HU204086B - Process for producing non-human mammal animal interferons, dna sequemces coding form them and pharmaceutical compositions comprising sand interferons - Google Patents

Abstract

The method of production of polypeptide with sequence of aminoacids of animal interferon according to the solution consists in cultivation of an organism of E.coli type or yeast type or a cellular culture of CHO cellular line. The microorganism or the cellular line is transformed by a replicable vector with ability of expression of DNA sequence coding the above-mentioned polypeptide. The obtained polypeptide is isolated. The isolated polypeptide can be used as an active substance in treatment of viral, malign, immunosuppressive or immunodeficiency states of animals.

Description

The present invention relates generally to methods and methods of using recombinant DNA technology (DNA) recombinantly for the production of animal interferons, the various products and uses thereof.

More particularly, the present invention relates to the production of DNA recombination expression carriers for the isolation and identification of DNA sequences encoding interferons of non-human mammalian origin, operably linked to the expression affecting promoter sequences. The present invention also relates to the production of host cell cultures used for DNA recombination technology, including the production of appropriate transformed microorganisms and vertebrate cell cultures which are thus capable of expressing the above DNA sequences.

The invention also relates to the conversion of the novel end products produced into a pharmaceutical composition for use in the prophylactic or therapeutic treatment of animals.

All methods of the invention which are useful in the preparation of said DNA sequences, expression vehicles, host cell cultures, final products and compositions containing the latter are provided.

The invention is based in part on DNA sequences and amino acid sequences wherein the DNA sequences encode a series of bovine interferon alpha. The sequence includes 3'- and 5'-flanking sequences which allow for in vitro binding to expressing carriers. Thus, it will be possible to develop DNA recombination methods capable of producing sufficient amounts of animal interferons to determine their biochemical properties and biological activity, thus opening the way to their efficient production for practical use.

References to the prior art and to the discussion of particular issues are referred to herein with increasing Arabic numerals from 1 through. We will provide you with a compiled bibliography at the end of the description.

Interferon components have been isolated from tissues of animals of various phylogenetic stages (1,2, 3). Exposure studies with these interferons have shown varying degrees of antiviral activity in the host animal (3,4,5,6). It has also been shown that these interferons are not always specific to the particular animal species. Thus, tissue-derived bovine interferon preparations have shown antiviral activity on both monkey cells and human cells (7). Similarly, interferons of human origin have been shown to be efficacious in cells of inferior animal species (see 7).

The consequence of this mutual effectiveness is that there is a significant degree of agreement between the individual interferons in terms of amino acid composition and amino acid sequence. So far, however, this explanation has remained theoretical, since the amount and purity of animal interferons that can be recovered were not sufficient to carry out unambiguous experiments to characterize the structure and biological properties of the purified components compared to other human interferons (8,9,10,11, 12).

In spite of the low levels of interferon available and their poor purity, a causal relationship has nevertheless been established between interferon in10 and the antiviral activity in the host animal.

Thus, it is desirable to produce animal interferons in good production and high purity in order to provide effective efficacy studies in 15 animals and thus bring us closer to the industrial use of interferons in the treatment of animals for viral infections, malignancies and immunization disorders. In addition, the production of isolated animal interferons provides an opportunity for their elimination of azo20 and, consequently, can serve as a basis for their classification and comparative studies with appropriate human interferons (see 8-20).

Studies with interferons of animal origin have so far been limited to the use of crude formulations, since these interferons were only available in very small quantities, but nevertheless have important biological effects. It is expected that they may also be used for animal infiltration purposes in combination with vaccine and / or antibiotic treatment. Therefore, interferons of animal origin have great promise in medical and economic terms.

It has been found that the use of DNA recombination technology can be a very effective method of providing the greater amount of animal interferon required for medical and industrial use. The materials thus produced are expected to be glycosylated, which is considered to be a characteristic of the naturally occurring material 40, and are likely to have biological activity which makes them suitable for treating a variety of viral, cancerous, and immune-related conditions or diseases in animals.

DNA Recombination Technology Has Come to a relatively advanced stage Molecular biology professionals can easily link various DNA sequences together to create new DNA capable of producing abundant amounts of exogenous protein product in 50 transformed microbes. General methods are available for in vitro linking of various "gymnastic" or "sticky" DNA fragments. In this way, effective expression vehicles are formed with which certain organisms can be transformed to efficiently synthesize the desired exogenous product. However, the path of synthesis is somewhat convoluted from the point of view of each product and science has not yet reached a level where performance can theoretically be predicted.

Indeed, he who predicts without a proper experimental basis

EN 204 086 Β the risk, since the procedure may prove to be unusable in reality.

An essential element of DNA recombination technology remains the plasmid, which is a double-stranded helix of extra-chromosomal DNA in bacteria and other microbes, often in multiple copies per cell. Information encoded in plasmid DNA is required for reproduction of the plasmid in newer cells and, in general, for the transfer of one or more secretory characteristics, such as antibiotic resistance in bacteria, which ensures that growth of clones of the host cell containing said plasmid is favored. medium.

The utility of plasmids is based on the fact that they can be cleaved in a specific manner by one or the other restriction endonuclease (or restriction enzyme). Each restriction enzyme recognizes different sites in the DNA in your plasmid. In this way, heterologous genes or gene fragments can be inserted into your plasmid by attaching their ends to the cleavage site or to the adjacent rearranged end. In this way, expression carriers capable of replication are formed.

Recombination of DNA is carried out outside the cell, however, the resulting "recombinant" replication-expressing carrier or plasmid can be introduced into cells by a process called transformation, and large amounts of the recombinant expression carrier can be obtained by propagating the transformed cells.

Furthermore, if the gene is inserted correctly in relation to the portions of the plasmid that regulate transcription and translation of the information encoded in the DNA, the resulting expression carrier can be used to actually produce the polypeptide sequence encoded by the inserted gene. This process is called an expression.

The term starts in the region known as the promoter, which is recognized and bound by ribonucleic acid (RNA) polymerase. During the transcription phase of the expression, the DNA is unwound and serves as a template for the synthesis of the messenger ribonucleic acid from the DNA sequence. In contrast, the messenger RNA will be translated into a polypeptide having the amino acid sequence encoded by the messenger ribonucleic acid (mRNA). Each amino acid is encoded by a nucleotide triplet or "codon" and together form the structural gene (or structural gene), i.e., the portion that encodes the amino acid sequence of the polypeptide produced. The translation starts at a start signal (this is usually ATG (adenine-thymine-guanine), which is converted to AUG in the resulting messenger ribonucleic acid). The so-called stop codons determine the end of the translation and consequently stop the production of additional amino acid units. If necessary, the proteins are obtained by lysis of the host cell from microbiological systems and the product is appropriately purified from other proteins.

In practice, DNA recombination technology can express fully heterologous polypeptides, a so-called direct expression, or a heterologous polypeptide that is linked to a portion of the amino acid sequence of a homologous polypeptide. In the latter case, the desired biologically active product is sometimes rendered inactive within the linked homologous / heterologous polypeptide and will only be biologically active after cleavage in the extracellular environment (21,22).

Cell or tissue cultures used for genetic and cell physiological studies are known. Methods are available to maintain constant cell lines. (These cell lines are produced by sequential transfer of isolated normal cells.) For research purposes, cell lines are maintained by propagation on a solid support, in a liquid medium, or in a suspension containing the carrier nutrients. The increase in size seems to be purely mechanical (see generally 23.24).

The present invention is based on the discovery that DNA recombination technology can be used to efficiently produce animal interferons in such a form and in such amounts that the resulting interferons can undergo the efficacy assays that are a prerequisite for their practical use. All forms of the product can be used for the prophylactic and therapeutic treatment of animals, especially viral infections, malignant tumors and conditions of insufficient immunity. Possible forms of the product include oligomeric forms which may be glycosylated and allelic variants. The products are produced using genetically modified microorganisms or cell cultures. As a result, it has become possible to produce and isolate interferons of animal origin more efficiently than before.

One important aspect of the present invention is to genetically direct a microorganism or cell culture to produce, in isolable amounts, a typical animal interferon, the bovine interferon produced in a mature form by the host cell.

The present invention includes processes for the preparation of interferons of animal origin. The present invention also relates to the production of replicable DNA expression carriers for the expression of gene sequences encoding interferons of animal origin. The present invention also relates to the production of microorganism strains or cell cultures transformed with the above expressive carriers, and to fermentation cultures containing these transformed strains or cell cultures, which cultures are capable of producing interferons of animal origin.

Accordingly, the present invention encompasses various methods of producing interferon gene sequences, DNA expressing carriers, microorganism strains and cell cultures, as well as fermentation of the above microorganisms and cell cultures.

Methods for the production of EN 204086 Β cultures.

In some host systems, vectors can be developed to produce the desired animal interferon selected by the host cell in mature form. It is believed that interferon containing the signal sequence from the 5 'flanking region of the gene is delivered to the cell wall of the host organism, where the signaling portion that facilitates this migration is cleaved during the selection of the mature interferon product. This allows isolation and purification of the mature interferon that you want to produce without the need to eliminate contaminants from the host cell protein or cell debris ·

A preferred embodiment of the present invention relates to the production of bovine interferon, a typical representative of animal interferons, by direct expression of mature interferon.

By "mature animal interferon" is meant that microbiologically or cellular animal-derived interferon is not associated with a "signal" peptide or peptide precursor that directly precedes the translation of the animal-derived interferon ribonucleic acid. The first amino acid of the mature animal interferon of the invention is methionine (this is due to the ATG start signal codon being inserted before the structural gene) or when the methonin is cleaved inside or outside the cell, the usual 30 first amino acids are located at the front.

Mature animal interferon may also be prepared according to the present invention with a conjugated protein other than a conventional signal polypeptide, wherein the conjugated product is specifically cleaved within or outside the cell (see 21).

The end thereof may also be obtained by direct expression of the mature animal interferon without the need to cleave any excess polypeptide.

This is particularly important when the signal peptide is not efficiently removed by the host, where the expression vehicle expresses the mature interferon together with the signal peptide. The mature interferon thus produced is isolated and purified to such an extent that it can be used to treat viral infections, malignancies, or immune-related disorders.

The animal interferons of the present invention are otherwise produced in the animal body and may be animal 50 alpha (leukocyte), beta (fibroblast) and gamma (immune) interferons analogous to the human interferon nomenclature. All three types were identified in animal models. Bovine interferon has been shown to be composed of animal-derived alpha interferon-like proteins, such as human 55 alpha interferon. The interferons tested are less similar in protein content to human alpha interferons than either animal interferons or human alpha interferons. However, bovine 50-derived beta interferon-building proteins are quite different from human beta-interferon-forming proteins.

The invention also provides inter-species and inter-family hybrid interferons using common restriction sites on the genes of various animal interferons and recombinant portions using methods known per se (see 57).

The animal interferons of the present invention include those produced in cattle, dogs, horses, felines, goats, sheep and pigs Preferably, carnivorous animals such as cattle, sheep and goats. The interferons of the present invention are used as antiviral and antineoplastic agents in said host animal. For example, bovine interferons may be used to treat respiratory disease in cattle, either in combination with known antibiotics where the antibiotics are present as a therapeutic component or with vaccines where the vaccines are for prophylaxis. Other uses include cattle as well as goats, sheep, pigs, horses, dogs and cats. In dogs, cats, horses and birds in particular, the antitumor activity of the appropriate interferons is of greater economic importance.

Interferons of animal origin can be prepared as follows, with reference to bovine interferon as a typical example:

Claims 1. Cattle tissues, such as bovine pancreatic tissue, are frozen into a powder and subjected to treatment to digest RNA and proteins. Upon precipitation, high molecular weight bovine DNA is obtained.

