CS273182B2 - Method of expressive vector production capable to give mature human leucocytic interferon in transformed strain of escherichia coli by means of expression - Google Patents

Abstract

A method of production of an expressive vector capable in a transformed Escherichia coli strain of producing mature human leukocytic interferon with a partial amino acid sequence Cys-Ala-Trp-Glu-Val-Val-Arg-Ala-Glu-Ile-Met-Arg-Ser with 165 to 166 amino acids and with the amino acid Asp, Glu or Val in position 114 or, if methionine is linked to the N-terminal of the first amino acid of the abovementioned interferon, with 166 to 167 amino acids and with the amino acid Asp, Glu or Val in position 115, based on the fact that a) an E. coli vector is engineered containing an E. coli trp promoter, operator and trp leader of the ribosome binding point, b) a DNA fragment is prepared coding mature human leukocytic interferon, selected from a group consisting of human leukocytic interferon (LeIF) A, B, C, D, F, H, I and J, c) the abovementioned DNA fragment human leukocytic interferon is inserted in the direction of the fibre from the E. coli trp promoter, operator and trp leader of the ribosome binding point, whereby an E. coli expressive vector is acquired able, by expression in E. coli, to mature human leukocytic interferon.

Description

The present invention relates to the field of recombinant ONA technology, i.e., the methods used in said recombinant ONA technology and the products obtained by these methods.

In particular, the invention relates to a method for preparing a replicable expression vector comprising ONA sequences comprising a sequence encoding mature human leukocyte interferon with a partial amino acid sequence of Cye-Ala-Trp-Glu-Val-Val-Arg-Glu-Ile-Met-Arg-Ser, having 165 to 166 amino acids and having amino acid Asp, Glu or Val at position 114 or, if the N-terminus of the first amino acid of the above interferon is methionine, ee 166 to 167 amino acids and having amino acid Asp, Glu or Val at position 115, by constructing a first ONA sequence encoding the aforementioned mature interferon and operably linked to the aforesaid capable of effecting the battery expression of the aforementioned interferons.

Human leukocyte interferon (LelF) was first discovered and prepared in the form of very crude precipitates by Isaacs and Lindenmann (Proc. R. Soc. B 147. 258-2B7 (1957) and U.S. Pat. No. 3,699,222). Since then, efforts have been made to clean and characterize this material. These efforts have led to the preparation of relatively homogeneous leukocyte interferons derived from normal or leukemic donor leukocytes (German Patent Publication No. 2,947,134). These interferons form a family of proteins known to have the ability to confer viral reactivity on their target cells.

Furthermore, interferon may inhibit cell proliferation and modulate the immune response. These properties of interferons have inspired their clinical use as therapeutic agents for the treatment of viral infections and malignancies.

Leukocyte interferons have been purified to a substantially homogeneous state / see Rubinstein et al., Proc. Nati. Acad. Sci. USA 76: 640-644 (1979); Zeon et al., Ibid., 76, 5601-5605 (1969)], and their reported molecular weights ranging from about 17500 to about 21000. The specific activity of these preparations is remarkably high, 2 x 10 to 1 x 10 units per mg but the yields of cell culture methods are discouragingly low. Thus, advances in working methods for determining protein sequences have allowed the determination of partial amino acid malts in these interferons (see Zeon et al.). Science 207,527 (1980); Left and coworkers, Why. Nati. Acad. Sci. USA 77; 5102-5104 (1980)]. The clarification of the glycosylation of the various leukocyte interferons is not yet complete, but it is already clear that the differences in gylcosylac between the members of the group do not in themselves explain the spectrum of observed molecular weights. In addition, leukocyte interferons apparently differ in amino acid composition and order, and the amino acid identity is in some cases less than 80%.

While the isolation of interferons from donor leukocytes has provided material sufficient for partial characterization and limited clinical studies 8 with homogeneous leukocyte interferon, this source is totally inadequate for obtaining interferons in the amounts required for its wide-scale clinical trial and for its extensive use for prophylactic and / or therapeutic purposes. Indeed, recent clinical trials using intarfanones derived from human leukocytes to test their antineoplastic and antiviral effects have in principle been limited to raw preparations (less than 1% purity) and the long time required to produce sufficient amounts of epole at unrealistic price levels has critically delayed research front.

The advent of ONA recombination technology has enabled controlled microbial production of an enormous range of useful polypeptides. Bacteria modified by this technology are already available to produce such polypeptide products such as eomatostatin, A and B chains of human insulin and human growth hormone / see Itakura et al. 198, 1056-1063 (1977); Goaddal ae coworkers. Nature 281, 544-548 (1979)], More recently, ONA recombination technology has been used for bacterial production of proinaulin and thymoeine alpha 1, and many authors report finding DNA coded for human leukocyte rs, q

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CS 273 182 B2 tare interferon and that the resulting proteins had the activity of leukoeytar interferon (Nagata et al., Nature 284, 316-320 (1980); Mantei et al., Gene 10,

1-10 (1980); Tanlguchi and coworkers. Nature 285, 647-549 (1980)],

A powerful feature in ONA recombination technology is the plasmid, the chromosome O proeto loop of double-stranded ONA found in bacteria and other microbes, often in multiple copies in a single bacterial cell. The information encoded in the plasmid DNA incorporates the information needed to reproduce the plasmids into the daughter cells (the so-called replicon) and usually one or more selection characteristics, such as antibiotic resistance bacteria, which allows the host cell clones containing the plasmid that is of interest may be recognized, selected and preferably grown in a selective growth environment. The usefulness of bacterial plasmids lies in the fact that Js can be specifically cleaved by one or the other of a reetric endonuclease or restriction enzyme, each of which recognizes a different cleavage site on the plasmid DNA. After cleavage, foreign (hetarologic) genes or gene fragments can be introduced into the plasmids either by direct attachment at the cleavage site or by attachment at the reconstructed ends adjacent to the cleavage site. DNA recombination takes place outside the cells, but the resulting recombined plasmid ls can be introduced into bacterial cells by a method known as transformation and cultivation of the resulting transformant can produce large quantities of rhombined plasmids containing heterologous gsn. In addition, depending on where the gene is suitably introduced with respect to plasmid portions; which directs the transcription and translation of the encoded ONA information, the obtained expression capable carrier can be used to actually produce the polypeptide sequence for which the built-in gene is encoded; this procedure is referred to as expression in the description of the invention.

Expression is initiated in a region known as a promoter, which is recognized and to which it attaches RNA-polymase. In some cases, as in the case of the tryptophan (trp) promoter, which is preferably used in the practice of the method of the invention, the promoter regions are overlapped by the operator regions to form the ε combined promoter-operator. Operators are DNA sequences; which are recognized by so-called repressor proteins, which serve to regulate the frequency of transcription initiation at a specific promoter. Polymerose ee moves along the DNA strand, transcribes the information contained in the DNA coding strand and its 5 'end to the 3' end of the ribonucleic acid information (m-RNA), and this information is immediately translated into a polypeptide sequence having such an amino acid sequence; for which the respective DNA is encoded. Each amino acid is encoded by a single nucleotide triplet, or codon, for which the term structural gene is used in the description of the invention and which designates that part of the DNA in which the amino acid sequence of the expressed peptide product is encoded. After the RNA polymerose binds to the promoter, it first transcribes the nucleotides by encoding the riboaomal binding site, then initiates translation or the start signal (usually the ATG codon, which becomes AUG in the resulting m-RNA), and then translates the nucleoside codons within the structural itself. gene. At the end of the structural gene, they transcribe the so-called stop codons, and the polymase may then form another sequence of m-RNA which, due to the presence of the stop signal, remains untranslated by ribosomes. Ribosomes bind to the m-RNA-prepared binding site, in bacteria usually after m-RNA is formed, and produce the encoded polypeptides themselves, starting with the translation start signal and ending with the aforementioned etop signal. The desired product is only formed when the ribosome binding site coding sequences are positioned correctly relative to the AUG initiation codon and all remaining codons follow the initiation codon in the correct phase. The resulting product can be obtained by lysing the host cell and separating it from other bacterial proteins by a suitable purification method.

The inventors realized that applying recombinant DNA technology (i.e., inserting interferon genes into microbial expression carriers and expressing them under the control of microbial gene regulatory elements) would be the most effective way to ensure

CS 273 182 B2, p

Bi • in large amounts of leukocyte interferon, which despite being not glycosylated, as in the human leukocyte product, could be clinically used to treat a wide range of viral and neoplastic diseases.

According to the invention, the first leukocyte gene can be prepared by the following sequence of operations:

1) Using homogeneous purified human leukocyte intarferon partial amino acid sequences, sets of synthetic ONA test samples are constructed whose codons in total represent all possible nucleotide combinations capable of encoding said partial amino acid sequences.

2) Bacterial colony flasks containing complementary ONA (cDNA) from induced mRNA are prepared. Ono-induced mRNA, radiolabeled, hybridized to plasmid cDNA from this flask. Hybridizing mRNAs are eluted and tested for their ability to translate to interferon using an oocyte assay. Colony plasmid DNAs found to induce interferon activity in this method and further tested for their ability to hybridize to test samples prepared as described above under 1).

3) Simultaneously 8 as described above in section 2), cDNA derived from induced mRNA in plasmids was used to generate an independent colony transformed bank. The test samples of part 1) are used to initiate aynthsis of radiolabeled single stranded cONA, which is used as hybridization test samples. Synthetic assay samples hybridize to the induced mRNA template and extend reverse transcription to produce induced, radiolabeled cDNA. Colony banks clone that hybridize to the radiolabeled cDNA obtained above are further tested to confirm the presence of the entire length of the interferon coding sequence. gene. The jackets of the full length interferon gene can be used as such for any putative partial chain gene fragment obtained in the above sections.

1) and 2).

4) The full-length interferon gene obtained as described above is made using synthetic DNA so as to remove any leader sequence that could prevent microbial expression of the mature polypeptide and to ensure appropriate placement in an expression-capable carrier relative to the initiator. start signals and cribosome binding milling of the microbial promoter. The expression of the interferon obtained is purified to a degree allowing confirmation of its character and determination of its activity.

5) The interferon gene fragment prepared as described above is used as such for screening, hybridization, for the preparation of other partially hooological leukocyte interferon species.

