DD246318A5 - New genetic sequences generated by the coded type I interferon peptides and these organisms producing them - Google Patents

Abstract

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

Description

Berlin, 3 · '3 · 1986 ~ ^Λ ~ AP C 12 Ν / 279.375-4

  ·. 65 649 11

Process for the preparation of novel human interferon peptides ; ___

Field of application of the invention

The present invention relates to a process for the preparation of novel type I interferon peptides and to recombinant DNA processes for the production of these peptides and products which are required in these processes, for example gene sequences, recombinant DUA molecules, expression vehicles and organisms.

The interferon peptides prepared according to the invention are used as medicaments, for example for an anti-tumor treatment or for an anti-viral treatment.

Characteristic of the known technical solutions

Interferon is a term coined to describe a selection of proteins that are endogenous to human cells and characterized by having partially overlapping and partially divergent biological activities. These proteins modify the body's immune response and are believed to contribute to effective protection against viral diseases. The interferons have been classified for example in three classes, namely O6, ß - and "p-interferon.

Of ß> - and / "" - interferon only one subtype is known in human organism (eg S. Ohno et al., Proc. Hatl.

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Acad. Be. 7_§, 5305-5309 (1981); Gray et al., Nature 295 , 503-508 (1982); Taya et al., EMBO Journal 1/8 , 953-958 (1982)). In contrast, various subtypes of voma-interferon are described (eg, Phil. Trans. R. Soc., Lond., 299 , 7-28 (1982)). The mature a-interferons differ from each other here - by a maximum of '23% divergence and are about 166 amino acids long. Also noteworthy is a report on an α-interferon which has a surprisingly high molecular weight (26,000 as determined by SDS polyacrylamide / gel electrophoresis) (Goren, P. et al., Virology 130 , 273-280 (1983)). This interferon is called IFN-a 26K. He was found to have the highest specific antiviral and anticellular activity to date.

The known interferons appear to be active in a variety of diseases, but have little or no effect on many other diseases (e.g., Powledge, Bio / Technology, March 1984, 215-228 "Interferon On Trial"). Interferons also have side effects. For example, in tests of the recombinant α-interferon anticancer effect, it has been found that doses of about 50,000,000 units considered safe by the Phase I results are acute states of confusion, inability to perform joint pain, severe fatigue, loss of appetite , Loss of orientation, epileptic seizures and liver toxicity. In 1982, the French government banned IFN-a testing after cancer patients treated with it suffered fatal heart attacks. At least two cardiac deaths have also been reported in recent trials in America. It has become increasingly clear that at least some of the side effects, such as fever and ailments, are directly related to the interferon molecule itself and are not due to impurities.

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Due to the high hopes aroused by interferon and stimulated by the desire to find new interferon-like molecules with reduced side effects, it is desirable to have new such compounds available.

Object of the invention

The aim of the invention is the provision of new interferons, which are particularly suitable for anti-tumor treatment or antiviral treatment and which do not have the disadvantages of known interferons.

Explanation of the essence of the invention

The invention has for its object to find new interferons with the desired properties and processes for their preparation. ;

According to the invention, novel type I human interferon peptides encoded by a DNA molecule are prepared, the coding sequence being recognized under stringent conditions allowing homology greater than 85 % with the inserts in the Pst-1 cleavage a) of the plasmid E76E9 in E. coli HB 101, deposited with the DSM under the number DSM 3003 or b) of the plasmid P9A2 in E. coli HB 101, deposited with the DSM under the number DSM 3004 or with degenerate variations of this insert hybridized,

They are prepared by transforming a suitable host organism with genetic information encoding the interferon peptides as described above, expressing this information to produce the corresponding interferon in the host organism and

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thus obtained interferon-Pep.tid is isolated from this host organism.

According to the invention, certain novel type I interferons are provided which may contain a leader peptide and their N-glycosylated derivatives (referred to herein as omega-interferon or IFN-omega) containing 168 to 174 "preferably 172 amino acids" and which have a divergence of from 30 to 50%, preferably from 40 to 48%, over the hitherto known subtypes of the o6> interferon and a divergence of approximately 70 % with respect to β-interferon and, on the one hand, show similar effects as α-interferons, on the other hand do not possess many therapeutic disadvantages of these known substances

The compounds prepared according to the invention are novel interferons in completely pure form, their non-glycosylated and glycosylated forms, the gene sequences coding for them as well as recombinant molecules containing these sequences. The invention furthermore relates to expression vehicles such as plasmids containing said gene sequences, as well as various host organisms, such as microorganisms or tissue culture, which allow the production of the new interferon by fermentation or tissue culture method.

A preferred subject of the invention are omega-interferons as well as the corresponding gene sequences of the following formula:

Cys Asp Leu Per 5 gins Asn His Gly Leu 10 leu Ser bad Asn Thr 15 leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG Val Leu Leu His 20 gins mead bad bad He 25 Ser Per Phe. Leu Cys 30 leu GTG CTT CTG CAC CAA ATG AGG AGA ATC .TCC CCT TTC TTG TGT CTC Lys Asp · bad bad 35 Asp Phe bad Phe Per 40 gin. Glu mead Val Lys 45 GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA ' GGG Ser gin Leu Gin 50 lys Ala His Val mead 55 Ser Val Leu His Glu 60 Meters AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG Leu gin Gin He 65 phe Ser Leu Phe His 70 thr Glu bad Ser Ser 75 Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT Ala Trp Asn mead 80 Th r Leu Leu Asp gin 85 leu His Thr Gly Leu 90 His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT gin gin Leu Gin 95 His Leu Glu Thr Cys 100 leu Leu gin Val Val 105 GIy CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA Glu Gly Glu Ser 110 Ala Gly Ala He Ser 115 Ser Per Ala Leu Thr 120 leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG bad bad Tyr Phe 125 gin Gly He bad Val 130 Tyr Leu Lys Glu .Lys 135 lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA Tyr Ser Asp Cys 140 Ala Trp Glu Val Val 145 Arg mead Glu He mead 150 lys TAC AGC GAC tg :. ' GCC TGG GAA GTT GTC AGA • ATG GAA ATC ATG AAA Ser Leu Phe Leu 155 Ser Thr Asn mead gin 160 GIu bad Leu bad Ser 165 lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA Asp bad Asp Leu 170 GIy Ser Ser GAT AGA GAC CTG GGC TCA TCT

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In position 111, the sequence GGG (coding for GIy) can be replaced by GAG (coding for GIu). The preferred molecules also include derivatives that are N-glycosylated at amino acid position 78.

For example, the two omega interferons mentioned above may be the leader peptide of the formula

Meta Leu Leu Phe Pro Leu Leu Ala Ala Leu VaI Met Thr Ser Tyr Ser Pro VaI GIy Ser.Leu GIy included

 Legend to the drawings:

Figure 1: Restriction map of clone E76E9 ^ - Figure 2: Restriction map of clone P9A2 Figure 3: DNA sequence of clone P9A2 Figure 4: DNA sequence of clone E76E9

Figure 5: Genomic Southern blot analysis using the DNA of clone P9A2 as a sample

Figure 6: Construction of the expression clone pRHW12

Figure 7: Amino acid and nucleotide differences between type I interferons

Figure 8a: List of unique nucleotide positions of the IFN-aA gene

Figure 8b: List of unique nucleotide positions of the IFN-omegal gene

Figure 8c: List of singular nucleotide positions of the

IFN-ctD gene

Figure 9: Schematic representation of the synthesis of interferon subtype-specific samples.

Figure 10: Detection of interferon subtype specific mRNAs Figure 11 ": DNA sequence of the IFN-omegal gene Figure 12: DNA sequence of the IFN-pseudo-omega2 gene Figure 13: DNA sequence of the IFN-pseudo-omega3 gene Figure 14: DNA sequence of the IFN pseudo-omega4 gene Figure 15: Corrected listing of the IFN-omega gene

sequences

Figure 16: Homologies of the signal sequences Figure 17: Homologies of the "mature" protein sequences Figure 18: - Homologies of the 4 DNA sequences to each other

According to the invention, the novel omega interferons and the DNA sequences coding therefor are obtained as follows:

A human B lymphoma cell line, ζ.Β .. Namalwa cells (see G. Klein et al., Int. J. Cancer Γ0, 44 (1972)) can be obtained by treatment with a virus, e.g. Sendai virus, stimulated for the simultaneous production of α- and ß-interferon.

In this case, if the mRNA formed from stimulated Namalwa cells is isolated, it can serve as a template for the cDNA synthesis.To increase the yield of interferon-specific sequences in the cloning process, the mRNA preparation becomes

  separated by a sugar density gradient corresponding to the different length of the individual mRNA molecules. Advantageously, the mRNA is collected in the region around 12S (about 800 to 1000 bases in length of the mRNA). In this area, the mRNA specific for α- and β-interferons sediments. The mRNA from this gradient region is concentrated by precipitation and dissolution in water.

The preparation of a cDNA library follows "essentially methods known from the literature" (see, for example, E. Dworkin-Rastl,

\ 0 MB Dworkin and P. Swetly, Journal of Interferon Research 2/4, 575-585 (1982)). The mRNA is primed by the addition of oligo-dT. Subsequently, the cDNA is synthesized for 1 hour at 45 ^° C. by addition of the four deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and the enzyme reverse transcriptase in a suitably buffered solution. , By Chlöroformextraktion and chromatography on a .Gelsäule, for example via Sephadex G50, the cDNA / mRNA hybrid is purified. The RNA is by alkali treatment (0.3 mol NaOH at 5O ⁰ C, 1 hour) hydrolyses, and the cDNA, after neutralization with acid Natriumaze-acetate solution präzipi-.tiert with ethanol. After addition of the four deoxynucleoside triphosphates and E. coli DNA polymerase I in a corresponding solution, the double-strand synthesis is carried out, the cDNA also acts as a template and as a primer by hairpin formation at its 3 'end (6 hours at 15 ⁰ C) (see A Eftratiadis et al., Cell 7: 27 * 9 (1976)). After phenol extraction, Sephadex G50 chromatography and ethanol precipitation, the DNA is subjected to treatment with the single strand-specific endonuclease S1 in a suitable solution. The hairpin structure as well as non-double stranded cDNA are degraded. After chloroform extraction and precipitation with ethanol, the double-stranded DNA (dsDNA) is separated by size on a sugar density gradient. For the following steps of cloning, it is preferred to use only dsDNA of size 600 bp and above to monitor the truth.

To increase the probability that only clones are obtained which contain the complete coding sequence for the new interferons. dsDNA greater than 600 bp in length is concentrated from the gradient by precipitation with ethanol and dissolution in water.

In order to propagate the resulting dsDNA molecules, they must first be introduced into an appropriate vector and subsequently into the bacterium E. coli. The vector used is preferably the plasmid pBR322 (F. Bolivar et al., Gene 2: 95 (1977)). This plasmid consists essentially of a replicon and two selection markers. These confer resistance to the host on the antibiotics ampicillin, and tetracycline (Ap ^r , Tc ^r ). The gene for the β-lactamase (Ap ^r ) contains the recognition sequence for the restriction endonuclease PstI. PBR322 can be cut with PstI. The overhanging 3'-ends are extended with the enzyme terminal deoxynucleotide transferase (TdT) with presentation of dGTP in appropriately buffered solution. At the same time, the dsDNA is also extended with the enzyme TdT using dCTP at the 3 'ends. The homopolymer ends of plasmid and dsDNA are complementary and hybridize when plasmid DNA 'and dsDNA are mixed in the appropriate concentration ratio and under appropriate conditions of salt, buffer and temperature (Telson et al., Methods in Enzymology 68_, 41-50 (1980)). ,

E. coli from strain HB101 (genotype F-, hsdS20 (rB, mB) recA13, ara-14, proA2, lacY1, galK2, rpsL20 (Smr), xyl-5, mtl-1, supE44, lambda) becomes Reception of the recombinant vector dsDNA molecules prepared by washing with a CaCl ₂ solution. Competent E. coli HB 101 are mixed with the DNA, and after incubation at ⁰ ° C, the thus obtained plasmid DNA by heat shock at 42 ⁰ C for 2 minutes transformed (M. Dagert et al., Gene 6_, 23-28 (1979)). On-

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Finally, the transformed bacteria are plated on tetracycline-containing agar plates (10 μg per ml). Only E. coli HB 101 can grow on this agar and have taken up a vector or recombinant vector molecule (Tc ^r ). Recombinant vector dsDNA molecules give the host the genotype Ap ^s Tc ^r , since the introduction of the dsDNA into the β-lactamase gene has destroyed the information for the β-lactamase. Clones are transferred to agar plates containing 50 μl of ampicillin. Only about 3% grew, which means that 97% of the clones contained the insertion of a dsDNA molecule. Starting with 0.5 μg dsDNA, more than 30,000 clones were obtained. 28,600 clones were individually transferred to the wells of microtiter plates containing nutrient medium, 10 μg / ml tetracycline and glycerol. Once the clones had grown therein, the plates are kept for storage at -70 ⁰ C (cDNA library).

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

The insert of the clone pER33 (E. Rastl et al., Gene 2J ₁ , 237-248 (1983) - see also EP-A-0.115.613), which is the gene for. Contains IFN-a2-Arg. By nick translation, this piece of DNA is radiolabeled,

32 using DNA polymerase I, dATP, dGTP, dTTP and α-P-dCTP. The nitrocellulose filters are first pretreated under suitable, non-stringent hybridization conditions without addition of the radioactive sample and then submerged for about 16 hours. Addition of the radioactive sample hybridized. Thereafter, the filters are also under non-stringent

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Conditions washed. Due to the low stringency of the hybridization and washing not only interferon-2-Arg-containing clones are detected, but also other interferon-containing clones whose sequences can differ considerably from those of the previously known interferon-α. After drying, the filters are exposed to X-ray film. Blackening significantly above background levels indicates the presence of a clone with interferon-specific sequences.

.'0 Since the radioactivity signals of different quality, the positive clones or the positive-suspicious clones are grown on a small scale. The plasmid DNA molecules are isolated, digested with the restriction endonuclease PstI and electrophoretically size fractionated on an agarose gel (Birnboim et al., Nucl.

Acid. Res. 1, 1513 (1979)). The DNA in the agarose gel is transferred to a nitrated cellulose filter according to the method of Southern (EM Southern, J. Mol. Biol. -98 , 503-517 (1975)). The DNA of this filter is hybridized with the radioactive, iFN-gene-containing, denatured sample. The positive control is plasmid 1F7 (deposited with the DSM under DSM number 2362) which contains the gene for interferon-a2-Arg. The autoradiogram shows that clone E76E9 and clone P9A2 contain a sequence which hybridizes under non-stringent conditions to the gene of interferon-2-Arg. To further characterize the dsDNA inserts of clones E76E9 and P9A2, the plasmids of these clones are made on a larger scale. The DNA is digested with various restriction endonucleases, such as

with AIuI, Sau3A, BgIII, Hinfl, Pstl and Haelll. The resulting fragments are separated in an agarose gel. By comparison with corresponding size markers, e.g. the fragments resulting from digestion of pBR322 with the restriction endonuclease Hinfl or Haelll, respectively

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the sizes of the fragments are determined. By mapping according to Smith and Birnstiel (H.O. Smith et al., Nucl. Acid. Res., 3: 2387-2398 (1967)), the order of these fragments is determined. Surprisingly, it can be deduced from the restriction enzyme maps determined in this way (FIGS. 1 and 2) that the inserts of the clones E76E9 and P9A2 are hitherto unknown interferon genes, namely those of the omega interferons.

This information about the omega interferons is used

The resulting fragments are ligated into the dsDNA form (replicative form) of the bacteriophage M13 mp9 (J. Messing et al., Gene 19: 269-276 (1982)) and "0" to digest the cDNA insert with appropriate restriction endonucleases Sanger et al., Proc Natl Acad., 7, 5463-5467 (1977)). The single-stranded DNA of the recombinant phage is isolated after binding a synthetic Oligomers are synthesized in two separate sets of second-strand syntheses using the large fragment of DNA polymerase I from E. coli (Klenow fragment), and each of the four partial reactions undergoes one of the four dideoxynucleoside crystals (ddATP, ddGTP, ddCTP, ddTTP). This results in statistically distributed chain terminations at those sites where the template DNA contains the base complementary to the particular dideoxynucleoside triphosphate, and additionally uses radiolabelled dATP the synthesis reactions, the products are denatured and the single-stranded DNA fragments separated in size in a denaturing polyacrylamide gel (F. Sanger et al., FEBS Letters 81, 107-111 (1978)). Thereafter, the gel is exposed to X-ray film. From the autoradiogram, the DNA sequence of the recombinant M13 phage can be read. The sequences of the inserts of the various recombinant phages are processed by means of suitable computer programs (R. Staden, Nucl. Acid. Res. 10, 4731-4751 (1982)).

, ₁₂ . , - 216 3 H

Figures 1 and 2 show the strategy of sequencing. Figure 3 shows the DNA sequence of the insert of clone P9A2, Figure 4 shows that of clone E76E9. The not coding. DNA strand is shown in 5 '-> 3 ¹ direction, together with the amino acid sequence derived therefrom.

The isolated cDNA of clone E76E9 for omega (Glu) interferon is 858 base pairs long and has a 3 ¹ untranslated region. The region coding for mature omega (GIu) interferon ranges from nucleotide 9 to nucleotide ^"1 P 524. The isolated cDNA of clone P9A2 for omega (Gly) interferon is 877 base pairs in length, wherein the omega for mature Interferon coding sequence ranges from nucleotide 8 to nucleotide 523. The 3 ¹ untranslated region extends in the case of P9A2 to the poly A segment.

The mature omega-interferon-encoding DNA sequences are completely contained in clones E76E9 and P9A2. They begin at the N-terminal end with the amino acids cysteine-aspartic acid-leucine. Surprisingly, the two mature omega interferons are 172 amino acids long, which is significantly shorter than the previously known interferon. e.g. 166 (or 165) amino acids deviate at a-Interfero'nen. Surprisingly, the two omega interferons have a potential N-glycosylation site at amino acid position 78 (asparagine-methionine-threonine).

A comparison of the DNA of clones E76E9 and P9A2 shows a difference. The triplet in clone E76E9 coding for amino acid 111 is GAG and encodes glutamic acid, while this triplet is in clone E'9Ä2 GGG and encodes glycine. The two omega Interferbn proteins therefore differ in one amino acid and are termed omega (Glu) interferon (E76E9) and omega (Gly) interferon (P9A2).

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The comparison of the two omega-interferons with the previously known human α-interferon subtypes gives the following picture:

omega alpha i j length of the protein · j in amino acids 172 166 ( ⁺ > ; potential N-glycosylation site at position 78

'Ό +) Interferon alpha A has only 165 amino acids

++) Interferon alpha H has a potential N-glycosylation site at position 75 (D. Goeddel et al., Nature 2: 0, 20-26 (1981)).

E. coli HB 101 with the plasmid E76E9 and E. coli 101 with the plasmid P9A2 have been deposited with the German Collection of Microorganisms (DSM Göttigen) under the number DSM 3003 or DSM 3004 on 3 July 1984.

For example, to demonstrate that the newly discovered clones produce an interferon-like activity, clone E76E9 is cultured and the lysate of the bacterium is checked for growth in a plaque reduction assay. The bacterium was expected to produce interferon-like activity (see Example 3).

Further, to prove that the two newly-found interferons are members of a new interferon family, all DNA from Namalwa cells was isolated and digested with various restriction endonucleases.

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This allows the number of genes encoded by die.cDNA ¹ s of clones P9A2 and E76E9 to be estimated. For this purpose, the obtained DNA fragments are separated on agarose gel according to the method of Southern (EM Southern et al., J. Mol. 5 Biol. 9J3, 503-517 (1975)), placed on nitrocellulose filter and under relatively severe conditions hybridized with radiolabelled specific DNA of clone P9A2.

The results obtained after hybridization with DNAs from plasmid P9A2, 10 and pER33 are shown in Figure 5a:

Here, the individual lanes are labeled with letters indicating the differently digested DNA samples, • (E = EcoRI, H = HindIII, B = BamHI, S = SphI, P = PStI, C = CIaI). The left half of the filter was hybridized with the interferon a gene probe ("A") and the right half hybridized with the cDNA insert of clone P9A2 ("0"). The set of bands / hybridizing to either the α-interferon gene probe or the new interferon gene probe is different. For the two different samples, no cross-hybridization could be detected by judging the corresponding lanes.

