FI90667B - Process for Preparation of New Human Type I Interferon Protein - 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

i v 9 0 667

Method for the preparation of a novel human type I interferon protein - Pörfarande för framställning av nytt interferonprotein av human type I

The present invention relates to novel type I interferons as well as recombinant DNA methods for the production of these peptides and products required in these methods, for example gene sequences, recombinant DNA molecules, expression vehicles and organisms.

Interferon is a term formed to describe a set of proteins that are endogenous to human cells and are characterized by having partially overlapping and partially different biological activities. These proteins modify the body's own immune response and are believed to contribute to effective protection against viral diseases. For example, interferons are divided into three classes, namely, o-, fl-, and γ-interferons.

Only one subtype of each of β- and γ-interferon is known in the human body (e.g., S. Ohno et al., Proc. Natl. Acad. Sci. 28, 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 α-interferon 11e have been described (e.g., Phil. Trans. R. Soc. Lond. 299. 7-28 (1982)). The mature α-interferons then differ by up to 23% in divergence and are about 166 amino acids in length. Of further note is the knowledge that α-interferon has a surprisingly high molecular weight (26,000, determined by SDS-polyacrylamide / gel electrophoresis) (Goren, P. et al. Virology 130, 273-280 (1983)). This interferon is called IFN-α 26K. It has been found to have the highest specific antiviral and anti-cellular activity to date.

Known interferons appear to be effective in a variety of diseases, but have little or no effect in several other diseases (e.g., Powledge, Bio / Technology, March 1984, 215-228, "Interferon On Trial") .In addition, interferons have side effects. For example, when studying the Anticancer effect of recombinant α-interferon, it was found that doses of around 50 million units, which were considered safe based on the results of the first phase of studies, caused acute confusion, even joint pain leading to disability, very severe tiredness, loss of appetite, decreased appetite, epileptic seizures and hepatotoxicity In 1982, studies with IFN-α were banned by the French government after fatal heart attacks had been reported in cancer patients treated with IFN-α, and short-term studies in the United States have reported at least two cardiac deaths. It has become increasingly clear that at least some of the side effects, such as fever and nausea, are directly related to the interferon molecule itself and not due to impurities.

Awakening of interferon due to the high desire, which has been increased by the desire to find new interferon-like molecules with fewer side effects, it is an object of the present invention to find and prepare such new substances.

The present invention therefore relates to certain novel type I interferons which may contain a leader peptide and their N-glycolysed derivatives (referred to herein as omega interferons or IFN omega) containing 168 to 174, preferably 172 amino acids and having is to the hitherto known subtypes of α-interferon. . compared to a divergence of 30-50%, preferably 40-48%, and which, on the one hand, have interferon-like effects and, on the other hand, do not have many of the therapeutic disadvantages of these known substances.

The invention thus relates to novel interferons in completely pure form, their non-glycosylated and glycosylated II.

3,90667 forms, the gene sequences encoding these, and recombinant molecules containing these sequences. The invention further relates to expression vehicles, such as plasmids, containing said gene sequences, as well as to various host organisms, such as microorganisms or tissue cultures, which allow the production of a novel interferon by enzymatic or tissue culture methods.

A preferred aspect of the invention are omega interferons and corresponding gene sequences of the following formula: 4,90667 5 10 15

Cys Asp Leu Pro Gin Asn His Gly 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

Val Leu Leu His Gin Met Arg Arg lie 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 Glu Met Val Lys Gly

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 Val Met Ser Val Leu His Glu Met

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

65 70 75

Leu Gin Gin lie Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala

CTG CAG CAG ATC TTC AGC CTC TTC CAC AC A GAG CGC TCC TCT GCT

80 85 90

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

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

95 100 105

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

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

110 115 120

Glu Gly Glu Ser Ala Gly Ala lie Ser Ser Pro Ala 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 Gly lie Arg Val Tyr Leu Lys Glu Lys Lys

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

140 145 150

Tyr Ser Asp Cys Ala Trp Glu Val Val Arg Met Glu lie 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 Glu Arg Leu Arg Ser Lys

TCC TTG TTC TTA TCA AC A AAC ATG CAA GAA AGA CTG AGA AGT AAA

170.: Asp Arg Asp Leu Gly Ser Ser

- ..: GAT AGA GAC CTG GGC TCA TCT

II

5,90667

At position 111, the sequence GGG (encodes a Gly amino acid) may be replaced with the sequence GAG (encodes a Glu amino acid). Preferred molecules also include derivatives that are N-glycosylated at amino acid position 78.

For example, both of the aforementioned omega interferons may contain a leader peptide of the formula

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

Explanations of 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 probe clone P9A2 DNA

Figure 6: Construction of expression clone pRHW12

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

Figure 8a: List of individual nucleotide sites in the IFN-αA gene

Figure 8b: List of single nucleotide sites in the IFN omegal gene 6,90667

Figure 8c: List of individual nucleotide sites of the IFN-αD gene

Figure 9: Schematic representation of the synthesis of specific probes for the interferon subtype

Figure 10: Detection of mRNA specific for interferon subtype

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 IFR pseudo-omega3 gene

Figure 14: DNA sequence of the IFN pseudo-omega4 gene

Figure 15: Corrected list of IFN omega gene sequences

Figure 16: Signal sequence homology

Figure 17: Homology of "mature" protein sequences

Figure 18: 4 DNA sequence homology

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

The human B lymphoma cell line, for example Namalwa cells (see

G. Klein et al., Int. J. Cancer 10, 44 (1982), can be activated to simultaneously produce interferon α and β by treatment with a virus, e.g., Sendai virus. If mRNA formed from stimulated Namalwa cells is then isolated, then this can serve as a template for cDNA synthesis. In order to improve the yield of the interferon-specific sequence of 7,90667 in cloning, the mRNA preparation is separated in a sugar density gradient based on the different lengths of the individual mRNA molecules. Preferably, mRNAs of about 125 in length (mRNA of about 800 to 1000 bases in length) are assembled. In this region, α- and β-interferon-specific mRNAs are sedimented. The mRNAs obtained from this gradient region are concentrated by precipitation and dissolution in water.

The cDNA library is prepared essentially by methods known from the literature (see, e.g., E. Dworkin-Rastl, M.B. Dworkin, and P. Swetly, Journal of Interferon Research 2/4, 575-585 (1982)). mRNA into the medium by the addition of oligo-dT-1.

The cDNA is then synthesized in a buffered solution for 1 hour at 45 ° C, respectively, by the addition of four deoxy-nucleoside ditriphosphates (dATP, dGT, dCTP, dTTP) and the enzyme reverse transcriptase. The cDNA / mRNA hybrid is purified by extraction with chloroform and chromatography on a gel column, for example a Sephadex G50® column. The RNA is hydrolyzed by pretreatment (0.3 moles of NaOH at 50 ° C, 1 hour) and the cDNA is mixed with ethanol after neutralization with acidic sodium acetate solution. After the addition of four deoxynucleoside triphosphates and E. coli DNA polymerase I to the corresponding solution, double-stranded synthesis is performed, with the cDNA acting simultaneously as a template and primer to form a hairpin structure at its 3 'end (6 hours at 15 ° C) (see A. Eftratiadis et al., Cell 7, 279 (1976)), after phenol extraction, Sephadex G50® chromatography, and ethanol precipitation, the DNA is treated in a suitable solution with a single-stranded endonuclease11a SI. In this case, the hairpin structure and the cDNA that does not react into a double strand are degraded. After chloroform extraction and precipitation with ethanol, double-stranded DNA (dsDNA) is separated by size in a sugar density gradient. In the following, for the cloning step, only dsDNA of 600 bp or more is best used, in order to obtain with greater probability only clones containing the sequence completely encoding the new interferon. dsDNA, 8,90667 with a length of more than 600 bp, is concentrated from the gradient by mixing with ethanol and dissolving in water.

To add the resulting dsDNA molecules, these must then be inserted into the corresponding vector and then into E. coli. The vector used is preferably plasmid pBR322 (F. Bolivar et al., Gene 2, 95 (1977)). This plasmid consists essentially of a replicon and two screening markers. These confer host resistance to ampicillin and tetracycline antibiotics (Apr, Tcr). The β-lactamase (Apr) gene contains the restriction endonuclease PstI recognition sequence. pBR322 can be digested with PstI. The 3 'ends of different lengths are extended with terminal deoxynucleotide transferase (Tdt) enzyme by first adding dGTP to a correspondingly buffered solution. Similarly, dsDNA is also extended at the 3 'ends by the enzyme Tdt using cCTP. Homopolymer ends of plasmid and dsDNA are clumpy and hybridize when plasmid DNA and dsDNA are mixed at appropriate concentration ratios and under appropriate salt, buffer, and temperature conditions (T. Nelson et al., Methods in Enzymology 68, 41-50). (1980)).

E. coli strain HB 101 (genotype F-, hsdS20 (r-8, mB) recA13, ara-14, proA2, LacY1, galK2, rpsL20 (Smr), xyl-5, mtl-1, sup 44, lambda- ) is prepared for reception of a recombinant vector dsDNA molecule by washing with a CaCl 2 solution. Components of E. coli HB 101 cells are mixed with DNA and, after incubation at 0 ° C, the plasmid DNA thus obtained is transformed by heat shock at 42 ° C. For 2 minutes (M. Dagert et al., Gene 6, 23-28 (1979)). The transformed bacteria are then plated on a tetracycline containing 11 e agar. 11 e (10 ug / ml). Only E. coli HB 101 that has taken up a vector or recombinant vector molecule (Tcr) can grow on this agar. Recombinant vector dsDNA molecules confer the host genotype ApsTcr, as information about β-1actamase is destroyed when the dsDNA is introduced into the β-lactamase gene. Clones are transferred to agar plates containing 9,90667 50 ug / ml ampicillin. In this case, only 3% increase, which means that 97% of the clones contain an insert of dsDNA molecule. Starting with 0.5 ug of dsDNA, more than 30,000 clones are obtained. Of these, 28,600 clones are transferred individually to microtiter plate membranes containing medium, 10 μg / ml tetracycline and glycerin. After the clones have grown in these, the plates are kept for storage at -70 ° C (cDNA library).

To search for clones containing a new interferon gene in the cDNA library, the clones are transferred to nitrocellulose filters after thawing. These filters are in tetracycline-containing nutrient agar. After the growth of bacterial colonies, the bacterial DNA is attached to a filter.

Preferably, an insert of clone pER33 (E. Rasti et al., Gene 2JL, 237-248 (1983) - see also EP-A-0.115.613) containing the INF-α2-Arg gene is used as a probe. By Nick translation, this piece of DNA is radiolabeled using DNA polymerase I, dATP, dGTP, dTTP, and α-32p_ (jcpp. Nitrosel lul oosa filters are first pretreated in suitable, no under stringent hybridization conditions without the addition of a radioactive probe and then hybridized for about 16 hours by the addition of a radioactive probe, after which the filters are also washed under non-stringent conditions.According to interferon-α2-Arg-containing clones, other interferon-containing clones are obtained due to the low stringency of hybridization and washing. whose sequences may differ from those of hitherto known interferon αs.After drying, the filters are drained by X-ray.The darkening, which is clearly higher than the background level, indicates the presence of a clone with interferon-specific sequences.

Because of the different quality of the radioactivity signals, positive clones or clones suspected of being positive are grown on a small scale. Plasmid DNA molecules are isolated, treated with restriction endonuclease 1 PstI, and electrophoretically separated on an agarose gel (Birnoboim et al., Nucl., Acid. Res. 1513 (1979)). The agarose gel DNA is transferred by the method of Southern (E.M. Southern, J. Mol. Holii. 98., 503-517 (1975)) to a nitrocellulose filter. The DNA of this filter is hybridized with a radioactive IFN gene-containing denatured probe. Plasmid 1F7 (deposited in the DSM collection under DSM number 2362) containing the interferon α2-Arg gene is used as a positive control. From the autoradiogram, it is clear that clones E76E9 and P9A2 contain a sequence that hybridizes under non-stringent conditions to the interferon-ot2-Arg gene. For further characterization of the dsDNA inserts of clones E76E9 and P9A2, plasmids from these clones are prepared on a larger scale. DNA is treated with various restriction endonucleases, such as the endonucleases AluI, Sau3A, BglII, Hinfl, PstI and Haelll. The corresponding pieces are separated on an agarose gel. The sizes of the pieces are determined by comparison with corresponding size markers, for example, the amounts obtained by treatment of plasmid pBR322 with restriction endonucleases H1fl or Haell1.

The mapping method of Smith and Brinsteil (H.O. Smith et al. Nucl. Acid. Res. 3_, 3287-2398 (1967)) determines the order of these pieces. From the restriction enzyme maps thus obtained (Figures 1 and 2), it can surprisingly be concluded that the inserts of clones E76E9 and P9A2 are hitherto unknown interferon genes, namely omega interferon genes in both cases.

This information on omega interferon is utilized in the treatment of the cDNA insert with appropriate restriction endonucleases. The corresponding fragments are ligated into the dsDNA form of the bacterial M13 mp9 (replicative form) (J. Messing et al., Gene 1, 9, 269-276 (1982)) and sequenced. by the dideoxy method (F. Sanger et al., Proc. Natl. Acad. Sci. 74, 5463-5467 (1977)). Recombinant phage single-stranded DNA is isolated. After binding of the synthetic oligomer, the synthesis of double strands is performed in four separate batches of n. 90667 using large pieces of DN A polymer I from K. Coli (Klenou piece). One of the four dideoxynucleoside triphosphates (ddAlP, ddGTP, ddCTP, ddTTP) is added to each of these four subreactions. This results in a statistically distributed chain break at the sites where the base medium DNA has a base complementary to the currently used dideoxynucleoside triphosphate. In addition, radiolabeled dATP is still used. After completion of the synthesis reaction, the products are denatured and single-stranded DNA fragments are separated by size on a denatured polyacrylamide gel (F. Sanger et al., FEBS Letters 87, 107-111 (1978)). The gel is then exposed to X-ray film. The recombinant M13 phage DNA sequence can be read from the autoradiogram. The insert sequences of the various recombinant phages are processed using suitable computer programs (R. Staden, Nucl. Acid. Res.

10, 4731-4751 (1982)).

The sequencing strategy is shown in Figures 1 and 2. Figure 3 shows the DNA sequence of the insert of clone P9A2 and Figure A shows the corresponding sequence of clone E76E9. The uncoded DNA strand 'is indicated in the 5' -> 3 'direction together with the f' 'amino acid sequence derived therefrom.

; The isolated sDNA of the clone E76E9 omega (G lu) - i nt e r f e ron i 1 le: is 858 base pairs long and has a 3 'non-translated region. The region encoding mature omega (Glu) interferon ranges from nucleotide 9 to nucleotide 524. The isolated cDNA for clone P9A2 for omega (G ly) interferon 1 is 877 base pairs ·. ** · long, giving the mature omega interferon coding sequence: extending from nucleotide 8 to nucleotide 523. In the case of P9A2 clones, the 3 'untranslated region extends to Up to the A-section,

The DNA sequences encoding the prepared omega interferon in 12,90667 clones are E76E9 and P9A2 complements. They start at the N-terminus with the amino acids cysteine-aspartic-leucine. Surprisingly, both mature omega interferons are 172 amino acids in length, which is clearly different from the length of interferons known to date, for example α-interferons have 166 (or 165) amino acids. Surprisingly, both omega interferons have a potential N-glycolysis site at amino acid position 78 (asparagine-methionine-threonine).

Comparison of clones E76E9 and P9A2 reveals a difference. The triplet encoding amino acid 111 in clone E76E9 is GAG encoding glutamic acid, whereas this triplet in clone P9A2 is GGG and encodes glycine. Both omega interferon proteins thus differ by one amino acid and are called omega (Glu) interferon (E76E9) and omega (Gly) interveron (P9A2).

When both omega interferons are compared with the hitherto known human α-interferon subtypes, the following picture is obtained: omega alpha f: Protein length at amino acids 172,166 ^ +). ·: Potential N-glycosylation site 78 - (++) +) Interferon alpha A contains only 165 amino acids ++) Interferon alpha H contains one potential N-Gly cosylation site at position 75 (D. Goeddel et al. Nature 290. 20- 26 (1981)).

