CS274401B2 - Method of polypetide production with sequence of animal interferon's amino acids - 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 the field of recombinant DNA technology, to compositions and methods utilizing such technology in the discovery of a large class of animal interferons, their production, various products of such manufacture, and their use.

More particularly, the present invention relates to the isolation and identification of DNA sequences encoding animal interferons and the construction of recombinant DNA expression vectors containing DNA sequences operably linked to expression promoter sequences and thus constructed expression vectors. In another aspect, the present invention relates to host culture systems such as various microorganisms and vertebrate cell cultures transformed with such expression vectors to direct expression of the above DNA sequences. In another aspect, the invention relates to compositions and methods for converting new end products of such expression, for example, pharmaceutical compositions that are useful in the prophylactic and therapeutic treatment of animals. In addition, the present invention relates to various processes useful in the production of the above DNA sequences, expression vectors, host culture systems, end products and components thereof, and the arrangement thereof.

The present invention is based on the discovery of DNA sequences and derived amino acid sequences encoding a number of bovine alpha interferons, including their 3- and 5-contiguous sequences, facilitating their in vitro binding to expression vectors. These discoveries have further enabled the development of means and methods for producing sufficient amounts of animal interferons by recombinant DNA technology. This in turn made it possible to determine their biochemical properties and their biological activity, so that their efficient production for commercial / biological use is possible.

A. Animal interferons

Interferon components were isolated from tissues of various phylogenetic species lower than humans. Efficacy studies of these interferons have shown that these interferons exhibit varying degrees of antiviral activity in the respective host animal. It has also been shown that the effect of these interferons is not always specific. For example, preparations of bovine interferons isolated from tissues had antiviral activity on monkey and human cells. Similarly, human interferons have been found to be effective on various cells of phylogenetically lower species.

This species interactivity is undoubtedly a consequence of the high degree of homologous conservation of interferons in both amino acid composition and sequencing. So far, however, this explanation has remained theoretical, since the amount and purity of the animal interferons obtainable were insufficient to carry out unambiguous characterization and experimentation on the biological properties of the purified components, as well as comparing them with some of their human counterparts.

Despite these low amounts and low purities, a causal link has been found between interferon and antiviral activity in the respective host animal. Thus, it has been desirable to produce animal interferons in high yields and purities in order to begin and successfully conduct experimental animal bioassays that would lead to commercial use in the treatment of animals for viral infections and for malignant, immunosuppressive and immunodepressive conditions. In addition, the production of isolated animal interferons would allow their physical and biological characterization. This would lay the basis for categorization and subsequent comparative studies with human counterparts of interferon particles.

Studies previously conducted with the animal interferons of the present invention are limited to the use of rather crude preparations due to their low availability, but indicate their very important biological functions. The class of animal interferons not only has therapeutic antiviral activity, but is effective as a prophylactic addition in vaccine and / or antibiotic treatment. Both clinically and commercially, they are very promising.

It seems that the application of recombinant DNA technology could be the most efficient way of obtaining the appropriately large amounts of animal interferons needed to be clinically and commercially available. Whether or not the materials so produced include glycosylation, which is considered to be characteristic of natural material, they are likely to exhibit bioactivity permitting their clinical use in the treatment of viral, neoplastic and immunosuppressive conditions or animal diseases.

B. Recombinant DNA technology

Recombinant ONA technology has recently reached a certain degree of development. Molecular biologists are able to recombine different DNA sequences quite easily. This creates new DNA entities capable of producing abundant amounts of exogenous protein products in transformed microbes. There are general means and methods for in vitro ligation of various DNA fragments (which can be aligned or overlapping one strand). This provides potential expression vectors useful for the transformation of particular organisms. Tin will affect the synthesis of the desired exogenous products by such organisms. However, on the basis of an individual product, this path remains largely unfinished. Science has not yet reached a stage where it can be regularly predicted that success can be achieved. Those who achieved successful results achieved it without the proper experimental bases and with considerable risk.

The plasmid, the extrachromosomal loop of double-stranded DNA found in bacteria and other microbes, often found in a cell in many copies, remains an essential element of recombinant DNA technology. The information that is encoded in the plasmid DNA is that which is required for the reproduction of the plasmid in the daughter cells (i.e., the origin of replication) and usually such information as one or more phenotypic selection characteristics, such as in the case of antibiotic resistance bacteria . This yields clones of the host cell containing the appropriate plasmid. These are then preferably cultured in a selective medium. The use of plasmids is based on the fact that they can be cleaved by one or another restriction endonuclease or restriction enzyme, whereby the respective restriction endonucleases or restriction enzymes recognize different sites on the plasmid DNA.

In this way, it is possible to insert heterologous genes or gene fragments into the plasmid by joining into the cleavage site to the reconstructed ends adjacent to the cleavage site. The so-called replication expression vectors are produced. DNA recombination occurs outside the cells. However, the resulting recombinant replication expression vector or plasmid can be introduced into cells by a method known as transformation. By cultivating the transformant, large quantities of recombinant vectors are then obtained. In addition, if the gene is appropriately inserted into those portions of the plasmid that control the transcription and translation encoded in the information DNA, the resulting expression vector can be used to actually produce a polypeptide sequence that is encoded by the inserted gene. This process is called expression.

Expression begins in a region known as a promoter. This is recognized and bound by RNA polymerase. In the transcriptional phase of expression, DNA develops; it acts as a template in the initial synthesis of information RNA (mRNA) from a DNA sequence. The information RNA is then translated into a polypeptide that has an amino acid sequence that is encoded in the mRNA by translation. Each amino acid is encoded by a nucleotide triplet codon. These then collectively represent the structural gene, i.e., the portion that encodes the emino acid sequence of the polypeptide product that results from expression. Translation starts at the start signal site (usually ATG, which becomes AUG in the resulting information RNA). The so-called stop codons define the end of translation and thereby the production of the next amino acid unit. This resulting product can be obtained by digesting (if necessary) host cells in microbial systems and isolating the product from other proteins by appropriate purification procedures.

In practice, recombinant DNA technology can express a fully heterologous polypeptide (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 putative bioactive product is sometimes bioneactive until the fused homologous / heterologous polypeptide is cleaved in the extracellular environment.

C. Cell culture technology

The cultivation of cells or tissues for the study of genetics and cell physiology is well known to those skilled in the art. There are means and methods for storing permanent cell lines prepared by successful serial transfers from isolated normal cells. For use in research, such cell lines are maintained on a solid support in a liquid medium or by culture in a suspension containing nutrients. Large-scale work is now annoyed only by mechanical problems.

The present invention is based on the discovery that recombinant ONA technology can be used to successfully produce animal interferons, preferably directly and in sufficient quantities to carry out clinical trials that are a necessary necessity prior to commercial approval. The product is suitable in all its forms for the prophylactic or therapeutic treatment of animals, particularly viral infections and malignant, immunosuppressive or immunodeficiency conditions. Product forms are various oligomeric forms, which may include glycosylation, as well as allelic variations of individual members or groups. These products are produced by systems of microorganisms or cell cultures that are controlled by genetic engineering methods. Thus, there are now opportunities to isolate and scramble animal interferons more effectively than ever possible. One important factor of the present invention, in its most preferred embodiment, is the ability to genetically control a microorganism or cell structure to produce a representative animal interferon, bovine interferon, in isolatable amounts produced by the host cells in maturation form.

