arg DNA and the other half I

I with interferon DNA isolated from plasmid P9A2. IN

After the hybridization, both filters are washed at room temp

In 35 rator four times with a solution consisting of 2 x SSC I

41 DK 175194 B1 and 0.1% SDS, and then twice at 65 ° C with a solution consisting of 0.2 x SSC and 0.01% SDS. Then, dry the filters and expose to a Kodak X — Omat S— film.

Example 5

Preparation of expression plasmids _pRHW 12 and pRHW 11_

Introduction: Preparation of the expression plasmids is illustrated in FIG. 6 (not scaling), all restriction enzyme orders are also performed according to the manufacturer's instructions.

a) Preparation of Plasmid pRHW 10 15 µg of plasmid P9A2 is digested using 100 units of restriction endonuclease Avail (New England's Biolabs). Thereafter, the enzyme is inactivated by heating to 70 ° C and fractionating the obtained fragments by size of a 1.4% agarose gel with 20 TBE buffer (TBE buffer: 10.8 g / l Tris base, 5.5 g / l boric acid, 0.93 g / l EDTA). The bands containing the entire cDNA insert are electroeluted and purified on an elution tube (Schleicher & Schuell). 20 µg are obtained and 6 µg are further digested with the restriction endonuclease Sau3a (20 units total 100 µl solution).

The fragments are separated by 2% agarose gel TBE buffer. After staining with ethidium bromide (EtBr), the 189 bp DNA piece was electroeluted and purified as described above (= fragment b in Fig. 6).

The corresponding DNA fragment is isolated from the expression plasmid pER33 (E. Rastl-Dworkin et al. Gene 21 For this purpose 50 µg pER 33 to 35 are charged

I DK 175194 B1 I

I 42 I

In digestion, digest with the restriction enzymes EcoRI and PvuII I

And fractionate the obtained fragments by size

In the case of a 1.4% agarose gel in TBE buffer. It 389 bp I

In long DNA pieces containing the Trp promoter, it ri- I

At 5 bosomal binding sites, and the start codon, I is electroeluted

In and cleaned over an elutip column. It thus provides

Then, in clipped fragments, digest with Sau3A, I

I and the desired 108 bp fragment are obtained by means of I

I of agarose gel electrophoresis, electroelution and elutip- I

In 10 column purification with a yield of about 100 ng (= frag- I

I ment c i f ig. 6). IN

In 20 ng of fragment b is joined over 18 l

For hours at 14 ° C with 20 ng of fragment c in a volume of 1

In 40 μΐ using 10 units of T4 ligase in an

In solution containing 50 mM Tris / Cl pH = 7.5, 10 mM I

In MgCl2 # 1 mM DTT and 1 mM ATP. The enzyme I is then charged

Destroy by heating to 70 ° C and leave to provide

You brought DNA slices with HindiII to a total volume of 50 I

lul. IN

In 10, 10 µg of the expression plasmid pER103 I is let

(E. Rastl-Dworkin et al., Gene 21, 237-248 (1983))

In arises with HindIII in a total volume of 100 μΐ. After I

For two hours at 37 ° C, 1 volume of 2 x phosphate I is added

In sepa buffer (20 mM Tris / Cl ph 9.2, 0.2 mM EDTA) together with I

In one unit of calf intestinal phosphatase (CIP). After 30 minutes I

At 45 ° C, an additional unit of CIP is added and incubated

You cook for another 30 minutes. It thus in-

In pathway DNA is purified by double phenol-I

In traction, single chloroform extraction and precipitation at I

In the addition of 0.3 M sodium acetate (pH = 5.5) and 2.5 room-I

In catch ethanol. Next, it is dephosphorylated to prevent

To prevent the vector from being restored at the next process step. IN

In 18 hours at 14 ° C 100 I are bonded together

In the linearized pER 103, and they ligated the fragment

In 35 ter b and c (after digestion with HindIII) in a solution I

43 DK 175194 B1 of 100 µl containing ligase buffer with T4 DNA ligase.

200 µl of prepared E. coli HB 101 (E. Dworkin et al., Dev. Biol. 76, 435-448 (1980)) is mixed with 20 µl of the above mixture and incubated for 45 minutes on ice. Then, DNA recording is stopped by a heat shock of 42 ° C for two minutes. The cell suspension is incubated for an additional ten minutes on ice and then transferred to LB agar (10g / l tryptone, 5 g / l yeast extract, 5 10 g / l NaCl, 1.5% agar) containing 50 mg / l ampicillin . The plasmids found in the provided 24 colonies are isolated by a method described by Birnboim and Doly (see Nucl. Acid. Res. 7, 1513-1523 (1979)). After digestion with various restriction enzymes, a plasmid exhibits the desired structure. This is designated pRHW 10 (see Fig. 6).

b) Preparation of plasmid pRHW 12

Ca. 10 µg of plasmid pRHW 10 is cut with BamHI.

Then the Klenow fragment of DNA polymerase I and the four deoxynucleoside triphosphates are added and incubated for 20 minutes at room temperature. The linearized plasmid having straight ends is purified by phenol extraction and precipitation and then cut with 25 restriction endonuclease Nco I in a volume of 100 µl.

The largest fragment is recovered by agarose gel electrophoresis, electroelution, and elutip purification. Fragment a) (see Fig. 6) is obtained from 4 µg Avall fragment containing the P9A2 cDNA insert 30 (see above) by digestion with NcoI and Alul to obtain about 2 µg fragment a).

Finally, fragment a) is joined and added thereto twice with BamHl / NcoI digested pRHW 10 in a volume of 10 µl, using 10 ng of each DNA.

35 When joining a fulfilled BamHI cutting site to

I DK 175194 B1 I

I 44 I

In a DNA cut with Alul, a BamHI-I is again formed

In the cutting site. IN

The above-mentioned mixture is transformed with I

In pretreated E. coli HB 101 as described above. By I

In 5 of the 40 colonies provided, one is selected which one gets you

In the designation pRHW 12. I

In the plasmid, isolate and EcoRI / BamHI insert I

In the piece, sequence analysis is performed according to Sanger's method (F. I

In Sanger et al., Proc. Nat- Acad. Sci 74, 5463 - 5467 I

I 10 (1979)). It contains the expected sequence.

I c) Preparation of plasmid pRHW 11 I

In the Process, Example 5b follows. 1 µg of plasma I

In mid pRHW 10 is digested with BamHI. The sticky ends of I

In the DNA obtained is blunted by Klenow-I

In the DNA polymerase I fragment and the 4 deoxynucleoside I

In triphosphates, the linearized DNA I is then cut

I with Ncol. The large fragment is recovered by aga-I

In rose-gel electrophoresis, electroelution and Elutip column- I

In chromatography. IN

The NcoI-AluI fragment from clone E76E9 isolates I

In Example 5b. 10 ng of this vector fragment and 10 l

In the cDNA fragment, one binds in a volume of I

I 10 μΐ under suitable conditions using a unit T4-I

In 25 ligase · After transformation of the DNA mixture into E. coli I

In HB 101 and scanning the provided 45 colonies I

On LB plates containing ampicillin, one is selected

In the clone, which is named pRHWll. The clone is cultivated and you

In isolating plasmid DNA. This structure is confirmed

You know that it contains restriction endonuclease section I

In places of Alul, EcoRI, HindlH, Ncol, Pstl. IN

I 35 I

D) Expression of interferon activity of E. coli HB 101 containing plasmid pRHW 12_ 100 ml of the bacterial culture is incubated in M9 minimal medium containing all amino acids except tryptophan (20 µg / ml per amino acid ), 1 µg / ml thiamine, 0.2% glucose and 20 µg / ml indole (3) -acrylic acid (IAA), tryptophan operon inductor, up to an optical density of 0.6 at 600 nm. The bacteria are then centrifuged (ten minutes, 7000 rpm), washed once with 50 mM Tris / Cl pH 10 = 8, 30mM NaCl and suspended in 1.5 ml of this buffer. Incubate 30 minutes with 1 mg / ml lysosyme on ice, then freeze and thaw the bacteria five times. Cell residues are removed by centrifugation for one hour at 40,000 rpm. The centrifugate is sterile filtered and assayed for interferon activity by a plaque reduction test using human A549 cells and Encophaolo myocarditis virus.