E.coli HB 101 with plasmid E76E9 and E.coli 101 with plasmid P9A2 were deposited on 03.07.1984 in DSM collection li 13 90667 (Deutschen Sammlung för Mikroorgariismen, Gottingen) under number DMS 3003 and DSM 3004, respectively.

To demonstrate that the newly discovered clones produce interferon-like activity, for example, clone E76E9 is cultured and the bacterial lysate is examined after growth in a plaque test (Plaque-Reduktions-Test). As expected, the bacterium produced interferon-like activity (see Example 3).

To demonstrate that each newly discovered interferon is a member of a new interferon family, DNA assembled from Namalva cells was further isolated and treated with various restriction endonucleases.

In this way, the number of genes encoded by the CDNs of clone P9A2 and E76E9 can be estimated. To this end, the obtained DNA fragments are separated on an agarose gel by the method of Southern (EM Southern et al., J. Mol. Uiol. 98, 503-517 (1975)), applied to nitrocellulose filters and hybridized under relatively high conditions to radiolabeled specific DNA of clone P9A2: with n: *.

Figure 5a shows the results obtained by hybridization with DNA from plasmid P9A2 and pER33:

Individual traces are then marked with letters referring to the different DNA probes treated (E = EcoRI, H = H ind III, B = BamHI, S = SpHI, PsPstI, CsCl2). The left side of the filter was hybridized to the interferon-α gene probe ("A") and the right side to the cDNA insert of clone P9A2. with ("0") · The band group that hybridizes to either the α-interferon gene probe or the new interferon gene probe is different. No cross-hybridization could be detected with these two different probes, 1 * 90667 when corresponding traces were evaluated.

Figure 5b depicts the cDNA of clone P9A2 and the fragment utilized for hybridization. Cleavage sites for individual restriction enzymes are given (P = PstIf S = Sau3A, AsAluI). The probe comprises only two of the three possible PstI fragments. The hybridization pattern shows only one hybridizing fragment of about 1300 base pairs (bp) in length that belongs to a homologous gene. A smaller 120 bp long piece had passed out of the gel. The band belonging to the 5 'part of the gene cannot be detected because the probe does not contain this region. At least six different bands can be detected in this Pst I trace. This means that there must be some other genes in the human genome that are closely related to the new sequences. If these genes have one or more PstI cleavage sites, then it can be expected that at least three other genes can still be isolated.

These genes are best isolated by hybridizing a human gene library where they are in a plasmid vector, phage vector, or cosmid vector (see Example 4e).

It should be noted at this point that the omega interferons according to the invention are not only two ready-made interferons described individually, but also each modification of these po i y pep t ids which does not affect the IFN omega activity. These modifications include, for example, shortening of the molecule at the N- or C-terminus, exchange of amino acids for other groups without substantially changing the activity, - chemical or biochemical binding of the molecule to other molecules that are inert or active. The latter modifications may be, for example, hybrid molecules formed from one or more omega interferons and / or known α- or β-interferons.

15 90667

In order to compare the differences in amino acid and nucleotide sequences of new interferons, in particular omega (Glu) and ornega (Gly) interferon, with the amino acid and nucleotide sequences already published for ai nterfer one i 1 le and β-mterfe-Ron (C. Weissrnann et al. ai., Phil. Trans. R. Soc. London 299, 7-28 (1982), A. Ullrich et al., J. Molec. Biol. 156, 467-486 (1982), T. Taniguchi et al. , Proc. Nat. Acad. Sci. 77, 4003-4006 (1980); K. Tokodoro et al., EMBO J. 3, 669-670 (1984)), the corresponding sequences are arranged in pairs and the differences at individual sites are calculated.

The results shown in Figure 7 show that the DNA sequences of clones P9A2 and E76E9 are closely related to the sequences of type I interferon (e.g., α- and β-interferon). On the other hand, it appears that the amino acid sequence differences between the individual α-interferons and the new sequences are greater than 41.6% and less than 47.0%> The differences between the new sequences and the individual «interferons and β-interferon sequences are of the order of about 70?» . In view of the results of Example 4, which showed the existence of a whole group of closely related genes, and the proposed designation for interferons (J. Vilceck et al., J. Gen. Virol. 65, 669-670 (1984)), it is believed that clones P9A2 and E76E9 cDNA inserts encode ·. ·: a new class of type I interferons called • omega interferons.

In addition, it is shown that the expression of the omega interferon gene occurs analogously to the expression of the type I interferon gene. with. To this end, the Sl-Mapping method (A.J.

Berk et al., Cell 1 E, 721 (1977)) based on the transcription of individual members of the multigenic families of α- and omega-interferons: see Example 7 and show that omega-1 mRNA expression can be induced by viruses. .

. Because the transcripts of such a gene family differ only; the hybridization of a few bases, only about 1000 bases, alone is not a sensitive enough feature to distinguish between different IFN mRNAs.

To this end, mRNA sequences are prepared from 9 α-interferon, omega-1 interferon and 8-interferon. The bases that are specific for the top sequence are then marked in capital letters. Such specific locations can be easily found using a simple computer program. The hybrid large antibody starting from a specific site can then completely hybridize to a given subtype of mRNA. No other mRNAs can hybridize at subtype-specific sites. Once the hybridization probe is labeled radioactively at its specific 5 'end, only those radioactive labels are protected against single-stranded specific nuclease (preferably S1 nuclease) that have hybridized to the interferon subtype mRNA for which the probe was constructed.

This principle is not limited to interferon, but rather can be used for each group of known sequences with the specific sites depicted in Figure 8.

·· '· The above-mentioned specific sites of the subtype are in most cases no case, in which case cleavage of the cDNA by restriction endonucleases is not suitable for the preparation of subtype-specific hybridization probes.

Y; The probes used in this example have therefore been prepared by extending the radiolabeled oligonucleo-; tid, which is complementary to the specific site of the omega 1-in t er f e ron mRNA ....

: Figure 10 shows that, as expected, omega-1 mRNA

: is inducible in Namalu / a and NC37 cells.

The invention therefore relates not only to gene sequences encoding specifically administered omega interferons, but also to modifications that can be readily and routinely obtained by mutation, digestion, transposition or addition. The invention includes any sequence that encodes and is degenerate with respect to the described human omega interferons (i.e., having the biological activity spectrum described herein); those skilled in the art will be able to degenerate the DN A sequences of the coding regions. Also included in the invention is any sequence that encodes a polypeptide having the IFN-Omega activity spectrum and that hybridizes to these sequences (or portions thereof) under stringent conditions (e.g., conditions that select above 85 S, preferably greater than 90% homology).

Hybridizations are performed in 6 x SSC / 5 x Denhardt's solution / 0.1% SDS at 65 ° C. Stringency is determined at the growth stage. Thus, 0.2 x SSC / 0.01%, 5DS / 65 ° C conditions are suitable for the selection of sek sequences with a homology of about 85 μm or more, and suitable for the selection of DNA sequences with a homology of about 90% or more. conditions 0.1 x SSC / 0.01% SDS / 65 ° C.

Examination of the cosmid library under these conditions (0.2 x SSC) -: · yields a few that hybridize to the IFN-omegal probe: cosmid. Sequence analysis of the restriction enzyme fragments isolated from these yields the authentic IFN-omegal gene: · .: (see Figure 11) and three other closely related genes named IF Np s eu do-omega 2, IFN-pseudo-omega3 and IFN- pseudo-omega4 (see Figures 12-14). These are also the subject of the present invention and their. . encoded peptide. These objects are set out in claims 43 to 52.

DNA comparison gives pseudogens approximately 85% homology to the IFN-omegal gene (interferon-omega-one gene). 1 · The IFN-omegal gene further indicates that in transcription ib 90667 contains mRNA information about a functional interferon protein, i. encodes a 23 amino acid long signal peptide of the formula

Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Vai Met Thr Ser Tyr Ser Pro Vai Gly Ser Leu Gly followed by a finished 172 amino acid long IFN-omega1.

Interferon-omega genes can be introduced under certain conditions into any organism that gives high yields. The person skilled in the art is best acquainted with the most suitable hosts and vectors; examples are given in EP-A-0.093.619.

Prokaryotes are considered particularly good for expression. For example, E. coli K 12, strain 294 (ATCC No. 31,466) is suitable. Other suitable strains are in E. coli X1776 (ATCC No. 31,537). In addition to the above strains, E. coli W 3110 (F ”, lambda”, prototroph, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcenses can also be used. bacteria, and various peeudomonas.

Plasmid can generally be used with these hosts. vectors, replicons, and controls that are derived from species compatible with the host cells.

-: In addition to the site of replication, the vector usually contains recognition sequences that allow phenotypic selection of the transformed cells. For example, E. coli: is usually transformed with the plasmid pBR 322, which is derived from E. coli (I b) (Bolivar, et al., Gene 2, 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance and therefore makes it possible to identify transformed cells in a simple manner. Plasmid pBR322 or other plasmids must also contain promoters or, for this purpose, must be modified to contain promoters which can be used by microorganisms to express their own proteins. The promoters most commonly used in the production of recombinant DNA include the beta-lactamase (penile 11inase) and lactose promoter systems (Chang et al., Naure 275, 615 (1978); Itakura et al.,

Science 198, 1056 (1977); Goeddel et al., Nature 281, 544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res., 4057 (1980); EP-A-0.036.776). Although the above are the most common promoters, other microbial promoters have been developed and used in addition to them. For example, the IFN-Omega gene sequence can be used under the control of the Leftuard promoter (P1) of a lambda bacterial phage. This promoter, which can be directed, is one of the particularly well-known promoters. Control is possible with a lambda repressor from which adjacent restriction sites are known.

The temperature-sensitive allele of this repressor gene can be inserted into a vector containing the complete IFN omega sequence. If the temperature is raised to 42 ° C, the repressor is inactivated and the promoter is expressed at its maximum concentration. The mRNA produced under these conditions:; : the sum must be large enough to obtain a cell which:: from the new synthetic ribonuclein ha about 10 ° ί is derived from the P promoter. In this way it is possible; develop a clone bank in which the functional IFN-omeqa .. sequence is located in the vicinity of the ribosome binding site at switch 1 evi1 distances from the lambda-P promoter. These clones can then be examined and selected · at the highest yields.

. . ·. Expression and translation of the IFN omega sequence may also occur under the control of other regulatory systems that may be "homologous" to the organism in their non-transformed form. Thus, for example, the chromosomal DNA of lactose-dependent E. coli contains a lactose or Lac operon, through the formation of the enzyme beta-galactosidase, which allows the breakdown of lactose.

Lac control elements can be obtained from lambda-plac5 bacterial phage that infect E. coli. The phage Lac operon can be obtained by transduction from the same bacterial species. The regulatory systems that can be used in the method of the invention can be obtained from organism-specific plasmid DNA. The Lac promoter-operator system can be induced by IPTG.

Equally, other promoter-operator systems or portions thereof may be used: for example, an arabinose operator, a colicin E 1 operator, a galactose operator, an alkaline phosphatase operator, a trp operator, a xylose A operator, a tac promoter, and the like.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures can also be used. Saccharomyces cerevisiae is the most commonly used eukaryotic microorganism, although a number of other species are commonly available. For expression in Saccharomyces yeast: for example, the plasmid YRp7 (Stinchcomb et al. Natur 282, 39 (1979); Kingsman et al., Gene 1, 141 (1979); Tschumper et al., Gene 1, 157 ( 1980)) and plasmid YEp 13 (Bu / ach et al., Gene 8, 121-133 (1979)). Plasmid YRp7 contains the TRP1 gene, which can act as a sel-. · As a thiomarker for a yeast mutant that is unable to grow in tryptophan-containing medium; for example, ATCC No. 44076.

. * ‘The presence of TRP1 damage as a hallmark of the yeast host genome - may be an effective aid in demonstrating transformation ·. when cultivated without tryptophan. The situation is exactly the same in the case of plasmid YLp11, which contains the yeast gene *: LEU 2, which can be used to supplement LLU-2-minus-Mutant m. Promoter sequences suitable for the yeast vector li 21 90667 include the 5 'protection region of the ADH I gene (Ammerer G., Methods of Enzymology 101, 192-201 (1983)), 3-Phosphoglycerate Kinase (Hitzeman et al., J. Biol Chem. 255, 2073 (1980)) or other glycolytic enzymes (Kawasaki and Eraenkel, E3BRC 106, 1107-1112 (1982)), such as enolase, glyceraldehyde-3-phosphate, dehydrogenase, hexokinase, pyruvate, decarboxylase, decarboxylase, , glucose-6-phosphate isomerase, phosphoglucose isomerase and glucokinase. Using appropriate expression plasmids, the terminal sequences associated with these genes can also be used at the 3 'end of the sequence expressed in the expression vector to polyadenylate and terminate the mRNA.

Other promoters that also have the advantage of growth-controlled transcription include the alcohol-dehydrogenase-2 gene, isocytochrome C, acid phosphatase-degrading enzymes that bind to nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate promoter regions of the enzymes responsible for use. Promoters regulated by the yeast Mating Typ Locus, for example promoters of the genes BAR1, MFct1, STE2, STE3, STE5, can be used in temperature-controlled systems using temperature-independent side mutations. (Rhine PH.0., Dissertation,

University of Oregon, Eugene ,. Oregon (1979), Herskowitz and Oshima, The Molecular Biology of the Yeast Saccharomyces,; part 1, 181-209 (1981), Cold Spring Harbor Laboratory).

These mutations affect the expression of yeast resting Mating Typ cassettes and thus indirectly Mating Typ dependencies on promoters. In general, however, all plasmid vectors containing * are suitable. "a promoter compatible with yeast, the original replication and termination sequences.

In addition to microorganisms, cultures of multicellular organisms are also suitable host organisms. In principle, any such culture can be used, regardless of whether it is a culture of a vertebrate animal or an invertebrate animal. However, the greatest interest is in vertebrate cells, and in recent years vertebrate cells have been grown in culture (tissue culture) by a routine method (Tissue, Culture, Academic Press, Kruse, & Patterson, eds. (1973)). Examples of such useful host cell lines include 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 upstream of the gene to be expressed, together with each required ribosome binding site, an RNA splicing site, a polyadenylation site, and transcriptional termination sequences.

When used in mammalian cells are mostly viral material must be subjected concerns an expression vector's control functions. Commonly used promoters are, for example, from Polyoma Adenovirus 2 virus and particularly often Simian Virus 40 (SV 40) virus. Particularly useful are the early and late final promoters of the SV 40 virus, as both can be readily obtained from the virus in a piece that still contains SV 40 virus replication sites "(Fiers et al., Nature 273, 113 (1978)). Also smaller: : and larger pieces of SV 40 virus can be used, provided that they contain a sequence of about 250 bp in length, extending from the HindIII intersection to the Bql 1 intersection in the virus-r ep 1 ikaa t iok section. it is possible and often advisable to use promoter or control sequences which are usually associated with the desired gene sequences, provided that these control sequences are compatible with the host cell systems.

The replication site can be constructed with either the corresponding vector. by constructing an exogenous site, * "for example from SV 40 virus or other viral sources (e.g. Polyoma, Adeno, VSV, PBV, etc.) or can be generated by chromosomal replication mechanisms of host cells. If the vector is integrated with the chromosome of the host cell , the latter measure is usually sufficient.