2. The high molecular weight DNA is partially digested.

3. The resulting DNA fragments are fractionally sized to give 15-20 kilobase pair fragments.

4. The resulting fragments are cloned using the λ Charon 30 phage vector.

5. The mature lambda phage particles infecting the vectors produced in this manner are enveloped using an invitrolambda wrapping system to generate phage kits. This is multiplied on bacterial cells by about 10 to ⁶ fold. The phage is cultured to an apparent confluence on bacteria and screened for hybridization using a radioactive human interferon probe (assay sample).

6. The appropriate DNA is isolated from the Idons, mapped by restriction analysis and analyzed by Southem hybridization. Restriction fragments containing bovine interferon genes were subcloned in plasmid carriers and their sequences were determined.

7. DNA of known sequence is then in vitro

EN 204086 Β so that it can be inserted into the appropriate expression carrier. This expressing carrier is then transformed into a suitable host cell, which in turn is cultured in a suitable medium to express the desired bovine interferon product.

8. The bovine interferon produced in this manner contains 166 amino acids in its mature form, including the first cysteine and the precursor sequence of 23 amino acids. The resulting interferon is extremely hydrophobic and is expected to have a molecular weight of about 21409. It has similar characteristics to human leukocyte interferons (8,9,10,11) and is found to be approximately 60% homologous to human leukocyte interferon.

Execution example

A. Micro-organisms and cell cultures

1. Bacterial strains and promoters Escherichia coli strain K-12 294 was used in our experiments (25). This strain is deposited under ATCC 31446 in the American Type Culture Collection. However, various other microbial strains such as the known Escherichia coli strains, such as Escherichia coli B, Escherichia coli XI776 (ATCC 31537), Escherichia coli W 3110 (F, λ, proto) (ATCC 27325), Escherichia coliDP 50 , deposited on March 5, 1982), Escherichia coli JM83 (ATCC 39062, deposited on March 5, 1982), or other microbial strains deposited with and obtainable from recognized microbial collections . An example of such an institution is the American Type Culture Collection (ATCC) and, for microorganisms, see its catalog (see also 26.26a). Examples of such other microorganisms include: Bacilli, e.g. Bacillus subtilis; and other enterobacteria, e.g. Salmonella typhimurium and Serratia marces cens. Plasmids capable of replication and production of heterologous gene sequences are used for microorganisms.

For example, the beta-lactamase and lactose promoter systems are advantageously used to initiate and maintain the production of heterologous polypeptides by microbiology. Details of the preparation of these promoters can be found in the literature (27,28). More recently, a system based on the tryptophan operon has been developed. This is the so-called trp promoter system, the production of which is disclosed in Goeddel et al. (12) and Kleid et al. (29).

Many other microbiological promoters have also been produced and utilized. Details of their nucleotide sequences are available in the literature (30), which allow one of ordinary skill in the art to ligate them within plasmid vectors.

Yeast strains and yeast promoters

The expression system of the invention may also use the plasmid YRp7 (31,32,33), which is capable of selection and replication in both Escherichia coli and the yeast strain of Saccharomyces cerevisiae. For selection in yeast, the plasmid (31, 32,33) contains the TRP1 gene, which complements (enables growth in the absence of tryptophan) yeast, which contains mutations in a gene on the yeast chromosome IV (34).

One valuable strain is RH218 (35), which is deposited without restriction in the American Type Culture Collection under accession number ATCC 44076. However, it is understood that any strain of Saccharomyces cerevisiae that contains a mutation that renders the cell a TRP1 type provides an efficient environment for expression of a plasmid containing the expression system for plasmid production. For example, another strain that can also be used is pep4 (36). This tryptophan auxotrophic strain also has a mutation point in the TRP1 gene,

When inserted at the 5 'end of a non-yeast gene, the DNA sequence (promoter) flanking the 5' end of a yeast gene (e.g., alcohol dehydrogenase 1) can facilitate expression of a foreign gene in yeast when inserted into the plasmid used to transform yeast. In addition to the promoter, proper expression of the non-yeast gene in yeast also requires a second yeast-derived sequence located at the 3 'end of the non-yeast gene in the plasmid to allow proper transcription and polyadenylation in the yeast. This promoter, like other promoters, is suitable for use in the present invention.

In a preferred embodiment of the method of the invention, the 5 'end of the yeast 3-phosphoglycerate kinase gene is located upstream of the structural gene, followed by DNA that terminates by signals of polyadenylation such as TRP1 (31,32, 33). ) or the PGK (37) gene.

Given that the 5 'end of the yeast flanking sequence (linked to the yeast's 3' end DNA) (see above) may promote the expression of foreign genes in yeast, it seems likely that the 5 'end flanking sequences of any high-yielding yeast gene can be used for the production of gene products. Because in some circumstances yeast expresses glycolytic enzymes (38) up to 65% of its soluble protein content, and since this high level appears to be a consequence of the high production of each of the messenger ribonuldic acids (39), any other glycolytic gene 5 'end flanking sequences may be used for such expression purposes. Examples of such enzymes are enolase, glycerol aldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphorus fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate,

EN 204086 Β Merase or Glucokinase. Any of these 3 'flanking sequences for these genes can be used for proper termination and mRNA polyadenylation in such an expression system - see above. For example, genes encoding acid phosphatase (40) as well as those containing mutations in the 5 'end flanking region, usually due to the presence of a TY1 transfer element (41), may be expressed in large amounts.

It is believed that all of the above genes are transcribed by yeast RNA polymerase H (41). It is possible that the RNA polymerase I and IH promoters, which transcribe genes for Rihosomal RNA, 5S RNA and tRNA, are also useful in these expression constructs (41,

42).

Finally, many yeast promoters also contain transcriptional regulators, which can be turned on or off by changing the culture conditions. Examples of such yeast promoters include the genes that produce the following proteins: alcohol dehydrogenase Π, isocytochrome c, acid phosphatase, nitrogen metabolism-degrading enzymes, glycerol aldehyde-3-phosphate dehydrogen, responsible for the utilization of maltose and galactose (3-9).

Such a regulatory range is very useful for regulating the production of protein dishes, especially if the resulting product is toxic to yeast.

It should also be possible to link the regulatory region of one of the 5 '30 flanking sequences to a 5' flanking sequence that contains a promoter derived from a highly expressed gene. This results in a hybrid promoter, and this is probably because the regulatory region 35 and the promoter appear to be physically separate DNA sequences.

Cell culture systems and cell culture vectors

Cell culture (tissue culture) of vertebrates that have become routine in recent years 40 (see 43). The COS-7 cell line of monkey kidney fibroblasts may be used as a host for the production of animal interferons (44). . In addition, the expression vector is also required to have an origin of replication and a promoter upstream of the gene, together with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Although these essential elements of SV40 will be utilized throughout the description, it will be understood that the invention, which is discussed in connection with preferred embodiments 55, is not limited to these sequences. For example, replication origins of other viral vectors (such as Polyoma, Adno, VSV, BPV, etc.), as well as cellular origins of DNA replication, which may be non-integrated, may be used. 60

B. Vector systems

Direct expression of mature bovine interferon in Escherichia coli

In order to directly express bovine 5-derived interferon in the form of a mature interferon polypeptide (i.e., without a signal sequence) in Escherichia coli, a plasmid containing a promoter fragment and a translation initiation signal must be linked to a well-defined fragment of animal genomic DNA. contains a coding range.

Expression in yeast

In order to express a heterologous gene, such as DNA for animal interferon, in yeast, a plasmid vector containing four components must be constructed.

The first component is the portion that permits transformation of both Escherichia coli and yeast and therefore must contain a selectable gene from each organism, such as the ampicillin resistance gene from the Escherichia coli strain and the TRP1 gene from the yeast. . This first component also requires an origin of replication from both organisms in order to remain as plasmid DNA in both organisms. Examples of such are the E. coli origin from pBR322 and the arsl origin from the yeast chromosome H1.

The second component of the plasmid is a 5 'end flanking sequence derived from a highly expressed yeast gene that promotes transcription of a downstream structural gene. An example is the 5 'end flanking sequence derived from the yeast 3-phosphoglycerate kinase (PGK) gene.

The third component of the system is a structural gene having the structure of an ATG translation start signal and a translation stop signal. The isolation and construction of such a gene will be described below

The fourth component is a yeast DNA sequence containing the 3'end flanking sequence of a yeast gene and contains the appropriate signals for transcription termination and polyadenylation.

3. Expression in mammalian cell culture

The synthesis of immune interferon in a mammalian cell culture is based on the development of a vector capable of autonomously replicating and expressing a foreign gene under the control of a heterologous transcription unit. Replication of this vector in tissue culture can be accomplished by providing an origin of DNA replication (derived from SV 40 virus) as well as a helper function (T antigen) so that the vector is introduced into a cell line expressing this antigen endogenously (46, 47). The SV40 late promoter prevents the interferon structural gene and provides for transcription of its gene.

A vector useful for reaching the expression consists of pBR322 sequences which provide an selectable marker for selection in Escherichia coli (resistance to ampicillin) and E. coli.

HU 204 086 Β

It consists of DNA origin of replication. These sequences are derived from plasmid pML-1 (46) and span the region between the EcoRI and BamHI restriction sites. The SV40 origin is derived from a Pvu b-Hindin fragment of 342 base pairs that spans this region (48, 49) (both ends converted to EcoRI). These sequences, in addition to containing viral DNA origin of replication, encode the promoter of both early and late transcription units. The orientation of the SV40 origin region is such that the promoter of the late transcription unit is located closest to the gene encoding interferon.

The invention is illustrated by the figures.

Figure 1 illustrates Southem hybridization of (a) human, (b) bovine, and (c) porcine DNA genomes where Eco RI was digested and hybridized to human leukocyte interferon A / D hybrid at different formamide concentrations. with a 570 bp EcoRI fragment containing ³² P isotopes. Hybridization in the presence of 20% formamide provides the clearest picture of bovine and porcine leukocyte interferon genes.

Figure 2 shows the fecal mutant Southem hybridization of four different bovine genomic DNA phages digested with EcoRI, BamHI or Hindin and hybridized with the ³² P isotope labeled human leukocyte gene probe. Clone 83 provides two fragments of hybridization for each restriction enzyme.

3a. Figure 1A shows a portion of the nucleotide sequence from the 1.9 kb plasmid p83BamHI subclone and the deduced amino acid sequence for bovine leukocyte interferon encoded therein. The signal peptide is represented by amino acid residues S1-S23.

3b. Fig. 3A shows the nucleotide sequence and the deduced amino acid sequence of another leukocyte interferon (alpha 2) derived from the 3.2 kb plasmid p67EcoRI subclone.

3c. Figure 3A shows the complete mature nucleotide sequence for the third bovine interferon (alpha 3) from the 3.5 kb p35EcoRI-BamHI subclone, together with the deduced amino acid sequence.

3d. Figure 4A shows the fourth bovine leukocyte interferon nucleotide sequence from the 2.9 kb plasmid p83EcoRI-BamHI subclone and the deduced amino acid sequence. The signal sequence is represented by the amino acid residues S1S23. The mature protein contains 172 amino acid residues. It should be noted that the elongation of the six amino acid residues is due to the nucleotide base exchange at position 511, which allows six additional translatable codons to be located in-phase before the next stop signal.

Figure 4 compares the amino acid sequences of BoIEN alpha 1, alpha 2, alpha 3, and alpha 4 with 11 known human leukocyte interferons. The amino acid residues remaining in all human leukocyte interferons, in all bovine leukocyte interferons, and in the case of bovine alpha 1 and alpha 4 interferons are those where homology to the majority of human leukocyte interferons is indicated.

Figure 5 schematically depicts the construction of the plasmid expressing pBoEFN-alpha 1 bovine leukocyte interferon. The starting materials are the expression vector pdeltaRIsrc trp and the BamHI fragment from the 1.9 Kb plasmid p83BamHI subclone.

A6. Figure 1B shows Southem hybridization of bovine DNA digested with EcoRI, Hindin, BamHI, GblH orPvuH. Hybridization is performed with radioactive material from BoIFN-alpha 1 or BoIEN-alpha 4 gene fragments. Each IFN gene favors a specific subset of BoIEN alpha genes for hybridization.

