CH685299A5 - Highly purified human interferon prepn. - Google Patents

Abstract

Isolation of genetic material (DNA) contg. the nucleotide sequence for interferon in human cells involves cultivating cells producing interferon when exposed to an inducer of interferon, then exposing the cells to an inducer, extracting messenger RNA from the induced cells, purifying the RNA, transcribing it into DNA and cloning the DNA in a suitable vector. Detection of the different genetic sequence expressed in a human cell when it is induced to produce interferon involves effecting a differential hybridisation of the bacterial cloves first with DNA from induced-cells RNA (the inducer is double stranded RNA in the above described procedure) and second with identical DNA derived from uninduced-cell RNA. Engineering a bacterial strain to produce interferon polypeptide involves introducing a cloned interferon DNA obtd. as described above into a vector carrier. The highly purified interferons can be produced on a large scale for use as antiviral and antitumour agents.

Description

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description

Interferon is an important body-made antiviral protein that also inhibits the growth of tumor cells. Due to its species specificity, human interferon is required for use in human medicine. The limited amounts of interferon that are formed in tissue culture cells and fresh leukocytes are not sufficient for clinical use on a large scale. Introducing the genetic information for human interferon into bacteria could allow the production of a polypeptide with interferon activity. It is known that such methods have been developed for human growth hormone and for insulin.

The two types of interferon, namely leukocyte interferon (IFN-a) and fibroblast interferon (IFN-ß) are apparently encoded by different mRNAs; see. Cavallieri et al., Proc. Nati. Acad. Sei., USA, Volume 74 (1977), pages 4415 to 4419. The isolation of these mRNAs in pure form has not yet been successful. This also appears to be all the more difficult since the host cell is only able to synthesize the interferon mRNA if the cell is exposed to suitable exogenous factors, for example a viral infection or special synthetic polynucleotides such as poly- (rl: rC). Even then, the cell produces only the smallest quantities. Accordingly, it has been proposed to use an mRNA extract from host cells which had previously been induced to form interferon, and to have this mRNA translated in vitro in a cell-free system with all components, in particular the natural amino acids, in order in this way Obtain protein preparation with interferon activity. However, the interferon mRNA represents a very minor part of the mRNAs to be translated, and accordingly the protein preparation finally obtained with interferon activity was very heavily contaminated by other proteins.

Therefore, the use of such mRNA preparations as starting material for direct conversion into double-stranded DNA, which is to be cloned after insertion into a suitable vector, would involve screening difficulties which would be practically insurmountable.

The invention is therefore based on the object of largely overcoming the difficulties mentioned in the production of interferon.

While it has only been possible to isolate human fibroblast interferon β1, which has a molecular weight of about 20,000, using known methods, in connection with the present invention it has been possible to isolate a new, highly active substance with a molecular weight of about 23,000, which as Interferon ß2 was called.

The present invention thus relates to human fibroblast interferon β2, as defined in claim 1.

The invention further relates to a method for producing human fibroblast interferon β1 and β2, as defined in claim 2. Particular embodiments of this method are defined in claims 3 and 4. The invention further relates to the interferons obtained in this way and to the use of interferons for the manufacture of pharmaceutical preparations as defined in claims 7 and 8.

The method according to the invention is preferably used for the production of highly purified human fibroblast interferons β1 and β2.

It is to be considered an advantage that some of the stages listed do not have to be carried out in exactly the order given. This applies in particular to stage f), which relates to the production of the cDNA samples.