However, the genes can best be expressed in the expression plasmid pER103 (E. Rastl-Dvi / Orkin et al., Gene 21, 237-248 (1983) and EP-AQ.115-613) deposited with the DSM collection under number DSM 2773 on December 20, 1983. ) because this vector contains all the regulatory elements that result in high expression of the cloned gene. According to the invention, the short EcoRI / BamHI fragment characteristic of plasmid pBR322 is thus replaced by a DNA sequence of the formula

EcoRI Sau3A

gaattcacgct GATC GC TAAAAC ATTGTGC AAAAAGAGGG TTGAC TTTGCC TTCGCGA 59 ^ mRNA- start Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RBS Hindlll

Cys Asp TGT GAT C - IFN-omega gene->

Sau3A 1 - · In order to achieve the object according to the invention, the procedure according to Figure 6 is thus carried out, for example, as follows: I. Preparation of the necessary individual pieces of DNA:

Pala a

To prepare fragment a), the Avail plasmid containing the IFN-Omega gene is treated with a restriction endonuclease,

for example, plasmid P9A2. After chromatography and purification of the resulting cDNA insert, this is further treated twice with r es t r ik t ioendonuk 1 eaa se i 11 a NcoI and AluI

and then isolated by chromatography and electroelution 24 90667. This piece contains most of the corresponding omega interferon gene. Thus, for example, the omega (Gly) interferon gene of clone P9A2 has the following structure: 5 10 15

His Gly Leu Leu Ser Arg Asn Thr Leu c | CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 |

Ncol 20 25 30

Vai Leu Leu His Gin Met Arg Arg Ile Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73 35 40 45

Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Vai Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 50 55 60

Ser Gin Leu Gin Lys Ala His Vai Met Ser Vai Leu His Glu Met AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 65 70 75

Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 80 85 90.-Ala "rp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His * ,: ®CC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253 95 100 105 - '· Sln Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Vai Vai Gly •.-CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 110 115 120

Glu Gly Glu Ser Ala Gly Ala Ile 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 Ile Arg Vai Tyr Leu Lys Glu Lys Lys - 1 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 li :: 25 90667 140 145 150

Tyr Ser Asp Cys Ala Trp Glu Or Val Arg Met Glu lie Met Lys TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 155 160 165

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

Asp-Asp Arg Leu Gly Ser Ser GAT AGA GAC GGC CTG TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 530 589 648 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATAC TATTTATATTCTTATATAACAAATGTTTGCC 707 766 802 TTTACATTGTATTAAGATAACAAAACATGTTCAGfct

AluI

Pala b:

To prepare fragment b), plasmid P9A2 is treated with the restriction endonuclease Avail. After chromatography and purification of the resulting cDNA insert, this is reprocessed with restriction endonuclease Sau3A and the desired 189bp piece is isolated by chromatography and electroelution.

; It has the following structure: Z6 90667 5 10 15

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

IgAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 42

Sau 3A | Ncol 20 25 30

Val Leu Leu His Gin Met Arg Arg lie Ser Pro Phe Leu Cys Leu GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 87 35 40 45

Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly AAG GAC AGA AGA GAC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 132 50 55 60

Ser Gin Leu Gin Lys Ala His Vai Met Ser Vai Leu His Glu Met ACG CAG TTG CAG AAG GGC CAT GTC ATG TCT GTC CTC CAT GAG ATG 177

Leu Gin Gin Ile CTG CAG CA | g a te 189

Sau 3A | ':: Pala c: -; To prepare piece c), plasmid pER33 (see

E. Rastl-Dworkin et al., Gene 21, 237-248 (1983) and EP-A-Q.115.613) twice the restriction enzymes EcoRI and PvuII. The 389 bp piece obtained after fractionation and purification of the agarose gel, containing the Trp promoter, ribosomal binding site and start codon, is then treated with Sau3A. Wanted . A 108bp long piece is obtained from an agarose gee1i electro for ees in,. !! after electroelution and Elutip column purification.

'' It has the following structure: li 27 90667

EcoRI | Sau3A

gaa t tcac gc tEATCGCTAAAACATTGTGCAAAAAGAGGGTTGAC TTTGCCTTCGCGA 59 ^ jtiRNA-ku Met ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RES Hindlll

Cys Asp TGT gat c 123

Sau3A

Liquefaction of pieces b and a:

Pieces b and c are ligated with T4 ligase and, after destruction of the enzyme, cut with HiridIII. This ligated piece has the following structure:

Hindin Sau3A Ncol ajAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100: ·. CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAkqct t; Sau3A Hind IIil This piece of DNA needed to make plasmid pRHWIO Λ *! Alternatively, it can also be formed using two synthetically prepared oligonucleotides: 28 90667

An oligonucleotide of the formula 5'-AGCTTAAAGATGTGT-3 'is retained at its 5' end without being phosphorylated.

An oligonucleotide of the formula 5'-GATCACACATCTTTA-3 'kinase is treated at the 5' end with a polynucleotide kinase and ATP.

When both oligonucleotides hybridize, the following short piece of DNA is obtained: 5'-AGCTTAAAGATGTGT 3 '3'-ATTTCTACACACTAGp 5' This gives a 5 'extension typical of HindIII at one end and typical of S au 3A at the other end, *; 5 1 - Continued.

"Returning b) de fos fory1 oidaan using calf intestinal fos - '* ·,. phosphatase. Piece b) and the piece described above are brought together: and joined together by T 1 ligase.

Since the ligase needs at least the 5 '- f os f aa 11 ip i t oi: end of the nuclei, only a synthetic piece of DNA can be joined with piece b) or also two synthetic pieces at its Sau3A ends. Since the lengths of the two pieces obtained are different, they can be distinguished by selective isopropano 1 isoprecipitation.

:: The thus purified piece is phosphorylated f-polynucleotide -; kinase and MP.

li 29 90667 II. Preparation of expression plasmid a) Preparation of plasmid pRHWIO:

PERl03 expression plasmid (E. Rastl-Dworkin et al.f, Gene 21, 237-284 (1983) and EP-A-0115613 ΟςΜ stored in the collection DSM number 2773) is linearized with Hind III and then treated with calf intestinal phosphatase again with. After isolation and purification of the DNA thus obtained, it is dephosphorylated and then ligated with the ligated pieces b and c (after their HindIII treatment). E. coli HB 101 is then transformed with the resulting ligation mixture and cultured on LB agar with 30 ug / ml ampicillin. The plasmid having the desired structure is labeled pRHW 10 (see Figure fa).

After its replication, it is used as an intermediate in the preparation of other plasmids.

b) Preparation of plasmid pRHW12:

A Klenow fragment of DNA polymerase I and four deoxynucleo- cells are added to plasmid pRHWIO cut with BamHI. "siditriphosphate. The linearized plasmid with blunt ends obtained after incubation is purified and cut '·' 'then Neo I-en t cause mi 1 la. The larger piece obtained' · ag ar oos igee 1 ie lek t ro f orees in, after electroelel elution and Elutip purification, are ligated with piece a. Thereafter, the ligation mixture is transformed with E. coli HB 101 and cultured on LB agar with 50 ug / ml ampicillin. The plasmid having the desired structure is named pRHW12 (see Figure 6), which expresses the desired omega (G ly) interferon. 1 l of the bacterial culture thus obtained (optical density: 0.6,: 600 nm) contains, for example, I x 10 ^ international mter-____: pheron units.

3D 90667 c) Construction of plasmid pKIIWli:

A Klenow fragment of DNA polymerase I and 4 deoxynucleoside triphosphate are added to plasmid prHWIO cut with BamHT. The linearized straight-ended plasmid obtained after incubation is purified and then digested with NcoI. The larger fragment obtained after agarose gel electrophoresis, electroelution, and Elutip bundling is ligated with fragment a obtained analogously from plasmid E79E9, in which only the GGG codon encoding the 111th amino acid (Gly) has been replaced by the GAG codon (Glu). The resulting ligation mixture is then transformed with f. Coli HB 101 and cultured on LB agar. The plasmid having the desired structure is named pRHW11 (see analogous to Figure 6), which expresses the desired omega (G1u) interferon.

Transformation of cells 1 can be accomplished by a number of methods. This can be done, for example, with calcium, in which case either the cells are washed in magnesium and the DNA is added to the cells suspended in calcium, or the cells are treated with a combination of DNA and calcium phosphate. In the next gene expression, the cells are transferred to media that - · · select the transformed cells.

Once the host has been transformed, the gene is expressed or fer-; : mentored or cultured cells under conditions with IFN-omega ;; is expressed, the product can usually be extracted by known chromatographic separation methods to give: a material containing omega interferon having -. There are no Leader or Tailing sequences or these. The IFN omega can be expressed in the N-terminus of the Leader sequence (Pre-IFN omega), which can be removed from some host cells. If this is what a dj can do, a ready-made IFN-Omega is needed. : to obtain a Leader polypeptide (when present) by cleavage ____. off. Alternatively, the IFN-omega clone can be modified with li

Ji 90667 so that the microorganism directly produces the finished protein instead of Pre-IFN-Omega. For this purpose, the precursor sequence of the yeast Mating-Pheromon-MF-a 1 fa-1 can be used, which allows the correct "production" of the fused protein11 and the separation of the product into the medium or plasma state. The DNA sequence of a functional or mature IFN-Omega can be linked to MF-alpha-1 at a putative cathepsin-like cleavage site (after Lys-Arg amino acids) from the start codon ATG at position 256 (Kurjan, Herskowitz, Cell 30, 933-943 (1982)).

Based on their biological spectrum of activity, the new interferons according to the invention can be used in all kinds of treatments which also use known interferons. These include, for example, herpes, rhinovirus, suitable AID5 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 anti-tumor or anti-viral therapy is required, and in cases of immunosuppressive:. features. The dosage and dosage amounts may be the same as in clinical trials of IFN-α materials to date, for example about (1-10) x 10 6 units per day and for preparations with a purity of more than 1%, for example up to 5 x 10 6 units per day.

• In the case of a substantially homogeneous IFN omega produced by bacteria ** ', for example, for parenteral use, 3 mg of IlN omega may be dissolved in 25 ml of 5-p Rosen 11 human salum albumin. then through a bacteriological filter and the filtered solution is aseptically filled into 32 90667 aseptically 100 vials, each containing 6 x 10 6 units of pure IFN omega suitable for parenteral application. Before use, bottles are best stored refrigerated (-20 ° C). The substances according to the invention can be formulated in a known manner so as to obtain pharmaceutically usable substances. The polypeptide of the invention is then mixed with a pharmaceutically acceptable carrier. Ordinary carriers and their formulations have been described by E.W. Martin in: Remingtom's Pharmaceutical Sciences, which is incorporated herein by reference. A measured amount of carrier is mixed with omega interferon to provide suitable pharmaceutical agents that can be effectively used by the recipient (patient). Parenteral administration is preferred.

The following examples illustrate the invention in detail. Example 1 Finding clones specific for the IFN sequence a) Preparation of a cDNA library

MRNA obtained from cells stimulated with Sendai virus1a is used as a starting material for the preparation of a cDNA library by methods known from the literature (E. Dworkin-Rast1 et al., Journal of Interferon Research Vol. 274, 575-585 (1982)).

The 30,000 clones obtained are transferred one by one to the wells of microtiter plates. The following medium is used for colony growth and freezing: n “90667 10 g Tryptone 5 g Yeast extract 5 g NaC 1

0.51 g Na citrate x 2 H 2 O.

7.5 g K2HPO4 x 2 f ^ O 1.8 g KH2PO4 0.09 g MgSO4 x 7 ^ 0 0.9 g (NH4) 2504 44 g glycerin 0.01 g Tetracycline x HCl ad 1 1 H2O

Microtiter cells containing individual clones are incubated overnight at 37 ° C and then stored at -70 ° C.

b) Hybridization probe

The starting material for the hybridization probe is the recombinant plasmid pER33 (E. Duforkin-Rastl et al., Gene 21, 237-248 (1983)). This plasmid contains a region encoding the mature interferon IFN-α2arg and 190 bases of the 3 'untranslated region. 20 ug of plasmid pER33 and 30 units of HindIII restriction endonuclease are incubated for 1 hour at 37 ° C in 200 ul of reaction solution (10 mM Tris-HCl pH 7.5, 10 mM Mg Cl 2 · 4. 1 mM dithiothreitol (OTT)). The reaction is stopped by adding 1/25 vol. 0.5 M ethylenedinitrilotetraacetic acid (EDTA) and heating for 10 minutes at 70 ° C. Add 1/4 vol. 5 x buffer (80? Glycerol, 40 mM tris -acetic acid pH = 7.8, 50 mM EDTA, 0.05% sodium dodecyl (5DS), 0.1% bromine phenolic blue) and the pieces formed are separated by electrophoresis on a 1% agarose gel / gel and running buffer ( TBE): 10.8 g / l-trishydroxymethylaminomethane (tris-base), 5.5 g / l tris-hydroxymethylaminomethane (tris-base), 5.5 g / l boronic acid, 0.93 g / l EDTA /. incubate in 0.5 ug / ml ethidium bromide solution, then make the DNA bands visible under UV light and remove the IFN gene-containing DNA fragment (approximately 800 bp 34 90667 long). the gel area containing it is cut off. Using voltage, DNA is electroeluted in 1/10 x IBL buffer. The DNA solution is extracted once with phenol and four times with ether and the DNA is mixed from the aqueous solution at -20 ° C by adding 1/10 vol. 3 M sodium acetate (NaAc) pH 5.8 and 2.5 vol. ethanol. After separation by centrifugation, the DNA is washed once with 70% ethanol and dried in vacuo for 5 minutes. DNA is taken up in 50 μl of water (about 50 μg / μl). This DNA is radiolabeled by ε · maniatis et al. (Τ · Maniatis et al., Method modification, Molecular Cloning, ed. CSH). 50 [mu] l of incubation solution contains: 50 mM Tris pH = 7.8, 5 mM MgCl2, 10 mM mercaptoethanol, 100 ng pER33 inert DNA, 16 [mu] g DNassel, 25 [mu] mol dATP, 25 [mu] mol dGTP, 25 [mu] mol dllP, 20 μl of α 32 P-dCTP (> 3000 Ci / mMol) and 3 units of DN A polymer a si 1 (E. coli). Incubation was performed at 140 ° C for 45 minutes. The reaction is stopped by adding 1 vol. 50 mM EDTA, 2% SDS, 10 mM Tris pH = 7.6 and heating at 70 ° C for 10 minutes. DNA is separated from unbound radioactivity by chromatography on a Sephadex G-100 column in TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). The specific activity of the radiolabeled probe is about 4 x 10 ^ cpm / μg.

c) Screening of the IFN gene-containing insert from the clones 'The microbes frozen in the microbes are thawed: ·'; (A). 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 te t rasy k 1 ii ni-HC 1) a correspondingly sized piece of nitro se 11u1ose filter piece is placed on top. . (Schleicher und Senile, BA 85, pore size 45 μm). Using individual microbes, individual clones are transferred to a n-rose cellulose filtrate. Bacteria: grow overnight at 37 ° C into colonies of about 5 mm in diameter. To destroy bacteria and DNA. for denaturation, place nitrocellulose filters on top of the label with a Whatman li 35 90667 3MM filter moistened with the following solutions: 1) 8 minutes with 0.5 M NaOH, 2) 2 minutes with 1 M Tris pH = 7.4, 3) 2 minutes with 1 M Tris pH = 7.4 and 4) 4 minutes 1.5 M NaCl, 0.5 M Tris pH = 7.4. The filters are air dried and then kept at 80 ° C for 2 hours. The filters were pretreated for 4 hours at 65 ° C in a hybridization solution consisting of: 6 x SSC (1 x SSC corresponds to 0.15 M NaCl; 0.015 M trisodium citrate; pH = 7.0), 5 x Denhardt's solution (1 x Denhardt's solution corresponds to 0.02% PVP (polyvinylpyrrolidone); 0.02%

Picoll (mp .: 40,000 D); 0.02% BSM (Bovine Serum Albumine) and 0.1% SDS (sodium dodecyl sulfate). Approximately 1 x 10 6 cpm / filter in the probe prepared in b) is denatured by boiling and the hybridization solution is added. Hybridization occurs for 16 hours at 65 ° C. The filter is washed at 65 ° C 4 times for 1 hour with 3 x SSC / 0.1 5 SDS solution. The filters are air dried, covered with a Sara Wrap cover, and exposed to Kodak X-OmatS film.