The present invention encompasses animal interferons so produced and compositions and methods of making the same. The present invention also relates to replicative DNA expression vectors carrying gene sequences that encode animal interferons in a form capable of being expressed. Further, the present invention relates to microorganism strains or cell cultures that are transformed with the expression vectors described above, and to fermentation media containing such transformed strains or cultures capable of producing animal interferons.

In a further aspect, the present invention relates to various processes useful for preparing the above-mentioned interferon gene sequences, ONA expression vectors, microorganism strains, cell cultures and specific arrangements thereof. The invention also relates to the preparation of fermentation media of the above microorganisms and cell cultures. In certain host systems, such vectors may be derived to produce the desired animal interferon, which is secreted from the host cell in maturation form. The interferon, which contains a signal sequence derived from the 5-flanking region of the gene of interest, appears to be transported to the cell wall of the host organism where the signal portion cleaves off during secretion of the mature interferon product. This arrangement allows the isolation and purification of the desired maturation interferon without the need for procedures that eliminate contaminants of the intracellular host protein or cell debris.

In addition, the present invention specifically relates to the production of bovine interferon, which herein represents a group of animal interferons produced by direct expression in maturation form.

Expression of the mature animal interferon means the production of animal interferon by either microbial or cell culture. This animal interferon is not accompanied by a signal peptide or peptide sequence that directly accompanies the translation of the animal interferon mRNA. Thus, according to the present invention, a mature animal interferon is obtained which has methionine as the first amino acid (due to insertion of the ATG initiation signaling codon at the front of the structural gene) or methionine as the first amino acid is cleaved intracellularly or extracellularly. The maturation animal interferon according to the invention may also be produced together with a conjugated protein other than a conventional signal polypeptide. The conjugate is specifically cleaved in an intracellular or extracellular environment.

Finally, the mature animal interferon can be produced by direct expression without the need for cleavage of any extraneous excess polypeptide. This is particularly important when a given host does not remove or ineffectively remove a signal peptide in which the expression vector for the expression of the mature interferon is in association with the signal peptide. The parent interferon so produced is isolated and purified to be suitable for the treatment of viral, malignant, immunosuppressive or immunodeficiency conditions.

Animal interferons of the invention are interferons that are endogenous to animal organisms. These include (according to nomenclature analogous to human interferons) animal alpha (leukocyte), beta (fibroblast) and gamma (immune) interferons. All three series were identified in animal models. According to the example of bovine interferon, the animal alpha series consists of a family of proteins as in humans. These interferons are less homologous to the corresponding human alpha interferons than any of these animal interferons with each other or any of the human alpha interferons with each other. The Bovine Beta Council consists of a group of proteins that differ from human proteins. According to the present invention, particles and group hybrid interferons are obtained by taking advantage of conventional restriction sites in the genes of various animal interferons and recombining the corresponding portions by known methods.

The animal interferons of the invention are interferons that are normally endogenous to the following animal groups: birds, dogs, cattle, horses, cats, goats, sheep, fish and pigs. Particularly according to the invention, interferons of even-toed ungulates such as calves, sheep and goats are obtained. The interferons of the invention are used as antiviral and antitumor agents in the respective host animal. For example, bovine interferons could have practical application in the treatment of calves airways, either in conjunction with (known) antibiotics as a therapeutic component or with vaccines as a prophylactic component. Use for classes as described above could be extended to other cattle, goats, sheep, pigs, horses, dogs, cats, birds and fish. In horses, dogs, cats and birds, it can be expected that the antitumor effect of the corresponding interferons would be of particular commercial importance.

The following principle, which is described with respect to bovine interferon as a class representative, can be used in obtaining the animal interferons of the invention.

1. Bovine tissue, such as bovine pancreatic tissue, has been converted to freeze-dried powder; it was processed to yield bovine ONA (high molecular weight) upon cleavage of RNA and protein materials.

2. High molecular weight DNA is partially randomly cleaved due to the location of the genes.

3. The resulting DNA fragments are size fractionated. This yields fragments that contain from 15,000 to 20,000 bp (= base pairs, preferably nucleotide pairs).

4. The resulting fragments of step 3 were cloned using the? T Charon 30 phage vector.

5. The vectors thus prepared are packaged in vitro in infectious phage particles containing rDNA. This yields a phage bank. This is increased to about 10-fold by bacterial cell proliferation. The phage is sown into confluence on the lawn of bacteria. Hybridization is probed with a radioactive human interferon probe.

6. The corresponding DNA is isolated from the appropriate clones, the restriction map is assembled and analyzed by Southern hybridization. Restriction fragments that contain bovine interferon genes are subcloned into plasmid vectors. Then they sequenced.

7. The sequenced DNA is then adapted for in vitro insertion into an appropriate expression vector, which is used to transform the respective host cell. This is then grown in the culture medium to express the desired product, bovine interferon.

8. The bovine interferon thus produced contains 166 amino acids in mature (maturation form) and 23 amino acids in sequence. It has a very hydrophobic character. Its molecular weight was calculated to be about 21,409. It has similar characteristics to human leukocyte interferons. It was found to be about 60% homologous to human leukocyte interferon.

Accordingly, the present invention provides a method for producing polypeptides having an amino acid sequence of animal interferons by culturing an E. coli or yeast microorganism or a CHO cell line cell culture. The microorganism or cell line is transformed with a replicable vector capable of expressing the DNA sequence encoding said polypeptide. The conjugated polypeptide is then recovered.

Description of preferred arrangements. A. Microorganisms / cell cultures

1. Bacterial strains / promoters

This work uses inter alia the E.coli K-12 strain 294 (end A, thi, hsr, _k hsm ⁺ ) (25). This strain is deposited in the American Type Culture Collection, ATCC No. 31,446. However, various other microbial strains, including known strains of E. coli, such as E. coli 8, E. coli X 1776 ( ATCC No. 31,537), E.coli W 3110 (F-, ^- , prototrophic) (ATCC No. 27,325), E.coli DP 50 SuPF (ATCC No. 39,061, deposited

5 March 1982), E.coli JM83 (ATCC No. 39,062, deposited 5 March 1982) or other microbial system. Many of them are stored and (potentially) available from recognized microorganism storage institutions such as the American Type Culture Collection (ATCC) - see ATCC catalog list. Other microorganisms include, for example, Bacilli, such as Bacillus subtilis and other enterobacteria. These include, for example, Salmonella typhimurium and Serratia marcesans, which utilize plasmids that can provide replication and expression of heterologous gene sequences.

Examples of preferred uses for initiating and maintaining microbial production of heterologous polyppeptides include the beta lactamase and lactose promoter systems. A system based on the tryptophan operon, the so-called trp promoter system, has recently been developed. Details concerning the design and construction of this system have been published by Goeddel et al. and Kleid et al. The discovery and exploitation of numerous other microbial promoters and details regarding their nucleotide sequences have been reported. This allows skilled artisans to functionally ligate them with plasmid vectors.

2. Yeast strains / yeast promoters

A plasmid YRp7 that is capable of selection and replication in both E. coli and Saccharomyces cerevisiae yeast can also be used for the expression system of the invention. For yeast selection, plasmid TRP1 contains a gene (tryptophan-1 gene) that complements (allows growth in the absence of tryptophan) yeasts containing mutations in this gene found on chromosome IV of yeast. One useful strain is the RH218 strain deposited with the American Type Culture Collection (ATCC No. 44,076). However, it is to be understood that any Saccharomyces cerevisiae strain that contains a mutation that provides a terminal trp1 could be an effective medium for expression of a plasmid containing an expression system. An example of another strain that can be used is pep4-1. This tryptoan auxotrophic strain also has a mutation in the TRP1 gene.