Figure 5b shows the cDNA of clone P9A2 and the fragment used for hybridization. The interfaces of some restriction enzymes are shown (P = PStI, S = Sau3A, A = AluI). The sample comprises only two of the possible three PstI fragments. The hybridization pattern shows only one hybridizing fragment of about 1300 base pairs (bp) belonging to the homologous gene. The

 50 smaller 120 bp fragment had leaked from the gel. The B'ande associated with the 5'-part of the gene can not be detected because the sample does not contain this region. At least 6 different bands can be detected in this Pstl track

become. This means that some other genes related to the new sequences must be present in the human genome. If one or more PstI sites are present in these genes, it can be expected that at least 3 additional genes can be isolated. , ,

These genes can preferably by hybridization from a human gene Bibliothe-k in - are included einem- PLA Smid vector, phage vector or cosmid vector, iso- / ¹ O lines (see Example 4e).

It should be noted at this point that the omega interferons according to the invention are not just the two mature interferons which are described in detail, but also any modification of these polypeptides. tide, which leaves the IFN-omega activity unaffected. These modifications include e.g. Truncation of the molecule at the N- or C-terminal end, replacement of amino acids with other residues, whereby the activity is not significantly altered, chemical or biochemical bonds of the molecule to other molecules that are inert or active. The last-mentioned modifications may, for example, be hybrid molecules of one or more omega interferons and / or known α- or β-interferons.

The differences between the amino acid and nucleotide sequences of the new interferons, in particular the omega (GIy) and the omega (GIy) interferon, in comparison to the amino acid and nucleotide sequences already published for the α-interferons and for the β-interferon ( C. Weissmann et al., Phil. Trans Soc R. London 299 , 7-28 (1982), A. Ullrich et al., J. Molec. Biol. 156 , 467-486 (1982); Taniguchi et al., Proc Nat Acad., 7, 7, 4003-4006 (1980); K. Tokodoro et al., EMBO J. 3: 669-670 (1984)), the corresponding sequences become pairwise arranged and counted the differences at individual positions.

From the results shown in Figure 7, it can be seen that the DNA sequences of clones P9A2 and E76E9 are related to the sequences of type I interferons (e.g., α and β interferons). On the other hand, it is shown that c is the difference in amino acid sequences between the single α-interferons and the new sequences greater than 41.6% and less than 47.0%. The differences between the new sequences as well as those of the individual α-interferons and β-interferon are in the order of about 70%. Taking into account the results of Example 4, which demonstrates the existence of a whole set of related genes, and taking into account the proposed nomenclature for the interferons (J-Vilceck et al., J. Gen. Virol. 669-670 (1984)), it is believed that the cDNA inserts of clones P9A2 and E76E9 encode a new class of type I interferon, referred to as interferon-omega.

Furthermore, it is demonstrated that the omega-interferon gene expression is analogous to that of an interferon-Typl gene. For this purpose, the transcription of the individual members of the multigene families of α and omega interferons is investigated based on the Sl-mapping method (AJ Berk et al., Cell 12, 721 (1977)) (see Example 7) and shown - the expression omega-1 mRNA is virus-inducible. Since the transcripts of such a gene family differ only by a few bases of about 1000, hybridization alone is not a sufficiently sensitive distinguishing feature to distinguish the different IFN mRNAs.

For this purpose, the mRNA sequences of 9 a-interferons, interferon-omega-1 and ß-interferon are shown. Uppercase letters indicate those bases which are specific to the uppermost sequence. Such specifics can be easily found through a trivial computer program. A hybridization sample,

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which emanates from a specific site, can only hybridize perfectly with the mRNA of a particular subtype. All other mRNAs can not hybridize at the specific site of the subtype. When the hybridization probe is radiolabeled at its specific 5 'end, only those radiolabels are protected against digestions with a single-stranded specific nuclease (preferably SI nuclease) which interacts with the interferon subtype mRNA for which the probe constructs have -wac, hybridized ..

This principle is not limited to interferon but may be applied to any group of known sequences that have the specific sites described in Figure 8. '

The specific locations of the above-mentioned subtypes are '-''most cases, no restriction sites, so that the cutting of the cDNA ¹ s is not suitable with Restrictions endonucleases to produce subtype-specific hybridization probes. The samples used in this example were therefore prepared by extension of a 5'-end radiolabeled oligonucleotide complementary to the mRNA of interferon-omegal above the specific site.

Figure 10 shows that omega-1 mRNA is expected to be inducible in Namalwa and NC37 cells.

The invention therefore not only gene sequences which code specifically for the specified omega-interferons, but also modifications which can be easily and routinely obtained by mutation, degradation, transposition or addition. Any sequence coding for the described human omega interferons (i.e., having the biological activity spectrum described herein) and degenerate in comparison to those shown is

also included; Those skilled in the art will be able to degenerate DNA sequences of the coding regions. Similarly, any sequence encoding a polypeptide having the spectrum of activity of the IFN-omega and which hybridizes to the sequences (or portions thereof) shown under stringent conditions (for example, conditions greater than 85%, preferably greater than 90%, homology select).

The hybridizations are carried out in 6 χ SSC / 5 χ Denhardt's Lö. 10 sung / 0.1% SDS at 65 ⁰ C. The degree of stringency is set in the washing step. Thus, for a selection on DNA sequences with about 85% or more homology, the conditions are 0.2 χ SSC / 0.01%, SDS / 65 ° C and for selection on DNA sequences with about 90% or more Homology the conditions 0.1 χ SSC / 0.01% SDS / 65 ° C suitable.

Examination of a cosmid library under these conditions (0.2 χ SSC) yields some cosmids hybridizing with an IFN-omegal probe. Sequence analysis of restriction enzyme fragments isolated therefrom yields the authentic 'IFN-omegal gene (see Figure 11), as well as three other related genes known as IFN-pseudo-omega2, IFN-pseudo-omega3, and IFN-pseudo-omega4 (see Figures 12-14). These are a further object of the present invention and the peptides encoded by them. This article is characterized in claims 43 to.

The DNA comparisons reveal an approximately 85% homology of the pseudogenes to the IFN-omegal gene (interferon omega one gene).

Furthermore, the IFN-omegal gene shows that upon transcription, the mRNA contains the information for a functional interferon protein, i.e., it becomes a 23 amino acid long signal peptide of the formula

Met Ala Leu Leu Phe Pro Leu Leu Ala AIa Leu VaI Met Thr Ser Tyr Ser Pro VaI GIy Ser Leu. Gly

encoded by the mature 172 amino acid IFN-omegal followed.

.5 Interferon omega genes can be introduced into any organism under conditions which result in "high yields." Suitable hosts and vectors are well known to those skilled in the art, for example EP-A-0 093 619.

In particular, prokaryotes are preferred for 'expression. For example, E. coli K 12, strain 294 (ATCC No. 31,446) is suitable. Other strains that are suitable include Έ. coli X1776 (ATCC No. 31,537). Like the aforementioned strains, E. coli W 3110 (F **, Lambda ', Prototröph, ATCC No. 27325), bacilli such as Bacillus sübtilis, and other Enterobacteriaceae such as Salmonella typhimurium or Serratia marcensens and various pseudomonads can also be used.

In general, plasmid vectors containing replicons and "control sequences derived from species compatible with the host cells can be used in conjunction with these hosts. The vector usually carries recognition sequences next to a replication site, which make it possible to phenotypically select the transformed cells. For example, E. coli is usually transformed with pBR322, a plasmid derived from E. coli species (Bolivar, et al., Gene 2: 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance, providing simple means to identify transformed cells. In addition, the pBR322 plasmid or other plasmids must themselves contain promoters or must be modified to contain promoters,

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which can be used by the microbial organism to express their own proteins. The promoters most commonly used in the production of recombinant DNA include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275 , 615 (1978); Itakura et al., Science 198); JLO56L (1977); Goeddel et al., Nature 281 , 544 (1979)) and tryptophan (trp) promoter systems (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980); EP-A-036 776). While these are the most commonly used promoters, other microbial promoters have also been developed and used. For example, the gene sequence for IFN-omega can be used under the control of the Leftward promoter of bacteriophage lambda (P-). This promoter is one of the promoters known to be particularly strong, which is controllable. Control is possible through the lambda repressor of which adjacent restriction sites are known.

A temperature-sensitive allele of this repressor gene can be inserted into a vector containing a complete IFN-omega sequence. When the temperature is raised to 42 ^° C., the repressor is inactivated and the promoter is expressed to its maximum concentration. The sum of the mRNA produced under these conditions should be sufficient to obtain a cell containing about 10% of its new synthetic ribonucleic acid derived from the P, promoter. In this way, it is possible to establish a clone bank in which a functional IFN-omega sequence is placed adjacent to a ribosome binding site at varying distances to the lambda P _L promoter. These clones can then be checked and selected with the highest yield.

The expression and translation of an IFN-omega sequence may also proceed under the control of other regulatory systems which may be considered to be "homologous" to the organism in its untransformed form. Thus, e.g. chromosomal DNA

from a lactose-dependent E. coli, a lactose or lac operon, which enables the lactose degradation by release of the enzyme beta-galactosidase.

The Lac controls can be obtained from bacteriophage lambda plac5, which is infectious for E. coli. The lac operon of the phage can be derived by transduction from the same bacterial species. Regulation systems that find use in the inventive method. can be derived from plasmid DNA inherent in the organism. The lac promoter-operator system can be induced by IPTG.

Other promoter-operator systems or parts thereof may be used as well: for example, arabinose operator, colicine egg operator, galactose operator, alkaline phosphatase operator, trp operator, xylose A operator, tac promoter, and the like.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures may also be used. Saccharomyces cerevisiae is the most widely used among eukaryotic microorganisms, although a number of other species are commonly available. For expression in Saccharomyces , for example, the plasmid YRp7 (Stinchcomb et al., Nature 2J32, 39 (1979), Kingsman et al., Gene 7, 141 (1979), Chumper et al., Gene 10, 157 (1980)), and the Plasmid YEp13 (Bwach et al., Gene 8_, 121-133 (19.79)) is commonly used. The plasmid YRp7 contains the TRPl gene, which provides a selection marker for a yeast mutant unable to grow in tryptophan-containing medium; for example, ATCC No. 44076.

The presence of TRPl damage as a characteristic of the yeast host genome then provides an effective means of detecting the transformation by using no tryptophan

is cultivated. Similarly, the plasmid YEpl3 contains the yeast LEU 2 gene, which can be used to complement a LEU 2 minus mutant. Suitable promoter sequences for yeast vectors include the 5 'flanking region of the genes of ADH I (Ammerer G., Methods of Enzymology 101 , 192-201 (1983)), 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Biol. Chem. 255 , 2073 (1980)) or other glycolytic enzymes (Kawaski and Fraenkel, BBRC 108 , 1107-1112 (1982)) such as enolase, glyceraldehyde-3-phosphate, dehydrogenase, hexokinase, Pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase. When concentrating suitable expression plasmids, the termination sequences associated with these genes can also be used in the expression vector at the 3 'end of the sequence to be expressed in order to provide polyadenylation and termination of the mRNA.

Other promoters that also have the advantage of growth-controlled transcription are the promoter regions of the genes for alcohol dehydrogenase-2, isocytochrome C, acid phosphatase-degrading enzymes coupled with the nitrogen metabolism discussed above. mentioned glyceraldehyde S-phosphate dehydrogenase and enzymes responsible for the processing of maltose and galactose. Promoters that are regulated by the yeast mating type locus, for example promoters of the BARI, MFaI , STE2 , STE3 , STE5 genes, can be used in temperature-regulated systems through the use of temperature dependent siv mutations. (Rhine PH.D., Thesis, University of Oregon, Eugene, Oregon (1979), Herskowitz and Oshima, The Molecular Biology of Yeast Saccharomyces , part I, 181-209 (1981), Cold Spring Harbor Laboratory). These mutations affect * the expression of quiescent mating type cassettes of yeasts and thereby indirectly the mating type dependent promoters. In general, however, any plasmid vector containing a yeast-compatible promoter, original replication and termination sequences is suitable.

In addition to microorganisms, cultures of multicellular organisms are also suitable host organisms. In principle, each of these cultures can be used, whether from vertebrate or invertebrate animal cultures. However, there has been a great deal of interest in vertebrate animal cells, so that the proliferation of vertebrate cells in culture (tissue culture) has become a routine method in recent years (Tissue, Culture, Academic Press, Kruse and Patterson, Editors (1973). ). Examples of such useful host cell lines are VERO and HeLa cells, Golden hamster ovary (CHO) cells and W138, BHK, COS-7 and MDCK cell lines. Expression vectors for these cells usually contain (if necessary) a replication site, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding site, RNA splicing site, polyadenylation site, and transcriptional termination sequences.

When used in mammalian cells, the control. functions on the expression vectors often made of viral material. For example, the promoters commonly used are derived from Polyoma Adenovirus 2, and more commonly from Simian Virus 40 (SV 40). The early and late end promoters of the SV 40 are particularly useful because both are easily obtained from the virus as a fragment that also contains the SV 40 viral replication site. (Fiers et al., Nature 273 , 113 (1978)). Also, smaller or larger fragments of the SV 40 can be used, provided they contain the approximately 250 bp sequence that extends from the HindIII site to the BglI site in the viral replication site.

In addition, it is also possible and often advisable to use promoter or control sequences normally linked to the desired gene sequences, provided these control sequences are compatible with the host cell systems.

24 6 31

A replication site may either be provided by appropriate vector construction to incorporate an exogenous site, such as SV40 or other viral sources (e.g., polyoma, adeno, VSV, PBV, etc.), or may be provided by the host cell's chromosomal replication mechanisms. If the vector is integrated into the host cell chromosome, the latter measure is usually sufficient.

However, the genes may preferably be expressed in an expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21, 237-248

(1983) and EP-A-O.115-613 - deposited with the DSM under the number DSM 2773 on December 20, 1983), since this vector contains all regulatory elements which lead to a high expression rate of the cloned genes. According to the invention, therefore, in the plasmid pBR322, the shorter plasmid EcoRI / BamHI fragment was replaced by the DNA sequence of the formula

EcoRI Sau3A

gaattc acgct GATC GCTAAAACATTGTGCAAAAGAGGGTTGACTTTGCCTTCGCGA

imRNÄ-Start Met

-> 0 accagttaactagtacacaagttcacggcaacggt aaggagg ttta agct taaag atg

RBS _ HindIII. Cys Asp

TGT GAT C IFN-omega gene >

Sau3A

replaced.

In order to achieve the object according to the invention, as shown in FIG. 6, the procedure is, for example, as follows:

I. Preparation of Required Individual DNA Fragments:

- .25 -

246 31

Fragment a

For the preparation of fragment a), a plasmid containing a gene. for IFN-omega, e.g. the plasmid P9A2, digested with the restriction endonuclease Avail. After chromatography and purification of the resulting cDNA rlnser.ts this is digested with the restriction Endonucleasen Ncol and AIuI two times and then isolated by chromatography and electroelution. This fragment contains most of the corresponding omega interferon gene. For example, the omega (GIy) interferon gene of clone P9A2

following structure:.

5 10 15

His GIy Leu Leu Ser Arg Asn Thr Leu

C I CAT GG C CTA CTT AGC AGG AAC ACC TTG Nc ο I

20 25 30

VaI Leu Leu His Gin Met Arg Arg He Ser 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 GIu Met VaI Lys GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG

50 55 60

Ser GIn Leu GIn Lys Ala His VaI Met Ser VaI Leu His GIu 'Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 16

65 70, 75

Leu Gin Gin He Phe Ser Leu Phe His Thr GIu Arg Ser Ser AIa CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT

80 85 ". 90

AIa'Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr GIy Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT

95. 100 105

Gin Gin Leu Gin His Lei Giu Thr Cys Leu Leu Gin VaI VaI GIy CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA

, 26 -

110 115 120

GIu GIy GIu Ser Ala GIy Ala lie Ser Ser Pro Ala Leu Thr 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 Arg VaI Tyr Leu Lys GIu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388

14540 145 150

Tyr Ser Asp Cys AIa Trp GIu VaI VaI Arg Met GIu He Met Lys TAC AGC GAC TGT GCC TGG GAA GTT. GTC AGA.ATG GAA ATC ATG AAA 433

.10 155 160 165

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

170 Asp Arg Asp Leu GIy Ser Ser

GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530

TAACACTTGCACATGTGACTCTGGTCAATTCAAAGACTCTTATTTCGGCTTTAATCAC 589

AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648

AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707

TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 7 66

TTTACATTGTATTAAGATAACAAAACATGTTC AGbt 802

• Alul

Fragment b:

For the preparation of fragment b), the plasmid P9A2 is digested with the restriction endonuclease Avail. After chromatography and. Purification of the resulting cDNA insert is

this with the Restrictionsendonuclease Sau3A further digested and the desired 189bp long fragment using Chromatogra-. phie and electroelution isolated. This has the following structure:

246 31

- "5 10 15

Asp Leu Pro Gin Asn His Glu Leu Leu Ser Arg Asn Thr Leu j GATCTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG

Sau3A

ncol

20 25 30

VaI Leu Leu His Gin Met Arg Arg He He-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 GIu Met VaI Lys GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG

50 55 60

Ser GIn Leu GIn Lys Ala His VaI Met Ser VaI Leu His GIu Met ACG CAG TTG CAG AAG GGC CAT GTC ATG TCT GTC CTC CAT GAG ATG

Leu Gin Gin Hey

CTG CAG CA Jq ate .

Sau3A |

Fragment c

For the preparation of Fragment'c) the plasmid pER33 (see E. Rastl-Dworkin et al., Gene 2J ₁ , 237-248 (1983) and EP-AO.115.613) with the restriction enzyme EcoRI and PvuII digested twice. The 389 bp fragment obtained after agarose gel fractionation and purification, which contains the Trp promoter, the ribosomal binding site and the start codon, is then digested with Sau3A. The desired 108bp fragment is obtained by agarose gel electrophoresis, electroelution and Elutip acid purification. This has the following structure:

- 28 - 24 6

EcoRI | Sau3A

gaattca cgct JGATC GCTAAAACATTGTGCAAAAGAGGGTTGACTTTGCCTTCGCGA '59

JmRNA start. · · · Met

ACCAGTTAACTAGTACACAAGTTCACGGCAACGGT AAGGAGG TTT AAGCTTA AAG ATG .5 RBS HindIII

Cys Asp

TGT gat c .-. · - -

Sau3A

Ligation of fragments b and c:

, 10 Fragments b and c are ligated and ligated with T4 ligase

Destruction of the enzyme cut with HindIII. This ligated fragment has the following structure:

HindIII Sau3A Ncol

a JAGCT TAAAG ATGTGTGATC TGCCTCAGAA CCATGGC CTA CTTAGCAGGA '50

ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200

A GATCA CACA TCTTTA chases t Sau3A Hindlll!

Alternatively, this may be for the preparation of the plasmid

pRHWlO necessary DNA fragment can also be generated by the use of two synthetically produced oligonucleotides:

- ²⁹ - 24ö 3 1

The oligonucleotide of the formula

5'-AGCTTAAAGATGTGt-S '. remains dephosphorylated at its 5 'end.

The oligonucleotide of the formula 5'-GATCACACATCTTTA-S '

is kinased by T. polynucleotide kinase and ATP at the 5'-end ^

When hybridizing both oligonucleotides, the following short DNA fragment is obtained:

5'-AGCTTAAAGATGTGt 3 '

3'-ATCTACACACTAGp. 5 '

This results in one end of the typical Hindlll 5'-overhang and at the other end of the typical Sau3A 5'-overhang.

Fragment b) is dephosphorylated using calf intestinal phosphatase. Fragment b) and the fragment described above are brought together and ligated together by T. ligase.