Example 2

Southern hybridization to detect other recombinant plasmids IFN-oene-pj

From positive or positively believed colonies, grow 5 ml cultures in L-broth (10 g / l tryptone,: /. [5 g / l yeast extract, 5 g / l NaCl, 20 mg / l tetracycline x HCl): · "; overnight At 37. Plasmid DNA is modified by Briboim and

By the method of Doly (Nucl. Acid Res. 7., 1513 (1979)) and isolated. The cells are centrifuged separately from 1.5 ml of suspension (Eppendorf centrifuge) and resuspended at 0 [deg.] C. in 100 [mu] l of a lysozyme solution consisting of: 50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl pH = 8.0 and 4 mg / ml lysotime. Incubate for 5 minutes at room temperature, then add 2 volumes of ice-cold 0.2 M NaOH, 1% SDS solution and incubate for another 5 minutes. Then - add 150 μΐ of ice-cold potassium acetate 1 solution pH = 4.8 and incubate for 5 minutes. The precipitated cell constituents 56 9 0 6 6 7 are separated from the centrifuges. The DNA solution is extracted with 1 volume of phenol / chloroform (1: 1) and the DNA is mixed by adding 2 volumes of ethanol. After separation of the centrifuged soot, the pellet is washed once with 70% ethanol and dried for 5 minutes in vacuo. DNA is dissolved in 50 μl of Iran (TL) buffer. A 10 μl aliquot is treated for 1 hour at 37 ° C with 10 units of PstI restriction endonuclease in 50 μl of reaction (10 mM Tris-HCl pH 7.5, 10 mM MqCl 2, 50 mM NaCl, 1 mM OTT). 1/25 volume of 0.5 H LDTA and 1/4 volume of 5 x buffer (see Example Ib)) are added and then heated for 10 minutes at 70 ° C and then the DNA is separated electroelectrically on a 1-prose agar gel ( TBE buffer). The agarose gel DNA is transferred by the method of Southern (E.M. Southern, J. Mol. Biol. 9J3_, 5U3-517 (1975)). The DNA of the gel is denatured by incubating the gel for 1 hour together with a 1.5 M NaCl / 0.5 M NaOH solution, after depression the solution is neutralized for one hour with a 1 M Tris x Hai pH = 8 / 1.5 M NaCl solution. The DNA is transferred to a solution of 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate, pH = 7.0) in a nitrocellulose-1 o portion of 11 e. After complete transfer (approximately 16 hours), the filter is briefly rinsed with 6 x SSC. in buffer, then air-dried and finally baked: 2 hours at 80 ° C. Filter precursor 1 for four hours •: - for 6 x 5SC / 5 x Denhardt's solution / 0.1% SDS (see example: · le) -1 solution at 65 ° C. Approximately 2 x 10 cpm of the hybridization probe (see Example Ib) is denatured by heating. At 100 ° C and then the hybridization is added to the solution.

The hybridization time is 16 hours at 65 ° C. Then the filter. . wash 4 x 1 hour at 65 ° C with 3 x SSC / 0.1% S D S solution. After air-drying, the filter is covered with a • ’Hinge Wrap and exposed with Kodak X-0matS.

Example 3 'Demonstration of interferon activity in clone E76E9 li 3, 90667 A 100 ml culture of clone E76D9 is grown in L-broth (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 5 g / l glucose, 20 mg tetracycline x HCl pro 1) at 37 ° C to an optical density of Agoo = 0.8. The bacteria are separated by centrifugation for 10 minutes at 7,000 rpm, washed once with 10 mM Tris x HCl pH 8.0, 30 mM NaCl and resuspended in 1.5 ml of growth solution. Incubate at 0 ° C for half an hour with 1 mg / ml lysozyme, after which the bacterial suspension is frozen and thawed five times. Cell debris is pelleted by centrifugation at 40,000 rpm for one hour. The supernatant is sterile filtered and tested for interferon activity. Plaque assay with V3 cells and Vesicular Stomatitis virus 11a (G.R. Adolf et al., Arch. Virol. 72., 169-178 (1982)) are used as a test. Surprisingly, the clone produces up to 9,000 IU of interferon / liter of output.

Example 4

Genomic Southern Blot to determine the number of genes containing new sequences a) Isolation of DNA from Namalwa cells: 400 ml of Namalwa cell water is centrifuged in a JA21 centrifuge at 1000 rpm to pellet the cells. / Min. Obtained: The pellets are gently washed by resuspending these in NP40 buffer (NP40 buffer: 140 mM NaCl, 1.5 mM MgCl 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 the cell walls are lysed by adding 1 ml of 10% NP40 solution. Allow to stand for 5 minutes in an ice bath, after which the intact cells are pelleted by centrifugation at 1000 rpm. and the supernatant is discarded. The nuclei are resuspended in 10 ml of a solution of: 50 mM Tris / Cl pH = 8.0, 10 mM *. **: EDTA and 200 mM NaCl, followed by the addition of 1 ml of 2U to remove proteins. -prosenltistu SD5- 11 uoota. The resulting viscous solution is extracted twice with an equal volume of phenol (saturated 10 mM Tris / Cl pH = 8.0 buffer 11a) and twice with chloroform. The DNA is precipitated by adding ethanol, separated by centrifugation, and then the resulting DNA pellet is washed once with 70% ethanol. Dry in vacuo for 5 minutes and dissolve in 6 ml of TE buffer (TE buffer: 10 mM Tris / Cl pH = 8.0, 1 mM EDTA). The concentration of DNA is 0.8 mg / ml.

b) Restriction endonuclease treatment of DNA from Namalwa cells

Restriction endonuclease treatments are performed under the conditions specified by the manufacturer (New England Biolabs).

1 pg of DNA is treated with 2 units of the appropriate restriction endonuclease in a volume of 10 at 37 ° C for 2 hours or more. EcoR1, HindIII, BamHI, SphI, PstI and ClaI enzymes are used as restriction endonucleases. 20 ug of DNA is used in each treatment. To determine the completeness of the treatment, always separate • · 'at the beginning of the reaction. 10 μl (aliquot) and 0.4 μg of lambda phage DNA is added.

. . ·. After 2 hours of incubation, these aliquots are controlled by agarose gel electrophoresis and the completeness of the treatment is assessed by the pattern of stained lambda phage DNA pieces *.

: After performing this control, the reactions are stopped by adding EDTA to a final concentration of 20 mM and heating the solution for 10 minutes at 70 ° C. DNA is precipitated by the addition of 0.3 M NaAc pH = 5.6 and 2.5 volumes of ethanol. Incubate for 30 minutes at -70 ° C, after which the DNA is pelleted in an E·pendorf centrifuge, washed once with 70% ethanol and dried. The resulting DNA is taken up in 30 Risa TE buffer.

li 39 90667 c) Gel Electrophoresis and Southern Transfer The treated DNA probes are fractionated by size on a 0.8% agarose gel in TBE buffer (10.8 g / l Tris base, 5.5 g / l boric acid, 0.93 o / l EDTA). For this, 15 μl of the DNA probe is mixed with 4 μl of loading buffer (0.02% 5D5, 5 x TBE buffer, 50 mM EDTA, 50% glycerin, 0.1% bromophenol blue), heated briefly at 70 ° C and placed in reserved gel pools. Lambda DNA cut with EcoRI and HindIII is placed in the corresponding pool to serve as a marker of DNA size. Gel electrophoresis is performed for 24 hours at a voltage of about 1 V / cm. The DNA is then transferred by the Southern method to a nitrose cellulose filter (Schleicher und Schuell BA85) using 10 x SSC (1 x ^ SC: 150 mM trisodium citrate, 15 mM NaCl, pH = 7.0) as transfer buffer. The filter is dried at room temperature, then heated for 2 hours at 80 ° C to bind DNA to the filter.

d) Hybridization probe

20 μg of plasmid P9A2 is digested with Avall to release a fragment of approximately 1100 bp containing the entire cDNA insert. This piece of DNA is re-cut with the enzymes Sau3A and AluI and the largest piece of DNA is isolated after agarose gel electrophoresis (see Figure 5b) by electroelution: and Elutip-py 1 flash chromatography. The DNA thus obtained

(1.5 μg) is dissolved in 15 μl of water.

20 ug of plasmid pER33 is digested with HindIII I, in which case this IFN-α2-Arg expression plasmid (E. Rasti.-Dworkin et al. Gene 21, 237-248 (1983)) is cleaved twice. The smaller piece of DNA contains the interferon-α2-Args gene: and is similarly isolated in the case of plasmid P9A2.

Both DMAs are Nick-translated by P.W.J. Rigby'n et al.

40 9 0 6 6 7 (J. Mol. Biol. 113, 237-251 (1977)). Nick translation is performed in each case using 0.2 μg of DNA in 50 μl of a solution consisting of: 1 x Nick buffer (1 x Nick buffer: 50 mM Iris / Cl p H = 7.2, 10 mM MgSO 4). , 0.1 mM DTT, 30 pg / ml BSA), 100 μMol dATP, dGTP and d Γ TP, 150 μCi α- β-dCTP (Arnersham, 3000 Ci / mMol) and 5 units DNA polymerase I (Boehringer -Mannheim, Nick-tra Is aatio-1 aatu). After 2 hours at 1 ° C, the reaction mixture is stopped by adding an equal volume of EDtA solution (AO mMol) and the unreacted radioactive material is separated by G 50 column chromatography on a TE buffer. The specific radioactivity remaining is about 100 x 10 cpm / μg DNA.

e) Hybridization, and the au torad ioq ra f ia N it rose 1 cellulose filter is cut in two halves. Each half contains an identical progeny of Namalvi / α DNA which, as mentioned in Example Aa, has been treated with restriction enzyme 11. The filters are prehybridised for 2 hours at 65 ° C in a solution consisting of: 6 x SBC, 5 x Denhardt's solution (1 x Dernhardt's solution: 0.02 S bovine serum albumin (BSA), 0.02 % polyvinylpyrrolidori (PVP), 0.02% Ficoll A00), 0.5% SDS, 0.1 mg / ml denatured calf DNA and 10 mM EDTA.

: Hybridization is performed in a solution of: 6 x SBC, 5 x Denhardt's solution, 10 mM EDTA, 0.5% SD9 and about 10 x 10u cpm nick-translated DNA, for 16 hours at 65 ° C. Half of the filter is hybridized to interferon-u2-Arg-DNm: L1a and the other half to interferon-DNA isolated from plasmid P9A2. After hybridisation: both filters are washed four times at room temperature with a solution of 2 x SSC and 0,1? Ό · - SDS and then twice for A5 minutes at 65 ° C with a solution of 0,2 x SSC and 0.01% SDS.

The filters are then dried and exposed to Kodak X-0mat S-film.

41 90667

Example 5

Preparation of expression plasmids pRHW 12 and pRHW II Preliminary note:

The preparation of expression plasmids is shown in Figure 6 (scale not correct). All restriction enzyme procedures are performed according to the enzyme manufacturer's data.

a) Preparation of plasmid pRHW 10 100 pg of plasmid P9A2 is treated with 100 units of restriction endonuclease Avail (New Englands Biolabs). After treatment, the enzyme is inactivated by heating to 70 ° C and the resulting pieces are size fractionated on a 1.4% agarose gel with TBE buffer (TBE buffer: 10.8 g / l Tris base, 5.5 ° / l boric acid, 0.93 q / l EDTA).

The band containing the entire cDNA insert is electroeluted and purified on an E. lutip column (Schleicher 4 Chuell). A 6 μg aliquot of the resulting 20 pg is re-treated with the restriction endonucleus si11a Sau3a (20 units in total with 100 μl of solution). Pieces are separated in 2% agarose igee 111 I in TBE buffer. After staining with ethidium bromide (EtBr), an 189 bp 'piece of DNA is electroeluted and purified as described above. * (= Piece b in Figure 6).

In order to insert a promoter, a ribosomal binding site and a start codon into the interferon gene, they are isolated. the corresponding DNA fragment from the expression plasmid pER 33 (E. Rasti-Oworkin et al., Gene 21, 237-248 (1983)). For this purpose, 50 .mu.g of plasmid pER 33 are treated twice with the restriction enzymes EcoRI and PvuII and the pieces obtained are fractionated: **: by size on a 1-4. TBE buffer. A 389 bp piece of DNA containing the Trp promoter, ribosomal binding site and start codon is electroeluted and purified in a Eiutip strain. The piece thus obtained is then treated with Sau3A and the desired 108 bp piece is obtained after agarose-qee1ι electrophoresis, electroelution and Elutip column purification in a yield of about 100 ng (= piece c in Figure 6).

20 ng of piece b and 20 ng of piece c are ligated for 18 hours at 14 ° C in a volume of AO pI using 10 units of T4 ligase in a solution of: 50 mM Tris / Cl pH = 7.5, 10 mM MgCl 2, 1 mM DTT and 1 mM ATP. The enzyme is then destroyed by heating to 70 ° C and the resulting DNA is digested with HindIII in a total volume of 50.

The 10 pg expression plasmid pER 103 (E. RAst 1-Dwork in et al., Gene 21, 237-248 (1983)) is linearized with HindIII in a total volume of 100. The mixture was kept for 2 hours at 37 ° C, followed by addition of 1 vol 2 x phosphatase slpuskuria (20 mM Tris / Cl pH = 9.2, 0.2 mM FDTA) with one unit of calf intestinal phosphatase (C1P).

Keep at 45 ° C for 30 minutes, then add another unit of CIP phosphatase and incubate for another 30 minutes. The DNA thus obtained is purified by extraction twice with phenol, once with chloroform and precipitation by adding 0.3 M sodium acetate (pH = 5.5) and 2.5 vol. ethanol. It is then de phosphorylated to prevent re-ligation of the vector in the next step.

100 ng of non-irradiated plasmid pER 103 and ligated pieces b and c (after H ind 111 treatment) are added to 100 μl of a solution containing ligase buffer and ligated with 1 N DNA DNA for 18 hours at 14 ° C. .

· Mix 200 μl of competent E. coli HB 101 bacteria li m. 90667 (E. Dworkin et al., Dev. Biol. 7 £, 435-448 (1980)) and 20 μl of ligation and incubated for 45 minutes on ice. The 0NA migration is then completed by heat shock (2 minutes at 42 ° C). The cell suspension is incubated for a further 10 minutes on ice and then the suspension is applied to LB agar (10 q / l tryptone, 5 g / l yeast extract, 5 g / l waCl, 1.5% agar) containing 50 mgl ampicillin. The plasmids contained in the resulting 24 colonies are isolated by the method of Birnboim and Doly (see Nuc. Acid. Res. 7 * 1513-1523 (1979)). After treatment with various restriction enzymes, the plasmid has the desired structure. This is named pRHW 10 (see Figure 6).

b) Preparation of plasmid pRHW 12

Approximately 10 ug of plasmid pRHW 10 is digested with BamHI enzyme 11. A Kleno fragment of DNA polymerase I and 4 deoxynucleoside triphosphates are then added and incubated for 20 minutes at room temperature. The resulting linearized and straight-ended plasmid is purified by phenol extraction and blending, followed by digestion with restriction endonuclease IcoI in a volume of 100. Larger:: The piece is recovered by agarose gel electrophoresis, electroelution and Elutip purification. Piece a) (see Figure 6) is obtained by NcoI and AluI treatment of 4 ug of an Avall piece containing the P9A2 cDNA insert (see above).

. . This gives about 2 .mu.g of a).

- - In the last ligation step, piece a) and the BamHI / NcoI concept Ity pRHW 10 added to it in a volume of 10 [mu] l, each time using 10 [mu] g of DNA, are ligated. The BamHI site is again filled by ligation. BamHI cleavage site in AluI-cleaved DNA.

The resulting ligato mixture is transformed as described above with competent t.coli HB 101 bacteria. Of the 40 colonies obtained, one is selected and named pRHW

«4 90667 12.

The plasmid is isolated and sequenced with the EcoK1 / BamHI insert by the method of Sanger (F. Sanger et al., Proc. Nat. Acad.

Sci _74, 5463-5467 (1979)). This has the expected sequence.

c) Preparation of plasmid pRHW 11 This is done analogously to Example 5b. 1 μg of plasmid pRHW 10 is treated with Bamh1 enzyme. The sticky ends of the resulting DNA are flattened with a DNA polymerase I Klenow fragment and 4 deoxynucleoside triphosphates, after which the linearized DNA is cleaved with NeoI. The larger piece is recovered by agarose gel electrophoresis, electroelution, and E1utip-day chromatography.