Result: 1 1 of the bacterial culture produced contains 1 x 10 8 IU of interferon (A. Billian, 20 Antiviral Res. 4, 75-98 (1984)).

Example 6

Differences in amino acid and nucleotide sequences for type I interferons_ 25 a) Comparison of the amino acid sequences

The pairwise comparison of the amino acid sequences is made to begin the comparison with the first cysteine residue in the final α-interferon and at the first cysteine residue in the amino acid sequences encoded by the cDNA insertions in plasmids P9A2 and E76E9. Both sequences are called in FIG. 7 IFN-ω, since the obtained values could not detect any differences between the specific sequences belonging to P9A2 and the E76A9 cions. The only correction was the insertion 35 of an empty space at position 45 of interferon aA, which

I DK 175194 B1 I

I 46 I

You were counted as a mistake. When ω-interferon sequence is part I

In the comparison, this is performed taking into account I

In the usual 166 amino acids. This value is given in FIG. IN

I 7 along with values for the other 6 amino acids that I

In the 5 clones P9A2 and E76E9 encode. You get a percentage increase

In the divisions of division of the calculated differences with numbers- I

In light 1.66. An additional amino acid thus corresponds to an I

In percentage of 0.6. For the six additional amino acids of IFN-I

In ω this gives 3.6%, which is already recognized in percent- I

In the 10th century. IN

When compared with β-interferon, you start

I at the 3rd amino acid of the finished β-interferon, and with I

In the first amino acid of the final α-interferon or I

In the first amino acid for which plasmids P9A2 and I

In 15 E76E9 codes. The longest comparative structure for o- I

Thus, in interferon versus β-interferon, it exceeds 162 I

In amino acids, and there are two additional amino acids both for α-I

interferon and for β-interferon. These are counted as error I

I and are shown separately in FIG. 7, but is recognized in percent- I

In the 20 numbers. Comparison of β-interferon with amino acid I

In the sequences belonging to clones P9A2 or E76E9, I-I

You are carried in the same way. However, a total of 10 I occurs

In extra amino acids. IN

B) Comparison of Nucleotide Sequences I

I Sequence sequences for comparison

analogous to Example 6a). The first nucleotide of the I

In finished α-interferon DNA, the first nucleotide that I

You belong to the triplet that encodes cysteine in it

In 30 g of α-interferon. The first nucleotide in plasm I DNA

In the mites P9A2 or E76E9 is also the first nucleotide in I

In the codon of cysteine-1. The first nucleotide in DNA from I

In β-interferon, the first nucleotide of the third is tri- I

In the spot. The comparison runs over a total of 498 nucleotides, I

35 when comparing the individual DNAs of the α interferons with I

B1 DNA from β-interferon, and over 516 nucleotides, when the DNA sequences of the individual α-interferons or β-interferon are compared with the sequences of plasmids P9A2 and E76E9. The absolute error number is indicated in the left-hand part of the table in FIG. 7, followed by the corresponding percentage.

Example 7 10 Virus Inducible Expression for ω-1 mRNA and NC37 Cells

5000 Ci / mMol) and 10 units of polynucleotide kinase for a total volume of 10 µl (70 mM Tris / Cl pH = 7.6, 10 mM

MgCl2 / 50 mM DTT) and leave to stand for one hour at 37 ° C. Thereafter, the reaction is stopped at a temperature of 10 ml for 20 minutes to 70 ° C. The radiolabeled oligonucleotide obtained is hybridized with 5 µMol M13pRHW 12 ssDNA (see Fig. 9) for a total volume of 35 µl (100 mM NaCl) for one hour at 50 ° C.

Cool to room temperature and add chopped translation buffer, the 4-deoxynucleoside triphosphates and 10 units of Klenow polymerase to a total volume of 50 µl (50 mM Tris / Cl pH = 7.2, 10 mM MgCl 2, 50 µg / ml BSA , 1 mM per nucleotide). The polymerization is carried out for one hour at room temperature and then quenched by heating for five minutes to 70 ° C.

Upon the reaction, a partially double-stranded circular DNA is obtained. This is then cut into a total volume of 500 µl with 25 units of Avail, using the buffer prescribed by the manufacturer. This cuts the double-stranded region into uniform sizes.

I DK 175194 B1 I

I 48 I

H see. Then, the reaction is stopped at a 5-minute I

In heating to 70 ° C. IN

b) Preparation of RNA from Virus-Infected Cells I

In 5 x 100 cells (0.5 x 10 6 / ml), 48-72 I are treated

For hours with 100 μΜοΙ dexamethasone - the control sample contained I

There is no dexamethasone. For the use of interferon induc- I

In tion, the expression cells are suspended in a serum-free medium

dium at a concentration of 5 x 106 / ml and infected with I

10 2 ^ ® units / ml Sendai virus. Aliquote parts of liquid I

In the above cell culture precipitate is tested in Plaque reduction I

the sample (Example 5b) for IFN activity. The cells in- I

harvested six hours after virus infection using I

centrifugation (1000 g, 10 minutes), washed with 50 ml of I

15 NP40 buffer (Example 4a) is suspended in 9.5 ml of ice cold I

NP40 buffer and lysed by holding on ice for five minutes

ter with 0.5 ml of 10% NP40. Cell nuclei are removed at I

centrifugation (1000 x g, 10 minutes), and centrifugate I

is adjusted with 50 mM Tris / Cl, 0.5% sarcosine and 5 mM EDTA I

H 20 at pH = 8 and stored at -20 ° C. RNA is isolated from one-I

In the trifugate by extraction once with phenol, once I

I with phenol / chloroform / isoamyl alcohol and once with I

In chloroform / isoamyl alcohol. The aqueous phase is transferred to 4 L

In ml 5.7 molar CsCl chloride and centrifuged in a SW-ro-I

In 25 rotors (35 rpm, 20 hours) to free the extract of DNA I

In and residual proteins. The RNA precipitate is suspended in 2 ml of I

In TE pH = 8.0 and precipitated with ethanol. The precipitated RNA I

is then dissolved in water to a concentration of 5 mg / ml. IN

C) Detection of interferon-ω mRNA I

0.2 µl of the hy-I prepared according to Example 7b

The bridging probe coincides with 20-50 µg of the I

In Example 7c, RNA prepared by the addition of I

In ethanol. In a control experiment is used instead of I

In 35 cellular RNA transfer RNA (tRNA) or RNA originating I

49 E. 175194 B1 from E. coli, transformed with the plasmid pRHW 12 (Example 5).

The obtained precipitate is washed salt-free with 70% ethanol, dried and dissolved in 25 μΐ 80% formamide (100 5 mM PIPES pH = 6.8, 400 mM NaCl, 10 µM EDTA). then

If the probes are heated for five minutes to 100 ° C to denature the hybridization probe, immediately set the temperature to 52 ° C and incubate at this temperature for 24 hours. After hybridization, 10 is placed on ice and 475 µl of S1 reaction mixture (4 mM) is added

Zn (Ac) 2 »30 mM NaAc, 250 mM NaCl, 5% glycerine, 20 µg ss calf thymus DNA, 100 units of S1 nuclease). It is digested for one hour at 37 ° C, after which the reaction is stopped by ethanol precipitation.

The precipitate is dissolved in 6 µm formamide buffer and separated on a 6% acrylamide gel containing 8 M urea, essentially as by probes for DNA sequencing reactions (F. Sanger et al., Proc. Nat.

Acad. Sci. 74, 5463-5467 (1979)).

20 For autoradiography, the dried gel and one are used

DuPont Cronex X film using a Kodak Lanex Regular Intensifier and operating at -70 ° C.

Explanation_to_fig._10 25 Rows A to C are for control.

