SE464088B - MANUFACTURING INTERFERON FROM HUMAN GENERAL MATERIAL - Google Patents

Description

The present invention relates to a method in which a specific fragment of human genetic DNA, extracted from human blood cells, is produced in a bacteriophage vector and used directly to program the synthesis of IFN in bacteria which have been cultured and thereby results in the production of interferon. With this method, there is thus no longer a need for loan trace pairings and reproduction of the entire length of complementary DNA (cDNA) fTånmRNA. According to the present invention, a suitable portion of DNA is taken directly from an readily available human cell, such as a white blood cell, and reproduced in an x-phage vector. The desired IFN gene is not disturbed by introns and can thus direct IFN synthesis in bacteria. High IFN activity was obtained by infecting appropriate bacteria, such as E. coli, with a recombinant phage containing introduced human DNA material with the IFN-31 gene or the IFN-β gene.

IFN was recovered and purified from the bacterial lysate resulting from such an infection. The phages were found to be suitable vectors for introducing human DNA fragments into bacteria; The A Charon 4A phage was particularly suitable and led to high production of IFN. The invention further relates to the introduction of such reproduced, human, genetic DNA fragments into plasmid expression vectors, which are provided with strong promoters. The procedure can be performed with different IFN-U genes as well as IFN-3 1 genes, provided that they lack introns.

In one embodiment, DNA was fragmented from an adult by partial Eco-RI digestion and reproduced in Aßharon 4A. The thus obtained clone, designated clone C15, with introduced human DNA material of about 17 kb, was identified as containing a gene for fibroblast interferon IFN-B 1. Restriction mapping showed that this gene placed on a 1.8 kb Eco RI fragment was not disturbed by intron. This human genetic DNA fragment can direct the synthesis of active human IFN-β1 in E. coli and in other suitable bacteria. A high IFN activity can be obtained from phage lysate. Under certain conditions, an activity of the order of 107 U / liter was recovered from the phenylates. The lysates are most advantageously subjected to chromatography, preferably on Cibacron Blue Sepharose or similar columns, and the interferons thus obtained have the same immunological properties and species specificity as IFN produced from human fibroblasts.

Similar results were obtained with IFN-α, whose gene was identified in clone 18-3 containing a 13 kb human gene fragment. In a further embodiment, the human gene DNA fragment containing the IFN-31 or IFN-β gene was introduced into a plasmid such as the plasmid pBR322 containing the recA promoter of E. coli. Cultures of E. coli transformed by these recombinant plasmids produced large amounts of IFN-β or IFN-β when the recA promoter was introduced with nalidixic acid. This IFN-β1 gene fragment was then cut off adjacent to the code sequence of the first methionine of the fully developed IFN-31 and bound to the ribosomal binding site by E. coli or lac promoter. This lac promoter was modified to contain the RNA polymerase binding site of the tryptophan promoter. With this hybrid tryp-lac promoter, the genetic IFN-B 1 DNA was able to produce 108 U / liter IFN-6 1 in E. coli minicell strain p679-54. Similar satisfactory yields of IFN-β C were obtained when the IFN-β C gene was appropriately bound to the tryp-lac promoter. In all cases, the IFN activity was recovered from bacterial cells by lysis with lysosome and 30% propylene glycol and then purified for phage lysate on a hydrophobic chromatographic support. For IFN-31, a preferred carrier is Cibacron Blue Sepharose. Elution can be performed using substances such as ethylene glycol or preferably propylene glycol. Further purification of IFN-31 or Q C can be performed by immunoaffinity chromatography or monoreproducing antibody columns or by other conventional techniques.

Thus, it is clear that human gene DNA fragments can be used for high yield production of human IFN in E. coli or any other suitable host bacterium. As mentioned above, the method of the present invention comprises introducing a fragment of human gene DNA into a suitable vector such as a phage, infecting a bacterial culture with such phages, resulting in lysis of the bacterium and recovering the resulting interferon content of lysate. The invention relates to the novel phages obtained by introducing human gene DNA and in particular to recombinant A-type phages such as clone C15, which results in IFN-5 and 1813, which results in IFN-ac. The invention further relates to a process for the production of interferon by introducing human gene DNA into a suitable phage, recovering a suitable DNA section from the phage, introducing it into expression as a plasmid in a suitable bacterium, culturing it and recovering the desired interferon. Other and further objects of the invention will become apparent from the following detailed description made for a non-limiting purpose.

An important advantage of the present invention is that genes from healthy and diseased individuals can be compared in terms of their ability to produce IFN, which allows us to study genetic defects in IFN production.