Figure 7 is a restriction map of genomic bovine DNA from three phage recombinants that hybridize to the human interferon gene probe. Black rectangles indicate the location and orientation of each BoIFN beta gene fragment. Restricted sites marked with an asterisk serve as a reference for restriction analysis.

Figure 8 is a more refined restriction image of the three genes shown in Figure 7. The dashed part corresponds to the signal sequence and the dotted part corresponds to the mature sequence.

9a, 9b. and 9c. Figures 3A and 4B show the nucleotide sequences for the BilEN beta 1, 2 and 3 genes and the deduced amino acid sequences.

A10. Figure 3B compares the amino acid sequences of the three BoIFN-beta with the HuEFN-beta amino acid sequences.

Figure 11 is a Soutbem hybridization image obtained by re-hybridizing the hybridization product of Figure 6 with a BillFN-beta 1 gene assay under conditions where only a single hybridizing fragment appears with human genomic DNA and HuIFN. beta gene (9) in an analogous experiment.

A12. Figure 1B illustrates the method used for expression of all three BoIFN-beta genes under the control of the Escherichia coli trp operon.

Figure 13 compares the deduced amino acid sequences of BoIEN gamma, HuIFN gamma and rat IFN gamma.

14a-14e. Figures 1 to 8 show the complete nucleotide and deduced amino acid sequences of porcine IFN-alpha 1, porcine IENbeta 1, porcine IFN-gamma, feline IFN-beta 1, and rabbit IFN-gamma genes.

Figure 15 shows the homologies between IFN-? Human, bovine, porcine, rabbit, mouse, and rat. The letters have the following meanings according to the standard notations for amino acids:

Asp D Asparaginsavlle Ϊ Isoleucine

Thr T TreoninLeu L Leuein

HU 204086 Β

SerS SerinTyr YTirozin

GIuEGIutaminsavPheFFeml-alanine

ProPProIinHisHHisztidin

Gly G GlicinLys KLizin

AlaAAlaninArgRArginin

Cys C CysteineTrp W Tryptophan

ValVvalmGInW Glutamine

MetMMetioninAsnNAszpargin

The following is a detailed description of the method of the present invention for the production of various animal interferons of the invention by DNA recombination technology. In particular, four generally applicable methods for the production of specific bovine leukocyte interferons are discussed. The process of the invention is described in relation to a bacterial system.

A. Isolation of bovine DNA

In order to assemble a set of genes of animal origin, high molecular weight DNA is isolated from animal tissue by modifying the method of Blin and Stafford (50), randomly fragmenting the gene focuses, and size fractionation. In this way, 15-20 kilobase size rounds are obtained for cloning into the lambda phage vector (51).

Frozen tissue such as bovine pancreas is ground to a fine powder in liquid nitrogen and solubilized for 3 hours at 50 ° C in a mixture of 0.25 M ethylenediaminetetraacetic acid, 1% sarcosyl, and 0.1 mg K proteinase (25 ml / ml). g tissue). The resulting viscous solution was de-proteinated by extraction with three phenols and one chloroform, dialyzed against 50 mM tris-hydrochloride (pH-8), 10 mM ethylenediaminetetraacetic acid and 10 mM sodium chloride and digested with 0.1 mg / ml of ribase for 2 hours at 37 ° C. application. After extraction with phenol and ether, the DNA is precipitated with two volumes of ethanol and washed with 95% ethanol, lyophilized and dissolved in TE buffer overnight at 4 ° C, adjusting to a final concentration of 12 mg / ml. The composition of the TE buffer solution is 10 mM Tris hydrochloride (pH 7.5), mM Ethlenediaminetetraacetic acid.

The resulting DNA preparation is longer than 100 kb as determined by electrophoresis on a 0.5% neutral agarose gel.

B. Partial digestion of bovine DNA with endonuclease and size fractionation

Aliquots of bovine DNA (0.1 mg) were digested at 37 ° C with 1.25, 2.5, 5 and 10 units of Sau3A for 60 minutes in 1 ml of a reaction mixture containing 10 mM tris hydrochloride, pH 7.5. containing mM magnesium chloride and 2 mM dithiothreitol. The incubation was stopped by adding ethylenediaminetetraacetic acid to a concentration of 25 mM. The mixture was extracted with phenol and ether, adjusted with 0.3 M sodium acetate (pH 5.2) and precipitated with 3 volumes of ethanol.

The ADNS buffer is redissolved at 68'C, resuspended at 20'C for 6 and 22 hours using a Beckman SW 27 centrifuge rotor at 27,000 rpm, with a 10-40% linear sucrose gradient (51). The 0.5 mL fractions were analyzed on a 0.5% gel using Eco RI digested Charon 4A (51a) DNA as the molecular weight standard. Fractions containing the 1520 kilobase DNA fragments were pooled, precipitated with ethanol and redissolved in TE buffer.

C, Compilation of the bovine genome DNA kit

The 15-20 kilobase DNA is cloned into the lambda Charon 30 A vector (52) which has G-A-T-C sticky ends. These are constructed to remove two internal Bam HI fragments of the phage. The Charon 30 A vector was grown in Escherichia coli DP 50 SupF strain (ATCC 39061, deposited on March 5, 1982) on NZYDT medium, then concentrated by precipitation with polyethylene glycol and purified by centrifugation using a CsCl density gradient (53). Phage DNA is prepared by extracting the purified phage twice with phenol, once with phenol and ether, and then concentrating the DNA with ethanol precipitation.

The final fragments of Charon 30 A vector were treated with 50 micrograms of phage DNA in 0.25 ml of 50 mM Tris hydrochloride (pH 8), 10 mM magnesium chloride and 0.15 M sodium chloride for 2 hours at 42 ° C. chloride, then fully digested with BamHI, phenol, and ether and pelleted by centrifugation using a gradient of 10% to 40% as described above. The 32 kilobase fractions containing the heat-treated end fractions were combined and precipitated with ethanol.

The purified purified Charon 30A portions (6 micrograms) are again heat-treated at 42 ° C for 2 hours, 0.3 micrograms of 15-20 kilobase bovine DNA and 400 units of T4 phaginucleotide li3 are added and the mixture is incubated overnight for 12 '. C at 0.075 ml in a reaction mixture containing 50 mM tris hydrochloride (pH 7.5), 10 mM magnesium chloride, 20 mM dithiothreitol and 50 micrograms) of bovine serum albumin. The ligated DNA mixture is then introduced into mature lambda phage particles in vitro using a lambda wrapping system (54).

The known method (54) uses ultrasonic extract (SE), freeze-thawed digest (UP), protein A, buffer A and M1 in this system.

Aliquots of 3 microliters of the alligated DNA mixture were incubated with 15 microliters of Apuffer solution, 2 microliters of Ml buffer, 10 microliters of SE and 1 microliters of protein A for 45 minutes at 27 ° C. The FIL was melted on ice for 45 minutes, 0.1 volume of Ml buffer solution was added, centrifuged for 25 minutes at 35,000 rpm at 4 ° C, and aliquots of supernatant fluid (0.075 ml) were added to the above reaction mixture. Incubate for another 2 hours

204086 ° C, then determine the titer of a small aliquot of the coating reaction mixture using the DP 50 SupH strain as described above.

This procedure provides a total amount of about l.lxlO ⁶ independent recombinant bovine DNA. The remainder of the mixture used to coat the mature lambda phage into particles was amplified by plate lysis (52), plated on DP 50 SupF strain at a density such that 10,000 TF units per 15 cm NZYOT agar plate.

D. Screening of bovine phage kit for bovine interferon genes

The strategy for identifying phage recombinants carrying bovine interferon genes is to test the homology of the nucleotides using radioactive probes from cloned human leukocyte (8, 9), fibroblast (12) and immune (55) interferon genes. Hybridization conditions were determined from Southern blots of genomic animal DNA (56).

5 to 5 micrograms of the high molecular weight DNA produced from human placenta, bovine pancreas, and porcine jaw as described above was completely digested with EcoRI, electrophoresed on 0.5% agarose gel, and transferred to nitrocellulose paper (56). ^{A 32} P isotope-labeled DNA probe is prepared from a 570 bp EcoRI fragment containing the protein coding region of mature human leukocyte interferon A / D at the Bgl Π restriction site (57). We do the usual operations for this.

Each nitrocellulose filter was pre-hybridized at 42 ° C overnight in the following composition: 5xSSC (56), 50mM sodium phosphate (pH 6.5), 0.1mg / ml ultrasound salmon sperm DNA, 5x Denhardt solution (59), 0.1% sodium dodecyl sulfate, 0.1% sodium pyrophosphate and 10%, 20% or 30% formamide. Hybridization is then carried out with a sample of 100x10 ⁶ beats / min in a solution of the same composition but containing 10% sodium dextran sulfate. (60) After incubating overnight at 42 ° C, the filters are washed four times with 2xSSC and 0.1% SDS at room temperature and once with 2xSSC and 0.1% SDS at room temperature, then 2xSSC solution once and then overnight exposed to Kodak XR-5 X-ray film using Dupont Cronex amplification filters. As shown in Figure 1, several hybridization bands are readily recognized in digested bovine and porcine DNA when 20% formamide is present in the hybridization.

This result provides evidence for the presence of more leukocyte interferon genes in bovine and porcine animals, similar to those previously reported for human leukocyte interferon (12, 61). Therefore, the same hybridization conditions are used to select the interferon genes from the bovine DNA pool.

500,000 recombinant phages were plated on DP 50

The SupF strain is 10,000 plaque forming units per 15 cm plate density and two parallel nitrocellulose filters are prepared for each plate according to Benton and Davis (62). Filters were hybridized with human leukocyte interferon (LelF) gene assay as described above. Ninety-six parallel hybridized patches are obtained which give strong signals on repeated screening.

The bovine gene pool is now screened for fibroblast and immune interferon genes. Assay samples were prepared from the following fragments: a 502 bp Dbal-Bglin fragment containing the complete mature human fibroblast interferon gene (12); the 318 bp Alul fragment (containing amino acids 12-116) and the 190 bp Mbo Π fragment (containing 99-162 amino acids) from the mature coding region of the human immune interferon gene (55). 1,2xl0 ⁶ recombinant phage hybridization of a total of 26 bovine fibroblast and 10 bovine immune interferon obtained occasionally.

E. Characterization of the recombinant phage

Phage DNA was prepared as described above from 12 recombinants that hybridize to the human leukocyte interferon assay. Each DNA was digested individually using EcoRI, BamHI and Hindin, electrophoresed on a 0.5% agarose gel, and the hybridization sequence was mapped using the South mode (56). DNA digested separately from ldons 10, 35, 78 and 83 are compared in Figure 2. For each phage, the size of the restriction fragments observed and the corresponding hybridization pattern can be distinguished and do not overlap. This indicates that each of the four phages carries a different bovine interferon gene. Furthermore, digestion of clone 83 with each of the three enzymes results in two separate hybridization bands in each case, indicating that this recombinant can carry two closely linked interferon genes.

F. Subcloning of bovine leukocyte interferon genes

Restriction fragments of three of the recombinant phages which hybridize to the human leukocyte gene probe are subcloned into the multiple restriction enzyme cloning site pUC9322. Plasmid pUC9 is derived from pBR322 by first deleting the 2067 base pair EcoRI-PvuH fragment containing the gene conferring resistance to tetracyldin, and inserting the 425 bp Haell fragment derived from the phage mP9 derivative of M13 (62a). Haell at position 2352 (relative to pBR322).

The Haell fragment from mP9 contains the N-terminal coding region of the Escherichia coli lacZ gene, which contains a sequence of amino acids 4 and 5 of the beta-galactosidase containing several sequences of CCA AGC TTG GCT GCA GGT CGA CGG ATC CCC GGG.

HU 204086 Β hairy restriction enzyme cloning site inserted.