Row A: 20 µg tRNA

Row B: 10 µg RNA from pER 33 (E. coli expression strain for interferon-o2-Arg) Row C: 1 ng RNA from pRHW 12 (E. coli expression strain for interferon-ω-Ι) Row D: 50 ug RNA from untreated Namalwa cells Row E: 50 ug RNA from virus-infected Namalwa cells Row F: 50 ug RNA from Namalwa cells, pretreated with dexamethasone and infected with virus 35 Row G: 20 ug RNA from untreated NC 37 cells

I DK 175194 B1 I

I 50 I

In Row H: 20 µg RNA from virus-infected NC 37 cells I

In Row I: 20 µg RNA from NC 37 cells pretreated with I

dexamethasone and infected with virus I

In Row M: Scale marking (pBR 322 cut with Hinfl). IN

I 5 Rows B and C show that the expected signal is only I

You can detect when a ωΐ-specific RNA exists among I

In the RNA molecules. Further, it appears that even a large I

In excess of false RNA does not give background signal (see I

In row B). Furthermore, tRNA, used as I, also does not yield

In 10 hybridization partners, some signal (see row A). IN

In rows G to I, induction of ωΐ-specific I shows

In RNA in virus-infected NC 37 cells. The pretreatment with I

In dexamethasone this effect intensifies. IN

In Rows D to F, one shows that in large and large

15 le gets the same result with Namalwa cells. Induction I

however, with ωΐ-specific RNA is not as strong as with NC 37-I

In the cells. The result is parallel to the result of measure I

You linger for interferon on the centrifugate over the I

In cell sediment. IN

I The ratios of interferon-cal gene expression are I

So, as one might expect from an interferon typel gene. IN

In Example 8 I

I Isolation of the gene encoding IFN-ωΙ and Be- I, respectively

In related genes: _ I

I a) Cosmid study I

I A human cosmid bank (human DNA, male) cloned I

I in cosmid vector pcos2 EMBL (A. Ponstka, H.-R. Rockwitz, I.

In A.-M. Frischauf, B. Hohn, H. Lehrach Proc. Natl. Acad. IN

In Scl. 81, 4129-4133 (1984)) having a complexity of 2 x 1

I 106) is searched for IFN-ω- and related ge- I, respectively

You down. E. coli DH1 (rK “* rec.A; gyrA96, sup- E) I

I 35 is used as the host organism. I was then prepared

51 DK 175194 B1

Mg ++ cells (= "plating bacteria"). E. coli DH1 grows overnight in K-herb (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl), supplemented with 0.2% maltose · The bacteria are centrifuged and transferred to a 10 mM MgSO -5 solution until ODggo = 2 · 5 ml of this cell suspension is incubated for 20 minutes at 37 ° C with 12.5 x 10 10 colony forming units of packed cosmide 20.

10 volumes of LB are then added and the slurry is kept for one hour at 37 ° C to obtain expression of the kanamycin resistance supplied by the cosmid. Then the bacteria are centrifuged, suspended in 5 ml LB and ironed out in 200 μΐ aliquots on nitrocellulose filters (BA85, Schleicher and Schvill, 132 mm average) on LB agar (1.5% in L herb) plus 30 µg / ml kanamycin. 15 10,000 - 20,000 colonies are grown per filter. The colonies are replicated on additional nitrocellulose filters stored at 4 ° C.

A group of the colony filters is treated as in Example 1c), i.e. the bacteria are denatured, whereby single strand DNA can be fixed to nitrocellulose. The filter is pre-washed for four hours at 65 ° C in a 50 mM Tris / HCl, pH = 8.0, 1 M NaCl, 1 mM EDTA, 0.1% SDS solution. Then the filter is incubated for two hours at 65 ° C in a 5 x Denhardt's (see Example 1c), 6 x SSC, 0.1% SDS solution and then hybridized for 24 hours at 65 ° C with approx.

50 x 10 6 cpm notch-translated, denatured IFN-ωΙ DNA (HindIII-BamHI insert in clone pRHW12), see FIG.

6) in the same solution. After hybridization, the filter is washed 3 x 10 minutes at room temperature in a 2 x SSC,

30 0.01% SDS solution and then 3 x 45 minutes at 65 ° C

in a 0.2 x SSC, 0.01% SDS solution. The filter is dried and exposed to Kodak X-Omat S film using an amplifier film at -70 ° C. Positively responsive colonies are located on the replica filter, scraped off and resuspended in L-herb + kanamycin (30 µg / ml). From this suspension-

I DK 175194 B1 I

I 52 I

In sion, some yl are plated on LB agar + 30 µg / ml kanamycin. IN

The resulting colonies are replica-plated on an I

In nitrocellulose filters. These filters are hybridized as I

As described above, labeled with IFN-ωΙ DNA. From each I

In 5 hybridizing colony, the cosmid is isolated by a method I

In described by Birnboim & Doly (Nucl. Acids Res. 7, 1513 I

(1979)). With this cosmid DNA preparation, I is transformed

E. coli DH1, and the transformants are selected on LB-I

In agar + 30 µg / ml kanamycin. The transformants are tested again

I-labeled with IFN-w1 DNA for positive reaction.

In germinating clones. Each site selects a clone from it

original isolated if cosmid is produced in larger I

H scale (Clewell, D.B. and Helinski, D.R., Biochemistry I

9, 4428 (1970)) - Three of the isolated cosmids carry I

15 names cos9, coslO and cosB. IN

I b) Derived cloning of hybridizing fragments in PPC8 I

1 µg of each of the cosmids cos9, cos10 and cosB I

cut with HindiII under those of the manufacturer (New I

20 England Biolabs) recommended conditions. The fragments I

is electrophoretically separated on 1% agarose gel in TBE buffer and I

is transferred onto nitrocellulose filter by Southern's method (Example 4c). Both filters are hybridized as described in Example 4d) with notch-translated ωΙ DNA, washed and exposed. Approximately 20 µg of each cosmid is used for cutting with HindIII and the resulting fragments I are gel electrophoretically separated. The fragments that hybridized with ωΙ DNA in the pre-I experiment are electroeluted and purified over elutip columns (Schleicher + Schiill). These 30 fragments are linked to HindIII linearized, de-phosphorylated pUC8 (Messing, J., Vieira, J., Gene 19, I 269-276 (1982)). E. coli JM101 (supE, thi, (lac-proAB), I [phD tra36, pro AB, lac q Z M15] (e.g., fa. L. L. Bio-I chemicals) is transformed with this reaction mixture.

I 35 The bacteria are smeared on LB agar containing 50 µg / DK 175194 B1 • 53 ml ampicillin, 250 µg / ml 5-bromo-4-chloro-3-indolyl - & - D-galactopyranoside (BCIG, Sigma) and 250 µg / ml isopropyl - $ - D-thiogalacto-pyranoside (IPTG, Sigma). A blue staining of the colonies formed shows that the insertion of a 5 piece pUC8 has failed. From some white clones, small-scale plasmid DNA is isolated, cut with HindIII and separated on 1% agarose gel. The DNA fragments are transferred onto nitrocellulose filters and hybridized with 32ρ_ω_ £ »« ν as above. Based on cos9 and cos10, a derived clone is selected from each site, designated pRHW22 and pRH57, respectively. Starting from cosB, two fragments are hybridized with the ωΐ probe and designated pRH51 and pRH52 respectively.

C) Sequence analysis

The DNA inserted into pUC8 is excised from the vector portion by cutting with HindIII and subsequent gel electrophoresis. This DNA, ca. 10 µg, are bound together using T4 DNA ligase in 50 µl reaction solution, the volume is increased to 250 µl with notch translation buffer (Example 4d) and finally the DNA is broken down by ice cooling using ultrasound (MSE 100 Watt Ultrasonic Disintegrator, greatest performance at 20 kHz, five times 30 seconds). Thereafter, the ends of the fragments are repaired by the addition of 1/100 volumes of 0.5 mM dATP, dGTP, dCTP and dTTP, respectively, together with 10 units of Klenow fragment of DNA polymerase I for two hours at 14 ° C. The obtained DNA fragments having straight ends are separated by size of a 1% agarose gel. Fragments of 30 sizes between 500 and 1000 bp are isolated and cloned using the phage vector M13 mp8, which is cut with SmaI and dephosphorylated. Single strand DNA recombinant phages are isolated and subdivided according to Sanger's method (Sanger F. et al., Proc. Natl. Acad. Sci 74, 5463-5467 (1976)).