Preparation of human gene set: High molecular weight DNA was prepared from peripheral blood cells of an adult with 5-thalasemia. DNA solutions (4009) were digested separately for 30 minutes with 100, 200 or 300 units of Eco RI, then heated to 65 ° C for 10 minutes and applied together on a 30-40% sucrose gradient as described. DNA fragments between 10 and 20 kb were ligated with T4 DNA ligase as described by Mory et al. (1980, Mol. Biol. Reports 6, 203-208) to Å Charon 4A DNA arms prepared by Eco RI digestion according to Maniatis et al. (1978, Cell 15, 687-701). In vitro packaging was done as described by Hohn and Murray (1977, PNAS 74, 3259-3263) by mixing several 8 μl solutions of the compound reaction (0.5 μg vector DNA and 0.125 μg human DNA with 50 μl extract of E.coly BHB2688 (0205 recA '(Aimm 434 clts b2 red 3 Eam4 Sam7) m] and ßuaeseo [Nzos vec / l' (in imm 434 aus bz rea s nam 15 sam 7) / i). Per and recombinant DNA was obtained 3x105 pfu, compared to 108 pfu / U g intact A DNA, a total of 40 x 40 464 os 4 amounts of 1.8x106 recombinant phage were diluted in 6 ml of 10 mM Tris-HCl pH 7.5. 10 mM MgSO 4, 0.01% gelatin After the addition of 0.1 ml of chlorosorm and the removal of debris by microfuge centrifugation, the phages were amplified 10-fold by coating on E.coli DP50 (Leder et al., 1977, Science 196). , 175-177) at 4x103 pfu / 15 cm plate. For filtration, 2x104 pfu were inoculated on 15 cm plates and double nitrocellulose filters were lifted in sequence as indicated by Benton et al., (1977, Science 196, 180-182): This work was performed under P3-containing conditions.

Preparation of test material of IFN-β, cDNA: Poly A + from fibroblast FS11 cultures of human foreskin) induced 4 hours of 100 ug / ml poly (rI) :( rC), 50 ug / ml cycloheximide (with 2 pg / n fl actinomycin During the last hour)) were prepared and the two peaks of interferon mRNA were fractionated by sucrose gradient centrifugation as described by Neissenbach et al., (1980 PNAS 77, 7152-7156; Neissenbach et al., 1979, Eur. J. Biochem. 98 1-8).

This 11S mRNA (1.5 11g) was used to prepare reverse transcriptase RNA-cDNA hybrids from avian myeloblase fifi intoxication (J. Beard) and oligo (dT) as before Weissenbach et al., (1980, PNAS 77, 7152- 7156). The RNA-cDNA hybrids were directly reproduced according to Zain et al., / 1979, Cell 16, 851-861), but using dCTP for tailing the hybrids and dG-extended, Pst I-cleaved pBR322 as a vector (Chang et al., 1978, Nature 275, 617-624) A total of 12,500 tetr amps transformants of E.coli MM294 were obtained as exposed by Stenlund et al., (1980, Gene 1.0, 47-52 Coal hybridization was performed with ÉZp eDNA test samples transmitted either from 11S mRNA (6 pg) of induced FS11 cells or from total poly A + RNA (3.7 μg) of non-induced cells. using a synthetic complementary primer ("primer"), (see Narang et al., 1979, Methods Enxymol. 68, 90-98), 5'GAGATCTTCAGTTTC 3 ', which includes the Bg1 II site of the IFN-81 sequence. With 32 P-5 'end-labeled (Houghton et al., 1980, Nucl.Ac. Res. 8, 1913-1931) starting material, an expected 640 nucleotide long cDNA was noted only when induced RNA was used as a template. To prepare the batches, 20 pmol of starting material with each RNA were cooled in 4 μl of 0.4 M KCl at 30 ° C for 60 minutes, after which they were incubated in 25 l with 200 μCl each of 32 P-α-dATP and dCTP (both 400 Ci / mmol), 0.5 mM each of dGTP and TTP, 50 mM Tris-HCl hP 8.3, 4 mM MgCl 2, 5 mM dithiotretol, 50 ng / ml actinomycin 0, 4 mM pyrophosphate and 30 units reverse transcribed for 2 hours at 3706. After adding 11 g of E.coli DNA, the reaction mixture was treated with 0.3 N NaOH for 60 minutes at 7000, neutralized and filtered through Sefadex G-50 in 50 mM Tris-HCl pH 8, 0.1 M NaCl , 10 mM EDTA, 0.5% dodecyl sulfate.

From each RNA, 5x106 cpm cDNA was obtained. Colonies grown on nitrocellulose filters were fixed and DNA was denatured as described by Thayer (1979 Anal. Biochem. 98, 60-63). Each filter was prehybridized with 10 ml of 6xSET (SET = 150 mM NaCl, 2 mM EDTA, 30 mM Tris-HCl pH 8 ) 10xDenhardt's solution (Denhardt 1966, Biochem. Biophys. Res. Conm 10 15 20 25 30 35 40 464 088 23, 641-646). 0.1% Na pyrophosphate, 0.1% dodecyl sulphate, 50 ng / ml denatured E. coli DNA for 4 hours at 6706. Filts were hybridized at 6700 for 14-18 hours in the same solution at 0.5x10 6 cpm / ml of each of the two 32 P cDNA test samples, 10 μg / ml in A and 2.5 119 / ml in a C. The fibers were washed for one hour at 6706 in prehybridizing solution, then 3 times for 30 minutes at 6700 with 3xSET, 0, 1% pyrophosphate, 0.1% dodesy sulfate, then 60 minutes at 2500 with 4xSET, see Maniatis (1978, Ceii 15, 687-701). The fibers were dried and subjected to Agfa Curix X-ray film with intensifying fibers for 1-2 days at -7000.

