NL8100558A - RECOMBINANT DNA TECHNIQUE FOR THE PREPARATION OF A PROTEIN LIKE A HUMAN INTERFERON; GEN FOR THE EXPRESSION OF SUCH PROTEIN; SUB-UNIT OF SUCH A GEN; METHOD FOR THE PREPARATION OF SUCH A GEN OR SUCH SUB-UNIT; PLASMIC RECOMBINANT; METHOD FOR THE PREPARATION THEREOF; CELL CONTAINING SUCH A GEN, SUB-UNIT OF IT, OR PLASMIC COMBINANT; METHOD FOR PREPARING SUCH A CELL; PROCESS FOR PREPARING A PROTEIN; SO PREPARED PROTEIN. - Google Patents

Description

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Recombinant DNA technique for the preparation of a human interferon-like protein; gene for the expression of such a protein; subunit of such a gene; method for the preparation of such a gene or such a subunit; plasmid recombinant; process for the preparation thereof; cell containing such a gene, subunit thereof or plasmid recombinant; method for the preparation of such a cell; method for the preparation of a protein; protein thus prepared.

The invention relates to a recombinant DNA technique for the preparation of a human interferon-like protein.

Interferon is a natural molecule, which is prepared by 5 cells in response to a viral infection and certain other stimuli (see eg Isaacs, A., and Lindenmann, J., Proc. Roy. Soc. B., (1957), 147 258-267).

When exposed to cells, this molecule makes the cells resistant to virus infection. Interferon is therefore a known anti-virus agent (see, e.g., Isaacs, A. et al., (1957),

Proc. Ray. Soc. B., 147, 268-273).

Since the initial observation of anti-virus activity, a number of other properties have been attributed to interferon (see, e.g., Stewart, WE, II, Interferon I, (1979), Acade-15 mie Press, I. Gresser (Ed.)), Including a potential role in inhibiting cancer cell growth in vivo (see, e.g., Paucker, K., et al., (19S2), Virology, 7, 324 - 334; Gresser, I., et al., (1370) Ann. NY Acad Sci., 173, 634-639; Wellstedt, H., et al., (1S7S), Lancet, 245-247; Slamgren, H., et al. (1976), Acta.

20 fled. Scand., 133, 527-532; Werigan, T.C., et al., (1978) New

Engl. J. Wed., 295, 961-987; and Werigan, T.C. at al., (1973),

New Engl. J. Wed. 299, 1449-14533.

Interferon vertcont also has a remarkable degree of species-8100558-2 specificity (see, e.g., Tyrell, DAJ, (1959), Nature, 184, 452-453, and Gresser, I., et al., (1974), Nature, 251 , 543-545), which means that for the treatment of a human disease, preference is given to interferon derived from human cells.

For a detailed overview regarding interferon, see Texas Reports on Biology and Medicine, 35, 1979, published at the University of Texas Medical Branch, Galveston, Texas, U.S.A., CS. Baron and F. Diazani, Editors).

As for conventional sources, tissue culture cells are the only practical source of material. A tissue culture cell-based method is considered to be labor intensive, unreliable and expensive and generally results in low yields.

The aim of the invention is to provide means for the preparation of a protein with proteins resembling human interferon, by recombinant DNA techniques in bacteria, which means are cheaper and more reliable and also offer the possibility to prepare modified forms of interferon . Furthermore, higher yields can be obtained.

Interferon is a glycoproteins with a molecular weight of about 20,000. The amino terminal polypeptide sequences of human and murine interferons are known (see, e.g., Knight, E., Jr., et al., (1980), Science 207, 525 - 526; Zoon, KC, et al., 25 (1980). ), Science, 207, 527; and Taira, H., et al. (1980), Science, 207, 528 - 529).

Fig. 1 shows selected regions of these sequences, along with the corresponding oodon capabilities. Of the 13 amino acids known for human fibroblast FS-4 interferon, three are common (underlined) with the mouse interferons A and B, obtained from Ehrlich ascites tumor cells, while only one amino acid is common between human fibroblast and lympho- blastoid interferons. Indeed, on the DNA level, similarity between human fibroblast interferon and mouse interferons 35 A and B (indicated by dots) appears to be more likely than between two human proteins.

The region corresponding to the five amino-terminal residues of human fibroblast interferon clearly shows a minimal number of permutations of coding oligonucleotide over a sufficiently long sequence to be reasonably specific for an individual mRNA in a higher eukaryote cell C, e.g. Montgomery, O. L, et al., C19783, Cell, 14, 673-680]. The codons for the serine and leucine residues are deduced by first selecting those triplets that most likely exhibited maximum homology to the analog mouse codans. The uncertainties now remaining in this and the other triplets were then further reduced by acting on known frequencies of codon usage in human genes Csee e.g. Grantham, R., et al., C1980), Nucl. Acids Res., 8, r47 - rS2). It was therefore possible to reduce the number of possible coding sequences to two, the only difference being the third remainder of the serine codon, which showed no clear distinction in this position. On this basis, the sequences of two oligodeoxyribonucleotides CIFIA and 16} were deduced, which were expected to specifically initiate the synthesis of interferon cDNA when incubated with reverse transcriptase and polyA-mRNA C3 'terminal polyadenylated mRNA], extracted from " induced "cells.

An additional factor in predicting the structure of interferon mRNA-specific primers was the assumption that a G residue in DNA can form a moderately stable base pair with a U residue in RNA (or a T residue in DNA). Thus, when the primers contain a G-residue at certain positions instead of the correct A-residue, a degree of specific initiation could still be expected (see eg Powers, GJ, et al., C1S75], JACS, _97 , 875-8843.

The oligonucleotides CIFIA and 133 were synthesized and radiolabelled 32 with β-phosphate at their 5 'endpoints, hybridized to mRNA preparations from which human fibroblasts, either "quasi-induced" or induced to the production of interferon by cp (priming) with interferon and subsequent superinduction - 4 - using poly (I): poly (C) and cycloheximide, and incubated with the enzyme reverse transcriptase with the conventional deoxynucleoside triphosphate substrates Only IFIA gave a transcript of about 150 nucleotides specific for mRNA of induced fibroblasts The nucleotide sequence of this negative strand transcript was determined according to known methods and is shown in Figure 2.

The top strand in Figure 2 is the sequence of the DNA transcript. This DNA sequence defines the sequence of the 5 'end 10 of the human fibroblast interferon mRNA, shown as the lower strand in Figure 2. The mRNA follow-up in turn defines the protein follow-up, starting at the first AUG, of the amino terminal region of the putative interferon precursor molecule.

A second oligodeoxynucleotide CIFII] was synthesized, which was complementary to part of the particular sequence as shown in 32 in Figure 3. C PJ IFII was hybridized as above to cDNA, prepared according to known procedures from total mRNA of induced and quasi-induced fibroblast cells and an above-produced transcript. When the products of this reaction were fractionated based on dimensions using denaturing gel electrophoresis, a particular radioactive transcript of about 700 nucleotides was obtained from cDNA from induced, but not quasi-induced cells.

After native horny electrophoresis, the interferon specific product was approximately 850 base pairs in length, which approximately corresponds to the size of the complete interferon mRNA molecule. This size difference can be explained by assuming that the single-stranded interferon cDNA, as it also acts as a template for I-II, also self-initiates thanks to the looping back of the 3 'end . This transcript is then extended to the position of the IFII-initiated transcript where it is halted, thereby obtaining a full-length double-stranded interferon 35 (ie a gene encoding interferon), where 81 0 0 5 5 8-5 in the 3 'end of the self-initiated transcript and the 5' end of the IFII-initiated transcript are not covalently linked. The size of the interferon specific product will therefore vary with the type of gel system.

The interferon was eluted from native gels and followed by known methods leading to elucidation of the gene follow-up coding for the amino endpoint of the mature fibroblast interferon. An extended version of IFII, IFIII. (see Figure 4) was also prepared and the above procedure repeated. The same results were obtained.

This follow-up largely verifies the predictions used in the synthesis of IFIA. From a comparison of Figures 1 and 5, it can be seen that the actual mRNA sequence that specifies the amino endpoint of the mature interferon polypeptide is identical to that on which the structure of IFIA was based, except for only one nucleotide difference in the codon for the fifth amino acid of the amino terminus of the mature polypeptide. Therefore, out of a total of seven questionable nucleotide positions, six were correctly anticipated in the case of IFIA.

(See, e.g., Houghton, M., et al., (1980), Nucl. Acids Res. J3, 1913-1931.)

An additional oligodeoxynucleotide primer, IFIV (see Figure 4), corresponding to an area near the end of the freshly determined sequence was then prepared. To repeat the above procedure, further information on the sequence from which another primer, IFV (see Figure 4) was obtained. Fig. 4) was deduced and synthesized. The procedure was then repeated again. In this manner, the entire gene-encoding coding for the mature fibroblast interferon polypeptide (see Figure 5) was determined step-by-step. (See, e.g., Houghton, ΙΊ., Et al., (1930), Nucl. Acids Res. 8_, 2385-2834.3

In Figure S, the double stranded gene sequencing encoding the 5 'untranslated mRNA sequencing and the protein-encoding mRNA sequencing is shown.

An illustrative diagram of the basic procedures, uses 8100558 t

B

for cloning the interferon to bacterial plasmids, is shown in Figure 7.

The end products of these procedures were recombinants containing interferon genes encoding the mature inter-5 feron polypeptide and the 3 'untranslated interferon mRNA sequence.

In addition, these recombinants contain 1 molecule of IFIII primer that preceded the mature interferon gene sequence, and also a synthetic Hind III switch molecule at both ends of the gene.

A partial QNA nucleotide sequence of one of these inter-10 feron gene recombinants is shown in Figure 8, which shows that during the cloning procedures [probably as a result of the S1 nuclease treatment), the 5 µ-terminal nucleotide of IFIII was lost. went. It was also observed that the mRNA triplet coding for the tyrosine residue at the amino acid position 30 in the mature protein was 15 UAU, instead of the UAC triplet previously determined by direct follow-up of reverse transcripts [see Fig. 53.

This "silent" nucleotide alteration is likely due to gene polymorphism and means that both types of genes will lead to the expression of identical interferons.

In order to express the interferon gene in bacteria, it was transferred from pBR 322 (see eg Bolivar, F., et al., (19773, Gene, 2.95 - 1133 in the Hind III site of the so-called pWT 2x1 expression plasmids). The latter series of plasmids contains the promoter follow-up, which is responsible for the transcription of the tryptophan operone, and allows for all three possible translation phases (see eg published British patent application 2.052.5163. Therefore, if the interferon gene is in these plasmids being inserted into the correction orientation relative to the transcription will only lead to the gene's reliable expression 30. From the follow-up shown in Figure 8, it was predicted that pWT 221 would provide the "open" translation phase required for the reliable expression of the mature interferon polypeptide, although the latter would be linked to a 16-residue amino terminal peptide, resulting from the expression of the short-35 t The trp E gene sequence and the Hind 8100558 contained in the pWT 2x1 plasmids

V

III switch molecules also from the expression of the Hind III link and the IFIII primer, used in the initial cloning of the interferon gene. When the interferon gene was transferred to the pWT 2x1 plasmids and the trp promoter was derepressed by incubation in the absence of tryptophan and in the presence of 3/3-indole acrylic acid, indeed only antivirus activity was detected in the case of the pWT 221 derivative, as expected.