A Neo I-AluI fragment is isolated from clone E76E9 according to Example 5b. 10 ng of the vector portion and 10 ng of the cDNA portion are then ligated in a volume of 10 under appropriate conditions and using one unit of T4 ligase. The resulting DNA mixture is transformed into E. coli HB 101 and the resulting 45 colonies are selected with LB-ma1joI containing ampicillin α, after which one clone is named pRHW 11. After culturing the corresponding clone, plasmid DNA is isolated. The structure of this is demonstrated by several specific restriction endonuclease cleavage sites (Alul, EcoRI, r: HindIII, NcoI, PstI).

d) Expression of interferon activity in E. coli HB 101 bacteria containing plasmid pRHW 12 100 ml of bacterial culture is incubated to an optical density of 0.6 (600 nm) in M9 minimal medium containing all amino acids except tryptophan (20 μg / ml - - per amino acid), 1 ug / ml thiamine, 0.2% glucose and 20 ug / ml indole- (3) -acrylic acid (IAA) containing the tryptophan operon inducer. It is then pelleted

II

90667 bacteria are centrifuged (10 minutes, 7,000 rpm), washed once with 50 mM Tris / Cl pH = 30, 30 mM NaCl solution and then suspended in 1.5 ml of the same buffer.

It is then incubated for 30 minutes with 1 mg / ml lysozyme, after which the bacteria are frozen and thawed five times. Cell debris is removed cent r.i f Ugo ima 1 la 1 hour 40,000 rpm. The supernatant is sterile filtered and tested for interferon activity in a plaque assay using human A549 cells and enke fa 10o myocarditis viruses.

Result: 1 L of prepared bacterial culture contains 1 x 10 6 international units of interferon (A. Billiau, Antiviral Res. 4, 75-98 (1984)).

Example 6

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

The amino acid sequence is compared in pairs by listing the first cysteine group of the mature α-interferon with the first cysteine group of the amino acid sequences encoded by the inserts of plasmid P9A2 and E76E9 c D N A - ·. ·. Both '/ ·' sequences are shown in Figure 7 as IFN omega, as no differences could be detected between the specific sequences of P9A2 or E76E9 clones based on the values obtained. The only correction performed was the insertion of a gap at position 45 of αA-interferon. *, Which was considered an error site. When the reference ... was the sequence of the omega interferon, the comparison was performed taking into account the other 166 amino acids. This value is. shown in Figure 7 together with 6 other amino acids encoded by clones P9A2 and E76E9. Percentages. . the differences are obtained by dividing the differences obtained by 1.66. One additional amino acid thus represents a number of 0.6 in percent. For the 6 additional amino acids of IFN-Omega, 3.6% are obtained, which are already included in the percentage of 46,90667.

Comparison with g-interferon is performed by listing amino acid 3 of mature β-interferon with the first amino acid of mature α-interferon or the first amino acid encoded by plasmids P9A2 and E76E9. The longest reference structure of α-interferon with g-i nt er fe ron thus extends over 162 amino acids, giving 2 1 amino acids for both α-interferon and g-interferon11e. These are considered error sites and are shown separately in Figure 7, but still included in the percentage, The listing of g-interferon with the amino acid sequences of clones P9A2 or E76E9 is performed in a similar manner. However, a total of 10 additional amino acids are obtained.

b) Comparison of nucleotide sequences

The listing in the comparison queues takes place according to Example 6b. The first nucleotide of the mature α-interferon DNA is the first nucleotide of the triplet encoding the mature u-interferon cysteine. The first nucleotide of the DNA of plasmids P9A2 or E76E9 is likewise the first nucleotide of the cysteine-1 codon:, the first nucleotide of the g-interferon DNA is the first nucleotide of the 3rd triplet. Comparison - a total of more than 498 nucleotides when comparing the individual DNA of interferon-α with the DNA of interferon-β, and more than 516 nucleotides when comparing the DNA sequences of individual α-interferons or g-interferons with plasmids P9A2 and E76E9. The absolute number of error locations is given in the left part of the table in Figure 7 and then the corresponding percentages in parentheses.

. Example 7

Virus-inducible expression of omega-1 mRNA and NC37 cells: * '90667 a) Synthesis of an omega-interferon-specific hybridization probe .10 picomoles of oligonucleotide d (TGCAGGGC TGCTAA) and 12 picomoles of A-gamma (specific activity:> 5000 Ci / mMol) and 10 units of polynucleotide kinase are mixed in a total volume of 10 (70 mM Tris / Cl pH = 7.6, 10 mM MgCl 2, 50 mM DT Γ) and allowed to stand for one hour at 37 ° C. The reaction is then stopped by heating at 70 ° C for 10 minutes. The resulting radiolabeled oligonucleotide and 5 picomoles of M13pRHW 12 ssDNA (see Figure 9) are hybridized by standing for one hour at 50 ° C in a total volume of 36 (100 mM NaCl).

After cooling to room temperature, Nick's translation buffer, 4 deoxynucleoside triphosphates and 10 units of Klenow polymerase are added to a total volume of 50 (50 mM Tris / Cl pH 7.2, 10 mM MgCl 2, 50 μg / ml BSA). , 1 mM per nucleotide). The polymerization is carried out by standing for one hour at room temperature, after which the reaction is stopped by heating at 70 ° C for 5 minutes.

The reaction yields partially double-stranded circular DNA. This is then treated with 25 units of Avall enzyme in a total volume of 500 HI, using; the buffer described by the manufacturer. Double-stranded areas intersect. . then become equal pieces. After the reaction; is stopped by heating for 5 minutes at 70 ° C.

b) Preparation of RNA from virus-infected cells *: 100 x 10 6 cells (0.5 x 10 6 / ml) are treated for 48-72 hours with 100 μmol dexamethasone and the control does not contain dexa-II metazone. For interferon induction, expression cells are suspended in serum-free medium at 5 x 10 6 / ml and infected with 210 units / ml of endai virus. Aliquots of cell culture supernatants are assayed for IFN activity in * 8 90667 p 1 (Example 5). Six hours after virus infection, cells are harvested by centrifugation (1000 g, 10 minutes), washed in 50 ml of NP40 buffer (Example 4a), resuspended in 9.5 ml of ice-cold NP4Q buffer, and lysed by adding 0.5 ml of 10- prcsent NP40 buffer for 5 minutes on ice. The nuclei are removed by centrifugation (1000 xq, 10 minutes), after which the supernatant is adjusted to pH 8 with 50 m M fris / Cl, 0.5% Sarcosine solution in 5 m M EDTA and stored at -2 ° C. C; C.

To isolate total RNA from the supernatant, it is extracted once with phenol, once with phenol / chloroform / isoamyl alcohol, and once with chloroform / isoamyl alcohol 11a. The aqueous phase is adjusted to 4 ml of 5,7-mol arlse 11a CsCl-Po 1 ster-1 solution and centrifuged in a SW40 rotor (35 rpm, 20 hours) to remove DNA and residual proteins from the extract. . The resulting RNA pellet is resuspended in 2 ml of TE pH = 8, D buffer and seeded with ethanol.

The precipitated DNA is then dissolved in water at a concentration of 5 mg / ml.

c) Detection of interferon-omeqa mRNA The hybridization probe prepared according to Example 7b and 20-50 μg of the RNA prepared according to Example 7c are co-precipitated by adding ethanol. In the control experiment, it is added tRNA instead of 11 ara RNA. . (transfer RNA) or RNA derived from E. coli transformed with plasmid - - pRHW 12 (Example 5).

The resulting pellets are washed desalted with 70% ethanol, dried and dissolved in 25 μl of 80% formamide (100 mM PIPES pH = 6.8, 400 mM w a Cl, 10 mM EDTA).

The probes are then heated for 5 minutes at 100 ° C to denature the hybridization probe, immediately adjusted to 52 ° C, and incubated at this temperature for 24 hours. After hybridization, the probes are placed on ice · ’. ··; and 475 μl of S1 reaction mixture (4 mM Zn (Ac) 2, 30 μl of 49 90667 mM NaAc, 250 mM MaEl, 5% glycerin, 20 μg ss of calf DNA, 100 units of S1 nuclease) is added. Treat for one hour at 37 ° C, after which the reaction is stopped by stirring with ethanol.

The pellets are dissolved in 6 μl of formamide buffer and separated essentially as probes with DN A-sec v / en sso in 11 reaction with 6% acrylic inide containing 8 M urea (F. nger et al. , Proc. Nat. Acad. Sci. 74, 5463-5467 (1979)).

For autoradiography, the dried gel is exposed to DuPont Cronex X-ray film using Kodak Lanex Regular intensifiers at -70 ° C.

Explanations for Figure 10 Traces A to C show controls.

Trace A: 20 μς tRNA

Trace B: 10 ug of RNA from pER 33 (p. Coli expression strain for interferon-2-Arg) Trace C: 1 ng of RNA from pRHW 12 (E. coli expression strain for interferon-omega 1): Trace D: 50 pg RNA from untreated Namalwa-::: cells: Trace E: 50 μ g RNA from virus-infected

From Namalu / a cells Trace F: 50 ug of RNA dexamethasone i 1 la from pre-treated and ... virus-infected Namalvi / a- cells 5u 90667 Trace G: 20 pq RNA from untreated NC 37 cells Trace H: 20 μg of RNA from infectious RNA viruses from NL '37 cells Trace I: 20 μg of RNA from dexamethasone-11 and virally infected NC 37 cells Trace M: Size test ( pBR 322 digested with H IN f I).

Traces B and C indicate that the expected signal can only be detected when omega-specific RNA is present in the RNA molecules. It further turns out that even a large excess of false RNA does not cause any background signal (see trace B). The tRNA used as a hybridization component also does not generate a signal (see footnote A).

Traces G-I show the induction of omegal-specific RNA in viral-infected NC 37 cells. Pretreatment with dexamethasone then confirms this effect.

j ‘Traces D to F give essentially the same results as. : what is obtained from Nama1wa - so 1ui1la. However, induction with omega 1-specific RNA is not as potent as in NC 37 cells. This result is consistent with interferon ni- ters measured from all cell supernatants.

This expression of the interferon ni-omega 1 gene behaves as expected for the basic type I interferon gene.

- - '. Example 8

The gene encoding omega 1 - i n t e r f e ron ia, or close relatives. ·: Isolation of genes: I!.: Si 90667 a) Cosmid screening

Human DNA (mind) from the human cosmid bank cloned into the cosmid vector pcos2 EMBL (A. Ponstka, H.-R. Rocku / itz, A.-M. Fnschauf, B. Hohn, H. Lehrach Proc. Natl. Avad.

Sci. (1, 4129-4133 (1984)) with a complexity of 2 x 10 2) examined IFN omega or related genes. E. coli DH1 (r ^ -, Π) κ +> rec.A was used as the host; gyrA96, sup.E) strain. First, MG ++ cells (= "plating Bacteria") were prepared. E. coli DH1 grows overnight in L-broth (10 g / l tryptone, 3 g / l yeast extract, 5 g / l NaCl) supplemented with 0.2% maltose. The bacteria are separated by centrifugation and taken up in 10 mM MqSO 4 solution at a density OD 2+ = 2. 5 ml of this cell suspension and 12.5 x 10 6 colony forming units of packaged cosmid are incubated for 20 minutes at 37 ° C. Then add 10 butter. The LB solution and suspension are maintained for 1 hour at 37 ° C to express humidified-mediated kanainycin resistance. The bacteria are then separated by centrifugation, resuspended in any LB solution and applied in 200 μl aliquots to nitrocellulose filters (BA85, Scti le ie her und Schöll, diameter 132 mm) which are LB-aqann (1.5% agar in L-broth). ) with 20 ug / ml •:: kanamycin, on. About 10,000 - •:: 20,000 colonies grow per filter. Colonies are replicated on new nitrocellulose filters stored at 4 ° C.

A number of colony filters are treated as described in Example le), i. the bacteria are denatured and single-stranded - · DNA is attached to nitrocellulose. The filters are prewashed for 4 hours at 65 ° C in 50 mM Tris / HCl, pH = 8.0, 1 M NaCl, 1 mM EDTA, 0.1% SDS. The filters are then incubated for 2 hours at 65 ° C in 5 x Denhardt's solution (see

'. Example 1e), 6 x 5SC, 0.1% in DS solution and hybridized; 2 hours at 65 ° C in the same solution using about 50 x 10 μm nick-translated, denatured IFN-omegal-: DNA (Hind-III-BamHI insert of clone pRHW12, see Figure; ··; 6). After hybridization, the filters are washed first for 2 x 10 x 90667 minutes at room temperature in 2 x SSC, 0.01% SQS solution and then for 3 x 45 minutes at 65 ° C for 0.2 x SSC, 0.01 % In SDS-1 solution. The filters are dried and exposed to Kodak X-0mat 5 membranes using amplification at -70 ° C. Positively reacting colonies are localized in a replica filter, detached and resuspended in L-broth containing kanamycin (30 μl / ml). A few ml of the aged suspension is plated on LB agar containing 30 μg / ml kanamycin. The resulting colonies were rep1 i ka-ma 1 j a t aan for nitrocellulose filters. These filters are hybridized to P-labeled IFN-omegal DNA as described above. The cosmid was isolated from the hybridized colonies in each case by the method described by Birnboim & Doly (Nucl. Acids Res. 1513-1979). These cosmids were transformed into coli DH1 and transformed with LB agar containing 30 ug / ml kanamycin. Positively reactive clones from the transformants were again examined with cP-radiolabeled IFN-omegal DNA. One clone from each of the original isolates is selected and its cosmid is prepared on a larger scale (Clewell, D.B. und Helinski, L). ^. , Biochemistry 9, 4428 (1970)).

The three isolated cosmids have the names cos9, cos10 and |; c o s B.

b) Subcloning of hybridizing fragments in PUC8

Under the conditions recommended by the manufacturer (New England Biolabs), 1 μg of cosmids cos9, cos10 and cosB Hind 111 enzyme were cut in each case. The pieces are electrophoretically separated on 1% acjarose gels in TBE buffer and transformed by the Southern method on nitrocellulose filters (Example 4c). Both filters are hybridized to nick-translated omegal DNA as described in Example 4d); 11a, washed and exposed. Approximately 20 μg of each cosmid is cut with HindIII enzyme and the resulting pieces are separated by gel electrophoresis. In a preliminary experiment, pieces that hybridize to omegal DNA are electroeluted and purified 55 90667

On Elutip columns (Schleicher + Schull). These fragments are ligated with HindIII-1inearized de-phosphorylated pUC8 (Messing, J., Vieira, J.f Gene 19, 269-276 (1982)). E. coll JM101 (supE, thi, (Iac-proAB), F ', traD36, pro AB, Lac q 1 M15 / (e.g. PL Biochemica1 s) is transformed with a ligase solution. The bacteria are plated on LB agar containing 50 yug / ml injected 11 μg, 250 μg / ml 5-bromo-4-chloro-3-indolyl-β-D-Qala ctopyranoside (BCIG, Sigma) and 250 pg / ml isopropyl-BD-thiogalactose. pyranoside (IPTG, Sigma) The blue staining of the colonies formed indicates the absence of an insert in pUC8 Plasmid DNA is isolated from a few white clones on a small scale, cut with HindIII and separated on a 1% agarose gel. starting from cosmids cos9 and cos10, one sub-clone is selected for each of them, to which pRHW22 and pRH57 are given, and two pieces of DNA that hybridize well to the omegal probe are created and given the names p RH51 and pRH52.

c) Sequence analysis. - The DNA inserted into pUC8 is separated from the vector part by digestion of Hind III enzyme and its residues by gel electrophoresis.

; About 10 of this DNA in 50 of the reaction solution using T1 DNA ligase, the volume is adjusted to 250 silicon with nick translation buffer (Example 4d) and then sonicated under ice-cooling (MSE 100 Watt Ultrasonic Disintegrator), max. power at 20 kHz, five times for 30 seconds.