The above-described application of ONA recombination technology techniques resulted in microbial production, in high yields and high purity, of a group of homologous leukocyte interferons (non-glycolised) in the form of mature polypeptides not accompanied by substantially the corresponding sequence or any part thereof. These interferons can be directly expressed, isolated and purified to a degree of purity suitable for their use in the treatment of viral and malignant diseases in animals and humans. The microbial expression of the products of this group so far demonstrated efficacy in in vitro testing, and in the first tests of this kind also in in vivo testing, including the first mature leukocyte interferon produced in a microbial fashion.

The term mature leukocyte interferon used in the description of the invention refers to a microbially (for example bacterially) produced glycosyl-free interferon molecule. The mature leukocyte interferon is immediately expressed in the method of the invention starting with the tlanslacl (ATG) initiation signal before the first amino acid codon of the natural product. Thus, the mature polypeptide may contain as the first amino acid in its sequence the methionine (for which it encodes ATG) without substantial

CS 273 182 B2 changes in nature. On the other hand, the microbial host can process the translation product by omitting the initial methionine. The mature leukocyte interferon may be expressed together with a conjugate protein of a different nature than the conventional leader protein, said conjugate being specifically cleaved in an intra- or extracellular environment (see British Patent No. 2,007,676 A). Subsequently, the mature interferon may be produced in conjugation 8 microbial signal peptides that transport the conjugate to the cell wall where the signal peptide is cleaved and the mature polypeptide is secreted. The term expression of mature leukocyte interferon refers to bacterial or other microbial production of an interferon molecule lacking glycosyl groups or amino acid sequences that are directly involved in mRNA translation of the human leukocyte interferon genome. _x

The specific leukocyte interferon proteins disclosed herein are defined by the determined DNA gene (see Figures 3 and 8) and by the deduced amino acid sequence (see Figures 4 and 9). It is understood that these specific interferons, although indeed all Include in the leukocyte interferon protein family, there are natural allelic variations that occur on a case-by-case basis. These variations can be demonstrated by a difference (or differences) in the total amino acid sequence, or by omission, substitution, insertion, inversion or addition of amino acid (or amino acids) with said sequence. For each leukocyte interferon protein of the present invention, designated LelF A to LelF symboly, said allelic variations are included within the scope of said labels or of the terms defining these interferons.

The preferred method of the invention is illustrated in more detail in the following detailed description and in the accompanying figures 1 to 9.

Figure 1 shows two amino acid sequences that are common to all species of interferons isolated from human leukocytes and purified to a homogeneous state, designated T1 to T-13. All possible mRNA sequences encoding these peptides are shown as corresponding DNA sequences. Breeds A, T, G, C and U refer to nucleotides containing the base adenine, thymine, guanine, cytosine and uracil. The letter N denotes any of the indicated nucleotides A, 'G, C and U. P ₀ lynucleotides are shown to be read from the 5' (left) to 3 '(right) direction and where double-stranded (dv) is shown DNA, on the other hand, is read from the bottom or from the non-coding strand.

FIG. 2 shows an autoradiogram showing FIG. hybridization of potential LelF 32 plasmids with synthetic P-labeled deoxyoligenucleotides

Figure 3 shows the nucleotide sequences (coding strands) of the eight gene fragments that have been isolated as possible candidates for use in the expression of leukocyte interferons, and are indicated by the letters A to H. The translation initiation ATG codons and tripletlets of each LelF are underlined; Stop codons or stop triplets are followed by non-translated regions at position 3 '. The full length incorporated gan for LelF A has omitted one codon found in the other drawn genes, as shown in the third A line of Figure 3. At position 5' the untranslated regions precede the leader sequences. In fragment E, after meeting the isolation, the entire protein-leading vaccine sequence is included, but the entire gene for the putative mature LelF E is included. In fragment G, the entire coding sequence is missing after isolation.

Figure 4 shows a comparison of eight LelF protein sequences determined by nucleotide sequences. Amino acids use the one-letter abbreviations recommended by the IUPAC-IUB Biochemical Nomenclature Commission: A alanine; C «cysteine:

and aspartic acid; E glutamic acid; F phenylalanine; G and glycine: H and histidin I and isoleuain; K lysine; L leucine; M; methionine; N and asparagine; P and proline;

Q and glutamine; R and arginine; S and serine; T-threonine: V and valine; W and tryptophan; and Y = ro tyrosine. The numbers indicate the location of the amino acids (S ee refers to the signal peptide).

CS 273 182 B2

A dash at position 44 of the 165 amino acid sequence of interferon LelF A is introduced to align said LelF A sequence with the 166 amino acid sequence of other LelFs. The LelF e sequence was determined neglecting the axtranuclaotide (position 187 in Figure 3) in its coding region. Asterisks indicate the termination codons that are in phase. The amino acids common to all LelF (except for the LelF E pseudogen) are also evident. Underlined residues are amino acids that are also present in human fibroblast interferon.

Figure 5 shows maps of restriction endonucleases of eight types (A to H) of cDNA cloned by LelF intarferons. Hybridized plasmids were constructed using the dC: dG terminal residue method (Goeddel DV and co-workers, Nature 287, 411-416 (1980)). In this method, cDNA inserts were excised using a PstI screening endonuclease. The lines at the end of each cDNA insert represent adjacent horapolymeric dC: dG terminal residues. PvuII, EcoRI and BglII cleavage sites are also indicated in the figure. Shaded regions of the figure indicate the coding sequences of mature LelF; The hatched regions indicate sequences encoding a signal peptide; the free regions represent the 3 ^Z and 5 ^Z non-coding sequences.

Figure 6 schematically depicts the construction of the gene encoding direct microbial synthesis of mature LelF A. The cleavage sites and residues formed (Pst I ', etc.) are indicated. Abbreviation b.p. indicates a pair of bases.

Figure 7 schematically depicts a raster map of two gene fragments used to express mature leukocyte interferon LelF B. The codon sequences indicated are the coding end strands obtained by digestion with the restriction enzyme Sau3a in the two cases.

Figures B and 9 show the DNA and amino acid sequences (with respect to the respective single-letter amino acid abbreviations as described above in Figure 4) of the five LelF proteins (including the lad species). The asterisk in Figure 9 indicates the termination codon of the respective DNA sequence and the hyphen indicating the deletion or gap in the sequence.

A, Used microorganisms

The method of the invention comprises the use of two microorganisms: E. coli xx 1776 as described in U.S. Patent No. 4,190,495, and E. coli K-12 strain 294 (terminated A, thi, hsr, hsmjp; Each of these microorganisms is deposited with the American Type Culture Collection (ATCC) under the designation ATCC Nos. 31537 and 31446. All work on 8 recombinant DNAs was performed in accordance with the binding guidelines of the National Health Authority (NIH). ) in USA.

Most preferably, the method of the invention is described using E. coli microorganisms, including not only E. coli strains x 1776 and E. coli K-12 strain 294 mentioned above, but also other known E. coli strains, such as E. coli. coli B, or other microbial strains, many of which are stored and available in known collection institutes, such as the American Type Culture Collection; see also German Patent Publication 2,644,432. Other microorganisms include, for example, Bacilli strains, such as e.g. Bacillus subsilis, and other snterobacterial microorganisms including, but not limited to, Salmonella typhimurium and Serrata marcescans strains that utilize plasmids and are capable of replicating and expressing the heterologous gene sequences contained therein.

As a host organism for the production of interfaronic proteins by expressing genes encoded therein, under the control of a yeast promoter, yeasts such as Sacharomyces carevisiae can also be used advantageously.

B. Source and Purification of Leukocyte Intarferon mRNA (LelF mRNA)

LelF mRNA can be obtained from human leukocytes, usually from leukocytes patients with chronic myeloid leukemia; these leukocytes can be induced to produce interferon virus by the Sendai or Newcastle disease virus, as described, for example, in German Patent Publication No. 273 182 82 Novelty 2,947,134. A particularly preferred source, also used in this work, is the cell line designated KG-1 obtained from patients with acute myeloid leukemia. This cell line, described by H.P. Koeffler and D.W. Goldem in Science 200, 1153 (1978), readily grows in culture medium containing RPMI (Rosewsll Park Memorial Institute) 1640 plus 10% FCS (fetal talc serum) inactivated by heating, 25 mM HEPES buffer (N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid) and 50 µg / ml gentamicin, and are subcultured to one to three distributions twice a week. Cells from previous culture medium! is frozen with 10% OMSO (dimethylsulfoxide). KG-1 cells are deposited with the American Type Culture Collection under the designation ATCC No. CRL 8031.

KG-1 cells are induced to produce leukocyte interferon mRNA by Sendai or Newcastle disease virus as described by Rubinsteins et al. (See Proc. Nati. Acad. Sci. USA 76, 650-644 (1979)]. Cells are harvested 5 hours after induction and RNA is prepared as described (Chirgwin et al., Blochemistry 18, 5294-5299 (1979)). In the same way, RNA is isolated from uninduced cells. Using oligodeoxythymidine (dT) -cellulose chromatography and sucrose gradient ultracentrifugation, the 12S fraction of poly (A) mRNA, as described by Grsen et al. (Arch. Biochem. Biophys. 172, 74-89 (1976)) and Okinura et al. /Sheet. Biochsm. Biophys 188, 98-104 (1978)]. The mRNA thus obtained has an interferon activity of 8000 to 10,000 units per microgram in an oocyte assay using the Xanopus Laevls frog (see Cavalieri et al., Proc.). Nati. Acad. Sci USA 74, 3287-3291 (1977)].

C. Preparation of banks for colony containing LsIF cONA sskvsncs

Using 5 µg mRNA 9 with standard procedures, prepare! double stranded cDNA / see Wicksns et al., 3. Biol. Chsm. 253, 2483-2495 (1978) by Gosddsl and 9 Colts, Naturs 281, 544-548 (1979)]. The cDNA obtained is fractionated according to size by electrophoresis on a 6% polyacrylamide gel (PAGE), and 230 ng of the sizing material is isolated by electroelution. v rozmez! from 500 to 1500 b.p. For a portion of this cDNA (100 ng) ss, the deoxycytidine (dC) residues are bound to the end by the method described by Chang and co-workers in Nature 275, 617-624 (1978), the product is annealed with 470 ng of plasmids pBR322. attached dssoxyguanidine (dG) residues (see Bolivar et al., Gana 2, 95-113 (1977)) and the plasmids thus modified are used to transform E. coli x 1776, yielding about 130 tetrycycline-resistant and ampicillin-sensitive transformants, per ng of cONA.