If a 5-flanking DNA sequence (promoter) from a yeast gene (for alcohol dehydrogenase 1) is placed on the 5 ' side of the non-yeast gene, it may promote expression of the foreign gene in yeast (after placement into a plasmid that is used to transform yeast). In addition, the promoter of appropriate expression by the non-yeast gene in yeast requires that the second yeast sequence be located at the 3-terminus of the non-yeast gene on the plasmid. This allows for appropriate polyadenylation and termination of transcription in yeast. This promoter as well as others can be advantageously used in the present invention - see above. In a preferred embodiment, the 5-flanking sequence of the yeast PGK (3-phosphoglycerate kinase) gene is placed against a structural

CS 274401 D2 gene is followed by DNA which contains termination-polyadenylation signals, for example TRP1 gene or PGK gene.

Since the yeast 5-flanking sequence (along with the 3-yeast termination DNA) (see above) can promote the expression of foreign genes in yeast, it seems likely that the 5-flanking sequence of any high-expression yeast gene could be used to express important gene products. Since in some circumstances up to 65% of their soluble protein is expressed as glycolytic enzymes, and since this high level appears to be due to the production of high levels of individual mRNAs, it would be possible to use 5'-flanking sequences of any other glycolytic gene enolases, glyceraldehyde-3-phosphate dehydrogenases, hexokinases, pyruvate decarboxylases, phosphofructokinases, glucose-6-phosphate isomerases, 3-phosphoglycerate mutases, pyruvate kinases, triosophosphate isomerases, phosphoglucoisomerases and glucokinase. Any 3-contiguous sequence of these genes can be used for appropriate termination and mRNA polyadenylation in such an expression system - see below. Some other genes that show high expression are genes for the production of acid phosphatases and those that show high levels of expression products due to mutation in the 5-flanking regions (mutants that increase expression) - usually due to the presence of TY1 transportable element.

All of the above genes are transcribed by yeast RNA polymerase II. It is conceivable that RNA polymerase I and III promoters that transcript ribosomal RNA, 5S RNA and tRNA acid genes may also be useful in such expression constructs.

Finally, many yeast promoters also contain transcriptional control, so that they can be turned off or on by changing the culture conditions. Some examples of such yeast promoters are genes that produce the following proteins: alcohol dehydrogenase II, isocytochrome-c, acid phosphatase, degradative enzymes associated with nitrogen metabolism, glyceraldehyde-3-phosphate dehydrogenase, and enzymes that are responsible for the use of meltose and galactose . Such a control strand would be very useful in controlling the expression of a protein product - especially when the product is toxic to yeast. It would also be possible to insert a control region of one 5'-flanking sequence with a 5'-flanking sequence that contains a promoter from a gene that shows high expression. The result would be a hybrid promoter. This would be possible because the control region and the promoter are apparently physically different DNA sequences.

3. Cell culture systems / cell culture vectors

In recent years, the proliferation of vertebrate cells has become routine. COS-7 monkey kidney fibroblast lines may be used as hosts for the production of animal interferons. However, the experiments described in detail herein may be performed in any cell line capable of replicating and expressing a compatible (compatible) vector, e.g.

WI38, ΒΗΚ, 3T3, CHO, VERO and HeLa cell lines. The expression vector further requires that the origin of replication and the promoter be placed at the front of the gene (to be expressed) along with the necessary ribosomal binding sites, RNA splicing sites, polyadenylation site, and transcription termination sequences. Although these essential elements of SV40 will be used herein, it is to be understood that, although the present invention is described herein in terms of a preferred embodiment, the preferred embodiment is not designed to be limited to said sequences. For example, origins of replication of other viral vectors (eg, Polyoma, Adeno, VSV, BPV, etc.) can be used as well as cellular origins of DNA replication that can function in an unintegrated state.

B. Vector systems. 1. Direct expression of mature bovine interferon in E. coli

The method used to obtain direct expression of bovine interferon in E. coli as a maturation interferon polypeptide (minus signal sequence) comprises combining a plasmid containing a promoter fragment and a translation initiation signal with an engineered animal genomic DNA fragment containing a coding region for maturation interferon .

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CS 274401 Β2

2. Expression in yeast

In order to express a gene, such as animal interferon DNA, in yeast, it is necessary to construct a plasmid vector that contains four components. The first component is the part that allows transformation of both E.coli and yeast. Thus, this portion must contain a selectable gene from each organism, such as the ampicillin resistance gene from E. coli and the TRP1 gene from yeast, and this component also needs to maintain the origin of replication from both organisms as plasmid DNA in both organisms, such as E. coli origin from pBR322 and arsl origin from yeast chromosome III.

The second component of the plasmid is a 5-flanking high-expression sequence from a yeast gene that transcribes a downstream structural gene, such as a 5'-flanking sequence from the yeast PGK (3'-phosphoglycerate kinase) gene.

The third component of this system is a structural gene, which is constructed to contain both ATG initiation and termination (translation) termination signals. Isolation and construction of such a gene is described herein above.

The fourth component is a yeast DNA sequence comprising a 3-flanking sequence from the yeast gene, which contains the appropriate transcription termination and adenylation signals.

3. Expression by mammalian cell culture

The strategy of synthesizing immune interferon by mammalian cell culture is based on the development of a vector capable of both autonomous replication and expression of a foreign gene under the control of heO in a terologic transcriptional unit. Tissue culture replication of this vector can be accomplished by obtaining an origin of DNA replication and a helper function (T antigen) by introducing the vector into a cell line endogenously expressing the antigens. The SV40 promoter precedes the interferon structural gene and provides for transcription of the gene.

A useful vector for obtaining expression consists of the pBR322 sequence. It represents a selectable marker for selection in E. coli (ampicillin resistance) as well as E. coli origin of DNA replication. These sequences are derived from plasmid pML-1 and include the EcoRI and BamHI restriction sites. The SV40 origin is derived from the 342 bp PvuII-HindIII fragment encompassing this region (both ends being converted to EcoRI ends). These sequences, in addition to containing the viral origin of ONA replication, encode a promoter for both the upstream and downstream transcriptional units. The orientation of the SV40 origin region is such that the promoter of the later transcriptional unit is located adjacent to the gene encoding the interferon.

The invention is illustrated by the accompanying drawings, in which:

Figure 1 shows Southern hybridization of a) human, b) bovine, and c) porcine genomic

DNA digested with EcoRI and hybridized at various concentrations of formamide 570 bp with EcoRI frag3 with a 2 (labeled p) containing the human leukocyte interferon hybrid coding region. Hybridization in 20 percent formamide yields the clearest multigene sample of the bovine and porcine leukocyte interferon group.

Giant. 2 shows Southern hybridization of four different phage recombinants of bovine genomic DNA digested with EcoRI, BamHI and HindIII and hybridized with a P-labeled human leukocyte gene probe. Clone 83 provides two hybridization fragments, each with a restriction enzyme.

Giant. 3a depicts a portion of the nucleotide sequence from the plasmid subclone p83BamHI1, 9 kb, and the derived amino acid sequence encoded therein by bovine leukocyte interferon. The signal peptide is an amino acid residue from S1 to S23.

