BG60445B2 - New genetic sequences, encoding interferon peptides of type i, and organisms producing them - Google Patents

Abstract

The invention relates to interferon of type I, as well as to the recombinant methods for the production of these peptides, and also to different genetic sequences, recombinant DNA-molecules, means for expression and host organisms.

Description

It is an object of the present invention to provide new type I interferons as well as recombinant DNA methods for the preparation of these peptides and products that are required in this process, for example gene sequences, recombinant DNA molecules, expression agents and organisms.

Interferon is a term used to describe selected proteins that are endogenous to human cells and are characterized by having partly overlapping and partly extending biological activities. These proteins modify the overall immune response and are thought to contribute to significant protection against viral diseases.

The interferons are, for example, divided into three classes, namely alpha, beta, and gamma interferons. From the beta and gamma interferons in the human body, only one subtype is known so far (np.S. Ohno et al., Proc. Natl. Acad.Sci. 78. 5305-5309 (1981); Gray et al., Nature 295, 503 -508 (1982); Taya et al., EMBO Journal 1/8, 953-958 (1982) In contrast, different subtypes have been described by alpha interferons (Phil. Trans. R. Soc. Lond., 299, 7- 28 (1982)) Mature alpha interferons differ in that they have a maximum of 23% divergence and are about 166 amino acids long. Also noteworthy is a message about alpha interferon having surprisingly high molecular weight (26,000, determined by sodium dodecyl sulfate). polyacrylamide gel electrophoresis / SDS-PAGE / (Goren, P. et al. Virology 130, 273-280 (1983). This interferon is called Interferon alpha 26 K. It has been found to exhibit the highest specific antiviral and anti-cellular activity to date. activity.

However, the known interferons active in various diseases show little or no effect at all in many other diseases (Powledge, Bio / Technology, March 1984, 215-228 'Interferon On Trial'). Interferons exhibit side effects. For example, when tested for the anticancer effect of recombinant οό-interferon, it was found that doses of about 50 million units, which were considered safe after phase 1 studies, caused acute viral conditions, joint pain leading to disability, very severe fatigue , anxiety and loss of orientation, seizures and liver intoxication. In 1982, the French government banned trials with interferon because of cancer patients who had been treated with fatal heart attacks. Two deadly heart attacks have also been reported in short-term trials in America. In doing so, it is clear that at least one part of the side effects, such as fever and malaise are directly interdependent with the interferon molecule itself and cannot be attributed to impurities.

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The purpose of this study is to find and produce such new substances that are close to the interferon molecule, but with reduced side effects.

The present invention therefore concerns certain novel Type I interferons which may contain a leader peptide and their N-glycosylated derivatives (hereinafter referred to as omegainterferon or interferon-omega), which contain 168 to 174 (preferably 172) amino acids and exhibit a divergence of 30- 50% (predominantly 40-48%) of the previously known subtypes of ^^, interferon, and one divergence of approximately 70% relative to β of interferon.On the one hand, they exhibit similar effects to obinterferons and, on the other hand, do not have many therapeutic disadvantages. zi known substances.

The subject of the present invention are both novel interferons in completely pure form, their non-glycosylated and glycosylated forms, their coding gene sequences (sequences), and recombinant molecules containing these sequences. The subject of the invention are further expressing agents, such as plasmids containing the above mentioned sequences, as well as various host organisms, such as microorganisms or tissue cultures, which support the production of new interferons during fermentation or tissue culture.

An object of this invention is the omegainterferons, as well as the corresponding gene sequences of the following formula:

5 10 15 5 Cys Asp Leu Pro Gin Asn His Gly Leu Leu Sir Arg Asn Thr Leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 20 25 30 10 Vai Leu Leu His Gin Met Arg Arg lie Sir Pro Phe Leu Cys Leu GTG CTT CTG . CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Vai Lys Gly 15 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 50 • 55 60 Sir Gin Leu Gin Lys Ala His Vai Met Sir Vai Leu His Glu Met 20 AGC CAG TTG CAG AAG GCC CAT GTC  ATG TCT GTC CTC CAT GAG ATG 65 • 70 75 Leu Gin Gin lie Phe Sir Leu Phe His Thr Glu Arg Sir Sir Ala 25 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 80 85 90 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 30 95 100 105 Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Vai Vai Gly CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 35 .-- 110 115 120 Glu Gly Glu Sir Ala Gly Ala He Sir Sir Pro Ala Leu Thr Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 40 125 130 135 Arg Arg Tyr Phe Gin Gly He Arg Vai Tyr Leu Lys Glu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 140 145 150 45 Tyr Sir Asp Cys Ala Trp Glu Vai Vai Arg Met Glu lie Met Lys TAC AGC GAC TGT GCC ^? GG GAA GTT GTC AGA ATG GAA ATC ATG AAA 155 • 160 165 50 Sir Leu Phe Leu Sir Thr Asn Met Gin Glu Arg Leu Arg. Sir Lys TCC TTG TTC TTA tca ' ACA AAC; ATG CAA GAA AGA CTG AGA AGT AAA 170 • - 55 Asp Arg Asp Leu Gly Sir Sir - GAT AGA GAC CTG GGC TCA TCT

At position 111, the sequence GGG (encoding Gly) may be replaced by GAG (encoding Glu). Preferred molecules also contain derivatives which at amino acid 78 are Nglycosylated. For example, the two mentioned omega-interferons contain a leader peptide of the formula

Met Ala Leu Le Phe Pro Leu Leu Ala Le Ala Leu Vai Met Thr Ser Tyr Ser Pro Vai Gly Ser Leu Gly

Legend to the images:

Figure 1: Restriction map of branch E76E9; Figure 2: Restriction map of branch P9A2 ', Figure 3: DNA sequence of clone P9A2 ' Figure 4: DNA sequence of clone E76E9; Figure 5: Genomic Southern blot analysis using

clone P9A2 as probe;

Figure 6: Construction of the expression clone pRHW12 · Figure 7: Amino acid and nucleotide differences between

Type I interferons ·

Figure 8 a: Enumeration of single nucleotide positions of interferon-A gene

Figure 8 b: Enumeration of single nucleotide positions of interferon-omega gene ·,

Figure 8 c: Enumeration of single nucleotide positions of the interferon-D- gene

Figure 9: Schematic representation of the synthesis of specific

probes for interferon subtypes ^

Figure 10: Documenting specific m RNAs for interferon subtypes *

Figure 11: The DNA sequence of the interferon gene -

omega 1;

Figure 12: DNA sequence of the interferon pseudo-omega 2 gene

Figure 13: DNA sequence of the interferon pseudo-omega 3 gene;

Figure 14: DNA sequence of the interferon pseudo-omega 4 gene;

Figure 15: Corrected enumeration of interferon-omega gene sequences;

Figure 16: Homology of signal sequences *

Figure 17: Homology of mature protein sequences;

Figure 18: Homology of 4 DNA sequences together,

According to the invention, novel omega interferons and their DNA coding sequences (sequences) are prepared as follows:

Human B-lymphoma cell line (eg Namalwa cells) (see G.Klein et al., Int.J.Cancer 10, 44 (1972) is challenged to simultaneously produce ab-and / 3-interferon by virus treatment Sendai virus, mRNA isolated from Namalwa stimulated cells can serve as a cDNA synthesis matrix To enhance the use of interferon-specific sequences in the process, the mRNA preparation is separated by sucrose gradients corresponding to different lengths of the individual RNA Molecules It is advantageous to collect mRNAs with a range of 12 S (approximately 800-1000 bases per m P In this range, the specific for <* and / 1-interferon m RNA are precipitated. M RNAs from this range are concentrated by precipitation and dissolution in water. The preparation of a cDNA library follows essentially the methods known in the literature (E. Dworkin-Rastl,

Μ.B. Dworkin and P.Swetly, J. of Interferon Research 2/4, 575-585 (1982). Oligo-dT (primer) was added to the mRNA. Finally, cDNA was synthesized by addition of deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP) and reverse transcriptase enzyme for one hour at 45 ° C. The cDNA / mRNA hybrid was purified by chloroform extraction and chloroform extracted gel column (eg Sephadex G 50). The RNA was hydrolyzed by alkaline treatment (0.3 Mol NaOH, 50 ° C, 1 hour) and the cDNA was neutralized with ethanol after neutralization with acidic sodium acetate solution. After the addition of the four deoxynucleoside triphosphates and DNA polymerase I of E. coli in the respective solution, double strand synthesis was carried out, where the cDNA served as a template and embryo by fork at its 3 'end (6 hours, 15 ° C) (Eftratiadis et al. ., Cell 7,279 (1976)). After phenolic extraction, chromatography on Sephadex G 50, and precipitation with ethanol, the DNA in a suitable solution was treated with single chain specific SI endonuclease. This breaks down the fork-shaped structures as well as the double-stranded cDNAs. After chloroform extraction and ethanol precipitation, the double stranded DNA (dsDNA) is separated by a large amount into a sugar gradient. For the next cloning step, only dsDNA of 600 base pairs (bp) or more is preferred to increase the likelihood of obtaining only clones that have the complete coding sequence for new interferons. Double stranded DNA longer than 600 bp is concentrated by gradients by precipitation and dissolution in water. In order to increase the resulting double stranded DNA molecules, they must then be inserted into the corresponding vector and finally into E. coli. The vector used is predominantly plasmid pBR322 (F.Bolivar et al., Gene 2, 95 (1977). This plasmid consists essentially of one replicon and two selective markers. They confer on the host antibiotic resistance ampicillin and tetracycline (Ap ^h , Tc. The β-lactamase (Aβ) gene contains a recognition sequence for restriction endonuclease PstI. At the same time, double stranded DNA also hit The homopolymers of plasmid and dsDHK are complementary and hybridize, with plasmid DNA and ds DNA being mixed at appropriate concentrations and under favorable salt, buffer and temperature conditions (T. Nelson et al., Methods in Enzymology 68, 41-50 (1980).

E.coli strain HB 101 (genotype ^{F:, hsdS20, (rB,} mB) recA13, ara14, pro-A2, lacYl, galK2, rpsL20 (Smr), xyl- 5, mtl-1, supE44, lambda-) is prepared to receive the recombinant vector ^ DNA molecule by washing with CaCl2 solution. The competent E. coli HB 101 was mixed with DNA and, after incubation at 0 ”C, the plasmid-DNA thus obtained was transformed with thermal shock at 42 * C for 2 minutes (M.Dagert et al., Gene 6: 23-28 (1979 )). Finally, the transformed bacteria were seeded on tetracycline-containing agar plates (10 μg / ml). Only E. coli HB 101 that have received a vector or recombinant vector molecule (Tcg) can grow on this agar. The recombinant vectorDNA molecules attach the host genotype AβTcG to the host because, by providing double stranded DNA in the beta-lactamase gene, the beta-lactamase information is destroyed. The clones were transferred to agar plates containing 50 μg / ml ampicillin. Only 3% grow, meaning that 97% of the clones contain the insertion of a single ds DNA molecule. Based on 0.5 µg ds DNA, more than 30,000 clones are obtained. 28,600 of these were transferred individually to wells in microtiter plates containing medium, 10 μg tetracycline and glycerin. Once the clones have developed, the plates are stored at -70 ⁱⁿ C (cDNA library).

To search the cDNA library for new genes containing interferon clones, the latter were transferred after thawing them on nitrocellulose filters. These filters lie on tetracycline-containing agar. After the bacterial colonies have grown, the DNA of the bacteria is fixed on the filter.

The probe primarily serves the insert of the clone pER33 (E. Rastl et al., Gene 21, 231-248 (1983) - see also EP-A-0.115.613), which contains the interferon gene-oC2-Arg. In nick translation, this region is radiolabelled using DNA polymerase I, dATP, dGTP, dTTP and 32P-dCTP. The nitrocellulose filters are first pre-treated for hybridization under mild, non-stringent conditions without the addition of a radioactive probe and then, after the addition of the latter, hybridize for 16 hours. The filters are then washed under non-stringent conditions. The low severity of hybridization and washing allows the capture not only of interferon-c2-C2-clones but also of other interferon-containing clones whose sequences may differ significantly from the previously known oC-interferons. After drying, the filters are exposed on an X-ray film. A blackout that differs sharply from the background indicates the presence of a clone with interferon-specific sequences.