35 The single sequences are combined into a total sequence by

I DK 175194 B1 I

I 54 I

I I

With the help of EDB (Staden, R., Nucl. Acids Res. 10, 4731 - I

In 4751 (1982)). IN

I d) Sequence of the clone pRH57 (IFN-ωΙ) I

In the sequence shown in FIG. 11. This 1933 bp long I

In fragment, the gene contains interferon-ωΐ. The region, I

encoding protein comprises nucleotides 576 to 1

I 1163. The sequence is completely identical to the sequence I

I from the cDNA clone P9A2. Nucleotide sections 576 to 674 I

I 10 codes for a 23 amino acid signal peptide. The so- I

The called TATA box is located in an interferon type I gene I

At a distinct distance from the start codon ATG (positions I

I 476-482). In the gene are some signal sequences for pol I

lyadenylation at the transcription (ATTAAA in posi- I

In the 15 tions 1497 - 1502 and 1764 - 1794 respectively; AATAAA i

In positions 1729 - 1734 and 1798 - 1803, respectively), where- I

I of the first is used in clone P9A2. IN

I e) Sequence of the derived clone pRHW22 (IFN-pseudo- (n2) I)

In FIG. 12 reproduces the 2132 bp sequence which I

belongs to the Hindi 11 fragment from the cosmid cos 9 and as I

You hybridize with the ωΙ DNA probe. An open reading is seen

In the frame from nucleotide 905 to nucleotide 1366. It corresponds to I

The running amino acid sequence is reproduced. The first 23 amino- I

In 25 acids, what is found in the signal peptide of type I

In whip type I interferon. The next 131 amino acids show I

I up to amino acid 65 similar to ω-1 interferon, and that is I

Notably, tyrosine is the first amino acid in the I

fully developed protein. IN

Following amino acid position 66, the sequence of I follows

In a potential N-glycosylation site (Asn-Phe-Ser). From I

at this site, the amino acid sequence differs from the sequence I

Seen from the sequence belonging to a type 1 interference

I on. However, it can be proved that by appropriate insertions I

In 35 and consequent displacements of the protein reading frame I

55 DK 175194 B1

Can achieve similarity to IFN-ωΙ (see Example 9). Seen from type I interferon, the isolated gene is a pseudogenic: IFN-pseudo-ui2.

F) Sequence of the derived clone pRH51 (IFN-pseudo-M3)

An approximately 3,500 bp HindIII fragment originating from cosmid B and hybridizing with the ωΐ probe is partially subdivided (Fig. 13). An open reading frame exists from nucleotide positions 92 to 394. The first 10 23 amino acids exhibit a signal peptide's characteristics. The subsequent sequence begins with tryptophan and is similar to IFN-ωΙ until amino acid 42. Then the sequence becomes different from IFN-ωΙ and ends after amino acid 78. On insertion, the sequence can be altered to show a large homology to IFN-ωΙ (Example 9). The gene is designated IFN-pseudo-u> 3.

g) Sequence of the insert in pRB52 (XFN pseudo-d> 4) ___ 20 A 3,659 bp Hind fragment isolated from cosmid B and hybridizing with ωΙ DNA is shown in FIG. 14th

An open reading frame whose translation product shows partial homology to IFN-ωΙ exists between nucleotide positions 2,951 and 3,250. After a 23 amino acid long signal peptide, the following amino acid sequence begins with phenylalanine. The homology to IFN-1 is broken even after the 16th amino acid, resumes at the 22nd amino acid and ends with the 41st amino acid. An optional translation was possible for amino acid 77. Analogously to Examples 8f) and 8g), a good homology to IFN-ωΐ can also be provided here by the insertions (Example 9). The pseudogen isolated here is referred to as IFN-pseudo-o »4.

35

I DK 175194 B1 I

I 56 I

In Example 9 I

In Utilizing the Genes of 4 Members of the United Nations Family I

In FIG. .15 shows the IFN-ωΙ genes for IFN-I

In 5 pseudo-u> 4 together with the amino acid transliteration. For I

In order to achieve better conformity, empty plates are introduced

You look in the individual genes. These are denoted by points. There you

No bases are removed. The base numbering also includes I

In the empty seats. The amino acid translation of IFN-ωΙ bi- I

In 10, {e.g. at positions 352 - 355: "C.AC" codes for I

In His). In the pseudogens, only one amino acid I is translated

re where the translation is unambiguous. It can by this

In comparison, it is immediately seen that the four isolated genes are I

You related to each other. Thus, e.g. it po- I

At the tenth N-glycosylation site (nucleotide positions I

In 301 to 309) in all four genes. IN

In addition, except in IFN-pseudo-u> 4, I exists

At nucleotide positions 611 to 614, a triplet constituting I

In a stop codon that, at IFN-ωΙ, ensures that a full I

In 20 wrapped protein with a length of 172 amino acids you become

finished. This arrangement exists at IFN-I

In pseudo-ta2 (nucleotide positions 497 to 499), and at I

IFN-pseudo-α> 4 (nucleotide positions 512 to 514) early

You give stop codons. IN

I 25 The degree of kinship between the genes and the ami I, respectively

In the acidic translations it is possible to calculate by the arrangement I

In FIG. 15. FIG. 16 gives the DNA homology between member I

In the mares of the IFN- <i> family. By pairwise comparison of I

In positions you do not count these, where in the one I

In 30 or both partners, an empty space is in the position. IN

In the comparison, a homology of about 85% between I

IFN-u> 1 DNA and the sequences of the pseudogens. IFN pseudo-I

I o) 2 DNA is approximately 82% homologous to IFN-pseudo-u3 I DNA

In IFN-pseudo-ω4, respectively. FIG. 17 shows the result of I

In comparing the signal sequences and FIGS. 18 the result I

57 DK 175194 B1 of comparisons of the "fully developed" proteins.

The latter are between 72 and 88%. However, this homology is substantially larger than that found between IFN-ωΙ and IFN-αs and IFN-β (Example 6). The fact that the 5 individual members of the IFN-w family are more distant from each other than those in the IFN-a family can be explained by the fact that three of the four isolated IFN-u genes are pseudogenic and are obviously no longer subject to the selection pressure that functional genes are.

10

Example 10 Fermentation

Inventory: A single colony of the bacterial strain HB10l / pRHW14 on LB agar (with 25 mg / ml ampicillin) is transferred by grafting to tryptone soybean (OXDID CM129) with 25 mg / ml ampicillin and shaken at 37 ° C and 250 ° C. / min to an optical density (546 nm) of approx. 5 is reached (logarithmic growth phase). 10 wt% sterile glycerol is added, the culture is filled into sterile vials each containing 1.5 ml and frozen to -70 ° C.

Pre-culture 25 The culture medium contains 15 g / l Na2 HPO4 χ 12H2O, 0.5 g / l NaCl; 1.0 g / l NH 4 Cl; 3.0 g / l KH2 PO47 0.25 g / l

MgSO4 x 7H2? 0.011 g / l CaCl 2; 5 g / L casein hydrolyzate (Merck 2238); 6.6 g / l glucose monohydrate; 0.1 g / l in picillin; 20 mg / l cysteine and 1 mg / l thiamine hydrochloride. 4 portions of 200 ml of this medium are transferred to 1000 ml Erlenmeyer flasks, each seeded with 1 ml of a stock culture of HBl01 / pRHW14 and shaken for 16-18 hours at 37 ° C and 250 rpm.