In a further advantageous embodiment of the method according to the invention, steps c) and f) are only carried out on fractions of the mRNAs which are extracted from the cells, specifically from the induced or non-induced cells. In this regard, the fact can be exploited that mRNAs contain a poly-A part which enables the separation of the mRNA from the total RNA by binding to oligo-dT cellulose. The subsequently eluted mRNA fraction is then advantageously fractionated by a sucrose gradient centrifugation. The various bands are then separately translated in a suitable cell-free system, for example a reticulocyte lysate. Each of the translation products is then examined to see if it is precipitated by an anti-interferon serum. The bands of the mRNA, the translation products of which give a positive reaction in this immunoprecipitation test, are retained for carrying out the two steps c) and f) described above. Another aspect of this method can be seen in the fact that two different mRNAs which code for interferon can be isolated from human fibroblast cells if these cells are induced according to step a). The smaller mRNA sediments at 11 S and delivers a protein with a molecular weight of 20,000 by translation in a cell-free system. This is selectively precipitated by antibodies which are produced against one of the interferons, which can be obtained from these cells and purified. This protein is human interferon (IFN) -ßl. The larger mRNA sediments at 14 S and yields a protein with a molecular weight of 23,000. This protein is precipitated by antibodies against a less purified fibroblast interferon preparation. This protein is called human interferon-β2. Fractions of mRNA for both proteins are used in step c). This enables the production of IFN ß1 or IFN ß2 in practically pure form. The

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Hybridization (stages g, h, i) allows precise identification of individual E. coli colonies which contain one of the two interferon cDNAs for IFN β1 or β2.

It is obvious that those colonies that only hybridize with “induced” cDNA samples and not with “non-induced” cDNA samples represent only those colonies that contain a modified vector with the cDNA that the interferon -mRNA corresponds because a) the DNA that cannot be hybridized with the “uninduced” cDNA sample does not correspond to any of the mRNAs that are normally produced by an uninduced cell and b) the only difference between the two Samples is that the "induced" cDNA sample contains a cDNA from one of the interferon mRNA's, but the other sample does not.

These cDNA samples can be produced by customary methods, in particular by transcription with a reverse transcriptase. Preferably a high copy number bacterial vector such as the pBR322 plasmid is used. In order to obtain the later expression of the vectors modified by the interferon cDNA in a microorganism, one can use a set of vectors as described by Patrick Charney, et al. In the article "Bacteriophage Lambda and Plasmid Vectors Allowing Fusion of Cloned Genes in each of the three translational phases », Nucleic Acids Res., Vol. 5 (12), 1978, pp. 4479 to 4494.

According to a further important step of the method according to the invention, the vector modified above, whatever its (translation) reading frame may be, can be used for the extraction of interferon mRNA from the RNA mixture which is formed by the induced cells. At this stage, these RNAs are brought together with such a vector modified by the interferon cDNA, which has previously been fixed on a support. The union (of m-RNA's and modified vector) takes place under conditions that allow hybridization. The stuck (bound) mRNA is then eluted from the fixed DNA vector. In this way, human interferon nRNA (either IFN-ß1 or IFN-ß2) is obtained in a highly purified form.

The high degree of purification results from the fact that the translation product in a suitable in vitro system in any case essentially consists of a single polypeptide with interferon activity, which is precipitated by the antibodies against human interferon.

The invention thus also enables the recovery of the purified mRNAs, which normally contain about 900 nucleotides for IFN-ß1 and about 1300 nucleotides for IFN-ß2. The corresponding cDNA's are obtained by transcription of the mRNAs.

It is obvious that each of the hybridization steps of the method according to the invention is preceded, if necessary, by a denaturation of the possible double-stranded nucleic acids in order to ensure that no double-stranded nucleic acids (which could induce interferon activity in cells) remain in the purified mRNA's if these are used for in vitro production by translation of practically pure protein with interferon activity.

From the pure mRNAs and cDNAs obtained, it is readily possible for the person skilled in the art to produce human fibroblast interferon β1 and β2 by known methods. These interferons can be formulated into pharmaceutical preparations in the usual way.

The invention is further explained on the basis of the figures and the table.

Flg. 1 shows the fractionation of mRNA on a sucrose gradient and the translation of these mRNA fractions derived from induced cells into Xenopus laevis oocytes for the production of human interferon and their translation in reticulocyte lysates for the production of specifically immunoprecipitable proteins. This method allows the separation of the mRNAs for IFN-ß1 and IFN-ß2.

Fig. 2 illustrates the result of differential hybridization of the DNA's of the same bacterial colonies with the two aforementioned cDNA samples from induced and non-induced cells.

3 illustrates a purification of interferon IFN β2-mRNA by hybridization with immobilized DNA from the bacterial clone A341, detected by translation in a reticulocyte lysate.