Giant. 3b shows the nucleotide sequence and deduced amino acid sequence of the second bovine leukocyte interferon (o (2) plasmid subclone p67EcoRI3.2 kb).

Giant. 3c shows the complete maturation nucleotide sequence and deduced amino acid sequence of the third bovine leukocyte interferon (? 3) from the plasmid subclone p35EcoRI-BamHI3.5 kb.

CS 274401 82

Giant. 3d shows the nucleotide sequence and deduced amino acid sequence of the fourth bovine leukocyte interferon from the plasmid subclone p83EcoRI-BamHI2.9 kb.

The signal is amino acid residue S1 to S23. The maturation protein consists of 172 amino acid residues. It should be noted that the last extension of the six amino acid residues is attributed to a change in nucleotide base at position 511. This allows 6 additional translation codons before the termination signal.

Giant. 4a, 4b and 4c compare the amino acid sequences of BoIFN- <Z1, <Z2, o3, and <Z4 with the sequences of 11 known human interferons. The amino acids that are retained for all human leukocyte interferons, all bovine leukocyte interferons and position are also listed. in most human leukocyte interferons due to bovine eyes and << 4.

Giant. 5 is a schematic diagram of the construction of the bovine leukocyte interferon expression plasmid pBoIFN-X.ltrp55. Starting materials are the trp expression vector pdeltaRIsrc and the BamHI fragment from the plasmid subclone p83BamHI, 9 kb.

Giant. 6 shows Southern hybridization of bovine DNA digested with either EcoRI or KindIII, BamHI, BglII or PvuII with a radioactive probe derived from BoIFN-Z1 or BoIFN- <Z4 gene fragments. Each IFN is preferentially hybridized with different subgroups of BoIFN genes.

Giant. 7 shows a restriction map of genomic bovine DNA inserts from three phage recombinants that hybridize to human fibroblast by a human interferon gene probe. The location and orientation of IFN- / 3 beef is indicated by a black rectangle. Restriction sites, which are marked with an asterisk, indicate that this is partial information in the restriction map.

Giant. 8 shows a detailed restriction map of the three genes shown in Figure 7; the shaded portion indicates the signal sequence, the shaded portion indicates the maturation sequence.

Giant. 9a, 9b and 9c describe the nucleotide and deduced amino acid sequences for the BoIFN- / 31, 2 and 3 genes.

Giant. 10 compares the amino acid sequences of the three BoIFN-ft genes with human IFN- / 3.

Giant. 11 is a Southern blot of FIG. 2 hybridized with a probe of the BoIFN- / 31 gene under conditions in which only one hybridization fragment would generally be evident when analogous experiments with the human genomic DNA and the human IFN- / 3 gene are performed.

Giant. 12 schematically describes a strategy that was used to express all three BoIFN- / 3 genes under the control of the E. coli trp operon.

Giant. 13 shows a comparison of the derived amino acid sequences of BoIFN- / ¹ , HuIFN-, and murine IFN- / ⁴ . at

The following detailed description illustrates the invention with respect to the preparation of various animal interferons by recombinant DNA technology and discloses generally applicable methods for preparing particularly bovine leukocyte interferons. The method of preparation is described on a bacterial system.

A. Isolation of bovine DNA

For the purpose of constructing an animal gene bank, high molecular weight DNA is isolated from animal tissue by modifying the procedure of Blin and Stafford. This DNA is randomly frag.

is assembled with respect to gene placement and is size fractionated to yield 10,000 to 20,000 bases (nucleotides) for cloning into a lambda phage vector.

The frozen tissue, such as bovine pancreas, is ground to a fine powder in liquid nitrogen. It is then transferred to a solution (solubilization) containing 0.25M EDTA, 1% Sarcosyl and 0.1 mg / ml proteinase K (25 ml / gram tissue) for three hours at 50 ° C. Proteins are removed from the obtained viscous solution by three phenol extractions and one chloroform extraction. The residue was dialysed in 50 mM Tris-HCl (pH = 8), 10 mM EDTA, and 10 mM sodium chloride, and then cleaved for two hours at 37 ° C with heat treated pancreatic ribonuclease (0.1 mg / ml).

After extraction with phenol and ether, the DNA is precipitated with two volumes of ethanol, washed with 95¾ ethanol, lyophilized and redissolved overnight at 4 ° C in TE buffer (1 mM Tris-HCl (pH = 7.5), 1 mM EDTA) to final a concentration of 1-2 mg / ml. According to 0.5¾ neutral agar gel electrophoresis, the final DNA had a length of more than 100 kilobases (kb; kilonucleotides)

Partial endonuclease digestion of bovine DNA and size fractionation

0.1 mg of bovine DNA is digested at 37 ° C for 60 minutes with 1.25, 2.5, 5 and 10 units of Sau3A in a reaction (1 ml) containing 10 mM Tris-HCl (pH-7.5), 10 mM magnesium chloride and 2 mM dithiothreitol. Incubation is stopped by adding EDTA to 25 mM concentration. This is followed by phenol extraction, ether extraction, addition of sodium acetate to 0.3 M concentration (pH = 5.2) and precipitation with three volumes of ethanol. DNA is redissolved in TE buffer at 68 ° C and sedimented with a 10 to 40¾ linear sucrose gradient in a Beckman SW 27 SW at 27,000 rpm for 22 hours at 20 ° C. Fractions (0.5 ml volume) are analyzed on 0.5¾ gel using Eco R1 digested Charon 4A DNA as a molecular weight standard. Those fractions containing 15 to 20 kb DNA fragments are pooled, precipitated with ethanol and redissolved in TE buffer.

C. Construction of a bovine genomic DNA bank

Cloning of 15-20 kb bovine DNA yielded a lambda Charon 30 A vector with G-A-T-C misaligned ends, which were obtained by removing two internal Bam HI fragments from the phage.

Chron 30 A is cultured in E. coli strain DP 50 SupF (ATCC No. 39061, deposited March 5, 1902) in NZYDT medium, concentrated by precipitation with polyethylene glycol and purified by gradient centrifugation. Phage DNA was prepared from purified phage by two times phenol extraction and then one phenol-ether extraction. It is concentrated by precipitation with ethanol.

To prepare Charon 30A end fragments, 50 µg of phage DNA was annealed for 2 hours at 42 ° C in 0.25 ml of 50 mM Tris-HCl (pH 8), 10 mM magnesium chloride and 0.15 M sodium chloride, completely digested with Bam HI, extract with phenol and ether and sediment in a 10 to 40¾ sucrose gradient as described above. Fractions containing the 32 kb annealed portions of the phage were pooled and precipitated with ethanol.

The purified Charon 30 A portions (6 micrograms) were annealed again at 42 ° C for two hours, combined with 0.3 µg of 15-20 kb bovine DNA and 400 units of phage T4 polynucleotide ligase, and incubated overnight at 12 ° C in 0.07 ml reaction mixture containing 50 mM Tris-HCl (pH = 7.5), 10 mM magnesium chloride, 20 mM dithiothreitol and 50 microorganisms / ml bovine serum albumin. The ligated DNA is then packaged into the mature lambda phage particles using an in vitro lambda packaging system.