As the radioactive signals are of different quality, the positive and doubtfully positive clones are grown on a small scale. Plasmid DNA molecules were isolated, digested with restriction endonuclease PstI, and separated by molecular weight electrophoretically on agarose gel (Brackl et al., Nucl.Acid. Res. 7, 1513 (1979)). Agarose gel DNA was transferred to a nitrocellulose filter by the Southern method (EMSouthern, J. Mol.Biol., 98, 503-517 (1975)) .The DNA of these filters was hybridized with a radioactive, denatured probe containing an interferon gene. . Plasmid 1F7 (deposited in the German microbial collection under DSM 2362), which contains the interferon oC-2-Arg gene, is a positive control. The autoradiography clarifies that clone E76E9 and clone P9A2 contain a sequence that hybridizes with the interferonx * 2-A ^ gene under non-stringent conditions. In order to better characterize the double stranded DNA regions of clones E76E9 and P9A2, their plasmids are produced in large quantities. DNA is digested with various restriction endonucleases such as Alul, Sau3a, BgHI, Hinfl, PstI and Haelll. The resulting fragments were separated into an agarose gel. Comparison with the corresponding size markers determines the volume of these fragments. (For example, determine the size of fragments that are obtained upon degradation of pBR322 with restriction endonuclease Hinf1 or Nae3. In Smith and Birnstiel mapping (HOSmith et al., Nucl.Acid. Res. 3, 2387-2398 (1967)). The restriction enzyme maps thus obtained (FIGS. 1 and 2) are surprisingly derived from the insertion of clones E76E9 and P9A2 into the contents of previously unknown interferon genes, namely omega genes. -interferon.

This omega-interferon information is used to degrade the cDNA region with suitable restriction nucleases. The resulting fragments were ligated into the double-stranded (replicative) form of the bacteriophage M13 mp9 (J. Massing et al., Gene 19, 269-276 (1982)) and sequenced by the Dideoxy method of Sanger (F.Sanger et al., Proc. Natl. Acad. Sci. 74, 5463-5467 (1967) The single stranded DNA of the recombinant phage was isolated. After binding of one synthetic oligomer, double strand synthesis proceeds in four different directions using a large DNA polymerase1 fragment from E. coli (Klenow fragment). To each of the four partial reactions was added one of the four deoxyribonucleotide triphosphates (ddATP, ddGTP, ddCTP, ddTTP). This results in statistically separated strand breaks at the place where the complementary base of the deoxyribonucleotide triphosphate is located in the template DNA. In addition, radiolabeled dATP is used. After synthesis, the products were denatured and single stranded DNA fragments separated by size into a denatured polyacrylamide gel (F.Sanger et al., FEBS Letters 87, 107-111 (1978)). The gel is then exposed to an X-ray film. The DNA sequence of the recombinant phage M13 is selected from the autoradiogram. The insert sequences of the different recombinant phages are processed with a suitable computer program (R.Staden, Nucl.Acid.Res. 10, 4731-4751 (1982)). FIG. 1 and 2 show the sequencing strategy. FIG. 3 shows the sequence of clone P9A2, FIG. 4 - the one of branch E76E9. The non-encoded DNA strand is shown in the 5 '-> 3' direction together with the derivative amino acid sequence.

The isolated cDNA of E76E9 clone for omega (O1i) -interferon is 858 pairs of bases long and contains one 3 'untranslated region.

The region encoding the mature omega (Glu) interferon extends from nucleotide 9 to nucleotide 524. The isolated to DNA of clone P9A2 for omega (Gly) -interferon is 877 pairs long, of which sequence is sufficient for encoding the mature omega-interferon. from nucleotide 8 to nucleotide 523. In the case of P9A2, the 3'-untranslated region reaches the poly-A segment. The DNA sequences encoding mature interferons are contained entirely in clones E76E9 and P9A2. They begin at the N-terminus with cysteine-aspartic acid-leucine. Surprisingly, both omega-interferons are 172 amino acids long, which clearly deviate from previously known interferons: e.g. 166 (165) amino acids at <X, interferons. The two omega-interferons also have one potential N-glycosylation site at amino acid position 78 (asparagine-methionine-threonine).

Comparison of clones E76E9 and P9A2 shows one difference. The amino acid encoding triplet 111 in E76E9 is GAG and encodes glutamic acid, while the triplet in P9A2 is GGG and encodes glycine. The two omega-interferon proteins differ by one amino acid and are designated as omega (O1i) -interferon (E76E9) and omega (Gly) -interferon (P9A2), respectively.

A comparison of the two omega-interferons with the previously known subtypes of human alpha-interferons gives the following picture.

omega alpha

Protein length in amino acids

172 166 (*)

Potential N-glycosylable 78 - (♦♦) sites

*) Interferon alfa-A consists of only 165 amino acids **) Interferon alfa-H contains one potential N glycosylation site at position 75 (D.Goeddel et al., Nature 290, 20-26 (1981)).

E.coli HB 101, with plasmid E76E9 and E.coli 101 with plasmid P9A2, have been deposited in the German Microbial Collection (DSM-Göttingen) under DSM 3003, respectively. DSM 3004 on July 3, 1984.

To demonstrate that the newly invented clones produce interferon-like activity, clone E76E9, for example, is cultured and bacterial lysis is tested after growth in a plaque-reducing assay. The bacteria produce the expected interferon-like activity. (See example 3)

Further, to prove that the newly discovered interferons are a new family of such, all DNA from Namalwa cells is isolated and digested with various restriction endonucleases. Through this, the amount of genes encoded by the cDNA of the clones can be estimated

E76E9 and P9A2. The DNA-containing fragments are then separated into agarose gel by the method of Southern (E .; Southern et al., J. Mol.Biol. 98, 503-517 (1975)), transferred to nitrocellulose filters and at relatively strict conditions were hybridized with radiolabeled specific DNA for clone P9A2.

The results obtained after hybridization with DNA containing plasmid P9A2 and pEIZ3- are shown in FIG. 5a:

There are single traces here, indicated by letters indicating the different digested DNA probes (E = EcoRI, H = HindIII, B = BamHI, S = Sphl, P = Pstl, C = Clal). The left half of the filter was hybridized with the interferon-alpha ('A') gene probe and the right half with the P9A2 clone cDNA insert ('0'). The assortment of tapes that are hybridized with the interferon-alpha probe and that of the new interferon is different. Cross-hybridization cannot be performed between the two different probes, judging from their respective traces.

In FIG. 5b shows the cDNA of clone P9A2 and the fragment used for hybridization.

The sites of action of individual restriction enzymes (P = Pstl, S = Sau3a, A = Alul) are shown. The probes cover only two of the possible three PstI fragments. The hybridized sample shows only one hybridization fragment, approximately 1300 pairs in length, belonging to the homologous gene. The smaller one

A 120 bp fragment was leaked from the gel. The 5'-end of the gene strand cannot be detected because the probe does not contain this region. At least 6 different types of chains can be found in these traces of PstI. This means that individual other genes that are related to the new sequences must be present in the human genome. Since these genes present one or more regions that are truncated by PstI, it could be expected that at least three additional genes could be isolated.

These genes can preferably be isolated by hybridization from a human gene library contained in a plasmid, phage or cosmid vector. (See Fig. 4e).

It should be recalled at this point that according to the invention, the omega interferons are not only two mature interferons, which are described separately, but all modifications to those polypeptides that contain the interferonomeg activity unchanged. These modifications include, for example, abbreviations of the C and N terminus of the molecule, replacement of amino acids with other residues in which activity does not change significantly, chemical or biochemical linkages of the molecule to other molecules that are inert or active. The so-called modifications may for example concern hybrid molecules with one or more omega interferons or those with known alpha and beta interferons.

In order to be able to compare the differences in the amino acid and nucleotide sequences of the new interferons, incl. omega Glu and Gly interferons, with respect to alpha and beta interferons, the corresponding sequences are arranged in pairs and the differences in single positions are counted. (S. Weiss; dk et al., Phil.Trans.R.Soc. London 299, 7-28 (1982); A.Ulrich et al., J. Mol.Biol. 156,467-486 (1982); T. Taniguchi et al., Proc.Nat.Acad.Sci 77,4003-4006 (1980); K.Tokodoro et al., EMBO J. 3, 669-670 (1984).

From the illustrated in FIG. 7 results indicate that the DNA sequences of clones P9A2 and E76E9 are related to the type I interferon sequences (alpha and beta interferons). On the other hand, the difference in amino acid sequences between single alpha 16 interferons and novel sequences is reported to be greater than 41.6% and less than 47.0%. The differences between the new sequences as well as those of single alpha interferons and beta interferons are in most cases about 70%. Considering the results of Example 4, which proved the presence of a whole series of related genes and examining the proposed nomenclature for interferons (J. Vilcek et al., J. Gen. Vir. 65,669-670 (1984)), inserts of clones E76E9 and P9A2 encode a new class of type1 interferons, which are referred to as omega-interferons.

Further, it has been demonstrated that expression of the interferon gene omega is similar to that of type T interferons. Individual members of the multigene family of alpha and omega interferons have been tested, based on SI mapping methods (AJBerk et al. Cell 12, 721 (1977)) and the expression of omega-1-mRNA has been shown to be virus-inducible. Since the transcripts of such a gene family differ in number of bases (about 1000), hybridization alone is not a sufficiently sensitive feature to distinguish the individual m RNAs of interferons.

The m RNA sequences of 9 alpha-interferons, interferon-omega 1 and beta-interferons are presented. Capitalization specifies those bases that are specific to the above sequences. These specific positions are easily found with the help of a trivial computer program. A hybridization probe originating from a specific position can only be hybridized perfectly with m mRNA of a particular subtype. All other mRNAs cannot be hybridized to the subtype-specific site. When the hybridization probe is radiolabelled at its specific 5 'end, then only the labeled sites that are hybridized to a probe engineered specifically for a given mRNA subtype remain protected against single-strand specific degradation (mainly SI nuclease).

This principle is not limited to interferon, it is repeatedly applied to each group of known sequences having the specific positions described in Figure 8.

The specific sites mentioned above are in most cases not restriction (tear) positions, so cutting of k DNA with restriction endonucleases is not suitable for producing subtype-specific hybridizing probes. Therefore, the probes used in this example are lengthened with a radiolabeled (5 'end) oligonucleoside that is complementary to m interferon-omega mRNA.

It is apparent from Figure 10 that omega-1-mRNA is expected to be inducible in Namalwa and NC37 cells. It is therefore an object of the invention not only gene sequences specifically encoding omega-interferons, but also modifications that can be obtained slightly and routinely through mutations, degradation, transposition or complementarity. Any sequence that encodes the human omega-interferons described herein (i.e., has a biological activity spectrum as described herein) and which degenerates from that shown, is therefore included herein. Those skilled in the art are able to degenerate the DNA sequences of the coding regions. Also included here is any sequence that encodes an activity spectrum polypeptide such as that of omega-interferons and hybridizes to the sequences shown (or a portion thereof) under stringent selectivity conditions of more than 85%, and preferably more than 90 % homology.

lb

Hybridizations were performed in 6 x SSC / 5 x Denhardt solution / 0.1% SDS at 65 ^with C. The degree of rigor was determined in the washing step. For a single DNA sequence selection of approx. 85% homology conditions are 0.2 x SSC / 0.01% SDS, 65 ^β C, and for homology 90% or more - 0.1 x SSC / 0.01% SDS / 65 ^e C.

Examination of a cosmid library under these conditions (0.2 x SSC) yielded several hybridized with an interferon-omega 1 cosmid probe. Sequence analysis of the restriction enzyme fragments thus isolated gave the authentic interferon gene omega 1 (see Figure 11), as well as three related ones. genes designated as interferon-pseudo-omega-2, interferon-pseudo-omega-3 and interferon-pseudo-omega-4. (see FIGS. 12-14). They are a further object of the present invention as well as the peptides encoded by them. They are characterized in claims 43-47.

The DNA comparison gives a round 85% homology to the interferon-omega-1 gene.

The interferon-omega-1 gene further indicates that transcription produces m RNA that contains information about a functional interferon protein, i. E. a signal peptide of the formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Ala Leu Val Met Thr Ser Tyr Ser Pro Val Gly Ser Leu Gly is encoded, followed by a mature containing 172 amino acids protein of interferon-omega.

The interferon-omega genes can, under certain conditions, be introduced into any organism, resulting in high yields. Suitable hosts and vectors, e.g. specified in EP-A-0.093.619.