Since the ligase requires at least one 5'-phosphate-containing end> only the synthetic DNA piece can be linked to fragment b) 'or else 2 synthetic fragments at their Sau3A ends. Since the two resulting fragments differ in length, they can be separated by selective isopropane precipitation. The thus purified fragment is phosphorylated by T. polynucleotide kinase and ATP.

II. Preparation of the expression plasmids a) Preparation of the plasmid pRHWIO:

The expression plasmid pER103 (E. Rastl-Dworkin et al., Gene 21, 237-284 (1983) and EP-A-0.115.613 deposited with the DSM under DSM number 2773) is linearized with HindIII and then treated with calf intestinal phosphatase , After isolation and purification of the DNA thus obtained, it is dephosphorylated and then ligated with ligated fragments b and c (after their HindIII digestion). Subsequently, E. coli HB 101 is transformed with the resulting ligation mixture and cultured on LB agar plus 50 μg / ml ampicillin. The plasmid containing the desired structure was named pRHW 10 (see FIG. 6), which after its replication served as an intermediate for the preparation of further plasmids.

bj Preparation of the plasmid pRHW12:

To the cut with BamHI plasmid pRHWIO are added the Klenow fragment of DNA polymerase I and the 4 Deoxynucleosidtr! Phosphates. The obtained after incubation linearized, blunt ends provided _w ith plasmid is purified and then cut with NcoI. The larger fragment, which is recovered by agarose gel electrophoresis, electroelution and Elutip purification, is ligated with fragment a. Subsequently, the ligation mixture is transformed with E. coli HB 101 and cultured on LB agar plus 50 μg / ml ampicillin. The desired structure-containing plasmid was named pRHW12 (see Figure 6), which expresses the desired omega (Gly) interferon.

For example, contains 1 1 of the resulting bacterial culture (optical density: 0.6 be

 Units of interferon.

(optical density: 0.6 at 600 nm) 1 χ 10 International

24ö 31 8

, c) Preparation of the plasmid pRHWII:

To the BarnHI-cut plasmid pRHW10 is added the Klenow fragment of DNA polymerase I and the 4 deoxynucleoside triphosphates. The linearized, straight-ended plasmid obtained after incubation is purified and then cut with NcoI. The larger fragment, which is obtained by means of agarose gel electrophoresis, electro-elution and Elutip purification, is incubated with the fragment a obtained analogously from the plasmid E79E9, in which

Once the GGG codon coding for the 111th amino acid (GIy) has been replaced by the GAG codon (Glu), the ligation mixture obtained is then transformed with E. coli HB 101 and cultivated on LB agar desired structure having plasmid received the name pRHWll (see Figure 6 analogous), which expresses the desired omega (Glu) interferon.

Transformation of the cells with the vehicles can be achieved by a variety of methods. For example, she can. calcium, either by washing the cells in magnesium and adding the DNA to the calcium suspended cells or exposing the cells to a coprecipitate of DNA and calcium phosphate. Upon subsequent gene expression, the cells are transferred to media which selects for transformed cells.

After host transformation, expression of the gene, and fermentation or cell culture under conditions where iFN-omega is expressed, the product can usually be extracted by known chromatographic separation techniques to yield a material containing IFN-omega with or without a leader and includes tailing sequences. IFN-omega can be expressed with a leader sequence at the N-terminus (Pre-IFN-omega), which may be expressed by some host

cells can be removed. If not, cleavage of the leader polypeptide (if present) is required to obtain mature IFN-omega. Alternatively, the IFN-omega clone can be modified so that the mature protein is produced in the microorganism directly instead of the pre-IFN-pmega. In this case, the precursor sequence of the yeast mating pheromone MF-alpha-1 can be used to ensure correct "maturation" of the fused protein and excretion of the products into the growth medium or periplasmic space. The DNA sequence for functional or mature IFN-omega can be labeled with MF-alpha-1 at the putative cathepsin-like (Lys-Arg) interface at position 256 from the ATG initiation codon (Kurjan, Herskowitz, Cell 3_0_, 933-943 ( 1982)),

Based on their biological spectrum of action, the novel interferons according to the invention can be used for any type of treatment for which the known interferons are also used. These include, for example, herpes, rhinovirus, opportunistic AIDS infections, various cancers and the like. The novel interferons may be used alone or in combination with other known interferons or biologically active products, for example IFN-alpha, IL-2, other immunomodulators and the like.

IFN-omega may be administered parenterally in cases where antitumor or antiviral treatment is required and in cases where immunosuppressive properties are demonstrated. Dosage and dosage rates may be similar to those currently in clinical trials for iFN-a materials, e.g. approx. (1-10) χ '10 units daily and for preparations that are more than 1% pure, up to, for example, 5 χ 10 units daily.

246 31

For example, for a convenient dosage form with a substantially homogeneous, bacterially produced IFN-omega, 3 mg IFN-omega may be dissolved in 25 ml of 5% human serum albumin when used parenterally. This solution is then passed through a bacteriological filter and filtered Dissolve the solution aseptically on 100 vials, each containing 6 χ 10 units of pure IFN-omega suitable for parenteral administration. The vials are preferably stored in the cold (-20 ⁰ C) before use · The substances according to the invention may be formulated in known manner to obtain pharmaceutically acceptable agent, wherein the polypeptide of the invention is mixed with a pharmaceutical acceptable carrier · Common carriers and their formulation is described by E.W. Martin in Remington's Pharmaceutical Sciences, to which reference is expressly made. IFN-omega is mixed with an appropriate amount of the excipient to provide suitable pharmaceutical agents suitable for effective use in the recipient (patient). Preferably, a parenteral administration is carried out.

embodiment

The following examples, which are not intended to limit the invention, describe the invention in detail.

example 1 Find IFN sequence-specific clones

a) Preparation of the cDNA library

mRNA from Sendai virus-stimulated cells is used as the source. 5 used to prepare a cDNA library according to methods known from the literature (E. Dworkin-Rastl et al., Journal of Interferon Research VoI 2/4, 575-585 (1982)). The

accumulated 30,000 clones are transferred individually in the wells of microtiter plates. The following medium is used to grow and freeze the colonies:

10 G tryptone 5 G Yeast Extract 5 G NaCl 0.51 G Na citrate χ 2 H ₂ O 7.5 G K ₂ HPO ₄ χ 2 H ₂ O 1.8 G KH ₂ PO ₄ 0.09 G MgSO ₄ χ 7H ₂ O 0.9. G (NH ₄ J ₂ SO ₄ 44 ' G glycerin 0.01 G Tetracycline x HCl 1 1 H ₂ O

ad

The microtiter plates with the individual clones are incubated overnight at 37 ° C and then stored at -70 ⁰ C.

b) hybridization probe . ·

The starting material for the hybridization probe is the recombinant plasmid pER33 (E. Dworkin-Rastl et al., Gene 21, 237-248 (1983)). This plasmid contains the coding

24 6 31

Region for the mature interferon IFN-a2arg plus 190 bases of the 3 'untranslated region. 20 μg p'ER33 are incubated with 30 units HindIII restriction endonuclease in 200 μΐ reaction solution (10 mM Tris-HCl pH-7.5, 10 mM MgCl ₂ , 1 mM dithiothreitol (DTT), 50 mM NaCl) for 1 hour at 37 ⁰ C. , The reaction is terminated by the addition of-1/25 volume of 0.5 M Äthylendinitrilotetraessigsäure (EDTA) and heating to 7O ⁰ C for 10 minutes. After addition of 1/4 vol 5 χ buffer (80% glycerol, 40 mM Tris-acetic acid pH = 7.8, 50 mM EDTA, o., O5% Natriumdo.decylsulfat (SDS), 0.1% Bromophenol Blue) are the The resulting fragments were size-separated electrophoretically in a 1% agarose gel [gel and running buffer "(TBE): 10.8 g / l tris-hydroxymethylaminomethane (TrisBase), 5.5 g / l boric acid, 0.93 g / l EDTA] After incubation of the gel in a 0.5 μg / ml ethidium bromide solution, the DNA bands are visualized in UV light and the gel area containing the IFN gene-containing DNA piece (about 800 bp in length) By applying a voltage, the DNA is electroeluted in 1/10 χ TBE buffer The DNA solution is extracted once with phenol and four times with ether and the DNA by addition of 1/10 vol 3 M sodium acetate (NaAc) pH- 5.8 and 2.5 vol of EtOH from the aqueous solution at -20 ⁰ C precipitated. After centrifugation, the DNA is washed once with 70% ethanol and dried for 5 minutes in a vacuum. D The DNA is taken up in 50 μΐ of water (approx. 50 ug / ul). This DNA is radiolabeled by nick translation (modified according to T. Maniatis et al., Molecular Cloning, ed. CSH). 50 μΐ incubation solution contains: 50 mM Tris pH = 7.8, 5 mM MgCl ₂ , 10 mM mercaptoethanol, 100 ng pER33 insert DNA, 16 pg DNasel, 25 μM dATP, 25 μM dGTP, 25 μM dTTP, 20 μα a ³² P-dCTP (> 3000 Ci / mmol) and 3 units of DNA polymerase I ( E. coli) . The incubation was carried out at 14 ⁰ C for 45 minutes. The reaction is stopped 7.6 solution and heating at 7O ⁰ C for 10 minutes by addition of 1 vol of 50 mM EDTA, 2% SDS, 10 mM Tris pH =. The DNA is separated by chromatogra-

246 31

phie over Sephadex G-IOO in TE buffer (10 mM Tris pH = 8.0, 1 mM EDTA) of unincorporated radioactivity. The radioactively labeled sample has a specific activity of about 4 × 10 cpm.

5.c) Screening of clones for JFN gene-containing inserts

The bacterial cultures frozen in the microtiter plates are thawed (a). A piece of nitrocellulose filter of the appropriate size (Schleicher and Schüll, BA 85 / 0.45 μΐη pore size) is placed on LB agar (LB agar: 10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15.g / l Bacto agar, 20 mg / l tetracycline HCl). With a stamp adapted to the microtiter plates, the individual clones are transferred to the nitrocellulose filter. The bacteria grow overnight at 37 ° C to approximately 5 mm diameter colonies. To destroy the bacteria and denature the DNA, place the nitrocellulose filters sequentially in a stack of Whatman 3MM filters soaked in the following solutions: 1) 8 minutes to 0.5 M NaOH, 2) 1 minute Tris pH = 7.4 3) 2 minutes 1 M Tris pH = 7.4 and 4) 4 minutes 1.5 M NaCl, 0.5 M Tris pH = 7.4. . The filters werden'luftgetrocknet and then "held 2 hours at 80 ⁰ C. The pretreatment of the filter was carried out for 4 hours at 65 ⁰ C in the hybridization solution consisting of 6 χ SSC (1 SSC χ corresponds to 0.15 M NaCl, 0.015 M trisodium citrate Denhardt's solution (Ix Denhardt's solution corresponds to 0.02% PVP (polyvinylpyrrolidone), 0.02% Ficoll (MW: 40,000 D), 0.02% BSM (bovine serum albumins) and 0 , 1% SDS (sodium dodecylsulfate) Ca 1 χ ΙΟ ⁶ cpm per filter of the sample prepared in b) are denatured by boiling and added to the hybridization solution Hybridization is carried out for 16 hours at 65 ° C. The filters are washed at 65 ° C. four times 1 hour with 3 χ SSC / 0.1% SDS The filters are air dried, covered with Saran Wrap and exposed on Kodak X-OmatS film.

" ³⁷ - 246 31

Example 2

Southern transfer confirming IFN gene-containing recombinant plasmids

Of the positive or suspect positive cells, 5 ml cultures in L-Broth (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 20 mg / l tetracycline χ HCl) overnight at 37 ° C bred. The plasmid DNA is modified according to Birnbo.im and DoIy (Nucl. Acid Res. "], 1513, (1979) isolated.) The cells of 1.5 ml of suspension are centrifuged off.

'10 ffers Eppendorf centrifuge) and at ^{0 0} C in 100 μΐ Lyso-. zymlösung consisting of 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH = 8.0 and 4 mg / ml lysozyme resuspended. After 5 minutes incubation at room temperature, 2 volumes of ice cold 0.2 M NaOH, 1% SDS solution are added and incubated for a further 5 minutes. Then 150 .mu.l of ice-cold potassium acetate solution pH = -4.8 are added and incubated for 5 minutes. The precipitated cell components are centrifuged off. Extract the DNA solution with 1 volume of phenol / CHCln (1: 1) and precipitate the DNA by adding 2 volumes of ethanol. After centrifuging, the pellet is washed once with 70% ethanol and dried in vacuo for 5 minutes. The DNA is dissolved in 50 μί (TE) buffer. 10 μΐ thereof are dissolved in 50 μΐ reaction solution (10 mM Tris-HCl pH = 7.5, 10 mM MgCl ₂ , 50 mM NaCl, 1 mM DTT) with 10 μl

pstl Restrlktionsendonuklease for 1 hour at 37 ° C digested. After addition of 1/25 volume of 0.5 M EDTA and 1/4 volume of 5 χ buffer (see Example Ib)) is heated for 10 minutes at 70 ⁰ C and then the DNA in a 1% agarose gel (TBE buffer) separated by electrophoresis , The DNA in the agarose gel is transferred to a nitrocellulose filter by the method of Southern (EM Southern, J. Mol. Biol. 9, 503-517 (1975)). The DNA in the gel is denatured by incubation of the gel with a 1.5 M NaCl / 0.5 M NaOH solution for one hour. Then, about 1 hour with a χ χ _M Tris HCl pH = 8 / l, NaCl 5 M solution neutralized. The DNA

246 31

is transferred to the nitrocellulose filter with 10 χ SSC (1.5 M NaCl, 0.15 M sodium citrate, pH = 7.0). After completion of the transfer (about 16 hours), the filter is rinsed briefly in 6 χ SSC buffer, then air-dried and finally baked at 80 ⁰ C for 2 hours. The filter is pretreated for 4 hours with a χ SSC / 5 χ Denhardt's solution / 0.1% SDS (see Example Ic) at 65 ^° C. Approximately 2 x.10 cpm the hybridization probe (see Example Ib) are introduced by heating to 100 ⁰ C, and then denatured in the hybridization solution. The hybridization time is 16 hours at 65 ^° C. Subsequently, the filter is washed for ⁴ × ¹ hour at .65 ° C. with a 3 × SSC / 0.1% SDS solution. After air drying, the filter is covered with Saran Wrap and exposed to Kodak X-OmatS film.

Example 3 Detection of Iriterferon Activity in Clone E76E9

A 100 ml culture of clone E76E9 is dissolved in L-broth (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 5'g / l glucose, 20 mg tetracycline χ HCl per 1) at 37 ° C grown to the optical density Ag ₀₀ = 0.8. The bacteria are centrifuged off for 10 minutes at 7000 rpm, washed once with a 50 mM Tris χ HCl pH = S ₇ O, 30 mM NaCl solution and resuspended in 1.5 ml of washing solution. After incubation with 1 mg / ml lysozyme at 0 ^° C. for half an hour, the bacterial suspension is frozen and thawed five times. The cell debris are pelleted by centrifugation at 40,000 rpm for one hour. The supernatant is sterile filtered and tested for interferon activity. The test used is the plaque reduction test with V3 cells and vesicular stomatitis virus (GR Adolf et al., Arch. Virol. 22, 169-178 (1982)). Surprisingly, the clone produces up to 9000 IU of interferon per liter of starting culture.

Example 4

Genomic Southern blot to determine the number of genes associated with the new sequences

a) Isolation of DHA from Namalwa cells

400 ml of a Namalwa cell culture are centrifuged at 1000 rpm in a JA21 centrifuge to pellet the cells. The resulting pellets are gently washed by resuspending them in NP40 buffer (NP40 buffer: 140 mM HaGl, 1.5 mM IgCl ₂ , 10 mM Tris / Cl pH = 7.4) and pelleted again at 1000 rpm. The resulting pellets are resuspended in 20 ml of NP40 buffer and destroying the cell walls with 1 ml of a 10% UP40 solution added. After standing for 5 minutes in an ice bath, the intact cell nuclei are pelleted by centrifugation at 1000 rpm and the supernatant discarded. The cell nuclei are resuspended in 10 ml of a solution consisting of 50 mM Tris / Cl pH = 8 x 0, 10 mM EDTA and 200 mM ITaCl, followed by addition of 1 ml of 20% SDS to remove the proteins. The resulting viscous solution is extracted twice with the same amount of phenol (saturated with 10 mM Tris / Cl pH = 8.0) and twice with chloroform. The DUA is precipitated by addition of ethanol, separated by centrifugation, then the DNA pellet obtained is washed once with 70 % ethanol, dried in vacuo for 5 minutes and in 6 ml TE buffer (TE buffer: 10 mM Tris / Cl pH = 8.0, 1 mM EDTA). The concentration of the DNA is 0.8 mg / ml.

b) Restriction endonuclease digestion of DNA from Namalwa cells

The Restriction endonuclease digestions are each according to the according to the manufacturer

specified conditions. 1 μg DNA is digested with 2 units of the appropriate Restriction-Endonuclea.se in a volume of 10 μΐ at 37 ⁰ C for 2 hours or longer. EcoRI, HindIII, BamHI, Sphl, PstI and CIaI are used as restriction endonucleases. 20 μg of DNA are used in every digestion. In order to check the completeness of the digestion, 10 μΐ (aliquots) are separated off each time the reaction is started and 0.4 μg lambda phage DNA is added. After incubation for 2 hours, these aliquots are monitored by agarose gel electrophoresis and the completeness of digestion is assessed by a pattern of the stained lambda phage DNA fragments.

After carrying out this control, the reactions are stopped by adding .EDTA to a final concentration of 20 mM and heating the solution to 7O ⁰ C for 10 minutes. The DNA is precipitated by addition of 0.3 M NaAc pH = 5.6 and 2.5 volumes of ethanol. After incubation for 30 minutes at -70 ⁰ C the DNA is pelleted in an Eppendorf centrifuge, washed once with 70% ethanol and dried. The resulting DNA is taken up in 30 μM TE buffer.

c) gel electrophoresis and Southern transfer

The digested DNA samples are sized according to their size in a 0.8% agarose gel in TBE buffer (10.8 g / l Tris base, 5.5 g / l boric acid, 0.93 g / l EDTA). fractionated. For this purpose, 15 μΐ of the DNA sample with 4 μΐ loading buffer (0.02% SDS, 5 χ TBE buffer, 50 mM EDTA, 50% glycerol, 0.1% bromophenol blue), briefly heated to 70 ⁰ C and applied in the provided troughs of the gel. Lambda DNA cut with EcoRI and HihdIII is applied in a suitable well and serves as a marker for the size of the DNA. Gel electrophoresis will last 24 hours

246 3

carried out at about 1 V / cm. Subsequently, the DNA is brought to the method of Southern on a nitrocellulose filter (Schleicher and Schuell, BA85), in this case, as a transfer buffer 10 χ SSC (1 χ SSC: 150 mM trisodium citrate, 15 mM NaCl, pH = 7.0 ) used. After drying the filter

at room temperature, this is heated for 2 hours at 80 ⁰ C to bind the DNA to this.

d) hybridization probe

20 μg of plasmid P9A2 are cut with Avail, releasing an approximately 1100 bp fragment,

, which contains the whole cDNA insert. This DNA fragment is again cut with Sau3A and AluI and the largest piece of DNA after agarose gel electrophoresis (see Figure 5b), isolated by electroelution and Elutip column chromatography. The DNA thus obtained (1.5 μg) is dissolved in 15 μl of water. ,

20 μg of the plasmid pER33 are cut with HindIII, whereby this expression plasmid is cut twice for IFN-α2-Arg (E. Rastl.-Dworkin et al., Gene 2_1, 237-248 (1983)). The smaller DNA fragment contains the gene for interferon-a2-Arg and is isolated in the same manner as in the plasmid P9A2.

  Both DNAs are prepared according to the method described by P.W.J. Rigby et al. (J. Mol.