FIG. 4 shows that DNA from the bacterial clone A341 is complementary to an mRNA with approximately 1300 nucleotides, which occurs in human cells only after induction of interferon synthesis.

Table I shows that this mRNA, after translation in the Xenopus laevis oocytes, provides biologically active interferon which inhibits the growth of a virus in human cells.

1a shows the separation of two mRNAs coding for human interferon activity (dotted line and stars). The separation was accomplished by centrifugation in a tube containing a sucrose gradient that separates mRNAs according to their size. The mRNA peak around fraction 6 on the abscissa is larger (14S) than the mRNA peak around fraction numbers 10-12 (11S). Interferon activity was measured by injecting each of the RNA fractions into frog (Xenopus) oocytes and measuring the activity of the human interferon secreted by these oocytes.

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To illustrate which proteins are encoded by these two human interferon mRNA peaks, each fraction was added to rabbit reticulocyte lysates with radioactive methionine so that the proteins synthesized by each RNA fraction are radioactive. An antiserum produced in rabbits using partially purified interferon from human fibroblasts (interferon-beta) was used to immunoprecipitate the radioactive proteins made in response to each mRNA fraction. The proteins which were immunoprecipitated in this way were separated by gel electrophoresis on a polyacrylamide gel with SDS, which separates by size (from top to bottom in FIG. 1 b). Autoradiography shows the proteins recognized by the antiserum (antibody).

For comparison with the proteins which were produced with RNA from fibroblasts which were not induced to produce interferon (s), it is shown in FIG. 1b that the mRNA fractions synthesize two proteins of different sizes from (i) fibroblasts induced for interferon production, marked with 23 (molecular weight 23,000 daltons) and 20 (20,000 daltons), which are absent when the fibroblasts do not produce interferon beta. The size of these proteins was determined by comparison with molecular weight marker proteins shown on the right in Fig. 1b. The amount of each of these two proteins in Fig. 1b produced from each of the mRNA fractions in Fig. 1a was quantified by scanning the X-ray film of the autoradiography. The amount of each protein is expressed as the protein area represented by solid lines in Figure 1a. It is shown that the amount of protein of 23,000 daltons (closed circles) is proportional to the amount of larger mRNA that encodes human interferon activity and that is found around fraction 6 in Fig. 1a. In contrast, the amount of 20,000 dalton protein (open circles) is proportional to the amount of smaller mRNA that encodes human interferon activity and that is found around fractions 10-12.

This experiment 1) shows that there are two mRNAs in human fibroblasts that have been induced to produce interferon that encode human interferon activity, 2) that the larger of these mRNAs for a protein of 23 KDa and the smaller of these mRNAs for another 20 KDa protein encodes 3) that both proteins, the 23 KDa and 20 KDa proteins, are immunologically related to proteins found in partially purified human interferon beta. The smaller mRNA and the 20 KDa protein were called interferon-beta-1, while the larger mRNA and the 23 KDa protein were called interferon-beta-2.

Figure 2 illustrates how cDNA clones corresponding to mRNAs found only in human fibroblasts induced to interferon activity are identified. The figure shows an autoradiography of two nitrocellulose filter disks with a diameter of 5 cm, onto which DNA from 15 different colonies of the bacterium E. coli were adsorbed. These E. coli colonies each contain a different cDNA clone derived from human fibroblasts induced to produce interferon. (In this experiment, the mRNA originated from the fractions in FIG. 1a which have the size for coding the 23 KDa protein). A total of 2000 colonies, each with a different cDNA due to the cloning procedure, were examined, but the figure shows results from only 15 selected colonies. In order to identify which E. coli coionies contain a cDNA clone that corresponds to an mRNA that only occurs in induced human fibroblasts, a method was used, which is referred to as differential hybridization. Each of the two filter disks, which are identical, was hybridized with a different radioactive cDNA: the left filter disk (Ni) was hybridized with cDNA from RNA (from fraction 6 of a gradient as in FIG. 1 a), which was obtained from non-induced human fibroblasts ( Ni cDNA). In contrast, the right filter disk was hybridized with cDNA from RNA derived from induced fibroblasts (i-cDNA).