Of the 3,500 filtered coioniae, 14 preferentially hybridized to initiated cDNA from induced mRNAs while 130 also hybridized to initiated cDNA of non-induced mRNAs. DNA (0.3 pg) of piasmides from the first group was hybridized to films (Kafatos et al., 1979, Nuci. Ac. Res. 7, 1541-1552) with the 5 'end 326 cpm) in 6xSSC, 10xDenhardts solution at 35 ° C -labeled starting material itself (0.3 pmoi, 4x10 for 21 hours, followed by washing with 6xSSC (900 mM NaCl, 90 mM Na citrate) at 35 ° C.

A cion I-6-5 was highly positive and DNA sequence analysis (see Maxam et al., 1980, Methods Enzymoi. 65, 499-560) showed that it contained about 370 nucleotides from the 3 'side of the IFN-5. the sequence. An additional 50,000 cions were filtered, prepared as before by introducing dC-extended ds cDNA from sucrose gradient-purified induced mRNA into Pst I mode of pBR322: the IFN-51 ions were found to be 0.16% (80 cions) in comparison. with 0.65% IFN-β 2-ions.

IFN-51 cion I-6-5 described above and derived from FS-11 mRNA was used to filter the human array.

Isolation of the genetic kion: IFN-61 cion Piasmid DNA from IFN-61 cDNA ions was labeled by nick translation according to Rigby et al., (1977, J. Moi. Bioi. 113, 237-251) to 2x208 cpm / U 9 DNA and hybridized at 106 cpm / ml to the human gene set. The preparation of the filters, the DNA denaturation and the hybridization processes were carried out as described above. Piaque phage that gave positive hybridization were once again coated at a density of 1-2x102 pfu on 9 cm piatas and refilled. This procedure was repeated 3 times until more than 95% of the piaque on the plate was hybridized to the IFN-61 cDNA. The phage cion C15 was isolated from such a piaque.

Phages are allowed to grow in flowing cultures as described by Biattner et al., (1977, Sciense 196, 161-169). E. coli DP50 is grown overnight in 1% tryptone, 0.5% NaCl, 0.5% yeast extract, 0.2% maitose, 0.25% MgSO 4, 0.01% diaminopimelic acid, 0.004% thymidine Approx. 0.3 ml of the culture was mixed for pre-adsorption with 0.3 ml of 10 mM MgSO 4, 10 mM CaCl 2 and 107 phage for 20 minutes at 3700. The cultures were then diluted in a 2 liter coif with 500 ml of preheated medium containing 1% NZ-amine. , 5% yeast extract, 0.5% NaCl, 0.1% casamino acids, 0.25% MgSO 4, 0.01% diaminopimeic acid, 0.004% thymidine and incubated with good aeration for 15-18 hours at 37 ° C. After complete analysis, the cell debris was removed by centrifugation for about 15 minutes at 7,000 rpm in a Sorvaii GSA rotor. From the prepared lysate, the phages were precipitated with 7% polyethylene chloride 6,000 and purified by two consecutive CsCl gradients. Phage C15 DNA was purified with phenol and its structure was analyzed by restriction enzyme digestion, horizontal agarose gel electrophoresis in 20 mM Tris base, 10 mM Na acetate, 1 mM EDTA with 0.5 μg / ml ethidium bromide, Transferred to nitroceil (Southern, 1975, J. Moi. Bioi. 98, 503-517) and hybridization to nick-translated IFN-51 cDNA as above. Eco RI fragments of C15 DNA were subscribed to in the Eco RI site of pBR322 for precise restriction mapping of piasmid DNA according to established methods, see Boiivar (1979, Methods Enzymoi. 68 245-267).

Analysis of interferon activity in phage lysate: Prepared phage IFN-β C15 lysates, prepared as above, were analyzed against phosphate buffered saline (PBS) for 7 hours. A 10 ml solution was applied to a 0.2 ml solution of Cibacron Biue Sepharose CL6B, (Pharmacia Fine Chem.). The solution was washed with 10-15 ml of 1 M NaCl, 0.02 M Na phosphate buffer pH 7.2 and then with 10 ml of 50% propylene glycol in the washing solution. Fractions (0.5 ml) were collected and pooled at 400; in some cases 0.1% human serum albumin was added for stabilization. The interferon activity was analyzed, as described by Weissenbach et al., (1979, Eur, J. Biochem. 98, 1-8) by diluting each fraction in series in a 95-well micropiatta. Each compartment in each well received 2-3x104 FS11 human fibrobiasters in 0.1 ml Minimum Essentiai Medium (Gibco), 5% fetal serum (FCS), 0.5% Gentamycin. After 18 hours at 3700, the medium was removed and vesicular stomatitis virus (VSV) was added in an amount of 1 pfu / cell in medium with 2% FCS. Inhibition of the cytopathic effect was noted 30-40 hours after infection, compared to IFN-β NIH standard G023-902-527.