The components of this system - sonic extract (SE), frost lysate (FTL), protein A with buffers A and M1 - were prepared as described. Aliquots of the ligated three microliter DNA mixture were incubated with 15 microliters of Buffer A, 2 µl Buffer M1, 10 µl SE and 1 µl protein AO for 11 minutes at 27 ° C. During 45 minutes, FTL in ice melted, combined with 0 1 volume of M1 and centrifuged at 35,000 rpm at 4 ° C for 25 minutes. 0.075 ml of the supernatant was added to the above reaction mixture. After an additional two hours incubation at 27 ° C, a small portion of the packaging reaction is titrated to the DP 50 SupF strain - see below. A total of about 1.1.1 (/ independent bovine recombinant DNA) was obtained. The remainder of the wrapping mixture was propagated by plate-lysate by screening recombinants on DP 50 SupF at a density of 10,000 PFU (plaque forming units) on a 15 cm NZYDT agar plate.

D. Screening of phage banks of bovine interferon genes

The strategy used to identify phage recombinants carrying bovine interferon genes is to detect nucleotide homology by radioactive probes prepared from cloned human leukocyte, fibroblast and immune interferon genes. The hybridization conditions are given by Southern blots of genomic animal DNA. Five micrograms of each of the high molecular weight DNA (prepared as described above) from human placenta, bovine pancreas, and porcine submaxillary glands were completely digested with EcoRI, then electroplated on 0.5% agar gel and transferred to nitrocellulose paper. The β-labeled ONA probe was prepared from a 570 bp EcoRI fragment containing a protein encoding the mature leukocyte interferon (A / D hybrid) coding region at a Bgl II restriction site according to standard procedures reported in the literature. Each nitrocellulose filter is prehybridized at 42 ° C overnight in 5 x SSC (sodium citrate in saline), 50 mM sodium phosphate (pH = 6.5), 0.1 mg / ml sonicated salmon sperm DNA, 5 x Denhardt solution, 0.1% sodium dodecyl sulphate, 0.1% sodium phosphate containing either 10, 20 or 30% formamide and then hybridized with a labeled probe at 100.10 pulses per minute in the same solution containing 10% sodium dextran sulfate. After incubation overnight at 42 ° C, the filters were washed four times in 2 x SSC, 0.1% SDS (sodium dodecyl sulphate) at room temperature, once in 2 x SSC, and then exposed to Kodak XR-5 X-ray film overnight. Dupont Cronex contrast filter. The highest number of hybridizations was detected in bovine and porcine DNA grafts when hybridization was performed with 20% formamide. This result demonstrates the multigenicity of the leukocyte interferon genes in cows and pigs analogously to that previously described in human literature. The same hybridization conditions were used to screen for interferon genes in a bovine DNA bank.

500,000 phage recombinants were plated on a DP 50 SupF plate at a density of 10,000 PFU / 15 cm plate. Copies of each nitrocellulose paper sheet were prepared according to the method of Benton and Davis. The filters were hybridized with a human LelF gene probe as described above. Ninety-six hybridized plaques were obtained which gave strong signals during repeated sresning.

Fibroblast and immune interferon genes were also screened in a bovine bank. The probes were made with a 502 bp Xba I - Bgl III fragment containing the full-length maturation human fibroblast interferon gene, a 318 bp Alu I fragment (containing amino acids 12 to 116) and a 190 bp Mbo II fragment (containing amino acids 99 to 162) from the maturation coding region of the human immune interferon gene. Hybridization 1,2. A total of 26 bovine fibroblasts and 10 bovine immune interferon clones gave 10 ° phage recombinants.

E. Characterization of phage recombinants.

Phage DNA was prepared as described above from 12 recombinants that were hybridized with a human leukocyte interferon probe. Each DNA was digested separately and in combination with EcoRI, Bam HI and Hind III; the product was subjected to electrophoresis on a 0.5% agar gel. Hybridization sequences were mapped by Southern method. A comparison of the self-digested DNAs from clones 10, 35, 78 and 83 is shown in Figure 2. For each phage, the observed restriction fragment sizes and the corresponding hybridization matrices were found to be different and not overlapping. That is, each of these four phages carries a different bovine interferon gene. In addition, it was found that cleavage of clone 83 by each of the above three enzymes yielded in each case two discrete hybridization bands; that is, this recombinant can carry two closely linked interferon genes.

F. Subcloning of bovine leukocyte interferon genes.

Restriction fragments from three phage recombinants that are hybridized with the human leukocyte gene probe were subcloned at the cloning site of the multi-restriction enzyme pBR322 derivative, pUC9. Plasmid pUC9 was derived from pBR322 by first removing the 2067 bp EcoRI-PvuII fragment containing the tetracycline resistance gene, then inserting the 425 bp Haell fragment and the phage M13mP9 into the Haell site of the resulting plasmid at position 2352 (relative to p8R322 notation). . The Haell fragment from the mp9 phage contains the N-terminal coding region of the E.coli lacZ gene, into which a multi-restriction enzyme cloning site having the sequence CCA AGC TTG GCT GCA GGT CGA CGG ATC CCC GGG was inserted between the 4th and 5th amino acid residues of β-galactosidase . Insertion (insertion) of the foreign DNA fragment into these cloning sites interrupts the conti11

CS 274401 Blusion between the lac promoter and the lacZ gene. This changes the phenotype of the transformed plasmid JM83 from lac ⁺ to lac.

The above fragments are: a) a 1.9 kb Bam HI fragment and a 3.7 kb EcoR1 fragment from clone 83 (which corresponds to non-overlapping segments of the same recombinant), b) a 3.5 kb BamHI-EcoR1 fragment from clone 35 and c) 3.2 EcoR1 fragment from clone 67. In all cases, 0.1 µg of the appropriately digested vector was ligated with a ten-fold molar excess of the purified fragment, transformed into E. coli strain 3M83 (ATCC No. 39,062, deposited March 5, 1982) and Sieve on M9 plates containing 0.04 mg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside and 0.2 mg / ml ampicillin. White colonies that purportedly carry the DNA insert at the restriction site interrupting the lacZ gene coding region in pIC15 were transferred to 5 ml LB (culture medium) with 0.02 mg / ml ampicillin, cultured for several hours at 37 ° C and then tested for insertion. fragment by plasmid DNA miniprep procedure.

G. DNA sequence of the bovine leukocyte interferon gene on clone 83.

The ONA sequence from the Bam HI site of plasmid p83BamHI1, a 9 kb (1.9 kb fragment subclone of clone 83) was determined by Maxam-Gilbert chemistry, as known in the literature.

This sequence is shown in Figure 3. The longest open reading frame encodes a 189 amino acid polypeptide with significant homology to human leukocyte interferons (Figure 4). Analogously to human proteins, bovine leukocyte interferon consists of a hydrophobic signal peptide of 23 amino acids, which precedes the mature amino acid protein of 166 amino acids with an identical sequence: ser - leu - gly - cys. The four cysteine residues at positions 1, 29, 99 and 139 are precisely retained in these portions. The homology of bovine and human interferon is compared in Table 1. As expected, bovine protein is substantially less homologous (of about 60 O with any human protein as opposed to mutual homology of human proteins to each other (greater than 80%)).

The ONA sequences and deduced amino acid sequences of the other three bovine leukocyte interferon genes that appear on the plasmid subclones p67EcoRI3.2 kb, p35EcoRI-BamHI3.5 kb and p83EcoRI3.7 kb are shown in Figures 3b, 3c and 3d, respectively.