Since each numbered spot on one of the two filter disks contains only one and unique cDNA clone, this can either contain a cDNA from an mRNA that is only present in induced fibroblasts or a cDNA from an mRNA that also contains in non-induced fibroblasts is. In the second case, the bacterial colony cDNA will hybridize to both i- and N.i.-radioactively labeled cDNAs used as probes. In the first case, DNA from the bacterial colony will only hybridize with the radioactive cDNA from i.

It is shown that the two DNAs of the bacterial colonies hybridize only with the i-cDNA: these are identified by arrows (numbers 5 and 13). The other bacterial colonies near numbers 1, 3, 6, 9, 11, 12 and 15 hybridize to both i and N.i. cDNA probes. We conclude that E. coli colonies 5 and 13 each contain a cDNA clone that corresponds to an mRNA that is only present in induced fibroblasts. We named the clone in colony number 13 as clone A341.

Figure 3 shows that the cDNA clone called A341 (in Figure 2) contains cDNA that is complementary to the mRNA encoding the 23 KDa protein produced by the antiserum (antibody) against human interferon (this protein is IFN-ß-2, see Fig. 1) is recognized.

The figure shows an autoradiography of a polyacrylamide gel electrophoresis in SDS which separates the radioactive proteins which have been synthesized in rabbit reticulocyte lysates from various mRNAs by decreasing size (from top to bottom). The band marked “dead” shows the proteins that were produced from the entire RNA from induced human fibroblasts. Many pro

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Lines with many different sizes are produced. The band marked "341" shows the protein produced from the mRNA purified by hybridization with the cDNA clone A341 (isolated from the DNA of the bacterial clone A341 shown in Fig. 2). The hybridization selection procedure used to obtain this mRNA ensures that only mRNA that can hybridize and is complementary to one of the two DNA strands of the A341 clone cDNA (and therefore identical in sequence to the other) Strand of A341 cDNA) is selected and purified from the large mRNA mixture that is contained in the “total” RNA. It is shown that this mRNA corresponding to the A341 cDNA only produces a protein whose size is 23 KDa compared to the marker proteins (left band m). This protein is exactly the same size as the 23 KDa protein (labeled as IF), which is immunoprecipitated by the antibodies against human interferon. As a control, we used RNA that was selected by hybridization with an unrelated DNA, namely the pBR plasmid from E. coli. The band labeled Pbr shows that the 23 KDa protein is not produced by this control mRNA.

It can be concluded that the A341 cDNA contains a sequence that is identical to that of the mRNA that is the IFN-β-2 protein. The experiment also shows that the hybridization selection procedure used (hybridization of the total RNA with A341 cDNA, which was immobilized on a nitrocellulose filter square, followed by washing and elution of the hybridized RNA) yields pure IFN-β-2 mRNA. This is shown by the fact that this mRNA selected by hybridization only codes for a protein from the large mixture that was synthesized with the total RNA. (Note that the "total", "341" and Pbr bands contain all of the RNA in response to rabbit reticulocyte lysates and immunoprecipitation was not used).

Figure 4a shows that cDNA A341 actually corresponds to an mRNA found only in human fibroblasts that have been induced to form interferon. The figure shows an autoradiography of 3 nitrocellulose filter strips (1, 2, 3), onto each of which an agarose gel was blotted after electrophoresis of the RNA extracted from (i) and non-induced (ni) human fibroblasts induced for interferon formation. Gel electrophoresis separates RNA by decreasing size (from top to bottom). The positions of the 28S, 18S, and 5S RNA markers are shown. The nitrocellulose strip number 1 was hybridized with radioactive cDNA of the cDNA clone A341. It is shown that this cDNA hybridizes with an RNA whose size was determined as 14S by comparison with the RNA markers, which is found only in i RNA and not in ni RNA. The strip marker 2 was hybridized with pbR-DNA as a control: no RNA could be detected. Strip number 3 was another control hybridized to cDNA made from total fibroblast RNA. It can be seen that band n hybridizes with many RNAs, even more RNAs than in band i, proving that RNA is present in band n, although the A341 cDNA does not hybridize with this RNA. This shows that hybridization of the A341 cDNA with the induced 14S RNA is very specific.