Antiviral activity was also measured by reduction of 3 H-uridine incorporation after VSV infection as previously described (Weissenbach et al., 1979. Eur. J.

Biochem. 98, 1-8). Antiserum to IFN-3 was obtained from hares and analyzed as before (Weissenbach et al., 1979, Eur. J. Biochem. 98, 1-8). For the neutralization test, 0.1 ml of antiserum (titration 104 U / ml) of a non-immune serum was mixed with 0.2 ml of 50% suspension of protein A-Sepharose beads (Pharmacia Fine Chem.) In PBS, under 1 hour at 3700. The beads were washed twice with PBS and 0.1 ml of bacterial interferon (100 U / ml) was added. After 2 hours at 37 ° C, the beads were centrifuged and the supernatants were analyzed for inhibition of 3 H-uridine inforivates in VSV-infected cells.

Gene ion isolation: IFN acion 18-3 Two short monofilament oligonucleotides, chemically synthesized as above, were used to directly filter the human gene set. The oligonucleotides are complementary to conventional sequences of IFN- (X genes and had the sequence 10 '5' CTCTGACAACCTCCC 3 'and 5'CCTTCTGGAACTG 3'. The oligonucleotides were used as after 5 'labeling, either directly or as starting material for cDNA synthesis on RNA from Sendai virus-induced human leukemic cells. .

The hybridizations were performed in plastic-tight cooking containers with a minimum volume of liquid.

Hybridization with the 15 base starter units was performed as follows: First, prehybridization in 6xSSC, 10XDenbardts at 37 ° C for 4 hours; then hybridization in 6xSSC, 1DxDenhardts at 3700 for 18 hours and washes, 3-4 times, 30 minutes each in 6xSSC at 3706 or until nothing else could be counted in the wash. For hybridization with the 13 base starter units, the hybridization and washing temperatures were lowered to 3006. A total of 16 phages gave positive hybridization and were further analyzed by restriction mapping and DNA sequence analysis. Clone 18-3 was shown to carry the 3 IFN gene of which one had a sequence identical to sDNA clion (XC by GoeddeI et al., (1980, Nature 290, 20-25). Clone 18-3 was purified as expression for clone C15 above Expression of clone 18-3 in A bacteriophage and after insertion into expressing piasmid vector is described in Result 6 RESULTS 1. Structure of genetic IFN-3 1 C15 clone: This phage clone was isolated from a set of human DNA which was depleted by Eco RI and reproduced in the arms of 1 Charon 4 A. The ion was strongly hybridized to two different IFN-5 1 cDNA samples, each independently produced by mRNA fractions of two strains of human fibrobias. DNA in Eco RI showed, in addition to the two A-arms, four fragments with 12; 2.6; 1.84 and 0.6 kioio bases (Fig. 1A) .Transmission to nitrocellulose using the method of Sothern (1975, J. MoI (Bio1. 98, 503-517) and hybridization with nick-translated DNA from the IFN-31 I-6-5 cDNA cion indicated that the 1.84 kb fragment contains IFN-β. 51 genes. This Eco RI fragment was reproduced in the Eco RI site of pBR322; Restriction analysis of this 1.84 kb subcion with Bgl II, Pvu II, Pst I, Hinc II and Taq I, and hybridization with batch II and full-length IFN-β1 cDNA samples, allowed the conclusion that the coding sequence for IFN-β preprotein (Hinc II site) starts about 350 nucleotides from Eco RI site (Fig. 1A). The distance between the different restriction sites in the genetic DNA is the same as in the cDNA sequence within the experimental phase (Table II 1). There were no unexpected restriction sites within the IFN-B1 coding region, which indicates the absence of any measurable interfering sequence.