Biol. 113 , 237-251 (1977)) nick-translated. The nick translation is carried out in each case with 0.2 μg of DNA in a solution of 50 μΐ, consisting of 1 × Nick buffer (1 × Nick buffer: 50 mM Tris / Cl pH = 7.2, 10 mM MgSO ₄ , 0 , 1 mM DTT, 50 μg / ml BSA), 100 μΜοΙ each dATP, dGTP and dTTP,

32

150 μίί α-P-dCTP (Amersham, 3000 Ci / itiMol) and 5 units of DNA polymerase I (Boehringer-Mannheim, nick translation quality) performed. After 2 hours at 14 ⁰ C, the reaction by adding the same amount of an EDTA

_ 42 -

Solution (40 mmol) stopped and the unreacted radioactive material separated by GSO column chromatography in TE buffer. The remaining specific radioactivity is approximately 100 χ 10 cpm / g of DNA.

e) hybridization and autoradiography

The nitrocellulose filter is cut in half. Each half contains an identical set of traces of Namalwa DNA treated with the restriction enzymes mentioned in Example 4a. The filters are suspended in a solution containing 6 χ SSC, 5 χ Denhardt's (1 χ Denhardt's: 0.02% bovine serum albumin (BSA), 0.02% polyvinylpyrrolidone (PVP), 0/02% Ficoll 400), 0.5 % SDS, 0.1 mg / ml denatured calf thymus DNA and. 10mM EDTA prehybridized for 2 hours at 65 ° C. The hybridization is carried out in a solution containing 6 χ SSC, 5 χ Denhardt's, 10 mM EDTA, 0.5% SDS and approximately 10 χ 10 cpm nick translated DNA for 16 hours at 65 ° C. One half of the filter is hybridized with interferon-a2-Arg DNA and the other half with interferon DNA isolated from plasmid P9A2. After hybridization, both filters are washed four times at room temperature with a solution consisting of 2 χ SSC and 0.1% SDS and then washed twice at 65 ° C for 45 minutes with a solution consisting of 0.2 χ SSC and 0.01% SDS , The filters are then dried and exposed to a Kodak X-Omat S film.

Example 5

Preparation of expression plasmids pRHW 12 and pRHW 11

Preamble:

The preparation of the expression plasmids is shown in Figure β

2 4 6 31

(not to scale), and all Restrictionsenzymverdauungen are performed according to the instructions of the enzyme manufacturers.

a) Preparation of the plasmid pRHW 10

100 μg of the plasmid P9A2 are digested with 100 units of the restriction endonuclease Avail (New England Biolabs). After Digestion- DAS is inactivated enzyme by heating to 7O ⁰ C and the resulting fragments "of a l, 4% agarose gel with TBE buffer (TBE buffer: 10.8 g / l Tris base,

10; 5.5 g / L boric acid, 0.93 g / L EDTA) according to their size. The band containing the entire cDNA insert is electroeluted and purified on an Elutip column (Schleicher & Schuell). 6 μg of the 20 μg obtained are further digested with the restriction endonuclease Sau3a (20 units in a total of 100 μl solution). The fragments are separated by means of 2% agarose gel in TBE buffer. After staining with ethidium bromide (EtBr), the 189 bp piece of DNA is electroeluted and purified as described above (= fragment b in Figure 6).

In order to link the interferon gene with a promoter, a ribosomal binding site and a start codon, the corresponding piece of DNA is isolated from the expression plasmid pER 33 (E. Rastl-Dworkin et al., Gene 2JL, 237-248 (1983)). For this purpose, 50 μg of pER 33 are digested twice with the restriction enzymes EcoRI and PvuII and the fragments obtained are fractionated according to their size on a 1.4% agarose gel in TBE buffer. The 389 bp piece of DNA containing the trp promoter, the ribosome binding site and the start codon is electroeluted and purified on an Elutip column. The resulting fragment is then digested with Sau3A and the desired 108 bp fragment is obtained by agarose gel electrophoresis, electro-elution and Elutip column purification in a yield of approximately 100 ng (= fragments c in Figure 6).

_AA 246 3-1

20 ng of fragment b are mixed with 20 ng of fragment c in a volume of 40 μΐ using 10 units. T4 ligase in a solution containing 50 mM Tris / Cl _{Λ ρΗ} = 7.5. 10 mM MgCl ₂ , 1 mM DTT and 1 mM ATP, ligated at -14 ° C for 18 hours. Subsequently, the enzyme is destroyed by heating to 7O ⁰ C and cut the DNA obtained with'Hindlll in a total volume of 50 μΐ.

10 μ9 of the expression plasmid pER 103 (E. Rastl-Dworkin et al., Gene 2 \ _ < 237-248 (1983)) is linearized with HindIII in a total volume of 100 μΐ. After 2 hours at 37 ° C, 1 volume of 2 χ phosphatase buffer (20 mM Tris / Cl pH = '9.2, 0.2 mM EDTA) is added along with one unit of calf intestinal phosphatase (CIP). After 30 minutes at 45 ° C, add another unit of CJP and incubate again for 30 minutes. The resulting DNA is purified by phenol extraction twice, chloroform extraction once, and precipitation by the addition of 0.3 M sodium acetate (pH = 5.5) and 2.5% ethanol. It is then dephosphorylated to prevent religation of the vector in the next ligation step.

100 ng of the linearized pER 103 and the ligated fragments b and c (after HindIII digestion), a solution of 100 are in · μΐ which ligase containing buffer was added and at 14 ⁰ C for 18 hours with T. DNA ligase ligated.

200 μΐ of competent E. coli HB 101 (E. Dworkin et al., Dev. Biol. 1S, 435-448 (1980)) are mixed with 20 μΐ of the ligation mixture and incubated on ice for 45 minutes. Thereafter, the DNA uptake is completed by a heat shock of 2 minutes at 42 ° C. The cell suspension is incubated on ice for an additional 10 minutes and finally plated on LB agar (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 1.5% agar) containing 50 mg / l ampicillin , The plasmids contained in the obtained 24 colonies

are isolated by the method of Birnboim and Doyy (see Nucl. Acid. Res., 1, 1513-1523 (1979)). After digestion with various restriction enzymes, a plasmid has the desired structure. This was named pRHW 10 (see Figure 6).

b) Preparation of the plasmid pRHW 12

.Ca. 10 ng of the plasmid pRHW 10 are mixed with BamHI. .geschnitten ,. Subsequently .The Klenow fragment of DNA Pοlymerase I and the 4 deoxynucleoside phosphates added tr! And incubated for 20 Minu ^th at room temperature. The obtained linearized and straight-ended plasmid is purified by phenol extraction and precipitation and then cut with the restriction endonuclease Ncol in a volume of 100 μΐ. The larger fragment is recovered by agarose gel electrophoresis, electroelution and EIutip purification. The fragment a) (see Figure 6 ^ is obtained by digesting 4] ig Avali fragment containing the P9A2 cDNA insert (see above) with NcoI and AluI to yield approximately 2 \ xg of fragment a) obtains.

In the last ligation step, fragment a) and the BamHI / NcoI doubly digested pRHW 10 are ligated in a volume of 10 μΐ, using 10 ng per DNA. The ligation of a filled-in BamHI site to Alul cut DNA again yields a BamHI site.

The resulting ligation mixture is transformed with competent E. coli HB 101 as described above. Of the 40 colonies obtained, one is selected, this receives the name ρRHW 12th

The plasmid is isolated and the EcoRI / BamHI insert is sequenced by the method of Sanger (F. Sanger et al., Proc. Nat. Acad., 7, 5463-5467 (1979)). This has the expected sequence.

c) Preparation of the plasmid pRHW 11

This takes place analogously to Example 5b. 1 μ9 of the plasmid pRHW 10 is digested with BamHI. The sticky ends of the DNA obtained are made blunt by means of the Klenow fragment of DNA polymerase I and the deoxynucleoside triphosphate, then the linearized DNA is cut with Ncol. The larger fragment is using. Agarose gel electrophoresis, electroelution and Elutip column chromatography.

The Ncol-Alul fragment is isolated from clone E76E9 analogously to Example 5b. Subsequently, 10 ng of the vector portion are ligated with 10 ng of the cDNA portion in a volume of 10 μΐ under suitable conditions and using 1 unit T4 ligase. After transformation of the. Obtained DNA mixture in E. coli HB 101 and selection of the obtained 45 colonies on ampicillin-containing LB plates, a clone is selected, which receives the name pRHW 11. After culturing the corresponding clone, the plasmid DNA is isolated. Their structure is evidenced by the presence of several specific restriction endonuclease cleavage sites (AluI, EcoRI, HindIII, Ncol, PstI).

d) Expression of interferon activity by E. coli HB 101 containing the plasmid pRHW 12

100 ml of the bacterial culture are minimal medium up to an optical density of 0.6 at 600 nm in M9, all amino acids except tryptophan (20 ug / ml per amino acid), 1 ug / ml thiamine, 0.2% glucose and 20 ug / ml indole (3) -acrylic acid (IAA) containing the inducer of the tryphtophan operon. Subsequently, the bacteria are pelleted by centrifuging (10 minutes at 7000 rpm), washed once with 50 mM Tris / Cl pH = 8, 30 mM NaCl and finally

· - 47 -

suspended in 1.5 ml of the same buffer. After incubation for 30 minutes with 1 mg / ml lysozyme on ice, the bacteria are frozen 5 χ and thawed. The cell debris is removed by centrifugation at 40,000 rpm for 1 h. The supernatant is sterile filtered and assayed for interferon activity in the plaque reduction assay using human A549 cells and encophalo myocarditis virus. Result: 1 liter of the bacterial culture produced contains 1 × 10. International. Units of interferon. , (A. Billiau, Antiviral Res. 4_, 75-98 (1984)).

Example 6

Compilation of differences in amino acid and nucleotide sequences of Typl interferons

a) Comparison of the amino acid sequences

The pairwise comparison of the amino acid sequences is obtained by listing the first cysteine residue of the mature α-interferon with the first cysteine residue of the amino acid sequences encoded by the cDNA inserts of plasmids P9A2 and E76E9. Both sequences are shown in Figure 7 ^a ls IFN-omega, since no differences between the specific sequences of the · P9A2 or E76E9 clones could be found in the values determined. The only correction made was an insertion of a gap at position 45 of interferon-aA, which was counted as a defect. If the sequence of the omega interferon is a partner, the comparison was carried out considering the usual 166 amino acids. This value is shown in Figure 7 together with the additional 6 amino acids encoded by clones P9A2 and E76E9. The percentage differences are calculated by dividing the

differences obtained by the number 1.66. An additional amino acid thus represents the percentage number 0.6. This gives 3.6% for the 6 additional amino acids of IFN-omega, which are already included in the percentage number.

Comparisons with the β-interferon are made by listing the 3rd amino acid of the mature β-interferon with the first amino acid of the mature α-interferon, or the first amino acid, = encoded by plasmids P9A2 and E76E9. The longest comparison structure of an alpha interferon with beta interferon thus goes over 162 amino acids, giving 2 additional amino acids each for the alpha interferon and beta interferon. These are counted as defects and are shown separately in Figure 7 but are included in the percentage number. The listing of the β-interferon with the amino acid sequences of clones P9A2 or E76E9 is performed in the same manner. But this gives a total of 10 additional amino acids.

b) Comparison of nucleotide sequences

The list of the sequences to be compared is carried out analogously to Example 6b. The first nucleotide of mature alpha interferon DNA is the first nucleotide of the cysteine-encoding triplet of mature alpha interferon. The first nucleotide of the DNA of plasmids P9A2 or E76E9 is also the first nucleotide of the codon for cysteine-1. The first nucleotide of the DNA of β-interferon is the first nucleotide of the third triplet. The comparison is made over a total of 498 nucleotides, when the individual DNAs of the α-interferons are compared with the DNA of the β-interferon, and over 516 nucleotides, when the DNA sequences of the individual α-interferons or β-interferon with those of the plasmids P9A2 and E76E9 are compared. The absolute number of defects is given in the left part of the table in Figure 7 and then in brackets the corresponding percentages.

Example 7 Virus-inducible expression of omega-1 mRNA and NC37 cells

a) Synthesis of an omega-interferon-specific hybridi

10 pmol of the oligonucleotide d (TGCAGGGCTGCTAA) are incubated with 12 pmol of gamma- ³² P-ATP (specific activity:> 5000 Ci / mmole) and 10 units of polynucleotide kinase in a total volume of 10 μl (70 mM Tris / Cl pH = 7, 6, 10mM MgCl ₂ , 50mM DTT) and incubated for one hour at 37 ° C

ditched. Subsequently, the reaction is stopped by heating for 10 minutes at 7O ⁰ C. The radiolabeled Obligonucleotid obtained is mixed with 5 pmol of ssDNA M13pRHW 12 (see Figure 9) in a total volume of 35 μΐ (100 mM NaCl) hybridized by one hour of standing at 50 ⁰ C.

After cooling to room temperature, nick translation buffer, the 4 deoxynucleoside triphosphates and 10 units of Klenow polymerase are added to a total volume of 50 μΐ (50 mM Tris / Cl pH = 7.2, 10 mM MgCl ₂ , 50 μ 9 / ηη 1 BSA, 1mM per nucleotide). The polymerization is carried out by standing at room temperature for one hour, then the reaction is stopped by heating to 70 ⁰ C for five minutes.

The reaction gives a partially double-stranded circular DNA. This is then cut in a total volume of 500 μΐ with 25 units of Avail, using the buffer described by the manufacturer. Here, the double-stranded regions are cut to uniform sizes. The reaction is then stopped by heating to 70 ^° C. for 5 minutes.

b) Preparation of RNA from virus-infected cells

'''"" fiii ι ii _o

100 × 10 ⁶ cells (0.5 × 10 ⁶ / ml) are treated with 100 μl of dexamethasone for 48 to 72 hours - the control contains no dexamethasone. For the purpose of interferon induction, the expression cells are suspended in serum-free medium at a concentration of 5 × 10 / ml and infected with 2 units / ml of Sendai virus. Aliquots of cell culture supernatants are assayed for IFN activity in the P 1-aq-ue-Reduction -As say (Example 5b). The cells were harvested 6 hours after (3 _he virus-infection (by centrifugation 1000 g, 10 minutes), washed (50 ml in NP40 buffer Example 4a), resuspended ice cold NP40 buffer to 9.5 ml by addition of 0 5 ml of 10% NP40 is lysed on ice for 5 minutes, after removal of the nuclei by centrifugation (1000 g, 10 minutes), the supernatant with 50 mM Tris / Cl, 0.5% sarcosine and 5 mM EDTA to pH = 8 is set and stored at -20 ⁰ C. for the isolation of total RNA from the supernatant is extracted once with phenol, once with phenol / chloroform / isoamyl alcohol and einmal.mit chloroform / isoamyl alcohol extracted. the aqueous phase is applied to a 4 ml 5, 7 molar CsCl pad and centrifuged in a SW40 rotor (35 krpm, 20 hours) to free the extract of DNA and remaining proteins The resulting RNA pellet is resuspended in 2 ml TEpH = 8.0 and ethanol The precipitated RNA is then added at a concentration of 5 mg / ml dissolved in water.

c) detection of interferon-omega mRNA

0.2 μΐ of the hybridization sample prepared according to Example 7t> are precipitated together with 20-50 μg of the RNA prepared according to Example 7c by addition of ethanol. In the control experiment, RNA (tRNA) or RNA derived from E. coli transformed with the plasmid pRHW 12 (Example 5) is added instead of the cellular RNA.

24 6 31

The resulting pellets are washed salt-free with 70% ethanol, dried and dissolved in 25 μM 80% formamide (100 mM PIPES pH = 6.8, 400 mM NaCl, 10 mM EDTA). The samples are then heated for 5 minutes at 100 ^ ⁰ C to denature the hybridization · sample immediately adjusted to 52 ° C and incubated for 24 hours at this temperature. After hybridization, the samples are placed on ice and 475 μL Sl reaction mixture (4 mM Zn (Ac) ₂ → 30 mM NaAc, 250 mM NaCl, 5% glycerol, 20 μg ss calf thymus DNA, 100 units of Sl After 1 hour of digestion at 37 ° C, the reaction is stopped by ethanol precipitation.

The pellets are dissolved in 6 μM formamide buffer and separated essentially as samples from DNA sequencing reactions on a 6% acrylamide gel containing 8 M urea (F. Sanger et al., Proc. Nat. Acad 74 , 5463-5467 (1979)).

To. Autoradiography, the dried gel is exposed to a DuPont Cronex X-ray film using the Kodak Lanex Regular Intensivators at -7O ⁰ C.

Legend to Figure 10

Lanes A to C represent the controls.

Lane A: 20 μg tRNA

Lane B: 10 μg RNA from pER 33 (E. coli expression strain for interferon-a2-Arg)

Lane C: 1 ng RNA from pRHW 12 (E. coli expression

Strain for interferon-omega 1)

Lane D: 50 μg RNA from untreated Namalwa cells

- 52 - 24 6 3 1-β

Lane E: 50 μ s RNA from virus infected Namalwa cells

Lane F: 50 μg RNA from dexamethasone pretreated and virus-infected Namalwa. 5 cells

Lane G: 20 μ s RNA from untreated NC 37 cells.

Lane H: 20 μ9 RNA from virally infected NC 37

cell

Lane I: 20 μg RNA from with. Dexamethasone reserved. infected and virus-infected NC 37- '

cell

Lane M: large mark (pBR 322 cut with

Hinfl).

Lanes B and C show that the expected signal can only be detected if an omega-specific RNA is under the RNA molecules. Further, it is shown that even a large excess of the counterfeit RNA does not elicit a background signal (see lane B). Furthermore, the tRNA used as hybridization partner also gives no signal (see lane A).

Lanes G to I show the induction of omega-specific RNA in virus-infected NC 37 cells. Pretreatment with dexamethasone enhances this effect.

Lanes D to F basically show the same result obtained with Namalwa cells. However, induction with omega-specific RNA is not as strong as with NC37 cells. This result is in parallel with the interferon titers measured in each cell supernatant.

^DJ £ <r ο -i: j

This behavior of interferon-omegal gene expression is thus as expected from an interferon type I gene.

Example 8

Isolation of the gene encoding IFN-omegal or related genes:

a) cosmid screening

A human cosmid library (human DNA (male) was cloned in the cosmid vector pcos2 EMBL (A. Ponstka, H.-R. Rockwitz, A.-M. Frischauf, B. Hohn, H. Lehrach Proc. Natl. Acad. Sci. 81, 4129-4133 (1984)) with a complexity of 2 χ ΙΟ ⁶ ) was searched for IFN-omega or related genes.

E. coli DHl (RA, m + _v, rec.A; gyrA96, sup.E) as wurdd

Wirt'verwendet. Initially, Mg cells (= "plating bacteria") were produced. E. coli DHI grows overnight in L-Broth (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl) Supplemented with 0.2% maltose. The bacteria are centrifuged and centrifuged in a 10 mM MgSO 4. Solution added to an ODgQQ = 2. 5 ml of this cell suspension

, 20 with 12.5 × 10 colony forming units of packaged cosmids for 20 minutes at 37 ⁰ C incubated. Subsequently, 10 vol LB are added and the suspension was maintained for 1 hour for expression of kanamycin resistance conferred by the cosmid at 37 ⁰ C. The bacteria are then removed by centrifugation, resuspended in 5 ml of 2.5 LB and streaked in 200 μL aliquots on nitrocellulose filters (BA85, Schleicher and Schuell, 132 mm diameter), which are incubated on LB agar (1.5% agar in L-broth) plus 30 μg / ml kanamycin. Approx. 10,000 to 20,000 colonies grow per filter. The colonies are replica-plated on additional nitrocellulose filters stored at 4 ° C.

One set of the colony filters is processed as described in Example Ic), ie the bacteria are denatured and the single-stranded DNA is fixed to the nitrocellulose. The filters are pre-washed solution at 65 ⁰ C in a 50 mM Tris / HCl, pH = SzO, 1 M NaCl, 1 mM EDTA, 0.1% SDS for 4 hours. The filters are then incubated in a 5 χ Denhardt's (see Example Ic), 6 χ SSC, 0.1% SDS solution for 2 hours at 65 ⁰ C and with about 50 χ 10 cpm nicktranslated, denatured IFN-omegal DNA (HindIII -BamHI insert of the clone pRHW12, see Figure 6) hybridized for 24 hours at 65 ° C in the same solution. After hybridization, the filters are first placed for 3 χ 10 minutes at room temperature in a 2 χ SSC, 0.01% SDS solution and then 3 χ 45 minutes at 65 ⁰ C in a 0.2 χ SSC, 0.01% SDS Solution washed. The filters are exposed to Kodak X-Omat getrocknet.und on S film using an intensifying screen at -7O ⁰ C. Positively responding colonies are localized on the replica filter, scraped off and resuspended in L-broth + kanamycin (30 μg / ml). From this suspension, several μΐ are plated on LB agar + 30 μg / ml kanamycin. The resulting colonies are replica-plated on nitrocellulose filters. These filters are as described above.