FIG. 4b shows two strips (β1 and β2) onto which the entire RNA from the induced fibroblasts was blotted. The strip designated β2 was hybridized with the A341 cDNA. The size of the RNA detected by the A341 cDNA was determined to be 1.3 kilobases (kb) by comparison with RNAs of known sizes (not shown). The strip designated β1 was hybridized to a cDNA clone that identified as the 20 KDa protein corresponding to the IFN-β1 protein (as in Fig. 1b) using the methods illustrated in Figs. 2 and 3 for IFN-β2 has been. The IFN-β1 mRNA is shown to be 0.9 kb in size.

Table 1 shows that mRNA that was purified by hybridization selection using the A341 cDNA (as in FIG. 3) encodes interferon activity. The purified RNA was injected into Xenopus oocytes and the proteins secreted in the medium in which the oocytes (oocyte supernatant) were incubated were examined for IFN activity by means of two experimental methods. In experiment 1, the proteins produced were added to human fibroblasts which were infected with vesicular stomatitis virus (VSV). An antibody to the VSV G protein was used in a radioimmunoassay to measure the amount of virus produced by the cells. It is shown that with the oocyte supernatant of the non-injected oocytes corresponded to the arbitrary amount of viruses produced by the human fibroblasts in the 7445 cpm radioimmunoassay. With an interferon standard of 100 units per ml, this amount decreases as expected up to 700 cpm. With the protein produced by the purified RNA, which is identical to a cDNA clone of IFN-ß2 (in this case E474 corresponds to the 5 'position of the IFN-ß2 mRNA), the amount of virus was reduced to 1920 cpm, almost so deep as with the IFN standard. As a control, we used RNA that was purified with an unrelated plasmid cDNA, and this control resulted in only a non-significant virus reduction compared to the uninjected oocyte control.

In experiment 2, the proteins from the supernatant oocyte were also added to the human fibroblasts, but we measured the induction of 2'-5'-oligoadenylate synthetase, which is an enzyme activity induced by IFN in cells. In this case the radioactive [32P] -A2'p5'A product will be higher the more IFN there is in the assay.

It is shown that human fibroblasts exposed to 100 units per ml IFN standard give 10,800 cpm compared to only 1,700 in fibroblasts, which extracts (in incubation5

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medium lysed oocytes) were not exposed to injected oocytes. The RNA hybridized with a pool of IFN-β2 cDNA plasmids gives 4700 cpm, which allows to calculate that the extracts of the oocytes injected with this IFN-β2-RNA give an equivalent of 30 U / ml IFN activity. The RNA of the plasmid DNA of the control gave no activity that was higher than that of the uninjected oocyte extract. Note that the oocyte extract was diluted 15 times so that the IFN produced by IFN-ß2-specific mRNA is higher and corresponds to 450 U / ml. This table shows that the pure IFN-ß2 mRNA obtained by hybridization with clones of the IFN-ß2 cDNAs produces a protein with the biological activity of interferon. This proves that the IFN-β2 protein (as defined in Figure 3 by the same hybridization selection) has this biological activity.

table

Hybridization and translation of β2-interferon mRNA

Trial 1

Trial 2

V.S.V. virus yield

Radioimmunoassay,

cpm

Oligo isoadenylate synthetase induction [32P] -A2'p5'A, cpm calculated IF titer

Oocyte supernatant (diluted 1:10)

Oocyte extract (diluted 1:15)

not injected

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1700

with RNA which hybridized to IF-ß2 plasmid (= A341)

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4700

30 U / ml with RNA which hybridizes to an unmodified plasmid (= pBR322)

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1500

0

Interferon standard 100 U / ml

700

10800

Note: In experiment 1, DNA from clone E 474 was used, while for experiment 2 a pool (mixture) of IF-ß2 DNA plasmids was used.