The insertion of a foreign DNA fragment into these cloning sites interrupts the lacpromotor and lacZ gene continuity, resulting in a plasmid-transformed JM83 phenotype on lac ⁺ - 5 lac.

The above fragments are:

(a) a 1.9 kilobase BamHI fragment and a 3.7 kilobase EcoRI fragment from Id 83 (corresponding to non-overlapping segments of the same recombinant);

(b) a 3.5 kb BamEH-EcoRI fragment from Idon 35; and (c) a 3.2 kilobase Eco RI fragment from clone 67. In each case, the properly digested vector is 15

0.1 micrograms were ligated with purified fragments in a ten-fold molar excess, transformed into Escherichia coli strain JM83 (ATCC39062, deposited March 5, 1982), and then 0.04 mg / ml of 5-bromo-4-ldoro-3-indolyl-beta. Plated onto M9 (63) plates containing D-galactoside and 0.2 mg / ml ampicillin. White colonies suspected of carrying DNA insertion at one of the restriction sites, thereby interrupting the coding region of the lacZ gene on pUC9, were transferred to 25 ml of LB medium containing 0.02 mj / ml ampicillin and cultured for a few hours at 37 ° C. C and screened for the inserted fragment by the plasmid DNA minipreparation procedure (64).

G. The DNA sequence of the leukocyte interferon gene in clan 83 is 30

The 1.9 kb BamHI site DNA sequence of the 1.9 kb p83BamHI (i.e., 1.9 kb subclone of clone 83) was determined by the Maxam-Gilbert chemical method (65) and the results are shown in Figure 3. The longest open reading frame 35 encodes a 189 amino acid polypeptide that is substantially homologous to human leukocyte interferons (Figure 4). Similar to human-derived proteins, bovine leukocyte interferon contains a 23 amino acid hydrophobic signal peptide 40 which precedes a mature protein containing 166 amino acids with the same sequence (serule-gly). Cysteine residues 1, 29.99, and 139 are precisely conserved for each type of interferon. Table 1 compares pairwise bovine and human interferons for homology. As expected, bovine protein is substantially less homologous (approximately 60%) to individual human proteins than is homologous to each other (80% fe10).

The three additional bovine leukocyte interferon genes in the 3.2 kb p67EcoRI, 3.5 kb p35EcoRI-BamHI and 3.7 kb p83EcoRI subclones have the DNA sequences and the amino acid sequences derived therefrom as shown in Figures 3b, 3c. and 3d. shown.

As shown in Table 1, while the BoIEN-alpha 2 and 3 genes encode peptides that differ only slightly from the BoIEN-alpha 1 peptide, 20 the BoIEN-alpha 4 protein is as different from the other bovine peptides as any of the two bovine peptides. and human leukocyte interferon.

To make sure that the alpha 4 gene encodes as many cellular protein classes as the other BoIEN-alpha genes, we digest genomic bovine DNA with various restriction endonuclease enzymes and then hybridize with radioactive DNA fragments (alpha 1 genes). 612 bp AvaH fragment, Figure 6) and the protein coding regions for the alpha 30 4 gene (EcoRI-XmnI fragment from pBoENEN-alpha 4 trp 15b). Hybridization is performed under very stringent conditions (50% formamide) which does not allow cross-hybridization between the two genes.

As shown in Figure 6, each gene is preferably hybridized to form a specific sequence of bovine DNA fragments. These results together clearly indicate that there are two distinct groups of bovine leukocyte interferon peptides, and it is believed that a typical member is alpha 1 protein or alpha 4 protein.

Table 1

Pairwise comparison of coding sequences for bovine and human LEN alpha genes

alphal alpha2 alpha 3 alpha 4 Alfa alfabet BoIEN-alpha 1 94 92 54 61 62 BoIEN-alpha 2 96 91 53 61 64 BoIEN-alpha 3 100 96 45 BoIEN-alpha 4 52 48 51 54 54 Huie-alpha 56 52 29 81 Huie-alfabet 43 39 48 70 Huie-Alfacam 61 57 52 70 65 HuIFN-alpha 52 48 48 74 61 HuIFN-alfaF 61 57 52 70 65 HuIFN-alfaH 56 52 52 74 74 HuIFN-alphal 61 57 52 70 65 Huie-subspecies 56 52 48 61 57 HuIFN-alpha 52 48 48 83 70

HU 204086 Β

Table 1 (continued)

Differences in the coding sequences of bovine and human IFN-alpha genes in pairs! Compare

Alfacam Alfa alfaF alfaH alphal subspecies Alfa BoIFN-alpha 1 63 64 61 64 63 61 65 BoIFN-alpha 2 BoIFN-alpha 3 64 63 62 63 63 64 64 BoIFN-alpha 4 58 55 56 58 56 54 54 HuIFN-alpha 81 83 82 83 81 80 86 HuIFN-alfabet 81 77 81 83 80 79 81 HuIFN-Alfacam 81 89 86 94 92 83 HuIFN-alpha 65 83 81 80 78 84 HuIFN-alfaF 100 65 83 89 85 83 HuIFN-alfaH 74 83 74 84 84 84 Huie alphal 100 65 100 74 91 81 Huie-subspecies 91 70 91 65 91 80 HuIFN-alpha 74 91 74 91 74 65

The numbers in the table represent percent homology.

When the two parts of Table 1 are combined, a diagonal from left to right between the first column of the BoIFNalpha 1 gene is observed. The left part below the diagonal corresponds to the 23 amino acid precursor, the right part above the diagonal corresponds to the 166 amino acid mature protein.

A, B, C, etc. is human leukocyte interferon A,

B, CSTB.

H. Direct expression of mature BoIFN-alpha 1 in Escherichia coli

The construction of the direct expressing plasmid is shown in Figure 5. The 1.9 kb plasmid p83BamHI subclone was digested to completion with Ava Π and the 612 base pair fragment of bovine leukocyte interferon gas was electrophoresed on a 6% polyacrylamide gel and subjected to electroelution. About 1.5 micrograms of this fragment is digested with Fnu4H, extracted with phenol and ether and finally precipitated with ethanol. The resulting Fnu4H adhesive ends were supplemented with blunt ends by treatment with 6 units of DNA polymerase I (Klenow fragment) for 30 minutes at 12 ° C. The 20 microliter reaction mixture contains the following components: 20 mM tris hydride (pH 7.5), 10 mM magnesium chloride, 4 mM dithiothreitol and 0.6 mM dATP, dGTP, dCIP, and dTTP. After extraction with phenol and ether, the DNA was digested with PsU and subjected to electrophoresis on a 6% gel. The resulting 92 base pair blunt-ended PsŰ fragment, which spans the first nucleotide of the bovine mature leukocyte interferon coding region, is recovered from the gel by electroelution.

The remainder of the mature coding region is isolated as follows. The BamHI insert from the 1.9 kb plasmid p83BamHI subclone was partially digested with 14 units of PsU. Digestion was carried out for 10 minutes at 37 ° C in a 45 microliter reaction mixture containing 10 mM tris hydrochloride (pH 7.5), 10 mM magnesium chloride, and 2 mM dithiothreitol. After extraction with phenol and ether, the desired partial 1,440 bp fragment of Psü-BamHI, starting at nucleotide 93 of the coding region of the nucleus, is separated from a 6% polyacrylamide gel.

Plasmid pdeltaRIsrc is a derivative of plasmid pSRCexlo (66), from which EcoRI sites near the trp promoter and away from the src gene are removed by DNA polymerase (67) insertion of the AATTATinAcTatATat which is synthesized by the phosphor triester ester (68). Plasmid pdeltaRIsrc (20 micrograms) was completely digested with EcoRI, extracted with phenol and ether and finally precipitated with ethanol. The plasmid is then digested for 30 minutes at 100C with 100 units of S1 nuclease in 25 mM sodium acetate (pH 4.6), 1 mM zinc chloride and 0.3 M sodium chloride. In this way, the blunt end of the ATG sequence is created. After extraction with phenol and ether and precipitation with ethanol, the DNA is digested with BamHI, electrophoresed on a 6% polyacrylamide gel, and the large vector fragment (4300 base pairs) is recovered by electroelution.

The expression plasmid was constructed by ligating together 0.2 micrograms of vector, 0.02 micrograms of a 92 base pair, blunt-ended PsŰ fragment and 0.25 micrograms of 1400 base pair partial Psfl-BamHI fragment using 400 units of T4 DNA ligase. The ligation was performed overnight at 12 'C and the resulting product was transformed with Escherichia coli strain 294 (ATCC 31446) to confer resistance to ampicillin. Plasmid DNA was prepared from 96 colonies and digested with Xba and Pst I. Of these plasmids, 19 contain the desired 103 bp fragment of Xba I-Ps and 1050 bp Ps. DNA sequence analysis confirms that many of these plasmids contain an ATG initiation codon that

HU 204086 is located at the start of the bovine interferon coding region. One of these plasmids, pBolen-alpha Itrp55, was selected for further studies.

I. Direct expression of another group of mature bovine leukocyte interferon (shark-4) in Esckerichia coli

An ATG initiation codon was placed upstream of the mature coding region by enzymatic extension of the CATGTGTGACTTGTCT synthetic DNA primer. The heptadecamer is phosphorylated with T4 polynucleotide kinase and gamma- ³ * P ATP as described previously (12). An amount of 250 pmol of primary is combined with approximately 1 microgram of Hindi fragment of bovine leukocyte interferon (this fragment contains amino acid residues S20 to amino acid residue 102) in 30 microliters of water for 5 minutes, followed by 25 units of Escherichia coli DNA. The reaction product was digested with HgiAT and the resulting 181 base pair, blunt-ended HgiAT fragment was separated from a 6% northern acrylamide gel.

The complete gene for the mature peptide behind the trp promoter was constructed by enzymatically ligating the above fragment with a 508 bp HgiA-PsfT fragment containing the carboxyl-terminal portion of the peptide and one with the EuENEN-gamma expression plasmid, pIEN-gamma-trp48 Plasmid -13 (55), previously digested with EcoRI, was extended with PstI using Klenow DNA polymerase, digested and finally isolated on a 6% polyacrylamide gel. Transformation of the resulting mixture into Escherichia coli strain 294 identified a number of clones in which the EcoRI uptake site between the original expression vector trp promoter-ribosome binding site and the full coding region of mature bovine interferon (pBoIFN-alpha4trp15) was recovered. '

J. False cloning of bovine fibroblast interferon genes

The human interferon beta DNA probe hybridized to one phage recombinants six cleaned, separated from the dezoxiribonukleinsavjukat fen- ² as described below appears for further analysis. Restriction mapping, together with Southem hybridization analysis, shows that the six isolates contain three distinct regions of the bovine genome and thus form a multi-gene BoEEN-beta type. The results obtained are summarized in the restriction maps shown in Figure 7. In order to obtain a more detailed restriction map and nucleotide sequence for each type of recombinant, the hybridizing fragments are subcloned into plasmid vectors.

Thus, two 5-kb BglH fragments of lambda-beta 1 phage and two 5-kb BamHI fragments of lambda-beta 2 are cloned separately into plasmid pBR322 at the BamHI site, the overlap of 4.5 lambda-EcoRI-XhoI phage lambda-3 and A fragment of 1.4 kb PstI-Hpal is inserted into pUC9 (which is free of EcoRI-SalI sites) and pLeIF87 (10) (which is free of Hpal-Psu sites).

K. DNA sequences of three separate bovine fibroblast interferon genes

Figure 8 depicts the three, bovine IFN-beta genes

Shows a restriction map of the subwonder type O. Individual genes are readily distinguished by the presence of individual cleavage sites. The coding regions of the peptide, as well as the sequences immediately upstream and downstream of them, are determined by the single maxima-Gilbert half-chemistry method of the gene, as shown in Figures 9a, 9b. and 9c. 4A. Nucleotide homology to the human fibroblast interferon gene (12) can be deduced from the correct reading frame and the entire amino acid sequence of each bovine gene product which contains a 21 amino acid hydrophobic marker peptide followed by a mature amino acid residue of 185 amino acids. Bovine protein gels differ from each other (Table 2, Figure 10) with an even greater ϊ (about 60%) difference from the human-derived peptide.