Prokaryotes are particularly preferred for expression. E.g. strain 294 (ATCC No. 31446) of E. coli K12 is suitable. The E. coli W 3110 strains (F, Lambda, prototroph, ATCC) are also used

No 27325), bacilli such as Bacillus subtilis and others from

Enterobacteriaceae such as Salmonella typhimurium, Serratia marcensens and various pseudomonads.

Generally, plasmid vectors that include the replicon and the control sequence and are compatible with the host cells are used in conjunction with that host. The vector normally carries, in addition to the replication position, recognition sequences that allow the transformed cells to be phenotypically selected. E.g. E. coli is usually transformed with pBR 322, a plasmid derived from E. coli (Bolivar et al., Gene 2, 95 (1977)). pBR322 contains ampicillin and tetracycline resistance genes, and it simply provides a means of identifying transformed cells. pBR322 and other plasmids should also contain promoters or be modified to contain promoters that can be used by the microbial organism to express its own proteins. The promoters most commonly used in the preparation of recombinant DNA include beta-lactamase (penicillinase) and the lactose promoter system (Chang et al., Nature 275, 615 (1978); Itakura et al., Science 198, 1056 (1977) ; Goedel et al., Nature 281, 544 (1979)) and the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res., 8, 4057 (1980); EP-A0,036,776). While these are the most commonly used promoters, other microbial promoters are grown and used. The interferon omega gene sequence may e.g. to be placed under the control of the Leftward bacteriophage promoter Lambda. This promoter is one of the particularly well-known promoters that can be targeted. Direction is possible through a lambda repressor, which knows the boundary sites for restriction degradation.

A temperature-sensitive allele from this repressor can be inserted into a vector that contains the complete interferon-omega sequence. If the temperature rises to 42 ° C, the repressor is inactivated, the promoter is started to its maximum concentration. The amount of m RNA produced under these conditions should be sufficient to produce a cell that contains about 10% of the new synthetic amino acids originating from the PL promoter. Thus, it is possible to establish a clone bank in which the functional interferon-omega sequence will be located adjacent to the ribosome binding site and at varying distances from the lambda-βb promoter. These clones are then tested and selected with highest yield. The expression and translation of an interferon-omega sequence can also proceed under the control of another regulatory system that can serve as a homolog of the organism in its untransformed form. For example, the chromosomal DNA of a lactose-dependent E. coli contains a lactose or Lac operon, which, by synthesizing the beta-galactosidase enzyme, provides for the breakdown of lactose.

Lac control elements can be obtained from the bacteriophage Lambda-plac 5, which is infectious to E. coli. LacoperonBG phage can be transmitted by transduction from the same type of bacteria. Regulatory systems that are empirically established can originate from plasmid DNA that is inherent in the body. The lac promoter-operator system can be induced by IPTG.

Other promoter-operator systems or parts thereof that can be used with the same success are: arabinose operator, colchicine E1-operator, alkaline phosphatase operator, tryptophan operator, xylose-A-operator, tac-promoter and others.

In addition to prokaryotic, eukaryotic microorganisms can also be used, such as molds, e.g. Saccharomyces cerevisiae is the most widely used among eukaryotic microorganisms, although many others can be found. For expression in Saccharomyces cerevisiae, plasmid YRp7 is commonly used (Stinthcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschumper et al., Gene 10,157 (1980)) and plasmid YEpl3 (Bwach et al., Gene 8, 121-133 (1979)). Plasmid YRp7 contains TRP1gen, which is a selectable marker for a mold mutant that cannot grow in tryptophan-containing medium, eg ATCC No. 44076.

The presence of a TRP-1 defect as a trait in the host genome provides effective help in demonstrating transformation, since it can be cultured without tryptophan. Similarly, plasmid YEp 13 contains the mold gene LEU-2, which can be used to supplement a LEU-2 negative mutant. Appropriate promoter sequences for mold vectors (Hitzeman et al., J. Biol.Chem. 255 2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 108, 1107-1112 (1982)) such as enolase, glycerin-aldehyde- 3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphatisomerase, phosphoglucoisomerase, and glucokinase. Suitable expression plasmids can be included in the concentration together with the termination sequences associated with these genes at the 3'-end of the expression vector. Thus, polyadenylation and termination of the m RNA can be predicted.

Other promoters that have the advantage of controlling transcription through growth conditions are the promoter regions of the alcohol dehydrogenase-2 gene, isocytochrome-6 acid phosphatase, degrading enzymes that are related to nitrogen metabolism, the aforementioned glycerol-aldehyde hydrodehyde-aldehyde hydrodehyde-aldehyde hydrodehyde-aldehyde hydrodehyde the enzymes responsible for the processing of maltose and galactose. Promoters that are regulated by loci of the Hefe-Mating type (accompanying mold), e.g. BAR I, MF alpha-1, STE 2, STE 3, STE 5 gene promoters can be inserted in temperature-controlled systems using temperature-dependent gray mutations (Rhine PhD Thesis, University of Oregon, Eugene, Oregon ( 1979), Herskovitz and Oshima, The Molecular Biology of the Yeast Saccharomyces, Part 1, 181-209 (1981), Ccld Spring Harbor Laboratory). These mutations affect the expression of resting Mating-type cassette cartridges and thus the indirectly dependent Mating-type promoters. Generally, any plasmid vector containing a mold-compatible promoter, original termination and replication sequences is suitable.

In addition to microorganisms, cultures of multicellular organisms are also suitable hosts. In principle, any crop, whether derived from invertebrate or vertebrate animals, is applicable. However, animal cells from vertebrates are of most interest, so their reproduction in tissue cultures in recent years has been a routine method (Tissue Culture, Academic Press, Kruse and Patterson Editors (1973). Examples of such cell lines are

- 23 VERO and He La cells, Hamster Ovary (CHO) cells, W138, BHK, COS-7 and MDCK. Expression vectors for these cells contain a simple replication site, a promoter that is localized before the gene to be expressed. , in conjunction with the required ribosome binding site, PHK-splicing region, polyadenylated region, and terminating transcription (termination) sequences. When using mammalian cells, the control functions on the expression vectors are usually from viral material. E.g. commonly used promoters originate from polio adenovirus-2 and very often from Simianvirus 40 (SV 40), early and late end promoters of SV 40 are especially used as they are easily obtained from the virus as a fragment that also contains the viral replication region of SV 40 (Fiers et al., Nature 273, 113 (1978)) Also, larger or smaller fragments of SV 40 can be used provided they contain an approximately 250 bp long sequence sufficient to locate breaking Hind 111 to that of Bgl 1 in the viral replication site. In addition, it is possible and often recommended to use promoter or control sequences that are normally associated with the desired gene sequences, provided that these control sequences are compatible with the host cell system. A replication site can be secured or exogenously, by a suitable vector construction, e.g. from SV 40 or other viral source (Polyoma, Adeno, VSV, PBV, etc.) or through the chromosomal replication mechanisms of the host. If a vector integrates into the host chromosome, in most cases this is sufficient. Genes can advantageously 'die down' in the expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21,

237-248 (1983) and EP-A-0.115-613 - implemented in DSM under DSM number 2773 on December 20, 1983), since this vector contains all the regulatory elements leading to a high expression of the cloned gene. According to the invention, in plasmid pBR322, the intrinsic EcoRi / BamH1 fragment is replaced by a DNA sequence of the formula:

EcoRI Sau3a gaattcacgctGATCGCTAAAACATTGTGCAAAAAGAGGGTT GACTTTGCCTTCGCGA 59 mRNA START

ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAA

Met

GGAGGTTTAAGCTTAAAG ATG 116

RBS Hindlll

Cys Asp

TGT GAT C Interferon-omega gene

Sau3a

In order to achieve the object of the invention, this according to Figure 6 is carried out as follows:

I. Obtaining the required single DNA fragments: Fragment a:

To obtain fragment a), a plasmid containing an interferon gene is an omega e.g. P9A2 ,. was digested with restriction endonuclease Avail. After chromatography and purification of the resulting k DNA inserts, the latter are treated twice with restriction endonucleases

Ncol nAlul and were isolated by chromatography and eluting. This fragment contains most of the relevant omega interferon gene. Thus, the OMera- (Gly) interferon gene of clone P9A2 shows the following structure:

20 c His Gly 1 CAT GGC ... Ncol Leu CTA Leu CTT 25 Sir AGC Arg AGG Asn AAC Thr ACC Leu TTG 30 28 Vai Leu Leu His Gin Met Arg Arg lie Sir Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC. TTG TGT CTC 73 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Vai Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 50 55 60 Sir Gin Leu Gin Lys Ala His Vai Met Sir Vai ' Leu His Glu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 65 70 75 Leu Gin Gin He Phe Sir Leu Phe His Thr Glu Arg Sir Sir Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC AC A GAG CGC TCC TCT GCT 208 80 85 90 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 95 * 100 105 Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Vai Vai Gly CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298

Glu Gly G1U 110 Sir Ala Gly Ala lie Sir 115 Sir Pro Ala Leu Thr 120 Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 125. 130 135 Arg Arg Tyr Phe Gin Gly He Arg Vai 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 Sir Asp Cys Ala Trp Glu Vai Vai Arg Met Glu He Met Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 155 160 165 Sir Leu Phe Leu Sir Thr Asn Met Gin Glu Arg Leu Arg Sir Lys TCC TTG TTC -TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478

170th Asp Arg Asp Leu Gly Ser Ser

GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530

TAAC AC TTGC AC ATGTG AC TC TGG TCAATTCAAAAGAC TC TTATTTCGGC TTTAATC AC 589 ²⁵ AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 AAAAGACTTAGGTTCAGGGGCATCATTTATTATTATTATTAT

TTTTC TTAC ATTTTATC ATATTTATAC TATTTATATTC TTATATAAC AAATGTTTGCC 766

TTTACATTGTATTAAGATAACAAAACATGTTCAGfct 802

Alul

- fragment b:

To obtain fragment b), plasmid P9A2 was digested with restriction enzyme Avail. After chromatography and purification of the resulting cDNA slice, it was further treated with restriction enzyme Sau3A and the desired 189bp long fragment _; is isolated by chromatography and elution. It shows the following structure:

_s 1 Asp Leu (GAT CTG BaiZd) Pro CCT 5 Gin CAG Asn AAC His Gly -Leu 10 Leu CTT Sir AGC Arg AGG Asn AAC 15 Thr ACC Leu CAT GGC CTA TTG 42 NCOI 20 25 30 JO Val Leu Leu His Gin Met Arg Arg lie Sir Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 87 15 35. 40 45 * Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 132 20 50 55 60 5er Gin Leu Gin Lys Ala His Val Met Sir Val Leu His Glu Met ACG CAG TTG CAG AAG GGC CAT GTC ATG TCT GTC CTC CAT GAG ATG 177

Leu Gin Gin lie

CTG CAG C / jg ate 1θθ

Sau3A

- '2B Fragment c:

To obtain fragment c) plasmid pER33 (see E. RastlDworkin et al., Gene 21 (1983) and EP-A-0.115.613) was digested twice with EcoRI and Pvull restriction enzymes. After agarose gel fractionation and purification, the resulting 389 bp fragment containing the Trp promoter, ribosome binding site and start codon was treated with Sau3a. The resulting fragment, 108 bp long, was obtained by agarose gel electrophoresis, electroelution and purification in an Elutip column. It shows the following structure:

EcoRI Sau3a gaattcacgctGATCGCTAAAACATTGTGCAAAAAGAGGGTT GACTTTGCCTTCGCGA 59 mRNA START

ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAA

Met

GGAGGTTTAAGCTTAAAG ATG 116

RBS Hindlll

Cys Asp

TGT pier c

Sau3a

The fragments b and c are ligated with T4 ligase and after the enzyme destruction is sewn with HindIII. These ligated fragments show the following structure:

Hindlll aAGCTTAAAG CCATGGCCTA

Sau3a

ATGTGTGATC

Ncol

TGCCTCAGAA

CTTAGCAGGA 50

ACACCTTGGT

GAATCTCCCC

GCTTCTGCAC

CAAATGAGGA

TTTCTTGTGT 100

CTCAAGGACA CAGGAGATGG

GAAGAGACTT

CAGGTTCCCC

TAAAAGGGAG 150

CCAGTTGCAG

CCTCCATGAG

AAGGCCCATG

TCATGTCTGT

ATGCTGCAGC 200

AGATCACACA TCTTTAagct t Sau3a Hindlll

Alternatively, for the production of the pRHWIO plasmid, the necessary DNA fragments can be obtained by the application of two synthetically synthesized oligonucleotides:

The oligonucleotide of the formula

5-AGCTTAAAGATGTGT-3 'which remains dephosphorylated at its 5'end.