In a second similar experiment approximately 1000 tetracycline-resistant ampicillin-sensitive strain E. coli K-12 strain 294 was obtained per ng of cDNA. In this case, cDNA fractionation by molecular size and electroelution yielded a material ranging in size from 600 up to 1300 bp, which is used to bind terminal dC.

Preparation of synthetic oligonucleotides and their use

Knowing the amino acid sequences of the different tryptic fragments of human leukocyte interferons allows to design eynthetic deeoxyoligonucleotides complementary to different regions of the LelF mRNA. Two tryptic peptides T1 and T13 were chosen, since they have an amino acid sequence requiring only 12 or possibly 4 undecamers to synthesize. sequence (see Figure 1). Four sets of deoxyoligonucleotide test samples for each sequence are synthesized, each containing a cell of three (T-1A, 8, C, D) or one (T-13A, B, C, D) oligonucleotide. Said complementary. 11-base deoxyoligonucleotides are synthesized chemically using the phosphfortrieater method (see Crea et al., Proc. Nati. Acad. Sci. USA 75, 5765-5769 (1978)] In a T-13 series, four individual samples are prepared, in a T-1 series, twelve samples in four groups of three samples each as shown in Figure 1.

Four individual test samples in the T-13 series and twelve T-1 test samples

CS 273 182 B2 prepared in four groups of three primers in each a and I will use radiolabeled single stranded cDNA for information synthesis for use in hybridization assays. Oako sample mRNA was used with either SendSi virus induced 12S 'RNA from KG-1 cells (8000 units IF activity per µg) or total poly (A) mRNA from uninduced leukocytes (activity less than 10 units per µg). ³² P-labeled cDNA was prepared from the primers using known reaction conditions (see Noyes et al., Proc. Nati. Acad. Sci. USA 76, 1770-1974 (1779)]. Reactions were performed in 60 microliter buffers of 20 mM Trie-HCl (pH 8.3 hydrochloride), 20 mM potassium chloride, 9 mM anhydrous magnesium chloride and 30 mM 1-mercaptoethanol. ug of each primer (i.e. a total of 12 ug _y primers Tl series and a total of 4 ug _y for T-13 series), 2 / ug induced 12S fraction mRNA (or 10 .mu.g of uninduced poly (a) mRNA), 0.5mM dATP, dCTP, dTTP

200 y.uCi (/ ¹³ P) dCTP (Amersham supplier, 2000-3000 Ci / mole) and 60 transcriptase reverse units (Bethesda Research Laboratories supplier). The desired product was separated from the unincorporated marker gel filtration on a 10 ml Sephadex ^R- G-50 column and the product was heated at 70 ° C for 30 minutes with 0.3 N sodium hydroxide solution to decompose the RNA and then neutralized with hydrochloric acid. . Hydridizations are performed as described by Kafatos and co-workers in Nucleic Acids Res. 7, 1541-1552 (1979).

5. Identification of clones pL1 to pL30

To prepare 1 / µg plasmid DNA from each of the 500 individual transformants

E. coli K-12 strain 294 (see section C) ss will use a rapid method of isolating the plasmid described by Birnboim and co-workers in Nucleic Acids Res. 7, 1513-1523 (1979). Each DNA sample obtained was denatured and then applied in triplicate to a nitrocalulose filter according to the procedure described by Kafatos and co-authors (see above).

The above three sets of nitrocellulose filters containing 500 plasmid samples were hybridized

a) induced cDNA containing a T-1 set of primaris

b) induced cDNA containing T-13 primers, and

(c) uninduced cDNA prepared from the use of both sets of primers.

Clones are considered positive when hybridizing more strongly on one or both of the cONA-induced samples than on the total non-induced sample. Thirty positive clones (pL1 to pL30) were selected from the initial 500 plasmid samples for further analysis.

F. Identification of clones pL31 to pL39. Isolation of plasmid # 104 containing the LelF gene fragment

Transformants of E. coli x 1776 microorganisms were short-listed by the Grunstein and Hogness colony hybridization method (see Proc. Nati. Acad. Sci. USA 72, 3961-3965 (1975) (using P-labeled induced mRNA as a test sample) (see Lillenhaug et al., Biochemistry 15, 1858-1865 (1976)). Unlabeled mRNA from uninduced cells is ee emitted with a test sample at a ratio of 200: 1 to compete with the uninduced mRNA present in the ³² P-labeled preparation. Hybridization of the labeled mRNA occurs preferably with colonies containing induced sequences. Three types of transformants are obtained:

1) 2 to 3% colonies hybridized to ³² P-mRNA very strongly;

2) 10% colonies hybridized significantly less than in Group 1), and

3) a residue that does not give a detectable hybridization signal.

Positive colonies (Groups 1 and 2) are screened for the presence of interferon-specific sequences by an assay that involves interferon mRNA hybridization specifically to plasmid DNA. First, 60 strongly positive colonies (from Group 1) were cultured individually in 100 ml of M9 broth supplemented with tetracycline (20 µg / ml), diaminopimylic acid (100 µg / m), thymidine ( 20 µg / ml) and d-biotinam (1 µg / ml). M9 broth contains 1 liter of sodium hydrogen phosphate (6 g), potassium dihydrogenphosphate (3 g), sodium chloride (0.5 g) and ammonium chloride (1 g), after sterilization in an autoclave, 1 ml of sterile 1M magnesium sulphate solution is added. (anhydrous) and 10 ml of sterile 0.01 M anhydrous calcium chloride solution. DC-link cultures of ten and six of the above-mentioned DC-link groups isolated plasmid DNA as described by Clswell et al. In Biochemistry 9, 4428-4440 (1970). Ten µg of plasmid DNA from each group is digested with HindIII, the mixture is digested and covalently bound to DBM-paper (diazobenzyloxymethyl filter paper). Deden / µg of purified mRNA from induced cells hybridizes to the cleaved DNA bound to OBM-paper. Unhybridized mRNA is removed by washing, specifically hybridized ss servujs β mRNA is tested with Xsnopus lasvis oocytes. In this test, all six groups were negative. Of the 59 weakly positive colonies (group 2), five groups of 10 colonies and one group of 9 colonies were formed, and from these groups plasmids were prepared as described above and evaluated as above. Of the six test groups, one (K 10) hybridized to interferon mRNA and exhibited levels significantly higher than baseline at each time of testing. In order to identify a specific interferon cONA clone, plasmid DNA was prepared from 9 K10 colonies and tested individually. Two of the above-mentioned plasmids (number 101 and number 104) bound interferon mRNA significantly above baseline levels.

Bgl II restriction fragment containing 260 b.p., labeled with radioactive P as described by Taylor and co-workers in Biochim. Biophys, Acta 442, 324-330 (1975) and used as a test sample for independent selection on 400 E. coli 294 transformants using an in situ screening method (see Grunstein and Hogness, Proc. Nati. Sci. KDA 72, 3961-3965 (1975)]. Nine colonies (pL31 to pL39) were identified that hybridized to a different degree in this assay.

The above 260 bp labeled fragment. was used in an independent screen for 4000 E. coli 294 transformants in the same manner. 50 colonies were identified that hybridized to the test sample to different degrees in this assay. One of them contained a LelF G fragment, one LelF H fragment, and one contained a fragment designated as LelF H1, an obvious allelic variant of LelF H. The hybrid plasmids obtained in this manner were designated as pLelF H, and so on.

G. Isolation and sequence determination of the first LsIF gsnu and full-length chain

From all 39 potential LelF cONA clones, plasmid DNA was prepared and re-selected with the same 260 bp DNA sample using the hybridization procedure described by Kafatos et al. (Supra). Three plasmids (pL 4, pL 31, pL 34) gave very strong hybridization signals in this screening, four (pL 13, pL 30, pL 32, pL 36) hybridized slightly and three (pL 6, pL 8,, pL 14) hybridized poorly to the test sample.

The above 39 potential LelF cDNA recombinant plasmids were also subjected to selective screening using synthetic radiolabeled undecamers.

P (groups of individual T-1 primers or individual T-13 primers) directly as hybridization assay samples. The hybridization conditions were chosen so that perfect pairing of the bait for detectable hybridization signals could be achieved (see Wallace et al.). Nucleic Acids Ree. 6, 3543-3557 (1979)]. Thus, plasmid DNA was prepared from 39 clones by the ethane-dardium lysate procedure (see Clswsll et al., Supra), which were purified by column chromatography on Biorad Agarose A-50 agarose. Samples (3 µg) of each preparation were linearized with Eco RI, de-alkaline and applied to 2 separate nitrocellulose filters at a dose of 1.5 µg per application (according to Kafatos and co-workers, cited above). Individual synthetic9

CS 273 182 B2 codon deoxyoligonucleotide primers and pooled primers were phosphorylated with (4 ^ ³² P) -ATP as follows: 50 pmol oligonuclaotide and 100 pmol labeled (γ ³² Ρ) -ΑΤΡ (supplier New England Nuclaar, activity 2500 Ci / mmol ) was added to 30 microliters of a buffer composed of 50 mM Tris-HCl, 10 mM anhydrous magnesium chloride and 15 mM / 3-mercaptoethanol. Two units of polynucleotide kinase T4 were added to the mixture and after 30 minutes heating at 37 ° C, the resulting ³² P labeled primers were purified by column chromatography from 10 ml Sephadex ® G-50. Hybridizations were then performed using 10 ⁵ cpm of T-13C primer or 3 χ 10 ⁶ cpm of linked T-10 primers. Hybridizations were performed at 15 ° C for 14 hours in 6 x SSC (1 x SSC 0.15 M sodium chloride and 0.015 M sodium citrate, pH 7.2) and 10 x Dsnhardt's solution (0.2% bovine sarumalbumin, 0.2% polyvinylpyrrolidone (0.2% Ficoll), as described by Wallace and coworkers (cited above) · Filters were washed for 5 minutes (3 times) at 0 ° C in 6 x SSC, dried and ex32 plated on film sensitive to X-rays. The results obtained with β-labeled fused primers T-13C and with primary T-1C are shown in Figure 2.