Head culture:

I DK 175194 B1 I

I 58 I

In the fermentation medium, 10 g / l (NH4) contains 2HPO4, 4.6 l

In g / 1 K2HP04 x 3H20? 0.5 g / l NaCl; 0.25 g / l MgSO 4 x I

I 7H 2 O; 0.011 g / L CaCl 2; 11 g / l glucose monohydrate; 21 I

In g / l casein hydrolyzate (Merck 2238); 20 mg / l cysteine; 1 I

5 mg / l thiamine hydrochloride and 20 mg / l 3-B-indolacrylic acid. IN

In 8 liters of sterile medium in a fermentation container of 14 liters I

total volume (height: radius = 3: 1) is inoculated with 800 l

ml of pre-culture. IN

The fermentation runs at 28 ° C, 100 rpm. (Effigas- I

I 10 Turbine), an air supply of lvvm (volume / volume / mi-I

In Nut) and an initial Ises pH of 6.9. During fermentation I

You decrease the pH and then keep constant at 6.0 with 3 N I

In NaOH. When reaching an optical density (546 nm) of 18 l

I to 20 (usually after 8.5 - 9.5 hours of fermentation)

In 15, under other conditions, it is cooled to 20 ° C, at room temperature

You set the pH with 6N H2SO4 to 2.2 and I without air supply

You stir for one hour at 20 ° C and 800 rpm. (without air supply- I

In progress). The now inactivated biomass is centrifuged in I

In a CEPA laboratory centrifuge type GLE at 30,000 rpm, I

I 20 is frozen to -20 ° C. and stored. IN

Example 11 I

Purification of Interferon-W-Gly I

I 25 I

Partial purification

In All processes are performed at 4 ° C. IN

In 140 g of biomass (E. coli HB101 transformed with I

In the expression clone pRHW12) is suspended in 1150 ml of 1% I

In 30 acetic acid (pre-cooled to 4 ° C) and stir for 30 minutes

I ter. The pH of the suspension is adjusted with 5 M NaOH to 1

In 10.0 and stir another two hours. Then you set

I with 5 M HCl to pH 7.5, stir for a further 15 minutes and I

I centrifuge (4 ° C, 1 hour, 10,000 rpm, J21 centrifuge

In 35 joint (Beckman), JA10 rotor). IN

59 DK 175194 B1

The clear centrifugate is transferred to a 150 ml CPG (controlled pore glass) column (CPG 10-350, pore size 120/200) at 50 ml / hour, washed with 1000 ml 25 mM Tris / -1M NaCl, pH 7.5 and elute with 25 mM Tris / lM KCNS / 50% ethylene glycol, pH 7.5 (50 ml / h).

The protein peak containing the interferon activity is collected overnight dialyzed against 0.1 M Na phosphate, pH = 6.0, and 10% polyethylene glycol 40,000, and the resulting precipitate is centrifuged (4 ° C, 1 hour, 10,000 rpm). , J21 centrifuge, JA20 rotor) (see Table 1).

b) Further Purification

The dialyzed and concentrated CPG eluate is diluted with buffer A (0.1 M Na phosphate pH 6.25 / 25% 1.2-15 propylene glycol) 1: 5 and transferred to a Superloop injection device (Pharmacia) and from there. at 0.5 ml / min to a buffer A equilibrated monoS 5/5 (Pharmacia, cation exchanger) column. The elution is performed at 0.5 ml / min over 20 ml linear gradient from 0 to 1 M NaCl in buffer 20 A. The throughput and fractions of 1 ml are collected and tested by a CPE reduction test for interferon activity. The active fractions are pooled.

TABLE 1 25

Volume Biological Test Protein total U / mg Yield _ (ml) U / ml + U total (mg / ml) (mg) _%

Vol. 1150 15,000 17.3x106 3.6 4140 4180 100 DL 2200 <600> 1.3x6 0.74 1628 <600_ <5 30 Eluate 124.3 170000 21.0x106 16.8 2088 10000 121 after dialysis 41 300000 12, 3x106 12.6 516.6 23800 71 + CPE reduction test: A549 cells, EMC virus 35 U: Units based on interferon-α2 (see Example 2 in

I DK 175194 B1 I

I 60 I

In EP-A-0,115,613; E. coli HB101 transformed with I

In expression clone pER 33) as standard I

In DL: Throughput. IN

I 5 I

In Example 12 I

In Characterization of HuIFN-ωΙ I

In A. Antiviral activity on human cells

In B. Antiviral Activity on Monkey Cells I

I 10 C. Growth-inhibiting activity on human Burkitt's lymph- I

In phom cells (cell line Daudi) I

In D. Anti-growth activity on human cervical carcinoma cells I

I (Cell line HeLa) I

In E. Stability of acid

In 15 F. Serological characterization. IN

I A * Antiviral activity on human cells I

In Cell Line: Human Lung Carcinoma Cell Line A549 (ATCC I

I CCL 185 I

In 20 Viruses: Mouse encephalomyocarditis virus (EMCV)

In Test System: Inhibition of the Cytophatic Effect (All I

In titrations are performed four times)

I A partially purified preparation of HuIFN-ωΙ with an I

At a protein content of 9.4 mg / ml, titrate in an appropriate formula

In thinning in said test system. The composition showed anti-I

In viral action with a specific activity of 8300 IU / mg I

I based on reference Go-23-901-527. IN

I 30 I

I 35 I

61 DK 175194 B1 B. Antiviral activity on monkey cells

Cell line: GL-V3 monkey kidney cells (Christofinis G. J., J. Med. Microbiol. 3, 251-258, 1970)

Virus: Vesicular Stomatitis Virus (VSV) 5 Sample System Plaque Reduction Test (all titrations four times)

The preparation described in Example 12A showed a specific antiviral activity of 580 U / mg in said test system.

10 C. Anti-growth activity on human Burkitt'oe lymphoma cells (Daudi cell line) __

Human Burkitt's lymphoma cell line Daudi was grown in the presence of various concentrations of HuIFN-ωΙ. Cell density is determined after two, four and six days of incubation at 37 ° C; untreated cultures were control. All experiments were performed in triplicate.

The following figure shows that IFN-ω exhibits a pronounced inhibition of cell growth at a concentration of 100 anti-20 viral units (IU, see Example 12A) per day. ml; at a concentration of 10 IU / ml a partial temporary inhibition could be observed (the following symbols are used in the figure: o Control, ° 1 IU / ml, Q 10 IU / ml, ^ 100 IU / ML): 25 30 35

I DK 175194 B1 I

I 62 I

I! Χο4ΤΠ i; i ~: · I

I '7 j

I 5 0.5- | '| ': ff / I

I oi- | : ·; | f / 'I

I J- o.i- 'fiTi .. ·; ; IN

I 5? r i. · i I

I o 'I

Η

I 15 S I

I »'i: I

I o i I

I: · »'! . i, · I

I jl '*: ϊ I

· • '- r -: -: - r i; “T” I

I 20 ·: ·! 0. i 2 ·: 4 ·! e: I

DAYS I

In D. Growth-inhibiting activity on human cervical carcinoma cell-I

I clay (HeLa cell line) _ I

The human cervical carcinoma cell line HeLa is cultured

together with the following proteins or protein mixtures; IN

In HuIFN-ωΙ (see Example 12A) 100 IU / ml I

In HuIFN-γ (see Example 12A) 100 IU / ml I

Human tumor necrosis factor (HuXFN),> 98% pure # I

In 30 manufactured by Genentech Inc., San Francisco, I

In the United States (see Pennica D. et al. Nature 312, 724-729; I

In 1984) 100 ng / ml I

All binary combinations of said proteins I

I with concentrations as above. IN

In each of the 3 cm plastic tissue culture dishes I was placed

In two cultures (50,000 cells / dish) and six I are incubated

63 DK 175194 B1 days at 37 ° C and thereafter, cell density was determined · Treatment of cells with HuIFN-ωΙ or HuIFN-f had little effect on cell growth, whereas HuIFN-γ showed a distinct cytostatic effect.

5 Combinations of IFN-γ with IFN-ωΙ showed synergistic cytostatic / cytotoxic effect.

The following figure shows the results of the experiment:! i i ππττπ— · —I — I — γτττγπ-, 10 ·. '·!.! i ;:? ti '·: i C =! : ϊ i S j / -Ή. »·).! · R · .; i · 4 · Κ I f: j; 'iI ^ if: iCT'nnrij »r, l: -to i ::.!: = 1» ·: ιΐΐ, ΐ; -j {J |:,. ilM. 1: Mi ri (L · 'tf:' *. * *. I '* .i * *:!' '' J * '1 · ·. * * *. ·. · * Ί: * * ··' * ϊ | '· *: I * ·· *! · * j · j * ij ί ·· ΐ “Γ ·· *** ·: *; 15 M.! ·" ") r: r ~ - 15 - ;: »R; ~! SG isaasi -:!; · * H r-bS: l afe _ iil · 'i.;'. 1: i ·" · -; Γ1> T ^ _yj5rF ^ 7r ^ · Γ " ~ - · Γ ~ ^ · ^ ΓΤ ~ Γ ~ 'ί' · 'ιΓ73Γ7 · i · J 1 file ντ ·': •: -7 ir: i ί i mm

: p · - ί i- · -I i j. ri} V | -: Γ; 1 Γ: ν; ^: j! ': i Γ Lrl.p -T

2o t -vhner? · 3 5 10 30 60 100 25 10 4 x cells / scale The following symbols are used in the figure above: C untreated control, T HuTNF, O HuIFN-ωΙ, G HuIFN-γ.