The oligonucleotide of the formula

5'-GATCACACATCTTTA-3 'which is phosphorylated at the 5'-end by T4-polynucleotidkinase and ATf

Hybridization of the two oligonucleotides yields the following short DNA fragment:

5'-AGCTTAAAGATGTGT 3 '

3'- ATTTCTACACACTAGp 5 '

It contains a typical Hindlll region at one end and Sau3a at the other.

Fragment b) is dephosphorylated by calf phosphatase. Fragment b) and the fragment described above are mixed and bound by T4 ligase.

Because the ligase requires at least one 5'-phosphate-containing end, only a synthetic fragment with fragment b) or two synthetic fragments at their Sau3a ends can be bound. As the resulting two fragments differ in length, they can be separated by precipitation with isopropanol. The currently purified fragments are phosphorylated with T4-polynucleotidkinase and ATf.

II. Preparation of expression plasmids

a) Preparation of plasmid pRHWIO:

The expression plasmid pER103 (E. Rastlorkorkin et al., Gene 21,237-284 (1983) and EP-A-0.115.613 registered in DSM under No. DSM 2773) was linearized with HindIII and treated finally with calf phosphatase. After isolation and purification of the DNA thus obtained, it is dephosphorylated and ligated with the bound fragments b and c (after digestion with HindIII). Finally, the resulting ligation mixture was transformed with E. coli 101 and cultured on LB agar plus 50 μg / ml ampicillin. The plasmid exhibiting the desired structure has been designated pRHWIO (see Fig. 6) and serves after its replication as an intermediate for the production of subsequent plasmids.

B) Preparation of plasmid pRHW12:

A Klenow-fragment of DNA polymerase I and the four deoxynucleoside triphosphates were added to the PAMHI-cut plasmid pRHWIO. The linearized and equilibrated ends obtained after incubation were purified and cut with Ncol. The larger fragment obtained after agarose gel electrophoresis, electroelution and purification through an Elutyp column is ligated with fragment a). The ligated mixture was finally transformed with E. coli HB101 and cultured on LB-arap plus 50 μg / ml ampicillin. The plasmid obtained with the desired structure is designated pRHW12 (see Fig. 6) and expresses the desired OMera- (Gly) interferon.

1 of the bacterial culture thus obtained (optical density: 0.6 at 600 nm) contains, for example, 1x10 ^ international units of interferon.

Preparation of plasmid pRHWll:

Klenowfragment of DNA polymerase I and the four deoxynucleoside triphosphates were added to the cut-off BamHI plasmid pRHWIO. electroelution and purification through an Elutyp column was ligated with a similarly derived from plasmidE79E9 fragment a) in which the amino acid (Glu) GGG codon cGAG codon coding for glycine was exclusively replaced. Finally, the resulting ligation mixture was transformed with E. coli HB101 and cultured on LB-arap. The plasmid having the desired structure is referred to as pRHW11 (see Figure 6), which expresses the desired OMera- (Glu) interferon.

The transformation of cells by different means can be accomplished by many methods. For example, with calcium, where either the cells are grown in the presence of magnesium and then suspended in a medium containing calcium and DNA is added, or the cells are provided with calcium phosphate DNA coprecipitate. Following expression, cells are transferred to media that are selectable for the transformed cells.

After successful host transformation, gene expression, and fermentation or cell culture, under conditions under which interferon-omega is expressed, the product is »typically extracted by known chromatographic separation methods to obtain interferon-omega containing material with or without leading and ending sequences. The interferon-omega can be expressed with one leading sequence at the N-terminus (pre-interferon-omega) that is withdrawn from the host. When this happens, cleavage of the leading polypeptide (if present) is required to produce mature interferon-omega. Alternatively, the interferon-omega clone may be modified so that the mature protein is produced directly into the microorganism instead of pre-interferon-omega. In this case, the precursor sequence of mold Mating is used

- ό3 MF-alpha-1 pheromone to obtain proper maturation and to ensure the excision of products in the washing medium or in the periplasmic space. The DNA sequence for the functional or mature interferon-omega can be linked via MF-alpha-Ι to the putative cathepsin-like cleavage region (after Lys Arg) at position 256 of the ATG initiation codon (Kurjan, Herskowitz, Cell 30,933-943 (1982).

Based on the spectrum of their biological activity, newly invented interferons can substitute in any respect for the treatment of known interferons. This includes herpes, rhinovirus, opportunistic AIDS infections, various cancers and the like. The new interferons can be used alone or in combination with other known interferons or biologically active products, for example interferon-alpha, interleukin-2, other immunomodulators and the like.

Interferon-omega can be administered parenterally when antitumor or antiviral treatment or immunosuppressive properties are required. The dosage may be similar to that obtained in clinical trials for interferon-alpha (eg (1-10) x 106 units / day and for preparations cleaner than 1% to 5x107 units / day.

A suitable dose of one basic, homogeneous interferon-omega of bacterial origin, with parenteral administration, is 3 mg of interferon-omega in 25 ml of 5% human serum albumin. This solution was passed through a bacteriological filter and separated aseptically into 100 vials, each containing 6x1 Ob units of interferon-omega for parenteral administration. The vials are stored mostly at -20 ° C.

The substances obtained by the invention can be formulated in a known manner to obtain a pharmaceutically useful agent, wherein the polypeptide is mixed with an acceptable, pharmaceutical carrier. The carrier materials used and their formulations are described by E.W. Martin in Remington's Pharmaceutical Sciences, from which the imprint was taken. Interferon-omega is mixed with a fixed amount of carrier to obtain the desired pharmaceutical agent that is suitable for effective administration to patients. The parenteral form of administration is generally accepted The following examples, which are not limited by the invention, describe it in detail.

Example 1

Finding specific clones for interferon sequences.

a) Preparation of the k DNA library cDNAs from Sendai virus-stimulated cells are used as a starting tool in the methods known in the literature for the preparation of the k DNA library. (E.Dworkin-Rastl et dl., J. Interferon Res., Vol. 2/4, 575-585 (1982)) The resulting 30,000 clones were transferred to wells of microtiter plates. The following medium is used for the growth and freezing of colonies:

3b

10 Mr Rücker Trypton 5 Mr Rücker Yeast extract 5 Mr Rücker NaCl 0.51 Mr Rücker Na-Citrate x 2 H2O 7.5 Mr Rücker K2HPO4 x 2 H2O 1.8 Mr Rücker KH2PO4 0.09 Mr Rücker MgSO4 x 7 H2O 0.9 Mr Rücker (NH4) 2SO4 44 Mr Rücker Glycerin 0.01 Mr Rücker Tetracycline x HC1 ad 1 1 H2O

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

B) Hybridizing probe

The recombinant plasmid pER33 (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983)) is used as the starting material for the hybridization probe. The plasmid contains the coding region for the mature IFN-alpha-2 arg interferon plus 190 bases of 3 'untranslated elastomer. 20 μg of pER33 was incubated with 30 units of Hind 111 restriction enzyme in 200 μl of reaction solution (10MM TrisHCL pH 7.5, 10mM MgC12, 1mM Dithiotreitol (DTT), 50mM NaCl) for 1 hour at 37C. adding 1/25 vol. 0.5 M and heating at 70 ^with C for 10 min. After adding 1/4 vol. 5 x buffer (80% glycerol, 40 mM Tris-acetic acid pH = 7.8, 50 mM EDTA, 0.05% SDS, 0.1% bromophenolblau) The resulting fragments were separated by size in

1% agarose gel by electrophoresis (gel and electrolyte buffer (TBE): 10.8 g / l Tris-hydroxymethylaminomethane (Trisbase), 5.5 g / l boric acid, 0.93 g / l EDTA). After incubation of the gels in ethidium bromide solution, where the DNA strands become visible under ultraviolet light, the gel region containing the interferon gene DNA is excised (ca 800 bp in length). By applying voltage, the DNA is electroeluted in 1/10 x TBE buffer. The DNA solution was extracted once with phenol and 4 times with ether. DNA was precipitated from the washing solution with the addition of 1/10 volume 3M sodium acetate (NaAc), pH 5.8 and 2.5 vol. EtOH at -20 ° C. After centrifugation, the DNA was washed with 70% ethanol and dried for 5 min in vacuo. The DNA was dissolved in 50 μl of H ₂ O (ca. 50 μg / ml). This DNA is radiolabelled by the method of Maniatis et al., Molecular Cloning, ed. CSH via Nick-translation. A 50 µl incubation solution contains: 50 mM Tris pH = 7.5, 5 mM MgCl _2> 10 mM mercaptoethanol, 100 ng DNA segment from pER33, 16 µg deoxyribonuclease I, 25 µmol dATP, 25 µmol dGTP, 25 µmk dTTP, 20 µm dTTP alpha 32 p-dCTP (> 3000 Cl / mMol) and 3 units of DNA polymerase I (E.coli). The incubation was carried out at -14 ° C for 45 minutes. The reaction was stopped by the addition of 1 vol. 50 mM EDTA, 2% SDS, 10mM Tris pH = 7.6 and heating at 70 ° C for 10 min. Unbound radioactivity was separated by chromatography over Sephadex G100 in TE buffer (10 mM Tris pH = 8.0, 1 mM EDTA). The radiolabelled probe shows a specific activity of approx. 4 x 10 ⁷ cpm / mcg.

c) Screening of clones on inserts containing interferon genes

The bacterial cultures frozen in microtiter plates were thawed (a). Appropriate size nitrocellulose filter sheet (Schleichter and Schull, BA 85, 0.45 µm pore size was applied to LB agar (LB agar: 10g / l Tripton, 5g / l yeast extract, 5 g / 1 NaCl, 15 g (1 Bacto arap, 20 mg / 1 tetracycline HCI) With a multichannel device fitting the microtiter plate, the individual clones were transferred to a nitrocellulose filter Bacteria were transferred at 37 ° C to form colonies of about 5 mm in diameter. bacteria and DNA denaturation, nitrocellulose filters are placed one after another on a pile of Whatman Zmm filters impregnated with the following solution : 1) 8 min. On 0.5 M NaOH; 2 min IM Tris pH = 7.4; 2 min IM Tris pH = 7.4; 4 min 1.5 M NaCl; 0.5 M Tris pH = 7.4. The filters are air-dried and kept at 80 ° C. The pre-treatment of the filters is 4 hours in a 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 solution (1x p- Denhardt's p corresponds to 0.02% PVP (polyvinylpyrrolidone); 0.02% Ficoll (M.t. 40,000D); 0.02% BSA (bovine serum albumin) and 0.1% SDS (sodium dodecyl sulfate). About 1 x 10 ⁶ cpm / filter from the probe manufactured according to b) are denatured on boiling and added to the hybridization solution. The hybridization was continued for 16 hours at 65 ° C. The filters were washed at 65 ° C. <3 x SSC / 0.1% SDS four times, the filters were air-dried, coated with Saran Wrap and exposed on a Kodak X-OmatS film.