From Table 1, it can be seen that while the BoIFN-α2 and BOIFN-oC3 genes encode peptides with only minor differences relative to BoIFN-c0, BOIFN-c <4 differs from any other bovine peptide as much as any bovine leukocyte interferon differs from human leukocyte interferon.

To ensure that the α 4 gene is derived from the same broad class of cellular proteins as other BoIFN α-genes, genomic bovine DNA was digested with several restriction endonucleases and hybridized with radioactive DNA fragments representing the coding regions of the α / 1 protein (612 bp Avall fragment) and the o4 (Eco-RI-XmnI fragment of pBoIFN-o64trp15) genes in 50¾ formamide, which does not allow hybridization of the two genes. Preferably, each gene hybridizes to a different series of bovine DNA fragments. These results clearly demonstrate the existence of two different groups of bovine leukocyte interferons, where the α 1 and α 4 proteins can be considered as their representatives.

Table 1

Comparison of coding sequences of bovine and human eye interferons

etl o62 </ 3 J.4 ciA <ZB e £ C oíD oíF << H dl CÍJ <ZK BoIFN- target 94 92 54 61 62 63 64 61 64 63 61 65 BoIFN oí 2 96 91 53 61 61 64 63 62 63 63 64 64

T a h at 1 k and 1 - continuation

Zl Z2 Z3 Z4 FOR ZB zc ZD ZF ZH Zl Z3 ZK BoIFN- Z3 100 ALIGN! 96 45 BoIFN-Z 4 52 50 52 54 54 58 55 56 58 56 54 54 HuIFN- ZA 56 52 39 81 81 83 82 83 81 80 86 HuIFN-, / B 43 39 48 70 81 77 81 80 79 79 81 HuIFN- J.C 61 57 52 70 65 81 89 86 94 92 83 HuIFNIC D 52 48 48 74 61 65 83 81 80 78 84 HuIFN- ZF 61 57 52 n 70 65 100 ALIGN! 65 83 89 86 83 HuIFN-ZH 56 52 52 74 74 74 83 74 84 84 84 HuIFN- Zl 61 57 52 70 65 100 ALIGN! 65 100 ALIGN! 74 91 81 HuIFN-Z J 56 52 40 61 57 91 70 91 65 91 80 HuIFN- ZK 52 48 48 83 70 74 91 74 91 74 65

The numbers indicate percent homology.

The lower left half refers to a 23 amino acid presection.

The upper right half refers to the mature amino acid protein of 166 amino acids.

A, 0, C etc. means human leukocyte interferons A, B, C etc.

H. Direct expression of maturation BoIFN-Z1 in E.coli.

The construction of the direct expression plasmids is summarized in FIG. 5. The plasmid subclone p83BamHI, 9 kb was completely digested with Ava II. The 612 bp fragment containing the bovine leukocyte interferon gene was isolated by 6% polyacrylamide gel electrophoresis and electroelution. Approximately 1.5 µg of this fragment was digested with Fnu 4 H, extracted with phenol and ether and precipitated with ethanol. The resulting Fnu4H overlapping ends are made up to the blunted ends with six units of DNA polymerase I (Klenow fragment) at 12 ° C for 30 minutes in 20 microliters containing 20 mM Tris-HCl (pH = 7.5), 10 mM magnesium chloride, 4 mM dithiothreitol and 0.1 mM dATP, dGTP, dCTP and dTTP. After extraction with phenol and ether, the DNA was digested with PstI and then subjected to electrophoresis on a 6% gel. The resulting blunt-ended Pst1 fragment containing 92 bp (bp) starting from the first nucleotide coding region of the bovine leukocyte interferon coding region is electroeluted from the gel.

The remainder of the maturation coding region is isolated as follows. Three micrograms of Bam HI insert from 1.9 kb pO3 BamHI was partially digested with 14 PstI units for 10 minutes at 37 ° C in a 45 microliter reaction containing 10 mM Tris-HCl (pH = 7.5), 10 mM magnesium chloride and 2 mM dithiothreitol, moss. is extracted with phenol and ether. The desired 1440 bp partial PstI-BamHI fragment, starting at nucleotide 93 of the maturation coding region, is isolated on a 6¾ polyacrylamide gel.

The plasmid pdeltaRIsrc is a derivative of plasmids pSRCex16, in which the EcoRI site, in addition to the trp promoter and away from the src gene, is removed by DNA polymerase I and complemented with the oligonucleotide AATTATGAATTCAT (synthesized phosphotriester method as reported). Twenty micrograms of pdeltaRIsrc is completely digested with EcoRI, extracted with phenol and ether and precipitated with ethanol. The plasmid was then digested with 100 units of nuclease S1 at 16 ° C for 30 minutes in 25 mM sodium acetate (pH = 4.6), 1 mM zinc chloride, and 0.3 M sodium chloride. Creates

CS 274401 02 thus aligned ends with the ATG sequence. After phenol-ether extraction and ethanol precipitation, the DNA was digested with Bam HI, electrophoresed on a 6% polyacrylamide gel, and a large vector fragment (4300 bp) was isolated by electroelution.

The expression plasmid was prepared by ligation of a 0.2 / µg vector, 0.02 µg of the 92 bp blunt-ended Pst1 fragment, and 0.25 micrograms of the 1,400 bp partial Pst1-BamHI fragment along with 400 T4 DNA ligase units overnight at 12 ° C. This plasmid was used to transform E. coli strain 294 (ATCC # 31446) into ampicillin resistance. Plasmid DNA was prepared from 96 colonies and digested with XBA I and Pst I. Ninety of these plasmids contained the desired 103 bp XbaI-PstI and 1050 bp Pst I fragments. DNA sequence analysis confirmed that some of these plasmids have the ATG initiation codon correctly positioned at the beginning of the bovine interferon coding region. One of these plasmids - pBoIFN- cCltrp55 was selected for further study.

I. Direct expression of the second class of mature bovine leukocyte interferon (<Z4) in E. coli

The ATG initiation codon is placed at the front of the maturation coding region by enzymatic extension of the synthetic DNA primer, CATGTGTGACTTGTCT. This heptadecameree phosphorylates the T4 poles by 32 nucleotide-kinase and / · -P ATP as previously described in the literature. 250 picomoles of primer were combined with approximately 1 microgram of a 319 bp HindIII fragment containing amino acid residues S20 to 102 in 30 microliters of water, boiled for five minutes, and then set at 25 ° C for 3 hours with 25 units of E. coli DNA polymerase I ( Klenow fragment). The product of this reaction is cleaved with HgiAl. The resulting 101 bp blunted HgiAI fragment is isolated from a 6¾ polyacrylamide gel.

The entire maturation peptide gene was assembled downstream of the trp promoter by enzymatic ligation of the above fragment with the 508 bp HgiA-PstI fragment containing the terminal carboxy-portion of the peptide and the HuIFN-expression plasmid, pIFN - / < Klenow DNA polymerase, digested with PstI and finally isolated on a 6¾ polyacrylamide gel. After transformation of the resulting mixture into E. coli 294, several clones were identified which had a restored EcoRI recognition site between the trp promoter-ribosome binding site of the original expression vector and the complete coding region of the mature bovine interferon pBoIFN-? 4trp15.