32

hybridized with P-labeled IFN-omegal DNA.

From each hybridizing colony, the cosmid was isolated by the method described by Birnboim & Doyl (Nucl. Acids Res., 7, 1513 (1979)). E. coli DH1 was transformed with this cosmid DNA preparation and the transformants were selected on LB agar + 30 μg / ml kanamycin. The transformants were again tested with ³ P-radiolabelled IFN-omegal DNA for positive-reacting clones. One clone each starting from the original isolate is selected and its cosmid produced on a larger scale (Clewell, DB and Helinski, DR, Biochemistry 9_, 4428 (1970)). Three of the isolated cosmids are named cos9, cos10 and cosB. ,

b) Subcloning of hybridizing fragments into PUC8

In each case 1 μg of the cosmids cos9, cos10 and cosB were cut with HindIII under the conditions recommended by the manufacturer (Hew England Biolabs). The fragments were electrophoresed on 1 % agarose gels in TBE buffer and transferred to Southern on a Hitrocellulose filter (Example 4c). Both filters are hybridized with nick-translated omegal-DUA as described in Example 4d), washed and exposed. About 20 μg of each cosmid are cut with HindIII and the resulting fragments are separated electrophoretically. The fragments hybridizing in the preliminary experiment with omegal-DHA are electroeluted and purified over Elutip columns (Schleicher + Schull). These fragments are ligated with HindIII-linearized, dephosphorylated pUC8 (Messing, J, Vieira, J, Gene T9, 269-276 (1982)). E. coli JM101 (supE, thi, (lacproAB), £ "F ^f, traD3 *>, pro AB, lac Z q 1115_7 is transformed with the Ligasereaktionslösung · The bacteria are on IiB agar containing 50 / ug / ml ampicillin , 250 μg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (BCIG, Sigma) and 250 μg / ml isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma) · A blue colouration of the resulting colonies indicates the lack of an insert in pUC8 · In some white clones, the pasmid DBA's were isolated on a small scale, cut with HindIII and separated on 1 % agarose gels · The DNA fragments were seeded on to Efcrc cellulose

32

filter and hybridized as above with ^ P-omega-DHA. Starting from cos9 and cos10, a subclone was selected in each case and designated pRHW22 or pRH57 · Yon cosB were subcloned two DHA fragments which hybridized well with the omegal sample and designated pEH51 or pRH52.

- 56 -. 2 46 3 I S

c) Sequence analysis

The DNA inserted into pUC8 is separated from the vector portion by sections with HindIII and subsequent gel electrophoresis. This DNA, about 10 \ ig, is ligated using _T4 DNA ligase in 50 μΐ reaction solution, the volume to 250 μΐ reacted with nick-translation buffer (Example 4d) and then broken under ice cooling by means of ultrasound (MSE 100W ultrasonic disintegrator, max Output at 20 kHz, five times 30 seconds). Thereafter, the ends of the fragments are repaired by addition of 1/100 vol per 0.5 mM dATP, dGTP, dCTP and dTTP and 10 units Klenow fragment of DNA polymerase I for two hours at 14 ° C. The resulting straight ended DNA fragments are size separated on a 1% agarose gel. Fragments of sizes between 500 and 1000 bp were isolated and subcloned into the SmaI-cut, dephosphorylated phage vector M13mp8. The single-stranded recombinant phage DNA is isolated and sequenced according to the method developed by Sanger (Sanger, F. et al., Proc. Natl. Acad., 7, 543-5467 (1976)). The assembly of the individual sequences into the total sequence is carried out by means of a computer (Staden, R., Nucl. Acids Res. IC), 4731-4751 (1982)).

d) Sequence of Subclone pRH57 (IFN-omegal)

The sequence is shown in Figure 11. This 1933 bp fragment contains the gene for interferon-omegal. The protein coding region comprises nucleotides 576 to 1163. The sequence is completely identical to that of the cDNA clone P9A2. Nucleotide section 576 to 674 encodes a 23 amino acid signal peptide. The TATA box is located in a distance characteristic of interferon type I genes before the start codon ATG (positions 476-482). The gene has some transcriptional polyadenylation signal sequencing sera (ATTAAA at positions 1497-1502, and 1764-1796 respectively, AATAAA at positions 1729-1734 and 1798-1803, respectively), the first of which was used in clone P9A2.

e) Sequence of Subclone pRHW22 (IFN-pseudo-omega2)

Figure 12 shows the 2132 bp sequence of the Hindi II fragment hybridizing with the omegal DNA probe. Cosmid cos 9 reproduced. The result is an open reading frame from nucleotide 905 to nucleotide 1366. The amino acid sequence derived therefrom is reproduced. The first 23 amino acids are similar to those of the signal peptide of type I type interferon. The following 131 amino acids show similarity to omegal interferon up to amino acid 65, with tyrosine being the first amino acid of the mature protein.

Amino acid position 66 is followed by the sequence of a potential N-glycosylation site (Asn-Phe-Ser). From this point the amino acid sequence will be different from that of a type 1 interferon. However, it can be shown that it is possible to produce similarity to the IFN-omegal by suitable insertions and the resulting displacement of the protein reading frame (see Example 9). Therefore, the isolated gene is a pseudogene: IFN-pseudo-omega2, as seen from the point of view of type I interferons.

f) Sequence of Subclone pRH51 (IFN-pseudo-omega3)

The one, from the Cosmid B originating, approximately 3,500 bp and with the omeg'al sample hybridizing HindIII fragment was partially sequenced (Figure 13). The result is an open reading frame from nucleotide position 92 to 394. The first 23 amino acids show the characteristics of a signal peptide. The subsequent sequence begins with tryptophan and shows similarity to IFN-omegal to amino acid 42. Thereafter, the deduced sequence becomes different from IFN-omegal and ends after amino acid 78. The sequence can be changed by transpositions so that they are then large Homology to IFN-omegal (Example 9). The gene is called IFN-pseudo-omega3.

- 58 -. - ^:! ο ο w w

g) Sequence of the Insert of pRH52 (IFN-pseudo-omega4)

The 3659 long, isolated from cosmid B and hybridizing with omegal DNA sequence of the HindIII fragment is shown in Figure 14. "An open reading frame whose translation product partially shows homology to IFN-omegal is located between nucleotide positions 2951 and 3250. After a signal peptide containing 23 amino acids, the further amino acid sequence begins with phenylalanine, the homology to IFN-I being interrupted after the 16th amino acid , continues at the 22nd amino acid and ends with the 41st.

Amino acid. A possible translation would be possible up to the amino acid 77. Analogous to Example 8f) and 8g), a good homology to IFN-omegal can also be produced here by introduction of insertions (Example 9). The pseudogen isolated here is called IFN pseudo-omega4.

Example 9 Evaluation of genes for 4 members of the IFN-ornega family

In Figure 15, the genes for IFN-omegal to IFN-pseudo-omega4 are listed together with the amino acid translation. To better fit gaps in the individual genes are inserted, which are represented by dots. No bases are left out. The base numbering includes the gaps. The amino acid translation of IFN-omegal is retained (e.g., at position 352-355: "CAC" encodes His). In pseudogenes, translation into an amino acid occurs only where it is clearly possible. Based on this listing, it is immediately apparent that the 4 isolated genes are related to each other. For example, e.g. obtained the potential N-glycosylation site (nucleotide positions 301 to 309) in all 4 genes.

- 59 - I - '-> ii

Also, except for IFN pseudo-omega4 at nucleotide positions 611 to 614, there is a triplet that is a stop codon and that terminates a mature protein of 172 amino acids in IFN-omegal. However, IFN pseudo omega2 (nucleotide positions 497 to 499) and IFN pseudo omega4 (nucleotide positions 512 to 514) have premature stop codons in this arrangement.

The degree of relationship between the genes and the amino acid translations can be calculated from the arrangement in Figure 15. Figure 16 shows the DNA homologies between the members of the IFN-omega family. In the pairwise comparison, those positions in which one of the two partners or both have a gap are not counted. The comparison gives a homology of approximately 85% between IFN-omegal DNA and the sequences of the pseudogenes. IFN pseudo omega2 DNA is approximately 82% homologous to the DNAs of IFN pseudo omega3 and IFN pseudo omega4, respectively. Figure 17 shows the results of the comparisons of the signal sequences and Figure 18 shows the comparisons of the "mature" proteins. The latter range between 72 and 88%. However, this homology is significantly greater than that between IFN-omegal and IFN-α's and IFN-β (Example 6). 'The fact that the individual! Members of the IFN-omega family are more distant from each other than those of the IFN-a family, can be explained by the fact that three of the four isolated IFN-omega genes are pseudogenes and open. bar no longer subject to that selection pressure as functional genes.

24 6 3 13

Example 10 fermentation Stairimhaltung:

A single colony of strain HB101 / pRHW12 on LB agar (containing 25 mg / ml ampicillin) is inoculated into tryptone soy broth (OH) ID CM129) with 25 mg / ml ampicillin and incubated at 250 revolutions per minute (rpm). and 37 ^° C. until an optical density (546 nm) of approximately 5 is reached (logarithmic growth phase). Then 10 weight percent of sterile glycerol is added, the culture filled in 1.5-ml portions in sterile vials and frozen at -70 ⁰ C.

preculture:

The culture medium contains 15 g / l Ha ₂ HPO ₄ χ 12H ₂ Oj 0.5 g / l NaCl ₅ 1.0 g / l M ₄ Gl; 3.0 g / l KH ₂ PO ₄ ; 0.25 g / l MgSO ₄ .7H ₂ O; 0.011 g / l CaGl ₂ ; 5 g / l casein hydrolyzate (Merck 2238); 6.6 g / l glucose monohydrate; 0.1 g / l ampicillin; 20 mg / 1 cysteine and 1 mg / 1 thiamine hydrochloride. 4 2 200 ml of this medium in a 1000 ml. Erlenmeyer flask are inoculated with 1 ml of a stock of HB101 / pRHW1 ¥ and shaken for 16 to 18 hours at 37 ⁰ C and 250 revolutions per minute ·

Hauptkulturt

The medium for the fermentation contains 10 g / l (HH ₄ ) ₂ HP0 ₄ ; 4.6 g / l K ₂ HPO ₄ χ 3H ₂ O; 0.5 g / l HaGl; 0.25 g / l MgSO ₄ .7H ₂ O; 0.011 g / l CaGl ₂ ; 11 g / l glucose-honohydrate; 21 g / l casein hydrolyzate (Merck 2238); 20 mg / l cysteine, 1 mg / l thiamine hydrochloride and 20 mg / l 3-β-indoleacrylic acid · 8 liters of sterile medium in a fermenter with 14 liters total volume (height: radius = 3: 1) are inoculated with 800 ml of the IT culture

The fermentation is carried out at 28 ° C, 1000 U / min. (Effigas turbine), a ventilation rate of lvvm (volume / volume / minute) and a starting pH of 6.9. During the fermentation, the pH decreases and is then kept constant at 6.0 with 3N NaOH. If an optical density (546 nm) of 18 to 20 is reached (usually after 8.5 to 9.5 hours fermentation time) is cooled under otherwise identical conditions to 20 ⁰ C and then with 6N H ₂ SO ₄ (without aeration) the pH adjusted to 2.2 and stirred for 1 hour at 20 ⁰ C and 800 U / min, (without aeration). The thus inactivated biomass is then in a CEPA laboratory centrifuge type GLE at 30 000 U / min, centrifuged and frozen at -20 ⁰ C and stored. ,

Example 11

Purification of interferon-omgega-Gly

a) Partial cleaning . _

All stages are carried out at 4 ⁰ C.

140 g. Biomass (E. coli HBlOl transformed with the expression clone pRHW12) are resuspended in 1150 ml of 1% acetic acid (pre-cooled to 4 ° C) and stirred for 30 minutes. The suspension is adjusted to pH = 10.0 with 5 M NaOH and stirred for a further 2 hours. After adjustment with 5 M HCl to pH = 7.5, the mixture is stirred for a further 15 minutes and then centrifuged (4 ° C., 10,000 rpm for 1 hour, J21 centrifuge (Beckman), JA10 rotor).

The clear, supernatant solution is applied to a 150 ml controlled pore glass (CPG 10-350, Mesh Size 120/200) column at 50 ml / h, with 1000 ml of 25 mM Tris / IM NaCl, pH 7, 5 and washed with 25 mM Tris / lM KCNS / 50% ethylene glycol, pH = 7.5 (50 ml / h).

3 j 3

The protein peak containing interferon activity was GESAM melt, dialyzed against 0.1 M Na-phosphate, pH = 6.0 and 10% polyethylene glycol 40,000 overnight and the resultant precipitate collected by centrifugation (4 ⁰ C, 1 hour, 10,000 u / min, J21 Centrifuge, JA20 rotor) (see Table 1).

b) Further cleaning

The dialyzed and concentrated CPG eluate is diluted 1: 5 with buffer A (0.1 M Na phosphate pH = 6.25 / 25% 1,2-propylene glycol) and assayed 0 with a "Superloop" injection device (Pharmacia) , 5 ml / min applied to the MonoS 5/5 (Pharmacia, cation exchanger) equilibrated with buffer A column. Elution is carried out by 20 ml of a linear gradient from 0 to 1 M NaCl in Buffer A also at a flow rate of 0.5 ml / min. The run and the 1 ml fractions were collected and tested for interferon activity by a CPE reduction assay. The active fractions were collected.

Table 1

Volume (ml) Biological test U / ml ⁺ U total 17.3x10 ⁶ Protein (mg / ml) total (mg) U / mg From prey% raw 1150 15000 <l, 3xlO ⁶ 3.6 4140 4180 100 DL 2200 <600 21, OxIO ⁶ 0.74 1628 <600 <5 eluate 124.3 170000 12.3xlO ⁶ 16.8 2088 10000 121 after 41 dialysis 300000 12.6 516.6 23800 71

⁺ CPE reduction test: A549 cells, EMC virus U: units based on interferon-a2 (see Example 2 of EP-AO.115.613: E. CoIi HBlOl transformed with the ex

press clone pER 33) as standard DL: run

Example 12 Characterization of HuIFN-omegal

A. Antiviral activity on human cells

B. Antiviral activity on monkey cells

C. Antiproliferative Activity on Human Burkitt's Lymphoma Cells (Daudi Cell Line)

D. Antiproliferative Activity on Human Cervical Carcinoma Cells (Cell Line HeLa)

E. Acid stability

F. Serological characterization

A. Antiviral activity on human cells

Cell line:. Human lung carcinoma cell line A549

(ATCC CCL 185)

Virus: Mouse Encephalomyocarditis virus (EMCV) Test system: Inhibition of the cytopathic effect

(all titrations were done four times)

A partially purified preparation of HuIFN-omegal with a protein content of 9.4 mg / ml was titrated in a suitable dilution in said test system. The preparation showed an antiviral activity with a specific activity of 8300 IU / mg based on the reference standard Go-23-901-527.

B. Antiviral activity on monkey cells

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

Virus: Vesicular stomatitis virus (VSV) Test system: Plaque reduction test (all titrations fourfold).

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

C. Antiproliferative Activity on Human Burkitt ¹ s Lym phom Cells (Daudi Cell Line)

The humae Burkitt ¹ s lymphoma cell line Daudl was grown in the presence of various concentrations of HuIFN-omegal. After two, four and six days of incubation at 37 ° C, the cell density was determined; Untreated cultures served as controls, all cultures were set up in triplicate. The following figure shows that IFN-omega shows a marked inhibition of cell proliferation at a concentration of 100 antiviral units (IE, see Example 12A) per ml; at a concentration of 10 ΙΕ / ml, a partial, transient inhibition was observed (following symbols are used in the following figure: O control, * 1 IU / ml, C 10 IU / ml, "^ 100 IU / ml).

! Λ

D. Antiproliferative Activity on Human Cervical Carcinoma Cells (Cell Line HeLa)

The human cervical carcinoma cell line HeLa was cultured in the presence of the following proteins or protein mixtures:

HuIFN-omegal (see Example 12A) 100 IU / ml HuIFN-gamma (see Example 12A) 100 IU / ml Human Tumor Ne'Rrose Factor (HuIi ¹ N),> _98% pure, manufactured by Genentech Inc., San Franzisco, USA (see Pennica D. et al., Nature 312 , 724-729, 1984) 100 ng / ml

All binary combinations of said protein concentrations are as above

Each 2 cultures were seeded in 3 cm plastic tissue culture dishes (50,000 cells / dish) and incubated at 37 ° C for six days; then the cell density was determined. Treatment of cells with HuIFN-omegal or HuINF-gamma had little effect on cell growth, while HuIFN-gamma showed a marked cytostatic effect. Combinations of IFN-gamma with IFN-omegal showed synergistic cytostatic / cytotoxic effects.

The following figure shows the result of the experiment:

ii i ι ii,! 1 I! · .- Ml · - .:: I: -ii   T- ; t: m .. ··· -; · ι :::; L- .-..-. ι :::.: "  · "- ι  '.-! : ·.. "Ι ί · ·  :! ·: Γ Li " >. : I! i ii ...,. .- ...:; ι: , ! , !. ^: ·.; · | · Ϊ́'ί ^: · |. · Γ · | 'ί; i -I'I Ir ₁ : : j f ·; ^: -! i. ^: t ^: · :; ·: J..i •;. |.   .: ·-Γ.ί... t i -V- · .- ::::. | :: 'r [- ilili ι:  Wed: ·· -   .VirrLT '·' '· *'; --- , , , I. ^: ί i ί " '.:' 'ι.' ;:; : ..:; ·· :: · (·. ·: :::. ι. :! , ρ - · η hl • Γ ί. "  rlüiili: · ι : !! ^; V ί ..-:: · '! ^: ^! ί-, Λ -ii '. ¹ "'>^; 1 ^: I ;;;.: ; .. r, -.-, · .-.:;) -. .Τ + ΟΓ " 'I I ! .: ''. ''" : i:! ' , [ .ι · - i ]: ':'' 1 - -t I I.; : ":::; :; ί · ..: i · ·; :!: -.Ir '. >) <: < "ΐ" * t ° ^u m £ m  i ' I · · ·: ¹ μ ::. π I - / ::: - - J- ._: :: T .. :.; '.: .: ..... .. ... - '·' -. '. * I " · - · · Η.Ι: : __ ': ^.-': '. ί ^;    : 1:., .1 ι :! , Ff.Li •; -; 1 ' : . : ..:!: I: ι. · ·: -: r.ri: - rl. :; \ \\ ύ - · i -; - ι-: ί ·: • j-. · :. '. :: ι ι ι. ! , -_> I.j .. "· Ί ! LlJ'l. - ii, ~ .:}. ^: i ".!:. ·· -H-tv γ · ~ "ί ;: j -: ^-:.;  T-ili " : '.-.  ":.] IMF - i-Lili 'JZ'. ι .1 ^ :: ^ V Kr'rf. " ".-JI .: ' : ::.; ir "* '" ι -ί- · ^ -: Ε - i · hit : IV.'X \ i .:. '; Γ-Γτ.-ν-ΐτ.ΐ-Ξ " :. · (: "• · · ψ1ζί ι2 I - .: · · ·. '.'-. L: · - ":': I ^ M kr? i: · ··:: r lit J: l ~. ~ - ~ :: irl ^: .l ·   ; ,  ~ · - , : .- L: ~ .z.z :: tr.i '' :: " 'Ti .: .t .. ι ;. :; j .: r:; ir; r :: , - ..; - τ: · ::.! , , _. ._ ......., : \ -. ": - |:: r .: r. .r Τ'-Γ'- "": . ;, '; · ... ι .; '..  τ ··. ·:! ... '. ·.! , Μ. ;. ::!. ·;: |.   · Ι ·:. - I - I  rl · _;. · .; , ·; i \. ': ..'. '. ': f:' i '' ^:: P-i-i ΊΊ i ii-i: -:! · :.