Fig. 9 (in which Veros = control cells, not infected with 10 EMC virus (Encephalomyocarditis virus); Std. A = interferon standard A; Std. B = interferon standard B; 221 = extract of recombinant, containing it introduced into pWT 221 interferon gene; 231 * extract of recombinant, containing the interferon gene introduced in pWT 231; 231 + Std. B = extract of recombinant, containing the interferon gene, introduced in pWT 231 to which interferon standard before extraction B was added; and EMC = control cells infected with EMC virus) shows a typical result obtained when the bacterial extracts are analyzed for antivirus activity in an in vitro system. In this system, the samples were diluted in 0.5 log steps and applied to monolayers of

African green monkey cells (Veros). After overnight incubation, the cells were tested with EMC virus before staining the cells, with samples containing interferon activity found to protect the vero cells from the cytopathic effect (cpe) of the virus. By comparing the obtained results with known interferon standards, the activity level could be quantitatively determined.

No activity was observed in extracts of the pWT 211 or pWT 231 interffer recombinants (only the result of gene gene with the pWT 231 recombinant is shown in Figure 9), while the observed activity of extracts of pWT 221 recombinants is equivalent. in a yield of about 10 international reference units of interferon per dm bacteria (ie equivalent g to 10 units of an international standard preparation of inter-35 terfaron, one unit being defined in terms of those 8 i 0 0 5 5 8 5 8 - 10 15 20 25 30 amount, leading to a 50% reduction in virus replication in tissue culture. It is likely that significant interferon activity is lost during the extraction procedure because only a yield of about 30 % was obtained when a native fibroblast interferon standard was added to the pWT 231 recombinant before extraction CO Note that the observed toxic effect v The recombinant extracts in the antivirus analyzes are due to the presence of Triton X-100, which was used in this particular extraction process.} It was now necessary to add the DNA coding for the non-interferon amino acids to the amino terminus of the linked interferon poly peptide. This can be conveniently performed using the plasmid pWT 501 (see eg British patent application 80 36080}. The plasmid pWT 501 has a molecular length of 3805 bp Cbase pairs} and the restriction map thereof is shown in Fig. 10. This plasmid are produced by a method in which pWT 221 is restricted using Hha I, the trp promoter-containing fragment is isolated, this fragment is incubated with DNA polymerase I, the resulting blunt-ended fragment is ligated to Hind III linkages , the ligated material using Hind III is subjected to restriction, the trp promoter-containing fragment is isolated, this fragment is ligated to Hind III-cut, bacterial basic phosphatase-treated pAT 153 (see eg Twigg, AJ, and Sherratt, D., (1980], Nature 283, 216 - 218], E. coli K.-12 HB101 is transformed (genotype gal, lac, ara, pro, arg, strr, roe A, r., M; see e.g. Boyer, H.W., and Roullard-Dussoix, K K D., J. Mol. Biol., 41., 459 - 472] using the resulting plasmid and selected for ampicillin resistance and tetracycline resistance. A small Hind III / Taq I restriction fragment containing the trp promoter can be isolated from this plasmid. The Taq I cleavage site in question is in the DNA sequence corresponding to the ribosome binding site in the mRNA region, which encodes the 0 5 5 8 35 9 trp leader polypeptide, which occurs just before the initiating methylion triplet. . This fragment can be linked to a Sac I / Hind III restriction fragment of the cloned interferon gene, which contains the entire mature protein coding sequence, preceded by only six base pairs as shown in Figure 11. Since the amino-terminal residue of the mature interferon appears to be methionine, the joining process can be carried out in such a way that a chimeric DNA is obtained which after transcription contains an artificial ribosome binding site using this methionine codon as initiating codon for translation.

The pooled molecule was then cloned into E. coli K-12 HB101 using pAT 153 as a plasmid vector. This method is shown in Figure 11.

Another important consequence of the above method is that the resulting expression plasmid lacks the trp diluent region which plays an important role in regulating the transcription of the trp operon Czie e.g. Niozzari, G.F., and Yanofsky, C. (1378), J. Bacteriol., 133, 1457-1466).

Recombinants containing the required pooled molecule in one orientation or the other were then analyzed for their ability to produce interferon. They were found to produce significantly higher levels, from roughly six-fold Cin both orientations, extractable antivirus activity compared to the recombinants containing the interferon gene introduced in pWT 221. As expected, the size of the interferon product (about 19,000 Daltons) was smaller than that obtained using pWT 221 as an expression plasmid (about 24,000 Daltons). This is shown in Fig. 12 and is fairly consistent with the expected nature of the interferon polypeptides produced in both cases (in Fig. 12i. A = recombinant, which contains the joined molecule in right orientation. Cg. I.e. transcription proceeds from the trp- promoter to the tetracycline resistance gene in the plasmid); b = recombinant, which contains the assembled molecule in left-hand orientation (i.e. transcription proceeds from the trp promoter away from the tetracycline resistance gene); c = 8100558 10 - transformant, which contains pAT 153 only d = recombinant containing the interferon gene, but the trp promoter is missing f * expression recombinant, consisting of the interferon gene j and g 8 expression recombinant introduced in pWT 221, consisting of from the interferon gene introduced into pWT 211]. Figure 12 also shows the abundance of 19,000 daltons interferon polypeptide over the 24,000 daltons product prepared in the pWT 221 recombinant. This increase in expression of the interferon gene can be attributed to the removal of the trp thinner region.

The Artificial ribosome binding sequence contains six nucleotides between residues of the Taq I cleavage site and the ATG initiator codon. The original plasmid (pWT 501] contains only five nucleotides (of different sequence) in this analog region (see Fig. 113. Therefore, further changes in the size and follow-up of this particular region in the interferon recombinant may increase the efficiency of the ribosome. binding and thus improve the yield of the interferon polypeptide.

It is expected that the yield of interferon-like polypeptide can be further improved by increasing the number of interferon genes and / or trp promoters in elK plasmid. This can be done according to known methods.

Also, the use of bacterial fermenters, in which high cell densities can be obtained, will of course increase the yield of interferon activity per dm of bacteria.

In addition to being able to protect the Vero cells from the cpe of EMC virus, the interferon polypeptide produced in bacteria also protects V3 African green monkey cells from infection by vesicular stomatitis virus (VSV) in vitro. In addition, the bacterial interferon polypeptide as well as the native fibroblast interferon has also been shown to enhance the natural killing activity of human lymphocytes in vitro.

The genetically engineered interferon-like protein is primarily intended for use in the treatment of diseases and will be used in effective amounts, optionally with a pharmaceutically acceptable carrier 8100558-11. The dosages used in studies using native human interferons varied considerably, e.g. from 10 to 10 international reference units per day for different periods Csee e.g. Scott, G.M., and Tyrall, D.A.J., C19S0J, Brit. Med. J., 250, 1555-582). The preferred treatment regimen is expected to vary according to the type and severity of the disease. The interferon polypeptide prepared from bacteria may, in some cases, exhibit aberrant activities and pharmacokinetic properties compared to native fibroblast interferon.

The interferon recombinant DNA has also been used to examine human gene banks for the presence of clones containing the chromosomal intsrferon gene. It was accomplished by first extracting the Hind III restriction fragment containing the interferon gene from the pWT 221 recombinant plasmid, radiolabelling it and then hybridizing it to platelets prepared by transfection of bacteria with a human chromosomal DNA / phage. hybrid library. The plates specifically binding the interferon gene probe were purified and jT recombinant DNA was prepared therefrom. The fibroblast (or / 3) interferon gene present in these molecules was then monitored and the monitoring is shown in Figure 13.

An uninterrupted gene follow-up, identical to that shown in Figure B, was observed, followed immediately by a follow-up corresponding to the 3 'untranslated interferon mRNA sequence, which was previously determined (see, e.g., Taniguchi, T. et al. (1980) Gene, 11,11-15, and Derynck, R. et al. (1980, Nature, 285, 542-547).

This observation shows that there are no intervening sequences (introns) within this particular fibroblast interferon gene in the genome.

In addition, short gene sequences upstream and downstream of the interferon gene were also established. The upstream flanking region may be part of the RMA polymerase initiation / promoter monitoring that controls transcription of the interferon gene in human fibroblasts (see e.g.

8100 558 12 -

Houghton, M., et al. (1981), Nucl. Acids Res. £, No. 2).

It appears that bacteria cannot reliably express eukaryotic genes containing introns and thus an implication of the above is that it should be possible to induce bacteria for the production of human interferons by transforming them with chromosomal genes encoding interferon. These chromosomal genes could be isolated from readily available human gene banks, and such an approach has advantages over first isolating the interferon mRNA from induced eukaryotic cells, synthesizing the cDNA gene in vitro, transforming bacteria, etc. it appears that interferon genes generally do not contain introns, because a leukocyte interferon gene also lacks them (see, e.g., Nagata, S., et al, (1980), Nature 287, 401-408), such an approach could be applied to different interferon genes.

By using the cloned interferon gene, ie cDNA gene, as a probe for homologous sequences in total genomic DNA, restricted with a variety of restriction enzymes, the presence of one predominant type of fibroblast interferon gene could be detected observed (see, e.g., Houghton, M., et al., loc cit). However, there was some evidence for the presence of other genes that were partially homologous to the probe (ibid). Therefore, the possibility persists that other interferon genes could be isolated (from restriction digestions of total genomic DNA or directly from human chromosomal gene banks) using the present particular cloned interferon gene. Furthermore, now that specific oligonucleotides have been successfully used to detect bacterial recombinants containing interferon genes (see 11 below), it is feasible to use selected oligonucleotides for the detection and isolation of various interferon genes are present in human chromosomal gene banks or are present in a restriction digest of total genomic DNA. The present interferon gene sequence shows e.g. certain similarities to 81 0 0 5 5 8 - 13 - human leukocyte genes Csee e.g. Taniguchi, t., Et al., (1980) Nature 285,547; and Streuli, M., et al. (1980}, Science, 209, 1343 - 13473. Using an oligonucleotide probe common between the fibroblast and leukocyte interferon gene sequences, it is possible to identify both gene types from, for example, a to isolate human gene library Similarly, other partially homologous interferon genes could be obtained by using other oligonucleotides homologous to different regions of the present cloned interferon gene.

Such an approach would be more effective than simply using the entire cloned firoblast interferon gene as a probe for partially homologous genes, due to a reduction of the "background" in such experiments using specific oligonucleotides.

Common sequences between different interferon genes indicate that they may encode a common function displayed by the different interferon polypeptides. The gene follow-up coding for the 50-60 amino acids approximately at the carboxyl endpoint of the molecule may therefore be of particular significance and it is noted that the product of a recombinant containing such a sub-gene or part thereof may be of special value . Similarly, other sub-genes (whether they show any homology to other interferon gene sequences or not) may also have special value.

It is also noted that the fibroblast interferon gene follow-up may be limited with respect to expression in bacteria due to the use of codons, which may be infrequently used in bacterial mRNA molecules. It is therefore evident that by altering certain interferon gene codons so that they will still encode the same amino acid, but will be translated more efficiently by the bacterial translation equipment, the overall yield of interferon cai will be improved. The logic involved in deciding which codons, if any, would benefit from an institution can be based on observed codon usage in prokaryotic mRNA molecules (see 8100558 14 - eg Grantham, R., et al., loc cit. 3.

It was also noted that clones of the interferon gene, which additionally contains the DNA for the prepeptide signal follow-up, may lead to some benefit. For example, the interferon product can be isolated in the peri-plasmic space, optionally after splitting the signal follow-up. In addition, the conformation of the interferon polypeptide may more closely resemble the natural state than that obtained by direct expression of the gene sequence encoding the mature interferon alone. Therefore, the activity of the interferon product may be greater.

Alternative approaches to interferon production by genetic engineering include the introduction of the required interferon gene into eukaryotic cells, e.g. mouse L cells or yeast cells.