Example a) Purification of the two interferon mRNAs from human diploid fibroblasts

RNA is extracted from monolayer cultures of the human fibroblast line FS11 (isolated at the Weizmann Institute of Science). These diploid cells from the foreskin of a normal 8-day-old boy are selected from 15 separate isolates for their ability to produce high interferon titers. Alternatively, cultures of a clone of human SV 80 cells are used. The cultures in Eagle's minimal medium with 10% fetal calf serum are incubated in 2.2 liter glass roller bottles or 22 x 22 cm plastic trays in an atmosphere of 5% carbon dioxide and 95% air at 37 ° C. 3 days after the confluence of the cells, cultures are induced to produce interferon by mixing with 100 (ig / ml poly (rl: rC) and 50 ng / ml cycloheximide for 3½ hours (this blocks the synthesis of proteins by the host) Actinomycin D (which blocks the synthesis of cellular RNA) is added in an amount of 1 ng / ml, 1 hour later the cells are lysed with buffered Nonidet-P40 detergent (wetting agent) and the cytoplasmic RNA is washed with a phenol -Cresol mixture extracted according to Kirby (1965) The mRNAs are isolated from the total RNA by binding them to oligo-dT-cellulose, since they themselves contain poly A. The mRNA fraction is then fractionated by sucrose gradient centrifugation The fractions containing interferon mRNA are identified by microinjection into Xenopus laevis oocytes according to NKB Raj and PM Pitha, (Proc. Nati. Acad. Sci. USA, Vol. 74 (1977), pp. 1483 to 1487) 24 up to 40 hours later the antiviral activity of the interferon released into the oocyte incubation medium is determined. Antiviral activity is determined by treating FS11 cells with dilutions of the oocyte medium. The cells are infected with the vesicular stomatitis (VSV) virus. Inhibition of the cytopathic effect of the virus is observed. The interferon titers are calculated by comparison with a known solution according to the last effective dilution. The fractions containing interferon mRNA are furthermore obtained by translation in a reticulocyte lysate and subsequent immunoprecipitation of the product by the method of Weissenbach et al., (Eur. J. Biochemistry, Vol. 98, (1979), pp. 1 to 8) identified.

1a shows the two peaks of the interferon mRNA activity, determined by injection into oocytes. Figure 1b shows the lines obtained by immunoprecipitation of the translation products with the anti-interferon serum. The two arrows show the two polypeptides of molecular weight

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23,000 (23K) and 20,000 (20K). The sucrose gradient fractions encoding the 23 K and 20 K im-precipitated polypeptides are shown in Figure 1a. It can be seen that they correspond to the two peaks of the interferon mRNA activity.

Interferon activity is also detected in the translation products of the reticulocyte lysates by determining the induction of (2'-5 ') oligo-isoadenylate synthetase in human cells. Based on the two methods, it can be determined that the larger interferon mRNA peak codes for the 23K polypeptide, while the smaller interferon mRNA peak codes for the 20K polypeptide. Both interferon mRNAs are isolated in this way and used for cloning in E. coli.