; * - The ends of the fragments are then repaired by adding 1/100 butter. each of the following: 0.5 mM dATP, dGTP, dCTP, and dTTP, and 10 units of Klenou piece of DNA polymerase I over 2 hours at 14 ° C. The resulting DNA fragments with straight ends are separated by size on a 1% agarose gel. Pieces between 500 and 1000 bp in size are isolated and subcloned into the SmaI-cut, dephosphorylated 5,90667 phage vector MlimpO. Single-stranded DMA of recombinant phage is also isolated and sequenced by the method developed by Sanger (Sanger, F. et al., Proc, Natl. Acad. Sci. 74, 5463-5467 (1976)). The assembly of individual strands into a complete sequence is done by computer ($ taden, R ,, Nucl. Acid Res. 10, 4731-4751 (1982)).

d) The sequence of the subclone pRH57 (iFN-omeqal) is given in Figure 11. This 1933 bp long fragment contains the interferom-omega1 gene. The protein coding region comprises nucleotides 576 to 1163. The sequence is completely identical to the sequence of the cDNA clone P9A2. Nucleotide fragment 576-674 encodes a 23 amino acid signal peptide. The TATA-Box is located at a characteristic distance from the type I interferon gene11 e before the start tissue ATG (positions 476-482). The gene has some sequences in transcription (issa at positions 1497-1502 or 1764-1796; AATAAA at positions 1729-1734 or 1798-1803), the first of which was used in clone P9A2.

e) Sequence of subclone pRHW22 (IFN-pseudo-omeqa2): Figure 12 is re-administered with the omega1 DNA probe. a 2132 bp sequence of the hybridizing HindIII fragment from cosmid cos9. The open reading frame is located from nucleotide 905 to nucleotide 1366. The amino acid sequence derived therefrom is reissued. The first 23 amino acids are similar to the amino acids of a typical type I interferon signal peptide. The next 131 amino acids up to amino acid 65 are similar to those in omegal interferon, with the first amino acid of the mature protein being found to be tyrosine.

Amino acid position 66 is followed by the sequence of the potential N-glycSolysis site (Asn-Phe-Ser). From age, the amino acid sequence is different from that of type 1 interferon.

li 90667

However, it can be shown that with the help of suitable inserts and the consequent shift in the protein reading frame, similarity to IFN-omegal is obtained (see Example 9). The gene isolated for type I interferon is thus a pseudogene: IFN-pseudo-omega 2.

f) Sequence of aliclone pRH51 (IFN-pseudo-omeqa3)

The HindIII fragment from cosmid B, which is approximately 3,500 bp in length and hybridizes to the omeoal probe, was partially sequenced (Figure 13). The open reading frame extends from nucleotide position 92 to position 394. The first 23 amino acids have the characteristics of a signal peptide. The next sequence then begins with tryptophan and is similar to IFN-omegal up to amino acid 42. The following sequence is then different from the IFN-omegal and ends after amino acid 78. The sequence can be altered by insertions so that it then has strong homology to IFN-omegal (Example 9). The gene is named IFN-pseudo-omega3.

q) Sequence of the pRH52 insert (IFN-pseudo-omeqa4): Figure 14 shows a 3659 long Hindi 11 fragment sequence isolated from cosmid B and hybridizing to omega 1 DNA.

I The open reading frame, whose translation product has partial homology to IFN-omegal, is between nucleotide positions 2951 and 3250. After a 23 amino acid signal peptide, the next amino acid sequence begins with phenylalanine. Homology with IFN-1 ends already after amino acid 16, - continues from amino acid 22 and ends at amino acid 41. Possible translation up to amino acid 77 would be possible.

By analogy with Examples 8f) and 8g), good homology to IFN-omegal can now also be achieved by inserting inserts (Example 9). The isolated pseudogen is called 56 90667 IFN-pseudo-omeya4.

Example 9 Evaluation of the genes of member 4 of the IFN-omeqa family

Figure 15 lists the genes of omegal interferon-pseudo-omega4 interferon listed below together with the amino acid composition. To improve compatibility, individual genes are spaced with dots. No base is omitted. Spaces are included in the numbering of bases. The amino acid translation of IFN-omegal is included (e.g., positions 352-355: "C.AC" encodes a His amino acid). Pseudogenes are involved in amino acid translation only when this is unambiguously possible. From this listing, it can be immediately seen that these four isolated genes are closely related to each other. Thus, for example, potential N-glycolysis sites (nucleotide positions 301-309) have been obtained for all four genes.

Apart from IFN-pseudo-omega4, there is also a triplet at nucleotide positions 611-614, which shows a stop codon and which omega 1 interferon11a terminates the mature, 172 amino acid long protein. However, in this arrangement, the stop codons for IFN-pseudo-omega2 (nucleotide positions 497-499) and IFN-pseudo-omega4 (nucleotide positions 512-514) are located.

The degree of relatedness between genes or amino acid translations can be calculated from the arrangement shown in Figure 15. In the figure; 16 are re-administered DNA homologies between members of the IFN-omega family. In the case of a pairwise comparison, the places where the stallion is spaced in one or both components are not included. The comparison gives a holology between IFN-omegal DNA and pseudogene sequences of about 85%.

. : IF N-β seudo-omega 2-DN A is approximately 82% homologous to IFN-pseudo-omega3 or IFN-pseudo-omega4 DNA.

li 57 90667

Figure 17 shows the results of the comparison of the subunit sequences and Figure 18 the results of the comparison of the "finished" proteins. The latter are between 72 and 88%. However, this homology is substantially greater than the homology between IFN-omegal and α-m interferons and β-interferon (Example 6). The fact that individual members of the IFN-omega family are more distant from each other than members of the IFN-α family can be explained by the fact that three of the four isolated IFN-omega genes are pseudogenes and appear to be under no more selective pressure than functional genes.

Example 10 F ermentation

Getting a position:

A single colony of strain HBl01 / pRHW14 on LB agar (with 25 mg / ml ampicillin) is inoculated into a tryptone-protective buffer (OXDID CM129) containing 25 mg / ml ampicillin and shaken at 250 rpm. (Rpm) at 37 ° C until an optical density (546 nm) of about 5 (logarithmic growth step) is obtained. Thereafter, 10% by weight of sterile glycerin is added, the culture is filled in 1.5 ml aliquots into sterile ampoules and frozen at -70 ° C.

Esivil.jely:

The composition of the culture medium is: 15 g / l Na 2 FIPO 2 x 12 H 2 O; 0.5 g / l NaCl; 1.0 q / l NH 3 Cl; 3.0 o / l KH 2 O 2; 0.25 g / l MqSO 4 x 7H 2 O; ‘.J 0.011 a / l CaCl 2; 5 g / l casein hydrolyzate (Merck 2238); : 6.6 o / l glucose monohydrate, 0.1 g / l ampicillin; 20 mg / l cysteine and 1 mg / l thiamine hydroxide. 4 x 200 ml of this medium, each in a 1000 ml Lrlenmeyer flask, is inoculated with 1 ml of HB 101 / pRHW14 carrier: · · and shaken for 16-18 hours at 37 ° C and 250 rpm.

it 90667 Main crop:

The composition of the fermentation medium is: ID g / l 4.6 q / l K 2 HPO 4 x 3H 2 O; 0.5 o / l MaCl; 0.25 g / l MgSO 4 χ 7H 2 O; 0.011 g / l CaCl 2; 11 a / l glucose monohydrate; 21 g / l casein hydrolysis (Merck 2238); 20 mg / l cysteine, 1 mg / l thiamine hydrochloride and 20 mg / l 3- (3-indolacrylic acid).

8 liters of sterile medium are inoculated with 800 ml of preculture in a fermentor with a total volume of 14 liters (height: radius = 3: 1).

The fermentation takes place at 28 ° C at 1000 rpm. (E ffigas turbine), with an aeration rate of 1 vvm (v / v / min) and an initial pH of 6.9. During fermentation, the pH decreases and is then kept constant at 6.0 with 3N sodium hydroxide. When the optical density (546 nm) is reached 18 to 20 (usually after a fermentation time of 8.5 to 9.5 hours), it is otherwise cooled to 20 ° C under the same conditions and then the pH is adjusted to 2.2 with 6N sulfuric acid (without aeration) and stirred. one hour at 20 ° C and 800 rpm. (without aeration). The biomass thus inactivated is then separated by centrifugation 11a in a CEPA laboratory centrifuge (type GLE) at 30,000 rpm.

and frozen at -20 ° C and stored.

-: Example 11 ·· - · I nt e r f e ron i-ome ga - Purification of G ly a) Partial purification ':: All steps are performed at 4 ° C.

; ; 140 g of biomass (E. coli HB 101 transformed with the expression clone pRHW 12) are resuspended in 1150 g of 1% ethanic acid (es l jiitluly Le l t y 4uL ’) and mixed for 30

II

59 90667 minutes. The suspension is adjusted to pH 10.0 with 5 M sodium hydroxide and the violin is stirred for 2 hours. Adjust to pH 7.5 with 5 M hydrochloric acid and then stir for a further 15 minutes and then centrifuge (4 ° C, 1 hour 10,000 rpm. J21 centrifuge (Beckman), JA10 rotor).

The clear supernatant is applied to a 150 ml CPG (controlled pore glass) column (CPG 10-350, Mesh Size 120/200) at a rate of 50 ml / h, washed with 1000 ml of 25 mM Tris / 1M NaCl, pH 7.5, buffer and eluted with 25 mM Tris / 1M KCNS / 50SJ ethylene glycol, pH = 7.5 (50 ml / h).

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

b) Further cleaning

The dialyzed and concentrated CPG eluate is diluted with buffer A (0.1 M Na-phosphate pH = 6.25 / 25? 1,2-propylene glycol); 1: 5 and passed through a "Super loop" ionizer (Pharmacia) at a rate of 0.5 ml / min. to buffer A equilibrated MonoS 5/5 (Pharmacia, cation exchanger) column. Elution f uses a 20 ml linear gradient from 0 to 1 M NaCl in buffer A also at a flow rate of 0.5 ml / min. The flow-through and 1 ml fractions are collected and tested for interferon activity using the CP E reduction test. The active fractions are combined.

r ~ -------------- 60 90667

table 1

Volume Biological test Protein Total U / mg Yield (ml) U / ml + U total (mg / ml) (mg)%

Unrefined 1150 15000 17.3x106 3.6 4140 4180 100 DL 2200 600 l, 3x106 0.74 1628 600 5

Eluate 124.3 170000 21.0x106 16.8 2088 10000 121

After dialysis, 41,300,000 12.3x10 6 12.6 516.6 23800 71 + CPE reduct * · iofest: A549 cells, EMC virus U: Unit when interferon-2 is used as standard (see Example 2 in EP-A -0.115.613: E. Coli HB101 transformed expression clone1 la pER 33) DL: Flow-through

Example 12:: Characterization of Hu IF N-omega 1 A. Antiviral activity against human cells B. Antiviral activity against monkey cells C. Reproductive activity against human Burkitt's lymphoma cells (cell line Daudi):: D. Reproductive activity against human cervix against carcinoma cells (cell line HeLa) E. Acid stability 61 90667 F. Serological characterization A. Antiviral activity against human cells

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

Virus: Mouse encephalomyocarditis is virus (EMCV)

Testing systems: Inhibition of cytopathic effect (all titrations were performed four times)

A partially purified Hu IFN-omega1 preparation with a protein content of 9.4 mg / ml was circulated at an appropriate dilution in said test system. The preparation had an antiviral effect with a specific activity of 8300 Ie / mg calculated from the reference standard Go-23-901-527.

B. Antiviral activity against monkey cells

Cell line: GL-V3 monkey kidney cells (Chirstofinis G.J., J. Med. Microbiol. .3, 251-258, 1970). Virus: Vesicular Stomatitis Virus (VSV)

Test system: Plaque reduction test (all four titrations) · The preparation described in Example 12A had a specific antiviral activity in said test system: 580 U / mg.

C. Reproductive activity in human Burkitt lymphoma cells (Daudi cell line): The human Burkitt lymphoma cell line Daudi was grown in the presence of various HuIFN omegal concentrations. Cell densities were determined after two, four, and six days of incubation at 62,90667 at 37 ° C; untreated cultures were used as controls. All cultures were three in parallel. The following figure shows that UN-omega clearly inhibits cell proliferation at a concentration of 100 antivirals per unit (IE, see Example 12A) per ml; At a concentration of 10 IU / ml, partial, transient inhibition was observed (the following symbols are used in the following figure: O control, · 1 It / ml, Q10 IU / ml, ^ 100 IU / ml): i · 'j · f *: ·; | 'II j? ::! Ϊ i.

'X · I' i V ·! CO i, „I, O '| · H; : -Mi i l! ' · · · '·' J ':' H; R; : r ~ ~ r ··:; · o. ) and 4. e: ·! * *! ! · V:: '. ! speck

II

63 90667 D, Reproductive activity against human cervix carcinoma omaso 1 u.j en (cell, and HeLa)

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

Hu IF N-omega 1 (see Example 12A) 100 IU / ml HuIFN gamma (see Example 12A) 100 IU / ml Human Tumor Necrosis Factor (HuIFN), purity i 98 8, manufactured by Genentech Inc., San Fanzisco, USA (see Pennica D. et al., Nature 312, 724-729; 1984) 100 ng / ml

All binary combinations of said protein concentrations were used as above for two cultures in 3 cm plastic tissue culture dishes (50,000 cells / dish) and incubated for six days at 37 ° C; cell density was then determined. Treatment of cells with HuIFN-omegal or HuIFN-gamma11a had only a minor effect on cell growth, whereas HuIFN-gamma11a had a clear cytostatic effect. The combination of IFN-gamma and IFN-omegal had synergistic cytostatic / cytotoxic effects.

The result of the experiment is shown in the following figure: 64 90667 ..... II TTTrrrr— "i — i — πτπη-. Ill1 {its i-SHi ··: ·» * *? ·:? * · ΜίiH-h »'Ι ; = ί;! ^ Ι! ϊ · Τ · Μι f! η: r'n-rptn ^ ih ^ fTnnimT [: 1 ^^ ψΠΊι ^^ ίΤΤΓi.is s ΤμΓη riiill MCE iH mi; uiiiipu Miifpte Ίί'Μά iiiiiiliHgii ': !! -ΓΡιΊ:?: Ji, ιΊΤΤ il;):!: Ρ ^ Π ^ ΜϊΤπΐΙτΓΤ ίΠΤί · ΜΓΠΐϊ ;. i rtJI; rl?

ΑμΙ & 3 ffcvi * Hi; jj: i; :: M ΙίΙφΤίΤIVH?!

.ΔΧΧi1! Tir!; I! -'- 'T: i:! Iff Tl: a lii »i ύ ^ isiiit: i r * .t vTnTtTKFTrfrLim

iM '"' i“ j / i 'I 1 ^ .1: ¾} λbfcj :: \ r fp 4K · ftlifÄf'-kl-iT

'Am MMMimSMtmFfeteKfifgfcHg mSI? TOT iffffc ψΐΓίΐ up C «^ 4? H’jgp Ά tjäfl? .CTmPt ^ ΚΓ7.Η! ΠψΠ ^ ΤΓι7Μ: rU-t «T« f.

n .'- finr-mzyzτ: χϊ. fi * ΚαΤ ^ ΛκΙ® · * ,: ίο & sff f. «- r · .ISTiyr ~ τρ + γϊ ~ τπ * η * ττττΎ -τΗγϊγρ ^ τ -TT ^ uiyd: bl μΡλ · γ -I-iTfaipas l ^ TT ; 1 · i ί-ii i: 'ΪΤ-iaii.lr,. ~ I.. ·. -.:1 ^ ΓΠΤΤ

'' F: ", · 'Γ!" ("; -I-'ί, m:' · ΓΙ Μ · Γ ·: ϋ 'cia ·!;:! ::; ί.] T-Ίλ!: I_ .L_tU.JT

1 ^^ ΤΤ-ί-Γ'ΠΤΤί .1Wit ItU- ^ J

3 g 10 30 60 100

10 _ <* * CELL / BOWL

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

F, Acid Stability • To study the acid stability of HyIF N-omega 1, the dilution of the preparation mentioned in Example 12A in cell culture medium (U / min.I 1640 with 10% fetal calf serum) was adjusted to pH 2 with hydrochloric acid. 24 hours at 4 ° C and then neutralized with sodium hydroxide. This sample was titrated in antiviral test a 6b 90667 (see Example 12A); it had 75% of the biological activity of the control incubated at neutral pH. IFN-omegal must therefore be considered acid stable.