Partial Eco RI digests of C15 DNA showed that the 0.6 kb Eci RI fragment is paced between the 1.84 and 2.6 kb Eco RI fragments. A Bam HI site was present in 2.6 kb, but not in the other Eco RI fragments of the inserted human DNA material. 1.84 kb The Eco RI fragment, which hybridizes to the IFN-61 cDNA, was found on a 9.6 kb Bam HI fragment of C15 DNA. As shown in Fig. 1, this fragment can come only from the Bam HI site of the right A-arm (5.1 kb from Eco RI), leaving 4.5 kb for the Eco RI-Bam HI segment of the human inserted the material and it is indicated that the 1.84 kb Eco RI fragment must be placed next to the right Å arm. The gene orientation (Fig. 1A) was determined as follows. When C15 DNA was cut with Pvu II and hybridized to full length or to 3 'half length of IFN-81 cDNA, a 0.66 kb fragment was found which hybridizes only to the 5' end of IFN-61. But then 1 The 84 kb Eco RI moiety was cut with Pvu II, the 5 'end of IFN-β was found on a smaller 0.54 kb fragment. Since no Pvu II site was found in the 0.6 kb Eco RI portion of the human introduced material, the 0.66 kb Pvu II fragment must end in the A right arm and thus the 5 'end of the IFN-61 is closest to the Right arm. . The structure shown in Fig. 1A is supported by a number of other restriction mapping experiments using Hind III, Sma I and Bgl II digests. Thus, the coding sequence for the IFN-β1 gene in clone C15 was introduced approximately 1,775 nucleotides away from the strong A PL promoter that controls transcription to the left (Williams et al., 1980, Genetic Engineering Vol II, pages 201-281, Plenum Corp.), and in the right waiting direction. This, together with the absence of measurable introns, led us to investigate whether interferon can be produced in E.coli infected with C15 recombinant phage. 2. Recovery of biologically interferon-active material from lysate of E.coli infected with IFN-B, C15 phage: Phage from clone C15 were grown in 500 ml cultures of E.coli DP50 as described previously. 10 ml of clarified lysate was dialyzed and loaded onto a small Cibacron Blue-Sepharose column (0.3 ml) and the fractions were analyzed for inhibition of cytopathic effect of vesicular stomatitis virus (VSV) with an IFN-B standard as reference. In an experimental derphage titer in the lysate was 1.3x10 10 phages / ml, the interferon active pattern shown in Fig. 2 was observed. A total of 7,500 units of IFN activity was recovered in the fractions eluted with 50% propylene glycol - 1 M NaCl from the column. . These would correspond to 0.75x106 units / liter lit. In the crude lysate, however, the measurable activity was much lower, which makes it probable that certain material in the lysate prevents correct analysis of the IFN activity. In a reconstruction experiment, a standard human IFN-B was added to a wild type A Charon 4A lysate and the activity was measured to only 1/10 of the constituent. When this mixture was passed through a Blue-Sepharos column, 75% of the constituent activity was recovered. During a control run, lysates from a wild-type Å Charon 4A phage were analyzed and no IFN activity was found (Fig. 2).

The chromatographic behavior of IFN from lysate of clone C15 may vary. In an experiment where the phage titer was 3x10 11 pfu / ml, the IFN activity was high, but was not maintained on the Blue-Sepharose column. In this experiment, the total activity recovered from the coefficient amounted to 70-80,000 units IFN for an input batch of 10 ml of lysate (7-8x106 units / liter). The effluent fractions were reabsorbed on a second Biue-Sepharose column: this time the activity on the column was maintained but was again eluted by washing the column with PBS alone. Human IFN-β should normally be mediated from Biue-Sepharos by hydrophobic solvents alone (Knight et al., 1980, Science 207, 525-526). Thus, standard human IFN-β was mixed with the same phage lysate and 30% of the activity was not applied to the cohort and the recovery of the activity was only 25%. This presupposes that components thereof have presumably destabilized the interaction of IFN-β, especially that produced by bacteria, with Biue-Sepharose. Although no interferon was retained on the coion, over 90% of the protein of the Tysate was removed from the active fractions, viika had a specific activity of 4-5x105 U / ml protein. This partial purification was essential to remove the chemicals which masked the activity of the crude lysate. In one experiment, the cion C15 IFN activity was also recovered after chromatography of the lysate on carboxymethyl-Sepharose (not shown). 3. Properties of interferon produced in E. coli infected with cion C15 The interferon activity recovered after the chromatography step reduced the 3 H-uridine incorporation into VSV-infected human cells treated with actinomycin D (Weissenbach, 1979, Eur. J. Biochem. 98, 1-8). The titration curve obtained for bacterial IFN-β was similar to that of standard human IFN-β (Fig. 3). To test the immunological properties of bacterial IFN, the deivis purified product was mixed with rabbit anti-IFN-B serum (inactive on IFN-o) eggs with non-immune rabbit serum linked to protein A-Sepharose. After centrifugation, the supernatant was analyzed for IFN activity: anti IFN-β serum removed α11 interferon activity, while the activity was maintained when non-immune serum was used (Fig. 3). Bacterial IFN (20 U / ml) was similarly neutralized after being mixed only with anti IFN-8 (20 U / ml) on the ce11 cultures and analyzed by inhibiting VSV cytopathic effect.

The species specificity of bacterial interferon was analyzed by comparing different cell types. When human IFN-β product of kion-infected E. coli showed less than 10% activity on monkey kidney cells BSC-1 and ox MDBK cells compared to human cells. No detectable antiviral activity is obtained on mouse L egg A9 cells (Table 2. The same result is obtained with 6,000 U / ml of bacterial IFN produced by cion C15.