The fact that bovine fibroblast interferon is composed of multiple genes is also confirmed, as shown in Figure 11, by hybridizing the Southem hybridization product with a radioactive probe derived from pBoENENbeta 1 trp as described below. , 415 bp EcoRIPvul fragment. As soon as below. As shown in FIGS. 1A to 4B, this experiment demonstrates the presence of a further homologous IEN-beta gene. Less hybridizing bands may in fact represent genes with more distant similarity which, in turn, encode more than one distinct beta-interferon.

Table 2

Homologue of bovine beta interferons and human beta interferon coding sequences in pairs! Compare

beta 1 beta 2 beta 3 Hu beta bETA 138 (83) 138 (83) 84 (51) beta 2 20 (95) 146 (88) 92 (55) beta 3 20 (95) 19 (90) 87 (52) Hu beta 16 (76) 17 (81) 16 (76)

HU 204086 Β

For each pair, the number of identical amino acids in the coding sequences is indicated. The lower left compares the 21 amino acid signal peptide, while the upper right compares the 166 amino acid mature beta interferons. For each pair, first the total number of identical amino acids is given, followed by the homology expressed as a percentage.

L. Direct expression of three bovine interferon beta strains in Escherichia coli

Because the three bovine interferon beta genes share many of the same DNA sequences and restriction sites (see Figure 8), a general method for expressing all three genes can be developed. Given that in each case the same DNA sequence encoding the first five amino acids and containing the two Alu sites, two additional synthetic oligonucleotides containing the ATG translation initiation codon were designed to restore the first 4 amino acids of the mature bovine interferon beta. codons and creates an EcoRI sticky end for insertion downstream of the trp promoter. The construction of the expressing plasmids is shown schematically in Figure 12. By ligating the synthetic oligomers to the 85 base pair Alul-Xhol fragment from the BoIFN beta subclone plasmids, digestion with EcoRI and Xhol enzymes yields a 104 base pair fragment flanked by EcoRI and Xhol sticky ends.

The entire coding system is then embedded in the trp producing vector, ligating the 104 bp fragment with the approximately 700 bp XhoI-PstI fragment, which encodes the remainder of each BoIFN beta protein, and the plasmid pIEN-gamma tr48-13 (55). , from which the internal EcoRI-PstI fragment was previously removed. In the resulting plasmids, pBoIFN-beta 1 trp, pBoIEN-beta 2 trp, and pBoIFN-beta 3 trp are in all cases under the control of the Escherichia coli trp operon for the transcription and translation of the interferon beta genes.

M. Characterization and subcloning of the bovine immune interferon (BoIFNgamma) gene

Ten phage recombinants hybridized with the human interferon gamma probe are purified and DNA is prepared therefrom as described above. Each of the ten DNA samples gives characteristic hybridization bands in the Southem hybridization assay. Lambda-gamma 4 and lambda-gamma 7 clones are selected for further analysis because of their different hybridization band patterns. Restriction analysis of the two clones shows that their DNA sequences overlap. The overlapping region contains the Xbal, EcoRV and Ncol restriction sites. Analysis of the DNA sequences of the two clones shows that in general the gene structure is similar to that of the human immune interferon gene (70), with the lambda-gamma 7 clone containing the fourth exon coding sequence and the lambda-gamma clone 4 containing those sequences. which encode the first three exons of the bovine interferon gamma gene, the human interferon gamma gene

Based on homology to DNA sequences. The deduced amino acid sequence of BoIEN gamma is compared in Figure 13 to that of HuIFN gamma (55) and murine IEN gamma.

In order to assemble the entire bovine IFN-gamma gene on a contiguous DNA segment, we isolate the 3,000 bp BamHINcol fragment, which encompasses the first three exons of the bovine IENgamma lambda-gamma gene, consisting of 4 clones and 2500 base pairs of N fragments that encircle the last exon of the lambda gamma 7 clones. These two DNA fragments were then cloned into the BamHIHindm vector from plasmid pBR322 by triple ligation.

N. Expression of bovine IFN-gamma gene in mammalian system

In order to obtain an intron-free version of BoIFN-gamma for expression of the gene in a prokaryotic system such as Escherichia coli, the gene is fragmented into large amounts in animal cell expression system to obtain a significant amount of specific mRNA. A 550 base pair BamHIHindHI fragment spanning the entire bovine IFN-gamma gene was inserted into an SV40 vector for expression in COS cells (44).

In particular, the BamHI-HindIII fragment of the bovine IEN gamma gene is cloned into a 2800 bp SV40 plasmid vector, pDLAR1. [This plasmid vector is prepared from an HBV (hepatitis B virus) antigen-expressing plasmid, pHBs348-L, by deleting the EcoRI site upstream of the SV40 selective origin of replication for late transcription. . The plasmid pHBe348-L expressing plasmid was constructed by cloning the HBsAG gene encoding the HBsAG obtained by digestion of HBV with EcoRI and BglII (71) at the EcoRI and BamHI sites (72). . Plasmid pML is a derivative of plasmid pBR322 which has a deletion that removes sequences that inhibit plasmid replication in monkey cells (72). a fragment is inserted into the EcoRI site of plasmid pRI-Bgl. The origin can be inserted in both orientations in a fragment. Because this fragment encodes both early and late SV40 promoters outside the origin of replication, expression of HBV genes can be effected by control of any promoter, depending on the orientation between BamHI and SalI sites (plasmid pHBs348-L expresses HBs expressed by late promoter control). It represented). The above fragment was introduced by triple ligation in the presence of a 600 base pair HindIII-SalI convertor fragment derived from a plasmid pBR322. Transfection of the resulting plasmid into COS cells can efficiently express bovine IEN gamma under the control of the SV40 late promoter.

HU 204086 Β

COS cells so infected are prepared mRNA + polyA and used to produce cDNA by conventional methods (55). The cDNA clones that hybridize to the bovine IFN-gamma probe are isolated. For further analysis, the cDNA Idon with the longest PstI insert was selected. DNA sequence analysis of this cDNA gene showed that all intron sequences predicted by the bovine IEN-gamma genomic clone were correctly removed.

The cDNA was digested for expression in the Escherichia coli strain by the primary mating method described above.

0. Isolation of rabbit and porcine IFN-gamma cDNA

The IEN-gamma cDNA was used as a hybridization probe to isolate rabbit and porcine IFN-γ complementary DNA sequences. Rabbit and porcine RNA was isolated from appropriate cultures of rabbit and porcine spleen cells induced with phytohemagglutinins (10 pg / ml) and phorbol12-myristate-13-acetate (10 μg / ml) overnight [Berger et al., Biochemistry 18,5143 (1979)]. Single samples of maternal and porcine RNA were then run through oligo (dT) cellulose to enrich the mRNA. Double-stranded cDNA libraries made from 2 pg samples of rabbit and porcine mRNA were used to construct cDNA libraries in the Xgt10 bacteriophage vector [Huynth et al., "Practical Approaches in Biochemistiy", edited by IRL, Oxford, England (1985). ], 140 hybridizing clones (or about 140) are obtained by recombinant screening of 1.5.10 ⁵ phage from the rabbit cDNA library, while only 16 positive hybridizing clones are obtained by recombinant screening from 1.6.10 ⁶ phage cDNA library. DNA restriction mapping shows that most of these Xgt10 recombinant phages contain an EcoRI insert ranging in size from lkb to 1.5kb. Two phages (lambdaR gamma7 and lambda Ryl3) from the spleen cDNA library to M13mpl9 [Norrander et al., Gene 26,101 (1983)] subdonate and analyze their sequences by the dideoxy chain method (62a).

The longest cDNA clone rabbit IFN-gamma (lambdaR gamma7) and the longest porcine IFN-yklónt (lambda gamma P 14), and these are predicted ammosav- ^guide sequences based on the human IFN-gamma cDNA, the DNA sequence homology, 14e and 14c, respectively. There are no nucleotide differences between the two rabbit IFN-gamma cDNA clones subjected to sequence analysis with the poly A length extension at the 3HuENEN-alpha terminus. Comparatively, two porcine IEN-gamma cDNA clones have the same DNA sequences, 5'- except for the length of coding regions.

P. Amino acid sequence of IFN-gamma 5

Figure 15 shows the sequence homology of the IEN-gamma protein in several mammalian species. The signal sequences consist of amino acids in human, porcine and rabbitIFN gamma, while the mouse and rat signal sequences contain 23 amino acids due to deletion at codon 19. These are animal

The signal sequences of IFN-gamma are named by their structural homology to the native human IEN-gamma. This range of amino acids has features in common with the secreting protein signal sequences having a 10 amino acid hydrophobic core; this -7.-Ü is -16. amino acids. The aseptic peptidase recognition sequence is expected to be (Cys / Ser) -Tyr- (Cys-Gly), which corresponds to -21.-23. with a carcass after the space. The anhydride chain polypeptide is identical to the mature human, bovine and porcine IFN-gammánál as 143 amino acids in length, while the rabbit IFN-gamma is a 144 amino acid-amino acid added to the carboxy-terminal end of ^one fifth The mouse and rat IEN gamma has only 133 and 134 amino acids, respectively. Amino acid sequence comparison shows that the 9 amino acids found in human, bovine, and porcine ΙΕΝ-γ are deleted in the mouse and rat IEN gamma. ElD, except for mouse and rat IFN-gamma, which have cysteine at their C-terminal end (which may not be present in vivo), none of the other mature IEN-gamma described herein contain any cysteine. Therefore, no disulfide linkage is required to maintain the complete structure of mature IFN-gamma, although full-length recombinant murine IEN-gamma readily forms an internal dimeric disulfide bridge (which does not alter specific activity). Rabbit IEN gamma contains three possible N-glycosylation sequences [Asn-X- (Thr / Ser)] [Struck et al., J. Biol. Chem., 253.5786 (1978)], whereas the second five IEN gamma have only two such sites in their mature protein. Mouse IEN gamma is the only IEN gamma that has an additional glycosylation site within the peptide signal. The position of these sites does not appear to be preserved among the various IFN gamma.

The generic amino acid sequence homology is greater than 60% of the human, bovine, porcine, and rabbit IEN gamma, while the homology is between the mouse or rat IEN gamma and any of the other four IEN gamma presented herein. less than 40%. Homology in mouse and rat IFN gammaTotal 85%.

> Q. IFN-gamma synthesis of bovine, porcine and rabbit E. coli

The IEN-gamma of bovine, porcine, or rabbit (the method used to express DNA inserts directly in E. coli) is as follows. Using synthetic ¹ oligonucleotides, an ATG codon, the codon for the starter netionin, or rabbit IEN gamma putative amino terminus. Protein end of XbaI is terminated before the ATG initiation codon. The transgenic restriction fragments containing the IEN gamma complete mature coding sequence are constructed by cleavage with Xba I and a second restriction enzyme, each of which has the unique IFN cleavage site. -cDNA located in the 3'-non-coding region of Mons, namely, these Dral are cattle

HU 204086 Β

IFN-gamma cDNA, Sspl for porcine IEN-gamma cDNA, and Nsil for rabbit IEN-gamma cDNA. The resulting restriction fragments are inserted between the Xba I and Pst I sites of the pIEN-beta3 trp expression plasmid (see above), of which the Xba I site is located downstream of the trp leader ribosome binding site.

High-level expression of bovine, porcine, and rabbit IFN-gamma is obtained due to transcription from the strong trp promoter and because the trp leader ribosome binding sequence is located at an optimal translation distance from the ATG initiation codon of the inserted coding sequence. Analysis of bacterial extracts containing porcine, bovine, and rabbit IFNgamma by SDS gel electrophoresis reveals the presence of IEN gamma as the major band on the protein gel with an apparent molecular weight of 17-18 kilodaltons.