0. Subcloning of bovine fibroblast inteferon genes.

at

Six phage recombinants that hybridize with the human IFN- / 3 DNA probe are purified. Their DNA is isolated as described above for further analysis. Restriction mapping together with Southern-hybridization analysis show that 6 isolated recombinants contain three different regions of the bovine genome, so that they include the multigen (multigagen) BoINF- /) grouping.These results are summarized by the restriction maps shown in Figure 7. In order to obtain a more detailed restriction map and nucleotide sequence for each additional class of recombinants, the hybridization fragments were subcloned into plasmid vectors. Thus, the 5 kb BglII phage A / 31 fragment and the 5 kb Bam HI fragment I / 12 were individually cloned into pBR322 at the Bam HI site; overlapping with 4.5 kb EcoRI-Xhoi and 1.4 kb PstI-HpaI phage fragments. 3 were inserted into pUC9 and pLeIF87, respectively.

K. DNA sequences of genes of three different bovine fibroblasts.

Figure 8 shows restriction maps of all three types of IFN- / 3 bovine genes that have been subcloned. They can be easily distinguished by unique cleavage sites. The peptide coding regions and sequences in both the lower and upper strands of each genome were determined by Maxam-Gilbert chemistry and are shown in Figures 9a, 9b and 9c. Nucleotide homology with the sequence intended for the human fibroblast inteferon gene predicts the correct reading form and the complete amino acid sequence of the product of each bovine gene; includes a 21 amino acid hydrophobic signal peptide followed by a 185 residue maturation protein. Bovine proteins differ from each other (Table 2, Fig. 10), but even greater are differences between bovine and human peptides (approximately 60 · <).

The multigenic nature of the bovine fibroblast inteferon was further demonstrated by Southern blot rehybridization, a radioactive probe prepared from a 415 bp EcoRI-Pvul fragment derived from pBoIFN- / IIItrp (described below). This experiment demonstrates the existence of other homologous IFN-? Genes. Less hybridized bands may in fact represent more distantly related genes, which would then encode more distal [beta] -interferons.

Table 2

Comparison of homology of coding sequences of bovine / 3-interferon (IFN- / 3) and human / 3-interferon (HuIFN- /?)

/ 91 / 12 Hu /? / 31 138 (04) 138 (82) 84 (52) / 32 20 (94) 146 (88) 92 (55) 20 (94) 19 (90) 87 (52) Hu / 4 16 (76) 17 (01) 16 (76)

The table lists the number of identical amino acids for each pair of coding sequences. Signal peptides of 21 amino acids are compared in the lower left part of the table, maturation INF- / 3 interferons of 166 amino acids are compared in the upper right part of the table. The first number represents the total number of identical amino acids of the pair in question, and the parenthesis indicates the percent homology. '

L. Direct express of three IFN-β in E. coli

Since the three bovine IFN- / 3 genes have many common DNA sequences and restriction sites (see Figure 0), a scheme for the expression of all three genes is possible. Since the DNA sequences encoding the first five amino acids, which contain two Alu sites, are identical in all cases, two complementary synthetic oligonucleotides have been designed that include the ATG translation initiation codon, restoring the codons of the first four amino acids of the bovine IFN- / 3 and produce EcoRI overhangs for insertion behind the trp promoter sequence The construction of the expression plasmids is schematically shown in FIG. 12. Ligation of synthetic oligomers to the B5 bp Alul-Xhol fragment, which is derived from BoIFN- / 3 subcloned plasma, followed by EcoRI and Xhol cleavage. a 104 bp fragment flanked by overhanging EcoRI and XhoI termini was generated. The complete code is then inserted into the trp expression vector by ligating a 104 bp fragment with an approximately 700 bp XhoI-Pst fragment encoding the residue of the BoIFN- / 3 protein and the plasmid pIFN-βΓ48-13 from which the internal EcoRI-Pst1 fragment was removed. All resulting plasmids, pBoIFN- / 31trp, pBoIFN- / 42trp and pBoIFN- / 53trp, carry out appropriate transcription and translation of IFN- / 3 genes under the control of the E. coli trp operon.

M. Characterization and subcloning of the bovine immune interferon gene (BoIFN- ¹ )

Ten phage recombinants that were hybridized with the human IFN-J7 * probe were purified. DNA was prepared as described above. All ten DNA samples give Southern blot analysis specific hybridization bands. The clones Afi and λ f-Ί were selected for further analysis because they differ in the pattern of the hybridization bands. Restriction mapping of the two clones shows that their DNA sequences overlap each other. The overlapping regions contain restriction sites XbaI, EcoRV and NcoI. DNA sequence analysis of the two clones shows that the gene structure is generally similar to the human immune interferon gene and further that 1/7 contains the sequence encoding the fourth exon and 2/4 contains the sequence encoding the first three exons of the bovine IFN- gene relative to ONA homology. sequence with the human IFN- / gene. In Figure 15, the amino acid sequence derived for BoIFN- / is compared to the amino acid sequence of HuIFN- / and mouse IFN- /.

To assemble the full length bovine IFN- / gene on a continuous DNA segment, the 3000 bp BamHI-NcoI fragment bridging the first three exons of the 2/4 bovine IFN- / gene was isolated and the 2500 bp NcoI-HindIII fragment bridging the last exon derived from. The two fragments were then cloned into a BamHI-HindIII vector derived from plasmids pBR322 by ligation of three portions.

N. To obtain an intron-free version of BoIFN- / to express this gene in a prokaryotic system such as E. coli, an expression gene (at high levels) was prepared in the animal cell expression system to obtain sufficient amounts of specific mRNA .

The 5500 bp BamHI-HindIII fragment bridging the full length bovine IFN- / gene was inserted into the SV40 vector for expression in COS (monkey kidney cells producing SV-40 ⁺ antigen) cells. The bovine IFN- / BamHI-HindIII gene fragment was cloned into 2800 bp of the SV40 plasmid vector pOLdeltaR1 ((derived from the HBV antigen expression plasmid pHBs34B-L by enzymatically omitting the EcoRI top strand site from the SV40 origin of replication selectively downstream). was constructed by cloning the 1986 bp fragment obtained by digesting HBv with EcoRI and BglII (HBv bridges the gene encoding HBsAg) to the plasmid pML at EcoRI and BamHI sites (pML is a derivative of p8R322 having omitted elimination sequences that inhibit plasmid replication in monkey cells). The resulting plasmid (pRI-Bg1) was then linearized with EcoRI The 348 bp fragment, which represents the SV40 origin region, was inserted into the EcoRI site of pRI-Egl The origin fragment can be inserted in any orientation as the fragment encodes both earlier and later SV40 promoters in addition to the origin of replication, this could occur x expression of HBV genes under the control of any promoter depending on this orientation (expression of pHBs34B-L representing HBs occurs under the control of a later promoter) between the BamHI and SalI site by ligation of three portions in the presence of a 600 bp HindIII-SalI converter fragment derived from pBR322.

Transformation of the resulting plasmids into COS cells results in efficient expression of bovine IFN- / ¹ under the control of the SV-40 later promoter.

A plus mRNA poles were prepared from transfected COS cells and used to prepare cDNA by standard procedures reported in the literature. CDNA clones hybridized with a bovine IFN- / gene gene probe were isolated. The cDNA clone with the longest PstI insert was selected for further analysis. DNA sequence analysis of this cDNA clone shows that all intron sequences of the genomic bovine IFN- / clone are correctly deleted.

For expression in Ecoli, it was prepared by the cONA primer-dependent method described above.