Example 2

Confirmation of recombinant plasmids containing interferon genes by Southern method transfer

- 3d Cultures are grown from the positive and doubt positive reacting colonies and allowed to stand in 5 ml L-broth (10g / l Trypton, 5g / l yeast extract, 5 g / 1 NaCl, 20 mg / 1 tetracycline x HCl) at 37 ° C. Plasmid DNA was modified by Birnboim and Doly (Nucl.Acid Res. 7, 1513 (1979)). Cells of 1.5 ml of suspension were centrifuged (Eppendorf centrifuge) and resuspended at 0C in 100 μl of lysozyme containing 50 mM glucose, 10 mM EDTA, 25 mM TrisCH1 pH = 8.0 and 4 mg / ml Lysozym . After 5 min of incubation at room temperature, 2 volumes of ice-cold 0.2 M NaOH, 1% SDS were added and incubated for another 5 min. Then 150 μl of potassium acetate pH = 4.8 was added and 5 more were incubated. The precipitate containing fraction of the cell fraction was centrifuged. The DNA solution was extracted with an equal volume of phenol / chloroform (1: 1) and the DNA was precipitated by the addition of 2 volumes of ethanol. After centrifugation, the precipitate was washed once with ethanol and dried for 5 min in vacuo. The DNA was dissolved in 50 μl of TE buffer. 10 µl of this was dissolved in reaction solution (10 mM Tris-HCl pH = 7.5, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT) and digested with 10 units of Pstl-restriction enzyme for 1 hour at 37 ° C. After the addition of 1/25 vol. parts of 0.5 M EDTA and 1/4 vol. portions 5 x buffer (see Example 1b), the mixture was heated at 70 ° C for 10 min and finally separated into a 1% agarose gel (TBE buffer) by electrophoresis. DNA was transferred from the gel to a nitrocellulose filter by the Southern method (EM Southern, J. Mol.Biol., 98, 503-517 (1975)). The DNA in the gel was denatured with a solution containing 1.5 M NaCl / 0.5 M NaOH. Finally, neutralize for 1 hour with IM Tris x HCl pH = 8 / 1.5M NaCl. The DNA was transferred to a nitrocellulose filter with 10 x SSC (1.5 M NaCl, 0.15M sodium citrate pH = 7.0. After transfer (approx. 16 hours), the filter was rinsed briefly in 6 x SSC buffer, dried air and stand for 2 hours at 80 ° C. The filter is pre-treated with 6 x SSC / Denhardt pp / 0, 1% SDS (see Example 1 s) at 65 ° C. About 2 x 106 cpm of the hybridization probe (see ex. 1b) are denatured by heating at 100 ° C and added to the hybridization solution. Hybridization is continued for 16 hours at 65 ° C. Finally, filter® is washed 4 times at 65 ° C with 3 x SSC / 0.1%. SDS filters are air dried ha, they are covered with Saran Wrap and exposed on a Kodak X-OmatS film.

Example 3

Demonstration of interferon activity in clone E76E9

EOmL of clone E76E9 was grown on L-broth (10 g / 1 Trypton, 5 g / 1 yeast extract, 5 g / 1 NaCl, 5 g / 1 glucose, 20 mg tetracycline x HCl for 1) at 37 ° C to optical density A ^ oq = O, 8. Bacteria were centrifuged at 7000rpm for 10 min, washed with a solution containing 50 mM Tris x HCl pH = 8, 30 mM NaCl and resuspended in 1.5 ml washing solution. After incubation with 1 mg / ml lysozyme solution for half an hour, the bacterial suspension is frozen and thawed 5 times. The cell debris was removed by centrifugation at 40 OOOrpm for 1 hour. The supernatant was filtered sterile and tested for interferon activity. A plaque-reducing assay with US cells and vesicular stomatitis virus was used (G. R. Adolf et al., Arch.Virol, 72, 169-178 (1982). A surprising clone produced up to 9,000 units of interferon per liter of starting culture.

Example 4

Genomic Southern blot to determine the number of genes that are assigned to the new sequences.

a) Isolation of DNA from Namalwa cells

400 ml of Namalwa cell culture were centrifuged at 1000 rpm. in a JA21 centrifuge. The resulting precipitate was washed thoroughly and resuspended in NP40 buffer (140mM NaCl, 1.5mM MgCl2, 10mM Tris-Cl pH = 7.4) and again precipitated at 1000 rpm. The resulting precipitate was dissolved in 0 20 ml KP40 buffer and 1 ml of 10% NP40 solution was added to destroy the cell walls. After a 5-minute stay in an ice water bath, the intact cell nuclei were precipitated by centrifugation at 1000 rpm and the precipitate discarded. The cell nuclei were dissolved in 10 ml of a solution consisting of 50 mM Tris / Cl pH = 8.0, 10 mM EDTA and 200 mM NaCl and finally 1 ml of 20% SDS was added to remove the proteins. The resulting viscous solution was extracted twice with an equal amount of phenol (saturated with 10 mM Tris / Cl pH = 8) and twice with chloroform. The DNA was precipitated by ethanol addition, separated by centrifugation, and the resulting DNA precipitate washed once with 70% ethanol. Dry for 5 min in vacuo and dissolve in 6 ml of TE buffer. The DNA concentration is 0.8 mg / ml.

B) DNA digestion of Namalwa cells with restriction endonucleases.

Restriction digestion is performed according to the manufacturer's (New England Biolabs) conditions. 1 μg of DNA was digested with 2 units of suitable restriction endonuclease in a volume of 10 μl at 37 ° C for 2 hours or longer. EcoRI, HindIII, BamHI, Sphl, PstI and Clal are used as restriction enzymes. 20 mcg of DNA was added to each digestion. To control the completeness of digestion at the start of the reaction, 10 μl (aliquots) were separated and mixed with 0.4 μg of Lambda-phage DNA. After two hours of incubation, aliquots were monitored by agarose gel electrophoresis and the digestion completeness was determined using a Lambda-phage DNA staining witness. After the control, the reaction was stopped by adding EDTA to a final concentration of 20 mM and 10 minutes heating the solution at 70 ° C. The DNA was precipitated by the addition of 0.3 mM NaAc pH = 5.6 and 2.5 parts by volume of ethanol. After incubation at -70 ° C for 30 minutes, the DNA was precipitated in an Eppendorf centrifuge, washed once with 70% ethanol and dried. The resulting amount of DNA was dissolved in TE buffer.

c) Gel electrophoresis and Southern transfer

The digested DNA probes were separated by size in 0.8% agarose gel in TBE buffer (10.8 g / l TrisBase, 5.5 g / l boric acid, 0.93 g / l EDTA). Here, 15 µl of the DNA probe was mixed with 4 µl of loading buffer (0.02% SDS, 5 x TBE buffer, 50mM EDTA, 50% glycerol, 0.1% bromophenolblau), heated briefly at 70 ° C and is placed in the intended wells of the gel. Lambda DNA that will be cut with EcoRI and HindIII is inserted into the appropriate well and serves as a marker for DNA size. Gel electrophoresis was carried out for 24 hours at 1 V / cm. Finally, the DNA was transferred by the Southern method onto a nitrocellulose filter (Schleicher & Schuell, BA85) using 10 x SSC (1 x SSC: 150 mM trisodium citrate, 15mM NaCl, pH = 7.0) as the transfer buffer. After drying the filter at room temperature, it is heated for 2 hours at

80 ° C to attach DNA to it.

d) The hybridizing probe mcg of plasmid P9A2 is treated with Avail which releases a fragment of 1100 bp containing the entire cDNA slice. This section of DNA was again sectioned with Sau3a and Alul and the largest DNA strand was isolated by agarose gel electrophoresis (see Fig. 5b), electroelution, and Elutip column chromatography. The DNA thus obtained (1.5 μg) was dissolved in 15 μl of water.

µl of plasmid pER33 is cleaved with HindIII, npM which cleaves this expression plasmid for interferon-alpha 2-α 2-fold (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983)). The smaller DNA fragment contains the interferon-alpha-2-arg gene and is isolated in the same way as plasmid P9A2.

The two DNAs are translated in isolation by the method provided by PWJRigby et al., (J. Mol.Biol. 113,237-251 (1977)). Nickel translation was performed with 0.2 µg DNA in 50 µl solution containing 1 x Nick buffer (1 x Nick buffer: 50 mM Tris / Cl pH = 7.2, 10 mM MgSO4 0.1 mM DTT, 50 μg / ml BSA), 100 μmol / ml dATP, dGTP, dTTP, 150 mkCi alpha-3 ² P-bSTR (Amersham, 3000 Ci / mMol) and 5 units of DNA polymerase I (Boehringer-Mannheim, Nick-Translation Quality ). After 2 hours at 14 ° C, the reaction was quenched by the addition of the same amount of EDTA solution (40mMol) and the free radioactive material was separated by Sephadex G50 column chromatography in TE buffer. The remaining specific activity carries about 100 x 10 ^{6 C} P ^M / ^μg DNA.

e) Hybridization and autoradiography

The cellulose filter is cut in two halves. Each half contains an identical set of Namalwa-DNA traces that have been treated with the aforementioned restriction enzymes (see Example 4a). filter »is hybridized to a solution containing 6 x SSC, 5 x Denhardt (1 x Denhardt: 0.02% bovine serum albumin (BSA), 0.02% polyvinylpyrrolidone, (PVP), 0.02% Ficoll 400), 0, 5% SDS, 0.1 mg / ml denatured veal thymus DNA and 10 mM EDTA for 2 hours at 65 ° C. Hybridization was performed in a solution containing 6 x SSC, 5 x Denhardt, 10 mM EDTA, 0.5% SDS, and about 10 x 10 cpm Nick-translated DNA for 16 hours at 65 ° C. One half hybridizes with interferon-alpha-2α ^ DNA and the other hybridizes with interferon DNA isolated from plasmid P9A2. After hybridization, the two filters were washed at room temperature four times with a solution containing 2 x SSC and 0.1% SDS, and twice 45 min at 65 ° C with a solution consisting of 0.2 x SSC and 0p01% SDS. Finally, the filters are dried and exposed to a Kodac X-Omat S film.

Example 5

Preparation of expression plasmids pRHW 12 and p RHW 11

Preliminary explanation:

The production of expression plasmids is shown in Figure 6 (not in scale), further all restriction digestions were performed according to the instructions of the enzyme manufacturers.

Preparation of plasmid pRHWIO

100 μg of P9A2 plasmid were digested with 100 units of the Avail (New England Biolabs) restriction enzyme. After degradation enzimytse inactivated at 70 ^is C and the resulting fragments are fractionated by size in 1.4% agarose gel in TBE buffer (TBE buffer: 10,8 g / 1 Tris-base, 5,5 g / 1 boric acid,

0.93 g / 1 EDTA). The resulting strips containing the entire cDNA insert were electroeluted and purified through an Elutypcolumn. Of the 20 μg obtained, 6 μg were further digested with restriction enzyme Sau3a-20 units in a total of 100 μl solution. The fragments were separated with 2% agarose gel in TBE buffer. After staining with ethidium bromide (E1Br), the 189 bp long strand of DNA was electroeluted and purified as described above. (= fragment b in FIG. 6)

To supply the interferon gene with a promoter, ribosome binding site, and start codon, the corresponding DNA region was isolated from the expression plasmid pER33 (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983). 50 µg of pER33 was digested twice with EcoRI and Pvull and the resulting fragments were separated by size into a 1.4% agarose gel and TBE buffer The long 389 bp DNA strand, whose Tp promoter contains the ribosome binding site and start codon, was is eluted and purified through an Elutyp column, thus treating the purified fragment with Sau3a and the desired 108 bp fragment obtained agarose gel electrophoresis, electroelution, and purification in an Elutyp column in a yield of about 100 ng (= fragment from FIG. 6).

ng of fragment b was ligated with 20 ng of fragment with a volume of 40 µl by applying 10 units of T4-ligase in solution containing 50mM Tris / Cl pH = 7.5, 10 mM MgC12, 1 mM DTT and 1 mM. 18 hours at 14 ° C. Finally, the enzyme was destroyed by heating at 70 ° C and the resulting DNA was digested with HindIII in a total volume of 50 µl.

µg of the expression plasmid pER193 (E. Rastl-Dworkin et al., Gene 21, 237-248 (1983) linearized with HindIII in a total volume of 100 µl. After two hours at 37 ° C, 1 volume of 2 x phosphatase buffer ( 20 mMTris / Cl pH = 9.2, 0.2 mM EDTA) with one unit of calf phosphatase (CIP) After 30 min

4b at 45 ° C, a further unit of CIP was added and incubated for another 30 min. The DNA thus obtained was purified by double phenolic extraction, single extraction with chloroform and precipitated by the addition of 0, 3M sodium acetate (pH = 5.5) and 2.5 volumes of ethanol. Finally, it is dephosphorylated to prevent the vector from following in the next steps.

100 ng of linearized pER103 and the ligated fragments b and c (after digestion with HindIII) were transferred into 100 μl of p-containing ligase buffer and ligated at 14 ° C for 18 hours with T4 DNA ligase.