b) Cloning of interferon B2 cDNA in E. coli

The purified mRNA from induced cells contains about 1 to 3% of the mRNA for the polypeptide with a molecular weight of 23,000. It serves as a template for the synthesis of cDNA by reverse transcriptase from bird myeloblastosis virus (AMV). Oligo-dT is used as the starter. After the RNA has been eliminated by alkali treatment, the second strand of DNA can be synthesized using reverse transcriptase or DNA polymerase I. Single stranded DNA is hydrolyzed away with nuclease S1 and the 3 'ends of the DNA are extended with nucleotide terminal transferase using dGTP as the substrate. The plasmid DNA, which was extended in a corresponding manner with terminal transferase and dCTP, is hybridized with this dG-extended human cDNA and introduced into E. coli DP 50. Transformed bacterial colonies are identified (selected) by application to agar plates containing Luria broth, diaminopimelic acid, thymidine and tetracycline. Colonies continue to be tested on similar agar plates containing ampicillin as the only antibiotic. The ampicillin-sensitive, tetracycline-resistant bacterial colonies are grown on a nitrocellulose filter on an agar plate of the type described above with 10 ng / ml tetracycline. Over 2000 of the transformed colonies obtained are transferred to other nitrocellulose filters, which are also placed on agar plates of the type described above. Each of the duplicate colonies is assigned to its respective origin or parent colony, usually by common numbering. As soon as the colonies have reached a diameter of 3 to 5 mm, the filters (starting colonies and duplicates) are transferred to a stack of filter papers, first with 0.5 N sodium hydroxide solution, then with 0.15 molar sodium chloride solution and 0.1 N sodium hydroxide solution have been impregnated in order to release the corresponding DNAs in situ. The filter papers are neutralized and dried. To detect the bacterial colonies that contain the interferon DNA sequences, the filter papers are hybridized with two different [32P] cDNA samples. A cDNA sample is prepared by reverse transcriptase the mRNA from the sucrose gradient fraction of induced cells (arrow 23K of FIG. 1). The second sample is prepared in the same way from the corresponding fraction of the non-induced cell preparation. Both cDNA samples are synthesized with the four highly radioactive [32p] deoxynucleoside triphosphates as substrates and fragmented calf thymus DNA as starters. In this way, a statistical representation of the mRNA sequences in the cDNA samples is achieved. The hybridization is carried out for 18 hours at 62 to 64 ° C. in a 0.9 molar NaCl-0.09 molar sodium citrate buffer with a pH of 7.0. The starting colonies are hybridized with the cDNA samples of the induced cells and the duplicate colonies with the cDNA samples of the non-induced cells (or vice versa). After thorough washing, the filters are placed on X-ray film and the bacterial colonies are found which hybridize only with the “induced” cDNA, but not with the “non-induced” cDNA. In this way, 20 different bacterial colonies were isolated from a total of more than 2000 transformed colonies examined. These 20 bacterial colonies contain multiple copies of a plasmid in which DNA sequences are inserted which are derived from human mRNA, which is only expressed after the cells have been induced to produce interferon by poly (rhrC).

An example for explaining this method is given in FIG. 2 on the basis of 15 pairs of alkali-treated colonies (starting colonies and duplicate colonies) on their nitrocellulose filters, whose DNAs have been hybridized with [32P] cDNAs which are derived from the mRNA fractions 23K from 1, which were either derived from poly (rl) :( rC) for inducing interferon production (i) cells or from non-induced (ni) cells. The arrows show (two) colonies, especially colonies 5 and 13, which contain the induced sequences.

Colony No. 13 is called E. coli DP50 / A341.

c) Isolation of interferon mRNA from human fibroblasts

Interferon mRNA (and the detection of the presence of interferon cDNA sequences in the plasmid DNA of clone A341) is obtained as follows:

A 500 ml culture of this bacterial clone is used to produce 50% plasmid DNA. After prior denaturation, this DNA is covalently cultured on diazobenzyloxymethyl cellulose powder according to the method of Aldwine et al., Proc. Nati. Acad. Sci. USA, Vol. 74 (1977) p. 5350. Similarly, plasmid pBR322-DNA (which contains no human DNA sequences) is bound to cellulose, mRNA from human fibroblasts containing poly-A which induces interferon formation

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have been hybridized with the two DNA Celiulose preparations in 50 percent formamide at 52 ° C. Then eluted with pure formamide at 70 ° C. The RNA recovered after elution is translated in the cell-free reticulocyte system (FIG. 3). The resulting translation product of the mRNA selected on the A341 DNA cellulose is essentially the polypeptide with a molecular weight of 23,000. However, no human interferon mRNA is isolated from the pBR 322 DNA cellulose. In comparison to the translation products of the human mRNA before hybridization to A341 DNA cellulose, it was found that the cloned A341 DNA is only to a small extent complementary to the mRNA of the mixture. The product of the mRNA bound to A341 DNA cellulose is immunoprecipitated by the human fibroblast interferon antiserum.

The interferon mRNA can also be isolated by a similar method to that described above, but in which the piasmid A341 DNA is bound to nitrocellulose filter, then m-RNA is hybridized to it and finally eluted by boiling in water for 1 minute .

The activity of this purified mRNA for coding for biologically active human interferon could be determined by injection into Xenopus laevis oocytes and subsequent determination of the inhibition of virus multiplication in human cells which were exposed to the oocyte incubation medium; see. Table I. The interferon activity of the purified β2 mRNA can also be demonstrated by induction of (2'-5 ') oligoisoadenylate synthetase in human cells by the oocyte translation products; see. Table I.