E. Acid Stability 30 To test the acid stability of HuIFN-ωΙ, a dilution of the preparation mentioned in Example 12A was placed in cell culture medium (rpm in 1640 with 10% fetal calf serum) and the pH was adjusted to a value of HCl with 2, incubated for 24 hours at 36 ° C and neutralized with NaOH. This sample was titrated

I DK 175194 B1 I

I 64 I

By means of an antiviral test (see Example 12A), the H

I showed 75% biological activity compared to a con I

In trolls incubated at neutral pH. Thus IFNojI can H

You are referred to as acid stable. IN

I 5 I

In F. Serological characterization I

I For the purpose of determining HuIFN-ωΙ serolo- I

In gic properties compared to HuIFN-α2 preincubates

In the test (diluted to 100 IU / ml with equal volume)

In 10 fold of solutions of monoclonal antibodies or poly

In lyclonal antisera 90 minutes at 37 ° C, then i

You agree the antiviral activity of the samples. The following ta- I

I bel shows that HuIFN-ωΙ can only be neutralized by an anti-I

In serum against leukocyte interferon at relatively high concentrations

In 15 tion, but not by antibodies that act against HuIFN-α2 I

In or HuIFN Island. HuIFN- ^ l is therefore neither serological I

In related to HuIFNct2 or HuIFN-β: I

I 20 I

I 25 I 1

I 35 I

30 I

65 DK 175194 B1

TABLE

Antiserum Mono- Dilution Neutralization of

Count «antibodies yg / ml_HuIFN-a2_HuIFN-ωΙ 5 EBI-11) 1 + 10 +++ 100 +++ 1000 - 0 EBI-33 ·) 1 +++ 10 10 +++ 100 +++ 1000 +++ 0 L3B72) 100 +++ 0 1000 +++ 0 15 Danger anti-leukocyte IFN ^ 1: 50,000 +++ - 1: 5,000 +++ 0 1: 500 +++ + 1: 50 - ++ + 20 Rabbit Anti-

HuIFN-a2 1: 1,000 +++ 1: 100 +++ 0 1: 10 - 0 Sheep anti-HuIFN-84 ^ _1: 50 _ = _ 0_ 1) = see EP-A-0.119.476 2) = A Berthold et al. in Arzneimittelforschung 35, 364-369 (1985) 3) = Research Reference Reagent Catalog no. 30 G-026-502-568 4) = Research Reference Reagent Catalog no.

G-028-501-568

Research Resources Branch, National Institute of Allergy and Infectious Diseases, Bethesda, 35 Maryland, USA.

Symbols: - not tested, 0 no neutralization, + some neutralization, +++ complete neutralization.

Claims (37)