200 µl of competent E. coli HB 101 (E.Dworkin et al., Dev. Biol. 76,435-448 (1980) was mixed with 20 µl of the ligation mixture and incubated for 45 min on ice. DNA uptake was interrupted by heat shock. for 2 min at 42 ° C. The cell suspension was incubated for another 10 min on ice and transferred onto LB-agar (10g / l Trypton, 5 g / 1 yeast extract, 5 g / 1 NaCl, 1.5% agar), containing 50 mg / ml ampicillin The plasmid contained in the resulting 24 colonies was isolated by the method of Birnboim and Doly (Nucl.Acid.Res.7, 1513-1523 (1979)). After digestion with various restrictases, the plasmid was obtained The desired structure is denoted by p RHWIO (see Fig. 6).

B) Preparation of plasmid pRHW12

About 10 μg of plasmid pRHWIO were digested with BamHI. Finally, a maple fragment of DNA polymerase1 and the four deoxynucleotide triphosphate were added and incubated for 20 min at room temperature. The resulting linearized and straight-edged plasmid was purified by phenolic extraction and precipitation and finally treated with restriction enzyme Ncol in a volume of 100 μl. Fragment a) (see FIG. 6) was prepared by grinding a 4 μg Avall fragment containing an insert of P9A2-cDNA (see above) with Ncol and Alul, yielding about 2 μg fragment a).

In the final ligation step, fragment a) and the addition of twice treated with BamHI and Ncol pRHWIO were bound in a volume of 10 µl and 10 ng of DNA was added. Ligation of a single BamHI cut site and the Alul cut DNA yields a BamHI cut site again.

The resulting ligation mixture was transformed with competent E. coli HB101 as described above. From the 40 colonies obtained, one is selected which contains the designation pRHW12.

Plasmid was isolated and the EcoRI / BamHI insert was sequenced by the Sanger method (F.Sanger et al., Proc. Nat.Acad.Sci 74, 54635467 (1979)). It has the expected sequence.

c) Preparation of plasmid pRHW 11

Following is analogous to Example 5 b). 1 µg of plasmid pRHW was digested with BamHI. The adhesive ends of the resulting DNA were aligned using a Maple Fragment of DNA polymerase I and the four deoxynucleotide triphosphates. Finally, the linearized DNA was cut with Ncol. The larger fragment was obtained by agarose gel electrophoresis, electroelution and purification in an Elutyp column.

The Ncol / Alul fragment was isolated from E76E9 analogously to example 5b. Finally, 10 ng of the vector ends were ligated with 10 ngjDT cDNA ends in a volume of 10 µl under appropriate conditions and the use of 1 unit of T4 ligase. After transformation of the resulting DNA mixture into E.coli HB 101 and selection of the resulting 45 colonies on ampicillin-containing LB petrits, one clone was selected that contained the designation pRHW 11. After growing the respective clone, plasmid DNA was isolated. Its structure is evidenced by the presence of many specific restriction sites (Alul, EcoRi, HindIII, Ncol, PstI).

d) Expression of interferon activity by E. coli HB101 containing plasmid pRHW 12.

100 ml of bacterial culture was adjusted to an optical density of 0.6 at 600 nm in M9 minimum medium containing all amino acids except tryptophan (20 μg / ml for amino acid), 1 μg / ml thiamine, 0.2% glucose and 20 μg / ml indole (3) acrylic acid (IAA), an inducer of the tryptophan operon, and incubated. Finally, the bacteria were precipitated by centrifugation (10 min / 7000 rpm), washed once with 50 µM Tris / Cl pH = 8, 30 mM NaCl and resuspended in 1.5 ml of the same buffer. After 30 minutes incubation with 1 mg / ml lysozyme on ice, the bacteria were frozen and thawed 5 times. The cell debris was removed by one-hour centrifugation at 40,000 rpm. The supernatant was sterile filtered and interferon activity tested for plaque reduction assay using human A549 cells and encephalomyocarditis virus.

Result: 1 l of bacterial culture produced contains 1 x 106 international units of interferon (A. Biliau, Antiviral Res, 4, 75-98 (1984)).

Example 6

Comparison of differences in amino acid and nucleotide sequences of type I interferons

a) Comparison of amino acids

The pairwise pairing of amino acid sequences consists of matching the first cysteine residue of the mature alpha interferon with the first amino acid residue of the amino acid sequence encoded by the cDNA insert of plasmids P9A2 and E76E9. The two sequences are shown in FIG. 7 as interferon-omega since no difference was found between the specific sequences of P9A2 and E76E9 at the given values. The only adjustment made is the insertion of a gap at position 45 of interferon alfa-A, which is considered to be a defect. When a partner is a sequence of omega-interferons, the comparison is made taking into account the ordinary 166 amino acids. This significance is presented in Figure 7 together with the additional amino acids encoded by clones P9A2 and E76E9. The percentage differences are obtained by dividing the number listed to 1.66. The additional amino acids are represented by a factor of 0.6, which gives 3.6% for the six additional amino acids of interferon-omega, when calculated as a percentage. Comparison with beta-interferon starts from the third amino acid of beta-interferon and the first amino acid of mature alpha-interferon encoded by plasmids P9A2 and E76E9. The longest compared structure of one alpha-interferon with one beta-interferon is 162 amino acids, which gives 2 additional amino acids for alpha and beta interferons. They are reported as defects and are presented separately in Figure 7 but are included in the percentage. The listing of betainterferons with the amino acid sequences of clones P9A2 and E76E9 is carried out in the same manner. This gives a total of 10 additional amino acids.

B) Comparison of nucleotide sequences

The enumeration of the compared sequences follows analogously to Example 6a. The first nucleotide of the mature alpha-interferon is the first nucleotide of the cysteine-coding triplet of the mature alpha-interferon. The first nucleotide from the plasmid P9A2 or E76E9 DNA is also the first nucleotide of the cysteine-1 codon. The first nucleotide of beta-interferon DNA is the first nucleotide of the third triplet. The comparison follows a total of 498 nucleotides when aligning single DNA alpha interferons with beta-interferons and more than 516 nucleotides when comparing the DNA sequences of single alpha or beta interferons with those of plasmids P9A2 and E76E9. The absolute number of errors is given in the left part of Fig. 7, and in brackets are the corresponding percentages.

Example 7

Virus-induced expression of omega-1-mRNA and NC37 cells

a) Synthesis of an omegainterferon-specific hybridization probe pMol of oligonucleotide d (TGCAGGGCTGCTAA) was mixed with 12 pMol gamma- ² P ATP (specific activity:> 5000 Ci / mMol) and 10 units of polynucleotide kinase (10) 70 mM Tris / Cl pH = 7.6, 10 mM MgC12, 50 mM DTT) and left for one hour at 37 ° C. Finally, the substitution was stopped by heating at 70 ° C for 10 min. The resulting radiolabeled oligonucleotide was hybridized at 50 ° C for one hour with 5 pMol M13pRHW 12 ssflHK (see Figure 9) in a total volume of 35 μl (100mM NaCl).

After cooling to room temperature, a buffer is added

- IO for short translation, the four nucleoside triphosphates and 10 units of Maple polymerase to a total volume of 50 µl (50 mM Tris / Cl pH = 7.2, 10 mM MgCl ₂ , 50 mcg / ml BSA, 1 mM per nucleotide). The polymerization was carried out for one hour at room temperature and quenched for five minutes at 70 ° C.

The synthesis partially yields double stranded circulating DNA. It is digested with 25 Avail units in a total volume of 500 µl using the buffer described by the manufacturer. In this case, the double-stranded areas are degraded to a uniform size. Synthesis was stopped by heating for 5 minutes at 70 ° C.

B) Production of RNA from virus-infected cells

100 x 10 ⁶ cells (0.5 x 10 ⁶ / ml) were treated with 100 μMol dexamethasone for 48 to 72 hours - the control did not contain dexamethasone. For interferon induction, expression cells were suspended in serum-free medium at a concentration of 5 x 10 ⁶ / ml and infected with 21 ° Sendai virus units. Aliquots of the cell cultures were assayed in a plaque-reducing interferon activity assay (Example 5b). Cells were harvested 6 hours after virus infection by centrifugation (100Og / Yumin), washed in 50 ml IP40 buffer (Example 4a), dissolved in ice-cold NP40 buffer and lysed by adding 10% NP40 for 5 min. ice. After removal of the nuclei by centrifugation (1000g, 10MHH), the supernatant was adjusted to pH = 8.0 with 50mM Tris / Cl, 0.5% Sarkosin and 5mM EDTA. The whole RNA of the supernatant was extracted once with phenol, once with chloroform / phenol / isoamyl alcohol and once with chloroform / isoamyl alcohol. The aqueous phase was loaded onto bl ml of a 5.7 molar gradient of CsCl and centrifuged with a SW 40 rotor. (35krpm, 20 hours) to release the DNA extract from the remaining proteins. The resulting RNA precipitate was resuspended in 2 ml TE pH = 8 and precipitated with ethanol. The precipitated LNA was dissolved in water at a concentration of 5 mg / ml.

c) Demonstration of interferon-omega mRNA.

0.2 ml of the hybridization probe prepared according to Example 7b were precipitated with 20-50 μg of RNA prepared according to 7c by the addition of ethanol. In the control experiment, transfer RNA (m RNA) or RNA transformed with pRHW 13 E. coli was added instead of cellular RNA.

The resulting precipitate was desalted by washing with 70% ethanol, dried and dissolved in 25 μl of 80% formamide (100 mM PIPES pH = 6.8, 400 mM NaCl, 10 mM EDTA). Finally, it was heated at 100 ° C for 5 min to denature the hybridization probe, then cooled immediately to 52 ° C and incubated at this temperature for 24 hours. After hybridization, the samples were placed on ice and 475 µl of reaction mixture (4mM Zn (Ac) 2, 30 mM NaAc, 250 mM NaCl, 5% glycerol, 20 µg calf thymus DNA, 100 SI units) were added. at 37 ° C, the reaction was stopped by ethanol precipitation. The precipitate was dissolved in 6 μl of formamide buffer and, similarly to probes obtained from DNA sequencing, was separated into a 6% acrylamide gel containing 8M urea (F.Sanger et al., Proc. Nat. Acad. Sci., 74, 5463-5467 ( 1979).

For autoradiography, the dried gel was transferred to a DuPont Cronex X-ray film using a Kodak Lanex Regular intensifier at -70 ° C.

- b2 The legend to Fig. 10

Traces A through C represent the controls

Trace A: 20 μg of tRNA

Trace B: 10 µg RNA from pER33 (E. coli-expressing interferon alfa-2-arg strain)

Trace C: 1 ng RNA from pRHW 12 (E.co11-expressing strain for interferon omega 1)

Trace D: 50 μg of RNA from untreated Namalwa cells

Trace E: 50 μg of RNA from Namalwa virus infected cells

Trace F: 50 μg of dexamethasone treated and Namalwa-infected cells

Trace G: 20 μg of untreated NC 37 cells.

Trace H: 20 μg of virus-treated NC 37 cells

Trace I: 20 mcx of dexamethasone treated and vir 37 NC cells infected

Track M: Size marker (pBR 322 treated with Hinfl).

Traces B and C show that the expected signal is established only when the omega-specific RNA is in the form of an RNA molecule. It is further shown that the excess of fake acid does not produce a background signal (trace B). The mRNA used as the hybridizing partner also produces no signal (trace A).

Traces G to I show the induction of omega-specific RNA in virus-infected NC 37 cells. Dexamethasone pre-treatment enhances this effect.

Traces D through F show the same effect obtained with Namalwa cells. The induction of omega-specific RNA is not as strong as that of NC 37 cells. This result is parallel to the interferon titer measured in the extracellular fluid.

- 33 This reference to the expression of the interferon-omega gene is as expected from a type I interferon gene.