Thus, the present invention demonstrates that the IFN-81 gene isolated directly from Eco RI fragments of human gene DNA by reproduction in Charon 4A can be expressed and accumulate significant amounts of IFN activity in kuituriysate.

The activity produced is kiart fibrobiast interferon (ß) as shown by its immunological properties and its species specificity. The activity is given by the bacterial culture only in response to infection by IFN-B1 C15 phage ions. The IFN activity produced by clone 15 in E. coli is similar to human IFN-B with its chromatographic properties on Cibacron Blue Sepharose. The chromatographic step is essential to recover high activity (up to 7x106 U / liter) from bacterial lysate. Interferons - thus provide the first examples of biologically active protein that can be produced by bacteria while directing a piece of human DNA taken directly from the gene for the individual. 4. single IFN-α, 18-3 kian This clone was identified by direct hybridization of short oligonucleotides, which were chemically synthesized to be complementary to the sequences of IFN-d.

From a total of 0.5x106 gene units, 16 clones were positive at hybridization. The Eco RI fragments that hybridize to the oligonucleotides were sequenced and clone 18-3 was shown as in Fig. 1B to contain a 2 kb Eco RI fragment 18-33 (inserted material in Fig. 1) in which the gene for, 1FN mRNA which gave gives rise to the IFN-cxc clone reported by Goeddel et al., (1981, Nature 290, 20-25). Clone 5-1 represents an overlapping DNA fragment. Together, clone 5-1 and clone 18-3, in addition to IFN-α C, carry two other IFN-γ genes, designated d-CZ and α-C4 (Fig. 1B). 5. Expression of IFN genetic fragment under the control of recA promoter The recA promoter is termed one of the strongest E. coli promoters. Normally, it is strongly pushed back through the product by the lex gene, even when it is present on a dopant dasmid such as pBR322. It is induced in the presence of damaged DNA to cleave its own repressor and undergoes induction to very high levels (Sancar and Rupp, 1979, PNAS 76, 3144-3148). The standard inducers for recA are nalidixic acid (which blocks DNA synthesis) or mitomycin C (which crosslinks DNA).

A plasmid, pDR1453 containing the reproduced recA gene, was used to isolate the promoter on a TaqI-BamHI fragment of about 1 kilobase. This was combined with pBR322 which had been opened with ClaI and BamI to plasmid RAP-1. The TaqI-ClaI compound restores the ClaI site in this case and allows the plasmid to be uniquely opened at the third code site of the recA gene. RAP-2 was constructed by cleaving RAP-1 with Eco RI and ClaI, filling in the adhesive ends and reassembling which restores the RI site. When RAP-2 is opened at the RI site, foreign DNA can be introduced at the third encoding of the recA protein.

The RAP-1 vector was used to indirectly express the interferon activity by 1 genetic fragment from the human gene set. The IFN-31 gene and several isolated IFN-Q genes of the α C subclass produced strong interferon activity (as determined by standard antiviral assay) when introduced into this plasmid. The RI fragments of DNA on which these genes are located (Figs. 1A and B) were subcloned into the RI sites of the RAP-1 plasmid. The plasmid was placed in a recA + host, the bacteria were grown and induced with naidixic acid, the cells were harvested, analyzed by lysozyme using 30% propylene glycol and the extract analyzed for antiviral activity. The level of activity is clearly dependent on the induction with the aid of naidixic acid, the optimum being 50 119 / m1 (Table 11 3). Interestingly, in some cases, the genetic fragment produces activity in both possible directions relative to the recA promoter. The IFN-β 1 and IFN-β genes that showed activity were α11a piaced on the DNA Eco RI fragments of 1.8-2 kio bases, so that the distance from the promoter to the ATG start code of the interferon gene is in the order of magnitude of a 100-ta1 nucleotides.

These experiments show that the promoter works as expected and it allows rapid determination of which interferon-like genes from the gene set may be active. High expression capacity of the IFN-61 gene under the control of the tryp-1ac hybrid promoter E 'E Piasmiden PLJ3 (Johnsrud, PNAS, 1978, 75, 5314-5318) contains two 1ac promoters each 95 baS'PaP1ang, separate of 95 unrelated nucleotides. This 285 nucleotide long fragment is inserted into the Eco RI site of the piasmid PMB9. The 95 base-pair Lac sequences contain the operator and promoter sequences and are secreted before ATG is converted to galactosidase. A coding DNA sequence with an ATG initiator, inserted at the correct distance from the ribosomal binding site of the β-galactosidase gene, should be properly expressed in E. coli cells. The 285 base pair Eco RI fragment was reproduced to 11 pBR322 at the Eco RI site; the recombinants were filtered by growing the cells on plates containing 40 g / ml X-gal and 15 g / ml tetracycline. Only positive colonies were pooled and DNA was purified by CsCl gradient centrifugation. Two orientations of the 1ac promoter are expected; against the ampicillin gene or against the tetracycline gene of pBR322. Since we are only interested in having the Eco RI site piaced between the 1ac ribosomal binding site and the pBR322 sequence, this site was protected by binding RNA polymerase to p1asmid DNA, while the outer site was opened by Eco RI restriction, Kïenow's fragments of DNA were polymerized and reunited. After transformation of MM294 E. coli cells, piasmids were isolated and the distance between the Eco RI site and the Bam HI site of pBR322 was measured. The piasmids containing a 375 base pair fragment were those in which the 1ac promoters were directed against the tetracycline gene, while those containing 670 base pairs (375-295 base pairs) had the 1ac promoters facing the ampicillin gene. genes.