Significant hybridization of plasmid DNA from clone 104 was found to be associated with a group of linked primers T-1C and primer T-130; ais not detectable hybridization with other undersamers. Oak is shown in Figure 2, many of the 39 potential LsIF plasmids (pL 2; 4, 13, 17, 20, 30, 31, 34) also hybridize to both of these test samples. However, restriction andonuclease analysis revealed that only the nucleate of these plasmids, plasmid pL31, also contained an internal BglII fragment of 260 b.p. By digestion of pL31 with the restriction enzyme PstX, the size of this cDNA insert was found to be approximately 1000 b.p.

In the latter Pst1 insert of plasmid pL31, the nucleotide sequence was analyzed by the Mexam-Gilbert chemistry (see Msthods Enzymol.).

65, 499-560 (1980) and by the didaoxyd chain termination procedure (see Sm th, Msthods Enzymol. 65, 560-580 (1980) / after subcloning the Sau 3a fragments into the M13 vector. The DNA sequence is shown in Figure 3 under the designation A. The corresponding translational reading of the DNA construct can be predicted from the information available about the amino acid sequence of the interferon protein, the known molecular weight range of LelF and the relative occurrence of etop-triplets in three possible constructs. , and the identified nucleotide sequence allows, in turn, to predict the amino acid sequence of the entire LelF, including the presequence or signal peptide. The first ATG translation initiation codon was found at a distance of 60 nucleotides from 5 ^{of the} terminus sequence, and is followed by 188 codons later by a TGA termination triplet; then there are 342 untranslated nucleotides at 3 ^Z. ends, followed by the poles (A) of the sequence. The putative eignal peptide (probably involved in the secretion of mature LelF from leukocytes) is 23 amino acids in length. The mature 165 amino acid LelF has a calculated molecular weight of 19390. The inventors have designated LelF encoded by plasmid P131 as LelF A, the sequence data (see A 'in Figure 4) shows that tryptic psptides T1 and T13 of LelF B correspond to amino acids 145-149; optionally 57 to 61, LelF A interferons.

H. Direct expression of mature leukocyte interferon A (LelF A)

I. General

The following method for expressing LelF A directly in the form of mature interfaron polypeptides is a variant of the method used previously for the preparation of human growth hormone (Goeddsl et al., Nature 281, 544-548 (1979)) to the extent that it involves a combination of synthetic (N-terminal) and complementary DNA, as shown in Figure 6, the Sau3a restriction endonuclease cleavage site is preferably located between codons 1 and 3 of LelF A interferons. Two synthetic deoxyCS 273 182 82 oligonucleotides have been identified that incorporate the ATS translation initiation codon, restoring the codon for translation. amino acid 1 (cysteine) and forms the capture (sticky) EcoRI end. These oligomers were linked to a 34 b.p. fragment. between Sau3a - Avall plasmid milling pL31. The product obtained by 45 b.p. It was ligated to other DNA fragments and thus an 865 bp eynthetic-natural gene was constructed which encodes LelF A and which is bound by EcoRI and PstI restriction sites. This gene was inserted into plasmids pBR 322 m with its EcoRI and PstI restriction sites to give plasmids pLsIF A1.

2. Construction of a tryptophan control element (containing the E. coli trp promoter and operator, and a trp main ribosome binding mill, but lacking an ATG translation initiation sequence)

The plasmid pGMI nsss tryptophan (trp) opsron to E. coli having Ls1413 deletion (see Miozzari et al., Bacteriology 133, 1457-1466 (1978)) and therefore expresses a fusion protein comprising the first 6 amino acids of the trp sequence leader and approximately the last third of trp E polypeptides (hereinafter referred to as LE), and also express the trp D polypeptide as a whole, all under the control of the tryptophan promoter-operat system. The plasmid (20 µg) was digested with the restriction enzyme PvuII, which cleaved the plasmid at five sites. The gene fragments are then combined with EcoRI linkers (consisting of a self-complementary oligonucleotide having the sequence pCATGAATTCATG) that contain the EcoRI restriction milling needed for later cloning into plasmids containing the EcoRI site. For DNA fragments (20 ~ g) obtained from pGMI was treated with 10 units T ₄ DNA ligase in the presence of 200 pmoles of a 5 * -fosforylovaného synthetic oligonuklsotidu pCATGAATTCATG and in an environment of 20 .mu.l _of T4 DNA ligase buffer (20 mM Tris, pH 7.6, 0 , 5 mM ATP, 10 mM anhydrous magnesium chloride, 5 mM dithiothreitol) at 4 ° C overnight. The solution was then heated to 70 ° C for 10 minutes to stop the coupling. The linkers were cleaved by digestion with EcoRI endonuclease, the resulting fragments, now with EcoRI ends, were separated using PAGE Sclotrophors (5% gsl) and the three largest fragments were isolated after prior staining with ethidium bromide and detection in ultraviolet light by cutting the appropriate portion of the gel layer. containing the desired fragment. Each of said gsl fragments were placed together with 300 ml of 0.1 x TBE buffer (TBE buffer containing 10.8 g Tris-bass, 5.5 g boronic acid and 0.09 g ethyl disodium diamine-stearic acid disodium salt (NagEDTA) in a liter of water ) into a dialysis bag and subjected to slaktroforsse at 100 V for one hour in 0.1 x TBE buffer. The aqueous solution was removed from the dialysis bag, extracted with phenol, extracted with chloroform, adjusted to a 0.2 M solution with sodium chloride, and the ONA was recovered after precipitation with ethanol in an aqueous medium. The resulting gsn with EcoRI capture kits containing the trp promoter-operator is identified by the method described below, which calculates the insertion of the fragments into tetraoycline-sensitive plasmids, which becomes tetracycline resistant after said fragment incorporation.

Plasmid pBRHI / see Rodrigusz et al., Nuclsic Acids Res. 6, 3267-3287 (1979) / expresses ampicillin resistance and contains gsn for tetracycline resistance, but since it is not associated with a promoter, it subsequently expresses said resistance. Plasmid Js is therefore sensitive to tetracycline. By introducing the promoter-opsator system into the EcoRI site of the plasmid cleavage site, the es plasmid becomes resistant to tetracycline.

To this end, the plasmid pBRHI was digested with EcoRI reetric endonuclease, the enzyme removed by phenol extraction followed by chloroform extraction, and after precipitation of the DNA, the DNA was recovered in an aqueous medium. The DNA molecule obtained, using separate T ₄ DNA ligase, in separate reaction mixtures, binds to each of the three ONA fragments obtained as described above, as described above. The resulting DNA contained in the reaction mixture was used to transform the respective E. coli K-12 strain 294 using standard techniques (see Ksrshfisld et al., Proc. Nati. Acad. Sci.

USA 71, 3455-3459 (1974)]. The bacteria are then plated on LB (Luria-Bsrtani) agar plates containing 20 µg / mol ampicillin and 5 µg / ml tetracycline. Colonies of tetraoycline-resistant bacteria are selected, plasmid DNA is isolated and the presence of the desired fragments is isolated.

CS 273 182 B2 was confirmed by restriction enzyme analysis. The plasmid obtained was designated as pBRHtrp.

By digesting the hepatitis B viral genome with the EcoRI and BamHI enzymes, a product is obtained in a conventional manner, which is incorporated into EcoRI and BamHI restriction milling of plasmid pGH6 (see Goeddel et al., Nature 281, 544 (1979)) to produce plasmid pHS32. The resulting plasmid pHS32 was digested with XbaI, extracted with phenol, then extracted with chloroform and ethanol precipitated. The digested plasmid ss is then treated with 1 microliter of E. coli DNA polymase I, Klenow fragment (Boehringer-Mannheim) in an environment of 30 microliter of polymerase buffer (i.e. 50 mM potassium phosphate, pH 7.4, 7 mM anhydrous magnesium chloride and 1 mM (3-mercaptoethanol) containing 0.1 mM dTTP and 0.1 mM dCTP for 30 minutes at 0 ° C and then 2 hours at 37 ° C. In this reaction, two of the 4 nucleotides complementary to the 5 'protruding end of the XbaI cleavage site are added:

5 'CTAGA - 5' CTAGA -3 'T - 3' TCT Two nucleotides, dC and dT, 'are inserted and the end is formed with two 5' protruding nucleotides. This linear residue of the plasmid pHS32 (isolated after phenol extraction, chloroform extraction and ethanol precipitation in aqueous medium) was cleaved with the restriction enzyme EcoRI. The large plasmid fragment was separated from the smaller EcoRI-XbaI fragment by PAGE electrophoresis and electroelution was isolated. This ONA fragment of plasmid pHS32 (0.2 µg) was ligated, under conditions similar to those described above, to an EcoRI-Taq1 fragment of the tryptophan operon (0.01 µg) obtained from pBRHtrp.

In the above procedure of binding the fragment from plasmid pHS32 to the EcoRI-Taq1 fragment, the Taq1 protruding end binds to the XbaI remaining protruding end, although its bases are not completely paired according to Watson and Grick's principle:

- CTAGA - __TCTAGA +

-AGC TCT -AGCTCT A portion of the ligand reaction mixture thus obtained is transformed into E. coli 294 cells, then the cells are heat treated and inoculated onto LB agar plates containing ampicillin. Selection yields 24 ampicillin resistant colonies, which are cultured further on 3 ml of LB broth (Lakria-Bertani) and plasmid and isolated, Seet from these plaamides has an XbaI cleavage site regenerated by E. coli catalysed mismatching and DNA replication:

-TCTAGA- -TCTAGA-AGCTCT- = -AGATCT It has also been found that these plasmids are cleaved by both the EcoRI and Hpa I enzymes and provide the expected cleavage fragments. One of these plasmids, designated pTrp14, was used to express histologic polypeptides as described below.