F. Serological characterization

To compare the serological properties of HuIFN-omegal and HuIFN-α2, samples (diluted to 100 IU / ml activity) were preincubated with equal volumes of monoclonal antibodies or polyclonal antiserum solutions for 90 minutes at 37 ° C; the antiviral activity of the samples was then determined. The following table shows that HuIFN-omegal can only be neutralized with anti-leukocytic interferon antiserum at relatively high concentrations, but not with antibodies already directed against HuIFN-α2 or HuIFN-beta. Thus, HuIFN-omegal is not serologically closely related to HuIFN-β2 or HuIFN-beta: 66 90667

Antiserum Dilution HuIFN-α2 Hu IFN-omega 1 monocl. neutralization of antibody [mu] g / ml EBI-11) 1 + 10 +++ 100 +++ 1000 - 0 EBI-3X) 1 +++ 10 +++ 100 +++ 1000 +++ 0 L3B72 ^ 100 + ++ 0 1000 +++ 0

Sheep-ant i-leukosy y 11 i -1 FN'5 ^ 1: 50,000 ++ + 1: 5 000 +++ 0 1: 500 +++ + 1:50 - +++

Rabbit anti- HuIFN α2 1: 1,000 ++ +: 1: 100 +++ 0 1: 10 0

Sheep anti-

HuIFN-g4) l: 50 - 0. - = k t s. EP-A-0.119.476: 7) '= A. Berthold et al. Arzneimittel Forschung 3F5> 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.

Il ·: 67 90 667

Symbols: - not tested, 0 no neutralization, + partial neutralization, +++ complete neutralization.

Claims (35)