These IFN-gamma were purified to homogeneity from E. coli cell extracts by adsorption, ion exchange and size exclusion chromatography. Purified proteins are routinely stored at 4 ° C in a concentration of 0.55.0 mg / mL in 20 mM Tris-HCl and 0.5 M NaCl, pH 8.0, after sterile filtration. Protein concentration was determined by a Bradford dye binding assay using bovine serum albumin as a standard. These IFN gamma were judged to be 98% pure by SDS PAGE with both Coomassie and Silver staining. Endotoxin levels in each formulation are below 0.1 ng / mg [unit (EU) / mg]. Size exclusion chromatography on Pharmacia Superose 12 columns under non-denaturing conditions suggests that bovine IFN-gamma, porcine IFN-gamma and rabbit IEN-gamma are present in solution at 25.8 kD, 31.6 kD and 21.0 kDa, respectively. kD with apparent molecular weight. However, they appear as a monomer in the 4M guanidine hydrochloride solution. These values are in good agreement with the apparent molecular weights obtained by SDS PAGE. All three species still carry the initiating methionine (literally) at its N-terminus.

R. Estimation of the number of IFN-gamma genes in bovine, porcine and rabbit genomes

The number of IEN gamma genes in the various mammalian genomes is estimated by Southem blot analysis. Bovine, porcine, and rabbit chromosomal DNA was digested with various restriction enzymes, fractionated by agarose gel electrophoresis, and transferred to nitrocellulose. Hybridization is performed under either stringent or non-stringent conditions with a single radiolabeled 600 bp AvaU fragment derived from the bovine IFN-gamma cDNA clone.

The appearance of only one or two hybridizing bands for digestion with different restriction enzymes as haploid genome indicates only the presence of an IFN-gamma gene copy. The IEN gamma of bovine, porcine, rabbit, human, and mouse, respectively, are all single copy genes.

S. Bacterial plasmid. for the expression of porcine IFNalpha 1

Two synthetic nucleotides are MCDLP

5'-AATTCATGTGCGACCTGCC

GTACACGCTGGACGGACT-5 'was designed to incorporate an ATG translation start codon, to restore codons for the first four amino acids of mature porcine IEN-alpha, and to form EcoR1 and Ddel sticky ends. The ligation of these oligomers to the 150 kp partial Ddel-Ncol fragment from the porcine IFN-alpha genomic clone and the 550bp Ncol-Ahaül fragment generates a 720 bp synthetic natural gene, which is the IEN-alp 1 encodes with EcoRl and AhalH sites. The insertion of this porcine IFN-alpha 1 flanked by EcoR1 and AhalH into your pIFN-beta 3 plasmid (Leung et al., BioTechnology2,458 (1984)) converts the pTrpPoA1 expression plasmid into the EcoRI and PstI sites, in which porcine IFNalpha under the control of the E, coli trp operon.

T. Preparation of bacterial extracts

Overnight cultures of LB medium containing 0.02 mg / ml ampicillin or 0.005 mg / ml tetracycline were inoculated into 50 mlM9 medium (63) at a 1: 100 dilution. M9 medium contains 0.2% glucose and 0.5% casinoic acid mixture with the appropriate antibiotic. Then, shake at 37 ° C until nA550 = 1.0. Samples (10 ml) were centrifuged and the gelled material was immediately frozen in an ethanol-dry ice bath. The frozen pellet is suspended in 1 ml of 7M guanidine, incubated on ice for 5 minutes, and diluted in PBS for assay.

Alternatively, the frozen pellet is lysed in 0.2 ml of a solution of 20% sucrose, 100 mM tris hydrochloride, pH 8.0, 20 mM ethylenediaminetetraacetic acid and 5 mg / ml. ml of lysozyme. After 20 minutes on ice, 0.8 mL of the following solution was added: 0.3% Triton X-100, 0.15 M Tris hydrochloride, pH 0.8, 0.2 M ethylenediaminetetraacetic acid, and 0.1 µM PMSF (phenylmethylsulfonyl fluoride). The lysate is centrifuged for 15 minutes at 19,000 rpm, and the supernatant fluid is assayed after dilution with PBS.

U. Interferon assays

Bovine interferon activity was determined by assay based on inhibition of cytopathic effect (CPE) in 96-well microtiter plates as follows.

1. Add 100 µl of cell suspension containing 10% fetal calf serum to each well of a 96-well microtiter plate (8 rows x 12 columns). The cell concentration is adjusted to give a monolayer flowing overnight.

Shake the slides gently on a shaking table for 2.10 minutes to evenly distribute the cells. The next day -

HU 204086 Β

3. Add 80 µl of additional medium to each well in the first column,

4. Into one of the cavities in the first column, 20 pl of the sample whose interferon activity is desired to be determined.

5. Mix the sample and the medium in the well by aspirating 100 µl of material into a 100 µl pipette and releasing it. The suction and release are repeated several times.

6. Transfer 100 pl of the contents of a cavity of the first column into a second cavity horizontally from the above cavity.

7. Stir as described in point 3 above.

8. Transfer 100 µl of the contents of one cavity again to the next column until a total of 11 transfers have been made.

9. Dispense 100 µl of the contents of the cavity in column 12. This procedure is used to reconstitute a double dilution series.

10. Each titration plate shall contain the appropriate standard (National Institute of Health) samples.

11. Plates in carbon dioxide. incubate at 37 ° C for 24 hours.

12. Each microtiter plate contains wells containing 100 µl of cell suspension and 100 µl of medium, which are considered as control of cell growth. There are also wells in each microtiter plate containing 100 µl of cell suspension, 100 µl of medium and 50 µl of virus suspension. These form a virus-induced cytopathogenic control group.

13. Except for the wells used for cell growth control, all wells were inoculated with 50 µl of a venous suspension. The multiplicity of infection used is the amount of vFlu that elicits a 100% cytopathic effect in the respective cell line within 24 hours.

14. Incubate the plates for 24 hours at 37 ° C in carbon dioxide.

15. Remove the liquid from the plates and stain the cells with 0.5% crystal violet.

Vol

Allow the cells to stain for 2-5 minutes.

16. Rinse cavities in tap water and allow to dry.

17. The interferon titer of the sample is the reciprocal of the dilution at which 50% of the cells remain viable.

18. The activity of each sample is normalized using the conversion factor. This factor is calculated using the following 10 formulas:

NIH standard titer / titration factor observed in the assay (see 69).

Extracts from 15 strains of Escherichia coli294 (ATCC 31446) transformed with pBOIFN-alpha 1 trp 55 show significant activity in the bovine kidney cell line (MDEK) infected with the VS virus (Ihdiana strain). However, no such activity was found in similarly infected stone20 descending cell lines: monkey kidney (CROP), human cervical carcinoma (HeLa), rabbit (Rk-13) or mouse (L929). Extracts used as a control of Escherichia coli strain 294 transformed with pBR322 showed no activity on MDEK 25 cells.

Table 3 summarizes the antiviral activity of BoIFN-alpha I, which was tested in vitro on various infected animal and human cell lines. ABoEN-alpha 1 is readily distinguished from human leukocyte interferons by the fact that it does not appear to exert an antiviral effect on human cells by its activity on bovine cells when infected with VS virus.

Table 4 shows interferon activity measured in extracts of Escherichia coli W3110 transfected with the expressing plasmids pBoIEN alpha 4 trp15, pBoEFN35 beta 1 trp, pBoIFN beta 2 trp and pBoIFN beta 3 trp. Of particular note is the finding that bovine fibroblast interferon is approximately 30-fold more active on bovine kidney cell line than human anion cell line, whereas there is an inverse relationship with human fibroblast interferon (12).

cell Line Interferon preparation liters, units / ml VSV EMCV LelFAstandard 640 SO MDBK SzarvasmarhaLeukocitalFN 300,000 SO Control extract <40 SO LelFAstandard 650 1500 HeLa SzarvasmarhaleukocitalF <40 <23 Control extract <40 <23 Mouse IFN standard 640 1000 L-929 SzarvasmarhaleukocitalFN <20 <31 Control extract <20 <31

HU 204086 Β

Continuation of Table 3

cell Line Interferon preparation Titer, units / ml VSV EMCV Rabbit EFN standard 1000 SO RK-13 Bovine leukocyte IFN <60 SO Control extract <60 SO LelF A standard 1500 Vero Bovine Leukocyte EFN <12 Control extract <12

MDEK = bovine kidney cells

VERO = African monkey kidney cells

HeLa- human cervical carcinoma cells

RK-13 = renal cells

NA- not applicable because the virus does not reproduce well on the cell.

Table 4

Iterferon activity in extracts of Escherichia coli

Escherichia coli IFN-beta activity ΠΝ-beta activity 294 strains trans- (units / 1 culture) (units / 1 culture) for shaping utility VSV-MDEK WISH-VSV plasmid pIEN-alpha 1 l.OxlO ⁸ no data available pEFN beta 1 ⁸ 2,2xl0 6.5x10 ⁶ pIFN beta 2 l.lxlO ⁸ 3.5x10 ⁶ pEFN beta 3 ⁸ 6,0xl0 2.0x10 ⁷

Bacterial extracts are prepared and assayed for their interactions with the bovine kidney MDEK cell line and human amniotic WISH cell line using VSV for infection as described in a published procedure (Weck et al., 1981).

Similarly, biological assays demonstrate the following activities.

Tables 5

interferon cell Line Activity in E.coli extract porcine -IFN-alfalfa MDBK 2.5.10 ¹⁰ units / L 8.33.10 ⁵ units / ml / OD setrtés -EFN-gamma PK-15 1.2.10 ¹⁰ units / l 3.10 ⁵ units / ml / OD IFN-gamma-rabbit RK-13 1.58.10 ⁸ units 8.10 ⁴ units / ml / OD

V. Preparation of Pharmaceutical Compositions The compounds of the present invention can be formulated into pharmaceutical compositions by methods known per se. The animal interferon product is then admixed with one or more pharmaceutically acceptable carriers. Suitable carriers and types of pharmaceutical compositions are known in the art. The pharmaceutical compositions of the present invention contain an effective amount of interferon protein together with a suitable amount of a carrier, and the compositions may be administered by known means, for example parenterally.

One of ordinary skill in the art will recognize that the animal interferons of the present invention have natural allelic variants. These variants are characterized by the fact that the sequences differ in one or more amino acids, or deletions, substitution, insertions, inversions, or additions that affect one or more amino acids in the sequence. The invention encompasses allelic variants.

Although preferred embodiments are described herein, it is to be understood that the invention is not limited thereto, but that the scope of the claims is defined by the claims.

Bibliography

1. Rinaldo et al., Ihfection and hnmunity,

24.660 (1976).

HU 204086 Β

2. Pulton and Rosenquist, Am. J. Vet. Res., 57, 1497 (1976).

3. BabrickésRouse.Interfectionandlmmunity, 75, 1567 (1976).

4. Todd et al., Infection and Immunity, 5, 699 (1976).

5. Ahl and Rump, Infection and Immunity, 14, 603 (1976).

6. Babrick and Rouse, Interviroloty, 8,250 (1977).

7. Tovey et al., J. Gene. Virol., 56, 341 (1977).

8. Goeddel et al., Nature, 287,411 (1980).

9. Goeddeling, Natur., 290.20 (1981).

10. Yelverton et al., Nucleic Acids Research, 9,731 (1981).

11. Gutterman et al., Annals of Int. Med., 95,399 (1980).

12. Goeddel et al., Nucleic Acids Research, 5,4057 (1980).

13. ip et al., Proc. Natl. Acad. Sci. (USA) 75, 1601 (1981).

14. YTaniguchi et al., Proc.NaÜ. Acad. Sci. (USA), 75,3469 (1981).

15. Bloom, Natura, 1980, 289,593.

16. Sonnenveld et al., Cellular Immunol., 49, 285 (1978).

17. Fleishmann et al., 1979, Infection and Immunity 26,248.

18. Blalock et al., Cellular Immunology, 49, 390 (1980).

19. Rudin et al., Proc. Natl. Acad. Sci. (USA), 77.5928 (1980).

20. Crane et al., J. Natl. Cancer Ihst., 61, 871 (1978).

21, 2007676A published British patent application.