From the Eco RI 1.84 kb subcion of the IFN-31 genetic fragment, we cut a HincII-Bg1II fragment containing the pre-interferon sequence. The complete IFN-β1 protein contains at its NH2 side end a methionine which can be used as an initiator code in E. coli. There are two A1uI sites near this ATG code separated by 13 nucleotides, one A1uI site is 8 nucleotides in front of ATG while the other is located 10 15 20 25 30 35 40 12 464 088 2 nucleotides after ATG. A partial AIuI digestion was performed on the HincII-BglII fragment and fragments of about 510 base pairs in length were isolated from agarose gels. The vector containing the Iac promoters facing the tetracycline gene was restricted by Eco RI, the site was filled in by DNA was polymerized and cut off again with BamHI.

Upon association of the A1uI-Bg1II fragment with the liquid Eco Ri-BamHI vector, the Eco RI site was restored. In order to be able to distinguish these ations containing the ATG code (13 nucleotides longer), the obtained ions are subjected to restriction with Eco RI and Pst1; the ions containing the expected 151 base-pair fragments were sequenced. Thereby, the Eco RI site and the distance between the ribosomal binding site were reopened and ATG was shortened by Bai 31 uptake and reunion.

E. coli cells transformed with these piasmids were seeded for IFN-β production.

One kion, L-11, was found to produce approximately 3x106 U / liters of IFN-3. The lac promoter region of this kion was cut off with HpaHI, ie 17 nucleotides before the starting point of mRNA. To remove the IFN-51 gene, Sau3a enzyme was used, the DNA was only 3 nucleotides beyond the IFN-31 secretion code. This HpaII-Sau3a 1ac-IFN-β1 fragment was inserted between the C1aI and HindIII sites of a tryp promoter piasmide, which was prepared as follows. A 180 nucleotide long TaqI-TaqI fragment from -21 to 11 -201 before start of tryp mRNA was removed from the tryp plasmid pEP121-221 and reproduced in the C1aI site of pBR322. We vaïde out the orientation where the tryp promoter fragment is oriented clockwise. This procedure restores a C1aI site at position -21 of the tryp promoter. Following association to the Iac-IFN-51 fragment, a hybrid promoter tryp-Iac is formed before the IFN-81 gene. This piasmid TL-11 was used to transform E. coli MM294 or E. coli miniceii strain p679-54. These bacteria produce 108 U / liters of IFN-β1 during fermentation to a 00650 out of 10, which demonstrates that the IFN-β1 genetic DNA fragment is highly active (Fig. 4).

TABLE 1: Restriction sites in IFN-B1 of cDNA and the gene Restriction_Space between sites sites Calculated from * Measured from ** t 81 cDNA sequence 61 genetic DNA nucleotides nucleotides Taq I-Pvu II 255 250 Hinc II -Pvu II 204 210 Hinc II-Bg] II 570 570 Pvu II -Bgi II 366 360 Pst I -B91 II 363 360 * From Taniguchi et al, 1980 Gene 10 (11-15).

** Measured from 1.84 kb Eco RI fragments The Hinc II and Taq I sites taken into account are those closest to the 5 'end of the gene. 10 15 20 25 30 35 13 0 TABLE 2: Species specificity for bacterial interferon produced by IFN-31 C15-k1on Interferon titer, U / m1 Interferon-kä11a Human Monkey Ox Mouse Mouse FS11 BSC-1 MDBK L A9 ce11er ce11er ce11er ce11er ce11er Cells 1. Human Cells FS11 1,500 32 32 4 4 2. E. coli infected with C15 clone 1,000 32 32 4 4 Bacterial IFN was used after chromatography of the C15 lysate on Cibacron B1ue-Sepharose as in Fig. 2.

TABLE 3: IFN-β and β -activities obtained from genetic fragments in the recA-plasmid RAP-1 Experiment Ki 011 iwajidlan ïF fi Hffftivity 1. RAP-1 (1833) -IFN-d GC - Naidixic acid 32_ 2. RAP-1 (1833) -IFN-ac right orientation 1000 RAP-1 (631) -IFN-B1 right orientation 3000 RAP-1 (631) -IFN ¥ ß1 opposite orientation 750 Kion RAP-1 (1833) contains the Eco RI fragment with the IFN-ac gene (Fig. 1B).