A method for producing recombinant DNA molecules encoding a novel human type I interferon protein containing 168 - 174 amino acids, interferon-pseudo-omega2 (Figure 12), interferon-pseudo-omega3 (Figure 13), or interferon pseudo-omega4 (Fig. 4), characterized by the B lymphocell line cDNA under the required conditions, which allow a homology above 85%, by hybridization with inserts a) of the plasmid E76E9 E.coli HB 101 bacterium stored in the DSM collection under number DSM 3003 or b) in the plasmid P9A2 E. coli HB 101 bacterium, stored in the DSM collection under number 3004, at the Pst-1 intersection point is isolated and finally the desired DNA molecule is replicated. 2. A method according to claim 1, characterized in that, under the required conditions, a homology above 90% is obtained. Method according to claim 1 or 2, characterized in that the coding sequence of the DNA molecule is insert a) into the plasmid E76E9 E.coli HB 101 bacterium, stored in the DMS collection under number DMS 30003 or b) in P9A2 E The coli HB 101 bacterium, stored in the DSM collection under number DSM 3004, at the Pst-1 intersection or in the degenerate variations of these inserts. Method according to claims 1-3, characterized in that the DNA molecule contains the sequence 1i. 05 90667 j \; T GAT 1 'T G CCT CAG AAA -' A'i 'C d c: I A 1 1 Αλ, ι. ·· ·; a- I; 'j (j ait CTG CAC CAA ATG AGG AGA Ai "i";' act A ic; c AAG A / a; AAA AGA GAC TTC AGG TAG ccc '' AG GAG ΑΊΑ - A AAA; .a. '' ACC aaC T'l G CAG AAG GCC A AT Ai C ATG 'T GTC Cl' Ά.Τ T, r ΑΊΑ: · 'TG AAG CAG ATC TTC AGC CTC TIC CAC ACA GAC GC · ..; i' C ·; T ac GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC AC i GGA '' * '' ΆΊ. CAG CAA CTG CAA CAC CTG GAC ACC TGC TTG CTG CAG CTA GIG; c, / \ GAA GG A GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTC, Aa'a TTG -AGG M '-> G TAC TTC CAG GGA ATC CGT GTC TAG CTG ΛΑΛ GAC AA.G AAA A TAG AGC GAC TGT GCC TGG GAA G'i'T GTC AGA ATG GAA ATC AC C CAA C TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA ATG AGA ACT AAA GAT AGA GAC ATG GGC TCA TCT 1 'T. For removal according to patent claims 1 - 3, K ann _ ro: · .nate thereof , that the DNA molecule according to claim 4 of claim 1 is in the nucleotide ID of the nucleus G. b. For testing according to patent tk rave t 1 or 2, may be signed; DNA -mc Leky Ien des towers contain 1 for years of DNA sequence encoding the conductive peptide, but · ATM GCC CTC CTG TTV GCT CTA CTG GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC 7. slotted end assembly according to claim 1, c h nn · vecknat in that the coding DNA mo leky Ien is IFN omega in gene vai.sor Me 1 A r 86 90 667 17 GATCTGGTAAACCTGAA GCAAATATAG AAACCTATAGGGCCTGACTTCCTAC ATAAAGTAAGGAGGGTAAAAATGG 7:06 a.m. AGGCTAGAATAAGGGTTAAAATTTTGCTTCTAGAACAGAGAAAATGATTTTTTTCATAT 13:05 ATATATGAATATATATTATATATAC ACATATATACATATATTCACTATAGTGTGTATAC 194 ATAAATATATAATATATATATTGTTAGTGTAGTGTGTGTCTGATTATTTAC ATGCATAT 25 3 AGTATATACACTTATG ACTTTAGTACCCAGACGTTTTTCATTTGATT AAGCATTC ATTT 312 GTATTG ACACAGCTGAAGTTTACTGGAGTTTAGCTGAAGTCTAATGCAAAATTAATAG A 3 71 TTGTTGTCATCCTCTTAAGGTCATAGGG AGAAC ACACAAATGAAAAC AGT AAAAG AAAC 4:30 a.m. TG AAAGTAC AG AG AAATGTTC AG AAAATG AAAACC ATGTGTTTCCT ATT AAA AGCC ATG 4 89 CATAC AAGCAATGTCTTCAGAAAACCTAGGGTCC AAGGTTAAGCCATATCCC AGCTC AG 548 TAAAGCCAGGAGCATCCTCATTTCCCA ATG GCC CTC CTG TTC CCT CTA CTG 5 99 GCA GCC CTA GTG ATG ACC AGC TAT AGC CCT GTT GGA TCT CTG GGC 64 4 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 G AC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 779 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT G AG ATG 824 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG 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 CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 95 9 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 100 4 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 109 4 TCC TTG TTC TTA TCA ACA AAC ATG CAA CTG GAA AGA AGA AGT AAA 1139 GAT AGA GAC CTG GGC TCT TCA TGA AATGATTCTCATTGATTAATTTGCCAT 1190 ATAACACTTGCACATGTGACTCTGGTC AATTCAAAAGACTCTTATTTCGGCTTT AATCA 24:04 9 CAGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTT 1308 AAAAAGACTTAGGTTCAGGGGCATC AGTCCCTAAG ATGTTATTTATTTTTACTC ATTTA 13:06 7 TTT AAC ATTCTTACATTTTATCAT ATTT ATACTATTTATATTCTTATAT AAATGTTTGC 14:02 6 CTTTACATTGTATT AAGATAACAAAACATGTTCAGCTTTCCATTTGGTTAAATATTGTA TTTTGTTATTTATTAAATTATTTTCAAACAAAACTTCTTGAAGTTATTTATTCGAAAAC 1485 1544 1603 CAAAATCCAAACACTAGTTTTCTGAACCAAATCAAGGAATGGACGGTAATATACACTTA CCTATTCATTCATTCCATTTACATAATATGTATAAAGTGAGTATCAAAGTGGCATATTT TGGAATTGATGTCAAGCAATGCAGGTGTACTCATTGCATGACTGTATCAAAATATCTCA 1662 1721 1780 TGTAACCAATAAATATATACACTTACTATGTATCCCACAAAAATTAAAAAGTTATTTTA AAAAAGAAATACAGGTGAATAAACACAGTTTCTTTCCGTGTTGAAGAGCTTTCATTCTT ACAGGAAAAGAAACAGTAAAGATGTACCAATTTCGCTTATATGAAACACTACAAAGATA 1839 1898 1933 AGTAAAAGAAAATGATGTTCTCATACTAGAAGCTT li 87 90 667 8. A method according to claim 1, characterized in that the coding DNA molecule is an IFN pseudo-omega-gene whose formula is AAGCTTGAGCCCCCAGGGAAGCATAACCACATGAACCTGAATFAATATATT 51 CTAGAAGGAGGGAAGCACCAGAGAAGTTCTTTCACTAATAACCATCAACGTCTTCTGTG 110 AATCAAATATCAAACAAAGATAGTCCTAAAAAGTTTAATTTCCAGAGATAGGTAATTTC 169 CTAACTGAATACAGAAACCCATAGGGCCCAGGGATCCTGATTTCCTATGCAAAATGGAG 228 GGTAAAACTGGAGGCTAGGATCTGGGCTAAAAGTATATACTTCTAACAGTAGCACAAAG 28 7 ATGTTTCTCATCTGATTGATCAATATTCATTTGGATTGATATATCTTAAGTTTACTGGG 346 AATATTGAACATCCATTGCAAAAATCAAGAGTGTAGAGTGATGACCTCCTTTTAGGTCA 408 TATAGAACAAGGTTTTTCAACCCCCATCCATGGACCGGGGTACTGGTCCTGGCCTGGTA 464 GGAACAGGGCCGCACAGCAGGAGGCAAGCAGGCCAACCAACAAGCATTAACGCCTGAGC 523 TCTGCCTCCTGTCAGATCAGCAGTGGCATTGGATTCTCAAAAGAGCAGGAACCCTATTG 582 TG AAAACT ATTTGTGCTCCATTAAAGCC ATCC 818 ATAGATAGAATGTCTTCATAGAACCTAGGATCCAAGGTTCTATGAAGACCTCAGCTCAA 877 CCAGGCCAAAAGCATCCTGATTTCTCA ATG GCC CTC CTC TTC CCT CTA CTG 928 GCA GCC CTA GAG GTG TGC ACG TGT GGC TCT TCT GGA TCT CTA GGA 973 TAT AAC CTG CCT CAG AAC CAT GGC CTG CTA GGC AGC 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 TTC AAC TTC TCA CAC AAA GCG CTC CTC TGC TGC 1198 ATG GAA CAT GAC CTT CCT GGA CCA ACT CCA CAC TTT ACG TCA TCA 1243 GCA GCT GGA ACA CCT GGA GAC CTG CTT GGT GCA GGA G AT GGG AGA 12 88 AGG AGA AGC TGG GGG CAG TGG GTG GGC GAG ATT TCT ACA CTG GCC 1333 AGG AGG TTG CAG TAT TTC GAA TCC ATC TCT AGC TGA AAGAGAAGAAA 1380 TAAAGATTGTGCCTGGGAAGTTGTCAGAGTGG AAATCATGAGATCCTTTTCATCCACAA 14 3:09 a.m. GTTTGCAAGAAAG ATTG AGAAGTAAGG ATG AAG ACCTGGGCTCATCATGAAATGATTCT 14 98 CATTGACTAATCTGCCATATCACACTTGTACATGTGA CTTTGGATATTCAAAAAGCTCA 155 7 TTTCTGTTTCATCAGAAATTATTGAATTAGTTTTAGCAAATACTTTATTAATAGCATAA 1619 AGCAAGTTTATGTCAAAAACATTCAGCTCCTGGGGCATCCGTAACTCAGAGATAACTGC 16 75 CCTGATGCTGTTTATTTATCTTCCTTCTTTTTTTTCATGCCTTGTATTTATGATATTTA 17 3 4 88 9 0 6 6 7 TATATTTTATATTTTCATCTTCACATCGTATTAAAATTTATAAAACATTC ACTTTTTCA 17:09 3 TATTAAGTTTGCATTTTGTTTTATT AAATTC ATATC AAAG AAAACTCTGT AAATGTTTC 185 2 TATTCTAAAAACAATGTCTACTTTCTCTTTTTGTAAACCAAATTG AAAATATGGTAAAA 1911 TGTATTAACTCATTCATTTCATTCCTATTATATGTATAAATTG AGTAAATGGCAAACTG 19:07 0 TGGGGTTTTCTTAAAGAAATACAGGTGAATAAAGCAAACACAGTTTCTCTCAGTCTAAG 2 0 2 9 AGGGAAAGAGACGTAAAAACAGGACAAATATTTATATTATTTCAATTATGTTAAATGCT 2:00 a.m. 8:08 a.m. ACAAAGAGAAGTAAAGAAAAGTGATGTTCTCACATCAGAAGCTT 2132 9. A method according to claim 1, characterized in that the coding DNA molecule is an IFN pseudo-omega gene, the formula of which is CCATG CATAGCAGG CTT TGC CAC TGT GGC CCT GTT GGA TCT CTG AGC 16 0 TGG GAC CTG CCT CAG AAG CAT GGC CTA CTT AGC AGG AAG ACC TTG 205 GCA CTT CTG GGC CAA ATG TGC AGA ATC TCC ACT TTC TTG TGT CTC 250 AAG GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA 2 95 GTC AGT TGC AGA AGG CCC CGG CCC TGT CTG TCC TCC ATG AGA TGC 3 40 TTC AGC AGA TCT TCA GCC TCT TCC CCA CAG AGT GCT CCT CTG CTG 3 85 CCT GGA ACA TGA CCCTCCTGGACCAACTCCACACTGGACTTCATCTGCAGCTGGA ATGCCTGGATGCTTGCTTAGGGCAGACAAAAAGAGAGGAAGAATCTGTGGGGGTGATTG 440 4 99: GGGCCCTACACTGGCCTTGAGGACGTACTTTCAGGGAATGCATGGGAATCCAGGGAATC 5:05 a.m. 8 'TACCTGGAGGAGAAGAAATACAGTGACTGTGCTTGGGAGGTTGTCAGAGTGG AATCGTG 617 AAATCCTTCTCTTCATCATCAACAAACTTGCAAGAAGGACTGAGAAGTAAGG ATGAAGA 6 76 CC TGGGTTCATCCTGAAATTATTCTCATTGATTAATCTGCCATATC ACACTTGCACATG 7 3 5 TGTCTTTGGTCATTTCAATAGGTTCTTATTTCTGCAG 772 10. The method according to claim 1, characterized in that the DNA molecule is an IFN pseudo-omega-gene whose formula is Li 89 90667 AAGCTTTGGGCATGACCTAGTAGGTGACTCTT j2 AGTTGGAGTGGTCAGTTGTAAGGCTCTTGCTCAGTCACGTGGCTCCCCTGTATTTCCCC 91 ACGTTTGCAGCCGTGCTCCTTCTCAATGCTATGAAAGTGTGGGCTCCTCTCCC ACTGG A 1 5 0 GTGCTGACTGTAGCTTGTATCTTGGCACTCCCAGGCTGTACATAACAGCTCTG AGGTGA 2 0 9 TCTCAGGGTTAATGTTTTCTCCCCGACTTGGAGGCCATTGAAGGAAGGGACCTTAGTAG 2 6 8 TAGTTGTAACTGAGGGTCTTTTGCTTGTCTCCTGGGGGCTCCACCCCAGAGATGCAGGT 3 2 7 GAGC AATC ACTCAGTGCATTCAGCTTGGGATGGGGGTGTCTGTGCTGTGGGCCC AAGAC AGGGGTTCCCTGCCTGGTGATGTGAGGGGTGGGTGGTTGACCAATGGCAG ACAGACTGG 3 86 4 4 5 4 5:00 a.m. CCTCCTCTCTTGGGTTGACAGCAGCTTGTTGGAGGTATGCATAAGGCACTTGGGGTCTT GCTCCTTCATTAGTTCCAAGGTAGCTGGGGTAGTACCACTGCAG AGGCAGTGTTAGACA GGCTTTCTGTTACCCCTGGGGTCTCCACCTCCTAGAAATGTGAAGTCATGTTAATGGG A 56 3 6 2 2 6 81 GTGTTTAGCCAGAGGAGTGGGGTGGCTGCATGCTGGTGTAAGTATGGG ACTTCACTTCT TGGGAAAC AGGGAGGTGGAATCTTACCAGCAGGAGACTGTTCTCCTCACGATGTGTAAC 7 4:00 a.m. CTGCAGTGTGCTGGAAGTTTAGGTGAC TGTGGCATG ATGTTAGCTTGT AAACAAAG AGC TTCAGACTCTTTGTCTCTTCCCCAGACCCAAGGCAGCAAGGATAAAAGCTGCTGCTGTG 9 79 8 5 8 917 GCAGTGGCAGAGGTAGGATGGTTGTGGGAGCCTCTCCCCAGGGAAACTCTAGTCAACTA CAAGTGG ATATGCTCAGCCATGGGTAGGGTGACTGTTCTGCAGTCATGGGCAGGGGGCC 97 6 TGCTCCTG AAG AATAGGGACAGGGATTCTCAGGGAAGAGGGGCTGG ACTCCTCTTC AT A10 3:05 a.m. TAGTGGCGATGATGTGCTGGAGGTGCCAGTGTAGTGAATAGGCCTTTGTTCCTTCCCC A 109 4 GCCAG AGGGCTGCTGGGGCTGTCCCACTGCAACGGTCATGGTGG ATGGGTTATGGGTTG 115 3 ACTGTGGGATTTCTTTCTTGGAGAAATGCTGGACTGCCTGATTG AGG AGACG AGGCAAG 1212th GG AAGAGATGCCTGTGCTGGAGTCTCTGGTCAGGGGGGGGTGTGCCACAGAGGGGGGGGGGAGAGGAGGGGGGGAGGAGGGGGGGGGGGGGGGGGGGGGGGGGGGG CAAGAGCTGTGAGAAAGCAAAGATAGCAACCCACCCTTCTCACTGGGAGCTCTGTTCCA GGGAGATGCAGAGCTGCCATTGCTCAATAGCCCCAGCTGGTAGCTGCAGACCCAGGCCT 1448 1507 • 1566 GGCAGACCCACCCAGTGAGCAGATAGGGGATTAGGGACCCACATAACACACAGTCTGGC -CACTTTTCCATAGGGCTGCTGAATATGCTGGGGGTCCAATCCAGACCATAGTCACCTCA CATTTTTCAGTACCTGAAGATATCCACAGTGAAGGCTATGAAACAGTGAAGATGGGGAC 1625 1684 1743 CTGCCCCTGCCTCTGGACCTCTGTTCCAGAGAGGTACAACCTGTTGCCTCCGACATACA TGCAGGAGGTGGCTGGAGACCCGGTGGATATCCCTCCCACTGAGGAGAAGCAGCATCAG GGAATCAGGTGAAGAAACAGTCTGGCCACTTTTTGGTAGAGCAGCTGTGCTTGCTGGGG 1802 1861 1920 90 90 667 GTCTGCTACCACCCCCAGCAAAAAGAATGGCATTTGCAAGAATGGCTAAGGCTGCTAAA CAGCAACAATGGCAACCTACCATTCCCTTTGGAGCGCCATCCCAGGGATATTCG AAACT GCTGTCCACTAGAAAACAGTGGTGGAGGTGACTGGAGACCCCAGTGGAGAGTTTCACCT 19:07 9 20:03 20:09 8 GGTGAAAAGAAACAGGATTTGGGATCGACATGAATAACCAATCTGACTGCTTCCCCGTA 7G AGCTGCTGG ACTGTGCTGGGTGGCTGCTCC AGTCCCTAGCTGCCTTGG ACTCCCG AG A 2156 ACCCAAAGGCTCCAATAGCTAAGATTGTGAAAGAGCAAAGATGGCAGCCCACCCCCTGC 2 215 CACAGGGACGTCCATGTCAGGGAGGTATGCGGCTGCTACCAGTGTCTGGCTGGATTCCC 2 2 7 4 AAGTCGAGTGGGTCTTACCCTGAGACAGGCCATGGAAGGTGGGCCTGTCATTGTCACTG 2:03 a.m. 3:03 a.m. CCCAGCGCCCTGGATGAAACCCCTTTCCTAGGGGTATGTATAGGGGTCTAGCGTCCTGC 2 3 9 2 TTGGCTGGAGTTATAGCTTCTTTTGTGGGGAGGCCTGGGTATCTAAGGCTCCAGGGTAC 2451 CCATGCATGCGAGAGCGGCTGCTCTGCTGAACCCTACGTAGCCCTGCATGTCAGACTAA 2510 ATGCCCTGGTAGAGTGGGTTCACTAGGAGATCTCCTGACCTGAGGATTGCAAAGATCTG 256 9 TGGGAGAAGCGTGGGTCCCCAGGGCTGCTCACTTACTCACCACTTCCCTGGGCAGG AGA 2628 GGCTCCCCTGGCTCTGTGTCATCCTGGGGGGGCAGTTGTCCTGCCTTACTTTGCTTTAT 2:06 a.m. 87 TCTCCATGGGTCAAGTTGTTTTCTTGAGTCTCAATGTGTGCACCTGGTTTTTTCAGTTG 2746 AAGGTGCTGTATTTACTTGCCCCTTCCATTTCTCTCCATGAGAGTGGCACACACTAGCA 2805 GGTTCCAGTCGGCCATCTTGCAACCCCTGAAAACTATTTGTTTCCAGCTATAAGCC TO 286 4 GAGAGAACCTGGAGTGGCATAAAAAGAATGCCTCGGGGTTCATCCCGACCCCAGCTCAG 2923 CTAGGCCAGCAGCACCCTCGTTTCCCA ATG GTC CTA CTG CTT CCT CTA CTG 297 4 GTG GCC CTG CTG CTT TGC CAA TGT GGC CCT GTT GGA TCT CTG GGC 3 019 TTT GAC CTG CCT CAG AAC CGT GGC CTA CTT AGC AGG AAC ACC TTG 306 4 GCA TTC TGG GCC AAA TGC AGA ATC TCC ACT TTC TTG TGT CTC A 310 9 GAC AGA AGA GAC TTC AGG TTC CCC CTG GAG ATG TGG ATG GCA GTC 315 4 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 32 4 4 GGA ACA TGA CCCTCCTGGACCAGCTCCACACTGGATTTCATCAGCAGCTCGAATAG 3:03 a.m. 00 CCTGGAGTCTTGCTTAGGGCAGGCAACAGGAGAGGAAGAATCTGTGGGGGTGATTGGGA 3:03 a.m. 5:09 a.m. CCCT AC ACTGGCCTTG AGG AGGTACTTCC AGGG AATCC ATGGG AATCC AG AG A ATCT AC 3418 CTGAAAGAGAAGAAATACAGTGACTGTGCTTAGGAGGTTGTCAGAATGGAATCATGAAA 347 7 TCCTTCTCTTCATCAACAGACTTGCAAGGACTGAGAAGTAAGGATGAAGACCTGGGGTC 3:05 a.m. 3:06 a.m. TGCTTTACTCTTTCTTATTTTCTTCCTCTTCCTTACTATGTGTTTATTTCTTCTTTTTC 3:05 a.m. 95 TAGTTCCTTAACTTGTAAGTAGTTCACTTGGTTTGAGGTCTTTCTTCTTTTTTAATATA 365 4 AGCTT 3659 II si 90667 11. A method according to claim 1 or 10, characterized in that the recombinant DNA molecule is replicated in a vehicle capable of being replicated in prokaryotes or eukaryotes. Method according to claim 11, characterized in that as a vehicle a plasmid or an expression vehicle capable of being replicated in microorganisms or in mammalian cells is used. 13. A method according to claim 12, characterized in that vehicle is used a) a plasmid E76E9 in the E. coli HB 101 bacterium stored in the DSM collection under Nunun DSM 3003 or b) a plasmid P9A2 in E. coli HB 101 the bacterium, stored in the DSM collection under number DSM 3004. A process for the preparation of novel type I interferon in humans, wherein the divergence of the finished interferons compared to the female alpha interferons is 30-50%, and their N-glycolated derivatives, characterized in that the genetic information in the recombinant DNA molecule is expressed and the desired interferon isolated. 15. A method according to claim 14, characterized in that the divergence of the finished interferon relative to beta interferon is furthermore about 70%. Method according to claims 14 and 15, characterized in that the divergence of the finished interferon in relation to human alpha interferons is 40-48%. 17. A method according to claims 14-16, characterized in that the finished interferons contain conductive peptidine. 92 SO 667 18. Process according to claims 14-17, characterized in that the finished interferons contain 172 amino acids. 19. A method according to claim 18, characterized in that the final interferon contains the amino sequence 5 10 1.5 Cys Asp Leu Pro Gin Asn His Gly Leu Leu Sor Arq Asn Thr], or 20 25 JO Vai Leu Leu His Gin Met Arg Arg Ile Ser Pro Phe Leu Cys Leu 35 40 45 List Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu With Vai Lys Gly 50 55 60 Ser Gin Leu Gin Lys Ala His Vai With Ser Vai Leu His Glu With 65 70 75 Leu Gin Gin Ile Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala 80 85 90 Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His 95 100 105 Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Vai Vai Gly 110 115 120 Glu Gly Glu Ser Ala Gly Ala Ile Ser Ser Pro Ala Leu Thr Leu 125 130 135 Arg Arg Tyr Phe Gin Ile Arg Vai Tyr Leu List Glu List List 140 145 150 Tyr Ser Asp Cys Ala Trp Glu Vai Vai Arg With Glu Ile With List 155 160 165 Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Arg Ser Lys 170 Asp Arg Asp Leu Gly Ser Ser li 93 9 0 6 6 7 and at its amino acid site 78 N- glycolized derivatives. 20. A process according to claim 18, characterized in that an interferon according to claim 19 is prepared which at site 111 contains Glu amino acid instead of Gly amino acid, and at its amino acid site 78 N-glycosylated derivatives. 21. A process according to any one of claims 14 to 20, characterized in that interferons are prepared which contain a conductive peptidine, the formula of which is Met Ala Leu Leu Phe Pro Leu Leu Ala Ala Leu Val Met Thr Ser Tyr Pro Val Gly Ser Leu Gly 22. A method for producing prokaryotes or eukaryotes containing genetic information according to claims 1 to 10, characterized in that a vehicle containing a recombinant DNA molecule is transformed with prokaryote or eukaryote. 23. A method according to claim 2, characterized in that the vehicle additionally contains for expression the necessary replication and control sequences. A method according to claim 2 or 3, characterized in that mammalian cell line or E.coli is used as prokaryote or eukaryote. 25. A method according to claim 4, characterized in that as prokaryote is used E. coli, whose DSM number is 3003, or E. coli, whose DSM number is 3004. 94 90667 The method of claim 3, characterized in that the replication and control sequences required for expression of plasmid pER103 (DSM No. 2773, Fig. 6) are used. 27. A method according to claim 26, characterized in that, as IFN-omegal expression plasmid, plasmid pBR322 is used, which instead of the short characteristic EcoRI fragment characteristic of the plasmid contains a DNA sequence of the formula EcoRI Sau3A a§5ttcacgctGATCGCTAAAACATTGTGGGTA ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116 RBS Hindlll Cys Asp TGT GAT C - IFN-omega-gene -> Sau3A 28. A method according to claims 26 and 27, characterized in that, as a prokaryote, the E. coli HB 101 bacterium is used and as the vehicle plasmid pRWH11 and pRWH12. 29. A method according to claim 28, characterized in that as the vehicle, expression plasmid pRHW11 is used, wherein the IRN omega gene has a DNA sequence of the formula: TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 Ncol GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 7 3 AAG G AC AGA AGA G AC TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT G AG ATG 16 3 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA G AG CGC TCC TCT GCT 20 8 GCC TGG AAC ATG ACC CTC CTA G AC CAA CTC CAC ACT GGA CTT CAT 25 3 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 29 8 It 95 90667 GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 3 4 3 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AA AG AG AAG AA A 3 88 TAC AGC CAG TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG ΑΛΑ 43 3 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 478 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 5 30 TAACACTTGCA CATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 5 89 6 4:08 a.m. AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAAC 7 0 7 7 6 6 AAATGTTTGCC TTACATTGTATTAAGATAACAAAACATGTTCAG et 8 0 3 Alu I 30. A method according to claim 28, characterized in that as the vehicle, expression plasmid pRHW12 is used, wherein the IRN omega gene has a DNA sequence of the sequence TGT GAT CTG CCT CAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 28 Ncol GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 7 3 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 16 3 CTG CAG CAG ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 20 8 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 25 3 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 29 8 GAA GGA GAA TCT GCT GAG GCA ATT AGC AGC CCT GCA CTG ACC TTG 34 3 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 43 3 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA AGT AAA 47 8 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTG ATTAATTTGCCATA 5 30 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACT CTTATTTCGGCTTTAATCAC 5:08 a.m. 6:04 a.m. AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 8 AAAAG ACTTAGGTTCAGGGGCATCAGTCCCTAAG ATGTTATTTATTTTTACTC ATTTAT 70 7 ATTCTTACATTTTATCATATTTATACTATTTATATTCTT AT TT AT AAC AAATGTTTGCC TTTACATTGTATTAAGATAACAAAACATGTTCAGet 766 802 A] u 96 90 667 31. Preparation Method for Plasmid pRHW10, characterized in that plasmid pER103 (DSM No. 2773, see Fig. 6) is joined in situ HindIII by ligase reaction a Defragment, whose formula is HindIII Sau3A Ncol CAAATGAGGA GAATCTCCCC TTTCTTGTGT LOO CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGaTcTa 32. Preparation method for the expression plasmid pRHW10, characterized in that the larger fragment of the plasmid's pRHW10 obtained with restriction endonuclease-treated BamHI enzyme, by filling the intersection of DNA polymerase-Klenow fragments and 4 deoxynucleoside triphosphate triphosphate triphosphate triphosphate in plasmid pERl03-HindIII intersection (DSM 2773, picture 6 only) is timed DNA fragment having the formula II 97 90667 HindIII Sau3A NcoI aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 50 ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAaqct t Sau3A HindIII is joined by ligase reaction to a fragment obtained by treating the clone P9A2 (DSM # 3004) ACC TTG 28 Ncol GTG CTT C TG 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 GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343 AGG AGG TAC TTC CAG GGA ATC CGT GTC TAC CTG AAA GAG AAG AAA 388 TAC AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433 TCC TTG TTC TTA TCA ACA AAC CAA AGA GAA CTG AGA AGT AAA 478 ACA GAT GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC 530 589 648 AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAACAAATGTTTGCC 707 7 6 6 8 TTTACATTGTATTAAGATAACAAAACATGTTCAGct 02 AluI 98 90667 and for replication, the resulting plasmid 1 E, coli HB 101 bacterium is transformed and cultured. 33. Preparation method for expression plasmid pHWR11, characterized in that the larger fragment of the plasmid's pHRW10 obtained with restriction endonuclease-treated BamHI enzyme, by filling the intersection of DNA polymerase I-Klenow fragments and 4 deoxxynucleoside triphosphate triphosphate triphosphate triphosphate which in the plasmid pERlO3 (DSM 2773, Figure 6) Hindi11-stä1le inside pouring a DNA fragment which has the formula HindIII Sau3A NcoI aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA 5:00 a.m. ACACCTTGGT GCTTCTGCAC CAAATGAGGA GAATCTCCCC TTTCTTGTGT 100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG 150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC 200 AGATCACACA TCTTTAact to Sau3A HindIII is connected by ligase reaction to a fragment obtained by treating the clone E76E9 (DSM 3003) AvaII fragment with NcoI and Alul enzymes and whose formula is II 99 90 667 c CAT GGC CTA CTT AGC AGG AAC ACC TTG 2 8 Ncol GTG CTT CTG C AC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 7 3 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 16 3 CTG CAG CAG ATC TTC AGC CTC TTC CAC AC A GAG CGC TCC TCT GCT 208 GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 25 3 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298 GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 34 3 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 4 33 TCC TTG TTC TTA TCA ACA AAC ATG GAA CAA CTG AGA AGA AAA AGT 47 8 GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 5:30 a.m. TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCTTATTTCGGCTTTAATCAC AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 5 89 64 8 70 7 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT TTATTCTTACATTTTATCATATTTATACTATTTATATTCTTATATAAC AAATGTTTGCC 7 66 TTTACATTGTATTAAGATAACAAAACATGTTC AGct 802 Alul 84 and for replication, the resulting plasmid is transformed into E. coli HB 101 bacterium and cultured. 34. Preparation of plasmids E76E9 and P9A6, characterized in that the iFN-omegal coding sequence according to claims 4 and 5 for cDNA-in-obtained from Sendal virus-induced Namalwa or NC37 cells by hybridization of plasmid pER34 encoding IFN-alpha2 (Arg) is isolated and eventually replicated in plasmid pPR322. loo 90667 35. A process for preparing a Met derivative according to claims 19 and 20, wherein the plasmid pHRW11 or pRHW12 transformed E. coli HB 101 is cultured and that the in situ formed Met derivative is isolated . Il