Example 8

Isolation of interferon-omega encoding genes as well as related genes.

a) Cosmid screening

Human Cosmid Bank (Human DNA (Male)) Cloned into the Cosmos Vector pcos2 EMBL (A.Ponstka, H.R.Rockwitz, A.-M.Frischauf, L.Horn, H.Lerach, Proc. Natl.Acad.Sci 81 , 4129-4133 (1984) with a complex of 2 x 10 ⁶ is searched for interferon-omega- or related genes, E. coli DHI (r ^ -, m +, rec. A, gyrA96, sup.E) is used as a host. .Mg ++ - 'planting bacteria' cells are directly produced. E.coli DHI develops through fasting in L-broth (10 g / l Trypton, 5 g / l yeast extract, 5 g / l NaCl) supplied with 0.2% The bacteria were centrifuged and brought to OD6oo ⁼ 2 with 10 mM solution of MgSO4. 5 ml of this cell suspension was incubated with cosmid packing 12.5 x 10 6 colony-forming units Finally, 10 vols of LB were added and the suspension was left for 1 hour at 37 ° C for expression of the kanamycin-mediated resistance to the bacteria, then the bacteria were centrifuged, resuspended in 5 ml of LB and were applied in portions of 200 μl on a nitrocellulose filter (BA85, Schleicher & Schuell, 132 mm diameter), which was applied to LB-arap (1.5% agar in L-broth) plus 30 μg / ml kanamycin. About 10000-20000 colonies grow for the filter. The colonies were transferred to another cellulose filter and stored at 4 ° C. One batch of filters containing colonies was treated as described in Example 1 c), i. bacteria were denatured and single stranded DNA was fixed on cellulose, filters were pre-washed at 65 ° C for 4 hours in 50 mM Tris / Cl, pH = 8.0, 1 M NaCl, 1 mM EDTA, 0.1% SDS. Finally, the filters were incubated in a solution of 5 x Denhardt (see Example 1c), 6 x SSC, 0, 1% SDS for two hours at 65 ° C and hybridized to the same solution with 5 x 106 with Nickel-translated, denatured interferon-omega DNA. 1 (HindIII-BamHI clone insert pRHW12, see FIG. 6) for 24 hours at 65 ° C. After hybridization, the filter was washed 3 times x 10 min at room temperature in 0.2 x SSC, 0.01% SDS solution and 3 times x 45 min at 65 ° C, in 0.2 x SSC, 0.01% SDS solution, filters are dried and exposed to Kodak X-Omat S film using a reinforcement film at -70 ° C. Positively responsive colonies were localized on the replication filter, scraped, and resuspended in L-broth + kanamycin (30 μg / ml). From this suspension several µl. were weighed on LB-arap + 30 μg / ml. The resulting colonies were again plated on a nitrocellulose replication filter. These filters were hybridized as described above with 32P-labeled DNA for interferon omega I. From this hybridized colony, a cosmid was isolated by the method of Birnboim and Doly (Nucl.Acid.Res. 7, 1513 (1979)). With this cosmid DNA preparation, E. coli DHI is transformed and the transformants are selected on LB-arap + 30 μg / ml kanamycin. One clone is selected from the parent isolate and is produced on a larger scale (Clewell, DB & Helinski, DR, Biochemistry 9, 4428 (1970)). Three of the isolated cosmids bear the names cos9, cosl0 and cosB.

B) Subcloning of the hybridized fragments into PUC8

More than 1µg of cosmids cos9, cos 10, and cosB are treated with HindIII under conditions recommended by the manufacturer (New England Biolabs). The fragments were separated by electrophoresis in 1% agarose gel in TBE buffer and transferred by Southern to

- bb nitrocellulose filter (Example 4c). The two filters were hybridized as described in Example 4d with Nick-translated omega I DNA, washed and exposed. About 20 μg of each cosmid was cut with HindIII and the resulting fragments separated by gel electrophoresis. The DNA fragments hybridized in the previous experiment were electroeluted and purified through an Elutyp column (Schleicher & Schuell). These fragments were ligated with HindIII linearized, dephosphorylated pUC8 (Messing, J., Vieira, J., Gene 19, 269-276 (1092)). E. coli JM101 (supE, thi, (lac-pro-AB), [F ', traD36, proAB, lac q Z M15] (np. Fa. PLBiochemicals) was transformed into a ligase reaction solution. The bacteria were seeded on LB-arap containing 50 μg / ml ampicillin, 250 μg / ml 5-Bromo-4-Clor-3-indolyl-beta-Dgalactopyranoside (BCIG, Sigma) and 250 μg / ml Isopropyl beta-Dthiogalactopyranoside (IPTG, Sigma). of the resulting colonies shows the lack of insert in pUC8 From a few white clones, the plasmidDNA was isolated in small amount, treated with HindIII and separated on a 1% agarose gel The DNA fragments were transferred to a nitrocellulose filter and hybridized with the above ³² Romega-1 DNA Selection a, each subclone originating from cos 9 and cos10 and labeled with pRHW22 and pRH57, respectively. From cosB, two well hybridized with omega-1 probe DNA fragments were subcloned and designated pRH51 and pRH52 respectively.

c) Sequence analysis

DNA incorporated into the PUC8 was separated from the vector portion by HindIII treatment and gel electrophoresis. This DNA (approx. 10 μg) was ligated under the action of T4 DNA ligase in a reaction solution, the volume of the solution was brought to 250 μl with Nick translation buffer (Example 4d) and finally ultrasound degraded with ice (MSE 100) Watt Ultrasonic, Disintegrator, max feed at 20 kHz, 5 times 30 seconds). Then the edges of the cut pieces are repaired with the addition of 1/100 vol. 0.5 mM dATP, dGTP, dCTP and dTTP as well as 10 units of Maple DNA polymerase I fragment for 2 hours at 14 ° C. fragments between 500 and 1000 bp in size were isolated and subcloned into dephosphorylated Smal-treated phage vector M13. Single-stranded recombinant phage DNA was isolated and sequenced by the Sanger method (Sanger et al., Proc.Natl.Acad.Sci 74, 5463-5467 (1976)). Comparison of single sequences to the common sequence is achieved with a computer (Staden, R., Nucl.Acid Res. 10 47314731 (1982)).

d) Subclone sequence pRH57 (interferon omega-

The sequence is shown in Figure 11. This 1933 bp long fragment contains the interferon omega-1 gene. The protein coding region comprises proteins 576 to 1163. The sequence is completely identical to that of the cDNA clone P9A2. The nucleotide region 576-674 encodes a signal peptide 23 amino acids long. The TATA region lies at a distance from the ATG start codon, which is characteristic of type I interferon genes (positions 476-482). The gene has a polyadenylation signal transcription sequence (ATTAAA at positions 1497-1502, respectively 17641796; ATAAA at positions 1729-1734, or 1798-1803), which is used first in clone P9A2.

e) Subclone sequence pRHW22 (interferon pseudo-omega-2)

Figure 12 shows a 2132 bp long sequence of hybridized HindIII fragments of an omega-1 probe from

- b7 cosmids cos9. It provides an open reading frame from nucleotide 905 to nucleotide 1366. The resulting amino acid sequence is given. The first 23 amino acids are similar to the signal peptide of type I interferons. The following 131 amino acids show up to 65 amino acids similar to omega-interferon, with tyrosine being remarkable as being the first amino acid of the mature protein. At amino acid position 66 follows a potential N-glycosylation site (Asn, Phe, Ser). From this site, the amino acid sequence differs from that of type I interferons. It has been shown that, through favorable insertions and the resulting displacements of the protein framework, it resembles the omega-1 gene (see Example 9). In terms of type I interferons, the gene isolated here is a pseudogene: interferon-pseudo-omega2.

f) Subclone sequence pRH51 (interferon pseudo-omega 3)

A HindIII fragment originating from cosmid B 3500 bp long and hybridized with the omega1 probe was partially sequenced (Fig. 13). An open reading sequence from nucleotide positions 92 to 394 is obtained. The first 23 amino acids show the signal peptide features. The following sequence begins with tryptophan and shows similarity to interferon-omega 1 to amino acid 42. A sequence other than interferonomega follows! to amino acid 78. The sequence can be altered by insertions to exhibit great homology to interferon-omega 1. (Example 9). The gene is referred to as interferon-pseudo-omega 3.

- b8 -

g) Insert sequence of pRH52 (interferon pseudo-omega-4).

A sequence of HindIII fragments, 3659bp long and omega 1 hybridized with DNA, was isolated from the cosmid shown in FIG. An open reading frame, whose translation point shows partial homology to interferon-omega 1, is located between nucleotides 2951 and 3250. Following a signal peptide, 23 amino acids long begins with a phenylalanine further amino acid sequence. Homology with interferon 1 is terminated after 16. amino acid, resumes at 22. and ends at 41. amino acid. A possible translation to ΊΊ would be possible. amino acid. Similarly to Examples 8f and 8g, good homology to interferon-omega 1 can be obtained by inserting insertions. The pseudogene thus isolated is referred to as interferon-pseudo-omega 4.

Example 9

Alignment of genes for the four members of the interferon-omega family.

In Fig. 15, the genes from interferonomega1 to interferon-pseudo-omega4 are stacked together with their amino acid translation. For better match, gaps are inserted into single genes, which are represented by dots. No bases were missed. Base numbering includes gaps. The amino acid translation for omega 1 is preserved (e.g., heading 352-355: CAC encoding His). In pseudogenes, translation into amino acids is only possible where this is unambiguous. Based on this presentation, it is clear that the four isolated genes are related to each other. For example, a potential N-glycosylation & 9 site (nucleotide positions 301-309) in all four genes is obtained. Also in interferon-pseudo-omega 4, there is a triplet of nucleotide positions 611 to 614, which is a stop codon and which in interferon-omega 1 completes a protein of 172 amino acids in length. Undoubtedly, this arrangement features premature stop codons at interferon pseudo-omega2 (nucleotide positions 497-499) and interferon pseudo-omega4 (nucleotide positions 512-514).

The degree of kinship between genes, respectively. the amino acid sequences can be calculated from the alignment given in FIG. 15. Figure 16 gives the homology between the DNAs of the interferon-omega family members. In addition, when comparing pairs, those positions where one partner or both have a gap are not calculated. The comparison gives a homologous roundness of 85% between interferon-omega1 and pseudogenes. Interferon-pseudo-omega2 DNA is homologous to interferon-omega-3 DNA, respectively. interferon-omega4 about 82%. Fig. 17 shows the results of the comparison of signal rotations, and Fig. 18 - those from the comparison of mature proteins. The latter range between 72 and 88%. This homology is significantly greater than the homology between interferon-omega 1 and interferon-alpha and interferon-beta (Example 6). The fact that the individual members of the interferon-omega family differ further from each other, and the interferon-alpha family explains that three of the four isolated interferon-omega genes are pseudogenes and often cannot be the basis of selection prints as functioning genes.

Example 10 Fermentation

Storage of strains:

A single colony of HB101 / pRHW12 strain on LB-arap (25 mg / ml ampicillin) was transferred onto tryptone soy broth (OXDID CM129) with 25 mg ampicillin, shaking at 250 rpm at 37 ° C until optical density was reached. about 5 (546nm). 10% by weight of sterile glycerin was added, the culture was poured into 1.5 ml portions of sterile ampoules and frozen at -70 ° C.

Preliminary (primary) culture

The culture medium contains 15 g / ml Na2HPO4 χ 12H2O; 0.5 g / l NaCl; 1.0 g / l NH ₄ C1; 3,0 g / l KN ₂ PO ₄ ; 0.25 g / l MgSO ₄ x 7H ₂ 0; 0.01 lg / Ι CaCl ₂ ; 5 g / Ι casein hydrolyzate (Merck 2238); 6.6 g / l glucose monohydrate; 0.1 g / l ampicillin; 20 mg / l cysteine and 1 mg / l thiamine hydrochloride. 4 x 200 ml of this medium was placed in 1000 ml Erlenmeyer flasks and each was injected with 1 ml of HB101 / pRHW14 culture and incubated with shaking for 250 rpm for 18 hours at 37 ° C.

Main culture:

The fermentation medium contains 10g / l (NH4) ₂ HPO4; 4.6 g / 1 K ₂ HPO ₄ x 3H ₂ O; 0.5g / l NaCl; 0.25 g / l MgSO ₄ x 7H ₂ O; 0.011 g / l CaCl ₂ ; lgg / l glucose monohydrate; 21 g / l casein hydrolyzate (Merck 2238); 20 mg / l cysteine, 1 mg / l thiamine hydrochloride and 20 mg / l 3-beta-indolacrylic acid. 8 liters of sterile medium were placed in a fermentor with a total volume of 14 l (height: radius = 3: 1) and inoculated with 800 ml of primary culture.

the fermentation was carried out at 28 ° C, 1000 rpm (EffigasTurbine), aeration 1 wm (volume / volume / minute) and initial pH = 6.9.

During fermentation, the pH drops and is maintained at 6.0 with 3M NaOH. When optical density (546nm) of 18-20 was reached (usually after 8.5-9.5 hours of fermentation), the mixture was cooled to 20 ° C under the same conditions and the pH was adjusted to 2.2 with 6N H2SO4 (without aeration) and stirred for 1 hour at 20 ° C at 800 rpm. without aeration. The biomass thus inactivated is centrifuged at 30,000 rpm (GEP-CEPA centrifuge, frozen and stored at -20 ° C.