22. Wetzel, American Scientist, 65,664 (1980).

23. Microbiology, 2nd Edition, Harper and RowPublishers, Inc., Hagerstown, Maiyland (1973), especially all22 and tracking pages. 40

24. Scientific American, 245.66. and subsequent pages).

25, 2,055,382A. British Patent Publication.

26. 26 44 432 German Federal Republic 45 publications

26a-eder et al., Science, 796, 175 (1977).

27. Chang et al., Natura, 275,617 (1978).

28. Itakura et al., Science, 795, 1056 (1977). 50

29. European Patent Application Publication No. 0036776.

30. Siehenlist et al., Naturre, 1979, 282.39.

32. Kingsman et al., Gene, 1979, 7.141.

33. Tschumper et al., Gene, 70,157 (1980). 55

34. Mortimer et al., Microbiological Reviews, 44,519 (1980).

35. Miozzari et al., Journal of Bacteriology, 754.48 (1978).

36. Jones, Genetics, 55, 23 (1977). 60

37. itzeman et al., J. Bioi. Chem., 1980, 255, 12073.

38. HHess et al., J. Adv. Enzyme Reg., 7, 149 (1968).

39. Holland et al., Biochemestry, 77, 4900 (1978).

40. Bostian et al., Proc. Natl. Acad. Sci. (USA), 77,4504 (1980).

41. The Molecular Biology of Yeast (August 11, 1810, 1981), Cold Spring Harbor Laboratory, Cold Spring

Harbor, New York.

42. Chambon, Ann. Port. Biochemistry, 44, 613 (1975).

43. Tissue Culture, Academic Press, Kruse and Pat15, (1973).

44. Gluzman, Cell, 25,175 (1981).

45. Goeddel et al., 1979, Nat. 257, 544.

46. Lusky et al., Natura, 295.79 (1981).

47. Gluzman et al., Cold Spring Harbor

Symp. QuantBiol., 44, 293 (1980).

48. Fiers et al., Natura, 1975, 275,113.

49. Reddy et al., Science, 200,494 (1978).

50. Blin and Stafford, Nucleic Acids Research, 5,2303 (1976).

51. Maniatis et al., Cell, 75, 687 (1978). 51a Blattner et al., Science, 796, 161 (1977).

52. Rimm et al., Gene, 72,301 (1980).

53. Blattner et al., (1978) Procedures for the Use of Charon Phages in Recombinant DNAResearch, Research Resources Branch, National Institute of Allergy and Infectious Diseases, Bethesda, Maiyland.

54. Blattner et al., Science, 202, 1279 (1978).

55. Gray et al., Nature, 295, 503 (1982).

56. Southern, J. Mol. Biol., 95,503 (1975).

57. Weck et al., Nucleic Acids Research, 9, 6153.

58. Taylor et al., Biochem. Biophys. Acta, 442, 324 (1976).

59. Denhardt, Biochem. Biophys. Gap. Comm., 25, 641 (1966).

60. Wahl et al., Proc. Nat. Acad. Sci., 76, 3683 (1979).

61. Nagata et al., Natura, 257,401 (1980).

62. Benton and Davis, Science, 796,180 (1977). 62aMessing et al., Nucleic Acids Research,

9.309 (1981).

63. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

K Bimbóim et al., Nucleic Acids Research, 1979, 7.1513.

>. Maxam and Gilbert, Proc. Nat. Acad. Sci., 74, 560 (1977).

i. McGrath and Levinson, Natura, 295, 423 (1982).

HU 204086 Β

67. Itakura et al., Science, 1987, 1056 (1977).

68. Crea et al., Proc. Natl. Acad. Sci., 75, 5765 (1978).

69. Stewart (1979) The Interferon System, Springer Verlag, New York, pages 17 et seq.

70. Gray and Goeddel, Natur., 298,859 (1982).

71. Animated Virus Genetics (Publisher: Fields et al.), Chapter 5, page 57, Academic Press, New York (1980).

72. Lusky and Botchan, Naturre, 1981, 293.79.

Claims (106)