Plasmid pHGH 107 (see Goeddel et al., Nature 281, 544 (1979)) contains a human growth hormone gene consisting of 23 amino acid codons produced from synthetic DNA fragments and 163 amino acid codons obtained from complementary DNA generated by reverse transcription of information RNA for human growth hormone. This gene, although lacking the human growth hormone pre-sequence codons, contains an ATG translation initiation codon. The gene was isolated from 10 µg of plasmid pHGH by a procedure comprising first treating with the restriction enzyme EcoRI and then attaching the dTTP and dATP to the obtained fragment by treatment with the Klenow fragment of E. coli ONA polymerase I as described above. The resulting plasmid is isolated by phenol extraction, chloroform extraction, and digested with ethanol, then digested with BamHI.

The resulting fragment containing the human growth hormone (HGH) gene was isolated by PAGE electrophoresis followed by electroelution. The resulting DNA fragment also contains the first 350 nucleotides of the structural gene for tetracycline resistance, but lacks the tetracycline promoter-operator system, so that when it incorporates this fragment into a plasmid capable of expression in the following procedure, plasmids containing the insert can be determined by renewal tetracycline resistance · Since the EcoRI end of the fragment is complemented by the Klenow polymerase I procedure, the fragment has one blunt and one sticky end, which ensures it is correctly oriented when it is later incorporated into a plasmid capable of expression ·

Next, an expression capable plasmid pTrp 14 is prepared so that it can receive the HGH gene-containing fragment obtained as described above. For this purpose, plasmid pTrp14 was digested with the restriction enzyme XbaI, and the resulting sticky ends of the fragment were complemented by the Klenow polymerase I procedure using dATP, dTTP, dGTP and dCTP triphosphates. After extraction of the resulting mixture with phenol, extraction with chloroform and ethanol precipitation, the DNA obtained is treated with BamHI and the resulting large plasmid fragment isolated by PAGE electrophoresis and electroelution. Said fragment derived from pTrp 14 has one blunt and one sticky end which provides it with the correct orientation when recombined with the above described fragment containing HGH gsn.

The fragment containing HGH gsn and the latter fragment pTrp 14 AXba-BamHI were combined and ligated together under conditions similar to those described above. The added XbaI and EcoRI ends are ligated together with blunt binding ends to restore both XBaI and EcoRI cleavage sites:

complemented with XbaI end complemented with EcoRI end of HGH gene initiation

-TCTAG

AATTCTATG — TCTAGAATTCTATG-s *

-AGATC

TTAAGATAC -AGATCTTAAGATAC

Xbal EcoRI

This construction also restores the resistance of the gene to tetracycline. Since plasmid pHGH 107 expresses tetracycline resistance from the upstream promoter of the HGH gene (lac promoter), this resulting construction of a plasmid designated pHGH 207 allows expression of the tetracycline resistance gene under the control of the tryptophan promoter-operator. The ligation product mixture is transformed into E. coli 294 and resistant colonies are selected after cultivation on LB agar plates with soil containing 5 µg / ml tetracycline.

The pHGH plasmid was digested with the restriction enzyme EcoRI and PAGE by electrophoresis, followed by electroelution to isolate a fragment of 300 b.p. containing! the trp promoter, operator, and tryptophene main ribosome binding site, but lacking ATG translation initiation sequences. This DNA fragment is cloned into the EcoRI cleavage site of plasmid pLelF A. Expression-capable plasmids containing the aforementioned modified trp regon (the E. coli trp operon from which the attenuation sequence is omitted to control the expression levels controllable) can be cultured to predetermined stages. levels on the broth containing the additional tryptophan in an amount sufficient to suppress the promoter-operator system, then tryptophan is removed, the system is derepressed, and the desired product is expressed.

This procedure is described in more detail below and shown in Figure 6. Plasmid pL 31 (250 µg) was digested with the restriction enzyme PstI and gel electrophoresis at 6%.

CS 273 182 B2 acrylic gel gel is used to insert the inserted fragment of 10000 b.p. About 40 µg of the desired inserted fragment is obtained from the gel by electroelution and is divided into 3 aliquots for further digestion by enzymatic digestion:

a) About 16 µg of this fragment was partially digested with 40 units of BglII endonuclease for 45 minutes at 37 ° C, and the reaction mixture was purified by PAGE electrophoresis on a 6% gel, yielding about 2 µg of the desired fragment of 670 b.p.

b) Second sample (8 µg) of 1000 bp Pst fragment. cleaved with Avall and BglII endonucleases; gel chromatography (PAGE) yielded one µg of said fragment of 150 b.p.

c) Per 16 µg of 1000 b.p. ee acts by the enzymes Sau3a and Avall; after electrophoresis on a 10% polyacrylamide gel, about 0.25 µg (10 pmol)) of the 34 b.p. fragment was obtained.

The two following deoxyoligonucleotides are synthesized by the phosphoryriester method,

5'-dAATTCATGTGT (fragment 1) and 5'-dGATCACACATG (fragment 2). Fragment 2 is phosphorylated, 39 as follows: 200 microliters (about 40 pmol) of (γ P) -ATP (supplier Amereham, activity 5000 Ci / mol) is dried and resuspended in 30 microliters of a buffer consisting of 60 mM Tris-HCl (pH δ), 10 mM anhydrous magnesium chloride and 15 mM β-mercaptoethanol, and containing 100 pmol of said DNA fragment and 2 units of T4 polynucleotide kinase. The mixture was heated at 37 ° C for 15 minutes, then 1 microliter of a 10 mM ATP solution was added, the reaction was allowed to proceed for an additional 15 minutes at this temperature, and then inactivated by heating the mixture at 70 ° C for 15 minutes. 100 pmol of 5'-OH fragment 1 and 10 pmol of Sau3a-Avall fragment of 34 b.p. were added to the reaction mixture. and bind the fragments together in 50 microliters of medium consisting of 20 mM Tris-HCl (pH 7.5), 10 mM anhydrous magnesium chloride, 10 mM dithiothreitol, 0.5 mM ATP, in the presence of 10 units of T4 DNA ligase, at 4 ° C for 5 hours. The mixture was resolved by PAGE on 6% gel and electrophoresed to yield the product by 45 b.p. About 30 ng (1 pmol) of the latter product by 45 b.p. was combined with a 0.5 µg (5 pmol) Avall-BglII fragment of 150 b.p. and 1 µg (2 pmol) of the BglII - PstI fragment of 670 b.p. Ee binding is performed by treatment with 20 units of T4 DNA ligase at 20 ° C for 16 hours. The ligase is inactivated by heating at 65 ° C for 10 minutes and the mixture is digested with the enzymes EcoR1 and PstI to remove gene polymers. Purification of the mixture by electrophoresis on 6% PAGE e gives about 20 ng (0.04 pmol) of 855 b.p. Half (10 ng) of this product was ligated into plasmid pBR 322 (0/3 / Ug) between its EcoR1 and PstI cleavage sites. Transformation of this plasmid into E. coli 234 yields 70 tetracycline-resistant and ampieilin-sensitive transformants. Plasmid DNA isolated from 18 of these transformants was digested with EcoR1 and PstI. 2 of these 18 plasmids contained a 16 EcoR1-PstI fragment of 865 b.p. One µg of one of these fragments, pLelF A1, was digested with EcoR1 and ligated to the 300 bp EcoR1 fragment prepared as described above. (0.1 µg) containing the trp promoter and the major ribosome E binding site, coli. The trp promoter-containing transformants were identified using a P-trp assay in conjunction with the Grunetein-Hogness bacterial colony screening method. The asymmetrically located XbaI cleavage site in the trp grament allows the determination of recombinants in which the trp promoter is oriented in the direction of the LalF A gene.

I, In vitro and in vivo efficacy of interferon LelF A

Prepare for determination of interferon activity. extracts as follows:

Culture 1 ml was grown in L broth containing 5 / ug / ml tetracycline to the absorbance values at 550 nm _(G50) of about 1.0 / SS then culture was diluted into 25 mL of M9 culture medium containing 5 / ug / ml tetracycline . When _AggQ reaches 1.0, 10 ml samples are taken and centrifuged, and the obtained cells are suspended in 1 ml solution containing 15% sucrose, 50 mM Tris-HCl (pH 8.0) and 50 mM EOTA (ethylandiaminetetraacetic acid). . One mg of lysozyme is added to the suspension, and after standing for 5 minutes at 0 ° C, the cells are disrupted by ultrasound. The samples are centrifuged for 10 minutes (15,000 rpm) and the supernatant is collected

5 * “^

CS 273 182 B2 determines intarferon potency by comparison with interferon LelF standards using the cytopathic potency inhibition (CPE) assay. LelF with a specific activity of 4 x 10 ⁸ units / mg is used to determine the number of interferon (IF) molecules per cell.

As shown in Table 1, clone pLsIF A trp 25, in which the trp promoter is incorporated in the desired orientation, gives high levels of activity (at 2.5 x 10 ° units per liter) in this assay. Oak shows in Table 2, the interferon produced by E. coli K-k2 strain 294 / pLeIF A trp 25 behaves as authentic human interferon LelF; is stable to acidic environment! o pH 2 and its activity is neutralized by rabbit antibodies of human leukocyte interferons. Said interferon has an apparent molecular weight of about 20,000.

The in vivo efficacy of interferon requires the presence of macrophages and natural killing (NK) cells, and its mode of in vivo efficacy appears to be by stimulating these cells. It is therefore possible that the interferon produced by E. coli 294 / pLeXF A 25, although it has antiviral activity in cell culture assays, may not be effective in infected animals. In addition, the in vivo antiviral activity of bacterially produced, non-glycosylated LelF A interferon could be different from glycosylated LelF derived from human leukocytes. Therefore, the biological efficacy of bacterially synthesized interferon LelF A (2% purity) was compared to that of human leukocyte-coated interferon (LelF, purity 8%) in the lethal encephalomyocarditis (EMC) viral infection of stoat (see Table 3).

Table 1. Intarferon activity of E. coli extracts

E. coli K-12 cellular IF efficiency Počat LelF strain 294 density units / ml of molecules transformed (number of cells culture per cell plasmid per ml) pLalF A trp 25 3.5 χ 10 ⁸ 36 000 9 000 pLelF A trp 25 1.8 χ 10 ⁹ 250 000 12 000

Table 2. Comparison efficiency of extracts from bacteria E. coli 294 / pLaIF A25 with standard interfsronam LelF ^x Extract Interferon efficiency (units / ml) without modification at pH 2 in the presence of rabbit anti-human leukocyte antibodies E. coli 294 / pLeIF A trp 25 extract 500 500 10 LelF standard 500 500 10 ^{x / f} Extract of bacteria E. coli 294 / pLeIF A trp 25 o intarferonové 250 000 activity

Note per ml, as described in Table 1, was diluted 500-fold with a minimum baseline broth to a specific activity of 500 units / ml.