30 50 100

CELL / SHELL

The following symbols are used in the figure above; C untreated control, T HuTNF, O HuIFN-omegal, G HuIFN-gamma.

- 67 - / 4 6 3 I c

E. Acid stability

To examine the acid stability of HuIFN-omegal a diluent mentioned in Example 12A preparation in cell culture medium (U / min.I 1640 with 10% fetal calf serum) was adjusted by means of HCl to pH 2, incubated 24 hours at 4 ⁰ C, and then neutralized with NaOH. This sample was titered in the antiviral assay (see Example 12A); it showed 75% of the biological activity of a control incubated at neutral pH; IFN-omegal is thus to be designated as acid-stable.

F. Serological characterization

To determine the serological properties of HuIFN-omegal compared to HuIFN-a2, samples (diluted to 100 ΙΕ / ml) were pre-incubated with equal volumes of solutions of monoclonal antibodies or polyclonal antisera for 90 minutes at 37 ° C; then the antiviral activity of the samples was determined. The table below shows that HuIFN-omegal can only be neutralized by an antiserum against leukocyte interferon at a relatively high concentration, but not by antibodies directed against HuIFN-a2 or HuIFN-beta. HuIFN-omegal is therefore not serologically related to either HuIFN-a2 or HuIFN-beta:

Antiserum dilution neutralization of

monocl. Antibodies ] xg / TRl HuIFN-a2 HuIFN-omegal

EBI-1

-1 ¹⁾

EBI -3

L3B7 ²⁾

Sheep anti-Leukocyte -I FN

3)

Rabbit anti-HuIFN-a2

Sheep anti-HuIFN-ß ⁴⁾

10

100

1000

10

100

1000

100

1000

1:50 000

1: 5,000

1: 500

1:50

1: 1 000 1: 100 1:10

1:

O O O

O O

= see EP-A-O.119.476

= A., Berthold et al. in Arzneimittelforschung 35 , 364-369 (1985)

= Research Reference Reagent Catalog No. G-026-502-568 · = Research Reference Reagent Catalog No. G-028-501-568 Research Resources Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.

Symbols: - not tested, O no neutralization, + partial neutralization, +++ complete neutralization

2 4 6

Patent application in the GDR No. AP C12N / 279.375.4 Case 12/010-Fl

Preliminary remarks; see example 1 ^o '

The construction scheme for the IFN-omegal yeast expression vector (see picture) is not to scale.

The starting plasmid pES103 is obtained by introducing the approximately 1450 bp ADHI promoter fragment (BamHI-XhoI) from the plasmid AXalI (see G. Ammerer: Methods Enzymology 101 , 192-201 (1983), Academic Press) in pUC 18 (see Pharmacia PL, # 27-4949-01).

The starting plasmid pJDB207 (see VD B eggs: Gene cloning in yeast, Ed. R. Williamson: Genetic engineering Vol. 2_, 175-203 (1981), Academic Press, London) was used in E. coli RR1 under DSM number 3181 in the German Collection of Microorganisms, Grisebachstraße 8, D-3400 Göttingen, deposited by the same applicant on December 28, 1984.

Enzyme reactions were carried out according to the manufacturer's instructions, other techniques are standard techniques and can-. nen z. In the book "Molecular cloning - a laboratory manual" by T. Maniatis (Cold Spring Harbor, 1982).

In the yeast transformation, the following yeast media were used:

YNB + CAA medium (1 liter):

6.7 g YNB without amino acids 5 g casamino acids water

3897j ·

YPD medium (1 liter): 10 g yeast extract 20 g Bacto-Peptone water

χ minus leu drop out solution (100 ml): 0.25 g tryptophan 0.20 g arginine 0.10 g histidine 0.60 g isoleucine 0.40 g lysine 0.10 g methionine 0.60 g phenylalanine 0.50 g Threonine 0.12 g adenine 0.24 g uracil water Sterile filtration

Medium for yeast culture:

100 ml autoclaved YNB + CAA medium or YPD medium 4 ml 50% glucose 4 ml 50 χ minus leu drop out solution

Sorbitol plates (600 ml): 109.2 g sorbitol 12 g glucose

4, 02 g YNB 15 g agar water

Autoclave and cool to 45 ^° C., then add the drop-out solution and plate

-71- 246

Topagar (1 liter):

g sorbitol 20 g glucose

6, 7 g YNB 25 g agar water autoclave

SED (25 ml):

12.5 ml 2 M sorbitol 1.25 ml 0.5 M EDTA 11.25 ml dist. Water 192.75 mg DTT (Dithiothreitol) Sterile filtration

SCE (25 ml):

12.5 ml 2 M sorbitol 5 ml 0.5 M Na ₃ citrate 0.5 ml 0.5 M EDTA 7 ml sterile, dist. water

CAS (25 ml):

12.5 ml 2 M sorbitol

2.5 ml of 100 mM CaCl ₂

0.25 ml of 1 M Tris pH 7,

9.75 ml sterile, dist. water

PEG solution (20 ml):

4 g PEG 4000 (polyethylene glycol, SERVA) 2 ml 100 mM CaCl ₂

0.2 ml 1 M Tris pH 7, 17.8 ml dist. Water sterile filtration

SOG (5 ml):

2.5 ml 2 M sorbitol

1.7 ml of YPD

0.3 ml 100 mM CaCl ₂ 12.5 μΐ 50 χ minus Leu drop out solution

0.5 ml of dist. Water sterile filtration

Example ή3 , '

Construction of a yeast expression vector for IFN-omegal

a) Preparation of the yeast λDHI promoter fragment

80 μg of the plasmid pES103 are digested twice in 500 μl solution with BamHI and Xhol. The approximately 1400 base pair (bp) promoter fragment is separated from the vector via a 1% agarose gel, isolated from the gel by electroelution and precipitated. The fragment is taken up in 40 μM TE buffer (10 mM Tris, 1 mM EDTA, pH 8).

b) Preparation of the vector pJDB207

10 μg pJDB207 are cut in 100 μl solution with BamHI and thereby linearized. In order to avoid subsequent religation, the 5'-terminal phosphate groups are removed by treatment with calf intestinal phosphatase (Calf intestine phosphotase, CIP). The linear form is separated on a 1% agarose gel from any uncut plasmid, isolated therefrom by electroelution and precipitated. The vector DNA is dissolved in 20 μM TE buffer.

- 73 - 2 i 6: m c

c) expression vector for IFN-omegal

50 μg of plasmid pRHW12 (Example 5) are linearized in 600 μΐ solution with HindIII. By adding the Klenow fragment of DNA polymerase I (10 units) and the four deoxynucleoside triphosphates (25 μΜ each) and incubating at room temperature, the 5 'overhanging ends are converted to straight ends. The linear form of the plasmid is purified by agarose gel electrophoresis followed by isolation. The fragment is dissolved in 50 μΐ TE buffer.

To generate ends compatible with the promoter fragment, Xhol linkers must be attached to the ends of the linear pRHW12. 3 μΐ Xhol linkers (0.06 OD ₀₁ ^, Pharmacia PL, # 27-7758-01, formula dCCCTCGAGG)) are phosphorylated in 20 μΐ using 5 units of T4 polynucleotide kinase and rATP. After inactivation of the enzyme by heating for 10 minutes at 70 ⁰ C 5 μΐ of this solution are combined with 10 μΐ linear pRHW12 and a total of 20 μΐ reaction solution with T4-DNA ligase ligated (16 hours at 14 ⁰ C). The ligase is inactivated by 10-minute treatment at 70 ⁰ C, and the reaction volume with 1 χ medium buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl ₂₎ brought to 150 μΐ.

By treatment with 100 units of Xhol, the Xhol specific 5 'overhangs are released. The Xhol-terminated, linear pRHW12 is purified by agarose gel electrophoresis and electroeluted from the gel. 5 μΐ promoter fragment (point a) are added before precipitation. After precipitation, the DNA molecules are taken up in 14.5 μΐ TE buffer and ligated for 16 hours at 14 ° C after addition of the ligase buffer and 5 units of T4 DNA ligase. After heat inactivation of the enzyme, the volume of the solution is brought to 200 μΐ with 1 χ medium buffer and the DNA cut with BamHI. The desired DNA is obtained by agarose gel purification and dissolved in 20 μM TE buffer.

d) Ligation of the fragments

The final expression vector is obtained by ligating 5 μΐ of the BamHI fragment (promoter and IFN-omegal gene) with 1 μΐ of linearized pJDB207 vector (yeast 2μ terminator and yeast 2μ origin of replication, Leu2 marker, E. coli origin of replication, ampicillin resistance gene) in total 10 μΐ solution in the presence of 1 unit T4 DNA ligase.

e) transformation

10 μΐ competent E. coli HBlOl cells are transformed by addition of 5 μΐ ligase solution and plated on LB agar with 100 μg / ml ampicillin.

Twelve of the resulting clones were picked at random and the plasmids contained therein were isolated on a mini scale (1.5 ml cultures). After cutting these plasmids with various restriction enzymes and agarose gel electrophoresis, a plasmid having the desired construction was selected and designated ρY-JDB-HuIFN-omegal.

f) Yeast Transformation ·

As preculture 25 ml of YPD + minus Leu drop out solution are inoculated with a yeast colony (strain GM3-C2). The culture is grown overnight at 30 ^° C. 100 ml YPD + minus Leu drop out solution are inoculated with 100 μΐ of the preculture and at 30 ⁰ C up to a 0D ^ _nn of 1

₇ 600 nm

grow (about 4x10 cells / ml). The yeast cells are centrifuged off (5 minutes at 50O0 rpm). The pellet is resuspended in 10 ml of SED and incubated at 30 ^° C. for 10 minutes. The cells are again centrifuged off (6 minutes at 2500 rpm). The cells are resuspended in 10 ml of SCE.

- 7s-

Are added 100 μΐ Glusulase (B-glucuronidase, arylsulfatase, Boehringer-Manhheim) and incubated for 30 minutes at 30 ⁰ C. This creates the spheroplasts. The spheroplasts are centrifuged off (5 minutes at 2000 rpm). The pellet is washed once with CAS and resuspended in 500 μΐ of GAS.

120 μΐ spheroplasts are mixed with 6 μg of plasmid DNA (pY-JDB-HuIFN-omegal) and incubated for 15 minutes at room temperature. After addition of 1 ml of PEG solution, the mixture is incubated for a further 15 minutes at room temperature and then the cells are centrifuged off (5 minutes at 2000 rpm). The pellet is resuspended in 150 μΐ SOG and incubated for 30 minutes at 30 ⁰ C. Thereafter, 5 ml Topagar + 250 μΐ 50 χ minus Leu drop out solution are added and the cells plated on sorbitol plates. After 2 to 4 days of incubation at 3O ⁰ C, colonies develop.

g) He'e-ze lly sate

25 ml YNB + CAA medium + 1 ml 50% glucose + 1 ml 50 χ minus leu drop out solution are inoculated with a transformed yeast colony. The incubation beträgt.zumindest for 40 hours at 30 ⁰ C. 13 ml of this culture are centrifuged at 4500 rpm for 5 minutes. The pellet is resuspended in 0.5 ml of 2 M guanidinium hydrochloride while vortexing. After adding 2 ml of glass beads (diameter: 0.4 to 0.5 mm), shake vigorously for 10 minutes at 4 ° C. Thereafter, the supernatant is transferred to an Eppendorf vessel and incubated at 0 ⁰ C. The glass beads are washed once with 0.5 ml of 7 M guanidinium hydrochloride, and the washing solution is combined with the first supernatant. The solution is centrifuged for 1 minute in an Eppendorf centrifuge. The supernatant is tested for interferon activity in the CPE reduction assay.

Yield: 1 χ 10 units IFN-omegal / l yeast culture

Claims (7)