O. Preparation of bacterial extracts.

Overnight cultures in LB broth containing either 0.02 mg / ml ampicillin or 0.005 mg / ml tetracycline are inoculated at a 1: 100 dilution into 50 ml M9 medium containing 0.2 percent glucose, 0.5% casamino acids and the appropriate compound, and cultured at 37 ° C with shaking to ΑΑ θ = 1.0. Ten milliliters of sample are centrifuged and immediately frozen in a dry ice / ethanol bath. The frozen pellets are resuspended in 1 ml of 7M guanidine, incubated for 5 minutes on ice and diluted with PBS (phosphate buffered saline) for testing. Alternatively, the frozen pellets are decomposed by the addition of 0.2 ml of 20¾ sucrose, 100 mM Tris-HCl (pH = 8.0), 20 mM EDTA and 5 mg / ml lysosome. After 20 minutes in ice, 0.8 ml of 0.3¾ Triton (reionogenic detergent) X-100, 0.15 M Tris-HCl (pH = 0.0), 0.2 M EDTA and 0.1 mM PMSF (phenylmethylsulfonyl fluoride) are added. The lysate is purified by centrifugation at 19,000 rpm for 15 minutes. The supernatant is assayed after dilution with PBS.

P. Interferon Assays; the effectiveness of bovine interferon is tested by the cytopathic effect inhibition (CPE) assay in 96-well microtiter plates as follows:

1. 100 µl of cell suspension in medium containing 10 percent fetal calf serum is added to each well of the plate (B rows x 12 columns). The cell concentration was adjusted so that one liquid layer was obtained the next day.

2. Shake the plates slightly for ten minutes. This will result in a uniform distribution of cells.

Next day:

3. Add 80 µl of additional copper to each well of the first column.

4. Add 20 µL of sample to be assayed for interferon activity.

5. Mix sample and medium in the well by withdrawing 100 µl of pipette several times from the contents of the well and adding it to the well again.

6. Remove 100 µl of content from the first column well and transfer horizontally to the second column well.

7. Continue as (in the third column) in step tri.

0. Continue to transfer 100 µl of well contents to subsequent columns until all columns are filled (i.e., eleven relocations occur).

9. Remove 100 microliters of content from the well of the twelfth column. This procedure resulted in a double dilution series.

10. Each test plate contains appropriate NIH standards.

11. Incubate plates at 37 ° C in CO 2 for 24 hours.

12. Each test plate contains wells with 100 µl cell suspension and 100 µl medium (cell growth control) and wells with 100 µl cell suspension, 100 µl medium and 50 µl viral suspension (virus-indicated cytopathogenic control).

13. Add 50 microliters of virus suspension to all wells except control cells. The multiplicity of infection is given by the amount of virus that causes 100¾ cytopathic effect of the respective cell line within 24 hours.

14. Incubate plates at 37 ° C for 24 hours

15. Remove the liquid from the plates and stain cells with 0.5¾ crystal violet. Cells stain for two to five minutes.

16. Rinse wells with water and dry.

17. The titre of interferon in the sample is a breakthrough dilution value for 50% living cells.

18. The activity of all samples shall be normalized by the conversion factor of the reference units, calculated as follows:

actual titer of NIH standard

- = conversion factor of ref.

observed titer in test (NIH - National Institute of Health)

Extracts prepared from E. coli strain 294 (ATCC No. 31446) transformed with pBoIFN- (K, ltrp55) showed significant activity on VS virus (MDBK) cell line (strain

Indiana) but not on monkey kidney (VER), human cervical carcinoma (HeLa), rabbit kidney (RK-13) or mouse (1929) cell lines. Control extracts prepared from strain 294 transformed with pBR322 did not show activity on MDBK cells. Table 3 summarizes the in vitro antiviral activity of BoIFN-4 for the various animal and human cell lines tested. BoIFN-oC / 1 is readily distinguishable from human leukocyte IFN by being clearly anti-virally inactive in human cells compared to its anti-viral activity in bovine cells (VS virus was used). Table 4 shows the levels of interferon activity obtained in extracts prepared from E. coli W3110 transformed with the expression plasmids pBoIFN-βtrtr5, pBoIFN- / 3trtr, pBoIFN- / 4trtr, and pBoIFN-βtrtr. Of particular note is the observation that bovine fibroblast interferons are approximately 30 times more effective on bovine kidney cell lines than human anion cell lines, while an inverse relationship has been found for human fibroblast IFN.

Table 3

cell line prepared by IFN titr (units / ml) VSV EMCV MDBK LelF A standard 640 ON bovine leukocyte IFN 300 000 ON control extract 440 ON HeLa LeiF A standard 650 1 500 bovine leukocyte IFN Z 40 4 23 control extract 4 40 4 23 L-929 mouse IFN standard 640 1 000 bovine leukocyte IFN 4 20 431 control extract 4 20 4 31 RK-13 rabbit IFN standard 1 000 ON bovine leukocyte IFN <60 ON control extract 4 60 ON VĚRO LelF A standard 1 500 bovine leukocyte IFN 4 12 control extract 4 12

MDBK - bovine kidney cells

VERO - African green monkey kidney cells HeLa = human cervical carcinoma cells RK-13 = rabbit kidney cells

Na = not applicable, since the virus does not replicate well in the respective cells

Table 4

Interferon activity in extracts of E. coli

E.coli 294 transformed effect: IFN activity (units / liter of culture) MDBK-VSV IFN- / J activity (units / 1 culture) WISH-VSV pIFN- <Zl 1.0. 10 ⁸ at not specified pIFN- / i 1 2.2. 10 ⁸ 6.5. 10 ^é pIFN -> 2 1.1. 10 ⁸ 3.5. 10 ⁶ PIFN- / J 3 6.0. 10 ⁸ 2.0. 10 ⁷

Bacterial extracts were prepared and tested for interferon activity using bovine kidney cell lines (MDBK) and human amnion cell lines (WISH) against VSV according to a previously published procedure (Wech et al 1981).

The compounds of the invention may be formulated by known methods. Pharmaceutically useful compositions are thus prepared which comprise the animal interferon products of the invention in admixture with an acceptable carrier (s). Suitable binders and their formulations have been previously described in the literature. Such compositions comprise an effective amount of an interferon protein of the invention together with a suitable binder. Thus, acceptable compositions are prepared that are suitable for effective administration by known methods, for example, parenterally, to a host.

It is to be understood that animal interferons that are encompassed within the scope of this invention exist in natural allelic variations. These variations may represent the difference (s) in the amino acid (s) in the sequence or omission, substitution, insertion, inversion or addition of the amino acid (s) in the sequence. All such allelic variations are included within the scope of the invention.

It is to be noted that the present invention is not designed to be limited by the particularly suitable arrangements, but is limited only by the scope of the appended subject matter of the invention.

Claims (1)

SUBJECT OF THE INVENTION CLAIMS 1. A method for producing polypeptides having an amino acid sequence of an animal interferon, which comprises culturing an E. coli or yeast microorganism or a CHO cell line cell line transformed with a replicable vector capable of expressing a DNA sequence encoding said polypeptide and recovering the polypeptide. 2. Method according to point 1, characterized se that se isolates polypeptide glycosylation. 3. Method according to point 1, characterized se that se isolates polypeptide 4. Method according to point 1, characterized se that se isolates polypeptide without natural in mature form. type of bovine interferon. 10% 20% 30% abc abc abc