Kion RAP-1 (631) contains the Eco RI fragment with the IFN-B1 gene (Fig. 1A).

The fragments are introduced at the beginning of the recA gene. Orientation is given with respect to the recA promoter orientation.

Explanatory text to the figures: Fig. 1A: Schematic structure of the IFN-31 clone C15 DNA a): Schematic map of A Charon 4A. The two arms are shown in solid lines. b): Structure of the human DNA material introduced into the C15 cion, the blackened area shows the Eco RI fragment which hybridizes to the IFN-31 cDNA. c): en dubbe1 Iinje. The single line is deiar of the right arm of A. PL is the left promoter of A. d): IFN-61 mRNA position. The upper scales are for a) and b). The lower scale for c) and d). For details see the text.

Schematic structure of the IFN ac 18-3 Magnification of the blackened fragment from b shown as Fig; 1B: Restriction map of the introduced material in two x Charon 4A k1ones is displayed.

The IFN-ac gene is denoted by piïen a1fa-c *. The other two IFN-α genes are present near IFN11c. The inserted material shows the Eco RI 2 kb fragment Fig. 2: F19. a; Fig. 4: 464 us in .M 18-33, which contained the IFN-γ C genes and was reproduced in the recA-piazemide, as explained in the text. Symbols for the restriction enzyme sites are shown.

Analyzes of interferon produced by the genetic clone IFN-B 1_CJ5 on Bïue-Sepharosê Râïsat of E. coli DPSÛ infected with phage C15 were fractionated as indicated in the description and the IFN activity was analyzed in each fraction (1 '- 0). The protein concentration was measured in the same fractions (0- --O). A para11e11 chromatography and analysis of ivsat from the .Charon 4A phage are shown G3 --- CD.

Titration of generic clone IFN-61 C15 interferon A fraction of phage C15 IFN from the column in Fig. 2 (approximately 103 U / ml) was diluted 1:40 and then serially diluted in the micropiate to analyze IFN by diluting VSV RNA synthesis. Go - toe. Human standard fibrobiast interferon 25 U / mï analyzed paraïe fl t (0 ----- o). KontroH without VSV (CI!) And with VSV without IFN (yet) is displayed. The two spaces to the right show residual activity of the phage C15 IFN (at final dilution 1:40) after adsorption on immobilized anti IFN-3 or non-immune IgG, as described previously.

Ti11 growth and IFN production in E. coli containing piasmid TL11 The TL11 piasmid contains a hybrid tryp1ac promoter and the coding sequence for fully human IFN-β1, which is derived from the human genetic fragment as described in the text previously. Bacterial cultures are grown in a 1-liter New Brunswick fermenter and at the indicated times the bacteria are allowed to undergo analysis of lysozyme-propylene glycol and IFN activity was assayed in human cells activated with VSV. The IFN activity is calculated per m1 of bacterial kit.

Claims (10)

“S 464 us PATENT CLAIMS Process for the production of human interferon of fibroblast type (B) and of leukocyte type (a), characterized in that human genomic DNA encoding interferon of fibroblast type B1 and leukocyte type ac and lacking introns is introduced into a lambda phage and either that suitable bacteria are infected with such a phage recombinant or that from such a phage a DNA segment containing the IFN-B1 gene or the IFN-α gene is recovered, which is introduced into an expression plasmid of a suitable bacterium, that the bacteria are cultured and that the interferon is recovered. A method according to claim 1, characterized in that the phage is a lambda-Charon 4A, resulting in a clone designated C15. A method according to claim 1, characterized in that the phage is lambda-Charon 4A, resulting in clone 18-3. A method according to any one of claims 1-3, characterized in that the bacterium that is infected is E.coli. 5. A method according to claim 4, characterized in that the phage is lambda-Charon 4A and the bacterium is E.coli DP-50. A method according to claim 1, characterized in that a human is recovered from such phages by DNA fraction, introduced into a plasmid expression vector of a suitable bacterium and that the bacterium is cultured to produce interferon. A method according to claim 1, characterized in that DNA is introduced into a plasmid provided with a strong promoter, which is capable of directing the synthesis of human interferon B1 or human interferon and in a suitable bacterium. Method according to claim 7, characterized in that the plasmid used is pBR322 and that the inserted gene is IFN-B1 or the IFN-α gene. The method of claim 8, characterized in that the plasmid is pBR322 containing the recA promoter for E. coli. Modification of a method according to claim 1 or claim 6, characterized in that the human genomic DNA fragment containing the IFN-αC or IFN-β1 gene is removed from the lambda phage or from the plasmid, cut off next to the code for the first the methionine of the complete IFN associated with the ribosomal binding site of the E. coli lac promoter, which is modified to contain the RNA polymerase binding site of the tryptophan promoter, thereby obtaining a hybrid tryp-lac promoter, that the plasmid returns introduced into the bacterium and that the bacterium is cultured to produce interferon.