Mouse L cells can be transformed with exogenous DNA and this can be done with human interferon genes in a number of ways. E.g. a chromosomal interferon gene containing the entire DNA sequence required for correct RNA polymerase initiation could be cloned into the mouse cells, thus allowing transcription and subsequent expression. On the other hand, an interferon gene lacking this promoter follow-up could conveniently be linked to another stretch of eukaryotic DNA containing a promoter, e.g. the thymidine kinase gene used as a basis for transformant selection (see eg Wigler, M., et al., (19791, Cell, 1S, 777-785). In this way, transcription of the interferon gene via the linked gene would follow-up expired.

It is also possible that transformation of L cells with an interferon gene that lacks a promoter follow-up to. expression of the DNA results from accidental insertion into certain regions of the genome.

On the other hand, certain plasmids reside in yeast cells and interferon genes could be introduced into such plasmids and suitably modified versions thereof to obtain expression of the heterolayer DNA and yield interferan polypeptide.

8 1 0 0 5 5 8 - 15 -

Interferon prepared by such methods may be more biologically active and effective than interferon prepared in prokaryotes that do not have certain important modifications. the eukaryotic protein can be applied. Also, the yield of interferon may be higher in these eukaryotic systems.

Thus, in a first embodiment, the invention provides a gene for the expression of a protein having properties similar to that of human interferon, comprising a coding strand and a complementary strand, the coding strand of which contains the following sequence: 5 'GGC.CAT .ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.TTC.GAA.-GCC.- TTT. GCT. CTG. GCA. CAA. CAG. GTA. GTA. GGC. GAC. ACT. GTT. CBT. GTT. GTC. -AAC.ATG. ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.TTC.-TCC.-ACT. ACA. GCT. CTT. TCC. ATG, AGC. TAC. AAC. TTG. CTT. GGA. TTC.CTA.CAA.-AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.-AGG. CTT. GAA. TAC. TGC. CTC. AAG. GAC. AGG. ATG. AAC. TTT. GAC. ATC. CCT. GAG. -GAG-. ATT. AAG. CAG. CTG. CAG. CAG. TTC. CAG. AAG. GAG. GAC. GCC. GCA. TTG.ACC.-ATC. TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.-TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT .GAG.AAC.CTC.CTG.GCT.AAT.-GTC. TAT. CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA. GAA. AAA. CTG. -GAG -.- AAA. GAA. HOLE. TTC. ACC. AGG. GGA. AAA. CTC. ATG. AGC. AGT. CTG. CAC. CTG. -AAA.AGA. TAT. TAT.GGG.AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.-ACT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.- TAC.TTC.ATT.AAC.AGA.CTT. ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.-AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC.ATT.CTT.CAA .CCA.-GCA. GAT.GCT.GTT.TAA.GTG. ACT. GAT.GGC.TAA.TGT.ACT. GCA.TAT.GAA.AGG.-ACA.CTA.GAA. GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.GTT.ATT.TTT.ATT.-TAT.TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.TTA.TTT.TTG .GTG.CAA.AAG.TCA.-ACA.TGG.CA 3 '

or a sub-unit thereof or an equivalent thereof. (It is noted that the so-called coding strand has the same meaning as the mRNAJ

The above corresponds to the entire mRNA coding sequence plus the upstream and downstream gene sequences obtained by isolating the gene from a chromosomal gene bank 8100558 16 - and sequencing CEngels: "sequencing"].

According to a further embodiment, the invention provides a gene, the coding strand of which contains the following sequence: 5 'GGC. CAT. ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.-5 TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT.- GTT.CGT.GTT.GTC.AAC.ATG. ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.-GCT.CTC.CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC, ATG.AGC.-TAC.AAC. TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.-TGT.CAG.AAG.CTC.CTG.TGG.CAA7TTG.AAT.GGG.AGG.CTT.GAA.TAC .-10 TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG.GAG.ATT.- AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.-ATC, TAT.GAG.ATG.CTC.CAG.AAC, ATC.TTT.GCT.ATT.TTC.AGA.CAA.-GAT.TCA.TCT.AGC.ACT.GGC.TGG. AAT.GAG.ACT.ATT.GTT.GAG.AAC.-CT C.CT G.GCT.AAT.GTC .TAT. CAT.CAG.ATA.AAC.CAT.CTG.AAG.ACA.-15 GTC.CTG.GAA.GAA. AAA.CTG.GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.- AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.- ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.GCC.-TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC .TTC.-ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.-20 AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA. ATT.GCT.TCA.AGC.ATT.- CTT.CAA. CCA. GCA.GAT.GCT.GTT.TAA.GTG, ACT.GAT.GGC.TAA.TGT.- ACT. GCA.TAT.GAA.AGG.ACA.CTA.GAA.GAT, TTT.GAA.ATT.TTT.ATT.-AAA.TTA. TGA.GTT.ATT.TTT.ATT.TAT.TTA.AAT.TTT.ATT.TTG.GAA.-AAT.AAA.TTA.TTT.TT G.GTG.CAA.AAG.TCA.ACA.TGG.CAG. TTT.TAA.-25 TTT.CGA.TTT.GAT.TTA.TAT.AAC.CA..3 'or a subunit or equivalent.

This is consistent with the above with some additional downstream follow-up.

The invention also provides a gene, the coding strand of which contains the following sequence; 5 'GGC.CAT.ACC.CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.-CCT.TTC.GAA.GCC.TTT.GCT.CTG.GCA.CAA.CAG.GTA.GTA .GGC.-GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.-CTC -CAA.ATT.GCT.CTC.CTG, TTG.TGC.TTC.TCC .ACT.ACA.GCT.-35 CTT.TCC.ATG.AGC.TAC.AAC, TTG.CTT.GGA.TTC.CTA.CAA.AGA.- 8 1 0 0 5 5 8 5 - 17 - AGC.AGC .AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.-AAT.GGG.AGG.CTT.GAA. TAT.TGC.CTC.AAG.GAC.AGG.ATG.AAC.-TTT.GAC.ATC.CCT. GAG. GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.-CAG.AAG. GAG.GAC.GCC. GCA.TTG. ACC.ATC.TAT.GAG.ATG.CTC.-CAG.AAC.ATC.TTT.GCT. ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC.-ACT.GGC.TGG.AAT. GAG.ACT.ATT.GTT. GAG.AAC.CTC.CTG.GCT.-AAT.GTC.TAT.CAT.CAG.ATA.AAC.CAT.CTG.AAG.ACA.GTC.CTG.-GAA ·; GAA.-AAA. CTG. GAG. AAA. GAA. HOLE. TTC. ACC. AGG. GGA. AAA. -CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.-ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.-GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.- TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.-AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT .GCT.-TCA.AGC.ATT.CTT.CAA. CCA. GCA. GAT.GCT.GTT.TAA.GTG.ACT. -GAT.GGC.TAA.TGT. ACT. GCA. TAT.GAA.AGG.ACA.CTA.GAA. GAT.-TTT.GAA. ATT.TTT.ATT.AAA.TTA.TGA.GTT. ATT.TTT. ATT. TAT.-TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.TTA.TTT.TTG.GTG.CAA.-AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.CGA.TTT. GAT.TTA. TAT.-AAC.CA .. 3 "

I.Q

25 20 25 30 or a subunit thereof.

This forms a polymorphic form of the above.

Certain particular parts of such genes are also embodiments of the invention.

Thus, in a particular further embodiment, the invention provides a gene. of which the coding string contains the following sequence: 5 'TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG.GCA.-CAA.CAG.GTA.GTA.GGC.GAC.ACT. GTT.CGT.GTT.GTC.AAC.ATG.-ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.TGC.-TTC.TCC.ACT.ACA.GCT. CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.-GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG.AAG.-CTC.CTG.TGG. CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.-AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG.GAG.ATT.AAG.-CAG. CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC.GCA.TTG.ACC.-ATC.TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA .- 35 81 0 0 55 8 18 f CAA. GAT.TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.-GAG.AAC.CTC.CTG.GCT.AAT.GTC.TAT.CAT.CAG, ATA.AAC. CAT.-CTG.AAG.ACA.GTC.CTG.GAA.GAA. AAA. CTG. GAG.AAA.GAA.GAT.-TTC. ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.-AGA.TAT. TAT.GGG.AGG. ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.-GAG.TAC.AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.-ATC.CTA.AGG.AAC.TTT. TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.-TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.-GAC.TGG.ACA. ATT.GCT.TCA.AGC. ATT.CTT.CAA. CCA.GCA.GAT.-GCT. GTT-. TAA. GTG. ACT. HOLE. GGC. TAA. TGT. ACT. GCA. TAT. GAA. -AGG.ACA.CTA.GAA. GAT.TTT.GAA. ATT.TTT.ATT.AAA.TTA.TGA.-GTT.ATT.TTT.ATT.TAT.TTA.AAT.TTT.ATT.TTG.GAA.AAT.AAA.-TTA.TTT.TTG.GTG.CAA. AAG.TC 3 'or its equivalent.

Optionally with the addition of one or two A residues at the 3 'end, this forms the gene sequence, which corresponds to the entire mRNA sequence.

In another particular embodiment, the invention provides a gene, the coding strand of which contains the following sequence: 5 'ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG.-TGC. TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.-CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.TGT.CAG .-AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.- CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG .GAG.ATT.-AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC.GCA.TTG, -ACC.ATC.TAT.GAG.ATG.CTC.CAG.AAC.ATC .TTT.GCT.ATT.TTC.-AGA.CAA.GAT.TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.-GTT. GAG.AAC.CTC.CTG.GCT.AAT.GTC.TAT. CAT.CAG.ATA.AAC.-CAT.CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG, AAA.GAA.-GAT.TTC.ACC.AGG.GGA.AAA.CTC. ATG.AGC.AGT.CTG.CAC.CTG.-AAA.AGA.TAT.TAT.GGG.AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC.-AAG.GAG.TAC.AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.-GAA, ATC.CTA.AGG.AAC.TTT. TAC.TTC.ATT.AAC.AGA.CTT.ACA.-GGT.TAC.CTC.CGA.AAC.TGA. 3 '8100558 - 19 - or equivalent.

This is consistent with the gene sequence encoding the prepeptide and the mature protein.

The invention also provides a gene, the coding 5 strand of which comprises the following sequence: 5 'ATG.AGC.TAC, AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.- AGC.AAT.TTT .CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.-GGG.AGG.CTT.GAA.TAC.TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.-GAC .ATC.CCT.GAG.GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.-10 AAG.GAG.GAC.GCC. GCA.TTG.ACC.ATC.TAT.GAG.ATG.CTC.CAG.- AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC.ACT.-GGC.TGG. AAT.GAG.ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.AAT.-GTC. TAT. CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.GTC.CTG.GAA.-GAA.AAA.CTG. GAG.AAA.GAA. GAT.TTC.ACC.AGG.GGA.AAA.CTC.-15 ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT. TAT.GGG.AGG.ATT.- CTG. CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.GCC.-TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA, AGG.AAC.TTT.TAC .-TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA. 3 "or equivalent.

This is consistent with the gene sequencing encoding the mature protein.

Subunits of such genes are also embodiments of the invention.

The invention also provides a method for the preparation of a gene or an equivalent thereof or a subunit thereof according to any of the first three embodiments mentioned above, wherein a molecule is used as probe with a gene or an equivalent thereof or a subunit thereof according to any one of the above-mentioned embodiments for isolating a human chromosomal interferon gene from human chromosomal DMA.

It is noted that although single-stranded or double-stranded DNA may initially be used as a probe, a single-stranded form will be present at the hybridisazic stage.