1) transforming the tissue, preferably pancreatic tissue, of the animal into a frozen powder, removing the RNA and proteins and then recovering the DNA by precipitation; 1. A method for producing DNA encoding interferons from non-human mammals by extracting DNA from the tissues of said animal, partially digesting the DNA, size fractionating the resulting DNA fragments, cloning these fractions into vectors, generating vector sets from the vectors, and hybridizing to the vector set. by screening the vector set with a labeled probe made of DNA encoding interferon fragments of human or other mammalian origin, the appropriate DNA is then isolated and mapped from the hybridized Idons and re-isolated after further cloning. 2) partially digesting the high molecular weight DNA; A process according to claim 1, characterized in that Method according to claim 1 or 2, characterized in that the vector set is screened with a labeled probe consisting of DNA encoding human interferon fragments. 3) fractionating the resulting DNA fragments by size and recovering the 15-20 kilobase pair fragments; 4. Method for producing DNA encoding bovine interferons according to any one of claims 1 to 3, characterized in that the starting DNA is obtained from bovine tissues, preferably pancreas. 4) cloning the resulting fragments using the λ Charon phage vector; With 5 DNA isolates. The method of producing DNA encoding bovine leukocyte interferons according to claim 4, characterized in that the vector set is screened with a radioactive probe consisting of DNA encoding human leukocyte interferon fragments of human or other mammalian origin. 5) encoding the vectors thus produced into infectious mature lambda phage particles using an in vitro lambda envelope system, thereby generating phage sets, propagating the phage set, cultivating on bacterial fields, and encoding human or other mammalian interferon fragments. Using a radioactive probe made of DNA, hybridization screened, the appropriate DNA was isolated from the hybridized Idons, mapped by restriction analysis, and analyzed by Southern hybridization, and re-isolated after further cloning and sequencing. 6. The method of claim 5, wherein the vector set is screened with a labeled probe made from DNA encoding human leukocyte interferon fragments. A method according to claim 5 or 6 for the production of DNA encoding a bovine leukocyte interferon having the sequence of Figure 3a or selected in triplets of this sequence according to natural degeneration, characterized by isolating and mapping the DNA and then repeating it. after cloning, the DNA having the sequence described in the present disclosure is isolated. The method of claim 5 or 6 encoding a bovine leukocyte interferon encoded by bovine leukocyte interferon. 1A, or the sequence selected in the triplets of this sequence, corresponding to natural degeneration, characterized in that after isolating and mapping the DNA and then re-cloning, the DNA of the sequence is isolated. The method of claim 5 or 6 encoding a bovine leukocyte interferon encoded by 3c. 1A or a sequence selected in the triplets of this sequence according to natural degeneration, characterized in that after isolation and mapping of the DNA and subsequent cloning, the DNA of the sequence referred to herein is isolated. The method according to claim 5 or 6, encoding a bovine leukocyte interferon, 3d. 1A, or the sequence selected in the triplets of this sequence, corresponding to natural degeneration, characterized in that after isolation and mapping of the DNA and subsequent cloning, DNA of the sequence referred to herein is isolated. 11. The method of claim 4 for producing DNA encoding bovine fibroblast interferons, wherein the vector kit is screened using a labeled probe consisting of DNA encoding human or other mammalian fibroblast interferon fragments. 12. A11. The method of claim 1, wherein the vector set is screened with a radiolabelled probe formed from DNA encoding human fibroblast interferon fragments. The method of claim 11 or 12, encoding a bovine fibroblast interferon encoding a bovine fibroblast interferon. 3A, or triplets of this sequence for the production of DNA of the sequence selected according to natural degeneration, characterized in that after isolation and mapping of the DNA, and after repeated cloning, the sequences mentioned in the prior art19. HU 204086 Β DNA Dissociation. The method according to claim 11 or 12, encoding a bovine fibroblast interferon encoding a bovine fibroblast interferon. 1A, or having the sequence altered in the triplets of this sequence according to natural degeneration, characterized in that after isolation and mapping of the DNA, and subsequent re-cloning, the DNA of the sequence referred to herein is isolated. The method according to claim 11 or 12, encoding a bovine fibroblast interferon encoding a bovine fibroblast. 1A, or having the sequence altered in the triplets of this sequence according to natural degeneration, characterized in that after isolating and mapping the DNA and then re-cloning, the DNA of the sequence described herein is isolated. 16. The method of producing DNA encoding bovine immune interferons according to claim 4, wherein the vector set is screened with a labeled probe consisting of DNA encoding fragments of human or other mammalian animal immune interferons. 17. A16. The method of claim 1, wherein the vector set is screened with a DNA sample encoding human immuno-interferon fragments using an aldtottradioactive probe. 18. A process for the production of DNA encoding porcine interferons according to any one of claims 1 to 3, characterized in that the parent DNA is obtained from porcine tissues. 19. A18. The method for producing DNA encoding porcine leukocyte interferon according to claim 1, characterized in that screening of the phage set is carried out with a labeled probe consisting of DNA encoding human or other animal leukocyte interferon fragments. 20. A19. The method of claim 1, wherein the vector kit is screened with a labeled probe formed from DNA encoding human leukocyte interferon fragments. 21. A19. A method according to claim 14 or 20, encoding a porcine leukocyte interferon, 14a. 1A to generate DNA of the sequence selected from natural sequence degenerate, characterized by isolating and mapping the DNA and then, after re-cloning, deleting the DNA of the sequence referred to herein. 22. A18. A method of producing DNA encoding porcine fibroblast interferons according to claim 1, characterized in that the vector set is screened using a labeled probe consisting of DNA encoding fragments of human or other mammalian animal fibroblast interferons. The method of claim 22, wherein the screening of the phage set is performed with a labeled probe formed from human fibroblast interferon coding DNA. 24. A22. or a method according to claim 23 or 23, encoding a porcine fibroblast interferon; 5A, or by sequencing the DNA selected in the triplets of this sequence according to natural degeneration, characterized by isolating and mapping the DNA and, after re-cloning, isolating the DNA of the sequence referred to herein. 25. A18. A method for producing DNA encoding porcine immune interferons according to claim 1, characterized in that the vector kit is screened using a labeled probe consisting of DNA encoding human or other mammalian fibroblast interferon fragments. 26. A25. The method of claim 1, wherein the vector set is screened with a radioactive probe formed from DNA encoding fragments of human immune interferon. 27. A25. A method according to claim 14 or 26 which encodes a porcine immune interferon. 5A, or having the sequence altered in the triplets of this sequence according to natural degeneration, characterized in that after isolation and mapping of the DNA, and after repeated cloning, the sequences mentioned in the prior art are sequenced. 28. A method according to any one of claims 14 to 14 which encodes a feline fibroblast interferon. 1A, or having the sequence altered in the triplets of this sequence according to natural degeneration, characterized by isolating and mapping the DNA and, after re-cloning, deleting the DNA of the sequence referred to herein. 29. The method of producing rabbit interferon-encoding DNA according to any one of claims 1 to 5, wherein the starting DNA is obtained from rabbit tissues. 30. The method of claim 29 for producing DNA encoding rabbit immune interferon, 31. The method of claim 30, wherein the vector set is screened with a labeled probe formed from DNA encoding human fibroblast interferon fragments. 32. The method of claim 30 or 31, which encodes a rabbit immune interveron. 5A to generate DNA having the sequence altered in the triplets of the sequence SEQ ID NO. 33. A method of producing vectors encoding interferons of non-human mammals in a bacterial, yeast, or mammalian host cell, characterized by the use of any one of claims 1-3. claim 34. The method of claim 33 for producing vectors capable of expressing bovine interferon DNA in an aforementioned host cell, comprising inserting a DNA prepared according to claim 4. 35. The method of claim 34, for producing vectors capable of expressing bovine leukocyte interferons in said host cell, comprising inserting a DNA prepared according to claim 5 or 6. 36. The method of claim 35, wherein The DNA encoding the bovine leukocyte interferon having the amino acid sequence shown in FIGS. 37. The method of claim 35, wherein The DNA encoding the bovine leukocyte interferon having the amino acid sequence shown in FIGS. 38. A35. claim-based procedure 3c. The DNA coding for leukocyte interferon in the deer moiety having the amino acid sequence shown in FIG. 1A is constructed to insert a DNA prepared according to claim 9 into said host cell. 39. The method of claim 35, wherein the method of claim 3d. The DNA encoding the bovine leukocyte interferon having the amino acid sequence shown in FIGS. 40. The method of claim 34, for producing vectors capable of expressing DNA encoding bovine fibroblast interferons in any of said host cells, comprising inserting a DNA prepared according to claim 11 or 12. 40 that the vector set is screened with a labeled probe consisting of DNA encoding leukocyte interferon fragments of human or other animal origin. 41. The method of claim 40, wherein The DNA encoding the bovine fibroblast interferon having the amino acid sequence shown in FIGS. 42. The method of claim 40, wherein the method of claim 9b. The DNA encoding the deer fibroblast interferon having the amino acid sequence shown in FIGS. 43. A40. The method of claim 9c. The DNA encoding the bovine fibroblast interferon having the amino acid sequence shown in FIGS. 44. The method of claim 34 for producing vectors capable of expressing bovine immune interferons in said host cell comprising inserting a DNA prepared according to claim 16 or 17. 45. The method of claim 33 for producing vectors capable of expressing porcine interferons in said host cell, comprising inserting the DNA of claim 18. 46. The method of claim 45, wherein said DNA encoding porcine leukocyte interferons is expressed in a host cell, comprising inserting a DNA prepared according to claim 19 or 20. 47. The method of claim 46, which is a process according to claim 14a. The DNA encoding the porcine leukocyte interferon DNA of the amino acid sequence shown in FIGS. 48. The method of claim 45 for producing vectors capable of expressing porcine fibroblast interferons in said host cell comprising inserting the DNA prepared according to claim 22 or 23. 49. The method of claim 48, wherein The DNA encoding the porcine fibroblast interferon having the amino acid sequence shown in FIGS. 50. The method of claim 45, for producing vectors capable of expressing porcine immune interferons in said host cell comprising inserting a DNA prepared according to claim 25 or 26. 50, optionally one of 20 HU 204086 Β is inserted into a vector capable of expression in the host. 51. The method of claim 50, wherein the method of claim 14c. The DNA encoding the porcine immune interferon of the porcine amino acid sequence shown in Figure 1A is capable of expressing a vector capable of expressing in said host cell a DNA prepared according to claim 27. 52. The method of claim 33 for producing vectors capable of expressing DNA encoding rabbit interferons in said host cell, comprising inserting a DNA prepared according to claim 29. 53. The method of claim 52 for producing vectors capable of expressing rabbit immune interferon DNA in any of said host cells, comprising inserting a DNA prepared according to claim 30 or 31. 54. The method of claim 53 is a process according to claim 14e. The DNA encoding a rabbit immune interferon having the amino acid sequence shown in FIGS. 55. A 33-35. A method according to any one of claims 1 to 4 encoding a bovine leukocyte interferon HU 204086 Β A vector capable of expressing DNA in a said host cell under the control of a tryptophan operon, comprising: 1. digesting a 1.9 kb plasmid p83BamHT subclone with AvaH to a 612 base pair containing the bovine leukocyte interferon gene. isolating the fragment, supplementing it with Emi4H-formula, at the non-sticky ends, digesting the resulting DNA-tPstI and isolating the 92 base pair, blunt-ended PstI fragment; 2. partially digesting the same 1.9 kb plasmid p83BamHI subclone with PstI, isolating a 1440 base pair PstI-BamHI fragment; Plasmid pdelta Rfsrc 3 was digested with EcoRI, treated with S1 nuclease to create blunt ends, and the resulting DNA was digested with BamHI and the 4300 bp large fragment was isolated and the fragments obtained in steps 1, 2, 3 were ligated together , transforming the E. coli strain with the ligation mixture and selecting the plasmid containing the DNA coding for bovine leukocyte interferon, as described herein, based on analysis of plasmid DNAs of the transformed E. coli strains. 56. A method of producing transformed host cells capable of expressing interferons in non-human mammals, which comprises transforming with a primer vector according to claim 33. 57. The method of producing transformed prokaryotic host cells according to claim 56, wherein said prokaryotic host cell is transformed. 58. The method of claim 56 for the preparation of transformed yeast host cells, which comprises transforming a yeast host cell. 59. The method of producing transformed mammalian host cells according to claim 56, wherein the cells of a mammalian tissue culture are transformed. 60. Az56-59. Methods for producing bovine interferons according to any one of claims 1 to 5, characterized in that they are transformed with a vector produced according to claim 34. 61. The method of claim 10 for the production of transformed host cells expressing bovine leukocyte interferons, which comprises transforming with a vector according to claim 35. 62. A61. The method of claim 3a, wherein The bovine leukocyte interferon expressing transformed host cells having the amino acid sequence of FIG. 1A is characterized in that it is transformed with a vector prepared according to claim 36. 63. A61. The process of claim 3b. The bovine leukocyte interferon expressing the amino acid sequence shown in FIG. 1A is capable of producing transformed host cells which are transformed with a vector according to claim 37. The transformed host cell of claim 64 is cultured. 64. The method of claim 3 for producing transformed bovine leukocyte inter30 ferron-expressing transformed host cells having the amino acid sequence shown in Figure 3c, which is transformed with a claim 38 constructed vector. 65. A61. A method according to claim 1 to 3. The bovine leukocyte interferon expressing the amino acid sequence shown in FIG. 1A is capable of producing transformed host cells which are transformed with a vector of claim 39. 66. The method of claim 60 for producing transformed host cells capable of expressing bovine fibrohlast interferon, which comprises transforming with a vector produced in accordance with claim 40. A transformed host cell prepared according to claim 67, The method of claim 67, wherein the method of claim 9a is used. The bovine fibrohlast interferon having the amino acid sequence shown in Figure 1A is capable of producing transformed host cells which are transformed with a vector according to claim 41. The transformed host cell of claim 68 is cultured. 68. The method of claim 66, wherein the method of claim 9b. The bovine fibrohlast interferon having the amino acid sequence shown in Figures 1 to 4 is expressed to produce transformed host cells, which is transformed with a vector according to claim 42. The transformed host cell of claim 69 is cultured. 69. A66. the claim-based procedure in FIG. 5A to produce transformed host cells expressing bovine fibrohlast interferon having the amino acid sequence of FIG. 70. The method of claim 60 for the production of transformed host cells capable of expressing bovine immune interferons, wherein said vector is a vector prepared according to claim 44. 71. Az56-59. A method for producing transformed host cells capable of expressing porcine leukocyte interferons according to any one of claims 1 to 3, comprising transforming with a vector produced according to claim 45. 72. The method of producing transformed host cells capable of expressing porcine leukocyte interferons according to claim 71, which comprises transforming with a vector produced according to claim 46. 73. The method of claim 72, wherein 5A to produce transformed host cells expressing porcine leukocyte interferons of the amino acid sequence of FIG. 74. A71. The method of claim 1 for producing transformed host cells capable of expressing porcine fibroblast interferons, which comprises transforming with a vector of claim 48. 75. The method of claim 71, wherein 1 to 3, which comprises transforming with a vector produced according to claim 49, which is capable of expressing porcine fibrohlast interferon. 76. The transformed host cell of claim 71 capable of expressing porcine immune interferons Characterized in that it is transformed with a vector according to claim 50. 77. The method of claim 76, wherein the method of claim 14c. The porcine amino acid sequence of FIG. 1A is capable of producing transformed host cells expressing immune interferon, which is transformed with a vector of claim 51. 78. A method of producing transformed host cells capable of expressing rabbit interferons according to any one of claims 1 to 5, which comprises transforming with a vector produced according to claim 52. 79. The method of claim 78 for the production of transformed host cells capable of expressing rabbit immune interferons, which comprises transforming with a vector of claim 53. 80. The method of claim 79, which is a process according to claim 14e. Fig. 6A to produce transformed host cells capable of digesting immune interferons of rabbit according to claim 54, characterized in that it is transformed with a vector according to claim 54. 81. A method of producing interferons from non-human mammals comprising culturing a transformed host cell of claim 56 in a suitable medium and isolating the resulting interferon from the culture medium. 82. The method of claim 81, wherein the transformed prokaryotic host cell of claim 57 is cultured. 83. The method of claim 81, wherein the transformed yeast host cell of claim 58 is cultured. 84. The method of claim 91, wherein the transformed mammalian host cell of claim 59 is cultured. 85. A 81-84. The method of any one of claims 1 to 3, wherein the transformed host cell of claim 60 is cultured. 86. The method of claim 85, wherein the transformed host cell of claim 61 is cultured. 87. The method of claim 86, the method of claim 3a. A method for producing a bovine leukocyte interferon having the amino acid sequence of FIG. 6A, wherein the transformed host cell of claim 62 is cultured. 88. The method of claim 86, the method of claim 3b. A method for producing a bovine leukocyte interferon having the amino acid sequence shown in FIG. 6A, wherein the transformed host cell of claim 63 is cultured. 89. The method of claim 86, the method of claim 3c. The bovine leukocyte interferon having the amino acid sequence of FIG. 90. The method of claim 86, the method of claim 3d. A method for producing a bovine leukocyte interferon having the amino acid sequence shown in FIG. 6A, wherein a transformed host cell according to claim 65 is cultured. 91. The method of claim 85 for producing bovine fibroblast interferons, wherein the transformed host cell of claim 66 is cultured. 92. The method of claim 91, wherein The bovine fibroblast interferon having the amino acid sequence shown in FIG. 93. A91. The method of claim 9b. The bovine fibroblast interferon having the amino acid sequence shown in FIG. 94. The method of claim 91, wherein the method of claim 9c. The bovine fibroblast interferon having the amino acid sequence shown in FIG. 95. The method of claim 85 for producing bovine immune interferons, wherein the transformed host cell of claim 70 is cultured. 96. A 81-84. A method for producing porcine interferons according to any one of claims 1 to 3, characterized in that a transformed host cell according to claim 71 is cultured. 97. The method of producing porcine leukocyte interferons of claim 96, wherein the transformed host cell of claim 72 is cultured. 98. The method of claim 97, wherein A method for producing a porcine leukocyte interferon having the amino acid sequence of Figure 1c is characterized in that a transformed host cell according to claim 73 is cultured. 99. The method of claim 96 for producing porcine fibroblast interferons, wherein the transformed host cell of claim 74 is cultured. 100. The method of claim 99, which is a process according to claim 14b. A method for producing porcine leukocyte interferon having the amino acid sequence shown in Figure 1A is characterized in that a transformed host cell according to claim 75 is cultured. 101. The method of claim 96 for producing porcine immune interferons, wherein the transformed host cell of claim 76 is cultured. 102. The method of claim 101, wherein the method of claim 14c. A method for producing a porcine immune interferon having the amino acid sequence of Figure 1c is characterized in that a transformed host cell according to claim 77 is cultured. 103. A81-84. A process for the preparation of rabbit interferons according to any one of claims 1 to 3, characterized in that HU 204086 Β transformed host cell cultures. 104. A103. A method of producing rabbit immune interferons according to claim 1, wherein the transformed host cell of claim 79 is cultured. 105. A104. The method of claim 14e. A method for producing a rabbit immune interferon having the amino acid sequence shown in FIG. 106. A method of preparing an antineoplastic, antiviral or immune interferon animal pharmaceutical composition comprising the process of any one of claims 81-105. The animal interferon produced according to any one of claims 1 to 6, mixed with auxiliaries customary in the manufacture of a medicament, to form a pharmaceutical composition.