The leukocyte interferon standard (from Wadley Institute), pre-titrated against the NIH leukocyte interferon standard, was also diluted to a final concentration of 500 U / ml. Aliquots of 1 ml were treated with hydrochloric acid to make

CS 273 182 B2 by concentrating IN to pH 2, samples incubated for 52 hours at 4 ° C, neutralized by addition of potassium hydroxide solution, and IF activity determined using a standard CPE inhibition assay. 25 µl aliquots of 500 units / ml (not treated) samples were incubated with 25 µl of rabbit anti-human leukocyte interferon antibody at 37 ° C for 60 minutes, centrifuged at 12000 x g for 5 minutes, and the supernatant tested.

Table 3. Antiviral activity of various LelF preparations against EMG viral infection of stoat

Treatment interferon Survival Units of serum platelet formation / ml day 2 day 3 den 4 Control AND E (bacterial 0/3 10) 3x10 ^ ¹⁰ ) proteins) 0  3 0 10 ⁴ 1200 () 3.4x10 ^ 0 J 0 in Bacterial 3/3 • 0 0 0 LelF A 0 0 0 0 0 0 Standard LelF 3/3 0 0 0 0 0 0 0 0 0

Male ostriches with an average weight of 713 g and who had no antibody to the EMC virus prior to infection were used for testing. Animals were infected intramuscularly with a dose of 100 x LDgQ EMC virus (found in mice). In the control group, the stoat died 134, 158 and 164 hours after infection. Treatment of animals with 10-unit interferon was administered by intravenous route at intervals of -4, +2, 23, 29, 48, 72, 168 and 240 hours, based on the time at which infection was performed. The bacterial leukocyte interferon used was a fraction from column chromatography of E. coli 294 / pLeIF A 25 with a specific activity of 7.4 x 10 6 units / mg protein. Oako control bacterial proteins were used from the equivalent fraction of column chromatography of E. coli 294 pBR 322 lysate at twice the total protein concentration. Oako standard leukocyte interferon was used interferon induced by chromatography to specific activity of 32 x 10 units per mg protein.

Stoats in the control group showed progressive lethargy, loss of balance, flaccidity and paralysis of the hind limbs, and watering of eyes beginning about 8 hours before death. Interferon-treated stoats did not show any of these abnormalities; they remained active at all times and did not develop viremia. One of the control group, who had not developed viremia within 4 days, died at least from untreated animals (164 hours after infection), but showed high virus titers in the heart and brain post mortem. Interstates treated with interferon did not develop antibodies to the EMC virus as determined at 14 and 21 days after infection. These results show that the arithiviral efficacy of LelF preparations in infected animals can only be attributed to interferon, since the contaminating proteins in bacterial preparations and in human leukocyte preparations are quite different. These results further suggest that glycosylation is not required for the in vivo antiviral activity of interferon LelF A.

CS 273 182 B2

Isolation of other cDNAs for other leukocyte interferons

PstI DNA was excised from fully characterized plasmids containing interferon LelF A cDNA, isolated by electrophoresis and labeled with radioactive P. The radiolabeled DNA obtained was used as a test sample for the selective screening of other E. coli 294 transformants obtained by a standard procedure as described in C, using the in situ colony screening method of Grunstein and Hogness (see above). Colonies are isolated which hybridize in varying amounts with the test sample. by digesting these colonies and the 10 hybridizing colonies described in Part G with PstI, plaemid DNA was isolated and characterized by three different methods. Said Pst1 fragments were first characterized on the basis of samples obtained by digestion with restriction snducleases, enzymes Bgl II, Pvu II and Eco RI. This analysis made it possible to characterize at least eight different types (LsIF A, LsIF B, LsIF C, LelF D, LeiF E, LeiF F, LelF G and LeiF H) shown in Figure 5, which approximately shows the location of the various restriction sites in relation to The deden of these fragments, LelF D, is probably identical to the 9 DNA fragment described by Nagata and collaborators in Nature 284, 316-320 (1980).

Second, some of the DNAs were tested using the hybridization selection assays described by Clevelands and co-workers in Cell 20, 95-105 (1980), based on the ability to selectively remove LalF mRNA from KG-1 cellular RNA containing cell RNA. poly-A chains. In this paste, LelF A, B, C, and F were positive. Third, the latter Pst fragments were inserted into an expression-capable plasmid, E. coli 294 was transformed with said plasmid, and the fragments were expressed. All of the expression products thought to be pre-interferons were positive in the CPE assay for interferon activity, although only marginal efficacy was found for the LelF F fragment. In addition to this characterization, a sequence was determined for all LelF types described.

K, Direct expression of second mature leukocyte interferon (LelF B)

The sequence of the first fourteen nucleotides of the isolated fragment containing the mature interferon LelF B gene was found to be the same for both types A and B. Therefore, a fragment bearing the trp promoter-opsator, ribosome binding site and LsIF A initiation signal was isolated from pLelF A 25. The B) gene, and this fragment was combined with the remainder of the B-sskvencs to form an expression plasmid. The maps of the protruding restriction sites of the Pst fragment of plasmids pL4 (a plasmid containing the interferon LelF 8 gene terminated by the Pst residues shown in Figure 5) and plasmids pLelF A25 are shown in Figures 7a and 7b.

In order to obtain the Sau3a to Pst1 fragment containing approximately 950 b.p. of the sequence shown in Figure 7a, several operations have to be performed since one or more of the intervening Sau 3a restriction sites are present, i.e.:

1. After digestion, the following fragments are isolated:

(a) a fragment from Sau3a digestion ground to an EcoRI site containing 110 b.p.;

b) a fragment from the EcoRI site to the Xba site containing 132 b.p.;

c) a qd fragment of the Xba site to the Pst site containing more than 700 b.p.

2. Fragments 1a) and 1b) are ligated together and the Sau 3a and Xba end groups are cut off with Xba and BhlII to prevent self-polymerization caused by these terminals (the relevant Sau3a site is within the BglII site; BglII is cleaved to cut) Sau 3a catch end). The 242 b.p. fragment was isolated.

3. The products obtained by processes 2) and 1c) gg bind to each other and, in order to prevent spontaneous polymerization, the end groups are cut off with the enzymes PstI and BglII. A fragment from Sau3a to P9t1 of the threshing containing about 950 b.p., shown in Figure 7a, is isolated. This

CS 273 182 82 fragment contains that part of the LelF B gene that is not common to β LelF A.

4. Isolate a fragment containing approximately 300 b.p. from plasmid pLelF A25 ss. (from HindIII to Sau3a), containing the trp promoter-operator, ribosome binding site, ATG start signal, and cysteine codon for LelF A.

5. A fragment from Five to H nd XII containing approximately 3600 bp is isolated from plasmid pBR 322, which fragment contains a replicon and a coded tetracycline but not ampoule resistance.

6. The fragments obtained in steps 3, 4 and 5 are ligated in triplicate and the resulting ss plasmid is transformed into E. coli K-12 strain 294.

Perform minimal search engine sorting of transformants / by the procedure of Birnboim and colleagues, Nucleic Acids. Res. 7, 1513-1523 (1979)] and plasmid samples were digested with EcoRI endonuclease. Three fragments were identified by digestion which were identified as 1) a fragment containing the EcoRI-EcoRI trp promoter; 2) an internal EcoRI to EcoRI fragment of plasmid pL4, and 3) a fragment of plasmid pL4 containing an initiation signal for translation of the protein to EcoRI.

Bacterial extracts from the clones prepared as described above showed a typical value of about 10 x 10 6 units of interferon activity per liter in CPE assays at _{55 55} θ »= 1. The deden representative clone prepared by this procedure was designated E. coli 294 / pLeIF B trp 7 .

L. Direct expression of other mature leukocyte interfarons (LelF C, D, F, Η, I and □)

As with LelF A, other full-length gene fragments containing other types of LsIF interferons can be engineered and placed in expression carriers designed for their expression. By means of complete sequence determination by conventional methods, it is determined which restriction site lies sufficiently close to the first amino acid codon of the mature interferon type to allow the procedure described in Part H above to express mature interferon LelF A, i.e., elimination of the restriction pruning precision and codon replacement for N -terminal amino acids lost by elimination of presequence by binding of a synthetic DNA fragment. If this method fails, the following procedure can be used which, in brief, consists in cleavage of the fragment containing the sequence just before the point at which the codon for the first amino acid of the mature polypeptide begins. The procedure is that of

1. Double-stranded DNA converts into single-stranded DNA in the region surrounding this point;

2. The complementary length of the single-stranded DNA primer is hybridized to the single-stranded region formed in (1), the 5 'end of the primer lying on the opposite side of the nucleotide adjacent to the intended cleavage site:

3. That portion of the second strand removed in step 1) that lies 3 ^Z away from the primer, restores the reaction with DNA polymerase in the presence of deoxynucleotides triphosphates containing adsine, thymine, guanine, and cytosine;

4. Remaining length of single-stranded DNA that projects above the intended cleavage site is removed by digestion.

A short strand of synthetic DNA with an ATG translation initiation signal can then be ligated to the 3 terminus of the coding strand as its end, for example by binding to the engineered mature interferon gene using sticky ends, and inserted into a plasmid capable of expression. under the control of the promoter and its associated ribosome binding site.

In a manner similar to that described in Section K above, gene fragments encoding LelF C and LelF D are appropriately arranged for direct bacterial expression. The expression strategy for these other leukocyte interferons includes in each case the use of

CS 273 182 B2 fragment approximately 300 b.p. (HindIII to Sau3a), containing the trp promoter-operator, ribosome binding site, ATG initiation signal, and cysteine codon of interferon LelF A from plasmids pLelF A25. With this fragment, ee will combine gene fragments from other interferon genes encoding their respective amino acid sequences outside the initial cysteine that is common to all. Each plasmid thus produced was used to transform E. coli K-12 strain 294. The following ligation was used to construct the appropriate genes.