The invention further provides a method for the preparation of a gene or its equivalent or a subunit thereof according to any of the above embodiments, other than the first three thereof, wherein poly A mRNA is isolated from induced fibroblast cells, single-stranded cDNA is synthesized using oligo dT primer and reverse transcriptase, and double-stranded ONA is synthesized therefrom using reverse transcriptase or E. coli ONA polymerase I and, optionally, a primer. Preferably, a primer is used in the third step of this method. Generally, the reaction mixture is fractionated after the second stage and / or after the third stage. Self-initiation is preferably hindered after the second stage. The product can be identified after cloning by using a primer to examine the colonies.

The invention also provides a method for the preparation of a gene or its equivalent from a subunit thereof according to any of the above embodiments, other than the first three thereof, wherein mRNA is isolated from induced fibroblast cells, single-stranded cDNA is synthesized using a specific primer and reverse transcriptase and double-stranded DNA is synthesized therefrom using reverse transcriptase or E. coli DNA polymerase I and optionally a primer.

This can be considered a modification of the above method.

The invention also provides a method for the preparation of a gene or an equivalent thereof or a subunit thereof having a portion that is common to or related to a portion of a gene or an equivalent thereof or a subunit thereof according to any of the first three of the above-mentioned embodiments, wherein a molecule is used as probe with a sequence corresponding to at least part of a common or related portion for the isolation from human chromosomal DNA of a human chromosomal gene.

The invention also provides a plasmid recombinant comprising 8100558-21 sen plasmid vector into which a gene or subunit or equivalent thereof has been introduced into an insertion site to enable the plasmid recombinant to translate into the correct phase for the mRNA corresponding to the introduced gene or its subunit or its equivalent and a bacterial promoter upstream of and adjacent to the site of entry, such that the inserted gene or its subunit or its equivalent is under bacterial promoter control.

It can be considered advantageous to insert multiple DNA fragments. It may also be desirable to use multiple bacterial promoters. In a preferred embodiment, at least one trp promoter is used. It is particularly advantageous to use a plasmid recombinant that lacks at least part of the DNA follow-up responsible for transcription dilution. A particular example of a preferred plasmid recombinant comprises a modified derivative of pWT 501. In certain cases an advantage can be gained from the fact that the introduced DNA also encodes an initiation codon and / or at least part of a ribasome binding site. .

The preparation of such a plasmid recombinant by a method in which the DNA is introduced at the insertion site of a suitable plasmid vector also forms an embodiment of the invention.

In a particular case, such a method may include: the isolation of a trp promoter containing approximately 150 bp Hind III fragment from pWT 501, * self-ligation; partial digestion with Taq Ij treatment with S1 nuclease; treatment with E. coli DNA polymerase I; restriction with Hind III; isolation of a trp promoter containing about 100 bp fragment; ligation to an approximately 700 bp fragment obtained by treatment of a recombinant plasmid of pWT 231 containing a gene or a subunit thereof or equivalent, with Sac I, S1 nuclease, E. coli DNA polymerase I and Hind III; restriction with Hind III; and ligation to 35 Hind III restrained bacterial basic phosphatase-treated pAT 153.

The invention further provides a cell which contains such a gene or a subunit thereof or an equivalent thereof or such a plasmid recombinant introduced therein.

In a preferred case, the cell is an E. coli K.-12 HB101 cell.

The preparation of such a cell by a method of introducing the DNA or the plasmid recombinant into a cell is also an embodiment of the invention.

In a further embodiment, the invention provides a method of preparing a protein having properties similar to that of human interferon, wherein such a cell is cultured and recovered expressed protein.

The invention is further elucidated with reference to the following.

Cl) Synthesis of the oligodexy nucleotides.

The six oligonucleotides CpApGpGpTpTpGpTpApGpCpTpCpApT CIFIA), CpApGpGpTpTpGpTpApApCpTpCpApT (IFIB), CpTpCpTpTpTpCpCpApTpG CIFII), CpApGpCpïpCpTpTpTpCpCpApTpG (IFIII), GpApGpGpApGpApTpTpApApG (IFIV) and CpCpTpGpGpApApGpApApA (IFV), were synthesized according to a conventional 20 triëstermethodologie cSee e.g. Hsiung, H.M., et al, (1979), Nucl. Acids Res. 6_, 1371-1355), and according to the process shown in FIG.

14 Reaction scheme, depicting N (3isobujγ, cbz or Abz. The fully protected oligonucleotides were unblocked with 2% (w / v) benzenesulfonic acid, 0.1 PI tetraethylammonium fluoride 25 in tetrahydrofuran (THF / pyridine / h ^ O (8 / 1/1 volume ratio), followed by treatment with ammonia The unblocked oligonucleotides were purified by high pressure liquid chromatography (HPLC) on "Partisil 10 SAX", microscopic size silica of which a derivative was made with quaternary ammonium groups (Whatman).

(2) Phosphorylation of the oligonucleotides.

Each synthetic oligonucleotide (120 pmol) was spiked with 200 32 pmol 5 '- C jf' - P) ATP (Radiochemical Center, Amersham, Buckinghamshire, England; specific activity greater than 5000 Ci / mmol) and 35 units polynucleotide kinase (EC 2.7. 1.78), (1 unit is the 32 amount which catalyzes the production of one nmol of acid insoluble β-8100558 5-23 after incubation at 37 ° C for 30 minutes according to Richardson, CC, (1972), Progress in Nucleic Acids Research , 2, 815), incubated in a total volume of 20 ydm, containing 50 mM tris-BC1 (tris- (hydroxymethyl) aminomethane hydrochloride), pH 9.0, 10 mil MgC1, 0.1 mM EDTA (ethylenediamine tetraacetic disodium salt) ), 0.1 mil spermidine and 1 mM dithiothreltol CDTT). After SO minutes at 37 ° C when most of the primer molecules were labeled 3, the mixture was either diluted to 200 ydm and adjusted to 1% (w / v) sodium dodecyl sulfate (SDS) and 10 mM EDTA, and since 10 15 20 25 3 little oligonucleotide was present, 5 µg / cm yeast tRNA, shaken with an equal volume of water saturated phenol and shaken again after addition of an equal volume of chloroform, centrifuged (10,000 x g 2 min) and the resulting aqueous phase extracted as above and extracted twice with an equal volume of chloroform and then concentrated by ethanol precipitation (adjusting the NaCl concentration to 200 mM, adding 2.5 volumes of absolute ethanol and leaving overnight at -20 ° C) , CIFIA and IB), or simply CIFII, III, IV and V) heated at 100 ° C for 5 minutes.

(3) Isolation of poly A - mRNA.

3 20 dm roller bottles from human fibroblasts (FS-4 (derived from human foreskin cells) or 17/1 (derived from human embryo lung cells)) were run with homologous interferon overnight (60 international reference units / cm) before to be superinduced for 5 hours with poly (I): poly (C) (30 '; yg / cm), and cycloheximide (12 µg / cm). Kwasi-induced cells did not receive poly (I): poly (C). Cells were removed from the glass surface by treating kart with a phosphate buffered saline (PBS) solution containing 1.25 mg / cm trypsin (EC 3.4.4.4), 0.5 mg 3 per cm EDTA, 2 µg / cm cycloheximide and collected in an ice-cold RPMI 1S40 (Searle modification) medium (Flow Laboratories) containing 2 µg / cm cycloheximide and 10% (v / v) calf scrum.

After washing the cell pellets (1000 x g, * 5 minutes) with R.P.M.I.

3 IS40, containing 2 µg / cm cycloheximide, the cells were lysed 8) 00558 24 by homogenization in 0.2 M tris-HCl, pH 9, 50 mM NaCl, 3 10 mM EDTA, 0.5% ( w / v) SOS, 1 mg / cm heparin and then de-proteinized with an equal volume amount of phenol equilibrated in the above buffer (without heparin), followed by 5 extractions with an equal volume amount of a buffered phenol / CHClg -mix (1 iv / v). After ethanol precipitation and dissolution, the RNA was selectively precipitated with 3 M NaCl and polyadenylated mRNA isolated by oligo (dT) cellulose chromatography (see, eg, Aviv, H., and Leder, P., (1972), Proc. Nat. Acad Sci, 10 USA, 69, 1408-1412).

(4) Reverse transcription of fibroblast mRNA using IFIA and IB.

32 75 pmol of 5 'C PJ ^ -labelled oligonucleotide (IFIA or IB) was incubated with 22.5 µg poly (A) containing fibroblast mRNA 15 for 1 hour at 25 ° C in 36 µm ^ 0.4 M KC1 to allow primer to hybridize to complementary follow-up in the mRNA.

This was then diluted with transcription mixture to obtain a final solution containing 0.5 mM each of dATP, dCTP, dGTP and dTTP, 50 mM tris-HCl, pH 8.4 mM

MgCl2, 60 mM KC1, 5 mM DTT, 0.01% (v / v) Triton X-100 (nonionic detergent, isoctylphenoxypolyethoxyethanol), 0.22 yM IFIA or IB, 3 ^ 3 50 yg / cm actinomycin D .67 yg / cm poly A mRNA, 20 units / om rat liver ribonuclease inhibitor> (1 unit is the amount that gives 2'5 a 40% inhibition of 5 mg pancreatic ribonuclease according to Shortman, K., (1961), Biochim Biophys Acta. 51, 37) (see, e.g., Gribnau, AAM, et al., 0.969), Arch. Biochem. Biophys., 130, 3 48-52), and 100 units / cm AMV (avian myeloblastosis virus) reverse transcriptase (1 unit is the amount that takes up 1 nmol 30 dTMP in an acid-precipitable product after incubation for 10 minutes at 37 ° C according to Houts, GE, et al. (1979), J. Virol., 29, 517). Incubation was continued for 60 minutes at 37 ° C, after which the mixture was extracted with phenol and chloroform and concentrated by ethanol precipitation (see 35 above (2)).

8100558 - 25 - (53 Analysis of the reverse transcription products obtained with IFIA and IB.

The ethanol precipitated products were dissolved in water and incubated in the presence of 0.1 H NeQH and 1 mM EDTA 5 for 30 minutes at 37 ° C. An equal volume of 10 M urea, 25% Cw / v3 sucrose, 0.05% (w / v3 of both xylene cyanol and bromophenol blue was then added and the mixture was heated to 90 ° C for 90 seconds before applying to a 12.5% Cw / v) polyacrylamide gel containing 7 M urea. Czie bv

IQ Maxam, A., and Gilbert, W., C1977), Proc. Wet. Acad. Sci. USA, 74, 550 - 5643. After electrophoresis, the gel was examined autoradiographically to determine the position of the labeled molecules.

The labeled product of about 150 nucleotides and produced only by mRNA from induced cells was eluted from the gel slice 15 and its follow-up determined C see e.g. Maxam and Gilbert, loc cit). The follow-up is shown in fig. 2.

(6) Preparation of cONA from fibroblast polyA mRNA.

cDNA was prepared by the following mixture for 2 hours

incubate at 37 ° C: 0.5 mM dCTP, dGTP, dTTP, 0.15 mM J ~ 3HJ

20 dATP (167 mCi / mmol3, 5 mM DTT, 50 mM tris-HCl, pH 8.10 mM MgCl 3, 3 z 3 0.01% Cv / v) Triton X-100, 50 µg / cm actinomycin D, 20 units / cm rat liver ribonuclease inhibitor, 50 mM KCl, 20 µg / cm3 oligoC CdT) ^ 2 µg, 60 µg / cm3 polyA mRNA and 100 units / cm3 AMV reverse transcriptase. After incubation, the mixture was extracted with phenol 25 and chloroform and ethanol precipitated C see top bottom (23 3.