1. Recombinant DNA molecules, containing a II coding sequence for novel type I human interferon II proteins containing 168-174 amino acids, for interferon pseudo-ti> 2 (see Figure 12), interferon - II ron-pseudo-u> 3 (see Figure 13) or interferon-pseudo-II ω4 (see Figure 14), characterized in that the co-II sequence under such stringent conditions that only II has a homology of more than 85% can be detected, II 10 hybridizes with a) insertion into the Pst-1 intersection site of II plasmid E76E9 in E. coli HB101 deposited by DSM with II deposition number DSM 3003, b) in plasmid P9A2 in E. II coli HB 101 deposited by DSM with deposition number A DNA molecule according to claim 1, characterized in that the stringent conditions only allow II to detect a homology of more than 90%. IN A DNA molecule according to claims 1 and 2, characterized in that the coding sequence is present as an I insert at the Pst-1 intersection site a) in the I I plasmid E76E9 in E. coli HB101 deposited by DSM med. I in deposit number DSM 3003, b) in plasmid P9A2 in E. I in coli HB 101 deposited by DSM with deposit number I in DSM 3004, or is a degenerate variant of these I in 25 insertions. IN DNA molecule according to claims 1 to 3, characterized in that the vehicle is a plasmid capable of replication in prokaryotes or in eukaryotic cells. A DNA molecule according to claim 4, characterized in that it is an expression vehicle, which can undergo replication in microorganisms or in mammalian cells. In DK 175194 B1 The DNA molecule of claim 5, characterized in that it contains the sequence TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 45 5 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 90 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 135 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 180 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 225 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 270 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 315 10 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 360 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 405 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 450 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 495 GAT AGA GAC CTG GGC TCA TCT 516 15 1 2 3 4 5 6 7 8 9 10 11 The DNA molecule of claim 5, knowing 2 characterized in that the DNA molecule of claim 6 at 3 position 332 contains nucleotide A instead of 4 nucleotide G. DNA molecule according to claim 5, characterized in that it is the plasmid E76E9 in E. coli 7 HB101 deposited by DSM with deposit number DSM 8 3003 or the plasmid P9A2 in E. coli HB 101 deposited 9 by DSM with deposit number DSM 3004. 10 The DNA molecule of claims 6 and 7, characterized in that it further contains a DNA peptide coding sequence of the formula ATG GCC CTC CTG TTC CCT CTA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC I DK 175194 B1 II 68 I 10. Interferon-IOL gene II characterized in that it has the formula GATCTGGTAAACCTGAA GCAAATATAGAAACCTATAGGGCCTGACTTCCTACATAAAGTAAGGAGGGTAAAAATGG 17 II 76 II 135 II AGGCTAGAATAAGGGTTAAAATTTTGCTTCTAGAACAGAGAAAATGATTTTTTTCATAT ATATATGAATATATATTATATATACACATATATACATATATTCACTATAGTGTGTATAC ATAAATATATAATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT II 194 253 II 312 II AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCATTTGATTAAGCATTCATTT GTATTGACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAAATTAATAGA TTGTTGTCATCCTCTTAAGGTCATAGGGAGAACACACAAATGAAAACAGTAAAAGAAAC II 371 430 II 469 II TGAAAGTACAGAGAAATGTTCAGAAAATGAAAACCATGTGTTTCCTATTAAAAGCCATG CATACAAGCAATGTCTTCAGAAAACCTAGGGTCCAAGGTTAAGCCATATCCCAGCTCAG 548 II TAAAGCCAGGAGCATCCTCATTTCCCA ATG GC £ CTC C TG TTC CCT CTA CTG 599 II GCA GCC CTA GTG ATG ACC AGO TAT AGC CCT GTT GGA TCT CTG GGC 644 II TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 689 II GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 734 II AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 779 II AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 824 II CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 869 II GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 914 II CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 959 IIG AA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 1004 II AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 1045 II TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 1094 II TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 1139 II GAT GAC CTG AGA GGC TCT TCA TGA AATGATTCTCATTGATTAATTTGCCAT ATAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCA 1249 1190 II II II AAAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTA CAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTT 1308 1367 1426 II I TTTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGC CTTTACATTGTATTAAGATAACAAAACATGTTCAGCTT TCCATTTGGTTAAATATTGTA 1485 TTTTGTTATTTATTAAATTATTTTCAAACAAAACTTCTTGAAGTTATTTATTCGAAAAC In 1544 I I I I CAAAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA 1603 CCTATTC ATTC ATTCCATTT AC TO AATATGTATAAAGTG AG TAT CA AAGTGGCA TATTT 1662 I I I I TGGAATTGATGTCAAGCAATGCAGGTGTACTCATTGCATGACTGTATCAAAATATCTCA 1721 TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA 1780 I I I I AAAAAGAAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT 1839 ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA 1898 I I AGTAAAAGAAAATGATGTTCTCATACTAGAAGCTT 1933 In DK 175 194 B1 11. Interferon-glycol-gene characterized in that it has formula 51 AAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATGAATATATT CTAGAAGGAGGGAAGC ^ æAGAGAAGTTCTTTCACTAATAACCATCAAGGTCTTCTGTG 110 AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAG 169 228 287 GGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG ATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGG AATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCA 346 405 464 TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA GGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC TCTGCCTCCTGTCAGATCAGCAGTGGCATTGGATTCTCAAAAGAGCAGGAACCCTATTG 523 582 641 TGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGAGAATCTAATGCCTCTG AAAGCATTCCCTCCCTGACCCCATTTTTCGTGGAAAAATTATCTTCCACCAAACTGGTG GCCAAAAGGTTGTGGATGCTGATATAGAAGACATGTAAATGAAAACAATAAATGGAATT 700 759 818 AAAAATTTAGAGAAATGCTCAGAAAAATGAAAACTATTTGTGCTCCATTAAAGCCATGC ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTC AGCTCAA 877 CGAGGCCAAAAGCATCCTGATTTCTGA ATG GCC CTC CTC TTC CCT CTA CTG 928 GCA GCC CTA GAG GTG TGC AGC TGT GGC TCT TCT GGA TCT CTA GGA 973 TAT AAC CTG CCT CAG AAC CAT GGC CTG CGGGGG AGG AGA ATC TCT CCC TTC TTG TGT CTA 1063 AAG GAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG GTG GAA GTC 1108 AGC CAG TTG CAG AAG GCC CAG GCT ATG TCT TTC CTC TAT GAT GTG 1153 TTA CAG CAG GTC TTC AAC TAC CAC AAA GCG CTC CTC TGC TGC 1198 ATG GAA CAT GAC CTT CCT GGA CCA ACT CCA CAC TTT ACG TCA TCA 1243 GCA GCT GGA ACA CCT GGA GAC CTG CTT GGT GCA GGA GAT GGG AGA 1288 AGG AGA AGC TGG GGG CAG TGG GTG GGC TCT ACA CTG GCC 1333 TTG AGG AGG TAT TTC CAG GAA TCC ATC TCT ACC TGA AAGAGAAGAAA 1380 TAAAGATTGTGCCTGGGAAGTTGTCAGAGTGGAAATCATGAGATCCTTTTCATCCACAA 1439 GTTTGGAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGAAATGATTCT 1498 CATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCA 1557 TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA 1619 AGCAAG TTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC 1675 CCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTA TATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTCACTTTTTCA 1734 1793 1852 TATTAAGTTTGCATTTTGTTTTATTAAATTCATATCAAAGAAAACTCTGTAAATGTTTC In DK 175194 B1 70 I I I I I I TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTGAAAATATGGTAAAA 1911 TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG 1970 2029 TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG I I I I I I AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT 2088 2132 In ACAAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 12. Interferon pseudo-a) 3 gene, characterized in that it has the formula CCATG 5 II CATAGCAGGAATGCCTTGAGAGAAGCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG 64 II CTAGGCCAGCAGCACCCTCGTTTCCCA CCT CCT CCT CCT CCT CCT CCT CCT CCT CCT GGA TCT CTG AGC 160 II TGG GAC CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 205 II GCA CTT CTG. GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TGT CTC 250 II AAG GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 295 II GTC AGT TGC AGA AGG CCC CGG CCC TGT CTG TCC TCC ATG AGA TGC 340 II TTC AGC AGA TCT TCA GCC TCT TCC CCA CAG AGT GCT CCT CTG CTG 385 II CCT GGA ACA TGA CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA 440 II ATGCCTGGATGCTTGCTTAGGGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG 499 II GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC 558 II TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG 617 II AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA 676 II CCTGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG 735 II TGTCTTTG GTCATTTCAATAGGTTCTTATTTCTGCAG 772 II 13, Interferon-pseudo-ω4 gene , characterized in that it has the formula II AAGCTTTGGGCATGACCTAGTAGGTGACTCTT 32 II AGTTGGAGTGGTCAGTTGTAAGGCTCTTGTCGTCGTGTGTGTGTGTGTGTTTCCCCC 91 II ACGTTTGCAGCCT TATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA 209 I I TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG 268 I I TAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT 327 I I GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC 386 I I AGGGGTTCCCTGCCTGGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTGG 445 I I CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT 504 I I GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA 563 I I GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA 622 * In DK 175194 B1 GTGTTTAGCCAGAGGAGTGGGGTGGCTGCATGCTGGTGTAAGTATGGGACTTCACTTCT 681 TGGGAAACAGGGAGGTGGAATCTTACCA6CAGGAGACT6TTCTCCTCACGATGTGTAAC 740 CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAAACAAAGAGC 799 TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 858 GCAGTGGCAGAGGTAGQATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA 917 CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGCAGGGGGCC 976 TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGGACTCCTCTTCATA 1035 TAG TGGCGATGATGTGCTGGAGGTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCCA 1094 GCCAGAGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTGAGGAGACGAGGGAAG 1212 GGAAGAATGCCTGTGCTGGAGTCTCTGGTCAGGTGGCTCTGCCACAGAGGAGAGGTGAC 1271 CACGAGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330 TGGGGATCTGGAGGAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGG 1389 CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGGAGACCCAGGCCT 1507 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566 CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625 CATTTTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684 CTGCCCCTGCCTCTGGACCTCTGTrCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG 1802 GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1861 GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920 CAGCAACAATGGCAACCTACCATTCCCr rTGGAGCGCCATCCCAGGGAXATTCGAAACT 1979 GCTGTCCACTAGAAAACAGTGGTGGAGGTGAGTGGAGACCCCAGTGGAGAGTTTCACCT 2038 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAACCAATCTGACTGCTTCCCCGTA 2097 GAGCTGCTGGACTGTGCTGGGTGGCTGCTCCAGTCCCTAGCTGCCTTGGACTGCCGAGA 2156 ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC 2215 CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGGCTGGATTCCC 2274 AAGTCGAGTGGGTCTXACCCTGAGACAGGCCATGGAAGGTGGGCCTGTCATTGTCACTG 2333 CCCAGCGCCCTGGATGAAACCCCTTTGCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC 2392 TTGGCTGGAGTTAIAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAAGGCTCCAGGGTAC 2451 CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA 2510 ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 2569 TGGGAGAAGCGTGGGTCCCCAGGGCTGCTCACTTACTCACCACTTCCCTGGGCAGGAGA 2628 GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTXACTTTGCTTTAT 2687 TCTCCATGGGTCAAGTTGTTlTCTTGAGTCTCAATGTGTGCACCTGGTTTiTrCAGTTG 2746 AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGAGAGTGGCACACACTAGCA 2805 GGTTCCAGTCGGCCATCrTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAA GCCATT 2864 In DK 175194 B1 II 72 II GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2923 II CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 II GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT GGA TCT CTG GGC 3019 II TTT GAC CTG CCT CAG AAC CGT GGC CTA CTT AGC AGG AAC ACC TTG 3064 II GCA TTC TGG GCC AAA TGC AGA ATC TCC ACT TTC TTG TGT CTC AAG 3109 II GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 3154 II ATT TGC AGA AGG CCC AGG CTG TGT CTG TCC ATG AGA TGC TCC TTC 3199 II AGC TCT AGA TCT TCA GCC TCC CAG AGC CCA CTG CTG CCT GCT CCT 3244 II GGA ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG CCTGGAGTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGGGTGATTGGGA 3359 3300 II II II CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAATCCAGAGAATCTAC 3418 3477 3536 II II TCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGACCTGGGGTC TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTT CTTCTTTTTC 3595 I I TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA 3654 I I AGCTT 3659 Transformed host organism, characterized in that it contains the genetic information according to claims 1 to 10. II. Host organism according to claim 14, known - II characterized in that the genetic sequence is contained in a vehicle that can undergo replication in the II host organism. The host organism of claim 14, characterized in that it is a mammalian cell line I 25 or E. coli. A host organism according to claim 14, characterized in that it is E. coli with DSM No. 3003 I or E. coli with DSM No. 3004. II 18. Host organism according to claim 17, characterized in that: that the vehicle additionally contains II for the expression necessary replicon and control sequences. In DK 175194 B1 Host organism according to claim 18, characterized in that the vehicle contains the expression and control sequences required for expression from plasmid pER103 with DSM No. 2773 (see Figure 6). A host organism according to claim 19, characterized in that it is transformed with an expression plasmid according to claim 21, 22 or 23. 21. Expression plasmid for interferon-ω, a derivative of the plasmid pBR322, characterized in that the plasmid pBR322, instead of its own shorter EcoRI / BamHI fragment, contains a DNA sequence of the formula EcoRI Sau3A go into c ^ mRHA Start With ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAfl ATG 116 RBS Hindlll Cys Asp TGT fiÅX. C-jIH ^ -omega-Gen- »Sau3A Expression plasmid pRHW12 according to claim 21, characterized in that the interferon-o> gene displays a DNA sequence of the formula TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 Ncol GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 I DK 175194 B1 II 74 II TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 II GAT AGA GAC CTG GGC TCA TCT TGAAAT6ATTCTCATTGATTAATTTGCCATA 530 II TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTA ATCAC AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 589 I I 648 I I 707 I I AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766 I I I I 802 TTTACATTGTATTAAGATAACAAAACATGTTCASCi Alu I I I I 10 Expression plasmid pRHWII according to claim 21, characterized in that the interferon-6> gene displays a DNA sequence of formula II TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 II Ncol II GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 II AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 II AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 II CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 II GCC TGG AAC ATG ACC CTC CIA GAC CAA CTC CAC ACT GGA CTT CAT 253 II CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 II GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GGA CTG ACC TTG 343 II AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 II TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 II TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 II GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 II TAACACTTGCACATGTGACTCTGG TCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 589 I I I I AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707 In In. TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766 I I TTTACATTGTATTAAGATAACAAAACATGTTCASst 802 I I Alul I DK 175194 B1 24. Plasmid pRHW10, a derivative of pBR.322, characterized in that a DNA fragment of the formula Hindi II Sau3A Ncol has been inserted into the plasmid pER103 with DSM No. 2773 (see Figure 6). a ^ SCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAaoet ·. t Sau3A Hindlll E. coli HB 101, characterized in that it is transformed with a plasmid according to claims 12, 22, 23 or 24. Human type I interferon proteins, characterized in that they are encoded by a DNA molecule according to any one of claims 1 to 10. Human interferons according to claim 26, characterized in that the mature interferons show a divergence of 30 to 50% against human ot interferons. Type I human interferons according to claim 28, characterized in that a) the mature interferons exhibit a divergence of 30 to 50% against human α-interferons and a divergence of about 70% to β-interferon, and b ) they have a length of 168-174 amino acids; as well as N-glycosylated derivatives thereof. __ _ ____ I DK 175194 B1 I I 76 I Interferons according to claim 28, characterized in that they exhibit a divergence of 40 I to 48% against human ot interferons, and N-I I glycosylated derivatives thereof. Interferons according to claims 26 to 29, characterized in that they contain a leader I peptide. IN 31. Interferons according to claims 26 to 29, characterized in that they have 172 amino acids. HI 10 32. Interferon according to claim 31, characterized in that it has the amino acid sequence II 5 10 15 II Cys Asp Leu Pro Gin Asn His Gly Leu Len Ser Arg Asn Thr Leu II 20 25 30 II Val Leu Leu His Gin net Arg Arg Ile Ser Pro Phe Leu Cys Leu II 35 40. 45 II List Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Net Fall List Gly II 50 55 60 II Ser Gin Leu Gin Lys Ala His Fall With Ser Val Leu His Glu Met I 65 70 75 II Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala II 80 85 90 II Ala Trp Asn Met Thr Len Leu Asp Gin Leu His Thr Gly Leu His I 95 100 105 II Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Val Val Gly II 110 115 120 II Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu Thr Leu I 125 130 135 I Arg Arg Tyr Phe Gin Gly Ile Arg Val Tyr Leu Lys Glu Lys Lys II 140 145 150 II Tyr Ser Asp Cys Ala Trp Glu Val Val Arg With Glu Ile With List II 155 160 165 II Ser Leu Phe Leu Ser Thr Asn With Gin Glu Arg Leu Arg Ser Ser II II 170 II Asp Arg Asp Leu G ly Ser Ser I I and in amino acid position 78 N-glycosylated derivatives I I thereof. In DK 175194 B1 Interferon according to claim 31, characterized in that the interferon according to claim 32 in position 111 contains the amino acid Glu instead of Gly, and in amino acid position 78 N-glycosylated derivatives thereof. Interferons according to claims 30 to 33, characterized in that they contain a leader peptide of the formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly. A method for producing interferon proteins according to claims 26 to 34, characterized in that a suitable host organism is transformed with genetic information encoding interferon proteins according to claims 26 to 34; this information is expressed to produce the interferon in question in the host organism; and the resulting interferon protein is isolated from this host organism. The method of claim 35, characterized in that the information is contained in any DNA molecule of claims 1 to 10. The method of claim 36, characterized in that, after the transformation, the host organism is defined according to one of claims 15 to 20. A method according to claim 35, characterized in that the interferon is defined according to claims 32 to 34. A method according to claim 35, characterized in that the host is used as E. coli HB 101 and as a plasmid a plasmid according to claims 21, 22 or 23. In ω 175194 B1 II 78 II 40. ω-Interferon which may be prepared by the method of claim 35 or 39. I A composition suitable for antitumor or II antiviral treatment, characterized in that II contains, in addition to one or more inert carriers and / or diluents, an active amount of II interferon peptide according to one of claims 26 to 34. I Use of ω-interferon according to claims 30 I I to 34 to prepare a mixture suitable for antitumor or I I 10 antiviral treatment. IN 43. A method for producing the plasmid I pRHW10, characterized in that a DNA II fragment of formula II HindIII is inserted into the plasmid II pER103 with DSM No. 2773 (Figure 6) in the HindIII II site by ligase reaction. Sau3A NcoI II aAGCTTAÅAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 II ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 II CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 II CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 II AGATCACACA TCTTTAaact t II Sau3A Hind II and to the thus obtained plasmid is transformed II for replication in E coli and grown. In DK 175194 B1 44. A method of producing the expression plasmid pRHW12, characterized in that, by restriction endonuclease digestion with BamHI, filling sites by the Kleene 5 Now fragment of DNA polymerase I and the 4 deoxy nucleoside triphosphates and a subsequent cutting obtained with NcoI larger fragment of the plasmid pRHW10, which in the Hindu I site of plasmid pER103 with DSM No. 2773 (see Figure 6) contains the DNA fragment of the formula: HindIII Sau3A Ncol a CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAaact by P CTT AGC AGG AAC ACC TTG 28 Ncol GTG CTT CTG CAC CAA ATG AGG AGA A TC TCC CCT TTC TTG TGT CTC 73 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 I DK 175194 B1 II 80 II TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 II TCC TTG TTC TTA TCA ACA AAC ATG GAA CAA CTG AGA AGA AGT AAA 478 II GAT GAC CTG AGA GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC II 530 589 II 64 B II AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC II 707 766 II TT TACATTGTATTAAGATAACAAAACATGTTCAGct 802 I I Alul I I and the plasmid thus obtained is transformed I I for replication in E. coli and grown. IN 45. A method of producing the expression plasmid pRHWII, characterized in that II, in the restriction endonuclease reaction with II BamHI, fills cut sites by the Kle II II fragment of DNA polymerase I and the 4 deoxy II nucleoside triphosphates and a subsequent cut with II Ncol obtained larger fragment of plasmid pRHW10, which II 20 in Hindu I site of plasmid pER103 with DSM No. 2773 II (see Figure 6) contains the DNA fragment of formula II HindiII Sau3A Ncol II aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 II ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 II CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG XAAAAGGGAG 150 II CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 II AGATCACACA TCTTTAaact t II Sau3A, HindIII In DK 175194 B1 by ligase reaction inserted it by digesting Avail fragment of the clone E76E9 (DSM # 3003) using NcoI and Alul obtained fragment of formula C CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 NCOl GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 CTG CAG CAG ATC TTC AGC CTC TTC CAC AGA GAG CGC TCC TCT GCT 208 3GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 TAC AGC GAC TGT GCC TGG GAA GT GTC AGA ATG GAA ATC ATG AAA 433 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 589 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707 TTATTCTTACATTTTATCATATTTATACTATTT ATATTCTTATATAACAAATGTTTGCC 766 TTTACATTGTATTAAGATAACAAAACATGTTCAGCt 802 Alul and the plasmid thus obtained is transformed for replication in E. coli and cultured. The synergistic mixture according to claim 41, characterized in that it further contains γ-interferon. The synergistic mixture of claim 41, characterized in that it further contains human tumor necrosis factor.