Example 11

Purification of interferon-omega Gly

a) Partial purification

All steps were carried out at 4 ° C.

140 g of biomass (E. coli HB101 transformed with expression clone pRHW12) was dissolved in 1150 ml of pre-cooled to 4 ° C acetic acid and stirred for 30 min. The suspension was adjusted to pH = 10.0 with 5M NaOH and stirred for at 2 o'clock. After fixing the pH at 7.5 with 5M HCl, it was stirred for a further 15 minutes and centrifuged (4 ° C, 1 hour, 10,000 rpm, centrifuge J21, Beckman, JAlO-rotor).

The clear supernatant was transferred onto a CPG (glass, controlled pores) column (CPG 10-350, Mesh Size 120/200), washed with 50 ml / h of 50 mM Tris / IM NaCl, pH = 7.5, for 50 ml / h, and pH = 7.5 and eluting with 25mM Tris / 1M KCNS / 50% ethylene glycol, pH = 7.5 (50ml / h).

The peak containing interferon activity was collected, dialyzed against 0.1 M Na-phosphate, pH = 6.0 and 10% polyethylene glycol 40,000 overnight, and the resulting precipitate was centrifuged. (4 ° C, 1 hour, 10,000 rpm, J21centrifuge DA20 rotor (see Table 1).

2 shelves I

About him (ml) Biological test Protein (mg / ml) total. (mg) U / mg He is dooo * in / ml ⁺ In total nenpe- tu <Χ ρ gr ρ μ · 1150 15000 17, 3x10 ⁶ 3.6 4140 4180 100 DL 2200 <600 <1, 3x10 ^b 0.74 1628 <600 <5 The eluate 124,3 170000 21,0x10 ⁶ 16.8 2088 10000 121 followed by dialide 41 300000 12, 3x10 ^b 4 12.6 516,6 23800 71

+ CPE-reduction assay: A349 - cells, EMC virus U-Units derived from interferon-alpha 2 / see. Example 1 of EP-A-O. 5/11/06: E. coli HB 101 Transformed with Expression Clone PEP 33 as Standard

PL-Overflow column

B) Further purification

The dialyzed and concentrated CPG eluate was diluted 1: 5 with buffer A (0.1 M sodium phosphate pH = 6.25 / 25%

1,2 propylene glycol and transferred onto a 'Superloop' (Pharmacia) injection device at a rate of 0.5 ml / min on a buffered A MonoS 5/5 column (Pharmacia, cation exchanger). Elution was achieved by a 20 ml linear gradient of 0.0 to 1 M NaCl in buffer A at a flow of 0.5 ml / min. Collect fractions of 1 ml and test for interferon activity with a CPE-reduction test. The active fractions were collected.

Example 12

Characterization of human interferon-omega!

A. Antiviral activity on human cells

B. Antiviral activity on monkey cells

C. Antiproliferative activity on human Burkittlimphoma cells (Daudi cell line)

D. Antiproliferative activity on human cervical carcinoma cells (He-La cell line)

E. Acid stability

F. Serological characteristics

A. Antiviral activity on human cells

Cell line: Human lung carcinoma cell line A549 (ATCC CCL 185)

Virus: Mouse encephalomyocarditis virus (EMCV)

Test system: Retention of cytopathic effect (all titrations were performed 4 times).

A partially purified preparation of human interferonomega1, with a protein content of 9,4 mg / ml, is titrated at appropriate dilutions in the aforementioned test system.

The preparation shows an antiviral activity with a specific activity of 8300 units against the reference standard Go-23-901527.

B. Antiviral activity on monkey cells

Cell Line: GL-V3 Monkey Cell Line (Christofinis G.J., J.Med. Microbiol. 3, 251-258, 1970)

Virus: Vesicular Stomatic Virus (VSV)

Test system: Plaque reduction test (all titrations were performed 4 times)

The preparation described in Example 12A shows a specific antiviral activity of 580 E / mg in the above test system.

C. Antiproliferative activity on human Burkittlimphoma cells (Daudi cell line)

The human Burkitt-lymphoma Daudi cell line is grown in the presence of different concentrations of human interferonomega1. After a three-, four- and six-day incubation at 37 ° C, cell density was determined; untreated cells serve as controls. All crops are pledged three times in parallel. The following illustration shows that interferon-omega shows marked suppression of cell proliferation at a concentration of 100 antiviral units (IE, see Example 12A) per ml. At a concentration of 10 IU / ml, partial transient suppression was observed. (The following symbols are used in the following figure: O-Control, £ 2D 11E / ml, n-10 1E / ml, ▼ 100 1E / ml).

- 65 15

D. Antiproliferative activity on human cervical carcinoma cells (He-La cell line)

The human cervical carcinoma line He-La is grown in the presence of the following proteins, respectively. protein mixes: Human interferon-omega! (see Example 12A) 100 IU / ml

Human interferon gamma (see Example 12A) 100 IU / ml

Human tumor necrosis factor (Hu TNF), = 98% purified, manufactured by Genetech Inc., San Francisco, USA (see Pennica D. et al., Nature 312, 724-729, 1984) 100ng / ml.

All binary concentrations of the aforementioned protein concentrations as above.

Two cultures were plated in 3 cm tissue culture plates (50,000 cells for plates) and incubated for six days at 37 ° C. Finally, cell density is determined. Treatment of cells with HuTNF or HuINF-omega has little effect on cell growth, whereas HuIFN-gamma has a pronounced cytostatic effect. The combination between interferon-gamma and interferon-omega shows synergistic cytostatic and cytotoxic activity.

The following figure shows the results of the study.

The following symbols are used: C-untreated control, THuTNF.O- HuIFN-omega, G-HuIFN-gamma.

• · · · · 1 i IT Lil ¹ Uni- ί and '..i • I st · s: * : _7t. ·. · i—: iG ·: = Ξϊ.-1 :: ϊγΞ •• IT · if L1: * - .: ::: 1 -:? L-L *. .:. 1 - .: i: • · ... ·. TLiLTiTrf Μ · us Lu • . · 1 ..G-: I l: •: '~~ .4 = 4 tg:: : - ..- ί7λ; : 1 Γί · «· '·. t ··. nr. : i :. bt-i 4: 1- ·! K · -4 - τ'τ ί , 1 ... · -. · .-. ..4 -W: 1 - »- ♦. ♦ •. : LIT :.: .1; ::: i l; i ·. ·  ·  : * r • T TTI L! LiTL -: 1: ::. .ι.— i -? ·· · : ΛΙ. : ·. t • Li i'l ψί- i '! . r ^: .'T •. *: · .1 <4 · - · ^; ·· 'L: I . ·. ! :: ... • · and ·. · , Τ Τ ·: t- U • - .. 1 r 1 1 l l i: i i · ·; ·:. •. . . '. ..: l ·! . 1 -. > ιΤ + Ο ' : :: ..:. i. ··; ··; *; · : i i: I *. 1. -, -. ::: -: : -. . · I: · :: | 1 - · 1 Tllii ...; i -.1 : | · Ί Ί Γ : L4 T-

I

E. Acid stability

To study the acidic stability of human interferon-omega1, dilute the preparation described in Example 12A in a cell culture medium (RPMI 1640, 10% fetal calf serum) and adjust to pH 2 with hydrochloric acid. It is incubated for 24 hours at 4 ⁰ C and neutralized with sodium hydroxide. This sample was titrated in an antiviral test (see Example 12A); it shows 75% of the biological activity of an incubated in neutral pH control. Interferon-omega1 is designated acid-stable.

F, Serological characteristics

To determine the serological properties of human interferon'-omega1 compared to human interferon alfa2, samples (diluted to 100 IU / ml) were preincubated for 90 min at 37 ° C with equal volumes of solutions of monoclonal antibodies or polyclonal antisera. Finally, the antiviral activity of the samples is determined. The following table shows that human interferon-omega is neutralized at a relatively high concentration by only one antiserum against human leukocyte interferon, but not by antibodies directed against human interferon-alpha2 or human interferon-beta. Therefore, HuIFN-omega 1 is not serologically related to either Ni11 ^; 1H-alpha2 neither with HuIFN-beta.

Publication of the Patent Office of the Republic of Bulgaria

1113 Sofia, Dr. Γ Blvd. M. Dimitrov 52-B

Expert: Art. Stefanova Editor: E. Sinkova

Cf. 37292

Circulation: 40 CS

A

 ". . GU f, r, - - »· d-> flushing Neutralize from ek /: dream about Λπ τ ·· '·, κπ ο pg / ml HuIFN-a2 HuIFN-omegal

EEI-1 ^1} 1 + - 10 ++ + - 100 +++ - 1000 - Oh EBI-3 ¹⁵ 1 ++ + - 10 +++ - 100 +++ - 1000 + ++ 0 L3B7 ²⁾ 100 +++ 0 1000 +++ 0 Sheep-aip- _w " _T ," -IFN ³ ' 1:50 000 + + + leucopitus. 1: 5,000 ++ + 0 1: 500 +++ + 1: 50 - +++ Zaeipso-anti HuiFN-a2 1: 1 000 + + + - 1: 100 +++ 0 1:: 10 - 0 Sheep-anti 'Λ \ Util FN-B 1: .50 - 0

ⁿ = see EP-A-0.119.476 · 1 = A. Berthold et al. and Arzneimittelforschung 35,

364-369 (1985) = Research Reference Reagent Catalog no. 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, Maryland, USA.

h estestusan h neutralization

Hes neutralization + partial +++ complete neutralization

Claims (10)

Claims A recombinant human DNA molecule comprising a coding sequence for novel human type I interferon proteins consisting of 168-174 amino acids, for interferon-pseudo-omega2 (see Figure 12), interferon-pseudo-omega3 (see Figure 13) or interferon-pseudo-omega4 (see Figure 14), characterized in that the coding sequence is hybridized under stringent conditions that allow a greater than 85% homology to be recognized with insertions in the Pst-Ι shear region: a) plasmid E76E9 in E. coli 101 deposited with DSM under DSM 3003 or b) P9A2 amide in E.coli HB 101 deposited with DSM 3004. A DNA molecule according to claim 1, characterized in that, under stringent conditions, it is possible to recognize a homology greater than 90%. A DNA molecule according to claims 1 and 2, characterized in that the coding sequence is present as an insert in the shear region of Pst-Ι: a) of plasmid E76E9 in E. coli 101 deposited in DSM under number DSM 3003 or b ) of plasmid P9A2 in E. coli HB 101 deposited with DSM under number 3004. or degenerate variants of this insert. A DNA molecule according to claims 1 to 3, characterized in that the carrier is a plasmid that is replicable in prokaryotes and eukaryotes. A DNA molecule according to claim 4, characterized in that it is an expression agent replicable in microorganisms or mammalian cells. A DNA molecule according to claim 5, comprising the sequence: - a. - TGT GAT CTG Art CAG AAS CAT GGC CTA CTT AGC AGG AAC ACC TTG 45 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 90 AAG GAC AGA AGA GAC TTS AGG TTC CCC CAG GAG ATQ GTA AAA GGG 135 AGC CAG TTG CAG AAG GCC CAT GTC: ATG TCT GTC CTq CAT GAG ATG 180 CTG CAG CAG Automatic telephone exchange TTS AGC STS TTC CAC ACA GAG CGC TCC TCT GCT 225 GCC TGG AAS ATG ACC STS HUNDRED 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 GAA GGA GAA TST GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 360 AGG AGG TAS TTS CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 405 TAS AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 450 mc TTG tts TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 4 9 5 1 GAT AGA GAC CTG GGC TCA TCT 516 ' The DNA molecule of claim 5, wherein the DNA molecule of claim 6 contains nucleotide A instead of nucleotide G at position 332. A DNA molecule according to claim 5, characterized in that it is a) plasmid E76E9 in E. coli 101 deposited in DSM under number DSM 3003 or b) plasmid P9A2 in E. coli HB 101 deposited in DSM under number 3004. A DNA molecule according to claims 6 and 7, further comprising a DISK sequence of formula ATG GCC CTC CTG TTC CCT STA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC _? encoding the lead peptide · 10. The interferon-omega gene! with the formula