The DNA was collected by centrifugation, dried and dissolved in 200 µm 0.3 N NaOH. After incubation for 1 hour at 50 ° C, it was neutralized and eluted as the excluded peak of a G50 CM3 Sephadex, particulate cross-linked modified dextranpo-30 polymer (Pharmacia], column C25 x 0.5 cm3 equilibrated in 50 mM

NaCl / 0.1% Cw / v3 SOS. The peak fractions were collected and concentrated by ethanol precipitation.

(7) Transcription of cDNA with oligonucleotides IFII, III, IV and V.

The 5 'C P3-labeled oligonucleotide C3 yM] was pre-incubated with full-stranded cDNA (440 µg / cm 3, derived from 8100558 26 total polyA mRNA, for 2 hours at 25 ° C in the presence of 0 ° C). 4 M KCl This was then incubated for 3 hours at 37 ° C in a final solution, which is now 0.01% (v / v) Triton-X-100, 50 mM Tris-HCl, pH 3.80 mil DTT, 10 mil magnesium acetate, 0.5mM each of dATP, dCTP, dGTP and dTTP, 80mM KC1, 1000 unit-3 32 den / cm AMV reverse transcriptase, 5'T P) -labelled oligonucleotide ( 0.12 yM) and single stranded cDNA (17.6 yg / cm]. After extraction with phenol and chloroform, the aqueous phase was passed through a G50 (M3 Sephadex column in 50 mM NaCl, 0.1% (w / v) SDS and the excluded fraction was precipitated with ethanol, dissolved and electrophoresed.

(83 Analysis of transcripts produced by IFII, III, IV and V.

The transcripts were either treated as above, ie for the transcripts produced by IFIA and IB, except using a 5% (w / v3 polyacrylamide gel in 7 M urea), or they were prepared and electrophoresed by native 1.4% (w / v3 agarose gels (see, e.g., Sharp, PA, et al., (19733, Biochemistry, 12, 3055 - 30633, which were then autoradiographed. The interferon genes were eluted from selected agarose gel discs by by stirring a 19G, then a 21G hypodermic needle (Gillette3 and the gel fragments for 3 hours at 25 ° C in 10 mM Hepes, N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulfonic acid (Ultrol3, pH 7 , 0.1 mM EDTA and 0.02% (v / v) Triton X-100 After centering (700-xg, 5 minutes) through a glass wool stopper, placed on a GF / C and a GF / B filter paper (Whatman), the filtrate was extracted with phenol and chloroform and then precipitated with ethanol.

The DNA was then monitored as noted above. (English: "sequenced".) 30 (93 Preparation of interferon genes' for cloning.

(a) Preparation of single stranded cDNA from tgtalAgolyA mRNA, geq / 3 cDNA was synthesized in a 20 cm incubation containing components described above under (6). After extraction with phenol 35 and chloroform, the water phase was chromatographed on a 8100558 - 27 -

Sephadex G50 CM) column C28 x 4 cm) from which the cONA was eluted in the excluded group in 50 mM NaCl, 0.1% Cw / v) SOS. The cDNA was precipitated with ethanol and then recovered, dissolved in 0.4 ml of 0.2 N NaOH and incubated at 50 ° C for 30 minutes before centrifugation through a basic sucrose gradient as described below.

(b) Zuiyering_yan_interferon_enkelstrengs_cDNA:

The above cDNA was divided into two equal aliquots and each was centrifuged at 36,000 rpm at 2 ° C for 24 hours in a Beckmann SW 41 rotor through a 5-20% (w / w) isokine 3.

sucrose gradient (C11 cm) containing 0.2 N NaOH, 100 mM NaCl, 10 mM

3 contained EDTA. 0.5 cm fractions were collected by upward displacement using 50% Cw / v) sucrose and corresponding fractions from both gradients were collected, neutralized and precipitated with absolute ethanol. The precipitates were collected by centrifugation, washed in absolute ethanol and then dissolved in 100 µm H 2 O. The fractions containing long interferon cONA transcripts were identified by analysis of the obtained products after initiating small amounts of each fraction with CPI (III) and reverse transcriptase as described above under C7) and CS). The major fractions leading to the synthesis of an interferon DNA molecule of about 700 nucleotides in length were collected in preparation for a large-scale synthesis of double-stranded interferon genes.

Cc) inhibition_of_the_self_to_start_bringing §

Before carrying out a large-scale preparation of double-stranded interferon genes, the self-triggering activity of the purified interferon single-stranded cONA C when incubated with reverse transcriptase) was reduced by incubating the cONA in 1 for 3 hours at 37 ° C. 5 cm ^, containing 150 mM sodium cacodylate, pH 7.2, 1 mM 2-mercaptoethanol, 1 mM CcCl2, 100 yg / cm ° gelatin, 20 yg / crrP cONA, 1.2 mM rATP and SCO units / cm ^ terminal transferase CEC 2.7.7.31), Cl unit is c: e amount which absorbs 1 mmole of dATP in an acid precipitable product in 1 hour at 37 ° C using dCpA) as a primer according to Bollum, Ri, et al., C1974J, flethods in Enzymology, 29, 70).

After extraction with phenol and chloroform as described above, the water phase was adjusted to 0.3 N NaOH and incubated at 50 ° C for 1 hour. The mixture was then neutralized and chromatographed on a Sephadex G50 Cmedium (column C20 x 2 cm) and the cDNA eluted in the excluded fraction in 50 mM NaCl, 0.1% (w / v) SDS, It was then precipitated with ethanol and dissolved again in HjO.

The result of this method is the addition of a single ΑΓΊΡ residue to the original 3'-hydroxyl endpoint of the cDNA. The alkali treatment also gives a mixture of 2 'and 3' phosphate residues to this endpoint that can no longer participate in a self-initiated reverse transcriptase reaction. Thus, when double-stranded genes are made using an oligonucleotide to initiate specific transcription on the single-stranded cDNA, the level of contaminating self-initiated products is reduced and the remaining single-stranded cDNA can subsequently be processed by S1 nuclease CED 3.1.4.21 ) are reduced.

The self-initiating activity can also be severely inhibited by preincubation of the cDNA in appropriate buffer mixtures with either reverse transcriptase or endpoint transferase in the presence of the four dideoxynucleoside triphosphates. Alternatively, the cDNA can be incubated with endpoint transferase and deoxy-adenosine triphosphate, thereby obtaining a polyCdA tail at the 3'-hydroxyl endpoint which also effectively inhibits self-triggering activity.

Cd) §ynthesis_of_double-stranded_interfergn; genes_under_application of_IFIII as primer:

Interferon single-stranded cDNA was purified by centrifugation through a basic sucrose gradient and its self-triggering activity was reduced as described above, 10 µg of this material was then prepared at 25 ° C for 2 hours 8 1 0 0 5 5 8 3 - 29 -

incubated in 0.36 cm, containing 0.4 µl K.C1 and 575 prnol of IFIII

previously incubated with polynucleotide kinase CEC 2.7.1.78] 32 and er- "P) ATP, also as described above. Double-stranded genes were then produced by continuing for 3 hours Λ

Incubate at 37 ° C in 1.8 cm, which is now 0.01% Cv / v] Triton X-1CD

50 mil tris-HCl, pH 8, 20 mil DDT, 10 mM magnesium acetate, 0.5 mM

3 dATP, dCTP, dGiP and dTTP, 1000 units / cm reverse transcriptase, 80 mil KCl, 11.1 µg / cm3 cDNA and 0.32 µM C32P] IFIII. After extraction with phenol and chloroform, the genes were eluted from a Sephadex G50 [medium] column C20 x 2 cm] in 50 mil NaCl, 0.1%

Cw / v] SDS. Yeast tRNA was added as carrier C3 µg / cm] and the NaCl concentration adjusted to 0.2 M before precipitating with 2.5 volumes of absolute ethanol. After standing at -70 ° C for 30 minutes, the precipitate was collected by centrifugation and the dried pellet redissolved in 100 µm H 2 O.

Ce] Iyiyering_van_dubbelstrangs_interferon; genes:

The DNA was sedimented through a 5-20% Cw / w] linear sucrose gradient containing 10 mil tris-HCl, pH 7.5. 0.8 M NaCl, 8 mil EDTA

3 contained. A 5.2 cm gradient was centrifuged in a Beckmann 20 SW S5 rotor at 42,000 rpm for 18 hours at 2 ° C, after which 3 0.2 cm fractions were collected and precipitated with ethanol. The DNA was collected by centrifugation, redissolved and then small amounts were electrophoresed through a 5% Cw / v] polyacrylamide gel containing 7M urea, which was then examined by autoradiography. The fractions showing the 700 nucleotide interferon DNA molecule were collected. Cf] Preparation_of_interferon genes_that_Hind_III_links_on_de

The above collected gradient fractions were first treated with S1 nuclease by incubation for 30 minutes at 37 ° C in 50 µm containing 0.1 mM ZnSCL, 150 mM NaCl, 25 mM sodium acetate, pH 4 , S, and 120 units / cm S1 nuclease Cl unit is the amount of 10 µg nucleic acid dissolved at 45 ° C in 10 minutes according to Vogt. V.M., C1973], Eur. J. Siochem., 33, 192].

3 3

After addition of 0.5 µm 10% Cw / v] SDS, 1 µm 0.5 M EDTA and 1 µm0 0.5 M tris-HCl, pH 8.3, the mixture was extracted with 8100558-30-phenol and chloroform and the aqueous phase was precipitated with ethanol.

At this stage, there was a total amount of 0.24 µg double-stranded DNA.

In order to ensure that blunt ends were present for the subsequent ligation with Hind III switch molecules.

recovered the DNA from ethanol and collected at 14 ° C for 20 minutes

incubated in 50 ydm containing 50 mM tris-HCl, pH 7.8, 10 mM

MgCl-, 1 mM 2-mercaptoethanol, 0.2 mM dATP, dCTP, dGTP and dTTP 3 and 40 units / cm of E. coli DNA polymerase I (EC 2.7.7.73, (1 ββητ 10 is the amount 10 nmol total nucleotides in an acid-precipitable fraction over 30 minutes at 37 ° C using poly-d (AT) as a primer according to Richardson, CC, et al., (19643, J. Biol. Chem., 239, 2223 The DNA was then extracted in the usual manner, precipitated with ethanol and then the pellet obtained by centrifugation dried.

2.5 yg Hind III link (Collaborative Research3 was phosphorylated by first incubating for 30 minutes at 37 ° C in 3 50 ydm, containing 50 mM tris-HCl, pH 7.8, 10 mM MgC 2, 10 mM 2- mercaptoethanol, 0.25 mM Cl 2 PJATP (20 mCi / ymol 3, 50 µg / cm 3 BSA 20 (bovine serum albumin 3 and 100 units / cm 3 polynucleotide kinase.

In order to ensure complete phosphorylation, the mixture was incubated for an additional 30 minutes at 37 ° C after the addition of 10 µm containing 50 mM tris-HCl, pH 7.6, 10 mM MgCl 3, 3.75 mM rATP and 500 units / cm3 polynucleotide kinase. The mixture was then extracted with phenol and chloroform and precipitated with ethanol after the addition of yeast tRNA to 10 µg / cm.

Ligation of the phosphorylated Hind III links to the interferan genes was accomplished by dissolving the interferon gene pellet at 3 in 40 ydm containing 50 mM tris-HCl, pH 7.8, 10 mM, 30 MgCl_, 1 mM rATP .20 mM DTT, 20 µg / cm3 phosphorylated link and Δ 3 about 480 units / cm of T4 DNA ligase (EC 6.5.1.13, (1 unit is the amount that converts 1 nmol PP. Into («< // 3 ~ P3-ATP catalyzed in 20 minutes at 37 ° C according to Weiss, B., et al., (19683, J. Biol, Chem., 243, 45433. This was then continued for 16 hours at 25 ° C. ° C incubated.