246 Claims - Case 12/010 1. A process for the preparation of novel human interferon peptides of type 1, characterized in that a suitable host organism containing a coding sequence for the new interferon peptides, wherein the coding sequence is characterized in that the coding sequence under stringent conditions, which allow a homology greater than 85% to be detected, with the inserts in the Pst-1 cleavage a) of the plasmid E76E9 (deposited in E. coli HB 101 in the DSM under the number DSM 3003) or b ) of the plasmid P9A2 (deposited in E. coli HB 101 in the DSM under the number DSM 3004) or hybridized with degenerate variations of these inserts, is transformed, this information is expressed to produce the corresponding interferon in the host organism and the resulting interferon peptide is isolated from this host organism. 2 i 6 65 7 0 7 5 Leu Gin Gin VaI Phe Asn Phe Ser His Lys Ala Leu Leu Cys Cys TTA CAG CAG GTC TTC AAC TTC TCA CAC AAA GCG CTC CTC TGC 1198 80 85 90 Met Glu His Asp Leu Pro Gly Pro Thr Pro His Phe Thr Ser Ser ATG GAA CAT GAC CTT CCT GGA CCA ACT CCA CAC TTT ACG TCA TCA 1243 95 100 105 Ala Ala GIy Thr Pro GIy Asp Leu Leu GIy Ala GIy Asp GIy Arg GCA GCT GGA ACA CCT GGA GAC CTG CTT GGT GCA GGA GAT GGG AGA 1288 HO 115 120 Arg Arg Ser Trp Gly Gin Trp Vai GIu Gly Ser Thr Leu Ala AGG AGA AGC TGG GGG CAG TGG GTG ATT GAG GGC TCT ACA CTG GCC 1333 125 130 Leu Arg Arg Tyr Phe Gin Glu Ser He Ser Thr TTG AGG AGG TAT TTC CAG GAA TCC ATC TCT TGA ACC AAGAGAAGAAA 1380 TAAAGATTGTGCCTGGGAAGTTGTCAGAGTGGAAATCATGAGATCCTTTTCATCCACAA 1439 GTTTGCAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGAAATGATTCT 1498 CATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCA 1557 TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA 1619 AGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC 1675 CCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTA 1734 TATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTCACTTTTTCA 1793 TATTAAGTTTGCATTTTGTTTTATTAAATTCATATCAAAGAAAACTCTGTAAATGTTTC 1852 TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTGAAAATATGGTAAAA 1911 TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG 1970 TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG 2029 AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT 2088 ACAAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 2132 λ 4 Figure 13 (IFN pseudo-omega 3) CCATG 5 CATAGCAGGAATGCCTTCAGAGAACCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG 64 -20 -16 Met VaI Leu Leu Leu Leu Leu CTAGGCCAGCAGCACCCTGGTTTCCCA ATG GTC CTC CTG CTT CCT CTA CTC 115 -10 -5 -1 VaI AIa Leu Pro Leu Cys His Cys GIy Pro VaI GIy Ser Leu Ser GTG GCC CTG CCG CTT TGC CAC TGT GGC CCT GTT GGA TCT CTG AGC 160 2. The method according to claim 1, characterized in that the coding sequence under stringent conditions has a homology of more than 90%. 3 ' sample Illustration 216 a i xSau3A * I  fl ClI , AAGCTTAAAS AIO TCTl , TTCCAATTTC TAC ACA CIA g) j, "HMIJISMU , Hind / SiilA [GAT CTO CCT CAO AAC CAT CGC __ - ^ - COA CTC TTC CTA CCO - -wai - Ir. - " _t Me * l X Hind III I II xHindlll x Bam Hl, Klenow - fill in xNcol TT "Xl.I ligase Figure 7 A »lno acid differences m β »r * O * M« ο ^ u f> φ (^ γ * · ¹ O ^ Min ^ r ^ fn ^ ffTin α3 (Νθ · "·" 0ι (βσνθ
vr * rir> r * - \ oiAvoP + + + + + + + + + / O m * O * ntf% tn ^ OtHfn- / r ^ ui * - S »- · - O #wf» in t * "
(N CN ^ ^ * «™ Γ »CD Kl Λ η * ^ Q * r <r o 05 (M (N (N OS ^ O) ν · m ^ r * - is Γ * in ρ ·· · - ^ cn O - ^G -Q -        = · ^ Ί ί δ Figure 8a 130 140 150 IFN αΑ gaggagtttgGCaaccagttccaAaaggct IFN αΒ gaggagtttgatgataaacagttccagaag IFN aC gaggagtttgatggcaaccagttccagaag IFN ctD gaggagtttgatggcaaccagttccagaag IFN ctF gaggagtttgatggcaaccagttccagaag IFN ctG gaggagtttgatggcaaccagttccagaag IFN ctH gaggaatttgatggcaaccagttccagaaa IFN otK gaggagtttgatggccaccagttccagaag IFN ctL gaggagtttgatggcaaccagttccagaag IFN omegal gagatggtaaaagggagccagttgcagaag IFN beta gagattaagcagctgcagcagttccagaag ~ 69 Figure 8b 340 350 360 IFN omegal GgggCaaTtAGcaGcCctGCActgaccTtg IFN oA ctgatgaaggaggactccattctggctgtg 3. The method according to claims 1 and 2, characterized in that the coding sequence as an insert in the Pst-1 interface a) of the plasmid E76E9 (deposited in E. coli HB 101 in the DSM under the number DSM 3003) or b) of the plasmid P9A2 (deposited in E. coli HB 101 in the DSM under the number DSM 3004) or as a degenerate variation of these inserts. r>. η lur -if! - 3Γ ^ Γ? ζ 4. The method according to claim 3, characterized in that the coding sequence is the formula TGT GAT CTG CCT CAG AAC 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 TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 135 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 180 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 225 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 270 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 315 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 360 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 405 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 450 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 495 GAT AGA GAC CTG GGC TCA TCT 516 having. 5 10 15 Phe Asp Leu Pro Gin Asn Arg GIy Leu Leu Ser Arg Asn Thr Leu TTT GAC CTG CCT CAG AAC CGT GGC CTA CTT AGC AGG AAC ACC TTG 3064 20 25 30 AIa Phe Trp AIa Lys Cys Argue Ser Thr Phe Leu Cys Leu Lys GCA TTC TGG GCC AAA TGC AGA ATC TCC ACT TTC TTG TGT CTC AAG 3109 35 40 45   30 Asp Arg Arg Asp Phe Arg Phe · Pro Leu GIu Met Trp Met Ala VaI GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 3154 50 55 60 ITe Cys Arg Arg Pro Arg Leu Cys Leu Ser Ser Met Arg Cys Phe ATT TGC AGA AGG CCC AGG CTG TGT CTG TCC TCC ATG AGA TGC TTC 3199 , 65 70 75 Ser Arg Ser Ser AI Ser Ser Pro GIn Ser Ala Pro Leu Leu Pro AGC AGA TCT TCA GCC TCT TCC CCA CAG AGC GCT CCT CTG CTG CCT 3244 - ΛΛΟ - GIy Thr GGA ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG 3300 CCTGGAGTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGGGTGATTGGGA 3359 CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAATCCAGAGAATCTAC 3418 CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA 3477 TCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGACCTGGGGTC 3536 TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC 3595 TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA 3654 AGCTT 3659 Figure 15 MALLFPLLAALVMTSYSPVGSLGCOLPQNH ATGGCCCTCCTGTTCCCTCTACTGGCAGCCCTAGTGATGACCAGCTATAGCCCTGTTGGATCTCTGGGCTGTGATCTGCCTCAGAACCAT HALLFPLLA .A LEVCSCGSSGSLGYNLPQNH ATGGCCCTCCTCTTCCCTCTACTGGCAGCCCTAGAGGTGTGCAGCTGTGGCTCTTCTGGATCTCTAGGATATAACCTGCCTCAGAACCAT MVLLLPLLYALPLCHCGPVGSLSWDLPQNH atggtcctcctgcncctctactcgtggccctgccgcttlgccactgtggccctgttggctctgagctgggacctgcctcagaaccat mvll-lvllvalllcqcgpvgslgfdlpqnr atggtcctactgcttgttctactggtggccctgctgctttgccaatgtggccctgttggatctctgggctttgacctgcctcagaaccgt IO 20 30 40 50 60 70 80 90 GLLSRNTLVLLHQMR-RISPFLCLKORROFR GGCCTACTTAGCAGGAACACCTTGGTGCTTCTGCACCAAATGAGGAGAATCTCCCCTTTCTTGTGrCTCAAGGACAGAAGAGACTTCAGG GLLGRNTLVLLGQMRR 1 SPFLCLKORSDFR GGCCTGCTAGGCAGGAACACCTTGGTGCTTTTGGGCCAAATGAGGAGAATCTCTCCCTTCTTGTGTCTAAAGGACAGAAGTGACTTCAGA GLLSRNTLALLGQMCRISTFLCLKORRDFR GGCCTACTTAGCAGGAACACCTTGGCACTTCTGGGCCAAATGTGCAGAATCTCCACTTTCTTGTGTCTCAAGGACAGAAGAGACTTCAGG GLLSRNTLA LGQM RISTFLCLKORRDFR GGCCTACTTAGCAGGAACACCTTGGCA.TTCTGGGCCAAATG..CAGAATCTCCACTTTCTTGTGTCTCAAGGACAGAAGAGACTTCAGG 100 UO 120 130 140 150 160 170 180 fpqemvkgsqlqkahvmsvlhemlqqifsl ttcccccaggagatggtaaaagggagccagttgcagaaggcccatgtcatgtctgtcctccatgagatgctgcagcagatcttcagcctc fpqekvevsqlqkaqamsflydvlqqvf l ttcccccaggagaaggtggaagtcagccagttgcagaaggcccaggctalgtctttcctctatgatgtgttacagcaggtcttcaa.ctt fplem ogsqlqkapalsvl hemlqhifsl ttccccctggagatg.tggatggcagtcagttgcagaaggccccggccctgtctgtcctccatgagatgcttcagcagatcttcagcctc fplem ogshlqkaqavsvlhemlqqifsl ttccccctggagatg. tfgatggcagtcatttgcagaaggcccaggctgtgtctgtcctccatgagatgcttcagcagatcttcaucctc 190 200 210 220 230 240 250 260 270 FHTERSSAAHNMTLLOQLHTGLHQ.Q LQ Ή LE TTCCACACAGAGCGCTCCTCTGCTGCCTGGAACATGACCCTCCTAGACCAACTCCACACTGGACTTCATCAGCAACTGCAAC. ACCTGGA HTKRSSAAWNMT-FLD Q LHTLRHQQLE HLE .CTCACACAAAGCGCTCCTCTGCTGCATGGAACATGACCTTCCTGGACCAACTCCACACTTlACGTCATCAGCAGCTGGAAC. ACCTGGA FPTECSSAAWNMTLL OQ LHTGLHLQLE CLE TTCCCCACAGAGTGCTCCTGTGCTGCCTGGAACATGACCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTuGAAT.GCCTGGA FPTERSSAAWNMTLLOQLHTGFHQQLE SLE TTCCCCACAGAGCGCTCCTCTGCTGCCTGGAACATGACCCTCCTGGACCAGCTCGACACTGGATTTCATCAGCAGCTCGAATAGCCTGGA 280 290 300 310 320 330 ^- 340 350 3CÜ TCLLQVVGEGESAG AISSPALTLRRYF GACCTGCTTGCTGCAGGTAGTGGGAGAAGGAGAATCTGCTGGG GCAAH AGCAGCCC TGCACTGACCT TGAGGAGGT ACTTC TCLVQEMGEGE AG VIEGSILALRRYF GACCIGCTTGGTGCAGGAGATGGGAGAAGGAGAA- ^ GCTGGGGGCAGrGGGTGATTGAGGGCTCTACACTGGCCTTGAGGAGGTATTTC SCLGQTKREE. ESVG VIG PTLALRTYF GTCTTGCTTAGGCAGACAAAAGAGAGGAAGAATCTGTGGGG GTGATTGGGG.CCCTACACTGGCCTTGAGGACGTACTTT SCLGQATGEEESVG VlG PTLALRRYF GTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGG GTGATTGGGA. CCCTACACTGGCCTTGAGGAGGTACTTC 370 380 390 400 410 "420 430 440 450 QGIR VYLKEKKYSOCAWEVVRMEIMK CAGGGAATCCGT GTCTACCTGAAAGAGAAGAAATACAGCGACTGTGCCTGGGAAGTTGTCAGAATGGAAATCATGAA Q IH LYLKEKK "DCAWEVVRVEIMR CAGGA. ATCCAT CTCTACCTGAAAGAGAAGAAATAAA. .GATTGTGCCTGÜGAAGTTGTCAGAGTGGAAATCATGAG QGMH IYLEEKKYSDCAWEVVRVE VK CAGGGAATGCATGGGAATCCAGGGAATCTACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGlGGAA.TCGTGAA QGIH IYLKEKKYSOCA'EVVRMEIHK CAGGGAATCCATGGGAATCCAGAGAATCTACCTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAA.TCATGAA 460 470 480 490 500 510 520 530 540 SLF LSTNMQERLRSKDROLGSS * ATCCTTGTTC ... TTATCAACAAACATGCAAGAAAGACTGAGAAGTAAAGATAGAGACCTuGGCTCATCTTGA S F SSTSLQERLRSKOEDLGSS · ATCC ... TTT ... TCATCCACAAGTTTGCAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGA SFSSSSTNLQEGLRSKOEDLGSS · ATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGACCTGGGTTCAI CCTGA SFS SSTOLQ GLRSKOEDLGSAL ATCCTTCTCT ..TCATCAACAGACTTGCAA ..GGACTGAGAAGTAAGGATGAAGACCTGGGGTCTGCTTTA 550 560 570 580 590 600 610 Figure 16 ..i 4 O Upper right part: number of identical bases / total compared bases. Lower 1 Inker part: percent homology η 7 ο c Ο7ΊΓ0 η ι: ο Figure 17 Right upper part: number of identical amino acids of a total of 23 amino acids. Left lower part: percent homology. , 5 10 15 Trp Asp Leu Pro GIn Asn His Glu Leu Leu Ser Arg Asn Thr Leu TGG GAC CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 205 20 25 30 Ala Leu Leu GIy GIn Met Cys Arg He Ser Thr Phe Leu Cys Leu GCA CTT CTG GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TGT CTC 250 35 40 · -45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Leu GIu Met Trp Met AIa AAG GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 295 " 50 55 60 ^Va l ^Ser Cys ^Ar 9 ^{Ar <} 3 ^{Pro Ar} 9 ^{Pro C} Y ^{S Le} u Ser Ser Met Arg Cys GTC AGT TGC AGA AGG CCC CGG CCC TGT CTG TCC TCC ATG AGA TGC 34 0 65 70 "75 Phe Ser Arg Ser Ser AIa Ser Ser Pro GIn Ser Ala Pro Leu Leu TTC AGC AGA TCT TCA GCC TCT TCC CCA CAG AGT GCT CCT CTG CTG 385 Pro GIy Thr · · CCT GGA ACA TGA CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA 4 40 ATGCCTGGATGCTTGCTTAGeGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC 499 558 617 TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA CCTGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG 676 735 772 TGTCTTTGGTCATTTCAATAGGTTCTTATTTCTGCAG - λό% - '2 4 6 3 3 Figure 14 (IFN-pseudo-omega4) AAGCTTTGGGCATGACCTAGTAGGTGACTCTT 3 2 AGTTGGAGTGGTCAGTTGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCCCC 91 ACGTTTGCAGCCGTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCCACTGGA 150 GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG 209 268 327 TAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC AGGGGTTCCCTGCCTGGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTGG 386 44 5 504 CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA .563 GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA GTGTTTAGCCAGAGGAGTGGGGTGGCTGCATGCTGGTGTAAGTATGGGACTTCACTTCT 622 681 740 TGGGAAACAGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAAACAAAGAGC TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 799 8 58 917 GCAGTGGCAGAGGTÄGGATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA "CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGCAGGGGGCC 9 76 TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGGACTCCTCTTCATA 1035 TAGTGGCGATGATGTGCTGGAGGTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCCA 1094 GCCAGAGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTGAGGAGa ^- CGAGGCAAG 1212 GGAAGAATGCCTGTGCTGGAGTCTCTGGTCAGGTGGCTCTGCCACAGAGGAGAGGTGAC 1271 CACCAGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330 ¹ TGGGGATCTGGAGCAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGg 1389 CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1507 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566 CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625 CATTTTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684 CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG 1802 GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1861 GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920 CAGCAACAATGGCAACCTACCATTCCCTTTGGAGCGCCATCCCAGGGATATTCGAAACT 1979 GCTGTCCACTAGAAAACAGTGGTGGAGGTGACTGGAGACCCCAGTGGAGAGTTTCACCT 20 38 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAACCAATCTGACTGCTTCCCCGTA 2097 GAGCTGCTGGACTGTGCTGGGTGGCTGCTCCAGTCCCTAGCTGCCTTGGACTCCCGAGA 2156   ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC 2215 CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGGCTGGATTCCC 2274 AAGTCGAGTGGGTCTTACCCTGAGACAGGCCATGGAAGGTGGGCCTGTCATTGTCACTG 2333 CCCAGCGCCCTGGATGAAACCCCTTTCCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC 2392 TTGGCTGGAGTTATAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAAGGCTCCAGGGTAC 2451 CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA 2510 ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 2569 TGGGAGAAGCGTGGGTCCCCAGGGCTGCTCACTTACTCACCACTTCCCTGGGCAGGAGA 2628 GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT 2687 TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG 2746 AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGAGAGTGGCACACACTAGCA 280 5 GGTTCCAGTCGGCCATCTTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAAGCCATT 2864 GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2923 -20 -16 Met Vai Leu Leu Leu Vai Leu Leu CTAGGCCAGCAGCACCCTCGTT.TCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 , 20 -iO · -5 -i VaI Ala Leu Leu Leu Cys Gin Cys GIy Pro VaI GIy Ser Leu GIy GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT GGA TCT CTG GGC 3019 5 10 15 Tyr Asn Leu Pro GIn Asn His GIy Leu Leu GIy Arg Asn Thr Leu TAT AAC CTG CCT CAG AAC CAT GGC CTG CTA GGC AGG AAC ACC TTG 1018 20 25 30 VaI Leu Leu GIy GIn Met Arg Arg He Ser Pro Phe Leu Cys Leu GTG CTT TTG GGC CAA ATG AGG AGA ATC TCT CCC TTC TTG TGT CTA 1063 35 '40. 45 Lys Asp Arg Ser Asp Phe Arg Phe Pro Gin GIu Lys VaI GIu VaI AAG GAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG GTG GAA GTC 110 8 50 55 60 Ser GIn Leu GIn Lys Ala.Gin Ala Met Ser Phe Leu Tyr Asp VaI AGC CAG TTG CAG AAG GCC CAG GCT ATG TCT TTC CTC TAT GAT GTG 1153 5 10 .15 Cys Asp-Leu Pro Gin Asn His Giu Leu Leu Ser Arg Asn Thr Leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 6 89 20 25 30 ' VaI Leu Leu His Gin Met Arg Arg He He Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 734 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin GIu Met VaI Lys GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 7 79 50 55 · 60 Ser GIn Leu GIn Lys Ala His Vai Met Ser Vai Leu His Giu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 824 65 70 75 Leu Gin Gin He Phe Ser 'Leu Phe His Thr GIu Arg Ser Ser AIa CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 8 69 80 85 90 AIa Trp Asn Met Thr Leu Leu Asp-Gin Leu His Thr Gily Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 914 246 3) 8 46 3) 95 100 105 Gin Gin Leu Gin His Lei Giu Thr Cys Leu Leu Gin VaI'VaI GIy CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 959 110 115 120 GIu GIy GIu Ser Ala GIy Ala He Ser Ser Pro AIa Leu Thr Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 1004 125 130 135 Arg Arg Tyr Phe Gin Gly Arg VaI Tyr Leu Lys GIu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 1049 140 145 150 Tyr Ser Asp Cys AIa Trp Glu VaI VaI Arg Met GIu He Met Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 1094 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Giu Arg Leu Arg Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 1139 170
Asp Arg Asp Leu GIy Ser Ser GAT AGA GAC CTG GGC TCT TCA TGA AATGATTCTCATTGATTAATTTGGCAT 1190 ATAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCA 1249 cagaattgactgaattagttctgcaaatactttgtcggtatattaagccagtatatgtt 13 ö 8 AAAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTA 1367 TTTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGC CTTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA 1426 1485 1544 TTTTGTTATTTATTAAATTATTTTCAAACAAAACTTCTTGAAGTTATTTATTCGAAAAC CAAAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA CCTATTCATTCATTCCATTTACATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT 1603 1662 1721 TGGAATTGATGTCAAGCAATGCAGGTGTACTCATTGCATGACTGTATCAAAATATCTCA TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA AAAAAGAAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT 1780 1839 1898 ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA AGTAAAAGAAATATGATGTTCTCATACTAGAAGCTT 1933 O,} Λ ; ί q Figure 12 (IFN-pseudo-omeqa2) AAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATGAATATATT 51 CTAGAAGGAG'GGAAGCACC AGAGAAGTTCTTTC ACTAAT AACC ATCAACGTCTTCTGTG 110 AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC 169 CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAATGGAG 228 GGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG 287 ATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGG 346 AATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGGATGACCTCCTTTTAGGTCA 405 TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA 464 GGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC 523 TCTGCCTCCTGTCAGATCAGCAGTGGCATTGGATTCTCAAAAGAGCAGGAACCCTATTG 582 TGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGAGAATCTAATGCCTCTG 641 AAAGCATTCCCTCCCTGACCCCATTTTTCGTGGAAAATTATCTTCCACCAAACTGGTG 700 GCCAAAAGGTTGTGGATGCTGATATAGAAGACATGTAAATGAAAACAATAAATGGAATT 7 59 AAAAATTTAGAGAAATGCTCAGAAAATGAAAACTATTTGTGCTCCATTAAAGCCATGC 818 ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAA 877 -20 -16 " Met Ala Leu Leu Phe Pro Leu Leu CCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC CCT CTA CTG 9 28 , - ₁₀ -5 -1 Ala Ala Leu Glu VaI Cys Ser Cys Gly Ser Ser Gly Le Lei GIy GCA GCC CTA GAG TGG TGC AGC TGT GGC TCT TCT GGA TCT CTA GGA 973 "5 IFN aB ctgatgtacgaggactccatcctggctgtg IFN aC ctgatgaatgaggactccatcctggctgtg IFN aD ctgatgaatgtggactccatcttggctgtg IFN aF ctgatgaatgtggactccatcttggctgtg IFN ccG ctgatgaatgtggactctatcctgactgtg IFN aH ctgatgaatgaggactccatcctggctgtg IFN aK ctgatgaatgaggacttcatcctggctgtg IFN aL ctgatgaatgaggactccatcctggctgtg IFN beta 'aggggaaaactcatgagcagtctgcacctg - <\ C0 " 200 210 220 tcttcaacctcttTaCcacaaaAgaTtcat tcttcaatctcttcagcacaaaggactcat ccttcaacctcttcagcacaaaggactcat ccttcaatctcttcagcacagaggactcat ccttcaatctcttcagcacaaaggactcat ccttcaatctcttcagcacaaaggactcat ccttcaatctcttcagcacaaagaactcat ccttcaatctcttcagcacagaggactcat ccttcaatctcttcagcacagaggactcat tcttcagcctcttccacacagagcgctcct tctttgctattttcagacaagattcatcta  Κ Λ - Figure ^ Μ13 pRHW12 Hind IH / BamHi fragment of PRHW12 Hi3mp9 _χ Primer eitessioo by Klenoi Palymerase Figure 10 M ₁ A BC DEF GH IM Figure 11 (IFN-omegal) GATCTGGTAAACCTGAA 17 'GCAAATATAGAAACCTATAGGGCCTGACTTCCTACATAAAGTAAGGAGGGTAAAAATGG 7 6 AGGCTAGAATAAGGGTTAAAATTTGTCTTCTAGAACAGAGAAATATTTTTTCATAT 135 ATATATGAATATATATATATATACACATATATACATATATTCACTATAGTGTGTATAC 194 ATAATATATEATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT 253 AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCATTTGATTAAGCATTCATTT 312 GTATTGACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAATTAATAGA 3 71 TTGTTGTCATCCTCTTAAGGTCATAGGGAGAACACACAAATGAAAACAGTAAAAGAAAC 430 TGAAAGTACAGAGAAATGTTCAGAAATGAAAACCATGTGTTTCCTATTAAAAGCCATG 4 89 CATACAAGCAATGTCTTCAGAAAACCTAGGGTCCAAGGTTAAGCCATATCCCAGCTCAG 548 -20 -16 Met Ala Leu Leu Phe Pro Leu Leu TAAAGCCAGGAGCATCCTCATTTCCCA ATG GCC CTC CTG TTC CCT CTA CTG. 599 , -10 -5 -1 Ala Ala Leu VaI Met Thr Ser Tyr Ser Pro Vai GIy Ser Leu GIy GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC 644 5 ' t
S .1 t s 100 bp t
A 5 10 "15 Cys Asp Leu Pro Gin Asn His Giu Leu Leu Ser Arg Asn Thr Leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 20 25 30 VaI Leu Leu His Gin Met Arg Arg He He Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 9 35 40 45 ^L Y ^S Asp Arg Arg Asp Phe Arg Phe Pro Gin GIu Met VaI Lys GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 50 55 60 Ser GIn Leu GIn Lys Ala His Vai Met Ser Vai Leu His Giu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 65 70 75 Leu Gin Gin let marriage Ser Leu Phe His Thr GIu Arg Ser Ser Ala 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 GIy Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 95 100 105 Gin Gin Leu Gin His Lei Giu Thr Cys Leu Leu Gin VaI Vai GIy CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 110 115 120 GIu GIy GIu Ser Ala GIu Ala He Ser Ser Pro AIa Leu Thr Leu GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 3 125 130 135 Arg Arg Tyr Phe Gin Giy He Arg VaI Tyr Leu Lys Giu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 3Ό 140 145 150 Tyr Ser Asp Cys AIa Trp GIu VaI VaI Arg Met GIu He Met Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Giu Arg Leu Arg Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 503 170