Restriction mapping of the A341 plasmid DNA (analysis of the A321 plasmid DNA by restriction enzymes) shows that it contains an insert from human DNA of about 900 nucleotides in the Pst section. The A341 DNA also hybridizes to three fragments of the human genome cut by Eco R1 nuclease. These fragments are separated from each other by agarose gel electrophoresis. Hybridization to agarose gel electropherograms of mRNA from human fibroblasts further shows that A341 DNA is complementary to RNA sequences that are only expressed in cells that have been exposed to the interferon inducer poly (rl: rC); see. Fig. 4a. Even treating the cells with poly (rl: rC) for one hour leads to the accumulation of an approximately 1300 nucleotide long RNA which hybridizes with A341 DNA. This represents the IFN ß2 mRNA.

The above values show that the bacterial clone E. coli DP50 / A341 contains an insert of about 900 nucleotides from human cDNA sequences in the Pst site of its pBR322 plasmid, which are complementary to a human interferon mRNA. Several clones prepared in a similar manner were obtained. FIG. 4b shows that clones for IFN-β2 hybridize with the larger, approximately 1300 nucleotide unit long mRNA, while clones for IFN-β1 hybridize with the smaller, approximately 900 nucleotide unit long mRNA.

The method can be used to produce clones of interferon DNA of different types (a, β, 7) from human cells.

Claims (1)

Claims 1. Human fibroblast interferon β2, characterized in that it is a single polypeptide with a molecular weight of about 23,000. 2. A method for producing human fibroblast interferon ß1 and ß2, characterized in that (a), a culture of fibroblast cells is exposed to an exogenous factor, which induces the synthesis of interferon mRNA in the cells mentioned, (b) the mRNAs formed in the induced cell cultures and also mRNAs are extracted from non-induced control cultures of the same host cells, (c) double-stranded cDNAs derived from the mRNA extracted from the induced culture are synthesized, said cDNAs are inserted into vectors, microorganisms are transformed with the modified vectors obtained, and the said microorganisms are cultivated under suitable conditions in order to induce a selective development of microorganism colonies containing the vector mentioned, so-called starting colonies, (d) duplicate colonies of the starting colonies are formed, (e) the DNAs of both the starting and duplicate colonies are released in situ, (f) cDNA samples of the mRNAs of both the induced culture and the non-induced control culture are synthesized, the corresponding mRNAs being used as templates, thereby producing induced cDNA and non-induced cDNA, (g) the DNAs of the starting colonies on the one hand and of the duplicate colonies on the other hand are each hybridized with the above-mentioned induced and non-induced cDNA samples, (h) recover the DNAs that hybridize with the induced cDNA samples but not with the non-induced cDNA samples, and (i) the mRNAs previously extracted in process step (b) are contacted with individual DNAs from process step (h) which, after insertion in a vector, were immobilized on a support under conditions which are suitable for permitting hybridization and subsequently the fixed ones mRNAs are eluted from the immobilized DNA vector in essentially pure form, the mRNAs thus translated are translated in order to determine which are in human fibroblast interferon 8th 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 685 299 A5 ß1 or ß2 are translatable and thus the corresponding DNAs from step (h) which code for human fibroblast interferon ß1 or ß2 are identified, (j) the interferon DNAs identified in this way from process step (h) are introduced into microorganisms so that cultivable cells are formed, (k) the cells are cultivated and the human fibroblast interferon β1 or β2 is extracted from them and purified. 3. The method according to claim 2, characterized in that the exogenous factor from process step (a) is double-stranded RNA. 4. The method according to claim 2 or 3, characterized in that the mRNAs obtained in step (b) are fractionated by sucrose gradient centrifugation. 5. Human fibroblast interferon β1 obtained by the method according to one of the claims 2 to 4, characterized in that it is a polypeptide with a molecular weight of approximately 20,000. 6. Human fibroblast interferon β2 obtained by the method according to one of claims 2 to 4, characterized in that it is a single polypeptide with a molecular weight of about 23,000. 7. Use of human fibroblast interferon ß1 according to claim 5 for the manufacture of a pharmaceutical preparation with antiviral and antitumor activity. 8. Use of human fibroblast interferon ß2 according to claim 1 for the manufacture of a pharmaceutical preparation having antiviral and antitumor activity. 9