8 1 0 0 55 8 - 31 -

These conditions gave an approximately 100-fold molar excess of link to DNA.

After heating the mixture for 5 minutes at 65 ° C

the DNA was restrained with Hind III by adding 3 3

gene 600 µm of a mixture containing 400 units / cm Hind III CEC

3.1.23.213. Cl unit is the amount of 1 µg-AA digested in 50 µm 3 at 37 ° C in 15 minutes], 3 mM DTT, 167 µg / cm 3 gelatin, 83 mM tris-HCl, pH 7, 5.83 mil NaCl, 17 mM MgCl 2 and 8 mM

2-mercaptoethanol. After incubation for 2 hours at 37 ° C, the mixture was brought to 0.2% Cw / v3 SDS and 20 mM EDTA and the DNA was extracted with phenol and chloroform and yeast tRNA was added 3 to the final aqueous phase to a concentration of 5 yg / cm. This was then chromatographed on a Sephadex G150 superfine column C50 x 0.7 cm3 where the double stranded genes were eluted in the excluded fraction using 50 mM NaCl, 0.1% Cw / v3 SDS and 5 µg. / cm yeast tRNA. The latter was then precipitated with ethanol after the 3 tRNA concentrations were adjusted to 10 µg / cm. The recovered DNA was estimated to contain approximately 0.125 µg double-stranded genes.

Cg3 §rgn genes:

The DNA was then dissolved in 50 ydm H 2 O and 20 ydm was electrophoresed by a native 1.4% Cw / v3 agarose gel C20 x 14 x 0.5 cm 3 for 2 hours at 120 V. DNA between 600 and 1000 bp in length was then eluted from the gel in the following manner: the appropriate slice was excised and passed through a Gillette 21 G needle. The latter was washed with 4 cm3 of 10 mM Hepes, pH 7.5, 0.1 mM EDTA, 0.02% Cv / v3 Triton X-100, 65 µg / cm3 yeast tRNA and the disc frozen at -70 ° C.

It was then thawed and agitated on a rotary mixer overnight at room temperature, converge at 25 ° C3 before filtering through a Whatman 52 paper. The filtrate was adjusted to 0.1 M ammonium acetate, 2 mM magnesium acetate, 0.02% 3

Cw / v] SDS and bound to a 1 cm column of disthylaminoethyl CDEAE3 52 cellulose. This was then washed three times with 1.5 'cnT3 aliquots of the above buffer before the DNA was 8100558 3 - 32 - eluted with 0.5 cm aliquots of 1.1 M NaCl, 0.1 M ammonium acetate, 2 mM magnesia acetate, 0.02% (w / v) SOS, 0.02 mM EOTA. The DNA eluted with the 2nd and 3rd aliquots which were then collected, supplemented with 5 µg yeast tRNA and then precipitated with ethanol. The precipitate was collected by centrifugation, washed 3 with absolute ethanol, dried and dissolved in 20 µm water. 4 µm was taken for liquid scintillation counting and the residue (about 8 mg) dried by vacuum desiccation.

(10) Clones of the interferon genes in X1776 using 10 pBR 322 as a plasmid vector.

The vector was prepared as follows: 10 µg of the plasmid pBR 322 was restrained with Hind III, extracted with phenol and chloroform and precipitated with ethanol. The recovered linear plasmid was then incubated in 25 udm containing 20 mM tris-HCl, pH 7.5, 0.1% (w / v) 3 SOS and 0.7 mg for 30 minutes at 65 ° C. / cm bacterial basic phosphatase (EC 3.1.3.1), before extracting three times with phenol and chloroform, twice with ether and reprecipitating with ethanol. The recovered precipitate was then dissolved in 100 µm water.

20, The dried gene pellet (see above (9Xg)) was then dissolved in 10 µm containing 50 mM. tris-HCl, pH 7.8, 10 mM MgCl2, 1 mM rATP, 20 mM DTT, 10 µg / cm3 of the treated 2 vector DNA and about 16 units / cm of T4 DNA ligase. This mixture was then incubated overnight at 15 ° C for 3

25 which was diluted to 100 µm with 10 mM tris-HCl, pH 7.5, 1 mM

EDTA, 0.1 M NaCl and was used to transform E. coli K.-12 X1777 according to known methods (see, e.g., Curtiss, R., III, et al., (1976), Recombinant Molecules: Impact-on Science and Society (RFBeers, Jr., and EGBassett (Eds.), 45-56). In this way, a total of 370 ampicillin (100 µg / cm) -resistant transformants were obtained, which were then sensitive to 10 yg / cm3 tetracycline were tested.62% were found to be tetracycline sensitive and were therefore identified as recombinants. 160 of the latter were then grown on millipore filters, placed on culture plates to identify interferon gene recombinants by Colony hybridization with (8100558 5 32 32). f ~ P3IFIA and CT - ^ P) IFIV.

C113 Research of recombinants.

I.Q

15 20 25 30

Recombinants were grown on nitrocellulose filters (9 cm diameter) until the colonies reached 2 or 3 mm in diameter. After the filters were lifted from the plates, they were placed on a Whatman No.1 filter paper pad soaked in 0.5 N NaOH for 7 minutes, then on a pad soaked in 1 M tris-B £ for 2 minutes. 1, pH 7.5, and then on a pad, soaked in 0.5 Niris-HCl, pH 7.5, 1.5 M NaCl for 7 minutes. The filters were then dried by placing them on a vacuum manifold for about 5 minutes and finally washing with 100 cm absolute ethanol before heating at 80-85 ° C for 2 hours.

The filters were pre-incubated in 3 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.63, containing 0.02% Cw / v) BSA, 0.02% at 25 ° C for 2 hours. Cw / v) Ficoll copolymer of sucrose and epichlorohydrin) and 0.02% (w / v) polyvinylpyrrolidone before liquid was discharged and they were placed on a filter paper to air dry for several minutes. The oligonucleotides IFIA and IFIV were radiolabelled with if-P) ATP and polynucleotide kinase as described above and then suspended in three times SSC. 300 µm C-containing 0.85 pmol of each ice (- P) oligonucleotide Convex 1 µCi / pmol) was uniformly applied over each filter (standing on a non-absorbent surface) and left for 5 minutes to settle. weeks. The filters were immersed in light paraffin and incubated at 25 ° C for 2 or 3 days. They were then de-oiled, rinsed in chloroform, air-dried and washed four times in 3 x SSC at 25 ° C (20 minutes per wash) before washing twice in 2 x SSC at the same temperature. After gently drying with blotting paper, the filters were enclosed in a polyethylene bag and examined by radiographic examination at -70 ° C in a Kodak normal booster cassette.

The autoradiogram showed the presence of five colonies 8 100 558 34 - which were significantly darker than the rest. Plasmid DNA was prepared from these by known methods. See e.g. Birnboim, H.C., and Doly, J., C19793, Nucl. Acids Res., 711, 1513-1523] and subsequent restriction and nucleotide follow-up analysis confirmed that each of these recombinants contained one fibroblast interferon gene. , which encoded the mature interferon polypeptide and the 3 'untranslated mRNA follow-up.

C12) Transfer of the interferon gene into pWT 2x1 expression plasmids, Plasmid DNA was prepared from one of the above interferon recambinants by centrifugation of a clarified lysate through density gradients containing ethidium bromide C see e.g. Katz, L., et al., C1973), J. Bacteriol. 114, 577 - 591, and Wensink, P.C., et al., C1974], Cell, 3.315-325]. 3 µg was then restrained with Hind III and the extracted DNA was electrophoresed through a native 5% Cw / v] polyacrylamide gel. The interferon gene Congeer Feather 730 bp] was visualized 3 by staining with 1 µg / cm ethidium bromide and viewing under UV light C254 nm], allowing excision of a small gel slice containing the gene. This slice was dispersed 3 through the opening of a 1 cm plastic syringe, which was then washed with 4 volumes by volume, relative to the gel slice, AGEB C0r5 N ammonium acetate, 10 mM magnesium acetate, 0.1% Cw / v] SDS and 0.1 mM'EDTA), which was then mixed with gel suspension and gently shaken at 37 ° C for 16 hours. Yeast tRNA was added to a final concentration of 25 µg / cm. before the mixture was centrifuged through a Whatman 52 paper in a 2 ml syringe and then the paper was washed with AGEB. The collected filtrate was then diluted with H 2 O 5 times before chromatography followed on a 1 cm column of DEAE 52-clululose as described above under C9] Cg]. The eluate C1 cm total] 3 was extracted twice with 1 cm isoamyl alcohol, saturated with 1/5 x AGEB containing 1.1 H NaCl Com to remove the ethidium bromide] before precipitating with ethanol after the addition of 8100558 3 - 35 5 yg yeast tRNA. The DNA was collected and dissolved in 50 ydm H 2 O (about 0.2 yg DNA).

Subsequently, 4 ydm 2 was ligated (in a final volume of 10 ydm 2 1 with 80 mg of pWT 211, pWT 221 or pWT 231, which had been pre-restrained with Hind III and then treated with basic phosphatase to ligate the parent plasmid. (see above (10)) to a minimum, each ligation was then used to transform E. coli K-12 HB101 using known methods (see, e.g., Emtage, JS, et al., (1980), Nature 283, 171-174) Transformants were grown on L-agar plates containing 100 µg / cm ampicillin (in fact, carbenicillin sodium (Beecham), which is a derivative which can be considered an equivalent, was used; carbenicillin is oC-carboxybenzylpenicillin) and plasmid DNA was prepared from 6-12 colonies in each case By limiting Pst I (EC 3.1.23.31) and analyzing the products by gel electrophoresis, interferon recombinants could be identified as well as the orientation of the inte rferon gene relative to the tryptophan promoter sequence. For each of the three pWT 2x1 plasmids, a recombinant containing the interferon gene in the required orientation for expression was selected and tested for its ability to produce biologically active interferon.

(13) Induction of expression plasmids containing the interferon gene.