Asp Arg Asp Leu GIy Ser Ser GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 555 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 613 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 672 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 731 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 790 TTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTAT 649 TTTGTTAT. 857 Fig! Idling 5 - 9 d A a) l A I 'I O I EHBSPCMEHBSPC b) P9A2 • 5 10 15 Cys Asp Leu Pro Gin Asn His GIy Leu Leu Ser Arg Asn Thr Leu TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 20 25 30 VaI Leu Leu His Gin Met Arg Arg He Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 9 35 40 45 Ί0 ^L ys Asp Arg Arg Asp Phe Arg Phe Pro Gin GIu Met VaI Lys GIy AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 50 55 60 Ser GIn Leu GIn Lys Ala His VaI Met Ser VaI Leu His GIu Met-AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 65 70 75 Leu Gin Gin He Phe Ser Leu Phe His Thr GIu Arg Ser Ser AIa CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 80 85 90 AIa Trp Asn Met-Thr Leu Leu Asp Gin Leu His Thr GIy Leu His GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CA "T 95 100 105 Gin GIn Leu Gin His Leu Glu Thr Cys Leu Leu Gin VaI Vai GIy CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA HO 115 '120 GIu GIy GIu Ser Ala GIy Ala lie Ser Ser Pro AIa Leu Thr Leu GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 125 130 135 Arg Arg Tyr Phe Gin GIy He Arg VaI Tyr Leu Lys GIu Lys Lys AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 140 145. 150 Tyr. These are: Asp Cys Ala Trp Glu VaI VaI Arg Met GIu He Met Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Giu Arg Leu Arg Ser Lys TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 502 170
Asp Arg Asp Leu GIy Ser Ser GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 554 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 613 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 672 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 731 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 790 TTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATÄTTGTAT 849 TTTGTTATTTATTAAATTATTTTCAAAC-poly-A 8 77 Figure 4 (Ε76Ε9): CTCTGGGC 5 10 15 Phe Asp Leu Pro GIn AsnXArg GIy Leu Leu Ser Arg Asn Thr Leu 20 25 30 AIa Phe Trp AIa Lys Cys Arg He Ser Thr Phe Leu Cys Leu Lys 35 40 45 Asp Arg Arg Asp Phe Arg Phe Pro Leu GIu Met Trp Met Ala VaI 50 55 60 ^ He Cys Arg Arg Arg Arg Leu Cys Leu Ser Ser Met Arg Cys Phe 65 70 75 Ser Arg Ser Ser AI Ser Ser Pro GIn Ser Ala Pro Leu Leu Pro GIy Thr
having. illustration 1 UJ co LU ο-ο cn ο o O O ο o (D O O IO O O O O CO O O CM O Otiti Figure 2 Oh Oco O O in ο o O O CO O O § ^H ti I! cn - <5Ί - 246 3 :. Figure 3 (Ρ9Α2): TCTGGGC 5 10 15 Trp Asp Leu Pro Gin Asn His Giu Leu Leu Ser Arg Asn Thr Leu 20 25 30 AIa Leu Lei GIy GIn Met Cys Arg He Thr Thr Phe Leu Cys Leu 35 40 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Leu GIu Met Trp Met Ala 50 55 60 VaI Ser Cys Arg Arg Arg Pro Cys Leu Ser Ser Met Arg Cys 65 70 75 Phe Ser Arg Ser Ser Al Ser Ser Pro GIn Ser Ala Pro Leu Leu Pro GIy Thr
having. 30. The method according to claim 11, characterized in that in the produced IFN pseudo-omega4 peptide, the amino acid sequence of the formula Met Vai Leu Leu Leu Vai Leu Leu -10 -5. -1 VaI AIa Leu Leu Leys Cys GIn Cys GIy Pro VaI GIy Ser Leu GIy 5 10 15 Tyr Asn Leu Pro GIn Asn His GIy Leu Leu GIy Arg Asn Thr Leu 20 25 30 VaI Leu Lei GIy GIn Met Arg Arg He He Pro Phe Leu Cys Leu 35 40 45 Lys Asp Arg Ser Asp Phe Arg Phe Pro Gin Glu Lys Vai GIu VaI 50 55 60 Ser GIn Leu GIn Lys Ala Gin Ala Met Ser Phe Leu Tyr Asp VaI 65 70 75 Leu Gin Gin VaI Phe Asn Phe Ser. His Lys AIa Leu Leu Cys Cys 80 85 90 Met Glu His Asp Leu Pro Gly Pro Thr Pro His Phe Thr Ser Ser 95 100 105 Ala Ala GIy Thr Pro GIy Asp Leu Leu GIy Ala GIy Asp GIy Arg 110 115 120 Arg Arg Ser Trp GIy GIn Trp VaI He GIu GIy Ser Thr Leu AIa 125 130 Leu Arg Arg Tyr Phe Gin Glu Ser He Ser Thr having. 29. The method according to claim 10, characterized in that in the produced IFN pseudo omega3 peptide, the amino acid sequence of the formula Met VaI Leu Leu Leu Pro Leu Leu -10 -5 -1 VaI AIa Leu Pro Leu Cys His Cys GIy Pro VaI GIy Ser Leu Ser 5. 10 15 Cys Asp Leu Pro GIn Asn His GIy Leu Leu Ser Arg Asn Thr Leu 20 25 30 VaI Leu Leu His Gin Met Arg Arg He He Pro Phe Leu Cys Leu 35 4U 45 Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin GIu Met VaI Lys GIy 50 55 60 Ser Gin Leu Gin Lys Ala His Vai Met Ser Vai Leu His Giu Met 65 70 75 Leu Gin Gin He Phe Ser Leu Phe His Thr GIu Arg Ser Ser Ala 80 85 90 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gily Leu His 95 100 105 Gin Gin Leu Gin His Lei Giu Thr Cys Leu Leu Gin VaI Vai GIy 110 115 120 GIu GIy GIu Ser Ala GIy Ala He Ser Ser Pro AIa Leu Thr Leu 125 130 135 Arg Arg Tyr Phe Gin Giy He Arg VaI Tyr Leu Lys Giu Lys Lys 140 '145 150 Tyr Ser Asp Cys AIa Trp GIu VaI VaI Arg Met GIu He Met Lys '155 160 165 Ser Leu Phe Leu Ser Thr Asn Met.Gin GIu Arg Leu Arg Ser Lys 170 · Asp Arg Asp Leu GIy Ser Ser and its N-glycosylated derivative at amino acid position 78. 26. A process for the preparation of an interferon according to claim 24, characterized in that the interferon prepared in accordance with claim 25 in position III contains the amino acid Glu instead of Gly, and of its in amino acid position 78 N-glycosylated derivative. 27. A process for the preparation of the interferons according to claims 24 to 26, characterized in that they are the leader peptide of the formula Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu VaI Met Thr Ser Tyr Ser Pro "VaI GIy Ser Leu GIy contain. 28. The method according to claim 9, characterized in that in the produced IFN pseudo omega2 peptide, the amino acid sequence of the formula Met Ala Leu Leu Phe Pro Leu Leu -10 -5 -1 Ala Ala Leu Glu VaI Cys Ser Cys Gly Ser Ser Gly Ser Leu GIy 5. The method according to claim 3, characterized in that the coding sequence according to claim 4 instead of the coding for the amino acid 111 GGG codon contains the GAG codon. 6. The method according to claim 3, characterized in that the coding sequence a) in the plasmid E76E9 (deposited in E. coli HB 101 in the DSM · under the number DSM 3003) or b) in the plasmid P9A2 (deposited in E. coli HB 101 in DSM under number DSM 3004). 7. The method according to claims 1 to 6, characterized in that the coding sequence additionally coding for the Leader Pe.ptid DNA sequence of the formula ATG GCC CTC CTG TTC CCT CTA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG contains GGC. 8. The method according to claims 3 and 7, characterized in that the coding for IFN-omegal sequence the
formula GATCTGGTAAACCTGAA 17 GCAAATATAGAAACCTATAGGGCCTGACTTCCTACATAAAGTAAGGAGGGTAAAAATGG 76 AGGCTAGAATAAGGGTTAAAATTTTGCTTCTAGAACAGAGAAAATGATTTTTTTCATAT 135 ATATATGAATATATATTATATATACACATATATACATATATTCACTATAGTGTGTATAC 194 ATAAATATATAATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTACATGCATAT 253 AGTATATACACTTATGACTTTAGTACCCAGACGTTTTTCATTTGATTAAGCATTCATTT 312 GTATTGACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAAATTAATAGA 3 71 TTGTTGTCATCCTCTTAAGGTCATAGGGAGAACACACAAATGAAAACAGTAAAAGAAAC 430 TGAAAGTACAGAGAAATGTTCAGAAAATGAAAACCATGTGTTTCCTATTAAAAGCCATG 489 CATACAAGCAATGTCTTCAGAAAACCTAGGGTCCAAGGTTAAGCCATATCCCAGCTCAG 548 TAAAGCCAGGAGCATCCTCATTTCCCA ATG GCC CTC CTG TTC CCT CTA CTG 599 GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC 644 TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 689 GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 734 AAG GAC AGA AGA GAC TTC AGG TTC CGC CAG GAG ATG GTA AAA GGG 779 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 824 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TGC TTG CTG CAG GTA GTG GGA 959 GAA GGA GAA TCT GCT GXG GCA ATT AGC AGC CCT GCA CGC CGC TCC TCT GCT 869 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 914 CAG CAA CTG CAA CTG ACC TTG 1004 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 1045 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 1094 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT 1139 AAA GAT GAC CTG AGA GGC TCT TCA TGA AATGATTCTCATTGATTAATTTGCCAT ATAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCA 1190 1249 1308 CAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTT AAAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTA 1367 TTTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGC CTTTACATTGTATTAAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA 1426 148 5 1544 TTTTGTTATTTATTAAATTATTTTCAAACAAAACTTCTTGAAGTTATTTATTCGAAAAC CAAAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA CCTATTCATTCATTCCATTTACATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT 1603 166 2 TGGAATTGATGTCAAGCAATGCAGGTGTACTCATTGCATGACTGTATCAAAATATCTCA 1721 TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA 1780 AAAAAGAAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT 1839 ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA 1898 AGTAAAAGAAAATGATGTTCTCATACTAGAAGCTT 1933 wherein X represents the nucleotide A or G. 9. The method according to claim 1, characterized in that
the sequence coding for IFN pseudo-omega2 is the formula AAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATGAATATATT 51 CTAGAAGGAGGGAAGCACCAGAGAAGTTCTTTCACTAATAACCATCAACGTCTTCTGTG AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC 110 169 228 CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAG GGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG ATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGG 287 346 405 AATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCA TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA GGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC 464 5 23 582 TCTGCCTCCTGTCAGATCAGCAGTGGCATTGGATTCTCAAAAGAGCAGGAACCCTATTG TGAAGTGCAGATGCGAAGGATCTAGGTTGTGGTCTCCTAATGÄGAATCTAATGCCTCTG 641 AAAGCATTCCCTCCCTGACCCCATTTTTCGTGGAAAAATTATCTTCCACCAAACTGGTG GCCAAAAGGTTGTGGATGCTGATATAGAAGACATGTAAATGAAAACAATAAATGGAATT 700 759 818 AAAAATTTAGAGAAATGCTCAGAAAAATGAAAACTATTTGTGCTCCATTAAAGCCATGC ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAA 877 CCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC CCT CTA CTG 928 GCA GCC CTA GAG GTG TGC AGC TGT GGC TCT TCT GGA TCT CTA GGA 973 ACT AAC CTG CCT CAG AAC CAT GGC CTG CTA GGC AGG AAC ACC TTG 1018 GTG CTT TTG GGC CAA ATG AGG AGA ATC TCT CCC TTC TTG TGT CTA 1063 AAG GAC AGA AGT GAC TTC AGA TTC CCC CAG GAG AAG GTG GAA GTC 1108 AGC CAG TTG CAG AAG GCC CAG GCT ATG TCT TTC CTC TAT GAT GTG 1153 TTA CAG CAG GTC TTC AAC TTC TCA CAC AAA GCG CTC CTC TGC 1198 ATG GAA CAT GAC CTT CCT GGA CCA ACT CCA CAC TTT ACG TCA TCA 1243 GCA GCT GGA ACA CCT GGA G CTG CTT GGT GCA GGA GAT GGG AGA 1288 - SC - AGG AGA AGC TGG GGG CAG TGG GTG ATT GAG GGC TCT ACA CTG GCC 1333 TTG AGG AGG TAT TTC CAG GAA TCC ATC TCT ACC TGA AAGAGAAGAAA 1380 TAAAGATTGTGCCTGGGAAGTTGTCAGAGTGGAAATCATGAGATCCTTTTCATCCACAA 1439 GTTTGCAAGAAAGATTGAGAAGTAAGGATGAAGACCTGGGCTCATCATGAAATGATTCT 1498 CATTGACTAATCTGCCATATCACACTTGTACATGTGACTTTGGATATTCAAAAAGCTCA 1557 TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA 1619 AGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC 1675 CCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTA 1734 TATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTCACTTTTTCA 1793 TATTAAGTTTGCATTTTGTTTTATTAAATTCATATCAAAGAAAACTCTGTAAATGTTTC 1852 TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTGAAAATATGGTAAAA 1911 TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTGAGTAAATGGCAAACTG 1970 TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG 2029 AGGGAAAGAGACGTAAAACAGGACAAATATTATTTTCAATTATGTTAAATGCT 2088 ACAAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 2132 having. 10. The method according to claim 1, characterized in that the coding for IFN pseudo omega3 sequence the formula CCATG 5 CATAGCAGGAATGCCTTCAGAGAACCTGAAGTCCAAGGTTCATCCAGACCCCAGCTCAG 64 CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTC CTG CTT CCT CTA CTC 115 GTG GCC CTG CCG CTT TGC CAC TGT GGC CCT GTT GGA TCT CTG AGC 160 TGG GAC CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 205 GCA CTT CTG GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TGT CTC 250 AAG GAC AGA AGACAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 295 GTC AGT TGC AGA AGG CCC CGG CCC TGT CTG TCC TCC ATG AGA TGC 340 TTC AGC AGA TCT TCA GCC TCT TCC CCA CAG AGT GCT CCT CTG CTG GGA ACA TGA CCT 385 CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA 440 ATGCCTGGATGCTTGCTTAGGGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC 499 558 617 TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGGAATCGTG AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGGATGAAGA 6 76 CCTGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATCACACTTGCACATG 735 TGTCTTTGGTCATTTCAATAGGTTCTTATTTCTGCAG 7 72 having. 11. The method according to claim 1, characterized in that
the sequence coding for IFN pseudo-omega4 has the formula ' AAGCTTTGGGCATGACCTAGTAGGTGACTCTT 3 2 AGTTGGAGTGGTCAGTTGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCCCC 91 ACGTTTGCAGCCGTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCCÄCTGGA GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTGAGGTGA 150 209 268 TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG TAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT 3 27 386 GAGCAATCACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCCAAGAC AGGGGTTCCCTGCCTGGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAGACAGACTGG CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT 445 504 563 GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAGAGGCAGTGTTAGACA GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGGA GTGTTTAGCCAGAGGAGTGGGGTGGCTGCATGCTGGTGTAAGTATGGGACTTCACTTGT 622 681 740 TGGGAAACAGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC CTGCAGTGTGCTGGAAGTTTAGGTGACTGTGGCATGATGTTAGCTTGTAAACAAAGAGC TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 799 858 917 GCAGTGGCAGAGGTAGGATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA CCAGTGGATATGCTCAGCCATGGGTAGGGTGACTGTTCT GCAGTCATGGGCAGGGGGCC 976 TGCTCCTGAAGAATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGGACTCCTCTTCATA 1035 TAGTGGCGATGATGTGCTGGAGGTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCCA 1094 GCCAGAGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGGATGGGTTATGGGTTG 1153 ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTGAGGAGACGAGGCAAG 1212 GGAAGAATGCCTGTGCTGGAGTCTCTGGTCAGGTGGCTCTGCCACAGAGGAGAGGTGAC 1271 CACCAGGAACTGCGTGGAGAACAGTGTAGCCACTCTTCTGTGAGGCAGTTGCTCTGTTT 1330 TGGGGATCTGGAGCAGCCGCTATTCCTTACAGATTCCCAGAGCCTGGAGACAGCAAGGG 1389 CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA 1448 GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1507 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC 1566 CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA 1625 CATTTTTCAGTACCTGAAGATATCAACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1684 CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA 1743 TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG 1802 GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1861 GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA 1920 CAGCAACAATGGCAACCTACCATTCCCTTTGGAGCGCCATCCCAGGGATATTCGAAACT 1979 GCTGTCCACTAGAAAACAGTGGTGGAGGTGACTGGAGACCCCAGTGGAGAGTTTCACCT 2038 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAACCAATCTGACTGCTTCCCCGTA 2097 GAGCTGCTGGACTGTGCTGGGTGGCTGCTCCAGTCCCTAGCTGCCTTGGACTCCCGAGA 2156 ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC 2215 CACAGGGAGCTCCATGTCAGGGAGGTATGAGGCTGCTACCAGTGTCTGGCTGGATTCCC 2274 AAGTCGAGTGGGTCTTACCCTGAGACAGGCCATGGAAGGTGGGCCTGTCATTGTCACTG 2333 CCCAGCGCCCTGGATGAAACCCCTTTCCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC 239 2 TTGGCTGGAGTTATAGCTTCTTTT GTGGGGAGGCCTGGGTATCTAAGGCTCCAGGGTAC 2451 CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA 2510 ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 2569 TGGGAGAAGCGTGGGTCCCCAGGGCTGCTCACTTACTCACCACTTCCCTGGGCAGGAGA 2628 GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT 2687 TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG 2746 AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGAGAGTGGCACACACTAGCA 2805 GGTTCCAGTCGGCCATCTTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAAGCCATT 2864 GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2923 CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT GTT CTA CTG 2974 GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT GGA TCT CTG GGC 3019 TTT GAC CTG CCT CAG AAC CGT GGC CTA CTT AGC AGG AAC ACC TTG 3064 GCA TTC TGG GCC AAA TGC AGA ATC TCC ACT TTC TTG TGT CTC AAG 3109 GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 3154 ATT TGC AGA AGG CCC AGG CTG TGT CTG TCC TCC ATG AGA TGC TTC 3199 AGC AGA TCT TCA GCC TCT TCC CCA CAG AGC GCT CCT CTG CTG CCT 3244 GGA ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG 3300 CCTGGAGTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGGGTGATTGGGA 3359 CCCTACACTGGCCTTGAGGAGGTACTTCCAGGGAATCCATGGGAATCCAGAGAATCTAC 3418 CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA 3477 TCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGACCTGGGGTC 353 TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC 3595 TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA 3654 AGCTT 3659. 12. The method according to claims 1 to 11, characterized in that the coding sequence is contained in an expression vehicle which is replicable in microorganisms or mammalian cells. 13. The method according to claim 12, characterized in that the coding sequence is contained in a plasmid which is replicable in prokaryotes or in eukaryotes. 14. The method according to claim 13, characterized in that the host is a mammalian cell line or E. coli. 15. The method according to claim 14, characterized in that the host E. coli with the DSM no. 3003 or E. coli with the
DSM No. 3004 is. 16. The method according to claim 15, characterized in that the vehicle additionally contains the replicon and control sequences required for the expression. 17. The method according to claim 16, characterized in that the vehicle contains the required for the expression Replikon- and control sequences of the plasmid pER103. 18. The method according to claim 17, characterized in that the plasmid pBR322 instead of the plasmid itself shorter
EcoRI / BamHI fragment the DNA sequence of the formula BH ~ EcoRI Sau3A
gaattc acgct GATCG CTAAAACATTGTGCAAAAGAGGGTTGACTTTGCCTTCGCGA 5 9 ^ mRNA Start, Met ACCAGTTAACTAGACACAAGTTCACGGCAACGGT AAGGAGGT TT AAGCTT AAAG ATG 116 RBS HindIII
Cys Asp TGT GAT C IFN-omega gene ^ Sau3A contains. 19. The method according to claim 18, characterized in that
the IFN-omega gene is the DNA sequence of the formula TGT GAT CTG CCT CAG AAC CAT G GC CTA CTT AGC AGG AAC ACC TTG 28 ncol GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG. ATG 163 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT GCT GXG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 ^ TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 TCC TTG TTA TTC TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 GAT AGA'GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 589 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707 TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766 TTTACATTGTATTAAGATAACAAAACATGTTC AGCT 802 AluI >
where X is the nucleotide A (pRHW12) or G (pRHWll) represents. -S-5 · - 2 4 6 3 J β 20. A process for the production of new type I human interferons according to claims 1 to 19, characterized in that the mature interferons have a divergence of 30 to 50% compared to the human α-interferons. 21. A process for the preparation of new type I human interferons according to claims 1 to 19, characterized in that a) the mature interferons have a divergence of 30 to 50% compared to the human α-interferons and a divergence of about 70% to β-interferon, and b) 168 to 174 amino acids in length, and of their N-glycosylated derivatives. 22. A process for the production of interferons according to claim 21, characterized in that they have a divergence ~ of 40 to 48% compared to the human α-interferons, and of other N-glycosylated derivatives. 23. A process for the production of interferons according to claims 1 to 22, characterized in that they contain a leader peptide. 24. A process for the preparation of interferons according to claims 1 to 22, characterized in that they contain 172 amino acids. 25. A process for the preparation of an interferon according to claim 24, characterized in that it has the amino acid sequence 7.85-7100: Figure 18 ύ ι ύ Upper Right Tell: Number of Identical Amino Acids / Number of Total Amino Acids Compared. Lower left part: percent homology. .la in. "Ί9 s ~ .i - 6 ADHI promoter χ Hin dill fill in HuIFNcjl tr ρ ρ / ο + Xhol linker, ligase xXhoI χ Bam HI HuIFNcol · HEH χ Bam HI χ Xhol ADHI p. Ligase χ Bam HI ADHI p. HuIFNtJl E = Eco RI H = Hindin B = Bam HI X = Xhol Sm = SmaI K = KpnI S = SphI Ss = SsII P = PstI An ^ Amniiriin Rpsistenz AD HI-prom · Ap ^r