Interferon LelF C

The following fragments were isolated from the plasmids pLelF C:

a) fragment from Sau 3a to Sau 96 by 35 b.p.j

b) a fragment from Sau 96 to PstI of more than 900 b.p.

It was isolated from the plasmids pLelF A-25 as described above in section K4).

c) 300 bp HindIII to Sau 3a fragment;

It was isolated from the pBR 322 plasmids as described in Section K (5) above.

d) a fragment of PstI to HindIII of about 3500 b.p.

Construction of the gene

(1) Weigh fragments (a) and (c), digest the product with Bgl II and Hind III and isolate the fragment by about 335 b.p.

2) Fragmentation of fragments 1) + b) + d) is performed in triplicate and the resulting plasmid is transformed with E. coli.

A typical clone obtained by this method was designated E. coli K-12 strain 294 / pLeIF C trp 35.

Interferon LelF O

The following fragments were isolated from plasmids pLelF 0:

(a) a 35 bp Sau 3a to Ava II fragment;

b) a fragment from Ava II to Bgl II of 150 b.p.j

c) a fragment from BglII to PstI of approximately 700 b.p.

It was isolated from the plasmids pLsIF A25

d) a fragment from HindIII to Sau3a of 300 b.p.

It was isolated from pBR 322

e) a fragment from HindIII to PstI of approximately 3600 b.p.

Construction of the gene

1) Weigh the fragments a) + b), cut the product with the enzyme BglII and after purification yield a product of 185 b.p. (1).

(2) Binding of fragments (1) + (d), excision of the HindIII and BglII enzymes and purification of the product of approximately 500 b.p. (2),

3) The 2) + c) + a) fragments were ligated and the obtained plasmid transformed with E. coli.

A typical clone prepared in this way was designated E. coli K-12 strain 294 / pLeIF D trp 11.

Interferon LelF F

The LsIF F-containing fragment can be constructed for direct expression by preferably using the amino acid sequence 1 to 13 of interferon LelF B, which is exactly the same as that of interferon LelF F, for reconstitution. From the pHGH 207 plasmids described above, of Pst I and Xba I digestion enzymes by digestion and subsequent isolation at 19

CS 273 182 B2 right fragment about 1050 b.p. (a) containing the trp promoter and having the desired configuration at its ends. The second fragment (b) is isolated as a larger fragment from the mixture obtained by digesting the plasmids pHKY 10 with PstI and BglII endonucleases. Fragment a) comprises approximately half of the gene encoding ampicillin resistance; fragment b) contains the remainder of this gene and the entire tetracycline resistance gene except the linked promoter. Fragments a) and b) were ligated with T4 ligase and the product was digested with XbaI and BglII to remove dimerization products. This results in fragment c) containing the trp promoter-operator and genes for tetracycline and ampicillin resistance.

By digesting the plasmids pLsIF F with the enzymes Avall and BglII, fragment d) having approximately 580 b.p. and containing codons for amino acids 14 to 166 of interferon LelF F.

The digestion of the plasmids pLelF B by the XbaI and Avall enzymes yielded a fragment of 49 b.p. which encodes amino acids 1 to 13 of interferon LelF F.

Fragmentation of fragments c), d) and e) is performed in triplicate in the presence of T4 ligase. The cohesive ends of the respective fragments are such that the ss fragments correctly encircle to form a composite plasmid which the nsss gene for tetracycline resistance under the control of the trp promoter-operator, together with the gsns for the mature interferon LelF F. said resistance to their cultivation on tetraoycline-containing agar plates. A typical clone prepared in this way was designated E. coli K-12 strain 294 / pLeIF F trp 1.

Interferon LelF H

The full length LelF H gene for the expression of mature leukoeytar interferon LelF H can be constructed as follows:

1) Plasmid pLsIF H was subjected to < tb > digestion with Haell and Rsal, and a fragment of 816 b.p. extending from the signal for peptide amino acid 10 to 3 'of the non-coding region.

2) The fragment is dsnaturated and subjected to reparative synthesis using the Klenow fragment of DNA polymerase I (see Klenow et al.). Why. Nati. Acad. Sci. USA 65, 168 (1970)], using the synthetic deoxyribooligonucleotide δ'-dATG TGT AAT CTG TCT as primer.

3) The resulting ee was digested with Sau3a and the 452 b.p. fragment was isolated. representing the amino acids of interferon 1 to 150.

4) The plasmid pLelF H was digested with the enzymes Sau3a and PstI and the resulting fragment of 500 b.p. was isolated; obtaining a gene encoding amino acids from 150 to the end of the coding sequence.

5) The fragments isolated in steps 3) and 4) are ligated together to form a fragment

166 met. aep stop

ATG TGT ... - .... GAT TGA

Sau3a encoding 166 amino acids of interferon LelF H.

6) The plasmid pLelF A trp 25 was digested with the restriction enzyme XbaI, terminated with the capture ends with DNA polymerase I and the product digested with PstI endonuclease. The resulting large fragment of ss is isolated and ligated with the product obtained in step 5). It obtains an expression-capable plasmid which, upon transformation into E. coli K-12 strain 294 nsbo into another host bacterium, can express the mature interferon LelF H.

Interferon LelF I

Recombinant human phase A gsnom of Charon 4A, constructed by Lawns and coworkers (see Cell 15, 1157 (1978)), was screened for leukocyte interferon

CS 273 182 B2 genes using the procedures described by Lawn and coworkers (see above) and Maniatie and coworkers (see Cell 15, 687 (1978)). A radioactive LelF assay, obtained from a cONA clone of interferon LelF A, was used to screen approximately 500,000 sand, of which six LelF genomic clones were selected. After repeated plaque sorting and purification, one of these clones, HL1F2, was selected for further analysis.

Using the method described above, other test samples may be advantageously used to isolate the other leuco-yoyl interferon proteins of the invention.

Construction of the LelF X interferon gene:

1) EcoRI fragment of λ HLelF 2 clone 2000 b.p. was subcloned into plasmid pBR 325 at its EcoRI restriction site. The digested plasmid containing LelF I was digested with EcoRI and the 2000 b.p. fragment isolated. For this EcoRI fragment of 2000 b.p. The dAATTCTGCAG deoxy oligonucleotide (EcoRI-PstI converter) is ligated, the resulting product is digested with PstI and a fragment of 2000 b.p. containing petl ends. This fragment was digested with the enzyme Sau 96 and a fragment of 1100 b.p. was isolated which had one PstI and one Sau 96 end.

2) Plasmid pLelF C trp 35 was digested with PstI and XbaI and the larger fragment isolated.

3) Small XbaI - PstI fragment from plasmid pLelF C trp 35 was digested with XbaI and Sau96 and the XbaI - Sau 96 fragment of 40 b.p. was isolated.

4) Ligate the fragments isolated in steps 1); 2) and 3) to generate an expression capable plasmid pLelF I trp 1.

Interferon LelF O

To construct a plasmid capable of expressing LelF 3:

1) Plasmid pLelF 3 contains a HindIII fragment of 3.8 kilobaeich human genomic ONA. which comprises the LelF O gene sequence. From this plasmid, the .alpha.-Rsal fragment of 760 b.p.

2) The plasmid pLsIF trp 7 was digested with HindIII and DdeI and isolated the HindIII-DdeI ee fragment by 340 b.p.

3) Plasmid pBR 322 was digested with PstI, the fragments were terminated with a sticky end by incubation with 8 DNA Polymerase I (Klenow fragment) and digested with HindIII. A large fragment of approximately 3600 b.p. is isolated.

(4) Ligate the fragments isolated in steps (1), (2) and (3) to ligate to produce an expression plasmid-pLelF 0 trp 1.

M. cleaned

The leukocyte interferon content of bacterial extracts can be increased by sequential purification:

A polyethyleneimine 3-precipitation in which most of the cellular proteins, including interferon, remain in the supernatant.

2. Ammonium sulfate fractionation in which the interferon falls out of a 55% saturated ammonium sulphate solution.

3. Suspending the ammonium sulfate pellets in a buffer composed of 0.06 M potassium phosphate and 10 mM Tris-HCl, pH 7; and dialysis against 25 mM Tris-HCl, pH 7.9 (interferon activity remains in solution).

4. Chromatography of the supernatant obtained above, at a pH adjusted to 8.5, on a cellulose OEAE column (linear gradient elution with 0 to 0.2 M sodium chloride solution in 25 mM Tris-HCl, pH 8.5).

5. Adsorption on agarose (Cibachrome Blue Agarose) or hydroxyapattle and elution

CS 273 182 B2

1.5 M potassium chloride solution or 0.2 M phosphate solution.

O

6. Molecular sorting on a Saphadax G-75 column.

7. Catax-chromatography on CM-cellulose in 25 mM ammonium acetate solution at pH 5.0, eluting with an ammonium acetate gradient (up to 0.2 M ammonium acetate solution) ·

By the above purification procedures, a material with a purity of more than 95% is obtained.

The crude material can also be purified by molecular size chromatography, ravarani phase high-pressure liquid chromatography (RP-8), or affinity chromatography on immobilized anti-interferon antibodies.

Alternatively, the material obtained in step (4) above may be applied to a compound containing the same. a monoclonal antibody, prepared as described by Milstainam in Scientific American 243, 66 (1980), and interferon eluting with 0.2 M acetic acid mixtures,

0.1% Triton solution and 0.15 M sodium chloride solution.

In an alternative preferred embodiment, the leukocyte interferon produced as described above can be purified by the following purification operations:

Frozen cell pellets containing expressed isocyte interferon and are broken either manually or in a suitable grinding apparatus. partially thawed cells and suspended in 4 volumes of buffer A containing 0.1 M Trisbaae solution adjusted to pH 7.5 to 8.0 10% (w / v) sucrose solution, 0.2 M sodium chloride solution, 5 mM EDTA solution, 0.1 mM PMSF solution, and 10 to 100 mM anhydrous magnesium chloride solution. The suspension is maintained at about 4 ° C.

The slurry is then passed through a homogenizer at about 400 psig and then again at a pressure of less than about 200 psig. The homoganizer effluent from both passages is collected while cooling the ice bath.