3 150 cm cultures of each of the three pWT 2x1 interferon gene clones were grown in L broth (luria broth: 1% (w / v).

bactotrypton, 0.5% (w / v) bactogist extract, 0.5% (w / v) NaCl), 0.2% (w / v) glucose, 0.004% (w / v) thymine, pH 7) ampicillin to a 0.D. ", -" of about 0.3. The bacteria were peeled-aOQ nm tar by centrifugation, washed and then resuspended in induction medium (42 mfl ^ ^ HPO ^, 22 mN KH ^ PO ^, 6.6 mH NaCl, 18.7 mH NH Cl, 0.1 mM CaCl2, 1.0 mN NgSQ ^, 1 yg / cm ^ vitamin B1, 0.2% (w / v) glucose, 0.5% (w / v) casamino acids missing tryptophan (Qifoo), 20 yg / crrf5 3/3- indole acrylic acid and 100 µg / cm 13 ampicillin). After incubation for 4 hours at 37 ° C (at which time the 0.D.flnn approximately 8100558 5 36-10 15 20 25 30 30 was 1.0), the cells were centrifuged and extracted as described below. (14) Extraction of interferon from bacteria. The following operations were performed at 2 ° C: the bacterial pellets were resuspended in 1.2 cm PBS containing 2% (w / v) HSA (human serum albumin). An equal volume amount of 50% (w / v) sucrose, containing 0.1 M tris-HCl, pH 8, and 1% (w / v) 3 HSA was then added, followed by 0.8 cm of a fresh 3 3 lysozyme solution (10 mg / cm in PBS). After 15 minutes, 0.8 cm varv 0.5 M EDTA, pH 8.5, was added and the cells were left for an additional 10 minutes. 4 cm 0.6% (v / v) Triton X-100 was then added and after mixing for 10 minutes, the extract was exposed to sound waves (English: "sonicated") to decrease the viscosity and ensure complete lysis, then centrifuged at 50,000 rpm in a Beckman-Ti 50 rotor for 2 hours. The supernatant was dialyzed against PBS, cleared by centrifugation (10 min at 10,000 rpm) before being analyzed for interferon activity by following the protection given to Vero cells against it. cpe of EMC virus in an in vitro microplate analysis system (see, e.g., Dahl, H., and Degre, M., (1972), Acta. Path. Microbiol. Scan., 1380, 863). An alternative extraction method involves simply exposing the induced bacteria in 10 mM tris-HCl, pH 8.0 to sound vibrations, followed by centrifugation at 15,000 x g for 30 minutes and collecting the supernatant. (15) Construction of modified expression recombinants. The plasmid pWT 501 contains a 150 bp Hind III fragment containing the trp promoter and part of the gene sequence encoding the trp leader polypeptide. This fragment was recovered from a native 5% (w / v) polyacrylamide gel and self-ligated using T4 DNA ligase (10). The cancemers were then partially digested with Taq I before first treating with S1 nuclease, then E. coli DNA polymerase I and finally Hind III (9) (f). In this way, the required Taq I cleavage 81 0 0 5 5 8 35 - 37 - within the sequence corresponding to the ribosome binding site in the mRNA region encoding the trp leader polypeptide, results in a fragment of approximately 100 bp after the Hind III restriction. This fragment was isolated from an 8% Cw / v) native polyacrylamide gel C12) in preparation for ligation to the Sac I / Hind III fragment of the interferon gene. The latter fragment was obtained by first unlocking the interferon gene cloned in pWT 231 with Sac I. After treatment with S1 nuclease, then with E. coli DNA polymerase I, the DNA was restrained with Hind III and the resulting approximately 700 bp fragment recovered from a 5% Cw / v) native polyacrylamide gel, 10

The two fragments were then ligated together by incubating at 25 ° C for 16 hours in a final volume of 20 ydm, containing 50 mfl tris-HCl, pH 7.8, 10 mfl NgCl, 15 1 mM ATP, 20 mM DTT, approximately 480 units / om3 T4 DNA ligase, 3 3 0.5 µg / cm of the Hind III / Taq I trp fragment and 5 µg / cm of the 20

Sac I / Hind III fragment of the interferon gene. After restriction with Hind III, the pooled molecule was transformed into E.coli K-12 HB101 using pAT 153 as the vector. The latter had been previously restrained with Hind III for treatment with bacterial basic phosphatase (CEO 3.1.3.1) to decrease the level of non-recombinant transformants (CIO). Plasmid DNA was prepared from transformants according to known methods. Csee e.g. Katz, et al., And Wensink, et al., Loc cit), after which this was analyzed by restriction enzyme classification and also by nucleotide monitoring. In this manner, various recombinants containing the required pooled molecule were identified. These were then induced to produce interferon as described above C13) with the proviso that the cc

O

The tration of 3/3 indole acrylic acid was adjusted to 2.5 µg / cm.

To prepare radioactive interferon, bacteria were induced as described, but in the absence of casamino acids and 3 in the presence of 5 µg / cm 3,3-indoalacrylic acid. At the end of the 3 induction period, 1 cm aliquots were incubated for another 35 minutes with 5 yCi of a "C) -amic acid mixture. The 8100558-38 bacteria were then centrifuged and the pellet lysed by adding 50 µm of a mixture of 10% (v / v) glycerol, 5% Cw / v) 2-mercaptoethanol, 3% Cw / v) SDS, 62.5 mM tris-HCl, pH 6.8, 0.01% Cw / v) bromophenol blue and heating at 90 ° C for 2 minutes. Samples were then electrophoresed by 12.5% Cw / v) polyacrylamide gels by known methods C see e.g. Laemmli, U.K., C1970), Nature, 227, 680 - 685). The gel was then dried and autoradiographed to reveal the interferon polypeptide (see Figure 12).

10 8100558

Claims (43)

A gene for the expression of a protein having properties similar to that of human interferon, comprising a coding strand and a complementary strand, the coding strand containing the following sequence: 5'GGC. CAT. ACC. CAT. GGA. GAA. AGG. ACA. TTC. TAA. CTG. CM. CCT. -TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT.-GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.-GCT.CTC. CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.-TAC.AAC.TTG. CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG.-TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC .-TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC.CCT.GAG.GAG.ATT.-AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG.ACC.-ATC.TAT.GAG.ATG.CTC.CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.- GAT. TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.GAG.AAC.-CTC.CTG.GCT.AAT.GTC.TAT. CAT.CAG. ATA.AAC. CAT.CTG.AAG.ACA.-GTC.CTG.GAA.GAA.AAA.CTG, GAG.AAA.GAA.GAT.TTC.ACC.AGG.GGA.-AAA.CTC.ATG.AGC.AGT.CTG. CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.-ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.GAG.TAC.AGT.CAC.TGT.GCC.-TGG.ACC, ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC .TTC.-ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.-AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT .GCT.TCA.AGC.ATT.CTT.-CAA.CCA.GCA.GAT.GCT.GTT.TAA.GTG.ACT.GAT.GGC.TAA.TGT.ACT.- GCA. TAT.GAA.AGG.ACA.CTA.GAA.GAT.TTT.GAA.ATT.TTT.ATT.AAA.- TTA.TGA.GTT.ATT.TTT.ATT.TAT.TTA.AAT.TTT.ATT.TTG , GAA.AAT.-AAA.TTA.TTT.TTG.GTG.CAA.AAG.TCA.ACA.TGG.CA 3 'or a subunit thereof or equivalent. The gene of claim 1, the encoding string of which contains the following sequence: 5'GGC.CAT.ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.CCT.-TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.GAC.ACT.-GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.-GCT.CTC. CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.- 8 1 0 0 55 8 TAC. AAC. TTG. CTT .GGA.TTC. CTA. CAA. AGA. AGC. AGC. AAT. TTT .CAG.-TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.-TGC.CTC.AAG.GAC.AGG.ATG.AAC.TTT. GAC.ATC.CCT. GAG. GAG.ATT.-AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG. GAG.GAC.GCC. GCA.TTG. ACC. -ATC. TAT. GAG.ATG.CTC.CAG.AAC.ATC.TTT, GCT. ATT, TTC.AGA.CAA. - HOLE. TCA.TCT.AGC.ACT.GGC.TGG, AAT.GAG.ACT.ATT.GTT.GAG.AAC.-CTC.CTG.GCT.AAT.GTC. TAT. CAT.CAG.ATA.AAC. CAT.CTG.AAG.ACA.-GTC.CTG.GAA.GAA.AAA.CTG. GAG.AAA.GAA. GAT.TTC.ACC.AGG.GGA.-AAA.CTC.ATG.AGC, AGT.CTG.CAC.CTG.AAA.AGA. TAT.TAT.GGG.AGG. -ATT.CTG.CAT.TAC.CTG.AAG.GCC.AAG. GAG.TAC.AGT.CAC.TGT.GCC. -TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.TTC.-ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC. TGA.AGA.TCT.CCT.-AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT.GCT.TCA.AGC.ATT.CTT.-CAA. CCA. GCA.GAT.GCT.GTT. TAA.GTG. ACT. GAT.GGC.TAA.TGT.ACT.- GCA. TAT.GAA.AGG.ACA.CTA.GAA. GAT.TTT.GAA.ATT.TTT.ATT.AAA.-TTA.TGA.GTT.ATT.TTT.ATT.TAT.TTA.AAT.TTT.ATT.TTG.GAA.AAT.-AAA.TTA.TTT. TTG.GTG.CAA.AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.-CGA.TTT. CAT.TTA, TAT.AAC.CA ... 3 "or a subunit or equivalent. Gene according to claim 1 or 2, the coding strand of which comprises the following sequence: 5'GGC. CAT.ACC. CAT.GGA.GAA.AGG.ACA.TTC.TAA.CTG.CAA.-CCT.TTC.GAA.GCC.TTT.GCT.CTG. GCA.CAA.CAG.GTA.GTA.GGC.-GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.ACC.AAC.AAG.TGT.CTC.-CTC.CAA.ATT.GCT.CTC. CTG.TTG.TGC.TTC.TCC.ACT.ACA.GCT.-CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.-AGC.AGC.AAT. TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA.TTG.-AAT.GGG.AGG.CTT.GAA.TAT.TGC.CTC.AAG.GAC.AGG.ATG.AAC.-TTT. GAC.ATC.CCT.GAG.GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.-CAG.AAG.GAG.GAC.GCC.GCA.TTG.ACC.ATC.TAT.GAG, ATG.CTC .-CAG.AAC.ATC.TTT.GCT.ATT.TTC.AGA.CAA.GAT.TCA.TCT.AGC.-ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.GAG.AAC.CTC .CTG.GCT.-AAT.GTC.TAT. CAT.CAG.ATA.AAC. CAT.CTG, AAG.ACA.GTC.CTG, -GAA.GAA. AAA.CTG. GAG. AAA.GAA.GAT.TTC.ACC.AGG.GGA.AAA.-CTC.ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA.TAT.TAT.GGG.AGG.- 8100558 - 41 ATT.CTG . CAT.TAC.CTG.AAG, GCC.AAG.GAG.TAC.AGT.CAC.TGT.-GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.- TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC.CTC.CGA.AAC.TGA.-AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.GAC.TGG.ACA.ATT .GCT.-TCA.AGC.ATT.CTT.CAA.CCA.GCA.GAT.GCT.GTT.TAA.GTG.ACT.-GAT.GGC.TAA.TGT.ACT. GCA.TAT.GAA.AGG.ACA.CTA.GAA.GAT.-TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.GTT.ATT.TTT.ATT.TAT.-TTA.AAT.TTT. ATT.TTG.GAA.AAT. AAA.TTA.TTT.TTG.GTG.CAA.-AAG.TCA.ACA.TGG.CAG.TTT.TAA.TTT.CGA.TTT.GAT.TTA.TAT.- AAC.CA..3 'or a subunit thereof. The gene according to any of claims 1 to 3, the encoding strand of which comprises the following sequence: 5 'TTC.TAA.CTG.CAA.CCT.TTC.GAA.GCC.TTT.GCT.CTG.GCA.-CAA .CAG.GTA.GTA.GGC.GAC.ACT.GTT.CGT.GTT.GTC.AAC.ATG.-ACC.AAC.AAG.TGT.CTC.CTC.CAA.ATT.GCT.CTC.CTG.TTG. TGC.-TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.CTT.-GGA.TTC.CTA.CAA.AGA.AGC.AGC.AAT.TTT.CAG. TGT.CAG.AAG.-CTC.CTG.TGG.CAA.TTG.AAT.GGG.AGG.CTT.GAA.TAC.TGC.CTC.-AAG.GAC.AGG.ATG.AAC.TTT.GAC.ATC. CCT.GAG.GAG.ATT.AAG.-CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC.GCA.TTG.ACC.-ATC.TAT.GAG.ATG.CTC.CAG. AAC.ATC.TTT.GCT.ATT.TTC.AGA.-CAA.GAT.TCA.TCT.AGC.ACT.GGC.TGG.AAT.GAG.ACT.ATT.GTT.-GAG.AAC.CTC.CTG. GCT.AAT.GTC.TAT.CAT.CAG.ATA.AAC. CAT.-CTG.AAG.ACA.GTC.CTG.GAA.GAA.AAA.CTG.GAG.AAA.GAA.GAT.-TTC.ACC.AGG, GGA.AAA.CTC.ATG.AGC.AGT.CTG. CAC.CTG.AAA.-AGA.TAT.TAT.GGG.AGG.ATT.CTG. CAT.TAC.CTG.AAG.GCC.AAG.-GAG.TAC.AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.GAA.-ATC.CTA.AGG.AAC.TTT. TAC.TTC.ATT.AAC.AGA.CTT.ACA.GGT.-TAC.CTC.CGA.AAC.TGA.AGA.TCT.CCT.AGC.CTG.TGC.CTC.TGG.-GAC.TGG.ACA. ATT.GCT.TCA.AGC.ATT.CTT.CAA.CCA.GCA.GAT.-GCT.GTT.TAA.GTG.ACT.GAT.GGC.TAA.TGT.ACT.GCA.TAT.GAA.-AGG. ACA.CTA.GAA.GAT.TTT.GAA.ATT.TTT.ATT.AAA.TTA.TGA.-GTT «ATi,! Ίi.A ii.ιAT.TTA.AAT.1iT.Aii, iTG.GAA.AAi .AAA.-TTA.TTT.liG.GiG.CAA.AAG.TC, .3 'or equivalent. 8100558 42 - 5 5. AT G.AGC.TAC.AAC.TTG.CTT.GGA.TTC.CTA.CAA.AGA.AGC.-AGC.AAT.TTT.CAG.TGT.CAG.AAG.CTC.CTG.TGG.CAA. TTG.MT.-GGG. AGG. CTT. GM. TAC; TGC. CTC. AAG. GAC. AGG. ATG. AAC, TTT. -GAC.ATC.CCT.GAG.GAG.ATT.AAG.CAG.CTG.CAG.CAG.TTC.CAG.-MG. GAG. GAC. GCC. GCA, TTG. ACC. ATC. TAT. GAG. ATG. CTC .CAG.-AAC. ATC.TTT. GCT. ATT.TTC. AGA. CM. GAT.TCA.TCT. AGC, ACT.-GGC.TGG.AAT.GAG.ACT.ATT.GTT.GAG.AAC.CTC.CTG.GCT.AAT.-GTC. TAT. CAT. CAG. ATA. AAC. CAT. CTG. AAG. ACA, GTC. CTG. GM. -GAA. MA. CTG. GAG. AAA. GAA. HOLE. TTC. ACC. AGG. GGA. AAA. CTC.-ATG.AGC.AGT.CTG.CAC.CTG.AAA.AGA. TAT.TAT.GGG.AGG.ATT.-CTG. CAT1 TAC. CTG. AAG. GCC. AAG. GAG. TAC. AGT. CAC .TGT.GCC.-TGG.ACC.ATA.TGC.AGA.GTG.GAA.ATC.CTA.AGG.AAC.TTT.TAC.-TTC.ATT.AAC.AGA.CTT.ACA.GGT.TAC. CTC.CGA.AAC.TGA. 3 "or equivalent. 5. ATG. ACC.MC, AAG.TGT. CTC.CTC. CAA. ATT.GCT. CTC.CTG.TTG.-TGC.TTC.TCC.ACT.ACA.GCT.CTT.TCC.ATG.AGC.TAC.AAC.TTG.-CTT.GGA.TTC.CTA.CAA.AGA.AGC.AGC. AAT.TTT.CAG.TGT.CAG.-MG. CTC. CTG. TGG. CAA. TTG. AAT. GGG. AGG. CTT. GAA. TAC. TGC. -CTC. AAG. GAC. AGG. ATG. MC. TTT. GA C. ATC. CCT. GAG. GAG. ATT. -: 10 15 20 25 30 AAG.CAG.CTG.CAG.CAG.TTC.CAG.AAG.GAG.GAC.GCC.GCA.TTG.-ACC.ATC.TAT.GAG.ATG.CTC.CAG.AAC. ATC.TTT.GCT.ATT.TTC.-AGA. CAA. HOLE .TCA.TCT. AGC. ACT. GGC. TGG. MT. GAG. ACT .. ATT. · -GTT. GAG.AAC.CTC.CTG.GCT.AAT.GTC.TAT. CAT.CAG.ATA.AAC.-CAT. CTG. MG. ACA. GTC. CTG. GAA. GM. AAA. CTG. GAG.AAA, GAA. -GAT.TTC.ACC.AGG.GGA.AAA.CTC.ATG.AGC.AGT.CTG.CAC.CTG.-AAA.AGA.TAT. TAT.GGG.AGG, ATT.CTG. CAT.TAC.CTG.AAG.GCC.-AAG.GAG.TAC.AGT.CAC.TGT.GCC.TGG.ACC.ATA.GTC.AGA.GTG.-GAA.ATC.CIA.AGG.AAC.TTT. TAC.TTC.ATT.AAC.AGA.CTT.ACA.-GGT.TAC.CTC.CGA.AAC.TGA ... 3 'or equivalent. Gene according to one or more of claims 1 to 3, the encoding strand of which comprises the following sequence: Gene according to one or more of claims 1-3, the coding strand of which comprises the following sequence: Subunit of a gene according to one or more of claims 8100558 35 - 43 - siss 1-6. The gene or equivalent thereof or a subunit thereof, according to any of claims 1 to 7, essentially as described herein. A method for the preparation of a gene or an equivalent thereof or a subunit thereof, according to any of claims 4-8, wherein polyA mRNA is isolated from induced fibroblast cells, single-stranded cDNA is synthesized using oligo dT primer and reverse transcriptase and double-stranded DNA is synthesized therefrom using reverse transcriptase or E. coli DNA polymerase I and optionally a primer. A method for the preparation of a gene or an equivalent thereof or a subunit thereof, according to any of claims 4-8, wherein mRNA is isolated from induced fibroblast cells, single-stranded cDNA is synthesized using a specific primer and reverse transcriptase and double-stranded DNA is synthesized therefrom using reverse transcriptase or E. coli DNA polymerase I, and optionally a primer. The method of claim 9 or 10, wherein a primer is used in the third step. A method according to any of claims 9-11, wherein the reaction mixture is fractionated after the second stage and / or the third stage. The method of any of claims 9-12, wherein self-initiation after the second stage is inhibited. The method of any of claims 9-13, wherein the post-cloning product is identified by using a primer to examine the colonies. A method according to a Gf more of claims 9-14 for the preparation of a gene or an equivalent thereof or a subunit thereof, according to one or more of claims 4-8, essentially as described herein. A gene or an equivalent thereof or a subunit thereof according to one or more of claims 4-8, prepared by a method according to one or more of the following claims: 8100558 • 5 - 44 - • 5 - 44 - 10 15 20 25 30 claims 9-15. A method for the preparation of a gene or an equivalent thereof or a subunit thereof according to one or more of claims 1-3, wherein a molecule is used as probe with a sequence of a gene or an equivalent thereof or a subunit thereof according to any of claims 1 to 8 or 16 for isolating a human chromosomal interferon gene from human chromosomal DNA. A method according to claim 17 for the preparation of a gene or an equivalent thereof or a subunit thereof, according to one or more of claims 1 to 3, essentially as described herein. A gene or equivalent thereof or a subunit thereof as claimed in one or more of claims 1 to 3, prepared by a method according to claim 17 or 18. A method for the preparation of a gene or an equivalent thereof or a subunit thereof having a portion in common with or related to a portion of a gene or an equivalent thereof or a subunit thereof according to any of claims 1-3 wherein a molecule is used as probe with a sequence corresponding to at least part of the common or related part for the isolation from human chromosomal DNA of a human chromosomal gene. A method for the preparation of a gene or an equivalent thereof or a subunit thereof having a part in common with or related to a part of a gene or an equivalent thereof or a subunit thereof according to one or more of claims 1 to 3, essentially as described here. Gene or equivalent thereof or subunit thereof having a portion in common with or related to a portion of a gene or an equivalent thereof or a subunit thereof according to any of claims 1-3, prepared by a method according to claim 20 or 21. A plasmid recombinant, comprising a plasmid vector having introduced into an insertion site a gene or a subunit 8100558 35 - 45 - thereof or an equivalent thereof according to any one of claims 1 - 8, 18 or 19, which plasmid recombinant translation is in the correct phase. allows the mRNA corresponding to the introduced gene or subunit thereof or equivalent thereof and has a bacterial promoter upstream of or adjacent to the insertion site so that the inserted gene or subunit or equivalent thereof is under bacterial promoter control. The plasmid recombinant according to claim 23, comprising a plurality of genes or subunits thereof or equivalents introduced therein according to any one of claims 1 to 8, 16 or 19. Plasmid recombinant according to claim 23 or 24, comprising a plurality of bacterial promoters. Plasmid recombinant according to one or more of claims 23 to 25, comprising at least one trp promoter. Plasmid recombinant according to one or more of claims 23 to 26, which lacks at least part of the DNA follow-up responsible for transcription dilution. Plasmid recombinant according to one or more of claims 23 to 27, comprising a modified derivative of pWT 501. Plasmid recombinant according to one or more of claims 23 to 28, comprising a plasmid vector having a gene or a subunit thereof or an equivalent thereof according to one or more of claims 1 to 8, 16 or 19 encoded therein, which also encodes a initiation codon and / or at least part of a ribosome binding site. Plasmid recombinant according to one or more of claims 23 to 29, essentially as described herein. 31. A process for the preparation of a plasmid recombinant according to any of claims 23-30, comprising the introduction of a gene or a subunit thereof or an equivalent thereof according to any one of claims 1-8, 16 or 19 into an insertion pair of a suitable plasmid vector. A method according to claim 31, comprising: isolation of a trp promoter containing approximately 150 bp Hind III fragment from pWT 35 501 self-ligation partial digestion with Taq Ij treatment with 81 0 0 55 8 5-46-S1 nuclease treatment with E. coli DNA polymerase I; restriction with Hind III; isolation of a trp promoter containing about 100 bp fragment; ligation to an approximately 700 bp fragment obtained by treatment of a recombinant plasmid of pWT 231 containing a gene or a subunit thereof or equivalent according to any one of claims 1 to 8, 16 or 19 with Sac I, Sl nuclease, E. coli DNA polymerase I and Hind III; restriction with Hind III; and ligation to Hind III restrained bacterial basic phosphatase treated pAT 153. 10 15 20 25 30 The method of claim 31 or 32, essentially as described herein. 34. Plasmid recombinant according to one or more of claims 23 - 30, prepared by a method according to one or more of claims' 31 - 33. A cell comprising a gene or a subunit thereof or an equivalent thereof as claimed in any one of claims 1 to 8, 16 or 19 or a plasmid recombinant according to any of claims 23 to 30 or 34 inserted therein. The cell of claim 35, which is an E. coli K.-12 HB101 cell. The cell of claim 35 or 36, essentially as described herein. A method for the preparation of a cell according to any one of claims 35-37, comprising introducing a gene or a subunit thereof or an equivalent thereof according to any one of claims 1-8, 16 or 19, or a plasmid recombinant according to any of claims 23 to 30 or 34 in a cell. The method of claim 38, essentially as described herein. The cell of any one of claims 35 to 37, prepared by a method of claim 38 or 39. A method for the preparation of a protein having properties similar to that of human interferon, wherein a cell is cultivated according to one or more of claims 35-37 or 40 and recovered expressed protein. 35 8100558 - 47 The method of claim 41, essentially as described herein. 43. Protein with properties similar to that of human interferon prepared by a method according to claim 41 or 42. 8 1 0 0 55 8