2. Polyethyleneimine is slowly added to the homogenate to a concentration of about 0.35% and allowed to stand for about 30 minutes. The solids were collected by centrifugation or filtration. This step is carried out under constant temperature control or sufficiently fast so that the temperature of the supernatant (or filtrate) is still below 10 ° C. The supernatant (filtrate) is concentrated by ultrafiltration to approximately 1/10 of the original volume. Small particles or haze in the retained thickened solution can be removed by filtration through a suitable filter, such as a microporous membrane.

3. The clarified solution is applied directly to the monoclonal antibody-containing column at a flow rate of 5-8 cm / hr. (e.g. 25 to 40 ml / h on a 2/6 cm diameter column). After application of the solution, the column is washed with approximately ten times the volume of a column of mM Tris-HCl pH 7.5 to 8.5, containing 0/5 M sodium chloride solution and a surfactant solution such as Triton X-100 0.2; equivalent substances. After washing, the column is rinsed ten times the column volume of a solution containing 0.15 M sodium chloride and a surfactant such as Triton X-100 (0.1% solution) or equivalent. The column was then eluted with a 0.2 M acetic acid solution containing a surfactant such as Triton X-100 (0.1% solution) or equivalent. The Bilkine fraction from the β column by the monoclonal antibody (determined by UV abeorbance or other suitable analytical method) is collected and the pH is adjusted to approximately 4.5 with 1N sodium hydroxide solution or 1.0 M Tris-base solution.

4. Combine intarferon fractions are loaded onto a cation exchange column such as a CM 52 cellulose column (Whatman brand) or equivalent thereof previously equilibrated with a suitable buffer, such as a 50 mM ammonium acetate solution at pH 4.5. After interferon deposition, the column is washed with the buffer used to equilibrate until the UV abeorbance of the afluant reaches a level such that the ee from the column consumes some of the desired protein. The column is then eluted with 25 mM sodium acetate solution containing 0.12 M sodium chloride or another combination that optimizes the recovery of interferon and provides, after lyophilization, a product having satisfactory properties such as appearance and solubility.

CS 273 132 B2

The monoclonal antibodies used in the preferred purification of the invention described above can be prepared by the procedures described by Staohalin and co-workers in Proc, Natl. Acad. Sci. USA 78, 1848-1852 (1981), Monoclonal antibodies prepared from ascites fluid are ee purified and covalently bound to Affigel-10 as described below:

Each of the five Balb / c female mice is inoculated with 5-10 x 10 6 hybridoma cells from the medium growth phase of the strain. About 5 10 10 ³ viable cells obtained from mouse production fluid are inoculated intraperitoneally with another group of ten or more mice. Ascites fluid was collected (2x to 4x) from each mouse of the latter group. Up to three cell transfers and subsequent removal of ascites fluid from one mouse group to another can be performed. The ascites fluid collected from the mice at each cell transfer is collected.

Cells and their debris are removed from the ascites fluid by centrifugation at low speed (500-100 times g) for 15 minutes. Centrifugation is then continued at 18,000 rpm for 90 minutes. The supernatant is frozen and stored at -20 ° C.

After freezing, additional fibrin and fine precipitated material are removed by centrifugation at 35,000 rpm for 90 minutes. All batches of ascites fluid from each transfer are tested for specific antibody activity by determining the solid phase antibody binding (see Stachelin et al., Supra, supra) and the appropriate proportions are pooled.

Protein concentrations in the pooled solutions and e are determined by approximation on the basis that 1 mg of protein at 280 nm exhibits an abrosance of 1.2 in a 1.0 cm cuvette. Ascites fluids with high antibody levels contain 30 to 35 mg protein per ml.

This amount is equivalent to 4-7 mg specific antibody / ml. The ascites fluid at this concentration was diluted with PBS (0.01 M sodium phosphate solution at pH 7.3 and 0.15 M sodium chloride) to a concentration of 10-12 mg protein per ml.

To each 100 ml of this dilute solution, 90 ml of a saturated ammonium sulfate solution was slowly added at 0 ° C, with vigorous stirring. The slurry was cooled with ice for 40 to 60 minutes and then centrifuged for 15 minutes at 4 ° C at 10,000 rpm. The supernatant is decanted off and the centrifugate is dried well. The protein pellets were dissolved in a solution containing 0.02 M Trie-HCl (pH 7.9) and 0.04 M sodium chloride (buffer I). The DC protein solution is dialysed for 16-18 hours at room temperature against 100 volumes of said buffer I with at least one buffer change. The dialysed solution is centrifuged at 15,000 rpm for 10 minutes to remove insoluble material. By determining the absorption at 280 mm ss, 30-35% of the original amount of total protein present in the ascites fluid is recovered.

The solution containing 30 to 40 mg protein per ml is then applied to a DEAE-cellulose column equilibrated beforehand with Buffer I. A column layer of at least 100 ml is used for each gram of protein loaded. The antibody was eluted from the column by gradient elution with sodium chloride solution containing 0.02 M Tris-HCl, pH 7.9, with a linear gradient from 0.04 M to 0.5 M sodium chloride. The pooled protein fractions salified with a solution between 0.06 to 0.1 M sodium chloride were concentrated by precipitating the protein with an equal volume of ammonium sulfate solution saturated at room temperature and centrifuging the product. The obtained protein pellets are dissolved in a solution containing 0.2 M sodium bicarbonate (pH about 8.0) and 0.3 M sodium chloride (buffer XX) and the solution is dialysed at room temperature against the same buffer, which is changed three times, is centrifuged for 15 minutes at 20,000 times g to remove a small amount of insoluble material, and the protein concentration is adjusted to 20-25 mg / ml with buffer II. Said antibody solution is used to prepare an immunoadsorbant as follows.

Affigel-10 (available from BioRad Laboratories, Richmond, California) is washed on sin23

CS 273 182 B2 a triplicated glass filter three times with ice-cooled isopropanol and then three times with ice-cold distilled water. The gel slurry (about 50% ni in ice water) was transferred to plastic tubes and adsorbent sedimented by brief centrifugation and the supernatant was aspirated.

An equal volume (relative to the gel) of the purified antibody solution is added to the gel-filled tube and the tube is allowed to rotate about a perpendicular axis at 4 ° C for 5 hours to mix the contents. After completion of the reaction, the gel is centrifuged and washed twice with buffer III (0.1 M sodium bicarbonate solution and 0.15 M sodium chloride solution) to remove uncoupled antibody. Protein determination in the combined wash solutions showed that more than 90% of the antibody bound to the gel.

To block unreacted sites, the treated gel was mixed with an equal volume of 0.1 M ethanolamine hydrochloride solution (pH 8) and the tube was again rotated at room temperature for 60 minutes to mix the contents. The gslu slurry is washed until the reactants are removed with PBS and stored in PBS in the presence of 0.02% (w / v) sodium azide at 4 ° C.

N. Parenteral administration of interferons

LelF interfsrons can be administered parenterally to persons in need of anti-tumor or antiviral therapy and those who develop immunosuppressive conditions. The dosage and number of doses are parallel to those commonly used in clinical trials of human-derived interferons, e.g. about (1 to 10) x 10. in the case of materials of a purity greater than 1%, expediently up to this purity, for example 5 χ 10 ⁷ units per day.

□ as one example of a suitable dosage form for a substantially homogeneous bacterial interferon LelF, parenteral form can be mentioned: 3 mg of LsIF having specific activity per q, example 2 x 10 units / mg are dissolved in 25 ml of 5N human serumalbumin solution, filtered through a bacteriological filter and the filtered solution is aseptically dispensed into 100 vials, which then contain 6 x 10 units of pure interferon suitable for parenteral administration. The vials are preferably stored refrigerated (-20 ° C) prior to use.

The compounds of the present invention can be formulated by known methods into pharmaceutically acceptable compositions wherein the active polypeptide is used in admixture with a pharmaceutically acceptable carrier. Suitable formulas and their formulation are described, for example, in Remington's Pharmaceutical Sciences E.V., Martin, which is incorporated herein by reference. The pharmaceutical compositions of the present invention comprise an effective amount of an interferon protein together with a suitable amount of vshikula to permit effective administration of the preparation to the host. One of the preferred modes of administration is parental administration.

Claims (6)

A method of producing an expression vector capable of providing mature human leukocyte interferon LelF A, B, C, O, F, Η, I, or □ with the amino acid sequence from position 1 to 166 as shown in Figure 4 in a transformed strain of E. coli. and 9, characterized in that: a) constructing an E.coli vector containing the E.coli trp promoter, operator, and trp leader ribosome binding site, (b) preparing a DNA fragment encoding mature human leukocyte interferon, which is selected from the group consisting of human leukocyte interferon (LsIF) A, B, C, D, F, Η, I or J; and c) attaching the above DNA fragment of the human leukocyte interferon downstream of the E. coli trp promoter, operator and the trp leader ribosome binding site of the E. coli vector of step a) using a T ₄ DNA ligase. CS 273 182 B2 2. The method of item 1 for producing an expression vector capable of expressing mature human leukocyte interferon LelF A in the transformed E.coli strain with the amino acid sequence from position 1 to 166 as shown in Figure 4, characterized in that in step b ) prepares a DNA fragment encoding mature human leukocyte interferon (LelF) A. 3. The method of item 1 for producing an expression vector capable of providing mature human leukocyte interferon LeiF B, C, O, or F in the transformed E.coli strain with the amino acid sequence from position 1 to 166 as shown in FIG. that in step b) prepare! A DNA fragment encoding a mature human leukocyte interferon which is selected from the group consisting of human leukocyte interferon (LelF) B, C, D or F, 4. The method of item 1 for producing an expression vector capable of providing mature human Isukocyte interferon LelF H with the amino acid sequence from position 1 to 166 as shown in Fig. 4, characterized in that in step b ) prepares a DNA fragment encoding mature human leukocyte interferon (LelF) H. 5. The method of item 1 for producing an expression vector capable of providing mature human leukocyte interferon LelF I or 3 with the amino acid sequence from position 1 to 166 as shown in FIG. 9, as shown in FIG. 9, characterized in that: in step b) prepare! DNA fragment encoding mature human Isucocyte interferon (LelF) I or 3. 6. The method of claim 1, wherein the E. coli strain is E. coli K-12 strain 294 (ATCC 31446).